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conformational_heterogeneity_of_bax_helix_9_dimer_for_apoptotic_pore_formation

## Abstract: Helix α9 of Bax protein can dimerize in the mitochondrial outer membrane (MOM) and lead to apoptotic pores. However, it remains unclear how different conformations of the dimer contribute to the pore formation on the molecular level. Thus we have investigated various conformational states of the α9 dimer in a MOM model -using computer simulations supplemented with site-specific mutagenesis and crosslinking of the α9 helices. Our data not only confirmed the critical membrane environment for the α9 stability and dimerization, but also revealed the distinct lipid-binding preference of the dimer in different conformational states. In our proposed pathway, a crucial iso-parallel dimer that mediates the conformational transition was discovered computationally and validated experimentally. The corroborating evidence from simulations and experiments suggests that, helix α9 assists Bax activation via the dimer heterogeneity and interactions with specific MOM lipids, which eventually facilitate proteolipidic pore formation in apoptosis regulation.As a proapoptotic protein from the Bcl-2 family, Bax is a crucial executioner in the mitochondrial pathway of apoptosis 1-3 . In stressed cells, Bax from the cytosol is activated by the BH3-only proteins (such as Bim and Bid) and inserts into the mitochondrial outer membrane (MOM) [4][5][6][7][8][9] . Through multi-step conformational changes, Bax forms dimers and further oligomerizes into membrane permeabilizing pores [8][9][10][11] which subsequently release apoptotic mitochondrial proteins like cytochrome c and SMAC to the cytosol and lead to cell death. Nonetheless, the conformational transition of Bax after activation, involving the dimerization in the cytosolic region and/or the MOM, has not been fully understood. Upon activation, a symmetric dimer conformation with reciprocal binding of the BH3 region of one Bax to the groove of another Bax was first observed by X-ray crystallography using a truncated Bax protein 8 , and recently confirmed by crosslinking of intact Bax protein in the mitochondria 12 . While Bax dimerization has been confirmed in certain cytosolic domains, the MOM-anchoring domain -in particular, the helix α 9 (residue 169-189) -is found to form dimer interfaces that expand the oligomeric pore in the MOM 10,12,13 . In the soluble Bax, helix α 9 is buried in a hydrophobic groove formed by helices α 2, α 3, α 4, and α 5 11 . However, studies using Förster resonance energy transfer (FRET) and electron spin resonance (ESR) observed significant rearrangements of helix α 9 indicating its exposure to the cytosol before membrane binding 10,14 . In the activated Bax, helix α 9 functions as a membrane anchor to target Bax to the MOM, because its deletion could prevent Bax translocation to the mitochondria 15 . Consistently, mutations of Ser184 in helix α 9 could either abolish or enhance the Bax translocation 15 . While prior studies pointed out that helix α 9 would be a key to understand Bax activation and apoptosis regulation, its particular roles and relevant molecular mechanisms have not been fully understood.Recently we have discovered two distinct conformational states of the α 9 dimer in the MOM. The first state, termed the intersected α 9 dimer (Fig. 1A1), was based on the disulfide linkages of four cysteine mutants: I175C, G179C, A183C and I187C 12 . In this state, two α 9 helices intersect though the G 179 xxxA 183 motif with a right-handed crossing angle of ~40°1 6 . The other state is referred to as the parallel α 9 dimer (Fig. 1B1) with a right-handed crossing angle below 15°, according to the disulfide linkages of other four cysteine mutants: Q171C, A178C, T182C and L185C 12 . Although α 9 dimerization was believed to facilitate the lipidic pore 12 , the actual roles of these two dimeric states and their connections to Bax activation remain elusive or controversial. A number of key problems on the molecular level need to be studied. First, despite other mitochondrial targeting signals in Bax, helix α 9 by itself is sufficient to target the mitochondria 17 . However, what drives helix α 9 into the MOM, and how is helix α 9 stabilized in the membrane is currently unknown. Moreover, mitochondrial specific lipids are known to actively influence the structures and functions of the Bcl-2 family proteins including Bax 18 . It has been suggested that insertion of helix α 9 into the MOM and Bax pore formation are regulated by the membrane curvature and lipid compositions 19 . Then how does helix α 9 interact with various lipids in the MOM, especially the mitochondrion-specific ones? What is the significance of these interactions for helix α 9 dimerization and mitochondrial poration? In an attempt to gain molecular insight, we herein present the first systematic study of molecular dynamics (MD) simulations for the helix α 9 dimer in a model lipid bilayer, in tandem with experimental investigations of the helix α 9 dimer interface between intact Bax proteins activated in the native MOM. While MD simulations have been applied to study the stability of helix α 9 bound to the Bax groove in the aqueous solution , a large-scale simulation study of helix α 9 with atomic details in the membrane has never been reported. Also, it is required to further investigate the roles of Bax helix α 9 in apoptosis with a detailed membrane model to mimic the MOM lipid composition. Therefore, in this work we focus on the helix α 9 dimers in a MOM-mimicking lipid bilayer, using conventional all-atom MD and free-energy simulations for both qualitative and quantitative analyses. Simulations starting from the "static" intersected and parallel dimer models 12 were performed to understand the conformational dynamics, while in return, experiments with full-length Bax variants in native mitochondria were designed to validate the predictions from the simulations. Such synergy between simulations and experiments enable us to access structural and dynamic details of helix α 9 at various spatial and temporal scales. Building on good agreements between simulations and experiments, our work shines light on the molecular mechanism of Bax dimerization via helix α 9 interactions in the MOM, which has never been fully described before. In addition, our study establishes a unique approach for further investigations of the apoptotic MOM permeabilization induced by Bax and regulated by other Bcl-2 family proteins. ## Results The membrane environment is critical to stabilize the helix α9 dimers. We have compared the stabilities of the intersected and parallel α 9 dimers in a MOM-mimicking lipid bilayer versus an aqueous solution. Within the 200-ns MD simulations, both the intersected and parallel α 9 dimers appear stable in the membrane (Fig. 1), as shown by the small fluctuation around 1 of the backbone root-mean-square deviation (RMSD). Consistently, all the cross-linkable data 12 were reproduced by C β -C β ' distances along time evolution The second replicas reproduced the qualitative results (Supplementary Fig. 2). (Supplementary Fig. 1). In contrast, in solution the α 9 dimers became disordered during the simulations (A2 and B2 of Fig. 1). Compared to the starting conformations, the backbone RMSDs of both dimers in the solution dramatically increase in the first 50 ns and then fluctuate around 5 toward the end of the simulations (Fig. 1C). The helicity of the intersected dimer decreases from 87 to 54% due to melting in the terminal regions, giving rise (see Supplementary Fig. 3) to a separation greater than 27 for I187-I187′ pair in the C-termini. Similarly, the helicity of the parallel dimer drops from 87 to 70%, increasing Q171-Q171′ and T182-T182′ separations to 13 and 11 , respectively. Additionally, rotation of the individual α 9 helices in the parallel dimer was also observed in the solution, which increased the crossing angle between two monomers by over 10°. Likewise for a single α 9 monomer in the solution, a dramatic loss of helicity was observed in our simulations. Our MD simulations demonstrate that the membrane environment is essential to maintain the helicity of α 9, because α 9 remains helical in the membrane but becomes disordered in the solution. Combined with the previous experimental finding of helix α 9 being partially buried in a groove of the solvated Bax monomer 11 , we suggest a possible conformational transition of helix α 9 after Bax activation upon binding to a BH3 peptide or protein that displaces α 9 from the groove 5,8 . It is likely that an order-to-disorder transition occurs after helix α 9 becomes unbound to the groove of Bax, and a disorder-to-order transition follows when helix α 9 starts to insert into the MOM. α 9 adopts disordered conformations due to the exposure of its hydrophobic core to the cytosol or even membrane's charged periphery, while the membrane plays a key role in recovering the peptide helicity by providing a hydrophobic environment around α 9's hydrophobic core and charged/polar periphery around α 9's charged/ polar termini. In fact, many peptides such as melittin 23,24 are known to undergo similar disorder-to-order transitions when inserted to membranes. Therefore, the order (in groove)-to-disorder (in cytosol)-to-order (in membrane) transition may be a general pathway for α 9 targeting and insertion to the MOM during Bax activation. The intersected and parallel α9 dimers display distinct preferences toward anionic lipids. To understand how the membrane stabilizes the α 9 dimers, we have investigated the lipid distributions and dimer-lipid interactions. First, we analyzed (Fig. 2) the average anionic lipid distributions and diffusion coefficients in three 10-ns windows during the last 30 ns, and obtained converged results (Supplementary Fig. 4 and Supplementary Table 2) with clear lipid preference near each α 9 dimer. Despite the fact that neutral lipids account for ~75% of total lipids in the MOM, the anionic ones such as PS, PI, and CL have been known to facilitate Bax activation, insertion, and pore formation 25,26 . Starting with a membrane model that had a random distribution of neutral and anionic lipids, the anionic lipids were enriched around each α 9 dimer in the end. An area with a radial cutoff of 20 from the dimer centroid contains 24-26% of anionic lipids and 18-21% of neutral lipids on average (given its bulky tails, a CL counts as twice the area of other lipids). Within the radial cutoff of 20 from an α 9 dimer centroid, we calculated the stoichiometry of CL, PS, and PI, which is 2.0 ± 0.1, 2.7 ± 0.5, and 2.7 ± 0.5 per intersected dimer, and 3.7 ± 0.5, 1.0 ± 0.1, and 2.0 ± 0.1 per parallel dimer, respectively. Such evident discrepancy suggests a preference of the intersected dimer to PS and PI (that carry one negative charge under the neutral pH), in contrast to a preference of the parallel dimer to CL (that carries two negative charges). Second, we examined the protein-lipid interacting sites identified from the converged lipid distributions and found a structural basis for the lipid preference. Contacts between the α 9 dimer and the headgroups of PS, PI, and CL are found to involve the side chain, backbone, or the terminal groups (-COO − and -NH 3+ ) of amino acids (Fig. 3). Around the intersected dimer, PS and PI are able to form multiple polar contacts with each α 9 monomer: for example, PI contacts with W170 and Q171, and PS interacts with T169′ and W170′ (Fig. 3A1). However, CL only forms occasional, transient contacts (duration below 1 ns, Supplementary Fig. 5) with residues like K189′ (Fig. 3A2), which is insufficient to keep CL bound to α 9. This explains why more PS and PI lipids than CL dwell near the intersected dimer in our simulations. Around the parallel α 9 dimer, CL owing to its wide headgroup (~10 ) is able to span across a large region and form multiple long-lasting contacts with both monomers. However, since more terminal residues participate in the interfacial interaction between two monomers (Fig. 3B1), in the parallel dimer there are fewer residues available for binding PI and PS (Fig. 3B2). As a result, the presence of PI and PS in the vicinity of the parallel dimer is reduced. Therefore, it is the availability of the residues exclusive from the dimer interface and the distinct lipid head-group together that determine the intersected dimer's preference to PS and PI, and the parallel dimer's preference to CL. Our observation, which the α 9 dimer in different conformational states preferentially binds anionic lipids, is in line with a previous study showing that the α 9-membrane interaction is influenced by the presence of anionic lipids 27 . Further, while previous studies mainly focus on the role of CL and other anionic lipids on the activation of the BH3-only protein Bid or of Bax by a truncated Bid (tBid) 26,28 , our study describes a detailed picture of the α 9 dimerization that occurs downstream of Bax activation to link the BH3-in-groove Bax dimers into higher-order oligomers. Presumably anionic lipids play manifold roles in such processes. On one hand, they recruit a caspase-cleaved Bid (cBid) to the membrane and then activate tBid that in turn recruits and activates Bax 26 . On the other hand, our findings suggest that α 9 forms different dimeric states that interact with selective anionic lipids. Accordingly, Bax traps more anionic lipids into a microdomain via α 9 dimers (Supplementary Fig. 5), which may destabilize the membrane by concentrating the negative charges, or by increasing the membrane curvature (as PS generates positive curvature and CL leads to very negative curvature of the membrane 26 ). This membrane destabilization likely leads to collapse of the bilayer and formation of proteolipidic pores. Moreover, it is found that the binding affinity of cBid to the membrane without CL is similar to that to the membrane with CL as long as the total negative charge is maintained (e.g. by replacing CL with PS) 26 . However, the tBid conformational changes required for activation of Bax are impaired in the membranes that lack CL. Since the active tBid is enriched in the CL-rich domain where Bax can be recruited, the active Bax may first form the parallel dimer in the CL-rich domain and then convert into the intersected one that retains more PI and PS. In summary, the Rare PS lipids get close enough to the parallel dimer to form polar contacts (Supplementary Figs 5 and 6). α 9 dimerization may enhance the local Bax concentration to the membrane domains enriched with negatively charged CL, PS and PI, which will eventually stress the membrane and promote proteolipidic pore formation. The helix α9 dimer switches between multiple conformational states in the MOM. According to our conventional MD simulations, the α 9 dimers in the intersected and parallel states are stable in the MOM, but interact differently with anionic lipids, and their interconversion is likely related to the Bax activation. To further investigate the conformational transition pathway of the α 9 dimer, we employed metadynamics simulations to calculate the free-energy landscape of the α 9 dimer in the membrane, regarding two collective variables: the angles of rotation (α , β ) of backbone around the principal axis of the two helical monomers. The rotation angles in the starting intersected dimer are set to zero degree. The rotation directions are chosen along the smallest rotation angle to transit. We did not constrain the monomer-monomer separation or the crossing angle, and thus they were allowed to relax when the monomers rotated along the directions shown in Fig. 4. To ensure convergence, we performed two simulations to model the transition: one for the path from the intersected to the parallel dimer while the other for the reverse path. Consistent results have been obtained from these simulations, which confirm the convergence of our free-energy calculations (Supplementary Fig. 7). We then extracted the shortest transit pathway and identified the representative states (Fig. 4 and Supplementary Fig. 8), including four major low-energy states (I, II, IV, and VI) and two high-energy transition states (III and V). States I (α = 0°, β = 0°) and VI (α = ~70°, β = ~90°) correspond to the intersected and parallel dimer states respectively, which fit the crosslinking data from our previous study 12 . Our results suggest that the transition pathway from the intersected conformation toward the parallel one consists of three major steps (Fig. 4B). (i) The α and β rotation angles in the dimer increases to cause a slight increase in the monomer-monomer distance and a decrease in the crossing angle, which enables a transition from state I to II (α = ~30°, β = ~20°). (ii) State II converts into state IV (α = ~60°, β = ~40°), where the helix-helix crossing angle gets below 15° and T172 and T186 in one monomer approaches their counterparts in the other monomer at the dimer interface (Fig. 5A, hereafter the state IV is referred to as the iso-parallel state). There is an energy barrier of 3.1 kcal/mol (state III) when I175 and I175′ are very close. (iii) The monomers in iso-parallel state continue to rotate, resulting in state VI with Q171, A178, T182 and L185 in one monomer closer to their counterparts in the other monomer. Given the transition barrier as high as 6.2 kcal/mol (state V), this step is likely to be rate-limiting in the entire pathway. Our metadynamics simulations not only are consistent with prior crosslinking data 12 and conventional MD simulations on the stability of the α 9 dimer (Fig. 1C), but also provide valuable quantitative, mechanistic evidence that is not readily available via other approaches. First of all, the free-energy map (Fig. 4A) and the potential transition pathway (Fig. 4B) indicate multiple conformational states of the α 9 dimer in the MOM -which is more complex than a simple two-state model. The energy map indicates the conformational diversity of the dimer in the MOM with the intermediate states (including the blue areas not labeled in Fig. 4A) with lower free energies that are more stable than the others with higher free energies. Although the shortest transition pathway between the intersected and parallel states is presented in Fig. 4B, we are also aware of other pathways to connect the multiple dimer states, which can likely promote the conformational heterogeneity. Next, the intersected dimer is found 3.5 kcal/mol lower than the parallel one in free energy. Thus, in the MOM there could be slightly more intersected dimers, since it is thermodynamically more stable. However, the intersected and parallel α 9 dimers are likely to coexist, provided an overall barrier of just 12 kcal/mol between them. In addition, we have found the iso-parallel dimer interface as an intermediate state with a small crossing angle (~15 degree) and a moderate monomer-monomer distance. Because of the small energy difference between the iso-parallel (IV) and parallel (VI) states, it is likely that the iso-parallel state is also stable or at least metastable in the membrane. Supportively, the iso-parallel α 9 dimer still resembles its initial model from the metadynamics simulation after a 282-ns conventional MD simulation, with a final backbone RMSD as low as 2.2 . Therefore, combining our conventional and metadynamics simulations, we are able to support the previous experimental work 12 and further predict a new dimeric state. Recently, three compounds targeting at S184 in helix α 9 have been suggested as Bax agonists for potential treatments against the lung cancer 29 . The discovery of multiple conformational states proposes the helix α 9 dimer as a potential therapeutic target to treat diseases associated with excessive Bax activation or inhibition. To further our understanding towards this goal, we carried out experiments of the helix dimer in intact Bax proteins to validate the iso-parallel state and to show the possibility to perturb selective states. The simulation-predicted iso-parallel α9 dimer was validated in the mitochondria. To determine whether the iso-parallel α 9 dimer interface is actually formed by the active Bax proteins in the MOM, we made two single-cysteine Bax mutants T172C and T186C. The cysteine pairs that replace the T172-T172′ and T186-T186′ pairs are within disulfide-linkable distance according to the iso-parallel dimer model (Fig. 5A), but not in the disulfide-linkable distance of the intersected and parallel dimer models. We synthesized the [ 35 S] methionine-labeled mutant proteins in vitro, activated them by a BH3 peptide from Bax (BH3) or cBid, and targeted them to the mitochondria lacking endogenous Bax and Bak as before 12 . We then oxidized the resultant mitochondria to induce disulfide linkage between each mutant. We used non-reducing SDS-PAGE and phosphor-imaging to detect the radioactive Bax monomer and the potential disulfide-linked dimer. As expected from the iso-parallel α 9 dimer model, both single-cysteine Bax mutants formed disulfide-linked homodimers at the mitochondria (Fig. 5B,C). Of note, these in vitro synthesized single-cysteine Bax mutants could release cytochrome c from the Bax and Bak deficient mitochondria in a tBid-dependent manner (data not shown). Therefore, the iso-parallel α 9 dimer detected by the crosslinking is formed by the active Bax protein. The α9 dimer in different states can be selectively perturbed by mutations. Consistent evidence from both simulations and experiments has shown that the G179I and T182I mutations generate steric clashes in the interfaces to disrupt the intersected and parallel α 9 dimers, respectively 12 (Supplementary Figs 9 and 10). Likewise, as indicated by the simulations, A183I disrupts the iso-parallel α 9 dimer and increases the distance of the T186-T186′ pair beyond a cross-linkable range (Fig. 5A). To verify the simulation-predicted effect of the A183I mutation on the iso-parallel and other α 9 dimers, we made the A183I mutation into the T186C or A178C mutant that was used to detect the iso-parallel or parallel dimer in crosslinking experiments, respectively. In accordance to the MD simulations (Fig. 5A), the disulfide crosslinking of the T186C mutant was inhibited by the A183I mutation (Fig. 5C, comparing lane 6 to 4), providing further support to the iso-parallel dimer model. Unexpected from the parallel dimer model but expected from the predicted iso-parallel to parallel transition path, the A183I mutation also inhibited the parallel α 9 dimerization detected by the disulfide crosslinking of the A178C mutant (Fig. 5D, comparing lane 4 to 2). Also, we found that the contacts of the iso-parallel dimer to anionic lipids are less than they are around the intersected dimer (Supplementary Fig. 11). Taking the simulation and experimental evidence together, the iso-parallel α 9 dimer represents a conformational state formed during the Bax oligomerization, which intermediates the intersected and parallel states, and facilitate their interconversion. ## Discussion Connecting the evidence from simulations and experiments, we propose a possible molecular mechanism for the dynamic conformation and interaction of Bax helix α 9 to induce apoptotic pores in the MOM (Fig. 6). The cytosolic Bax protein is equilibrated between a fully folded state with the helix α 9 buried in the hydrophobic groove (as shown by the NMR structure) 11 and a partially unfolded state with α 9 released from the groove. Interactions The N-terminus and helix α 1 are shown as the green surface, while helices α 2 to α 5 in sky blue and helices α 6 to α 8 in deep blue, and helix α 9 is represented by yellow ribbon. After displaced by tBid from the groove formed by helices α 2 to α 5, helix α 9 loses helicity in the cytosol but then folds back within the MOM. The formation of the intersected and parallel α 9 dimers attracts different anionic lipids to the vicinity as indicated, which may increase membrane tension and curvature, thereby promoting proteolipidic pore formation to release cytochrome c. with a BH3-only protein such as tBid may shift the equilibrium to the partially unfolded state due to displacement of helix α 9 from the hydrophobic groove to an aqueous environment. Such an order-disorder transition drives α 9 to the MOM, where α 9 folds back to the helical conformation in a disorder-order transition with its hydrophobic core stabilized by the lipid tail region and the polar/charged termini stabilized by the lipid headgroup region. During the initial activation, Bax is likely targeted to a CL-rich domain in the MOM (usually near the outer-inner membrane contact site) where the activator tBid is likely located 33,28 . Through specific interactions, helix α 9 traps more anionic lipids that are wandered by. To remain stable in the membrane, helix α 9 likely first forms the parallel dimer that preferentially binds CL. Then the parallel dimer relaxes into the intersected dimer that is more stable and able to bind other anionic lipids like PS and PI. The parallel to intersected conformational transition can go through the intermediate iso-parallel state. The dynamics of the α 9 dimer conformation may result in the dynamics of anionic lipid distribution, which destabilizes the lipid bilayer to induce a proteolipidic pore that is lined by both Bax oligomers and lipid headgroups, and able to releases cytochrome c and other intermembrane space proteins to initiate apoptosis. In general, we have combined simulations and experiments to study the detailed conformations and dynamics of the Bax helix α 9 dimers in the MOM, a difficult task with any single approach alone. Corroborating evidence is obtained in the conformational stability of the α 9 dimer and mutants, the dimer-lipid interacting patterns, and the free-energy landscape to reflect the α 9 dynamics -suggesting a detailed mechanism of helix α 9 in the Bax-induced membrane poration, which may be facilitated by the interactions between multiple dimer conformational states and anionic lipids in the MOM. Our combined computational/experimental strategy will be useful to further explore the molecular mechanisms of the other membrane embedded/interacting helices in Bax activation and action, or even the mechanisms of other Bcl-2 family members in the MOM. ## Methods and Models Models. The intersected and parallel α 9 dimer models were generated in our previous study 12 : the intersected model was predicted ab initio by CATM program 16 ; the parallel model was obtained through a systematic search of the dimer conformational space directed by the disulfide-crosslinking data. These two models fit the disulfide-crosslinking data from the single-cysteine Bax mutants illustrated in Fig. 1. We used the membrane builder and MD simulator of CHARMM-GUI 30 to generate the protein-membrane and protein-solvent systems. The lipid bilayer consist of 46.5% L-α -phosphatidylcholine (PC), 28.4% L-α -phosphatidylethanolamine (PE), 8.9% L-α -phosphatidylinositol (PI), 8.9% L-α -phosphatidylserine (PS) and 7.3% cardiolipin (CL) in mole fraction 25 . All systems were solvated with explicit water in the TIP3P model in the periodic box. Counterions Na + and Cl − were added to keep the system charge neutral. Simulation setup. Summary of all the MD simulations is provided in Supplementary Table 1. Most simulations have two replicas, of which the longer one lasts 100-200 ns and a shorter one 50-150 ns. The NAMD package 31 with CHARMM36 force field has been used 32 . Both equilibration and production runs were performed in the NPT ensemble (310 K, 1 bar, Nose-Hoover coupling scheme) with a time step of 2 fs. The particle mesh Ewald (PME) technique was used for the electrostatic calculations. The van der Waals and short-range electrostatics were cut off at 12.0 with switch at 10.0 . The metadynamics simulations 33,34 were carried out in NAMD to study the transitions between the intersected and parallel α 9 dimers, with the assumption that the transition between two conformations is reversible. The sampling bias was applied to two collective variables -the angles of rotation (α , β ) of the backbone atoms around the principle axis of each monomeric helix.

chemsum

{"title": "Conformational Heterogeneity of Bax Helix 9 Dimer for Apoptotic Pore Formation", "journal": "Scientific Reports - Nature"}

fabrication_of_highly_ordered_sn_nanowires_in_anodic_aluminum_oxide_templates_by_using_ac_electroche

## Abstract: Sn and its nanostructures are one of the promising candidates to replace graphite in the anode of Lithium-ion batteries due to their higher capacity. One of the challenges, which limited the usage of Sn anodes for the Lithium-ion batteries, is Tin's high volumetric strain and its low cyclability. On the other hand, nanostructures show lower volume change during charge/discharge and as a result could address the cyclability issues. In this research, an alternating current (AC) electrochemical method is developed in order to facilitate the industrial scale production of Sn nanowires. The developed electrodeposition technique shows reliable controllability over chemical composition and crystalline structure of Sn nanowires. Also, the order structure of nanowires could be adjusted more accurately in comparison to conventional fabrication techniques. As a result, the Sn nanowires as well as Aluminum Oxide templates synthesized by using the developed electrochemical method are examined due to their morphology, chemical composition, and their crystalline structure in order to develop a practical relation between electrochemical composition of the solution and materials properties of Sn nanowires. The results show that the proposed electrodeposition method maintains a highly-ordered morphology as well as industrially acceptable controllability over crystalline structure of nanowires, which could be used to optimize the procedure for industrial applications due to low cost and simple experimental setup. ## Introduction Exceptional mechanical properties of nanowires (e.g. low volumetric strain) in comparison to thin films make them suitable for micro-electronic device applications, where cyclability and volume changes are great concerns which may lead to mechanical failure and decrease the efficiency of the devices (e.g. Lithium-ion batteries) . Tin (Sn) based anodes are thoroughly studied in order to increase the Li-ion batteries' capacities . Despite of large volumetric strains of Sn based anodes, during charge/discharge, which may lead to mechanical failure of anodes and reduce the cyclability of Lithium-ion batteries , alloying the anode material (i.e. Sn) with mechanically stable elements (e.g. Ni, Co, Cu, graphene, etc.) method is developed in order to achieve promising cycling performance and utilizing the higher capacity of Tin (Sn) simultaneously [12-14, 17, 23, 26]. Furthermore, nanostructured version of these Tin based alloys (e.g. nanowires, nanoparticles, etc.) could stabilize the volumetric strains more effectively, because of their small volume changes in comparison to thin films . Although, synthesizing the nanostructures in large volume for industrial applications, due to their expensive and time consuming production methods such as: surfactant based techniques or self-assembling of 0D nanostructures , limited their application for Lithium-ion batteries' anodes productions. On the other hand, novel fabrication techniques such as electrodeposition methods (e.g. direct or alternating current techniques) have shown more accurate controllability over materials morphology and chemical composition of nanostructures in comparison to conventional production techniques as well as their easier scalability for industrial applications . Electrodeposition of metallic alloys in the ordered self-assembled templates (e.g. Al 2 O 3 , TiO 2 , porous polycarbonate, etc.) is developed primarily to control the morphology of nanostructures . One of the main challenges, which limited the applicability of electrochemical method to fabricate highly-ordered nanowires, is that conventional direct current (DC) electrodeposition methods are suffering from practical difficulties related to producing a conductive self-assembled structure due to low conductivity of templates' materials . On the other hand, alternating current (AC) electrochemical deposition method is well designed to overcome the technical difficulties, related to removing the barrier layer of the anodic Aluminum Oxide (AAO) and coating this self-assembled template with conductive materials . As a result, the AC electrodeposition technique has a potential to pave the way for the industrial scale production of nanostructures with a highly controlled morphology as well as their chemical structure . In this research, AC electrodeposition technique has been deployed to fabricate highly-ordered Sn nanowires in Aluminum Oxide templates. Furthermore, a novel room temperature two-steps anodization technique, which is used to produce a highly-ordered template, as well as its morphological properties will be investigated in details. Finally, morphological properties as well as chemical structure of fabricated Sn nanowires will be studied in order to examine their crystalline structures as well as controllability of nanowires' chemical composition. All the experiments in this research are done at room temperature, which paves the way for its industrial scale generalizations due to technical difficulties related to costly temperature controlling systems . ## Two step anodizing Before anodizing of Aluminum samples (Merck KGaA, 99.95%, 0.3 mm thickness -annealed), electrodes are electropolished (A = 1cm 2 ) in HClO 4 60 wt. % solution. In fact, the electropolishing voltage and temperature are fixed at 2 V and room temperature (25 • C) respectively. Also, the electropolishing time is optimized at 5min in order to achieve a highly smooth surface without primary amorphous Aluminum Oxide. In both the first and second steps of anodization procedure, the electrodes are anodized in C 2 H 2 O 4 0.3M solution. Furthermore, anodization time, voltage, and temperature are fixed at 2h, 40 V and room temperature (25 • C) respectively for first and second steps of this anodizing procedure. Fabricated porous Aluminum Oxides after the first step of anodization are etched in a solution of 0.6M H 3 PO 4 85 wt. % -0.2M H 2 CrO 4 . The etching temperature and time are fixed at 60 • C and 30min respectively. The main challenge, in order to use Aluminum Oxide templates for electrodeposition purposes directly, is how to reduce the electrical resistance of Al 2 O 3 barrier layer, which prevents the electrical current flow through the thick insulative layer at the bottom of the pores (i.e. barrier layer) . In this research, the barrier layer thinning (BLT) procedure (i.e. reducing the second step anodization voltage gradually) is used to reduce the electrical impedance of the Aluminum Oxide layer at the bottom of the pores. In fact, in this BLT procedure, anodization voltage at the second step is decreased step-wise with a rate of 2 V min until reaching 30 V. After that, the voltage will be decreased with a rate of 1 V min to reach 6 V. Finally, the samples will be held in the anodization solution at the 6 V electrical potential for 10 minutes before removing them from anodization solution in order remove barrier layer as much as possible and increase the electrical conductivity of self-assembled templates. Additionally, the electrical impedance of the electrodes, before and after BLT procedure, are examined by using impedance spectroscopy. ## AC nanowire electrodeposition The AC electrochemical deposition technique is employed to reduce Sn 2+ ions in the pores of self-assembled Aluminum Oxide template. In all the experiments of this section, pH as well as Boric acid (H 3 BO 3 ) concentration, root mean square (RMS) voltage, and AC signal's frequency are fixed at 1, 0.5M, 10 V, and 200 Hz respectively. Also, the Tin Sulfate (SnSO 4 ) concentration is changed from 0.1M to 0.5M with step size of 0.1M, in order to examine the effect of concentration on crystalline structure as well as directional growth of Sn nanowires. As a result, chemical composition as well as crystalline structure of deposited nanowires are examined by using energy dispersive spectroscopy (EDS) and X-ray diffraction respectively. ## Materials characterization Samples' characterization, which was used to investigate morphology of self-assembled templates as well as Sn nanowires, was done with a field emission scanning electron microscope (FE-SEM) Quanta 3D FEG (FEI, Phillips, The Netherlands). In order to examine the pore sizes as well as morphology (i.e. order structure) of nanowires, ImageJ image processing software is used to obtain quantitative information on the average diameter of the self-assembled templates' pores' diameter and length, as well as diameter of the electrodeposited Sn nanowires. The crystalline structure of the Sn nanowire alloy was analyzed by X-ray diffraction using a Rigaku Ultima IV diffractometer with Co K radiation and operating parameters of 40 mA and 40 kV with a scanning speed of 1 • per minute and step size of 0.02 • . Finally, the impedance spectroscopy of the anodized samples were done by using a MultiPalmSens4 potentiostat in order to compare the electrical resistance of anodizied samples before and after barrier layer thinning procedure. ## Aluminum Oxide morphology The porous morphology of two steps anodized Aluminum Oxide is shown in fig. 1. According to fig. 1(a), which is analyzed by using image processing techniques, it could be understood that the average pore size of this self-assembled template is 60 nm. Furthermore, the order structure of this porous medium is analyzed by using the fast Fourier transform (FFT) technique in order to examine the spatial structure of pores and their deviation from honeycomb structure. As a result, according to fig. 1(b), the FFT result of this AAO microstructure shows 6 strong bright dots, which indicates that a perfect honeycomb structure is achieved after the second step of anodization. Additionally, in order to examine the aspect ratio of self-assembled pores of AAO, a cross-sectional FE-SEM microscopy is done to estimate the thickness of Aluminum Oxide after the second step of anodization (cref. fig. 2). As shown in fig. 2(a), the thickness of AAO template is about 33 µm. As a result, the aspect ratio of pores, which is defined as the ratio of thickness over diameter, could be estimated as 550. Also, this aspect ratio will be increased for nanowires after AAO dissolution because of nanowires' radial shrinkage due to compressive residual stresses . The wall thickness of pores in self-assembled AAO template is estimated as 50 nm, which is shown in fig. 2(b). This highly ordered structure after the second step of anodization is achieved because: the quantum dots are created on the electrode's surface after etching step, which could facilitate the directional growth of AAO as well as controlling of its diameter more precisely . ## Impedance spectroscopy of Aluminum Oxide template's barrier layer Impedance spectroscopy is done in order to examine the electrical resistance of barrier layer before and after the thinning procedure. Additionally, the impedance of barrier layer directly could be related to its thickness as : Where Z is the electrical impedance, j = √ −1 is the imaginary unit, ω is the frequency, a is frequency scattering factor, C bl is the barrier layer capacity, d bl is the barrier layer thickness, r is the relative electrical permittivity, 0 is the electrical permittivity of vacuum, and S is the surface area of the sample. The impedance magnitude versus frequency and its real part versus imaginary part (Nyquist plot) for before and after the barrier layer thinning procedure are shown in fig. 3(a) and fig. 3(b) respectively. The equations 1 and 2 are used to fit them into the Nyquist plots (cref. fig. 3) and as a result, the obtained values for barrier layer thicknesses before and after BLT procedure are 20nm and 5nm respectively. This calculation shows that the BLT procedure reduced the barrier layer thickness 4 times smaller, which could increase its conductivity and facilitate the AC electrochemical deposition step. Additionally, the electrical circuits equivalence of the Nyquist plots are extracted due to the fitted parameters which are shown in fig. 4. According to these electrical circuits, it could be understood that the second resistance/capacitance pair remained constant before and after BLT procedure. However, the electrical resistance of first resistance/capacitance pair is reduced by three orders of magnitude, which shows the electrical resistance is reduced after BLT procedure significantly (cref. fig. 4). ## Sn nanowires FE-SEM microscopy technique is used to investigate the morphology of Sn nanowires after dissolution of AAO template in 1M NaOH solution. In fig. 5, Sn nanowires are shown in two different resolutions, which show their long-range order structure (cref. fig. 5(a)) as well as the diameter of the nanowires (cref. fig. 5(b), 33 nm). As a result, according to fig. 5(b), it could be understood that the aspect ratio of the nanowires are increased by a factor of 2, which could increase their surface to volume ratio as well as their chemical reactivity for practical applications. Also, the EDS and X-ray diffraction techniques are used to examine the chemical composition and crystalline structure of Sn nanowires respectively (cref. fig. 6). According to fig. 6(a), there are some residual Al 2 O 3 due to presence of Aluminum and Oxygen peaks. These residual Aluminum Oxide could be eliminated by increasing the AAO template dissolution time. Furthermore, fig. 6(b) shows that Sn nanowire preferential growth direction could be controlled by changing Tin Sulfate concentration in the electrodeposition solution. In fact, solution's chemistry could be used as a controlling variable to optimize crystalline structure of Sn nanowires. Due to fig. 6(b), it could be observated that intensity peaks of direction (200) is maximized for concentration 0.5M. It means at 0.5M SnSO 4 concentration, the (200) direction is the most thermodynamically unstable direction in comparison to (211) direction , which will lead to preferential growth along the (200) direction. In fact, at the 0.5M SnSO 4 concentration the activity of the Sn 2+ ions are maximized and as a result of that it shows preferential growth. This preferential growth observed in X-ray diffraction pattern of Sn nanowires indicates that crystalline structure of Tin based nanostructures could be optimized by changing SnSO 4 concentration in the context of the proposed AC electrochemical method. Additionally, this oriented crystalline structure of Sn nanowires in (200) direction could maximize their efficiency as an anode for Lithium-ion batteries because of their maximum chemical activity as well as enhancing their cyclability due to minimized volumetric strain . ## Conclusions In this research, an AC electrochemical deposition technique is developed, which could be easily used to fabricate metallic nanowires with highly-ordered structure and reasonable controllability over the chemical composition as well as the crystalline structure. Additionally, this technique could facilitate the large-scale production of nanostructures due to its room temperature operating environment. As a result, this technique could be generalized to develop industrial scale coating facilities, which could be used in Lithium-ion battery production as well as other industries, such as: biomedical applications , oil and gas extraction plants , and nanoparticles technologies . Finally, these Sn nanowires' production technique should be optimized by using the proposed electrochemical synthesizing procedure, and be examined in assembled Lithium-ion batteries to accurately measure their capacity as well as efficiency in order to achieve higher cyclability.

chemsum

{"title": "Fabrication of highly ordered Sn nanowires in anodic Aluminum Oxide templates by using AC electrochemical method", "journal": "ChemRxiv"}

efficient_electrochemical_synthesis_of_a_manganese-based_metal–organic_framework_for_h₂_a

## Abstract: In this study Mn-DABDC (DABDC = diaminobenzenedicarboxylate, or 2,5-diaminoterephthalate) MOF was synthesised both via an electrochemical method, to make Mn-DABDC(ES), and via a conventional solvothermal approach, to make Mn-DABDC(ST). A Mn-BDC (BDC = benzenedicarboxylate) MOF was also prepared by a conventional solvothermal method for gas uptake capacity comparison. Investigation of the electrochemical synthesis parameters demonstrated that current density, electrolyte amount and reaction time were the most significant factors affecting crystal synthesis and product yield. The best conditions found for obtaining a crystalline MOF with high yield (93%) were 70 mA current, electrolyte 2.7 mmol/30 ml DMF and 2 h of reaction time. These optimized electrochemical conditions allow for a relatively fast MOF synthesis, important for reducing synthesis cost compared with conventional hydrothermal and solvothermal methods. The Mn-DABDC(ES) MOF sample was fully characterized to analyse its structure, thermal stability and surface area. The electrochemically synthesized MOF has high carbon dioxide uptake (92.4 wt% at 15 bar and 273 K) and hydrogen uptake (12.3 wt% at 80 bar and 77 K). This is the first amine-based manganese MOF synthesized electrochemically, and the method has excellent potential for reducing large-scale MOF production costs. ## Introduction Coordination chemistry has evolved as a most promising route to porous materials with precisely decorated interiors to obtain specific properties for versatile applications. 1 One of the most highly studied applications of metal-organic frameworks (MOFs) is the capture of CO 2 to address global energy and environmental issues. 2,3 Metal-organic frameworks have metal coordination sites bridged by organic ligands in highly ordered networks that often afford well defined structures, high crystallinity and large surface areas that can be used in catalysis, or gas separation applications. 4,5 Over the last two decades developments in MOF synthesis have enabled MOFs to become promising candidates for carbon dioxide capture, however there is often poor carbon dioxide gas selectivity from flue gas streams. 2,6 A notable development is in the functionalization of MOF structures, including selective functional group insertion within any given framework to serve specific end functions and impart desirable properties to the MOF materials. 2,7,8 Amine sites in particular show great affinity towards carbon dioxide and are known to be highly effective for CO 2 adsorption while also being amenable to use under dry or humid conditions. 7 We have recently reported the modification of a copper-based MOF during synthesis by doping with hexamethylenetetramine, resulting in the enhancement of carbon dioxide sorption over the unmodified framework. 9 In another study we reported that amine post synthetic modification on a Mn-DOBDC framework (DOBDC = 2,5-dihydroxyterepthalate) enhances water stability and carbon dioxide uptake of the MOF. 10 A major challenge in preparing MOFs for CO 2 capture applications is still the energy intensive, tedious and laborious conventional solvothermal process for MOF synthesis. Electrochemical synthesis of MOFs was first reported by BASF in 2005, using anodic dissolution to synthesise the copperbased framework HKUST-1. 11,12 Most subsequent examples using this method have focussed on Cu and Zn frameworks, although examples exploiting Al and Fe have been reported. Recent reports include Pirzadeh and co-workers, who electrochemically synthesized a Cu 3 (BTC) 2 metal-organic framework for CO 2 and CH 4 separation, 18 and, in a hybrid approach, Mitra et al. grew Cu-based MOFs onto modified thin-film electrodes to study their electrochemical properties. 19 The electrochemical MOF synthesis process has advantages over conventional MOF syntheses including the potential for shorter reaction times and lower energy consumption with a relatively simple equipment setup. 18 Perhaps the most attractive feature of electrochemical synthesis is the mild reaction conditions, since these reactions can be performed at ambient pressure and temperature. Despite these advantages, it is still an under-exploited approach, especially in the synthesis of functionalised framework materials. 20 This study demonstrates the synthesis of a new amine-functionalised Mn-DABDC MOF using electrochemical synthesis to cut synthesis costs, important for future scale-up. The prepared material was fully characterized to analyse its structure, thermal stability and surface area. For comparison, a Mn-BDC MOF that lacks amine functionalisation was also synthesized using a traditional solvothermal method to compare CO 2 and H 2 adsorption of these MOFs. ## Materials All the chemicals were purchased from Merck Sigma Aldrich and used as received. Synthesis of {Mn 2 (BDC) 2 (DMF) 2 } ∞ (Mn-BDC) Mn-BDC MOF was prepared using a conventional solvothermal method reported by Huiping et al. in 2016, with slight modifications. 21 Equimolar quantities (1 : 1) of Mn (NO 3 ) 2 •6H 2 O (287 mg, 1 mmol) and terephthalic acid (160 mg, 1 mmol) were dissolved in 10 ml DMF in a 50 ml beaker. The contents were ultra-sonicated at 45 °C for 2 hours then the solution was transferred to 23 ml Teflon vials. These were each sealed in a Parr autoclave and heated in an oven at 110 °C for 24 hours to yield white crystalline material. Crystals obtained were washed thrice with DMF then thrice with THF (5 ml for each wash). The resulting crystals were dried overnight at room temperature to get 83% yield (371 mg). The sample was activated in vacuum oven at 130 °C for 12 hours before further analysis. Electrochemical synthesis of {Mn 3 (DABDC) 3 (DMF) 4 } ∞ (Mn-DABDC(ES)) For electrochemical synthesis of Mn-DABDC, 2,5-diaminoterephthalic acid (588 mg, 3 mmol) was dissolved in 30 ml DMF. In another beaker, NaNO 3 (225 mg, 2.7 mmol) was mixed with 10 ml distilled water to serve as a conductive electrolyte for the reaction. These mixtures were combined and ultrasonicated for 1 hour at room temperature to ensure complete mixing of contents. Mn strips were prepared for reaction (10 cm long × 2 cm wide × 0.4 cm thick). Polishing was done with sandpaper (400 grit) to remove any oxide layer and washing with distilled water followed by ethanol. The electrochemical synthesis reaction was performed by dipping these Mn strips (3 cm depth) in the reaction mixture keeping them 2 cm apart. A direct current (DC) supply was then attached to the electrodes and the current adjusted to 70 mA. As the reaction proceeded, light brown crystals were observed in the solution. The reaction was performed at ambient temperature and pressure (i.e. 20-22 °C and 1 atm). After 2 h, the product was collected, filtered and washed with DMF three times and then three times with THF (5 ml for each wash). The product obtained was dried at 60 °C in the oven for 4 hours to obtain 93% yield (1.29 g). The sample was activated in a vacuum oven at 130 °C for 12 hours before further analysis. This electrochemically synthesised material is named Mn-DABDC(ES) throughout the manuscript. Note: a series of reactions were performed to optimize time of reaction, current density and electrolyte concentration to obtain the best Mn-DABDC(ES) MOF yield; details of this series are in the ESI and Fig. S1. † Solvothermal synthesis of {Mn 3 (DABDC) 3 (DMF) 4 } ∞ (Mn-DABDC(ST)) Samples of the Mn-DABDC framework were also prepared using the conventional solvothermal method to compare synthetic outcomes with the product obtained by electrochemical synthesis. Equimolar quantities (1 : 1) of Mn (NO 3 ) 2 •6H 2 O (287 mg, 1 mmol) and 2,5-diaminoterephthalic acid (196 mg, 1 mmol) were dissolved in 10 ml DMF. After ultra-sonication at 45 °C for 2 hours the solution was transferred to 23 ml Teflon vials and sealed in a Parr autoclave and heated in an oven at 120 °C for 22 hours to yield light brown crystals. Crystals obtained were washed thrice with DMF then thrice with THF (5 ml for each wash). The resulting crystals were dried overnight at room temperature to obtain 78% yield (377 mg). The sample was activated in vacuum oven at 130 °C for 12 hours before further analysis. This solvothermally synthesised material is named Mn-DABDC(ST) throughout the manuscript. ## Equipment and characterization Electrochemical syntheses were performed under constant current or voltage using a RIGOL DC Power supply and RIGOL millimeter DM3058E. Single crystal X-ray diffraction data for Mn-DABDC and Mn-BDC MOFs were collected on an Agilent SuperNova Dual Atlas diffractometer with Mo and Cu sources and a CCD detector. Data reduction and integration was performed using CrysAlisPro. Powder X-ray diffraction (PXRD) patterns were collected on an X'PertPro Panalytical Chiller 59 diffractometer using copper Kα (1.5406 ) radiation. A 2θ range from 5 to 40 degrees was used to record the diffraction pattern. A SHIMADZU IR Affinitt-1S spectrometer was used to obtain IR spectra. Thermogravimetic analyses (TGA) were performed using a PerkinElmer Pyris 1 TGA equipment. The temperature was increased from 25 °C to 700 °C at a heating rate of 5 °C min −1 under a flow of air (20 ml min −1 analyses were performed using a FlashSmart NC ORG elemental analyser. CO 2 adsorption experiments were performed on a Quantachrome Isorb-HP100 volumetric type sorption analyser. Samples were degassed at 130 °C under vacuum for 12 hours and then backfilled with helium gas prior to gas sorption studies. CO 2 sorption studies were performed at two selected temperatures, 273 K and 298 K, over a pressure range of 0.5-15 bar. H 2 adsorption studies were performed at 273 K and 77 K, over a pressure range of 0.5-80 bar. N 2 adsorption studies of prepared samples were conducted to analyse surface area and pore volume using a Quantachrome Nova 2200e at 77 K at a relative pressure of P/P 0 = 0.05-1.0. ## Results and discussion There are several known structures containing Mn(II) nodes and the BDC linker, the earliest being MOF-73, but these were made using, and consist of, different metal : linker ratios and solvents to our material; herein we have formed a new Mn-BDC framework. Briefly, our Mn-BDC framework crystallises in a monoclinic geometry with a = 13.4484( 4 FTIR spectra of the prepared materials confirmed the presence of representative functional groups indicative of Mn-BDC and Mn-DABDC MOF formation (Fig. S2 †). Sharp peaks representative of symmetric and asymmetric stretching of carboxylates bonded to Mn are observed at 1535 cm −1 and 1367 cm −1 in the Mn-DABDC sample. 3 Both samples contain a broad band at around 3250 cm −1 , which can be attributed to O-H stretching vibrations of adsorbed atmospheric water. 24,25 In addition to the C-H stretches in both samples around The PXRD patterns of the as-synthesized Mn-BDC, Mn-DABDC(ES), Mn-DABDC(ST), and those simulated from single crystal XRD are shown in Fig. 3 and 4. PXRD patterns indicate in all cases the formation of highly crystalline material. PXRD patterns for Mn-DABDC produced from solvothermal synthesis and electrochemical synthesis indicate the same framework is synthesized with both methods, and in almost all solid products produced during the electrochemical parameter optimisation the Mn-DABDC MOF phase was formed with no apparent secondary phases (see ESI and Fig. S2 †). Exceptions to this were the presence of a peak at 25°2θ indicating unreacted crystalline DABDC linker remaining when current density was too low for efficient conversion to product (Fig. S2 pattern S1 †), and the presence of small additional peaks, most notably around 11-12°and 25-27°2θ, in the sample with the lowest quantity of electrolyte (Fig. S2 pattern S4 †). There is also good agreement between the simulated and as-synthesized (optimised synthesis) PXRD patterns, indicating that the single crystals studied are representative of the bulk samples, which in the optimised syntheses exhibit good phase purity and absence of manganese dioxide. 26 The optimised product yield of Mn-DABDC obtained by electrochemical synthesis for 2 hours at room temperature was 93%, compared with only 78% obtained from the 22 hours, 120 °C solvo-thermal method. This improvement is possibly as a result of electrochemical delivery of metal ions from the manganese electrode at a rate determined by the electrolysis, combined with ready provision of nitrate counterions from the excess present as part of the electrolyte. Indeed, the nitrate ions can be recycled during the synthesis rather than having to be supplied stoichiometrically as part of the Mn(NO 3 ) 2 salt used in the solvothermal synthesis. These differences evidently have a marked impact on the reaction kinetics and hence may affect the resulting crystal size and defect content. The SEM images of Mn-BDC and Mn-DABDC are therefore presented in Fig. S5. † SEM results show a range of particle morphologies including flat hexagonal rods stacked on each other for Mn-DABDC(ES), a mixture of hexagonal rods and flake structures for Mn-DABDC(ST), and loose laminar rod-like structures for Mn-BDC. The surface roughness of Mn-DABDC(ST) visually appears greater than that of Mn-DABDC(ES). The electrochemically-synthesised crystallites are quite clearly larger than those formed in both solvothermal syntheses, with the largest Mn-DABDC(ES) rod diameters reaching ∼8 μm in contrast to 2-3 μm for Mn-DABDC(ST) and only 1-2 μm for Mn-BDC. Both changes in morphology and size of the electrochemically synthesised framework are consistent with a different crystal growth mechanism, a feature of interest for future study beyond the scope of this present work. Thermogravimetric analysis (TGA) was performed on Mn-BDC and Mn-DABDC (Fig. S4 †). Some weight loss was observed below 100 °C for both MOFs indicating there was little surface adsorbed moisture. There is a weight loss step between approx. 125-245 °C for both MOFs which we ascribe to the loss of coordinated DMF from the MOF structures. 6,30 There is prominent two-step DABDC linker degradation in the Mn-DABDC sample as the temperature increases above approximately 325 °C. No further weight losses were observed for Mn-DABDC above 560 °C, indicating residual metal oxide, while Mn-BDC MOF decomposed completely to the oxide at 425 °C, a notably lower temperature than Mn-DABDC. CO 2 and H 2 adsorption capacities of Mn-BDC, Mn-DABDC (ES) and Mn-DABDC(ST) The CO 2 adsorption capacity for both MOF materials was evaluated by monitoring pseudo equilibrium adsorption uptakes. Samples were first degassed at 130 °C for 12 hours. 200 mg of each sample was used for three consecutive adsorption-desorption cycles at 273 K and 298 K with adsorbate pressure ranging between 0.1 to 15 bar. The CO 2 capacities calculated at 273 K and 15 bar pressure were 11.5 mmol g −1 and 21 mmol g −1 for Mn-BDC and Mn-DABDC(ES), respectively. This trend also occurs for adsorption capacities recorded at 298 K (Fig. 5). Hydrogen uptake of Mn-DABDC(ES) MOF was 12.3 wt% at 80 bar pressure and 77 K (Fig. 6), and a pore size of 3.53 was calculated from gas sorption data. Moderate Q st values were calculated for both gases in Mn-DABDC(ES) (Fig. S6 †) which 7). A comparison can also be made against the solvothermallysynthesised Mn-DABDC(ST) material. The CO 2 uptake at 55 bar of Mn-DABDC(ES) is slightly higher at both temperatures than that of Mn-DABDC(ST) (an increase of 6.3% at 273 K and 4.5% at 298 K) and markedly higher than that of Mn-BDC (an increase of 130% at 273 K and 136% at 298 K). The H 2 uptake of Mn-DABDC(ES) is similarly slightly higher than that of Mn-DABDC(ST) (an increase of 5% at 77 K and 21% at 273 K) and again markedly higher than that of Mn-BDC (an increase of 61% at 77 K and 113% at 273 K). To put this work in a broader context, Table 1 provides a comparison of amine-based metal-organic frameworks for uptake of carbon dioxide and hydrogen. In previous studies, we reported amine-modification of Cu-BDC, a copper-based MOF, by doping the synthesis with hexamethylenetetramine (HMTA). 9 Despite a reduction in BET surface area from 708 to 590 m 2 g −1 , this modification afforded a 3-and 4-fold increase in 273 K CO 2 uptake over the unmodified Cu-BDC framework, at 1 and 14 bar respectively. We have also reported a post-synthetic modification approach, attaching ethylenediamine (EDA) to Mn-DODC, a manganese-based framework. 10 In that study, modification only reduced the BET surface area a small amount, from 1256 to 1203 m 2 g −1 , but again increased the 273 K CO 2 uptake, albeit by a smaller multiplier (see Table 1). However, it is notable in the present study that the incorporation of two primary amine groups per linker in Mn-DABDC (ES) results not only in the largest BET surface area of all three studies, 1453 m 2 g −1 , but in the highest overall CO 2 uptake of our amine-containing frameworks. At 273 K the CO 2 uptake of Mn-DABDC(ES) at 1 bar is 40.9 wt% and at 15 bar it is 92.4 wt%. These values surpass those of many related smallpore frameworks reported in the literature; some examples are given in Table 1. Given that the Q st values at zero loading for EDA-MnDOBDC and Mn-DABDC(ES) are, perhaps unsurprisingly, essentially the same (32 kJ mol −1 ) and most likely result from CO 2 binding to the primary amines in both cases, the improved performance of Mn-DABDC(ES) at higher pressure may be attributable in part to the greater surface area, and in part to the greater density of amine sites in the framework. ## Conclusions Mn-DABDC(ES) was successfully produced in good yield using electrochemical synthesis. Synthesis conditions were optimized to get a maximum product yield of 93%. Here, manganese metal cations were produced in situ using Mn electrodes, eliminating the need for the MOF-precursor metal salt as required in conventional solvothermal and hydrothermal MOF production approaches, since the counter-ions are transiently provided by the electrolyte solution and hence can be continuously recycled in the synthesis. SEM results revealed well-formed flat hexagonal rod-like crystals for Mn-DABDC(EC), larger than the rods produced for Mn-DABDC(ST) and Mn-BDC. The three MOF materials were tested for carbon dioxide and hydrogen gas uptake. Mn-DABDC(ES) demonstrated high carbon dioxide (92.4 wt% at 15 bar pressure and 273 K) and hydrogen uptake (12.3 wt% at 80 bar pressure and 77 K), a little higher than the respective CO 2 and H 2 uptake of the solvothermally synthesised Mn-DABDC(ST) material, with both outperforming the related Mn-BDC framework. These results are ascribed to the incorporation of basic amine groups into the organic ligand within the framework significantly enhancing electrostatic interactions between the framework and the guests, increasing gas sorption. The electrochemical synthesis has the following specific advantages over the traditional solvothermal synthesis: (i) the use of ambient temperature instead of 120 °C, (ii) the use of ambient pressure instead of high-pressure autoclaves, (iii) the use of mild reaction conditions that recycle the nitrate counterions, and (iv) a vastly reduced reaction time compared to the conventional solvothermal synthesis method. These advantages demonstrate a method of design and synthesis of new materials with high carbon dioxide and hydrogen uptake with the potential for cost-effective large-scale production in the future. CCDC 1948926 † contains the supplementary crystallographic data for the Mn-DABDC MOF structure and CCDC 2027762 † contains the supplementary crystallographic data for the Mn-BDC MOF structure.

chemsum

{"title": "Efficient electrochemical synthesis of a manganese-based metal\u2013organic framework for H₂ and CO₂ uptake", "journal": "Royal Society of Chemistry (RSC)"}

oxygen_transfer_in_electrophilic_epoxidation_probed_by_¹⁷o_nmr:_differentiating_between_o

## Abstract: Peroxide compounds are used both in laboratory and industrial processes for the electrophilic epoxidation of olefins. Using NMR-spectroscopy, we investigate why certain peroxides engage in this type of reaction while others require activation by metal catalysts, e.g. methyltrioxorhenium (MTO). More precisely, an analysis of 17 O NMR chemical shift and quadrupolar coupling parameters provides insights into the relative energy of specific frontier molecular orbitals relevant for reactivity. For organic peroxides or H 2 O 2 a large deshielding is indicative of an energetically high-lying lone-pair on oxygen in combination with a low-lying s*(O-O) orbital. This feature is particularly pronounced in species that engage in electrophilic epoxidation, such as peracids or dimethyldioxirane (DMDO), and much less pronounced in unreactive peroxides such as H 2 O 2 and ROOH, which can however be activated by transition-metal catalysts. In fact, for the proposed active peroxo species in MTO-catalyzed electrophilic epoxidation with H 2 O 2 an analysis of the 17 O NMR chemical shift highlights specific p-and d-type orbital interactions between the so-called metal spectator oxo and the peroxo moieties that raise the energy of the highlying lone-pair on oxygen, thus increasing the reactivity of the peroxo species. ## Introduction Electrophilic epoxidations are at the core of numerous processes, ranging from the industrial synthesis of propylene oxide to enzymatic oxygenase reactions. In organic synthesis, this ubiquitous transformation is commonly achieved by using stoichiometric epoxidation agents such as meta-chloroperoxybenzoic acid (mCPBA), dimethyldioxirane (DMDO), or oxaziridines. 9-14 H 2 O 2 and ROOH can also be used for epoxidation but they require a catalyst. The most commonly used catalysts are (i) organorhenium trioxides (especially methyltrioxorhenium, MTO) and group 6 metal dioxo compounds (Fig. 1) or (ii) early transition-metal alkoxides (e.g. Ti and V), that involve peroxo-species as key reaction intermediates. While the reactivity of oxidizing agents such as mCPBA or DMDO towards olefns is well established and exploited synthetically, the origin of their reactivity towards C-C double bonds has not been studied in detail. This question is particularly apparent when considering that other peroxides, such as H 2 O 2 or tBuOOH, are usually not reactive towards olefns, unless combined with metal catalysts. Recent work has shown that analysis of the 13 C NMR chemical shift tensor (CST) of metal alkyl compounds can give valuable insights into the electronic structure and the reactivity of ubiquitous reaction intermediates in organometallic chemistry and homogeneous catalysis. Considering the large chemical shift window of 17 O nuclei (around 1200 ppm), we reasoned that analysis of the 17 O NMR chemical shift tensor of oxidants would allow for a detailed understanding of the electronic structure and associated reactivity of these molecules. In addition, the quadrupolar coupling constant of 17 O (nuclear spin I ¼ 5/2) can provide valuable information on the charge distribution around the nucleus. In fact, 17 O NMR spectroscopy has been used to identify and study peroxo species as well as related compounds containing O-N bonds. The isotropic chemical shift d iso (eqn (1)) and the three principal components (d 11 $ d 22 $ d 33 ) of the CST contain a considerable amount of information on the electronic structure of NMR active nuclei. The corresponding shielding values (s, eqn (2)), can be decomposed computationally into diamagnetic (s dia ) and paramagnetic contributions, which also include contributions from spin-orbit coupling (s para+SO , eqn (3)). While the diamagnetic contributions, which arise from a molecule's electronic ground state, lead to shielding and are usually similar for all nuclei of a given kind independent of their chemical environment, the paramagnetic contributions, which give mostly rise to deshielding, originate from magnetically induced coupling of excited states to the ground state, by action of the angular momentum operator Li , as described in a 2nd order perturbation approach in eqn (4). 47 According to eqn (4), deshielding of a nucleus is expected along the direction i, if an occupied orbital on this nucleus can be "superimposed" onto a vacant orbital on the same nucleus rotated by 90 along the axis i (Fig. 2). Since the extent of deshielding increases with a decreasing energy gap between the two orbitals, the paramagnetic contribution to shielding is most strongly affected by frontier molecular orbitals (FMOs)energetically high-lying occupied and low-lying vacant orbitals. In this work, we make use of chemical shift to evidence specifc high-lying occupied and low-lying vacant orbitals in the aforementioned oxidizing agents, thereby probing their electronic structure and connection to the observed reactivities. As 17 O is a quadrupolar nucleus (I ¼ 5/2), the quadrupolar coupling, which typically complicates the interpretation of spectra by line broadening, holds information about the distribution of charges around the nucleus. The quadrupolar interaction is proportional to the electric feld gradient (EFG) tensor € V (eqn (5)), where e is the electron charge, Q is the quadrupolar moment of 17 O, ħ is the reduced Planck constant (ħ ¼ h/2p), Î is the nuclear spin operator and I the nuclear spin quantum number. V is a traceless second rank tensor (V 11 + V 22 + V 33 ¼ 0) where we follow the notation |V 33 | $ |V 22 | $ |V 11 | for the three principal components. The EFG tensor can be described by two independent variablesusually the largest principal component (V 33 ) and the asymmetry parameter h Q (eqn ( 6)). The quadrupolar coupling constant C Q arises from the interaction of the quadrupole moment of 17 O (I ¼ 5/2) with the EFG and is proportional to V 33 (eqn (7)). 38 Since the electric quadrupole moment Q of the 17 O nucleus is negative, V 33 and C Q have opposing signs. V 33 and hence C Q are indicative of how symmetric the EFG and thus the charge distribution around the nucleus is. This has been a valuable tool to assess local symmetry around quadrupolar nuclei (e.g. 17 O, 27 Al, 45 Sc). 38,41,42,46, ## CSTs of non-metal-based peroxides We calculated the chemical shift tensors (CSTs) of selected peroxides relevant to epoxidation reactions, as well as associated reduced compounds. We chose to investigate DMDO and mCPBA, two compounds showing activity towards electrophilic epoxidation, as well as H 2 O 2 and tBuOOH that do not participate in this transformation, unless activated by metal catalysts. In order to benchmark our calculations, we also experimentally determined the 17 O NMR chemical shift tensors of H 2 O 2 52 and acetone by solid-state 17 O NMR spectroscopy (see ESI † for experimental details). The measured and calculated chemical shifts are given in Table 1. Generally, a good agreement between calculated and experimental data (when available) is obtained. The oxygen atoms of the unsymmetric peroxides (tBuOOH and mCPBA) are labelled (O), for the oxygen bound to a carbon atom and (OH) for the oxygen connected to the hydrogen. A comparison of the isotropic chemical shifts given in Table 1 reveals that all peroxide species (H 2 O 2 , tBuOOH, mCPBA, and DMDO) show signifcantly more deshielded d iso values in comparison with H 2 O, tBuOH and mCBA, albeit less deshielded than carbonyl oxygens (e.g. mCBA (C]O) and acetone). While the differences among the various peroxides are less pronounced, the chemical shift (d iso ) of the oxygen atom which is transferred during epoxidation reactions is more deshielded for DMDO and mCPBA (OH) as compared to H 2 O 2 and tBuOOH (OH). A closer inspection of the principal components of the chemical shift tensor reveals that this is mostly due to the d 11 component of the CST which is signifcantly more deshielded in DMDO and the OH-oxygen of mCPBA (636 and 540 ppm) than in H 2 O 2 and tBuOOH (364 and 386 ppm). This highly deshielded component is accompanied by a signifcantly larger span U of the CST in the former compounds. ## Orientation of the CSTs In order to further understand these observed trends, we investigated the orientation of the 17 ## Orbital analysis of the CSTs We decided to further elucidate the origin of deshielding in the individual components of the CST in a Natural Chemical Shielding (NCS) analysis. 47,48, This analysis allows for a decomposition of the s ii /d ii components into diamagnetic (s dia ) and paramagnetic/spin-orbit (s para+SO) contributions (eqn (3)). The paramagnetic term can then be further decomposed into contributions of the various NLMOs (bonds and lone pairs) surrounding the nucleus of interest. Due to the orbital energy difference in the denominator of eqn ( 4), the orbitals contributing most strongly to s para+SO are the frontier molecular orbitals (FMOs) of the molecule with a non-vanishing coefficient on the investigated nucleus. Since the largest differences in the CST of the investigated peroxides originate from the s 11 /d 11 component of the CST, its orbital analysis will be further discussed (see Fig. S3 and S4 † for other components). The NCS analysis of the s 11 component of the various compounds (Fig. 4b) reveals, that the diamagnetic contributions to this component are essentially invariant throughout the whole series of compounds. The differences in d 11 result from the paramagnetic contributions, which are mainly affected by four different Natural Localized Molecular Orbitals (NLMOs). These correspond to two lone-pairs on oxygen (denoted as LP 'p' and LP 's'), as well as the two s-bonding orbitals (denoted as s(O-R) and s(O-O)). These four NLMOs are visualized for the case of hydrogen peroxide in Fig. 4a; for the other compounds they are shown in Fig. S11-S13. † Notably, the dominant contribution to the deshielding of s 11 /d 11 originates from the LP 'p' on oxygen in all cases given in Fig. 4, with the exception of mCPBA (O) (vide infra). The observation of the large deshielding perpendicular to the O-O axis originating from this lone pair indicates the presence of a lowlying vacant orbital, oriented perpendicular to both the lonepair and the direction of s 11 /d 11 ; i.e. oriented along the O-O bond (eqn ( 4)). This vacant orbital corresponds to s*(O-O), the lowest unoccupied molecular orbital with contribution of oxygen in all of the investigated peroxides. As the extent of b Note that the quadrupolar nature of 17 O can lead to inaccuracies in the determination of the chemical shift tensorssee ESI for a more detailed discussion of the experimental measurements. Additionally, the presence of solvents can also signifcantly impact the chemical shift. The importance of this "backdonation" was further explored by investigating transition state geometries where the olefn is perpendicular to the oxygen lone pair and hence co-planar with the dioxirane moiety in DMDO or the carbonyl group in mCPBA. The corresponding transition state (2nd order saddle point) energies for the epoxidation where the rotation around the reaction coordinate was restricted were found at 32.7 and 29.0 kcal mol 1 for DMDO and mCPBA, respectively. The ## Quadrupolar coupling parameters To complement the analysis of the 17 O NMR parameters we calculated the quadrupolar coupling parameters of the aforementioned peroxides. The respective 17 O C Q and h Q -values are reported in Table 2. For all investigated peroxide oxygens, the absolute magnitude of the quadrupolar coupling constant C Q (and V 33 accordingly) is signifcantly larger than for water. The orientation of the EFG tensor is similar in all of these peroxides, with the most positive component oriented along the O-O bond, and the most negative component oriented in the direction of LP 'p' (selected examples in Fig. 7a-c, see Fig. S14 † for other compounds). This orientation is hence indicative of a region of high electron density in the direction of LP 'p', and a region of depleted electron density in the direction of the O-O bond. The reversed sign of C Q observed for DMDO in contrast to other peroxides is due to the defnition of V 33 , which always corresponds to the EFG tensor component with the largest absolute value (indicated by a red arrow in Fig. 7). While the EFG tensor is similar in all peroxides, the negative EFG tensor component perpendicular to the O-O bond is slightly larger for DMDO by absolute value as compared to the positive EFG tensor component along the O-O bond. For the other investigated peroxides, the situation is reversed, leading to a change in sign of V 33 and hence of C Q . The large C Q value for peroxides in combination with the specifc orientation of the EFG tensor is consistent with the presence of a high-lying occupied orbital (LP 'p') oriented perpendicular to a low-lying vacant s*(O-O) orbital in these species. This observation parallels what is seen from the NCS analysis of the d 11 component of the CST. ## MTO-catalyzed epoxidation As both H 2 O 2 and tBuOOH are inactive in electrophilic epoxidation but are rendered active in the presence of transitionmetal catalysts, we investigated the process of activation and the properties of the proposed active species. We chose methyltrioxorhenium (MTO) as a prototypical catalyst because of its high efficiency in olefn epoxidation with H 2 O 2 (Fig. 1b), and the existence of detailed mechanistic studies (isolation of reaction intermediates, measurements of 17 O NMR parameters and computational studies). 15, We hence calculated the chemical shift tensors of MTO (Table 3 and Fig. 8c) and of the water and pyridine adducts of the corresponding mono-and bisperoxides (Table 3). The bisperoxo pyridine adduct will be discussed in more detail; the tensors are shown in Fig. 8a and b. 72 The CSTs and NCS analyses for other intermediates are provided in the ESI † (Table S1, Fig. S1 and Tables S2-S4, Fig. S8-S10, † respectively) and further commented below for comparison. ## CST of peroxo intermediates The measured and calculated chemical shifts of bis-and monoperoxo intermediates of the MTO-catalyzed olefn epoxidation are given in Table 3. Both the isotropic chemical shift (d iso ) and the three principal components of the CST are signifcantly more deshielded in the metal-peroxide compound by comparison with H 2 O 2 (Table 3, where cis/trans denotes the peroxo oxygen pseudo-cis/trans with respect to the methyl substituent). This observation suggests a change in the electronic structure of the peroxide oxygen atoms, which is likely connected to their increased reactivity towards olefns. Note the signifcantly larger deshielding found for the oxo-ligands as typically observed for metal-oxo compounds. 73,74 The CSTs of the peroxo oxygen atoms have similar orientations as shown in Fig. 8 (and Fig. S1 † for the water adducts and the monoperoxo species). For both peroxo oxygens, s 11 /d 11 and s 22 /d 22 are in the O-Re-O plane (defned by Re and the two peroxo O-atoms) whereas s 33 /d 33 is perpendicular to it. In contrast to H 2 O 2 , the most deshielded component of the metal peroxo species, d 11 , is no longer oriented perpendicularly to the O-O axis but is signifcantly tilted, while remaining in the peroxo O-Re-O plane. One can also note differences of chemical shifts observed for the cis and trans peroxo oxygens in the bisperoxo compounds, the former being slightly more deshielded than the latter for both d iso and d 11 . These values are not strongly affected by the apical ligand (pyridine vs. water), suggesting a similar electronic structure in all bisperoxo intermediates. For the monoperoxo species, the d iso of both peroxo oxygens are more similar, albeit slightly more deshielded than in the bisperoxo intermediates. ## Orbital analysis of the CSTs An orbital analysis reveals that the largest contribution to the paramagnetic deshielding of the s 11 /d 11 component arises from the LP 'p', as was also observed for non-metal-based peroxides (shown in Fig. 9 for the bisperoxo pyridine adduct). Note however, that a signifcant contribution of the s(O-Re) bond is also observed for the metal-peroxo species. The other components and peroxo species are given in Tables S2 10). In fact, the calculated molecular orbitals suggest that the antibonding combinations of the above mentioned dand p-bonds are the LUMO and LUMO+1 of the MTO bisperoxide, respectively. As observed for non-metal-based peroxides, the MTO-derived bisperoxide shows rather large C Q values (15.0 MHz and 15.2 MHz for the cis-and trans-oxygen, respectively, see Table S5 and b The monoperoxo species is proposed not to coordinate an additional water-ligand; in fact, coordination of water has little effect on the NMR parameters and the water-ligand dissociated upon optimizing the O-transfer transition state. 75 c Same data as shown in Table 1. perpendicularly to a low-lying s*(O-O) orbital. As observed for the CST, also the C Q values of the peroxo oxygen atoms are rather insensitive towards replacement of the pyridine ligand by water; the corresponding water adduct shows a C Q of 15.0 MHz on both oxygen atoms. These values differ more in the monoperoxo species, where C Q 's are equal to 17.2 and 13.0 MHz for the cis-and trans-oxygen in the pyridine adduct, respectively, consistent with a more dissymmetric structure (Table 3). ## Energetic considerations in oxygen-transfer reactions Transition state energy calculations were performed both for the attack of ethylene at the oxygen pseudo-cis to the methyl group and the oxygen pseudo-trans to it. The obtained geometries for the bis-peroxo pyridine adducts are shown in Fig. 11 (others are given as coordinate fles in the ESI †). The free energy barriers from the bis-peroxo pyridine adduct are 34.2 kcal mol 1 and 27.2 kcal mol 1 for transfer of the cis and trans oxygens, respectively. Notably, the more deshielded oxygen atom (pseudo-cis to the methyl ligand) is associated with a less favorable oxygen transfer step, consistent with a CST already close to what is observed in metal-oxo species, showing the connection between reactant and product. While in H 2 O 2 the dihedral angle (H-O-O-H) is calculated to be 114 with the two LP 'p' on the oxygen pointing away from each other, the lone pairs are coplanar in a peroxo metal complex, introducing again a maximized a-effect (similar to DMDO and mCPBA). In order to understand and quantify the effect of the LP 'p' backdonation into the olefn p*-orbital to the transition state energy, and hence probe the importance of the a-effect, a transition state (2nd order saddle point), where the attacking olefn is coplanar with the peroxo O-Re-O plane was calculated for the case of the oxygen pseudo-trans to the methyl group. The obtained free energy of activation was found to be at 31.6 kcal mol 1 and thus 4.4 kcal mol 1 higher in energy than for the perpendicular transition state. This is consistent with what has been found for the epoxidation with non-metal-based peroxides, again evidencing the importance of the backdonation of a high-lying LP 'p' on oxygenthe flled p*(O-O) orbitalinto the olefn p*(C]C) orbital. Similar trends are observed for the bis-peroxo water adduct with free energy barriers of 34.3 and 27.8 kcal mol 1 , for the transfer of the cis-and trans-oxygen, respectively. For the monoperoxo intermediate, the free energy barriers for O-transfer are typically slightly higher (>29.0 kcal mol 1 ) for both oxygen atoms, at the exception of the cis-peroxo oxygen of the pyridine adduct (26.8 kcal mol 1 ). These results suggest that the bisperoxo complex is possibly the more reactive species in water, and that in the presence of pyridine both mono-and bis-peroxo complexes are reactive. 72,75 Thus pyridine may have a dual role, i.e. as a phase transfer agent and as a ligand to accelerate catalysis. 17,76 Notably, for all metal-peroxo compounds, the easier oxygen transfer is associated with the peroxo oxygen with smaller d iso and d 11 , while the oxygen atom which is not transferred displays a larger oxo-character, evidenced by the larger d iso and d 11 . As for DMDO and mCPBA, the coplanarity of the two oxygen lone pairs LP 'p' in the MTO peroxo species maximizes the aeffect, raising the energy of the lone pairs thereby increasing their reactivity. In addition, the p-interaction of the peroxo moiety with the metal (Fig. 10) assists the oxygen transfer process in olefn epoxidation, during which a fully developed p(Re]O) bond is formed. Notably, the spectator oxo-ligand in the apical position in the bisperoxo Re complex is not innocent: this oxo-ligand interacts with the peroxo moiety via the metal d-orbitals involved in the pand d-interactions (Fig. 10). This interaction minimizes the stabilization of the peroxo LP 'p' by weakening the (stabilizing) dand p-bonds. In addition, the presence of the spectator oxoligand also provides a driving force for the formation of the metal-peroxo species in the catalytic cycle: formation of the peroxo species from MTO strengthens the bond of the apical "spectator" oxo-ligand as evidenced by a slight decrease of the Re]O bond length on going from MTO (1.69 ) to the monoperoxo (1.68 ) and then the bisperoxo (1.67 ) pyridine adducts. This effect is reminiscent of the spectator oxo effect discussed for metallacyclobutane formation from metal alkylidenes during the olefn metathesis reaction, as well as for the formation of metallacycle oxetane intermediates in the reaction of alkenes with metal oxo compounds. ## Conclusions Overall, peroxide compounds are associated with signifcantly deshielded 17 O chemical shifts that indicate the presence of low-lying vacant and high-lying occupied orbitals, corresponding to the s*(O-O) and the lone pairs on oxygens, associated with p(O-O) and p*(O-O), for both metal-based and non-metalbased peroxides. These specifc electronic features are particularly pronounced in peroxide species that engage in electrophilic epoxidation reactions (DMDO, mCPBA, and MTO bisperoxo), as evidenced by their remarkably large deshielding. This is due to the coplanarity of the oxygen lone-pairs in these peroxides which is induced by their strained cyclic structure or by H-bonding in the case of mCPBA. Both maximize overlaps and in fne raises the HOMO (a-effect) and increases reactivity towards electrophilic epoxidation. In metal peroxo species, this HOMO is further raised in energy by the presence of a spectator oxo-ligand in the apical position. In fact, this "spectator" oxo species participates in modulating the reactivity of peroxo intermediates in transition-metal-catalyzed oxidation processes; it is thus not surprising that such a moiety is ubiquitous in efficient epoxidation catalysts that use H 2 O 2 as a primary oxidant. The a-effect and the presence of a strained cyclic structure goes hand in hand with a weakening of the O-O bond. Both the high-lying lone pair of the flled p*(O-O) and the low-lying s*(O-O) orbital drive the observed reactivity in electrophilic epoxidation, in which these two orbitals interact with the p*(C]C) and p(C]C) orbitals of the olefn, respectively. Thus, 17 O NMR chemical shift provides a powerful descriptor to pinpoint key electronic features that are decisive for reactivity in oxidation chemistry (Fig. 12). ## Epilogue The frontier orbital interactions shown in Fig. 6 highlight the following observation: while an epoxidation with DMDO or mCPBA is typically thought of as an electrophilic epoxidation, with the oxidant acting as electrophile and the olefn acting as nucleophile, these molecular orbital interactions indicate that both substrates act as nucleophiles and electrophiles by exploiting the low-lying s*(O-O) orbital and the high-lying lone pair LP 'p' (O) induced by the a-effect. This is reminiscent of synergistic effects observed in transition metal chemistry, for example in olefn complexes or in oxidative addition processes (Fig. 13a and b). Considering that olefn epoxidation with DMDO or mCPBA is isolobal to epoxidations with oxaziridines and halogenation reactions by X 2 or NXS (X ¼ Cl, Br, I), a similar orbital picture can be anticipated for these "electrophilic" additions (Fig. 13c). ## Conflicts of interest There are no conflicts to declare.

chemsum

{"title": "Oxygen transfer in electrophilic epoxidation probed by ¹⁷O NMR: differentiating between oxidants and role of spectator metal oxo", "journal": "Royal Society of Chemistry (RSC)"}

exchange-biasing_in_a_dinuclear_dysprosium(iii)_single-molecule_magnet_with_a_large_energy_barrier_f

## Abstract: A dichlorido-bridged dinuclear dysprosium(III) singlemolecule magnet [Dy2L2(µ-Cl)2(THF)2] has been made using a diamine-bis(phenolate) ligand, H2L. Magnetic studies show an energy barrier for magnetization reversal (Ueff) around 1000 K. Exchangebiasing effect is clearly seen in magnetic hysteresis with steps up to 4 K. Ab initio calculations exclude the possibility of pure dipolar origin of this effect leading to the conclusion that super-exchange via the chloride bridging ligands is important. Individual molecules that show slow relaxation of magnetisation are known as single-molecule magnets (SMMs). 1 This field started in 1993, and SMMs have been proposed as possible media for high-density magnetic storage. 2 A key parameter to evaluate the performance of an SMM is the effective energy barrier to magnetization reversal (Ueff). Dysprosium(III) is particularly preferred as the high magnetic anisotropy arising from the 6 H15/2 state generates the highest values for Ueff when placed in strong axial crystal fields. In particular, two Dy-SMMs families have very high Ueff: the sandwich structures with biscyclopentadienyl ligands 3 and pentagonal bipyramidal complexes. 4 In the latter, the strong axial crystal field is normally defined by short coordination bonds to the ligands on the axial positions of the pentagonal bipyramid. 4,5 A feature common to many Dy-SMMs is loss of magnetization at zero field, which is attributed to the quantum tunneling of magnetization (QTM) under zero field. Interactions between spin centers can prevent such zero-field loss of magnetization, known as exchange-bias. This was first seen 7 in a dimer of [Mn4] SMMs and has more recently been seen in Dy(III) dimers. 8 Our aim was to combine high Ueff values with exchange biasing by making a dimer of highly axial Dy(III) ions. For this strategy to work, the best molecular design would have the local anisotropy axes of the two Dy(III) sites as close as possible to being coparallel. 9 The title complexes [RE2L2(µ-Cl)2(THF)2]• toluene (RE = Dy, 1; RE = Y, 2; 5% Dy@2, 3) were prepared by deprotonation of H2L (N-(2-pyridylmethyl)-N,N-bis(2'-hydroxy-3',5'-di-tertbutylbenzyl)amine) with NaH, followed by reactions with anhydrous RECl3 in THF (see ESI for details). Since they are isomorphous as confirmed by single-crystal X-ray diffraction, only the structure of complex 1 is discussed in detail (Table S1, ESI). Compound 1 crystallizes in space group C2/c and has a two-fold rotation axis passing through the two bridging chlorides (Figures 1 and S1, ESI). The Dy(III) ion has a seven-coordinate geometry completed by two phenoxide oxygen atoms and two nitrogen atoms from one tetra-chelated L 2-, two µ2 bridging Cland one THF molecule. The two Dy-O(PhO) bond lengths are 2.152(2) and 2.168(2) , much shorter than the Dy-OTHF (2.412(2) ), two Dy-N bonds (2.574(2) and 2.520(2) ) and Dy-Cl bonds (2.7880(6) and 2.7896(6) ) (Table S2, ESI). The local symmetry is not a regular polyhedron (as determined by Shape software, 10,11 see ESI Table S3), however the bond angle between the two short Dy-O(PhO) bonds = 149.62 (8) o and this defines the main magnetic anisotropy axis as calculated by CASSCF-SO, giving an angle between the two main anisotropy axes of ca. 68° (see below) Temperature-dependent direct-current (dc) susceptibility data of 1 were collected under 1 kOe applied field. At room temperature the χT value is 28.53 cm 3 mol -1 K, in good agreement with the expected value of 28.34 cm 3 mol -1 K for two Dy(III) ions (Figure S3, ESI). Upon cooling χT decreases slowly at first, and then more rapidly below 16 K, reaching 17.23 cm 3 mol -1 K at 2 K. The field-dependence of the magnetization reveals that the highest M value is 10.31 Nβ at 50 kOe and 2 K (Figure S4, ESI). Alternating-current (ac) susceptibility measurements with an oscillating field of 3.5 Oe were also performed. Under zero dc field, 1 exhibits clear temperature and frequency dependence of the ac susceptibility below 53 K, showing the typical slow magnetic relaxation of SMMs (Figures S5 and S6, ESI). The relaxation time (τ) at each temperature was extracted from a simultaneous fit of χ' and χ'' using the generalized Debye model. 12 The obtained parameters are summarized in Table S4, in which α values are always less than 0.07 in the temperature range of 8-53 K, indicating a narrow distribution of relaxation times (Figure S7, ESI). The α values found were converted into experimental uncertainties in the relaxation times for each temperature using the CC-FIT2 code. 12 We observe that relaxation on the timescale of our ac susceptibility experiments is dominated by a power-law temperature dependence characteristic of a two-phonon Raman process with log[C (s -1 K -n )] = -3.8  0.2 and n = 4.3  0.2 (Figure 2, Equation 1). At the highest temperatures there is an increase in the relaxation rate, likely indicative of a multi-phonon Orbach process with an exponential temperature dependence. Including the experimental uncertainties renders these parameters practically undefined with Ueff = 1000  1000 K and log[τo (s)] = -10  10. However, a Ueff value around 1000 K is independently supported by ab initio calculations, see below, and taking only the central relaxation rate, as has been the only approach in the literature prior to our new method, 12 a fit gives Ueff = 922  9 K, log[τo (s)] = -11.33  0.08 (Figure S8, ESI). The only higher Ueff value found for a polynuclear single-molecule magnets is in Dy2ScN@C80-Ih. 13,14 . To examine the influence of the exchange-bias, we studied the magnetic properties of compound 3, which contains a 5% concentration of Dy(III) ion doped into the isostructural diamagnetic yttrium dimer 2. At this dilution level, the paramagnetic material is dominated by isolated Dy(III) ions in the structure. 8a The dc magnetic susceptibility data and low temperature magnetisation data for 3 have very similar profiles to those of 1 (Figures S13-S15 Cycling the field between +10 and -10 kOe for 3 gives a hysteresis loop shaped like a butterfly at low temperatures (Figures 4 and S20, ESI). The loss of magnetization at zero field for the diluted sample clearly confirms that magnetic interactions between Dy(III) ions in the dimer shifts the quantum tunnel resonances away from zero field. The two steps at ca. +1000 and -300 Oe for 1 are missing after dilution (Figures 3, 4, S12 and S21), and thus are markers of the magnetic interaction between the Dy(III) ions in the dimer. To understand the local electronic structure of the Dy(III) ions, complete active space self-consistent field spin-orbit (CASSCF-SO) calculations were performed using MOLCAS 8.0. 15a Basis sets from the MOLCAS ANO-RCC library 15 were employed with the paramagnetic ion described using VTZP quality, the first coordination sphere with VDZP quality, and all other atoms with VDZ quality. To probe the single ion properties in the pure Dy compound 1, one of the two Dy(III) ions in the crystal structure was replaced with the diamagnetic Lu(III) ion, as this more closely resembles the electronic manifold of the neighboring Dy(III) ion than would Y(III) (as Lu(III) has filled 4d, 5p and 4f orbitals). As expected based on the structure with a pair of phenoxide donors at an angle of 149.6°, the crystal field stabilizes an almost pure |±15/2⟩ ground doublet. This state is well separated from the 1 st (534 K), 2 nd (868 K) and 3 rd (1053 K) excited states. While the 1 st and 2 nd excited states are fairly pure mJ = |±13/2⟩ and |±11/2⟩ functions respectively, the 3 rd excited state is only 67% |±9/2⟩ (Table S5, ESI), and thus magnetic relaxation via the Orbach process is likely to occur through this state, predicting Ueff = 1053 K (Figure S22, ESI); this is in agreement with experimental value of 1000 K. We can calculate the dipolar interaction between the two Dy(III) sites using the g-values and relative orientation of the g-frames from CASSCF-SO (Equation 2). 9.16 Owing to the two Dy(III) sites being related by a two-fold axis of rotation, the local anisotropy axes of the ions are not co-parallel, but rather the gz axes have an angle of 68.25° between them. This gives the interaction matrix (Equation 3), which can be implemented in PHI to simulate the low temperature magnetic behavior (Equation 4). 17 This purely dipolar interaction alone does not reproduce crossings or avoided crossings along any of the three main directions (Figures S23 and S24, ESI) corresponding to the steps in the hysteresis measurements, and instead predict a step only at zero field that is inconsistent with the experimental data. Therefore, there must be a non-zero superexchange interaction via the bridging chlorides. Compound 1 is EPR silent and therefore we were unable to directly measure the exchange interactions using EPR. 9.16 For comparison, we could estimate the Ising exchange parameter considering the system as a simple Ising dimer, Equation (5), 8e,18 giving JIsing = -1.88 cm -1 where Hcross is 1018 Oe from the first derivative of the magnetization, and g equals gz = 19.87. ## 𝐻 cross = −𝐽 𝐼𝑠𝑖𝑛𝑔 /2𝑔𝛽 (5) Substituting JIsing for Jxx in our simulations (because x in the molecular frame is the most magnetic direction, Figure S23) does not yield avoided crossings consistent with the QTM steps observed in the hysteresis measurements (Figure S25). 8d, 19 Combining the dipole interaction matrix with the Ising approximation (i.e. Equation 3 with Jxx replaced with JIsing) does predict an avoided crossing at ca. 1000 Oe, however, also predicts significant zero-field avoided crossing (contradicting experiment) and no evidence of the experimental feature at -300 Oe (Figure S26). As none of these models fully explain the magnetization data, we have started with the dipole interaction matrix and added a superexchange component in order to reproduce the observed QTM steps. By addition +0.5 and -0.2 cm -1 to Jxx, and Jyy, respectively, we find Zeeman simulations that predict avoided crossings consistent with the observed magnetization steps at ca. +1000 and -300 Oe (Figure 5). However, single crystal measurements at mK temperatures would be necessary to obtain accurate measurements of the low-lying magnetic states in 1 in order to verify the exchange model proposed here. ). The magnetic field along the molecular x-axis (left), y-axis (centre) and z-axis (right). Note that there is a small avoided crossing at zero-field between the two ground states, with a gap of 4.27×10 -3 cm -1 . The parameters used to fit the relaxation behaviour of 1 and 3 can be compared with those found for other seven-coordinate Dy-SMMs with O-donors in the axial positions. 20 For example, in regular pentagonal bipyramids 900 < Ueff < 1300 cm -1 ; log[τo (s)] = -11.63  0.57; log[C (s -1 K -n )] = -6.03  0.52; n = 4.1  1.0. Thus, while Ueff and n are very similar both τo and C are bigger in the exchange-coupled dimer (log τo = -11.33  0.08, log C = -3.41  0.06). This may also be due to the less regular coordination environment in 1. We have been able to switch off the zero-field loss of magnetisation in this high T SMM through exchangebiasing and this motivates us to improve the coupling strength while keeping the high anisotropy to construct better SMMs. ## Experimental Section Experimental Details can be found in the supplementary information.

chemsum

{"title": "Exchange-Biasing in a Dinuclear Dysprosium(III) Single-Molecule Magnet with a Large Energy Barrier for Magnetization Reversal", "journal": "ChemRxiv"}

redox-induced_umpolung_of_transition_metal_carbenes

## Abstract: Metal carbene complexes have been at the forefront of organic and organometallic synthesis and are instrumental in guiding future sustainable chemistry efforts. While classical Fischer and Schrock type carbenes have been intensely studied, compounds that do not fall within one of these categories have attracted attention only recently. In addition, applications of carbene complexes rarely take advantage of redox processes, which could open up a new dimension for their use in practical processes. Herein, we report an umpolung of a nucleophilic palladium carbene complex, [{PC(sp 2 )P} tBu Pd(PMe 3 )] ({PC(sp 2 )P} tBu ¼ bis[2-(di-iso-propylphosphino)-4-tert-butylphenyl]methylene), realized by successive one-electron oxidations that generated a cationic carbene complex, [{PC(sp 2 )P} tBu PdI] + , via a carbene radical, [{PCc(sp 2 )P} tBu PdI]. An EPR spectroscopic study of [{PCc(sp 2 )P} tBu PdI] indicated the presence of a ligandcentered radical, also supported by the results of reactions with 9,10-dihydroanthracene and PhSSPh. The cationic carbene complex shows electrophilic behavior toward nucleophiles such as NaH, p TolNHLi, PhONa, and PMe 3 , resulting from an inversion of the electronic character of the Pd-C carbene bond in [{PC(sp 2 )P} tBu Pd(PMe 3 )]. The redox induced umpolung is reversible and unprecedented. ## Introduction Transition metal carbene complexes, usually classifed as Fischer and Schrock type according to the polarity of the M] C carbene bond, 1 are among the most extensively studied organometallic species. 2 While the reactivity of the M]C carbene fragment is generally governed by the electronic properties of the metal center and the substituents of the carbene moiety, 3 its behavior in response to redox reactions has not been studied in detail. 4 For example, although the one-electron reduction of a Fischer-type carbene leading to a carbene radical anion, which upon a second one-electron reduction generates a dianion, is known, 5 the corresponding oxidation processes that could be applied to Schrock-type carbenes (Fig. 1) have not been reported so far. These electron transfer processes cause an umpolung of the original carbene's character, namely the inversion of the M]C carbene bond polarity, 6 and could open up new applications for these compounds in synthesis. The development of redox chemistry of transition metal carbenes and reactivity studies of the species generated under redox conditions are still in their infancy, 7 with only a few examples reported exclusively for Fischer carbenes. 8 Interestingly, Fischer type carbenes of group 9 metals in the +2 oxidation state exhibited a remarkable radical character, their redox noninnocent behavior being considered crucial to the asymmetric cyclopropanation of olefns catalyzed by Co II (porphyrins). 8d-i Pioneering work by Casey and later by Cooper et al. showed that Fischer type carbene complexes could be reduced to the corresponding radical and dianionic entities. 5 The latter reacted with CO 2 to form a malonate, indicating an umpolung from the electrophilic character of the Fischer type carbene to nucleophilic. 5b As mentioned above, the redox behavior of Schrock type carbenes has not been explored. We reasoned that palladium nucleophilic carbenes 9 represent good candidates for such a study given the higher stability of late transition metal carbene complexes when compared to that of their early metal counterparts. In addition, the combination between palladium, which can undergo two-electron processes (oxidative addition/ reductive elimination), and a redox active ligand, which can undergo sequential one electron processes, has only recently been investigated. 10 No examples in which a carbene carbon is the site of redox activity are known. On the other hand, although it is known that palladium carbene species generated from diazo compounds show unique properties and undergo novel transformations, their isolation and characterization is still challenging due to their highly reactive nature. 11 We previously reported the nucleophilic Pd(II) carbene complexes [{PC(sp 2 )P} H Pd(PR 3 )] (1: R ¼ Me; 2: R ¼ Ph) ([PC(sp 2 )P] H ¼ bis[2-(di-iso-propylphosphino)phenyl]-methylene) 9a,12 and [{PC(sp 2 )P} tBu Pd(PMe 3 )] (3, {PC(sp 2 )P} tBu ¼ bis[2-(di-iso-propylphosphino)-4-tert-butylphenyl]methylene), 9b in which the Pd-C carbene bonds are best described as ylide-like, M + -C type, as demonstrated by the reactivity of 1 toward MeI, HCl, MeOH and para-toluidine 9a and also by C-H activation reactions. 13 The strong nucleophilic character of 1 and 3 was also demonstrated by the rapid Lewis acid/base "quenching" reactions with B(C 6 F 5 ) 3 . 9b As indicated by DFT calculations, the HOMO of 1 is largely localized on the carbene carbon atom, therefore, an oxidation process to induce the loss of electrons might occur from that orbital. Consequently, the one-electron oxidation of 1 with 0.5 equivalents of I 2 afforded a stable radical complex, [{PCc(sp 2 )P} H PdI], which persisted in solution but dimerized in the solid state. Interestingly, the chloride and bromide congeners are monomers in both phases. 14 These results prompted us to study the oxidation of the radical species further to the corresponding cations, and therefore to realize an umpolung of a nucleophilic carbene. In order to prevent dimerization and also simplify the synthetic procedure, carbene complex 3 bearing bulky tert-butyl groups was employed instead of 1. 14 The good solubility provided by the t-Bu groups also facilitated reactivity studies. Herein, we report the successive one-electron oxidation of 3 leading to a cationic carbene complex, [{PC(sp 2 )P} tBu PdI][BAr F 4 ] (5), via a monomeric radical species [{PCc(sp 2 )P} tBu PdI] (4). Reactivity studies on both complexes are consistent with their unique electronic properties, and the umpolung of carbene complex 3 was undoubtedly demonstrated by the reactions of 5 with various nucleophiles. Notably, the electron transfer processes to generate the carbene, radical, and cationic species are reversible. ## Results and discussion Synthesis and characterization of palladium radical and cationic carbene complexes Slow addition of 0.5 equivalents of I 2 to the dark-brown solution of [{PC(sp 2 )P} tBu Pd(PMe 3 )] ( 3) in THF at 35 C instantly generated a dark-green solution, from which dark-green crystals of [{PCc(sp 2 )P} tBu PdI] (4) were isolated in 97% yield (Scheme 1). Complex 4 is silent by both 1 H and 31 P{ 1 H} NMR spectroscopy. The magnetic susceptibility measurement at 298 K using the Evans method gave an effective magnetic moment m eff of 1.85 m B , thus indicating an S ¼ 1/2 ground state. The X-band EPR spectrum recorded at 298 K revealed an isotropic signal typical for a carbon centered radical complex with a g value of 2; no hyperfne coupling was observed (Fig. S1 †). Complex 4 is stable toward water but highly sensitive toward oxygen. Upon exposing the dark-green solution of 4 to air, the color changed immediately to yellow. However, attempting to react 4 with a stoichiometric amount (1 or 0.5 equivalents) of pure oxygen only led to complicated mixtures of products. Interestingly, in contrast to radicals generated in situ from Fischer type carbenes, 4,5 complex 4 is thermally robust: no signifcant decomposition was observed after heating 4 in C 6 D 6 at 80 C for a week, likely a consequence of the radical delocalization over both phenyl rings and of the steric protection from the two tert-butyl groups present in 4. Cyclic voltammetry studies of 4 performed in a THF solution (Fig. 2) showed a quasi-reversible event at E 1/2 ¼ 0.38 V, assigned to [{PC(sp 2 )P} tBu PdI] + /[{PCc(sp 2 )P} tBu PdI], and an irreversible reduction event at E ¼ 2.03 V (vs. Fc/Fc + ), assigned to the reduction to [{PC(sp 2 )P} tBu PdI] . Treatment of the dark-green solution of 4 with an equivalent of [Cp 2 Fe][BAr F 4 ] in diethyl ether at 35 C instantly formed a dark-red solution, and the dark-red crystalline complex [{PC(sp 2 )P} tBu PdI][BAr F 4 ] (5) was isolated in 94% yield (Scheme 1). To the best of our knowledge, only two examples of nonheteroatom-stabilized cationic Pd(II) carbene complexes are known: complex A (Fig. 3) was prepared from a cationic precursor by triflate abstraction with di-p-tolyldiazomethane, 15 while compound B was synthesized by hydride abstraction from a PCHP pincer complex; 16 reactivity studies have not been reported for these species. In addition, cationic alkylidene complexes of other late transition metals are also rare. 17 Cationic Ir(III) alkylidene complexes generated either by chloride or hydride abstraction showed electrophilic reactivity toward PMe 3 and LiAlH 4 to form the phosphonium ylide and the alkyl species, respectively. 18 A cationic Pt(II) alkylidene complex synthesized by hydride abstraction also showed similar reactivity toward Lewis bases such as DMAP (4-dimethylaminopyridine). 19 It is worth mentioning that, unlike the synthesis of the previously reported cationic carbene complexes, the synthesis of 5 is based on a redox process. Due to its ionic nature, 5 is only soluble in ethers and chlorinated solvents. The 1 H NMR spectrum of 5 in CDCl 3 indicates a C 2 symmetric species. The 31 P{ 1 H} NMR spectrum shows one sharp singlet at d ¼ 72.04 ppm, which shifted to lower feld compared to that of 61.86 ppm in 3. The counter anion [BAr F 4 ] was confrmed by singlets at d ¼ 6.62 and 65.79 ppm in the 11 B{ 1 H} and 19 F{ 1 H} NMR spectra, respectively. The characteristic signal for the carbenium carbon was observed at d ¼ 284.47 ppm as a singlet in the 13 C{ 1 H} NMR spectrum, which is signifcantly shifted to lower feld compared to the corresponding values of 136.06 ppm for 3, 18.3 ppm for [Cy 2 P(S)CP(S)Ph 2 ]Pd(PPh 3 ), 9d and 189.6 ppm for the cationic carbene complex [P 2 C]PdCl]X (B, Fig. 3). 16 However, this value is close to that of 313.4 ppm for [(Trpy)Pd]C(p-Tol) 2 ][BAr F 4 ] (Trpy ¼ 3,4,8,9,13,14-hexaethyl-2,15dimethyltripyrrinato, A, Fig. 3). 15 The structures of 4 and 5 were unambiguously determined by single crystal X-ray diffraction studies. As shown in Fig. 4 and 5, both complexes contain square-planar palladium centers bound to the sp 2 hybridized backbone carbons (the sum of the angles at C is 359.8 for 4 and 359.7 for 5). The Pd-C carbene distance of 2.076(3) in 3 contracts to 2.022(3) in 4 and further to 1.968(3) in 5. The Pd-C carbene distance of 1.968 (3) in 5 is comparable to that of 1.999(4) in [P 2 C]PdCl][PF 6 ] (B, Fig. 3). 16 Although the Pd-C carbene bond in 3 is better described as a single bond, 9a,9b,14,20 the contraction observed for 4 and 5 can be attributed to the increased bond order between the metal center and the carbene carbon, achieved by the sequential removal of electrons from the p* antibonding orbital (vide infra). This trend also indicated a change of the electronic property of this atom from an anion in 3 to a cation in 5 via the radical in 4. Therefore, the isolation of 4 and 5 is remarkable and represents the frst example of successive oxidations of a transition metal nucleophilic carbene leading to a well-defned radical and cation, with all three complexes characterized by Xray crystallography. ## DFT calculations DFT calculations were performed on a model of the cationic carbene 5 (Fig. 6). In contrast to the antibonding character of HOMO observed for 3, a similar antibonding p type interaction was found for the LUMO of 5 and the SOMO of 4, resulting from the successive removal of electrons from the HOMO of 3 that is largely localized on the carbene carbon. The bonding component of this p bond was found in HOMO11 for 5. Therefore, consistent with the observed contraction of the Pd-C carbene distances from 3 (2.076(3) ) to 5 (1.968(3) ), the frontier molecular orbital analysis also indicates an increase of the Pd- A successive addition of electrons to the LUMO of 5 should regenerate the carbene radical 4 and, ultimately, the carbene 3. Treatment of the dark-red THF solution of 5 with KC 8 (1 eq.) at room temperature immediately led to a dark-green solution of 4, as confrmed by its reaction with 9,10-dihydroanthracene in C 6 D 6 to form 6 (vide infra). In the presence of PMe 3 , the further reduction of 4 with KC 8 (1 eq.) in THF also regenerated 3. Such reversible electron transfer among the carbene species 3, 4, and 5 allowed us to tune their electronic properties by redox methods (Fig. 1). 4 Accordingly, treatment of cation 5 with 1 eq. of carbene 3 in diethyl ether immediately generated a dark green solution containing carbene radical 4 and the cationic radical complex [{PCc(sp 2 )P} tBu Pd(PMe 3 )][BAr F 4 ] (Fig. S41 †), which were confrmed by their hydrogen atom abstraction reactions with 9,10-dihydroanthracene (vide infra). ## Reactivity of palladium carbene radical complex The carbene radical complex 4 is expected to undergo ligand centered radical-type reactions, such as hydrogen atom abstraction. 14 Despite this prediction, heating a C 6 D 6 solution of 4 with n Bu 3 SnH at 80 C for one week did not produce the hydrogen atom abstraction product, and the color of the solution remained dark green. However, 4 reacted slowly reacted with 0.5 equivalents of 9,10-dihydroanthracene under similar conditions to generate a bright yellow solution, from which the expected hydrogen abstraction product [{PC(sp 3 )HP} tBu PdI] ( 6) was isolated as an orange crystalline solid in 81% yield, after recrystallization from n-pentane (Scheme 2). The formation of the by-product anthracene was also confrmed by NMR spectroscopy. The 1 H NMR spectrum of 6 in C 6 D 6 showed C s symmetry as observed for its chloride analogue [{PC(sp 3 )HP} tBu PdCl]. 9b The benzylic proton of 6 was observed at d ¼ 6.41 ppm as a singlet, while the benzylic carbon was observed at d ¼ 58.35 ppm as a triplet ( 2 J PC ¼ 4.4 Hz) in the corresponding 13 C{ 1 H} NMR spectrum. Both values are slightly downfeld shifted compared to those of the chloride analogue (d ¼ 6.21 and 50.59 ppm for the 1 H and 13 C{ 1 H} NMR spectra, respectively). 9b In the 31 P{ 1 H} NMR spectrum, only one sharp singlet at d ¼ 51.25 ppm was observed. The crystal structure of 6 is analogous to that of [{PC(sp 3 )HP} tBu PdCl] (Fig. 7), in that both contain an sp 3 hybridized backbone carbon atom. The Pd-C backbone distance of 2.094(2) is also close to that of 2.078(2) in [{PC(sp 3 ) HP} tBu PdCl], 9b but longer than the Pd-C carbene distance of 2.022(3) for 4. Coupling of the radical in 4 with a thiyl radical occurs in the reaction with diphenyl disulfde (Scheme 2). Albeit slow, heating 4 with 0.5 equivalents of PhSSPh in C 6 D 6 at 80 C for 7 days formed [{PC(sp 3 )(SPh)P} tBu PdI] (7), which was isolated as a yellow waxy solid in 94% yield. The 1 H NMR spectrum of 7 in C 6 D 6 showed C s symmetry, indicating an sp 3 hybridized backbone carbon. No benzylic proton was observed and the quaternary backbone carbon showed a triplet at d ¼ 79.58 ( 2 J PC ¼ 6.1 Hz) in the 31 C{ 1 H} NMR spectrum, both facts being consistent with a C-S bond formation between the carbene radical of 4 and the [PhS]c radical. The 31 P{ 1 H} NMR spectrum showed only a sharp singlet at d ¼ 48.59 ppm. However, all attempts to crystallize 7 failed due to its high solubility in hydrocarbons. Therefore, complex 7 was treated with an equivalent of AgOTf in THF, 23 and the expected complex [{PC(sp 3 )(SPh)P} tBu Pd(OTf)] ( 8) was obtained as yellow blocks in 61% yield after recrystallization from diethyl ether. The crystal structure of 8 was determined by X-ray diffraction (Fig. 8), showing the sp 3 hybridized backbone carbon bound to a PhS group. The phenyl ring of the PhS group is pointing somewhat toward the triflate substituent with the corresponding dihedral angles between the plane of the phenyl ring and the plane defned by O, Pd, C backbone and S being 73.49 . According to the standard redox potential of PhSSPh (1.7 V vs. SCE, 2.1 V vs. Fc/Fc + ) 24 and the observed value for the [{PCc(sp 2 )P} tBu PdI]/[{PC(sp 2 )P} tBu PdI] + , redox couple (0.38 V vs. Fc/Fc + ), it is less likely that the carbene radical 4 was oxidized by PhSSPh to the carbene cation [{PC(sp 2 )P} tBu PdI] + which reacted with the [PhS] anion to form 7. Therefore, compound 7 was probably formed by direct radical coupling between the carbene radical and [PhSc] generated by the homolysis of PhSSPh. It is also interesting to note that the carbene complex 3 can be readily oxidized by PhSSPh. Treatment of 3 with an equivalent of PhSSPh in C 6 H 6 immediately generated a bright yellow solution at room temperature, from which the yellow crystalline solid of [{PC(sp 3 )(SPh)P} tBu Pd(SPh)] (9) was isolated in quantitative yield after recrystallization from n-pentane (Scheme 3). The 1 H NMR spectrum of 9 in C 6 D 6 also showed C s symmetry. No benzylic proton was observed and the corresponding quaternary backbone carbon showed a triplet at d ¼ 74.78 ( 2 J PC ¼ 3.2 Hz) in the corresponding 31 C{ 1 H} NMR spectrum. Only a sharp singlet at d ¼ 44.26 ppm was observed in the 31 P{ 1 H} NMR spectrum. The structure of 9 was further confrmed by X-ray diffraction studies. As shown in Fig. 9, complex 9 contains two PhS moieties, one bound to the sp 3 hybridized carbon backbone and the other to the palladium center. Unlike 8, the phenyl ring of the PhS group on the backbone carbon points away from the Pd center. When carbene 3 was treated with only 0.5 equivalents of PhSSPh in C 6 D 6 , compound 9 and unreacted 3 were observed by 1 H and 31 P NMR spectra; which upon the addition of another 0.5 equivalents of PhSSPh, the mixture led to 9. It is worth noting that a radical complex [{PCc(sp 2 )P} tBu PdSPh] (C) was not formed under these conditions, but it could be an intermediate species that generates 9 by rapid coupling with the [PhS]c radical. This intermediate, C, can be generated by an one-electron transfer from carbene 3 to PhSSPh, however, direct addition of PhSSPh across Pd + -C carbene moiety or oxidative addition to palladium followed by the migration of the PhS moiety to the carbene carbon to form 9, cannot be ruled out. ## Reactivity of palladium cationic carbene complex The cationic carbene complex 5 is expected to be electrophilic, therefore its reactivity toward various nucleophiles was studied. We had previously isolated complex 6 from a hydrogen atom abstraction reaction with the carbene radical 4, and thus assumed that a nucleophilic attack on the cationic carbene 5 by a H nucleophile would also produce 6. Treatment of a dark-red solution of 5 with an equivalent of NaH in THF at room temperature gradually generated a bright yellow solution within 24 hours. Both 1 H and 31 P{ 1 H} NMR spectra showed that the 6 was formed in quantitative yield together with the by-product Na[BAr F 4 ] (Scheme 4). Since the cationic Ir(III) metallacyclic alkylidene complex was reported to react with LiAlH 4 even at low temperature, 18b the slow reaction with 5 was attributed to the poor nucleophilicity of NaH, but also to its poor solublility in THF. (Dn 1/2 z 505 Hz) ppm, respectively. Upon cooling, complex 10 showed coalescence at 278 K, and further splitting to two sharp doublets at 238 K (Fig. 12). For 11, a slightly lower temperature (268 K) was required to reach coalescence and two sharp doublets were observed at 228 K (Fig. S34 †). Both 10 and 11 showed only one sharp singlet in the corresponding 31 P{ 1 H} NMR spectra at 338 K. Since a similarly dynamic behavior was not observed for 8 and 9, in which both backbone carbons are Although the nucleophiles H , p TolNH , and PhO afforded the expected products 6, 10, and 11, respectively, the reaction of 5 with PhCH 2 K did not lead to an isolable product. Also, the lithium salts MeOLi and PhOLi did not react with 5, probably due to their weaker nucleophilic character compared to that of the sodium salt PhONa. A neutral nucleophile such as PMe 3 also reacts instantly with 5 in diethyl ether to form a bright-yellow solution; yellow crystals of [{PC(sp 3 )(PMe 3 )P} tBu PdI][BAr F 4 ] ( 12) were isolated in quantitative yield by the diffusion of n-pentane into a fluorobenzene solution at room temperature. Compound 12 is only soluble in etheral and chlorinated solvents. Its 1 H NMR spectrum in CDCl 3 showed C s symmetry as was also observed for compounds 6-11. The protons of the PMe 3 group were observed at d ¼ 1.31 ppm as a doublet ( 2 J HP ¼ 11.5 Hz), which correlates with a doublet at d ¼ 13.86 ppm ( 1 J CP ¼ 54. 3 Hz) in the 31 C{ 1 H} NMR spectrum. In the 31 P{ 1 H} NMR, two sharp singlets at d ¼ 42.17 and 31.01 ppm were observed for the two i Pr 2 P phosphines and the PMe 3 on the backbone carbon, respectively. Both signals remain sharp when the temperature was lowered to 248 K, indicating a lower energy barrier for the rotation of PMe 3 group compared to those for the p TolNH and PhO groups in 10 and 11. In the 13 C{ 1 H} NMR spectrum, the backbone carbon resonance was observed at d ¼ 63.65 (dt, 1 J CP ¼ 21.5 Hz, 2 J CP ¼ 2.4 Hz) ppm, which is signifcantly shifted to higher feld compared to 284.47 ppm observed for 5, and close to the value of 58.35 ppm in 6. Thus, the strong donation from PMe 3 to the carbenium carbon effectively offsets the charge on this atom. The exclusive formation of 12 is consistent with its strong electrophilic character. Heating this compound at 60 C in CDCl 3 for 24 h did not lead to any decomposition. However, the cationic Ir(III) alkylidene hydride species [(C 5 Me 5 )HIr i Pr 2 P(Xyl)] [BAr F 4 ] generated by chloride abstraction was reported to react with PMe 3 to form a similar phosphonium ylide only as a kinetically favored product, which converts to the Ir(III) alkyl phosphine adduct at 45 C by migratory insertion of the hydride ligand into the alkylidene functionality. 18a The structure of 12 was also determined by X-ray diffraction studies (Fig. 13). Compound 12 contains an sp 3 hybridized backbone carbon bound to PMe 3 , with an average dihedral angle of 78.9 between the planes defned by P( 3 27 Other phosphines, such as Ph 3 P did not afford an isolable product probably because of steric crowding. A broad peak at d ¼ 72 ppm was observed in the 31 P{ 1 H} NMR spectrum of the reaction mixture of 5 and Ph 3 P, indicating that some interaction between the carbenium carbon and Ph 3 P occurred upon mixing, but a complicated mixture was formed after heating the reaction at 60 C. ## Conclusions In conclusion, the one-electron oxidation of the palladium carbene complex [{PC(sp 2 )P} tBu Pd(PMe 3 )] (3) with I 2 generated a monomeric radical carbene complex [{PCc(sp 2 )P} tBu PdI} ( 4), which upon a second one-electron oxidation with [Cp 2 Fe][BAr F 4 ] formed a cationic carbene complex, [{PC(sp 2 )P} tBu PdI][BAr F 4 ] (5). We were able to isolate and characterize for the frst time a whole series (Fig. 1) of an anionic carbene (3), a carbene radical (4), and a cationic carbene (5). Our studies show that: (1) transition metal carbene complexes possess a rich redox chemistry that allows the tuning of the electronic properties of the carbene carbon; (2) an umpolung of the M]C carbene bond can be accomplished by successive electron transfer processes; and (3) the electron transfers among the carbene, radical, and cationic species are reversible. ## Experimental All experiments are performed under an inert atmosphere of N 2 using standard glovebox techniques. Solvents hexanes, npentane, diethyl ether, and CH 2 Cl 2 were dried by passing through a column of activated alumina and stored in the glovebox. THF was dried over LiAlH 4 followed by vacuum transfer and stored in the glovebox. CDCl 3 was dried over 4 molecular sieves under N 2 , while C 6 D 6 was dried over CaH 2 followed by vacuum transfer, and stored in the glovebox. Complex 3, [Cp 2 Fe] [BAr F 4 ] and KC 8 were prepared according to literature procedures. 9b,28 p TolNHLi was prepared by deprotonation of p-toluidine with n BuLi, while PhONa was prepared from NaH and phenol. 1 H, 13 C{ 1 H}, 31 P{ 1 H}, 19 F{ 1 H} and 11 B{ 1 H} NMR spectra were recorded on a Bruker DRX 500 spectrometer. All chemical shifts are reported in d (ppm) with reference to the residual solvent resonance of deuterated solvents for proton and carbon chemical shifts, and to external H 3 PO 4 , BF 3 $OEt 2 , and CFCl 3 for 31 P, 11 B, and 19 F chemical shifts, respectively. Magnetic moments were determined by the Evans method 29 by using a capillary containing 1,3,5-trimethoxybenzene in C 6 D 6 as a reference. EPR spectrum of compound 4 was recorded on a Bruker EMXplus EPR spectrometer with a standard X-band EMXplus resonator and an EMX premium microwave bridge. Cyclic voltammetry was performed on a Metrohm Autolab PGSTAT-128N instrument. Elemental analyses were performed on a CE-440 Elemental analyzer, or by Midwest Microlab. Gaussian 03 (revision D.02) 30 was used for all reported calculations. The B3LYP (DFT) method was used to carry out the geometry optimizations on model compounds specifed in text using the LANL2DZ basis set. The validity of the true minima was checked by the absence of negative frequencies in the energy Hessian. Synthesis of [{PCc(sp 2 )P} tBu PdI] ( 4) Iodine (11 mg, 0.043 mmol) in 1 mL of THF was slowly added to a dark-brown solution of 3 (60 mg, 0.087 mmol) in 1 mL of THF at 35 C. Upon addition, a dark-green solution was formed immediately and stirred at ambient temperature for 30 min. All volatiles were removed under reduced pressure and the darkgreen residue was extracted with n-pentane (2 4 mL). After reducing the volume of the pentane solution to about 1 mL, the solution was stored at 35 C to give 4 as a dark-green crystalline solid; yield 62 mg (97%). Compound 4 is paramagnetic. Magnetic moment (Evans method, 298 K): m eff ¼ 1.85 m B ; EPR: g ¼ 2.0000. Anal. calcd for C 33 H 52 IP 2 Pd (744.04 g mol 1 ): C, 53.27; H, 7.04. Found: C, 53.05; H, 7.24. ## Reduction of 4 with KC 8 KC 8 (2.3 mg, 0.016 mmol) was mixed with 4 (12 mg, 0.016 mmol) and PMe 3 (32 mL, 0.032 mmol, 1 M in THF) in 1 mL THF at room temperature. The mixture turned dark-brown immediately. After stirring the reaction mixture for about 5 min, the volatiles were removed under reduced pressure and the residue was extracted with 1 mL of n-pentane. The solution was fltered through celite. Compound 3 was obtained as dark brown crystalline solid from this n-pentane solution at 35 C. 1 H and 31 P { 1 H} NMR spectra were identical with the previously reported data. 9b Yield 9 mg (80%). Synthesis of [{PC(sp 2 )P} tBu PdI][BAr F 4 ] (5) [Cp 2 Fe][BAr F 4 ] (83.2 mg, 0.079 mmol) in 3 mL of diethyl ether was slowly added to a dark-green solution of 4 (59 mg, 0.079 mmol) in 2 mL of diethyl ether at 35 C. Upon addition, a darkred solution was formed immediately that was stirred at room temperature for 15 min. After reducing the volume of the ether solution to about 1 mL, n-pentane (about 8 mL) was layered and the mixture was stored at 35 C overnight to afford compound 5 as a dark-red crystalline solid, which was washed with npentane and dried under vacuum; yield 119 mg (94%). 1 H NMR (500 MHz, CDCl 3 , 25 C): d ¼ 7.93 (td, 4 J HH ¼ 3.8 Hz, 3 J HP ¼ 1.5 Hz, 2H, ArH), 7.73 (m, 4H, ArH), 7.64 (s, 8H, ortho-Ar F H), 7.46 (s, 4H, para-Ar F H), 2.99 (m, 4H, CH(CH 3 ) 2 ), 1.39 (s, 18H, C(CH 3 ) 3 ), 1.36 (dt, 3 J HH ¼ 9.0 Hz, 3 J HP ¼ 9.5 Hz, 12H, CH(CH 3 ) 2 ), 1.26 (dt, 3 J HH ¼ 9.0 Hz, 3 J HP ¼ 8.0 Hz, 12H, CH(CH 3 ) 2 ) ppm; 13 Reduction of 5 with KC 8 KC 8 (2.6 mg, 0.018 mmol) and 5 (30 mg, 0.018 mmol) were mixed in 2 mL of THF at room temperature, which formed a dark-green solution immediately. After stirring the reaction mixture for about 5 min, all volatiles were removed under reduced pressure. The residue was extracted with 4 mL of npentane, fltered, and the pentane was removed under reduced pressure. The resulted green solid was mixed with 0.5 equiv. of 9,10-dihydroanthracene (2 mg, 0.010 mmol) in C 6 D 6 and heated at 80 C for 7 d to afford compound 6 (vide infra). Yield 12 mg (92%). Synthesis of [{PC(sp 3 )HP} tBu PdI] ( 6) 9,10-dihydroanthracene (3.6 mg, 0.020 mmol) and 4 (30 mg, 0.040 mmol) were mixed in 0.6 mL of C 6 D 6 and heated at 80 C for 7 d, during which time the dark-green solution slowly turned to bright yellow. All volatiles were removed under reduced pressure and the residue was extracted with 5 mL of n-pentane. After reducing the volume of the pentane solution to about 1 mL, the by-product anthracene precipitated as a white crystalline solid, which was fltered, and the solution stored at 35 C to give orange crystals. Further recrystallization from n-pentane at 35 C afforded 6 as analytically pure orange crystals; yield 24 mg (81%). 1 H NMR (500 MHz, C 6 D 6 , 25 C): d ¼ 7.46 (td, 4 J HH ¼ 4.3 Hz, 3 J HP ¼ 2.0 Hz, 2H, ArH), 7.42 (dm, 3 J HH ¼ 8.0 Hz, 2H, ArH), 7.22 (dm, 3 J HH ¼ 8.0 Hz, 2H, ArH), 6.41 (s, 1H, CH backbone ), 2.78 (m, 2H, CH(CH 3 ) 2 ), 2.60 (m, 2H, CH(CH 3 ) 2 ), 1.44 (dt, 3 J HH ¼ 7.0 Hz, 3 J HP ¼ 7.8 Hz, 6H, CH(CH 3 ) 2 ), 1.41 (dt, 3 J HH ¼ 7.5 Hz, 3 J HP ¼ 7.8 Hz, 6H, CH(CH 3 ) 2 ), 1.23 (s, 18H, C(CH 3 ) 3 ), 1.16 (dt, 3 J HH ¼ 7.5 Hz, 3 J HP ¼ 7.5 Hz, 6H, CH(CH 3 ) 2 ), 1.12 (dt, 3 J HH ¼ 7.5 Hz, 3 J HP ¼ 7. 3 Hz, 6H, CH(CH 3 ) 2 ) ppm; 13 Synthesis of [{PC(sp 3 ) (SPh)P} tBu PdOTf] (8) AgOTf (9.6 mg, 0.038 mmol) in 1 mL of THF was added to 7 (32 mg, 0.038 mmol) in 1 mL of THF at room temperature. The resulting orange slurry was stirred for 4 h, slowly turning to a pale yellow slurry. The volatiles were removed under reduced pressure and the residue was extracted with 5 mL of benzene ## Reaction of 5 with NaH NaH (0.6 mg, 0.024 mmol) and 5 (32 mg, 0.020 mmol) were mixed in 1 mL of THF at room temperature. The dark-red mixture was allowed to stir for 24 h, during which time a yellow solution was slowly formed. Removal of volatiles under reduced pressure led to a yellow solid. The 1 H and 31 P{ 1 H} NMR of the crude yellow solid in C 6 D 6 showed clean conversion to 6 and NaBAr F 4 . Synthesis of [{PC(sp 3 )(NH p Tol)P} tBu PdI} (10) p TolNHLi (3.3 mg, 0.029 mmol) in 1 mL of THF was slowly added to a dark-red solution of 5 (46 mg, 0.029 mmol) in 1 mL of THF at 35 C. The resulting brownish-green solution was then stirred at room temperature for 15 min. The volatiles were removed under reduced pressure and the residue was extracted with 5 mL of benzene and fltered. Removal of volatiles under reduced pressure gave a greenish-yellow residue, which was extracted with n-pentane (8 mL), fltered, and the volume of the pentane solution reduced to about 1 mL. Storing this solution at 35 C gave compound 10 as yellow crystals; yield 12 mg (49%).

chemsum

{"title": "Redox-induced umpolung of transition metal carbenes", "journal": "Royal Society of Chemistry (RSC)"}

cell-level_comparisons_between_literature_and_industry_for_lithium-sulfur,_lithium-ion,_lithium-oxyg

## Abstract: A set of 212 lithium-sulfur, lithium-ion, lithium-oxygen, and other next-generation experimental battery articles was reviewed, and 15 articles provided at least one cell-level performance metric and/or a complete set of information to calculate it. This subset was compared against 27 commercial technologies in key metrics across various energy storage applications using an Excel database tool. While many high cell-level specific energy batteries are reported in the literature, there is a lack of demonstrated high cell-level specific power batteries for any chemistry. Li-S applications are comparable to Li-ion in terms of specific power and generally have higher specific energy, but also exhibit lower cycle life. In future work and as more articles include cell-level information, this tool can be expanded to include thousands of data points, enabling development of new models via machine learning and other methods to predict relationships between battery structure and performance. Additionally, the tool would become more useful for industry experts to effectively search and sort through the vast amount of knowledge accessible in research journals. ## Introduction Battery technologies have helped to better the standard of living of millions of people through renewable energy , electric vehicle [1, , consumer electronic , medical , unmanned aerial vehicle (UAV) , military [3, and other applications. Batteries will continue transforming these sectors as systematic improvements are made in a range of key areas including specific energy [1,3, 23], specific power [1, , affordability (i.e. low-cost) [1, , cycle life [1,24, , amples of keywords include "lithium-sulfur", "lithium-air", "silicon anode", "flow battery", "specific energy", "specific power", and "cell-level." From a master list of articles, those that were experimental in nature were manually sorted from those that were theoretical, computational, or a review article, resulting in the 212 experimental articles considered for review. The experimental articles were manually evaluated to determine whether they reported or included enough information to calculate a cell-level metric. If sufficient information was included, experimental details from these articles were extracted and used to calculate cell-level metrics. Due to the differences in type of information reported and cell-design, each article required an individualized approach to make cell-level calculations (see for example, Table ??). It was also noted whether a paper with cell-level information made a claim to commercial potential of the battery and if a celllevel metric or set of metrics from the article was comparable to or exceeded that of a commercialized state-of-the-art solution. ## Visual Database via Radar Chart Cell-level performance of characteristic commercial technologies from the high-impact applications highlighted in this work were obtained from online datasheets, retail websites, and personal communications with companies. Experimental battery articles are compared with commercial solutions. ## Tool Features The tool offers versatile features to visually display the database information using sorting and filtering. A demonstration of the various features is provided in the supporting information. Best-in-Class sorting is a feature useful for identifying leaders in particular categories and can be helpful in evaluating the potential of ones own work in comparison with commercial technologies. In addition, it may provide insight for those looking to find available options or compare their products against competition. When evaluating whether or not a technology meets the needs of a particular application, custom filtering can help in comparing the oftentimes multiple minimum requirements involved. Data from multiple categories that is below a particular value can be excluded, resulting in a condensed and more complexly sorted list. There are 15 articles that report sufficient information to determine cell performance criteria such as specific energy and specific power. Within these 15 articles, some were even competitive with state-of-the-art commercial technologies in that a metric or combination of metrics was comparable with state-of-the-art technologies (e.g. high specific energy, high affordability). Because not all criteria are essential to know for each application, for simplicity, criteria can be manually added or removed. When the above two techniques (Best-in-Class Sorting and Custom Filtering) do not properly refine the data, technologies can similarly be manually included or excluded. ## Assessment of Next-Generation Literature This research reviews 212 experimental battery articles up until 2017 with a focus towards Li-S articles (Figure 1). Other types of batteries considered include lithium-air, silicon anode, lithium-ion, and flow batteries. Ones that either include enough information to calculate or directly report cell-level performance are shown in Table 1. The first set [27,46, represent articles that cite manufacturing potential, include a complete set of component information to make cell-level calculations, and do not directly report these metrics. The second set consists of those that directly report a metric at the cell-level and do not include a complete set of component level information to calculate cell-level metrics. The third set consists of articles that include a complete set of component level information and report a cell-level metric . There is a trade-off between specific energy and specific power, as well as a lack of representation above 100 W/kg for specific power in the literature. Arrows highlight which batteries are primary batteries (i.e. cycle life of 1). In the case of EV and RE applications, the two batteries highlighted are used in both applications. For the first set, cell-level performance was calculated from component-level information. The component-level information from each paper typically required an individualized approach to calculate cell-level performance due to cell configurations (e.g. coin cell vs. flow cell) and reported information (e.g. areal loading thickness vs. molarities) differing between papers. For the second set, some component-level information was not present that would be necessary to calculate a cell-level performance metric. This made it infeasible to make reasonable cost predictions. The percentage of experimental articles that contain some type of cell-level information and the importance of this type of information is consistent with observations by various groups . Including both comprehensive component-level information and cell-level metrics provides an opportunity for industry to easily make evaluations and comparisons of cutting-edge battery research and helps research groups narrow down key system limitations . This can help researchers compare potential for impact and high-priority gaps in knowledge. Quantities of materials such as electrolyte and lithium metal are often left unmeasured or unreported . By tracking and reporting these material types and quantities (e.g. "100 µL 1M LiTFSI in DME/DIOX (1:1) used as electrolyte" or "50 mg, 2 cm 2 dia. lithium metal foil used as anode"), failure mechanisms may be easier to elucidate because the performance of many next-generation chemistries change drastically based on parameters such as excess electrolyte or lithium . With an increase in reported information, these trends could be analyzed in broader ways through methods such as machine learning . Because processing methods and characterization parameters can vary drastically and the number of parameters are often extensive, many articles with such information are essential for reliable insights . This perspective is not meant to detract from the importance of understanding fundamental chemistries and cross-component and system-level challenges of next-generation energy storage technology. An interdisciplinary approach that balances basic studies of chemistry systems and battery components with a knowledge of practical energy storage application requirements is required for successful implementation of next-generation technologies. Due to lack of reported information and the subjective nature of storage characteristics and safety categories, for the purposes of this study, qualitative rankings are assigned based on chemical composition and material choice. For example, literature batteries that contain additives such as LiNO 3 are given Table 2: Summary of experimental data from industry. Six performance metrics -Specific energy (SE), specific power (SP), number of cycles to 80% capacity, affordability (energy affordability and lifetime energy affordability), safety, storage characteristics, and energy density (ED) -are given of characteristic technologies from commercial, start-up, and literature batteries. Safety and storage characteristics are assigned qualitative rankings based on the chemistry and the constituents of the cell/system. Commercial affordability data was obtained through online sources (see "Ref" column), whereas literature affordability data was estimated from raw material costs and complexity of processing. * BPS stands for Battery Power Solutions. lower safety rankings. ## Ragone and Radar Data Data is presented detailing Ragone plot comparisons within literature (Figure 2), among literature, start-ups, and commercial technologies (Figure 3), among various chemistries(Figure 4), and corresponding radar charts. In literature, very high specific energies can be achieved (Figure 2); however, many fall short of specific power values obtained in industry (Figure 3). Interestingly, when considering all secondary literature batteries (i.e. non-primary batteries), there is not a significant trade-off between specific energy and specific power; rather, a generally increasing trend is observed. This could be related to the amount of excess materials in the battery and the battery's ability to function well with an optimized amount of excess material or to a trade-off not seen in metrics other than cell-level specific energy and specific power. There are no start-up affordability data points due to lack of available information. This is to be expected, since estimating cost from components would require proprietary information from the, and the batteries are usually only available as evaluation cells or through private contracts. Li-S, Li-ion, and Li-O2 batteries each have examples of high specific energy (Figure 4). Several Li-S and Li-ion data points are near each other, with Li-S generally exhibiting higher cell-level specific energy. One of the major differences is that the cycle life of the Li-ion batteries is generally higher than the Li-S batteries (Table 2). The differences in shapes of the radar charts (Figures 5 and 6) illustrate that maximizing a single criteria does not necessarily produce the same kinds batteries. This supports the notion that multiple battery requirements change for a given application. Literature data is again noticeably lacking in terms of cell-level specific power. ## Conclusion A framework is provided to systematically integrate and compare experimental battery research data via a visual database tool aimed at helping to bridge the gap between science and industry. Cell-level data had limited availability in the literature. Reporting such information enables calculations of critical battery performance metrics including cell-level specific energy, cell-level specific power, and cell-level affordability estimates. Ragone and radar charts are used to compare technologies and identify trends across literature and industry. While great strides have been made towards higher specific energy batteries, this study reveals that high specific power batteries are lacking in the literature. Cycle life information is typically available in most experimental secondary battery publications; however, metrics such as energy retention (alternatively, self-discharge) are often not reported. Safety is more difficult to quantify but can be done to some extent by reporting the aforementioned information and evaluating the energy, power, and materials. Safety can be more rigorously evaluated through shock, short-circuit, and puncture tests. Additionally, and as application-specific focuses arise, research groups may consider studying and reporting other metrics such as temperature performance, energy efficiency, volumetric energy and power density, fast charge characteristics, and voltage stability. Ragone and radar charts supplied in the visual database tool are useful for planning and comparison purposes in both science and industry. The readers are encouraged to use these tools to inform their studies of high-impact battery chemistries. The visual database tool and relevant data are provided as an attachment for the reader. Funding: This work was supported through a BYU Office of Research and Creativities (ORCA) Grant.

chemsum

{"title": "Cell-level Comparisons between Literature and Industry for Lithium-Sulfur, Lithium-Ion, Lithium-Oxygen, and other Next-Generation Batteries", "journal": "ChemRxiv"}

iron_complexes_of_tetramine_ligands_catalyse_allylic_hydroxyamination_via_a_nitroso–ene_mechanism

## Abstract: Iron(II) complexes of the tetradentate amines tris(2-pyridylmethyl)amine (TPA) and N,N′-bis(2-pyridylmethyl)-N,N′dimethylethane-1,2-diamine (BPMEN) are established catalysts of C-O bond formation, oxidising hydrocarbon substrates via hydroxylation, epoxidation and dihydroxylation pathways. Herein we report the capacity of these catalysts to promote C-N bond formation, via allylic amination of alkenes. The combination of N-Boc-hydroxylamine with either FeTPA (1 mol %) or FeBPMEN (10 mol %) converts cyclohexene to the allylic hydroxylamine (tert-butyl cyclohex-2-en-1-yl(hydroxy)carbamate) in moderate yields. Spectroscopic studies and trapping experiments suggest the reaction proceeds via a nitroso-ene mechanism, with involvement of a free N-Boc-nitroso intermediate. Asymmetric induction is not observed using the chiral tetramine ligand (+)-(2R,2′R)-1,1′-bis(2-pyridylmethyl)-2,2′-bipyrrolidine ((R,R′)-PDP). ## Introduction The selective functionalization of C-H bonds is an area of considerable current research interest . The development of methods for catalytic C-H amination has attracted particular attention , given the significance of C-N bonds to the structures of biologically active natural products and pharmaceuticals. In this context there has been a renewed focus on the chemistry of acylnitroso species in recent times , in particular on α-hydroxyamination of carbonyl compounds via nitrosocarbonyl aldol reactions and allylic hydroxyami-nation of alkenes via nitroso-ene reactions . Several new developments in the related hetero-Diels-Alder reaction of acylnitroso species have also been reported recently . These methodologies generally involve in situ generation of the acylnitroso species, achieved using a variety of oxidants including vanadium- , manganese- , iron- , copper- , rhenium- , and rhodium- based reagents. The recent resurgence of interest in the nitroso-ene reaction builds on earlier work by Sharpless, Nicolas, Jørgensen and others. Sharpless reported allylic amination of 2-methyl-2hexene with N-(p-chlorophenyl)hydroxylamine using a molybdenum complex , a process that was made catalytic by adding excess N-phenylhydroxylamine . The combination of iron(II) phthalocyanines or iron(II)/iron(III) chloride and N-phenylhydroxylamine effect allylic amination reactions that are believed to follow a nitroso-ene mechanism. Similar reactions have been reported using copper salts and N-phenylhydroxylamine or N-Boc-hydroxylamine , presumably via oxidation of the hydroxylamine to a nitroso species which then undergoes the nitroso-ene reaction. Stemming from our interest in iron-catalysed hydrocarbon oxidation using systems inspired by the non-heme iron-dependent enzyme family , we have investigated the capacity of iron complexes of simple tetramine ligands to promote the reaction between an alkene and N-Boc-hydroxylamine. Herein we report that iron complexes of tris(2-pyridylmethyl)amine (TPA, 1) , N,N′-bis(2-pyridylmethyl)-N,N′-dimethylethane-1,2diamine (BPMEN, 2) and (+)-(2R,2′R)-1,1′-bis(2pyridylmethyl)-2,2′-bipyrrolidine ((R,R′)-PDP, 3) (Figure 1) catalyse the allylic amination of cyclohexene. Mechanistic investigations suggest the reaction proceeds via nitroso-ene reaction of the oxidised hydroxylamine and the alkene. ## Synthesis of metal complexes The tetramine ligands TPA (1), BPMEN (2) and (R,R′)-PDP (3) were synthesised following literature procedures , then combined with iron(II) triflate as previously reported to generate the complexes [Fe(TPA)(CH 3 CN) 2 ](OTf) 2 (FeTPA, 4) , [Fe(BPMEN)(OTf) 2 ] (FeBPMEN, 5) and [Fe(R,R′-PDP)(OTf) 2 ] (Fe(R,R′)-PDP, 6) . ## Allylic amination reactions As an extension of our previously reported iron-catalysed allylic oxidation of cyclohexene (7) , we wished to explore potential C-N bond formation at this position using iron catalysis. Combining cyclohexene (7, in excess) with N-Bochydroxylamine (8) as the nitrogen source and the iron complex FeTPA (4) or FeBPMEN (5) afforded a mixture of products: the allylic hydroxylamine 9 alongside the Fenton oxidation products alcohol 10 and ketone 11 , and a small amount of tertbutyl carbamate (12, Scheme 1). Initial reactions under an argon or air atmosphere returned product mixtures in the ratios shown in Table 1. Scheme 1: Allylic hydroxyamination of cyclohexene (7) using iron catalysts 4 and 5; i. 4 or 5 (10 mol %), BocNHOH (8), CH 3 CN, rt, 18 h; for yields see Table 1. Under an argon atmosphere, the allylic hydroxylamine 9 was produced in ~10% yield with either ligand; performing the reaction open to air lifted the yield of the allylic hydroxyamination product 9 as high as 40%, but also substantially increased yields of 10 and 11 (Table 1). Control experiments using just the metal salt or each of the ligands on their own returned trace quantities of product 9 and varying levels of Fenton-type pathways (Table S3, Supporting Information File 1), confirming that FeTPA (4) and FeBPMEN (5) are active agents in promoting allylic hydroxyamination of cyclohexene. The effect of catalyst loading was screened under an air atmosphere, since initial results indicated that better yields of 9 are obtained under air than argon. Thus cyclohexene (0.7 mL, 7 mmol, 100 equiv) was added to a solution of catalyst 4 or 5 (1-20 mol %) and BocNHOH (70 μmol, 1 equiv) in CH 3 CN (Table S4, Supporting Information File 1). Lowering the catalyst loading of FeTPA from 10 to 5 mol % led to a small a Each reaction was performed in triplicate; data are averages of at least three runs. b Yields determined by GC using single point internal standard method (Tables S1 and S2, Supporting Information File 1). c Yields are quoted relative to the initial amount of BocNHOH ( 8), limiting reagent for the hydroxyamination reaction of interest. Formation of products 10 and 11 is not dependent on hydroxylamine 8 so the combined yields for some entries in this table are more than 100%. increase in the yield of allylic hydroxylamine 9 with a significant decrease in the appearance of allylic oxidation products 10 and 11. The amount of FeTPA ( 4) could be further lowered to 2 and 1 mol %, bringing further small increases in the yield of 9. However increasing loading of FeTPA (4) to 20 mol % halts the amination reaction, returning only allylic oxidation products 10 and 11. In contrast, changing the catalyst loading of FeBPMEN ( 5) up or down from 10 mol % lowers yields of 9; at 1 mol % or 20 mol % loading of catalyst 5, increased levels of 10 and 11 are observed, but at 5 mol % catalyst 5, the yields of all three oxidation products are diminished. Clearly the competing hydroxyamination and Fenton reaction pathways are sensitive to the amount of catalyst used relative to BocNHOH; optimum catalyst loadings are 1 mol % for 4 and 10 mol % for 5. Nicholas and Kalita have reported that the addition of hydrogen peroxide can improve yields in their copper-catalysed allylic amination reactions using BocNHOH . Thus the addition of hydrogen peroxide (1:1 relative to BocNHOH) to reactions with 4 or 5 was investigated. Using a 1:1:1 ratio of cyclohexene:BocNHOH:H 2 O 2 with FeTPA (4) at 1 mol %, allylic hydroxylamine 9 was formed in only 4% yield, with the allylic oxidation products 9 and 10 predominant. This is not unexpected given the propensity of hydrogen peroxide to react directly with iron complexes to produce 10 and 11 via Fentontype pathways . We have previously observed solvent-dependent behaviour by non-heme iron complexes when mediating oxidation of cyclohexene in methanol versus acetonitrile as solvent . Using methanol as solvent in the allylic amination reactions with FeTPA (4, 1 mol %) and cyclohexene in excess, yields of allylic hydroxylamine 9 dropped: 9 was formed in 10% yield (vs 27% in acetonitrile), while yields of allylic oxidation products 10 and 11 were also lowered, to 25% and 38% respectively (vs 54% and 36% in acetonitrile). BocNH 2 (12) was not observed. Using FeBPMEN (5, 10 mol %) in methanol, 10 and 11 were formed but target compound 9 was not observed at all. Presumably with methanol as solvent, the oxidising power of iron:ligand system is partially redirected to oxidise the solvent. With a view to improving the synthetic potential of this reaction, the transformation was attempted at a 1:1 ratio of BocNHOH to cyclohexene. Thus BocNHOH (70 μmol), FeTPA 4 (1 mol %) and cyclohexene (70 μmol) were combined in acetonitrile and stirred for 18 hours at room temperature, open to the air. Allylic hydroxylamine 9 was formed in 6% yield; allylic oxidation products 10 and 11 were each observed in ≤1%. Reaction at 2:1 BocNHOH:cyclohexene did not significantly improve the yield of allylic amine 9 (7%). Similar results were obtained using FeBPMEN (5, 10 mol %) as catalyst, which yielded small amounts of 9 (8%) and 10 (2%) but not ketone 11. In their work using copper(I) iodide to catalyse similar reactions, Iwasa et al. conducted reactions at much higher concentrations of hydroxylamine and alkene (0.5 mmol BocNHOH and 0.75 mmol alkene in a total reaction volume of 1 mL) . With this in mind, the FeBPMEN (5) reaction was repeated at 10-fold higher concentration (i.e., 1:1 BocNHOH/cyclohexene in a total reaction volume of 1 mL). Under these conditions the yield of allylic amine 9 doubled relative to the more dilute 1:1 reaction, to 17%; 10 and 11 were not observed. ## Reaction using a chiral catalyst The chiral catalyst Fe(R,R′)-PDP (6) has been used previously to promote asymmetric C-H oxidation reactions . With a view to developing an asymmetric iron-catalysed allylic hydroxyamination reaction, catalyst 6 was prepared and used to effect conversion of cyclohexene 7 to hydroxylamine 9. This reaction afforded 9 in 13% yield, but only as the racemate: analysis by chiral GC (CP-Chirasil-Dex CB column) revealed two peaks with equal peak areas (t R = 8.6 and 8.8 minutes); the same peaks in the same ratio were observed using a reference sample of racemic 9. Scheme 2: Proposed mechanism for hydroxyamination of cyclohexene (7) by FeTPA (4) and FeBPMEN ( 5): (a) iron-mediated oxidation of BocNHOH (8) by O 2 affords the nitroso-species 13, which (b) undergoes an ene reaction with the alkene substrate; (c) an alternative disproportionation reaction to convert 8 to 13 can occur without involvement of O 2 and also generates BocNH 2 (12), observed as a byproduct at low levels (Tables 1, S3 and S4, Supporting Information File 1). The mechanistic evidence gathered to date suggests the formation of a free nitroso species and 'off-metal' reaction with the alkene. Several groups have recently reported efficient methods for the asymmetric hydroxyamination of carbonyl compounds using acylnitroso species generated in situ along with chiral oxazolines , N-oxides or amines as ligands or organocatalysts. In these nitrosoaldol contexts, the chiral agents induce asymmetry by virtue of their influence over the enolate reaction partner. Achieving asymmetric induction in the nitroso-ene reaction is a trickier proposition , although this has been demonstrated in an intramolecular context . ## Mechanistic studies Previous studies of iron-promoted allylic amination reactions with N-phenylhydroxylamine, and copper-catalysed reactions with N-Boc-hydroxylamine (8) return regio-and chemoselectivity profiles that are consistent with reaction via nitroso-ene mechanisms . Thus we hypothesised that the hydroxyamination reactions mediated by FeTPA (4) and FeBPMEN (5) follow a similar mechanism: iron-catalysed oxidation of 8 to generate the N-Boc-nitroso intermediate 13, which then participates in an ene reaction with cyclohexene (7, Scheme 2). Several experiments were conducted to test this hypothesis. Spectroscopic experiments evince interactions between N-Bochydroxylamine (8) and FeTPA (4) in solution. An acetonitrile solution of FeTPA ( 4) is a dark red-brown colour; adding BocNHOH prompts a colour change to deep purple (λ max = 550 nm, ε ≈ 300 L mol −1 cm −1 ). Monitoring the electronic spectrum between 400 and 900 nm (Figure S1, Supporting Information File 1), this absorption peak reaches a maximum when 1 equivalent of N-Boc-hydroxylamine (8) has been added, consistent with coordination of hydroxylamine 8 to the FeTPA complex (4). A similar result is observed using 1 H NMR: titrating a solution of hydroxylamine 8 in d 3 -acetonitrile with FeTPA (4) indicates coordination of hydroxylamine 8 to the complex 4 (Figure S2, Supporting Information File 1). Direct observation of the proposed Boc-nitroso intermediate 13 is difficult given its reactive nature. However BocNH 2 (12), the other product of the proposed disproportionation of N-Bochydroxylamine (8, Scheme 2c) is observed as a side product. When N-Boc-hydroxylamine (8) was treated with FeTPA (4) in the absence of an alkene partner, BocNH 2 (12) was isolated in increased yield (25%). This mirrors the results of Jørgensen and Nicholas who have observed an analogous iron-catalysed reduction of N-phenylhydroxylamine to aniline, and confirms that FeTPA (4) can mediate the conversion of BocNHOH to BocNH 2 . Nitroso species can be trapped by hetero-Diels-Alder reaction with dienes , and detection of the resulting hetero-Diels-Alder adducts used to confirm the formation of free nitroso intermediates. Thus trapping experiments were conducted using isoprene (14) to investigate the formation of a nitroso species in this reaction. First a 1:1 mixture of cycloadducts 15 and 16 was synthesised as a reference sample using Kirby's conditions for the hetero-Diels-Alder reaction (N-Boc-hydroxylamine (8), isoprene (14) and sodium periodate, Scheme 3a) . Then isoprene (14) and N-Boc-hydroxylamine (8) were combined in acetonitrile in the presence of FeTPA (4) or FeBPMEN (5). Cycloadducts 15 and 16 were formed, along with the allylic amination product 17 in a 1:1:3 ratio (Scheme 3b); the observation of 15 and 16 in this reaction confirms that a Boc-nitroso species 13 is formed in the FeTPA/ FeBPMEN-catalysed reaction. It is interesting to note that the nitroso-ene product is not generally observed under the Kirby conditions, as the allylic hydroxyamination product 17 is unstable in the highly oxidising environment rendered by sodium periodate . Observation of this product in the FeTPA-and FeBPMEN-mediated reaction of isoprene indicates the relative mildness of these conditions for nitroso formation. Nicholas et al. have reported a similar experiment to study the iron(II,III) chloride-catalysed reaction of N-phenylhydroxylamine with 2,3-dimethyl-1,3-butadiene, in which the Diels-Alder cylcoadduct was not observed, and only the allylic amination product was formed . Conversely Jørgensen and Johannsen report that N-phenylhydroxylamine and 1,3cyclohexadiene in the presence of iron(II) phthalocyanine do form the Diels-Alder cycloadduct . Although the outcomes with the different catalysts were contrasting, only one product was observed in each case. Cenini et al. have reported that both cycloadduct and ene product are formed in the ruthenium-catalysed reaction of nitrobenzene (an alternate route to a nitrosobenzene intermediate) with isoprene . As mentioned above, isoprene produces two different regioisomers in the hetero-Diels-Alder reaction with nitroso compounds, yet Cenini et al. only detected one isomer. From this observation they concluded that their Ru-catalysed amination reaction and the Diels-Alder reactions were occurring 'on metal' without generation of a free nitroso species . In contrast, the FeTPA-mediated reaction generates both BocNO cycloadducts of isoprene along with the amination product. Furthermore, the reaction with the chiral system 6 renders zero asymmetric induction. Thus we conclude that the Fe-TPA/BPMEN reaction involves the nitroso-ene reaction of a free nitroso intermediate. ## Conclusion FeTPA (4) and FeBPMEN ( 5) are established catalysts for the hydroxylation, dihydroxylation and epoxidation of hydrocarbon substrates [48, . In this study we have shown that they can also catalyse the allylic hydroxyamination of alkenes with N-Boc-hydroxylamine. Mechanistic investigations suggest the involvement of a free nitroso species which undergoes a nitroso-ene reaction with the alkene. The intermediacy of a free nitroso species means that asymmetric induction is not observed in reactions with the chiral catalyst Fe(R,R′)-PDP (6). ## Experimental General experimental All commercially available reagents were used without purification unless otherwise specified. Solvents for extraction and chromatography were distilled before use. Solvents for reactions were freshly distilled immediately prior to use. Tetrahydrofuran (THF) was dried over sodium wire and benzophenone. Dichloromethane and acetonitrile were dried over calcium hydride. Acetonitrile was degassed using three freeze-thaw cycles when it was to be used in an atmosphere of argon. Methanol (MeOH) was dried over magnesium methoxide. Alkenes used in allylic amination reactions were passed through a micro-column of neutral alumina immediately before use. Water was purified using a Millipore purification system. Analytical thin-layer chromatography (TLC) was performed using preconditioned plates (Merck Kieselgel 60 F254). Developed TLC plates were viewed using a UV lamp at a wavelength of 254 nm and visualised with a ninhydrin stain. Flash column chromatography was performed on Davisil Grace Davison 40-63 μm (230-400 mesh) silica gel using distilled solvents. Melting points were recorded on a Stanford Research Systems Optimelt automated melting system and are uncorrected. and are reported as (c mg mL −1 , solvent). Infrared spectra were recorded on a Bruker ALPHA FTIR spectrophotometer (ZnSe ATR). Gas chromatography was carried out on a Hewlett Packard 5890A and 5890 Series II Gas chromatographs with ChemStation software using HP1 (Crosslinked Methyl Silicone Gum) and CP-Chirasil-Dex CB columns, respectively. Both chromatographs were equipped with split/splitless capillary inlets and flame ionization detectors (FID). UV-vis spectra were recorded on a Varian Carey 4000 UV-vis spectrophotometer. Synthesis of iron complexes 4, 5 and 6 and N-Boc-hydroxylamine (8) Tris(2-pyridylmethyl)amine (TPA, 1) and N,N′-bis(2pyridylmethyl)-N,N′-dimethylethane-1,2-diamine (BPMEN, 2) were synthesised in good yields following literature procedures (Supporting Information File 1). (+)-(2R,2′R)-1,1′-Bis(2-pyridylmethyl)-2,2′-bipyrrolidine ((R,R′)-PDP, 3) was synthesised from commercially available (R,R′)-2,2′-bipyrrolidine L-tartrate trihydrate according to the procedure reported by White and Chen . Ligands 1-3 were combined with iron(II) triflate using literature protocols to generate [Fe(TPA)(CH 3 CN) 2 ](OTf) 2 (FeTPA, 4) , [Fe(BPMEN)(OTf) 2 ] (FeBPMEN, 5) and [Fe(R,R′-PDP)(OTf) 2 ] (Fe(R,R′)-PDP, 6) (Supporting Information File 1) . N-Boc-hydroxylamine (tert-butyl hydroxycarbamate, BocNHOH, 8) was prepared using a modified literature procedure (Supporting Information File 1) . ## Hydroxyamination reactions Acetonitrile was freshly distilled from calcium hydride; for reactions under argon (i.e., anaerobic conditions), the solvent was subjected to three freeze-thaw degassing cycles immediately before use. Stock solutions of iron complex (22.6 mmol L −1 ) and BocNHOH (8, 70 mmol L −1 ) in degassed acetonitrile were prepared under an atmosphere of argon. Acetonitrile (8.0 mL) was stirred under the required environment (argon or air) while iron complex stock solution (0.3 mL, 6.8 μmol) and cyclohexene (0.7 mL, 6.9 mmol) were added. Using a syringe pump, the BocNHOH stock solution (1.0 mL, 70 μmol) was added to the reaction mixture over 30 min. The reaction was stirred for 18 h after which time the solvent was removed in vacuo. The residue was dissolved in ethyl acetate and passed through a micro-column of silica to remove the iron complex. The sample was subjected to analysis by GC using n-decane as an internal standard and the single point internal standard method (Supporting Information File 1) . Each reaction was performed in triplicate and data presented above are the average of the three runs.

chemsum

{"title": "Iron complexes of tetramine ligands catalyse allylic hydroxyamination via a nitroso\u2013ene mechanism", "journal": "Beilstein"}

mechanism-based_design_of_quinoline_potassium_acyltrifluoroborates_(kats)_for_rapid_amide-forming_li

## Abstract: Potassium acyltrifluoroborates (KATs) undergo chemoselective amide-forming ligations with hydroxylamines. Under aqueous, acidic conditions these ligations can proceed rapidly, with rate constants of ~20 M -1 s -1 . The requirement for lower pH to obtain the fastest rates, however, limits their use with certain biomolecules and precludes in vivo applications. By mechanistic investigations into the KAT ligation, including kinetic studies, X-ray crystallography, and DFT calculations, we have identified a key role for a proton in accelerating the ligation. We applied this knowledge to the design and synthesis of 8-quinolyl acyltrifluoroborates, a new class of KATs that ligates with hydroxylamines at pH 7.4 with rate constants >4 M -1 s -1 . We trace the enhanced rate at physiological pH to unexpectedly high basicity of the 8-quinoline-KATs, which leads to their protonation even under neutral conditions. This proton assists the formation of the key tetrahedral intermediate and activates the leaving groups on the hydroxylamine towards a concerted 1,2-BF3 shift that leads to the amide product. We demonstrate that the fast ligations at pH 7.4 can be carried out with a protein substrate at micromolar concentrations. ## Introduction Potassium acyltrifluoroborates (KATs) are robust, bench stable compounds that undergo amideforming ligations with O-substituted hydroxylamines under dilute, aqueous conditions. KAT ligation has already found applications in protein PEGylation, hydrogel formation and modification, and post-polymerization modification. The reaction is exceptionally fast at lower pH, with rate constants >20 M -1 s -1 at pH 2, but becomes slower as the pH is increased. 8 At pH 7, amide formation can still proceed but at rates that are not suitable for reactions at micromolar concentrations. We have previously employed 2-pyridine-derived KATs as they were superior to aryl derivatives at pH 3-6, 4 with rate constants of ~10 M -1 s -1 at pH 3.6. These improved substrates, however, showed only modest reaction rates at physiological pH, limiting their use for many desirable applications of chemoselective ligations. 9,10 In this report we document the synthesis and rapid amide-forming ligations of 8-quinoline-derived KATs, which undergo KAT ligation at pH 7.4 with second order rate constants up to 4 M -1 s -1more than 300 times faster than aryl derivatives. This report also provides detailed rate constants of various KAT derivatives at both pH 7.4 and pH 3.8. DFT calculations on the protonated and unprotonated reaction pathways provide insights into the mechanistic basis for the role of a proton in accelerating the KAT ligation. Characterization of the pyridyl-and quinolyl KATs -including titrations and X-ray crystallography -confirm the unexpectedly high basicity of the quinolyl KATs, which translates to superior reaction rates at higher pH. ## Results and Discussion Synthesis of KAT substrates. While initially challenging to produce, synthetic access to KATs has expanded markedly in the past few years. 2, These advances have allowed us to prepare a broad range of KATs containing a diverse range of functional groups and structural features. Based on our previous finding of superior ligation rates with pyridyl KATs, which we attributed to protonation of the pyridine nitrogen under acidic conditions, we aimed to synthesize a series of pyridyl-and quinolyl KATs that contain a basic atom near the reactive KAT group. Exploration of a number of hypotheses led to the initial finding that 8-quinolyl KATs performed better at higher pH, and we elected to systematically study this class of compounds further -along with the aryl and pyridyl KATs -with regard to the role of electron-donating (-OMe) and withdrawing (-Cl) groups incorporated onto the aromatic rings. 20,21 Quinolines are typically less basic than pyridines (pKa = 4.9 for quinoline, pKa = 5.2 for pyridine), 22 but place the basic nitrogen three, rather than two atoms away from the acylboronate carbonyl. 23 Phenyl-and pyridyl KATs were synthesized from their corresponding halides using one equivalent of n-BuLi and reagent 1 each at -78 °C in THF according to previous reports. 11 Applying these conditions to 8-quinolyl halides, however, failed to yield any product and further optimizations were needed. Screening of reaction conditions showed that by conducting the lithium-halogen exchange at -110 °C in a solvent mixture of diethyl ether and THF (4:1 v/v) and allowing the reaction mixture to stir for one hour prior to the addition of reagent 1, followed by quenching at this temperature with aqueous KF and overnight mixing to form a precipitate provided access to the desired compounds (Scheme 2). Scheme 2. All reactions were performed in an oven-dried Schlenk flask. n-BuLi was added dropwise over 30 min via syringe pump at -78 °C for phenyl-and pyridyl KATs and at -110 °C for quinolyl KATs; the reactions were quenched with 3.0 equiv 6.5 M KF(aq) solution and the cooling bath was removed, allowing the reaction mixture to warm to rt. Despite these improvements, the reaction between the 8-lithioquinolines and reagent 1 was sluggish and remaining reagent 1 formed a hydrolyzed side-product 2 that must be separated from the desired KAT. This could be accomplished by washing the crude mixtures with acetone to extract 2, as 8-quinolyl KATs, unlike most phenyl-and pyridyl KATs, do not dissolve in acetone. The desired KAT can be isolated from the remaining crude mixture using DMF, leaving behind the residual inorganic salts. ## Kinetics of KAT ligations. With KAT substrates 3-5 in hand, we carefully evaluated the rates of their ligation with hydroxylamine 6. For the determination of rate constants for KAT ligation, we selected UV-vis spectroscopy, as KATs have unique absorption bands that can be used for accurate determination of their concentration. We established calibration curves in buffered solutions at various pH values used for kinetic studies over the concentration range of 0 -15 mM (Figure 1b). Quinolyl KAT 5c failed to dissolve above 0.25 mM in the buffers used for UV-vis measurements, therefore KAT 5d was synthesized and used instead. We selected 1:1 (v/v) mixtures of acetonitrile with aqueous buffer solutions for the measurement of the ligation kinetics to ensure solubility of all reagents. KAT ligations work well in purely aqueous conditions, but these substrates required an organic co-solvent to ensure homogeneity throughout the course of the reaction at the various pH ranges and concentrations studied. To improve solubility of the starting materials and products, we designed and prepared water-soluble hydroxylamine 6. Potassium acetate buffer was employed for ligations at pH 3.8, and potassium phosphate buffer was used for experiments at pH 7.4. The ligation reaction was initiated by mixing pre-buffered stock solutions of the KATs (15 mM) with hydroxylamine 6 (15 mM) to give an initial concentration of 7.5 mM in each reactant. UV-vis spectra were recorded over a period of 1000 s to monitor the consumption of KATs. A representative set of UV absorption profiles over time is illustrated in Fig. 1c and shows the decay of the KAT specific band as the ligation progress. Approximately 100 time points were recorded over the 1000 seconds of measurement. The concentration of the KATs was determined by Beer's law using the determined molar absorption coefficients. Initial studies determined that the reaction is single order with respect to each reactant and the reaction was treated as an overall second order reaction with equimolar amounts of KAT and hydroxylamine. Linear regression of the reciprocal concentration values against time was used to determine the apparent second order rate constants (Figure 1d). With the uncertainty in absorbance measurement being as low as 0.001 a.u. in terms of standard deviation from blank measurements, the number of time points enabled the reliable determination of the background absorption as well as a reasonable fit to the assumed second order kinetics. Only the later portions of the data deviated slightly from ideal second order behavior. All measurements were repeated three times (marked in color) to calculate the averages and standard deviations of the rate constants. increase in rate with a methoxy substituent, and an 8.3-fold increase with a chloro substituent. The same trend also held true within the quinolyl KAT 5 series. Electron-donating groups enhance the basicity of the nitrogen, which should lead to a higher proportion of the protonated forms. 20 This would account for the fact that methoxy-substituted pyridyl KAT 4c and quinolyl KAT 5d were the most reactive ones in their respective aryl subgroups at pH 7.4, but the slowest at pH 3.8. At pH 3.8, all of the KATs examined participated in the KAT ligation with second order rate constants higher than 1 M -1 s -1 , but the alkoxy-substituted pyridine 4c and quinoline 5d were slower than their analogs. This was consistent with the expectation that all pyridyl-and quinolyl KATs studied would be protonated at pH 3.8 and inductive substituent effects that modulate the electrophilicity of the KAT dominated. Overall, the most reactive KAT at pH 3.8 was chloropyridyl KAT 4b. This confirms that, as expected, more electron deficient KATs are more reactive overall, but that the presence of a proton near the KAT groups greatly accelerates the reaction and becomes the determining factor at higher pH regimes. This UV-vis method was able to determine the reaction rate constants of KATs with hydroxylamine 6 by monitoring the consumption of KATs, rather than the formation of the ligation product amide. To verify that the rate of product formation correlated with the reactivity of the KATs, and not just a difference in the formation of an initial adduct, a competition experiment was designed. KATs 4a, 4b, and 4c with relative reactivities of 3.34, 8.33 and 16.7 to the reference at pH 7.4, were selected for pairwise competition with a limited amount of hydroxylamine 7 and the final product distribution was measured by 1 H-NMR. The parallel competition outcomes were compared to modeled results based on the initial concentration of reactants and rate constants. The agreement of observed and predicted results are depicted in Figure 3 (see SI for model and parameters). ## Mechanism of KAT ligations. Our basic understanding of the mechanism of KAT ligation featured a nucleophilic addition of the hydroxylamine to the KAT carbonyl, forming a tetrahedral intermediate, followed by its breakdown (Scheme 3). At the outset of our studies, however, we had little insight into the specific bond-forming and bond-breaking processes or the role of protonation in accelerating the KAT ligation. leading to a C to N 1,2-BF3 migration. 26 1,2-migration of the tetracoordinated boron group to carbon, nitrogen, and oxygen atoms has been shown for B-MIDA (N-methyliminodiacetic acid) acylboronates. 27 These migrations proceeded via a tetrahedral intermediate, where a leaving group at the β-position of the boronate initiated the migration (Scheme 5). The stereochemistry between the leaving group and the boronate group has also been found to effect the migratory aptitude of the boronate group. 27 The computed reaction pathways shown in Scheme 4 led to the formation of an N-BF3 amide as the primary ligation product, which was also observed in real time mass spectrometry analysis of the reaction mixture (see Supporting Information Fig. 22). ## TS-c BF3 -Scheme 4. Computed KAT ligation reaction of KATs 4a and 5a. Reaction paths with the pyridine or quinoline being protonated were colored in brown. The vertical axes between 0 and -80 kcal/mol were truncated to accommodate the free energy of the reaction products. Sums of free energies of the hydroxylamine and (protonated) KATs were set to zero as references. Computations were performed at the def2/ma-TZVP level. 24,25 Scheme 5. Instances of 1,2-migration of boronate groups in B-MIDA acylboronates reported by Yudin. 27 [B] = B-N-Methyliminodiacetate. In the computed ligation pathway of quinolyl KAT 5a, quinoline protonation also favored the formation of a hydrogen bond-tethered intermediate I-c. The quinolinium proton was in vicinity to three basic atoms: the quinoline nitrogen, the hemiaminal oxygen, and the carbamoyl oxygen. Interaction between the proton and these atoms fixed the F3B-C-N-O dihedral angle (marked in orange), across the C-N bond between the KAT carbonyl and hydroxylamine nitrogen, close to an antiperiplanar 180°. This conformation would facilitate the concerted C-B bond / N-O bond cleavage towards the transition state. The activation barrier from intermediate I-c, formed from protonated KAT 5a, to TS-c, was found to be slightly higher than that of from I-b to TS-b. This corresponded to the slightly higher observed rate of 4a ligating at pH 3.8, where both KATs are mostly protonated. Nitrogen containing heteroaryl aldehydes are known to participate in reactions of neighboring groups. For example, pyridine-2-carbaldehyde and quinoline-8-carbaldehyde form hydrazones faster than their corresponding hydrocarbon variants, benzaldehydes and naphthaldehydes. Kool et al. proposed that an intramolecular proton donor could facilitate both the formation and breakdown of the tetrahedral intermediate. 28 Our findings suggest that a similar mode of rate acceleration was operative in KAT ligations, especially for quinolyl KATs 5 -with some qualifications. Quinolyl KATs had faster ligation rates than pyridyl KATs at neutral pH. This suggestedcounterintuitively -that quinolyl KATs were protonated to a greater extent than their pyridine counterparts, as pyridine (pKa = 5.2) is known to be more basic than quinoline (pKa = 4.9). To examine further, we performed titrations of the KATs with hydrochloric acid in aqueous acetonitrile to measure their basicities, and determined that the conjugate acid of quinolyl KAT 5a has a pKa of 5.96, while the conjugate acid of pyridyl KAT 4a has a lower pKa of 4.92. The inversion of the inherent basicity of pyridyl-and quinolyl KATs implies that the proximal KAT group facilitates the protonation of quinoline KATs, possibly due to the formation of an internal salt resulting in a zwitterionic species that does not require the potassium gegenion. To investigate this further, we grew crystals of various quinolyl-and pyridyl KATs from either pH 7.4 buffers or dilute HCl solutions and determined their structures by X-ray diffraction. In the case of quinolyl KAT 5a, crystals grown from either dilute HCl or from pH 7.4 buffer both delivered the protonated, zwitterionic form (Figure 4a). Notably, electronic rich quinolyl KAT 5d also crystalized as the protonated form, while the chloro derivative 5b afforded unprotonated potassium salts from the same buffer. In contrast, all pyridine KAT crystals were obtained as their potassium salts. The carbonyl group for the zwitterionic species 5a is almost coplanar to the aromatic ring while KAT 5b shows the highest torsional angle between the carbonyl group and its aryl ring (Figure 4b). The computational investigations indicated the importance of carbamoyl protonation during the N-O bond cleavage during KAT ligation. This is consistent with the observation that all KATs ligate faster at lower pH and follow specific acid catalysis. 31 The phenyl KATs 3a-c showed rate constants of ~3 M -1 s -1 at pH 3.8. For KATs 4 and 5, protonation is likely to both increase their electrophilicity and accelerate the N-O bond cleavage by protonation of the carbamate, increasing the ligation rates to around 10 M -1 s -1 at the same pH. The more basic quinoline KATs 5a-d will have higher protonated fractions at neutral pH than pyridyl KATs 4, and are more likely KAT Ligations on Folded Proteins at pH 7.4. At the outset of this project, we sought to identify KAT ligation partners that formed amide bonds under physiological conditions with rate constants of at least 1 M -1 s -1 , which would make it suitable for bioconjugations at micromolar concentrations and potential in vivo applications. Although detailed applications of the quinolyl KATs to these goals are beyond the scope of this manuscript, we have confirmed the benefit of higher ligation rates of quinolyl KATs for protein modification. According to our previously reported protocol, a sfGFP(S147C) mutant was expressed from chemically competent Escherichia coli BL21(DE3) cells in high yields and treated with the thiophilic hydroxylamine derivative. 4 A 100 μM solution of the sfGFP(S147C)-hydroxylamine adduct 8 was treated with quinolyl KAT 5d (5.0 equiv) at pH 7.4 in the same buffer system used for the kinetic measurements and was fully converted to the amide product 9 within 90 min. In contrast, the same experiment with pyridyl KAT 4a returned only unreacted protein after 90 min at pH 7.4 (Figure 5). The reaction mixture was passed through a size-exclusion column to elute the protein in MilliQ water prior to mass analysis (ESI-MS). ## Conclusion In summary, we have identified 8-quinolyl KATs as uniquely suited for rapid amide-forming ligations at pH 7.4. They exhibit second order rate constants of 1-5 M -1 s -1 at physiological pH, hundreds of times faster than previously examined KATs. The origin of the enhanced reactivity was traced -through kinetic studies, titrations, X-ray crystallography, and DFT calculations -to an unexpectedly high basicity of the quinolyl KATs and a key role for the proton in the mechanism of the KAT ligation. These findings were translated to ligation of a model protein at micromolar concentration in pH 7.4 buffer and pave the way for applications of KAT ligation on systems that require rapid ligations at physiological pH.

chemsum

{"title": "Mechanism-Based Design of Quinoline Potassium Acyltrifluoroborates (KATs) for Rapid Amide-Forming Ligations at Physiological pH", "journal": "ChemRxiv"}

in_silico_identification_and_docking-based_drug_repurposing_against_the_main_protease_of_sars-cov-2,

## Abstract: The rapidly enlarging COVID-19 pandemic caused by novel SARS-coronavirus 2 is a global public health emergency of unprecedented level. Therefore the need of a drug or vaccine that counter SARS-CoV-2 is an utmost requirement at this time. Upon infection the ssRNA genome of SARS-CoV-2 is translated into large polyprotein which further processed into different nonstructural proteins to form viral replication complex by virtue of virus specific proteases: main protease (3-CL protease) and papain protease. This indispensable function of main protease in virus replication makes this enzyme a promising target for the development of inhibitors and potential treatment therapy for novel coronavirus infection. The recently concluded α-ketoamide ligand bound X-ray crystal structure of SARS-CoV-2 M pro (PDB ID: 6Y2F) from Zhang et al.has revealed the potential inhibitor binding mechanism and the determinants responsible for involved molecular interactions. Here, we have carried out a virtual screening and molecular docking study of FDA approved drugs primarily targeted for other viral infections, to investigate their binding affinity in M pro active site. Virtual screening has identified a number of antiviral drugs, top ten of which on the basis of their bending energy score are further examined through molecular docking with M pro . Docking studies revealed that drug Lopinavir-Ritonavir, Tipranavir and Raltegravir among others binds in the active site of the protease with similar or higher affinity than the crystal bound inhibitor α-ketoamide. However, the in-vitro efficacies of the drug molecules tested in this study, further needs to be corroborated by carrying out biochemical and structural investigation. Moreover, this study advances the potential use of existing drugs to be investigated and used to contain the rapidly expanding SARS-CoV-2 infection. ## Introduction The ongoing and rapidly spreading outbreak of the Covid-19 pandemic, which is being caused by the highly contagious and pathogenic SARS-Coronavirus-2 (SARS-CoV-2), has endangered the global public health and alarmed the scientific community at an unprecedented magnitude. The novel SARS-CoV-2 was first reported to have emerged in the live wildlife market in the Wuhan region of Hubei province, where it has caused mystic pneumonia-like respiratory illnesses in the human population of the area (1,2). Later, the virus has made its way under extensive and fast-paced human travel to other geographic locations in the world such as Japan, Australia, Southeast Asian countries, Western European countries, Middle Eastern Countries, and finally to USA, Canada, and India. According to data presented by the World Health Organization (WHO), as of March 23, 2020, the virus has infected more than 693,224 people in more than 190 countries around the world including a staggering 33,106 deaths, with a cumulative mortality rate of >4.7% (3). Despite the instantaneous, collaborative, and monumental research efforts from the scientific community around the globe, no vaccine or therapeutic intervention could be developed to cure or mitigate the Covid-19 so far. The treatment of severely ill patients has been limited to the use of prophylactic and symptomatic management. In contrast, the prevention of disease has been only effective through strong and preemptive measures from health organizations and governmental authorities such as the sought outs for practicing social distancing, maintaining respiratory hygiene, and impositions of public curfews and state-wide lockdowns. Coronaviruses (CoVs), which belong to family Coronaviridae of viruses, constitute an essential class of pathogens for humans and other vertebrates (4). Before the current SARS-CoV-2 induced pathogenesis, only six of the CoVs were known to cause mild to severe illnesses in humans. HCoV-229E, HCoV-OC43, HCoV-NL63, and HKU1, which fall in genera alphacoronavirus, cause milder upper respiratory disease in adults, and sometimes can also cause severe infection in infants and young children. Whereas the betacoronaviruses like SARS-CoV (severe acute respiratory syndrome coronavirus; which has triggered an epidemic in China during 2002-03) and MERS-CoV (Middle East respiratory syndrome coronavirus; an etiological agent of middle East coronavirus epidemic of 2012) have potential to cause infection in lower respiratory tract along with cough & fever and triggers severe respiratory illness in humans (5,6). The causative agent of the current outbreak, SARS-CoV-2, also belongs to betacoronavirusesand is closely related to SARS-CoV with an overall genomic sequence similarity of >79% (7). The virion of SARS-CoV-2 is consists of crown-shaped peplomers, 80-160 nm in diameter, and consists of a ~30 kb long single-stranded RNA molecule of positive polarity with 5' cap and 3' Poly-A tail (8). The RNA genome is composed of at least six open reading frames (ORFs) of which the first ORF (ORF1a/b) makes up the 5'two-third and encodes two polypeptides pp1a and pp1ab both of which furthermore leads to the production of 16 nonstructural proteins (nsPs). Other ORFs that make up the remaining one-third of the viral genome give rise to the production of four main structural factors of the virion: Spike protein (S), Envelope protein (E), Membrane protein (M) and Nucleocapsid protein (N) (9). The virus uses the heterotrimeric Spike (S) protein, which consists of S1 and S2 subunit, on its surface to interacts with the ACE2 (angiotensin-converting enzyme 2) cellular receptor, abundantly expressed on many cell types in human tissues (10). Upon internalization into the cell, genomic RNA is used as a template for direct translation of two polyprotein pp1a and pp1ab which encodes a number of crucial nonstructural proteins (nsPs) including two proteases; Chymotrypsin-like protease (3CL pro ) or main protease (M pro ) -nsP5 and a papain like protease (P pro ) -nsP3, both of which processes the polypeptide pp1a and pp1ab in a sequence specific manner to produce 16 different nsPs (11,12). The papain protease processes the polyprotein to generate nsP1-4 while the M pro operates at as many as 11 cleavage sites by specifically recognizing the sequence Leu-Gln*Ser-Ala-Gly (* marks the cleavage site) to generate rest of the critical nsPs including helicase, methyltransferase, and RNA dependent RNA polymerase (RdRp) all of which play a critical role in the viral infection cycle by forming a replicationtranscription complex (RTC) (13). Therefore, the main protease constitutes a major and attractive drug target to block the production of nonstructural viral components and thereby to hamper the replication event of the virus life cycle. Additionally, no human protease with similar cleavage specificity is known to rule out the possibility of cellular toxicity upon the potential inhibition of main viral protease. In recent years drug repurposing screens have emerged as a resourceful alternative to fasten the drug development process against rapidly spreading emerging infections such as the one of SARS-CoV-2 (14,15). The approach of drug repurposing has successfully led to the discoveries of potential drug candidates against several diseases such as Ebola disease, hepatitis C virus, and zika virus infection (16)(17)(18)(19). In the present study, we have applied the approach of in silico virtual screening and protein-ligand docking of a spectrum of Food and Drug Administration (FDA) -approved antiviral drugs against SARS-CoV-2 M pro . To this end a recently elucidated X-ray crystal structure of SARS-CoV-2 M pro (PDB ID: 6Y2F) which have been shown to harbor an α-ketoamide as a potent inhibitor in the enzyme's active site, was chosen and screened for a number of FDA approved antiviral drugs to simulate the M pro -αketoamide interactions and thereby blocking the active pocket (20). The crystal structure of SARS-CoV-2 M pro in apo form (PDB ID: 6Y2E) and α-ketoamide bound form (PDB ID: 6Y2F) shows that the protein makes a crystallographic dimer composed of two monomers of identical conformations. Each protomer furthermore is made up of three domains. The interface of domain I and domain II form the active site of the protein, which is composed of a Cys 145 -His 41 dyad where α-ketoamide derivative 13b is bound (Fig. 1A). The uniquely globular domain III is linked to domain II through a linker region and deemed essential for the catalytic activity of this chymotrypsin-like protease (20,21). The α-ketoamide derivative 13b is shown bound in the active site and is stabilized by a number of interactions with the active site residues His 41 & Cys 145 and adjacent residues in substrate binding cleft such as Gly 143 and Ser 144 (20) (Fig. 1B). We have selected a number of existing drugs, most of which are reported to be used in humans for countering certain viral infections and screened them for binding in the active cleft of M pro . Our results have shown that some of the drugs occupied the active site of M pro with even increased binding affinity than that of the bound α-ketoamide 13b. Whilethe rest of the compounds has shown appreciable binding while holding most of the crucial active site determinants. We envisage that further in vitro examination of the inhibitory potential of these drugs on the catalytic activity of the main protease could lead the way to repurpose one or more of the tested FDA approved drugs in this study as a treatment therapy for SARS-CoV-2 induced disease. ## Phylogenetic analysis of SARS-CoV-2 genome To understand the evolutionary relationship between the previously known human coronavirus and the novel SARS-CoV-2, we have performed the phylogenetic analysis. For analysis, all the closely related and complete reference genome sequences of SARS-CoV-2 were downloaded from the NCBI GenBank database. A total of 50 genomes were considered for the study. MEGA 6.0 was used for multiple sequence alignment and construction of a phylogenetic tree and 1000 bootstrap replicates is performed using Neighbor-joining method (22). ## Molecular docking The recently elucidated X-ray crystal structure coordinates of SAR-CoV-2 M pro was downloaded from RCSB PDB (PDB ID: 6Y2F), having 1.75 resolution (20). In this structure the M pro was co-crystallized with the bound improved α-ketoamide (13b) inhibitor and multiple intermolecular interaction of ligand with the active site residues are characterized. To further identify the potent inhibitors for SAR-CoV-2 M pro among the FDA approved antiviraldrugs, we have downloaded more than 75 drug compounds from the PubChem chemical database. For molecular docking based drug repurposing, the download 3D structures of compounds and protein was prepared. The docking study was performed by AutoDock Vina, which uses a lamrackian genetic algorithm (GA) in combination with grid based energy estimation (23), to check the docking accuracy of software we have performed re-docking to co-crystal bound ligand.The main aim of this molecular interaction study was to identify the highly interacting drug with SAR-CoV-2 protein crystal structure and to propose the drug by in-silico repurposing method. All the interaction visualization analysis studies were performed by Discovery Studio Visualizer (DS), PyMol molecular visualization tool and LIGPLOT + (24,25). ## Genome sequence alignment and phylogenetic analysisof SAR-CoV-2 The sequence alignment of SAR-CoV-2 genome shows high similarity with the closely related reference genomes of other coronaviruses. The Blastn search of the complete genome of SARS-CoV-2 reveals that the most closely related virus available in GenBank is SL-CoVZXC45 ## Inhibitor binding cleft of M pro Coronaviruses uses a chymotrypsin like protease along with papain protease to process and cleaves its long polyprotein precursor into individually functional nsPs. Multiple sequence analysis of the main protease of SARS-CoV-2 with that of SARS-CoV reveals that amino acid sequence is conserved with a sequence identity of 96% (Fig. 3). The active site residues are thoroughly conserved and makes a catalytic Cys 145 -His 41 dyad. Additionally there are substrate binding subsites positioned in the active site groove of the protease. The specific subsite residues located in the enzyme active site are named as S1', S1, S2 , S3 and S4 depending on their relative position to the cleavage site and subsites P1', P1,P2, P3 and P4 in the polyprotein. In the M pro of SARS-CoV-2 active site region, the S1' residues are contributed by Cys145, Gly143 ans Ser144 which also serve as the oxyanion hole. The S1 residue is His163 while Glu166 & Gln189 located at the S2 position. Bulky Gln189 and Pro168 makes the S4 site (20) (Fig. 4A). The main protease recognizes and bind specific residues at each subsite of the peptide substrate to determine the initiation of proteolysis and production of nsPs for the formation of replication-transcription complex. ## Docking analysis The molecular docking based virtual screening of FDA approved antiviral drugs against the SARS-CoV-2 M pro revealed the strong interaction with higher docking energyand binding affinities. All the potential drugs docked with the independent conformation in the active site of protein where the co-crystal structure ligand (improved α-ketoamide 13b) bound. Molecular docking binding affinity of all the docked and analysed drugs with their binding energy ranking is shown in table S1 (Supplementary material). The molecular re-docking was also performed to check the docking accuracy of the software AutoDock Vina, and it was observed that the cocrystal bound ligand and re-docked ligand shows RMSD value of 0.51 , suggesting the high fidelity of docking method (Fig. 4B). In the present study, we focused on the top 10 docking results for further analysis as these drug compounds showing higher binding affinity ranging from (-10.6 to -7.9 kcal/mol). Although among the top 10 drugs,the top three drug compounds were showing binding affinity even higher than that of the improved α-ketoamide 13b compound (Fig. 5A-D) (Table 1). From the recently published studies for SARS-CoV-2 it was observed that virus binds with angiotensin-converting enzymes 2 (ACE2) receptors in the lower respiratory tracts of infected patients to gain entry into the lungs. The study reveals that SARS-CoV-2 main protease (M pro ) is the best drug target among coronaviruses (30). Interestingly, one of the most characterized and promising drug targetin against coronavirus infection is the main protease (M pro , also known as 3CL pro ) which has been co-crystallized with a bound ligand 'improved α-ketoamide 13b' in case of SARS-Cov-2 main protease (20,31). This crystal structure reveals that the α-ketoamide 13b is occupying the active site of the protein and making a number of hydrogen bonds and hydrophobic interactions with the active site residues as well as other substrate binding residues of the binding pocket. In the present study, we have screened more than 75 antiviral, anticancer, and anti-malarial drugs for the identification of potent drug molecules using drug repurposing virtual screening methods. Molecular docking studies have revealed that maximum of the screened drug compounds interact with SARS-CoV-2 M pro active pocket and also share same interacting amino acids residues. The SARS-CoV-2 bound ligand (improved-α-ketoamide) shows strong bond in interactions with surrounding amino acids within the region of 4 at different subsites with His164, Glu166, Gly143, His163, Cys145, His41, and Phe140 where it forms hydrogen bonds with active site His41 and also accept hydrogen bond from the backbone amides of Gly143, Cys145 and Ser144. This protein ligand interaction reveals a strong inhibition of virus protease (Fig. 5D) (20). The screening and molecular docking of at least 75 preexisting drugs we have carried out have shown to fit in the active site of protease in independent conformation and appreciable binding energy score (Fig. 6A-D). Further,we have analyzed and repurposing the top 10 drugs which showed higher or similar binding affinity as compared to the co-crystal bound ligand of SARS-CoV-2. The top 3 drugs that are exhibiting the interaction with same amino acid residues as of the α-ketoamide with themain protease are Lopinavir-Ritonavir showing binding affinity of (-10.6 kcal/mol) and Tipranavir (-8.7 kcal/mol), whereas Raltegravirhas binding affinity of (-8.3 kcal/mol), which is similar to improved-α-ketomaide 13b (-8.3kcal/mol). While the rest of the drug compounds have also shown good binding energy score, as presented in table1. The drug Lopinavir-Ritonavir is a combination product contains two medications lopinavir and ritonavir. This drug is mainly used for HIV-AIDS to control HIV infection by inhibiting the protease and help to decrease the amount of HIV in the body by promoting thefunction of body's natural immune system to work better (32,33).The enzyme SARS-CoV-2 M pro along with the papain-like proteases is essential for processing the polyproteins into various nonstructural protein by cleaving at specific sites, that are translated from the viral RNA. The interacting amino acids in the M pro enzyme active site were reported to be Leu, Gln, Ser, Ala, Glyalong with the Cys-His dyad which marks the cleavage site, similarly our in silico docking study shows that top screened drug Lopinavir-Ritonavir combination interacts with Glu166 (also form strong hydrogen bonding), Gln189, Leu167, Met165, Asp187, Met49, His41, Cys145, and Leu141(Fig. 5A). Interestingly, the binding energy score of Lopinavir-Ritonavir in protein-ligand docking was found to be even better than that of the docked α-ketoamide and the in silico inhibition constant (Ki) was obtained to be 16 nM. In silico inhibition constant (Ki), as obtained by docking is given in table 2 for top 10 drugs. Drug tipranavir or tipranavir disodium is another nonpeptidic protease inhibitor used in combination with ritonavir to treat HIV infection (34)(35)(36). In our study, the drug shows interaction with Gln192, Met165 (both form hydrogen bonding), Gln189, Asp187, Met49, Arg188, Ser46, Cys44, Thr25, and His41 in different conformation from that of α-ketoamide inhibitor (Fig. 5B). We hypothesizes that tipranavir or its other derivatives with even improved binding affinity in combination with ritonavir could serve as the potential protease inhibitor to counter SARS-CoV-2 multiplication in cell based assay. Another drug which has shown comparable binding affinity and binding energy with that of the docked α-ketoamide in M pro active site, theraltegraviris a characterized antiretroviral medication which work by inhibiting the integrase strand transfer and is used in combination with other drugs to relieve the HIV infection (37)(38)(39). In the present study, raltegravir drug shows interaction with His164, Arg188, Gln192, Glu166 (all residues were bonded with strong hydrogen bond), Met49, Met165, Phe140, Pro168, and Leu167. The drug shows four H-bonds with nearest interacting amino acids of SARS-CoV-2 M pro enzyme, which indicates good inhibition (Fig. 5C). This drug could also be used with other combinations like raltegravir and lopinavir for the treatment of COVID-19, if found producing desirable inhibitory effect against SARS-CoV-2 protease in biochemical activity assay or cell based assays. Additionally, other drugs which were screened and docked in the substrate binding cleft of the M pro , has shown good binding energy score which is comparably similar to the original compound in the protein crystal structure. Many of these drugs such as dolutegravir, letermovir & Nelfinavir are commonly used for treating different infections ranging from HIV to cytomegalovirus by employing different mechanism of action (40)(41)(42)(43)(44)(45). The identified repurposed drug and their interaction with binding amino acids in the M pro active site have been shown in table 1. After screening the different FDA approved drugs, the present study enabled us to understand the mode of interaction of approved antiretroviral drugs with new coronavirus SARS-CoV-2 main protease enzyme. However, we believe that all the drugs studied and screened for repurposing against COVID-19 in this study should furthermore be tested and their in vitro inhibitory potential needs to be investigated through robust biochemical proteolytic activity assays and other biophysical & structural studies. ## Conclusion From this study, we conclude that the repurposed drugs may be helpful for the treatment of novel coronavirus disease and can serve as potential drug candidates to curb the ongoing and everenlarging COVID-19 pandemic. Sinceall the drugs used in this study are of known pharmacokinetics standards and approved by FDA for human use they do not need to undergo specific long term clinical trials and therefore can fasten up the process of the therapeutics development. Our phylogenetic analysis of the available genomes of SARS-CoV-2 isolated from different sources also reveals that the virus is not showing any sign of mutation or diversification rapidly, therefore the repurposed drug combinations could be used against SARS-CoV-2 on pancommunity level.

chemsum

{"title": "In silico identification and docking-based drug repurposing against the main protease of SARS-CoV-2, causative agent of COVID-19", "journal": "ChemRxiv"}

peptidyl_acyloxymethyl_ketones_as_activity-based_probes_for_the_main_protease_of_sars-cov-2

## Abstract: The global pandemic caused by SARS-CoV-2 calls for a fast development of antiviral drugs against this particular coronavirus. Chemical tools to facilitate inhibitor discovery as well as detection of target engagement by hit or lead compounds from high throughput screens are therefore in urgent need. We here report novel, selective activity-based probes that enable detection of the SARS-CoV-2 main protease. The probes are based on acyloxymethyl ketone reactive electrophiles combined with a peptide sequence including non-natural amino acids that targets the non-primed site of the main protease substrate binding cleft. They are the first activity-based probes for the main protease of coronaviruses and display target labeling within in a human proteome without background. We expect that these reagents will be useful in the drug development pipeline, not only for the current SARS-CoV-2, but also for other coronaviruses. Although coronaviruses that infect humans have been known since 1966, 1 2 they have gained more global attention since the turn of the millennium, with the appearance of several strains that caused more severe symptoms and were more widely spread. Two outbreaks in 2002 and 2012, which were caused by severe acute respiratory syndrome coronavirus (SARS-CoV) 3 and Middle-East respiratory syndrome coronavirus (MERS-CoV), 4 respectively, were contained regionally. In December 2019, a novel coronavirus, later termed SARS-CoV-2 appeared in the province of Wuhan, China, 5 which led to a global pandemic that is currently ongoing. The clinical manifestations range from asymptomatic to fever and severe pneumonia known as covid-19, which in some cases can lead to death. 6 The virus has caused a worldwide crisis in healthcare, economy and society, and has painfully exposed the vulnerability of modern civilization. Currently, there is an ongoing global effort to develop vaccines and drugs that can eradicate SARS-CoV-2 and contain the pandemic. Unfortunately, the immune response against the virus is not well understood, and it is unclear whether infected people build up immunity against reinfection. This emphasizes the importance for the development of drugs that inhibit coronavirus replication. Coronaviruses -or coronaviridae -are enveloped, single stranded RNA viruses with a typical appearance of the solar corona caused by the viral spike protein that is part of the viral capsid. The replicase gene of the virus contains two overlapping open reading frames (ORFs) that are translated in two large polyproteins. 7 These polyproteins are proteolytically processed into 16 non-structural proteins (nsp1 to nsp16) by two viral proteases. Whereas the papain-like protease (PL pro ) performs three cuts to release nsp1-3, the main protease (M pro , also called 3CL pro for 3C-like protease) cuts at 11 sites, thereby liberating the most important protein products for replication. 7 8 Therefore, the M pro has attracted major interest as a potential drug target. M pro is a protease from the C30 family. 9 Its active site comprises a catalytic dyad formed by a cysteine and histidine residue. The substrate specificity is governed by a requirement for Gln in the P1 position, and preferences for large hydrophobic residues in P2 and small residues in P1'. 10 11 Recently, the Drag laboratory has reported a substrate library screen incorporating a large series of non-natural amino acids in a comparison of M pro from SARS-CoV and SARS-CoV-2. 12 In the past, information on substrate specificity has been instrumental in the design of active site directed M pro inhibitors -especially of peptidomimetics, 13 whereas crystallography has increased our structural understanding of the interactions between inhibitors and M pro for further optimization. 14 Since the start of the 2019 SARS-CoV-2 pandemic, several crystal structures of the M pro in complex with various inhibitors, such as peptidyl ketoamides, peptide aldehydes and peptidyl vinyl methyl esters, have been published. 15 16 17 In the past, activity-based protein profiling (ABPP) has been particularly useful for the study of proteases. 18 19 Activity-based protein profiling (ABPP) is a powerful technique to detect active enzymes in complex proteomes, such as cell lysates, whole cells or in vivo. 20 It relies on small molecules called activity-based probes (ABPs) that specifically form covalent bonds with active site residues of the target enzyme in a mechanism-based reaction. Although ABPP has been extensively applied for the characterization for the study of cancer-related enzymes 21 as well as enzymes from infectious bacteria, 22 application to viral infections has been much less investigated. 23 One of the underlying reasons may be the lack of ABPs specifically designed for viral targets, except for probes targeting the NS2B-NS3 protease of flaviviruses 24 25 and the smallpox virus protease K7L. 26 Here, we report the first ABPs for the Mpro of coronaviruses, exemplified by a probe for the Mpro of the recent SARS-CoV-2. It is based on a cysteine specific reactive electrophile and recent substrate specificity information combining natural and non-natural amino acids. We show that these probes detect the active form of the Mpro, are highly selective and can distinguish the protease within a human proteome. ## Results and discussion Probe design -As a warhead for the M pro ABPs we chose the acyloxymethyl ketone (AOMK) reactive electrophile, because it is selective for cysteine proteases and compatible with solid phase peptide synthesis. 27 To guide this warhead towards the active site cysteine of the SARS-CoV-2 M pro , we incorporated a peptide element matching the substrate specificity. Because the glutamine side chain may react with the ketone moiety of the AOMK in a cyclization reaction, as reported before for glutaminyl fluoromethyl ketones, 28 we also incorporated a citrulline as possible glutamine mimic, a dimethylated Gln and a His, which both have been used as P1 residue in M pro inhibitors. 13 For the P2-P4 position, we chose Abu-Tle-Leu, which is the recently reported optimal sequence for SARS-CoV-2 M pro . 12 However, for the P1 His molecules, a Thr-Val-Cha sequence was selected, because this was reported to be a potent sequence for the biologically similar SARS 3CL Protease. 29 As a detection handle, we chose a hexynoic acid, since it allows for click chemistry-mediated introduction of various tags. Probe synthesis -For the solid phase synthesis of the AOMK M pro ABPs, we first synthesized the necessary chloromethyl ketone (CMK) building blocks of properly protected glutamine, dimethyl glutamine (made from commercially available building block 1), citrulline and histidine, by activation of the carboxylic acid with isobutyl chloroformate, subsequent reaction with diazomethane and quenching with hydrochloric acid (Scheme 1A). The His CMK could not be made with the more standard trityl protecting group, not only because of the lability of the protecting group under acidic conditions, but also because of substitution of the chloride by the imidazole nitrogen in part of the product. Fortunately, with the more electron withdrawing Boc protecting group on the His side chain, CMK building block 3d was obtained in a satisfactory crude yield of 82%. For the construction of the probes, we took two different solid phase approaches. The probe with a P1 Gln side chain offered the opportunity to attach it by its side chain to a solid support. To this end, CMK building block 3a was first converted into an AOMK by reaction with dimethylbenzoic acid, before removal of the tert-butyl protecting group and coupling to the Rink amide resin (Scheme 1B). Elongation with the appropriate Fmoc-protected amino acids, capping with a hexynoic acid and cleavage from resin yielded probe 4. The compounds with a dimethyl Gln, Cit or His in the P1 position were made by loading the corresponding CMKs 3bd onto a semicarbazide resin, follwed by substitution with dimethylbenzoic acid, elongation and cleavage from the resin (Scheme 1C). Unfortunately, after cleavage from the resin and 6 concomitant cleavage of side chain protecting groups (where present), the compounds with a P1 His residue immediately hydrolyzed to hydroxymethyl ketones (HMKs) 7-8, probably assisted by the imidazole side chain (Scheme 1D). Evaluation of inhibition -Compounds 4-8, were evaluated an inhibition assay on purified SARS-CoV-2 M pro , and compared to the recently reported M pro active site inhibitor carmofur. 16 30 In short, the protease was incubated with the compound for 1 hour, after which a fluorogenic aminomethyl coumarin substrate 12 was added to measure residual activity. Compounds 4 and 6 showed clear inhibition of the protease, with probe 6 being most potent, but 5 and 8 did not show any activity (Figure 1A). Interestingly, probe 7 was still able to inhibit M pro to some degree, even without its ability to form a covalent bond with the protease, illustrating that a His is still accepted in the P1 position. To gain insight in the binding modes of the different probes, we performed covalent docking of the probe 4 -8 in complex with Mpro using the flexible side chain method of Autodock 4.2. 31 The P1 and P2 amino acid residues of probe 4 and 6 both dock well into the corresponding S1 and S2 pockets of the enzyme (Figure 1C-E). Moreover, they display good overlap with crystal structures of various different peptidomimetic inhibitors (Figure 1D and Figure S1). However, probe 5 with Cit in the P1 position does not fit well into the active site (Figure 1F). Repeated runs of docking did not result in placement of the P1 Cit into the S1 pocket, likely due to its larger side in comparison with Gln, which explains the lack of potency of probe 5 and confirms the importance of the S1 pocket for inhibitor design. In-gel fluorescence clearly shows that M pro is covalently modified by both 4 and 6 at concentrations as low as 200 nM (Figure 2A). Biotin blot was also able to detect the covalent probe-Mpro complex, albeit with slightly lower sensitivity. Overall, probe 6 with the dimethyl-Gln in the P1 position is more sensitive than probe 4, in line with the results from the inhibition assay. This may be attributed to the possible reversible formation of a cyclic hemiamidal by reaction of the glutamine side chain with the ketone (Figure 2B). We next investigated the capacity of the probes to label SARS-CoV-2 M pro in a complex proteome. To this end, prepared lysates of E. coli that were induced or not induced to express recombinant M pro . Because the click chemistry could potentially be problematic in more complex samples, we also synthesized a pre-clicked TAMRA version of probe 4 (probe 9; see Figure S2). Both probe 4 and 6 were able to label M pro selectively in the bacterial lysate, as the few other observed bands were caused by click chemistry background labeling as judged from a no probe control sample (Figure 2C). Labeling by probe 4 and 6 is largely prevented by the active site inhibitor carmofur and no substantial labeling was observed in the lysates of uninduced bacteria. Both results demonstrate the selectivity of the probes. For future application in infected human cells, we wanted to show that the probes do not show crossreactivity with human proteins. As we lack the facilities in our laboratory to work with live viruses, we spiked lysates of HEK293T cells with different amounts of purified M pro and labeled with preclicked probe 9. SDS-PAGE followed by fluorescent scanning revealed that M pro was detected with a sensitivity down to 0.07% of the total proteome content. No other substantial gel bands were detected, illustrating that probe 9 specifically labels M pro within a complete human proteome. ## Conclusion In conclusion, we designed and synthesized different ABPs targeting the protease M pro of SARS-CoV-2. They are based on cysteine reactive AOMK electrophiles and peptides resembling the substrate specificity of M pro . AOMKs have not been reported before as inhibitors for M pro . We have here demonstrated that they act as covalent, active site inhibitors. Admittedly, the P1 residues in this study are not yet optimal, as the side chain of Gln may form a cyclic hemiamidal and the GlnMe2 is generally less favorable for M pro . Hence, we anticipate that further optimization of the peptide element as well as the primed site leaving group will lead to AOMK probes and inhibitors with higher potency. Nevertheless, the here described probes are able to detect M pro in complex proteomes without background labeling of other

chemsum

{"title": "Peptidyl Acyloxymethyl Ketones as Activity-Based Probes for the Main Protease of SARS-CoV-2", "journal": "ChemRxiv"}

spectrally-selective_all-inorganic_scattering_luminophores_for_solar_energy-harvesting_clear_glass_w

## Abstract: All-inorganic visibly-transparent energy-harvesting clear laminated glass windows are the most practical solution to boosting building-integrated photovoltaics (BIPV) energy outputs significantly while reducing cooling-and heating-related energy consumption in buildings. By incorporating luminophore materials into lamination interlayers and using spectrally-selective thin-film coatings in conjunction with CuInSe 2 solar cells, most of the visible solar radiation can be transmitted through the glass window with minimum attenuation while ultraviolet (UV) radiation is down-converted and routed together with a significant part of infrared radiation to the edges for collection by solar cells. Experimental results demonstrate a 10 cm 3 10 cm vertically-placed energy-harvesting clear glass panel of transparency exceeding 60%, invisible solar energy attenuation greater than 90% and electrical power output near 30 W p /m 2 mainly generated by infrared (IR) and UV radiations. These results open the way for the realization of large-area visibly-transparent energy-harvesting clear glass windows for BIPV systems. T he role of renewable energy in addressing the challenges associated with implementing CO 2 emissions reduction, addressing the climate change and energy supply concerns, has been recognised globally. Photovoltaics (PV), the conversion of sunlight to electricity has been reported to be the fastest-growing technology for electricity generation 1 . One-fifth of the world's total energy consumption is delivered for civil applications, residential and commercial 2 . Developing energy-efficient buildings is of prime importance, and future building industry regulations will have energy-efficiency requirements that meet the growing demand on energy resources. Ideally, new energy technologies must be integrated into the existing and future infrastructure elements (e.g. buildings), and at the same time provide savings by reducing the energy consumption. Designing ''energy-saving and energy-producing solar windows'' provides a way of boosting building-integrated PV (BIPV) energy outputs dramatically and beyond what is possible currently, simultaneously with reducing the coolingand heating-related energy consumption. Various glazings and coated-type windows are in use worldwide as they offer thermal comfort improvements, energy savings, aesthetic value and ultraviolet (UV) protection. With the widespread use of glass panels covering vast areas of walls and roofs in modern buildings, significant economic benefits could be achieved if these glass surfaces had energy-generation capabilities. An economically-ideal energy-saving approach is to use window glazings that offer a combination of IR-range transparency blocking and energy-harvesting (due to the ability to efficiently convert the lighting-unrelated IR and UV solar energy into usable electric energy). It is likely that windows possessing these useful properties offer substantial economic and environmental benefits and would rapidly be recognized and marketed globally. It is well-known that industrial solar and wind farms require allocation of sizeable land areas in order to generate significant power. Conventionally, the power generation capability of green-energy PV installations is limited by the available roof area, especially for tall buildings. Wallmountable BIPV modules are starting to appear on the market, however, these mainly rely on thin-film photovoltaics of limited efficiency, and also cannot provide high optical transparency or energy savings related to solar-control properties. Solar energy contains strong IR radiation flux coming from direct, diffused, reflected and re-emitted radiation from illuminated surfaces. Therefore, by using high-efficiency photovoltaic solar cells, energy-harvesting windows can generate measurable electric power even in non-ideal illumination conditions. The development of principally new BIPV systems capable of high transparency and energy saving simultaneous with energy harvesting will bring the future goal of achieving net zero energy consumption in buildings closer to reality. The Luminescent Solar Concentrator (LSC) technology is currently being considered poten-tially suitable for engineering the PV windows of the future 6 , even though ensuring the high transparency in high-efficiency concentrators remains problematic for reasons of IR-luminophore limited availability. Organic dye-based LSCs have been employed to concentrate sunlight re-emitted at wavelength bands efficient for power generation by PV cells 4,5,7,8 . This type of concentrators commonly use dyes as luminescent organic species dispersed in optically clear polymer matrices 7,8 . However, it has been reported that when using organic dyes, the overlapping of absorption and emission bands reduces the concentration capabilities of LSC, which also contributes to their poor stability under long-term solar illumination exposure 5 . Moreover, most of LSC technologies and demonstrator samples featuring PV conversion of light concentrated by planar structures reported in the literature to date 9,10 , share the following major limitations that still prohibit product-level development of energy-harvesting windows and at the same time emphasize the need for further research in this field: . The concentrator-area scalability of all current LSC technologies is severely limited by the effects of re-absorption that reduce the propagation path-length of luminescence emissions within lightguiding structures; . No LSC structures reported so far in the literature were designed to enhance the visible transparency simultaneously with spectrally-selective harvesting of non-visible solar radiation using inorganic-only luminophores. This is despite the fact that over 50% of solar energy reaching the ground level is distributed spectrally outside the visible range. . The best power conversion efficiency of LSC devices reported to date remains within 2-4% if using Si cells. The sizes of most recently-reported high-performing concentrator panels never exceeded 100 mm 3 100 mm 10,11 . To the best of our knowledge, no literature sources reported on achieving these 2-4% efficiency figures in samples of significant visible-range transparency, and most luminophores used have either been not disclosed or their compositions implied a limited environmental stability . Also, no spectrally-selective IR-specific photon-trapping structures suitable for use in window systems or glazing-type applications have ever been reported. Glass structure, technology and materials. A window design reflecting and redirecting the invisible solar radiation (Fig. 1(a)) exhibits LSC capability that relies on the efficient absorption of UV and IR light across as broad spectral range as is possible. This can be achieved using an active luminescent functionalized interlayer that provides photo-induced re-emission of the absorbed photons. The glass structure is integrated to act as spectrally-selective concentrator in such a way as to provide efficient transport of the re-emitted and otherwise internally deflected photons towards its edges. Our solar concentration approach simultaneously enables electric power generation through PV cells attached to glass panel edges and maintains a maximized visible-range (400-700 nm) transparency. Concentrators of this type can be called ''hybrid'', since luminescence effects play only a partial role in energy harvesting functionality, together with multiple scattering and diffraction enabled by embedding quasi-random disordered arrays of luminophore particles inside polymer interlayers. Despite its contributions to optical losses within the concentrator structure 12 , multiple scattering effects can enhance light-trapping performance in planar-geometry structures . Our approach to the development of transparent hybridtype solar concentrators relies on the experimental optimization of electric energy output generated by the solar cells attached to the structure edges, by trialing a large number of luminescent material combinations. Some of these materials are presented in Table 1 and some others cannot be disclosed due to the confidentiality agreements. Generally, these materials are capable of converting the non-visible solar energy into near-IR emissions. Different ways of distributing the luminescent powders geometrically within interlayers have been trialed, including varying the particle sizes, epoxy loading concentrations, and layer thicknesses. Since a rather complex combination of several inter-related physical mechanisms and effects acting simultaneously was expected to lead (at least statistically) towards the increased IR illumination levels at sample edges, we chose to characterize the resulting structures' performance in terms of the actual energy harvesting output observed, in both the outdoor and solar-simulator experiments. Using PV cells attached to the sample edges was in this case the best practical way of detecting and measuring the total integrated contribution from all possible light redirection pathways present within the system structures, through measuring the electric output parameters such as open-circuit voltage (V oc ) and short-circuit current (I sc ). The relative contributions of different physical effects e.g. luminescence-assisted energy conversion versus multiple scattering or multiple diffraction events on powder particles is subject to our ongoing study. A thin film interference-coating was especially designed to achieve minimized transmission of UV and IR energy as well as fulfil durability requirements related to environmental-stability. Fig. 1(c, d) shows the modelled and measured transmission and reflection spectra of a metal-dielectric thin film, which was developed using an ebeam/thermal evaporator. The heat-mirror-film design was adjusted in terms of materials selection and number of layers deposited to ensure wide-band reflection of IR radiation and the associated ther-mal insulation performance. Multiple coatings of this type were deposited onto glass substrates following the methods described in 16 . Functionalized luminescent interlayers. To improve the light collection efficiency of light-trapping glass structures, we adopted an approach based on incorporating inorganic luminophore mixes into a transparent epoxy-based lamination layer, thus realizing a transparent, spectrally-selective, coating-assisted all-inorganic solar concentrator. The selection of inorganic luminophore types for developing the interlayers as well as the optimization of powder particle sizes, concentrations, and interlayer thicknesses were subject to application constraints imposed by our industry partner (Tropiglas Technologies Ltd). The principal requirements were related to ensuring a substantial visible-range sample transparency and clear, non-colorbiased glass appearance. These requirements limited the selection of potential luminophores to inorganic substances possessing photoluminescence excitation ranges within either the UV or near-IR ranges, which could be ground into fine (of the order of 1 mm) particles, and then incorporated effectively by either dissolution or ultrasoundassisted disperse suspension, into the interlayer host material (epoxy). The required transparency and visual clarity of the functionalised interlayers were the variables that primarily controlled the upper limits of powder concentrations and interlayer thicknesses used. Prior to undertaking the research activities described in this paper, we had evaluated the relative energy-collection performance of a large number of 20 3 20 3 6 mm samples of similar structure, which employed different inorganic luminophore mixes of various concentrations and layer thicknesses as well as some luminescent organic dyes (Lumogen F Red and others). After extensive experimentation with multiple high-performance commercial photoluminophore substances from a range of suppliers, we identified four inorganic compositions from three different manufacturers which, when used individually or mixed together in small concentrations (sub-1 wt% of each), led to substantial measured increases in the electric output from edge-mounted PV cells, when tested using a solar simulator beam directed at normal incidence (compared to a reference sample that did not contain luminophore materials within its epoxy-based interlayer). The beam of simulated sunlight was significantly smaller than the sample dimensions (20diam. vs 100 mm size), therefore some effective photon redirection mechanism within the glass structure (e.g. luminescence, Mie scattering promoting the wavevector deflection, diffraction events occurring on powder particles, or a combination of these) was crucial for increasing the photon flux reaching the side-mounted PV cells, as was evidenced by the significant dependency of electric output parameters on the interlayer type and composition. All samples constructed had interlayer thicknesses below 3 mm, to ensure large visible transmission and good visual clarity, despite some noticeable scattering and diffused transmission effects. Here, we report on the empiric optimisation of the pre-selected functionalised interlayer compositions in 100 3 100 3 18 mm 3 clear glass samples to maximise the electric power output in PV cells attached to structure edges to detect and convert the optical power deflected towards the edges of glass structures. The effective path length of the luminescence excitation was difficult to evaluate, since the multi-pass propagation through the luminophore-loaded interlayer assisted by the multiple coating reflections and scattering events was engineered to occur within the structures. Four different finely-ground luminophore powder types (described in Table 1) were selected (based on their performance characteristics such as excitation and emission spectral bands and quantum efficiency), and, in order to optimize the performance of the proposed energy-harvesting clear glass panel structure, various mixes were used to construct a number of concentrator prototypes of different interlayer chemistries. The luminophore powders and their mixes were primarily selected to enable improved energy harvesting performance by providing both the UV, short-wave visible and also near-IR wavelength regions for photoluminescence excitation, and special care was also taken to place the emission wavelength regions inside the near-IR band, where the solar cells used had good responsivity, and also within the high-reflectance regions of the spectrally-selective coating. The function of (Zn, Cd)S:Cu luminophore (c) was to convert a fraction of light from (300-550) nm band into (840-1040) nm. The main feature of this material and also of luminophore d, is related to the practical absence of overlapping between the excitation and emission wavelength bands, which effectively eliminates reabsorption-related energy losses. Emissions from all four phosphor types were within the high-responsivity band of the solar cell type used. Partial concentrations of luminophores (in wt%) were selected carefully in order to maintain the balance between optical clarity of the samples, reasonably high visible-range direct transmission, and enhancing the diffused transmission fraction together with material compatibility issues in terms of increasing scattering with increased number of powder types and concentrations used. Several energy harvesting clear glass samples were assembled with different functionalized interlayer properties, such as luminophore compositions, concentrations and thicknesses as shown in Table 2. We carried out a large number of interlayer property adjustment experiments in order to keep the visible-range sample transparency above 40% while trialing the use of various simple, binary and ternary luminophore powder mixes and different loading concentrations to develop the most suitable interlayer material system for the routing of incident radiation towards solar cells. Sample A of Table 2 was used as reference sample for benchmarking other samples' performance. Sample testing methodology and energy harvesting performance characterization. We designed the concentrator and interlayer characterization experiments in order to identify the bestperforming samples (in terms of energy conversion and routing capability). The electric output parameters of samples were measured when illuminating the glass energy-collecting areas using a normally-incident, 20 collimated (and also diffused) solar simulator beam spectrally equivalent to AM1.5G irradiation (Fig. 2(a)). A reference sample which intentionally did not incorporate any of the technology features designed to assist in energy routing towards glass edges, was selected for benchmarking the performance of all other samples. We define the ''Area Collection Gain'' parameter (ACG) to characterize the relative flux-deflection capability in our concentrator samples as follows: Parameter ACG (equation ( 1)) defines the way in which the reference sample is used and also describes the energy-routing performance differences between all samples measured through their electric output. Figure 2(b) summarizes the results of performance testing experiments performed with all 12 concentrator samples each containing a different interlayer type. The ACG measured in each sample varied depending on factors such as the luminophore chemistries used and Phosphor a was always used after dry grinding for several hours using Fritsch Pulverisette Premium Line 7 ball mill at 700 rpm to reduce the mean particle size to the vicinity of 1-2 mm. the concentrations of the powders, together with interlayer thickness. The goal was to search for an optimized interlayer type and properties, so as to deflect most of the non-transmitted incident optical power towards sample edges more effectively. For example, the results showed (as was somewhat expected) that the ability to deflect more radiation towards edges correlated inversely with visible transmission of samples. It is important to note that only the spectrum of direct transmission was measured, however diffuse transmission was also significant in our samples, as was evident from their visual appearance as well as from their light diffusion properties as illustrated in Fig. 3. As an example, the functionalized interlayer of sample C was made by mixing two luminophore powders a and b the excitation bands of which overlapped significantly which, in conjunction with a relatively large scattering observed in all samples containing powder b, resulted in a relatively weak light collection performance. The optimization of light scattering intensity and the related light diffusion effects on the light collection efficiency and samples transmission was made only empirically in our experiments, since multiple factors affecting concentrator performance (from the varying solubilities of various powders in polymer materials to the final particle or agglomerate size ranges available from grinding and homogenization processes) could not be realistically predicted computationally. Sample E, on the other hand, contained three different powders which provided a combination of wide excitation bands placed in the UV, blue and also the near-IR wavelength regions, a rather large Stokes shift of luminophore c, and the IR-range emission bands within the high responsivity wavelength range of CIS cells, showed considerably improved light concentration in comparison to most other samples. Luminophore c dissolved fairly well into our epoxies, had a rather small mean particle size of about 1 mm, and did not cause significant scattering at concentrations not exceeding about 0.05 wt%. The addition of other luminophores to the same sample increased scattering and decreased direct transmission in the visible range, while increasing the diffused transmission component. As shown in Fig. 2(b), the same ACG trends were seen across all samples in our batch, when using either the collimated-beam or diffused-beam illumination geometry. The effects of multiple scattering on weakly-absorbing luminophore powder particles distributed in a quasi-random, quasi-3D fashion inside our interlayers, on the character of light propagation through concentrator samples are rather complex and lead to an additional scattering-related loss mechanism. However, the disorder-induced light trapping also occurs, and the possible light-trapping mechanisms involving multiple scattering effects in disordered photonic systems were reported recently in . Additionally, the potential role of scattering effects in assisting the light trapping functionality in LSC systems requiring only modest flux gains has been recognized as early as 1981 5 . Fig. 3 shows that beam expansion and light diffusion effects are easily observable, as well as the appearance of fringe structure within a halo of diffused light formed due to multiple scattering on powder particles. The disorder-induced beam expansion effects and their role in assisting the light trapping in planar hybrid-type optical concentrators is subject to our ongoing study. The evaluation of the performance of LSC-type light concentration systems is commonly related to geometric flux gain measurements as well as to the measurements of the power conversion efficiency (overall system efficiency g) 4,5,10,11 . For concentrator systems designed to provide significant spectral selectivity and thus spectrally separating the energy-harvesting wavelength bands from the enhanced-transmission bands, the definition of flux gain can be adjusted to account for the limited spectral bandwidth available for radiation routing and flux concentration. Parameters measurable directly in all PV systems include the short-circuit current (I sc ) or its density J sc , open-circuit voltage (V oc ) and/or their product, or the output power I sc * V oc *FF. Within the framework of our experimental methodology, we define another figure of merit to characterize the energy routing (flux deflection) performance of concentrator samples assisted by spectrally-selective coatings, namely the Deflection Efficiency Factor (DEF), in the following way: First, we calculate the optical power (within the response bandwidth of PV cells used) of the normally-incident optical power, after the first coating-assisted reflection, that is Calculations according to equation (2) with the integration limits between 300 nm and 1220 nm show that the total optical power within the bandwidth of interest (for circular incident beam of 20 diameter) after its first coating reflection (P refl , opt ) was only a small fraction (26%) of the incident power. Figure 4(a) shows the effect of our highly-transparent spectrallyselective solar-control coating on the spectral modification of the spectral power density distribution of incident light after the first reflection off the coating. We integrated the product of standard AM1.5G spectral distribution and the coating's reflectivity function numerically. Second, we calculate the DEF as follows: This Deflection Efficiency Factor (DEF) quantifies the optical power within the spectral responsivity band of CIS cells that has actually reached the cell surfaces, as a fraction of the optical power within the same spectral band, within the incident-beam area, available after the first reflection off the spectrally-selective coating placed at the back surface of the samples. This definition emphasizes the capability of the samples to deflect the energy available, accounting for the effects of transmission, scattering and absorption which limit the possible energy flux available for trapping or re-direction within the structure of the samples and interlayers. This way of evaluating the performance of the concentrator samples can be used for all types of PV cells, coatings and illumination conditions used, as long as the beam is centered within the glass collection area at near-normal incidence conditions. Figure 4(b) shows a summary of the relative performance of our energy harvesting clear glass samples in terms of DEF as calculated using equation (3). Not surprisingly, significant dependency of DEF on the interlayer type was observed. ## Principal Results and Discussion As was expected, the radiation deflection performance trends quantified using the DEF (Fig. 4 showed a somewhat remarkable result of routing in excess of 20% of the total radiation energy reflected off its back coating towards the PV modules, considering that the input radiation was incident normally and multiple scattering and internal reflection events were required to propagate the photons incident onto the central region of glass collection area towards sample edges over a path length of several centimeters. In addition, the V oc generated by this sample (5.12 V) was approaching its saturation value (near 7.4 V for CIS modules of 100 mm length) despite not being directly illuminated by the light source. Performance comparisons were also made in outdoor illumination conditions using vertically-oriented glass concentrator samples. The performance differences between the different sample types (within this batch of 100 3 100 mm samples) were less pronounced compared to the results presented in Figs. 2 and 4, due to the increased direct (background) illumination reaching the CIS active areas, and the fact that the total area of CIS cells was as large as the glass collection area (which was in fact a design consideration for increasing the electric output of samples in lab conditions, where the background illumination of cells was quite easy to avoid), for reducing the ACG and DEF measurement errors. Somewhat surprisingly, sample J showed the best performance in peak outdoor illumination conditions (I sc 5 60.5 mA; V oc 5 7.4 V and thus P out 5 268.6 mW). When placed vertically, all samples intercepted a total flux of about 700 W/m 2 (7 W incident power), which brings the power conversion efficiency to g 5 3.8%. We believe that this result is outstanding for a glazing system-based concentrator of 55% visible transparency employing inorganic-only luminophore combination. The cells were able to partially collect some background solar illumination in addition to collecting the concentrated light from inside the glass structure. Since approximately 50% of the air-exposed cell areas (at top and one side of sample, which accounted for 14% of the total CIS area) were essentially shadowed from direct sunlight, and the other exposed cell areas received only a fraction of incident flux due to geometry, most of the output was generated through concentration effects. The results of interlayer optimization were used successfully to build up-scaled visibly-transparent glazing system concentrators of size 200 3 200 mm, however the detailed description of concentrator up-scaling results are beyond the scope of this article and will be reported separately. Fig. 5 shows typical I-V curves of our 100 3 100 mm and advanced 200 3 200 mm samples measured at ''average'' and peak outdoor illumination conditions, respectively. The performance characteristics of our energy-harvesting glass samples quantified in terms of the parameters used commonly in the LSC field (such as power conversion efficiency g, optical collection probability P defined by g/ g 0 as a ratio of wall-plug device efficiency to bare-cell efficiency, and geometric gain G ) are summarized in Table 3. All of 100 3 100 mm 2 samples had a small geometric gain of near unity (G 5 1.02), whereas the up-scaled 200 3 200 mm 2 sample had G 5 1.87. The wall-plug (power conversion) efficiency and photon collection probability of sample J both increased by almost 50% compared to sample B which relied on a blank (luminophore-free) epoxy interlayer and an identical structure and heat-mirror coating, due to adding an optimized concentration and mix of functional luminescent materials into its interlayer structure. Notable differences in the electric output stability versus the horizontal-angle orientation of these two samples were also seen, which were expected, because LSCtype collectors have an advantage of relative insensitivity to the incoming radiation flux direction. It is important to also note that, despite photoluminescence effects, other physical mechanisms including Mie scattering, multiple diffraction on a disordered array of powder particles and refractive-type coating-assisted geometric concentration, also contributed to energy collection at cells. The detailed analysis of relative contributions of all mechanisms leading to radiation flux re-direction and energy harvesting in visually-clear glass concentrators are subject to our ongoing studies. ## Conclusion We have proposed the use of all-inorganic spectrally-selective scattering luminophores in conjunction with spectrally-selective thin- film coatings and CuInSe 2 solar cells to realize visibly-transparent energy-harvesting clear laminated glass panels. Luminophore chemistries and concentrations have been optimized and incorporated into an optical epoxy lamination interlayer to maximize the power conversion efficiency of a 100 mm 3 100 mm vertically-placed energy-harvesting clear glass panel. Experimental results have demonstrated a transparency exceeding 55%, invisible solar energy attenuation greater than 90%, power conversion efficiency of 3.8%, a solar heat gain coefficient (SHGC) of 0.41, and a U-factor less than 1.8 W/(m 2 K). ## Methods Thin film coatings. Metal-dielectric coatings were manufactured by physical vapor deposition (PVD) techniques using sputtering targets and pellets of 99.99% pure materials purchased from AJA international, Inc. Chemically ultrasonically cleaned low iron borofloat glass cut-outs of size 100 3 100 3 6 mm were used as substrates. (KVE-ENT 200 hl) thermal/e-beam evaporator and (KVS-T4065) sputtering system, supplied by Korea Vacuum Tech, LTD, were used to deposit thin film layer sequences. The desired thin film characteristics were predicted using OptiLayer software package. The thin films transmission spectra were characterized using Agilent Cary 5000 UV-VIS-NIR and Beckman Coulter DU 640 B UV/Visible spectrophotometers. Functionalized interlayer formation. The laminated glass functional interlayer composites were made with various concentrations and combinations of luminophore powders as shown in table 1 and table 2. NOA61 UV-curable epoxy (Norland, Inc.) and WTS-80203 (H.K. Wan Ta Shing, Industrial Ltd.) were used as polymer host matrices. No significant epoxy performance differences were noted when using these epoxies interchangeably. Powders formed partially dissolved suspensions within the epoxy materials and were dispersed ultrasonically after mechanical stirring to de-agglomerate the suspended particles. Sonication of 100-150 ml epoxy volumes was performed for 5-10 minutes using Bandelin Sonopuls HD2200 fitted with 12 mm stainless steel horn, running at 10-20% of its transducer power. Interlayers were formed by pouring luminophore-loaded epoxy over glass sample surfaces manually. Layer thickness adapters were used at all sample corners during the liquid phase of the lamination process and then removed during layer solidification. Light curing system (ELC-410 Thorlabs, Inc.) was used as UV-blue light source for epoxy curing. Solar cells integration and electrical circuits. 5 W CuInSe 2 thin film solar cell modules of size 200 3 270 mm on 2 mm -thick glass substrates (AD Solar, Inc.) were cut into strips of size 98 3 25 mm using diamond-wheel glass cutter. The number of p-n junctions integrated along the cut-out lengths had to be made identical which was confirmed by making I sc and V oc measurements at full illumination to ensure uniform electric performance characteristics. Soldering connections to tabbing wires were made using ultrasonic soldering iron (PVLED Technology, Ltd.) and SB-220 (S-Bond, Inc.) active solder compound. Clear UV-curable epoxy identical to the interlayer host material was used as adhesive to attach the solar cell modules to each of the four laminated sample sides. All four cell modules were connected electrically in parallel using blocking diodes, since significantly non-uniform illumination conditions were expected to affect the system performance during outdoor operation of samples placed vertically. Device characterization. Indoor tests were performed using a solar simulator system (ScienceTech, Inc.) as described in Principal Results and Discussion. Care was taken to ensure that all samples were tested at normal incidence conditions at a fixed (100 mm) distance from the source's output aperture, and that the AM 1.5G beam was centered onto the glass surface. Background illumination level was minimized by using curtains and removing room lighting. Outdoor tests were performed in natural daylight conditions, and I-V characteristics were recorded manually (point by point) using 3700 Series Programmable DC Electronic Load (Array Electronic Co., Ltd.) after orienting the vertically-placed samples in the horizontal plane to obtain peak output. The ''peak'' conditions were observed typically for horizontal-plane sample orientation angles near the 45u flux incidence condition with respect to the Sun azimuth direction. 3.19% 34.71% 0.649 Geometric gain G 5 1.87; energy collection area of 400 cm 2 . varied slightly during each of the stages. The design of thin film coating and running the deposition processes was mainly accomplished by R.A., M.V. and M.N. M.V. and M.N. designed the electrical interconnections circuitry and performed the integration of solar modules. All four authors (R.A., M.V., K.A. and M.N.) worked on selecting the luminophore chemistries suitable for use in functionalised lamination interlayers and contributed to the fine-tuning of glass panels lamination processes. All authors participated in the design of both the indoor and outdoor solar energy harvesting performance characterisation experiments. The final data analysis was accomplished by R.A. and M.V. The figures and images included in the manuscript were generated throughout the research program, and all authors contributed actively to their contents. The research progress and the results achieved at each stage were supervised by K.A. from the intial stages; M.N. and K.A. reviewed the manuscipt after it had been written by R.A. and M.V., after which all authors again contrubuted to producing the final manuscript version.

chemsum

{"title": "Spectrally-selective all-inorganic scattering luminophores for solar energy-harvesting clear glass windows", "journal": "Scientific Reports - Nature"}

microwave-assisted_synthesis_of_<i>n,n</i>-bis(phosphinoylmethyl)amines_and_<i>n,n,n</i>-tris(phosph

## Abstract: A family of N, N-bis(phosphinoylmethyl)amines bearing different substituents on the phosphorus atoms was synthesized by the microwave-assisted and catalyst-free Kabachnik-Fields reaction of (aminomethyl)phosphine oxides with paraformaldehyde and diphenylphosphine oxide. The three-component condensation of N,N-bis(phosphinoylmethyl)amine, paraformaldehyde and a secondary phosphine oxide affording N,N,N-tris(phosphinoylmethyl)amine derivatives was also elaborated. This method is a novel approach for the synthesis of the target products. ## Introduction α-Aminophosphine oxides are of considerable importance as potential precursors of α-aminophosphine ligands . α-Aminophosphines play an important role in the synthesis of P(III)-transition metal complexes , which are often applied catalysts in homogeneous catalytic reactions . In addition, a few Pt, Ru and Au complexes incorporating phosphine ligands show significant anticancer activity . One of the most common synthetic routes to α-aminophosphine oxides is the Kabachnik-Fields (phospha-Mannich) reaction, where an amine, an oxo compound (aldehyde or ketone) and a secondary phosphine oxide react in a condensation reaction . However, only a few papers deal with the synthesis of α-aminophosphine oxides. (Phenylaminomethyl)dibenzylphosphine oxide was prepared by the three-component reaction of aniline, paraformaldehyde and dibenzylphosphine oxide , as well as by the reaction of (hydroxymethyl)dibenzylphosphine oxide and aniline . The condensation of butylamine, paraformaldehyde and di(p-tolyl)phosphine oxide to afford (butylaminomethyl)di(p-tolyl)phosphine oxide was also described . A microwave (MW)-assisted, catalyst-free method was elaborated by us for the synthesis of several (aminomethyl)phosphine oxides . As regards α-aminophosphine oxides with different P-substituents, only two different types were reported. Olszewski and co-workers synthesized chiral thiazole-substituted aminophosphine oxides 2 through the Pudovik reaction of alkylphenylphosphine oxides and the corresponding aldimine derivatives of thiazole 1 (Scheme 1) . Cherkasov and his group applied the Kabachnik-Fields reaction to synthesize a P-chiral aminophosphine oxide with a 2-pyridyl substituent 3 (Scheme 2) . Bis(aminophosphine oxide) derivatives were also prepared by the double Kabachnik-Fields reaction using primary amines , amino acids or aminoethanol as the amine component. To the best of our knowledge, only one example can be found for a bis(α-aminophosphine oxide) containing different P-functions that was prepared by the condensation of (octylaminomethyl)dihexylphosphine oxide, paraformaldehyde and di(ptolyl)phosphine oxide in the presence of p-toluenesulfonic acid in boiling acetonitrile (Scheme 3) . Furthermore, tris(α-aminophosphine oxide) derivatives have not been described in the literature up to now. In this paper, we report the efficient, catalyst-free and MW-assisted synthesis of N,N-bis(phosphinoylmethyl)amine and N,N,N-tris(phosphinoylmethyl)amine derivatives bearing different substituents on the phosphorus atoms. ## Results and Discussion Synthesis of N,N-bis(phosphinoylmethyl)alkylamines containing different substituents on the phosphorus atoms First, the (aminomethyl)phosphine oxide starting materials 5-7 were synthesized following our previous protocol . Thus, the MW-assisted Kabachnik-Fields reaction of primary amines (butyl-, cyclohexyl-or benzylamine), paraformaldehyde and di(p-tolyl)-or dibenzylphosphine oxide was carried out in acetonitrile at 100 °C for 1 h affording the products with excellent yields (Scheme 4). Then, (aminomethyl)diphenylphosphine oxide (9) was prepared through debenzylation of (benzylaminomethyl)diphenylphosphine oxide (8, Scheme 5). The reduction was carried out in the presence of a 10% palladium on carbon catalyst (Selcat Q), in methanol, at 75 °C for 3 h, and the (aminomethyl)diphenylphosphine oxide (9) was obtained in a yield of 47% after column chromatography. In the next step, (aminomethyl)phosphine oxides 5-7 were converted to bis(phosphinoylmethyl)amine derivatives bearing different substituents at the phosphorous atoms (Y 2 P=O) by reacting them with one equivalent of paraformaldehyde and diphenylphosphine oxide under MW conditions (Scheme 6). The three-component condensations were performed in the absence of any catalyst in acetonitrile as the solvent to over-come the heterogeneity of the reaction mixture. After an irradiation of 1 h at 100 °C, the mixed N,N-bis(phosphinoylmethyl)amines 10a,b, 11a,b and 12a,b were obtained in yields of 92-97% and their structures were confirmed by 31 P, 13 C and 1 H NMR, as well as HRMS measurements. Due to the two differently substituted phosphorous nuclei in the molecules, two signals were observed in the 31 P NMR spectra. The valuable intermediate 9 was then utilized in the synthesis of N,N-bis(phosphinoylmethyl)amines 13a-c (Scheme 7). The condensation of (aminomethyl)diphenylphosphine oxide (9), paraformaldehyde and various secondary phosphine oxides, such as diphenyl, di(p-tolyl) or dibenzylphosphine oxide, at 100 °C for 40 min led to the corresponding N,N-bis(phosphinoylmethyl)amines containing identical (13a) or different substituents on the phosphorus atoms (13b and 13c) in excellent yields (95-97%). ## Synthesis of N,N,Ntris(phosphinoylmethyl)amines Finally, N,N-bis(phosphinoylmethyl)amines 13a and 13b were reacted further with paraformaldehyde and a secondary phosphine oxide (diphenyl-, di(p-tolyl)-or dibenzylphosphine oxide) to afford the N,N,N-tris(phosphinoylmethyl)amine derivatives bearing identical (14) and different Y 2 P=O groups (15-17) (Scheme 8). The condensations were performed as mentioned above. The introduction of a third phosphinoylmethyl moiety into the bis-derivatives containing an NH unit (13a and 13b) required a longer reaction time (2 h) at 100 °C. In these cases, the conversion was 70-95%, and the corresponding N,N,Ntris(phosphinoylmethyl)amine derivatives 14-17 were isolated in yields of 27-77%. However, applying a higher temperature and/or longer reaction time, lead to decomposition. ## Conclusion In summary, we have developed an efficient, catalyst-free and MW-assisted method for the synthesis of N,N-bis(phosphinoylmethyl)amines and N,N,N-tris(phosphinoylmethyl)amines bearing different substituents on the phosphorus atoms by the Kabachnik-Fields reaction. This method is a novel approach for the synthesis of the target products. In all, thirteen new derivatives were isolated in high yields and fully characterized.

chemsum

{"title": "Microwave-assisted synthesis of <i>N,N</i>-bis(phosphinoylmethyl)amines and <i>N,N,N</i>-tris(phosphinoylmethyl)amines bearing different substituents on the phosphorus atoms", "journal": "Beilstein"}

combined_ionic_liquid_and_supercritical_carbon_dioxide_based_dynamic_extraction_of_six_cannabinoids_

## Abstract: The potential of supercritical CO 2 and ionic liquids (ILs) as alternatives to traditional extraction of natural compounds from plant material is of increasing importance. Both techniques offer several advantages over conventional extraction methods. These two alternatives have been separately employed on numerous ocassions, however, until now, they have never been combined for the extraction of secondary metabolites from natural sources, despite properties that complement each other perfectly. Herein, we present the first application of an IL-based dynamic supercritical CO 2 extraction of six cannabinoids (CBD, CBDA, Δ 9 -THC, THCA, CBG and CBGA) from industrial hemp (Cannabis sativa L.). Various process parameters were optimized, i.e., IL-based pre-treatment time and pre-treatment temperature, as well as pressure and temperature during supercritical fluid extraction. In addition, the impact of different ILs on cannabinoid extraction yield was evaluated, namely, 1-ethyl-3-methylimidazolium acetate, choline acetate and 1-ethyl-3-methylimidazolium dimethylphosphate. This novel technique exhibits a synergistic effect that allows the solvent-free acquisition of cannabinoids from industrial hemp, avoiding further processing steps and the additional use of resources. The newly developed IL-based supercritical CO 2 extraction results in high yields of the investigated cannabinoids, thus, demonstrating an effective and reliable alternative to established extraction methods. Ultimately, the ILs can be recycled to reduce costs and to improve the sustainability of the developed extraction process. † Electronic supplementary information (ESI) available. See ## Introduction Cannabis sativa L. is an annual herbaceous blossoming plant that has been used throughout history in the textile industry, for recreational purposes and in medical applications. It is regarded as one of the oldest cultivated plants, and one of the most essential crops for the progress of humankind. Although native to Eastern Asia, its extensive applications led to its global spread. 1 The medicinal properties of Cannabis sativa L. can be attributed to the many bioactive compounds present in the plant, such as terpenes, polyphenols, phytosterols, tocopherols, fatty acids, and, specifically, cannabinoids, which are terpenophenolic secondary metabolites. 2,3 It is important to mention that cannabinoids are not equally distributed in the plant. They are mainly found in the trichomes and in smaller to negligible amounts in the seeds, while roots contain none. 4 Presently, over 100 cannabinoids have been identified. 5 They are primarily encountered in their carboxylated form in the plant which constitutes a structure of 22 carbon atoms. So far, cannabinoids have been categorized into 11 subclasses: (1) (−)-Δ 9 -tetrahydrocannabinol (Δ 9 -THC), (2) (−)-Δ 8 -tetrahydrocannabinol (Δ 8 -THC), ( 3) cannabidiol (CBD), ( 4) cannabigerol (CBG), ( 5) cannabichromene (CBC), ( 6) cannabinol (CBN), ( 7) cannabinodiol (CBND), ( 8) cannabicyclol (CBL), (9) cannabielsoin (CBE), (10) cannabitriol (CBT) and (11) miscellaneous. The structures of cannabinoids from hemp investigated in this study are depicted in Fig. 1. 6 In terms of the biosynthesis of cannabinoids, CBGA is the main precursor for THCA and CBDA. 7 However, under high temperatures, both acids are prone to degrade into their respective decarboxylated analogues, Δ 9 -THC and CBD. 8 Δ 9 -THC and CBD are the most abundant cannabinoids present in cannabis plants. Δ 9 -THC is well-known as a psychoactive compound, which influences the central nervous and cardiovascular systems. Contrarily, CBD is non-psychoac-tive, but is regarded as a compound of enormous medical interest, as it has demonstrated numerous health benefits. It has been reported to have anti-inflammatory, antiepileptic and anticonvulsive properties, among many others. Excellent medicinal potential have been attributed to cannabinoids; thus, significant effort has been made in the past decades towards the research of the functions and mechanisms of cannabis-derived secondary metabolites in the human body. Due to the growing medicinal interest in cannabinoids over the years, scientists have undertaken efforts in the development of extraction methods for these valuable bioactive compounds. Traditionally, Δ 9 -THC and other cannabinoids have been isolated by solvent-based extractions, with hydrocarbons and alcohols delivering the highest yields. 12,13 Soxhlet extraction (SE) is also a commonly used technique, 14,15 which is characterized by shortcomings, namely, long extraction times and high temperature that may promote thermal degradation of the target compounds. 16 Other advanced extraction techniques, such as microwaveassisted extraction (MAE) allow higher yields, shorter extraction times, less solvent and reduced energy consumption. 14,17 Nevertheless, uneven heating and/or overheating may cause thermal degradation, and thus negatively impact the extraction efficiency. 18 Alternatively, the use of ultrasound-assisted extraction (UAE) achieves high yields in short times; 19 however, the distribution of ultrasound energy lacks uniformity and over time the power decreases, which can lead to inefficient use of the ultrasound-generated energy. 20 Supercritical fluid extraction (SFE) is an innovative separation technique, which has thus far been employed for extractions of valuable constituents from over 300 plant species. 21 Carbon dioxide is a widespread choice for SFEs due to its several advantageous properties, such as low reactivity, nontoxicity, non-flammability, affordability, availability, and recyclability. Additionally, its selectivity can be adjusted by modification of pressure and temperature, while product fractionation and recovery with high purity is feasible. Nevertheless, due to its low polarity, addition of small quantities of organic solvents (co-solvents or modifiers) is necessary to access more polar compounds, thereby expanding its extraction range. 22 The selection of an appropriate co-solvent is key for achieving optimum solubility of the bioactive compounds present in the plant. 23 Supercritical carbon dioxide has previously been used to assess the solubility of individual cannabinoids, for example, Δ 9 -THC, 24 CBD 25 and CBG. 25 Moreover, several extractions of cannabinoids from different parts of the cannabis plant, for instance, leaves, trimmings, buds, flowers and threshing residues, have been performed using ethanol as a co-solvent. Within the past years, ionic liquids have also emerged as alternative reaction media for the extraction of biomass that is regarded as a source of natural medicinally relevant complex compounds. Many different properties are attributed to ionic liquids, such as exceptional dissolution properties, high thermal stability and broad liquid range, to name a few. Furthermore, ILs display high tuneability, as the combination of different cations and anions leads to hydrophilicity or hydrophobicity and different polarity. 30 The dissolution and processing of lignocellulosic biomass is a particularly interesting application of ionic liquids (ILs), as they can directly dissolve and fractionate (ligno-)cellulose in an overall less energy intensive process. 31,32 The biomass dissolution capability of ILs is impacted by both their cation and anion, however, current publications suggest that anions have a more significant impact, since they play a role in breaking the many intermolecular hydrogen bonds. 30 Regarding the cation, imidazolium-based ILs were the most successful for the direct dissolution of cellulose, followed by pyridinium-and ammonium-based ones. 33 In addition, increasing the chain length of the cation had a negative influence on the dissolving capabilities of the ILs, as the viscosity increased, and the H-bond acidity decreased. As far as the anion is concerned, dissolving efficiency seems to be determined by the H-acceptor properties of the anion. In general, anions with weak H-bond basicity, for instance, [BF 4 ] − and [PF 6 ] − , could not successfully dissolve cellulose, while ionic liquids based on halide or acetate anions are typically the candidates of choice. 30,34 The growing research on ILs as solvents for lignocellulosic biorefinery also prompted innovations for the extraction of valuable ingredients from plant materials. 35 There are several aspects of ILs that are potentially advantageous for the extraction of highvalue compounds: apart from their unique solvent properties and potential environmental benefits, the ability of ILs to dissolve biomass can lead to a better, and higher, yielding access to valuable ingredients embedded in the biopolymers and contribute to a value-added biorefinery. 36,37 However, the recovery of natural products from ionic liquids is often more demanding than the mere extraction: many studies require extensive back-extraction with volatile solvents to actually isolate the valuable ingredients from ILs, thereby rendering the original solvent reduction less significant or even negating it altogether. The combination of non-volatile polar ILs with volatile nonpolar scCO 2 has several advantages for extractions, as well as for catalysis. Since scCO 2 is highly soluble in ILs, but ILs cannot dissolve in scCO 2 , it can easily penetrate the IL-phase. This allows the extraction of compounds from the IL-phase into the scCO 2 phase, taken into account that the organic compound of interest is soluble in scCO 2 . Ultimately they are transported into an extraction vessel in a pure, solvent-free and solid form. 38 Furthermore, ILs in the presence of CO 2 expand their applicability, as their melting point and viscosity decrease, thus, promoting mass transportation. 39 Consequently, the combination of ionic liquids with scCO 2 has found application in several catalytic processes, such as hydroformylations, hydrogenations or carboxylations of alkenes in IL-scCO 2 biphasic reaction media. In the IL-scCO 2 reaction systems, the reactants and products are carried by the scCO 2 and IL is used as a reaction media. 44,45 Additionally, it is demonstrated that IL-scCO 2 biphasic systems avoid cross-contamination of the extracted solute. 38,46 Until now, IL-based pre-treatment and subsequent SFE (IL-SFE) for natural products has not been described, although ideal conditions arise from the unique properties of both media. Hence, by comparing IL-scCO 2 extraction with the utilization of both applications individually or to traditional solvent-extraction, the IL-scCO 2 approach is preferable. To begin with, less additional preparation, e.g., filtration of the raw material and consequent evaporation of solvents or separation of IL from the organic solvent is required to obtain a solvent-free and solid extract (Fig. 2). Consequently, there is a lower chance of loss of product or impurities, due to less post processing steps. On the other hand, IL-SFE is performed without additional co-solvents, therefore it reduces further solvent consumption and leads to lower expenses. Ultimately, if chosen appropriately, the ionic liquid can be recovered and re-used to improve the sustainability of the extraction process. Recently, an investigation of the extraction of cannabidiol with the aid of ILs has been published; however, isolation of cannabidiol required tedious back-extraction with organic solvents or with an aqueous AgNO 3 solution. 47 To the best of our knowledge, no data has been reported thus far regarding a combined extraction process that takes advantage of the complementing properties. Herein, we present the first application of IL-SFE from industrial hemp of six cannabinoids (Δ 9 -THC, THCA, CBD, CBDA, CBG and CBGA). Several parameters during the IL-assisted pretreatment, such as time, temperature and dilution with H 2 O, were investigated. In addition, pressure and temperature during SFE were evaluated. Ultimately, the optimized process for 1-ethyl-3-methylimidazolium acetate ([C 2 mim][OAc]) was additionally performed with choline acetate ([Ch][OAc]) and 1-ethyl-3-methylimidazolium dimethyl phosphate ([C 2 mim][DMP]) to compare the extraction efficiency of the investigated cannabinoids. In addition, the developed extraction process is complemented by a simple ionic liquid recovering process without the usage of additional organic solvents. ## Results and discussion The focus of this research was the investigation and optimization of various parameters for the extraction of CBD, CBDA, Δ 9 -THC, THCA, CBG and CBGA from partially pre-dissolved hemp in various room-temperature ILs with supercritical CO 2 . The optimization was divided into three successive stages (Scheme 1). In the first stage, the pre-treatment conditions to digest and partially dissolve hemp using [C 2 mim][OAc] before SFE were investigated. The lignocellulosic composition of hemp hurds is reported to contain 43.0% cellulose, 24.4% lignin and 29.0% hemicellulose. 48 ILs are known to dissolve a variety of carbohydrates, e.g., cellulose, by combining strongly basic anions (e.g., Cl − or OAc − ) with various cations. In particular, [C 2 mim][OAc] was selected in this study as it was used to pre-treat various lignocellulosic biomasses 52 and it is known to effectively dissolve, hemicellulose 53 and lignin. 54 . Both ILs are liquid at room temperature, non-halogenated and hydrophilic. Moreover, both ILs have been reported for pre-treatment of biomass. 55,56 In addition, the positive rating of choline-based ILs in terms of toxicity and biodegradation renders them ideally suited for natural product extractions. 57,58 Pre-treatment with ionic liquid (Stage 1) Herein, the influence of temperature and time for the partial dissolution of Cannabis sativa L. in [C 2 mim][OAc] before the scCO 2 extraction is evaluated. Initially, the conditions to partially dissolve industrial hemp in [C 2 mim][OAc] were investigated in experiments 1-4 (Table 1). Therefore, the pre-treatments were carried out at S1 and S2 †). The cannabinoids CBD and CBDA are predominantly accumulated in industrial hemp compared to THC, THCA, CBG and CBGA, which are considered minor compounds. The pre-treatment with [C 2 mim][OAc] of industrial hemp at 25 °C and 70 °C indicated comparable cannabinoid yields. Increasing the time from 15 to 60 min at 70 °C in exp. 2 led to a small decrease of roughly 5% ∑(CBD) and 8% ∑(THC). However, similar ∑(CBD), but significantly more CBD (6.58 mg g −1 ) and less CBDA (6.3 mg g −1 ) at 60 min, was yielded in exp. 2 compared with exp. 4 (15 min), which led to 5.29 mg g −1 CBD and 8.8 mg g −1 CBDA, respectively ( p < 0.05, Fig. 3, Table S2 †). It was reported that an extraction process including [C 6 mim][NTf 2 ] at 60 °C and 50 min leads to high amounts of CBD and that the IL preserves CBD, 47 which correlates with the observations herein. In addition, the decarboxylation of cannabinoids at higher temperatures for longer times has been described before. 8 The IL [C 6 mim][NTf 2 ] was not utilized in this study, as the anion [NTf 2 ] − renders it is less suit- able to dissolve cellulose compared to the basic [OAc] − or [DMP] − and similarly, the longer alkyl side chain of the cation would be disadvantageous for this purpose. 59 Ultimately, [NTf 2 ] − was not considered for the extraction process, as it is hydrophobic and not mixable with H 2 O and thus, not suitable for the IL recovering process shown in here. A total time of 15 min instead of 60 min seems to be sufficient to release the investigated cannabinoids from the plant tissue with [C 2 mim][OAc] and hence, allows a significantly shorter pre-treatment time The highest cannabinoids yields were obtained at 70 °C for 15 min in exp. 4, namely 13.6 mg g −1 ∑(CBD), 0.513 mg g −1 ∑(THC)and 0.247 mg g −1 ∑(CBG) (Table 1). ## Ratio of ionic liquid to water (Stage 2) Optimization of temperature and time during the pre-treatment was performed with a constant ratio of 1 : 2 Here, the influence of several IL : H 2 O ratios was investigated and compared with the sole use of IL as well as pure H 2 O in the extraction vessel (Table 2 and Fig. 4). A decrease of water in the IL : H 2 O ratio from 1 : 2 in exp. 4 to 1 : 1 in exp. 5 led to a significant reduction of ∑(CBD) as well as ∑(THC) yield (Table 2) at 20 MPa and 70 °C. However, the significantly highest yield of CBD (7.45 mg g −1 ) of all performed IL-SFE was obtained under these conditions in exp. 5 ( p < 0.05) and additionally, low yields of CBDA (1.09 mg g −1 ) and no CBGA were extracted (Fig. 4, Table S2 † 60 Therefore, H 2 O was added to the IL after the initial pre-treatment. The addition of H 2 O resulted in a reduction of the mixture's viscosity, and thus improved mass transport. 60 It is reported that the viscosity of [C 2 mim][OAc] is reduced by 50% when mixed with 10 wt% H 2 O and that the IL is less viscous at higher temperatures. 61 Lower viscosity of the IL : H 2 O mixture led to higher yields, possibly due to the higher mobility of dissolved cannabinoids and better penetration of scCO 2 . An increase in carboxylated cannabinoids was observed by adding more water (Fig. 4). Furthermore, water is the only solvent without any negative impacts on the environment. Additionally, it is reported to have low solubility in scCO 2 62 and therefore less potential con- 0.260 mg g −1 ( p < 0.05, Table 2). In particular, the use of H 2 O alone tends to yield fewer neutral cannabinoids (Fig. 4), which verifies what has previously been reported; ILs preserve neutral CBD. 47 When comparing exp. 8 with exp. 4, even though the same total quantity of liquid was added in the high-pressure vessel, significantly less yields of ∑(CBD) by 12% and ∑(THC) by 27% are observed ( p < 0.05, Table 2, Fig. 4) in the sole water-based SFE extraction. Therefore, a pre-treatment with IL to liberate the cannabinoids from the plant tissue and subsequent dilution with H 2 O positively affects the yield. Ultimately, a reference scCO 2 extraction in the absence of both IL and H 2 O in exp. 9 (no pre-treatment) yielded 10.1 mg g −1 ∑(CBD), 0.355 mg g −1 ∑(THC), 0.196 mg g −1 ∑(CBG) at 70 °C and 20 MPa (Table 2). Thus, IL-SFE with [C 2 mim][OAc] : H 2 O 1 : 3 in exp. 6 and 1 : 2 in exp. 4, led to significantly higher yields of ∑(CBD, THC, CBG) than sole SFE ( p < 0.05). It has been reported that the cannabinoid yields during SFE can be enhanced by adding EtOH as a modifier. 26,28 In preliminary studies SFE with EtOH as a modifier at different temperatures and vol% EtOH as well as various conventional ethanolic extractions were carried out with another batch of industrial hemp. High yields of the targeted cannabinoids were obtained at 35 °C, 10 MPa and 120 min dynamic extraction with 10 and 20 vol% EtOH. In comparison to the performed conventional extraction, similar ∑(THC) yields, but less ∑(CBD) and ∑(CBG) were yielded ( p < 0.05, Table S3 †). All data is presented in the ESI. † The addition of EtOH as a co-solvent to IL-SFE would lead to the extraction of both IL and cannabinoids, thus, leading to impurities in the extract. In particular, IL-SFE does not require the use of a co-solvent to obtain cannabinoids in high yields, avoiding further solvent consumption. Hence, the highest extraction yields were obtained with a IL : H 2 O ratio of 1 : 3 in exp. 6, which achieved 15.6 mg g −1 ∑(CBD), 0.542 mg g −1 ∑(THC) and 0.335 mg g −1 ∑(CBG) (Table 2). ## SFE extraction parameterspressure and temperature (Stage 3) Apart from the optimization of pre-treatment conditions and the ratio of [C 2 mim][OAc] to H 2 O, temperature and pressure during SFE were investigated (Table 3). Initially, the pressure was reduced from 20 MPa in exp. 6 to 15 MPa in exp. 11 at 70 °C and led to a significant reduction by 17% ∑(CBD) to 13.00 mg g −1 , 16% ∑(THC) to 0.457 mg g −1 and 23% ∑(CBG) to 0.245 mg g −1 ( p < 0.05, Table 3). After further decreasing the pressure to 10 MPa in exp. 10, a significantly diminished yield of 3.66 mg g −1 ∑(CBD), 0.0885 mg g −1 ∑(THC), and 0.045 mg g −1 ∑(CBG) was observed (Table 3). Even though lower cannabinoid yields were obtained at 10 MPa and 70 °C in exp. 10, the extraction of neutral cannabinoids was favoured (Fig. 5). In literature, sole scCO 2 extractions yield neither CBD nor CBDA at 10 MPa at 70 °C for 120 min, 63 but SFE can be improved upon by adding EtOH 26 or by the pre-treatment with IL, as herein reported. In addition, the pressure was increased to 30 MPa at 70 °C in exp. 12, which resulted in comparable yields of ∑(CBD, THC, CBG) as IL-SFE at 20 MPa in exp. 6 (Table 3). It can be assumed that 20 MPa at 70 °C are sufficient to extract cannabinoids during IL-SFE. Furthermore, the temperature was lowered to 35 °C at 20 MPa during SFE in exp. 13. This led to comparable yields of ∑(CBD) and ∑(CBG), but significantly lower ∑(THC) yields (0.493 mg g −1 ) compared to 70 °C in exp. 6 ( p < 0.05, Table 3). This corresponds to literature data, where similar yields of ∑(CBD) were extracted during SFE at 35 °C and 70 °C at 50 MPa. 63 Lower temperatures are known to reduce the viscosity of H 2 O and additionally, have been reported to decrease the viscosity of [C 2 mim][OAc]. 61 Hence, the mixture is less pene- trable for scCO 2 to extract the target cannabinoids. In comparison of exp. 13 and exp. 6, the yields of decarboxylated cannabinoids decreased significantly (CBD by 28%; Δ 9 -THC by 16%; CBG by 33%) and similar yields of THCA and CBDA, but significantly more CBGA by 23% was obtained in exp. 13 ( p < 0.05, Table S3 †). A further decrease from 20 MPa at 35 °C in exp. 13 to 10 MPa in exp. 14 led to a slight reduction in ∑(CBD) by 8% and ∑(THC) by 5%, and significant reduction in ∑(CBG) by 23% ( p < 0.05, Table 3). Therefore, a combination of 35 °C and 10 MPa seems to affect the total cannabinoid yield negatively, but increasing the temperature to 70 °C at the same pressure further reduces the yields. Lower CBD and CBDA yields at 10 MPa at 70 °C compared with 35 °C during SFE have been described in literature. 63 Thus, 10 MPa at 70 °C during SFE seem to be unfeasible to extract cannabinoids from industrial hemp. At 20 MPa, the temperature seems to have a minor effect on the total yields of cannabinoids. Consequently, the optimum cannabinoid yields were obtained at 20 MPa and 70 °C in exp. 6 during supercritical CO 2 extraction. 4 and Fig. 6). ## Type of ionic liquid Ionic liquid assisted SFE with [Ch][OAc] in exp. 15 yielded comparable yields of ∑(CBD) (15.4 mg g −1 ) and ∑(THC) (0.535 mg g −1 ), but significantly more ∑(CBG) (0.401 mg g −1 ) than IL-SFE with [C 2 mim][OAc] in exp. 6 ( p < 0.05, Table 4). The change of cation does affect the yields of cannabinoids, however, the role of the cation during the dissolution of lignocellulose structure is not yet fully understood. 64 On the other hand, anions, such as [OAc] − , are described to effectively support the dissolution of cellulose by forming hydrogen bonds. 34 To investigate the influence of the anion in IL-SFE of cannabinoids from industrial hemp, the imidazolium-based IL [C 2 mim][DMP] was used in exp. 16. This resulted in a significant reduction of ∑(CBD) to 11.8 mg g −1 and total THC to 0.449 mg g −1 compared with the acetate-based ILs in exp. 6 and exp. 15 ( p < 0.05, Table 4). [C 2 mim][DMP] is described as effectively dissolving biomass, but has a high viscosity, 56,65 which could affect the extraction at supercritical conditions, due to the weaker penetration of scCO 2 . Nonetheless, phosphate based and acetate based IL-SFE yielded higher amounts of ∑(CBD, THC, CBG) compared with sole supercritical CO 2 extraction without IL pre-treatment (Fig. 6). The following mechanism can be proposed for IL-SFE. Firstly, the biomass is partially dissolved by breaking down the lignocellulose structure of the industrial hemp powder. This depends on the anion and cation of the ILs. 34,64 The cannabinoids are released from the plant tissues and the IL possibly stabilizes them. 47 Secondly, the water is added, which reduces the viscosity of the mixture 61 and lowers the solubility of the target cannabinoids. Due to the lower surface tension and higher mobility of cannabinoids, a higher mass transfer between the scCO 2 phase and the IL : H 2 O phase is generated. As reported the scCO 2 dissolves in ILs, however, neither the IL nor the H 2 O does dissolve in scCO 2 . 38,62 Finally, these synergic effects allow the scCO 2 to extract the targeted cannabinoids, due to better solubility in the supercritical phase without contaminating it with IL or H 2 O. Thus, no further organic solvents are necessary to purify the compounds from the IL phase and consequently, no additional work up is needed to obtain a solid and solvent free product (Fig. 2). Ultimately, IL-SFE was compared with reference solvent extraction (exp. 17-19). Ethanol is one of the most commonly used solvents to extract cannabinoids. 66 Herein, a conventional extraction for 2 h, at 70 °C, with EtOH in exp. 17, sufficiently extracted the investigated cannabinoids; however, employing H 2 O in exp. 19 alone under the same conditions, low yields of cannabinoids were obtained (Table 4). A control extraction in EtOH for 24 h was carried out in exp. 18 to investigate the influence of longer extraction times. Longer times at high temperatures seem to degrade carboxylated cannabinoids significantly, reducing CBDA by 52%, THCA by 65% and CBGA by 53% ( p < 0.05, Table S2 †). The decarboxylation of cannabinoic acids at high temperatures for longer times is described in literature. 8 However, it can be reported that the degradation over time does not affect the overall cannabinoid yields. By comparing the two-hour ethanolic extraction (exp. 17) with acetate based ionic liquid-SFE, several differences can be observed. Firstly, the yields of ∑(CBD) by SFE with [C 2 mim][OAc] in exp. 6 and [Ch][OAc] in exp. 15 are slightly higher but comparable to the 2 h ethanolic extraction (Table 4). Secondly, significantly more ∑(THC) with 4). Hence, the results underline the importance of appropriately selecting the IL cation and anion, as well as the optimal extraction parameters for IL-SFE to extract cannabinoids from industrial hemp. Ultimately, [Ch][OAc] based SFE yields high amounts of the investigated cannabinoids and also provides environmental and economic benefits. Not only is [Ch][OAc] biodegradable, but it is also considered relatively cheap (88 € for 25 g), easy to synthesize, as well as less toxic compared to other ionic liquids. 57,58,67,68 Furthermore, no co-solvents are applied during IL-SFE, which avoids additional solvent consumption and consequently leads to a purer, solid extract (Fig. S2 †). Ultimately, all three ILs were purified without any additional use of organic solvents. Neither water nor significant impurities were detected by NMR spectroscopic analysis for the purified ILs (Table S4 and Hence, the type of IL is of great importance and affects the cannabinoid yield significantly. However, not only the type of IL needs to be selected carefully, also the SFE parameters. In dependence of various parameters, e.g. IL pre-treatment temperature or the ratio of IL : H 2 O during SFE, it is possible to adjust the proportion of carboxylated and decarboxylated cannabinoids in the extracts. In addition, IL-SFE allows extracting cannabinoids in highest yields and, therefore, it can be reported as a novel competitive alternative to traditional extraction techniques or supercritical fluid extraction with co-solvents. Ultimately, the ILs can be recycled without additional usage of further organic solvents to reduce costs and improve the sustainability of the process. IL-SFE offers the opportunity to extract secondary metabolites from different natural sources without volatile organic solvents and the presented process has great potential for future industrial applications. ## Plant material The type III chemovar Futura 75 was cultivated in Austria, in the fields of Biobloom (Apetlon, Austria, 7°41′23.4″N 16°56′ 26.7″E), in September 2020. After the harvest, the plants (flowers, leaves and stems) were stored under mild conditions at 40 °C for 14 h. The samples were milled with a Fritsch Universal Pulverisette 19 mill through a 2 mm sieve (Fritsch, Oberstein, Germany). The dry matter was 94.73 ± 0.05 wt% (n = 3). A second batch of the same industrial hemp harvested in 2019 was used for the preliminary experiments, mentioned in section Results and discussion. The dry matter was 93.68 ± 0.03 wt% (n = 3). The hemp raw material was stored in the dark, at −20 °C, between experiments. ## Ionic liquid-supercritical fluid extraction For pre-treatment, a high-pressure vessel of approximately 50 mL (EV-3), produced by Jasco (Jasco Corporation, Tokyo, Japan), containing one input and one output connections on the lid, was used. The batch reactor was charged with 0. The SFE setup is presented in Fig. 7. All extractions were performed with a scCO 2 device manufactured by Jasco (Jasco Corporation, Tokyo, Japan). Liquid CO 2 (>99.995% purity; with ascension pipe; Messer GmbH, Vienna, Austria) was pressurized by two CO 2 -pumps (PU-2086, Jasco Corporation, Tokyo, Japan) with cooled heads (CF40, JULABO GmbH, Seelbach, Germany). An oven (CO-2060, Jasco Corporation, Tokyo, Japan) with a heating coil was used and was thermostated to the desired temperature. The vessel containing the IL pre-treated hemp was placed on a heating mantle set to a certain temperature and a stirring rate of 500 rpm and, subsequently, connected to the supercritical carbon dioxide (scCO 2 ) device. A back-pressure regulator (BP-2080, Jasco Corporation, Tokyo, Japan), a gas/liquid separator (HC-2086-01, Jasco Corporation, Tokyo, Japan), and a product collector (SCF-Vch-Bp, Jasco Corporation, Tokyo, Japan) were used to obtain the extracts. The conditions employed for the SFE of cannabinoids were based on literature data 63,69 and adapted for our purposes. ## Solvent-based extraction For comparison, conventional solvent extractions were performed in 30 mL Teflon screw cap vials. The hemp quantity used in each extraction was 0.2 g. Two extractions were performed in triplicate using 2 mL solvent, more precisely, H 2 O and EtOH, for 2 h at 70 °C and a third one, also in triplicate, using 10 mL EtOH for 24 h and 70 °C. 70 ## Ionic liquid recovering After extraction, the scCO 2 device was depressurized, the metallic extraction reactor was disconnected and brought to room temperature. The IL-water-hemp mixture (Fig. S3 †) was filtered to remove hemp particles, the water was evaporated in vacuo and the remaining ionic liquid was dried under vacuum (0.65 mbar) for 24 h. Afterwards 20 mg of purified IL (Fig. S4 †) were dissolved in chloroform-d 3 (Sigma Aldrich, St Louis, USA) and a 1 H-NMR was recorded with a 400 MHz Bruker Advanced Ultra Shield 400 spectrometer (Bruker, Billerica, USA). Spectroscopic data and NMR spectra are given in the ESI (Table S4 and Fig. S5-7 †) ## Cannabinoid quantification The determination of CBDA, CBD, CBGA, CBG, THCA, Δ 9 -THC, was carried out on a High-Performance Liquid Chromatography (HPLC) in a Dionex UltiMate© RSLC System, with DAD-3000RS Photodiode Array Detector (Thermo Scientific, Germering, Germany), on a Dionex Acclaim™ RSLC 120 C18 (2.2 µm, 120 , 2.1 × 150 mm, Bonded Silica Products: no. 01425071, Thermo Scientific, Germering, Germany). A mobile phase flow rate of 0.2 mL min −1 was employed and the oven temperature was set to 25 °C. As a mobile phase, H 2 O with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B) were used. The following gradient was carried out: 2 min of pre-equilibration at 70% B, 6 min hold at 70% B, 6 min from 70% B to 77% B, 18 min hold at 77% B, 0.5 min from 77% B to 95% B, 1.5 min at 95% B, 0.5 min from 95% B to 70% B, and 5 min at 70% B. 71 Acetonitrile was purchased from VWR Chemicals (Radnor, PA, USA) and formic acid from Merck (Darmstadt, Germany). All solvents for HPLC were of analytical grade. The cannabinoid standards CBD, CBDA, THCA, Δ 9 -THC, CBG and CBGA were provided by Medical Cannabinoids Research and Analysis GmbH (Brunn am Gebirge, Austria) in the course of previous joint research. A mixed cannabinoid stock solution (1 mg mL −1 ) in MeOH of the investigated cannabinoids diluted for calibration. ## Statistical analysis Statistical data analysis was performed with Origin 2021. Oneway ANOVA for multiple groups, followed by Tukey honestly significant difference (HSD) post hoc test at the 0.05 significance level, was carried out. ## Addendum The authors would like to point out that the focus of this study was the extraction of cannabinoids as a class, not THC specifically. Any THC extraction is purely incidental, and bound to be negligible, given that industrial hemp was used, which in the EU must have a THC content not in excess of 0.2%. The relevant EU law can be perused under: https://eur-lex. europa.eu/legal-content/EN/TXT/HTML/?uri=CELEX:32013R13 07&from=de. In particular, we refer to Article 32, paragraph 6. Additionally, the authors do hold a licence to for the purposes of research, in accordance with Austrian law, available under:

chemsum

{"title": "Combined ionic liquid and supercritical carbon dioxide based dynamic extraction of six cannabinoids from <i>Cannabis sativa</i> L.", "journal": "Royal Society of Chemistry (RSC)"}

improved_calibration_of_electrochemical_aptamer-based_sensors

## Abstract: Electrochemical aptamer-based (EAB) sensors support the real-time, high frequency measurement of pharmaceuticals and metabolites in-situ in the living body, rendering them a potentially powerful technology for both research and clinical applications. Here we explore quantification using EAB sensors, examining the impact of media selection and temperature on measurement performance. Using freshly-collected, undiluted whole blood at body temperature as both our calibration and measurement conditions, we demonstrate accuracy of better than ± 10% for the measurement of our test bed drug, vancomycin. Comparing titrations collected at room and body temperature, we find that matching the temperature of calibration curve collection to the temperature used during measurements improves quantification by reducing differences in sensor gain and binding curve midpoint. We likewise find that, because blood age impacts the sensor response, calibrating in freshly collected blood can improve quantification. Finally, we demonstrate the use of non-blood proxy media to achieve calibration without the need to collect fresh whole blood. Electrochemical aptamer-based (EAB) sensors are a specific class of aptasensors that, uniquely, support highfrequency 1,2 real-time molecular measurements 3,4 directly in complex biological media, including unprocessed, undiluted bodily fluids 5,6 . In this sensor architecture, a target-recognizing aptamer is modified with a redox reporter and covalently attached to a gold electrode using a self-assembled monolayer (Fig. 1A) 5,9 . Upon target binding, the aptamer undergoes a conformational change, producing an easily-measurable shift in electrochemical signal 1 . As these sensors include both a recognition element (the aptamer) and a signaling element (the redox reporter), they do not require washing steps or reagent addition. And because they maintain their signaling properties in even complex sample matrices, such as undiluted whole blood, they perform well even when placed in situ in the living body . Indeed, their ability to perform real-time molecular measurements in vivo even supports closed-loop feedback-controlled drug delivery 8,9 . Their utility for in-vivo measurements makes this class of sensors particularly promising for clinical health monitoring and wearable devices. While a number of electrochemical techniques 5, have been used to interrogate EAB sensors, converting the resulting signals into estimated target concentrations always relies on the use of a calibration curve. When applying square wave voltammetry, the most commonly employed interrogation technique, a calibration curve is produced by collecting voltammogram peak currents over a range of target concentrations (Fig. 1C). Of note, because EAB sensor signaling varies with applied square wave frequency, square wave voltammetry can be tuned to yield an increase ("signal-on") or a decrease ("signal-off ") in peak current upon target addition (Fig. 1B) 1, . To correct for drift and enhance gain during in vivo measurements, voltammograms are collected at two such frequencies and converted into "Kinetic Differential Measurement" (KDM) values. These KDM values are derived by subtracting the normalized peak currents seen at signal-on and signal-off frequencies, then dividing by their average (Fig. 1C) 16 . To generate a calibration curve, the averaged KDM values collected in vitro over a range of target concentrations are fitted to a Hill-Langmuir isotherm [Eq. 1, Fig. 1D] 17 : where n H is the Hill coefficient (a measure of binding cooperativity), K 1/2 is the midpoint of the binding curve, KDM is the KDM value observed at the applied target concentration, KDM min is the KDM value seen in the absence of target, and KDM max is the KDM value expected at saturating target. These parameters extracted from a calibration curve, then, allow translation of EAB sensor output into estimates of target concentration [Eq. 2, Fig. 1E]: EAB calibration curves thus depend on both the affinity of the aptamer, K 1/2 (which, in the case of a noncooperative aptamer, simplifies to K D , the dissociation constant), the cooperativity (or anti-cooperativity) of binding, n H , and the sensor's signal gain, KDM max . Given that both of these parameters are influenced by environmental factors, such as temperature 18,19 , pH 20 , and ionic strength 20,21 , careful selection of calibration conditions is required in order to accurate and precise measurements. Here, we examine the calibration of EAB sensors in detail, with the aim of improving the accuracy of in vivo measurements performed using them. Specifically, we investigate the impact of media temperature, age, and composition on the observed calibration curves obtained using square wave voltammetry, and assess the accuracy of in vitro measurements performed in freshly-collected, body temperature blood. As our test bed, we examine the response for a sensor detecting vancomycin, an antibiotic target 22 of strong interest for therapeutic monitoring . ## Results and discussion EAB sensors calibrated in fresh whole rat blood under the same conditions employed during the measurement achieve clinically useful performance. To demonstrate this, we applied parameters obtained from a calibration curve collected in fresh, body temperature (37 °C) rat blood (Fig. 2A) to measurements performed in fresh, body temperature rat blood dosed with known concentrations of vancomycin (Fig. 2B). Doing so, we achieve mean accuracy of 1.2% or better over the drug's 6 to 42 µM clinically relevant range 26,27 and 10.4% or better at all concentrations (Fig. 2C). Here, we define accuracy as the mean of the relative difference between the estimated and applied concentrations (100*(expected -observed)/observed). Likewise, we achieve precision of 14% or better in the clinical range, which we define as the coefficient of variation (100*population standard deviation/ population mean). This level of performance is more than adequate for many clinical applications. For example, Figure 2. Vancomycin-detecting EAB sensors calibrated using a calibration curve collected in matched media and temperature easily achieve clinically useful measurement accuracy. (A) Here we created a calibration curve by fitting the average response of four sensors to a Hill-Langmuir isotherm using data collected at 37 °C in freshly-collected rat blood. We derive KDM values by subtracting the normalized peak heights collected at 300 Hz from those collected at 25 Hz, and dividing by their average 16 . We indicate the clinical range of vancomycin in grey. Specifically, the clinical window of vancomycin ranges from 6 to 42 µM, which reflects the minimum target concentrations to achieve clinical effect, to the mean maximum peak concentrations concentrations 26,27 . The error bars shown here and in the following figures reflect the standard deviation of replicate measurements performed using independent sensors (K 1/2 = 73 ± 4 µM, n = 4). The error reported for these and all other K 1/2 values reflect 95% confidence intervals. (B) We then apply this calibration curve to quantify measurements performed using the same four sensors in 37 °C fresh rat blood to which 10, 20, 50, 100, or 300 µM vancomycin has been added (dotted lines, red lines). We indicate the clinical range of vancomycin in grey. (C) From these measurements, we calculate mean estimated concentration, standard deviation, mean accuracy (defined as 100*(expected -observed)/observed), and coefficient of variation (100*population standard deviation/population mean). We observe better than 10% accuracy for all challenges. Vol:.( 1234567890) www.nature.com/scientificreports/ accuracy of ± 20% over the therapeutic range is sufficient to achieve clinically relevant therapeutic monitoring of vancomycin 24,28 . This suggests that for measurements in the living body, applying a calibration curve captured in freshly collected, body temperature blood will yield clinically accurate and precise measurements of this drug. In the studies described above, we calibrated each individual sensor using a single, common calibration curve obtained by averaging the sensor's calibration curve with those of other vancomycin-detecting sensors. That is, each sensor was calibrated using a calibration curve to which it itself had also contributed. If, however, we instead use an "out-of-set" calibration curve (a calibration curve to which the sensor under investigation did not contribute, Fig. 3A), we see no significant change in accuracy and only a slight reduction in precision over the clinically relevant range of vancomycin concentrations (Table S1). Likewise, we do not see significant improvement in accuracy or precision over the clinical range if we calibrate each sensor using its own, individual calibration curve (Fig. 3B, Table S2). These observations suggest that sensor-to-sensor variation is not a major contributor to the level of accuracy and precision we achieve with this sensor at clinical target concentrations. Collecting calibration curves at the appropriate temperature is key to sensor calibration. Specifically, between room and body temperature, calibration curves differ significantly (Fig. 4A). Depending on interrogation frequency employed, this can lead to considerable under-or over-estimation of target concentrations. Interrogating at 25 and 300 Hz, for example, we observe up to a 10% higher KDM signal at room temperature than at body temperature over vancomycin's clinical concentration range. Consequently, applying a calibration curve collected at room temperature to those collected at body temperature causes substantial concentration underestimates (Figure S2). Interrogating using other square wave frequency pairs, likewise, can yield even more significant differences in KDM signal in the clinical range (Figure S3). These differences in sensor response occur because S3). (B) This occurs at least in part because the electron transfer rate from the redox reporter changes with temperature. Specifically, the peak charge transfer rate shifts toward higher frequencies at higher temperatures. To illustrate this, we plot here charge transfer versus frequency for a representative sensor in absence of target. We do so by determining square wave voltammogram peak current, and dividing it by its given interrogation frequency (additional sensor curves shown in Figure S1) 29 www.nature.com/scientificreports/ temperature changes can shift system properties, such as binding equilibrium coefficients and the electron transfer rate itself. For example, the electron transfer rate (indicated by the location of peak charge transfer, when plotting interrogation frequency versus charge transfer) increases with temperature for the vancomycin aptamer (Fig. 4B), as well as for EAB sensors against other targets (Figures S4-S6). This shift is great enough that it can affect the selection of signal-on and signal-off frequencies. For example, from room temperature to body temperature, 25 Hz changes from a weak signal-on frequency to a clear signal-off frequency (Figure S7). These results illustrate the importance of media temperature for both the accurate calibration of in vivo data, as well as the selection of the signal-on and signal-off frequencies used to perform KDM. Obtaining bovine blood from commercial vendors is often more convenient than collecting fresh blood samples each time one needs to generate a calibration curve. Unfortunately, however, calibration curves obtained in the commercially sourced and freshly-collected blood samples differ (Fig. 5A). Specifically, vancomycin sensors challenged in commercially sourced bovine blood yielded lower signal gain, which would lead to overestimated vancomycin concentrations (Fig. 5A). This difference in gain could arise from differences in the species source of the blood (bovine versus rat), how the blood was processed, or its age. Due to shipping, for example, commercially sourced blood is at least a day old by the time we use it. To determine whether blood age (time after collection) is an important calibration parameter, we titrated sensors in commercial bovine blood one day after collection and again using the blood from the same blood draw 13 days later. While the two samples yielded similar signals over the drug's clinical range, the older sample produced lower signal at concentrations above this range (Fig. 5B). To assess the source of these differences, we examined the normalized square-wave signals at 25 and 300 Hz (Figs. 5C-D). Doing so, we find that while we observe a marked signal decrease at target concentrations below 1 µM (Fig. 5C-D), KDM effectively corrects for this. That there is still a difference in the 1 and 14 day old blood calibration curves suggests that blood age itself can impact the EAB sensor response. Given this, we believe measurements performed in the living body are best calibrated using curves obtained in the freshest possible blood. Although it is the most accurate calibration matrix for in-vivo measurements, the collection of fresh blood for sensor calibration can be inconvenient, leading us to ask if there are other, more easily obtained media that Figure 5. (A) Titrations performed in fresh rat blood and commercially sourced, one-day-old bovine blood produce distinct calibration curves. (B) To determine whether this is driven by species-specific differences in blood, or the greater age of the commercially sourced bovine blood, we compared calibration curves obtained in a bovine blood sample 1 day (red, K 1/2 = 116 ± 15 µM) and 14 days (black, K 1/2 = 112 ± 9 µM) after it was collected. Doing so, we find that the KDM response does not change significantly in the clinical range. (C, D) Examining the signal at the two square-wave frequencies used to perform KDM (here, 25 Hz and 300 Hz) we see that, for the 14-day-old blood, there is a notable signal decrease at vancomycin concentrations far below the aptamer's dissociation constant. Given that no target-induced response should occur at these low concentrations, we believe this is an artifact due to time-dependent sensor degradation in the older blood. Vol:.( 1234567890 ), the titration yields greater signal gain than that observed in rat blood (Fig. 6A). Applying the resulting calibration curve, then, would lead to underestimation of vancomycin in rat blood. The PBS calibration curve, in contrast, exhibits higher response in the clinical range and a lower response above the clinical range. Thus, depending on the concentrations applied, this curve would lead to under-or overestimates of vancomycin in rat blood (Fig. 6B). A calibration curve collected in PBS with bovine serum albumin, however, reproduces the calibration curve obtained in fresh rat blood to a fair degree of accuracy (Fig. 6C). Enough so that, when we quantify the successive vancomycin additions in body temperature rat blood with this calibration curve, we obtain accuracies of better than 9% in the clinical range and better than 35% across all concentrations (Figure S8, Table S4). While poorer than the accuracy we achieve via calibration in rat blood, this calibration may still be acceptable in some applications. More broadly, these results suggest that buffered proxy systems can help guide development and application of sensors in vivo, and calibrate sensors when reduced accuracy is an acceptable tradeoff for improved convenience. ## Conclusions With the appropriate selection of media temperature, composition, and age, a vancomycin-detecting EAB sensor easily achieves clinically relevant accuracy. Specifically, when calibrated at the appropriate temperature in freshly collected blood, sensors achieve accuracy of better than 10% when challenged under those same conditions. This result holds for both sensors calibrated against themselves and for sensors calibrated against other sensors. In contrast, calibration curves collected at inappropriate temperatures or in aged blood samples deviated more significantly from those seen in fresh, body temperature blood. Thus, these conditions may produce substantial over-or under-estimates of target concentration. Finally, in some cases, it is possible to use simpler media, such as phosphate buffered saline with bovine serum albumin, as a convenient and proxy for freshly collected blood for applications in which reduced accuracy is an acceptable trade-off relative to improved ease of calibration. Here, we investigate whether simpler media can reproduce the response seen in freshly-collected rat blood. To do so, we compare titrations (KDM values derived from 25 and 300 Hz) collected in 37 °C freshly collected rat blood to (A) 37 °C Ringer's buffer with 35 mg/mL bovine serum albumin (BSA), (B) 37 °C phosphate buffered saline (PBS) with 2 mM MgCl 2 , and (C) 37 °C PBS with 2 mM MgCl 2 and 35 mg/mL bovine serum albumin. From these data we observe that calibration in 37˚C PBS with added BSA is a reasonable proxy for fresh rat blood. Of note, the close correspondence between the sensor's response in simple buffers and in whole blood indicate that the sensor does not respond significantly to the components naturally present in blood. Vol ## Materials and methods Electrode fabrication. We fabricated sensors by soldering a 5 cm long, 75 µm diameter, PFA-coated gold wire (A-M Systems, Sequim, WA) to a gold-plated pin connector (CH Instruments, Inc., Austin, TX) with 60/40 lead-selenium solder (Digikey, Thief River Falls, MN). Before soldering, we used a digital caliper and razor blade to isolate 3 mm of gold on both ends of the gold wire. One end forms the working electrode, and the other end must be exposed for successful soldering. To prevent breakage of the delicate wires, we coated the wire-solder interface with a coat of urethane conformal coating (MG Chemicals, Surrey CA). Aptamer preparation. We first thawed a 2 μL, aliquot of 100 μM aptamer (sequence below) modified with a 6-carbon thiol linker on the 5′ end and methylene blue on the 3' end (Biosearch Technologies, Novato, CA, dual HPLC purification, stored at − 20 °C). Because the manufacturer provides the DNA constructs in oxidized form, which does not effectively immobilize onto the gold surface, we must reduce the disulfide bond before deposition. To do so, we combined 2 μL of 10 mM Tris (2-carboxyethyl) phosphine (TCEP, Sigma-Aldrich, St. Louis, MO) with the aptamer sample for a 1 h thiol reduction in the dark at room temperature. We then diluted the sample with 96 μL 1X phosphate buffered saline (pH = 7.4). Here and elsewhere in our protocol, the 1X PBS is prepared by diluting 20X phosphate buffered saline (ChemCruz 20X phosphate buffered saline, Santa Cruz Biotechnology, Dallas, TX) to 1X using deionized water. , We quantified the concentration of the aptamer using the molar absorption coefficient at 260 nm provided by the supplier with UV-VIS spectroscopy (DU800, Beckman Coulter, Brea, CA). Using PBS, we then diluted the aptamer to 500 nM. ## Electrode preparation. For electrode cleaning and in-vitro characterization, our electrochemical cell contained a PFA-wrapped gold sensor working electrode, a platinum counter electrode (CH Instruments, Inc, Austin, TX), and an Ag|AgCl reference electrode (CH Instruments, Inc., Austin TX). We secured our working, counter, and reference electrodes in a cell vessel "shot glass" with a custom-fabricated Teflon lid fixture (See Fig. 1C). First, we electrochemically cleaned the electrodes in 0.5 M NaOH (Sigma-Aldrich, St. Louis, MO) by performing repeated cyclic voltammetry scans between − 1 and − 1.6 V (all potentials versus Ag|AgCl) at 1 V s −1 scan rate for 300 cycles (Table 1) using a CH Multipotentiostat (CHI1040C, CH Instruments, Inc.) 31 . We rinsed the electrodes in deionized water, then increased the surface area of the gold wire electrode by electrochemically roughening in 0.5 M H 2 SO 4 (Sigma-Aldrich, St. Louis, MO) using a previously-described procedure that involves repeatedly stepping the potential between 0 and 2.2 V using chronoamperometry (Table 2) 31 . This electrochemical procedure, repeated 50 times using the CH instrument software, yields a 2 to fivefold increase in microscopic surface area 31 . Sensor fabrication. Immediately after roughening, we rinse the electrodes thoroughly in deionized water and place them in the prepared aptamer solution for 1 h at room temperature in dark conditions 8 . We then rinsed the electrodes with deionized water and immersed them for 12 to 18 h at room temperature in a 10 mM 6-mercapto-1-hexanol (Sigma-Aldrich, St. Louis, MO) in 1X PBS to passivate the surface. Following a final rinse with deionized water, the sensor was ready for use. Blood collection. 12 mL of blood was collected from a single ~ 400 g adult male Sprague-Dawley Rat (Charles River Laboratory, Santa Cruz, CA). The rat was allowed to acclimatize to the colony for at least one week. The animal was placed under anesthesia using isoflurane gas and, following abolishment of toe pinch www.nature.com/scientificreports/ response, a horizontal incision was made below the rib cage. The incision was extended on each side to under the front legs, at which point vertical incisions were made through the ribcage. The diaphragm was then cut to reveal the heart and the septum pinned back using hemostats. A 20 mL syringe was prepared with 300 units of heparin in order to achieve a final concentration range of 20 units/mL (Sagent Pharmaceuticals, Schaumburg, IL). An 18G needle was attached to the top of the syringe and inserted into the left ventricle for blood collection (BD, Franklin Lakes, NJ). Very light pressure was placed upon the syringe in order to facilitate blood removal. The blood draw (15 mL total) was stopped when the flow of blood into the syringe finished after which point the animal was euthanized via isoflurane overdose. The Institutional Animal Care and Use Committee (IACUC) of the University of California at Santa Barbara approved our experimental protocol, which adhered to the guidelines provided by the American Veterinary Medical Association. Blood was transported immediately to a temperature-controlled bath at 37 °C, then used for experiments (Lauda Ecoline RE 106, Lauda-Brinkmann, Delran, NJ). This study adhered to ARRIVE guidelines, where applicable. Specifically, randomization, blinding, and exclusion criteria (ARRIVE guidelines 3, 4, and 5) are not applicable because we drew blood from a single animal. ## Measurements. We filled the electrochemical cell with PBS, rinsed the electrodes in deionized water, and secured them in the electrochemical cell's Teflon cap. Before each experiment, we collected three baseline cyclic voltammograms in PBS (− 0.1 to − 0.5 V versus Ag|AgCl, 0.1 V/s scan rate) to confirm successful aptamer deposition. To perform a titration, we moved the electrode cap to a vessel of the selected media held in a temperature bath at the selected temperature (Lauda Ecoline RE 106, Lauda-Brinkmann, Delran, NJ). For bovine blood experiments, we commercially sourced blood from Hemostat Laboratories (Dixon, CA). For Ringer's buffer experiments, we prepared Ringer's buffer at pH 7.4 using 154 mM NaCl, 5.64 mM KCl, 2.16 mM CaCl2, 11.10 dextrose, 2.38 NaHCO3, 2 mM Trizma®, and 35 mg/mL bovine serum albumin (heat shock fraction, protease free, Sigma-Aldrich, St. Louis, MO). In the selected media, we collected square wave voltammetry scans (approximately − 0.2 to − 0.4 V versus Ag|AgCl, 25 mV amplitude) at 10, 25, and 300 Hz first in absence of target. We selected these frequencies because they were the originally reported signal-off, non-responsive, and signal-on frequencies for this particular aptamer. We observed, however, that at body temperature, 25 Hz yields a reliable signal-off response, and thus use this as the signal-off frequency for this work. Upon addition of incremental target concentrations, we back pipetted the electrochemical cell 15 times, allowed the solution to rest for two minutes, then proceeded with the measurement. To perform a spiking experiment, we used a CH software macro to repeatedly collect and store square wave voltammograms. We collected a 10 min baseline with no target, then sequentially added 10, 20, 50, 100, 300 µM vancomycin. For each addition, we pipetted rapidly, while taking care not to create bubbles in the blood, then measured the resulting signal for 5 to 10 min. It is common to observe a downward drift in square wave voltammogram peak current, especially in complex media, such as whole blood. To correct for any peak signal loss, we applied a previously-described drift correction technique termed "Kinetic Differential Measurements" (KDM) 16 . Here, KDM denotes when normalized signal off-peak currents are subtracted from the normalized signal-on peak currents, then divided by the average of normalized signal-on and signal-off currents.

chemsum

{"title": "Improved calibration of electrochemical aptamer-based sensors", "journal": "Scientific Reports - Nature"}

enantioselective_assembly_and_recognition_of_heterochiral_porous_organic_cages_deduced_from_binary_c

## Abstract: Chiral recognition and discrimination is not only of significance in biological processes but also a powerful method to fabricate functional supramolecular materials. Herein, a pair of heterochiral porous organic cages (HPOC-1), out of four possible enantiomeric products, with mirror stereoisomeric crystal structures were cleanly prepared by condensation occurring in the exclusive combination of cyclohexanediamine and binaphthol-based tetraaldehyde enantiomers. Nuclear magnetic resonance and luminescence spectroscopy have been employed to monitor the assembly process of HPOC-1, revealing the clean formation of heterochiral organic cages due to the enantioselective recognition of (S,S)binaphthol towards (R,R)-cyclohexanediamine derivatives and vice versa. Interestingly, HPOC-1 exhibits circularly polarized luminescence and enantioselective recognition of chiral substrates according to the circular dichroism spectral change. Theoretical simulations have been carried out, rationalizing both the enantioselective assembly and recognition of HPOC-1. ## Introduction Porous organic cages (POCs) are newly emerging attractive crystalline molecular materials with great application potential in the felds of storage and separation, 1 sensing, 2 and catalysis. 3 Their advantages originate from the intrinsic and extrinsic voids together with the functional groups attached on POCs. Thus far, molecular POCs are mainly formed from the selfassembly of discrete reactive building blocks driven by dynamic covalent chemistry (DCC) including the reactions of boronic acid condensation, 4 imine condensation, 5 and alkyne/ alkene metathesis. 6 In particular, various amine-and aldehyde-decorated building blocks are available to assemble POC molecules with different cavities, dimensions, and topologies. 7 These discrete cage-like molecules obtained are engineered crystallographically to form porous assemblies (called POCs) through efficient supramolecular interactions, exhibiting huge adsorption capacity. In addition, the new application of POCs as a kind of unique synthon has been initiated by accommodating fne nanoparticles 8 and being fabricated into reticular frameworks 9 with the help of metal-coordination, covalent and hydrogen-bonding interactions. Chirality is vital to biological processes and widely exists in various biological structures at the molecular level, including polysaccharides, proteins and DNA. 10 Incorporation of chirality into artifcial functional materials provides new objectives towards chiral separation, 11 stereospecifc catalysis, 12 chiral recognition, 13 and unique chiroptical properties. 14 Selfassembly depending on chiral recognition and discrimination has been used to prepare chiral functional supramolecular materials through noncovalent interactions such as electrostatic interactions, 15 p-p interactions, 16 hydrogen bonding, 17 and metal-coordination bonds. 18 In quite recent years, DCCbased self-sorting of POCs has been achieved using the racemic mixtures of enantiomers as building blocks, generating a few homochiral and heterochiral cages. 19 It is worth noting that all the thus far obtained heterochiral POCs are made up of enantiomers from only one component. More complicated heterochiral systems derived from the enantiomers of two or more components still remain unreported, to the best of our knowledge. As a consequence, investigation of complicated heterochiral cages and the corresponding applications is surely of signifcance for developing POCs and chirality chemistry. Herein, we present the clean synthesis of heterochiral porous organic cages (HPOC-1) by enantioselective assembly of the enantiomer of a binaphthol-based tetraaldehyde with specifc enantiopure cyclohexanediamine (CA). The corresponding self-assembly reaction kinetics has been tracked using luminescence and nuclear magnetic resonance (NMR) spectroscopy, revealing the clean formation of heterochiral porous organic cages due to the enantioselective recognition of the CA enantiomer towards the enantiomeric binaphtholderived building block 5,5 0 -(6,6 0 -dichloro-2,2 0 -diethoxy-[1,1 0binaphthalene]-4,4 0 -diyl)diisophthalaldehyde (DBD). The crystal structures of the pair of heterochiral HPOC-1 enantiomers show precise sorting of two kinds of chiral building blocks. Interestingly, HPOC-1 displays circularly polarized luminescence (CPL). Furthermore, heterochiral HPOC-1 is able to enantioselectively recognize chiral substrates through circular dichroism (CD) spectral change due to the host-guest supramolecular interactions. ## Results and discussion The synthetic route to POCs from tetraaldehydes and CA created by Cooper's group has provided diverse [3 + 6] tubular organic cages. 20 Towards synthesizing more complex heterochiral POCs with two different kinds of enantiomers from more than one component, pure chiral binaphthol-bearing building blocks (DBD) shown in Scheme 1 were prepared to react with cyclohexanediamine (CA). Four sets of combination for these enantiomeric building blocks of two compounds, namely [(S,S)-DBD + (S,S)-CA], [(S,S)-DBD + (R,R)-CA], [(R,R)-DBD + (R,R)-CA] and [(R,R)-DBD + (S,S)-CA], are expected to generate four heterochiral organic cages. Unfortunately, only the reaction between (S,S)-DBD and (R,R)-CA or (R,R)-DBD and (S,S)-CA was able to deliver clean (S,R)-and (R,S)-HPOC-1, respectively, on the basis of MS and NMR spectroscopic data, Table S1 and Fig. S1-S11. † In order to understand the assembly process, timedependent NMR spectra were collected for the system of (S,S)-DBD in 2.0 mL CDCl 3 containing 1.0 mL TFA with the addition of 2.0 equiv. of (R,R)-CA and (S,S)-CA, respectively, Fig. S12. † (S,S)-DBD showed one typical singlet peak at d 10.26 ppm due to the aldehyde protons. In addition, methyl and methylene protons displayed peaks at d 1.16 and 4.15 ppm, respectively. Following the addition of (R,R)-CA for 1.0 min, the proton signal of the aldehyde quickly disappeared. Instead, a doublet peak started to appear at d 8.38 ppm due to the formation of imine bonds. After 20.0 min, the methyl proton signals moved towards high feld of d 0.27 ppm and methylene proton signals migrated to d 3.00 and 3.28 ppm. The larger high-feld movement indicates an increase in the density of the electron cloud around the methyl group, which further illustrates the location of ethoxy groups inside the cage molecule rather than outside the cage. After the proceeding of the reaction for 24.0 h, the 1 H NMR spectrum of the reaction mixture is overall consistent with that of the as-synthesized (S,R)-HPOC-1, implying the clean generation of the heterochiral POC. This is verifed by MALDI-TOF MS data with the observation of only one cage molecular ion peak at m/z ¼ 2495.6, Fig. 19a-d,f In addition, fluorescence spectroscopy was used to trace the reaction dynamics of (S,S)-DBD with (R,R)-CA and (S,S)-CA, respectively, with a molar ratio of 1 : 2 in CH 2 Cl 2 , Fig. 1a-c. There is a slight emission band of DBD at ca. 502 nm. After the introduction of (R,R)-CA for 5.0 min, an obvious emission band was observed at ca. 418 nm. As time went on, the maximum position of the emission band with gradually increased intensity slightly moved to 406 nm. After 60.0 min, the intensity remained constant. Such a phenomenon is also observed in the reaction system of (S,S)-DBD and (S,S)-CA, Fig. 1d-f. Differently, a shorter time of 30.0 min enabled a constant emission intensity at 406 nm maximum for the latter system. It is worth noting that the emission intensity at maximum for the former system was almost twice as big as that of the latter one. An enantiomeric fluorescence ratio (ef) value of 1.62 was therefore determined based on the division of maximum intensity of the former system by that of the latter one, confrming the higher enantioselectivity of (S,S)-DBD towards (R,R)-CA, rather than (S,S)-CA, to form pure [3 + 6] POC. When (R,R)-DBD was used, a similar phenomenon was found upon the addition of (S,S)-CA and (R,R)-CA, respectively, and the ef is calculated as 1.82, Fig. S16. † This value is similar to the ef of (S,S)-DBD. These results demonstrate the moderate enantioselectivity between these two chiral building blocks during the reaction. 13c,h Density functional theory (DFT) calculations were performed on (S,R)-HPOC-1 and (S,S)-HPOC-1 (assembled from (S,S)-DBD + (S,S)-CA) towards understanding the clean generation of the former heterochiral POC associated with enantioselective recognition between two kinds of enantiomeric building blocks. The simulated formation energy for (S,R)-HPOC-1 is 132.4 kcal mol 1 , much smaller than that for the latter species (71.5 kcal mol 1 ), hinting at the more favorable formation of the former cage from a thermodynamics perspective. In the present case, the optimized structure of (S,R)-HPOC-1 exhibits less variation in bond length for the cyclohexanediimine moiety than that of (S,S)-HPOC-1 (Fig. S17 and Table S2 †), indicating the more stable structure of the former POC. To further confrm this point, theoretical simulation was also carried out on another heterochiral POC, HPOC-2 derived from the reaction between DBD and 1,2-diphenylethylamine (DPA) enantiomers. The formation energy of (S,R)-HPOC-2 (137.3 kcal mol 1 ), derived from (S,S)-DBD and (R,R)-DPA, was revealed to be smaller than that of (S,S)-HPOC-2 (131.7 kcal mol 1 ), assembled from (S,S)-DBD and (S,S)-DPA, further supporting the present clean generation of heterochiral POCs due to the enantioselective assembly mechanism. These results agree well with the experimental fndings that [3 + 6] topological (S,R)-HPOC-2 has been cleanly generated according to NMR and MS data, Fig. S18 and S19. † However, the small enantiomeric fluorescence ratio of 1.17 for (S,S)-DBD toward (R,R)-DPA and (S,S)-DPA, Fig. 1f, indicates the weak selectivity of the enantioselective recognition process occurring in the generation of the latter heterochiral POCs in comparison with HPOC-1. To detect the structural information of the heterochiral cages, colourless single crystal enantiomers of HPOC-1 were surveyed using a single crystal X-ray diffraction instrument, Fig. 2a and Table S3. † Both enantiomers crystallize in the monoclinic system with a chiral P2 1 space group, and each unit cell is made up of two cage molecules. The detailed structures of these two compounds were described with (S,R)-HPOC-1 assembled from (S,S)-DBD and (R,R)-CA as a typical representative. As expected, it inherits the [3 + 6] cage structural characteristics, possessing three binaphthol segments and six chiral diamines via imine connection. The length of this cage is about 24.4 , Fig. 2a. Although the cage cavity is crowded due to the ethoxy side chains, the solvent-accessible surface is computed to be 32.4% using Platon software. 21 The Flack parameters for the resolved single crystal structures of (S,R)-HPOC-1 and (R,S)- HPOC-1 are 0.049(13) and 0.035(7), respectively, illustrating the enantiopurity of these two newly obtained POCs. As a result, the CD spectra were comparatively studied with reference to those of DBD, Fig. 2b and S20. † (S,S)-DBD shows a positive CD band from 240 to 260 nm and a negative band from 260 to 360 nm. The enantiomer exhibits mirror CD bands due to the completely inverse Cotton effect. After the formation of (S,R)-HPOC-1, three CD bands, namely a positive band in the range of 240-260 nm and two negative bands at 260-280 nm and 280-360 nm, respectively, were observed. The newly observed negative band at 260-280 nm might be the consequence of imine band formation from (R,R)-CA reacting with the (S,S)-DBD moiety. This is consistent with similar [3 + 6] POC analogues. 3b,5a,20 HPOC-1 in tetrahydrofuran (THF) displayed absorption bands at 295 and 357 nm, similar to those of DBD, Fig. S21. † The molar absorption coefficient of 3.9 10 4 L mol 1 cm 1 at 357 nm for the cage is bigger than that of DBD (2.0 10 4 L mol 1 cm 1 ). Upon excitation at 357 nm for DBD, a broad emission band with the maximum at 408 and 430 nm appears, Fig. S22, † corresponding to a low fluorescence quantum yield of 1.6% (calculated with quinine sulfate as the standard). In contrast, a strong emission band at 403 nm was observed for HPOC-1 under the same excitation conditions with the fluorescence quantum yield as high as 63.4% due to the formation of an imine bond, 13c,k exceeding that of most organic cage compounds. 3a,14b Additionally, transient fluorescence spectra showed that HPOC-1 has a higher fluorescence lifetime (1.67 ns) than DBD (1.09 ns), Fig. S23. † Since few POCs were revealed to show circularly polarized luminescence (CPL), a typical characteristic of the excited states of chiral systems with application potential in 3D display technology, optoelectronic devices and chiral sensing, 14d,f the present fluorescence heterochiral HPOC-1 enantiomers composed of the binaphthol moiety therefore inspire us to investigate their CPL properties. As shown in Fig. 2c, (S,R)-HPOC-1 in THF exhibited a broad positive emission in the range of 380-500 nm with the maximum at 425 nm. However, the CPL profle of (R,S)-HPOC-1 shows a mirror-image with that of (S,R)-HPOC-1. The CPL dissymmetry factor (g lum ) was calculated to be AE3.3 10 4 . This value is similar to that of analogous POCs such as 6M-2 and T-FRP 1. 14a,b Incorporation of two kinds of chiral building blocks into HPOC-1 provides a new heterochiral host for enantioselective recognition of chiral molecules in solution. Herein, CD spectroscopy was employed to monitor the potential interaction between heterochiral HPOC-1 and chiral small molecules since this technique is able to directly discriminate molecules with different absolute confgurations and ee quantitation according to visual signals, superior to fluorescence and NMR techniques. 13a,b,13j In the present case, a series of chiral small molecules, including carvone, 1-phenylethanol, limonene, tartaric acid and pinene, were screened to probe the chiral recognition potential of HPOC-1. Gradual introduction of D-carvone resulted in the continuous decrease of the CD signal of (S,R)-HPOC-1 at 250 nm, Fig. 3a. In good contrast, a tiny CD change was found upon the introduction of L-carvone, Fig. 3b and c. These results imply the existence of specifc interaction between (S,R)-HPOC-1 and D-carvone rather than L-carvone. In addition, upon increasing the D-carvone concentration from 0 to 240.0 mM, the CD signal at 250 nm for (S,R)-HPOC-1 solution decreased in a much faster manner than that of the control without POC molecules, supporting the presence of enantioselective recognition of this cage toward D-carvone, Fig. S24. † Such a phenomenon was also observed in the chiral recognition test using (R,S)-HPOC-1 towards L-carvone, Fig. 3d-3f. For the other four chiral substrates including 1-phenylethanol, tartaric acid, limonene and pinene, Fig. S25 and Table S4, † almost no obvious CD change was observed for (S,R)-HPOC-1, indicating the specifcity of the chiral recognition of this POC towards Dcarvone. These results imply the existence of specifc interaction between (S,R)-HPOC-1 and D-carvone rather than L-carvone. In addition, a titration experiment of carvone with (S,S)-DBD at three-times the concentration of (S,R)-HPOC-1 was carried out. As shown in Fig. S26, † the CD signal changes for the solution of monomer (S,S)-DBD upon adding D-and L-carvone are similar to those of the cage, indicating that this monomer can also enantioselectively recognize D-carvone rather than L-carvone. Similar enantioselective recognition phenomena of the cage and monomer towards carvone are due possibly to the presence of 1,1 0 -binaphthalene moieties. However, at the same concentration of 1,1 0 -binaphthalene moieties for the solution of (S,R)-HPOC-1 and (S,S)-DBD, the slope absolute value of the ftting line between the CD signal intensity and the concentration of chiral D-carvone for the cage (0.124) is much bigger than that of the monomer (0.075), indicating the better enantioselectivity of the former species. This most probably originates from the superiority of the cage-like molecular structure which provides stronger interaction with the chiral substrate. Similar to (S,R)-HPOC-1, no obvious CD change is observed for (S,S)-DBD upon adding 1-phenylethanol enantiomers (as a representative of the used chiral substrates except carvone), Fig. S27. † These results indicate that cage (S,R)-HPOC-1 has better chiral recognition properties than monomer (S,S)-DBD. To elucidate the monitored enantioselective recognition mechanism, molecular simulations were conducted on the (S,R)-HPOC-1 host and two enantiomeric analytes using DFT calculations. The optimized molecular structure of (S,R)-HPOC-1 bound with D-carvone shows that protons on the D-carvone point toward the face of the binaphthol moiety with atom-toplane distances of 2.620 and 3.046 (Fig. S28 †), respectively, indicating the presence of CH/p interactions between the cage and guest. This point was further supported by the observation of a proton signal shift in the 1 H NMR spectrum of a mixture of (S,R)-HPOC-1 and D-carvone in comparison with that of the control, Fig. S29. † The CH-p interaction may affect the p / p* transitions of the binaphthol moieties, leading to the change of the intensity of the CD peak at 250 nm. Furthermore, the binding energy of (S,R)-HPOC-1 and the D-carvone molecule is 102.5 kcal mol 1 , indicating the presence of binding interaction. Instead, a unstable state between (S,R)-HPOC-1 and the Lcarvone molecule is suggested by the high value of binding energy (807.3 kcal mol 1 ). In contrast to the obvious CD response of (S,R)-HPOC-1 towards D-carvone, we hypothesize that the non-enantioselective recognition effect of POC for Lcarvone might be attributed to the much weaker interaction strength between the host and chiral guest, as indicated by the absence of an optimized structure for (S,R)-HPOC-1 in the presence of L-carvone in a 1 : 1 ratio. However, the exact reason is still not clear at this stage. At the end of this section, it is worth noting that a series of CD spectra of (S,R)-HPOC-1 with the addition of a mixture of rac-carvone with different ee values were collected, Fig. S30a. † A good linear relationship between CD signal intensity at 250 nm and ee value was observed, Fig. S30b. † According to this standard working curve, it is easy to determine the enantiomeric excess of carvone. This point is further supported by the fact that the experimental results for three rac-carvone samples are consistent with the theoretical values, Table S5. † ## Conclusions In summary, enantioselective assembly of new heterochiral functional materials from two kinds of enantiomeric building blocks has been clearly established. The enantioselective recognition mechanism involved in the self-assembly of building blocks has guaranteed the clean formation of a pair of heterochiral porous organic cage enantiomers with circularly polarized luminescence property. This new POC possesses enantioselective recognition capability towards chiral substrates according to the circular dichroism data. This work no doubt provides a new perspective towards constructing heterochiral POCs and therefore should beneft the chemistry of chiral self-assemblies. performed theoretical calculations; J. Jiang, H. Wang, C. Liu and H. Ren linked experiments and analysis; and all the authors discussed and wrote the manuscript. ## Conflicts of interest There are no conflicts to declare.

chemsum

{"title": "Enantioselective assembly and recognition of heterochiral porous organic cages deduced from binary chiral components", "journal": "Royal Society of Chemistry (RSC)"}

a_boron-transfer_mechanism_mediating_the_thermally_induced_revival_of_frustrated_carbene–borane_pair

## Abstract: Chemists have designed strategies that trigger the conformational isomerization of molecules in response to external stimuli, which can be further applied to regulate the complexation between Lewis acids and bases. We have recently developed a system in which frustrated carbene-borane pairs are revived from shelf-stable but external-stimuli-responsive carbene-borane adducts comprised of N-phosphine-oxide-substituted imidazolylidenes (PoxIms) and triarylboranes. Herein, we report the detailed mechanism on this revival process. A thermally induced borane-transfer process from the carbene carbon atom to the Nphosphinoyl oxygen atom initiates the transformation of the carbene-borane adduct. Subsequent conformational isomerization via the rotation of the N-phosphinoyl group in PoxIm moieties eventually leads to the revival of frustrated carbene-borane pairs that can cleave H 2 . We believe that this work illustrates an essential role of dynamic conformational isomerization in the regulation of the reactivity of external-stimuli-responsive Lewis acid-base adducts that contain multifunctional substituents. T here have been many recent developments in the chemistry of frustrated Lewis pairs (FLPs) that have been of note, for example, the activation of H 2 mediated by main-group elements . In general, FLPs are transient and not shelf-stable species making their isolation challenging. Meanwhile, chemists have developed strategies that trigger the conformational isomerization of molecules in response to external-stimuli . These strategies can also be used to generate transient FLP species from classical Lewis adducts (CLAs) that act like their shelf-stable precursors . In 2015, we demonstrated a strategy to generate FLPs from shelf-stable CLAs (PoxIm•B 1 in Fig. 1) that are comprised of N-phosphine-oxide-substituted imidazolylidenes (PoxIms; 1) and B(C 6 F 5 ) 3 (B 1 ). Here, the revival of the FLP from the CLA is closely controlled by a thermally induced conformational isomerization of the N-phosphinoyl moiety 17, . In 2018, Stephan et al. reported a system to control the generation of FLPs from CLAs via a light-induced E/Z isomerization of (C 6 F 5 ) 2 B((p-Tol)S)C = CCH( t Bu) 31 . Nevertheless, such FLP revival systems, including external-stimuli-responsive conformational isomerizations, are still underdeveloped. Thus, clarifying the relationship between external-stimuli-responsive conformational isomerizations and the interconversion that occurs between frustrated and quenched Lewis pairs is of great importance. This would allow a significant expansion of different strategies to design and apply FLP species 19 . In our system that uses PoxIms, the revival mechanism has not been fully explained. A tentative mechanism in which a B(C 6 F 5 ) 3 moiety is repelled by the N-phosphinoyl group via a thermally induced isomerization from the syn to anti conformation had been proposed. In this case, the syn/anti conformation refers to the relative orientation of the carbene carbon atom and the N-phosphinoyl oxygen atom with respect to the N-P bond (Fig. 1a) 17 . Herein, we report the results of a combined experimental and theoretical mechanistic study that demonstrates the key role of a transfer step where the triarylborane (BAr 3 ) unit on the carbene carbon atom moves to the N-phosphinoyl oxygen atom (Fig. 1b). In this study, PoxIms with 2,6-i Pr 2 -C 6 H 3 , 2,4,6-Me 3 -C 6 H 2 , and 3,5-t Bu 2 -C 6 H 3 groups were studied and are herein referred to as 1a, 1b, and 1c, respectively. ## Results and discussion Effects of Lewis acidity. To explore the impact of the Lewis acidity of BAr 3 on the formation and reactivity of the carbene-borane adducts, the reaction between 1a and B(p-HC 6 F 4 ) 3 (B 2 ) was undertaken (Fig. 2a). Full consumption of 1a was confirmed after 20 min, resulting in the formation of two CLAs, i.e., 2aB 2 , which contains a N-phosphinoyl oxygen-boron bond, and 3aB 2 , which contains a carbene-boron bond, in 61% and 29% yield, respectively. Previously, we have reported that, even at -30 °C, 2aB 1 could be converted to 3aB 1 and that full identification of 2aB 1 could therefore be achieved using NMR analysis conducted at -90 °C17 . In the present case, 2aB 2 exhibited a longer life-time at room temperature than 2aB 1 , which enabled us to prepare single crystals of 2aB 2 by recrystallization from the reaction mixture at -30 °C. The molecular structure of 2aB 2 was unambiguously confirmed using single-crystal X-ray diffraction (SC-XRD) analysis. A set of (R a ) and (S a ) atropisomers of 2aB 2 was identified in the asymmetric unit of the single crystal. The molecular structure of (R a )-2aB 2 is shown in Fig. 2b and demonstrates a rare example of complexation-induced N-P axial chirality 29 . As the reaction progressed, 2aB 2 was converted to 3aB 2 and 4a; 2aB 2 was fully consumed within 6 h to afford these compounds in 75% and 25% yield, respectively. It should be noted that 4a is likely furnished via the migration of the Nphosphinoyl group from the nitrogen atom to the carbene carbon atom. However, in the absence of B 2 , this migration only proceeded to 9% at 100 °C, even after 25 h 30 . The formation of 4a was therefore promoted by the enhancement of the electrophilicity of the P center via the coordination of the N-phosphinoyl moiety to B 2 . Regeneration of B 2 was observed along with the production of 4a. The molecular structure of 3aB 2 was also confirmed by SC-XRD analysis (Fig. 2c). Comparison of the structural parameters between the solid-state structures of 3aB 2 and 3aB 1 shows their similarity. For example, the C1-B distances in 3aB 2 and in 3aB 1 are 1.710(3) and 1.696(3) , respectively. The interatomic distance of 3.257(3) between the O and B atoms in 3aB 2 suggests the absence of a specific interaction between these atoms, similar to that in 3aB 1 (3.234(3) ). Thermolysis of 3aB 2 at 60 °C for 3 h resulted in the generation of 4a and B 2 in 77% and 73% yield, respectively, with concomitant formation of [1a-H][HO(B 2 ) 2 ] in 4% yield (conversion of 3aB 2 = 81%; Fig. 3a). Although 2aB 2 was not observed via NMR analysis of this reaction at 60 °C, the formation of 4a and B 2 indicates the in situ regeneration of 2aB 2 (vide supra). The formation of [1a-H][HO(B 2 ) 2 ] can be rationalized in terms of a reaction between contaminated H 2 O and the FLP species regenerated from 3aB 2 via 2aB 2 . The regeneration of the FLP species from 3aB 2 was then clearly confirmed by treating 3aB 2 with H 2 (5 atm) at 22 °C, resulting in the formation of ) 2 ] (8%) and 1a (6%) (Fig. 3b). Under identical conditions, no reaction occurred when 3aB 1 was used 17 . At 60 °C, 5aB 2 was generated in 90% yield after 3 h, which is almost comparable with the production of 5aB 1 (89%) from 3aB 1 . Thus, the lower Lewis acidity of B 2 relative to B 1 allowed a more facile revival of the FLP species from 3aB 2 than from 3aB 1 . However, the lower Lewis acidity did not affect the progress of the heterolytic cleavage of H 2 by FLPs at 60 °C. Kinetic studies. To gain further insight into the reaction mechanism, the initial rate constants for the generation of 5aB 1 , k int [10 -5 s -1 ], from the reaction between 3aB 1 and H 2 in 1,2dichloroethane-d 4 (DCE-d 4 ) at 60 °C were estimated by varying the H 2 pressure from 0.5 to 5.0 atm (Fig. 4a). It should be noted here that when H 2 was pressurized at 5.0 atm, an excess of H 2 (ca. 0.3 mmol) with respect to 3aB 1 (0.010 mmol) was added to the pressure-tight NMR tube. The concentration of H 2 clearly influenced the progress of the reaction, suggesting that the heterolytic cleavage of H 2 by the FLP species is involved in the ratedetermining events. Next, the reaction between 3aB 1 and H 2 at 5.0 atm of pressure was monitored in DCE-d 4 whilst the temperature was varied from 50 to 80 °C (Supplementary Figure 27). Pseudo-first order rate constants, k obs [10 -5 s -1 ], of 2.95(2), 11.2(8), 46.4(4) and 183(2) were estimated for the reactions at 50, 60, 70, and 80 °C, respectively. Thus, the activation energy and pre-exponential factor obtained from the plot based on the Arrhenius equation, lnk obs = -(E a /R)(1/T) + lnA, are E a = 31.2 [kcal mol -1 ] and A = 3.3(36) × 10 16 [s -1 ] (Fig. 4b). Given the close relation between E a and ΔH ‡ , the values obtained for E a suggest that the formation of 5aB 1 via the reaction between 3aB 1 and H 2 only occurs at temperatures higher than 25 °C32 . Based on the results presented here and those previously reported 17 , the reaction between the carbene-borane adducts and H 2 to give [PoxIm-H][H-BAr 3 ] likely proceeds via the heterolytic cleavage of H 2 by the FLP species that are formed following the regeneration of the N-phosphinoyl oxygen-borane adducts. These steps are expected to be the rate-determining events because the concentration of H 2 (Fig. 4a), the steric bulk of the N-aryl group 17 and the Lewis acidity of the BAr 3 moiety (Fig. 3b) influence the reaction rates and/or the temperature required to initiate the reaction between the carbene-borane adducts and H 2 . Theoretical studies. Density-functional theory (DFT) calculations were carried out at the ωB97X-D/6-311G(d,p), PCM (DCE)// ωB97X-D/6-31G(d,p) for H 2 and 6-31G(d) for all other atoms level of theory (Fig. 5a). The relative Gibbs free energies with respect to [1a + B 1 ] (0.0 kcal•mol -1 ) are shown. During the transformation of 3aB 1 (-17.2 kcal•mol -1 ) to 2aB 1 (-9.8 kcal•mol -1 ), both of which were experimentally confirmed, the formation of an intermediate 2a′ B 1 (-7.7 kcal•mol -1 ) was predicted via a C-to-O transfer of B 1 in 3aB 1 . This distinctive boron-transfer process takes place via saddle 2 ] and 1a were also observed in 8% and 6% yield, respectively. b [1a-H][HO(B 2 ) 2 ] was also observed in 7% yield. c Results obtained using 3aB 1 are reproduced from ref. 17 37) 33,34 . This AIM analysis demonstrates that several noncovalent interactions, including π-π and H•••F interactions, exist between the 1a and B 1 moieties to stabilize TS1a. Two plausible mechanisms were evaluated for the FLPmediated cleavage of H 2 on the basis that the Lewis-basic center reacts with H 2 via cooperation with B 1 (Fig. 5b). One possibility is that the carbene carbon atom works as a Lewis base (path I; the right path in Fig. 5b) 35,36 , while the other is that the Nphosphinoyl oxygen functions as a Lewis base (path II; the left path in Fig. 5b) 37 . In path I, the heterolytic cleavage of H 2 takes places via TS4a (+11.4 kcal•mol -1 ), which arises from the insertion of H 2 into the reaction field around the carbene carbon and boron atoms in FLP-1aB 1 , affording 5aB 1 (-34.8 kcal•mol -1 ), a species more thermodynamically stable than 3aB 1 . In the optimized structure of TS4a (Fig. 5d), the dissociation of the H1-H2 bond (H1•••H2 = 0.84 ) occurs with the partial formation of the H2-C1/H1-B bonds (H2•••C1 = 1.83 /H1•••B = 1.49 ). Based on these results, the overall path from 3aB 1 to 5aB 1 via FLP-1aB 1 is substantially exothermic (ΔG°=-17.6 kcal•mol -1 ) and includes an overall activation energy barrier of +28.6 kcal•mol -1 required to overcome TS4a. In path II, which takes place via TS5a (a transition state for the insertion of H 2 into the O-P bond) and TS6a (a transition state for the cleavage of H 2 between the O and P atoms), a higher activation energy barrier of +32.7 kcal•mol -1 is predicted to yield intermediate 8aB 1 , which contains a P=O-H + and B-H − species. It should be noted that the potential energy of the optimized TS6a (-3633.288355 hartree) is almost identical to that of the optimized 7aB 1 (-3633.288363 hartree), which causes the reversed Gibbs energy levels as shown in Fig. 5b after the Gibbs energy correction and implementation of solvent effect. Therefore, the discussion on the activation energy barrier to overcome TS6a from 7aB 1 should be not essential. The subsequent transfer of H + from the Nphosphinoyl oxygen atom to the carbene carbon atom furnishes 5aB 1 , although the details of this process remain unclear at this point. The molecular structure of TS6a shows that the cleavage of the H1-H2 bond (H1•••H2 = 0.85 ) by the N-phosphinoyl oxygen and boron atoms occurs in a cooperative fashion (Fig. 5d). Given the experimental and theoretical results reported here, we conclude that path I is the more likely one. The impact of the N-aryl substituents on the activation energy barriers for the regeneration of [1 + B 1 ] was evaluated using calculations on 3bB 1 , which contains an N-2,4,6-Me 3 -C 6 H 2 group, as well as 3cB 1 , which contains an N-3,5-t Bu 2 -C 6 H 3 group. This afforded ΔG ‡ values of +28.3 and +32.8 kcal•mol -1 for 3bB 1 and 3cB 1 , respectively (Fig. 5a). These results are consistent with the experimental observations, i.e., that 3aB 1 −3cB 1 did not react in the presence or absence of H 2 under ambient conditions under the applied conditions. Furthermore, these results might rationalize the fact that temperature to induce the reaction between these CLAs and H 2 increases in the order 3aB 1 (60 °C) < 3bB 1 (80 °C) < 3cB 1 (120 °C) 17 . ## Conclusion. In summary, the reaction mechanism for the revival of frustrated carbene-borane pairs from external-stimuliresponsive classical Lewis adducts (CLAs), comprised of N-phosphine-oxide-substituted imidazolylidene (PoxIm) and triarylboranes (BAr 3 ), is reported based on a combination of experimental and theoretical studies. Remarkably, a transfer of the borane moiety from the carbene carbon atom to the Nphosphinoyl oxygen atom was identified as a key step in the heterolytic cleavage of H 2 by the regenerated FLP species. The optimized transition-state structure for this borane-transfer process was confirmed to include no bonding interactions between the carbene carbon/phosphinoyl oxygen and boron atoms, albeit that it is stabilized by intermolecular non-covalent interactions between the PoxIm and BAr 3 moieties. The heterolytic cleavage of H 2 takes place via the cooperation of the carbene carbon and the boron atoms, and exhibits a lower overall activation energy barrier than that of the path in which a combination of the Nphosphinoyl oxygen and boron atom mediates the H 2 cleavage. These results demonstrate the essential role of dynamic conformational isomerization in the regulation of the reactivity of shelf-stable but external-stimuli-responsive Lewis acid-base adducts by multifunctional Lewis bases. ## Methods Synthesis of 3aB 2 . PoxIm 1a (154.8 mg, 0.40 mmol) and B(p-HC 6 F 4 ) 3 (B 2 ) (183.4 mg, 0.40 mmol) were mixed in toluene (10 mL) at room temperature to furnish the yellow solution. Stirring this mixture for 4 h resulted into the precipitation of a white solid that was collected via removal of the supernatant solution. The obtained solid was washed with hexane (5 mL) and dried in vacuo to afford 3aB 2 as a white solid (230.2 mg, 0.27 mmol, 68%). A single crystal suitable for X-ray diffraction analysis was prepared by recrystallization from CH 2 Cl 2 /hexane at -30 °C. Synthesis of 5aB 2 . A solution of 3aB 2 (51.6 mg, 0.06 mmol) in CH 2 Cl 2 (3 mL) was transferred into an autoclave reactor, which was then pressurized with H 2 (5 atm). Subsequently, the reaction mixture was stirred at 60 °C for 4 h, before the solvent was removed in vacuo to give 5aB 2 as a white solid (51.8 mg, 0.06 mmol, >99%). A single crystal suitable for X-ray diffraction analysis was prepared by recrystallization from THF/hexane at -30 °C. Reaction between 1a and B 2 giving 2aB 2 . A solution of 1a (7.4 mg, 0.02 mmol) and B 2 (9.3 mg, 0.02 mmol) in CD 2 Cl 2 (0.5 mL) was prepared at -30 °C and then transferred into a J. Young NMR tube. The quantitative formation of 2aB 2 was confirmed at -90 °C by 1 H, 13 C, 19 F, and 31 P NMR analysis (Supplementary Figs. 5-8). A single crystal suitable for X-ray diffraction analysis was prepared by recrystallization from toluene/hexane at -30 °C.

chemsum

{"title": "A boron-transfer mechanism mediating the thermally induced revival of frustrated carbene\u2013borane pairs from their shelf-stable adducts", "journal": "Nature Communications Chemistry"}

palladium-catalyzed_asymmetric_allylic_alkylation_(aaa)_with_alkyl_sulfones_as_nucleophiles

## Abstract: An efficient palladium-catalyzed AAA reaction with a simple a-sulfonyl carbon anion as nucleophiles is presented for the first time. Allyl fluorides are used as superior precursors for the generation of p-allyl complexes that upon ionization liberate fluoride anions for activation of silylated nucleophiles. With the unique bidentate diamidophosphite ligand ligated palladium as catalyst, the in situ generated a-sulfonyl carbon anion was quickly captured by the allylic intermediates, affording a series of chiral homo-allylic sulfones with high efficiency and selectivity. This work provides a mild in situ desilylation strategy to reveal nucleophilic carbon centers that could be used to overcome the pK a limitation of "hard" nucleophiles in enantioselective transformations. ## Introduction Transition-metal-catalyzed asymmetric allylic alkylation (AAA) is a powerful tool for the enantioselective construction of stereogenic centers, enabling the elaboration of complex organic molecules and synthesis of pharmaceutical intermediates and bioactive natural products. 1 A variety of "soft" carbon nucleophiles (Nu-H with pK a <25) and heteroatoms have been used in AAA reactions, generating the corresponding stereogenic centers with good to excellent selectivity. 2 However, transition-metal-catalyzed allylic substitution reactions with "hard" nucleophiles (Nu-H with pK a > 25) is mainly limited to non-enantioselective transformations. 3 During the past decades, our group and many other research groups have made tremendous efforts to engage "unstable" nucleophiles, such as ketones, acyclic amides and nitrogen-contained heteroarenes, etc. in the palladium-catalyzed AAA reactions. 4,5 Despite these achievements, a great number of "hard" carbon nucleophiles are still not compatible in palladium-catalyzed AAA reactions. One important example of such unexplored carbon based nucleophiles for AAA reactions is a-sulfonyl carbon anion (pK a ¼ 29 for (methylsulfonyl)benzene, Fig. 1a). 6 Sulfone represents an important moiety that is widely spread in many biologically active compounds and pharmaceutical intermediates. 7 Moreover; sulfones can be converted into a wide range of other groups at the a position via traceless transformations. 8 Efficient utilization of a-sulfonyl carbon anion in the palladium-catalyzed AAA reaction would lead to chiral homo-allylic sulfones, which would provide promising opportunities for the exploration of chiral sulfone containing compounds (Fig. 1c). Previous exploration to build chiral homoallylic sulfones via AAA reactions were limited to special sulfone reagents, an additional electron-withdrawing group was usually needed to enhance the acidity of a proton (Fig. 1a). 9 For example, the use of the ester group allowed removal after the reaction of Krapcho demethoxycarbonylation; 10 however, Fig. 1 Representative methods for homo-allylic sulfones. besides the moderate yield (60-80%), the harsh reaction conditions for demethoxycarbonylation would rule out many useful functional groups. Therefore, exploration of efficient method to realize the direct asymmetric allylic reaction of simple alkyl sulfones under mild reaction conditions is highly in needed. To date, two elegant non-enantioselective reports on direct allylic alkylation of sulfones employed decarboxylation as the driving force. A thermodynamic decarboxylative Claisenrearrangement reaction reported by Craig et al. 11 under harsh reaction conditions (150 C) would restrict the diversity of functional groups (Fig. 1b). Another example is a palladiumcatalyzed intramolecular decarboxylative allylation of sulfonyl acetic esters with rac-BINAP as the ligated ligand (Fig. 1b). 12 Notably, the conditions developed by Tunge et al. still needed high reaction temperature or microwave conditions to get acceptable results. In most cases, the key nucleophiles were stabilized by both sulfonyl and phenyl or heteroatoms (pK a ¼ 23.4 for (benzylsulfonyl)benzene). 6 Herein, we report our endeavor and initial results on the palladium-catalyzed AAA reaction with simple a-sulfonyl carbon anion as the nucleophile (pK a > 25, Fig. 1c). ## Optimization of conditions Recently, our and other groups have found that phosphoramidite and diamidophosphite ligands could facilitate transition-metal catalyzed transformations via in situ deprotonation of pro-nucleophiles. 4l,13 Notably, the Sawamura group found that the chiral phosphoramidite ligated palladium catalyst can facilitate the asymmetric allylic alkylation at the "hard"a position of 2-alkyl pyridines without additives. 4l The above achievements inspired us to utilize these unique ligands and exploit a-sulfonyl nucleophiles in AAA reactions. We started our research with commercial compound 1a-1 as a donor, and tert-butyl cyclohex-2-en-1-yl carbonate 2a-1 as the model counterpart (Fig. 2a). Ligand L1 (Fig. 2c) which has proven to be a suitable ligand for palladium-catalyzed transformations involving a deprotonation mechanism was selected as the ligated ligand to test different conditions. 14, 15 We soon realized that in the absence of additional base, the reaction gave no desired results. The tert-butoxide presumably generated in situ from Pd-mediated ionization of 2a-1 is incompetent to efficiently deprotonate the sulfone 1a-1 or perhaps the carboxylate leaving group never lost CO 2 to form tert-butoxide. We surmised that addition of an external base could lead to deprotonation. Addition of LiHMDS, KHMDS and NaO-tert-Bu to facilitate the desired deprotonation failed to give acceptable results. Perhaps, these strong bases interfered with the ionization event of 2a-1 and the moisture sensitive nature of these strong bases makes the reaction hard to handle. We turned our attention to fnd mild conditions to generate the corresponding a-sulfonyl carbanion in a catalytic manner without additional stoichiometric base. Recently, our group and the Hartwig group found that allylic fluoride can be used as an excellent electrophilic precursor in transition metal-catalyzed asymmetric allylic alkylation, generating the nucleophile anion by in situ fluoride induced desilylation, respectively. 16 The strategy invoked a synergistic interplay of the fluoride leaving group to facilitate the generation of the electrophilic metal-allyl complex and delivery of a catalytically activated nucleophilic anion by desilylation. We sought to utilize this approach to engage asulfonyl 1a in palladium-catalyzed asymmetric allylic alkylation with allyl fluoride 2a (Fig. 2b). Encouragingly, using L1 gave the desired product 3a with good yield (83%) and excellent enantioselectivity (91% ee). When t-BuOMe was used as the solvent, the product 3a was obtained in lower 74% yield but better 94% ee. When L2 was used as the supporting ligand, the product 3a was obtained in 51% yield and 83% ee. Some other phosphoramidite ligands were also tested. L3 afforded the product in a moderate 72% ee with a poor 35% yield. On the other hand, L4 17 and L5, which were successful ligands in our previous palladium-catalyzed transformations, did not afford the desired results. Nevertheless, Sphos L6 afforded 3a in moderate 61% yield, thus this ligand was selected as the supporting ligand for the non-enantioselective transformations. The solvent effect was very important for this transformation and only ethereal solvents gave the desired sulfone 3a with acceptable results. Other solvents such as toluene and DCE gave trace amounts of the desired product. t-BuOMe was proved to be the optimal solvent for the enantioselective transformations. Active CpPd(h 3 -C 3 H 5 ) was another key factor for this reaction; other palladium sources such as commonly used Pd(dba) 2 gave poor results. ## Results and discussion To generate more elaborate chiral homo-allylic sulfones, we frst tested the scope of different sulfone donors with allyl fluoride 2a as the reaction counterpart (Fig. 3). Aryl sulfones bearing an electrondonating (3b) or an electron-withdrawing (3c) group were suitable in our system, giving good to excellent results. The substrates bearing halogen atoms, such as fluoro and chloro afforded corresponding products with good results (3e and 3f). Sterically hindered 2-naphthyl sulfone (3f) and bioactive 7-coumarinyl sulfone (3g) also gave rise to the desired products in good to excellent enantioselectivity. Additionally, different heteroaryl sulfones, 18 such as 2-pyridyl sulfone (3h), 4-pyridyl sulfone (3i), 1,3pyrimidinyl sulfone (3j) and benzothiazolyl sulfone (3k) all gave the desired products with excellent results (>94% ee). Besides the above aryl sulfones, simple alkyl sulfones were also tested with the optimized conditions, and the corresponding chiral products could be obtained with slightly diminished enantioselectivity compared to aryl sulfones (3l, 3m). Notably, sulfonamide which is a privileged functionality in modern drug discovery also could produce the corresponding homoallylic chiral sulfonamides with good results (3n, 3o). 19 An interesting anion shift was observed for the reaction with benzyl sulfone, which gave the expected product 3p in a good 63% yield and excellent 92% ee with a unseparated minor regioisomer 3p 0 . A similar shift is not exhibited in Tunge's decarboxylative allylations of benzyl alkyl sulfone. 12 Regrettably, vinyl and alkynyl sulfones did not give the desired products. The absolute confguration of 3h was determined by X-ray crystallography; the stereochemical outcome for all other homo-allylic sulfones was assigned by analogy. Next we turned our attention to the substrate scope of allylic fluorides. To make the detection and separation of product easier, 2-pyridyl sulfone 1h was selected as the standard nucleophile for most substrates (Fig. 4). Allylic fluorides bearing 2-naphthyl (4a), more sterically hindered 1-naphthyl (4b), electron-rich aryl (4c) and electron-defcient aryl (4d, 4e) all gave a range of chiral homo allylic sulfones with good yields and good to excellent ee values. Substrate bearing a chlorine atom, which is a good handle for further functionalization, was compatible with the reaction conditions (4f). Heteroarenes are also good choices for the reaction. Electron-rich indolyl (4g), thiophenyl (4h), and electron-defcient quinolinyl (4i) were all successfully employed to give the desired products with excellent results (>89% ee). Besides the different aryls, alkenyl and alkyl substituted allylic fluorides were also subjected to the optimized conditions. Alkenyl substituted acceptors gave the desired homo-allylic sulfones in good yield with slightly lower ee values compared to aryl substituted acceptors (4j, 4k). Simple benzyl substituted acceptor gave 57% yield but a poor 47% ee (4l). Added flexibility of the benzyl substituent could account for the compromised selectivity. A nitrogen-containing heterocyclic acceptor was also tested and delivered the desired product 4m in 66% yield and an excellent 91% ee. Besides the six membered all carbon cyclic acceptors, medium-sized rings such as sevenmembered cyclic acceptors also proved to be good substrates for this reaction, giving the desired products with excellent ee values (4n, 4o). Unfortunately, fve-membered acceptors are not suitable for our current conditions, as the active allylic fluoride preferentially eliminated HF thereby forming cyclopentadiene spontaneously. 20 To demonstrate the synthetic application of this transformation, the scale of the reaction was increased to 1.0 mmol (Fig. 5a). The palladium loading could be reduced to 2 mol% with 3 mol% of ligand L1 whereby the product 3a was obtained in 68% yield with 92% ee. Furthermore, the sulfone group in the products provides an enabling handle for further transformations (Fig. 5b). For example the sulfone group in 3a could be reductively cleaved with Na(Hg) to access chiral allylic methyl compound 5a in 70% yield. Notably, access to compound 5a is difficult by other methods. Here the sulfone donor acts as a formal methylation reagent. 21 Additionally, the sulfone group could be replaced by an ester group via a two-steps synthetic sequence in 82% yield (5b). Furthermore, the heteroaryl sulfonyl group in 3k could be used as a precursor for Julia-Kocienski olefnation whereby upon reaction with benzaldehyde the skipped diene 5c forms in excellent yield and geometric selectivity (5c) for the E isomer. 22 The 1,3-diene unit in 4j is a good counterpart for an intermolecular Diels-Alder reaction, which afforded fused cyclic compound 5d in 63% yield (dr ¼ 3.5 : 1) upon reaction with N-methyl maleimide. 23 ## Conclusions In conclusion, we realized the frst palladium-catalyzed AAA reaction with "hard" a-sulfonyl carbanions as the nucleophiles. This transformation provides a rapid entry to chiral homo-allylic sulfones that otherwise are challenging to obtain. The presence of a sulfone motif provides a powerful handle for subsequent structural elaborations. The "outer sphere" reaction pathway is presumed to be involved in this transformation, 24 however, another possible reaction pathway involved anionic Si-F species couldn't be ruled out. 25 Detailed mechanistic studies and application of this novel AAA reaction in the synthesis of biologically active compounds and analogy of natural products are ongoing.

chemsum

{"title": "Palladium-catalyzed asymmetric allylic alkylation (AAA) with alkyl sulfones as nucleophiles", "journal": "Royal Society of Chemistry (RSC)"}

improved_syntheses_of_halogenated_benzene-1,2,3,4-tetracarboxylic_diimides

## Abstract: The preparation of halogenated benzene-1,2,3,4-tetracarboxylic diimide derivatives is challenging because of the possibility of competitive incorrect cyclizations and SNAr reactivity. Here, we demonstrate that the direct reaction of benzene-1,2,3,4tetracarboxylic acids with primary amines in acetic acid solvent successfully provides a range of desirable ortho-diimide products in good yields. Furthermore, we demonstrate that sterically challenging N-derivatizations can be readily achieved under microwave reactor conditions, and that SNAr reactivity is only observed when excess amine is used. The halogenated diimides described here are attractive building blocks for organic materials chemistry. Aromatic diimides, also known as bis(dicarboximide)s, are the linchpin of a diversity of organic materials encompassing vivid pigments and dyes, thermally robust polymers, 4 and ntype electronic materials. 5,6 The electron-withdrawing nature of the cyclic imides lends itself to the creation of n-type semiconducting materials. 3 The annulation of aromatic rings with cyclic imides tends to lead to more significant LUMO-lowering effects than HOMO-lowering effects, thus resulting in red-shifted bandgaps and absorption profiles. Aromatic diimides derived from benzene (PMDI), naphthalene (NDI), and pyrene (PDI) (Figure 1a) have received significant research attention because of their facile syntheses and amenability to derivatization at both the core and imide N positions. While changes in Nfunctionalization are often exploited for tuning solid-state packing behavior and solubility profiles, core modification through substitution and metal-mediated cross-coupling reactions of halogenated aromatic diimides results in fine control over energy levels and electronic structure. 7,10 In recent years, researchers have developed new strategies for incorporating cyclic imides onto a growing number of aromatic scaffolds, resulting in interesting redox activity 14 and near-IR absorptions. 15 We recently began exploring the ortho-diimide structural isomer of the well-known pyromellitic diimide, which is known as mellophanic diimide (MDI), and have discovered that it has significant potential as a building block in the construction of compounds with properties of interest to organic materials chemists (Figure 1b,c). Core dichlorinated N,N'-dihexyl MDI (Cl2-MDI-Hex), for example, readily undergoes both SNAr and Pd-catalyzed substitutions with aromatic ortho dinucleophiles to lead to a range of highly chromophoric and electron-accepting hetero-and azaacene structures. 20,21 The development of MDI as a building block is further attractive because it is derived from 1,2,3,4-tetramethylbenzene, which is a byproduct of durene synthesis and a constituent of petroleum extract that has no significant industrial use. 22 With these observations in mind, we set out to establish generalizable synthetic methods for obtaining differently halogenated MDI derivatives. Results and Discussion. Conventionally, aromatic diimides are synthesized by condensation between the relevant aromatic cyclic dianhydride and an amine, a process which proceeds through an amide-carboxylic (amic) acid intermediate. Extending this approach to the synthesis of ortho aromatic diimides such as mellophanic diimide, however, is complicated by the possibility of incorrect cyclizations to yield 3,6-dicarboxyphthalimide byproducts. Two strategies were identified by Fang et. al for overcoming this challenge: 1) room-temperature amic acid formation followed by acetic anhydride-mediated dehydration and 2) high-temperature equilibration of the reaction mixture to reach the MDI thermodynamic product (Fig 2a). 18 While both of these methods were successful for synthesizing N,N'-diaryl MDI compounds, we found that they could not be reliably extended toward either N,N'-dialkyl-or corechlorinated MDIs because of competing nucleophilic aromatic substitution reactions and significant 3,6-dicarboxyphthalimide formation. Inspired by the mellitic triimide synthesis developed by Rose et. al, 23 we were previously able to obtain N,N'-dihexyl-4,5-dichloro-MDI (Cl2-MDI-Hex) after the 3-day solidstate dehydration of an ammonium carboxylate salt (Fig. 2b). 20 In further explorations, however, we found that this solid-state method could not be consistently extrapolated to differently halogenated benzene tetracarboxylic acids, and furthermore was not successful with more sterically demanding amines. As a result of further synthetic exploration, here we report our findings that the direct solution-phase reaction between benzene-1,2,3,4tetracarboxylic acids and primary amines is a widely generalizable method for synthesizing MDI derivatives of a variety of N substitutions and core halogenations (Fig. 3c). Synthesis. The commercially available 1,2,3,4-tetramethylbenzene was chlorinated, 20 brominated, 24 or iodinated 25 following literature procedures to yield intermediates 1-X (X = Cl, Br, or I) which were then subjected to exhaustive oxidation by 10 eq. of KMnO4 in tBuOH/H2O (1/1:v/v) to provide the halogenated benzene-1,2,3,4-tetracarboxylic acids. Although many KMnO4 methylarene oxidation procedures use pyridine as a co-solvent to improve reactant solubility, we have found that the use of tBuOH co-solvent reduces the equivalents of KMnO4 required to achieve full oxidation and furthermore is less prone to exotherm during the addition of KMnO4. Complete removal of reaction solvent prior to acidification of the carboxylate intermediate is important for avoiding the formation of t-butyl ester impurities. Our largest scale oxidation (20.0 g, 98.5 mmol of 1-Cl) proceeded smoothly to provide the tetraacid 2-Cl in 85% isolated yield. Although we initially followed our previously developed solid-state dehydration protocol for synthesizing 3-X-R, we were motivated by inconsistent yields and long reaction times to investigate a solution-phase method. Unexpectedly, simply heating the tetraacids 2-X with primary amines in acetic acid solvent followed, if necessary, by the precipitation of products with the addition of water or MeOH to the reaction mixture, provided MDIs 3-X-R in up to 93% isolated yield. In some cases, a small amount of additional product can be recovered by extraction of the aqueous filtrate with CH2Cl2. Although the reaction byproducts are often polar and soluble enough in AcOH to be removed during the filtration process, additional purification can be easily achieved by passing the crude product mixture through a SiO2 column. This method is successful with a variety of amines and there is no discernible trend between the identity of the halogen and the reaction yield. Hexylamine, aniline, and benzylamine react smoothly with 2-X (X = Cl, Br, and I) in good yields to produce a suite of 3-X-R compounds. Single crystal X-ray data for 3-Br-Ph confirms the MDI constitution of the products (Figure 3b). It is notable, as will be discussed below, that yields tend to be higher for the halogenated 3-X-R compounds than the nonhalogenated ones. Amino acid methyl esters can also be readily converted into MDIs, which suggests they may be interesting building blocks for self-assembling small molecules. 26,27 Attempts to perform imidization with 6-amino-1-hexanol, however, resulted in complex mixtures as a consequence of acetate ester formation with the free alcohol. With these results in hand, we attempted to install more sterically demanding Ngroups such as branched alkyl chains and 2,6-dialkylaryl groups since these are commonly used by organic materials chemists to manipulate crystal packing and solubility. Although 2ethylhexylamine reacted readily with 2-H to yield 3-H-EtHex, imidization of 2-H with the more sterically hindered nucleophiles such as (S)-1-phenylethan-1-amine and 2,6-diisopropylaniline proved to be more recalcitrant. We were unable to isolate any products after applying our standard reaction method, and heating (S)-1-phenylethan-1-amine with 2-H for 3 days at 110 °C resulted in only 11% formation of 3-H-1PhEt. These low yields for sterically hindered amines were also found when attempting these same reactions using our older solid-state approach (Figure 4). To overcome this slow reaction rate, we turned to microwave reaction conditions. Gratifyingly, reacting 2-Cl and (S)-1-phenylethan-1-amine at 200 °C for 24 hours led to 58% isolated yield for 3-H-1PhEt. Under these forcing conditions, an important consideration is whether nucleophilic aromatic substitution (SNAr) reactions at the aryl halides start to take place when X = halogen. In our trials, we did not observe significant levels of SNAr reactivity when 2.1 equiv of amine was reacted with tetraacid 2-Cl. However, when 12 equivalents of (S)-1-phenylethan-1-amine were reacted with 2-Cl, we did observe core-diaminosubstituted derivatives in the resulting product mixture. Mechanistic Insights. Our first attempted synthesis of 3-H-1PhEt under microwave conditions was performed at 200 °C for 2 hr of reaction time and resulted in 11% isolated yield. Both running the reactions for longer (200 °C for 24 hours) or with more equivalents of nucleophile (12 equiv. amine, 200 °C for 2 or 24 hours) improved the isolated yield, to 58% and 46%, respectively. It is worth noting that the excess amine approach is not feasible under microwave conditions when X = halogen because of the competitive SNAr reactions. From a mechanistic perspective, these observations suggest that in AcOH solvent, the reaction is likely taking place under overall equilibrating conditions. To gain more insight into the reaction process, we performed a 1 H NMR time-course study of the reaction between 2-H and (S)-1-phenylethan-1-amine in d4-acetic acid at 110 °C. Upon dissolving only 2-H, the 1 H NMR spectrum reflects the presence of a complex mixture of anhydrides corresponding to intermediates with or without symmetry around the central benzene ring. Although we cannot rule out cyclic anhydride formation, we believe that mixed acetic anhydride formation is more likely based on the solubility of the reaction intermediates. Upon addition of the amine and heating for 5 minutes, a number of different intermediates are detected by 1 H NMR spectroscopy and formation of the desired MDI product is first observable after 20 minutes of reaction time. After 19 hours, the mixture resolves itself into being primarily four species, three with symmetry around the benzene core and one without. After 11 days of reaction time, the desired product constitutes only 18% of the product mixture, which highlights the value of microwave reaction conditions for achieving higher yields on a reasonable time scale. The NMR spectrum of the reaction after a total of 31.5 days at 110 °C shows that the product distribution reaches a roughly 1:1:1 ratio of 3-H-1PhEt, 4-H-1PhEt, and 5-H-1PhEt (Figure 5c). We were able to tentatively assign the structure of the reaction intermediates by performing column chromatography on incomplete reaction mixtures and analyzing the filtrates collected during reaction workups. Of these, the most notable intermediates are dicarboxyphthalimides 4-X-R and 5-X-R, which correspond to the asymmetric and symmetric possibilities, respectively, for monophthalimide formation between the tetraacids 2-X and an amine. It is again worth noting that during the reaction, however, it is difficult to rule out whether mixed acetic acid anhydride derivatives are the dominant species. Liquid chromatography mass spectrometry analysis of crude reaction mixtures corroborates the 1 H NMR and preparatory observations of 4-X-R and 5-X-R as the primary reaction byproducts, but also reveal one other identifiable product corresponding to a diamidophthalimide species 6-X-R. We believe this isomer is more likely because the analogous diamido derivative of 4-X-R should be more likely to proceed to cyclize into 3-X-R. From a purely statistical perspective, intermediates 4-X-R and 5-X-R should be formed in a 3 to 1 ratio because both the 1-and 2-carboxamide derivatives of 2-X can dehydrate into intermediate 4-X-R, while only the 2-carboxamide can cyclize into intermediate 5-X-R. To add context to our understanding, we performed density functional theory calculations (M062X/6-31G(d)) with implicit acetic acid solvent to evaluate the energy landscape of the reaction for the reaction between methylamine and either 2-H or 2-Cl. In both cases, the formation of the less symmetric 4-X-Me is thermodynamically favorable compared to the formation of 5-X-Me. Interestingly, the transformation of 4-H-Me into 3-H-Me is found to be an uphill process by 3.5 kcal/mol, while the analogous conversion of 4-Cl-Me into 3-Cl-Me costs only 0.3 kcal/mol. Although these calculations do not account for the added complexities of mixed anhydride formation or R group identity, they do correlate with our experimental findings that 3-X-R formation is higher yielding when X = halogen. Taken together, it is reasonable to posit that the statistical advantage of monophthalimide formation balances against substrate-specific energetic differences to influence overall conversion into MDI products. In conclusion, we have developed methods for the preparation of a wide range of 5,6-dihalobenzene-1,2,3,4-bis(dicarboximides) from the direct solution-phase condensation of benzene-1,2,3,4-tetracarboxylic acids and primary amines in acetic acid solvent. The preparation of compounds with sterically demanding groups at the N atoms of the imides can be achieved readily under microwave conditions. Experimental and computational studies suggest that the reaction can take place under equilibrium conditions that are favored both statistically and thermodynamically to yield the desired ortho-diimide compounds instead of the symmetric dicarboxyphthalimide. Importantly, it is possible to avoid perturbing the aryl halide positions under these reaction conditions, which sets the stage for exploring a broad range of chemistries available for producing imide-decorated aromatic compounds with tailored form and function.

chemsum

{"title": "Improved syntheses of halogenated benzene-1,2,3,4-tetracarboxylic diimides", "journal": "ChemRxiv"}

the_periodic_transient_kinetics_method_for_investigation_of_kinetic_process_dynamics_under_realistic

## Abstract: Due to rising interest for the integration of chemical energy storage into the electrical power grid, the unsteady-state operation of chemical reactors is gaining more and more attention with emphasis on heterogeneously catalyzed reactions. The transient response of those reactions is influenced by effects on different length scales, ranging from the active surface via the individual porous catalyst particle up to the full-scale reactor. The challenge, however, is to characterize unsteady-state effects under realistic operation conditions and to assign them to distinct transport processes. Therefore, the periodic transient kinetics (PTK) method is introduced, which allows for the separation of kinetic process dynamics at different length scales experimentally under realistic operation conditions. The methodology also provides the capability for statistical analysis of the experimental results and therefore improved reliability of the derived conclusions. Therefore, the PTK method provides the experimental basis for modelbased derivation of reaction kinetics valid under dynamic conditions. The applicability of the methodology is demonstrated for the methanation reaction chosen as an example process for heterogeneously catalyzed reactions relevant for chemical energy storage purposes. ## Introduction The transition from a fossil-based energy sector to an energy supply based on renewables leads indispensably to fluctuations in the energy production . In order to dampen these fluctuations, buffer systems need to be integrated into the energy supply chain, which are based on storing electrical, chemical or physical energy. One attractive option for chemical energy storage is the Power-to-Gas processes (PtG), which allows to convert electrical into chemical energy by electrolysis yielding hydrogen. Subsequently hydrogen can be further converted with carbon oxides into methane, which can directly be stored and distributed in the existing natural gas grid . Due to cost and transport reasons efficient local PtG units are beneficial, allowing an implementation in proximity of the energy and carbon source . In the same vein cost-efficient local PtG units are equipped with small buffer capacities to dampen feed fluctuations, which limits the possibility to ensure a steady-state operation of the chemical reactor. Hence, fluctuations of the power supply propagate into the chemical reactor and an unsteady-state operation of the methanation reactor is necessary . Therefore, an efficient and safe operation of the methanation reactor is required under unsteady-state conditions, which needs a deep understanding of the underlying kinetic process dynamics at different length scales . The challenge becomes evident considering the fact that the underlying balance equations for describing the reactor behavior lead to a system of non-linearly coupled ordinary differential equations, even for the most simplifying assumptions. More realistic assumptions even lead to a system of partial differential equations, e.g. for distributed systems. On top of this the reactor has to be distinguished into three different scales, whose intrinsic dynamic behavior is rather independent of each other . For illustration the reactor dynamics at the reactor or macro scale is determined by the fluid dynamics and usually expressed by the residence time distribution (RTD). At the particle or meso scale diffusion processes between the bulk flow and the external catalyst surface, as well as within the porous system affect the dynamics. At the catalyst surface or micro scale, the sorption and surface reaction processes determine the dynamics. Therefore, the characteristic steps in heterogeneously catalyzed reactions, i.e. film and pore diffusion, ad-and desorption of reactants as well as surface reaction, can directly be linked to the length scales and dynamic behavior. Under realistic operating conditions the dynamic reactor response is an interplay of all those kinetic processes. This leads to a severe challenge for determination of the intrinsic dynamics at each individual length scale from the cumulative reactor response. Therefore, the scales are usually studied separately from each other, which requires sophistically designed experiments, though, in order to avoid mutual interaction. For the investigation of the processes at the catalyst surface under fluctuating feed gas mixtures several experimental methods already exist. Happel et al., Bennet and Biloen et al. developed the steady-state isotopic transient kinetic analysis (SSITKA) method , which is capable to determine the exchange rate and surface coverage of individual species . Therefore, the isotopic composition of the reactants is changed, while the catalyst surface is assumed to remain in a quasi-steady-state . Im-portantly, this assumption means that the reactions rates are unaffected by the transient change in isotopic composition and thus remain in steady-state. Additionally, an inert tracer is usually used to correct the residence time distribution of the reactants within the reactor . The capabilities and limitations of SSITKA as well as modeling approaches were summarized by Ledesman et al. comprehensively . Temporal analysis of products (TAP) measurements introduced by Gleaves et al. are capable to extract kinetic information by avoiding changes of the catalyst surface during the experiment . Therefore, small amounts of reactants compared to the number of active sites are pulsed into the system. The catalytic system experiences only small perturbations and the catalyst surface remains rather unchanged during the experiment . As these measurements are conducted with small catalyst particles under vacuum atmosphere only Knudsen diffusion determines the RTD. Thus, intrinsic kinetic data of the reaction network can be received, with the limitation that the system is far from realistic conditions. Another dynamic method introduced by Mori et al. exploits the benefits of single pulses applied to a reaction system. A defined number of molecules is pulsed into a carrier gas stream and the response signal is either analyzed with respect to the propagation in the falling edge or with respect to an internal standard. The latter was used by Friedland et al. to investigate the storage capacity of a catalyst under dynamic conditions . These methods, however, need welldefined conditions for meaningful results and are based on a limited degree of deflection of the catalyst from its original state for a small period of time. The chemical transient kinetics (CTK) method is based on a step-shaped change in the feed gas mixture and "track of the construction (build-up) or scavenging (backtransient) of the catalytically active phase as a function of time" . Therefore, the gas phase composition upon step-change is measured as function of time and linked to ad-and desorption processes between the catalyst surface and the gas phase. Since an internal standard is used as well, the RTD of the reaction system can be measured in-situ, which allows to separate the RTD and the dynamic processes at the catalyst surface in the measured signal . Furthermore, an external standard allows for derivation of quantitative information, as well . Finally, CTK provides the possibility to derive the coverage degree as function of time using the "surface atom counting technique" , which basically corresponds to the elemental balance of the gas phase. Interestingly, CTK has been applied successfully to CO hydrogenation reactions, such as Fischer-Tropsch (FT) synthesis , exhibiting the capability of deriving mechanistic insights and even information on the chain growth probability . The method, however, is demonstrated for ambient pressure only , which is identified as an important limitation by Athariboroujeny et al. , in particular for deducing the effect of operating pressure on the mechanistic pathways in FT reaction. Recently, Chen et al. applied a combination of CTK and SSITKA to deduce the mechanism of CO methanation and Fischer-Tropsch reaction for cobalt catalysts . Consequently, all methods described above are used to analyze the dynamic processes at the catalyst surface (micro scale) by eliminating the transport processes at macro and meso scale, but all are limited to model conditions (i.e. ambient operating pressure or below). An experimental methodology applicable to realistic conditions and capable to link the transient response of real reactors with the dynamic processes at all length scales, is thus still lacking. In the present contribution we therefore introduce the method of periodic transient kinetics (PTK), which allows the investigation of kinetic process dynamics at different length scales separately of each other. It is based on the CTK method recently emphasized by Raub et al. , but provides some important improvements. First of all, periodic step-changes are performed in order to enhance the reliability by statistical analysis of the limit cycle. Secondly, the PTK method is demonstrated at higher pressures, which allows to close the gap between model and realistic conditions in future. We focus on a detailed introduction of the experimental implementation and the quantitative evaluation approach of the molar flow rates derived from the measured gas phase composition as function of time. The applicability of the PTK method will be demonstrated for the methanation reaction as an example for heterogeneously catalyzed gas phase reactions but can also be transferred to more complex multiphase reactions, such as FT synthesis. Furthermore, the methodology is flexible to be applied to various reactor types. Since the present contribution focusses on the introduction of the methodology, it is thus based on comprehensive explanations of the relevant terms, as well as procedures for experimental conduction and data evaluation. We therefore structured the paper as follows: Because the terms used for unsteady-state reactor operation are often used inconsistently, we define and classify dynamic processes and the related terminology first. In section 3 we discuss the challenges related to the performance of dynamic experiments under realistic conditions and describe the developed methodology in detail, which comprises the experimental setup and procedure, as well as the respective model-based data evaluation. Finally, the methodology is demonstrated for two examples in the context of the methanation process in section 4, namely the CO2 adsorption on Al2O3 and the unsteady-state CO methanation reaction using a Ni/Al2O3 catalyst. The results of the latter example are briefly discussed in comparison to literature findings for illustration of the potential of the PTK methodology. ## Terminology In the field of dynamically operated chemical reactors and unsteady-state processes a variety of different terms are required, in order to precisely correlate the experimental observations with the underlying mechanisms. For sake of clarity those terms relevant to the PTK methodology introduced in the present study are briefly defined below. The PTK method can be classified as a forced unsteady-state process according to Matros et al. , since the unsteady-state behavior is induced by an external fluctuating input signal. In particular, a step-shaped change in the feed molar flow rate is used as input signal in the present study. According to the scope of the study, the system is investigated for examples in the context of the methanation reaction either by modeling and simulation or by experiments. Therefore, self-oscillating systems and other types and shapes of forced input signals are not in the focus here, but comprehensively summarized by different authors . A dynamic process is characterized by time dependent state variables, such as temperature or concentration, of the inlet and outlet streams, as well as of the reactor volume. Note that for forced unsteady-state reactor operation the inlet stream is usually used as the forced input, while the outlet stream is referred to as the reactor response. Each state variable can either be in steady-state or in unsteady-state, which is distinguished by the time derivative being zero or non-zero, respectively. Upon disturbance at time zero a state variable deflects from the initial state followed by a transient phase , which depends on the type of the disturbance and the dynamic behavior of the process. Therefore, the term 'unsteady-state' is defined strictly mathematical, while 'transient behavior' defines the trajectory of the variable in time. The type respectively periodicity of a temporal change is usually classified as follows (for illustration of different system behaviors see Table 1): An aperiodic signal is characterized by a transient change of a variable from an initial to a final steady-state. A periodic signal, instead, changes from the initial state to a second state and finally reaches the initial state again, which can be repeated multiple times. Note, that the response induced by a consecutive positive and negative step change, such as applied in the CTK method , is designated with the terms build-up and back-transient phase, respectively. The periodic operation allows the system to reach a limit cycle (LC), which is characterized by an invariant behavior for each period and which is particularly advantageous for the evaluation of the dynamic response . It has to be mentioned that the term LC refers to the response of a system only and characterizes its periodicity. The characteristics of a LC depends on the shape and cycle period 𝜏 (inverse frequency) of the input signal, as well as on the characteristic relaxation time 𝜏 res of the system, which can be determined from the system response . According to literature three types of the response being in a LC can be distinguished with respect to the 𝜏 /𝜏 res ratio. For large 𝜏 /𝜏 res ratios the response is in quasi-steady-state (qss), which means that the system response is fast compared to changes in the input signal. The system, thus, is nearly in a steady-state depending on the current input signal at each point in time. In the opposite case of small 𝜏 /𝜏 res ratios the relaxed steady-state (rss) is reached, which is characterized by fast changes of the forced input and a slow reaction by the inertial system. This leads to diminishing amplitudes of the response with increasing frequencies of the forced input. For high frequencies the system response even appears to be in steady-state, though it is still changing with time, since the response amplitude is smaller than the measurable threshold. Note that the apparent steady-state may differ from that under true steady-state conditions . The fulltransient state (fts) is reached for 𝜏 𝜏 res ⁄ ≈ 1 between both boundary cases, where the response depends strongly on the applied frequency. Interestingly, the information content is high in this region , which makes it attractive for fundamental research. The main challenges arise from the strong non-linear relation between input and response, which results in a high complexity of the experimental procedure and data evaluation. Furthermore, the applied frequencies to reach the fts region can be quite high, which requires a high temporal resolution of the analytical system and demanding switching for periodic input signals. ## Aperiodic step Response Literature [22, Periodic step Response Literature quasi steady-state (𝜏 ≫ 𝜏 res ) full transient-state (𝜏 ≅ 𝜏 res ) [47, relaxed steady-state (𝜏 ≪ 𝜏 res ) Table 1 illustrates the classification of forced input signals according to the periodicity and indicates references to the application in the context of the unsteady-state CO and CO2 methanation. The aperiodic step changes are mainly used to investigate the transient behavior of the reactants and to gain further insights into the underlying surface processes and reaction mechanisms . The forced periodic operation, though, has been investigated for various reaction systems in order to achieve higher conversion [28,29,32, or selectivity , while an increase in catalyst stability has been reported for different reaction systems, as well. With emphasis on methanation experimental studies on forced periodic operation are performed aiming at kinetic investigations , as well as process intensification for CO and CO2 methanation. In addition to that theoretical approaches are introduced for the evaluation of the higher frequency response . All these studies concentrate on the qss or fts region as the information content is high and experimental realization is feasible with reasonable effort. ## Challenges for unsteady-state experiments With emphasis on periodic step-shaped experiments for the methanation reaction following real effects contribute to the observed signal and have to be considered in design of experimental procedures and data evaluation. At the macro scale an ideal step change in the composition is hardly possible, due to the duration of the switching event between two compositions. Furthermore, the RTD in the piping between the switching valve and the catalyst bed, as well as between the catalyst bed and the detector of the analytics may deviate from ideal plug flow behavior and thus affects the intrinsic response of the catalyst bed. In particular, laminar flow profiles can be expected given by the small diameters of the piping in lab-scale equipment and thus small Reynolds numbers. Finally, complex real effects on the RTD within the catalyst bed render the separation of the dynamics at each characteristic length scale more difficult. Specifically, axial and radial gradients in composition, flow rate and pressure induce dispersion effects and thus deviation from ideal plug flow behavior. The pressure drop over the fixed-bed differs for different feed gas compositions, as well. For methanation, in particular, the chemical reaction also causes volume contraction and thus changes in the volumetric flow rate can occur. At the meso scale heat and mass transfer limitations between the fluid bulk and the external catalyst surface, as well as within the porous pellet, lead to the formation of pronounced temperature and concentration profiles, which superimpose the dynamic behavior of the processes at the micro scale. At the micro scale the main challenges arise from slow dynamic processes, such as catalyst deactivation, which have to be separated from the very fast processes involved in the chemical surface reaction. In addition to that chromatographic effects may occur at all scales upon step-changes in the inlet composition . ## Experimental Setup The experimental setup is adapted from the CTK method 45,46]. A flowsheet of the experimental setup is shown in Figure 1 and the corresponding 3D model in Figure 2. Note, that the colors given in brackets behind the described parts of the experimental setup below correspond to the colors in Figure 1 and Figure 2. The setup is equipped with two independent gas lines (red and blue). By means of a pressure actuated high speed valve (Fitok, BOSS-4C) (orange) one gas line is fed into the reactor (green), while the other is directly injected into the bypass line (grey). With the high speed valve the switching process takes less than 0.2 s, which is below the temporal resolution of the MS analytics (0.5 s, see below). This provides a nearly ideal step change (see Appendix, Figure S9). The gases H2 (5.0 purity, MTI), CO/Ar (90 vol.-% CO of 3.8 purity in Ar of 5.0 purity, Air Liquide), CO2 (4.8 purity, MTI), Ar (5.0 purity, MTI) and He (5.0 purity, MTI) are supplied via mass flow controllers (MFC) (EL-FLOW Prestige, Bronkhorst), without further purification. Note, that Ar is used as an internal standard (IS) for all experiments. Figure 1: Flowsheet of the experimental setup to investigate heterogeneously catalyzed gas-phase reactions under unsteady-state conditions (color code is used for explanation in the text and corresponds to Figure 2). All lines consist of 1/8" tubing (inner diameter 2 mm) in order to minimize the volume of the reactor periphery. The section between the switching valve and reactor exhibits a volume of 3 mL, only and is equipped with an additional analytic port directly at the reactor inlet (light green). This allows to measure the deviation of the switching event from an ideal step and thus the apparent RTD of the switching event, which is considered for the data evaluation (see Appendix, Figure S9). The experiments are conducted in a stainless-steel fixed-bed reactor (green) with an inner diameter of 4.5 mm and a total length of 30 cm, which results in a total volume of 7 mL. The reactor is equipped with a concentric 1/16-inch capillary containing a movable thermocouple (type K, TMH). The heating is accomplished by 4 heating cartridges (200 W Firerod, Telemeter Electronic) placed equally spaced inside an aluminum jacket, which provides an axial isothermal zone of around 10 cm. The catalyst particles are fractionated in a size range of 150 to 200 µm and are diluted with twice the amount of inert particles (200 µm, Al2O3, Sasol Puralox) of the same size. Therefore, internal heat and mass transfer limitations can be neglected, which was verified in our previous paper under identical conditions by applying the Mears, Anderson, and Weisz-Prater criteria . The catalyst bed with a total length of 2 cm is placed in the center of the isothermal section, which ensures isothermal conditions for temperatures up to 600 K with deviations of < ± 1 K. The packing is fixed inside the reactor by solid, non-porous glass particles (silica beads, häberle) of the same particle size and glass wool (silica wool, VWR). The total length of the packing sums up to around 10 cm. Non-porous glass is used, since it provides a small surface area for potential interaction with the gas species. Furthermore, solid glass particles reduce the gas holdup in the reactor, as well. RTD measurements are performed, which confirm minor deviation from ideal plug flow behavior within the packed fixed-bed reactor (see Appendix, Figure S9). The operating pressure is controlled by two back pressure regulators (violet, LF1, Equilibar). The working principle is based on an equilibrium of forces between a flowing gas and a pilot gas at a membrane, which ensures negligible pressure fluctuations even at low flow rates. One regulator is placed at the reactor outlet and the second in the bypass line. In order to reduce pressure spikes during the switching between both gas lines to about 50 mbar, the reactor inlet pressure is used as pilot pressure for the bypass-line. At the reactor outlet a mixture of H2 (5.0 purity, MTI) and Ne (5.0 purity, MTI) (yellow line) is dosed via MFCs (EL-FLOW Prestige, Bronkhorst). The external standard (ES) mixture containing Ne as ES and additional H2 is fed directly at the reactor outlet in order to reduce the residence time between outlet and analytics below the temporal resolution of the analytics. The tubing downstream the reactor is heated to 70 °C to prevent condensation of H2O. Note that the maximum H2O partial pressure is significantly below the vapor pressure, due to the low conversion and dilution with inert gas. The analytics consist of a process mass spectrometer (MS) (Cirrus 3-XD, MKS), which allows to analyze the mass-to-charge ratios 2 (H2), 4 (He), 15 (CH4), 18 (H2O), 20 (Ne), 28 (CO and CO2), 40 (Ar) and 44 (CO2) quantitatively with a temporal resolution of 0.5 s after calibration. The mass-to-charge ratios 26 (C2H6) and 43 (C3H8) are monitored qualitatively, as well. An auxiliary gas chromatograph (GC-2010, Shimadzu with RT-Q-Bond pre-column and molecular sieve 5A column), equipped with a thermal conductivity and flame ionization detector, is used to calibrate the MS results for CO, CO2, Ar, and hydrocarbons up to C3 under steady-state conditions. The analytics allow to minimize the error in the carbon mass balance based on MS measurements to < 5 % under steady-state and to < 10 % under unsteady-state conditions. Note, that higher hydrocarbons are negligible and not detected by means of MS, as indicated by steadystate experiments using the same catalyst and similar conditions previously reported . ## Implications of an internal and external standard The RTD of a reactor equipped with porous catalyst pellets is affected by transport processes at both the macro and the meso scale, since dispersion in the convective flow and diffusion in the porous structure contribute to the overall response. Since the RTD of the internal standard (IS) measured in-situ is assumed to be representative , it allows to provide each experimental data set together with the individual residence time behavior. If those transport phenomena are not selective towards certain components, the RTD is identical for all components 𝑖 and is just scaled with a proportional factor 𝐶 according to eq. ( 1), where 𝑛i refers to the measured molar flow rate of component 𝑖 as function of time. Hence, the obtained molar flow rate at the reactor outlet 𝑛̇𝑖 ,out of any compound 𝑖 can be plotted against 𝑛̇I S,out providing a linear dependency, if the above-mentioned assumptions are fulfilled. In other words, any deviation in experimental data from eq. ( 1) indicates compound selective effects in the system. For the example of heterogeneously catalyzed reactions, being in the focus of the present contribution, those effects are mainly caused by the interaction of compound 𝑖 with the solid surface. In particular, dissipation of species 𝑖 from the gas phase (𝑛̇𝑖 ,out /𝑛̇I S,out < 𝐶) indicates adsorption at the solid surface, while appearance (𝑛̇𝑖 ,out /𝑛İ S,out > 𝐶) may be caused by desorption. ## 𝑛̇𝑖 ,out (𝑡) 𝑛̇I S,out (𝑡) = 𝐶 = const. ( The ES is required to obtain the molar flow rates from MS data, in order to derive the material balance for all compounds 𝑖 quantitatively . The ES also allows to identify unwanted fluctuations in the volumetric flow rate, in particular during the switching event between two different feed gas compositions, which might cause pressure fluctuations at the reactor inlet. Therefore, the ES is dosed into the reactor outlet stream with a constant molar flow rate 𝑛Ė S and the molar fraction 𝑥 ES is measured over time by the MS, in order to derive the outlet molar flow rate 𝑛ȯ ut according to eq. ( 2). 𝑛̇𝑖 ,out (𝑡) = 𝛼(𝑡) 𝑥 𝑖,out 𝑝 STP 𝑉 ̇in,STP 𝑅 𝑇 STP (4) ## State space plot The graphical representation of eq. ( 1) is provided by the state-space plot, which displays the response of component 𝑖 as a function of that for the internal standard Ar in the present contribution. For heterogeneously catalyzed systems the state-space plot can unravel interactions between a gaseous component and the solid surface by sorption processes, for instance. Note that the state-space plot is not limited to specific types of transient signals and rather complements the typical plot of transient signals as function of time. ## SS II SS I does not change with time during. Therefore, the transient processes during the periodic experiments become more prominent in the state-space plot and are thus easier accessible. ## Derivation of the average limit cycle from experimental data The PTK methodology provides periodic data of the system response, since the input signal is periodic. Considering that the transient outlet molar flow 𝑛̇𝑖 ,out of component 𝑖 is equivalent to the system response, it provides the experimental data basis for further analysis. The key property of the LC is that each point within a period 0 ≤ 𝑡 ≤ τ of the duration 𝜏 is reached in each period 𝑁 𝑚 and that the corresponding response 𝑛i ,𝑚,out is identical. We also assume that each measured data is affected by noise. Therefore, the results obtained within the LC are averaged over a certain number of periods 𝑁 Θ according to eq. ( 5), in order to improve the signal-to-noise ratio by statistical analysis. The obtained average molar flow rate at the reactor outlet 𝑛̅ 𝑖,out thus corresponds to the average LC. In addition, the variance Δ𝑛̅ 𝑖,out 2 of the average LC can be calculated by eq. ( 6), which can further be used to determine confidence intervals of the LC. Δ𝑛̅ 𝑖,out Figure 4a shows the periodic response of the system in form of the CH4 outlet molar flow rate as an example. In total the response for 50 periods is shown of which the last 25 periods (in red) except the very last are used for averaging, since the activity loss over those periods is less than 3 %. Figure 4b depicts the corresponding average LC together with the standard deviation (red shaded area). ## Calculation of transient molar flow rates The deviation of the system response for component 𝑖 from the RTD can be evaluated quantitatively by calculating the transient molar flow rates, which are identical to the net surface flows defined in the CTK method . Therefore, the observed response in form of the measured outlet molar flow rate 𝑛i ,out of component 𝑖 during a transient process is assumed to be a superposition of two contributions according to eq. ( 7). The first contribution originates from the overall RTD, which results in the corresponding molar flow rate 𝑛̇𝑖 ,RTD . The second contribution quantifies the transient molar flow rate 𝑛i ,trans and thereby the deviation from the RTD during the transient process. Figure 5 visualizes this equation for CH4 as an example. It has to be mentioned that this approach requires the LC as necessary condition, since thereby 𝑛i ,trans corresponds to the deviation within one period only. If the LC is not achieved also previous deviations may accumulate in 𝑛̇𝑖 ,trans , which falsifies the analysis. Furthermore, it has to be mentioned that the transient molar flow rate is generally considered as a temporal source or sink term for each component, irrespective of the specific reason. Even though the main reason is probably the interaction of component 𝑖 with the catalyst surface by adand desorption, chemical reaction or irreversible processes may also contribute to 𝑛i ,trans . 𝑛i ,out (𝑡) = 𝑛i ,RTD (𝑡) + 𝑛i ,trans (𝑡) The contribution of the RTD for each component 𝑛i ,RTD is calculated by a function characterizing the RTD of the internal standard 𝐹 Ar and the outlet molar flow rates for each component at the steady-states for both feed gas mixtures 𝑛i ,out,ss,1 and 𝑛i ,out,ss,2 , according to eq. ( 8). Therefore, the function 𝐹 Ar is derived from the outlet molar flow rates of the internal standard Ar 𝑛̇A r,out normalized with the flow rates at both steady-states (see eq. ( 9)). 𝑛̇𝑖 ,RTD (𝑡) = (𝑛̇𝑖 ,out,ss,2 − 𝑛i ,out,ss,1 ) 𝐹 Ar (𝑡) + 𝑛i ,out,ss,1 (8) The steady-state is assumed to be achieved, if no temporal gradients in the signal occur, which corresponds to the mathematical definition of time derivatives being zero. Under real conditions, however, long term processes monotonical affecting the response, such as catalyst deactivation, have to be considered. Since the investigation of those processes do not require periodic experiments, due to their long term and irreversible nature, a certain but very small temporal gradient in the signal is acceptable, depending on the individual case. In our case steady-state conditions are reached for the last 5 s in each half period. Hence, the respective outlet molar flow rates are used for the calculation of 𝑛̇𝑖 ,out,ss,1 and 𝑛i ,out,ss,2 by averaging. The transient molar flow rates for each component can be applied in order to derive transient elemental balances, as shown for the carbonaceous species in eq. ( 10). For negligible formation of higher hydrocarbons (C2+), eq. ( 10) allows to calculate the amount of carbon stored at or released from the surface during the build-up and backtransient phase after integration over both half periods. Note, that the modification of carbon cannot be distinguished based on this equation without further surface specific analysis. In particular, various adsorbed carbon containing species may be present together with elemental carbon modifications and further carbon compounds . It is noteworthy, that the proper choice of the experimental conditions is of eminent importance for calculation and interpretation of the transient molar flow rates. Care must especially be taken regarding the signal-to-noise ratio expressed by the relative transient molar flow rate 𝑛̇𝑖 ,trans 𝑛̇𝑖 ,out ⁄ . In particular, the transient molar flow rate needs to be distinguishable from the total molar flow rate of the respective component despite of the errors in the measured data and the mass balance. The determination of the transient molar flow rate by the PTK method allows to link the analysis of the gas phase composition in continuous systems with the molar amount of adsorbed species at the surface measured with respective experimental operando methods (e.g. diffuse reflectance infrared fourier transform spectroscopy, DRIFTS) quantitatively using material balances as follows. The transient molar flow rate 𝑛̇ℎ ,trans of an element ℎ can be applied for calculation of the molar amount of substance of that element stored at the catalyst surface 𝑛 ℎ,surf , according to the elemental balance shown in eq. (11). The respective component balance (eq. ( 12)) requires to consider the amount of substance converted in a surface reaction 𝑅 𝑗,surf in addition to the transient molar flow rate 𝑛̇ℎ ,trans of component 𝑖, in order to calculate the amount of this component stored at the surface 𝑛 𝑖,surf . The stoichiometric coefficient 𝜈 𝑖,𝑗 describes the stoichiometry of the adsorbed species only and equals zero for non-adsorbed components. Eq. ( 12) therefore allows to deduce reaction kinetics under dynamic conditions, since it links model-based evaluation of experimental data with the transient surface reaction rates. The challenge is that the involved surface species and surface reactions depend on the specific reaction mechanism, which is not known a priori in most cases. Therefore, an iterative approach is meaningful by building up the complexity of the mechanism until the prediction of the model agree with the transient measurement results of gaseous and surface species. Eq. ( 13) connects the elemental balances with those of the components, using the number of atoms of element ℎ in component 𝑖 expressed by 𝛽 ℎ,𝑖 . Note that surface coverages can be obtained from the molar amount of substance stored at the catalyst surface, if the number of sorption sites is known. 𝑑𝑛 𝑖,surf 𝑑𝑡 = −𝑛i ,trans (𝑡) + ∑ 𝜈 𝑖,𝑗 𝑅 𝑗,surf 𝑗 (𝑡) 𝑛 ℎ = ∑ 𝛽 ℎ,𝑖 𝑛 𝑖 𝑖 and 𝑛̇ℎ = ∑ 𝛽 ℎ,𝑖 𝑛̇𝑖 𝑖 (13) ## Experimental procedure for demonstrating examples For the examples presented in this study the feed gas composition is switched 50 times either between H2/He (50/50 by volume) and CO2/Ar/H2/He (10/1/40/49 by volume) with a cycle period of 90 s for example I or between H2/He (40/60 by volume) and CO/Ar/H2/He (10/1/40/49 by volume) with a cycle period of 240 s for example II. Ar is chosen as the internal standard for the COx species and H2O, since it provides a similar diffusion coefficient and viscosity and is thus assumed to exhibit a representative RTD. The strong dilution with He is justified by the similarity to the physical properties of H2, the main component in the gas phase due to stoichiometric reasons. Therefore, the physical properties of all gas mixtures can be assumed to be rather similar, which provides the basis for a similarity in fluid dynamics in the system and thus in RTD. The total volumetric feed flow rate of 250 mLSTP min -1 was further diluted with the external standard mixture after the reactor outlet consisting of H2/Ne (245 mLSTP min -1 / 5 mLSTP min -1 ), which reduces the residence time between the reactor outlet and the analytics section to below the temporal resolution of the MS. The CO2 adsorption experiment of example I is conducted with 100 mg Al2O3 particles (Puralox, Sasol) with a particle size of 150-200 µm. The methanation experiments from example II are conducted with 50 mg of a 5 wt-% Ni/Al2O3 catalyst of the same size diluted with 100 mg Al2O3 particles (Puralox, Sasol) at a temperature of 556 K and a pressure of 2 bar, which provides almost full conversion of the carbon oxides in equilibrium . Furthermore, the reactor is operated under differential conditions in order to neglect the impact of concentration gradients by keeping the carbon oxide conversion below 20 % in all experiments . Catalyst aging, e.g. due to deactivation, is monitored based on the steady-states and can be ruled out in the reported experiments . ## Examples for method application 4.1 Example I: CO2 Adsorption For the example of CO2 adsorption at Al2O3 surface Figure 6a displays the observed system response in form of the measured outlet molar flow rate of CO2 𝑛̇C O 2 ,out . In addition, the theoretical response 𝑛̇C O 2 ,RTD is shown as well, which is expected from pure RTD according to eq. ( 8). Figure 6b depicts the transient molar flow rate 𝑛̇C O 2 ,trans , while Figure 6c shows the respective state-space plot for CO2. Due to the absence of catalytical active sites, the deviation of the CO2 response from the RTD can be attributed to interaction of CO2 with the solid surface by ad-and desorption processes. In Figure 6a a deviation in the build-up phase between the measured and theoretical CO2 response is observed, which causes a negative transient molar flow rate with a maximum of 2 µmol s -1 . In the state space plot the negative transient molar flow rate during the build-up phase causes a hysteresis below the linear relationship given by the dashed line. Note that a linear relationship would mean that the measured and the theoretical CO2 response is identical, indicating the CO2 signal being affected by RTD only. The negative transient molar flow rate is caused by a sink of CO2, which is thus removed from the gas phase due to adsorption at the surface. Furthermore, the maximum in transient molar flow rate in the early stage of the build-up phase indicates fast adsorption of CO2 at the alumina surface, which is still barely covered by adsorbent. With time the surface coverage increases resulting in the transient molar flow rate approaching zero towards the end of the build-up phase. During the back-transient phase, in contrast, the transient molar flow rate is positive also exhibiting a maximum in the early stage. Hence, the hysteresis in the state-space plot exhibits a higher value than expected from RTD. This observation indicates that CO2 desorbs from the solid surface into the gas phase, which is more pronounced for high surface coverages early in the back-transient phase. Further evaluation of the data can be performed by the integration of the transient molar flow rate during the back-transient phase, which yields the molar amount of CO2 desorbed. Therefore, the average back-transient phase and the last cycle is distinguished from each other and compared the sorption capacity measured volumetrically at an equal CO2 partial pressure and temperature (Triflex, micromeritics). After the last switch to the H2/He feed gas mixture the transient molar flow of CO2 is integrated over 180 s and sums up to 17 µmol, which is in reasonable agreement with the adsorption capacity of 24 µmol measured volumetrically. The deviation between the adsorbed amount determined by volumetric and flow methods, however, is in accordance with literature, where a lower adsorption capacity of CO2 during pulse experiments compared to volumetric measurements is reported . The reason is that equilibrium between the gas phase and the adsorbed species is not reached during flow experiments, while volumetric measurements are performed in order to ensure equilibrium conditions to be fulfilled. However, the amount of CO2 desorbing during the average back-transient phase amounts to 9 µmol and is thus significantly below the theoretical value and that of the last cycle. Hence, this difference between the average and the last cycle indicates incomplete desorption of CO2 during the half cycle period of 45 s. Note that CO2 is only weakly adsorbed at the surface, as found by temperature programmed desorption (TPD) experiments up to 400 °C (see Appendix, Figure S10), confirming the sorption process being completely reversible. Interestingly, the relative transient molar flow rate and thus the signal-to-noise ratio changes within each half-period and also differs for the build-up and back-transient phase. This becomes evident, if one considers that 𝑛̇𝑖 ,out follows the RTD and thus with time on the one hand. On the other hand, 𝑛i ,trans also changes with time depending on the underlying interaction mechanism with the catalyst. If ad-or desorption at the solid surface occurs, for instance, the surface coverage changes with time and therefore the sorption rate, as well. For illustration, the relative transient molar flow rate for one period is shown in Figure 7. Note that the time delay of the system amounts to 4 s, for which no meaningful data is available. It can be observed that the relative transient molar flow rate rises rapidly in the build-up phase and reaches a maximum within 10 s. During that phase the transient molar flow rate of CO2 is high, since CO2 In addition to that 𝑛Ċ O 2 ,trans is high in the early stage of the back-transient phase, due to high desorption rates given by the initially high surface coverages and decreases with time, as well. This gives raise to resolve slow processes in the interaction between gaseous species and the solid surface, which occur close to the steady-state. It is recommended to analyze and compare both half-periods in order to minimize the error in determination of the transient molar flow rates. ## Example II: CO methanation For the example of CO methanation the response in form of the measured outlet molar flow rate 𝑛̇𝑖 ,out for CO and CH4 are shown in comparison to the theoretical values 𝑛̇𝑖 ,RTD in Figure 8a, while the corresponding transient molar flow rates 𝑛̇𝑖 ,trans are displayed in Figure 8b. Note, that the CO signal is scaled in order to be displayed together with CH4. The comparison of the results shown in Figure 8 (a vs. b) exhibits that CO follows the RTD, though a significant transient molar flow rate is calculated. This discrepancy can be explained by the fact that the relative transient molar flow rate 𝑛̇C O,trans 𝑛̇C O,out ⁄ is small. We therefore focus on Figure 8b for further discussion. During the build-up phase (switch from H2/He to CO/Ar/H2/He) a significant negative transient molar flow rate for CO is observed shortly after the switching event, which indicates strong CO adsorption. At the same time a positive transient molar flow rate for CH4 indicates product formation and desorption into the gas phase. The interplay between both species can be evaluated by the transient molar flow rate of carbon species at the surface 𝑛̇C ,trans according to eq. ( 10). The negative value during the buildup phase indicates that the CO adsorbed at the surface is not only converted into CH4 but is also stored at the surface, which indicates that CO adsorption is a rather fast process. This is in accordance to Bundhoo et al., who reported a temporal delay of the formation of CH4 after the adsorption of CO on a Ni catalyst . During the back-transient phase the transient molar flow rate of CH4 exhibits positive values, while that for CO and overall carbon is positive at first and turns negative before approaching zero. The positive transient molar flow rate of CH4 is attributed to the hydrogenation of a highly reactive intermediates adsorbed at the surface and subsequent desorption of the CH4 formed, according to literature . Those intermediate are assigned as CHx species, which are converted into CH4 in the following two-step mechanism proposed by Bundhoo et al. : First, metallic sites at the catalyst surface are released by desorption or reaction of adsorbed species, which enhances the atomic hydrogen supply. In the following step the adsorbed reactive intermediate reacts with the available hydrogen to gaseous CH4. The positive transient molar flow rate of CO in the early stage of the back-transient phase indicates CO desorption from the surface, due to decreasing CO partial pressure in gas phase during the back-transient phase. Due to ongoing surface reaction further sorption sites become available with time, which leads to a (re-)adsorption of CO still available in the gas phase and therefore negative transient molar flow rates of CO. Obviously, this effect requires the surface reaction to be sufficiently fast, in order to release sorption sites, while CO is still present in the gas phase. The latter is governed by the residence time distribution of the reactor. The CO adsorbed in that later phase will also be converted into CH4 as long as sufficient H2 is available and thereby contributes to the positive transient molar flow rate of CH4 in that phase, as well. The transient molar flow rate of carbon corresponds to the carbon balance given in eq. ( 10), which is positive during the back-transient phase. This indicates that deposits of carbonaceous species formed during build-up phase are hydrogenated to CH4 due to the excess in hydrogen during the back-transient phase. ## Conclusion The PTK methodology is introduced in order to investigate the interplay of transient kinetic processes in heterogeneously catalyzed reactions under realistic conditions (i.e. elevated pressures). It allows to derive the molar flow rates between the gas phase and the catalyst surface quantitatively based on the transient reactor response with respect to gas phase composition and thus complements techniques focusing on analyzing the dynamics at the catalyst surface itself. In the contribution, the experimental procedure and equipment is described together with model-based evaluation of the experimental data. The strength of the PTK method relies on periodic step-shaped changes in feed gas composition, which induce a periodic reactor response, as well. Therefore, a limit cycle is reached for reversible interactions between the gas phase and the catalyst surface, which is evaluated statistically in order to provide reliable data for subsequent interpretation. In particular, the dynamic response with respect to the outlet molar flow rates of the reactive species present in the gas phase is evaluated in the limit cycle providing narrow confidence intervals. In order to distinguish the residence time behavior from transient processes related to interaction with the surface an internal standard is monitored and considered in result analysis. This approach provides the basis for evaluation of transient processes based on the material balances, exhibiting minor deviations only, and thus to resolve even small dynamic effects. Therefore, the presented PTK method is capable to link the transient behavior of single components to the dynamic response of the reactor. The evaluation procedure based on material balances of the catalyst surface and the overall reactor further provides the capability to deduce reaction kinetics valid under dynamic operation conditions from respective experimental results. The methodology is demonstrated for two examples in the context of the methanation reaction. The adsorption of CO2 at Al2O3 represents a non-reactive reversible process, while for hydrogenation of CO into CH4 sinks and sources exist for reactants and products, respectively. The results reveal that the dynamics of the ad-and desorption processes can be resolved and quantitatively evaluated in terms of molar flow rates. Such information allows to deduce the kinetics of those processes under reaction conditions, which is very important for modeling and simulation of chemical reactors operated under dynamic conditions. The second example shows that experimental data can be obtained, which provide the basis to even derive the kinetics of sink and source terms under dynamic conditions. Since the PTK method is not limited to both examples reported, it allows to derive kinetic information for various heterogenously catalyzed gasphase reactions operated dynamically under realistic reaction conditions. ## Symbol Description Unit Latin letters 𝐶 Constant 1 ## 𝐹 Step response 1 𝑁 Θ Evaluated number of periods in limit cycle 1

chemsum

{"title": "The periodic transient kinetics method for investigation of kinetic process dynamics under realistic conditions: Methanation as an example", "journal": "ChemRxiv"}

synthesis_and_electrocatalytic_her_studies_of_carbene-ligated_cu3-xp_nanocrystals

## Abstract: N-heterocyclic carbenes (NHCs) are an important class of ligands capable of making strong carbon-metal bonds. Recently, there has been a growing interest in the study of carbene-ligated nanocrystals, primarily coinage metal nanocrystals, which have found applications as catalysts for numerous reactions. The general ability of NHC ligands to positively affect the catalytic properties of other types of nanocrystal catalysts remains unknown. Herein, we present the first carbenestabilized Cu3-xP nanocrystals. Inquiries into the mechanism of formation of NHC-ligated Cu3-xP nanocrystals suggest that crystalline Cu3-xP forms directly as a result of a high-temperature metathesis reaction between a tris(trimethylsilyl)phosphine precursor and an NHC-CuBr precursor, the latter of which behaves as a source of both carbene ligand and Cu + . To study the effect of the NHC surface ligands on catalytic performance, we tested the electrocatalytic hydrogen evolving ability of the NHC-ligated Cu3-xP nanocrystals and found they possess superior activity to analogous oleylamine-ligated Cu3-xP nanocrystals. Density functional theory calculations suggest that the NHC ligands minimize unfavorable electrostatic interactions between the copper phosphide surface and H + during the first step of the hydrogen evolution reaction, which likely contributes to the superior performance of NHC-ligated Cu3-xP catalysts as compared to oleylamine-ligated Cu3-xP catalysts.Cu3-xP nanocrystal synthesis, characterization, and electrocatalytic HER studies will be discussed in detail, and density functional theory (DFT) calculations were carried out to elucidate the electronic effects of NHC ligands on catalysis. EXPERIMENTAL SECTIONMaterials and General Procedures. Reagents and solvents were purchased from commercial sources and used as received, unless otherwise noted. Benzimidazole (C7H6N2, 99%), 1-bromotetradecane (C14H29Br), copper(I) oxide (99.9%), potassium carbonate (K2CO3, anhydrous, 99%), and 1,4-dioxane were purchased from Alfa Aesar. Copper(I) bromide (98%) was purchased from Strem Chemicals. 1-octadecene (ODE, technical grade, 90%), oleylamine (technical grade, 70%), tris(trimethylsilyl)phosphine ((TMS)3P, 95%) and a phosphorus ICP standard (1000 ppm in 2% aqueous nitric acid) were purchased from Sigma-Aldrich. The copper ICP standard (1000 ppm in 2% aqueous nitric acid) was purchased from Perkin Elmer. Reactions involving air-or moisture-sensitive compounds were conducted under a nitrogen atmosphere by using standard Schlenk techniques. 1-Octadecene and oleylamine were degassed for 4 h at 105 °C and then overnight at room temperature prior to use. performance of the NHC-ligated Cu3-xP catalyst. Future studies should focus on leveraging the high synthetic tunability of the NHC surface ligands to confer better stability or improved activity. Specifically, the design of NHC ligands with variable electronic properties could be used to modulate their effect on electrocatalysis. ASSOCIATED CONTENT Supporting InformationThe Supporting Information is available free of charge on the ACS Publications website.Experimental details; UV-vis-NIR spectra; high-resolution TEM images; N 1s XPS spectrum; additional powder XRD patterns; TGA traces; EIS figures; double-layer capacitance figures; Tafel plots; controlled potential electrolysis figures; energy decomposition analysis from DFT; ligand density calculations (PDF) ## INTRODUCTION N-Heterocyclic carbene (NHC) ligands have significant value as a result of their structural diversity, chemical stability, and strong electron donation that leads to a high coordinating ability with metals. 1 While NHCs are now commonly employed as neutral L-type ligands for molecular complexes, they are also gaining increasing utility as ligands to support the steric stabilization of nanocrystals. And just as NHC ligands are widely utilized in molecular complexes for homogeneous catalysis as a result of their steric and electronic properties, 8 supporting NHC ligands are becoming of interest for heterogeneous nanocrystal catalysts. It has been demonstrated that NHC ligands have the ability to enhance nanocrystal catalyst stability and increase nanocrystal catalyst activity and/or selectivity. NHC-stabilized nanocrystals have been shown to display catalytic activity toward, for example, hydrogenations and semihydrogenations, 9,12-14 lactonization, 10 asymmetric arylations, 15 Buchwald-Hartwig aminations, 16 and cross-coupling reactions. 15 NHC-stabilized nanocrystals have also been shown to be competent electrocatalysts for CO2 reduction. 11,17 For example, Cao et al. investigated the use of NHC-stabilized Au nanocrystals as electrocatalysts for the reduction of CO2 to CO. Interestingly, the Au nanocrystal catalyst with surface-bound carbene ligands exhibited a higher Faradaic efficiency for CO2 reduction to CO than did bare (or ligand-less) Au nanoparticles; it was further shown that the NHC affected the mechanistic pathway of catalysis. 17 Despite the proven benefits of applying NHC ligands to nanocrystal catalysts, they have thus far only been supported on metal nanocrystal catalysts (e.g., Ru, Au, Pt, Pd, Cu). Therefore, the potential utility of NHC ligands as supporting ligands for other types of nanocrystal catalysts is currently unknown. Certain metal phosphide nanocrystals have emerged as an important class of catalysts for reactions such as hydrodesulfurization, hydrodeoxygenation, 22 and the hydrogen evolution reaction (HER). 23 Along these lines, self-doped copper phosphide (Cu3-xP) nanocrystals have been shown to be efficient Janus catalysts for overall electrochemical water splitting. 24,25 Synthetic methods for the preparation of colloidal Cu3-xP nanocrystals remain underdeveloped, however. Early reports utilized trioctylphosphine (TOP) to phosphidize zero-valent Cu nanocrystals under relatively high-temperature conditions (320 °C). 26 More recently, reactive phosphines, such as tris(trimethylsilyl)phosphine ((TMS)3P), have been introduced as the phosphide source, which helped to lower reaction temperatures to 120 °C. 27,28 Herein, we present a direct synthetic route to NHC-stabilized Cu3-xP nanocrystals to explore the effect of the carbene ligand when using the resulting nanocrystals as HER electrocatalysts. The direct synthesis of NHC-stabilized Cu3-xP nanocrystals circumvents the need for a post-synthetic ligand exchange for the installation of carbenes on the nanocrystal surface. To the best of our knowledge, this is the first example of a metal phosphide nanocrystal supported by a carbene ligand. The NHC-stabilized Preparation of (TMS)3P Stock Solution. Caution! (TMS)3P is a pyrophoric material that must be handled with care. In a N2 glove box, an ampule containing 1 g of liquid (TMS)3P was opened and the contents were added to a Schlenk flask containing 40 mL ODE (dried as mentioned above) to make a 0.1 M stock solution that was used for all subsequent reactions. ## Synthesis of NHC-Stabilized Cu3-xP Nanocrystals. In a typical Cu3-xP nanocrystal synthesis, a solution of NHC-CuBr (195 mg, 0.300 mmol) in 6 mL ODE was prepared. The mixture was subjected to vacuum and held for 1 h at 115 ˚C to remove any adventitious H2O. NHC-CuBr was completely dissolved in ODE after 1 h of stirring at 115 ˚C. Subsequently, the temperature was raised to 250 ˚C and 0.5 mL of the 0.1 M (TMS)3P stock solution was rapidly added into the NHC-CuBr solution. The solution immediately became black and turbid upon phosphine addition. After 2 min, the reaction was quenched by removing the heat and placing the flask in a room temperature water bath. To remove 1-octadecene and any unreacted precursors and by-products, the Cu3-xP nanocrystals were washed by splitting the crude reaction mixture into two 40-mL centrifuge tubes that were then filled to volume with acetone. The centrifuge tubes were sonicated for 10 min and centrifuged for 5 min at 6000 rpm. For long-term colloidal stability, particles were washed only once with acetone and redispersed in toluene or tetrachloroethylene (TCE). For electrochemical studies, an additional partial wash was performed in which the product of one centrifuge tube was redispersed in 5 mL of hexanes, washed with an additional 10 mL of acetone in the same manner as described above, and finally redispersed in 5 mL of hexanes. This hexanes suspension could then be dropcast directly onto a glassy carbon electrode for catalysis studies. Characterization. UV-vis-NIR spectroscopy was carried out on a Perkin-Elmer Lamba 950 spectrophotometer equipped with a 150mm integrating sphere, using a quartz cuvette for liquid samples. Spectra were taken in tetrachloroethylene to reduce solvent contributions to the spectrum in the NIR region. Powder X-ray diffraction (XRD) data was collected using a Rigaku Ultima IV diffractometer in parallel beam geometry (2-mm beam width) using Cu Ka radiation (l = 1.54 ). Samples were prepared by drop casting the nanocrystals onto zero-diffraction, single crystal Si substrates. X-ray photoelectron spectra (XPS) were obtained using a Kratos Axis Ultra X-ray photoelectron spectrometer with an analyzer lens in hybrid mode. High-resolution scans were performed using a monochromatic aluminum anode with an operating current of 6 mA and voltage of 10 kV using a step size of 0.1 eV, a pass energy of 40 eV, and a pressure range between 1-3 ´ 10 -8 Torr. The binding energies for all spectra were referenced to the C 1s core level at 284.8 eV. Transmission electron microscopy (TEM) analysis was performed on a JEOL JEM-2100 microscope at an operating voltage of 200 kV equipped with a Gatan Orius CCD camera. Samples for TEM analysis were prepared from dilute purified nanocrystal suspensions drop cast from hexanes dispersions onto 400 mesh carbon-coated copper grids (Ted Pella, Inc.). Thermogravimetric analysis (TGA) was performed on a TGA Q50 instrument with a heating rate of 10 °C•min −1 . 8-10 mg samples were used for all TGA studies, and samples were measured by TGA after one full wash and one partial wash, identical to the workup used for catalytic studies. More details on the ligand density calculations determined by TGA are provided in the Supporting Information. Fourier transform infrared spectroscopy (FT-IR) was performed on a Bruker Vertex 80 FT-IR spectrometer. Samples were prepared as a powder within a matrix of KBr. Inductively-coupled plasma optical emission spectroscopy (ICP-OES) was performed on an iCap 7400 ICP. All samples were digested with 2 mL of concentrated nitric acid and subsequently diluted to 25 mL with Millipore water in a volumetric flask. Phosphorus and copper ICP standards were prepared at different concentrations (0.1 ppm, 0.8 ppm, 2 ppm, 4 ppm, 10 ppm) to construct a five-point calibration curve from which the sample concentrations of Cu and P could be determined. NMR spectra were taken on a Varian VNMRS-600 in CDCl3. Electrochemical Methods. Electrochemistry experiments were carried out using a VersaSTAT 3 potentiostat in a three-electrode configuration electrochemical cell under an inert N2 atmosphere. A rotating disk electrode (RDE, glassy carbon insert, 0.196 cm 2 surface area) was used as the working electrode. The glassy carbon surface was polished with 0.05 µm Al2O3 polish powder and sonicated in Millipore water prior to use. A graphite rod, purchased from Graphite Machining, Inc. (Grade NAC-500 Purified, < 10 ppm ash level), was used as the counter electrode. The reference electrode, placed in a separate compartment and connected by a porous Teflon tip, was based on an aqueous Ag/AgCl/1.0 M KCl electrode (purchased from CH Instrument, Inc.). All potentials reported in this paper were converted to the reversible hydrogen electrode (RHE) by adding a value of (0.235 + 0.059 × pH) V. 0.5 M H2SO4 aqueous solution was used as the electrolyte and was purged with nitrogen thoroughly prior to electrochemical testing. Cyclic voltammograms for double layer capacitance (Cdl) measurements were taken over a 100 mV potential window centered around the open circuit potential (OCP) at scan rates of 10, 20, 30, 40, 50, and 60 mV/s. The capacitance current obtained from the current difference (Δi = iaic) at OCP was plotted against the scan rate. The slope is twice the value of the double layer capacitance. Controlled potential electrolysis (CPE) measurements to determine long-term stability and Faradaic efficiency were conducted in a sealed twochambered H cell where the first chamber held the working and reference electrodes in 40 mL of 0.5 M H2SO4 aqueous solution and the second chamber held the counter electrode in 25 mL of 0.5 M H2SO4 aqueous solution. The two chambers were both under N2 and separated by a fine porosity glass frit. CPE experiments were performed with a glassy carbon plate electrode (6 cm × 1 cm × 0.3 cm; Tokai Carbon USA) as the working electrode and a graphite rod as the counter electrode. The reference electrode was a Ag/AgCl/1.0 M KCl (aq.) electrode separated from the solution by a porous Teflon tip. Using a gas-tight syringe, 2 mL of gas was withdrawn from the headspace of the H cell and injected into a gas chromatography instrument (Shimadzu GC-2010-Plus) equipped with a BID detector and a Restek ShinCarbon ST Micropacked column. To determine the Faradaic efficiency, the theoretical H2 amount based on total charge flowed was compared with the GCdetected H2 produced from CPE. Electrochemical impedance spectroscopy (EIS) measurements were carried out at different overpotentials in the frequency range of 100 kHz -1 Hz with 10 mV sinusoidal perturbations. Experimental EIS data were analyzed and fitted with the ZSimpWin software using a two-time constant parallel model. All of the polarization curves presented herein were corrected for iR loss according to the following equation: where Ecorr is the iR-corrected potential, Emea is the experimentally measured potential, and Rs is the solution resistance extracted from the fitted EIS data. Density Functional Theory. Density functional theory (DFT) calculations at the B97/def2-SVP level of theory 29,30 were performed using the ab initio quantum chemistry software Q-Chem 31 to probe the electronic effects of L-type NHC and alkylamine ligands on proton binding to a Cu3P catalyst. ## RESULTS AND DISCUSSION Synthesis and Characterization. We previously reported that a metathesis reaction between bromo[1,3-(ditetradecyl)benzimidazol-2-ylidene]metal(I) (NHC-MBr, M = Ag, Cu) complexes and bis(trialkylsilyl) chalcogenides ((R3Si)2E, where E = S, Se) under ambient conditions yields monodisperse M2E nanocrystals, where the NHC ligands bearing long-chain alkyl substituents remain coordinated to the nanocrystal surface and provide excellent colloidal stability. 7 Here, we extended the use of the same NHC-CuBr precursor to the preparation of Cu3-xP nanocrystals by reaction with (TMS)3P. Initial attempts to synthesize Cu3-xP nanocrystals at room temperature resulted in an amorphous product, as revealed by powder X-ray diffraction (XRD). Therefore, the reaction temperature was raised to 250 °C and (TMS)3P was rapidly injected into a solution of NHC-CuBr in the high-boiling, non-coordinating solvent 1-octadecene. This resulted in the direct formation of Cu3-xP nanocrystals. The as-synthesized Cu3-xP nanocrystals (1) were confirmed by powder XRD to be phase-pure hexagonal Cu3-xP, with lattice parameters determined by a Le Bail fit of a = 6.9376( 14 00-002-1263, Figure 1a). The UV-vis-NIR absorption spectrum displays a characteristic LSPR band at ~1360 nm for the resulting nanocrystals (Figure S1), as expected due to copper vacancies in the structure. 27,28 Inductively coupled plasma optical emission spectroscopy (ICP-OES) analysis of the as-synthesized product confirms a substoichiometric average composition of Cu2.30P, consistent with the copper-poor composition of Cu3-xP, which is known to allow a wide range of compositions from 0.1 < x < 0.7. 32 Figure 1c illustrates a representative transmission electron microscopy (TEM) image of the quasi-spherical Cu3-xP nanocrystals. The nanocrystals have a mean diameter of 6.7 ± 1.1 nm (N = 300 counts), and high-resolution TEM (HR-TEM) reveals their apparent single-crystalline nature with a lattice spacing of d = 0.20 nm corresponding to the (300) planes of the expected hexagonal phase (Figure S2). Suspensions of 1 are colloidally stable under inert atmosphere, which can be ascribed to steric stabilization from legacy surface-bound NHC ligands, as evidenced by the presence of a N 1s peak from the NHC in the X-ray photoelectron spectrum (XPS) of the isolated nanocrystals (Figure S3), with further support from solution 1 H NMR spectroscopy (vide infra).The high-resolution XPS spectrum of the Cu 2p region confirms the presence of Cu + in the Cu3-xP nanocrystals with peaks centered at the expected binding energies of Cu 2p1/2 at 952.4 eV and 2p3/2 at 932.6 eV (Figure 2a). 25 Additionally, the high-resolution XPS spectrum of the P 2p region shows characteristic binding energies for P 3-(2p1/2 at 129.2 eV, 2p3/2 at 128.4 eV), with a small amount of oxidized P species (i.e., POx) observed at a binding energy of 132.5 eV (Figure 2b). 33 The oxidative stability of the Cu3-xP nanocrystals is in agreement with previously observed results. To further investigate the synthetic role of the NHC ligand, a control reaction was carried out in which the NHC-CuBr complex was replaced by CuBr in the presence of stoichiometric oleylamine, keeping the Cu:ligand molar ratio at 1:1. The reaction was otherwise performed identically. Interestingly, this control experiment yielded nearly amorphous copper phosphide nanoparticles (2) (Figure 1b, d), which also display a broad plasmon resonance feature in the NIR (Figure S1). Elemental analysis by ICP-OES of 2 reveals an average composition of Cu1.96P, deviating from the range of reported compositions for crystalline Cu3-xP. Transmission electron micrographs of the oleylamine-stabilized particles give a similar mean diameter of 5.5 ± 1.0 nm (N = 300 counts). This control experiment provides two insights. First, it demonstrates that copper phosphide nanoparticles (albeit quasiamorphous) can be synthesized using oleylamine as a ligand as long as it is not used in excess (i.e., Liu et al. observe the reduction of Cu + to Cu nanocrystals without phosphidation by (TMS)3P when oleylamine is used as a solvent in the absence of TOP 28 ). Second, it indicates that the NHC ligand plays a significant role in directing the crystallinity of the resulting product. Carbene-influenced nanocrystal growth is not wholly unprecedented; there exists evidence that nanoparticles synthesized in imidazolium-based ionic liquids nucleate and grow in the presence of carbene ligands formed in-situ by deprotonation of the imidazolium cation. In that vein, we previously observed that when imidazolium-based ionic liquids are used to synthesize nickel phosphide nanocrystals, phase-pure Ni2P forms, as compared to a mixture of phases that results from a different reaction pathway when using octadecene instead of the ionic liquid under otherwise identical conditions. 37 To explain the disparate degrees of crystallinity observed for 1 and 2, we noted that these two reactions visibly differ in the process of nucleation --while heating the oleylamine-CuBr mixture to the 250 ˚C reaction temperature, the solution transitions from a light-yellow color of dissolved oleylamine-CuBr at low temperatures to a dark blue-black color at temperatures greater than ~215 ˚C. Upon injection of the (TMS)3P precursor at 250 ˚C, the reaction mixture immediately turns black and becomes turbid, indicating the formation of Cu3-xP. We hypothesized that the color changes that precede the injection of (TMS)3P could be indicative of Cu nanoparticle formation induced by the reduction of Cu + by oleylamine. To test this hypothesis, we performed a control experiment in the absence of any phosphorus source, in which we heated the oleylamine-CuBr mixture to 250 ˚C and then quickly cooled the reaction mixture to room temperature. The electronic absorption spectrum of the product reveals a single, broad peak in the visible region centered at 700 nm (Figure S4), attributable to LSPR absorption of Cu nanoparticles. Powder XRD of the product confirms it is amorphous in nature (Figure S4). This result sheds light on the mechanism of formation of 2; injection of the very reactive (TMS)3P precursor phosphidizes amorphous Cu nanoparticles and, despite the high temperature injection, leads to a persistent quasi-amorphous phase of Cu3-xP. It is known that amorphous materials are often stabilized when containing glass-forming elements including B, C, Si or P, which may explain why structural reorganization to crystalline copper phosphide is not observed. 41 In contrast, no such cascade of color changes occurs prior to injection of (TMS)3P in the presence of the NHC ligand, indicating that copper nanoparticles do not form as intermediates. This difference is likely due to the robust Cu-C bond within the NHC-CuBr precursor (i.e., Boehme and Frenking calculated via DFT that the bond dissociation energy of the Cu-C bond of a CuCl complex of imidazole-2-ylidene falls between 67-73 kcal/mol), 42 and the absence of the primary amine reducing agent. Therefore, rather than proceeding through phosphidation of an amorphous Cu nanoparticle intermediate, the carbene precursor produces crystalline Cu3-xP directly through a metathesis reaction between NHC-CuBr and (TMS)3P to produce crystalline Cu3-xP and TMS-Br. Thus, the NHC ligand serves dual purposes, namely, it affords colloidal stability to the resulting nanocrystals and it circumvents the formation of zero-valent Cu intermediates, allowing crystalline Cu3-xP to be accessed directly. Thus, in general, using metal-carbene precursors to make metal pnictide or metal chalcogenide nanocrystals may prove to be beneficial for chemistries that are susceptible to proceeding through undesirable zero-valent intermediates. The carbene ligand used herein binds tightly to the nanocrystal surface but is more bulky than oleylamine. 3 To determine the density of ligands on the surface of 1 and 2, thermogravimetric analysis (TGA) was performed on isolated powders of both samples (Figure S5). The ligand density was determined to be ~1.0 ligands/nm 2 for 1 and ~2.5 ligands/nm 2 for 2 (see Supporting Information for calculation details). This finding demonstrates that, despite being a stronger binding L-type ligand, the steric bulk of the carbene prevents 1 from having a higher ligand density than 2. Due to the utility of Cu3-xP electrocatalysts for water splitting, 24,25,43 we aimed to test these carbene-stabilized Cu3-xP nanocrystals for HER to evaluate both their potential as electrocatalysts and the effect the NHC ligand has on catalysis. To evaluate the effect of the carbene on catalysis, we sought to compare the HER activity of carbene-stabilized Cu3-xP nanocrystals to oleylamine-stabilized Cu3-xP nanocrystals. However, since 2 differs both in the identity of the supporting ligand and the degree of crystallinity of the nanocrystals, a straightforward comparison of the HER activity of 1 and 2 cannot be cleanly made. Therefore, we synthesized crystalline, carbene-stabilized Cu3-xP nanocrystals and then performed a post-synthetic ligand exchange under forcing conditions by dispersing 1 in an excess of oleylamine at 85 °C for 2 h. The 1 H NMR spectrum of 1 prior to the ligand exchange clearly indicates the binding of the carbene to the nanocrystal surface, as the signature resonances of the NHC ligand are significantly broadened compared to the NHC-CuBr precursor (Figure 3a). The 1 H NMR spectrum of the ligand-exchanged Cu3-xP nanocrystals (3) indicates that ligand exchange is quantitative under these conditions, as no sign of the signature benzimidazole resonances remains in the aromatic region. Furthermore, the two peaks in the range of 5.26-5.46 ppm are diagnostic of a dynamic equilibrium of bound/free oleylamine on the surface of the nanocrystal (Figure 3b). 44,45 Interestingly, while oleylamine binding is a dynamic process on the NMR timescale, no resonances associated with a free carbene are observed in the 1 H NMR spectrum of 1, reiterating that the carbene binds more tightly to the nanocrystal surface than oleylamine. TGA analysis of 3 returns a higher ligand density than that of 1 or 2, at 3.3 ligands/nm 2 , which is not surprising considering the ligand exchange step was performed with a large excess of oleylamine, whereas the preparations of 1 and 2 maintained Cu:ligand ratios of 1:1. Importantly, the nanocrystals maintain their crystallinity after ligand exchange and show no statistically significant change in nanocrystal size, as verified by TEM analysis (Figure S6). Electrocatalytic HER Studies. To investigate the electrocatalytic HER activity of 1 and 3, detailed electrochemical measurements were performed in a three-electrode setup with N2saturated 0.5 M H2SO4 aqueous electrolyte. 1 and 3 were deposited onto glassy carbon electrodes by directly drop-casting the respective hexane suspensions without further modifications. In general, 1 exhibits better overall HER activity than 3 (Figure 4 and Table 1); that is, the overpotential to reach 10 mA/cm 2 current density is 0.45 V for 1 and 0.86 V for 3, and 1 shows a much lower Tafel slope of 85 mV/dec as compared to the 228 mV/dec for 3. The superior activity of 1 is correlated with a lower charge transport resistance (Rct), as revealed by the electrochemical impedance spectroscopy (EIS). Figures S8 and S9 show the EIS responses of 1 and 3, which are described by a two-time constant parallel model (Figure S7). In both cases, the Rct value decreases at larger overpotentials (Table S1), as expected for an enhanced HER rate with increased catalytic driving force, and 1 exhibits lower Rct values in comparison to 3. For instance, at an overpotential of 0.44 V, the Rct of 1 is only 19.7 W, whereas the Rct of 3 is 951 W at an overpotential of 0.55 V. The lower Rct and Tafel slope of 1 suggest that the HER kinetics are much more favored on the surface of 1 than 3. Double layer capacitance (Cdl) measurements were performed to obtain the electrochemical active surface area (ECSA) of the catalyst-modified electrodes. As shown in Figure S10, 1 displays a much higher Cdl (125.6 µF) than 3 (25.0 µF), indicating a larger ECSA. This is consistent with the fact that the surface ligand density of 3 is higher than 1, as mentioned previously. To achieve a more equitable comparison between the two electrocatalysts, we normalized the catalytic current by the ECSA, instead of the geometric surface area of the electrode (0.196 cm 2 ). As shown in Figure 4b, upon normalization by ECSA, the superior activity of 1 is maintained -there is still a 0.33 V overpotential difference at 3 mA/cm 2 ECSA that is not accounted for by the ECSA alone. Therefore, 1 displays higher HER activity than 3 not only because of its larger number of active sites, but also due to the higher intrinsic activity of individual sites. The superior HER activity of 1 suggests that the carbene ligands afford several benefits over oleylamine ligands for catalysis. First, the lower surface ligand coverage of 1 not only helps to expose more catalytically active sites, but also make the interfacial electron transfer between the electrode and the electrolyte more efficient. This is evidenced by a lower solution resistance (Rs) of 1 as compared to 3 (Tables S1, S2), which is affected by the electrical resistance of the catalyst layer. Second, it is also possible that the NHC ligands have a beneficial electronic effect at the nanocrystal 81.3 ± 0.5 72.1 ± 0.1 a ECSA = Cdl/Cs, Cs = 0.035 mF/cm 2 . 46,47 surface that helps to enhance the catalytic activity. Cao and coworkers previously reported on the enhanced electrocatalytic CO2 reduction activity of a NHC-functionalized Au nanoparticle catalyst. 17 Based on the different Tafel slopes of the carbenefunctionalized Au nanoparticles (72 mV/dec) and the parent Au nanoparticles (138 mV/dec), they concluded that the strong σdonation electronic effect from the carbenes alters the electron density at the gold surface, which induces a shift in the ratedetermining step for CO2 reduction. We observe a similar phenomenon here, where 1 exhibits a lower Tafel slope in comparison to 3 (85 mV/dec vs. 228 mV/dec, Figure S11). From this, we can deduce that for 1, the rate-determining step is likely the Heyrovsky or Tafel step, whereas for 3, the rate-determining step is likely the Volmer step. 48 However, it is noteworthy that the as-measured Tafel slopes might contain contributions from other sources that are not related to HER kinetics, such as the uncompensated resistance originating from the charge transfer between the electrode substrate and the catalyst. 49 Even so, such uncompensated contributions likely would not account for the large difference in the measured Tafel slopes of 1 and 3, maintaining that the rate-determining step of HER is different for these two catalysts. Controlled potential electrolysis (CPE) experiments were performed to investigate the long-term stabilities of the materials as well as their Faradaic efficiency (FE) for hydrogen production. For 1, the current response remains relatively steady over the course of 1 h under an applied potential of -0.59 V vs. RHE (Figures S12a), with slight fluctuations caused by the evolution of bubbles on the electrode surface. However, when comparing the Cdl and EIS responses before and after CPE, it is found that the ECSA of the catalyst was reduced from 46.4 cm 2 to 6.8 cm 2 which was accompanied by a decrease in Rct from 14.3 W to 7.5 W (Figure S12, Table S3). The FE for hydrogen production is 81.3 ± 0.5% for 1, calculated by comparing the total charge passed and the amount of H2 produced over the period of 1 h. The change in ECSA and Rct during electrolysis and the less-than-unity FE is potentially a consequence of partial surface ligand desorption, a phenomenon that has been observed for other electrocatalytic nanomaterials. 50,51 Indeed, the TEM image of the post-electrolysis 1 reveals sintering of the nanoparticles (Figure S14a). On the other hand, the FE of 3 is only 72.1 ± 0.1%. The Rct value is massively reduced after electrolysis (from 241 W to 11.3 W), along with a minimal decrease in the ECSA (from 1.9 cm 2 to 1.6 cm 2 ). Similarly, these behaviors are attributed to the loss of surface ligands, but to a larger extent in comparison to 1. Since the oleylamine ligands have a higher surface ligand density and do not bind as tightly as the carbene ligands, a greater loss of the oleylamine ligands is expected, which leads to a lower FE. TEM images of the ligandexchanged nanocrystals (3) following CPE also reveals sintering (Figure S14b). While recent studies of metal phosphide nanocrystal HER catalysts lack reports of Faradaic efficiencies, 24,52 we believe another potential source of Faradaic losses for these catalysts may originate from the reduction of surface oxidized species that form upon exposure to air. Indeed, XPS of 1 indicates the presence of POx species on the nanocrystal surface prior to catalysis (Figure 2b). Density Functional Theory Calculations. Density functional theory (DFT) calculations were performed to probe the electronic effects of NHC and alkylamine ligands on proton binding to a Cu3P catalyst. A model Cu9P3 cluster with one surface ligand (either 1,3-(dimethyl)benzimidazol-2-ylidene or methylamine, see Figure S15) bound to a copper atom was used to represent catalysts 1 and 3, respectively. The energies of proton binding to an adjacent surface site on these clusters were then calculated and the physical origins of catalyst-proton interactions were examined using the second generation absolutely localized molecular orbital energy decomposition analysis (ALMO-EDA, referred to as EDA in this text). Binding energies reveal that the proton prefers to bind at Cu-Cu bridge sites rather than P centers. When comparing the proton binding energies for NHC-and amine-ligated clusters, it was found that proton binding to the NHC-ligated cluster was favored by 29.7 kJ/mol (7.1 kcal/mol). EDA showed that cluster-proton interactions are dominated by charge transfer and polarization terms, with smaller, repulsive contributions from electrostatics (see Table S4). While charge transfer and polarization contributions are largely similar, electrostatic contributions are smaller for the NHC-ligated cluster than the amineligated cluster. This phenomenon likely arises as a consequence of stronger σ-donation from the NHC ligand to the Cu + center, which results in diminished electrostatic repulsion between Cu + and the proton. The calculations suggest that the weaker electrostatic repulsions lead to more favorable proton binding for NHC-bound clusters. During catalysis, more favorable proton binding should speed up the rate of the Volmer step for NHC-ligated nanocrystals. This result aligns well with our experimental Tafel analysis wherein 1 exhibits a low Tafel slope, indicating that the Volmer step is fast and is not likely the rate-determining step. In contrast, 3 displays a higher Tafel slope characteristic of a slow, rate-determining Volmer step. Therefore, the nature of the L-type ligand on the surface of our Cu3-xP nanocrystal catalysts could indeed be contributing an electronic influence on catalytic activity and the rate-determining step. ## CONCLUSIONS In conclusion, we report a new synthesis that directly produces crystalline, NHC-stabilized Cu3-xP nanocrystals. This represents the first example of a carbene-stabilized metal phosphide nanocrystal. The NHC-stabilized Cu3-xP nanocrystals exhibit better HER activity compared to their oleylamine-capped counterparts with respect to the onset of catalysis, Tafel slope, and overpotential to reach 10 mA/cm 2 current density. DFT calculations suggest that the NHC ligands change the electronics of the copper phosphide catalyst surface by reducing unfavorable electrostatics, which may contribute to the superior catalytic for 10 min and centrifuged for 5 min at 6000 rpm. An additional partial wash as described above was performed before dispersing the particles in TCE for UV-vis-NIR or in hexanes to be dropcast for powder XRD. Oleylamine-stabilized Cu3-xP nanocrystals via ligand exchange. NHC-capped Cu3-xP nanocrystals were synthesized as described in the main text and were then washed twice in 40 mL centrifuge tubes with acetone by sonication and centrifugation. Then, the product was redispersed in 5 mL of dry toluene. This suspension was added to 20 mL of oleylamine in a 100 mL three neck round bottom flask. The solution was then heated to 85 °C and was stirred for 2 h under a N2 atmosphere. The product was then washed in the same manner described in the main text prior to electrochemical testing and characterization.

chemsum

{"title": "Synthesis and Electrocatalytic HER Studies of Carbene-Ligated Cu3-xP Nanocrystals", "journal": "ChemRxiv"}

in-depth_lipidome_annotation_through_an_operatively_simple_method_combining_cross-metathesis_reactio

## Abstract: The in-depth knowledge of lipid biological functions calls for a comprehensive lipid structure annotation that implies implementing a method to locate fatty acids unsaturations, which remains a thorny problem. To address this challenge we have associated Grubbs' cross-metathesis reaction and liquid chromatography hyphenated to tandem mass spectrometry. Thus the pretreatment of lipids-containing samples by Grubbs' catalyst and an appropriate alkene generates substituted lipids through cross-metathesis reaction under mild, chemoselective, and highly reproducible conditions. A systematic LC-MS/MS analysis of the reaction mixture allows locating unambiguously the double bonds in fatty acid side chains. This method has been successfully applied at a nanomole scale to commercial standard mixtures consisting of 10 lipid subclasses as well as in lipid extracts from an in vitro model of corneal toxicity. Lipids are an essential class of metabolites playing a pleiotropic role in cells depending on their structures, the latter being actually very variable. According to the international LipidMaps consortium, molecules arising from isoprene subunits condensation define the sterols or prenol lipid class, while lipids resulting from ketoacyl subunits condensation belong to the classes of fatty acids (FA), glycerolipids (GL), glycerophospholipids (PL) and sphingolipids (SL). Within these latter classes, the structural diversity mainly originates from the nature of the fatty acids i.e. the chain length and the number and location of unsaturations. The location of double bonds in the FA chain is of prime relevance as in most cases it determines the biological activity of a given lipid. For example, -3 polyunsaturated FAs (PUFA) display antiinflammatory properties, while in contrast, -6 ones mainly act as pro-inflammatory metabolites. In ocular surface diseases, -3 PUFA has been shown to increase corneal wound healing and nerve regeneration through docosanoids biosynthesis. Indeed, the use of -3-based therapy has been purposed specifically in dry eye disease, both topically and per os. Regarding mono-unsaturated fatty acid (MUFA) species, a shift from Δ9 to Δ11 C=C isomers in phosphatidylcholines (PC) and phosphatidylethanolamines (PE) containing oleic acid (18:1), has been reported in the plasma of type 2 diabetic patients. Consequently, to clarify the role of lipids in physiological processes, it is indispensable to determine unambiguously the location of the double bond in FA chains of lipids. For two decades, mass spectrometry (MS) has been increasingly used to study lipids. Lipidomic analysis mainly uses liquid chromatography hyphenated to electrospray high resolution tandem mass spectrometry (LC-MS/MS). Indeed, LC-MS/MS analysis is highly suitable to identify polar head groups, and therefore lipid subclasses but also FA chains. However, LC-MS/MS alone can't determine position isomers when unsaturated FAs are involved, unless using sophisticated analytical approaches implementing ion activation technics, such as ultraviolet-photodissociation (UVPD) or ozone-induced dissociation (OzID). An online derivatization based on Paterno-Buchï (PB) photochemical reaction prior to MS analysis has also been successfully applied. Nevertheless, these online sample preparation methods need technical adaptations of the mass spectrometer to be routinely used: UVPD, an hybrid MS 3 CID/UV mass spectrometer, OzID, the introduction of ozone within the collision cell and PB, the use of an UV lamp inside the ion source. Regarding offline sample preparation, a derivatization step based on an epoxidation reaction has been proposed to identify double bond lipid isomers. Albeit very suitable for mono-unsaturated fatty acid (MUFA) species, this approach did not allow identifying double bond isomers in PUFA species readily. Furthermore, the above-mentioned methods implement harsh conditions that impede their chemoselectivity. In contrary, cross-metathesis (CM) is a highly chemoselective, functional group tolerant reaction using mild conditions, so it appeared to us particularly suitable for lipidomic analysis. Indeed, biologically occurring MUFA and PUFA species contain one or several but-2-en-1,4-ylidene moieties, these alkenes therefore belong with category II in the Grubbs' CM classification. They are thus expected to react readily with Ru carbene complexes such as the commercially available secondgeneration Grubbs' catalyst giving CM product easily. It can be anticipated that from the MS analysis of CM products the location of lipid double bonds would be easily and unequivocally determine (Scheme 1). In a previous study, double bond location was determined in FA species using CM and mass spectrometry. However, the method described in this study was designed for a use on pure FA samples at the mg scale, so it is sparsely adaptable to biological samples. The aim of our study was thus to explore the associating of CM and MS in order to devise a reproducible and routinely usable protocol allowing a universal identification of lipid isomers in biological samples, taking here, as example, a toxicological model of corneal damage. Scheme 1. Cross-metathesis reaction applied to PC (18:0/18:1 9). We have first hypothesized that lipids could react with the Grubbs catalyst of second-generation under very mild conditions using (Z)-but-2-ene-1,4-diyl diacetate as CM partner. Indeed, this symmetrical alkene is highly reactive and allows preventing formation of ethylene and ethylene CM adducts. A first assay was performed at the mg scale with a commercial phospholipid. Briefly, 6.3 µmol (5.0 mg) of PC (18:0/18:1 9) diluted in CH2Cl2 (0.5 ml) were treated at 40 °C for 2 hours with 5 equivalents of (Z)-but-2-ene-1,4-diyl diacetate in the presence of 5 mol% of Grubbs' catalyst. The reaction mixture was subsequently diluted (1/10,000) with MeOH and analyzed through direct infusion on a Synapt G2 (Q-ToF) high resolution mass spectrometer. The resulting mass spectrum displayed an intense peak at m/z 770.4953 corresponding to the [M+Na] + adduct PC (18:0/AcO-11:1 9), the distal chain transformed into undec-2-en-1-yl acetate giving no signal (SI Figure S1). Therefore, the C10-C18 moiety of the unsaturated FA chain has been replaced through CM by a 2-acetoxyethyl-1-ylidene moiety. Given that in a typical biological sample the overall content in phospholipids is of about tens of nmol for 10 6 cells, a scale down of our method was needed. However, at such a scale the concentration usually used in a CM reaction would require an amount of CH2Cl2 so small that it would anyway evaporate immediately at 40 °C. This led us implementing neat reaction conditions using circa one equivalent of the catalyst compared to the lipids and 50 equivalents of the CM partner. Using 2 nmol of the previous PC (18:0/18:1 9), CM was performed as described here after. Briefly, Grubbs' catalyst (0.1 M) was pretreated with 50 equivalents of (Z)-but-2-ene-1,4-diyl diacetate in CH2Cl2 at 40 °C for 2 hours to afford a Ru carbene bearing the CM partner. This solution was diluted, then an appropriate volume was added to 2 nmol of PC (18:0/18:1 9) and the solvent was subsequently removed under a flux of argon at 40 °C (SI Figure S2). The reaction mixture was then heated in a sealed tube at 40 °C for 2 hours, diluted in 100 µl of a CHCl3/MeCN/H2O/i-PrOH (20/30/10/30, v/v) mixture and 5 µL were injected in the LC-MS/MS system, the analysis being performed using previously described conditions. In positive ion mode, the MS extracted ion chromatograms (EIC) at m/z 748.5123 and m/z 788.618 showed a first peak at tR = 4.1 min and a second one at tR = 7.1 min corresponding to the [M+H] + of PC (18:0/OAc-11:1 9) and native PC (18:0/18:1 9), respectively (Figure 1A). The MS/MS spectra displayed an intense peak at m/z 184.07 characteristic of the PC polar head group (Figure 1B and 1C). In negative ion mode, the EIC at m/z 732.479 showed an intense peak assigned to the [M-CH3] ˗ PC (18:0/OAc-11:1 9) confirming information obtained under positive ion conditions. In addition, negative ion mode is required to identify the FA side chains. Indeed, the MS/MS spectrum displayed at m/z 283.26 a peak diagnostic of stearate in sn1 position and at m/z 241.1453, a second peak corresponding to 11-acetoxyundec-9-enoate (FA (OAc-11:1 9)) in sn2 position (Figure 1E). These product ions are indicative of an oleate in sn2 position and therefore allowed to unambiguously locate the double bond. On one hand, it has been long established that the Grubb's catalysts are poisoned by basic amines. On the other hand, among the phospholipid subclasses commonly encountered in biological samples, phosphatidylethanolamine (PE) and phosphatidylserine (PS), display basic primary amines functions. Accordingly, when a mixture of 20 commercially available standard phospholipids (SI Table S1) including PE and PS subclasses is submitted to our CM conditions, it gave a very poor yield of the expected CM products. Indeed, CM afford PC (18:0/OAc-11:1 9) with approximatively 20% yield when it is performed on a pure PC (18:0/18:1 9) solution and dramatically decreases to 5% when this PL is included in the standard lipid mixture (Figure 2B deleterious effect of basic amines, the lipid mixture was pretreated with 1M HCl solution to give innocuous ammoniums. CM performed under these conditions allowed to recover yields closed to those obtained with pure PC (18:0/18:1 9) whatever the PL considered (Figure 2C). Thus, HCl pretreatment proves essential for CM reactions performed on lipid of biological lipid mixtures. Our mixture of commercial lipid standards contains mono-and poly-unsaturated FA side chains representative of FAs commonly encountered in biological samples. While phospholipids such as PC (18:0/18:1 9) lead to a single CM product, all C=C bonds present in phospholipids containing PUFA chain are expected to react leading to several CM products. Indeed, when CM was performed on PC (18:0/20:4 5, 8, 11, 14) under the previous conditions, it led to 4 CM products: PC (18:0/OAc-16:4 5, 8, 11, 14) (C4), PC (18:0/OAc-13:3 5, 8, 11) (C3), PC (18:0/OAc-11:2 5, 8) (C2) and PC (18:0/OAc-9:1 5) (C1) (Figure 3A & 3B -SI Figure S3). Detecting simultaneously all possible CM products in the final reaction mixture is suitable as it allows locating all the double bonds and hence identifying phospholipids containing PUFA unambiguously. To ensure that the final reaction mixture contains all the CM products at concentrations letting optimal detection by mass spectrometry, a kinetic study was carried out by varying the temperatures with PC (18:0/20:4) and PC (16:0/20:4) (Figure 3D -3G). Whatever the reaction time, chromatographic peaks corresponding to C1 and C4 products exhibited the highest and the lowest AUC compared to C2 and C3 (Figure 3D & 3E). As expected, in the course of the reaction, C2, C3 and C4 decreased in favor of C1. Moreover, at 4 hours of reaction at 40°C C2, C3 and C4 displayed suitable AUC and thus represents the best compromise to identify all the C=C bonds position (Figure 3D and 3E). In contrast, CM carried out at 60°C mainly led to C1 products with a 4 times increased yield compared to 40°C, whatever the PC species (Figure 3F and 3G) while C2, C3 and C4 were sparsely detected in that case. min by calculating the ratio between peaks corresponding to the two fatty acid chains (SI Figure S4). On MS/MS spectra, an intense peak at m/z 184.07 corresponds to the PC polar head group (SI Figure S5). In negative ion mode, MS/MS spectrum exhibited peaks at m/z 337.31 and at m/z 297.21 due to respectively docos-13-enoate and 15-acetoxypentadec-13enoate. While m/z 337.31 is due to native PC (22:1/22:1 13/13), and m/z 297.21 associated to CM modified PC which allows pinpointing the C=C bond (Figure 4E -4G). Ring closure metathesis was also possible in that case, and interestingly PC Within PC subclass, CM has thus been successfully applied to identify isomers of lipid species commonly encountered in biological samples i.e., PL containing SFA/MUFA, SFA/PUFA and MUFA/MUFA side chains. The kinetic profiles we previously described for PC at 40 and 60°C were reproducibly observed for all lipid species whatever the phospholipid subclasses considered, i.e. PE, PS, PG and PI as well as with several sphingolipids and glycerolipids including Cer, SM, and DG, TG, respectively (Table S1 Figure S6-S25). Regarding the sensitivity of the method, CM was successfully applied on 10 pmol individual amount for species of the lipid standard mixture. We previously reported that benzalkonium chloride (BAK), a quaternary ammonium, have a major impact on the lipidome of human corneal epithelial (HCE) cell line, however we could not determine double bound position of lipids at that time. So this complex example appeared particularly relevant to assess the efficiency of our method. BAK is the more often encountered preservative in multidose eye drops which have long term indication for glaucoma and dry eye, two diseases deeply impacting quality of life. HCE cells exposed to BAK is also a well-established toxicological model of corneal damage, for which we previously demonstrated a striking decrease in PC species whatever the FA side chainsconsidered. So we were delighted to see that our method succeeded at fully determining the structure of the 5 following PC species revealing a homeostasis change following BAK exposure. Indeed, in PC (18:1/18:1), FA side chains were identified as an oleyl (18:1 9) and an octadec-11-enoyl (18:1 11) (SI Figure S26). In PC (16:1/18:1) FA chains were identified as palmitoleyl (16:1 9) and oleyl (18:1 9) in sn1 and sn2, respectively. PC (16:0/18:1) and PC (16:0/16:1) contain oleyl (18:1 9) and palmitoleyl (16:1 9) in sn2 position respectively. Finally, PC (18:0/20:3) contained an eicosatrienoyl (20:3 5 8 11) in sn2 position. Therefore, regarding PC subclass, our method has revealed BAK-induced changes mainly on lipid species containing -9 and -7 FA (Figure 5). It may thus be suggested that BAK induces ocular injury by targeting -9 and -7 FA. Indeed, stearoyl CoA desaturase (SCD), which is involved in the -9 FA metabolism, has been previously described as impacted by BAK exposure. To confirm these results, the investigation of enzymes involved in FA elongation and desaturation metabolism remains to be carried out. In summary, we have demonstrated that associating Grubbs catalyzed CM reaction and LC-MS/MS analysis allows an accurate identification of a broad scope of lipid isomers through an operatively simple and reproducible procedure. We have optimized conditions to obtain all the possible CM products enabling a full identification of MUFA and PUFA with a level of sensitivity allowing the analysis of biological samples. We are currently pursuing this study testing CM partners that could transfer an ionizable functional group allowing the identification of the distal moiety of FA chains. Furthermore, any sophisticated adaptation of the LC-MS system previously developed is needed to analyze complex biological lipid samples with our method. Regarding MS acquisition, both low-and high-resolution mass spectrometry are suitable and CM products exhibit same fragmentation patterns in MS/MS as native lipids facilitating identification. A remaining key challenge is the postacquisition of the big amount of data in the case of biological sample. The data processing strategy is currently in progress and will be the subject of a dedicated study.

chemsum

{"title": "In-Depth Lipidome Annotation Through an Operatively Simple Method Combining Cross-Metathesis Reaction and Tandem Mass Spectrometry", "journal": "ChemRxiv"}

shedding_new_light_on_an_old_molecule:_quinophthalone_displays_uncommon_n-to-o_excited_state_intramo

## Abstract: Excited state dynamics of common yellow dye quinophthalone (QPH) was probed by femtosecond transient absorption spectroscopy. Multi-exponential decay of the excited state and significant change of rate constants upon deuterium substitution indicate that uncommon nitrogen-to-oxygen excited state intramolecular proton transfer (ESIPT) occurs. By performing density functional theory (DFT) and time-dependent density functional theory (TDDFT) calculations, we found that adiabatic surface crossing between the S 1 and S 2 states takes place in the photoreaction. Unlike most cases of ESIPT, QPH does not exhibit tautomer emission, possibly due to internal conversion or back-proton transfer. The ESIPT of QPH presents a highly interesting case also because the moieties participating in ESIPT, quinoline and aromatic carbonyl, are both traditionally considered as photobases.ESIPT, where migration of a proton takes place within a molecule after photoexcitation, is a fundamental photophysical process that garnered decades of interest in varying fields of chemistry. Widespread attention to the phenomenon stems from the fact that molecules that undergo ESIPT may exhibit emission with a large Stokes' shift, which minimizes self-absorption and opens up possibilities of diverse applications 1 . Some examples utilizing this property include molecular sensors 2 , optical memory 3 , and white light sources 4 .Most research on ESIPT utilizes tautomer fluorescence for probing dynamics [5][6][7] and therefore lack of such emission, as is the case with our subject molecule QPH, poses challenge to investigation of ESIPT. Another problem is that it is difficult to recognize ESIPT at first sight if the tautomer is non-emissive. Possibility of fully non-radiative ESIPT has only recently been recognized by Yin et al. 8 and we suspect that this is the reason why ESIPT dynamics of QPH went unnoticed until now.QPH, or more commonly known as quinoline yellow, is a commercial dye with distinctive greenish yellow color. It has three possible tautomers (Fig. 1), of which the enaminone (E) form is confirmed to be the most stable one by NMR 9, 10 and calculation 11 . Unlike the ketoenol (K) and zwitterion (Z) forms, the bond connecting phthalone and quinoline rings is a double bond, making the molecule nearly coplanar 12 . An intramolecular hydrogen bond (IMHB) exists between hydrogen attached to the nitrogen of quinoline and the carbonyl group of the phthalone moiety. Previous researchers acknowledged possible contribution of the Z form in the ground state, whereas the K form does not appear in NMR and is believed to be too energetically unstable (~30 kJ/mol) to exist in the ground state.QPH has a long history of usage since it was discovered in 1882 13 . Because of its many desirable traits such as high solubility, significant resistance against photodegradation, and easy synthesis, QPH and its derivatives are used as coloring agents for various materials including polyester fibers, wools, paper, wax, paraffin, paints, and silk . Certification of QPH and its sulfonated derivative by the European Union and FDA allowed more diverse applications. Until now the majority of research on QPH has been limited to identifying potential cellular toxicity and developing assays for analysis. Especially the matter of its biocompatibility has long been a subject of debate . However, to the best of our knowledge, there exists only one photodynamics study of the molecule in pH 12 water conducted in the nanosecond scale 15 . Results in organic solvent and neutral pH water were not reported because the authors found no interesting signal. This most likely indicates that de-excitation dynamics of QPH is completed within sub-nanosecond scale. We hypothesized that QPH's notable stability against light may originate from its ultrafast relaxation to the ground state after photoexcitation and attempted to carry out femtosecond pump-probe spectroscopy in order to unravel the long-neglected picture of QPH photochemistry. In doing so we have unexpectedly discovered signs of intramolecular proton transfer occurring in the excited state. In the following sections, we report evidence of QPH displaying an unprecedented case of ESIPT between two groups known to be photobases (quinoline and aromatic carbonyl) and provide theoretical basis for its occurrence in QPH. ## Results Absorption and emission spectra. QPH is known for negative solvatochromism, i.e., absorption peaks shifting to shorter wavelengths in polar solvents 14,20 . It is a common behavior for many polar organic molecules that have environment-sensitive energy levels. Strong absorption below 460 nm gives QPH its characteristic yellow hue. Although emission in polar solvent is virtually nonexistent, we could observe weak photoluminescence in highly nonpolar solvents such as cyclohexane (Fig. 2). Even then, the emission signal is very low, reaffirming that significant non-radiative process is involved in relaxation dynamics of the molecule. A small Stokes' shift and near-mirror image symmetry of the absorption vs. emission peaks indicate that the emission is most likely fluorescence from the lowest singlet excited state 21 . ## Transient absorption spectroscopy. To probe the ultrafast relaxation process of QPH, we conducted femtosecond transient absorption measurement. Figure 3 shows transient absorption spectrum of QPH in cyclohexane excited with 400 nm pump and probed with supercontinuum light in the range of 480 to 660 nm. It is apparent from the picture that the decay kinetics cannot be represented by a single exponential curve, indicating the possibility of relaxation involving more than one excited state. We also take note of the fact that the spectra in the shorter timescales (Fig. 3c) show a "dynamic isosbestic point" near 590 nm, where absorbance remains the same regardless of the temporal evolution. It is generally regarded as a sign that a transient species is evolving into another over the course of time, rather than there being two unrelated excited species 22 . After rapid initial decay, the time profile shows a complex trace for tens of ps, after which the decay becomes nearly single exponential (Fig. S1). Almost no signal is found after 300 ps, indicating that most species has returned to the ground state. Lifetimes that best explain temporal behavior at four different wavelengths of 500, 530, 600, and 620 nm are acquired by global nonlinear fit (Fig. S1) and presented in Table 1. As the current decay profiles are not compatible with simple exponential fit, we truncated their middle part and divided them into a single exponential head (<2 ps) and a biexponential tail (>30 ps) for separate analysis. The initially populated state shows a strong negative signal below 580 nm, which may be interpreted as either disappearance of the ground state species or increased photon output due to stimulated emission 23 . Because the absorption spectrum of ground state QPH ends around 450 nm and the emission spectrum has peaks at similar wavelengths, it is most likely to be stimulated emission signal. Therefore the initially decaying species is presumably the lowest singlet excited state, which is an observable emissive state. From the rapid decay, we know that the emissive state lasts very short, which may explain the low quantum yield of the molecule. From the aforementioned isosbestic point, we assume that it evolves into a different excited state. The excited state absorption signal peaked at 610 nm lasts longer (until 300 ps), suggesting that at least two or more states are involved in the process. ## Deuterium isotope effect. To identify the evolutionary course of the excited state, we carried out comparative experiments using deuterium substituted QPH and unsubstituted QPH. As the hydrogen participating in IMHB (H 1 ) is known to be acidic 14 , we substituted it with deuterium (d-QPH) by following the protocols in the Methods section and acquired time profile at several wavelengths. The same procedure was carried out using water (h-QPH) and compared against QPH solution in cyclohexane to ensure that it was not the effect of hydrate formation. Overall spectral features of d-QPH remained similar to those of h-QPH, including stimulated emission and the isosbestic point, with their only difference being the decay rate. Comparison of the time profiles between d-QPH and h-QPH readily shows significantly slower kinetics of the former (Fig. 4). Lifetimes in Table 1 indicate that a primary kinetic isotope effect may be present in both τ 1 and τ 3 . Table 1. Lifetimes (in ps) of h-QPH and d-QPH and their ratios. τ 1 was determined from single exponential fitting of the sub-2 ps profile, while τ 2 and τ 3 were determined from biexponential fitting of over-30 ps profile. (*Standard error in parenthesis). Since isotopic mass change does not affect the force field of the molecule, we expect that only the nuclear motions involving the substituted atom will change 23 . In fact, deuterium substitution has been extensively used to verify ESIPT 6 . As the mass of H 1 hydrogen comprises a very small fraction of the mass of QPH, it is unlikely that single deuterium substitution can significantly affect any motion other than the N-H vibration. Since the vibrational frequency of an oscillator is inversely proportional to the square root of the reduced mass of the oscillator if the force constant remains unchanged, we expect the ratio τ D /τ H ( = k H /k D ) of Table 1 should be nearly equal to the square root of the ratio of the reduced masses between N-D and N-H, or {[(14 × 2)/(14 + 2)]/[(14 × 1)/ (14 + 1)]} 1/2 = (15/8) 1/2 = 1.37, which is actually in very good accord with our measured values for τ 1 and τ 3 , indicating that it is indeed the motion of hydrogen such as occurring in ESIPT that governs the relaxation of the excited state. Quantum calculation using DFT/TDDFT. Since ESIPT in systems like QPH is unexpected and rare as previously mentioned, we investigated the dynamics of ESIPT by DFT/TDDFT calculations. In order to enlist different QPH species involved in the reaction, we first define the optimized geometry of the singlet ground state (S 0 ) of the E tautomer as P E (Fig. 5(a)). This is the geometry where the HOMO, LUMO and LUMO+1 shown in the leftmost side of Fig. 6 constitutes a major MO for the S 0 , S 1 and S 2 states, respectively. Optimizing the first excited state at the Franck-Condon point P E yields P E-S1 (Figs 5(b) and 6), the state property of which differs markedly from that of the experimentally expected K tautomer, P K (Fig. 5(d)). This led us to suspect a missing link and search for other, more relevant local minima on the S 1 potential energy surface along the reaction coordinate. We eventually found another local minimum located between P E-S1 and P K , which is denoted P E-S2 (Fig. 5(c)) as it is associated with the S 2 state (see below). Each geometry possesses distinctive structural and electronic properties as described below. • The LUMO at P E-S1 is very similar to that at P E , indicating that the former is a relaxed conformation of S 1 after vertical excitation, hence the designation P E-S1 . The bond lengths of N 1 −H 1 and O • P K is the proton-transferred geometry (K form). Striking similarities between its LUMO and the LUMO+1 at P E and the LUMO at P E-S2 are apparent in Fig. 6. ## P E-S2 is the key intermediate geometry between P E and P K , as its tautomeric structure is similar to the geometry at P E but its LUMO is similar to that at P K . Also, the LUMO at P E-S2 lies lower in energy than LUMO at P E-S1 but higher than that at P K , making it a more likely candidate for a geometry immediately preceding proton transfer. Assigning the S 1 state at P E-S2 as the reaction intermediate leads us to two assumptions about the potential energy surface. First of all, because the electron density of the LUMO shifts from the quinoline ring at P E and P E-S1 to the phthalone ring at P E-S2 and P K , we may suspect the presence of an adiabatic surface crossing between the S 1 and S 2 excited states, which is also consistent with the similarity between the LUMO+1 at P E and the LUMO at P E-S2 (see also Figure S2 to observe apparent 'flip' in molecular orbitals). From the state property of each species, we deduce that the lowest singlet excited states of P E and P E-S1 are on a same adiabatic surface while those of P E-S2 and P K are on another adiabatic surface. In order to see the possibility of the nonadiabatic transition, we searched for a conical intersection of the S 1 and S 2 surfaces and found one at P E-S1/S2(CI) (Fig. 5(e)). Although the calculated value did not take the solvent effect into account, the transition is still shown to be energetically feasible when considering the excess excitation energy. Also the geometry at P E-S1/S2(CI) is quite similar to those at P E-S1 and P E-S2 (Fig. 5), which implies no additional barriers to the conical intersection. Another assumption is that the transition state (TS) from E to K is expected via the P E-S2 geometry. By placing H 1 midway between N 1 and O 15 , we found what appears to be a transition state (P E/K(TS) in Fig. 5(e)) that has a low energy barrier of ~0.13 eV (Fig. 6). Intrinsic reaction coordinate (IRC) path has been determined starting from P E/K(TS) by the steepest descent method, which led to minima that correspond to P E-S2 and P K (Fig. S3). Therefore P E-S2 is deemed the doorway geometry for the proton transfer. From the results above we construct a rudimentary potential energy surface diagram shown in Fig. 6. QPH photoexcited at the Franck-Condon point (P E ) will vibrate about the local minimum P E-S1 on the S 1 adiabatic surface, during which a nonadiabatic transition to the S 2 state occurs at P E-S1/S2(CI) , which leads to the ESIPT process toward the point P K via P E-S2 and P E/K(TS). We also note that we could not find a conical intersection between S 1 and S 0 of the K tautomer. Natural bond orbital (NBO) charge analysis reveals that N 1 becomes electrostatically less negative (−0.520 to −0.488) and O 15 more negative (−0.617 to −0.656) in the S 1 state at P E-S2 relative to those of the S 1 state at P E (Fig. S4). Such a change in electrostatic charge of atoms involved in IMHB should be a preceding sign of ESIPT. ## Discussions Molecules known for ESIPT typically display either a large Stokes'-shifted emission or dual emission 7 . Despite the fact that both characteristics are absent from the spectrum in Fig. 1, our experiment on deuterium substitution of H 1 proton verified the occurrence of ESIPT in QPH. Therefore we may conclude that after ESIPT, the K form of QPH undergoes a significant nonradiative decay. There are some notable examples for ESIPT that do not display tautomer emission. One is hypericin, for which it was speculated that either energy levels remain unchanged upon tautomerization or that tautomers are mixed in the ground state 24 . Another is 1-hydroxypyrene-2-carbaldehyde, for which intersystem crossing to a triplet state happens 8 . However, from aforementioned calculations and NMR results, we may rule out the possibility of the ground state tautomerization. For the latter possibility, we found the time profile of QPH insensitive to nitrogen purging (Fig. S5) and thus intersystem crossing to a triplet state seems unlikely. From our experimental results for deuterium substitution and the lack of conical intersection between S 1 and S 0 of the K tautomer, we suspect that the vibrational mode related to IMHB is involved when the K tautomer returns to the ground state. It has been discussed in depth that the presence of hydrogen bond is associated with enhanced internal conversion back to the ground state and that the isotope effect may appear if the associated hydrogen is deuterated . For ESIPT, some cases are known where enhanced internal conversion lowers photoluminescence quantum yield after tautomerization 6,29 . Another possibility is that ultrafast relaxation occurs via back proton transfer process 5 . Currently we cannot differentiate between the two possibilities and will leave it for future studies. It is also interesting to note that the biexponential global fit after 30 ps brings about a τ 2 component of ~15 ps that is seemingly unaffected by deuteration. This observation, along with the complex nature of kinetic traces before 30 ps, suggests that there might be another relaxation pathway other than ESIPT. Although IMHB is an important factor in facilitating excited state intramolecular hydrogen transfer, not every molecule with IMHB undergoes ESIPT. It has been well-established that ESIPT occurs readily in conjugated ketoenol system (O•••HO) or, similarly, N•••HN system and frequently reported, but ESIPT between heteroatoms are less known 30 . Especially, ESIPT from nitrogen to oxygen is a rare case, but not completely unheard of. Some molecules known for displaying N-to-O ESIPT include o-acetylaminoacetophenone 25 and 1-(acylamino)anthraquinone with appropriate functional groups 31 . More cases are known if dimerized molecules are considered 32 . However, what makes our case unique is that the proton donor of QPH is quinoline, which is better known for its photobasicity, meaning that it is more likely to accept a proton in the excited state 33 . The driving force for ESIPT has been traditionally explained by the change in electron density at atoms involved in ESIPT. Excited state tautomerization is usually accompanied by electron redistribution after vertical excitation 34 , and the resulting change in the relative acidity and basicity is explained by the change in electron density at 'heavy atoms' (nitrogen or oxygen) 35 . Calculations of electrostatic charge at atoms related to photoacidity have been presented as supporting evidence in several studies of established systems 33,36 . Results of our NBO calculation may be interpreted in similar way. In the transient P E-S2 state, the increased electron density renders carbonyl oxygen O 15 photobasic, and the decreased electron density makes N 1 photoacidic. Because acidity and basicity are only relative terms, we suggest that if the relative strengths differ, ESIPT between photobases may occur. Usually the magnitude of deuterium isotope effect is assessed by parameter k H /k D , whose value may lie anywhere between 1 and 50, depending on the shape of potential energy surface and the size of energy barrier 34,37 . Furthermore, the kinetic isotope effect may not appear at all when the system reaches the adiabatic limit. Generally, such lack of isotope effect is regarded as a sign of unsubstantial potential energy barrier, and happens in cases when the distance between heavy atoms involved in ESIPT is sufficiently short 5,29 . Since our system displays a clear isotope effect, the possibility of a barrierless potential energy surface may be excluded. Also the k H / k D value of 1.39 and 1.35 in our system is nearly identical to the square root of the reduced mass ratio between hydrogen and deuterium, suggesting the crucial role of H 1 vibration on the overall kinetics. To summarize, we conducted ultrafast transient absorption spectroscopy of QPH and observed signs of ESIPT. This study is the first report to observe the ESIPT of QPH that involves a very rare case of proton transfer from nitrogen to oxygen. Also, this study reports the first case of ESIPT between two traditionally known photobasic moieties, quinoline and aromatic carbonyl 35 . Calculated results show that an S 1 -S 2 adiabatic surface crossing occurs before undergoing ESIPT. ## Methods Quinophthalone (Quinoline Yellow 2SF, ≥97%, #01354) was purchased from Sigma-Aldrich and used without further purification. All solutions were prepared using ACS grade cyclohexane (Sigma-Aldrich, #179191) with 100 μM concentration. The d-QPH sample was prepared by sonicating the 1:2 volume mixture of QPH solution and D 2 O (Sigma-Aldrich, #151882) in the same vial. Then we waited for separation and took the upper layer of cyclohexane. We tested several experimental conditions and found that 15 minutes of sonication and 90 minutes of waiting for separation were enough to reproduce the results. The same procedure was adopted with HPLC grade water (JT Baker, #4218-03) to ensure that the change originates from the isotope effect. 1 H NMR spectrum of the solutions (Fig. S6) show that the majority of H 1 proton is deuterated by the protocol. Steady-state UV/Vis absorption spectra and photoluminescence spectra were recorded using Lambda 25 (Perkin-Elmer) and QM-3/2004SE (PTI), respectively. Femtosecond transient absorption spectroscopy was conducted using conventional pump-probe setup. The optical source was a regeneratively-amplified Ti:Sapphire laser (Spectra-Physics) with an average power of 0.65 mJ/pulse, pulse width of 130 fs FWHM, and repetition rate of 1 kHz at 800 nm output wavelength. Pump pulses were generated using a second harmonic generator (TP1A, Spectra-Physics), filtered down by neutral density filter and used at 1.5 mW. A continuum light, which was used as the probe pulse, was generated by focusing the 800 nm light using a UV-fused silica plano convex lens (CVI Melles Griot, f = 100 mm) onto a sapphire plate (WG31050, Thorlabs). Diameters of the pump and probe beams at the sample position were 390 μm and 295 μm, respectively. The IRF (~250 fs FWHM) was estimated by the cross-correlation between the pump with the probe. Solvent background subtraction and GVD-correction were done according to the known methods 38 . The time delay between the pump and the probe pulses was scanned using Daedal 404300XRMP delay stage (Parker). The pump pulses were modulated using an optical chopper (MC1000, Thorlabs) synchronized to the laser. The probe pulses of the signal were detected by photodiodes (2031, New Focus) after wavelength selection using a monochromator (250 IS/SM, Chromex, 600 grooves/mm grating blazed at 750 nm). Transient absorption signals were obtained using a lock-in amplifier (SR830, Stanford Research Systems). Temperature was controlled at 23 °C. Calculations were executed using the DFT and TDDFT methods at B3LYP/6-31 G(d) level of theory. Structure optimization was performed using quadratic approximation with gradient convergence tolerance of 0.0001 Hartree/Bohr (default value) using General Atomic and Molecular Electronic Structure System (GAMESS) 39,40 and Gaussian 09 41 . Transition state and conical intersection were acquired using the default method in GAMESS. It must be noted that the branching plane update method for CI search requires TAMMD approximation and therefore was applied only on this value. It is known that it gives a better intermediate geometry during photophysical processes 42 . NBO charge analysis was performed with the NBO version 3.1 included in Gaussian 09 43 . Molecular structures and orbitals were visualized with GaussView 5.0.8 44 and wxMacMolPlt 45 .

chemsum

{"title": "Shedding new light on an old molecule: quinophthalone displays uncommon N-to-O excited state intramolecular proton transfer (ESIPT) between photobases", "journal": "Scientific Reports - Nature"}

optical_characterization_of_non-thermal_plasma_jet_energy_carriers_for_effective_catalytic_processin

## Abstract: An argon plasma jet was sustained in open air and characterized for its chemical composition. The optically characterized plasma jet was used to treat industrial wastewater containing mixed textile dyes and heavy metals. Since plasma jet produces UV-radiations, the photocatalytic TiO 2 was used to enhance plasma treatment efficiency especially for degradation of dyes. Mixed anatase and rutile phases of TiO 2 (5.2-8.5 nm) were produced through surfactant assisted sol-gel approach. The emission spectrum confirmed the presence of excited argon, OH, excited nitrogen, excited oxygen, ozone and nitric oxide in the plasma jet. The spectral lines of excited Ar, NO, O 3 , OH − , N 2 , N + 2 , O, O + 2 and O + species were observed at wavelength of 695 The contamination of water bodies is generally caused by the release of pollutants into groundwater or into streams, lakes, estuaries, rivers and oceans. The polluting substances degrade the water quality and natural functioning of ecosystems 1,2 . In developing countries, the coliforms, pesticides, toxic metals and industrial effluents are the major sources of surface and subsurface groundwater pollution. The disposal of industrial effluents and heavy metals in water bodies raises the human health concerns, poisons the wildlife, and damages the longterm ecosystem 3 . Toxins in industrial effluents promote the reproductive failure, immune suppression and acute poisoning 4 . The bacterial strains in water release toxins in digestive tracts, which cause nausea, watery diarrhea, vomiting, renal failure and dehydration. The harmful bacteria are removed from water through antimicrobial treatment. In developed countries, strict regulations are imposed on industrial and agricultural operations to minimize the contamination of water bodies. Different methods are also being deduced to prevent the flow of pollutants into the water bodies and to remove the pollutants from wastewater 5 . With conventional water treatment techniques, such as chlorination, coagulation, adsorption, ultra-sonication, etc., it is difficult to eliminate all the harmful contaminants from the water. Recently, some advanced oxidation techniques like irradiation of high energy electrons, oxidation using ozone, ionizing radiations exposure, carbon absorption, plasma exposure and sonolysis have been practiced for the treatment of contaminated waters 4 . The non-thermal plasma jet is one of the best oxidation methods available for the treatment of polluted water. A non-thermal plasma jet is an electrically energized gas, which is produced by passing a gas through a strong electric field 6 . Instead of just gas heating, the bulk of electric field energy goes into the creation of energetic plasma species. These species include positive ions, electrons, negative ions, free radicals, electrically neutral gas atoms and or molecules and electromagnetic radiations. Being strong oxidizers, the plasma species strongly interact with the contaminated media and decompose the organic and inorganic compounds in the media. These plasma species also kill the bacterial endospores and vegetative cells. The highly energized photons, ozone, atomic oxygen, and free reactive oxygen radicals in the plasma damage the cells by charging the cell wall and reacting with macromolecules. Since membrane lipids at the cell surface are susceptible to the reactive oxygen species, the oxidized cytoplasmic membrane lipids release intracellular substances, which damage the cells. Moisan et al. 7 proposed some mechanisms of inactivation of microbial spores under non-thermal plasma exposures. These mechanisms are based on interaction of ultraviolet radiations with spore surface and volatilization of surface compounds, damaging of DNA by ultraviolet irradiation, and erosion or etching of spore surface with reactive oxygen radials. The reactive oxygen radials and nitrogen oxides in plasma jet not only kill the living organisms but decompose the organic and inorganic compounds as well 4,8 . If a suitable photocatalyst is added to the contaminated water, the plasma treatment of polluted water may be more effective. Titanium dioxide (TiO 2 ) is a well-known photocatalyst. Under suitable light exposure, it converts the pesticides, polymers, surfactants, aliphatics, aromatics, herbicides and dyes into water, mineral acids and carbon monoxide 9 . TiO 2 is a polymorphous material, which exists in anatase, rutile and brookite phases. All these phases show octahedral structures but differ in the arrangement of their octahedral units 10,11 . Anatase phase is the most prominent commercial phase of TiO 2 due to its better stability and photocatalytic activity as compared to rutile and brookite phases 4 . TiO 2 nanoparticles are also known for good surface acidity, good thermal and chemical stabilities and low toxicity potential 12 . Karami et al. 13 and Wang et al. 14 revealed that photocatalytic and semiconducting activities of anatase phased TiO 2 mainly depends on crystal structure, crystallite size, shape, active surface area and overall morphology. The specific optical and structural properties of TiO 2 nanostructures can be tailored through a deliberately chosen and well optimized method of synthesis. In many cases, sol-gel method is preferred over other methods when it comes to low cost production of nanomaterials, ceramics and glass. In this study, a sol-gel method was adopted to produce mixed anatase and rutile phases of TiO 2 nanoparticles. The photoactive TiO 2 was used to degrade the organic compounds in contaminated water under direct current plasma exposure. ## Materials and methods Preparation of TiO 2 photocatalyst. TiO 2 catalyst was produced by practicing a simple sol-gel technique. Hydrochloric acid was used as a surfactant. In a typical procedure, 45 ml of solution-I was obtained by dissolving 15 ml of deionized water in 30 ml of iso-propanol and stirring continuously at 80 °C. Then, solution-II was obtained by dripping 30 ml of titanium tetra iso-propoxide (TTIP) in solution-I under continuous stirring at 80 °C for 1 h. The water-acid mixture (1.5 ml of HCl or HNO 3 diluted with 50 ml of deionized water) was added to solution-II under stirring. The temperature was reduced from 80 to 60 °C to obtain solution-III. This solution was stirred continuously at 60 °C to obtain white thick precipitated solution, which turned into a transparent white sol after 3 h. To complete the process of hydrolysis and condensation, the sol was stirred further for 150 min. The resultant gel was annealed for 2 h at 300 °C and grinded into fine powder of TiO 2 12 . The synthesized TiO 2 powder was characterized for its surface morphology, particle size, crystallographic phases and band gap energy. The surface morphology was analyzed from SEM images of the sample, particle size and crystallographic phases were analyzed from XRD spectrum and band gap energy was determined from UV-visible spectrum of the sample. After characterizing the photocatalyst, it was used in degrading the pollutants in water under atmospheric plasma exposure. ## Plasma treatment setup. A plasma jet was sustained with DC voltage by flowing argon gas through open air and characterized for its chemical composition by using an optical emission spectroscopy technique. Figure 1 shows a schematic of DC plasma jet and associated optical emission spectroscopy diagnostic. The plasma jet is Ethical approval. This article does not contain any studies with human participants or animals performed by any of the authors. ## Results and discussion Characteristics of TiO 2 catalyst. Figure 2 shows XRD patterns of TiO 2 nanoparticles. The catalyst nanoparticles were composed of mixed anatase and rutile phases. The planes (101), (004), (020) and (121) of anatase phase were identified at 2θ of 25.5°, 38°, 48°and 54°, respectively. Similarly, XRD peaks at 2θ of 27.5°, 36° and www.nature.com/scientificreports/ 56° correspond to (110), ( 101) and ( 220) planes of rutile phase of TiO 2 . The plane (111) at 2θ of 42° reveals both anatase and rutile phases 15,16 . XRD pattern confirmed the crystalline nature of the nanoparticles. The particle size of the synthesized catalyst was determined using the Scherrer's formula: where, S is the particle size, K is shape factor or Scherrer constant and is usually equals to 0.89 for spherical shape, λ is the wavelength of X-rays, β is known as full width at half maximum height and θ is known as Bragg's angle. The particle size of catalyst varied from 5.2 to 8.5 nm. The particle size of HNO 3 stabilized nanoparticles remained slightly smaller than HCl stabilized nanoparticles. The surfactants found to be ineffective on phase transformation of TiO 2 nanoparticles, which mainly depends on the heat treatment 12,17 . The morphology of TiO 2 nanoparticles was assessed through scanning electron microscopy. The agglomerated spherical nanoparticles were observed in SEM images. Figure 3 shows a typical SEM image of TiO 2 nanoparticles. In some cases, the formation of agglomerates expands the boundaries between the nanoparticles by changing their shape and size 18,19 . The band gap energy of TiO 2 catalyst was determined using Kubelka-Munk equation and UV-visible spectrum of the catalyst 20,21 . The Tauc-Plot of the nanoparticles is shown in Fig. 4. The band gap energy of the nanoparticles was measured about 3.06 eV. The band gap energy of nanoparticles depends on particles 22 . The particle size dependent band gap energy of the catalyst is summarized in Table 3. The band gap energy of the catalyst decreased with an increase in particle size. The change in band gap energy might also be due to phase transformations (i.e. amorphous-anatase-rutile) or induction of charge from bulk to nanocrystals' surface 23,24 . Optical emission spectroscopy of plasma jet. Figure 5 shows a typical optical emission spectrum of argon plasma jet having OH, excited nitrogen and oxygen radicals from the air 25 . The emission spectrum confirmed the presence of excited argon, OH, excited nitrogen, excited oxygen, ozone and nitric oxide in the plasma jet. The energetic electrons in the jet excite and ionize the oxygen and nitrogen from the surrounding air. Oxygen molecules break into atomic oxygen to generate ozone through a three-body reaction. On the other hand, the nitrogen in its ground state gets excited due to multiple collisions with electrons as: The N 2 (C 3 � u ) state gets populated through electron impact excitation of N 2 (X 1 + g ) ground state and N 2 (A 3 + u ) metastable state. Other than electron impact excitation, the associate excitation, penning excitation, pooling reactions and transfer of energy among the colliding particles also populate N 2 (C 3 � u ) state 25 . This excited state decays into second positive system of nitrogen by emitting a characteristic photon of (0-0) band 6 as: The second positive system reacts with oxygen molecules to form oxygen radicals, nitrous oxide and ozone. Again, the excited N + 2 (B 2 + u ) state of nitrogen gets populated during direct impact ionization of nitrogen in the ground state N 2 (X 1 + g ) . The populated excited state decays into a first negative system by emitting characteristic photon of (0-0) band. The intensity of the emitted radiations is always proportional to the population density of the excited state. in the open atmospheric plasma jet was confirmed from the emission line intensities in optical spectrum at 695-740 nm, 254.3 nm, 307.9 nm, 302-310 nm, 330-380 nm, 390-415 nm, 715.6 nm, 500-600 nm and 400-500 nm, respectively 26 . The emission intensities ratios of the identified species and the second positive system of the nitrogen were found higher in the beginning of the plasma jet excitation. It reveals that the surrounding air quickly diffuses into the jet and the nitrogen concentration increases along the jet flow. Sretenović 27 characterized the free expanding plasma jet in an open atmosphere. The plasma jet was impinged onto the water surface and characterized for chemical species by generating FTIR spectra at the water-plasma interface. Figure 6 shows a typical FTIR spectrum they produced during water-plasma interaction in ambient air. FTIR absorption detection confirmed the presence of NO, N 2 O, NO 2 , HNO 3 and HNO 2 reactive species in the plasma exposed water. The formation of ozone was also noticed during water-plasma interaction both in ambient air and in nitrogen rich environment. Water quality parameters. The water quality was checked by determining TDS, pH, conductivity, hardness and color of the samples. Table 4 shows that pH of the water samples noticeably decreased after plasma treatment with and without using a catalyst. The catalyst did not show significant effect on pH of water during plasma treatment. The possible reduction in pH of treated water is referred to the formation of HNO 2 , HNO 3 and other active ions during water-plasma interaction. The hydrogen ion concentration in water increased with a decrease in pH and so does the water conductivity. Since conductivity depends on concentration of all the active www.nature.com/scientificreports/ ions present in the sample, pH by itself did not specify the water conductivity. Therefore, pH of water samples did not provide any information about other active ions affecting the conductivity of water. In fact, all the ions in the sample contribute to conductivity. The faster the ions travel towards the opposite electrodes, more conductivity they lead to. The electrical conductivity of treated water samples may also be affected by the type of intermediates formed during plasma-water interaction. The plasma treated samples showed maximum decrease in pH. Most of the untreated water samples were alkaline in nature, which started to neutralize on plasma treatment. There was no significant effect of plasma treatment on the total dissolved solid in water except sample X 4 . This sample exhibited a decrease in TDS by 140 points and 190 points after noncatalytic and catalytic plasma treatment. The hardness of water samples also decreased after plasma treatment. Water with a low pH was less hard, while water with a higher pH was harder or alkaline. The degradation of organic pollutants and dyes in particular increased in the presence of TiO 2 catalyst and reactive plasma species like Ar, NO, O 3 , OH, N 2 , N + 2 , O, O + 2 and O + . The presence of these reactive species was confirmed through optical emission spectroscopy. Some sulfates and phosphates were also detected in the water samples. The sulfate ions in water samples reacted with plasma generated OH radicals. The degradation of organic pollutants decreases with a decrease in availability of OH radicals. It reveals that for complete degradation of all organic pollutants, prolonged plasma treatment will be needed. Ghezzr et al. 28 reported that degradation of pollutants in water starts after 20 min of treatment time. The pollutants' degradation efficiency was measured about 95% after 60 min of noncatalytic plasma treatment. In the presence of TiO 2 catalyst, the same degradation efficiency was possible only after 30 min of treatment time. It is worth noting that the treatment time mainly depends on plasma intensity and the population of the reactive species. The plasma treatment also cause mineralization of water samples due to formation of chloride ions, sulphate ions and phosphate ions. Hu et al. 29 performed photocatalytic decomposition of dyes with TiO 2 catalyst. The role of inorganic ions in activity of TiO 2 for dye degradation was investigated. Each dye degraded differently depending on pH of the solution. The sulfate and phosphate ions in the water showed significant effect on dye degradation process. Ghezzar et al. 28 treated textile wastewaters of different pH values. The wastewater contained azo dyes. A gliding arc discharge plasma was used to treat the dye containing textile wastewaters in the presence of TiO 2 catalyst. They investigated the role of plasma treatment time and catalyst in degradation of azo dyes. The photocatalytic activity of TiO 2 was reported higher for the water samples of high pH. The degradation efficiency improved with an increase in treatment time. ## Study of heavy metals. The atomic absorption spectrophotometry of the untreated and plasma treated water samples was conducted for detection of heavy metals. Figures 7, 8, 9, 10 and 11 confirm the presence of Ni, Cd, Pb, Cr and Cu in the wastewater. Significant amount of heavy metals was detected in the samples. The heavy metals' content increased on plasma treatment due to mineralization of water samples. Some chloride ions, sulphate ions and phosphate ions also form during plasma treatment. Initially, the water samples were slightly alkaline, which started to neutralize on plasma treatment. Ke et al. 30 revealed that removal of heavy metals from wastewater is pH dependent. They used argon plasma discharge for removal of chromium through www.nature.com/scientificreports/ reduction process at plasma-water interface. The reduction efficiency was found higher for solutions with initial pH less than 2 or greater than 8. The reduction efficiency increased on addition of ethanol in the solution. The high reduction efficiency promotes the removal of heavy metals from plasma exposed solution in the form of sediments. www.nature.com/scientificreports/ As shown in Fig. 8, the removal of Cu in plasma and plasma/TiO 2 treated water samples was found higher than the untreated water. The removed metals settle at the bottom, which were removed through filtration. The residue of untreated water contained negligible amount of Cu. The removal of metals from treated water increases due to the of byproducts in the water during plasma exposure. The Pb removal efficiency of plasma treatment was significant higher. After plasma treatment, Pb was not detected in water samples. Similar trend was predicted for other metals. Icopini et al. 31 removed Cr from water samples of different pH values. The metal removal efficiency was reported higher for lower pH values. It was revealed that Cr containing samples would be neutral or positively charged when pH of the sample is low. In the presented work, pH of solution decreases on plasma treatment, which promotes the removal of metals. Cserfalvi et al. 32 tested an atmospheric gas discharge technique for determination of heavy metals in different solutions. For lower pH values, the sputtering of solution surface during plasma exposure and subsequent excitations within the solution were observed. Using electrolyte-cathode discharge spectrometry technique, they identified Ni, Pb, Cu, Zn, Mn and Cd metals in the aqueous solutions. The emission peak intensity and concentration of these metals depended on pH of solution and hydrogen ion concentration during plasma exposure. FTIR and XRD analysis of residue. Figure 12 shows FTIR spectra of untreated and plasma treated samples. FTIR analysis confirmed the presence of amines, hydroxyl groups, amides, esters, ethers, anhydrides and carboxylic acids in the sample. The N-H stretching of primary amines, aromatic amines and amides was observed in the wavenumber range of 3320-3520 cm −1 . Eithers with C-O-C linkage were observed in the wavenumber range of 1070-1240 cm −1 . Sulfates had SO 2 symmetric stretching in the wavenumber range of 1140-1200 cm −1 . Similarly, ketones with C-C=O group were identified in the wavenumber of 510-560 cm −1 . The reported results were inline with the findings of Tichonovas et al. 8 . They treated polluted water samples with a barrier discharge system. The plasma treated samples contained amides, amines, carboxylic acids and nitrates. The water samples were filtered to remove the solid residue. The residue was characterized for its chemical composition. Figure 13 www.nature.com/scientificreports/ to Pb. The peak at 47.3002 shows the presence of 220 plane of Si. Alite, ferrite and aluminate had similar peaks as described somewhere else 33 . Sharma et al. 33 treated polluted water with metallic nanoparticles. The nanoparticles were used to remove heavy metals for the wastewater. Several metals were identified and removed from industrial effluents collected from different industrial sites in India. Antibacterial activity of plasma species. As discussed earlier, the plasma jet contained some strong oxidizers, which can easy kill the bacterial endospores and vegetative cells. Other than the plasma born ultraviolet radiations, the ozone, atomic oxygen and free reactive oxygen radicals also damage the cells by charging the cell wall and reacting with macromolecules. Since membrane lipids at the cell surface are susceptible to the reactive oxygen species, the oxidized cytoplasmic membrane lipids release intracellular substances, which damage the cells. The ultraviolet radiations interact with spore surface and cause volatilization of the surface compounds and damaging of DNA. The reactive oxygen radicals apply the electrostatic forces by charging the cell wall and oxidize the spore surface. In this study, the effect of plasma on inactivation of different bacteria in water was investigated. The culture of Escherichia coli (gram positive) and Staphylococcus aureus (gram negative) was subjected to the plasma exposure. The efficacy of plasma treatment to inactivate the bacteria was determined by observing colony forming unit (CFU) counts before and after plasma exposure. Figure 14 shows photographically the plasma exposed regions of the bacterial culture. The plasma treated regions are marked with square boundaries. A colony counter was used to find the CFU/plate. Significant reduction in CFU was observed after plasma exposure. Roughly, 98% decay of both cultures was observed after treatment time of 5 min. Initially, without any plasma exposure, the effect of air on bacteria deactivation was observed. The air flow did not show any effect on bacterial CFU. Thereafter, bacteria cultures were exposed to plasma and CFUs were counted before and after plasma exposure in the marked area of the petri dish. Since bacteria have several protective layers surrounding the genetic nucleus, it was difficult to kill them in the unexposed areas. However, all the bacterial were dead in the areas directly exposed to plasma. For prolonged plasma exposure, the bacteria in the adjacent regions also started to deactivate. The effect of plasma treatment on Staphylococcus aureus was more pronounced than the Escherichia Coli. All the Staphylococcus aureus cells in the plasma exposed region were found dead after 5 min of treatment while some Escherichia Coli cells were still alive in the plasma exposed region. It is possible to neutralize all the cells by increasing the treatment time. ## Conclusions Catalytic plasma treatment of wastewaters was conducted in ambient air in the presence of TiO 2 catalyst. The catalyst nanoparticles were composed of mixed anatase and rutile phases with particle size in the range of 5.2-8.5 nm. The optical emission spectroscopy confirmed the presence of excited argon, OH, excited nitrogen, excited oxygen, ozone and nitric oxide in the plasma jet. The energetic electrons in the jet excited and ionized the oxygen and nitrogen from the surrounding air. The spectral lines of Ar, NO, O 3 , OH − , N 2 , N + 2 , O, O + 2 and O + species were observed at wavelength of 695-740 nm, 254.3 nm, 307.9 nm, 302-310 nm, 330-380 nm, 390-415 nm, 715.6 nm, 500-600 nm and 400-500 nm. These reactive plasma species degraded the organic pollutants and separated the heavy metals from the wastewater. The conductivity of the water samples increased while pH and hardness decreased on treatment. The atomic absorption spectrophotometry of the samples confirmed the presence of heavy metals, which were effectively removed through plasma treatment. FTIR analysis confirmed the presence of amines, hydroxyl groups, amides, esters, ethers, anhydrides and carboxylic acids in the samples. XRD analysis of the solid residue confirmed the presence of S, Alite (triclinic), ferrite, Ni, CdS, Si, SiO 4 , Ag, Pb, CdO, Cu, Cr 3 O 4 and Aluminate in the samples. On the antibacterial side, the effect of plasma treatment on Staphylococcus aureus was more pronounced than the Escherichia coli. Overall, 98% decay of both bacterial cultures was observed after plasma treatment for 5 min. These findings confirm that the reported plasma jet technique is effective for degradation of organic pollutants, inactivation of bacterial and separation of inorganic pollutants from the wastewaters.

chemsum

{"title": "Optical characterization of non-thermal plasma jet energy carriers for effective catalytic processing of industrial wastewaters", "journal": "Scientific Reports - Nature"}

facilitating_the_transmetalation_step_with_aryl-zincates_in_nickel-catalyzed_enantioselective_arylat

## Abstract: A method for the highly enantioselective construction of fluoroalkyl-substituted stereogenic center by a nickel-catalyzed asymmetric Suzuki-Miyaura coupling of a-bromobenzyl trifluoro-/difluoro-/monofluoromethanes with a variety of lithium organoborate in the presence of 1.0 equivalent of ZnBr2 was described. Preliminary mechanistic studies disclosed that reaction of lithium organoborate with ZnBr2 generated a zincate [Ph2ZnBr]Li, which facilitates the transmetallation step of the nickel-catalyzed cross-coupling reaction to enable high enantioselectivity. ## INTRODUCTION Over the past two decades, nickel-catalyzed asymmetric cross-coupling of secondary alkyl electrophiles with different nucleophiles has emerged as powerful methods for the construction of chiral tertiary carbon centers. Since the seminar work by Fu and coworkers in 2005, 5 a number of activated racemic alkyl halides such as a-bromoamides, 5 a-bromoketones, 6 benzylic bromides and chlorides, allylic chlorides 9 or 1-bromo-1-fluoroalkane 10 and unactivated racemic alkyl halides such as b-or g-ether, amide or sulfonyl-substituted alkyl bromides, and a-haloboronates 13 were effectively employed as the coupling partners, while the choice of nucleophiles was originally mainly focused on alkyl zinc halides. Only recently, the nickel-catalyzed asymmetric couplings of racemic alkyl halides were successfully extended to other nucleophiles such as alkyl-9-BBN, aryl Grignard reagents, aryl zinc halides, aryl/vinyl silicates vinyl/alkynyl indium/zirconium/aluminum reagents. 6, Figure 1. Ni-catalyzed asymmetric cross-coupling of racemic secondary alkyl halides. Organoboron reagents are one of the most widely studied and applied reagents that allows for the efficient construction of carbon-carbon and carbon-heteroatom bonds. Non-asymmetric couplings of secondary alkyl bromides with aryl boronic acids under nickel catalysis have been reported in early 2004. 23 Yet, mainly due to the slow transmetalation step of the aryl ## Ni/L1 Ar Fu and coworkers turned to alkyl-9-BBN and found that the reaction could be conducted at 5 o C-room temperature to ensure high enantioselectivity. 15 Nevertheless, alkyl boranes are generally air and moisture sensitive and should be prepared in situ by hydroboration of alkene before use, which hampered their widespread applications. In 2017, we discovered that the transmetalation step in nickel-catalyzed asymmetric Suzuki-Miyaura coupling of CF3O-substituted secondary benzylic bromide when easily available, air-insensitive lithium organoborate instead of aryl boronic acid was used as the nucleophile. 24 In this case, the reaction occurred smoothly at 0 o C to give the coupled product with a CF3O-stustituted stereogenic center with excellent enantioselectivity. Inspired by this discovery and considering the fact that fluoroalkyl groups including trifluoromethyl (CF3-), difluoromethyl (HCF2-) and monofluoromethyl (CH2F-) group are important structural motifs in refining the lead compound's selectivity and pharmacokinetics for new drug discovery, we envisaged that the same strategy might work if a fluoroalkylated secondary benzylic bromide was allowed to react with lithium organoborate. One main problem for the transition-metal catalyzed coupling reactions of fluoroalkylated secondary benzylic bromides is the fluoride elimination from the fluoroalkylated secondary benzylic metal species if the subsequent transmetalation step is too slow. The key for the success of such a coupling reaction, therefore, is to accelerate the transmetalation step. Herein, we report a nickel-catalyzed highly enantioselective coupling reaction for the construction of the optically active fluoroalkylated benzhydryl derivatives from easily available prochiral a-bromobenzyl trifluoro-/difluoro-/monofluoromethanes and lithium organoborate. The presence of ZnBr2 played a key role in promote the reaction by formation of a highly reactive zincate [Ph2ZnBr]Li, which facilitates the transmetallation step of the nickel-catalyzed cross-coupling reaction. ## RESULTS Screening of reaction conditions. Initially, we tried the reaction of prochiral trifluoromethylated benzylic bromide 1a and lithium organoborate 2a as a model reaction to optimize the reaction conditions. Surprisingly, the reaction did not take place at all when it was conducted in THF at 0 o C for 8.0 h using a combination of 20 mol% NiBr2•DME and 25 mol% L1 as the catalyst, which is the condition for the construction of trifluoromethoxylated stereogenic center (Eq 1). Notably, when 1.0 equivalent of ZnBr2 was used as additive, the reaction occurred after 8 h at 0 o C to afford the coupled product in 65% yield with 85:15 e.r. (Eq 1). As a comparison, we studied the reaction of other nucleophiles such as Grignard reagent phenylmagnesium bromide or phenyl zinc bromide. As summarized in equation 2-3, reaction of substrate 1a with phenyl magnesium bromide, under the identical conditions, mainly afforded the undesired defluorinated side product in 51% yield, while the reaction of substrate 1a with phenyl zinc bromide were slow and the formation of the coupled product was not observed. A quick further survey of the reaction conditions disclosed that a combination of NiBr2•DME with ligand L2 was the most efficient catalyst and the desired product 3a was obtained in 62% yield with 95.5:4.5 e.r. along with the undesired defluorinated side product 3a' in 5% yield when the reaction was conduct at -15 o C for 12 h (Scheme 2, entry 1). Switching the additive to ZnCl2 gave slightly inferior results, while using MgBr2 as additive was not effective at all (Scheme 2, entries 2-3). Further studied showed that reactions in DME or diglyme occurred in good yields with high enantioselectivity, while reactions in other solvents such as THF, DMA or DMF were less effective and reaction in toluene was completely shut down (Scheme 2, entries 4-8). Notably, using a combinaiton of DME/diglyme (v/v = 1/1) as the solvent gave slightly improved yield and enantioselectivity (Scheme 2, entry 9). The amount of ZnBr2 was Pyridine-oxazoline ligand with either an electron-donating group (-OMe) or an electron-withdrawing group (-CF3) at 5-position, as well as a methyl group at 3-position of the pyridyl moiety were less effective (Scheme 1, entries 13-17). Likewise, two commonly used dinitrogen ligands for nickel-catalyzed asymmetric coupling reaction were also ineffective under these conditions (Scheme 1, entries 18-19). ## Mechanistic investigation. During the optimization of the reaction conditions, it was found that addition of 1.0 equivalent of ZnBr2 dramatically accelerated the reaction rate. Presumptively, mixing lithium aryl borate with ZnBr2 might generate several different arylated zinc species that could accelerate the transmetallation step and the overall catalytic reaction. To probe which arylated zinc species was involved in the reaction, we did several control experiments (Eq 4). First, reaction of compound 1a with 3.0 equivalents of PhZnBr in the presence/absence of 3.0 equivalents of LiBr occurred under standard conditions in less than 5% yield of the coupled product. Likewise, reaction of compound 1a with 3.0 equivalents of Ph2Zn, again, gave the desired product in less than 5% yield. These results clearly excluded the possibility of the involvement of PhZnBr and Ph2Zn in the current reaction. Interestingly, addition of 3.0 equivalent of LiBr to the reaction of compound 1a with Ph2Zn led to full conversion of the starting material and gave the coupled compound 3a in 78% yield with 95.5:4.5 e.r. These experimental results suggest that an anionic zincate [Ph2ZnBr]might involve in the reaction, consistent with the observation from Ingleson and co-workers that mixing 2.0 equivalents of lithium aryl borate with ZnBr2 at room temperature generated an anionic [PhxZnBry] -(x + y = 3). 31 To gain more support about the formation of lithium zincate from lithium aryl borate with ZnBr2, we studied and compared the 13 C NMR spectra of the species generated from mixing lithium aryl borate with ZnBr2 and Ph2Zn with LiBr. As shown in Figure 2, mixing equimolar amount of Ph2Zn with LiBr at room temperature in THF-d8 for 0.5 h cleanly generated [Ph2ZnBr]Li, as evidence by a peak with a chemical shift at 161.0 ppm in 13 C NMR spectrum, which corresponds to the ipso carbon of the phenyl group in [Ph2ZnBr]Li. Likewise, the same species was formed after 0.5 h at room temperature for the reaction of 3.0 equivalvents of lithium phenyl borate 2a with ZnBr2. These results clearly suggest anionic arylated zincate [Ph2ZnBr]Li would facilitate the (Scheme 2, 3g-i). For example, reactions of both a-bromo-4-nitrobenzyl trifluoromethane and a-bromo-3-trifluoromethyl benzyl trifluoromethane with lithium phenyl borates 2a gave the corresponding products 3h and 3j in 53% and 75% yields with excellent enantioselectivities 96:4 and 97:3 e.r., respectively (Scheme 2, 3h, 3j). Notably, trifluoromethylated benzylic bromides with a halogen group such as chloride, bromide, fluorine, were compatible and reacted with lithium phenyl borates 2a to give the corresponding products 3k-m in 65%, 68%, and 52% yields, with 96:4, 95:5 and 96:4 e.r., respectively (Scheme 2, 3k-m). Furthermore, a-bromobenzyl trifluoromethyl with para-, meta-, and ortho-substituents are all compatible coupling partners, affording the desired products in good yields and high enantioselectivities. For example, both a-bromo-3,5-dibromide benzyl trifluoromethane and a-bromo-2-fluorine-4-cyano benzyl trifluoromethane reacted to afford compounds 3s, 3v in 58 and 71% yield with 96:4 and 98:2 e.r., respectively (Scheme 2, 3s, 3v). Previously reported method for the preparation of enantio-enriched benzhydryl trifluoromethane derivatives typically required to use optically secondary a-(trifluoromethyl)benzyl tosylates to react with various aryl boronic acids in the presence of a palladium catalyst. 29, Thus, the current method provided an alternative, more efficient method to access this family of compounds. Encouraged by the high enantioselectivity in nickel-catalyzed coupling of a-bromo-benzyl trifluoromethane with lithium aryl borates, we next tried to extend this reaction to other fluoroalkyl substituted benzyl bromides. After a quick screen of the reaction conditions, it was found that when a more sterically-hindered ligand L7 was used as the ligand and the reaction temperature was decreased to -40 o C, good to excellent enantioselectivities could be achieved (Scheme 2). For example, reactions of the construction of difluoromethyl-substituted stereogenic carbon center have been reported previously, the current method represents an attractive approach for the preparation of optically active difluoromethylated benzhydryl derivatives. On the other hand, reaction of monofluoromethylated substrates were much more challenging. After carefully screening of the combination of nickel salts and ligands, it was found that using a combination of NiBr2 Synthetic application. To showcase the applicability of the nickel-catalyzed asymmetric coupling reaction of prochiral trifluoromethylated benzylic bromide with lithium organoborate, we applied this protocol for the synthesis of trifluoromethylated mimic of an inhibitor for the histone lysine methyltransferase enhancer of Zeste Homolog 2 (EZH2). 40 As shown in Figure Due to the slightly acidic proton in the difluoromethyl group which allows it to act as a lipophilic hydrogen-bond donor, the difluoromethyl group (CHF2) was generally considered as a bioisostere for a hydroxy goup (-OH). Consequently, a difluoromethylated compound 6, which is a mimic of histamine H3 receptor, 42 was synthesized in 71% overall yield and 90:10 e.r. after four steps. ## CONCLUSION In summary, we developed a highly enantioselective nickel-catalyzed coupling of easily available a-bromobenzyl fluooalkanes with a variety of lithium aryl borates in the presence of stiochiometric amount of ZnBr2. Preliminary mechanistic studies disclosed that a highly reactive zincate [Ph2ZnBr]Li is generated, which facilitates the transmetallation step of the nickel-catalyzed cross-coupling reaction. Thus, the protocol may serve as a versatile, efficient, and convenient approach for the rapid access of chiral benzhydryl fluoroalkane derivatives. The application of the high reactive lithium aryl zincate [Ar2ZnBr]Li in other transition metal-catalyzed cross-coupling reactions are undergoing currently in our laboratory.

chemsum

{"title": "Facilitating the Transmetalation Step with Aryl-Zincates in Nickel-Catalyzed Enantioselective Arylation of Secondary Benzylic Halides", "journal": "ChemRxiv"}

ultrathin_fe-nio_nanosheets_as_catalytic_charge_reservoirs_for_a_planar_mo-doped_bivo₄_ph

## Abstract: The energy conversion efficiency of a photoelectrochemical system is intimately connected to a number of processes, including light absorption, charge excitation, separation and transfer processes. Of these processes, the charge transfer rate at the electrode|electrolyte interface is the slowest and, hence, the rate-limiting step causing charge accumulation. Such an understanding underpins efforts focused on applying highly active electrocatalysts, which may contribute to the overall performance by augmenting surface charge accumulation, prolonging charge lifetime or facilitating charge transfer. How the overall effect depends on these individual possible mechanisms has been difficult to study previously. Aiming at advancing knowledge about this important interface, we applied first-order serial reactions to elucidate the charge excitation, separation and recombination kinetics on the semiconductor|electrocatalyst interfaces in air. The study platform for the present work was prepared using a two-step Mo-doped BiVO 4 film modified with an ultrathin Fe-doped NiO nanosheet, which was derived from an Fe-doped a-Ni(OH) 2 nanosheet by a convenient precipitation and ion-exchange method. The simulation results of the transient surface photovoltage (TSPV) data showed that the surface charge accumulation was significantly enhanced, even at an extremely low coverage (0.12-120 ppm) using ultra-thin Fe-NiO nanosheets. Interestingly, no improvement in the charge separation rate constants or reduction of recombination rate constants was observed under our experimental conditions. Instead, the ultra-thin Fe-NiO nanosheets served as a charge storage layer to facilitate the catalytic process for enhanced performance. ## Introduction The photoelectrochemical (PEC) reaction is one of the most promising methods for solar energy conversion and storage and, therefore, has attracted tremendous research attention. 1,2 The key components include a semiconducting photo-absorber, and a co-catalyst to accelerate the surface redox reactions in the electrolyte. 3 Due to the complicated requirements of the high efficient light absorption/excitation, separation and transfer kinetics, the heterogeneous materials possess great advantages over single-component ones. For example, the co-catalyst on the semiconductors can display various effects on the charge reaction rates. Given the extremely sluggish surface redox reaction rate (ms-s) and the short charge lifetime (ps-ms), 10 how to manipulate the heteronanostructure or the interface between the semiconductor and the catalyst becomes critical for the high-efficiency charge separation/transfer rate. 6,11 Take BiVO 4 (E g ¼ 2.4 eV) as an example. It is an earth abundant n-type semiconductor that has been widely applied as a photoanode for water splitting or CO 2 reduction. 15,16 Generally, it suffers from slow charge separation/transport, slow electron mobility, 17 and poor water oxidation kinetics. 18 Various strategies have been proposed to address these issues, 19 such as (1) increasing the doping density by introducing Mo or W dopants, 20,21 or oxygen vacancies by hydrogen treatments; 22 (2) incorporating an SnO 2 underlayer to reduce interface charge recombination; 23 (3) fabricating heterojunctions for larger builtin electric felds; 24,25 (4) enlarging surface band bending by long time photocharging 26,27 or electrochemical treatments; 28 and (5) employing oxygen evolution catalysts (OECs) to lower the activation energy (E a ) and increase the charge transfer rate. For BiVO 4 /OEC heteronanostructures, crystalline NiOOH/FeOOH, Ni(OH) 2 , NiO, CoO, Co 3 O 4 , amorphous Co-Pi and NiFeO x have been successfully used, showing signifcant PEC performance enhancements. At least four possible functions of the OECs have been proposed. First, a typical catalyst can increase the charge separation/transfer, resulting in the decrease of surface recombination. 13,29 For example, by coating BiVO 4 with a FeOOH layer, researchers obtained a substantially increased hole collection at the solid|liquid junction, which is responsible for the high measured photocurrents. 18,30 For another example, an ultrathin CoO x (1 nm) catalyst layer allowed greater hole collection as opposed to faster kinetics. 31 By comparison, amorphous cobalt phosphate (Co-Pi: 30 nm) increased the charge transfer kinetics. 32 Second, the suppression of surface recombination led to a high photovoltage (band bending), causing faster surface reactions with higher photocurrent. 33 To this end, the Durrant group employed transient absorption spectroscopy (TAS) to demonstrate the retardation of electron/ hole recombination. 34 They did not observe any evidence of catalytic behaviours. When studying the CoPi, FeOOH or NiFeO x catalysts on BiVO 4 with the intensity modulated photocurrent spectroscopy (IMPS), 35 the photocurrent was found to be limited by fast surface recombination rate rather than surface catalysis. Third, charge separation/transfer can be intrinsically increased by the built-in electronic feld in heterojunctions (e.g., p-n junction). 36,37 Chang et al. introduced discrete p-type Co 3 O 4 co-catalysts on BiVO 4 to form a p-n heterojunction, which was shown to facilitate charge separation, increasing surface reactions and suppressing recombination at the interface. 38 Similarly, a Ni-doped CoO x uniform layer (ptype) increased the surface band bending with a cathodic V on shift and photocurrent increase. 39 This surface band bending enhancement also resulted in the reduction of surface charge recombination on the NiO/CoO x /BiVO 4 photoanode. 37 In addition, the hole-storage layer effect of ferrihydrite was suggested on BiVO 4 and Ta 3 N 5 photoanodes. 40,41 Despite these advances, it has been difficult to fully understand what the true causes are for the observed performance improvements at the semi-conductor|electrocatalyst interface. Therefore, charge behaviours at the semiconductor|electrocatalyst interface remain relatively poorly understood, presenting a challenge for further improvement of PEC systems. This interface is thus of great importance and has attracted signifcant research attention. To discern the thermodynamic and kinetic influences at this interface, the Boettcher group has successfully employed a dualworking-electrode (DWE) method to scrutinize the photovoltage and charge transfer differences between the adaptive and dense semiconductor|catalyst junctions in the electrolyte. 42,43 Their secondary working electrodes could be used to either probe or control the catalyst/electrolyte interface in situ, so that the electrochemical potential/current of the catalyst can be independently measured. 9 Separately, the Durrant group has measured a 3 rd order oxygen evolution reaction (OER) order with regard to surface hole concentrations on BiVO 4 under higher surface hole densities (>1 nm 2 ) based on photoinduced absorption analysis (PIA). 44 A 1 st order OER rate dependence on the hole concentration was found when the surface hole density was low (<1 nm 2 ). Results like these raise important questions concerning the detailed processes and their influence on the overall performance of photoelectrodes in PEC reactions. For instance, at low surface coverage, does a co-catalyst influence the system by changing the kinetics or surface energetics? How does the charge accumulated at the semiconductor|electrocatalyst interface contribute to the photoelectrochemical reactions? Similar questions were difficult to answer using existing methodologies. To correct this defciency, here we report a simple transient surface photovoltage (TSPV) analysis that can directly monitor the accumulated charges at the semi-conductor|air or semiconductor|electrocatalyst interface, especially under the open-circuit condition. We show that the technique is an important tool to advance our understanding of the interface charge phenomena. The merit of this TSPV method is the ability to individually study charge separation/transfer at the semiconductor|electrocatalyst interface with negligible redox reactions because the system is an open circuit. For this body of research, we chose crystalline ultrathin Fedoped NiO x (Fe-NiO) nanosheets as an oxygen evolution catalyst on planar Mo-doped BiVO 4 (Mo-BiVO 4 ) flms. The system was frst studied in air before in a contacting electrolyte, as our goal was to elucidate the charge separation kinetics. Different from the previously reported synthesis method of Ni(OH) 2 catalyst by plasma deposition 48 or in situ electrochemical decomposition, 13,14,30 we simply prepared ultrathin Fe-doped Ni(OH) 2 nanosheets through a precipitation and ion-exchange method. The catalyst was spin-coated onto the Mo-BiVO 4 flms and thermally converted to a discrete ultrathin Fe-NiO catalyst layer. Next, we applied the TSPV to investigate the surface charge accumulation kinetics on the semiconductor|electrocatalyst interface in air. Simulation of the kinetics was carried out, and we observed an apparent frst-order dependence of charge separation and recombination on charge concentrations. An increased surface charge accumulation was observed at the Mo-BiVO 4 /Fe-NiO interface, implying that the catalyst serves as a charge "reservoir", despite its relatively low loading. The Fe-NiO modifed Mo-BiVO 4 photoanode showed a signifcant overall enhancement for water oxidation in an alkaline electrolyte (1 M NaOH) with high charge transfer efficiencies. The charge separation and transfer efficiencies at the semi-conductor|electrolyte interface were also investigated during the PEC test with and without Fe-doped NiO catalyst, respectively. Following the previously reported methods, 13 Mo-doped BiVO 4 flms were prepared by a two-step process. Briefly, 30 mL of VO(acac) 2 in DMSO (0.5 M) was cast coated on the Bi flm (1 cm 2 cm) and dried in an oven, before being slowly heated in a muffle furnace to 450 C (heating rate at 2 C per minute) and maintained at 450 C for 4 h. The resulting brownish flms were soaked in 1 M NaOH solution for 20-30 min, rinsed with DI water to remove excess vanadate impurities, followed by post annealing at 500 C for 2 h. The obtained flms were denoted as undoped BiVO 4 . In the second step, a mixed solution containing MoO 2 (acac) 2 and VO(acac) 2 in a DMSO solution (Mo/V ¼ 5% in molar ratio) was cast on the BiVO 4 flms, slowly heated to 450 C and sequentially treated at 500 C for 2 h. After the impurities were removed using NaOH and DI water, the obtained flm was denoted as two-step Mo-doped BiVO 4 (2-Mo-BiVO 4 ). Single conversion process was used for homogenous 1-Mo-BiVO 4 with the same mixed Mo/V precursor solution. ## Materials Synthesis of ultrathin Fe-doped NiO nanosheets on 2-Mo-BiVO 4 25 mL of Ni(NO 3 ) 2 solution (0.1 M) was added into 1 M NaOH solution (6 mL) dropwise under vigorous stirring for 10 min. The resulting light greenish Ni(OH) 2 precipitate was centrifugated and washed using DI water several times, which was re-dispersed in 20 mL DI water. 300 mL of Fe(NO 3 ) 3 (1 M) was added to the above Ni(OH) 2 dispersion, allowing ion exchange under ultrasonic treatment for 2 h. The resulting brownish dispersion was centrifugated/washed in DI water several times to remove the excess ions. In the end, the obtained precipitation was denoted as Fe-doped Ni(OH) 2 . It was re-dispersed in DI water (120 mg mL 1 or 120 ppm), which was spin-coated onto 2-Mo-BiVO 4 flms. Typically, 50 mL of the Fe-doped Ni(OH) 2 dispersion was spread on a 2-Mo-BiVO 4 flm spinning at 3000 rpm for 60 s. After thermal annealing at 300 C for 2 h in air, the 2-Mo-BiVO 4 /Fe-NiO-120 flm was obtained. For the other loading amounts, the dispersion for spin-coating was diluted to 12 ppm, 1.2 ppm and 0.12 ppm, respectively. Structure, optical and photoelectronic characterization X-ray powder diffraction (XRD) was conducted on a Bruker X-ray diffractometer (D8 Advance, Cu K a , l ¼ 1.5418 ) in the range of 10 -70 . Scanning electron microscopy (SEM) was observed on a feld emission scanning electron microscope (FEI, Nova NanoSEM450). High resolution TEM image and electron diffraction were obtained on a transmission electron microscope (FEI Tecnai G2 F20) under 200 kV. The thickness/height image, photoconductive topology and Kelvin probe force microscopy were collected on a conductive atomic force microscope (C-AFM, Bruker Dimension Icon, coupled with AM 1.5G light) under ambient conditions, using conductive AFM probes (Bruker, PFTUNA and SCM-PIT, respectively). The Fedoped NiO samples (>80 cm 2 ) were dissolved into dilute HNO 3 ; after that, the surface catalyst loading amount was checked by inductive coupled plasma mass spectroscopy (ICP-MS, Agilent 7700). The XPS data were collected on a spectrometer (Thermo Scientifc Escalab 250Xi), and Raman spectra were collected on a Renishaw confocal Raman microscope (in Via Reflex) using a green laser (532 nm) in the range of 200-1000 cm 1 . The optical properties of the produced flms were measured in the transmission mode with a UV-vis spectrophotometer (Agilent Tech. Cary 5000). The transient surface photovoltage was investigated on a home-made capacitor-like spectroscope, 46,49 where a Quantel Nd:YAG nanosecond laser (Brilliant Eazy, BRILEZ/IR) was used as the excitation source (355 nm, 4 ns, spot area of 0.24 cm 2 ), coupled with a digital oscilloscope (Tektronix, TDS 3054C, 500 MHz) and pre-amplifer for recording. A sandwich structure of FTO|mica|BiVO 4 (on FTO) was assembled in a metal faradaic container, where a mica ($70 mm) was used as spacer. ## Photoelectrochemical and impedance measurements The photoelectrode was prepared by connecting a Cu wire with silver adhesive to a FTO substrate, encapsulated with insulated cross-linked rubber only with the active area exposed. The PEC measurements were carried out using a three-electrode confguration on a potentiostat (CHI 660E, Shanghai), with a counter electrode (Pt wire) and a reference electrode (Hg/HgO, in 1 M NaOH, 0.098 V vs. NHE) in an electrolyte solution (1 M NaOH, pH ¼ 13.5). The potential was converted to the reversible hydrogen electrode (RHE) scale following this equation: E ¼ E Hg/ HgO + 0.098 + 0.059 13.5. A standard simulated solar illuminator (AM 1.5G on Newport 94023, 100 mW cm 2 ) was chosen as the light source. The polarization J-V curves were recorded using a linear sweep technique with a scanning rate of 20 mV s 1 in the range of 0. ## Results and discussion To investigate the charge separation at the Mo-BiVO 4 /Fe-NiO interface, we start to prepare the Fe-doped NiO catalyst and Mo-BiVO 4 semiconductor separately before integrating them together. For the synthesis of the Fe-NiO catalyst, Ni(OH) 2 was freshly precipitated from a solution, which was converted to Fedoped nickel hydroxide under ultrasonic agitation. In Fig. 1 . This lattice increment of NiO after Fe doping agreed well with a previous report. 50 In Fig. 1(b), the binding energies of Fe2p and Ni2p electrons are shown, respectively. The Fe2p peak can be deconvoluted as Fe2p 3/2 , Fe2p 1/2 and a pre-2p 3/2 peak at 711.7 eV, 723.5 eV and 704.4 eV, respectively. This is in good agreement with a Fe 3+ state. 50 For the Ni2p peaks, two sets of Ni2p 3/2 , Ni2p 1/2 and their satellite peaks were shown at 855.6 eV, 873.2 eV and 861.3 eV and 879.2 eV, respectively, corresponding to a Ni 2+ in the Fedoped NiO product. 50 XPS showed that the element molar ratio of Fe/Ni was ca. 27%/73%. The higher Fe doping level may be due to the comparable radius of Fe 3+ (64.5 pm) and Ni 2+ (69 pm) with a six-fold coordination, 51 and/or large surface/volume ratio to release the lattice stress (strain). Thus, the preparation process can be demonstrated through three steps: a-Ni(OH) 2 precipitation as in eqn (1), Fe 3+ ion exchange in the precipitation as in eqn (2), and thermal dehydration to Fe(3+)-doped NiO in eqn (3): (1) For simplicity, we used Fe-NiO to represent the product of Fe The light absorption of the obtained brownish Fe-NiO on FTO was investigated in Fig. 1(c). The sample showed strong absorption between 300 and 550 nm. Using the Tauc plot (inserted in Fig. 1(c)), the indirect light absorption band can be calculated as 2.35 eV. Although the general undoped NiO had a wide bandgap ($3.6 eV), 52 the introduction of Fe dopants resulted in a narrower bandgap due to the less occupation of dbands of the Fe atoms than the Ni atoms. This observed indirect bandgap coincides with the one calculated by frst principles (2.26 eV for 25% doping). 53 The high resolution TEM image of the Fe-NiO revealed a highly crystalline structure with the zone axis of in Fig. 1(d), where the lattices separated by 2.06 and 2.38 were assigned to the (200) and (111) planes, respectively. The angle between these two planes was ca. 54 . Combining the HR-TEM image with the XRD pattern, we expect an ultrathin oriented flake morphology. Then, we prepared a Fe-NiO sample on a Si wafer from a dilute dispersion (1.2 ppm) for thickness evaluation. In Fig. 1(e), the AFM height image displays a typical 2-dimensional morphology with a width of 25-60 nm (size distribution shown in Fig. S2 †) and a thickness of 2.1-4.8 nm. Given that the distance between the (220) planes is ca. 1.5 , this thickness corresponds to 14-32 layers of nanosheets. When the ultrathin Fe-NiO nanosheets were deposited on the FTO substrate from various concentrations, they all displayed a highly catalytic activity as shown in Fig. 1(f). The current-potential J-V curves showed a dramatic current increase (e.g., 1 mA cm 2 ) when the applied potential was above 1.57 V (vs. RHE), with the increasing Fe-NiO loading amount. In Fig. S3, † the EDS mapping images of Fe, Ni and O were displayed, showing a uniform distribution of Fe and Ni in the electrocatalyst. Although the exact loading amount of the catalyst on FTO may be not strictly proportional to the content of the solid precursor in the suspension, the electrocatalytic performance showed a positive correlation to the precursor content. Compared with the other OER catalysts, such as NiCoO x , NiOOH, Ni(OH) 2 , NiFeO x , NiOOH, CoOOH, Co-Pi or NiO, 48,54 the overpotential (0.27 V for 0.1 mA cm 2 ) on ultrathin Fe doped NiO nanosheets is promising for practical OER applications. The planar Mo-doped bismuth vanadate flms were thermally converted from Bi flms (40 nm) on the FTO substrate, using VO(acac) 2 as the vanadium source and MoO 2 (acac) 2 as the doping precursor in DMSO as reported in the literatures. 13 The 2-Mo-BiVO 4 flm on the FTO substrate was characterized by Xray powder diffraction using a Cu target. In Fig. 2(a), the peaks marked with red "*" are all ascribed to the diffractions of (011), ( 112), (004), ( 121), ( 006), ( 204), ( 301) and (116) on a monoclinic BiVO 4 structure (JCPDS card, no. 83-1699). Based on the XRD pattern, the lattice constants (a ¼ 5.177 , b ¼ 5.123 , c ¼ 11.71 and g ¼ 90.20 ) were obtained, which were close to bare and one-step Mo-doped BiVO 4 (Fig. S4 and Table S1 †). The Modoping was additionally confrmed by the Raman and XPS spectra. In Fig. 2(b), the Raman spectrum of Mo-doped BiVO 4 displayed identical peaks at 325 cm 1 and 368 cm 1 , corresponding to the asymmetric and symmetric bending modes (d as and d s ) of the VO 4 3 tetrahedra, respectively. 55 And the peaks at 711 cm 1 and 826 cm 1 are assigned to the symmetric and antisymmetric stretching modes (n as and n s ) of V-O vibration, respectively. 21,55 Both the XRD pattern and Raman spectra revealed that a pure Mo-doped BiVO 4 phase was obtained, with no detectable impurities from the other structure or bismuth molybdenites. The surface oxidation state of 2-Mo-BiVO 4 was characterized by the XPS spectra (in Fig. 2(c)). The peaks at 158.9 and 164.2 eV correspond to the Bi4f 7/2 and Bi4f 5/2 electrons of Bi 3+ . 21 The peaks at 232.1 and 235.25 eV are assigned to the Mo3d 5/2 and Mo3d 3/2 electrons of Mo 6+ . 56 The peaks at 516.53 and 524.02 eV correspond to V2p 3/2 and V2p 1/2 electrons of V 5+ . 21 The calculated surface element ratios are 100/5.6/48.9 for Bi/Mo/V, indicating a surface defciency of V and Mo due to the soaping treatment in alkaline solutions. This is in good agreement with other literature reports. 21,57 We also used UV-vis absorption (Fig. 2(d)) to investigate the optical properties, where the band edge absorption was close to 510 nm and the indirect bandgap was $2.48 eV as determined by the Tauc plot. From the SEM image in Fig. 2(e), 2-Mo-BiVO 4 exhibited a planar morphology, with particle sizes ranging between 200 and 400 nm. From the inset cross-section image, the flm showed a rough surface and the thickness was ca. 130 nm. Next, the elements of the flm were analysed through EDS (Fig. 2(f)), showing the evidence of Mo, V and Bi from the flm with molar ratios of 100/4.3/111 for Bi/Mo/V. The values of the bulk flm are higher than those obtained by the XPS surface analysis, presumably due to the larger detect depth through EDS than that through XPS. Thus, the Modoped BiVO 4 are well prepared with good quality. With the well-prepared Fe-doped NiO OER catalyst and Modoped BiVO 4 semiconductor flm, we next studied charge separation, and recombination kinetics on the semiconductor/ catalyst interface by transient surface photovoltage spectroscopy (TSPV). Although TSPV has been used for the studies of charge separation in a qualitative fashion, the detailed charge kinetics has rarely been examined in a quantitative manner. The defciency was partially corrected by our recent study on the Cudoped CH 3 NH 2 PbI 3 perovskite flm (p-type) with the ITO substrate, where we applied a frst-order serial reaction system for the studies of charge separation at the perovskite/air interface. 46 Briefly, let us consider a n-type semiconductor as an example. Upon excitation by a laser pulse with nanosecond temporal resolution (Fig. 3(a)), the electron-hole pairs in the conduction and valence bands will be separated to the semiconductor/air interface due to the internal electric feld in the Schottky-type junction, which is regarded as the charge separation process. In the absence of an external circuit, ultimately the separated charges will be consumed through a recombination process. The charge separation/recombination processes (Fig. 3(b)) may be expressed as consecutive equations: 46 Since the charge separation (including the diffusion and drift) is sensitive to the initial excited charge pair densities, a frst-order charge separation may be applied to describe the separation process (rate constant: k sep ). Because the majority charge (electron in n-type semiconductor) density is much higher than the minority charge (hole) density, the recombination is expected to obey a quasi frst-order rate law relative to the hole concentration (rate constant: k rec ). According to the serial frst-order reactions theory in physical chemistry, 58 the intermediate density (accumulated charge) will display a maximum level at the time of t max (assuming k sep s k rec , other boundary conditions and theoretical calculations can be found in the ESI †): Where the transient accumulated charge (Q sep ) versus the time can be expressed as follows (assuming k sep s k rec ): 46 Where V corresponds to the measured surface photovoltage, C represents the capacity of the assembled TSPV detector (in Fig. 3(a)), and Q exc,0 is the apparent initial charge pair density. Based on eqn (6), we could simulate the apparent charge densities of the excited pairs (Q exc ), separated charge (Q sep ) and recombined charge (Q rec ), by varying the three parameters of k sep , k rec and Q exc,0 (Fig. 3(c)-(f)). When an increase in the charge separation kinetics constant (5 times of k sep in Fig. 3(d)), or a reduction of the charge recombination kinetics constant (0.2 times of k rec in Fig. 3(e)) is introduced, an increase of the maximum accumulated charge Q sep,max can be seen, together with a negative or positive shift of t max , respectively. Alternatively, when hole storage Q exc,0 increases, as shown in Fig. 3(f), the Q sep,max increases, but t max is unchanged. Therefore, the charge separation or recombination rate constant change can be easily identifed through the simulation and/or ftting the TSPV curves. Next, we obtained the TSPV spectra for doped, undoped BiVO 4 flms and Fe-NiO modifed 2-Mo-BiVO 4 flms in Fig. 3(g)-(i), respectively. The experimental data (black) were readily ft by simulated ones (red). The maximum separated charge accumulation displayed sensitivity to doping and surface modifcations. It was worth noting that the loading amount of the NiO on 2-Mo-BiVO 4 was proportional to the solid content of the catalyst precursor using EDS analysis (Fig. S5 †). And the SEM and EDS mapping images of the 2-Mo-BiVO 4 /Fe-NiO-12 were investigated in Fig. S6, † where all the elements were homogenously distributed in the detected region. Moreover, we found the highest Q sep on Fe-NiO modifed 2-Mo-BiVO 4 flms in Fig. 3(j), ) and smaller Q exc,0 . This indicates a difference in the mechanism between the ultra-thin crystalline Fe-doped NiO nanosheets and amorphous thick NiFeO x layer. It is worth noting that the consecutive-reaction hypothesis is based on the following assumptions, including high excitation rate and efficiency ($100%), fast bulk recombination rate and long lifetime (ns-ms) of separated charges. Additionally, for all the TSPV measurements, we used the same laser pulse power (7 mJ per pulse), therefore the theoretical Q exc,0 should be at the same level. It is noted that the calculated initial charge density (Q exc,0 ) is an apparent value and should be treated as such; it may be compared to the values obtained by transient absorption methods only in a qualitative fashion. 34,44 Moreover, the peak height of the separated charge (Q sep,max ) on the Fe-doped NiO modifed 2-Mo-BiVO 4 flm displayed 3 times storage as high as that of the bare one (in Table 1). A higher charge separation rate constant could be observed on thinner Fe-NiO application (1.2 or 0.12 samples in Table S2 †), which also corresponded to higher accumulated charge densities. Therefore, the increased Q sep,max could be attributed to the "charge storage" effect at the interface. To better understand the reason of these charge separation kinetics, we used the Kelvin probe force microscope (KPFM) to investigate the surface potential under dark conditions and the photoconductivity on bare and Fe-NiO-12 modifed 2-Mo-BiVO 4 flms under ambient conditions. In Fig. 4(a), the height image of the bare 2-Mo-BiVO 4 flm was shown, with the particle sizes ranging between 150 and 370 nm, consistent with that in the SEM image of Fig. 2(e). When the Fe-NiO-12 was incorporated on the bismuth vanadate surface (Fig. 4(b)), similar particles could be observed with a root-mean-square (RMS) surface roughness factor R q slightly increased from 19.2 nm to 20.7 nm. By using an Au flm (work function of 5.1 eV) as the standard, the surface potential of the Mo-doped BiVO 4 flms were measured between 4.6 V and 5.2 V vs. vacuum (corresponding to a 0.1 V to 0.7 V vs. NHE). The average surface potential was measured as 4.93 AE 0.09 V (0.43 V vs. NHE), which is slightly higher than the reported conduction band minimum (0.3 V). 25 a The relative value of the Q exc,0 were shown here (normalized with C as constant 1). When Fe-NiO was applied onto the surface, the potential (5.03 AE 0.09 V) ranged between 4.7 V and 5.3 V vs. vacuum (0.53 V vs. NHE). This suggests that the surface potential (or work function) of the flms are almost the same, due to the extremely low amount of Fe-NiO. Next, the Mott-Schottky method was applied in an electrochemical setting, and 2-Mo-BiVO 4 showed a flat band potential of 0.20 V vs. RHE (Fig. S9 †). The apparent discrepancy between the Mott-Schottky and Kelvin methods is probably due to the differences in the surface adsorbed species in an electrolyte and in air. It is surprising that the Mott-Schottky slope of the modifed sample is higher than that of the bare one. This may be due to the reduced contribution from surface state capacitance. The V FB of Fe-NiO modifed 2-Mo-BiVO 4 exhibits a negligible shift. For the Fe-doped NiO nanosheets, a negative slope and V FB at 1.41 V vs. RHE was obtained (Fig. S10 †), indicating that the Fe-NiO catalyst features holes as the majority carriers. A possible p-n heterojunction between BiVO 4 and Fe-NiO would facilitate charge separation at the semiconductor|electrocatalyst interface. We also investigated the electronic conductivity of bare and Fe-NiO-12 modifed flms, with the back illumination from the FTO side. In Fig. 4(e), the photoconductivity of bare 2-Mo-BiVO 4 showed random dark domains (0 to 40 nA). The average areal photocurrent density was estimated to be 1.19 nA, which was 20 times higher than the dark current (0.060 nA). After Fe-doped NiO was deposited on the 2-Mo-BiVO 4 surface, the photocurrent density slightly decreased to 0.69 nA, indicating that the Fe-NiO layer is less conductive than 2-Mo-BiVO 4 . Next, we used photoelectrochemical water splitting to test our understanding that the Fe-NiO nanosheets served as charge reservoirs in the combined system. Different from many other BiVO 4 studies which were carried out in phosphate or sulfate solutions, we chose an alkaline solution as the electrolyte because it is widely used in tandem confgurations. 59 In Fig. 5(a), we compared the photocurrent polarization curves of the undoped BiVO 4 , doped 1-Mo-BiVO 4 and 2-Mo-BiVO 4 flms. It was found that the bare BiVO 4 sample exhibited a poor water oxidation activity (<0.12 mA cm 2 with a positive onset potential at 0.55 V vs. [email protected] mA cm 2 ). When the Mo dopants were introduced, the photocurrent of 1-Mo-BiVO 4 was signifcantly increased to ca. 0.40 mA cm 2 at 1.4 V, and the onset potential was negatively shifted to 0.27 V. For the 2-Mo-BiVO 4 sample, the photocurrent further increased to 0.88 mA cm 2 at 1.4 V and the onset potential remained at 0.27 V. The performance of bare and Mo-doped BiVO 4 measured in alkaline solution is among the best of all the reports (Table S3 †). 31 Moreover, the photoelectrochemical stability of the Mo-doped BiVO 4 photoelectrode increased conspicuously with Fe-doped NiO (Fig. S11 †), which may be further improved by suitable conformal ALD coating. 60 Based on this, we further discussed charge separation and transfer in PEC confguration. The trend of the J-V performance on doped and undoped samples is consistent with the TSPV data: 2-Mo-BiVO 4 exhibited better charge separation and larger charge accumulation and, hence, better PEC performance. Next, in Fig. 5(b), when 2-Mo-BiVO 4 was chosen as a photoelectrode platform, the ultrathin Fe-doped NiO nanosheets were uniformly dispersed with prolonged ultrasonic treatment and applied onto the bismuth vanadate flms. Even with a precursor at 1.2 ppm (which is very low), the photocurrent already increased from 0.15 mA cm 2 to 0.18 mA cm 2 (0.7 V) in the range of 0.5-0.8 V vs. RHE. When the catalyst precursor concentration was increased to 12 ppm, the photocurrent increased dramatically to 0.55 mA cm 2 at 0.7 V and 1.65 mA cm 2 at 1.4 V. The loading amount of Fe-NiO on Mo-BiVO 4 was 0.11-0.12 mg cm 2 with the Fe/Ni molar ratio at 0.266/0.734, by re-dissolving in dilute HNO 3 and analyzed by ICP-MS. Further increase of the precursor concentrations to 120 ppm led to the decrease of the photocurrent to 1.03 mA cm 2 at 1.4 V. This is possibly due to the increased charge transfer resistance of the less electronically conductive Fe-NiO by the C-AFM measurements, or by the increased charge transfer resistance at the interface. 40 Another reason would be increased recombination of the thicker catalyst layers. Although the maximum of the accumulated charge Q sep,max on Mo-BiVO 4 /Fe-NiO was higher with the Fe-NiO-0.12 or Fe-NiO-1.2 catalyst (in Fig. S7 and Table S2 †) than that sample with the Fe-NiO-12 catalyst, extremely thin Fe-NiO-0.12 did not show obvious improvement in the J-V measurements, suggesting that not only the charge storage but also the catalytic sites contributed to the water oxidation reaction. On the other hand, this improvement was also not due to the surface passivation effect. Therefore, when the Fe-NiO modifcation is used, charge storage, fast kinetics and small transfer impedance need to be balanced consequently. We then applied charge separation and transfer efficiencies (h sep and h transf ) in the electrolyte to verify this speculation. We investigated bare and Fe-NiO-12 modifed 2-Mo-BiVO 4 sample in the presence of hydrogen peroxide (H 2 O 2 in 1 M NaOH) as a hole scavenger. By comparing the J-V curves (J H 2 O 2 ) in H 2 O 2 (Fig. S12 †) with the theoretical photocurrent (J abs ) by light absorption and 100% IPCE, we estimated the charge separation efficiency (h sep ¼ J H 2 O 2 /J abs ) at the semiconductor|electrolyte interface w/o Fe-NiO catalyst in Fig. 5(c). In the range of 0.5-0.77 V, the charge separation efficiency on 2-Mo-BiVO 4 /Fe-NiO-12 is higher than on bare 2-Mo-BiVO 4 . While in the range of 0.77-1.5 V, the charge separation on modifed Mo-BiVO 4 one is lower than on the bare one. This suggests that better charge separation is expected on 2-Mo-BiVO 4 /Fe-NiO-12 under the lower band bending conditions (<0.8 V) or slower surface redox reaction rates. The efficiencies of charge transfer to surface water molecules can be evaluated through h transf ¼ J OER /J H 2 O 2 , assuming that the faradaic efficiencies for water oxidation and H 2 O 2 oxidation are the same. Fig. 5(d) shows a higher charge transfer efficiency for 2-Mo-BiVO 4 with the Fe-NiO-12 catalyst than that without. For the bare 2-Mo-BiVO 4 sample, the transfer efficiency slightly increased from 13 to 39% (0.5 V to 1.4 V), suggesting over two thirds of the surface accumulated charges may be consumed by the recombination due to the slow water oxidation kinetics. With ultrathin Fe-NiO nanosheets modifcation, the charge transfer efficiency signifcantly improved to close to 99% ($1.2 V). This suggested that the higher applied bias (>0.8 V) contributed more to the surface charge transfer at the Fe-NiO nanosheets-modifed Mo-BiVO 4 photoanode rather than the charge separation at the Mo-BiVO 4 photoanode. The relationship between charge separation and transfer under open circuit and PEC conditions is highly complex. Generally speaking, further increases of charge separation and transfer are of great importance to the PEC performance. And the increased transfer efficiency possibly benefts from the charge storage effect at the semiconductor|electrocatalyst interface. As we have discussed above, through the TSPV, the Fe-doped NiO ultrathin nanosheet electrocatalyst on the Mo-doped BiVO 4 surface displayed a charge storage effect for a better charge separation. Conclusion was obtained by simulations of the kinetics under ambient air conditions without the electrolyte. To further confrm this speculation, we have analysed bare and Fe-NiO-12 modifed 2-Mo-BiVO 4 photoelectrodes in a 1 M NaOH electrolyte by electrochemical impedance spectroscopy (EIS) at 1.5 V vs. RHE and cyclic voltammetry (CV) between 1.1 and 1.6 V (vs. RHE). In Fig. 6(a) and (c), a typical Nyquist plot of bare 2-Mo-BiVO 4 were shown, where the capacitance (C SS ) and resistance (R SS ) of the surface state were calculated to be 6.42 10 6 F cm 2 and 19.39 U cm 2 , respectively. When the Fe-NiO electrocatalyst was loaded on the 2-Mo-BiVO 4 surface, in Fig. 6(b), we used the electrocatalyst to substitute the surface state circuit as Bisquert and Hamann have done for the Co-Pi coated Fe 2 O 3 photoanode. 61 The obtained capacitance (C cat ) and resistance (R cat ) of the Fe-NiO catalyst were 2.56 10 5 F cm 2 and 3.98 U cm 2 (Fig. 6(d)), respectively. The signifcant increase of C cat and reduced R cat was understood as a beneft for charge separation and the overall photoelectrochemical performance. Moreover, the CV curves of the Fe-NiO modifed 2-Mo-BiVO 4 showed an obvious current density in the window of 1.2-1.6 V vs. RHE (in Fig. S13 †), further confrming the surface capacitance behaviour of the Fe-doped NiO electrocatalyst. Combining the kinetic charge separation/transfer results at the solid/air interface and solid/liquid interface, we gained the following understanding on the charge separation and transfer processes. The p-type Fe-NiO nanosheets at the n-type 2-Mo-BiVO 4 surface form an interface that is more complex than a conventional p-n junction as evidenced by the slower charge separation after the Fe-NiO incorporation by TSPV. The increased hole storage at the interface acts as a reservoir possibly due to the Ni 2+ /Ni 3+ pair in the Fe-NiO nanosheets or on the semiconductor side. This surface accumulation of holes induces an internal electric feld to impede charge separation, resulting in an apparent slower rate than the bare semiconductor. Moreover, the thicker Fe-NiO nanosheets feature larger resistance/impedance for charge transport. Taken as a whole, we observed relatively low photocurrent on the 2-Mo-BiVO 4 flm modifed with Fe-NiO-120 nanosheets. For comparison, we have also checked thick catalyst layers (e.g., 90 nm), only to observe increased photocurrents during the frst scanning. The performance precipitated drastically during the following scans, due to the slow charge transfer to the electrolyte. Interestingly, the ultrathin Fe-NiO layer is permeable to the alkaline electrolyte and possess the fast charge transfer ability. The performance enhancement as observed in our experiments should be attributed to the ultrathin Fe-doped NiO nanosheets. They not only enable relatively high charge transfer efficiencies but also increase charge storage at the interface. Therefore, we understand this phenomena as the "adaptive" behaviours of the ultrathin nanosheets, 9 where the photogenerated holes are easily transferred to the redox pairs. By strong contrast, the thick catalyst would form a less "adaptive" junction due to its poor electrical and ionic conductivity, inducing a serious charge recombination and impeding charge transport. 6 The band structure is schematically illustrated in Fig. 7(a) and (b). The enhanced water oxidation performance is important evidence for the charge storage function of the ultrathin Fe-doped NiO nanosheets as water oxidation catalysts. ## Conclusion In this work, we have utilized transient surface photovoltage spectroscopy for the investigation of charge kinetics at the semiconductor|electrocatalyst interface. The Mo-doped bismuth vanadate flms have been prepared by the conversion of Bi metal flms through a two-step reaction. We used the ionexchange method for the synthesis of ultrathin Fe-doped NiO nanosheets, which could be conveniently applied onto the 2-Mo-BiVO 4 flms. On the 2-Mo-BiVO 4 /Fe-NiO samples, we found that charge separation to the surface led to charge accumulation and eventual annihilation following a frstorder consecutive reaction mechanism. A charge storage ($3 times) effect was confrmed on the interface between the ultrathin Fe-NiO nanosheet and the Mo-BiVO 4 surface, which signifcantly enhanced the photoelectrochemical performance. The fndings obtained from the planar semiconductor/electrocatalyst system should be easily applied to nanostructured photoelectrodes, which can further increase the photocurrent densities as reported by others. 13,14,21 Both the semiconductor|2D electrocatalyst and quantitative transient surface photovoltage analysis may be applied in photoelectrochemistry and other photoelectronic felds for broader impacts. ## Conflicts of interest There are no conflicts to declare.

chemsum

{"title": "Ultrathin Fe-NiO nanosheets as catalytic charge reservoirs for a planar Mo-doped BiVO₄ photoanode", "journal": "Royal Society of Chemistry (RSC)"}

a_practical_and_scalable_system_for_heteroaryl_amino_acid_synthesis

## Abstract: A robust system for the preparation of b-heteroaryl a-amino acid derivatives has been developed using photoredox catalysis. This system operates via regiospecific activation of halogenated pyridines (or other heterocycles) and conjugate addition to dehydroalanine derivatives to deliver a wide range of unnatural amino acids. This process was conducted with good efficiency on large scale, the application of these conditions to amino ketone synthesis is shown, and a simple protocol is given for the preparation of enantioenriched amino acid synthesis, from a number of radical precursors. ## Introduction Amino acids play a central role in the chemical and biological sciences. As primary members of the chiral pool, they are precursors to drugs, 1 chiral auxiliaries, 2 and catalysts. 3 In addition, they are fundamental building blocks for the construction of biomolecules. The use of peptides as therapeutic agents is attractive because they can display extremely diverse, potent, and selective biological activities. 4 However, there are signifcant challenges in peptide drug design, including low metabolic stability or poor physical properties. One proven strategy for overcoming these challenges involves substitution of the native residues with unnatural amino acids (synthetic mutagenesis). 5 Nitrogen-containing heteroaromatics are common in pharmaceuticals because they directly alter the solubility, metabolic stability, and binding affinity of the molecules that they comprise. 6 As such, heteroarene-containing unnatural amino acids are promising tools in the design of peptide therapeutics. Pyridine incorporation has a dramatic impact on the properties of amino acids and peptides. For example, azatyrosinea natural product that differs from the essential amino acid tyrosine by substitution of a single atom-displays potent antibiotic and antitumor properties (Fig. 1A). 7 Installation of the 3-pyridylalanine (3-pyr-Ala) residue in the gonadotropinreleasing hormone antagonist cetrorelix (Fig. 1B) was found to improve both aqueous solubility and receptor affinity, 8 and similar effects were observed in the development of other peptide hormones (not shown). 5b-d As part of a program centered on the catalytic functionalization of heteroaromatics, we target the development of impactful synthetic methods for the construction of novel b-heteroaryl a-amino acids through a radical conjugate addition mechanism. We have found that pyridyl halide activation via single electron reduction using photoredox catalysts 9 can be accomplished, and that the intermolecular reactivity of the resulting radical species can be dictated by the reaction conditions. 10,11 More specifcally, we found that pyridyl radicals display nucleophilic reactivity in aqueous DMSO, and they readily couple with electron-poor alkenes. We questioned whether this approach could be translated to heteroaryl amino acid synthesis through radical conjugate addition to dehydroalanine derivatives. There are a number of powerful methods for the synthesis of unnatural b-heteroaryl a-amino acids, including malonate (or enolate) alkylation, 12 cross-coupling of serine-derived organometallic reagents, 13 and reduction of dehydroamino acid derivatives. 14 However, strategies based on radical addition to DHA derivatives are unique due to the highly-chemoselective nature of radical species, and the broad functional group tolerance that results. 15 Alkyl radical addition to DHA has been effectively accomplished even in the complex setting of intact proteins. 16 While this is a highly attractive attribute, a radical approach to heteroaryl amino acids is currently unknown. Here, we describe the successful translation of our reductive heteroarene activation system to amino acid synthesis. ## Results and discussion Shown in Fig. 2 is a mechanistic picture that is consistent with our observations. Excitation of the photocatalyst [Ir(ppy) 2 (dtbbpy)]PF 6 ([Ir] 1+ ), followed by reductive quenching of the excited state by Hantzsch ester (HEH) gives rise to the [Ir] 0 (E 1/2 ¼ 1.51 V). 17 Stern-Volmer quenching studies indicated that Hantzsch ester is the most signifcant excited state quencher (see ESI for details †). Single electron reduction of halo pyridine I, followed by rapid mesolytic cleavage in polar solvents (X ¼ Br, I) 18 affords heteroaryl radical intermediate II, which exhibits nucleophilic radical behavior in aqueous DMSO. 10a It is possible that halopyridine reduction is assisted by protonation, as each catalytic turnover produces an nominal equivalent of Hantzsch pyridinium bromide (HEH + Br ). Hydrodehalogenation (HDH) of the arene is observed as a common byproduct, but this undesired pathway can be suppressed by limiting the solubility of the stoichiometric reductant, Hantzsch ester (HEH), in accord with our previous fndings. Radical conjugate addition (RCA) to dehydroalanine III and subsequent single electron reduction of the nascent radical IV would deliver the corresponding enolate V. The intermediacy of V is supported by the fact that the a-H amino acid product VI is produced in the presence of H 2 O as a cosolvent (regardless of H/D labeling of HEH). Conversely, when D 2 O is used as a cosolvent, complete deuterium incorporation is obtained at the a-position. As illustrated in Table 1, we identifed conditions that efficiently unite 2-bromo-5-hydroxypyridine with the indicated dehydroalanine derivative (readily accessed on 35 g scale from Boc-Ser-OMe) to give the protected azatyrosine 1 in 98% NMR yield (entry 1). These conditions employ 1 mol% of the photosensitizer [Ir(ppy) 2 (dtbbpy)]PF 6 (excited by irradiation with a commercial blue LED) and Hantzsch ester (1.5 equiv.) as a stoichiometric reductant in aqueous DMSO. Control experiments indicated that all of these components are necessary for the reaction (entries 2-4, 0% yield), and that use of the prototypical Ru(bpy) 3 2+ chromophore results in product formation, although with diminished efficiency (entry 5, 58% yield). Omission of water as a cosolvent was not well tolerated here (entry 6, 14% yield), a fnding that is in consistent with our previous observations. 10a We found that other aqueous solvent mixtures can be used (entries 7 and 8, 35% and 71% yield, respectively), and that this photoredox system is remarkably robust; an experiment using bourbon as solvent (open to air) afforded the desired product in 93% yield (entry 9). Importantly, protection of the phenol O-H function was not required under these mild radical conditions. Using the optimized protocol outlined above, we found that the heteroaryl halide scope of this transformation is broad (as shown in Table 2). Some reactions are complete in as little as 2 hours, but each experiment was conducted overnight (16 h) for consistency and convenience without negatively impacting the yields. Regiospecifc activation of each pyridyl position is possible via single electron reduction, and these conditions effectively delivered amino ester products from 2-and 3-iodopyridine (2 and 10), in 97% and 73% yield, respectively. Although less efficient, 4-iodopyridine also affords 4-pyridylalanine in useful yield (16, 34% yield), where reductive pyridine production is a signifcant alternative pathway. Methyl substitution is well-tolerated at all positions of 2-bromo pyridines, cleanly furnishing the corresponding pyridylalanines 3-6 in very high yield (93-97% yield). Reaction of 2-bromo-5-trifluoromethylpyridine ( 7) efficiently afforded product in 94% yield. Electron-donating groups are well-tolerated including amino (9, 71% yield), phenol (11, 67% yield), amide (12, 73% yield), and methoxy (17, 66% yield) groups. Dihalogenated pyridines can be programmed for regiospecifc radical formation and subsequent conjugate addition at any position, preserving 2-chloro-substituents in the presence of more reactive iodo-substituents. Coupling reactions of 2-chloro-3iodo-( 14), 2-chloro-4-iodo-( 18), 2-chloro-5-iodo-( 13), and 2chloro-3-methyl-4-iodopyridine ( 19) each gave single pyridylalanine products in good yield (73-83% yield). 2,5-Diiodopyridine is selectively activated at the more electrophilic 2position to afford the corresponding amino ester ( 8) as a single regioisomer in 74% yield. We found that halopyrimidines are also viable substrates in this process: 4-iodo-2-(methylthio) pyrimidine ( 15) and 4-bromodeazapurine (21) gave product in 80% and 95% yield respectively. This photoredox process is amenable to gram-scale preparation of heteroaryl amino acid synthesis, without the need for special equipment. We reacted 25 mmol of 2-bromopyridine with a slight excess (1.2 equivalents, 30 mmol) of the dehydroalanine substrate. In the presence of 1.0 equivalent of Hantzsch ester, in the presence of 1.0 equivalent of Hantzsch ester, and only 0.1 mol% (23 mg) of the iridium photoredox catalyst, the desired pyridylalanine derivative 2 was produced in 84% yield (8.0 g) after purifcation. As anticipated, selective unveiling of the amine and acid groups (in compound 2) using standard conditions went without issue. Hydrolysis of the methyl ester (2.0 equiv. of LiOH in THF/H 2 O) occurred with preservation of both Boc groups. Exposure of 2 to trifluoroacetic acid in CH 2 Cl 2 revealed the free amine as the TFA salt while leaving the methyl ester intact. Finally, sequential treatment of 2 with KOH in EtOH/H 2 O followed by direct acid-ifcation of the reaction mixture with HCl afforded the fully deprotected 2-pyridylalanine as the double HCl salt. Each of these processes occurred in high yield at room temperature (see ESI for details †). We conducted a brief evaluation of the scope of aminosubstituted alkenes with the expectation that this reaction template could be flexibly utilized to deliver other amino acid or amino-carbonyl substructures. We found that dehydroamino acid substrates with methyl-and phenyl-substituents in the bposition could be successfully employed, giving rise to products 22 and 23 in acceptable yield (66% and 54% yield, respectively) with modest diastereocontrol. Replacement of the a-imide group in the alkene starting material (a structural artifact of dehydroalanine synthesis via Boc 2 O-induced b-elimination) with an N-H aniline group or electronically diverse arylmethylamine groups was tolerated, although diastereoselectivity was low (25-28, 66-75% yield, #3 : 1 dr). These radical conjugate addition conditions directly translated to the synthesis of b-heteroaryl a-amino ketone derivatives 29-31, giving the desired products in 64-77% yield. These results are notable because they show the ability of this mild radical system to accomplish the formation of other of a-aminocarbonyl classes. We have demonstrated that this process is robust, scalable, and generally applicable for the synthesis of many heteroaryl amino acid and ketone derivatives. However, we recognize that the formation of products as racemic mixtures represents a main limitation of this method. To address this, we prepared the chiral tert-butyl oxazolidinone 32 that was described by Beckwith, 19 building on early work by Karady, 20 and Seebach. 21 In accord with early studies, we found that heteroaryl radical addition followed by diastereoselective protonation from the less hindered Re-face could be achieved with a variety of haloheteroarenes, furnished syn-products 33-36 with complete diasterocontrol (57-80% yield, >20 : 1 dr). Concurrent carbamate cleavage and hemiaminal hydrolysis of 36 under acidic conditions cleanly afforded the amino acid 37 with retention of stereochemical purity (98% yield, 97% ee) (Table 3). Other reducible radical precursors can be employed without modifcation of the reaction conditions to afford oxazolidinone adducts as single diastereomers. For example, the reaction of allyl bromide gives oxazolidinone 39 (42% yield). A redox-active N-hydroxyphthalimide ester 22 reacted to give 39 in high yield (86% yield). Finally, reducible fluorinated alky halides operate within this manifold, affording oxazolidinone adducts 40-42 with good efficiency (60-93% yield). Deprotection of two of these products would directly yield fluorinated amino acids which have been enabling tools in a number of biomedical applications. 23 For example, the difluorinated phosphonate L-pSer minic (deprotected 41) is an important tool in the study of kinase-dependent signal transduction. 23a Because chiral alkene 32 is easily accessible from cysteine (detailed in the ESI †), and both enantiomers of this starting material are commercial, this strategy would enable access to either enantiomer of the unnatural heteroaryl amino acids (Table 4). ## Conclusions In summary, we have described an efficient catalytic system for the preparation of unnatural a-amino acids. This protocol is effective for regiospecifc generation of a broad range of heteroaryl radicals, and intermolecular coupling with dehydroamino acid derivatives and a-aminoenones. We demonstrate that this photoredox system can be conducted on large scale using nearstoichiometric conditions with good efficiency. We also show that diastereoselective radical conjugate addition to a chiral alkene is a viable strategy to access enantioenriched products, and that this process allows utilization of a range of radical precursors. The application of these fndings to the synthesis of other valuable, highly complex products is a current aim of our program.

chemsum

{"title": "A practical and scalable system for heteroaryl amino acid synthesis", "journal": "Royal Society of Chemistry (RSC)"}

a_dnazyme-amplified_dna_circuit_for_highly_accurate_microrna_detection_and_intracellular_imaging

## Abstract: Biomolecular self-assembly circuits have been well developed for high-performance biosensing and bioengineering applications. Here we designed an isothermal concatenated nucleic acid amplification system which is composed of a lead-in catalyzed hairpin assembly (CHA), intermediate hybridization chain reaction (HCR) and ultimate DNAzyme amplifier units. The analyte initiates the self-assembly of hairpin reactants into dsDNA products in CHA, which generates numerous trigger sequences for activating the subsequent HCR-assembled long tandem DNAzyme nanowires. The as-acquired DNAzyme catalyzed the successive cleavage of its substrates, leading to an amplified fluorescence readout. The sophisticated design of our CHA-HCR-DNAzyme scheme was systematically investigated in vitro and showed dramatically enhanced detection performance. As a general sensing strategy, this CHA-HCR-DNAzyme method enables the amplified analysis of miRNA and its accurate intracellular imaging in living cells, originating from their synergistic signal amplifications. This method shows great potential for analyzing trace amounts of biomarkers in various clinical research studies. ## Introduction Isothermal nucleic acid amplifcation technologies have recently attracted widespread interest in clinical diagnosis due to their rapid and efficient accumulation of nucleic acid sequences at constant reaction temperature. 1,2 This amplifcation strategy provides an alternative tool for nucleic acid amplifcation considering the limitation of conventional polymerase chain reactions (PCRs), which always requires complex thermocycling. 3 Many enzyme-based isothermal amplifcation methods have thus been proposed, including rolling circle amplifcation (RCA), 4 loop-mediated isothermal amplifcation, 5 isothermal multiple displacement amplifcation, 6 single primer isothermal amplifcation, 7 and so on. Yet these exonuclease and polymerase enzymes tend to bring low stability and high cost into these different amplifcation systems. The complicated biological environment might also bring unexpected interference into these different molecular recognition and amplifcation processes. In addition, these fragile enzymatic systems always encounter product inhibition through the accumulation of pyrophosphate. Thus, it is highly desirable to implement enzyme-free isothermal DNA amplifcation procedures in highperformance biosensing research studies. DNAzymes are important nucleic acids that show fascinating catalytic functions. 8 They can mimic the functions of protein enzymes and catalyze a wide range of chemical reactions, including DNA ligation and cleavage. The multiple turnover rate of a DNAzyme turns it into an ideal signal amplifcation candidate for high-performance biosensing applications. The efficiency of DNAzyme amplifcation could be improved by integrating its functional sequence with other amplifcation means. 12,13 Besides DNAzymes, the catalytic DNA circuit, including the hybridization chain reaction (HCR) and catalyzed hairpin assembly (CHA), 17,18 is also emerging as a typical enzyme-free amplifcation strategy. The HCR mediates the target-initiated autonomous cross-opening of hairpin reactants for assembling long nicked dsDNA copolymers. 19,20 The generated dsDNA products could be utilized as versatile nanocarriers by encoding various functional DNA sequences or small molecules. CHA promotes the catalyzed hybridization of hairpins for assembling numerous dsDNA products without consuming the target. These approaches could be facilely conjugated with other amplifcation procedures to achieve an improved sensing performance. 24,25 For example, the RCA reaction can be encoded with tandem initiator sequences acting as CHA triggers, which then realize an extra CHA amplifcation without any interference in the initial RCA amplifcation. 26 All these nonenzymatic amplifcation methods have been applied for detecting different biologically important analytes (nucleic acids, 27,28 proteins, small molecules, 32 and metal ions 33 ) with different transduction approaches, such as fluorescence, 34,35 colorimetry, chemiluminescence (CL) 39 and electrochemical approaches. 40,41 MicroRNAs (miRNAs) are attractive post-transcriptional small RNA molecules 19-23 nucleotides in length, and can regulate the expression of given messenger RNAs (mRNAs) and the corresponding proteins. And their dysregulated expressions are closely related to cancer development, progression and therapy resistance, 45 which makes them a clinically crucial class of diagnostic and prognostic biomarkers. 46 MiRNA-21(miR-21) has been demonstrated to be an upregulated miRNA in many tumor types. 47,48 Thus the development of highly sensitive miRNA sensing platforms represents an urgent requirement for detecting low abundance miRNAs without elaborate separation and enrichment processes in the complex intracellular environment. 49,50 Both the catalytic DNA circuit and functional DNAzyme are utilized for intracellular miRNA imaging. 51,52 Yet these individual amplifers are always confronted with limited versatilities and unsatisfactory signal gains, which could be solved by their integration with other amplifcation strategies. The in-depth integration of these different amplifcation methods attracts more attention since a higher signal gain is expected to be realized in live cell analysis. Herein, we constructed an isothermal autonomous nucleic acid amplifcation system, consisting of CHA, HCR and DNAzyme amplifers, for high-performance biosensing applications. The analyte is translated by the CHA amplifer to assemble plenty of dsDNA products to realize the frst stage of amplifcation. And the generated CHA products are encoded with HCR trigger sequences for stimulating the subsequent HCR-involved cross-opening of hairpin reactants and for simultaneously activating the DNAzyme biocatalysts. In the ultimate DNAzyme amplifcation stage, the HCR-assembled DNAzyme then sustainably cleaves its substrates, leading to an amplifed fluorescence readout. By full use of these different amplifcation means, our isothermal amplifcation strategy realized the sensitive and selective detection of the analyte. The CHA-HCR-DNAzyme method could be considered as a general amplifcation module for realizing the amplifed analysis of miR-21 in an ideal buffer and for accurately localizing the target in living cells via fluorescence imaging. This approach could be extended to parallelly analyze more different biomarkers upon their integration with other recognition elements (aptamers) and multiple fluorescence transductions, and shows great potential for clinical diagnosis and prognosis. ## Results and discussion The principle of our isothermal cascading CHA-HCR-DNAzyme system is schematically illustrated in Fig. 1. The autonomous CHA-HCR-DNAzyme circuit is composed of CHA, HCR and DNAzyme amplifer units. All of the hairpins involved in the system are metastable (domains x and x* are complementary to each other), and can only be initiated by their corresponding triggers to form the energetically favored nicked long dsDNA nanowires analogous to alternating polymers. The product of upstream CHA should be able to trigger downstream HCR to integrate the efficient CHA-HCR-DNAzyme circuit. That is, the trigger of the HCR circuit should be carefully engineered and encoded into the hairpin reactants of the CHA system. In addition, the split DNAzyme of HCR reactants should also be reconstituted into DNAzyme subunits, of which the DNAzyme can only be activated by the concomitant assembly of DNA nanowires. The hierarchical hybridization acceleration character of this CHA-HCR-DNAzyme circuit contributes to the progressively sequential signal amplifcation. To start with, two hairpins, H 1 and H 2 , are involved in the upstream CHA amplifer (Fig. S1 †). the two separated DNAzyme segments of H 3 and H 5 for continuously integrating DNAzyme units. In the presence of Mg 2+ -ion cofactors, these newly assembled DNAzymes are activated to cleave a specifc ribonucleotide-containing substrate S whereby each end was attached with FAM and BHQ-1 fluorophores, respectively. The close proximity of these two fluorophores leads to the efficient quenching of FAM. And the DNAzyme-cleaved substrate triggers the continuous separation of FAM and BHQ-1, leading to a substantial fluorescence increase in the DNAzyme amplifcation stage. In summary, the cascading CHA-HCR-DNAzyme circuit can realize an enhanced amplifcation efficiency by implementing an exquisite integration of CHA, HCR, and DNAzyme schemes. The target-catalyzed successive CHA reaction amplifes the HCR trigger for further generating tandem DNAzyme nanowires with permanent activity. Each DNAzyme catalyses the cleavage of substrates to generate an amplifed fluorescence readout. The triple cascading amplifcation circuit was then systematically investigated as follows. The dual amplifer (CHA-DNAzyme or HCR-DNAzyme) and the triple amplifer (CHA-HCR-DNAzyme) were successively investigated for demonstrating the efficient biocircuit integration. As shown in Fig. 2A, both CHA-DNAzyme (curve c*) and HCR-DNAzyme (curve b*) systems show a signal readout for their dual amplifcation, while the triple amplifer (CHA-HCR-DNAzyme) shows a much higher fluorescence signal (curve a*), indicating a dramatic signal amplifcation efficiency of the triply amplifed system. What is more, the comparison of dual and triple amplifers also exemplifes the synergistic cooperation between CHA and HCR upon combining them, which realizes a relatively better sensing efficiency than that of them alone. Meanwhile, these three different DNA circuits show nearly no signal leakage without their corresponding initiators (Fig. 2B), demonstrating the metastable nature of the triply amplifed circuit. To further exhibit the upstream CHA-ampli-fed HCR reaction, atomic force microscopy (AFM) was carried out to investigate the supramolecular copolymer product of CHA-HCR-DNAzyme amplifer (Fig. 2C). Micrometer-long linear DNAs are obtained ($1.5 nm in height, a characteristic height of dsDNA nanochains) for the activated CHA-HCR-DNAzyme system (Fig. 2C inset). The partial bundling might be attributed to the cross-interactions between DNAzyme subunits. In contrast, only tiny spots are observed without any assembled products for the non-activated CHA-HCR-DNAzyme system (Fig. S3 †), suggesting that no undesired hybridization occurred between these hairpin components. The feasibility of the proposed strategy was further verifed by a 9% PAGE experiment (Fig. 2D). As compared to the H 1 + H 2 mixture (lane i), an obvious band of CHA product appeared upon its incubation with 50 nM initiator (lane ii). Similarly, the HCR mixture produces long dsDNA polymers upon its incubation with the 50 nM trigger (lane iv) while showing no spontaneous hybridization reaction without its trigger (lane iii). As expected, almost no background leakage is observed for the CHA-HCR-DNAzyme mixture without the trigger (lane v). Compared to the HCR control system (lane iv), the CHA-HCR-DNAzyme system generates a tremendous amount of highmolecular-weight HCR copolymers (lane vi), showing consistent efficiency with previous fluorescence assay (Fig. 2B). Both gel electrophoresis and AFM experiments demonstrated that the CHA-HCR-DNAzyme system proceeded with high efficiency as anticipated. Thus, the triply integrated amplifcation system could be utilized as a new high-performance sensing platform for a broad range of biosensing applications. Meanwhile, the high catalytic activity of the CHA-HCR-DNAzyme-assembled DNAzyme was also investigated by introducing an intact DNAzyme as a positive control (Fig. S4 †). For a deeper understanding of the effects of each hairpin on the reaction process, the one hairpin-excluded CHA-HCR-DNAzyme system was studied by a fluorescence experiment (Fig. S5 †). Despite the introduction of an initiator, no obvious signal difference was observed for the H 1 -, H 2 -, or H 4 -lacking system. This is reasonable for the efficient blockage of the CHAproducing HCR target or DNAzyme-assembling procedures. Yet the H 6 -lacking CHA-HCR-DNAzyme system retained the function of CHA-DNAzyme (slight fluorescence enhancement) even when the HCR process is blocked. Thus, all hairpin components are indispensable to the execution of their specifc functions as anticipated. All reaction procedures play crucial roles in the ultimate performance of our system. All of these hairpins need to be designed to realize a better signal to background ratio (S/B), e.g., H 1 was screened to ensure a better reaction performance (Table S3 and Fig. S6 †). As for DNAzyme, the present CHA-HCR-DNAzyme system (consisting of two DNAzyme-subunit-integrated hairpins) represents the optimized amplifer (Fig. S7 †). The amplifcation efficiency decreased after further integration of a DNAzyme subunit into the present triple amplifer, which is attributed to the steric hindrance of the newly introduced DNAzyme-subunit-grafted hairpins. In other words, the crosshybridization rate and completeness of HCR could be tremendously slowed down by the DNAzyme grafting fragments which could not be compensated by the doubly assembled DNAzyme. What's more, the concentration of hairpin reactants and DNAzyme substrate was appropriately optimized to guarantee a higher signal gain without undesired leakage (Fig. S8 †). Under the optimized conditions, the CHA-HCR-DNAzyme system was applied for DNA detection in vitro. The mixture of CHA-HCR-DNAzyme and substrate S was challenged with a DNA initiator of varied concentrations for 4 h. From the resulting fluorescence spectra (Fig. 3A), the absolute fluorescence change (DF) increases substantially with increasing concentrations of analyte (Fig. 3B). A quasi-linear correlation is obtained between DF and the concentration of the initiator ranging from 10 pM to 1 nM (Fig. 3B inset). Based on the traditional 3s/s calculation method, the limit of detection (LOD) was found to be 1.5 pM for the CHA-HCR-DNAzyme amplifer. In addition, the CHA-DNAzyme and HCR-DNAzyme amplifer controls were also used to analyze the same target of varied concentrations (Fig. S9 †). At a higher concentration of the same initiator I (50 nM), the integrated CHA-HCR-DNAzyme circuit revealed $1.7-fold higher amplifcation than that of the HCR-DNAzyme system, and $3-fold higher amplifcation than that of the CHA-DNAzyme system. Note that the CHA-HCR-DNAzyme system shows a much higher amplifcation capacity than the CHA-DNAzyme control system at a lower concentration range. This is reasonable since the amplifcation capacity of CHA, HCR and DNAzyme components could be more sufficiently realized for the integrated CHA-HCR-DNAzyme system, especially at the lower concentration of analyte. Besides sensitivity, the selectivity of the CHA-HCR-DNAzyme system was also examined by using one-, two-, and three-base mutant DNAs (I a , I b , and I c , Fig. S10 †). No obvious fluorescence enhancement was observed for these mutants, implying that the CHA-HCR-DNAzyme system affords high selectivity by realizing a clear single-base discrimination. The enzyme-free homogeneous CHA-HCR-DNAzyme realized the ultrasensitive detection of the DNA target within several hours which is substantially shorter than that of the autocatalytic DNAzyme system. 56 The integrated CHA-HCR-DNAzyme strategy shows a similar or even better sensing performance than that of the other nonenzymatic fluorescence microRNA detection strategies consisting of CHA, HCR or DNAzyme strategies (Table S2 †), thus showing great promise for the bioanalytical and clinical applications. The satisfactory sensitivity and robustness characters of the newly developed CHA-HCR-DNAzyme system are attributed to the synergistic signal amplifcation of these integrating CHA, HCR and DNAzyme components. The CHA-HCR-DNAzyme system can be applied as a general amplifer for various applications by further integrating an intermediate recognition/transduction element. Here, a sensing module hairpin H 7 is designed to include an miR-21-recognition function and thus consists of an miR-21 complementary sequence and the locked I sequence. As illustrated in Fig. 4, the miR-21 opens H 7 to expose the CHA initiator I (dark blue) that triggers the effective CHA-HCR-DNAzyme amplifcation. The fluorescence spectra of the extended sensing platform were acquired after the CHA-HCR-DNAzyme system was incubated with various concentrations of miR-21 (ranging from 10 pM to 50 nM) for 4 h (Fig. 5A). The relationship between fluorescence changes (DF) and miR-21 concentrations was formulated to acquire the calibration curve (Fig. 5B). A detection limit of 5 pM was obtained for the miR-21 analyte (Fig. 5B inset), demonstrating that this CHA-HCR-DNAzyme system could be adopted for a general amplifcation module for analyzing any other analyte with the incorporation of a recognition module. Apart from sensitivity, we further investigated the selectivity of our c), implying that the CHA-HCR-DNAzyme system affords high selectivity for analyzing miR-21 with singlebase mutation discrimination. Moreover, we also examined the selectivity of this system by choosing several representative interfering nucleic acids, including b-actin mRNA, son DNA and let-7a miRNA (Fig. S11 †). All of these interfering RNAs generate no obvious fluorescence response that is approximately equal to the blank control without an initiator. This indicates the high selectivity of our newly established CHA-HCR-DNAzyme-ampli-fed miR-21 detection strategy. Moreover, the performance of the miR-21-detecting system shows little interference in complex biological fluids, e.g., diluted 10% and 20% serum buffer (Fig. 5D). Having demonstrated the satisfactory amplifcation efficiency of the updated CHA-HCR-DNAzyme in an ideal buffer, we then challenged the present system for miR-21 imaging in living cells (Fig. 6). After transfection into MCF-7 cells via lipofectamine 3000, these hairpin reactants were further incubated with cells at 37 C for 4 h. Obviously, a signifcant green fluorescence image was observed in MCF-7 cells (sample a, Fig. 6A). As expected, the miR-21 inhibitor-pretreated MCF-7 cells show a much lower fluorescence readout (sample b, Fig. 6A), demonstrating that it is miR-21 that mediates the amplifed intracellular imaging procedure. Almost no fluorescence readout is observed for the H 4 -excluded CHA-HCR-DNAzymeimaging MCF-7 cells (sample c, Fig. 6A). Evidentially, the fluorescence readout of MCF-7 cells is ascribed to miR-21-specifc assembly of hairpin probes. The relative fluorescence intensity of the CHA-HCR-DNAzyme imaging system is acquired from the statistical analysis of large quantities of their respective living cells (Fig. 6B). To further emphasize the present miR-21imaging system in different cell lines, Hela cells were introduced as an indispensable control with low miR-21 expression and showed a weak fluorescence readout. MRC-5 cells were introduced as an important negative control since they were encoded with no miR-21 expression, and showed negligible fluorescence readout (Fig. S12 †). A low expression of miR-21 is observed in MRC-5 cells, as compared to MCF-7 cells. Clearly, the CHA-HCR-DNAzyme system can distinguish different cell lines based on their different endogenous miRNA expressions. The H 6 -excluded CHA-HCR-DNAzyme imaging system was also employed as a CHA-DNAzyme control, and showed a signifcantly reduced intracellular signal amplifcation performance (Fig. S13 †). And, almost no fluorescence signal was observed when the indispensable H 1 , H 2 or H 4 was excluded from the system. This result is consistent with that of fluorescence assay, indicating that all hairpin reactants are indispensable to the present intracellular CHA-HCR-DNAzyme circuit. ## Conclusions In conclusion, we have developed a versatile signal amplifcation platform based on the isothermal concatenated CHA-HCR-DNAzyme strategy for robust intracellular imaging applications. The upstream CHA recognizes the specifc initiator to produce numerous dsDNA strands with encoding HCR trigger sequences for stimulating the cross-opening of hairpin reactants in downstream HCR. The HCR-generated long dsDNA copolymers are encoded with tandemly integrated DNAzymes that cleave the fluorophore-labeled substrates to generate a tremendously amplifed fluorescence readout. This CHA-HCR-DNAzyme system realized the efficient transduction of an analyte through the multiple successive amplifcation cascades. The successive reaction accelerations and the multiple guaranteed recognitions make the present system a highly robust and promising sensing platform. And this sophisticated DNA circuit is facilely designed for recognizing and localizing other different analytes by further integrating an additional molecular recognition module. Specially, the molecularly engineered CHA-HCR-DNAzyme system was used for highly efficient and accurate intracellular miR-21 sensing. This versatile sensing platform could be utilized to detect trace amounts of intracellular biomarkers by introducing aptamers or other functional DNA sequences in clinical research studies. ## Experimental Materials 4-(2-Hydroxyethyl) piperazine-1-ethanesulfonic acid sodium salt (HEPES), magnesium chloride and sodium chloride were purchased from Sigma-Aldrich (MO, USA). The DNA marker, GelRed, fetal bovine serum (FBS) and Lipofectamine 3000 transfection reagent were purchased from Invitrogen (Carlsbad, CA). Dulbecco's Modifed Eagle Medium (DMEM) and Opt-MEM were purchased from HyClone (Logan, Utah, USA). MCF-7 and MRC-5 cells were obtained from Shanghai Institutes for Biological Sciences (SIBS). Trypsin was purchased from Genview (USA). All DNA primers were synthesized and HPLC-purifed by Sangon Biotech Co., Ltd. (Shanghai, China). The ribonucleobase (rA)-containing substrate was purchased from TaKaRa Bio. Inc. (Dalian, China), and was labeled at the 5 0 -and 3 0 -ends with the fluorophore/quencher pair (FAM/BHQ-1). Table S1 † depicts the sequences of all used oligonucleotides. ## Fluorescence assay To ensure that the hairpin reactants form the desired secondary structure, all hairpins need to be annealed at 95 C for 5 min, rapidly cooled and left to stand at 25 C for two hours at least in HEPES buffer (10 mM, pH 7.2, 1.0 M NaCl, 50 mM MgCl 2 ). Double-distilled ultrapure water was used in all experiments. The concentrations of hairpins involved in this system were optimized to get a better signal to background ratio. Then, different concentrations of target DNA were mixed with all the as-prepared hairpins (200 nM) and DNAzyme substrate (0.5 mM) for acquiring the fluorescence changes with an interval of 10 min at 25 C. As for the analysis of miR-21, the concentration of "helper" hairpin H 7 was 50 nM, and the concentration of any hairpin was consistent with DNA detection. Fluorescence measurements were conducted using a spectrophotometer with l ex ¼ 490 nm and l em ¼ 520 nm for FAM, respectively. ## Gel electrophoresis verication A 9% native polyacrylamide gel was prepared and rapidly transferred to a glue frame and polymerized for 40 min at 25 C. The gel was washed with water three times and soaked in 1 TBE buffer. The loading samples were prepared via a loading buffer and then added into these different notches of gel for electrophoresis. PAGE was run at room temperature at 120 V for 3 h. After staining in a newly prepared Gel-Red solution (20 min), the gel was transferred to a plate and was characterized by using a FluoChem FC3 (Protein Simple, USA) imaging system. ## Atomic force microscopy (AFM) imaging The newly cleaved mica surface (Structure Probe Inc., USA) was incubated in MgCl 2 (5 mM) for 10 min at room temperature, and then rinsed with ultrapure water for adsorbing these different DNA nanostructures. The hairpin mixture/product (with/without initiator I) of the CHA-HCR-DNAzyme system was diluted with HEPES buffer (10 mM, pH 7.2), and then deposited on the pretreated mica surface. The topological features of our CHA-HCR-DNAzyme mixture and products were characterized in tapping mode by using Multimode-8 AFM apparatus equipped with a NanoScope V controller (Bruker). ## Cell culture and imaging analysis Here the cell culture media are Dulbecco's Modifed Eagle Medium (DMEM) and Modifed Eagle Medium (MEM), which are respectively used for human breast cancer cells (MCF-7, Hela) and human embryo pulmonary fbroblasts cells (MRC-5). These culture media were further supplemented with 10% FBS and 1% penicillin/streptomycin for growing the corresponding cells at 37 C in a humidifed atmosphere containing 5% CO 2 . These different cells were digested with trypsin, re-suspended in 1 mL of new DMEM or MEM medium, and incubated in glassbottom culture dishes for 12 h before cell attachment. The miR-21-analyzing CHA-HCR-DNAzyme reactants were incubated in 200 mL of Opti-MEM for 5 min, and were then introduced into another 200 mL of Opti-MEM (containing 5 mL lipofectamine 3000) for 10 min. The old cell media were replaced with a new Opti-MEM medium (containing 50 mL FBS and the updated CHA-HCR-DNAzyme reactants) for incubating at 37 C for 4 h. Afterward, the cultured cells were washed three times with PBS and were transferred into 500 mL of freshly prepared medium for confocal laser scanning microscopy (CLSM) characterization. For the miR-21 inhibitor experiment, MCF-7 cells were transfected with an extra anti-miR-21 oligonucleotide to reduce the intracellular miR-21 content (1 h) and then were transfected and incubated with the CHA-HCR-DNAzyme system for 4 h. All cellular fluorescence images were acquired using a CLSM system (Leica TCS SP8). And these samples were excited at 488 nm with an accompanying emission ranging from 500 nm to 580 nm for the green channel of the FAM fluorophore. ## Conflicts of interest The authors declare no conflict of interest.

chemsum

{"title": "A DNAzyme-amplified DNA circuit for highly accurate microRNA detection and intracellular imaging", "journal": "Royal Society of Chemistry (RSC)"}

bottom-up_creation_of_an_artificial_cell_covered_with_the_adhesive_bacterionanofiber_protein_ataa

## Abstract: The bacterial cell surface structure has important roles for various cellular functions.However, research on reconstituting bacterial cell surface structures are limited. This study aimed to bottom-up create a cell-sized liposome covered with AtaA, the adhesive bacterionanofiber protein localized on the cell surface of Acinetobacter sp. Tol 5, without the use of the protein secretion and assembly machineries. Liposomes containing a benzylguanine derivative-modified phospholipid were decorated with a truncated AtaA protein fused to a SNAP-tag expressed in a soluble fraction in Escherichia coli. The obtained liposome showed a similar surface structure and function to that of native Tol 5 cells and adhered to both hydrophobic and hydrophilic solid surfaces. Furthermore, this artificial cell was able to drive an enzymatic reaction in the adhesive state. The developed artificial cellular system will allow for analysis of not only AtaA, but also other cell surface proteins under a cell-mimicking environment. In addition, AtaA-decorated artificial cells may inspire the development of biotechnological applications that require immobilization of cells onto a variety of solid surfaces. ## Introduction An artificial cell is a cell-like compartment that harbors various compounds and biological systems, thereby mimicking part of the cellular functions. Bottom-up creation of an artificial cell has been regarded as one of the approaches to understand the cellular functions that are too complex to interpret in conventional "top-down" studies 1 . Mimicking the cellular functions with defined molecules enables us to remove the complexity from a system, making it easier to interpret the dynamics or the behavior induced by the molecules. Furthermore, bottom-up creation of an artificial cell occasionally provides unexpected results that lead to new insights into biology and inspires researchers to develop new technologies 2,3 . To date, the liposome has been one of the most popular and cell-mimicking compartments used to create artificial cells. 3 Cellular functions such as uptake of substrates 4,5 , protein translocation 6 , phospholipid biosynthesis 7 , cell division and related processes 8,9 , membrane protein evolution 10,11 , and cascade reaction by a genetic circuit have been introduced into liposomes. Unlike liposomes, the surface of a living cell is structurally complex due to the presence of various proteins, including integral and peripheral membrane proteins, as well as cell appendages. These structures play important roles for cellular functions such as ligand recognition, cell-cell communication, motility, and adhesion. Nevertheless, bottom-up creation of an artificial cell that mimics bacterial cell surface structures is limited. More than 90% of environmental bacteria live in an adhesive state rather than a planktonic state 15,16 ; thus, adhesion to a solid surface is critical for the lifestyle of bacteria. Bacterial adhesion to solid surfaces has also an advantage in wide range of bioprocesses, most of which are conducted by enzymes, including bioproduction, bioremediation, and wastewater treatment, as adherent bacteria can be immobilized even under flow conditions, and can enhance their capability through high-density accumulation of cells 17 . Adhesion to a solid surface is achieved by various molecules, including exopolymeric substances produced by bacteria, or via the presence of cell surface appendages 17 . However, artificial cells harboring enzymes that adhere to solid surfaces by mimicking the bacterial cell surface have not yet been created. One of the reasons for this is the difficulty in synthesizing proteins on the bacterial cell surface, which mostly contains transmembrane or membrane interacting domains. In addition, natural bacterial cells use transport machinery to secrete and assemble proteins on the cell surface 18,19 . Although there has been some partial success 6,20 , full reconstitution of such complex molecular machinery in artificial cells is yet to be achieved. Trimeric autotransporter adhesin (TAA) is a cell appendage that mediates adhesion of Gram-negative bacteria to solid surfaces 21 . TAA forms fibers on the order of ten to hundreds of nanometers in length, composed of three polypeptides encoded by a single gene, i.e., a homotrimer. AtaA, a TAA discovered in a sticky Gram-negative bacterium, Acinetobacter sp. Tol 5, forms peritrichate nanofibers ≈225 nm in length and 4 nm in thickness on the cell surface 22 . Most of the TAAs reported to date exhibit specific adhesiveness to biotic surfaces such as extracellular matrix proteins on host tissues, whereas AtaA nonspecifically adheres to abiotic surfaces made of various materials, such as hydrophobic plastics, hydrophilic glasses, and metals. Furthermore, the adhesiveness mediated by AtaA is much higher than that mediated by YadA, which is the most wellstudied TAA. Due to this adhesive feature of AtaA, bacteria covered with AtaA fibers can be immobilized on the surface of various materials 23,24 . In this study, we aimed to create cell-sized liposomes covered with the adhesive nanofiber protein AtaA, thereby creating an artificial cell that adheres to various solid surfaces and can perform a reaction catalyzed by an encapsulated enzyme (Figure 1). Our strategy for the construction of surface-decorated artificial cells without the use of complex transport machinery allows for characterization and functional analyses of not only AtaA, but also other peripheral membrane proteins and cell appendages under cellmimicking environments in the absence of other cell surface components. ## Bacterial cell-size liposome was decorated with the adhesive nanofiber protein AtaA by the interaction between a SNAP-tag fused with the AtaA and benzylguanine (BG)-group on the liposome. Since βglucuronidase (GUS) is encapsulated within the liposome, the enzymatic reaction occurs on a solid surface. ## Decorating cell-sized liposomes with AtaA Natural bacterial cells use transport machinery to secrete and assemble huge cell appendages on the cell surface 18,19 . Full reconstitution of such complex molecular machinery in artificial cells is yet to be achieved. To cover artificial cells with AtaA, we combined chemical synthesis and protein engineering; i.e., we used the chemical reaction between a benzylguanine (BG) derivative-modified phospholipid and AtaA fused to the SNAP-tag (Figure 1). The SNAP-tag is a 20-kDa protein that forms a covalent bond with a BG derivative 25,26 . First, we designed and constructed a plasmid for the expression of a fusion protein of an AtaA fragment and the SNAP-tag in Escherichia coli. Membrane proteins and proteins with high molecular weights (> 60 kDa) are generally difficult to express in E. coli 27 . Hence, we assumed it difficult to express the full-length of AtaA in E. coli, because AtaA is a huge protein whose molecular weight is over 350 kDa and its C-terminal trans-membrane (TM) domain was embedded in the outer membrane. To decrease the molecular weight of AtaA while retaining its function and to enable its expression in the cytoplasm of E. coli, we deleted its signal peptide (AtaA1-58), Chead, Cstalk, and TM domains (AtaA2904-3630), which are not essential for its adhesive function 28 , yielding a truncated AtaA, NheadNstalk (NhNs)-AtaA (280 kDa) (Figure 2a). Because the GCN4 adaptor 29 assists in the trimerization of recombinant AtaA fragments 28 , the GCN4 adaptor sequence was connected to the leucine residue at the C-terminus of NhNs-AtaA, followed by the SNAP-tag and Strep-tag (8 amino acids). Because the NhNs-AtaA peptide trimerizes and the SNAP-tag is a monomer protein, the resulting chimera polypeptide should form a fusion protein of a trimer of truncated AtaA and three SNAP-tag molecules. This fusion protein was designated as NhNs-AtaA-SNAP. When NhNs-AtaA-SNAP was expressed in E. coli, more than half of the protein appeared in the soluble fraction, suggesting that a significant fraction of the expressed protein was folded properly (Figure S1). This is one of the largest fusion proteins that forms a complex quaternary structure (≈1 MDa when forming trimers) that is synthesized as a recombinant protein in the cytoplasm of E. coli. The bacterial cell size-liposomes (on average about 0.8 µm in diameter) were prepared by mixing BG-modified 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) 30 and Egg yolk phosphatidylcholine (Egg-PC) denoted as BG-liposome or using only Egg-PC denoted as EggPC-liposome as a control. For liposome decoration with NhNs-AtaA-SNAP, these liposomes were mixed with the supernatant of a cell lysate from E. coli BL21 (DE3) harboring a plasmid encoding NhNs-AtaA-SNAP (pNhNs-SNAP), denoted as BL21 (pNhNs-SNAP). Because these liposomes can be harvested by centrifugation, the decorated liposome should be obtained as precipitants. To confirm liposome decoration with NhNs-AtaA-SNAP, the precipitants were subjected to SDS-PAGE and subsequent immunodetection using anti-AtaA antiserum and anti-SNAP antibody. Signals were detected by both antibodies when BG-liposome was mixed with the supernatant of the cell lysate of BL21 (pNhNs-SNAP), but not when EggPC-liposome was mixed with the same lysate (Figure 2b). No signal was detected when BG-and Egg-PC liposomes were mixed with the supernatant of a cell lysate from E. coli BL21 (DE3), denoted as BL21 (WT). This result suggests that the SNAP-tag fused to a huge complex of truncated AtaA was functional. The liposome with NhNs-AtaA was further analyzed using fluorescence cytometry (FCM). All liposomes containing fluorescence dye Alexa Fluor 647 (AF647) in their aqueous phase for detection purposes were immunostained with anti-AtaA antiserum followed by an anti-rabbit IgG antibody conjugated to Alexa Fluor 488 (AF488). As shown in Figure 2c, the two-dimensional plot displayed the fluorescence signals of both AF488 and AF647 when BG-liposomes were treated with the cell lysate of BL21 (pNhNs-SNAP), whereas only the fluorescence signal of AF647 was detected from other liposomes. The results of the immunodetection and FCM analysis suggest that liposomes were decorated by NhNs-AtaA-SNAP via the covalent bond between the SNAP-tag and the BG-group. the primary antibody. c) Fluorescence cytometry (FCM) analysis of liposomes. The liposomes treated with the cell lysates were immunostained with anti-AtaA antiserum and anti-rabbit IgG conjugated to AF488. AF647 was encapsulated inside both liposomes. Orange, blue, and red dots represent liposomes treated with 10 mM Tris-HCl buffer (pH 9.0), the supernatant of the cell lysate from BL21 (WT), and the cell lysate from BL21 (pNhNs-SNAP), respectively. ## AtaA on the liposome forms the nanofiber To investigate whether NhNs-AtaA forms a nanofiber structure on the liposome, the size distribution of the decorated liposome was analyzed by dynamic light scattering (DLS). In the case of bacterial cells (Tol 5 and its DataA mutant), the size distribution by DLS analyses displayed a clear difference between the presence and absence of AtaA fibers (Figure 3a). Note that DLS analyses were performed under a condition where AtaA exhibit less adhesive activity (see Methods). The dominant size of Tol 5 cells was about 440 nm larger than that of DataA cells; this difference nearly corresponds to the size predicted from the length of native AtaA (225 nm × 2) 31 . Since the length of the NhNs-AtaA fiber was deduced to be about 180 nm, the size of the decorated liposome should be larger than a non-decorated one. In non-decorated BG-and EggPC-liposomes, their peak of the size distribution was 825 nm in diameter (Figure S2a). When a BG-liposome was treated with the cell lysate from BL21 (pNhNs-SNAP), the peak at 825 nm shifted to 1281 nm (Figure 3b); this difference nearly corresponds to the size predicted from the length of NhNs-AtaA (180 nm × 2). The peak shift did not occur when an EggPCliposome was treated with the cell lysate containing NhNs-AtaA-SNAP (Figure S2b). Furthermore, we attempted to directly observe the surface of the BG-liposome decorated with NhNs-AtaA-SNAP. Based on previous observations of Tol 5 cells using transmission electron microscopy (TEM), the NhNs-AtaA part of the fusion protein should be visible as a nanofiber on the BG-liposome 22,32 .. As expected, many fibrous structures were observed on the surface of BG-liposomes treated with the cell lysate containing NhNs-AtaA-SNAP, whereas no fibers were visible on non-decorated BG-liposomes (Figure 3c). The DLS results and TEM image provided evidence for the decoration of the BGliposome with NhNs-AtaA fibers, and demonstrated that we successfully created an artificial cell partially mimicking the bacterial cell surface structure without the use of membrane translocation machinery. In addition, the features of the observed nanofiber, which strongly resembles those of Tol 5 cells 22,32 , strongly suggests the formation of a trimer of NhNs-AtaA with adhesive function. ## Artificial cells exhibit adhesive functions To determine whether NhNs-AtaA fibers on BG-liposomes have an adhesive function, we subjected liposomes to the adherence assay using two 96-well plates with different physicochemical properties: one consisting of hydrophobic polystyrene (PS), and the other of hydrophilic glass; native AtaA fiber adheres to both of these surfaces 22 . Since all liposomes contained AF647 in their aqueous phase, the liposomes adhering to the surface could be evaluated by their fluorescence. To efficiently contact the liposomes, whose densities were close to that of water, with the bottom surfaces, the plates were weakly centrifuged, and then unbound (non-adhesive) liposomes were washed out. As a result, significant fluorescence signals were detected from both PS and glass-bottom plates with decorated BG-liposomes, and their fluorescence intensities increased with increasing protein concentration of cell lysate used for preparation of decorated BG-liposomes (Figure 4ab). Conversely, the increase in fluorescence intensity was not detected when EggPC-liposome was treated with the same cell lysate. To further confirm that the increase in fluorescence intensity could be attributed to the adhesive function of NhNs-AtaA, we inhibited the decoration of BG-liposome with NhNs-AtaA-SNAP in two different ways: one was the blocking of BG-groups on a liposome using SNAP-tagged GFP; the other was the inactivation of the SNAP-tag fused with NhNs-AtaA using SNAP-Surface Block, a compound that reacts with the SNAP-tag. Both of the inhibition treatments significantly decreased the fluorescence intensity on the plate surface (Figure S3). These results indicate that NhNs-AtaA-SNAP is coupled to the BG-liposome via the BG and SNAP-tag interaction and the NhNs-AtaA fiber exhibits adhesive features similar to those of native AtaA fiber. NhNs-AtaA-SNAP retained the functions of both AtaA and SNAP-tag. Finally, we examined if the constructed artificial cell can drive an enzymatic reaction inside a liposome adhering to the plate surface. As a model enzyme, β-glucuronidase (GUS) was encapsulated in BG-and EggPC-liposomes. These liposomes were treated with the supernatant of the cell lysate containing NhNs-AtaA-SNAP, placed into wells of the PS plates, and immobilized on plate surfaces following the adherence assay procedure described above. We then added TokyoGreen-βGlu 5 , a membrane-permeable substrate for GUS, which emits fluorescence only after hydrolysis to monitor the enzymatic reaction. A significant increase in fluorescence intensity was detected only from wells on which NhNs-AtaA-SNAP-decorated liposomes were immobilized (Figure 4c). This result indicates that GUS encapsulated in a liposome is active inside the decorated liposome immobilized on the plate surface. ## Discussion This study aimed to create, using a bottom-up approach, an artificial bacterial cell capable of adhering to solid surfaces. This was accomplished by assembling BG-modified cell-size liposomes and a truncated AtaA-SNAP fusion protein that exists in complexes as large as 1 M Da (trimer of 305 kDa). We did not use the protein secretion machinery (translocon) 18 or β-barrel-assembly machinery (BAM) complex 19 involved in the formation of appendages like AtaA in natural cells. The TEM image of the NhNs-AtaA-SNAP-decorated liposome shown in Figure 3c bears a striking resemblance to TEM images of Tol 5 22,32 . This is the first study to create a bacterial mimic of a cell surface structure from defined materials. One strategy for characterizing the extracellular part of a cell surface protein of interest is to investigate it directly at the cellular level. Although this strategy is useful, many other proteins are present on the cell surface, and their effects on the properties of the protein of interest are difficult to eliminate. In vitro analyses using a purified protein produced by its original strain or recombinant strains, typically after removing the transmembrane domain, is an alternative approach to characterizing the extracellular part of the cell surface protein of interest. Although this method gives useful information about molecular characteristics of the protein of interest, the actual functions and characteristics of the protein on the cell surface are difficult to realize due to uncontrolled orientation, direction, and localization. In this study, we synthesized a truncated AtaA recombinant protein without the signal peptide or transmembrane domain in the cytosol of E. coli, and succeeded in immobilizing it on the liposome surface by simply mixing the supernatant of an E. coli cell lysate with BG liposomes. Unlike isolated protein, the orientation of the recombinant protein fiber assembled on the liposome surface mimics its intact condition on the cell surface and its function on bacterial cells. As long as the extracellular part of cell surface proteins can be synthesized in the cytosol of an E. coli or other cells, this strategy should be applicable to other cell surface proteins for their characterization and functional analyses on the artificial cell membrane in the absence of other surface proteins. In the DLS analysis of the NhNs-AtaA-decorated liposome, a small peak was observed between 4000 and 6500 nm, which might correspond to a small fraction of liposome cluster. Native AtaA mediates autoagglutination as well as adhesion to solid surfaces, but these adhesive functions are lost in the condition of low ionic strength 33 . When preparing the sample for DLS analysis, Tol 5 cells were suspended in pure water to prevent the formation of cell aggregates. Conversely, a liposome decorated with NhNs-AtaA-SNAP was analyzed by DLS in 10 mM Tris-HCl buffer, a condition where cell aggregates are formed when using Tol 5 cells. However, the intensity of the peak observed on the decorated liposome was weak and the cluster size was small; under the same condition, the cell aggregates of Tol 5 were too large to be analyzed by DLS (data not shown). Therefore, the ability of NhNs-AtaA on the liposome to cause autoagglutination is thought to be quite low compared with native AtaA on Tol 5 cells. Although the mechanism of the difference between NhNs-AtaA and native AtaA remain unclear, the adhesive nature without autoagglutination of NhNs-AtaA might be convenient for the biotechnological application of functional liposomes. A mammalian cell specifically adheres to other cells and the extracellular matrix (ECM), namely, biotic solid surfaces via cell surface proteins such as cadherin and integrin. Artificial cells mimicking a mammalian cell surface were constructed by adding integrin to the liposome surface . These artificial cells exhibited adhesion to ECMcoated solid surfaces. In addition to specific adhesion to biotic surfaces, bacterial cells nonspecifically adhere to abiotic surfaces via the presence of cell appendages 17 . In particular, AtaA exhibits remarkably high adhesiveness, thereby immobilizing bacterial cells onto various solid surfaces . Unlike artificial cells mimicking the mammalian cell surface, those mimicking the bacterial cell surface can adhere to both hydrophobic and hydrophilic surfaces via NhNs-AtaA without ECM-coating of the solid surfaces. This feature should be beneficial for biotechnological applications that require immobilization of an artificial cellular system onto a variety of solid surfaces. Living cells (often modified genetically) immobilized onto solid supports have been used as whole cell catalysts for bioproduction, bioremediation, and wastewater treatment 17 , despite the risk of release of genetically modified organisms into the environment. These artificial cells may be used as an alternative in these processes. Artificial cells have the advantage that all of their extracellular and intracellular components are designed. In this study, we encapsulated GUS inside the artificial cell and employed the membrane-permeable substrate as a model system. GUS can be substituted with other enzymes including those that are difficult to handle with living cells such as membrane-associated enzymes and/or enzymes that exhibit cell toxicity. Membraneimpermeable substrates can also be used by forming nanopores on an artificial cell membrane, for example with α-hemolysin 4 . Therefore, artificial cells may be useful for developing new biotechnological applications encapsulating various chemical reaction systems, mimicking whole-cell catalysts. Unlike living cells, artificial cells do not replicate, but can still catalyze reactions of interest on a solid surface. These properties of artificial cells may be attractive in environments where the use of genetically modified organisms is prohibited. Although the artificial cell constructed in this study was robust enough to endure mixing with cell lysates, immobilizing to solid surfaces, and performing an enzyme reaction, further stabilization by introducing an artificial cytoskeleton, for example by incorporating DNA origami technology 37 , may give versatile catalytic activity under a wide range of conditions. In summary, using a bottom-up approach, we succeeded in constructing an enzymeencapsulating artificial cell that adhered to solid surfaces. This artificial cellular system is expected to reveal the properties of cell surface proteins without interference from other cell surface components, and to inspire the development of new biotechnological applications that require cell immobilization onto a variety of solid surfaces. ## Construction of plasmids The primers used in this study are listed in Table S1. A DNA fragment encoding AtaA59-325 was amplified from pDONR::ataA 22 by a PCR using the primer set AtaA59F/AtaA325R, digested with XbaI and BsaI, and ligated into the same site of pIBA-GCN4tri-His 29 , generating pIBA-AtaA59-325-GCN4tri-His. Subsequently, a DNA fragment encoding SNAP-tag was amplified from pSNAPf Vector (New England Biolabs Inc, Ipswich, MA) by a PCR using the primer set SNAPF/SNAPR. By using an In-Fusion HD Cloning Kit (Takara Bio, Shiga, Japan), this amplicon was fused to a DNA fragment amplified from pIBA-AtaA59-325-GCN4tri-His by an inverse PCR using the primer set HisF/GCN4R, generating pIBA-AtaA59-325-SNAP-His. To add a BglII site for further cloning, a DNA fragment was amplified from pIBA-AtaA59-325-SNAP-His by an inverse PCR using the primer set iPCR-BglII-F/iPCR-BglII-R and then self-ligated, generating Bacterial strains and culture conditions E. coli BL21 (DE3) and its transformant harboring the pNhNs-SNAP plasmid were grown at 37°C in Luria-Bertani (LB) medium. Acinetobacter sp. Tol 5 and its DataA mutant were grown at 28°C in LB medium. Ampicillin (100 μg/mL) was added for the E. coli transformant. For the production of the NhNs-AtaA-SNAP recombinant protein, E. coli transformant cells were grown to an optical density at 600 nm (OD600) = 0.5-0.7, and thereafter, 0.20 μg/mL anhydrotetracycline was added. After incubation at 18°C for 16 h, cells were harvested, resuspended in a buffer (25 mM Tris-HCl, 20 mM imidazole, 150 mM NaCl, pH 9.0), lysed by sonication, and centrifuged at 10,000 g for 10 min. To confirm production of NhNs-AtaA-SNAP in the E. coli strain, supernatant and pellet fractions were subjected to SDS-PAGE analysis. ## SDS-PAGE and immunodetection To examine the decoration of liposomes with NhNs-AtaA-SNAP, liposome suspensions were mixed with the same volume of SDS-sample buffer [0.125 M Tris-HCl buffer (pH 6.8), 4% (wt/vol) SDS, 10% (wt/vol) sucrose, 0.01% (wt/vol) bromophenol blue, 10% (wt/vol) 2-mercaptoethanol], heated at 100°C for 5 min, and subjected to SDS-PAGE. For immunodetection, the proteins were transferred to a PVDF membrane with a constant current of 100 mA for 90 min. The blotted membrane was blocked for 1 h at room temperature with a 5% (wt/vol) skim milk solution, and treated for 1 h at room temperature with anti-AtaA699-1014 antiserum 22 or anti-SNAP antibody (Medical & Biological Laboratories Co., Ltd, Nagoya, Japan) at a dilution of 1:10,000 or 1:2,000 in phosphate-buffered saline (PBS) containing 0.05% (vol/vol) Tween 20 (Calbiochem) (PBS-T), respectively. NhNs-AtaA-SNAP on the membrane was detected with a horseradish peroxidase-conjugated anti-rabbit IgG antibody (GE Healthcare) at a dilution of 1:10,000 in PBS-T, and visualized using EzWestLumi plus (ATTO). ## Preparation of liposomes Three hundred microliters of 50 mg/mL egg phosphatidylcholine (COATSOME NC-50 (EPC)) (Yuka-Sangyo Co., Ltd., Tokyo, Japan) dissolved in chloroform was rotated under vacuum in a round-bottom flask for 1 h. The lipid film was hydrated with buffer A (10 mM HEPES, pH 7.6, 50 mM potassium glutamate) supplemented with 25 µM Alexa Fluor 647, or buffer B (10 mM Tris-HCl [pH 9.0]) to obtain 300 µL of 50 mg/mL lipid solution. For the samples with GUS, 2 µM GUS was added to buffer A. GUS was produced and purified as described previously 41 . For the samples for electron microscopy observation, 1 mg/mL BSA was added to buffer B. The lipid solution was sonicated for 10 min and vortexed for 10 s. The lipid solution was further subjected to five rounds of freeze-thaw cycles. The liposome suspension was then extruded with a mini-extruder (Avanti Polar Lipids, Alabaster, AL, USA) using a 0.8 μm VCTP isopore membrane filter at room temperature. The prepared large unilamellar vesicle (LUV) was washed by adding 1,200 µL of buffer A or buffer B to 300 µL LUV solution prepared with buffer A or B, respectively; centrifuging at 20,000 g for 30 min; and replacing the supernatant with 1,200 µL of fresh buffer A or B. This washing step was repeated four times. BG-liposomes were prepared as follows. First, 14 µL of 2 mM BG-DSPE 30 dissolved in chloroform was rotated under vacuum in a glass micro test tube for 15 min, hydrated with buffer A or buffer B. This BG-DSPE solution was then added to a final concentration of 93 µM to the LUV solution and incubated at room temperature for 20 h followed by the four-times washing steps described above. ## Decoration of liposome with NhNs-AtaA-SNAP BG-and EggPC-liposome suspensions were mixed with 2.0 mg/mL of cell lysate extracted from either E. coli BL21 (DE3) or its transformant harboring the pNhNs-SNAP plasmid. After 1 h of incubation at 4°C on a rotary mixer, the liposome particles were precipitated by centrifugation at 15,000 g for 10 min to remove unbound NhNs-AtaA-SNAP and other proteins. The precipitated liposome particles were washed twice with 10 mM Tris-HCl (pH 9.0) buffer and resuspended in the same buffer. When inhibiting the decoration of NhNs-AtaA-SNAP to the liposome, BG-liposome suspension was incubated with a purified SNAP-GFP at a final concentration of 20 µM, or 2.0 mg/mL of the cell lysate containing NhNs-AtaA-SNAP was incubated with SNAP-Surface Block (New England BioLabs Japan Inc., Tokyo, Japan) at a final concentration of 1-100 µM in 100 µL of 10 mM Tris-HCl buffer (pH 9.0) for 1 h at 4°C by inversion mixing. ## Measurement of dynamic light scattering The size of a liposome or bacterial cell was measured by dynamic light scattering using Zetasizer Nano ZSP (Malvern Instruments, UK) equipped with a He-Ne laser (wavelength, 633 nm). Liposome suspensions were diluted to 50-fold in 10 mM Tris-HCl buffer (pH 9.0) for DLS measurement. Cells of Tol 5 and DataA mutant were harvested by centrifugation at 8,000 g, resuspended in deionized water, and adjusted to an OD660 = 0.05 with deionized water. Quartz cuvettes were filled with the samples and all the experiments were thermostatically controlled at 25°C. All the DLS measurements were made with a scattering angle of 173°. The results were given as diameters and the percentages correspond to intensity values. ## Fluorescence cytometry For fluorescence cytometry analysis, the liposome suspension was diluted 100-fold in 10 mM Tris-HCl buffer (pH 9.0). Liposomes were treated with anti-AtaA699-1014 antiserum 22 ## Electron microscopy The liposome suspension was diluted 50-fold in 10 mM Tris-HCl buffer (pH 9.0). The liposomes were adsorbed to carbon-coated copper grids (400 mesh) and were stained with 2% phosphotungstic acid solution (pH 7.0) for 10 s. Subsequently, the grids were performed with vacuum drying for 10 min. Grids were observed under a TEM (JEM-1400 plus; JEOL Ltd., Tokyo, Japan) at an acceleration voltage of 100 kV. Digital images (3296×2472 pixels) were taken with a CCD camera (EM-14830RUBY2; JEOL Ltd., Tokyo, Japan). ## Adherence and enzymatic assay Forty-five microliters of liposome suspension was placed into a 96-well polystyrene For the enzymatic assay by GUS encapsulated in a liposome adhering to the plate surface, liposome suspension was diluted 50-fold in 10 mM Tris-HCl buffer (pH 9.0) and 50 µL of the suspension was placed in a PS well plate. Liposomes were adhered to the plate surface as described above. As a substrate of GUS, 50 µL of 10 mM Tris-HCl buffer containing 10 μM TokyoGreen-β GlcU (GORYO Chemical, Inc, Sapporo, Japan) was added to each well after washing unbound liposomes. The hydrolysis reaction was detected as the fluorescence signal using the microplate reader at indicated time points. The excitation and emission wavelengths used were 485 and 535 nm, respectively.

chemsum

{"title": "Bottom-up creation of an artificial cell covered with the adhesive bacterionanofiber protein AtaA", "journal": "ChemRxiv"}

synthetic_investigation_of_competing_magnetic_interactions_in_2d_metal–chloranilate_radical_framewor

## Abstract: The discovery of emergent materials lies at the intersection of chemistry and condensed matter physics.Synthetic chemistry offers a pathway to create materials with the desired physical and electronic structures that support fundamentally new properties. Metal-organic frameworks are a promising platform for bottom-up chemical design of new materials, owing to their inherent chemical predictability and tunability relative to traditional solid-state materials. Herein, we describe the synthesis and magnetic characterization of a new 2,5-dihydroxy-1,4-benzoquinone based material, (NMe 2 H 2 ) 3.5 Ga 2 (C 6 O 4 Cl 2 ) 3 (1), which features radical-based electronic spins on the sites of a kagom é lattice, a geometric lattice known to engender exotic electronic properties. Vibrational and electronic spectroscopies, in combination with magnetic susceptibility measurements, revealed 1 exhibits mixed valency between the radical-bearing trianionic and diamagnetic tetraanionic oxidation states of the ligand. This unpaired electron density on the ligand forms a partially occupied kagom é lattice where approximately 85% of the lattice sites are occupied with an S ¼ 1 2 spin. We found that gallium mediates ferromagnetic coupling between ligand spins, creating a ferromagnetic kagom é lattice. By modulation of the interlayer spacing via post-synthetic cation metathesis of 1 to (NMe 4 ) 3.5 Ga 2 (C 6 O 4 Cl 2 ) 3 ( 2) and (NEt 4 ) 2 (NMe 4 ) 1.5 Ga 2 (C 6 O 4 Cl 2 ) 3 (3), we determined the nature of the magnetic coupling between neighboring planes is antiferromagnetic.Additionally, we determined the role of the metal in mediating this magnetic coupling by comparison of 2 with the In 3+ analogue, (NMe 4 ) 3.5 In 2 (C 6 O 4 Cl 2 ) 3 (4), and we found that Ga 3+ supports stronger superexchange coupling between ligand-based spins than In 3+ . The combination of intraplanar ferromagnetic coupling and interplanar antiferromagnetic coupling exchange interactions suggests these are promising materials to host topological phenomena. ## Introduction The discovery of new materials which host emergent phenomena lies at the intersection of condensed matter physics and synthetic chemistry. Certain lattice topologies, for example the kagomé lattice, which consists of corner sharing equilateral triangles, promote the creation of excitations which are different from those that occur in the building units of the lattice. This phenomenon is referred to as an emergent property a property in the collective which does not occur in the core unit. Beyond emergent electronic properties, many new magnetic phases arise in spin-based materials through exchange interactions governed by the lattice geometry and the active spin and orbital degrees-of-freedom dictated by the underlying chemistry. 1 Creating these materials from the ground up is a signifcant synthetic challenge which necessitates simultaneous control over both the local and extended structure and the electron flling of the frontier orbitals. The kagomé lattice is an especially promising structure to target as either antiferromagnet or ferromagnetic interactions within the lattice lead to emergent phenomena. Antiferromagnetic coupling of electronic spins on the kagomé lattice leads to magnetic frustration, which arises from the competing magnetic interactions that cannot be simultaneously satisfed. This magnetic frustration prevents the onset of magnetic ordering and results in a state known as a quantum spin liquid. 1 This state features an infnite number of degenerate magnetic ground states, and is predicted to host exotic fractionalized quasiparticles with applications in quantum computation 8 and high temperature superconductivity. 9 Alternatively, ferromagnetic coupling of electronic spins on a kagomé lattice leads to a different family of interesting electronic properties, and includes materials that are topological magnon band insulators, 4,5 exhibit skyrmionic excitations, 6 or host Dirac fermions. 7 Two-dimensional (2D) metal-organic frameworks (MOFs) are an attractive platform for the targeted design of such new emergent materials. In contrast to traditional solid-state materials such as metal oxides and minerals, which have inherently little chemical tunability, MOFs possess a high degree of modularity as they can be built up from molecular building blocks. This modularity encompasses the identity of the metal centre, the organic bridging ligand, and the interlayer spacing. In these systems, the unpaired spin density that gives rise to exotic electronic behaviour can reside on either the metal site 6,7, or on the organic ligand, expanding the number of viable synthetic targets. In these materials, the two primary components that dictate the electronic and magnetic properties are the intralayer interactions and the interlayer interactions. The former leads to the desired exotic properties of interest, while the latter often extinguishes the phenomena of interest. A prominent example is observed in antiferromagnetic kagomé materials, wherein the interlayer interactions often alleviate magnetic frustration. Judicious chemical design of both metal ion and organic ligand enables fne-tuning the electronic and magnetic properties of the intralayer kagomé lattice via a bottom-up chemical approach. Additionally, in 2D MOFs, the interlayer spacing in these materials can be modulated by intercalation of organic cations. We hypothesize that this will enable deconvolution of the magnetic behaviour as either the result of 2D interactions arising from the kagomé lattice or more complicated 3D interactions. This is an especially attractive feature of 2D MOFs over metal oxides and minerals. Towards this end, we synthesized a series of 2D honeycomb MOFs composed of 2,5-dichloro-3,6-dihydroxy-p-benzoquinone (chloranilic acid) and group 13 metals (gallium and indium). In this system, chloranilate (Fig. 1a) hosts a stable organic radical in its trianionic oxidation state, localizing unpaired spin density onto the vertices of the kagomé lattice (Fig. 1b). 21 This leads to the formation of an electronic spin based kagomé lattice on top of the structural honeycomb framework. In order to ensure the only unpaired spin density resides on the desired kagomé lattice sites, group 13 metals were chosen as the metal nodes for their diamagnetism and relative redox inertness. By preparing both the gallium and indium analogues, we are able to assess how the radial extent of the metal orbitals affect the strength of the spin-spin coupling mediated by metal-ligand superexchange. Finally, we modulate the interlayer spacing of the gallium chloranilate framework, enabling deconvolution of the inter-vs. intralayer magnetic interactions. ## Results and discussion We synthesized the frst 2D MOF of the series by reaction of Ga(NO 3 ) 3 $xH 2 O with chloranilic acid in dimethylformamide (DMF) and trace amounts of water at 130 C for 16 hours to produce green hexagonal crystals with the composition (NMe 2 H 2 ) 3.5 Ga 2 CA 3 (1). The structure of 1 was determined from Rietveld refnement of laboratory powder X-ray diffraction (PXRD) data. The PXRD pattern revealed the framework is isostructural with the known aluminum analogue (Fig. 1b) con-frming it maintains the desired kagomé lattice structure. 22 The Ga 3+ ions are octahedrally coordinated by three deprotonated chloranilate ligands to form the nodes of the honeycomb net. The pores of the honeycomb are 15.56 A in diameter and are flled with DMF solvent molecules and dimethylammonium cations formed from the decomposition of DMF during the reaction. Based upon structurally analogous frameworks and elemental analysis, these cations charge balance the anionic framework. The layers are eclipsed and are separated by an interlayer distance of 8.835(1) A. The layers stack in an ABAB pattern where neighboring layers are related by a mirror plane perpendicular to the c axis (Fig. S5 †). To assess the ligand oxidation state in 1, we performed vibrational spectroscopy as the C-C and C-O stretching modes are highly sensitive to the chloranilate ligand oxidation state. The frequency of the C-O stretching vibration should be largest in the CA 2 oxidation state and weaken as the ligand is reduced, whereas the C-C stretching vibration follows an opposite trend. After probing 1 using Raman spectroscopy, close inspection of the main band at 1453 cm 1 reveals an additional band at 1440 cm 1 (Fig. 2a). The closeness in energy of the two bands complicates assigning either as defnitively to the C-C or C-O stretch. However, either band, if assigned to the C-C stretch in 1, occurs at a much higher frequency than observed in the structural analogues (NMe 2 H 2 ) 2 Zn 2 (CA 2 ) 3 and (NMe 2 H 2 ) 1.5 (CoCp 2 ) 1.5 Fe 2 (CA 3 c ) 3 frameworks, which are isovalent in the dianionic and trianionic oxidation states of the ligand, respectively; 23 the remaining band assigned as the C-O stretch is also concomitantly much weaker in 1. Based on the observed vibrational frequencies and their near coalescence, it is evident the ligand is spontaneously reduced beyond a fully CA 3 c system. Comparison with fully reduced chloranilic acid (H 4 CA), which displays two C-C stretches at 1448 and 1500 cm 1 , eliminates the possibility that the bridging ligands in 1 are solely in the CA 4 state. Based on the aggregate of these data, we propose 1 hosts mixed valency between CA 3 c and CA 4 . To test our hypothesis of mixed valency, we investigated 1 by Fourier transform infrared (FTIR) spectroscopy. FTIR spectroscopy can often reveal low-lying intervalence charge transfer (IVCT) transitions in the near IR characteristic of delocalized mixed-valent species. 25 While the FTIR spectrum of 1 did not reveal any features characteristic of mixed valency, comparison of the spectrum of 1 to molecular compounds with chloranilate in well-defned oxidation states, namely (PPh 4 ) 3 [Ga(CA 2 ) 3 ] and [Ga(tren)] 2 (CA 4 )(BPh 4 ) 2 (tren ¼ tris(2-aminoethyl)amine), which feature chloranilate exclusively in exclusively the CA 2 and CA 4 states, respectively, allows for further characterization of the ligand oxidation state. The FTIR spectrum of 1 has two intense peaks closely spaced in energy at 1403 and 1383 cm 1 (Fig. S6 †). Both of these peaks are far weaker than would normally be assigned to the C-O double bond stretching vibration, and are much lower in energy than the C-O stretch at 1644 cm 1 in (PPh 4 ) 3 [Ga(CA 2 ) 3 ] supporting the absence of CA 2 . Additionally, the peak at 1383 cm 1 is considerably lower in energy than the C-C stretching mode in [Ga(tren)] 2 (CA 4 )(BPh 4 ) 2 (1467 cm 1 ). However, in the case of mixed valency of CA 3 c and CA 4 , there should only be one or two formal C-O double bonds per every three ligands. This change in bonding should significantly weaken (and strengthen) the C-O (and C-C) stretching vibrational mode. The aggregate of this data further supports our assignment of mixed-valent ligand oxidation states in 1 (Table 1). The absence of an IVCT band by FTIR spectroscopy motivated us to examine electronic absorption spectroscopy to further probe the ligand mixed valency in these frameworks. The diffuse reflectance data (Fig. 2b) collected for 1 featured many transitions across the energy range of inspection (7500-45 000 cm 1 ). The peak at $21 000 cm 1 was assigned as the p* / p* transition of CA 3 c and features a fne structure associated with the C-O vibrational modes which has been previously observed in molecular species containing CA 3 c . 26,27 The broad, intense electronic transition at 35 000 cm 1 is analogous to the p / p* transition of 1,2-dihydroxybenzoquinone and was likewise assigned to the same transition in chloranilate. 22,26 Of more immediate interest is the presence of a broad, lowintensity peak at 14 400 cm 1 which is tentatively assigned as an IVCT band. Due to the broadness, weak intensity, and relatively high energy of the peak, we assigned 1 as a weakly exchanging Class II Robin-Day mixed-valent material. 28 Similar low intensity features in the near IR were also observed in a structurally similar chromium(III) chloranilate framework and were assigned to an IVCT consistent with a localized electronic structure. 29 Conversely, strongly exchanging Class II and Class III Robin-Day mixed-valent chloranilate frameworks have lower energy and more intense IVCT bands. 29,30 The presence of an IVCT band in 1 is in contrast to mononuclear homoleptic gallium(III) complexes that are mixed-valent in the ligand oxidation state but lack an IVCT band in their electronic spectra. 31,32 In these complexes, gallium(III) is a poor bridging metal ion and does not facilitate strong electronic communication between the two oxidation states of the ligands. This suggests that interligand electronic communication is stronger in the framework than in corresponding molecular complexes. To test these assignments of the electronic transitions, we exposed 1 to air, leading to oxidation of the CA 3 c ligands to their CA 2 forms. Indeed, the ligands in 1 are oxidized to CA 2 , and as a result, the peaks assigned to CA 3 c and the IVCT disappear, concurrent with the appearance of a peak at 18 700 cm 1 , which is assigned to the p / p* transition of CA 2 (see Fig. S13 and S14 and ESI for extended discussion †). To quantify the degree of mixed valency in 1, we investigated its magnetic properties using SQUID magnetometry. Specifcally, variable-temperature dc magnetic susceptibility measurements allow us to directly assess the number of unpaired spins in the framework by quantitation of the hightemperature c M T value, thus elucidating the ratio of CA 3 c to CA 4 in 1. The 300 K c M T value of 1 is 0.96 cm 3 K mol 1 , which persists down to 100 K (Fig. 2c). This value is below the c M T value of 1.125 cm 3 K mol 1 expected for 3 uncoupled radical spins but above the c M T value of 0.750 cm 3 K mol 1 expected for 2 uncoupled radical spins. This c M T value corresponds to approximately 83% of the ligands being in the CA 3 c state, and 17% in the CA 4 , and further supports the ligand mixed valency suggested by our analysis of the vibrational and diffuse-reflectance data. Below 100 K, c M T rises and reaches a maximum value of 12.82 cm 3 K mol 1 at 2 K. This rise in c M T is attributed to ferromagnetic coupling between the ligand-based spins that does not however lead to magnetic ordering. To support the presence of signifcant ferromagnetic coupling in 1, we measured the magnetization of 1. At 1.8 K, the magnetization rapidly saturates (Fig. 2C, inset). By 0.15 T, the magnetization curve is no longer linear with feld, and by 0.50 T the material is fully saturated. We hypothesize the ferromagnetic coupling arises from intralayer coupling of spins within the same 2-D layer of the framework, as intralayer interactions are expected to be much stronger than interlayer interactions. This type of ferromagnetic interligand coupling was also observed in molecular complexes of Ga(III) tris-semiquinone complexes. 31,33,34 It was proposed that the empty 4p orbitals of Ga(III) mediated a ferromagnetic superexchange interaction between the radical bearing p* orbitals of the semiquinone ligands. 33 This pathway could also be responsible for the ferromagnetic coupling in 1. As noted above, competing magnetic interactions arising from interlayer and intralayer magnetic coupling often have dramatic effects on any potential exotic electronic behavior hosted by these materials. The synthetic modularity of this 2D framework yields an opportunity to investigate the nature of the interlayer magnetic coupling, and deconvolute it from intralayer magnetic interactions. We can probe the relative magnitude and nature of the interlayer coupling by modulation of the interlayer spacing by post-synthetic cation exchange. Based on literature reports of anilate MOFs synthesized with different alkyl ammonium cations without a distortion of the 2D lattice, we pursued the intercalation of bulky quaternary alkyl ammonium cations to expand the interlayer spacing without distorting the kagomé lattice. 35,36 Soaking 1 in a 0.15 M solution of NMe 4 OH in DMF at 75 C for 12 hours led to the isolation of (NMe 4 ) 3.5 Ga 2 CA 3 (2). Subsequently, soaking 2 in a 0.1 M solution of tetraethylammonium bromide in DMF at 75 C for 12 hours resulted in the formation and isolation of (NEt 4 ) 2 (NMe 4 ) 1.5 Ga 2 CA 3 (3). Comparison of the PXRD data across the series revealed a clear dependence of the (002) peak on cation metathesis, with a shift in the (002) peak from 10.21 to 8.82 to 8.61 in moving from 1 to 3 (Fig. 3a). These shifts in 2q point to changes in the interlayer spacing across the series, ranging from 8.835(1) A to 10.06(2) A to 10.155(4) A, from 1 to 3, respectively. These data suggest that as in isostructural frameworks with transition metal nodes, the NR 4 + cations reside between the Ga 3+ centres of neighbouring layers and act to modulate the interlayer spacing. 35,36 When this interlayer site is fully occupied, these sites account for two of the cations per formula unit; by elemental analysis, the remaining one and a half negative charges from the framework are charge balanced by tetramethylammonium ions we believe reside in the pore of the framework. Importantly, the incorporation of NR 4 + cations does not distort the 2D framework as the only affected peaks observed by PXRD are those associated with the c-axis, corresponding to the interlayer direction. Additionally, this cation metathesis process does not affect the ligand oxidation state, as the relevant vibrational modes observed by Raman and FTIR spectroscopy remain unchanged (see ESI Fig. S8-S10 †). To further probe the ligand oxidation states of 2 and 3, and to evaluate their magnetic properties, we collected variabletemperature dc magnetic susceptibility data. The 300 K magnetic moments measured for 2 and 3 resemble that for 1, further corroborating that post-synthetic cation exchange does not affect ligand oxidation state (Fig. 3b). Close inspection of the low-temperature (<50 K) c M T data reveals a divergence, leading to different maximum values of c M T at 3 K. To contrast the interlayer magnetic interactions across the series, the maximum c M T value (c M T max ) observed at low temperatures serves as a proxy for the overall strength of the ferromagnetic coupling in the material. The observed c M T max for 1 of 12.82 cm 3 K mol 1 dramatically increases upon interlayer expansion of 1.7 A, reaching a c M T max of 22.96 cm 3 K mol 1 in 2. Separating the layers further in 3 leads to a modest increase in c M T max to 25.20 cm 3 K mol 1 . For antiferromagnetic coupling between layers, c M T max should increase upon interlayer separation, as the magnetic coupling between layers weakens. Indeed, upon expansion of the interlayer spacing across the series, c M T max concomitantly rises, suggesting the presence of antiferromagnetic interlayer coupling. Though the enhancement of c M T max is more modest between 2 and 3 than it is from 1 to 2, it is important to note this change arises from an increased layer separation of only 0.10 A. Interestingly, the inset of Fig. 3b reveals c M T max varies relatively linearly with decreasing 1/R 3 where R is the layer separation, in the compounds. While the possibility of cation interaction cannot be excluded, these data demonstrate a clear trend with increasing layer separation. This observation supports the presence of antiferromagnetic dipolar coupling between the 2D kagomé layers. 37,38 We also sought to investigate the role of the diamagnetic metal in mediating radical-radical coupling by synthesizing the indium(III) analogue. However, the direct reaction of indium(III) nitrate hydrate and chloranilic acid in DMF and water leads to the formation of (NMe 2 H 2 ) 4 In 2 (CA 3 c ) 2 (CA 4 ), which hosts a different percentage of ligands in the radical state than 1 (see ESI for details †). In order to circumvent this obstacle, we targeted a tetramethylammonium based indium framework via direct reaction of the starting materials. Reaction of In(NO 3 ) 3 $xH 2 O, chloranilic acid, and 5 equivalents of NMe 4 OH in DMF produces (NMe 4 ) 3.5 In 2 (CA) 3 (4). Unlike (NMe 2 H 2 ) 4 In 2 (CA 3 c ) 2 (CA 4 ), 4 contains the same percentage ligands in their radical form as 1-3, allowing direct comparison of the magnetic behaviour of 2 and 4. The high temperature DC magnetic susceptibility data show the radical-bearing ligands are in the same percentage in 2 and 4 (see ESI †). At low temperatures, the magnetic behavior greatly differs, and c M T max is much greater for 2 than 4; the difference in c M T max is 15.5 cm 3 K mol 1 . We next performed electronic structure calculations to elucidate the microscopic origin of the enhanced spin-spin interactions (higher c M T max ) in the gallium compounds. Our density functional theory (DFT) calculations were performed using the Vienna Ab initio Software Package (VASP) with the projected augmented wave method. 39,40 The aforementioned compounds were modelled using the experimental structures as initial atomic confgurations without solvent molecules, i.e., as Ga 2 CA 3 (P6 3 /mcm symmetry) and In 2 CA 3 (P 3). 41 We achieved a CA 3 c (spin 1 2 ) confguration by electron doping the chloranilate anions and then imposing ferromagnetic spin alignment among these ligands within the 2D kagomé layers, which couple antiferromagnetically. The internal atomic positions of each structure was relaxed using the experimental volume. First, we found that the diamagnetic metal-oxygen bond lengths from the bidentate CA 3 c ligands were shorter for Ga 2 CA 3 (1.98 A) compared to In 2 CA 3 (2.16 A), consistent with the smaller ionic radius of Ga 3+ (187 pm) compared to In 3+ (220 pm). This leads to larger orbital overlap and enhanced charge density in the bonding region (indicated in Fig. S19 †), which initiates the superexchange path between sites, Ga-O-C-C-C-O-Ga; although the S ¼ 1 2 spin state is distributed about the chloranilate anion, it is predominately localized on the oxygen anions. In addition, the GaO 6 octahedral units are closer to ideal compared to the InO 6 owing to deviations of the intraoctahedral oxygen-metal-oxygen angles from 90 , which narrow the electronic bandwidth. As a result, the direct distance between coupled chlorinate anions and cations is signifcantly shorter in Ga 2 CA 3 (3.84 A) compared to In 2 CA 3 (4.049 A). Second, analysis of the electronic density-of-states reveals that in both compounds, the valence band is mainly comprised of the chloranilate anion states with chlorine 3p orbitals located below (from 5 to 3.5 eV) the oxygen 2p states (spanning 3 to 1.5 eV), whereas the conduction band consists of the main group s and p states. Although the empty gallium states are located at higher energy than those of indium (Fig. S19 †), they show a small overlap with the occupied oxygen 2p bands. This mixing is stronger for gallium compared to indium over the 4 to 2 eV energy range (Fig. S19 †). Furthermore, the Ga compound exhibits greater p-orbital occupancy (0.35e) than In (0.31e), demonstrating that there is stronger hybridization about the p states than s states (0.31e, 0.32e; Fig. S20 †). We therefore concluded that chelated diamagnetic metals favor ferromagnetic spin-spin coupling, owing to orthogonal symmetry of the p orbitals from the metal and oxide ligands consistent with trends reported for iminobenzosemiquinonato group 13 molecular complexes. 34,42 Moreover, because stronger spin-spin interactions arise when there are stronger hybridization (covalent interactions) among the active orbitals participating in superexchange, this coupling is greater for gallium owing to the greater exchange propagated by the orbital hybridization. Indeed, upon approximating the exchange interaction by fnding the energy difference between the FM and AFM confgurations (where a C ring has an opposite spin with respect to the other two rings), we found that energy difference was larger for Ga (40 K) compared to In (17 K). The observation of topological behaviour necessitates that each kagomé lattice site hosts a full S ¼ 1 2 spin, leading us to pursue post-synthetic chemical oxidation of the ligand to achieve a purely CA 3 framework. Previous work has demonstrated that honeycomb-type MOFs of chloranilate can successfully undergo single-crystal to single-crystal post-synthetic chemical reduction from a mixed-valent CA 2/3 c system to a fully CA 3 c system. 23 Thus motivated, we sought to treat the framework with an oxidant with the ability to oxidise CA 4 to CA 3 c , without effecting the oxidation of CA 3 c to CA 2 . Soaking 1 in a solution of ferrocenium hexafluorophosphate in acetonitrile results in oxidation of the CA 4 ligand to CA 3 c , as evidenced by vibrational and electronic spectroscopies. Peaks in the Raman spectrum grow in at 1390 and 1505 cm 1 (Fig. S29 †), which are consistent with the C-C and C-O stretches previously observed for CA 3 c . 23 However, a remnant Raman peak at 1445 cm 1 , suggests incomplete oxidation of the CA 4 ligand to the CA 3 c based framework. The IR peaks in 1 at 1405 and 1381 cm 1 undergo signifcant shifts to 1481 and 1361 cm 1 (Fig. S30 †), which is attributed to a strengthening of the C-O bond and weakening of the C-C bond, respectively, as the percentage of CA 3 c in the framework increases. Further, the oxidation of CA 4 to CA 3 c goes to completion as evidenced by the disappearance of the IVCT band at 14 500 cm 1 after post-synthetic oxidation (Fig. S31 †). Accurate quantitation of the magnetic properties of the oxidized material is precluded by the presence of excess ferrocenium ions in the pores of the framework. Ongoing work is focused on post-synthetic oxidation of 1-3 to the fully CA 3 c -based framework with the exclusion of paramagnetic cations convoluting our interpretation of the magnetic properties. ## Conclusions The bottom-up design of 2D materials with the potential to display exotic, emergent properties is an emerging challenge for synthetic chemists. Creating a pathway to realize these materials is a crucial step forward in an area dominated by traditional solid-state chemistry. The foregoing results demonstrate the synthesis of a series of 2D honeycomb MOFs using molecular building blocks geared towards accessing a kagomé lattice of unpaired electronic spins. The synthetic modularity of these 2D frameworks enables clear deconvolution of the inter-vs. intralayer magnetic interactions allowing the confrmation of ferromagnetic coupling within the kagomé plane and antiferromagnetic coupling between kagomé layers. Our data enabled us to establish the form of magnetic coupling within the layer and between the layers, thereby enabling us to assign intraplane ferromagnetic coupling interactions. The observation and confrmation of ferromagnetic coupling interactions within a kagomé plane suggests this may be a candidate for future study, in particular with the fully radical-bearing end member of the series. ## Conflicts of interest There are no conflicts to declare.

chemsum

{"title": "Synthetic investigation of competing magnetic interactions in 2D metal\u2013chloranilate radical frameworks", "journal": "Royal Society of Chemistry (RSC)"}

resolving_metal-molecule_interfaces_at_single-molecule_junctions

## Abstract: Electronic and structural detail at the electrode-molecule interface have a significant influence on charge transport across molecular junctions. Despite the decisive role of the metal-molecule interface, a complete electronic and structural characterization of the interface remains a challenge. This is in no small part due to current experimental limitations. Here, we present a comprehensive approach to obtain a detailed description of the metal-molecule interface in single-molecule junctions, based on current-voltage (I-V) measurements. Contrary to conventional conductance studies, this I-V approach provides a correlated statistical description of both, the degree of electronic coupling across the metal-molecule interface, and the energy alignment between the conduction orbital and the Fermi level of the electrode. This exhaustive statistical approach was employed to study singlemolecule junctions of 1,4-benzenediamine (BDA), 1,4-butanediamine (C4DA), and 1,4-benzenedithiol (BDT). A single interfacial configuration was observed for both BDA and C4DA junctions, while three different interfacial arrangements were resolved for BDT. This multiplicity is due to different molecular adsorption sites on the Au surface namely on-top, hollow, and bridge. Furthermore, C4DA junctions present a fluctuating I-V curve arising from the greater conformational freedom of the saturated alkyl chain, in sharp contrast with the rigid aromatic backbone of both BDA and BDT. Understanding charge transport through single molecules is a fundamental issue in molecular electronics . In recent years, increasing experimental and theoretical efforts have been devoted to the electronic characterization of a wide variety of single molecules. According to current understanding, structural details at the metal-molecule interface play a critical role in charge transport across a single-molecule junction. For example, the electronic conductance of the single-molecule junction is sensitive to metal-molecule contact configurations and molecular conformations . Therefore, to determine the single-molecule conductance, repeated formation and measurement of single-molecule junctions and statistical analyses of the individual molecular junctions have been routinely carried out using mechanically controllable break junction (MCBJ) 15,16 and scanning tunneling microscopy-based break junction (STM-BJ) methods 17 . For example, Li et al. demonstrated that alkanedithiol-molecular junctions sandwiched by Au electrodes feature three distinct conductance states at a fixed bias voltage 11 . With the aid of theoretical calculations, these three states have been assigned to a single-molecule junction with different contact configurations and trans/gauche conformations of the alkanedithiol. For the majority of single-molecule junction studies, structural identification of junctions have been performed by combining measured conductance at a fixed bias voltage and theoretical support of transport calculations at the low bias limit. This is because in BJ experiments at room temperature, the lifetime of a single molecule trapped between two electrodes is relatively short. The short life time of single-molecule junctions (< 100 ms 18,19 ) makes it difficult to routinely perform spectroscopic measurements such as surface enhanced Raman scattering (SERS) 20,21 and current-voltage (I-V) characteristics 4, . The I-V characteristics of single molecular junctions provide useful information on the molecule-metal contact configurations, such as the electronic coupling between the metal and the molecule (Γ ) and the energy difference between the Fermi level energy and the conduction-orbital (ε 0 ) . Despite the wealth of information contained in the I-V characteristics of single molecular junctions, these measurements still remain a challenge from both, the experimental point of view, and in terms of analysis and interpretation. For example, the influence of the voltage scan rate remains a matter of debate. Previous studies have typically employed I-V acquisition times of approximately 100 ms 22,26 . This time-span is comparable to the sub-second lifetimes of single-molecule junctions prepared in ambient conditions by means of STM-BJ. Hence, the reliability of the I-V measurements will certainly benefit from faster voltage scan rates. Furthermore, reducing the I-V acquisition times will reduce the structural instability caused by current-induced Joule heating effects 31 . In this study, we developed a robust statistical approach to obtain a detailed description of the metal-molecule interface in single-molecule junctions based on I-V measurements based on the STM-BJ method (Fig. 1a,b). A custom-made dataflow program was employed to control the STM in a semi-automated fashion, enabling the routine collection of I-V characteristics with reduced acquisition times up to 2.5 ms. To that end, a triangular voltage pulse was introduced into an otherwise typical STM-BJ procedure to collect the I-V characteristics of every junction formed. These experiments were repeated until a statistically significant dataset was obtained. In addition to the standard analysis in the conductance (G), statistical analysis of the I-V curve provides Γ and ε 0 , essential parameters needed to understand the metal-molecule contact configurations in molecular junctions. Combined analysis in G and both Γ and ε 0 enabled us to resolve the structural details of the metal-molecule interfaces at the molecular junctions. We applied this approach to the single-molecule junctions of 1,4-benzenediamine (BDA), 1,4-butanediamine (C4DA), and 1,4-benzenedithiol (BDT) (Fig. 1c). BDA and C4DA bind to the Au electrodes through the same functional groups, but have different molecular backbones; in contrast, BDA and BDT have the same rigid molecular backbone but different anchoring groups. Single-molecule junctions with NH 2 anchoring groups have well-defined conductance values 32 . Therefore, we first investigated the BDA molecular junctions, and then, extended our study to C4DA molecular junctions to understand the effect of molecular conformation on the I-V characteristics. Finally we demonstrated that, for the prototypical BDT junction 16, with a variety of metal-molecule contact configurations, our approach can capture not only electronic details but also resolve structural details in the molecular junctions based on statistical analysis in Γ and ε 0 with the support of ab initio charge transport calculations. ## Results and Discussion It proved difficult to take stable I-V measurements of the single-molecule junction at slow scan rates due to significant current fluctuation, most probably arising from variation in the metal-molecule contact configuration structure and molecular conformation. We checked bias-voltage-scan-rate dependence of the current fluctuation within the range of 4 to 400 Hz, where the current fluctuation displayed considerable scan-rate dependence for BDA molecular junctions (Figs S1 and S2). Because BDA contains a rigid benzene backbone, the current fluctuation is most probably due to effects arising from structural variations in the metal-molecule contact configuration. We found that a scan rate of 40 to 400 Hz was fast enough to obtain I-V curves without large current fluctuations. Figure 2a shows an example of an I-V curve of the BDA molecular junction. Two dimensional (2D) I-V histograms (Fig. 2b) were constructed from 1,000 I-V curves measured for the BDA molecular junctions, in which clear two band structures of I-V curves are apparent. The two bundles of the I-V curves, those of preferential molecular (low and high) conductance states, appear as current peaks at 290 and 450 nA (0.013 and 0.020 G 0 , where G 0 = 2e 2 /h) at 0.3 V in the current histogram (Fig. 2c). The lower conductance state with 0.013 G 0 is in good agreement with the molecular conductance of 0.01 G 0 reported in the literature 36 . The small difference in conductance values between 0.013 and 0.01 G 0 can be explained by the difference in the experimental conditions, such as the applied bias voltage for the charge transport. On the basis of peak positions in the current histogram, the I-V curves passing through the two current windows of 240-370 nA and 370-600 nA at 0.3 V are separated and averaged into two I-V curves, which are indicated by black dotted curves in Fig. 2b. Within the single channel transport model, the transmission probability of a single-molecule junction can be represented by where ε 0 and Γ L(R) are the energy of the conduction channel (orbital) and the electronic coupling energy between the molecule and the left (right) electrode, respectively 23,33,35 . Here, we set the Fermi level, E F , to zero. The current through the molecular junction is written by where n is the number of bridging molecules and f is the Fermi distribution function. When electronic temperature, T, is set to 0 K, Eq. 2 can be analytically evaluated as (3) 0 0 where Γ = Γ L + Γ R and α = Γ L /Γ R . Note that the temperature effect of the Fermi-Dirac distribution is several percent of I(V) at 300 K 35 . Curve fitting the bias of Eq. 3 for the two preferential conductance states (i.e., the two averaged I-V curves in Fig. 2b) reveals that the high and low conductance states correspond to a single conductance with a different number of n (i.e., the low state; n = 1, Γ = 85 meV, and ε 0 = 0.68 eV and the high state; n = 2, Γ = 75 meV, and ε 0 = 0.71 eV). For the BDA molecular junctions, the "single" conductance state and the corresponding set of Γ and ε 0 values were obtained by fitting statistically averaged I-V characteristics within a reasonable choice of n (See Table 1 and Supplementary Information, Section 2 for a further detail), which indicates that the single BDA junction displays a single conductance state with a preferential metal-molecule contact configuration 12,19,32 , Such preferential "NH 2 -Au" bonding and corresponding contact configuration has been revealed in the previous conductance measurements in combination with DFT-based transport calculations 37 ; in these, a BDA molecule is in contact with an undercoordinated Au atom (i.e., the on-top site).The "Au-NH 2 " contact configuration originates from a simple delocalization of the lone pair of electrons from the amine-nitrogen to the Au atoms, and the bonding is not strongly directional 32 . Therefore, the electronic properties of the BDA junction is relatively insensitive to the molecular orientation on the Au electrode. To clarify the effect of molecular backbone on the molecular I-V characteristics, we focused on C4DA, which has the same binding group, "-NH 2 ", as BDA but has a flexible molecular backbone that can adopt trans and gauche conformations 11,13 . In contrast to the rigid benzene backbone in BDA, C4DA is subject to conformational fluctuations in addition to bonding fluctuations (i.e., structural changes in the metal-molecule contact configuration). We extended our I-V measurement technique to C4DA molecular junctions. Figure 3a shows a typical I-V curve of the C4DA single-molecule junction, in which the significant current fluctuation, most probably due to the flexibility of the methylene backbone, is apparent 22 . Figure 3b shows a 2D I-V histogram constructed from 1,000 I-V curves for the C4DA molecular junctions. The histograms exhibit several faint band structures, which can be explained by formation of preferential conformers with trans and gauche conformations at the C4DA molecular junctions. Here, we focus on the one of the bands, whose current ranged between 15 and 25 nA at 0.3 V, covering the previously reported conductance values of single C4DA molecule junctions measured by STM-BJ 32 . In a similar manner as in the BDA-I-V measurement, the curves within the current window have been averaged, and these are indicated by the black line shown in Fig. 3b. The linear shape of the averaged I-V curve of the C4DA molecular junctions is remarkably dissimilar to the nonlinear curves of the BDA molecular junctions. To qualitatively discriminate the I-V characteristics and related electronic structures between the molecular junctions, we fitted the averaged C4DA-I-V curves with eq. 3 under a constraint condition of n = 1. A set of Γ and ε 0 values was determined to be Γ = 48 meV and ε 0 = 1.7 eV. The obtained ε 0 of 1.7 eV is substantially larger than the ε 0 of 0.7 V obtained for the BDA molecular junction. This remarkably large energy of 1.7 eV is caused by intrinsic molecular nature in the junctions, which is a wide HOMO-LUMO gap of the insulating alkane moiety in the C4DA junction. Statistical analysis of the I-V curves enabled us to capture molecular dependent I-V characteristics in a qualitative manner, which was used to assess the applicability of the present method. It is interesting to note that, despite both of the C4DA and BDA junctions have the same Au-N binding group, much lower electronic coupling strength was found for C4DA (48 meV) than BDA (75~85 meV). The electronic coupling strength depends on not only the local structure of the binding group but also electronic details in the molecular backbones (e.g., orbital delocalization in the molecular junction). The terminal N atom in BDA binds to a sp 2 -hybridezed carbon atom and the lone pair in the N atom is partly delocalized into the π -electron network in the molecular backbone. The resulting electronic interaction between the terminal binding group of N and the molecular backbone results in the larger coupling strength. On the other hand, the N atom in C4DA binds to a sp 3 -hybridized C atom and electronic interaction through the lone-pair in the N atom and the molecular backbone cannot be expected for C4DA. Thus, the electronic coupling of the molecular junction becomes smaller for C4DA. Such electronic interaction between the binding group and the molecular backbone has been demonstrated in rupture force measurements of BDA and C4DA molecular junctions sandwiched by Au electrodes, in which the BDA and C4DA junctions with the same Au-N binding group exhibited distinct rupture forces of the molecular junctions 38 . To allow characterization of the metal-molecule interface of single-molecule junctions, we applied our statistical I-V analysis to BDT molecular junctions. Since the pioneering MCBJ-I-V experiments of Reed et al., BDT is well-known as a prototypical molecule for electronic transport study of single-molecule junctions in the field of molecular electronics. Over the last decade, much research has been carried out, and a wealth of information on the variable transport properties of single molecule junctions has been accumulated, allowing a better understanding of the atomistic details at the junctions. The current understanding is that different "S-Au" bonding patterns, such as on-top, bridge and hollow adsorption-modes, and BDT-metal contact configurations at the molecular junctions, which have been demonstrated to display a variety of configurations, lead to differences in molecular conductance. One of the most challenging tasks is to identify the contact configurations and reliably characterize the corresponding charge transport properties and electronic structures at the molecular junctions. Figures S4, 4a,b show the typical I-V curves and 2D I-V histograms in two different current regimes. In contrast to the BDA junctions with a single "N-Au" bonding mode, the BDT junctions with multiple "S-Au" bonding modes features a variety of statistically significant I-V characteristics, as shown in Fig. 4a,b. To capture these complicated I-V characteristics, we developed an automated algorithm to fit each I-V curve with eq. 3 and extract the values of Γ and ε 0 . Figure 4c,d show 2D histograms of the fitted Γ and ε 0 values constructed from 1,000 individual I-V curves measured for the BDT molecular junctions; in these, three preferential distributions, H, M, and L, are noticeable. The histograms of each Γ and ε 0 (Fig. S5) value confirmed that there are preferential peaks-values for Γ = 31 and 126 meV and ε 0 = 0.63 eV for I-Vs in the large current regime (Fig. 4a) and peak values of Γ = 12 meV, and ε 0 = 0.65 eV for I-Vs in the small current regime (Fig. 4b). Curve-fitting and statistical analysis of the individual I-Vs revealed the existence of three preferential conductance states, H, M, and L, which can be recognized as band structures in the 2D I-V histograms (Fig. 4a,b). The current histograms at the bias voltage of 0.3 V revealed one current-peak at 7 nA in the small conductance regime (Fig. 4f) and three current-peaks at 20, 60, and 670 nA in the large conductance regime (Fig. 4e), which correspond to 0.3, 0.9, 2.6, and 29 mG 0 in order of conductance. A closer examination of the 2D I-V histograms revealed that additional fine structure appears in the large conductance regime, in which the M state is likely to split into two states. Based on the peak-positons in the current histograms, the I-V curves passing through the current windows of (L) 5~20, (M1) 20~40, (M2) 40~100 nA, and (H) 440~2200 nA at 0.3 V are divided into four groups to obtain statistically significant I-V characteristics. Averaged I-V curves within the windows are indicated by black dotted curves in Fig. 4a,b. Considering the number of bridging molecules (for further detail see Supplementary Information, Section 6 and Fig. S6), three preferential conductance states were identified for the BDT molecular junctions. By fitting statistically averaged I-Vs of each using eq. 2, Γ, ε 0 , and α were determined for the H, M, and L states (Table 1), which originate from distinct "Au-S" contact configurations in the BDT molecular junctions. To justify our experimental analysis and to assign the three conductance states, H, M, and L, to contact configurations of BDT, we calculated the I-V characteristics and properties of conductive molecular orbital (MO) for several model systems using nonequilibrium Green's function combined with DFT (NEGF-DFT) . We examined the three possible anchoring positions, i.e., hollow, bridge, and on-top, to the Au electrodes and determined the conformations of each junction model by standard DFT geometry optimization. The conductance values of hollow and on-top conformations were 0.024 G 0 and 0.009 G 0 , respectively. On the bridge type configurations, we found slightly different three stable structures, which are termed as (i) bridge, (ii) bridge-top, and (iii) tilted bridge. In the case of bridge-top, one anchoring point is the bridge site and the other site shifted slightly to the on-top position from the bridge site. The tilted bridge configuration is that the BDT molecule tilts by anchoring nonequivalent bridge sites of left and right electrodes. The schematic figures of these structures are given in Figs S9 and S10. Although the detailed structures of these bridge type conformations are different, all of the conformations have high conductance, e.g., (i) bridge, 0.22 G 0 ; (ii) bridge-top, 0.32 G 0 ; and (ii) bridge tilt, 0.27 G 0 , respectively (Table S3). The conductance of the bridge "family" is much higher than that those of the hollow or atop configurations. In Fig. 5, the structural models and the calculated I-V curves of on-top, hollow, and bridge configurations are plotted. From these results, we conclude that our calculations also show three distinct conductance regimes for BDT, and they can be assigned by anchoring sites, i.e., H is bridge, M is hollow, and L is on-top, respectively (Table 2). As described below, we chose the most conductive bridge type configuration, bridge-top, as the H state for further analysis. S1). The current windows for the I-V averaging in (a,b) were chosen based on the peak-currents and indicted by shaded areas in the current histograms. Next, we discuss the relationship of the assigned sites and Γ, obtained by fitting the experimentally observed I-V using eq. 2. We define the projected molecular orbital (PMO) by diagonalizing the molecular projected Hamiltonian (MPSH) and identify the conductive MO, whose energy ε a should be close to E F and whose coupling strength to electronic state of the electrodes, γ, is sufficiently large 42,43 . The value of γ is the imaginary part of the normalized self-energy to MPSH and was obtained for each PMO. Generally, the conductive MO is not the conduction channel state 43 . Thus (ε a , γ) is not equal to (ε 0 , Γ ), as defined by eq. 2. However, identifying conducting MO's is useful to check validity of our analysis via eq. 1. In addition, (ε a ,γ) is a good approximation that allows discussion of the tendency of γ as far as we can select suitable conductive MOs. When we rewrite (ε a ,γ) of the conductive MO's of the H state as (ε H ,γ H ) etc., the calculated values ε H , ε M , and ε L are − 0.75, − 1.09, and − 0.47 eV, respectively. Since the conductive MO energies of the three states are of the same order, analysis of the relationship between the contact configuration and Γ, as evaluated by I-V curves using eq. 1, is reasonable. The relative coupling strength γ γ / H L and γ γ / M L are 64 and 50, i.e., the correlation of conductance and Γ agree reasonably with that of conductance and γ. Towards the characterization of metal-molecule interface of single-molecule junctions, we developed a statistical approach for treatment of I-V measurements and analysis of single-molecule junctions measured by STM-BJ method under ambient conditions. For BDA, C4DA, and BDT molecules that are commonly used in the break junction-based-conductance measurements at a fixed bias voltage, we applied our statistical I-V-approach to determine the molecule dependent properties Γ and ε 0 in a qualitative manner within the single channel transport model. For BDA and C4DA, the molecule dependent ε 0 of 0.7 and 1.7 eV were obtained, which assessed applicability of the statistical I-V-approach. For BDT with a variety of metal-molecule contact configuration, three sets of statistically significant I-V characteristics with remarkable difference in the Γ values were captured and, by combining the result of first principle charge transport calculations, we identified the BDT junction-structures with on-top, hollow, and bridge sites-adsorption-configurations in the order of the molecular conductance. ## Methods I-V measurement of the molecular junctions. BDA, C4DA, and BDT were purchased from TCI Japan (Fig. 1c) and were used without further purification. The Au(111) substrate was prepared by thermal deposition of gold on mica at elevated temperature under high vacuum 44 . The sample for the I-V measurement was prepared by dipping the Au substrate into a 1 mM ethanol solution containing the target molecules. After evaporation of the solution, the substrate surface was washed with ethanol. We used a commercially available STM (Nanoscope V, Bruker, Santa Barbara, CA) operating at ambient conditions. Two current amplifiers, 1 μ A/V and 10 nA/V, were used to access wide molecular conductance ranges from 10 -5 to 10 1 G 0 . STM tips were prepared by mechanically cutting an Au wire (Nilaco, diameter ≈ 0.3 mm, purity > 99%). The I-V curves of the single-molecule junction were obtained by the following procedure (Fig. 1a,b). Firstly, an Au point contact (~10 G 0 ) was made between the STM tip and the sample surface. Secondly, the tip was withdrawn by 10 nm at a speed of 38 nm/s to break the Au contact and to make a nanogap between the Au electrodes, forming the molecular junction during current monitoring at a fixed bias voltage of 20 mV. Thirdly, the tip position was fixed and one I-V curve was recorded by scanning the bias voltage from 20 to 1000, -1000 mV, and back to 20 mV within a time period of 2.5~25 ms at constant tip-sample separation. Finally, the junction was broken by pulling the STM tip away from the substrate. To capture possible structural variation of the junction structures, we cycled the molecular junction making and breaking process and reformed the junction-structure after obtaining each I-V curve. This I-V measurement-scheme was performed though a signal access module III (Bruker, Santa Barbara, CA) using an external piezo driver (E-665 LVPZT-Amplifier, Physik Instrumente) and a data-acquisition-device with LabVIEW2014 (USB-6363, National Instruments). More than 1,000 I-V curves for the molecular junctions were collected for each molecule. The I-V curves of molecular junction were obtained by automatically removing I-V curves corresponding to Au-metallic junctions and vacuum gap formation. For the I-V measurement using the 1 μ A/V (10 nA/V) current amplifier, I-V curves with < 100 nA (< 5 nA) current at the bias of 1.0 V was classified as vacuum tunneling, while I-V curves with < 10,000 nA (< 100 nA) current at the bias of 0.2 V was classified as charge transport through an Au-metallic contact. Theoretical calculations. In this section, we survey the computational details used in the present first-principles calculations. The adsorption structures of BDT on Au electrodes were determined using a cluster model, where each side of the junction consists of 19 Au atoms and the cluster structure is taken to model the apex and the (111) surface of bulk electrodes. We fixed the two outermost Au layers with the structure of the bulk (clipped bulk), and the other atomic positions and the distance between the distance of the left and right electrodes were allowed to relax. We took hollow, bridge, and top adsorption sites as initial geometries and examined the tilt adsorption structure, i.e., the alignment of S-S axis of BDT molecule tilts to the surface. We used density functional theory (DFT) to carry out the calculations and the B3LYP exchange-correlation (XC) functional and LanL2DZP basis set for cluster model calculations. For the DFT calculations, Gaussian09 was used. To carry out transport calculations, we replaced the clipped bulk part of the cluster model with a c(5 × 5) bulk model by adding shortage Au atoms and then adding three more Au atomic layers to set up a scattering region. A periodic boundary condition was used, thus the unit cell was defined by a c(5 × 5) structure. We used the Perdew-Burke-Ernzerhof (PBE) XC functional for NEGF-DFT calculations. For the NEGF-DFT calculations, double-zeta plus polarization function basis set for all atoms in the molecule and a single-zeta plus polarization function basis set for the Au atoms were used. To check the validity of the evaluated conductance values and analysis, we also examined the XC using the local density approximation self-interaction correction (LDA-SIC). We confirmed that the PBE functional provides sufficient results for qualitative analysis in the present purpose, i.e., evaluation of the electronic coupling strength and identification of each conductance state (H, M, L). All the NEGF-DFT calculations were performed using the HiRUNE subroutine 39 and Smeagol 40,41 , which are both interfaced with the SIESTA package 45 . The electronic coupling strength and energy level of conductive molecular orbital (MO), which is qualitatively related to Γ and ε 0 , were evaluated directly by calculating projected MO's (PMO) and renormalizing the self-energy of each PMO. The details of the method, which is called the effective molecular projected state Hamiltonian (MPSH) approach, is given in ref. .

chemsum

{"title": "Resolving metal-molecule interfaces at single-molecule junctions", "journal": "Scientific Reports - Nature"}

general_synthesis_of_hierarchical_sheet/plate-like_m-bdc_(m_=_cu,_mn,_ni,_and_zr)_metal–organic_fram

## Abstract: Two-dimensional metal-organic frameworks (2D MOFs) are an attractive platform to develop new kinds of catalysts because of their structural tunability and large specific surface area that exposes numerous active sites. In this work, we report a general method to synthesize benzene dicarboxylic acid (BDC)-based MOFs with hierarchical 3D morphologies composed of 2D nanosheets or nanoplates. In our proposed strategy, acetonitrile helps solvate the metal ions in solution and affects the morphology, while polyvinylpyrrolidone (PVP) serves as a shape-control agent to assist in the nucleation and growth of MOF nanosheets. PVP also acts as a depletion agent to drive the assembly of the hierarchical sheet/plate-like M-BDC under solvothermal conditions. Further, we also demonstrate the flexibility of the proposed method using numerous coordinating metal ions (M ¼ Cu, Mn, Ni, and Zr). The potential of these MOFs for electrochemical glucose sensing is examined using the hierarchical sheet-like Ni-BDC MOF as the optimum sample. It drives the electrocatalytic oxidation of glucose over a wide range (0.01 mM to 0.8 mM) with high sensitivity (635.9 mA mM À1 cm À2 ) in the absence of modification with carbon or the use of conductive substrates. It also demonstrates good selectivity with low limit of detection (LoD ¼ 6.68 mM; signal/noise ¼ 3), and fast response time (<5 s). ## Introduction Metal-organic frameworks (MOFs) are porous crystalline materials formed by the coordination of metal ions with organic linkers. The metal clusters in MOFs act as joints, and the organic linkers act as struts to form an interconnected network via coordination bonds and intermolecular interactions. 5 The tunable pore geometries and flexible frameworks of MOFs have been exploited to develop new kinds of ultrahigh surface area materials for gas storage and adsorption, while the metal centers in MOFs have been examined as active sites to drive catalytic reactions. The combination of high surface area and catalytic activity of MOF architectures is promising as a platform in sensing applications, in part due to the numerous kinds of bonding interactions available to the analyte. 6,15,16 However despite these favorable attributes, the bulk three-dimensional (3D) morphologies of most MOFs (e.g., octahedra, dodecahedra, or polyhedra) limit mass or ion transport and reduce access to the active sites. Therefore, developing synthetic methods that enable effective morphological tuning of MOFs while still maintaining their catalytic properties and high surface area is desirable. Two-dimensional (2D) MOFs have unique physical and chemical properties that arise from electronic effects caused by their small thicknesses, large surface areas, and high surface-tovolume atom ratios. 20 2D MOFs have shown superior performance compared to bulk 3D MOFs in numerous applications. For example, 2D MOFs showed dramatically improved dye adsorption compared to 3D MOFs due to the improved diffusion and better access to interior Lewis acid sites. 19,21 In addition, the ease of access to the active sites of 2D MOFs could promote enhanced interactions between the active sites and target molecules in sensing applications, leading to high sensitivity. 22 2D MOFs have been synthesized using both topdown and bottom-up strategies. Top-down strategies typically include delamination, mechanical exfoliation, sonication exfoliation, and chemical exfoliation. Although these strategies can produce high quality single-or few-layer nanosheets, they are usually time-consuming and suffer from low yield. 26 In comparison, the bottom-up approaches including interfacial synthesis, three-layer synthesis, surfactant-assisted synthesis, modulated synthesis, and sonication synthesis are more straightforward and generate larger yields but are challenging to control the morphology precisely. Ultrathin 2D nanosheets can support enhanced electrocatalytic activity, but they tend to be less stable than 3D structures because they are prone to restacking. In this regard, the construction of hierarchical 3D MOF nanostructures is attractive because it may be engineered to maintain many of the aspects of 2D MOFs (i.e., large surface area, interconnected open pores, rich redox sites) but in a hierarchical framework that promotes stability for electrochemical applications. Previously, a hierarchical flower-like Ni-MOF [Ni 3 (OH) 2 (PTA) 2 (H 2 -O) 4 ]$2H 2 O (PTA ¼ p-benzenedicarboxylic acid) was prepared by the hydrothermal reaction between Ni 2+ and PTA at 150 C. The resulting Ni-MOF showed high electrocatalytic activity and good stability for glucose oxidation reaction. 30 In addition, hierarchical ZIF nest architectures were previously synthesized by the solvothermal reaction of zinc nitrate hexahydrate with 2-ethylimidazole and 5,6-dimethylbenzimidazole in a mixed solvent of methanol and aqueous ammonia. 31 Despite these achievements, it is still challenging to develop a generalized route for fabricating hierarchical 3D MOFs assembled from 2D nanostructures, such as nanosheets/nanoplates. Glucose sensing plays an essential role in the detection of diabetes. Enzymatic sensors display high sensitivity and selectivity toward glucose. However, natural enzymes suffer from some disadvantages, such as high cost, poor long-term stability, and complex immobilization process. Alternative glucose detection methods are being developed using sensors that employ light and acoustic waves, in addition to electrochemical methods. Among them, electrochemical glucose sensors are particularly attractive due to their simplicity, low cost, high sensitivity, and rapid response. Noble metals and their alloys are frequently utilized for electrochemical non-enzymatic glucose sensing owing to their high response and excellent selectivity. However, the expensive nature of these materials has impeded their large-scale applications. Therefore, there is a need to develop low-cost and high-performance catalysts for non-enzymatic glucose sensing. The direct utilization of MOFs as electrochemical glucose sensors is still limited due to the low electrical conductivity of pristine MOFs. Previously, Cu MOF-modifed electrode showed a relatively good electrocatalytic activity for glucose oxidation in the linear range of 0.06 mM to 5 mM with a sensitivity of 89 mA mM 1 cm 2 and a detection limit of 10.5 nM. 32 In another report, spheroidal Ni-MOF particles showed poor performance for electrochemical glucose sensing when utilized alone. However, their hybridization with carbon nanotubes (CNTs) yielded a higher sensitivity of 13.85 mA mM 1 cm 2 , a low detection limit of 0.82 mm, and a wide linear range of 1 mM to 1.6 mM. 33 Despite some success in preparing pure MOF electrodes, electrochemical glucose sensing with them is challenging because they have low sensitivity. Therefore, it is essential to create a hierarchical MOF architecture that enables control of size, structure, and composition. In this work, we report the general synthesis of hierarchical sheet/plate-like M-BDC nanosheets, where M ¼ Cu, Mn, Ni, and Zr, and BDC ¼ benzene-1,4-dicarboxylic acid. These MOFs are prepared in a solvothermal reaction by combining different metal nitrate precursors with the BDC ligand in the presence of polyvinylpyrrolidone (PVP) and acetonitrile as shape-directing agents. This polymer-solvent system promotes good solvation of the metal ions and leads to the formation of hierarchical sheet/plate-like M-BDC MOFs. When evaluated for electrochemical glucose sensing, the as-prepared hierarchical sheetlike Ni-BDC electrode exhibits superior electrocatalytic oxidation toward glucose in the range of 0.01 mM to 0.8 mM with a relatively high sensitivity of 635.9 mA mM 1 cm 2 , without any modifcation with carbon or the use of a conductive substrate. Furthermore, the hierarchical sheet-like Ni-BDC sensor has a relatively good selectivity with a low limit of detection (LoD) of 6.68 mM (signal/noise ¼ 3), and fast response time (<5 s). ## Chemicals Copper(II) nitrate trihydrate (Cu(NO 3 ) 2 $3H 2 O, 99.5%), nickel(II) nitrate hexahydrate (Ni(NO 3 ) 2 $6H 2 O, 99.5%), manganese(II) nitrate tetrahydrate (Mn(NO 3 ) 2 $4H 2 O, 99.5%), D(+)-glucose (C 6 H 12 O 6 , 99.5%), N,N-dimethylformamide (DMF), and acetonitrile (C 2 H 3 N, 99.8%) were purchased from Fujiflm Wako Pure Chemical Corporation (Japan). Benzene-1,4-dicarboxylic acid or terephthalic acid (H 2 BDC, 98%), uric acid (C 5 H 4 N 4 O 3 , $99%), and zirconium(IV) chloride (ZrCl 4 , 99%) were purchased from Sigma-Aldrich (Japan). Polyvinylpyrrolidone K-30 (PVP) (M w ¼ 40 000), maltose (C 12 H 22 O 11 , $99%), and L-ascorbic acid (C 6 H 8 O 6 , 99%) were purchased from Nacalai Tesque (Japan). ## General synthesis of hierarchical sheet/plate-like M-BDC (M ¼ Cu, Mn, Ni, and Zr) In a typical procedure, 0.3 g of the metal nitrate precursor was dissolved in a solvent mixture containing 10 mL of DMF and 30 mL of acetonitrile to form solution A. In a separate bottle, 0.3 g of H 2 BDC was dissolved in a mixture containing 30 mL of DMF and 10 mL of acetonitrile, followed by the addition of PVP into the mixture solution under stirring to form solution B. The optimized mass ratios of metal precursor to PVP are 1 : 3 for Cu-BDC and Ni-BDC, and 1 : 5 for Mn-BDC and Zr-BDC (i.e., 0.9 g of PVP was used to prepare Cu-BDC and Ni-BDC and 1.5 g of PVP was used to synthesize Mn-BDC and Zr-BDC). Next, 4 mL of solution A was mixed with 4 mL of solution B in a 50 mL vial, and then the mixture was sonicated for 2 min. The mixed solution was subsequently heated in an oil bath at 135 C for 24 h without stirring. The resulting precipitates were centrifuged at 14 000 rpm for 8 min, washed consecutively with DMF and methanol for three times each and then dried in air at 60 C. ## Characterization The morphological characterization of the MOF samples was performed with a scanning electron microscope (SEM, Hitachi SU-8000) operated at an accelerating voltage of 10 kV. The composition and crystal structure of the MOF products were checked by Xray diffraction (XRD, Rigaku RINT 2500X) with Cu-Ka radiation (l ¼ 0.15406 nm). Rietveld refnement analysis was carried out using EXPO2014 software. 34 The Fourier transform infrared (FTIR) spectra of the samples were collected by using a Thermo Scientifc Nicolet 4700 in the wavenumber region of 500 to 4000 cm 1 . Nitrogen (N 2 ) adsorption-desorption measurements of the MOF samples were carried out by using BELSORP-max (BEL, Japan) at 77 K. The specifc surface area and pore size distribution of the samples were calculated by employing Brunauer-Emmett-Teller (BET) and nonlocal density functional theory (NLDFT) methods, respectively. Before the BET measurement, each sample was degassed at 175 C for 20 h to remove adsorbed moisture. The post-cycling measurement of nickel concentration in the glucosecontaining NaOH solution was performed by atomic absorption spectroscopy using GBC SavantAA atomic absorption spectrophotometer (GBC Scientifc Equipment, Australia) at a wavelength of 232 nm. ## Non-enzymatic glucose sensing measurements The electrochemical measurements were performed with an electrochemical workstation (CHI-660, USA) using a threeelectrode system consisting of glassy carbon electrode (GCE) as the working electrode, platinum wire as the counter electrode, and Ag/AgCl as the reference electrode. The working electrode was prepared by drop-casting 5 mL of the MOF suspension onto a clean and polished glassy carbon electrode (GCE). The suspension was prepared by dispersing 5 mg of the MOF powder in 900 mL of isopropanol, followed by the addition of 100 mL of Nafon and subsequent sonication for 30 minutes. The electrolyte used was 0.1 M NaOH, and the glucose solutions were dissolved in 0.1 M NaOH at various concentrations (0.1-20 mM). The stability test was carried out by measuring the variation of the current response of the hierarchical sheet-like Ni-BDC electrode to 0.1 mM glucose for 6 consecutive cycles after 50 days of storage time. ## Synthesis and characterization of hierarchical sheet/ plate-like M-BDC MOFs The synthesis of the hierarchical sheet/plate-like M-BDC particles was performed in an oil bath at 135 C in the presence of both PVP and acetonitrile as structure-directing agents. The general synthetic procedure works for several different metals, but each type of M-BDC requires a different mass ratio of metal to PVP to achieve the hierarchical sheet/plate-like morphology (i.e., M : PVP ¼ 1 : 3 for Cu-BDC and Ni-BDC and 1 : 5 for Mn-BDC and Zr-BDC). SEM images of the optimized M-BDC MOFs are given in Fig. 1. The Cu-BDC sample displays a hierarchical architecture formed by the stacking of several square-like plates (Fig. 1a and b). In comparison, the optimized Mn-BDC product shows a hierarchical flower-like morphology which is assembled from nanoplates (Fig. 1c and d), whereas the optimized Zr-BDC sample has hierarchical plate-like morphology with lengths of $800 nm to 1 mm (Fig. 1e and f). In contrast, the optimized Ni-BDC product exhibits a hierarchical multilayered sheet-like structure (Fig. 1g and h). The hierarchical plate-like Cu-BDC exhibits major peaks at around 10.3 , 12.2 , 13.6 , 17.1 , 18.0 , 20.6 , 24.8 , 34.1 , and 42.1 , assigned to (110), (020), ( 111), (20 1), ( 111), ( 220), ( 402), and (242) planes of Cu-BDC, respectively (CCDC no. 687690) (Fig. 1i). 28 Rietveld refnement was carried out on the powder XRD (PXRD) pattern of the Cu-BDC product and the refned unit cell parameters are identifed to be a ¼ 11.32 A, b ¼ 14.33 A, and c ¼ 7.78 A. As shown in Fig. 1j, the Mn-BDC product exhibits two major peaks at around 9.86 and 10.4 , indexed to ( 111) and ( 202) planes of Mn-BDC, respectively (CCDC no. 265094), which is in good agreement with the report of Rosi et al. 35 The refned unit cell parameters for Mn-BDC are a ¼ 24.79 A, b ¼ 10.59 A, and c ¼ 17.42 A. The as-prepared Zr-BDC (more commonly known as UiO-66) sample displays strong peaks at around 7.2 , 8.8 , and 17.4 (Fig. 1k) which match well with the (111), (200), and (400) planes of Zr-BDC (UiO-66), respectively (CCDC no. 733458), in agreement with the work of Cavka et al. 36 The refned unit cell parameters for Zr-BDC are a ¼ 24.79 A, b ¼ 14.33 A, and c ¼ 7.78 A. The indexing of the powder XRD pattern of Ni-BDC (Fig. 1l) was performed using EXPO2014 software for generating the lattice parameters. 34 Rietveld refnement was performed based on a model which gives a closely resembled XRD pattern for Ni-BDC. The refned PXRD pattern can be assigned to monoclinic phase with space group P12 1 /n1 with unit cell parameters: a ¼ 12.98 A, b ¼ 11.38 A, and c ¼ 17.90 A (a ¼ b ¼ 90 and g ¼ 96.7 ). The details of the refned unit cell parameters of these M-BDC MOFs are given in Table S1. † FTIR measurements were carried out to further confrm the formation of M-BDC MOFs (Fig. 2). Fig. 2a and b display the FTIR spectra of pure PVP, the BDC ligand (terephthalic acid), and the as-synthesized M-BDC MOFs in the wavenumber regions of 4000-1800 cm 1 and 1800-525 cm 1 , respectively. The FTIR spectrum of the pure PVP shows a broad peak between 3200-3600 cm 1 , which can be assigned to the stretching vibration of O-H (Fig. 2a(i)). The IR bands between 2850-2950 cm 1 can be indexed to the asymmetric stretching vibration of CH 2 in the skeletal chain of PVP. The vibration band of C]O group is observed at 1642 cm 1 (Fig. 2b(i)), indicating the presence of carbonyl groups in PVP. 37 The peaks at 1462 cm 1 and 1371 cm 1 are assignable to the bending vibration of CH 2 , whereas the peak at around 1440 cm 1 is indexable to the bending vibration of O-H. The peak located between 1285-1295 cm 1 matches the stretching vibration of C-N in PVP. The deprotonation of H 2 BDC is confrmed by the shift of the C]O stretching vibration at 1700 cm 1 (Fig. 2b(ii)) to $1665 cm 1 for the M-BDC MOFs (Fig. 2b(iii)-(vi)). Furthermore, the M-BDC MOFs display sharp peaks in the regions of 1490-1600 cm 1 and 1350-1450 cm 1 , which match the asymmetric and symmetric stretching vibrations of the carboxyl group, respectively. 33 The separation between these two modes indicates that the COO of BDC ligand is coordinated to the metals through a bidentate mode. 38 The IR bands between 1080-1200 cm 1 in the FTIR spectra of M-BDC MOFs can be assigned to the C-O stretching vibration. The presence of the C-N stretching of aromatic amines is observed between 1293-1300 cm 1 , whereas the C-N stretching of aliphatic amines is located at around 1019 cm 1 . The bending vibration of C-N]O appears at around 675 cm 1 for M-BDC MOFs. 39 In addition, the IR bands between 750-880 cm 1 can be assigned to the C-H bending vibration. The IR band located at 740 cm 1 can be indexed to the metal substitution on benzene groups. 40 The strong bands at around 540 and 670 cm 1 are assigned to O-M(metal)-O vibrations. The presence of these new IR bands indicates the successful coordination of the metals with BDC ligands to form M-BDC MOFs. These FTIR results further confrm the successful formation of M-BDC MOFs. Time-dependent experiments were performed to study the growth mechanisms of M-BDC MOFs (Fig. 3). For Cu-BDC, square-like plate particles are readily observed within 2 h (Fig. 3a-1) and the stacking of these square plate-like particles is intensifed with the increase of reaction time from 4 to 24 h (Fig. 3a-2 to Fig. 3a-5), leading to the formation of hierarchical plate-like Cu-BDC particles. For Mn-BDC, microspindles are observed after 2 h (Fig. 3b-1) and 4 h (Fig. 3b-2) and slowly, plate-like particles are growing from these microspindles after 8 h, as seen in Fig. 3b-3. After 16 h, more plate-like particles are formed on these large spindle-like particles (Fig. 3b-4). Eventually, hierarchical plate-like Mn-BDC particles are achieved after 24 h of reaction (Fig. 3b-5). For Ni-BDC, bulk particles with irregular structure are obtained after 2 h (Fig. 3c-1). After 4 h, these bulk particles begin to self-organize into aggregated sheetlike particles (Fig. 3c-2) and this self-assembly process continues between 8-16 h (Fig. 3c-3 and c-4). As seen in Fig. 3c-5, well-defned hierarchical multilayered sheet-like Ni-BDC particles are successfully formed after 24 h. For Zr-BDC, the product obtained after 2 h consists mostly of aggregated bulk particles (Fig. 3d-1). After 4 h, these bulk particles separate into smaller aggregated nanoparticles with irregular structure (Fig. 3d-2). After 8 h, these aggregated nanoparticles become increasingly separated from each other (Fig. 3d-3) and slowly grow into plate-like particles after 16 h (Fig. 3d-4). Finally, uniform plate-like Zr-BDC particles are achieved after 24 h of reaction (Fig. 3d-5). The effects of the reaction temperature on the formation of M-BDC MOFs in the absence of PVP are shown in Fig. S1. † With the exception of Cu-BDC, all the other M-BDC MOFs cannot be formed at room temperature. Cu-BDC generates thin, highly interconnected square-like sheets with an average length of 2 mm at room temperature. Upon heating to higher temperatures between 55-95 C, the square sheet-like particles become more well-separated without a signifcant change in particle size. However, when the temperature is raised to 115 C, these sheets become stacked on top of each other. This stacking effect is further intensifed at 135 C. In contrast, Ni-BDC starts to precipitate at 75 C, showing irregular plate-like structures that assemble into 2D stacked structures at higher temperatures (i.e., 115 C and 135 C). Mn-BDC precipitates at 95 C and the product displays an aggregated bundle-like structure, and the increase of reaction temperature to 115 C leads to the separation of these bundles into individual nanorods and eventually, thick hierarchical microrods are achieved at 135 C. In comparison, Zr-BDC gradually transforms from agglomerated nanoparticles at 95 C to hierarchical spheres composed of small nanoparticles at 115 C and ultimately to hierarchical plate-like networks at 135 C. Based on these results, it is evident that the modifcation of reaction temperature alone is generally not sufficient for achieving the hierarchical 3D M-BDC MOFs. Therefore, in this work, we have employed PVP to promote the formation and growth of hierarchical sheet/platelike M-BDC MOFs. PVP is frequently used as a surfactant and shape directing agent in nanoparticle synthesis methods. 39 The pyrrolidone moiety of PVP has a highly polar amide group that reversibly interacts with polarizable ions and charged molecules and has been used to synthesize MOFs. 41 The concentration of PVP is also important for creating the hierarchical sheet/plate-like structures because PVP can generate depletion forces between larger particles including nanosheets, driving them to coagulate and self-assemble. 42,43 To identify the role of PVP, a series of control experiments were carried out by modifying the mass ratio of metal precursor to PVP (i.e., 1 : 1, 1 : 3, and 1 : 5) during the synthesis of each M-BDC MOF. Thick square plate-like Cu-BDC particles are obtained at a Cu precursor/PVP mass ratio of 1 : 1 (Fig. S2a †), whereas hierarchical square plate-like particles are achieved at an optimal ratio of 1 : 3 (Fig. 1a, b and S2b †). A further increase of the Cu precursor/PVP mass ratio to 1 : 5 yields aggregated nanoparticles as the product (Fig. S2c †). In comparison, the Mn-BDC product obtained at a Mn precursor/PVP mass ratio of 1 : 1 displays bulk dumbbelllike morphology (Fig. S2d †). The increase of the Mn precursor/ PVP mass ratio to 1 : 3 generates oval-like particles with an average size of 2 mm (Fig. S2e †). Finally, Mn-BDC particles with uniform sheet-like morphology are achieved at an optimized Mn precursor/PVP mass ratio of 1 : 5 (Fig. 1c, d and S2f †). For Ni-BDC, stacked square-like particles are observed at a Ni precursor/PVP mass ratio of 1 : 1 (Fig. S2g †), which are transformed into hierarchical sheet-like particles with the increase of the Ni precursor/PVP mass ratio to 1 : 3 (Fig. 1g, h and S2h †). In contrast, the Ni-BDC product achieved at a higher Ni precursor/ PVP mass ratio of 1 : 5 exhibits a net-like structure with many holes, as seen in Fig. S2i. † For Zr-BDC, hierarchical plate-like particles are readily observed at a Zr precursor/PVP mass ratio of 1 : 1 (Fig. S2j †), and the diameters of these plates are enlarged with a further increase of the Zr precursor/PVP mass ratio to 1 : 3 (Fig. S2k †). Eventually, a hierarchical structure assembled from well-separated plates is achieved at an optimized Zr precursor/PVP mass ratio of 1 : 5 (Fig. 1e, f and S2l †). In general, we can conclude that PVP facilitates the growth of hierarchical sheet/plate-like M-BDC, but excess PVP in the M-BDC growth solution leads to excessive stacking of the nanosheets or nanoplates to form bulk irregular crystals due to depletion attraction. The third set of control experiments involves the removal of acetonitrile from the growth solution of M-BDC with the metal precursor to PVP mass ratio fxed at the optimized ratio for each M-BDC sample. In the absence of acetonitrile, none of the M-BDC samples exhibit hierarchical sheet/plate-like morphology. The Cu-BDC product achieved in the absence of acetonitrile consists of stacked square-like particles with sizes between 500 nm to 2.5 mm (Fig. S3a †), whereas the Mn-BDC and Ni-BDC products consist of bulk crystals (Fig. S3b and c †). In contrast, the Zr-BDC sample obtained without acetonitrile is made up of aggregated quasi-cubic-like particles (Fig. S3d †). The above fndings highlight the important role of acetonitrile as the removal of acetonitrile causes poor solvation of the metal ions, which therefore interrupts the formation of hierarchical sheet/ plate-like M-BDC MOFs. The overall formation mechanism is proposed as follows. Acetonitrile serves primarily to improve the solvation of metal ions in solution. 44 DMF begins to decompose at $130 C in the presence of acid, generating carbon monoxide and dimethylamine molecules. The dimethylamine molecules have a relatively high pK a and deprotonate additional H 2 BDC linker molecules that may bond with metal precursors to form the MOF crystal. PVP plays an essential role in crystallization because it reduces the rate of crystal growth by forming weak hydrogen bonds with organic molecules, 45 and stronger pC] O/M bonds with metal cations and metal surfaces. 46,47 Therefore, PVP initially serves as a dynamic hydrophobic/ hydrophilic environment enabling reversible interactions to reduce the ionic mobility of the metal precursors and promote nucleation of MOF nanocrystals. During the growth phase, PVP tends to bond more strongly to one facet or more facets of the MOF crystal. Preferential binding promotes shape-control, which in the case of M-BDC generates hierarchical nanosheets/nanoplates depending on the metal. Shape-control is lost when only small amounts of PVP are used, or acetonitrile is omitted (Fig. S2 and S3 †), resulting in bulk crystals. PVP also plays a secondary role in assisting the formation of the hierarchical structures as mentioned above. N 2 adsorption-desorption measurements were used to characterize the specifc surface area and pore size distribution of the as-prepared M-BDC samples. Fig. 4a, b, and Table S2 † reveal that the trend in specifc surface area is in the order of Zr-BDC > Mn-BDC > Cu-BDC > Ni-BDC. Our M-BDC samples exhibit lower surface areas than some previously reported 2D MOFs, but this is likely due to the presence of PVP, which can lead to partial blocking of the internal space of the M-BDC crystals. 48 Nonetheless, BDC-based MOFs with surface areas lower than 100 m 2 g 1 have been reported previously. The pore volume of the as-synthesized Zr-BDC, Mn-BDC, Cu-BDC, and Ni-BDC samples are 1.914, 0.566, 0.308, and 0.207 cm 3 g 1 , respectively. The pore size distribution curves of the hierarchical sheet/plate-like M-BDC MOFs were calculated using the non-local density functional theory (NLDFT) method. The asprepared Cu-BDC, Mn-BDC, and Ni-BDC samples are mesoporous with mesopore peaks at 26.4, 26.3, and 13.7 nm, as shown in Fig. 4c-e, respectively. In contrast, Zr-BDC has a much higher surface area compared to the other M-BDC MOFs due to its microporous nature with a main peak at 2.67 nm (Fig. 4f). ## Non-enzymatic glucose sensing performance The hierarchical structure and exposed metal sites of the M-BDC architectures make them attractive for sensing applications. The as-prepared M-BDC samples were coated onto glassy carbon electrodes (GCEs) and used for electrochemical non-enzymatic glucose sensing. The glucose sensing measurements were carried out by using an electrochemical workstation with a three-electrode system. Fig. 5a displays the electrochemical responses of the hierarchical sheet/plate-like M-BDC electrodes to 5 mM of glucose in 0.1 M NaOH electrolyte in the potential range of 0.6 to 1.0 V. Among all the samples, only Ni-BDC shows a pair of asymmetric redox peaks in the CV curves with an anodic peak at around 0.63 V and a cathodic peak at 0.45 V, indicating the presence of redox reactions between Ni 2+ /Ni 3+ and OH with reversible faradaic mechanism to form NiOOH and Ni(OH) 2 . Although other M-BDC electrodes do not show any asymmetric redox peaks, the current densities of GCEs coated with the M-BDC samples are still much higher than that of bare GCE, as seen in Fig. 5a. Nonetheless, as only the Ni-BDC electrode displays a signifcant response to glucose, subsequent glucose sensing measurements will focus solely on this MOF. The electrochemical glucose sensing performance of the hierarchical sheet-like Ni-BDC was compared with that of bulk Ni-BDC. The bulk Ni-BDC (Fig. S3c †) was synthesized with a Ni precursor/PVP ratio of 1 : 3 at 135 C without acetonitrile. Compared to the hierarchical sheet-like Ni-BDC, the asymmetric redox peaks of bulk Ni-BDC show potential shift to the negative direction (0.64 V to 0.56 V) with increasing glucose concentration from 5 to 20 mM (Fig. S4 †). However, the current density remains more or less similar at $0.13 mA cm 2 . In comparison, the current density of the hierarchical sheet-like Ni-BDC ranges from $0.14 to 0.177 mA cm 2 with a potential shift to the positive direction (from 0.63 V to 0.67 V) using the same glucose concentration range (Fig. 5b). These results indicate the superior glucose sensing performance of the hierarchical sheet-like Ni-BDC compared to the bulk Ni-BDC, owing to the more accessible and increased active sites as well as the interconnected 3D structure which can provide interspaces for the diffusion of biomolecules, decrease the contact resistance, and enhance the mass or charge transfer rate at the interface between the electrode and the electrolyte. 55 The responses of hierarchical sheet-like Ni-BDC electrode to various concentrations of glucose (0.1, 1.0, 2.0, 5.0, 10.0 and 20.0 mM) were investigated by cyclic voltammetry (CV) (Fig. 5c). The current density of the Ni-BDC electrode increases with increasing glucose concentration, accompanied by a positive potential shift. These observations suggest that Ni 2+ and OH redox reactions are involved. The electrochemical glucose sensing mechanism of the hierarchical sheet-like Ni-BDC electrode is based on the possible redox reactions of Ni 2+ with 5d. Evidently, there is a linear correlation between the glucose concentration and the current density in the range of 0.01 mM to 0.8 mM with a correlation coefficient of 0.9973. The hierarchical sheet-like Ni-BDC sensor exhibits a relatively high sensitivity of 635.9 mA mM 1 cm 2 . The limit of detection (LoD) for glucose is determined to be 6.68 mM (S/N ¼ 3). Table 1 compares the electrochemical glucose sensing performance of various non-enzymatic catalysts with the asprepared hierarchical sheet-like Ni-BDC sensor. It can be observed from Table 1 that the hierarchical sheet-like Ni-BDC electrode shows higher sensitivity than non-enzymatic glucose sensors based on Ni(OH) 2 /Au, 60 Ni(OH) 2 nanoparticles/reduced graphene oxide (RGO), 61 GO x /p-NiO/n-Bi 4 Ti 3 O 12 , 66 NiCo layered double hydroxide (LDH) nanosheets/graphene nanoribbons, 67 and even some noble metal composites, such as Pt@carbon nano-onions, 64 Ni-Pd@activated carbon, 65 and PtPd/porous holey nitrogen-doped graphene. 68 Zhang et al. 33 previously compared the glucose sensing performance of bulk Ni-BDC with that of Ni-BDC/carbon nanotube (CNT) hybrid. They found that the bulk Ni-BDC did not show redox peaks during CV scans. However, the response was signifcantly increased when the Ni-BDC MOF was mixed with CNTs. In comparison, the assynthesized hierarchical sheet-like Ni-BDC sensor shows a higher sensitivity to glucose than this Ni-BDC/CNT hybrid even without the addition of carbon-based materials or the use of conductive nickel foam. Furthermore, compared with hierarchical flower-like Ni-BDC-SWCNT (single-wall carbon nanotubes)/GCE, 58 our hierarchical sheet-like Ni-BDC sensor exhibits superior sensitivity to glucose even without the addition of SWCNT. This indicates that the catalytic activity of Ni-BDC may be enhanced by tuning the morphology to a hierarchical 3D structure composed of 2D nanoarchitectures. Therefore, the good electrochemical glucose sensing performance of the Ni-BDC is largely attributed to its nanosheet-assembled hierarchical 3D structure, which can provide open pores and numerous accessible redox sites that are accessible due to the interconnected nature of the nanosheets and nanoplates. In clinical use, an electrochemical sensor must be able to distinguish between the target molecule and the interfering molecules in the sample test (e.g., blood, urine, saliva, etc.), especially in the case of non-enzymatic sensors. Therefore, the selectivity of the hierarchical sheet-like Ni-BDC electrode toward glucose was checked against other common interferents found in blood, such uric acid (UA), ascorbic acid (AA), and maltose, as shown in Fig. 5e. The amperometric responses of Ni-BDC to 0.1 mM of glucose, 5 mM of UA, 5 mM of AA, and 10 mM of maltose in 0.1 M NaOH at 0.63 V are largely different. Changes in current density of 50.99 and 19.8 mA cm 2 are observed after additions of glucose and UA into the electrolyte, respectively. If the response of the hierarchical sheet-like Ni-BDC to UA is compared with that to glucose, Ni-BDC has 100% selectively to glucose compared to only 38.88% for UA, implying that these two molecules can still be distinguished. In contrast, no signifcant changes in current density are observed after additions of AA and maltose, indicating the good selectivity of the hierarchical sheet-like Ni-BDC electrode toward glucose. The stability of the Ni-BDC electrode was examined by measuring its current response toward 0.1 mM glucose for 6 consecutive cycles after prolonged storing (50 days). As shown in Fig. 5f, the hierarchical sheet-like Ni-BDC electrode can retain 88% of its original value after 6 consecutive sensing cycles, even after prolonged storing, thus indicating its relatively stable sensing performance. The SEM images of the Ni-BDC electrode before and after this stability test (Fig. S5a and b †) reveal that the sheet-like structure is still maintained, however they become more crumpled in appearance. To further assess the stability of the as-prepared Ni-BDC electrode, we have carried out a leaching test on the glucose-containing NaOH solution after the sensing measurement to identify whether Ni metal has leaked into the solution. The atomic absorption spectroscopy (AAS) measurement reveals that the concentration of Ni metal in this solution is negligible (0.0042), indicating that the Ni metal in the Ni-BDC electrode has not been leaked into the solution. The XRD analysis of the Ni-BDC electrode after the glucose sensing test shows that the Ni-BDC becomes more amorphous. However, several weak peaks belonging to Ni-BDC can still be observed after the sensing test, as indicated by the circles on Fig. S5c, † indicating its respectable stability. ## Conclusions In summary, this work describes a general route to synthesize hierarchical 3D M-BDC (M ¼ Cu, Mn, Ni, and Zr) MOFs which are assembled from two-dimensional nanosheets/nanoplates in the presence of PVP and acetonitrile as shape-directing agents. The mass ratio of the metal precursor to PVP and the amount of acetonitrile strongly influence the formation of the hierarchical sheet/plate-like M-BDC MOFs. Acetonitrile helps maintain the solvation of metal precursor, while PVP assists in the nucleation and growth of the MOF crystals. Removal of either acetonitrile or PVP results in bulk MOF crystals. When employed for nonenzymatic electrochemical glucose sensing, only the hierarchical sheet-like Ni-BDC electrode shows a signifcant amperometric response toward glucose with a high sensitivity of 635.9 mA mM 1 cm 2 with a wide linear range between 0.01 to 0.8 mM. The limit of detection (LoD) of the hierarchical sheetlike Ni-BDC electrode toward glucose is around 6.68 mM (S/N ¼ 3). It is expected that this work will provide useful strategies for future synthesis of hierarchical 3D MOFs and promote the direct utilization of MOFs in other electrochemical applications. In addition, these MOFs can be utilized in the future as precursors for creating hierarchical metal oxides, carbons, and their hybrid materials. 14,69,70 ## Conflicts of interest There are no conflicts to declare.

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{"title": "General synthesis of hierarchical sheet/plate-like M-BDC (M = Cu, Mn, Ni, and Zr) metal\u2013organic frameworks for electrochemical non-enzymatic glucose sensing", "journal": "Royal Society of Chemistry (RSC)"}

radiocarbon_(_14_c)_concentration_of_local_pollution_in_street_trees_located_at_intersections

## Abstract: At large intersections, vehicles consume and generate a large amount of fossil fuel.Carbon derived from fossil fuels that do not contain radioactive carbon ( 14 C), i.e., dead carbon, is released in large amounts in the roadside air environment. By means of photosynthesis, street trees along the roadside assimilate both dead carbon, not containing radioactive carbon ( 14 C), and contemporary carbon, which includes radioactive carbon ( 14 C). Therefore, the concentration of radioactive carbon ( 14 C) in leaves of trees growing in heavily polluted environments decreases.Radioactive carbon ( 14 C) in the leaves of street trees that grow at heavily air-polluted intersection was measured. As a result, it was revealed that the decrease of the radioactive carbon ( 14 C) concentration in the leaves reflects the microscale local pollution at the intersection. ## Generation of radioactive carbon ( 14 C) Cosmic rays are permanently present in the upper layer of the Earth's atmosphere, and radioactive carbon ( 14 C) is generated by a nuclear reaction when cosmic-ray neutrons collide with the nuclei of nitrogen atoms in the atmosphere. 14 N + n (neutron) -> p (proton) + 14 C (1) The generated radioactive carbon ( 14 C) atom is rapidly oxidized to form stable carbon dioxide (CO2) gas molecules by covalent bonding. CO2, with a boiling point of −79°C, can behave as a stable gas molecule at every ground state unlike water that may exit in three physical states. In addition, due to the fluctuation of geophysical fluids such as planetary waves, CO2 is mixed well in the troposphere along with nitrogen, oxygen, and argon and is present universally in the atmosphere. A part of atmospheric radioactive carbon ( 14 C) is taken in the biosphere, together with stable isotopes (SI) 12 C and 13 C, by photosynthesis of plants. Unlike the stable isotopes (SI) 12 C and 13 C, the radioactive isotope (RI) 14 C undergoes β decay and is reduced to nitrogen atoms over time. 14 C -> 14 N + e -(β ray) The half-life of this β decay is 5,730 years. This indicates that after 5,730 years, 14 C in carbon decreases by 50% and so does the radioactivity of β rays. Since the radioactive intensity of β rays is always proportional to the ratio of radioactive carbon ( 14 C), the concentration of radioactive carbon ( 14 C) per unit carbon is expressed by the radioactivity of β rays. Fossil fuels, such as coal and petroleum responsible for air pollution, are produced from the stratum with the ages as old as tens of millions to hundreds of millions of years that is tens of thousands times longer than the half-life of 5,730 years. Therefore, the original concentration of radioactive carbon ( 14 C) present in fossil fuels has been now reduced to a totally undetectable level. The ancient carbon, whose β decay ended and the radioactive carbon ( 14 C) was lost, is called dead carbon. Most of the fossil fuel comprises dead carbon without this radioactive carbon ( 14 C), and β rays cannot be detected. In polluted areas such as at intersections wherein air pollution is significant, a large amount of dead carbon derived from fossil fuel is burned and discharged into the atmosphere as a result of 2 of 11 energy consumption by an overcrowded automobile traffic. Radioactive carbon ( 14 C) in the atmospheric environment is diluted by the generation of a large amount of dead carbon, and the β ray intensity of radioactive carbon ( 14 C) per unit carbon also decreases proportionally. Such an isotope effect is generally referred to as the Suess effect . 1.2 Mesoscale radioactive carbon ( 14 C) concentration distribution in leaves from the urban center to suburbs In our previous research, radioactive carbon ( 14 C) in street trees growing in the vicinity of general air pollution monitoring stations and automobile exhaust gas monitoring stations was measured. The results revealed that a strong negative correlation existed between the amount of sulfur oxides and nitrogen oxides measured at the stations and the radioactive carbon ( 14 C) concentration in the leaves . The concentration of radioactive carbon ( 14 C) in the leaves tends to increase gradually from the center of large cities to the less-polluted surrounding areas. The measurement results in Kyoto city is presented below as a specific example of this tendency. In the fall of 1996, fallen leaves from street trees were collected in the vicinity of several air pollution monitoring stations extending from the central part of Kyoto city to the surrounding suburbs, and their radioactive carbon ( 14 C) concentration was measured (Figure 1). The concentration of radioactive carbon ( 14 C) in leaves obtained from the central part of Kyoto city (1 to 4) was 0.220 to 0.228 Bq/gC, whereas that in the suburbs away from the center of the city ( 53 of 11 to 7) was between 0.238 and 0.242 Bq/gC (Table 1). The concentration of radioactive carbon ( 14 C) in the central part of Kyoto city was characteristically lower than that in the suburbs away from the center. In particular, the radioactive carbon ( 14 C) concentration of Jihaiminami station in the central part of the city was about 90‰ lower than that of the suburban Daigo station. In contrast to air pollutants such as nitrogen oxides, the concentration of radioactive carbon ( 14 C) in leaves in the center of the city tends to be lower than that in urban areas. This tendency indicates that the concentration of the dead carbon that hardly contains radioactive carbon ( 14 C) is comparatively high in the center of the urban district wherein the concentration of air pollution is high and low in the suburbs of the surrounding area wherein pollution is at a low level. Let us denote the concentration of carbon dioxide in the atmosphere not contaminated by dead carbon as (CO2). Radioactive carbon ( 14 C) comprises a certain proportion of this carbon, and there is a constant β radioactivity. At this time, the background concentration of the radioactive carbon ( 14 C) in the non-contaminated atmosphere, i.e., the radioactivity of β rays, is denoted as A. Since carbon dioxide generated from fossil fuels does not contain radioactive carbon ( 14 C), β rays are not detected at all. If the concentration of carbon dioxide generated by the combustion of dead carbon derived from fossil fuels is (C d O2), the concentration of the total carbon dioxide in the atmosphere increases to (CO2) + (C d O2). However, since radioactive carbon ( 14 C) in unit carbon is conversely diluted, the intensity of β-ray activity decreases. At this time, if the decrease in β ray radiation intensity per unit carbon is denoted as B, the following relation can be established. Therefore, the carbon dioxide concentration (C d O2) comprising dead carbon derived from fossil fuel is (A/B − 1)-times the concentration of carbon dioxide (CO2) of the unpolluted atmosphere. 1.4 Background concentration of radioactive carbon ( 14 C) in leaves The concentration of radioactive carbon ( 14 C) in the atmosphere, which rapidly increased following the atmospheric nuclear tests conducted in the mid-20th century, has gradually decreased over time due to its absorption by the ocean . To measure the background concentration of radioactive carbon ( 14 C) that is not affected by regional air pollution, it is appropriate to sample on remote islands considered to be non-polluted areas, as well as vessels in the open ocean and aircrafts. In order to obtain the background concentration of radioactive carbon ( 14 C) in tree leaves, tree leaves of C3 plants were sampled in the vicinity of the summit of a remote island in the northernmost part of the Japanese archipelago in late July 1996. The sampling site was on a slope near the summit (altitude of 440 m) of the Rebun mountain, Rebun Island, referred to as (north latitude of 45° 22′22′′ and east longitude of 141° 1′4′′). The results of the measurement of the radioactive carbon ( 14 C) in the leaves sampled from broadleaf birch trees of C3 plants growing on the slope showed that the background value was 0.246 ± 0.001 Bq/gC. ## Carbon dioxide concentration derived from fossil fuels in the center of major cities Carbon dioxide composed of dead carbon derived from fossil fuels is expected to exist at higher concentrations in urban areas compared to their surroundings. The radioactive carbon ( 14 C) concentration measured at the Jihaiminami station located in the central part of Kyoto, illustrated in As for the concentration of radioactive carbon ( 14 C) in street trees growing in the vicinity of the monitoring stations in the center of major cities other than Kyoto, namely, in Tokyo and Osaka, the β ray intensity decreased by at least 100‰ as compared to the background concentration in Rebun Island, showing a remarkable Suess effect . That is, in equation ( 3), if B ≦ 0.9 A, (C d O2) ≧ 0.111 (CO2). Pollutants measured at the general ambient air monitoring stations and automobile exhaust gas monitoring stations include nitrogen oxides, sulfur oxides, carbon monoxide, and suspended particulate matter. Since CO2 emitted from automobiles is basically non-toxic, it is not an object of monitoring in air pollution monitoring stations despite being discharged in significantly large quantities compared to other harmful pollutants. If CO2 is continuously measured at the monitoring stations, it could be expected that approximately 11% higher CO2 concentration would be recorded from monitoring stations located in the center of major cities in the daily average during the months when the tree grows, as compared to the non-polluted atmosphere. ## Research theme As stated above, dead carbon derived from fossil fuels in a mesoscale atmospheric environment is present in high concentrations in urban centers and in low concentrations in the surrounding suburbs. Apart from the general trend, it is expected that a strong contrast occur in the local density difference in the microscale atmospheric environment in the vicinity of intersections wherein main roads are intersected. To investigate how dead carbon originating from fossil fuels is spatially recorded in the leaves in microscale local polluted areas, measurements of radioactive carbon ( 14 C) were conducted in street trees at two intersections with severe air pollution. ## Leaf sampling from street trees In mid-October 1997, sampling was conducted to measure radioactive carbon ( 14 C) in the leaves obtained from 14 points at the Matsubarabashi intersection and the Yamatomachi intersection along the Annular Road 7 with serious air pollution in Tokyo (Figures 2 and 3). In order to eliminate the To measure the radioactive carbon ( 14 C) in leaves, the methanol method of liquid scintillation system was adopted . Methanol was synthesized from the elements in the sampled leaves; a toluene scintillator was added to the sample, which was then sealed in a 20 ml Teflon vial. β-rays were measured with a liquid scintillation counter (manufactured by Aloka) equipped for an external standard method. β-ray measurements were performed for 4,000 min or more per sample by the liquid scintillation system. ## Results of measurements The radioactive carbon ( 14 C) concentration at 14 points in Matsubarabashi intersection and Yamatomachi intersection measured by liquid scintillation method was from 0.208 Bq/gC to 0.238 Bq/gC (Table 2). With respect to the carbon isotope effect, i.e., the Suess effect, the permillage deviation was obtained from the following equation using the leaf radioactive carbon ( 14 C) concentration of the Rebun mountain, Rebun Island, as a background value. The isotope effect (Suess effect) across the 14 points ranged from −148‰ to −25‰. Furthermore, the ratio of CO2 concentration comprising dead carbon to the CO2 concentration in the uncontaminated atmosphere was determined from equation (3) in the calculation of the concentration of CO2 comprising dead carbon derived from fossil fuel in section 1.3. The proportion of CO2 concentration comprising dead carbon estimated at 14 points was from 173‰ to 25‰. In addition to the measurement results of the radioactive carbon ( 14 C) concentrations in the leaves at 14 points, the isotope effect (Suess effect) and the estimated proportion of CO2 concentration comprising dead carbon are shown in Table 2. ## Matsubarabashi intersection The Matsubarabashi intersection (35°35′44′′ north latitude, 139 ° 42′42′′ east longitude) in Ota Ward, Tokyo, is a grade-separated intersection of the Annular Road 7 and the National Route 1, and it is the earliest interchange in Japan equipped with ramps in the east and west (Figure 2). Furthermore, as for the street trees of three places (Matsubarabashi ④, ⑤, and ⑥) facing the Annular Road 7 other than the lower parts of overbridges, the isotope effect at Matsubarabashi ④ near the bus station located practically midway between the Matsubara bridge and the Shinmagome bridge was −111‰, which was slightly higher than that of under the overbridge. Matsubarabashi ⑥ is a point facing the inner tracks of the Annular Road 7, which is about 96 m southeast from the Matsubarabashi intersection, and the isotope effect in the street tree leaf was −90‰. Matsubarabashi ⑤ is a street tree facing the outer tracks of the Annular Road 7 line about 180 m southeast from the Matsubarabashi intersection, and the isotope effect was −90‰. Matsubarabashi ⑤ is distanced about 1.9 times farther from the intersection than Matsubarabashi ⑥ was but the isotope effects of the two points showed the same value. The Matsubarabashi intersection is equipped with ramps of about 50 m in diameter in the east and west. The isotope effect in the street tree leaves of Matsubarabashi ⑧, facing the ramp inner tracks on the east side, was −49‰. Matsubarabashi ⑨ is the site from where fallen leaves from broadleaf trees were collected from the ground near the central part of the west side ramp, and the isotope effect was −33‰. Although the ramp provides a structurally open space that is more ventilated than the office buildings and residential areas for the atmospheric environment around the intersection, the isotope effect of Matsubarabashi ⑧ and Matsubarabashi ⑨ showed characteristically higher values than in the 6 points facing the Annular Road 7. Among them, Matsubarabashi ⑨ and Matsubarabashi ② at the lower part of overbridge showed a difference in isotope effect of 94‰ despite the fact that the two are distanced as close as about 30 m; thus, the difference was significant. At this time, the difference in CO2 concentration generated by the combustion of fossil fuels was estimated to be 112‰. This is in contrast with the isotope effect of Matsubarabashi ③ located on the opposite bank of the Annular Road 7, similar to that about 30 m away across Matsubara bridge , which gave the same result as Matsubarabashi ②: −127‰. Radioactive carbon ( 14 C) in street trees was measured at three sites (Matsubarabashi ⑦, ⑩, and ⑪) in the residential environment at the hinterland of the roadside environment. Matsubarabashi ⑦ is a street tree in the residential area of approximately 65 m from the Annular Road 7 and approximately 26 m from the National Route 1, and the isotope effect in the leaves was −57‰. This was higher than that in the 6 points facing the Annular Road 7 and lower than that in the two points in the ramp. Matsubarabashi ⑩ is in a children's park located in the residential area furthest away from the Matsubarabashi intersection in the survey area and is a broadleaf tree in the environment with relatively good ventilation. The distance from the main roads is about 140 meters from the National Route 1 and about 235 m from the Annular Road 7 line, and the isotope effect in the leaves was −25‰. Matsubarabashi ⑪ is also a broadleaf tree planted in a children's park in the middle of a residential area, and the south side of the children's park is a site adjacent to Tokaido Shinkansen line and a relatively open environment. The distance from the main roads is about 70 meters from the Annular Road 7 and about 145 m from the National Route 1, and the isotope effect in the leaves was the same as in Matsubarabashi ⑩: −25‰. The isotope effect in the leaves of Matsubarabashi ⑩ and Matsubarabashi ⑪ in a small park in a residential area more than 70 m away from the main road was about 65%-123% higher than the isotope effect of Matsubarabashi ① to ⑥ facing the Annular Road 7. In addition, the difference in CO2 concentration comprising dead carbon is estimated to be between 74‰ and 148‰. ## Yamatomachi intersection The Yamatomachi intersection (35°45′41′′ north latitude, 139°42′20′′ east longitude) in Itabashi Ward, Tokyo, is a grade-separated road crossing wherein main roads from the lowest level, namely National Route 17, Annular Road 7, and Metropolitan Expressway Route 5 (Ikebukuro Line), intersect (Figure 3). At the Yamatomachi intersection, radioactive carbon ( 14 C) concentration in the leaves was measured in 3 street trees facing the Annular Road 7. The isotope effect in the leaves at these 3 points was between −135‰ and −102‰, which fell in the range of −148‰ to −90‰ measured at Matsubarabashi ①-⑥ facing the same Annular Road 7, and the presumed increase in CO2 concentration was form 156‰ to 114‰ (Table 2). Yamatomachi ① is a street tree facing the inner tracks (south side) of the Annular Road 7 and located about 23 m from the grade-separated road crossing, and the isotope effect in the leaves was −135‰. Yamatomachi ② is a street tree facing Yamatomachi ① across the Annular Road 7 and is located at the same distance of about 23 m from the grade-separated road crossing. The isotope effect in the leaves was −135‰, the same value as in Yamatomachi ①. Yamatomachi ③ is a street tree near the ground exit of the subway station facing the Annular Road 7 (north side), and the distance from the grade-separated road crossing is about 38 m. The isotope effect in the leaves was −102‰, 33‰ higher than in Yamatomachi ① and Yamatomachi ②, and the difference in estimated CO2 concentration consisting of dead carbon was 42‰. ## Conclusions The radioactive carbon ( 14 C) concentration in the street tree leaves in the surroundings of intersections records the influence of the daily average dead carbon during the months from spring to the fall when the trees grow. Moreover, the radioactive carbon ( 14 C) isotope effect (Suess effect) on local pollution quantitatively reflects the microclimatological trend of the micro-β-scale (20-200 m) over several months. The isotope effect in the leaves located at the lower parts of overbridges of the grade-separated road crossing, wherein the main roads overlap and contaminants tend to stagnate, is lower than −120‰, which is remarkably low among the measurement points (Matsubarabashi ①, ②, and ③ and Yamatomachi ① and ②). Among them, the isotope effects in the leaves located opposite one another across the Annular Road 7, which are thought to be influenced by the emissions from car traffic at the same level, show the same value (Matsubarabashi ② and ③ and Yamatomachi ① and ②). The isotope effect in the leaves facing the Annular Road 7 in sites other than the lower parts of overbridges at the grade-separated road crossing was from −111‰ to −90‰, showing a slightly higher isotope effect than that of the strongly contaminated lower part of overbridges (Matsubarabashi ④, ⑤, and ⑥ and Yamatomachi ③). The isotope effect in the leaves near the ramp part that forms a relatively ventilated open space in the roadside air environment was from −49 to −33‰, characteristically higher than in other sites facing the Annular Road 7 (Matsubarabashi ⑧ and ⑨). The isotope effects of near the ramp part and Matsubarabashi ②, approximately 30 m away from Matsubarabashi⑨ at the lower part of the overbridge, was −33‰ and −127‰, and the CO2 concentration composed of dead carbon was 34‰ and 146‰, respectively. Thus, a remarkable 4.3-fold concentration difference can be estimated. This indicates that the places with high and low levels of the micro-β-scale local contamination over several months exist side-by-side at a large intersection. Isotope effects in the leaves located in the residential environment in the hinterland of the highway show higher values than those of the lower parts of overbridges in the intersection and the area facing the Annular Road 7 (Matsubarabashi ⑦, ⑩, and ⑪). However, although it is in a residential environment wherein pollution is believed to be lower than at the roadside, Matsubarabashi ⑦, which is relatively close to the main road, showed lower isotope effect than Matsubarabashi ⑧ and Matsubarabashi ⑨ in the ramp section. A large intersection can be considered to be an actual field for diffusion experiments, wherein CO2, comprising dead carbon, is always generated by combustion of fossil fuel and a chemically stable CO2 tracer gas is released in large amounts. In other words, the measurement of radioactive carbon ( 14 C) in street trees can be a carbon isotope tracing method that actually diffuses CO2 tracer gas directly from automobile traffic in a roadside atmosphere without using a large-scale tracer system. Tracing dead carbon, which accounts for more than 80% of fossil fuels, is nothing less than tracking the diffusion of automobile emissions itself. Furthermore, radioactive carbon ( 14 C) measurements in street trees seem to lead to the estimation of microclimatological trends in microscale for local contamination by pollutants causing health hazards.

chemsum

{"title": "Radiocarbon ( 14 C) concentration of local pollution in street trees located at intersections", "journal": "ChemRxiv"}

metabolic_effect_of_drought_stress_on_the_leaves_of_young_oil_palm_(elaeis_guineensis)_plants_using_

## Abstract: The expansion of the oil palm in marginal areas can face challenges, such as water deficit, leading to an impact on palm oil production. A better understanding of the biological consequences of abiotic stresses on this crop can result from joint metabolic profiling and multivariate analysis. Metabolic profiling of leaves was performed from control and stressed plants (7 and 14 days of stress). Samples were extracted and analyzed on a UHPLC-ESI-Q-TOF-HRMS system. Acquired data were processed using XCMS Online and MetaboAnalyst for multivariate and pathway activity analysis. Metabolism was affected by drought stress through clear segregation between control and stressed groups. More importantly, metabolism changed through time, gradually from 7 to 14 days. The pathways most affected by drought stress were: starch and sucrose metabolism, glyoxylate and dicarboxylate metabolism, alanine, aspartate and glutamate metabolism, arginine and proline metabolism, and glycine, serine and threonine metabolism. The analysis of the metabolic profile were efficient to correlate and differentiate groups of oil palm plants submitted to different levels of drought stress. Putative compounds and their affected pathways can be used in future multiomics analysis. Palm oil, derived from the African Oil Palm (Elaeis guineensis Jacq.), is the most consumed edible oil in the World, with a global production of 83.96 million metric tons-palm oil and palm kernel oil-in 2020/2021 1 . This crop is highly dependent on water availability; therefore, drought stress could represent a high risk on the production yield. In the next few decades, the population growth and subsequently vegetable oil demands could lead to the unforeseen expansion of palm tree crops. However, limiting factors such as abiotic stresses are present in most potential farmable areas 12 . Water withhold directly affects the plant metabolism, given that defense mechanisms are promptly activated to reduce the implications of the stress. Usually, abiotic stress responses are related to crop growth, cell development, CO 2 fixation, photosynthesis capability, etc. 2 . Drought stress also induces the production and activation of compounds that modulate certain metabolites and pathways, e.g., cell homeostasis 3 . Metabolomics is a powerful tool to study applied stresses in plants due to the high capacity of compounds detection, identification, and pathway correlation through different methods . This technique is described as a "snapshot" of the studied organism, illustrating which compounds are present and their concentrations. The challenges faced on metabolomics analysis relies mainly on the complex biological matrices, which require different extraction and analytical techniques in order to detect, identify and/or quantify the highest possible number of metabolites 5 . The plant response to an environmental interaction such as drought stress is an enormous array of chemically altered metabolites. Metabolomics fits the abiotic stress study demand because metabolites are the most direct representation of the plant phenotype, since they are signatures of the biological and chemical activity 3 . Therefore, in order to lead stress tolerance studies in plants, there is a surging interest to observe the metabolite level changes after the abiotic stress 4,6 . Although many analytical techniques can be successfully employed in a metabolomics study, chemical separation and detection mainly resolves around nuclear magnetic resonance and mass spectrometry. Liquid chromatography is, in most cases, the choice adequate for polar phytochemical compound separation, even from complex matrices. Mass spectrometry offers a coupled technique (LC-MS) to detect and identify metabolites using high resolution and selectivity 7 . This tandem method is applied successfully to analyze a vast array of metabolites in plants, from different chemical classes-flavonoids, alkaloids, glucosinolates, organic acids, and others 4,5, . Discovering data patterns are a difficult task when done manually; therefore, a statistical treatment is necessary. The capability to organize and visualize high amounts of data comes from supervised classification methods, such as partial least square discriminant analysis (PLS-DA), which provides group separation based on their mass profile. Supervised methods bring the ability to reduce spatial components with no information loss, therefore metabolites detected and inserted in this model can be grouped through regression, which amplifies the discrimination between samples and visually defines groups with different treatments. Metabolic pathways can be further related to the grouped samples with the use of algorithms such as mummichog 11 to improve the biological meaning of the experiment. Young oil palm leaves were submitted to metabolic fingerprinting analysis using ultra-high-performance liquid chromatography-electrospray ionization-mass spectrometry (UHPLC-ESI-MS) for detection of polar compounds. Data analysis from MS spectra was performed through statistical visualization using PLS-DA, heatmap, and pathway activity analysis. Therefore, the aim of this study is to present a high-throughput untargeted method to identify droughtrelated metabolic pathways to improve the knowledge about oil palm response, which will be useful in further multiomics studies. ## Results and discussion Biochemical, morphophysiological responses and differential expression analysis: contextualization and data correlation. The current study derives from previous research activities on the characterization of the morphophysiological responses and analysis of differentially expressed genes of oil palm to drought stress 12 . Some results of these activities will be used in the future to corroborate and compare with the biochemistry of oil palm drought stress. Important parameters showed that non-irrigated plants were physiologically stressed and such stress could be responsible for metabolic changes. We have collected information regarding evapotranspiration and soil water potential, leaf gas exchange [net CO 2 assimilation rate (A), transpiration rate (E), stomatal conductance to water vapor (gs), and intercellular CO 2 concentration (Ci)], chlorophyll fluorescence [Fm, Fo, Y(II), Fv/Fm, Y(NPQ), and Y(NO)], pigment content, leaf relative water content and leaf temperature (including thermographic images). This data is not shown at this moment as it has been integrated to mRNA and miRNA transcriptome data for future studies. The drought-stressed plants suffered a gradual reduction in water content from the substrate, resulting in a fall of the soil water potential, evapotranspiration rate, and fresh biomass. The net CO 2 assimilation, stomatal conductance, and transpiration rates suffered a statistical reduction. The fall in net CO 2 assimilation and stomatal conductance rates, which led to a reduction or inhibition of the enzymatic activity, is the cause of this decrease in photosynthetic activity 13,14 . Therefore, the unbalance caused by the low water availability can directly affect the cellular metabolism given the excess or lack of essential metabolites needed for the plants' biochemical reactions. In a state of water deprivation, plants usually suffer function rates and photosynthetic efficiency alteration 15,16 . E. guineensis samples presented a linear decrease in chlorophyll concentration and factors related to chlorophyll fluorescence only after the 11th day of drought stress. These data led us to infer that some analyses are better for stress detection, depending on the level of sensibility. After irrigation interruption, many cellular metabolism alterations can be detected by high throughput phenotyping methods, depending on intensity, time of exposure, developmental stage, and species analyzed 17,18 . In this study, the metabolomics approach fits due to the drought sensitivity presented just a few days after the start of the water deprivation. ## Metabolic fingerprinting analysis. Metabolic fingerprinting is widely known as a powerful untargeted approach that correlates chromatogram profiles and the compound information within the MS peaks. The drought stress was studied by comparison of the metabolic profile in plants of three groups: control (irrigated) and stressed samples (7 and 14 days of water deprivation). In Fig. 1, a representative chromatogram of each group is shown. The data were acquired using UHPLC analysis and then treated with a "dissect" algorithm, where a list of compounds is created with averaged compound mass spectra making it possible to separate overlapping peaks. Based on the UHPLC gradient elution method, it is inferred that polar compounds are observed at 0-2 min, medium-polarity compounds at 2-6 min, and non-polar compounds at 6-10 min, all in the positive (UHPLC-ESI(+)-MS) and negative (UHPLC-ESI(−)-MS) ionization modes. A large number of chromatographic peaks after the dissect treatment was detected in both ionization modes, with an average peak count of 98 for UHPLC-ESI(−)-MS of drought samples, 96 for UHPLC-ESI(−)-MS control samples, 84 for UHPLC-ESI(+)-MS of drought samples, and 86 for UHPLC-ESI(+)-MS of control samples. ## Data analysis. In this study, a total of 32 chromatograms was acquired using UHPLC-MS, and then a manual comparison of spectra could easily lead to error. A series of chemometric methods were used to identify www.nature.com/scientificreports/ the metabolic differences among control and stressed plants. After data pre-processing, the statistic module of MetaboAnalyst was employed as the software for the analysis. MetaboAnalyst 4.0 is a web-based tool suite for comprehensive metabolomics data analysis, interpretation, and multi-omics data integration 19,20 . MetaboAnalyst supports a wide array of functions for statistical, functional, as well as data visualization tasks. Some of the most widely used approaches include supervised classification techniques-PLS-DA-and unsupervised models-clustering analysis and heatmaps; besides the correlation between metabolites and metabolic pathways, all presented in this study. ## Partial Least Square Discriminant Analysis (PLS-DA). To identify patterns and differentially expressed metabolites between the groups, the PLS-DA was applied as the multivariate separation method. This supervised method provides a robust regression technique based on labeled samples to optimize group separation by a component rotation 21 . PLS discriminant analysis was applied when comparing control, drought stress of 7 days, and drought stress of 14 days (Fig. 2). Both ESI(+)-MS and ESI(−)-MS datasets presented clear segregation between groups, showing that the metabolism is affected by water deprivation. The 7-day group was closer to the control group when compared to the 14 days group, indicating that metabolism changed gradually through time. Cross-validation is essential to ensure the model's robustness due either to the classificatory nature and inherent overfitting of the PLS analysis 21 . We used the leave-one-out cross-validation (LOOCV), and the Q 2 was evaluated on three components, resulting in the following values: Q 2 = 0.6866 and accuracy = 0.933 for ESI(+)-MS and Q 2 = 0.7830 and accuracy = 1.00 for ESI(−)-MS data, which represents a robust and reliable model. In a supervised classification model, R 2 and Q 2 are the accuracy parameters, where they range from 0 to 1 (higher means better accuracy) and R 2 represents the raw predictive accuracy. The Q 2 value is obtained when the PLS model is built on a training set against a test set, and usually a Q 2 value higher than 0.65 is considered substantial for the model predictability. The PLS-DA is a fitting-method for identifying metabolites differentially expressed through the variable importance in projection (VIP) value. A variable with a VIP value higher than one is potentially important in the model construction. In ESI(−)-MS, we found 1126 variables with VIP > 1. In ESI(+)-MS we observed 1069 variables with VIP > 1, and from those, 182 variables with VIP > 2. Hierarchical clustering heatmap. Figure 3 shows a heatmap generated using the top 50 variables showing the higher VIP values in each ionization mode analysis. The heat indicates the behavior of those variables throughout the samples. It is possible to confirm the metabolic trends observed on PLS-DA using heatmaps as multivariate cluster analysis. A gradient is observed in metabolic intensity, increasing in most cases from the control group (the blue area in the left) up to the 14 days of drought stress (the red in the middle). For example, m/z 565.2385 has a low intensity on the control group, a medium intensity at 7 days of stress, and a high at 14 days of stress. This trend indicates a mass production of defense metabolites as a plant mechanism to survive and keep its metabolic functions in the presence of abiotic stress. A few cases show an opposite trend, where metabolites went from a high intensity on control groups to a low one on the 14 days of the stressed group. For example, the detected ESI(−)-MS ions m/z 327. 9555 This heatmap cluster analysis shows that not only metabolite intensities can shift between groups with different treatments, those metabolites can be regulated according to the plants response to the stress applied. ## Metabolic pathway correlation. This metabolomics study ends on the pathways most affected by drought stress. A clear and objective understanding of the affected-pathways is a way to get the information required to develop multiple biotechnological applications, where the development of stress-tolerant genotypes is the final goal to increase productivity. This type of study could also be part of a combined multiomics integration approach, together with genomics, transcriptomics and proteomics studies. In recent metabolomics studies, many techniques have been applied in pathway correlation, from manual to automated methods 4, . Here, we used the mummichog algorithm 11,26 , based on over-representation analysis (ORA), to analyze UHPLC-MS data and predict enriched pathway activity, comparing the significant peaks of annotated metabolites. All samples from UHPLC-ESI(+)-MS and UHPLC-ESI(−)-MS were submitted to the "MS peaks to pathways" module of MetaboAnalyst. The pathway activity profile obtained is presented in Fig. 4, indicating the five most affected pathways in both ionization methods. In total, 176 and 85 metabolites from 42 pathways were significant upon applying the mummichog algorithm on UHPLC-ESI(+)-MS and UHPLC-ESI(−)-MS data, respectively. The "Supplementary material" (Tables S1 and S2) presents a list with all affected pathways. In the UHPLC-ESI(+)-MS analysis, the most affected pathways were: starch and sucrose metabolism; glyoxylate and dicarboxylate metabolism; alanine, aspartate, and glutamate metabolism; and arginine and proline metabolism. And the most affected pathways in the UHPLC-ESI(−)-MS were: starch and sucrose metabolism; glutathione metabolism; alanine, aspartate, and glutamate metabolism; and glycine, serine, and threonine metabolism. Table 1 indicates the annotated metabolites that ensured the importance of the affected pathways. The starch and sucrose metabolism was the most affected pathway in either analyses, ESI(+)-MS and ESI(−)-MS. This metabolic pathway has a role in photosynthesis, when sucrose and starch are converted from triose-phosphates during the CO 2 plant fixation, with strict governance between both processes. Synthesis of sucrose and starch occurs, respectively, at the cytosol and chloroplast, and the Pi-triose phosphate antiport system mediates the coordination 27 . Triosephosphate synthesis is affected by a slow sucrose production that results in low Pi available to the chloroplast, while a rapid sucrose production results in the removal of triose phosphate www.nature.com/scientificreports/ in excess. Morphologically, plants with deficient sucrose synthesis present reduced growth and tolerance to anaerobic-stress conditions 28 . Glyoxylate and dicarboxylate metabolism is an important abiotic stress-related pathway, providing a balance in metabolic disorders to improve tolerance 29 . The glutamic acid, indicated in Table 1 and present in both glyoxylate and glutathione metabolism, is vastly transported in phloem sap and plays a major role in many biosynthesis of other amino acids, chlorophylls, and tricarboxylic acid. The glutamate synthase (GS) isoforms GS1 and GS2 are described as pivotal enzymes used in genetically enhanced species to improve photorespiration capabilities 30 and response to energy supply 31 . The alanine, aspartate, and glutamate metabolism is considered a short catabolic pathway, where an alanine is converted into pyruvate, which was highly affected in our study. There are essential metabolic branches influenced by this pathway in mitochondrial multi-enzyme system, such as isoleucine, cysteine, methionine, and threonine synthesis, which clearly states its importance from a nutritional perspective 32 . The arginine and proline pathway is related to nitrogen metabolism in plants, essential for production of nucleic acids and proteins. Arginine is a precursor of polyamines and has a role in proline biosynthesis when glutamate is not available. The influence of drought stress is highly expected in this pathway, given that proline has the capability of protein protection and membrane structure in dehydration cases 33 , acting on redox status or as a scavenger of reactive oxygen species that could increase cellular solute concentration. Many studies on metabolites from glycine, serine, and threonine metabolism, looking for a better understanding of the chemical defenses against salt, cold, and drought stresses in plants, are available. For instance, some of them show that threonine metabolites are involved in plant growth and development, cell division, and phytohormones regulation 34,35 . ## Chemicals. Methanol UHPLC grade, acetonitrile LC-MS grade, methyl-tert-butyl-ether, formic acid LC-MS grade, and sodium hydroxide ACS grade were purchased from Sigma-Aldrich (Merck, USA). Water was obtained using a Milli-Q system (Millipore, USA). ## Plant material and growth conditions. The oil palm plants used were clones regenerated out of embryogenic calluses obtained from leaves of an adult plant belonging to the E. guineensis genotype AM33 12 . The AM33 genotype is a plant from a commercial field in the State of Pará, in Brazil. This field was established with seeds from a cultivar developed by Embrapa. Oil palm seeds produced and commercialized by Embrapa in Brazil are "Deli x La Mé", and the parentals came from progenies obtained from Dura and Tenera plants self-crossed. www.nature.com/scientificreports/ Plants were kept in black plastic pots (5 L), containing 1700g of a mix of vermiculite, soil, and a commercial substrate (Bioplant, Brazil) in a 1:1:1 ratio-on a dry basis-and fertilized using 2.5 g/L of the formula 20-20-20. Before starting the experiments, we screened the plants to standardize the developmental stage, size, and the number of leaves. The experiment was performed in a greenhouse at Embrapa Agroenergy (www. embra pa. br/ en/ agroe nergia), in Brasília, DF, Brazil (S-15.732°, W-47.900°). The plant material collection and methodology used in this study complied with relevant institutional, national and international guidelines and legislation. The main environmental variables (temperature, humidity, and radiation) fluctuated according to the weather conditions and were monitored throughout the experimental period from the data collected at a nearby weather station (S-15.789°, W-47.925°). Experimental design and drought stress. The experiment consisted of two treatments-control and drought-stressed plants-with four plants kept in a substrate in the field capacity (control), and six plants submitted to drought stress. The young oil palm plants were subjected to treatments when they were in the growth stage known as "bifid" saplings. Drought stress consisted of total suppression of irrigation for 14 consecutive days. At the end of this period, the substrate water potential, as measured by the water potential meter Decagon mod. WP4C (Decagon Devices, Pullman, WA, USA), was 0.19 ± 0.03 MPa (control) and − 13.61 ± 1.79 MPa (drought stress), while the relative water content of leaves was 90.50 ± 0.95% (control) and 49.18 ± 9.76% (stressed plants). Before the onset of drought stress, oil palm leaves had the highest gas exchange rates, as measured by infrared gas analyzer Li-Cor model 6400XT (Li-Cor, Lincoln, NE, USA). Under drought, leaf gas exchange rates in droughtstressed plants dropped to negligible values (data not shown). Leaf samples were collected at 7 and 14 days after the onset of the stress from four control plants and four stressed plants. Leaf samples with approximately 50 mg were collected for the metabolomics analysis; four replicates per plant. After harvesting, samples were immediately frozen in liquid nitrogen and stored at − 80 °C until metabolites extraction and analysis. ## Metabolites extraction. Each sample was ground in a ball mill (Biospec Products, USA) before solvent extraction. Metabolites were extracted using an adapted protocol from The Max Planck Institute 36 , called "Allin-One", which provides a polar fraction for secondary metabolite analysis, a nonpolar fraction for lipidomics and a protein pellet for proteomics; all obtained from the same plant sample. Each ground sample was added to a microtube and mixed with 1 mL of a methanol and methyl-tert-butyl-ether (1:3) solution at − 20 °C. After homogenization, they were incubated at 4 °C for 10 min. Each microtube was ultrasonicated in an ice bath for another 10 min. Then, 500 μL of a methanol and water (1:3) solution was added to the microtube before centrifugation (12,000 rpm at 4 °C for 5 min). Three phases were separate: an upper non-polar (green), a lower polar (brown), and a remaining protein pellet. Samples were transferred to fresh microtubes and vacuum-dried in a speed vac (Centrivap, Labconco, Kansas City, MO, USA) overnight at room temperature (~ 22 °C). ## UHPLC-MS. A total of 0.4 μL of the extract was then resuspended in 850 μL of methanol and water (1:3) solvent mixture and then analyzed by UHPLC-MS. The Nexera X2 UHPLC system (Shimadzu Corporation, Japan) was equipped with a reversed-phase Acquity UPLC BEH C8 column (1.7 μm, 2.1 × 150 mm) (Waters Technologies, USA). Chromatographic run parameters were: isocratic from 0 to 0.5 min (4% B), linear gradient from 0.5 to 10 min (34% B) and 10-15 min (100% B) and isocratic from 15 to 18 min (100% B). Solvent A was 0.1% formic acid in water (v/v), and solvent B was 0.1% formic acid in acetonitrile (v/v). The flow rate was set at 400 μL/min. High-resolution mass spectrometry (HRMS) was performed in a MaXis 4G Q-TOF MS system (Bruker Daltonics, Germany) using an electrospray source in the positive and negative ion modes (ESI(+)-MS and ESI(−)-MS). The MS instrument settings used were: endplate offset, 500 V; capillary voltage, 3800 V; nebulizer pressure, 4 bar; dry gas flow, 9 L/min, dry temperature, 200 °C; and column temperature, 40 °C. The acquisition spectra rate was 3.00 Hz, monitoring a mass range from 70 to 1200 m/z. Sodium formate solution (10 mM NaOH solution in 50/50 v/v isopropanol/water containing 0.2% formic acid) was directly injected through a 6-port valve at the beginning of each chromatographic run to external calibration. UHPLC-MS data was acquired by the HyStar Application version 3.2 (Bruker Daltonics, Germany), and data processing was done using Data Analysis 4.2 (Bruker Daltonics, Germany). This extraction method and UHPLC-MS analysis system has been optimized and used in recent studies from our group 4 and resulted in reliable results, therefore is replicated in the present work. ## Data analysis. The raw data from UHPLC-MS was exported as mzMXL files using DataAnalysis 4.2 software (Bruker Daltonics, Germany) and pre-processed using XCMS Online 37,38 for feature detection, retention time correction, and alignment of metabolites detected on UHPLC-MS analysis. Two datasets, one for the samples harvested at 7 days of drought and another for the samples harvested at 14 days, were created. Pre-processing done using optimized parameters based on Albóniga et al. 39 , which tunes feature detection to obtain a smaller data matrix but with a higher number of variables with an SD < 20%, which creates a more robust data processing. Peak detection was performed using centWave peak detection (Δ m/z = 25 ppm; mzdiff = 0.002; minimum peak width = 12 s; maximum peak width = 40 s) and mzwid = 0.02, minfrac = 0.16, bw = 1 were used for retention time alignment. Statistics analysis used an unpaired parametric t-test (Welch t-test). The processed data (csv file) was then submitted for analysis in the MetaboAnalyst 4.0 19,20 . Before multivariate analysis [partial least square discriminant analysis (PLS-DA), heatmap, and hierarchical cluster analysis (HCA)], all data variables were normalized by internal standard (sodium formate adduct, rT = 0.1 min; m/z 226.9522 in positive mode, m/z 316.9478 in negative) and scaled by the auto-scaling method. A PLS model was built with www.nature.com/scientificreports/ three groups to attempt the segregation between control (irrigated) and stressed samples (7 days and 14 days of drought). Internal validation-leave-one-out cross-validation (LOOCV)-was performed to ensure model robustness. The results described here were obtained at the MetaboAnalyst web tool in 4/14/2020. A heatmap was built using all samples and the following criteria: distance measure, Euclidean; clustering algorithm, Ward; standardization, autoscale; and top 25 features using t-test/ANOVA to retain the most contrasting patterns. The last step of the data processing was the use of the mummichog algorithm approach 11 in the MS peaks to pathways module of MetaboAnalyst. The criteria used on this analysis were: molecular weight tolerance, 5 ppm; primary ions enforced; p-value cutoff, 0.01; pathway library, Oryza sativa japonica (Japanese rice) from Kyoto Encyclopedia of Genes and Genomes (KEGG) . ## Conclusion Through an untargeted metabolomics method, different peak intensities between control and stressed groups were used as the main parameter to evaluate tolerance levels to water deficit and to screen drought tolerance in E. guineensis leaves. A high amount of metabolites and pathways were significantly affected by drought stress. We detected metabolites from a wide range of chemical classes using UHPLC-MS as a high-throughput untargeted method and putatively annotated 24 differentially expressed metabolites from the most affected pathways on ESI(+)-MS and ESI(−)-MS. These pathways were: starch and sucrose metabolism; glyoxylate and dicarboxylate metabolism; alanine, aspartate, and glutamate metabolism; arginine and proline metabolism; and glycine, serine, and threonine metabolism. Metabolic pathways and their respective compounds, presented in this study, corroborated with the clear metabolic response of E. guineensis, given that most of those pathways are known by their importance in response to abiotic stress, such as drought stress. These results implicate a more accurate and responsive multi-omics future study targeting enhanced crops with a higher tolerance to water deficit, resulting in an improved crop yield.

chemsum

{"title": "Metabolic effect of drought stress on the leaves of young oil palm (Elaeis guineensis) plants using UHPLC\u2013MS and multivariate analysis", "journal": "Scientific Reports - Nature"}

an_ultrasensitive_polydopamine_bi-functionalized_sers_immunoassay_for_exosome-based_diagnosis_and_cl

## Abstract: Early diagnosis and metastasis monitoring for pancreatic cancer are extremely difficult due to a lack of sensitive liquid biopsy methods and reliable biomarkers. Herein, we developed easy-to-prepare and effective polydopamine-modified immunocapture substrates and an ultrathin polydopamine-encapsulated antibody-reporter-Ag(shell)-Au(core) multilayer (PEARL) Surface-Enhanced Raman Scattering (SERS) nano-tag with a quantitative signal of the Raman reporter at 1072 cm À1 , which achieved ultrasensitive and specific detection of pancreatic cancer-derived exosomes with a detection limit of only one exosome in 2 mL of sample solution (approximately 9 Â 10 À19 mol L À1 ). Furthermore, by analyzing a 2 mL clinical serum sample, the migration inhibitory factor (MIF) antibody-based SERS immunoassay could not only discriminate pancreatic cancer patients (n ¼ 71) from healthy individuals (n ¼ 32), but also distinguish metastasized tumors from metastasis-free tumors, and Tumor Node Metastasis (TNM) P1-2 stages from the P3 stage (the discriminatory sensitivity was 95.7%). Thus, this novel immunoassay provides a powerful tool for the early diagnosis, classification and metastasis monitoring of pancreatic cancer patients. ## Introduction Pancreatic cancer is one of the most life-threatening malignancies worldwide, with a fve-year survival rate of lower than 5% due to difficulties in early diagnosis and metastasis monitoring because the pancreas is relatively hidden and lacks specifc biomarkers. 1 Traditional biomarkers such as carcinoembryonic antigen (CEA) and cancer antigen 19-9 (CA19-9) have improved the diagnostic accuracy of pancreatic cancer, 2 but their specifcity for pancreatic cancer is low because of high CA19-9 expression in benign pancreatic diseases and increased CEA expression in colorectal cancer. 3,4 Therefore, it is urgently required to establish new methods that improve the specifcity and sensitivity of pancreatic cancer diagnosis. As a "fngerprint" of their parental cells, exosomes, which are secreted vesicles 40-200 nm in diameter that are usually formed via the endosomal pathway and contain proteins, microRNAs and other non-coding RNAs, can reveal information about the metabolic state and degree of malignancy of parental cells. 5,6 Therefore, research on exosomes has increased with the aim of using these extracellular vesicles for the diagnosis, therapy and mechanistic study of cancers and other diseases. 7,8 Recent studies have reported two new biomarkers, glypican-1 (GPC-1) 9 and ephrin type-A receptor 2 (EphA2), that are expressed on exosome surfaces. 10 They then developed exosome-based nanotechnologies (nano-plasmonic nanohole arrays 11 and multichannel nanofluidic systems 12 ) and applied a new data analysis method (Machine Learning Algorithm 12 ) for sensitive and specifc diagnosis, classifcation and metastasis monitoring of pancreatic cancer. However, for the clinical application of these technologies, there are still some remaining challenges to solve: (1) more specifc and reliable exosomes or extracellular vesicle biomarkers need to be screened; (2) a sensitive detection method that requires only a small volume of bio-samples should be developed to replace traditional methods such as flow cytometry or enzyme-linked immunosorbent assay (ELISA); and (3) a simple, fast and effective pretreatment method for clinical bio-samples should be developed to avoid the current time-consuming high-speed ultracentrifugation steps for exosome enrichment. Based on our previous work on Surface-Enhanced Raman Scattering (SERS), in this study we developed an ultrasensitive SERS immunoassay that uses an ultra-small volume of serum for the exosome-based diagnosis, classifcation and metastasis monitoring of pancreatic cancer. As shown in Scheme 1, polydopamine (PDA) was self-polymerized 16,17 on glass slides and specifc antibodies (anti-MIF, anti-GPC1, anti-CD63, or anti-epidermal growth factor receptor (EGFR)) on the exosome surface were simultaneously encapsulated into the porous hydrophilic PDA layer. Then, exosomes derived from pancreatic cancer or healthy control samples were captured and enriched on the chip surface, followed by incubation with PDA encapsulated antibody-reporter-Ag(shell)-Au(core) multilayer (PEARL) SERS tags to form a "chip-exosome-PEARL tag" sandwich structure. The Raman spectrum was then scanned and the intensity of the Raman reporter at 1072 cm 1 was chosen as the quantitative signal. To our knowledge, this is the frst time that the self-polymerization of dopamine has been used to capture antibodies on a substrate in combination with PEARL SERS nano-tags to construct an immunoassay. Based on this ingenious design and synthesis, this approach provided strong SERS signals for the ultrasensitive detection of exosomes in an ultrasmall volume (2 mL) of clinical pancreatic serum samples, avoiding the time-consuming high-speed ultracentrifugation process. Furthermore, motivated by clinical needs, this liquid biopsy method distinguished metastatic tumors from nonmetastatic tumors, and P1-2 stages from P3 stage tumors, without the need of histopathological examinations. ## Creating SERS sensors with PDA chips and PEARL tags To develop sensitive and reliable SERS immunosensors for clinical pancreatic cancer diagnostics, we frst employed a selfpolymerizing PDA layer to simultaneously encapsulate and capture antibodies to increase the number of captured antibodies and maintain their bioactivity. The average thickness of the PDA layer is about 50-100 nm and the rough structure of the PDA surface (Fig. 1A and B) provided enough space for capturing antibodies. The PDA density (black dots) increased when the dopamine concentration increased from 16.5 to 66 mM (Fig. S1a †), resulting in an increased antibody capture efficiency (Fig. 1B and S1b †). The activity of captured antibodies was evaluated using Horseradish Peroxidase (HRP) as a model protein for capture due to its wide usage in commercial ELISA. As shown in Fig. S1c, † the activity of HRP decreased with increasing dopamine concentration, which suggested that more active sites of HRP were buried in the denser PDA layer. Finally, Raman spectroscopy was used to characterize the PDA surface. The optimum dopamine concentration was found to be 33 mM, as it generated the smallest interference Raman signal from PDA (Fig. S1d †). As one of the major concerns for this assay was quantitative accuracy, the reproducibility of the Raman signal was directly influenced by the homogeneity of the flm area. Glass slides with and without PDA modifcation both displayed signifcant "coffee ring effects", which showed the non-uniform adsorption of the SERS tag. In contrast, slides modifed with antibodies had no "coffee ring effect" (Fig. 1C), which indicated that the modifcation of both PDA and antibodies synergistically improved the homogeneity of modifed flms due to the homogeneous capture of exosomes and good distribution of SERS tags. Compared with other antibody capture methods, such as physical adsorption on polystyrene 96-well plates or chemical covalent modifcation on magnetic beads, 18,19 PDA encapsulation provided more biocompatible, mild and uniform surface modifcations for high antibody capture efficiency and a high sensitivity for detecting cancer derived exosomes, which is the frst essential factor for immunoassays. Secondly, the high sensitivity and stability of SERS tags play essential roles in the clinical application of SERS immunosensors. 20,21 A SERS tag with high brightness, stability, and targeting capability is typically composed of four parts, including SERS nanostructures with a high enhancement factor, signal molecules that provide Raman signals, a signal protective layer with nanostructures, and a functional layer having a recognizable ability at the outermost layer of the material. Therefore, we designed and prepared PEARL SERS tags. Gold nanoparticles were chosen as the core, and silver, the Raman reporter molecule, BSA, PDA and antibodies were consecutively assembled onto the gold nanoparticle surface using the self-polymerization reaction of dopamine under a weak alkaline environment to form an ultrathin (nanometer-thickness) protective and antibody encapsulating layer. The SERS tag has a distinct core-shell structure, with an approximately 1 nm-thick Ag shell and an approximately 3 nm-thick PDA shell, giving a total diameter of approximately 40 nm (Fig. 1D). As shown in Fig. 1E, the PEARL SERS tag had a very strong signal for the Raman reporter 4aminobenzenethiol (pATP), with the peak at 1072 cm 1 contributed by the breathing vibration of the benzene ring and that at 1582 cm 1 arising from the C-N symmetric stretching vibration, while the gold nanoparticles showed no signal except for the capillary scattering background signal. To show the brightness of this SERS tag, extreme Raman excitation conditions of 0.05 mW laser power and 10 ms acquisition time (averaged 100 times) were set, and the spectrum was recorded (Fig. S1f †). Although the laser power and acquisition time were very low and short compared with normal test conditions (8 mW and 1 s), these test results also showed a distinct SERS spectrum of pATP, which indicated that the SERS tag had excellent Raman intensity and great potential for detecting trace biomarkers. Importantly, PDA was not only used in the PEARL SERS tag as a protective shell to prevent oxidation of the Ag layer and the resulting decrease of the SERS signal, but also as an effective encapsulating reagent for detecting antibodies. The PDA thickness strongly influenced the stability and Raman intensity of the tags. 31 The Raman intensity of the tags dropped signifcantly when the dopamine concentration increased (Fig. 1F), and the SERS tags grew too large, resulting in the sedimentation of nanoparticles when the solutions were rested for a few minutes. Based on these observations, the optimal dopamine concentration for forming the encapsulation layer of the SERS tags was set at 0.83 mM and the optimal thickness of the PDA layer was 3 nm, which is thinner than the 6 nm PDA-SERS Au tag for bone cracks. 31 Our SERS tag with an ultra-thin PDA layer maintained the strong enhancement effect of Au-Ag nanomaterials and resulted in high SERS signal intensity. The PEARL tags were extremely stable and showed no decrease in the Raman signal for at least 6 months when stored at 4 C. ## Identication of candidate marker proteins on exosomes derived from pancreatic cells To realize the clinical potential of this immunoassay for serum samples from pancreatic cancer patients, we qualitatively characterized exosomes derived from pancreatic cancer (PANC-01) and healthy cells (HPDE6-C7) by TEM. Exosomes derived from pancreatic cells showed a typical phospholipid bilayer structure (Fig. 2A and B). The diameters of exosomes from HPDE6-C7 cells were approximately 100 nm, and they were smaller than that of PANC-01-derived exosomes (140 nm). The secretory ability of adenocarcinoma cells was stronger than that of healthy cells. Owing to polymorphisms and irregularities in cancer cells, 36,37 PANC-01 exosomes were less uniform than those from HPDE6-C7 cells. We further performed Nanoparticle Tracking Analysis (NTA) to quantify the number of exosomes. For NTA processing, exosomes were suspended in solution to prevent them from losing their biological functions and molecular structures. The distribution of particles smaller than 200 nm in diameter is shown in Fig. 2C. The concentration of exosomes was thus calculated to be 2.72 10 10 AE 2.05 10 9 particles per mL, and the average size was 123.46 AE 26.93 nm, which was in agreement with the exosome sizes previously reported using TEM. 38,39 To identify proteins commonly expressed on exosome membranes (such as CD9 and CD63) and specifc pancreatic cancer-derived exosome proteins (such as GPC1 and MIF), supermagnetic beads with the corresponding antibodies were used to capture exosomes (Fig. 2D), and then the exosome membranes were dyed with 3,3 0 -dioctadecyloxacarbocyanine perchlorate (DIO) and analyzed by flow cytometry. Goat antimouse IgG was used as the control sample. As shown in Fig. 2E, CD9 was expressed on 88.0% and 76.3% of exosomes from PANC-01 and HPDE6-C7 cells, respectively, CD63 was expressed on 89.4% and 83.0%, respectively, GPC1 was found on 97.0% and 0.832%, respectively, and MIF was found on 98.9% and 0.652%, respectively, indicating that there was a signifcant increase in exosomal GPC1 and MIF expression in PANC-01 cells compared with the healthy HPDE6-C7 cells. Meanwhile, CD9 and CD63 expressions were similar in the two groups. These results suggested that MIF and/or GPC1 expression might distinguish exosomes from pancreatic cancer cells and normal pancreas cells. This conclusion was consistent with previous studies that showed that GPC1 and MIF were dramatically overexpressed in the serum from pancreatic cancer patients, and thus could be used as biomarkers to distinguish early-stage cancer from benign disease and/or predict tumor metastasis or tumor burden. 9,40 Ultrasensitive exosome detection based on the chip-exosome-PEARL tag immunoassay Based on our developed PDA chips, PEARL tags and the iden-tifed pancreatic cancer exosome-specifc surface proteins mentioned above, we designed an exosome assay for pancreatic cancer (Scheme 1). Typically, 2 mL of PANC-01-or HPDE6-C7derived exosome solutions of different concentrations were dropped onto the PDA chips, followed by adding the PEARL SERS tag solution. The homogeneous encapsulation of antibodies on the PDA chip was found to contribute to the uniformity of the sample points on the chip. We designed four experimental groups using four different antibodies: anti-CD9, anti-CD63, anti-MIF and anti-GPC1. For each group, the antibodies on the PDA chips and the PEARL SERS tag were the same. For different antibody-based platforms, we dropped exosome solution onto different spots on the PDA modifed glass slide, not onto a single spot for all antibodies. The Raman peak at 1072 cm 1 was chosen as the quantitative signal, because it was one of the three strongest peaks in the spectrum and there was almost no interference from other impure peaks near the 1072 cm 1 peak. In PANC-01 exosomes, the intensities of the anti-CD9, anti-CD63, anti-GPC1 and anti-MIF groups were 1233, 3597, 2659 and 4455, respectively, while for HPDE6-C7 exosomes the respective intensities were 1240, 3414, 1024, and 648 (Fig. 3A and B). Interestingly, the CD63 intensity was higher than that of GPC1, which was not in accordance with the flow cytometry results. The reason for this discrepancy was that in flow cytometry, the membranes of captured exosomes were DIO-stained to facilitate counting the number of exosomes, while in the PDA chip, the exosomes were labeled with the PEARL tag, which was recognized by antigen epitopes on the exosomes. The number of CD63 antigens on each exosome membrane was larger than that of GPC-1, which resulted in stronger Raman intensity. Regardless, we observed that the intensities of anti-CD9 and anti-CD63 groups were similar, while there were signifcant differences between HPDE6-C7exosomes and PANC-01-exosomes in the anti-GPC1 and anti-MIF groups (Fig. 3B). Compared with flow cytometry, which requires large amounts of expensive antibodies, our PDA-SERS method only requires about one fortieth of the amount of antibody. Using our PDA-SERS method, a higher SERS signal and a larger signal difference between PANC-01-and HPDE6-C7derived exosomes were obtained using the MIF antibody than the GPC1 antibody, which was consistent with the fnding that MIF is more highly expressed on the exosomes from pancreatic cancer patients than those from healthy individuals. Moreover, MIF is markedly higher in exosomes from stage I pancreatic ductal adenocarcinoma patients who later developed liver metastases than from patients whose pancreatic tumors did not progress. 34,36,40,41 Exosomal MIF primes the liver for metastasis and may be a prognostic marker for the development of pancreatic ductal adenocarcinoma (PDAC) liver metastases. MIF is a well-known mediator of liver inflammation and fbrosis, 42 bone marrow cell recruitment to the liver, and liver metastasis. MIF tissue and plasma levels correlate with PDAC aggressiveness. 43,44 To our knowledge, this is the frst time that a sensitive and stable PDA-SERS methodology has been used in exosome research. Additionally, MIF-based exosome detection was performed for the frst time, except for using the conventional ELISA method. A recent study reported that exosomal GPC1 was a potential biomarker for diagnosing pancreatic cancer. 9 Unfortunately, the previously used GPC1 antibody is no longer commercially available. Thus, we suspect there are some differences between the GPC1 antibodies from the two different companies. We further used anti-MIF to capture PANC-01-derived exosomes at different concentrations (5.44 10 2 to 2.72 10 10 particles per mL), while the control sample was PBS without exosomes. The results showed that the SERS signal intensity increased with increasing exosome concentration (Fig. 3C and D). There was a good linear ft for log(intensity) and log(exosome concentration) between 5.44 10 2 and 2.72 10 4 particles per mL, with the limit of detection (LOD) being approximately 9 10 19 mol L 1 (S/N ¼ 3). There was only one exosome in a 2 mL exosome sample of 5.44 10 2 particles per mL. The LOD is three orders of magnitude lower than that of the most sensitive exosome detection methods currently reported, such as Au-Ag nanorods with an SERS reporter (LOD: 1200 exosomes), 18 super-hydrophobic surfaces decorated with nano-geometry-based photonic structures to detect exosomes on SERS (0.2 ng mL 1 ), 45 electrochemical impedance spectroscopy (9500 exosome particles per 50 mL) 46 and size exclusion chromatography with fluorescence detection (2.9 10 7 exosome particles per mL). 47 The MIF concentration of the PANC-01 exosome solution was also detected using a commercial Human MIF ELISA kit (argb1294; Arigo Biolaboratories, Hsinchu City, Taiwan). As shown in Fig. 4E, the detection limit of our PDA SERS tag method was almost 6-fold lower than that of the commercial ELISA kit, which was about 2.72 10 8 particles per mL. The excellent sensitivity of this PDA-SERS method undoubtedly results from the PDA on glass slides and the core-shell Au-Ag SERS nano-tags and has enough hydrophilic antibody binding sites and optimal protective function for Ag shells. Compared with the ultrastable silica shell protection method, which is one of the fnest protective modifcation methods, 48,49 the PDA shell has the advantages of being easily modifed, environmentally friendly, and having great biocompatibility. This supersensitive MIF SERS platform can analyze individual exosomes and distinguish pancreatic cancer derived exosomes from those of healthy cells, which is valuable for subcellular mechanistic research and for clinical supervision or therapy in pancreatic cancer. To compare the detection sensitivity with other antibodies, anti-GPC1, EGFR, CD63 and EpCAM SERS assays were also applied to test exosomes derived from PANC-01 cells at various concentrations (5.44 10 2 to 2.72 10 4 particles per mL). The results of these assays are summarized in Table S1. † The immunosensors based on anti-GPC1, EGFR and EpCAM all showed good linear ftting with R values (multiple correlation coefficient) of >0.99, P values (probability) <0.05 and a similar LOD as the anti-MIF immunosensor (Fig. S6a, b and d †). In contrast, the anti-CD63 immunosensor displayed slightly poorer linear ftting (R < 0.92 and P > 0.05) and had a higher LOD (Fig. S6c †). Validation of chip-exosome-PEARL SERS immunosensors in clinical serum samples from pancreatic cancer patients 71 serum samples from histologically diagnosed pancreatic cancer patients and 32 samples from healthy individuals were assayed using this immunosensor. Serum samples were diluted 3-fold with PBS, followed by fltration with a 0.22 mm flter. Then, 2 mL of diluted sample was added to the PDA chip encapsulated with the anti-MIF antibody and detected with PEARL SERS tags. For the control group 2 mL of PBS without serum was used, and the intensity acquired was subtracted from the intensity of the experimental groups. The results are shown as log(intensity) in Fig. 4A. The Shapiro-Wilk test showed that W control ¼ 0.806, P < 0.0001 and W experiment ¼ 0.916, P < 0.0001, indicating a non-normal distribution in both groups. A test of homogeneity of variances showed F ¼ 314.177, P < 0.0001. The comparison of the pancreatic cancer and healthy control groups was measured by two independent samples' non-parametric Mann-Whitney test, Z ¼ 6.257, P < 0.0001, which showed that there was a statistical difference. The intensities of the pancreatic cancer and healthy control groups (mean AE SD) were 3.77 AE 0.15 and 2.67 AE 0.80, respectively. In the pancreatic cancer group, the median was 3.7542 and the interquartile range was 0.25, while in the healthy control group, the median was 2.2785 and the interquartile range was 1.54. These results indicated that the MIF SERS-PEARL immunosensor distinguished the serum of pancreatic cancer patients from that of healthy individuals, and also provided proof for the clinical reference range, which makes it a promising method with sufficient basis for clinical application. Furthermore, we employed statistical methods to obtain more diagnostic information from the anti-MIF SERS-exosome immunosensor results, such as distinguishing different Tumor Node Metastasis (TNM) classifcation stages (if the patients' cancers had TNM staging), and metastasis from nonmetastasis according to their histopathological reports (Table S2 †). We divided the 41 pancreatic cancer samples with defned TNM stages (omitting those without TNM staging) into P3 and P1-2 subgroups and further compared their log(intensity). We classifed the pancreatic cancer samples into "metastasis" and "non-metastasis" groups; the former included metastases to the liver, hilum of the spleen, adrenal glands and lymph nodes, while the latter contained tumors that had infltrated into tissues around the pancreas, such as adipose tissue, nerves, and extra-pancreatic tissues, such as the duodenal submucosal layer and bile duct. These statistical results are shown in Table S3. † All Mann-Whitney test values for P1-2 and P3, and metastasis and non-metastasis groups were statistically signifcant and matched the histopathological reports. Surprisingly, this method could also discriminate between patients with P3-stage tumors and those with P1-2-stage tumors, meaning that it can supplement tumor staging to further realize accurate diagnoses. For comparison, serum samples of pancreatic cancer patients (n ¼ 22) and healthy controls (n ¼ 20) were also tested using the commercial Human MIF ELISA kit. As shown in Fig. 4B, only nine and eight serum samples gave positive results from patients and healthy controls, respectively. Thus, the comparison between our anti-MIF SERS immunosensor and a commercially available ELISA kit indicated that our analytical platform had signifcant advantages for analyzing small-volume serum samples. Along with MIF, GPC1 and EGFR were also highly expressed on exosome surfaces. 50,51 Therefore, to determine the most powerful antibody for accurate and sensitive diagnosis, anti-GPC1-and anti-EGFR-based chip-exosome-PEARL SERS immunosensors were also applied to the same serum samples of pancreatic cancer patients (n ¼ 34) and healthy controls (n ¼ 32). The Shapiro-Wilk test of GPC1 (Fig. 4C) and EGFR (Fig. 4D) and W/P values (Tables S4 and S5, † respectively) showed that the distribution of both experimental and control groups was nonnormal, similar to anti-MIF. The anti-GPC1 platform could distinguish healthy individuals from pancreatic cancer patients, but anti-EGFR could not. Furthermore, neither test could distinguish P3 from P1-2 tumors, nor could they distinguish the "metastasis" and "non-metastasis" subgroups. To estimate whether MIF, GPC1 and EGFR could constitute a more discriminatory panel for the clinical diagnosis of pancreatic cancer, TNM staging and metastasis, we performed a receiver operating characteristic (ROC) logistic regression (Fig. 5A) to determine the sensitivity, specifcity and accuracy (Table 1) of using exosome markers individually. The ability of the MIF-based immunosensor for discriminating between pancreatic cancer and healthy controls, metastasis and nonmetastasis, and P1-2 and P3 was much higher than that of GPC-1-and EGFR-based immunosensors. Notably, the MIF discriminatory sensitivity was 95.7% for early-stage pancreatic cancer (P1-2) versus P3, which further demonstrated the potential of MIF as a promising exosome marker for pancreatic cancer. The differential performances of the PDA-SERS (combined MIF-, GPC1-and EFGR-based platforms), and CEA-and CA19-9based ELISA assays (Fig. S8 †) are summarized in Table 2. As tumors develop, cancer cells can infltrate into the surrounding tissues, disrupting tissue homeostasis and causing organ dysfunction. As the tumor architecture deteriorates, cells can enter the circulatory system and relocate to remote organs, which is the primary cause of cancer-related deaths. Our MIF SERS-PEARL liquid biopsy platform was also able to distinguish metastasized tumors from non-metastasized ones without the need of tissue biopsy or MRI imaging. Thus, patients with and without metastases could be identifed and monitored throughout the following treatments, and this information could be incorporated when making treatment decisions. To our knowledge, this is the frst time that MIF-based liquid biopsy was used to differentiate tumors by stage and metastatic activity. Furthermore, only 2 mL of serum sample was required for SERS analysis, demonstrating that micro-volume detection can be realized. Additionally, our chip-based exosome-PEARL SERS immunosensor offered more intuitionistic ways to discriminate cancer patients from healthy individuals by Raman imaging techniques with tremendous progress in spectral acquisition speed, detection sensitivity, spatial resolution, and penetration depth. Briefly, the exosomes captured and SERS tag-labeled chip was fully scanned on its entire surface (7.2 1.8 cm 2 ) with a 200 mm step between each point. The anti-MIF-SERS chip containing eight pancreatic cancer samples and eight healthy samples was scanned in about 1 h. Then, the scanning results were analyzed by calculating the peak area of every point in the peak range (1045-1100 cm 1 ) and a color gradient was given to reveal the intensities. In this work, a brighter color in Raman imaging means more SERS tags, representing more antigens and exosomes. From the SERS images (Fig. 5B), we directly distinguished pancreatic cancer patients (bright spots) from healthy individuals (dark spots). Furthermore, the thresholds provided herein can serve as references for clinical applications. Fig. 5 (A) Receiver operating characteristic (ROC) curves were calculated for single exosome markers (MIF, GPC-1 and EGFR) (red: pancreatic cancer vs. healthy controls; purple: metastasis vs. nonmetastasis; and green: P1-2 vs. P3). AUC stands for the area under the curve. (B) Raman imaging scanning of the 7.2 1.8 cm chip containing serum samples from pancreatic cancer patients (P1-8) and healthy individuals (N1-8) tested using the anti-MIF platform. ## Conclusions PDA-modifed glass slides and an ultra-thin PDA layer encapsulated Au(core)-Ag(shell) SERS tag with a quantitative signal of the Raman reporter at 1072 cm 1 were constructed for the sensitive and specifc detection of pancreatic cancer-derived exosomes to clinically diagnose tumors and metastases. The MIF antibody-based PDA-SERS platform can detect exosomes in trace samples with the lowest detection limit down to one exosome, making it much more sensitive than previously reported methods. Clinical serum samples from pancreatic cancer patients and healthy individuals could be clearly differentiated using MIF-, GPC1-, and EGFR-based PDA-SERS methods, with the requirement of only 2 mL serum samples. Furthermore, the MIF-based method could distinguish metastatic tumors from those without metastases, and P1-2-stage tumors from those in the P3 stage, which could previously only be accomplished by surgical biopsy. Thus, this immune-based SERS analytical platform might be able to detect early cancerous lesions to improve therapeutic outcomes and patient lives. This study was designed to show the feasibility of the PDA-SERS method for diagnosing cancer. This method can be further expanded to simultaneous and multiplex target assays with high diagnostic accuracy by Raman imaging. Protein microarray and microfluidic chip technologies are also compatible with this PDA-SERS method for high throughput and fast liquid biopsy of exosomes, tumor-derived extracellular vesicles, circulating DNAs, and most other biologically relevant molecules. We believe that the clinical application of these liquid biopsy methods will greatly relieve the distress caused by histopathological tests, and will provide a promising future for early diagnosis and efficient therapy for cancer patients. ## Experimental Materials and methods Materials. Dopamine hydrochloride, N-(3-dimethylaminopropyl)-N 0 -ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, MES monohydrate, bovine serum albumin, Tween® 20, 4-aminobenzenethiol (pATP), trisodium citrate, hydrogen tetrachloroaurate (HAuCl 4 $3H 2 O), dopamine, silver nitrate, and other chemical reagents were from Sigma-Aldrich, United States. Sodium hydroxide and hydrogen peroxide solution were from Macklin Biochemical Co. Ltd., Shanghai, China. Concentrated sulfuric acid (98%) was from Sinopharm Chemical Reagent Limited Corporation, China. 3,3 0 -Dioctadecyloxacarbocyanine perchlorate was from Beyotime, Shanghai, China. RPMI 1640 Medium, DMEM, Fetal Bovine Serum (FBS), Phosphate-Buffered Saline (PBS), pH 7.4, Tris-HCl, pH 8.0, and trypsin-EDTA (0.05%) phenol red were from Thermo Fisher Scientifc, United States. Ethanol was from AoRui Biotechnology Company, Shanghai, China. Anti-CD9 antibody, anti-CD63 antibody, anti-MIF antibody, anti-GPC1 antibody, and goat anti-mouse IgG H&L (FITC) were purchased from ABCAM company, United States. AllMag® SM3-P100 superparamagnetic nanoparticles were from Shanghai Allrun Nano Science & Technology Co., Ltd, China. The PANC-01 cell line was from the cell bank of the University of Chinese Academy of Sciences. HPDE6-C7 was obtained from the American Type Culture Collection. Synthesis of SERS-labelled nanomaterials. The 18 nm gold nanoparticles were synthesized using Frens' protocol. 57 The Au-Ag core-shell nanocomposites were synthesized by the following steps: 600 mL of gold nanoparticle solution was put in a clean round flask with stirring; then 20 mL of 0.1 M ascorbic acid, 5 mL of silver nitrate (appropriate concentration), 100 mL of Tris-HCl buffer (50 mM, pH ¼ 8.5), 100 mL of pATP aqueous solution (appropriate concentration), and 200 mL of 1% BSA were added stepwise. After the mixture had reacted for 30 min, the solution was centrifuged at 6000 rpm for 15 min; then the supernatant was removed, and the precipitate was re-dispersed in 500 mL of Tris-HCl buffer. The re-dispersed solution was combined with 100 mL of 15 mg mL 1 antibody and 100 mL of dopamine solution (appropriate concentration), and the reaction lasted 1 h. After the reaction completed, the solution was centrifuged, and the precipitate was re-dispersed. The fnal solution was stored at 4 C until use. Modifcation of the polydopamine chip. Glass slides (24.5 76.2 mm 2 , 1-2 mm thick) were soaked in Piranha solutions for 2 h, and then washed with deionized water. Then, several glass slides were put into 20 mL of a dopamine hydrochloride solution of the appropriate concentration for 1.5 h, and then 20 mL of 1 M Tris-HCl (pH ¼ 8.0) with 15 mg antibody was added and reacted for approximately 1.5 h. The polydopamine chips were washed with PBS for further exosome detection. Cell culture. PANC-01 and HPDE6-C7 cells were cultured in RPMI 1640 and DMEM, respectively, with 10% FBS, at 5% CO 2 in culture bottles. The FBS used in this study was fltered through a 0.22 mm flter (Merck Millipore, Burlington, MA, USA) and then centrifuged for 16 h twice to make it exosome-free. Exosome separation. Culture medium from the two cell lines was collected and the ultracentrifugation process was performed in an ultracentrifuge (CS150FNX; Hitachi, Tokyo, Japan): the medium was centrifuged at 800 g for 5 min and 2000 g for 10 min to remove cellular debris; after fltering through a 0.22 mm flter to acquire the exosomes, the medium was centrifuged at 120 000 g for 4 h, and fnally the exosomes were diluted in PBS and centrifuged at 120 000 g for 4 h twice. The separated exosomes were then suspended in PBS to the desired concentration. For serum samples for transmission electron microscopy (TEM) characterization, the exosomes were diluted to the appropriate concentration, fltered through the 0.22 mm flter, and then ultracentrifuged at 120 000 g for 4 h and washed twice with PBS. Exosome detection using the SERS method. Before capture, the polydopamine chip was blocked with 0.05% BSA at 37 C for 30 min, and then it was washed with PBS and PBS-Tween20 (PBST). The original exosome solution from PANC-01 and HPDE6-C7 cells was diluted to the appropriate concentration, and then 2 mL of the exosome solution was dropped on the polydopamine chip, followed by incubation for 1 h at 37 C. After incubation, the chip was washed with PBS and PBST; then, 3 mL of PEARL was dropped on the sample to cover it, and the chip was incubated for 1 h at 37 C, and then washed with PBS and PBST. Raman signals were collected on a Horiba Jobin Yvon XploRA confocal micro-Raman system, and the excitation laser wavelength was 785 nm. Labspec software (version 6) was used to obtain the average Raman intensity of the samples and mapping images. The signal intensities of the different samples were obtained by averaging approximately 196 test point signals in a 250 250 mm 2 square region (testing step: approximately 19.2 mm, and 1 s for each point) with a laser power of 8 mW. The Raman mapping images of PDA chips were obtained by plotting the Raman peak areas in a 7.2 1.8 cm 2 oblong region (mapping step: 200 mm, and 0.1 s for each point) with a laser power of 80 mW. The Raman peak area was used to set the false color mapping scale and the scale value was set from 20 to 100. Patient samples. The serum samples of 71 patients diagnosed with pancreatic ductal adenocarcinoma and 32 samples from healthy volunteers were collected between December 2012 and August 2016 at Changhai Hospital, Shanghai, China, with written informed consent. All experiments on clinical samples were performed in accordance with the Guidelines for Care and Use of Laboratory Clinical Blood Samples of Changhai Hospital, Shanghai, China and were approved by the Medical Research Ethics Committee, Changhai Hospital, Shanghai, China. The average age of the pancreatic ductal adenocarcinoma patients was 60.08 AE 9.81 years, and there were 33 women and 38 men; for the healthy group, the average age was 50.25 AE 13.55 years, and it comprised 10 women and 22 men. Samples with complex tumors, including pancreatic cancer with other cancers were excluded from this study. Statistical analysis. Comparisons of measurement data among more than three groups were made by an LSD-t test, if the data met the homogeneity of variance (P > 0.05) and normality distribution (Shapiro-Wilk test P > 0.1) requirements. Comparisons between the pancreatic cancer group and the healthy group were made by two independent samples' nonparametric Mann-Whitney test. The test level for LSD-t and M-W was 0.05, while for the Shapiro-Wilk test it was 0.1. Receiver operating characteristic curves were obtained using Graph Prism 6.0. Sensitivity and specifcity results were calculated using IBM SPSS Statistics 21.0; the cut-off value was log(Raman intensity) where sensitivity (1 specifcity) was the max. The combined values of MIF, GPC1, and EGFR were calculated by the logistic regression method. Other statistical results or graphs were also from Graph Prism 6.0, IBM SPSS Statistics 21.0 and Origin 7.5.

chemsum

{"title": "An ultrasensitive polydopamine bi-functionalized SERS immunoassay for exosome-based diagnosis and classification of pancreatic cancer", "journal": "Royal Society of Chemistry (RSC)"}

the_first_pd-catalyzed_buchwald–hartwig_aminations_at_c-2_or_c-4_in_the_estrone_series

## Abstract: A facile Pd-catalyzed C(sp 2 )-N coupling to provide a range of 2-or 4-[(subst.)phenyl]amino-13α-estrone derivatives has been achieved under microwave irradiation. The reactions were mediated with the use of Pd(OAc) 2 as a catalyst and KOt-Bu as a base in the presence of X-Phos as a ligand. The desired products have been obtained in good to excellent yields. The nature and the position of the aniline substituent at the aromatic ring influenced the outcome of the couplings. 2-Amino-13α-estrone was also synthesized in a two-step protocol including an amination of 2-bromo-13α-estrone 3-benzyl ether with benzophenone imine and subsequent hydrogenolysis. ## Introduction Aminoestrones are of particular interest thanks to their diverse biological applications . There exist several aminated steroids in the literature, but the efficient generation of a C(sp 2 )-N bond on the aromatic ring A of estrone derivatives still remains a challenge. Aminoestrones substituted at C-2 or C-4 are mainly produced by the reduction or hydrogenation of the corresponding nitro derivatives . Classical nitration methods have, however, many drawbacks concerning elevated reaction temperatures, long reaction times, and poor yields. The introduction of amino or substituted amino groups onto ring A of estrone is fascinating from both organic chemical and biological points of view. Certain ring A-aminated estrone derivatives are described as inhibitors of estrogen biosynthesis. They are often synthesized via a three-step method including nitration, reduction, and functionalization of the amino group . This three-step protocol may be simplified to involve only one or two steps by the application of a Pd-catalyzed Buchwald-Hartwig amination. In recent years, extensive efforts have been made on the Pd(0)-catalyzed amination of aryl halides or triflates in order to achieve the efficient synthesis of substituted anilines . Buchwald et al. stated that the Pd source is determining in the amination step . They also found that X-Phos is an outstanding ligand with increased activity and stability compared to those based on BINAP . There are a number of literature methods with respect to microwave-assisted Buchwald-Hartwig couplings . Many publications have reported remarkable advantages of microwave-assisted syntheses, including shorter reaction times, higher yields and chemoselectivity . Concerning the aromatic ring A of estrone, the Pd-catalyzed Buchwald-Hartwig amination was carried out exclusively at position C-3, starting from the 3-triflate derivative . The C(sp 2 )-N cross-coupling of the triflate was achieved with benzophenone imine or benzylamine. The removal of the protecting groups resulted in 3-aminoestrone in high yields. Schön et al. developed two convenient protocols for the preparation of 3-aminoestrone using Pd(OAc) 2 and Pd 2 (dba) 3 as catalysts, X-Phos as a ligand, Cs 2 CO 3 as a base in toluene or DMF solvent under thermal heating or microwave irradiation . We recently described halogenations and Sonogashira couplings on ring A of 13α-estrone and its 3-methyl ether . The 13-epimer of natural estrone is a non-natural C-18 steroid containing cis junction of rings C and D . This coremodified compound differs from its natural 13β counterpart not only in the configuration of C-13, but also its more flexible conformation. Poirier et al. investigated the in vitro and in vivo estrogenic activity of 3,17-estradiol derivatives of 13α-estrone . The 13-epimers were shown to exhibit no significant binding affinity for estrogen receptor alpha and display no uterotropic activity. Nevertheless, certain 13α-estrone derivatives possess important biological activities including antitumoral effect . Thus 13α-estrone is a suitable compound for the development of biologically active steroids lacking estrogenicity. Literature reveals that besides the inversion of C-13, the introduction of an amino group onto C-2 or C-4 of estrone also leads to significant decreases in its binding affinity for nuclear estrogen receptors (ERα and ERβ) . Certain derivatives of 2-or 4-aminoestrone or their 3-methyl ether possess diverse biological activities, including enzyme inhibitory or antiproliferative properties 29,30]. The 17β-HSD1 enzyme is responsible for the reduction of estrone into 17β-estradiol, which may enhance the proliferation of tumor cells . Effective inhibition of 17β-HSD1 may result in an antitumor effect in hormone-dependent cancers . It is known that several 2-or 4-substituted estrone derivatives possess substantial 17β-HSD1 inhibitory action . The presence of a large lipophilic group on C-2 of estrone was found to be advantageous concerning the 17β-HSD1 inhibitory activity . Chin et al. reported that 2-bromoacetamidoestrone 3-methyl ether inhibits the 17β-HSD1 enzyme in an irreversible manner . Nevertheless, we proved that certain 4-halogenated 13α-estrone 3-methyl ethers are also effective inhibitors . Recently, we carried out the Pd-catalyzed C-C coupling of 2-and 4-iodo-13α-estrones as well as their 3-methyl ethers with p-substituted phenylacetylenes as terminal alkyne partners under microwave irradiation . The regioisomerism markedly influenced the reaction conditions. 2-Iodo isomers were transformed using Pd(PPh 3 ) 4 catalyst and CuI as a cocatalyst. Reactions of the 4-iodo counterparts could be achieved by changing the catalyst to Pd(PPh 3 ) 2 Cl 2 and using higher temperature. Additionally, the 2or 4-phenylethynyl derivatives were partially or completely saturated in order to get stereochemically different compounds for structure-activity determinations. The saturated derivatives contain a phenyl moiety at C-2 attached through an ethenediyl or ethanediyl linker. Of the synthesized 2-and 4-regioisomers, solely the 2-counterparts bearing a 3-OH group exhibited a substantial inhibitory effect against the 17β-HSD1 enzyme. Surprisingly, the enzyme inhibitory action did not depend on the hybrid state of carbon attached to C-2. From the pharmacological point of view it would be interesting to synthesize and investigate such 13α-estrone derivatives, bearing a lipophilic phenyl group attached to C-2 through an amino linker. In continuation of our studies with respect to cross-coupling reactions on ring A of 13α-estrone, here we disclose the development of a Pd-catalyzed C(sp 2 )-N coupling methodology for the transformation of 2-bromo-and 4-bromo-13α-estrone 3-methyl (1 or 3) as well as 3-benzyl ethers (2 or 4) with aniline or substituted anilines as reagents. To the best of our knowledge, there are no literature reports concerning the Pd-catalyzed 2-or 4-amination of the estrane core. ## Results and Discussion Based on recent literature results , we started to optimize the reaction conditions for the transformation of 2-bromo-13α-estrone 3-methyl ether (1) with aniline (Table 1). Since the Pd source has been shown to be crucial in the amination step, two Pd catalysts were investigated. Namely, Pd(OAc) 2 and Pd 2 (dba) 3 were used in the presence of X-Phos or BINAP as ligands. The literature data influenced the selection of the base. The arylation of anilines, escpecially of unsubstituted ones with o-bromoanisoles requires stronger bases such as NaOt-Bu or KOt-Bu . This is due to the deactivated, electron-rich nature of anisoles induced by the electron-donating methoxy group. Taking into account the above-mentioned observations [18, , couplings were performed in the presence of DBU, NaOt-Bu, KOt-Bu or Cs 2 CO 3 as the base. Toluene was chosen as a solvent, and the reactions were carried out under microwave irradiation or thermal heating. The solvent was selected a Reagents and conditions: 2-bromo-13α-estrone 3-methyl ether (1, 1 equiv), aniline (1.2 equiv). b Flash chromatography yield obtained under conventional heating (24 h, reflux temperature). c Flash chromatography yield obtained under microwave irradiation (10 min). on the basis of literature data reported for other Pd-catalyzed reactions of estrone derivatives . The pre-stirring of the reaction mixture without adding the aryl halide 1 was carried out at 60 °C for 5 min in a water bath, then aryl halide 1 was added and the mixture was irradiated in a microwave reactor at 150 °C for 10 min. The outcome of the couplings greatly depended on the nature of the Pd source, the ligand and the base. As summarized in Table 1, reactions with pre-catalyst Pd(OAc) 2 gave the desired aminoestrone 5 in low to high yields (Table 1, entries 1, 2, 4, 7-9) except when using Cs 2 CO 3 as the base (Table 1, entries 6 and 10). In the latter cases only dehalogenation of the starting aryl halide was observed in around 20-60% yield. The use of KOt-Bu ( After finding the best set of reaction conditions (Table 1, entries 2 and 4), the temperature was lowered to 100 °C (Table 1, entries 3 and 5) in order to suppress the dehalogenation side reaction. The efficiency of the couplings was found to be similar to that observed at higher temperature with improved yields. Nevertheless, reaction with KOt-Bu (Table 1, entry 3) proved to be slightly more efficient. In order to compare the efficiency and reaction time of thermal heating with microwave-irradiation conditions, all reactions of 1 with aniline were performed under both conditions (Table 1, Scheme 1: Pd-catalyzed aminations at C-2 or C-4 in the 13α-estrone series. Reactions were performed on a 0.25 mmol scale with 1.2 equiv of amine, 10 mol % Pd(OAc) 2 , 10 mol % X-Phos, at 100 °C, 10 min under microwave irradiation. Flash chromatography yields are reported. entries 1-18). As seen in Table 1, similar yields might be achieved, but reaction times differ considerably (10 min vs 24 h). On the basis of the optimization procedure discussed above, we selected microwave-assisted conditions at lower temperature (Table 1, entry 3) for further transformations. With the best reaction conditions in hand, the couplings at C-2 of starting compound 1 were extended to monosubstituted anilines bearing electronically different substituents at o, m or p positions (Scheme 1). As indicated in Scheme 1, all couplings proceeded with high yields. The best yields were achieved with nitroanilines, irre-Scheme 2: Two-step synthesis of 2-amino-13α-estra-1,3,5(10)-trien-17-one (13). spective of the position of the nitro group. Reaction of methylanilines led to slightly lower yields, indicating that the presence of the electron-donating methyl group is less advantageous over the electron-withdrawing nitro function. The coupling at C-4 of compound 1 with aniline under the same conditions yielded aminated derivative 10 in high yield. With an attempt to investigate the influence of the size of the 3-ether group, 2-or 4-bromo isomers of 3-benzyl ethers 2 or 4 were also submitted to C(sp 2 )-N couplings with aniline using the procedure elaborated above. Irrespective of the more bulky nature of the benzyl ether group compared to its methyl counterpart, compounds 2 and 4 were successfully aminated affording derivatives 6 and 11 without the need of changing the reaction conditions established for couplings at C-2. In continuation of our earlier work concerning the synthesis of 2-substituted 3-hydroxy-13α-estrone derivatives as potential enzyme inhibitors , here we were interested in the synthesis of 2-amino-13α-estrone (13). The efficient C(sp 2 )-N coupling method elaborated above proved to be suitable for the reaction of 2-bromo-3-benzyl ether 2 and benzophenone imine as an amine precursor (Scheme 2). The deprotection was achieved by hydrogenolysis using a Pd/C catalyst. The resulting newly-synthesized 2-amino-13α-estrone (13) itself may possess promising pharmacological properties or may serve as a key intermediate in the synthesis of biologically active 2-(subst.)amino-13αestrones. The structures of the newly synthesized phenylamino derivatives 5-13 were established through 1 H, 13 C, HSQC and/or HMBC measurements. ## Conclusion In conclusion, we have developed a convenient microwaveassisted one-step protocol for the facile and efficient preparation of 2-and 4-phenylaminoestrones 5-11. Our method affords the desired products in short reaction times in good to excellent yields. Thanks to the elaborated mild coupling procedure, the synthesis of 2-amino-13α-estrone 13 could be achieved in only two steps without the first, aromatic nitration step used extensively earlier. The newly synthesized amino derivatives of 13αestrone 5-13 may possess important biological activities without hormonal effect. ## Supporting Information Supporting Information File 1 Experimental procedures for compounds 5-13, their 1 H, 13 C NMR, MS, elemental analysis data and the copies of their 1 H and 13 C NMR spectra. [https://www.beilstein-journals.org/bjoc/content/ supplementary/1860-5397-14-85-S1.pdf]

chemsum

{"title": "The first Pd-catalyzed Buchwald\u2013Hartwig aminations at C-2 or C-4 in the estrone series", "journal": "Beilstein"}

fish_dna-modified_clays:_towards_highly_flame_retardant_polymer_nanocomposite_with_improved_interfac

## Abstract: Deoxyribonucleic Acid (DNA) has been recently found to be an efficient renewable and environmentallyfriendly flame retardant. In this work, for the first time, we have used waste DNA from fishing industry to modify clay structure in order to increase the clay interactions with epoxy resin and take benefit of its additional thermal property effect on thermo-physical properties of epoxy-clay nanocomposites. Intercalation of DNA within the clay layers was accomplished in a one-step approach confirmed by FT-IR, XPS, TGA, and XRD analyses, indicating that d-space of clay layers was expanded from ~1.2 nm for pristine clay to ~1.9 nm for clay modified with DNA (d-clay). Compared to epoxy nanocomposite containing 2.5%wt of Nanomer I.28E organoclay (m-clay), it was found that at 2.5%wt d-clay loading, significant enhancements of ~14%, ~6% and ~26% in tensile strength, tensile modulus, and fracture toughness of epoxy nanocomposite can be achieved, respectively. Effect of DNA as clay modifier on thermal performance of epoxy nanocomposite containing 2.5%wt d-clay was evaluated using TGA and cone calorimetry analysis, revealing significant decreases of ~4000 kJ/m 2 and ~78 kW/m 2 in total heat release and peak of heat release rate, respectively, in comparison to that containing 2.5%wt of m-clay.Layered silicate clays have been widely utilized to equip the pristine polymers with value-added properties, such as considerable mechanical strength, thermal durability, and gas impermeability 1,2 . The final properties of polymer/ clay systems dominantly depend on dispersion configuration of clay into polymer matrix and physico-chemical events at clay-polymer matrix interface 3 . Since the first attempt has been made to use layered silicate clays in construction of exfoliated nylon 6/clay nanocomposites by Toyota Company researchers 4 , several strategies have been developed to produce various exfoliated polymer/clay nanocomposites 5 . Nonetheless, due to the intense static forces among neighbouring platelets in the pristine layered silicate clays, complete exfoliation of platelets, and their further homogeneous dispersion in polymer matrices are still challenges to overcome 6,7 . Among various polymer-clay configurations, complete exfoliation of individual clay layers into the polymer matrices is of particular interest because it maximizes the interactions of clay layers with polymers matrix 8 . From the reactions kinetic viewpoint, to produce a completely exfoliated nanocomposite, theoretically, either polymerization reactions of monomers should be firstly initiated between clay galleries (which is so-called surface-initiated polymerization), and then is progressed to the bulk monomers, or polymerization rate between clay galleries should be faster than polymerization in bulk monomers, leading to the separation of clay layers 9,10 .Layered silicate clay materials inherently have a hydrophilic nature and as such their compatibility with most industrial polymers is poor and consequently incorporation of unmodified clay into polymers, not only, does not improve performance of the polymers, but also potentially could deteriorate intrinsic properties of the parent polymers 11,12 . Most commercially available clays have been modified through cationic exchange process of clay interlayer cations with ammonium cations consisting of long alkyl hydrophobic chains, which lead to the expansion of clay layers and consequently facilitate the penetration of polymer chains into clay layers 13,14 . Although these modifications improve the dispersion of clay into polymer matrices, the interface of the modified clay layers with polymer matrices are not usually taken into account and the interactions at interface remain as weak as van der Waals interactions 15,16 which could be accompanied with adverse plasticisation effects at clay-matrix interface 17 . Despite achievements in the exfoliation of clay layers into polymer matrices, it has been repeatedly reported that plasticisation has devastating effects on some mechanical properties of polymers, in particular glass transition temperature (T g ) of epoxy polymers. To reduce the plasticisation effects, strong interactions e.g., covalent bondings between clay modifiers with polymer matrix are inevitably required to be established at interface 18,19 . In the case of epoxy polymers, it has been proven that the alkyl ammonium modifiers can catalyse self homo-polymerization of epoxide groups within clay layers, facilitating the exfoliation process 20 . Nevertheless, there is no interaction between clay layers and the formed epoxy matrix leading to the profound plasticisation effects on T g of epoxy-clay nanocomposites. A common strategy to surmount the plasticisation effects at interface is coupling the hydroxyl groups of surface and edges of clay with polymer matrix through silane compounds 21 . The silane coupling agents can covalently react with polymer and reduce plasticisation effects 22 . Although it has been reported that clay treated with silanes can create a strong interface, a solvent process is required to achieve a highly individual layers dispersed into polymer matrix, which is not easily feasible in terms of its manufacturing 23 . Additionally, the amount of graftable hydroxyl groups on clay surface and edges are extremely limited and amount of silane grafted is low, in comparison to organic modifiers using cations exchange 24 . To overcome these challenges, modification based on cations exchange which appears to be more efficient in terms of its quantity, has to be formulated to create strong interactions between clay layers and polymer matrices, obtaning the desirable properties. During the past decades, there has been a significant interest in use of sustainable and renewable materials instead of conventional hazardous substances for development of high-performance materials . In this regard, a few approaches have been developed to modify layered silicate clays with natural compounds for polymer composites applications. Jin et al. coated montmorillonite with protein biopolymers extracted form soy plant using the pH change, leading to the exfoliation of clay layers into biopolymers 28 . Chitosan/clay nanocomposite is another example in which biopolymers are used to modify clay 29 . In the case of thermosetting epoxy/clay composites, Barua S. et al. reported a biocompatible epoxy/clay nanocomposite with enhanced mechanical properties for tissue engineering applications using modified bentonite with an oil derived from a specific plant 30 . Focusing on the role of interfacial physico-chemical interactions in mechanical properties of epoxy/clay nanocomposites, Yang L. et al. 31 reported a biomimetic approach using in situ polymerization of dopamine within clay layers. In this approach, cationic amine groups of polydopamine were exchanged with clay cations, and the hydroxyl groups of polydopamine were tasked to enhance the interfacial interactions through forming hydrogen bondings with an epoxy polymer. One of the most promising renewable materials, recently employed to enhance thermal performance of textile fabrics is DNA derived from fishing industrial waste . It has been reported that DNA can act as an intrinsically flame retardant on cotton fabrics and enhance fire retardancy of system 32, . In contrast to the conventional fire retardant materials which are commonly phosphorous or halogen based hazardous compounds 38,39 , DNA is a green and natural flame suppressant and retardant which can potentially be replaced with traditional fire retardant materials 37 . General structure of DNA consists of sodium phosphate backbone groups, deoxyribose unites, and nucleobases having hydrogen bondings together. The sodium phosphates groups can potentially act as a nucleophile intermediate in organic reactions e.g., reaction with epoxide rings. Inspired by these features of DNA, we hypothesized that if DNA can be intercalated within clay layers, interfacial interactions as well as thermal performance of epoxy/clay system may significantly be improved in comparison to those commercially modified. To verify this hypothesis, we have embedded DNA within the clay layers and subsequently incorporated such DNA modified clay layers into an epoxy matrix to produce epoxy/clay nanocomposites. Herein, structure, morphology, mechanical, thermal, and flammability performance of these newly developed nanocomposites, have been comprehensively investigated while focusing on the role of interfacial interactions between modified clay and polymer matrix. ## Results and Disccusions DNA-modifed clay characterizations. Figure 1 demonstrates how to change DNA structures, being able to cation-exchange with clay cations, leading to intercalation of DNA within clay layers. Although dispersion of DNA/water makes a solution with pH ~5.5, it has been reported that hydrogen bonding between nucleobases of DNA structure could be effectively dissociated at pH ~4 causing to form ammonium cations through its nucleobases; and at pH < 2, DNA structure will be hydrolysed, causing to break the phosphodiester bonds and consequently the bases will be broken off 40 . As shown in Fig. S1, maximum amount of 72 ± 6 mg DNA per gram of p-clay was obtained to be intercalated within clay layers at pH = 2, and its amount decreases significantly at higher pHs. Ability to disperse the pristine clay (p-clay) and clay modified with DNA (d-clay) in solvents, are also presented in Fig. 1. As shown, d-clay becomes suspended into organic phase (chloroform) instead of being at water phase, whereas p-clay remains in water phase, which preliminarily confirms a transition of hydrophilicity nature of p-clay into the organophilicity in d-clay. The d-clay was fully characterized using FTIR, XPS, XRD, and TGA analysis to find out its structural characteristics. In FTIR spectrum of p-clay, both of the peaks at 3620 cm −1 and 3420 cm −1 are ascribed to H− O− H stretching vibration bands of water molecules bonded to the Si− O surface on the clay. The stretching bands of Al− OH and Fe− OH are also appeared at below 916 cm −1 . The peak at 1635 cm −1 observed for p-clay can be attributed to the -OH deformation of water. The Si− O stretching vibration bands are observed around 1100 cm −1 . After modification of clay with DNA, obvious new peaks at around 1230 cm −1 , 1680 cm −1 , and 3200 cm −1 were appeared in FTIR spectrums of d-clay and DNA, as indicated in Fig. 2a. These peaks are related to the P-O, P = O, primary/secondary N-H stretching, respectively, showing presence of DNA characteristic peaks in d-clay structure. While a broad peak related to the hydroxyl groups of both hydrogen phosphate groups and nucleobases can be observed after 3200 cm −1 in FTIR spectrum of DNA. Moreover, an obvious peak at 1450 cm −1 denotes presence of C = C stretching bonds in nucleobase of DNA structure for both d-clay and DNA samples. As shown in Fig. 2b, the main XPS characteristic peaks of p-clay are Si2p, Al2p, O1s and Na1s which appear at 103, 74, 533 and 1072 eV, respectively. After modification of p-clay with DNA, the main Organophilicity of clays depends on wetting of modified clay by epoxy resin, which plays a significant role in dispersion quality in the matrix. The process of wetting of clays by epoxy resin consists of three types of wetting including adhesion wetting (W a ), immersion wetting (W i ), and spreading wetting (W s ). The work of dispersion (W d ) is the sum of these three aforementioned wetting terms which can be expressed as follows: Wetting and dispersion could be determined by the epoxy surface tension (γ LV ) and contact angle between epoxy and nanoclay (θ °). W a , W i , and W s are spontaneous when θ ° < 90°4 1,42 . Snap shots of epoxy droplet deposited on compacted discs of clay at different times duration (60 and 3600 seconds) are illustrated in Fig. 3. As shown, the angles formed between epoxy droplet and m-clay substrate are higher than that of d-clay. Aktas et al. 42 declared that the contact angle of epoxy droplet on Cloisite 25 A nanoclay reaches a stable state of ~42° with a decrease of 16% in initial volume of epoxy drop. In comparison, angles formed between epoxy droplet with m-clay and d-clay reached ~69° and ~59°, respectively. However, higher decrease in initial volume of epoxy drop could be seen for both m-clay and d-clay. It is postulated that d-clay shows better affinity towards epoxy droplet. In other words, epoxy droplet could easily be absorbed to d-clay disk. Such phenomenon could be analyzed through volume changes in epoxy droplet observed on the samples. As presented in Fig. 3, a faster decrease in droplet volume with elapsed time proves that the penetration of epoxy droplet to d-clay is much higher than that of for m-clay. In other words, a decrease of ~84% in epoxy volume on d-clay was observed after 3600 s; however, its counterpart, m-clay shows a decrease of ~78% in epoxy volume. It is hypothesized that prompt impregnation Interfacial interactions. Interfacial interactions between d-clay and m-clay with epoxy resin play a pivotal role in formation of different structures of epoxy-clay nanocomposites e.g., exfoliated/intercalated structures, which were studied by DSC and rheological analysis. Figure 4a depicts DSC thermograms of un-cured epoxy resin suspension containing various clays. As it can be seen, no curing reaction occurs during a dynamic heating of pure EP suspension and its nano-suspensions containing m-clays without hardener as evidenced by its thermogram which does not show any exothermic peak up to 150 °C, revealing that m-clays cannot have any effective interactions with the epoxy resin in this temperature range 43,44 . However, addition of 2.5 and 5 wt% d-clay in epoxy suspensions cause an exothermic peak to appear before 100 °C with enthalpies of − 12.3 and − 19.7 J/g, respectively. It is proposed that hydrogen phosphate groups intercalated between d-clay layers can react with the penetrated epoxy monomers into d-clay layers through ring opening of epoxide groups, schematically presented in Fig. 4d. These intra-gallery reactions could also facilitate diffusion of more epoxy monomers within the clay layers and are also responsible to expand the clay layers, inducing formation of exfoliated structures, before the extra-gallery reactions have been conducted by curing of the nanocomposites. The interfacial interactions arising from these intra-gallery reactions were also explored by studying the changes of rheological behaviour of nano-suspensions. In this regard, viscosity and shear stress versus shear rate flow curves for nano-suspensions containing various clays are illustrated in Fig. 4b and c, respectively. The rheology behaviors of samples were analyzed by Herschel-Bulkley's model according to the following equations: Where γ  is the shear rate (s −1 ), τ and τ c are, respectively, the shear stress and yield stress. The K and n are the flow consistency index and the flow index, respectively. Flow index determines the flow behavior. In other words, n < 1 for shear thinning behavior and n > 1 for shear thickening behavior could be observed in the nano-suspensions. The Herschel-Bulkley's model parameters were calculated and presented in Table 2. Epoxy nanocomposites containing 2.5 and 5%wt of d-clay and m-clay are named as EP-D2.5 and EP-D5, and EP-M2.5 and EP-M5, respectively. As illustrated in Fig. 4b and c and presented in Table 2, it is argued that addition of d-clay not only could increase the viscosity of epoxy resin but also promote shear-thinning behavior, steaming from interfacial interactions due to the intra-gallery reactions. In other words, compared with m-clay, DNA as a reactive modifier could physico-chemically involve and entangle with the epoxy chains, leading to a higher viscosity which could induce yield stresses in nano-suspensions. Compared with nanosuspensions containing 2.5 wt% m-clay, an increase of ~19 Pa in τ c is observed for suspensions reinforced with the same content of d-clay. Another extra reason behind such trend could be related to the temporary formation of hydrogen bonding between hydroxyl resulting in initial resistance toward shear stress with functional groups of DNA carbohydrates. Such phenomenon is more obvious at high contents of d-clay. To put it differently, compared with nanosuspension filled with 5 wt% m-clay, the addition of the same content of d-clay to epoxy shows an increase of ~23 Pa in τ c . It could be deduced that role of DNA as reactive modifier in increment of viscosity as well as τ c would be more effective in higher contents because interfacial interactions lead to decrease the possibility of agglomeration formation, which causes more d-clay to be involved in formation of network. Moreover, the same increasing trend is observed for flow consistency, whereas a decreasing trend could be detected for flow index. As viscosity behavior of the nano-suspensions also depends on nanoclay dispersion levels into epoxy matrix, another prerequisite condition for viscosity discussion is the relation of dispersion level with flow index. As discussed in literatures 45,46 , it was investigated that lower values of the flow index imply higher levels of uniform dispersion of nanoclay into polymer matrix. Therefore, compared with m-clay, d-clay is prone to be more-uniformly dispersed in epoxy system. It is assumed that dispersion of d-clays into epoxy suspensions could lead to delaminated structures by increasing the d-spacing of d-clay layers, resulting from intra-gallery reactions. Therefore, it is postulated that each individual platelet could efficiently restrict the mobility of epoxy chains, on the one hand, and promote shear thinning behavior, on the other hand. As presented in Table 2, the lowest values of n e.g., 0.76 and 0.72 are observed for the nano-suspensions containing 2.5 and 5 wt% d-clay, respectively, whereas dispersion of m-clays into epoxy resin could not induce the same shear-thinning performance. In other words, higher shear-thinning could be only seen when clay is modified with DNA based modifier. It is argued that although modification of clay with DNA could increase viscosity of nano-suspensions, we subscribe to the view that a significant decrease in the entanglement density of epoxy molecules could be obtained. It could imply that aligned d-clay could act like slippery agents, schematically presented in Fig. 4e. This phenomenon is in agreement with the observation reported in the literatures 47,48 . As clay concentration increases, the viscosity of epoxy resin increases, which is mostly accompanied by inducing heterogeneity in the system. Such heterogeneity is arising from agglomerations and micro-voids formed in epoxy resin while processing 49 . It is worth to consider that increasing the m-clay content into epoxy suspension from 2.5 wt% to 5 wt% makes the intercalation/exfoliation more and more difficult. As a result, the weakest shear-thinning tendency could be seen for nano-suspension containing 5 wt% m-clay. This means that it leads to a low alignment of clay layers, which causes nanosuspension to resist more against higher shear rates. Nanocomposites structure. In order to verify the hypothetical considerations discussed in DSC and rheological analyses, nano/micro-structures of epoxy nanocomposites containing d-clay were examined by XRD and TEM analysis. Figure 5 demonstrates XRD patterns of pure EP and epoxy nanocomposites containing d-clays. As shown, there is no peak in the XRD pattern of pure EP in the 2θ ° of 2°-10°, showing an amorphous structure for epoxy matrix. Therefore, if a XRD peak appears in this region for the nanocomposites, it should be related to the clay structure and its basal d-spacing in the nanocomposite. As can be seen for EP-D2.5 sample, this nanocomposite system shows no peak in its XRD pattern, demonstrating that initial d-spacing of dry d-clay which was ~1.9 nm, is completely expanded so that its 2θ ° becomes < 2° (equaled to d-spacing of > 4.4 nm). This finding reveals the present of exfoliated structures in this nanocomposite. In contrast, EP-D5 system has an obvious XRD peak reflection in 2θ ° = 3.1° which implies formation of the induced intercalated clay structures into epoxy nanocomposite at higher d-clay content with a basal d-spacing of ~2.8 nm. Details of nanocomposites structure were investigated by TEM observations as illustrated in Fig. 6. As depicted in Fig. 6a and d, the dark lines in these figures are related to the silicate nanolayers and the light sections are related to epoxy matrix. It was mentioned that penetrated epoxy monomers within semi-separated clays treated by DNA modifier, could enhance clay layers separation through intra-gallery reactions and consequently such phenomenon induce some semi-stacked clays to be partially exfoliated, as it can be seen from TEM of EP-D2.5 nanocomposite (Fig. 6b). Furthermore, the intercalated structures could also be observed for this nanocomposite. It is argued that incorporation of higher contents of clay could merely result in interacted structures. As shown in Fig. 6e, three individual intercalated ordered structures so-called "intercalated tactoids", could be detected for EP-D5 nanocomposite. However, compared with the EP-D5, EP-D2.5 possesses thin tactoids containing only a few clay layers. These small tactoids are uniformly and randomly dispersed in the epoxy resin, demonstrating that the clay modification based DNA is an effective approach to enhance both the exfoliation and dispersion of clay. In addition to such argument associated with dispersion, as reported in literatures , under an effective load, most of microcracks are initiated within the intra-layer of semi-stacked clay rather than at epoxy-clay interfacial region. This phenomenon proves that higher contents of d-clay, e. Mechanical and thermo-mechanical performance. The mechanical properties of epoxy nanocomposites containing various concentrations of m-clay and d-clay are shown in Fig. 7a and b. As it can be seen, the addition of m-clay has not improved the tensile strengths of epoxy matrix significantly and in fact the addition of 2.5 wt% of m-clay to epoxy resin (EP-M2.5) resulted in only ~5% increase in tensile strength, compared to pure EP. While, addition of 5 wt% of m-clay to epoxy matrix (EP-M5) not only did not increase the tensile strength but also led to a ~9% decrease. However, the inclusions of 2.5 and 5 wt% of d-clay in epoxy resin (EP-D2.5 and EP-D5) resulted in ~20% and ~8% increase in tensile strength of epoxy composites, respectively. This could be due to the improved dispersion of nanoclay as well as stronger filler-matrix physico-chemical interactions achieved through DNA modification of nanoclay. These interactions not only improve the epoxy monomer diffusion through faster intra-gallery reaction but also react and entangle with epoxy chains. This reinforcing mechanism will lead to the promoted strengths in epoxy nanocomposites. On the contrary, as evidenced by DSC and rheological analysis presented earlier, m-clays do not interact effectively and covalently with epoxy resin compared to d-clays. When it comes to moduli, it is argued that the moduli of nanocomposites could be improved by adding either m-clay or d-clay. It means that although interfacial adhesion could enhance nanocomposite properties, moduli are mostly controlled by some factors such as: (i) high aspect ratio of a single clay platelet, (ii) higher stiffness of fillers, and (iii) restriction of polymer chain mobility 11,19 . As illustrated in Fig. 7a, it is worthy to mention that nanocomposites containing d-clay still show higher moduli than those containing m-clay. This is possibly arising from higher exfoliation degree of d-clay in epoxy matrix, resulting in higher stress-transferring and shear deformation mechanism. Mostly, fracture toughness and critical strain energy release rate of epoxy systems reinforced with nanoclay have been improved through various mechanisms such as pull-out, bridging effect, and interface debonding being observed in morphology section 50 . Compared with EP-M systems, EP-D systems exhibit higher toughness which is due to the fact that d-clay layers, adhering perfectly to epoxy resin through covalent bonding, are capable of carrying and transferring the highest amount of stress applied to matrix, resulting in higher absorbent of fracture energy. As presented in Fig. 7b, fracture toughness of EP-D2.5 and EP-D5 increased by ~56% and ~66%, respectively, compared to the EP. Whereas, the inclusion of the 2.5 and 5 wt% of m-clay could lead to ~23 and ~30% increases in fracture toughness. The same trend could be also observed for critical strain energy release rate. According to earlier observations made by Miyagawa et al. 54 and Le Pluart et al. 55 , it has been hypothesized that higher intercalation degree of nanoclay might deflect crack more efficiently than exfoliated platelets due to the vulnerability to fracture. This leads to higher fracture toughness of EP-D5 in comparison to EP-D2.5, as TEM and XRD results of EP-D5 showed higher intercalation degree of d-clay into epoxy matrix compared to the EP-D2.5. Therefore, such enhancements confirm reinforcing potential of d-clay in high-performance epoxy nanocomposites, providing better stress-transfer. For comparison, Zaman et al. 11 reported that the addition of 2.5 wt% of clay treated by various reactive modifiers with different chains length having free amine-end groups result in ~36%, ~18%, and ~8% decrease in tensile strengths and ~21%, ~44%, and ~58% increases in fracture toughness of epoxy nanocomposites. It is believed that the length of surfactant molecule and its ability to react with matrix could affect mechanical properties. According to Wang et al. 27 , using a green approach in preparation of nanocomposites, the highest improvement of ~22% in tensile strength could be achieved for the epoxy systems reinforced with 1 wt% of Cloisite30B. Figure 7c illustrates the DMTA plots of storage modulus (E') versus temperature for various epoxy systems. Moreover, as presented in Fig. 7d, the tanδ, which is the ratio of the loss modulus to the storage modulus, gives insight into polymer chains movement in relation with the strength of the epoxy system. From temperature corresponding to the maximum value of tanδ, glass transition temperature (T g ) can be obtained. Moreover, crosslink density of the epoxy systems could be evaluated using following equation : Where v e is the estimation of crosslink density, and R is the universal gas constant. E r is storage modulus corresponding to the T r where T r is T g + 30, and E g is also defined as storage modulus corresponding to the T g −30. As it can be seen from Fig. 7c and data presented in Table 3, addition of 2.5%wt clay regardless of its modification increases storage modulus at T < T g which is glassy region of epoxy system. However, the storage modulus of EP-M2.5 system become lower than that of the EP at T > T g . In other words, the EP has higher storage modulus at its rubbery region in comparison to EP-M2.5. This is because of the formation of plasticity effect on epoxy matrix at the interface with m-clay, leading to a reduction in ability of load transfer from matrix to the m-clay, causing lower T g of EP-M2.5 in comparison to the EP 17 . As presented, T g of the EP decreases from 172 °C to 169 °C when incorporating m-clay into epoxy matrix, which is in agreement with other reports 22,59,60 . In contrast, storage modulus of EP-D2.5 is higher that of both pure EP and EP-M2.5 in both glassy and rubbery regions at all temperatures. However, this higher storage modulus is more conspicuous at glassy region, in comparison to the rubbery region. The T g of EP-D2.5 shows 6 °C and 9 °C increases, respectively compared to the pure EP and EP-M2.5. These increments not only do denote that no plasticity effect at interface of d-clay with epoxy is present, but also show that d-clay can establish a strong interface with epoxy, leading to the restriction of segmental chains motion. Moreover, the EP-D2.5 shows 0.35 mmol/m 3 increment in the crosslink density while a significant reduction was observed for the crosslink density of EP-M2.5, compared to the EP system. This means that delaminated/ exfoliated d-clay layers provide a higher surface available to be encountered with epoxy matrix, being able to have chemical bondings with the matrix, whereas a high crosslink density at interface of m-clay with epoxy matrix is effectively hindered by inducing the plasticity effect. Figure 8 shows fracture surfaces of the EP and its various nanocomposites. Although surface morphology of the PE is mostly smooth, it is possible to observe some approximately large fracture surfaces, as shown in Fig. 8a. On the contrary, when m-clay and d-clay are added into epoxy matrix, the crack propagates through matrix tortuously resulting in smaller fracture plates (Fig. 8b-e). This type of fracture is arising from crack deviation while applying load 61 . Moreover, compared with EP-M systems, the effect of d-clay incorporated into epoxy matrix (EP-D systems) on crack growth resistance via different mechanisms such as crack arrest, birding effect, and pull-out is more tangible (Fig. 8d). This phenomenon could be explained by effective interfacial interactions and homogenous dispersion, achieved by DNA modified clay. On the other hand, as m-clay does not have an effective adhesion to matrix as discussed above, the rejected m-clays from epoxy matrix are simply observable. Such occurrence causes reduction of mechanical performance, as discussed in pervious sections. Another consideration related to these reinforced epoxy composites is related to the poor levels of dispersion and micro-void formation at higher contents of nanoclay. In other words, the addition of higher contents of clay (e.g., 5 wt%) could result in agglomeration formation, causing lower filler/epoxy surface interactions. Although EP-D5 system possesses a few inevitable agglomerations, its morphology exhibits mostly disorderly congested d-clay (Fig. 8e). This means that it is highly likely that DNA modification could cause clay not only to be well-separated but also to be presented at least in congested forms instead of agglomerations. At higher loading of d-clay, it is hypothesized that they are prone to get closer. This could possibly lead to the formation of accumulation of intercalated clay instead of highly exfoliated one. Generally, as crack encounters nanoclay platelet, different scenarios can be assumed due to the micron-sized lateral dimension. The crack could bypass nanoclay platelets either by breaking them or pulling them out from matrix, as illustrated in Fig. 8d. In both conditions, the crack energy will be dissipated 62 . Therefore, when nanoclay is modified, the matrix could hold it tightly and restrict it from being easily pulled out. As a result, compared with m-clay, d-clay possessing strong interfacial bonding with matrix consumes crack propagation energy more and more. Another point related to taking advantage of DNA modified clay is that the possibility of interlayer delamination of clay under mechanical loading could decrease. In other words, the intercalated clay with enough d-spacing could lead to epoxy monomer diffusion. As epoxy monomer is diffused, elastic force applied by epoxy molecules cross-linking inside the clay galleries leads to exfoliation of clay layers i.e. swelling of clay galleries occurs 49,63 . Additionally, the ability of DNA modifier to react with epoxy through chemical bonding keeps clay layers to be firmly embedded within the matrix while load is applied. This will result in a more effective stress-transfer mechanism 64 . In contrast, despite the fact that epoxy monomer can also diffuse into stacked m-clay layers due to its initial d-spacing resulting from long alkyl chain quaternary ammoniums, its interactions with epoxy molecules still remains as weak as van der Waals forces, which can act like flaws in composites causing their premature failures and delamination, under mechanical loadings. Moreover, the higher chance of formation of agglomerates in m-clay could intensify such devastating effects and as such the properties of such composites will be similar to the micro-particle filled composites 49 . Thermal and flammability performance. We have investigated the effect of DNA as clay modifier and natural flame retardant on the thermal properties of epoxy-clay nanocomposites. In order to have a comprehensive evaluation, thermal performance of nanocomposites was examined by TGA and cone calorimetry to compare the thermo-oxidative degradation and flammability properties. Figure S2 and Fig. 9a show TGA thermograms of various epoxy nanocomposites at different heating rates under the air flow and the results are presented in Table S1 and Table 4. As it can be seen, thermo-oxidative behaviour of epoxy nanocomposites shows a multi-step degradation consisting of two main steps. Herein, we considered various parameters including T i and T max (temperatures corresponding to 5% weight loss and maximum degradation rate for each step, respectively), char yield at 850 °C, and total activation energy required for thermo-oxidative degradation (E 1 + E 2 = E total ), in evaluation of thermal properties using TGA analysis. Activation energy for each degradation step was calculated using Kissinger method 65 , as it is independent of any presumption on the degradation mechanism according to the following equation: Where β is heating rate, and C is constant. By plotting As expected, addition of 2.5%wt clay regardless of its modifier type enhances all thermal characteristics of epoxy matrix. Regarding the effect of DNA modifier on the thermal properties, 16 °C, 6 °C, and 19 °C increases in T i , T max,1 , and T max,2 are observed, respectively, for nanocomposite incorporated with 2.5 wt% d-clay when compared to that incorporated with 2.5 wt% m-clay. As shown in Table 4, char yield of the EP after thermo-oxidative process at 850 °C is insignificant (0.75%). The char yield increased upon incorporation of clay and the increase was more profound for EP-D2.5, compared to EP/M2.5. Moreover, E total of EP-D2.5 is ~172 kJ/mol which is ~10 kJ/mol higher than that of the EP-M2.5. These TGA results show a significant additional thermal stability effect on epoxy nanocomposites containing d-clay, resulting from DNA intercalated within clay layers. Combustion behaviours of samples were also examined by cone calorimetry as a useful tool in evaluating the flame retardancy performance under a forced-flaming combustion. Figure 9c and d display plots of heat release rate (HRR) and total heat release (THR) versus time, respectively. Using these plots, various parameters including THR, peak of heat release rate (PHRR), and time of reaching peak of heat release rate (t PHRR ) were extracted and summarized in Table 4. The results demonstrate that pure EP exhibits a high PHRR of 1542 kW/m 2 . The PHRR value of EP-M2.5 is 1298 kW/m 2 which is only 224 kW/m 2 lower than that of pure sample. While d-clay exhibits a notable flame retardant effect on epoxy system and the addition of 2.5%wt d-clay into the epoxy matrix results in a 322 kW/m 2 reduction in PHRR. Moreover, t PHRR of pure EP increases from 71 s to 87 s and 96 s for the EP-M2.5 and EP-D2.5, respectively. This shows that EP/D2.5 requires 9 s longer to reach its PHRR, in comparison to EP/M2.5. The mechanism behind this observation stems from intrinsically flame retardancy of DNA modifier, possessing phosphate groups which can act as a barrier in formation of char. Moreover, DNA can release ammoniac and carbon dioxide gas under heating conditions and reduce flammability of the system 33 . It was also observed that THRs of both nanocomposites show significant differences in comparison to the pure EP. However, THR of EP-D2.5 is ~4000 kJ/m 2 lower than that of the EP-M2.5. These remarkable reductions in PHRR and t PHRR values of EP-D2.5 could also result from the insulation barrier effect of a cohesive and compact char layers on postponing the oxygen diffusion and the escape of volatile decomposition compounds produced during the combustion 2,66 . This fact was further investigated by SEM observations on the char residues structures. As illustrated in Fig. 10, the char residues of the pure EP show a rickety surface having wide cracks. Although EP-M2.5 surface exhibits lower cracks in comparison to pure EP, it still has an incompact surface. On the other hand, EP-D2.5 shows a dense and fully compacted surface morphology without any cracks on its surface, leading to the lower efficiency of heat and volatiles transfer due to the obstructing effect, and consequently providing underlying epoxy matrix with an effective barrier 67 . Fire propagation was simply evaluated through the keeping a flame near the samples, which their photographs are presented in Fig. 10. As it can be clearly seen, the fire quickly propagates across the pure EP sample; while it encounters a delay for the nanocomposites. Moreover, an obvious slower fire propagation are observed for the EP-D2.5 in comparison to the EP-M2.5, confirming an additional fire resistivity effect of DNA-modified clay on flammability of epoxy polymer. We have compared the PHRR and THR values as well as mechanical performance of the epoxy-clay nanocomposite containing 2.5 wt% d-clay with the published reports in literatures in which epoxy nanocomposites contain both low and high nanofiller loadings. As shown in Table S2, a considerable low amount of d-clay can bring about acceptable figures in terms of improvements in both mechanical and flammability properties. This is while in other published reports mostly high loadings of nanofiller has led to only improvements in flammability per-Such deduction can be proved by comparing our results with results reported in literature. According to the data presented in Table S2, two different trends can be observed for comparison. The first trend is dealing with the case when the amount of nanofiller is approximately as same as the amount of d-clay e.g. 2.5 wt%. In this condition, the reported decrements in PHRR and THR are significantly lower than that of d-clay we are reporting herein. Considering the second trend, it can be said that more decreases in PHRR and THR can be seen for epoxy nanocomposites containing high amount of nano fillers which are usually destructive in terms of mechanical performance. In other words, at high nanofiller loadings, low mechanical performances are expected to be observed in various mechanical properties including tensile strength, tensile modulus, and fracture toughness. Mostly, the reduction in mechanical properties is attributed to poor dispersion and weak interfacial adhesion. However, in this study, through DNA modification, the aim is to improve mechanical performance and provide clay with reinforcing features through chemical interactions. The study presented here, demonstrates a balance between mechanical and flame properties. In other words, compared to other modified clays presented in current literatures, a small loading of DNA modified clay shows a great potential to enhance flame retardancy of epoxy composites while improving its mechanical performance. Moreover, in order to evaluate the contribution of DNA in overall flame retardency of the epoxy systems, PHRR, THR, and t PHRR values of epoxy composites containing neat clay, neat fish DNA were also obtained and their results are presented in Table S3. As it can be seen, addition of 2.5 wt% neat clay to epoxy (EP-N2.5 sample) can lead to insignificant decreases of ~4.6% and ~8.5% in PHRR and THR, respectively. Whereas the addition of the same amount of m-clay (EP-M2.5 sample) results in ~15.8% and ~25.7% reduction in PHRR and THR, respectively. Moreover, the PHRR and THP decrease by ~20.8% and ~31.2% for the epoxy nanocomposite containing 2.5 wt% d-clay (EP-D2.5) which demonstrates the highest flammability improvement in epoxy resin studied herein. This reveals the improved dispersion and barrier effect of clay as a result of DNA modification. In addition to this, two different amounts of neat DNA powder (0.2 and 2.5 wt%) are solely incorporated into the epoxy matrix to evaluate the effect of DNA agent on the flammability properties. It is worth to mention that 0.2 wt% DNA was calculated as maximum amount of DNA grafted within clay layers which obtained at pH = 2, as discussed in Fig. S1. Therefore, 0.2 wt% DNA was incorporated into epoxy matrix to compare with other epoxy systems. The results of flammability study of these samples show that the reduction in PHRR and THR are slightly lower than that of EP-M2.5 composite because the amount of DNA (0.2%) was much lower than that of m-clay (2.5 wt%). However, when the same amount of neat DNA powder (2.5 wt%) was used to produce the epoxy composite (EP-DNA2.5 sample), the highest improvement in both PHRR and THR were observed which are ~60.3% and ~51.1% decreases in PHRR and THR, respectively. This clearly confirms that DNA is an effective agent for improvement of flame retardency of epoxy polymer systems. Nonetheless, despite the role of DNA in improving the flammability performance of clay-epoxy nanocomposites, DNA to a great extent is incompatible and unprocessable with epoxy resin only which extremely hinder fabrication of epoxy-DNA composites with appropriate mechanical performance. Therefore, to take advantages of DNA performance, it should be grafted on a proper platform such as clay prior to incorporating into epoxy to provide composites with both improved mechanical and flame properties. In other words, both DNA and clay are set to cooperate with each other considerably through various mechanisms: (i) DNA can contribute to uniform clay dispersion as well as clay-matrix interactions leading to greater mechanical and flame properties, (ii) DNA agent can yield further improvement in flame properties via its functional groups having phosphorous and nitrogen, (iii) the reduction in mechanical properties which may be caused by DNA agent can be compensated by well dispersed clay. As a result, both flame and mechanical properties can be enhanced. ## Conclusions Waste DNA from fishing industry has a great potential for recovery and re-use as a source of flame retardant materials in nanocomposites while improving strength. Herein, for the first time we have shown that fish DNA can be used in modification of clay nanomaterials for preparation of epoxy nanocomposites with significantly improved mechanical and flammability properties. Based on the results obtained in this study, the following detailed conclusions can be drawn for the proposed application: • The results of epoxy droplet contact angle revealed ~44% increase in work of dispersion which is a critical factor in determination of epoxy resin compatibility and reactivity, and ~17% further decrease in penetration of epoxy droplet into DNA modified (d-clay), both in comparison to a commercially modified clay e.g., Nanomer I.28E. • The dispersion levels of d-clay into epoxy matrix studied by XRD and TEM analyses confirmed the outstanding role of DNA as a modifier and its remarkable influence on the well dispersed structures including intercalated/exfoliated structures arising from intra-gallery polymerization. • The rheological behaviours of epoxy-clay nanosuspensions, as another evidence for dispersion and interaction, proved the possibility of interactions between d-clay and epoxy monomers leading to formation of a network, which possesses high viscosity level and being resistance to shear rates. It was concluded that d-clay attached to epoxy chains could act as slippery agents to promote shear thinning behaviour. • Inclusion of d-clay into epoxy resin led to a significant improvement in tensile strengths, moduli and fracture toughness compared to composites containing m-clay. This phenomenon results from improved clay-matrix interfacial adhesion, better dispersion and more effective role of d-clay in consumption of crack energy through various mechanisms such as crack arresting, deviation, and pull out procedures as confirmed by SEM micrographs. Observation of ~3% increase in T g of epoxy/d-clay system versus ~1% decrease for epoxy/mclay system, both compared to pure epoxy system, demonstrates that plasticity effect of nano-clays on T g of epoxy nanocomposite was eliminated as a result of the effective interfacial interactions. • Contribution of DNA molecules to the considerable improvement of thermal stability and fire resistancy of epoxy-clay systems was approved by TGA and cone calorimetry results. This improvement is as a result of the formation of condensed char layers during combustion due to the release of effective suppressant agents during the decomposition of DNA structures. ## Methods Materials. Epoxy resin (diglycidyl ether of bisphenol A, D.E.R 332) and diethylenetriamine as curing agent were obtained from Sigma-Aldrich and used as received. The used pristine clay was sodium montmorillonite and the organoclay was a commercial product under the name of Nanomer I.28E, which were supplied by Nanocor Co., USA. DNA powder from herring sperm was supplied from Sigma-Aldrich and stored at below 8 °C. All the solvents used in this study were of analytical grade. ## Intercalation of DNA within clay layers (d-clay). To intercalate the DNA structures into clay layers, as-received DNA (2.00 gr) was dispersed into 200 ml DI water by stirring for 1 h, followed by adding 1.0 M HCl aqueous solution to adjust the pH to 2, 3, 4, and 5. The resultant solutions were stirred for further 3 h at 60 °C. In a separate beaker, pristine clay (2.00 g) was dispersed into 200 ml boiling water and stirred for 2 h before sonicated for 1 h in an ultrasonic bath. The dissolved and pH adjusted DNA solutions were added to the clay/DI water suspension and further stirred for 6 h to allow for the complete cation exchange process. The final mixtures were then filtered and washed several times with abundant DI water until no chloride detected by adding 0.1 N AgNO 3 solution. The obtained DNA-modified clays (d-clays) at various pHs were then vacuum dried at 60 °C prior to use. By measuring differences between initial pristine clay weight with various d-clays weight, amount of intercalated DNA at each pH can be calculated, which is presented in Fig. S1. It is found that the highest intercalation of DNA on pristine clay occurs at pH = 2. ## Epoxy-clay nanocomposites preparation. To take the full advantage of solvent properties in increasing the layers spacing in clay, fabrication of polymer nanocomposites were conducted according to the "slurry-compounding" process 22 , but with major modifications to simplify it and to assure that the clay concendoes not change during the process. As illustrated in Fig. 11, in a typical experiment, 1.00 g d-clay obtained at pH = 2 or Nanomer I.28E (m-clay) were dispersed in 100 ml acetone and stirred for 2 h, followed by sonication using an ultrasonic bath for 1 h to form a fine slurry before pouring the slurry into a high-pressure vessel and heating up to 100 °C for 12 h. This process facilitates the penetration of acetone between clay layers. After cooling to room temperature, proper amounts of epoxy resin were added to the clay/acetone slurry and stirred at 70 °C for 6 h. The mixture then was sonicated, for 30 min using a Hielscher UIP1000-230 ultrasonic processor operating at a frequency of 15 kHz to generate ultrasonic waves with an amplitude of 80 μ m peak-to-peak through the epoxy suspensions with an ultrasonic pulsing cycle of 2 s on and 2 s off, being kept in an ice bath. To completely remove the acetone, the epoxy mixtures were subjected to the vacuum at 60 °C for 24 h. Then, a stoichiometric amount of hardener was added to the compositions before applying the vacuum for 30 min to degasify the bubbles produced during mixing the hardener. The total mixtures were poured into a mould and finally the curing process was conducted at 70 °C for 6 h, followed at 120 °C for 2 h. A pure epoxy sample was also prepared using the same condition and considered as control sample and named pure EP sample. Epoxy nanocomposites containing 2.5 and 5%wt of d-clay and m-clay were named EP-D2.5 and EP-D5, and EP-M2.5 and EP-M5, respectively. For flammability comparisons, the epoxy systems containing 2.5% and 0.2% neat DNA powder as well as 2.5% neat clay were also prepared using the above-mentioned procedure, named EP-DNA2.5, EP-DNA0.2%, and EP-N2.5%, respectively. Characterizations. FT-IR spectra were recorded with KBr pellets containing the samples on a FTIR spectrophotometer of Bruker Optics. X-ray photoelectron spectroscopy (XPS) analysis was performed using an AXIS Nova spectrometer (Kratos Analytical Inc., Manchester, UK) with a monochromated Al K α source at a power of 180 W (15 kV × 12 mA) and a hemispherical analyser operating in the fixed analyser transmission mode. Survey spectra were acquired at a pass energy of 160 eV. The atomic concentrations of the detected elements were calculated using integral peak intensities and the sensitivity factors supplied by the manufacturer. XRD patterns were obtained using a PANalytical X'Pert Pro Diffractometer with Cu Kα radiation (λ = 1.54184 ), operated in 2°-10° (2ϑ°) at 45 kV and 30 mA with a step size of 0.033. The spreading of an epoxy droplet on compacted discs of clay, provided with a compaction pressure of 20 MPa, was analysed using KSV Model CAM101 Contact Angle Meter (KSV Instruments Ltd, Finland) equipped with an Olympus DP70 high resolution microscope at ambient temperature. A 4 μ L droplet of epoxy was poured onto compacted discs with diameter and thickness of 13 mm and 4 mm, respectively; and the amount of epoxy droplet penetrated to each clay substrate was evaluated by digital image analyser. DSC analyses were performed using a TA Q200 DSC instrument in high purity nitrogen atmosphere. The samples were heated up to 150 °C at the heating rate of 10 °C/min. From the exotherms obtained, the heat of reaction and the peak temperature were determined. Rheological evaluations were carried out using a TA DHR 3 rheometer with cone-plate geometry. A cone with a diameter of 40 mm and a tilt angle of 2° were utilized, and gap width was fixed to be 49 μ m. The range of shear rate, used in this experiment, was chosen to be between 0-1000 1/s. The nanosuspensions were located between the cone and plate and soaked for five minutes. Dynamic mechanical properties of the epoxy-clay nanocomposites were examined using a TA Instruments Q800 in the cantilever bending mode. The instrument was calibrated before use and the samples were prepared according to ASTM E1640 before being mounted on a single cantilever clamp. The DMA analysis were carried out from 25 °C to 250 °C at a heating rate of 2 °C/min and the frequency value of 1 Hz. TGA tests of various modified clay were carried out using a Perkin-Elmer TGA instrument at the heating rate of 10 °C/min under a steady nitrogen flow of 60 ml/min. While, TGA analyses of polymer nanocomposites were operated at various heating rates under an air flow of 100 ml/min. Flammability of the polymer nanocomposites were examined by cone calorimeter (Fire Testing Technology, UK) and measurements were performed at an incident heat flux of 35 kW/m 2 , according to the ISO5660 standard. The fracture surfaces of tensile samples were examined using a scanning electron microscope (SEM) operated at 25 kV. The fracture surfaces were gold-coated prior to microscopy observations. Transmission electron microscope (TEM) samples with specimens of approximately 80 nm in thickness were prepared using a Leica Ultracut UCT ultramicrotome at room temperature. Microtomed sections were imaged by a Philips TEM at 300 kV in bright field mode. Tensile tests were performed on dog-bone samples according to ASTM D638 Type I by using an Instron universal testing machine; cross-head speed 5 mm/min with a 30 kN load cell. Moreover, according to ASTM D 5045, fracture toughness was measured using the compact tension specimen (see Fig. S3) with dimensions of 48 mm × 48 mm width × 10 mm at 10 mm/min. An instantly propagating crack was designed for each specimen by tapping a razor blade to the samples because as mentioned in literature 11 it is the most economical approach to create a satisfactory sharp crack. To obtain statistically meaningful results, the tensile properties and fracture toughness of at least five specimens for each case were averaged and reported. Fracture toughness properties were shown as mode-l stress intensity factor (K 1C ) and critical strain energy release rate (G 1C ) according to following equations: Where P Q , B, W, a, E, and ν are the maximum load, the thickness, the width, crack length, Young's modulus, and Poisson's ratio, respectively. According to literatures, Poisson's ratio is considered 0.35 for DER 332 epoxy resin 68 .

chemsum

{"title": "Fish DNA-modified clays: Towards highly flame retardant polymer nanocomposite with improved interfacial and mechanical performance", "journal": "Scientific Reports - Nature"}

determination_and_stability_of_n-terminal_pro-brain_natriuretic_peptide_in_saliva_samples_for_monito

## Abstract: Heart failure (HF) is the main cause of mortality worldwide, particularly in the elderly. N-terminal pro-brain natriuretic peptide (NT-proBNP) is the gold standard biomarker for HF diagnosis and therapy monitoring. It is determined in blood samples by the immunochemical methods generally adopted by most laboratories. Saliva analysis is a powerful tool for clinical applications, mainly due to its noninvasive and less risky sampling. This study describes a validated analytical procedure for NT-proBNP determination in saliva samples using a commercial Enzyme-Linked Immuno-Sorbent Assay. Linearity, matrix effect, sensitivity, recovery and assay-precision were evaluated. The analytical approach showed a linear behaviour of the signal throughout the concentrations tested, with a minimum detectable dose of 1 pg/mL, a satisfactory NT-proBNP recovery (95-110%), and acceptable precision (coefficient of variation ≤ 10%). Short-term (3 weeks) and long-term (5 months) stability of NT-proBNP in saliva samples under the storage conditions most frequently used in clinical laboratories (4, − 20, and − 80 °C) was also investigated and showed that the optimal storage conditions were at − 20 °C for up to 2.5 months. Finally, the method was tested for the determination of NT-proBNP in saliva samples collected from ten hospitalized acute HF patients. Preliminary results indicate a decrease in NT-proBNP in saliva from admission to discharge, thus suggesting that this procedure is an effective saliva-based point-of-care device for HF monitoring.Heart failure (HF) is a pathophysiological condition that causes an inadequate blood supply to all the organs and apparatus. This is particularly due to the impairment of the heart's capacity to pump out blood or to fill one or both ventricles. HF is an increasingly common chronic cardiovascular disease and, according to the World Health Organization, the main cause of mortality and major morbidity worldwide, particularly in the elderly 1,2 .Approximately thirty million people worldwide are affected by HF 3 , however this does not include undiagnosed or misdiagnosed cases 4 . HF causes high mortality rates in elderly patients and is a heavy economic burden on national health services [5][6][7] .The diagnosis of HF in some patients is made more challenging due to nonspecific signs and symptoms 8, 9 , with possible risks to the patient's health and additional costs for the health services. Early diagnosis and therapy monitoring should therefore be improved to minimize the impact of HF on the population 10 .Biomarkers are commonly described as biochemical compounds that provide information on normal biological processes, pathogenic processes or responses to an exposure or intervention 11 . Several biomarkers have been considered for HF management [12][13][14][15][16] . Natriuretic peptides (NP), such as Brain Natriuretic Peptide (BNP) and the N-terminal proBNP (NT-proBNP), have been identified as gold standard biomarkers of HF by both European and American guidelines 17,18 . Increased plasma levels of circulating NP in patients with congestive HF are directly related to the severity of congestive heart failure, as classified by the New York Heart Association criteria 19 . Measuring the plasma or the serum concentrations of both BNP and NT-proBNP is therefore currently recommended to support the diagnosis of HF 13,20 . NT-proBNP has a very high prognostic power due to its correlation with the mortality, morbidity, and hospitalization rate of HF patients 18,21 . In addition, NT-proBNP shows additional advantages over BNP in diagnosing and assessing the severity of HF, such as a higher circulating concentration and longer stability 22 . Blood is generally regarded as the best body fluid to evaluate systemic processes through the determination of biomarkers, in which NT-proBNP is the gold standard biomarker for HF diagnosis and monitoring 16,23 . However, blood sampling can be stressful for patients due to its potential risks, such as transient discomfort, bruising, infection at the venipuncture site, and anemia 24 . Moreover, blood sample manipulation requires particular treatments, i.e. both in terms of sample analysis and disposal. Saliva analysis is an increasingly common alternative method to blood testing. Saliva (i.e. whole saliva) is an "ultra-filtrate" of blood and has gained importance as a potential source of clinical information because it reflects biological activity as well as a healthy or pathological status. Compared with blood, saliva samples can be easily and unobtrusively collected, even from critical subjects (e.g. children, the elderly, and the disabled). Non-invasive saliva sampling is suitable for the screening of a large population, and decreases psychological stress (especially if repeated sampling is needed), and health risks for patients and healthcare professionals . In addition, salivary diagnostics is being exploited in Lab-on-Chip (LoC) and Point-of-Care (PoC) devices 32,33 . However, there are currently no robust information on the salivary levels of NT-proBNP as HF biomarkers or reliable methods for its determination in saliva. In fact, BNP and NT-proBNP are usually quantified in blood or plasma by immunoassays, such as the Enzyme-Linked Immuno-Sorbent Assay (ELISA) , electrochemiluminescence immunoassay (ECLIA) and radioimmunoassay (RIA) 38 , immunoradiometric assay (IRMA) 39 , and fluorescent immunochromatographic assay (FICA) 40 . In addition, affinity chromatography and chromatography coupled to tandem mass spectrometry methods 41,42 can be used for NP determination in blood. ELISAs are the most common procedure for HF biomarker quantification, however commercially available immunoassay kits are generally intended to analyze cell culture supernates, serum, EDTA plasma, heparin plasma, and citrate plasma. One of the most widely used immuneassays for NT-proBNP quantification in plasma and serum sample is the Elecsys NT-proBNP II assay from Roche . This is an automated electrochemiluminescent immunoassay for NT-proBNP quantification in a concentration range of 10-35,000 pg/mL, with a detection limit (LoD) of 10 pg/ mL and limit of quantification (LoQ) of 50 pg/mL. In 2012, Foo et al. 47 used the Elecsys NT-proBNP II assay to validate their immunoassay in order to quantify NT-proBNP in saliva. Foo et al. used the NT-proBNP AlphaLISA kit from Perkin Elmer. This kit is sold for the quantitative determination of NT-proBNP in buffer, plasma, and serum, in a concentration range of 3.9-100,000 pg/mL, with a LoD of 3.9 pg/mL and a LOQ of 10.2 pg/mL. Foo et al. also investigated the assay performance characteristics of the NT-proBNP AlphaLISA immunoassay for saliva analysis in terms of recovery, intra-and inter-assay coefficient of variation, and LOD. Foo et al. found a % recovery of 85%, an intra-assay variation of 7.17% (± 0.75%), an inter-assay variation of 4.46% (± 0.59%), and an LoD of 16 pg/mL. However, they did not validate the Elecsys NT-proBNP II assay for saliva analysis specifically, and they did not investigate the NT-proBNP stability in saliva in relation to different storage conditions. In this study, we report the validation of an analytical method to quantify NT-proBNP in saliva samples based on a commercial ELISA kit designed for the quantitative determination of NT-proBNP in human serum/plasma. To the best of our knowledge, this is the first time that the stability of NT-proBNP has been investigated in saliva samples stored for up to 3 weeks at 4 °C (short-term stability study) and up to 5 months at both − 20 and − 80 °C (long-term stability study). The effect of the thaw/freezing cycle was also evaluated. Finally, we used our method to determine NT-proBNP in saliva samples collected from ten acute HF patients to highlight the potential difference in saliva NT-proBNP levels between hospital admission and discharge. The aim of our paper is not to correlate NT-proBNP levels in plasma and saliva, but to test and validate an analytical approach based on a plasma/serum NT-proBNP ELISA kit for its potential use in a saliva-based PoC. ## Results Assay validation for saliva analysis. In this study, an ELISA kit originally commercialized to determine NT-proBNP in human serum and EDTA plasma samples was validated for saliva samples. Linearity, matrix effect, sensitivity, recovery, intra-and inter-assay precision of the ELISA kit were evaluated using quality control samples (QCSs) prepared by spiking aliquots of pooled saliva samples (PSSs), collected from healthy volunteers, with a known amount of analyte. The assay response was evaluated following the procedure provided by the ELISA kit manufacturer. Method linearity was tested by analyzing both standard solutions (STDs) and QCSs at six concentration levels in the range of 1 -200 pg/mL, which were selected according to the NT-proBNP salivary levels previously reported by Foo et al. 47 . In this range and for eight calibration curves, the optical density (OD) of NT-proBNP linearly increased with the concentration of both STDs and QCSs (R 2 ≥ 0.9996 ± 0.0030 and 0.9987 ± 0.0020, respectively), as shown in Fig. 1. Calibration curves (y = mx + q) resulted in y = 0.0008x + 0.01884 and y = 0.0008x + 0.01438 for STDs and QCSs, respectively. A matrix effect was excluded by comparing, at a confidence level of 95%, the slopes of the calibration curves, obtained after subtracting blanks. The two-tailed p value (0.49) confirmed the null hypothesis that the slopes were statistically identical. The minimum detectable dose (MDD) resulted in 1 pg/mL (SD = 1 pg/mL). The intra-assay precision was evaluated by analyzing five QCSs of a known concentration, ten times each on the same plate, whereas the inter-assay precision was determined by testing the same samples in ten separate assays. Both parameters were expressed as a coefficient of variation (CV%) and were lower than 10% at each concentration level tested. Analyte recovery, determined from ten replicates of each of the five QCSs, ranged from 95 to 110%, with CV% lower than 10% for each concentration tested. The SOS device did not release any interferents and allowed for a recovery of NT-proBNP equal to 100% (SD = 2%, CV% = 10%) regardless of the concentration level tested (10, 50, and 100 pg/mL). ## Sample stability study. A short-term (T S ) stability study and a long-term (T L ) stability study were carried out to evaluate NT-proBNP stability in saliva samples. The T S study investigated analyte stability at 4 °C for up to 3 weeks (T 0 : collection day; T 1S : T 0 + 1 week; T 2S : T 0 + 2 weeks; T 3S : T 0 + 3 weeks), whereas the T L study investigated analyte stability at − 20 and − 80 °C for up to 5 months (T 0 : collection day; T 1L : T 0 + 1 month; T 2L : T 0 + 2.5 months; T 3L : T 0 + 5 months). T 0 was the same day for both T S and T L studies. More specifically, aliquots of PSS collected and prepared at T 0 were spiked with 10, 50, and 100 pg/mL of analyte (namely CL 1 , CL 2 , and CL 3 samples) and immediately analyzed for use as a reference value for both T S and T L studies. The effect of the thaw/freezing cycle was also evaluated at T 1L . Table 1 shows the results of the stability studies, which highlighted that NT-proBNP was not stable after 1 week at 4 °C. In such conditions, the concentration of NT-proBNP was lower than the MDD level at both 10 and 50 pg/mL, whereas the concentration of the target analyte measured in the sample containing 100 pg/mL was 13 pg/mL (CV% = 2%). Given these results, the stability of NT-proBNP at 4 °C was not studied for longer storage times. After 1 month (T 1L ), compared with NT-proBNP measured at T 0 , the mean recovery on the three concentration levels was 96% (CV% = 15%) and 87% (CV% = 13%) for samples stored at − 20 and − 80 °C, respectively. Satisfying results were also observed after 2.5 months (T 2L ), with a mean recovery of of 80% and a stable CV = 15% at − 20 °C, whereas a mean recovery of 91% was determined for samples stored at − 80 °C with a CV = 30%. After 5 months (T 3L ) recovery. No statistically significant changes (p value > 0.05) in the salivary level of NT-proBNP were observed after two consecutive freeze/thaw cycles. ## Preliminary clinical assessment. Saliva and blood samples were collected at admission and at discharge from ten patients hospitalized for acute HF at the Fondazione Toscana "Gabriele Monasterio", Pisa, Italy. On average, a patient was hospitalized for approximately six days. Compared with admission, HF patients at discharge showed significantly lower median (25th and 75th percentile) values of NT-proBNP in blood [3500 pg/ mL (1470-10,090 pg/mL) vs. 1200 pg/mL (560-3160 pg/mL), p value = 0.04]. Likewise, a significant reduction in the NT-proBNP concentration was observed in saliva [5 pg/mL (2-10 pg/mL) vs. 2 pg/mL (− 3 pg/mL), p value = 0.03]. There was an average decrease of about 40% in both saliva and blood. Figure 2 shows the box-plot for NT-proBNP measured in saliva and blood samples. ## Discussion Blood is one of the best biological fluids to assess systemic processes through the determination of specific biomarkers. Blood NT-proBNP is the gold standard biomarker for HF diagnosis and monitoring, and high levels of NT-proBNP in blood have been associated with cardiac (e.g. ejection fraction), renal (e.g. serum creatinine), and laboratory parameters (e.g. serum potassium and hemoglobin) 44,45 , as well as a higher NYHA class 46 . Our analytical approach is based on a sandwich enzyme immunoassay kit (the Biomedica Immunoassay) as a proof-of-concept method to obtain useful information on HF by monitoring NT-proBNP in saliva. In 2012, Foo et al. 47 evaluated an immunochemical assay for NT-proBNP quantification in saliva (NT-proBNP AlphaLISA kit, Perkin Elmer). Foo et al. used the commercial Roche assay to quantify NT-proBNP in plasma www.nature.com/scientificreports/ samples and to compare analyte levels in the two matrices. However, their approach to saliva analysis requires a pre-concentration step (3 kDa Amicon Ultra-0.5 Centrifugal Filter Devices at 14,000×g for 20 min) before performing the ELISA kit. This pre-concentration step provided a limit of detection (LoD) close to 16 pg/mL, recovery between 95 and 110% and intra-and inter-assay CVs below 10%. Unlike Foo et al., our analytical workflow requires no pre-concentration step and obtains a quantitative salivary recovery. We also obtained a higher NT-proBNP recovery (95-110% vs 85%) and a MDD of 1 pg/mL. Given that we do not need any pre-concentration step, fewer consumables (i.e. centrifugal filters) are required and instrumentation costs are lower. Table 2 compares the performance of the different assays referenced in this paper for plasma/serum and saliva analysis. Since it is not always possible to analyze samples immediately after collection, we also investigated the stability of NT-proBNP in saliva samples at different storage conditions commonly adopted by clinical laboratories. The simplest sample storage condition (i.e. 4 °C) could not be used for preserving NT-proBNP in saliva since a marked decrease in its levels was observed after 1 week of storage, probably due to the presence of microbial or proteolytic activities capable of degrading the peptide 48 . A longer storage time was obtained by storing saliva samples at − 20 °C and − 80 °C; however, we suggest that all measurements should be performed within 1 month. For example, after 1 month, the concentration of a sample stored at − 20 °C/− 80 °C and analyzed after 5 months was about 30% (CV% = 10%), which is lower than that of a sample stored at the same conditions but analyzed Table 1. Results on stability over time of NT-proBNP in different storage conditions, including the mean %recovery. The short-term stability (T S ) study investigated analyte stability at 4 °C for up to 3 weeks (T 0 : collection day; T 1S : T 0 + 1 week; T 2S : T 0 + 2 weeks; T 3S : T 0 + 3 weeks). The long-term stability (T L ) study investigated analyte stability at − 20 and − 80 °C for up to 5 months (T 0 : collection day; T 1L : T 0 + 1 month; T 2L : T 0 + 2.5 months; T 3L : T 0 + 5 months). An initial set of samples was analyzed immediately after the collection (T 0 ) to obtain the reference values, thus NT-proBNP values measured at T 0 in the table refer to this sample set. MDD was 1 pg/mL (SD = 1 pg/mL). Each experiment was performed in triplicate. a The analysis was not performed because the concentration of NT-proBNP was already lower than MDD at T 1S . b The analysis was not performed because the concentration of NT-proBNP was already lower than MDD at T 2S . www.nature.com/scientificreports/ after 1 month. Thus, the measurements of the saliva samples should be carried out at a constant storage time in order to minimize the bias due to the time difference between sampling and analysis. At − 20 and − 80 °C, the concentration of NT-proBNP in saliva was not affected by an additional thawing/freezing cycle. However, if saliva samples are used for a multi-parametric analysis, multiple aliquots should be prepared instead of stressing the samples with more than two freeze-thaw cycles. An improvement in an HF patient's health status is correlated with a significant decrease over time of the NT-prBNP in blood 45, . Even with the limited number of patients enrolled, the aim of our preliminary preclinical assessment was not to investigate a correlation between NT-proBNP salivary and blood levels, but rather to evaluate the possibility of monitoring the trend of NT-proBNP levels in saliva as an alternative indicator of disease progression. Interestingly, NT-proBNP levels in saliva showed a similar behavior to those in blood. A good agreement in the NT-proBNP concentration ratio was observed (Fig. 2), with a significant decrease of 30-40% from admission to discharge. The results of our approach therefore highlighted the potential role of saliva analysis for HF assessment through NT-proBNP monitoring, thus paving the way for future applications using dedicated salivary LoC and PoC devices. ## Study limitations Although NT-proBNP has already been shown to be a gold-standard biomarker for HF monitoring and saliva analysis has proven to be a powerful alternative matrix to blood, prior research studies on NT-proBNP determination in saliva samples are lacking. The strengths of this study include the use of saliva as an alternative matrix to blood, the study of NT-proBNP stability in saliva stored at different temperatures, as well as a preliminary evaluation of the trend of salivary NT-proBNP as an indicator of disease progression. Our study concerned the first phase of a preclinical assessment in which saliva was collected from HF patients only at admission (HF acute phase) and at hospital discharge. This limited number of samplings was initially necessary to understand if and how a saliva sampling could be incorporated into hospital routine. However, one of the main limitations of our study is the extremely low number of patients that it was possible to enroll due to the SARS-CoV-2 public health emergency. In addition, data obtained on NT-proBNP in saliva were compared with blood levels only, without taking into account other possible physiological parameters such as obesity or drug therapy. Nevertheless, although the number of HF patients enrolled was extremely limited (n = 10), these preliminary findings suggest the diagnostic value of salivary NT-proBNP for HF monitoring due to the correlation (p < 0.05) between the trends of NT-proBNP levels in both saliva and blood. It is well known how much inter-variability occurs in clinical studies involving the assessment of the clinical relevance of biomarkers. However, even simply considering the data on NT-proBNP values at the patient admission and discharge, we observed a significant difference in line with HF regression. ## Methods NT-proBNP standard solutions. Five lyophilized synthetic human NT-proBNP standard solutions supplied within the ELISA kit were reconstituted in 500 µL of ultrapure water (18.2 MΩ/cm type I ultrapure water, Elga PURELAB Classic) to obtain STDs at 0, 85, 340, 1360, 5420 pg/mL. STD solutions were left on an orbital shaker (80 rpm) at room temperature (22 ± 2 °C) for 10 min before use. These standards were then used to prepare QCSs and spike the saliva samples at the target concentration. Quality control samples (QCSs) and spiked PSSs were prepared by spiking samples at different concentrations using NT-proBNP standard solutions. Saliva sampling for assay validation and stability study. Saliva samples were collected from twenty nominally healthy volunteers according to the procedure described elsewhere 27 using the SOS device. They were then pooled to obtain a PSS which was used for the assay validation and stability study. The volunteers were asked to freely roll the swab in their mouths for about 2 min. Saliva was then recovered by centrifugation at 7000 rpm for 5 min at 4 °C. www.nature.com/scientificreports/ Analyte recovery from sampling device. The analyte recovery from the SOS sampling device was evaluated using PSSs spiked with 10, 50, and 100 pg/mL. An aliquot (1 mL) of each PSS was absorbed into three different swabs. The analyte recovery was calculated from the ratio between the average analyte concentration measured (C m ) in the samples recovered from the swabs and the spiked concentration (C s ). In addition, an aliquot (1 mL) of blank sample (milli-Q water, 18.2 MΩ/cm at 25 °C) was absorbed into another three different SOSs to evaluate the possible release of contaminants from the swab material. Procedure for NT-proBNP quantification in saliva. NT-proBNP was determined in saliva samples using the enzyme immunoassay for the determination of NT-proBNP in human serum/EDTA plasma, supplied by the Biomedica Immunoassay (Cat. No. SK-1204), following the assay procedure provided by the manufacturer. A wash buffer was prepared by diluting (1:20 v/v) the concentrate buffer supplied in the kit with ultrapure water. The sandwich enzyme immunoassay was as follows. First, an aliquot (50 µL) of STD, saliva, or QSC was pipetted in duplicate into the wells of the microtiter strips, which were pre-coated with polyclonal sheep anti NT-proBNP antibody. Subsequently, 200 µL of conjugate (sheep anti human NT-proBNP-HRPO) were added into the plates. The plate was then covered tightly by an adhesive strip provided within the kit, and incubated for 3 h at room temperature on a horizontal orbital microplate shaker set at 80 rpm for a gentle swirl. The NT-proBNP in the sample bound itself to the pre-coated antibody in the well and formed a sandwich with the conjugate (detection antibody). Each well-plate was then aspirated and washed five times with 300 µL of diluted wash buffer. In the washing step, all nonspecific unbound material was removed. Subsequently, 300 µL of Tetramethylbenzidine (TMB, Substrate) were pipetted into each well, and the plate was gently swirled again on the orbital shaker for 30 min at 80 rpm in a dark lab-made chamber. The change in color of the catalyzed enzyme in the substrate is directly proportional to the amount of NT-proBNP present in saliva. After the addition of 50 µL of 2 N sulphuric acid (Stop solution), the optical density (OD) was immediately determined at 450 nm and 630 nm. The readings at 630 nm were subtracted from the readings at 450 nm to correct for optical imperfections in the plate. A MultiSkan GO microplate (Thermo Scientific) reader was used to measure the OD. Assay validation for NT-proBNP quantification in saliva. Analytical figures of merit such as linearity, matrix effect, sensitivity, recovery, intra-and inter-assay precision were investigated to assess the performance characteristics of the ELISA kit for the analysis of NT-proBNP in human saliva. The validation was performed using STDs solutions supplied in the kit and QCSs prepared by spiking aliquots of PSSs with a known amount of analyte. The blank was also subtracted from the measurements. A PSS was freshly prepared every day of the analysis, which was then used for the assay validation. Linearity of the assay was evaluated in triplicate in three different ELISA kits by analyzing both STDs and QCSs at six concentration levels of NT-proBNP, in the range of 1-200 pg/mL. The matrix effect was evaluated by comparing, at a confidence level of 95%, the slopes 28,30 (reported with the corresponding standard deviation) of the calibration curves obtained from STDs and QCSs in the same concentration range. The minimum detectable dose (MDD) was determined by adding two standard deviations to the mean optical density value obtained for twenty replicates of unspiked PSS. The corresponding concentration at this level was calculated using the dedicated calibration curve. QCSs containing 5, 10, 50, 100 and 150 pg/mL of NT-proBNP were used to assess both analyte recovery and assay precision. Analyte recovery was evaluated by comparing the NT-proBNP concentration determined on ten replicates for each QCS with the expected value. Assay precision was evaluated by analyzing each QCS ten times with the same kit for intra-assay precision, and ten times with ten different kits for inter-assay precision. Sample stability study. Short-and long-term stability of NT-proBNP in saliva was evaluated at three concentration levels for up to 3 weeks at 4 °C and up to 5 months at both − 20 and − 80 °C, respectively. For this purpose, a PSS obtained at T 0 was divided into three main aliquots labelled as PSS short-term, PSS_long-term_20, and PSS_long-term_80. Each main aliquot was further divided into three aliquots that were spiked at different analyte concentrations: 10 pg/mL (CL 1 ), 50 pg/mL (CL 2 ), and 100 pg/mL (CL 3 ). All samples were prepared by weighting. . An initial set of samples was analyzed immediately after the collection (T 0 ) to obtain the reference values. The remaining samples of CL 1 , CL 2 and CL 3 from the PSS short-term were split into three aliquots and then stored at 4 °C. On the other hand, each corresponding sample from PSS_long-term_20 and PSS_long-term_80 were sub-aliquoted into four samples (three aliquots to perform the stability study over time, and one aliquot to investigate the freeze/thaw stability), split into three aliquots and then stored at − 20 and − 80 °C, respectively. A short-term (T S ) and a long-term (T L ) stability study were carried out to evaluate NT-proBNP stability in saliva samples over time. The T S study investigated analyte stability at 4 °C for up to 3 weeks (T 0 : collection day; T 1S : T 0 + 1 week; T 2S : T 0 + 2 weeks; T 3S : T 0 + 3 weeks), whereas the T L investigated analyte stability at − 20 and − 80 °C for up to 5 months (T 0 : collection day; T 1L : T 0 + 1 month; T 2L : T 0 + 2.5 months; T 3L : T 0 + 5 months). Aliquots of PSS collected and prepared at T 0 were spiked with 10, 50, and 100 pg/mL of analyte (namely CL 1 , CL 2 , and CL 3 samples) and immediately analyzed for use as reference values for both T S and T L studies. The effect of two thaw/freezing cycles was evaluated at T 1L . Figure 3 shows the experimental plan for the stability study. For the sake of simplicity, samples intended for investigating the freeze-thawing effect are not included. Whole saliva was collected at T A and T D between 8 a.m. and 10 a.m. Each subject was asked to refrain from oral hygiene, smoking, eating and drinking for at least 1 h prior to saliva collection. Each subject was also asked to drink water in order to rinse the mouth three times for at least one minute each time. After ten minutes, saliva was collected using a SalivaBio Oral Swab (SOS) (Salimetrics, cod: 5001.02 and 5001.05) according to the following procedure: (1) remove the SOS from package and place it in the subject's mouth, (2) ask the subject to roll the swab in the mouth for two minutes to collect saliva, avoiding chewing the swab, and (3) remove SOS from the mouth and place it into the container. Samples were kept at − 20 °C. Once the sample was available for analysis, the SOS container was thawed at room temperature and then subjected to centrifuge (7000 rpm, 4 °C, 5 min) to recover saliva. Saliva was then aliquoted (300 µL each) using a micropipette in 1.5 mL Eppendorf LoBind centrifuge tubes. ## Statistical analysis. The normally distributed variables were reported as mean ± standard deviation, whereas skewed variables were described by median with lower (25th percentile) and upper (75th percentile) quartiles. The difference between groups was assessed using a non-parametric test (signed-rank Wilcoxon test). A two-tailed p value of < 0.05 was considered statistically significant. The slopes of the calibration curves were compared with the statistical test described by Zar 50 at a confidence level of 95%. All data were analysed using GraphPad Prism v. 8.0 (GraphPad Software Inc., La Jolla, USA).

chemsum

{"title": "Determination and stability of N-terminal pro-brain natriuretic peptide in saliva samples for monitoring heart failure", "journal": "Scientific Reports - Nature"}

comparing_two_seized_drug_workflows_for_the_analysis_of_synthetic_cannabinoids,_cathinones,_and_opio

## Abstract: As the challenges faced by drug chemists continue to persist due to the presence of synthetic opioids, novel psychoactive substances, and other emerging drugs, laboratories are continuing to look for new analytical approaches or techniques to ease the burdens. These new solutions can range from simple changes in existing methods to better distinguish isomers to adoption and implementation of entirely new technologies for screening or confirmation. One barrier to making these transitions is lack of data to understand how, or even if, workflow changes will address the challenges. In this study, we attempt to compare, qualitatively and quantitatively, an existing analytical workflow for seized drug analysis to a new, experimental workflow to better understand the potential benefits and drawbacks. Using adjudicated and mock case samples containing synthetic cannabinoids, synthetic cathinones, and opioids, four forensic chemists were asked to analyze fifty samples using one of two workflows. The first was an existing workflow that employed color tests for screening alongside general purpose gas chromatography flame ionization detection (GC-FID) and general purpose gas chromatography mass spectrometry (GC-MS) analyses for confirmation. The second was an experimental workflow that combined direct analysis in real time mass spectrometry (DART-MS) for screening with class-specific (targeted) GC-MS methods for confirmation. At each step in the analysis scheme, chemists recorded the time required and as well as their interpretation of the results.Comparison of the workflows showed that screening by DART-MS required the same amount of time as color tests but yielded significantly more accurate, and specific, information. Confirmation using the general purpose GC-FID and GC-MS methods of the existing workflow required more than twice the amount of instrument time and data interpretation time while also presenting other analytical challenges that prevented compound confirmation in select samples. Use of targeted GC-MS methods simplified data interpretation, reduced consumption of reference materials, and addressed almost all the limitations of general purpose methods. While the experimental workflow is not yet validated for casework, this study shows how rethinking analytical workflows for seized drug analysis could greatly assist laboratories in reducing turnaround times, backlogs, and standards consumption. It also demonstrates the potential impact of being able to investigate workflow changes prior to implementation. ## Introduction Backlogs and analytical challenges continue to be major bottlenecks for forensic seized drug analysis. The increased prevalence of synthetic opioids, novel psychoactive substances (NPSs), and other emerging drugs, coupled with increased case submissions has led to a climb in turnaround times and backlogs in recent years . These novel compounds have also introduced a number of new analytical challengesso much so that over 80 % of laboratories reported limited analytical tools as one of their major challenges . Recent research efforts have focused on approaches to keep pace with the changing landscape, ensuring adequate standards are available, methodologies for differentiating isomeric or isobaric species, and tools for sensitive detection of small amounts of highly toxic compounds . To address these challenges laboratories may seek out new analytical capabilities that complement or replace their existing toolkit. New capabilities can include modifications to existing technologies, such as the adoption of new gas chromatography mass spectrometry (GC-MS) methods , or implementation of completely new technologies, such as DART-MS or Raman spectroscopy . When implementing new approaches or technologies, laboratories must estimate the improvements of changing their workflow. Improvements can be measured in overall analysis time (throughput), ease of analysis, or ability to obtain high-quality screening data (accuracy and reliability). The upfront and recurring costs of the change along with time required for procurement, method development, validation, and training, must also be considered. Oftentimes, the decision to change must be made without being able to tangibly measure the potential benefits or drawbacks of shifts in workflow, due to time and resource constraints. In some forensic disciplines, such as DNA analysis, the efficacy of different workflows has been studied, providing ability to make data-driven decisions . In this study, two different analytical workflows for seized drug analysis were compared to measure differences in time, data quality, safety, and simplicity. The workflows were compared using mock and adjudicated samples containing synthetic cannabinoids, synthetic cathinones, and opioids. The samples were given to four different practicing forensic chemists who were asked to analyze all samples using one of two workflows. The first workflow modeled existing practices at the Maryland State Police Forensic Sciences Division (MSP-FSD) and employed a combination of color tests, general purpose gas chromatography flame ionization detection (GC-FID), and general purpose gas chromatography mass spectrometry (GC-MS). The second workflow was developed to address many of the known limitations in the first workflow by leveraging direct analysis in real time mass spectrometry (DART-MS) for screening coupled with GC-MS methods developed for the targeted analysis of different drug classes. This study yielded tangible data to allow for direct comparison of the two workflows and better understand how changes to the existing laboratory protocols influence data quality, turnaround times, and requirements on the chemists. ## Study Design and Analytical Workflows For this study, the goal was to identify and quantify the differences in two analytical workflows for seized drug analysis, specifically targeting synthetic cannabinoids, synthetic cathinones, and opioids. To do this, 50 samples, (described in more detail in the next section) were created that span the range of complexities and compounds within the three drug classes that are commonly observed at MSP-FSD. A portion of each of the 50 samples was provided to four different chemists at MSP-FSD who were asked to analyze the samples using one of the two workflowsreferred to hereafter as the existing workflow and the experimental workflow. Each chemist analyzed half of the samples using the existing workflow and the remaining half using the experimental workflow. To simplify the process of recording times, samples were batched into groups of five and chemists analyzed one batch at a time. For each step in the workflow, chemists recorded the amount of time required to prepare, analyze, and interpret the data for the batch of samples. Chemists were also asked to provide their interpretation of the results after each analysis as well as an overall result of the controlled substance(s) present in each sample. Schematics of the existing and experimental workflows are provided in Figure 1. For the existing workflow, which reflects current procedures at MSP-FSD, a batch of samples was first screened using three color tests (Mayers, cobalt thiocyanate, Marquis ) to provide an indication of the type, or types, of compounds that may be present in the sample. Two separate methanolic extracts were then created for each sample, one for GC-FID analysis and the other for GC-MS analysis. Details regarding these methods are provided below. The resulting GC-FID data was used to compare retention times of compounds in the samples to known standards while the resulting GC-MS data was used to obtain mass spectra of compounds in a sample to compare to spectra of standards previously collected on the instrument. The methods used for GC-FID and GC-MS were general purpose methods designed to achieve reasonable detection of a wide range of controlled substances. In the experimental workflow, screening was completed using direct analysis in real time mass spectrometry (DART-MS) and was chosen because it produces more information-rich results than most other commonly deployed screening tools. It can often provide a near-complete chemical profile of a mixture and can identify the specific compounds, or group of isomeric compounds, in a sample. To leverage the higher fidelity screening information, confirmation was completed using a suite of targeted GC-MS methods. The methods were created to maximize retention time differences of similar compounds to reduce the number of pairs of compounds that could not be differentiated. Individual methods were created for synthetic cannabinoids, synthetic cathinones, and opioids. To investigate an approach to reduce consumption of reference materials, all methods were retention-time locked (where the carrier gas flow rate is adjusted to maintain consistent retention times of a lock column over the column's lifespan). This allowed for the analysis of only the lock compound with each batch, eliminating the need to run individual standards which were required for GC-FID analysis. For samples that contained compounds in multiple classes (i.e., dibutylone and fentanyl), analysis by multiple targeted methods was required. In addition, samples that were found by DART-MS to contain no controlled substances were concentrated, through the addition of more powder to the solution, and re-analyzed by DART-MS. If the concentrated sample also returned a negative result, the sample was reported as no controlled substances and no further analysis was completed. ## Case Samples For this study, a total of 50 samples were analyzed, the identities of which are provided in Table 1. Samples were created from either adjudicated case samples or standards purchased from Cayman Chemical (Ann Arbor, MI, USA) and Sigma-Aldrich (St. Louis, MO, USA). Samples were, largely, representative of commonly seen mixtures and ranged in complexity from simple, single compound samples to complex mixtures with drugs from multiple classes. Eight of the 50 samples contained no controlled substances. A total of 27 samples contained a single controlled substance, 10 contained two controlled substances (8 of which contained substances from multiple drug classes), and 5 contained three or more controlled substances. A total of 11 samples contained at least one synthetic cannabinoid, 19 samples contained at least one synthetic cathinone, and 22 samples contained at least one opioid. Once created, samples were divided into 2 mL GC-MS vials, each containing between 10 mg and 50 mg of powder. A set a vials was given to each chemist for analysis. Vials were labelled with only a number and the identity of the contents provided until the study was complete. Table 1. List of the 50 samples used in this study. Non-controlled substances in the samples are also listed, in italics. Sample numbers with a dagger ( † ) were created using one or more adjudicated case samples and sample numbers with an asterisk (*) were created using standards. Some samples were created using a mixture of both ( † *). Compound names with a double dagger ( ‡ ) are compounds that, when previously analyzed, were found to be insufficient concentrations to allow for confirmation. ## Color Tests Three color tests were completed (Mayers, cobalt thiocyanate, and Marquis) in disposable well plates. To complete a test, several drops of the appropriate reagent(s) were added to the well followed by a small amount (several milligrams) of sample powder after which the color change, if any, was observed. In addition to noting the color changes that occurred, chemists were also asked to provide an interpretation of each result, and record the time it took to complete the entire process for every batch of five samples. The Marquis reagent was created by combining 10 mL of 37 % formaldehyde with 100 mL of concentrated sulfuric acid. Cobalt thiocyanate reagent was created by dissolving 6.0 g of cobalt thiocyanate in 240 mL of water mixed with 360 mL of 0.1 M hydrochloric acid. The Mayer's reagent was created by dissolving 6.0 g of mercuric chloride in 600 mL of water followed by the addition of potassium iodide to dissolve the red precipitate. ## GC-FID GC-FID was employed to compare retention times of the controlled substances in the samples to reference materials. Analyses were completed on one of two Agilent GC systems (Agilent Technologies, Santa Clara, CA, USA) using methods that were validated for casework. Parameters for both methods are provided in Supplemental Table 1. Samples were prepared by dissolving 1 mg to 2 mg of material into approximately 1.5 mL of methanol. The solution was shaken by hand for several seconds then allowed to sit for several minutes so any undissolved particulates could settle. The supernatant was then transferred to another GC vial for analysis. All samples were analyzed with a single injection. Once compounds were preliminarily identified, reference materials (solutions containing known drugs) were analyzed using the same method to establish retention times for comparison. In addition to the suspected controlled substance, all isomers and similar compounds (compounds that have similar retention times) were also run. For each batch, reference materials were only run once, even if they were required for multiple samples. A list of reference materials run for each of the controlled substances in the study is provided as Supplemental Table 2. For a positive identification of a substance, the retention times of the sample and the reference material needed to be within ±1 % of one another and none of the other required reference materials, if applicable, had retention times within ±1 % of the sample. Overall identification of a substance required a positive identification from the GC-FID data and the GC-MS data, discussed in the next section. ## GC-MS (General Purpose) General purpose GC-MS was the second component of the confirmation process and was used to compare mass spectra from compounds in samples to those previously collected from reference materials. Analysis was completed on one of two Agilent GC-MS systems. There were three casework validated methods that chemists could use depending on which laboratory they were in as well as their preference and the suspected compounds in the sample. Method parameters for the three methods are provided in Supplemental Table 3. Sample preparation for GC-MS was identical to GC-FID. All samples were analyzed as a single injection. A cocaine positive control was run with each batch of samples for each method used. After analysis, all peaks in the chromatogram were searched against mass spectral libraries created in house, as well as the SWGDRUG library. Positive identification criteria included having an abundance of 200,000 counts or greater in the chromatogram along with an acceptable mass spectral match to a library entry. If any of these criteria were not met, or the GC-FID criteria were not met, an "insufficient" finding was made. ## DART-MS Sample screening using the experimental workflow was completed using DART-MS. The protocols used here have been discussed in detail elsewhere . Briefly, samples were prepared by dissolving approximately 1 mg of material into 1 mL of methanol containing tetracaine as an internal standard. Data was collected using a sequence-based approach with individual, 1 min data files collected for each sample. Within the 1 min datafile, the internal standard solution was analyzed once by itself followed by three analyses of the sample combined with the internal standard. All analyses were completed by dipping a clean glass microcapillary into the solution and placing it in the open-air sampling region. Measurements were made on one of two systems using identical methods. The systems consisted of DART-SVP ion sources (IonSense, Saugus, MA, USA) coupled to JEOL AccuTOF 4G-LCplus mass spectrometers (JEOL USA, Peabody, MA, USA). Helium was used as the DART gas source with a gas stream temperature of 400 ºC and operation in positive ionization mode. The mass spectrometer was also operated in positive ionization mode with an orifice 1 voltage of +30 V, a ring lens voltage of +5 V, an orifice 2 voltage of +5 V, and an ion guide voltage of +800 V. Spectra were collected from m/z 80 to m/z 800 at a rate of 0.4 s/scan. Upon completion of the sequence, the datafiles were automatically mass drift compensated using the m/z value for the protonated molecule of tetracaine (the internal standard). For each sample, an averaged mass spectrum of the three analyses was extracted, background subtracted, and saved as a centroided datafile. The centroided spectra were then analyzed using the "Search From List" feature within Mass Mountaineer (Diablo Analytical, Antioch, CA, USA) using an in-house created search list containing information for over 600 compounds of interest to seized drug analysis. Search parameters for peak identification included a minimum peak height threshold of 5 % relative abundance and a maximum m/z drift of ±0.005 Da (5 mDa) which was based on the mass tolerance of the instrument. For instances where multiple compounds produce the same m/z value, fragment ions were used to differentiate compounds, if possible. The tetracaine internal standard was used as a quality control compound, where the presence and correct m/z value of the protonated molecule was required for a datafile to be used. The time required to analyze every batch of five samples was also noted. ## GC-MS (Targeted Analysis) Confirmation was completed using a suite of targeted GC-MS methods. Preparation of samples was identical to that for the GC-FID and GC-MS methods described in the existing workflow above. The targeted methods were created using a previously published framework and were developed for each of the three compound classes investigated. Discussion on the development of the targeted methods is provided elsewhere , and the actual instrument methods are provided in Supplemental Table 4. All analyses were completed using an Agilent 7890/5977B GC-MS with helium as the carrier gas. The targeted methods were developed to maximize retention time differences between similar compounds within a reasonable runtime in order to minimize the number of compound pairs with overlapping retention time acceptance windows. The methods employed retention time locking to decrease consumption of reference materials. Using this approach, prior to running a batch of samples, the method was re-locked by analyzing the lock compound. A positive control was run with the batch of samples to confirm the locking was successful. If a sample contained compounds from multiple classes, repeat analyses were completed for all appropriate targeted methods. After analysis, the resulting data was interpreted by comparing both the retention time and the mass spectra for all peaks within a chromatogram. A retention time acceptance window of ±2 % for all methods and a ±1 % window for the retention time agreement of the lock compounds were used. A positive identification was defined as a chromatographic peak with a signal to noise ratio greater than 5:1 within the ±2 % acceptance window of the previously run reference material and with a minimum mass spectral match factor of 85 a.u. when compared to mass spectral libraries created in house or provided in the SWGDRUG Library (v 3.6). ## Comparison of Color Test to DART-MS for Compound Screening Analysis of the 50 samples by four examiners produced a total of 100 results per workflow to compare while also providing two independent analyses of each sample on each workflow. Comparison of the two screening techniques initially proved to be difficult because of the lack of comparable data. To address this challenge, a scoring system, outlined in Table 2, was created. Scores ranged from -1 to 4 and attempted to capture both the accuracy and specificity of the result, with more accurate and specific results receiving higher scores. For DART-MS, the result was the identified compound(s) that met the identification criteria. For color tests, the result was the chemists' interpretation of the color changes that occurred based on their expert knowledge and prior experience. If the result was inconsistent with the actual contents of the sample, a score of -1 was given. If the result was inconclusive (i.e. it could not be determined whether or not a controlled substance was present in the sample), a score of 0 was given. For results that were consistent with the contents of the sample, positive scores were given. A score of 1 was given to results that were accurate but the least specific, defined as those where only a class identification (i.e. the sample contains an opioid, synthetic cannabinoid, etc.) was possible for at least one of the controlled substances in the mixture. The next level of specificity was defined as the sub-class (i.e. fentanyl) or isomer group (i.e. AB-FUBINACA or one of its isomers). If the sub-class was identified for at least one controlled substance in a sample with multiple controlled substances, a score of 2 was given. A score of 3 was given if the sub-group was correctly identified for a sample containing a single controlled substance or for a sample where the sub-class or isomer group was correctly identified for all compounds in a sample containing multiple controlled substances. The most specific level of information was identification of the specific compound, which was given a score of 4. For samples containing multiple controlled substances, all controlled substances needed to be identified to obtain a score of 4. A score of 4 was also given when a sample that did not contain any controlled substances produced a result consistent with the absence of controlled substances. Correct identification of all compounds identified OR correct identification of a negative sample as negative for controlled substances This system was used to score all colorimetric and DART-MS results obtained by each of the four chemists. A complete list of scores is provided in the Supplemental Table 5 while the summary results are provided in For DART-MS, consistent results across chemists were obtained in all instances, except for Sample 42 where only one of the two chemists were able to detect low levels of FIBF and noscapine. There were no instances of a false positive or false negative identification. As expected, there were many instances where DART-MS produced only sub-class or isomer group information because of the fact isomeric compounds have identical base peaks and often have similar fragment ions. Given the lack of chromatographic separation, DART-MS is unable to differentiate these compounds from one another. When sub-class or isomer group information was obtained, it frequently consisted of a narrow of candidate compounds (five or fewer), though for the cathinones, the sub-class list (i.e. Cathinone at m/z 192) can encompass more than ten compounds. Given DART-MS is being used as a screening tool, this is not an issue as the chemist now has confidence in the type and class of compound(s) present in the sample. Chemists should be aware, however, that low-level compounds, especially those with low proton affinity, may be missed in a DART-MS analysis because of competitive ionization, as was the case in Samples 3 and 42, where heroin was not identified above 5 % relative intensity. DART-MS was able to correctly identify all eight of the samples that did not contain controlled substances as negative while color tests produced two false positives (discussed above) along with a single inconclusive result for one chemist (Sample 41). Confirmation of negative samples by DART-MS, completed by analyzing a concentrated sample, did not introduce any complications or produce any measurable signatures of carryover or contamination. The use of the internal standard eliminated the potential of false positive identification of noise peaks in spectra from samples that do not contain controlled substances or other easily desorbed and ionized species by providing a substantial base peak in all spectra. The lack of a base peak leading to false positive identification of noise peaks (because peak searching above a relative intensity threshold is often employed) is a common limitation in spectra that do not contain controlled substances. In addition to establishing the differences in accuracy and specificity produced by these two techniques, the time required for analysis was also measured. For both techniques, the time required for sample preparation, sample analysis, and data interpretation (for DART-MS), was noted by the chemists for each batch of five samples. For color tests, the average time per batch was 18.6 min while for DART-MS it was 20 min. This DART-MS analysis time was split up, roughly, as 5 min for sample preparation, 2 min for sequence preparation, 5 min for analysis of samples, and 8 min for data workup. In terms of sample consumption, color tests typically required more sample for analysis (approximately 5 mg versus 1 mg to 2 mg for DART-MS); though for most samples this difference would be negligible. From a potential exposure viewpoint, DART-MS presented a lower overall risk as handling of bulk powder is limited to only one transfer of material, unlike color tests which require multiple transfers of material. DART-MS only requires methanol to dissolve the sample, while color tests require the use of other, more hazardous, chemicals like formaldehyde and concentrated acids. While DART-MS provides a more information-rich, more accurate, possibly safer, analysis in roughly the same amount of time as color tests, it does require a large upfront investment in the technology which could present a barrier for adoption. However, color tests were found to be inconsistent and prone to differing results given the set of samples tested. The lack of class or compound specific results and the high frequency of inconclusive results obtained using color tests indicates that this approach would be ill-suited for inclusion in a workflow that utilized targeted or class-specific confirmation methods. The ability to obtain more granular and correct compound information from DART-MS is critical for use of targeted or classspecific confirmation methods. The benefits of DART-MS are not specific to the experimental workflow investigated here and can be realized when used alongside general purpose confirmation methods as well. ## Comparison of General GC-MS and GC-FID to Targeted GC-MS Because the technique used for confirmation in both workflows was identical, comparison of results was simplified. Overall, as expected, the results obtained from the existing workflow and the experimental workflow were largely similar. Because of differences in confirmation criteria between the two approaches, there were some differences regarding which compounds could be confirmed versus which compounds were identified but produced data that was insufficient for confirmation. Table 3 shows the summary of results obtained for the two workflows. Both workflows were found to have analytical limitations which presented as insufficient identifications. The existing workflow had ten samples with insufficient identifications while the experimental workflow had three samples. Insufficient identifications were caused by several factors including low chromatographic peak intensity, co-elution, and lack of inclusion on target compound panels. For the existing workflow, using general purpose GC-FID and GC-MS methods, there were several samples that had co-eluting peaksnamely acetyl fentanyl and FIBFwhich precluded the ability to confirm either when both were present in the sample. These two compounds were not sufficiently separated on the GC-FID method and did not provide sufficient separation to obtain clean mass spectra with the general purpose GC-MS methods. With the experimental workflow that used a targeted method developed specifically for opioid analysis detection and separation of these two compounds was readily achieved. An example of this is shown in Figure 3 for Sample 19. In addition to this, there was one sample (Sample 35) where co-elution of tramadol and mannitol precluded confirmation of tramadol for both workflows. Another limitation with the existing workflow was the inability to confirm dibutylone. When analyzing dibutylone on both GC-FID and GC-MS, there were other isomeric compounds that eluted well within the ±1 % retention time window of dibutylone and had mass spectra that were too similar to allow for differentiation. Using the targeted methods in the experimental workflow, however, provided sufficient separation to allow for confirmation of dibutylone. The general purpose GC-MS methods in the existing workflow use a minimum of 200,000 count peak abundance in the chromatogram for confirmation which lead to inability to confirm the identifies of compounds in seven samples (resulting in an insufficient identification). This limitation could be addressed by concentrating the sample, though care must be taken to ensure the major components in the sample do not saturate the detector. For the targeted method approach, there were two instances (Sample 2 and Sample 42) where controlled substances were present in the sample that were not part of the panels for any of the targeted methods and therefore could not be confirmed. While this resulting in incomplete confirmation of all substances in these two samples, it can be addressed by simply adding additional compounds to the panel(s). This process does require some time due to the need to complete replicate measurements of standards but is straightforward. This also highlights the potential need for a catch-all method that incorporates compounds outside of the classes that have targeted methods. Table 3. Summary results for the confirmatory analysis of the fifty samples using the existing and experimental workflows. Only controlled substances are listed. Compounds that were detected but could not be confirmed are listed as insufficient, and the reason for the insufficient designation is provided. A double dagger ( ǂ ) indicates that the compound was not at a high enough abundance in the GC-MS chromatogram for confirmation, a superscript RT ( RT ) indicates that there were multiple similar compounds with overlapping retention time windows which precluded confirmation, and compounds in parentheses indicate instances where co-elution precluded confirmation. A breakdown of these results is shown in Supplemental Table 6 and Supplemental Table 7. The biggest difference between the two confirmatory approaches occurred when comparing the time for analysis, summarized in Table 4. As expected, sample preparation for each of the instrumental techniques was almost identical, with GC-FID, general GC-MS, and targeted GC-MS all requiring approximately 10 min to prepare a batch of samples. However, because the existing workflow requires both GC-FID and GC-MS, the net time for sample preparation per batch is roughly twice as long. Instrument time was drastically different for the workflows, with the existing workflow requiring a total of 7728.8 min (128.8 hours) while the experimental workflow required only 2853.5 min (47.6 hours)inclusive of all samples, reference materials, and positive controls. Using the experimental workflow resulted in a 63 % reduction in time. A major driver for this difference is the large number of reference materials that are required for GC-FID analysis using the existing workflow due to lack of retention time locking, retention indices, or relative retention times. As shown in Table 4, the existing workflow required an average of 25.5 runs per batch, 19.0 of which, on average, came from GC-FID. GC-FID accounted for 68 % of the instrument runtime for the existing workflow. ## Sample If GC-FID were removed from the existing workflow, the time comparison between the two approaches becomes more similar. Comparing general purpose GC-MS runs to targeted GC-MS runs resulted in similar instrument runtimes per batch (116 min vs. 143 min, or 1.9 hours vs. 2.4 hours) and a similar number of runs (6.5 average vs. 7.4 average). These values are closer than were expected since samples containing multiple controlled substances needed to be analyzed on multiple targeted methods and because the opioid targeted method was significantly longer than the most commonly used general GC-MS method (35 min compared to 12.67 min). Part of what balanced the runtimes was that samples where no controlled substances were identified by DART-MS were not run on targeted GC-MS methods in the experimental workflow. It should be emphasized that using DART-MS as a stopping point for negative samples is something that would need to be thoroughly investigated prior to implementation in a real-world setting and may have too many limitations to be practical. In terms of data analysis, the general purpose GC-MS analysis and targeted method GC-MS analysis required a similar amount of analyst time, though the targeted method analysis was slightly faster. This is likely due to the use of a locked retention time lookup table where chemists entered the retention time of a peak in a sample and the possible compound(s) that fell within 2 % of that time were shown. Adding in the need to manually compare retention times to standards using GC-FID, the data interpretation component for the existing workflow was found to be almost twice as long as the experimental workflow. In terms of the amount of sample consumed and the risks to chemists, both confirmatory workflows were nearly identical. The existing workflow does require slightly more material since separate samples are created for GC-FID and GC-MS, but this difference is likely negligible for almost all cases. One potential challenge with the targeted method approach is that it requires different stationary phases (DB-200 and DB-5) which means laboratories would need at least two instruments to leverage such an approach. Alternatively, new methods would need to be developed. Table 4. Metrics for the GC-FID and GC-MS analyses for both workflows. A further breakdown of these results is shown in Supplemental Table 6 and Supplemental Table 7. ## Conclusions The results of this study demonstrate qualitative and quantitative gains that could be achieved by altering a seized drug workflow. Given the two workflows used here, it was found that screening of samples using color tests and DART-MS required approximately the same amount of time; however, the accuracy and specificity of the data obtained by DART-MS, on average, was superior. The use of DART-MS also eliminated false positives, which were observed with the color tests, and eliminated the need for toxic chemicals and acids. Though DART-MS was studied in combination with targeted GC-MS methods, the improved data quality and results it offers could benefit the existing confirmation workflow as well. While implementation of DART-MS has obvious advantages, the upfront and recurring costs as well as the time required to implement the technique should be considered. In terms of the confirmation processes studied, major improvements in analysis time were observed alongside some notable gains in analytical capabilities. Temporal benefits were largely driven by the use of a single confirmation tool (targeted GC-MS) in the experimental workflow instead of a dual-technique confirmation. The use of locked retention times provided further instrument time reductions due to the reduced analysis, and consumption, of reference materials. Ongoing work includes investigating the potential benefits of other approaches, such as relative retention times and retention indices, that could reduce the frequency of which reference materials are run. Interestingly, even with the need to analyze a sample on multiple targeted methods, instrument time of the experimental workflow was not substantially greater than the GC-MS analysis of the existing workflow. An obvious downside to the use of targeted methods is the need to have a panel of compounds, which for this study, was limited to only compounds within the particular drug classes. Adding more commonly coobserved compounds to the method is simple though it does require some time. The targeted methods also highlighted how class-specific methods designed for enhancing separation can address limitations presented by general purpose methods. This was observed for multiple compounds (acetyl fentanyl, FIBF, dibutylone, and α-PVP) in the sample set. The use of different chromatographic thresholds for confirmation can also lead to differences in the number of compounds that can be identified. While implementation of targeted methods may be appealing, they do require the use of an informationrich screening tool. Success of the targeted methods was largely due to the fact that DART-MS provided comprehensive and specific results to enable accurate identification of nearly all controlled substances in the samples. This approach would not have been successful had color tests been used as the screening tool. Another possible use for targeted GC-MS would be to supplement existing general purpose confirmation methods in cases where sufficient separation of compounds is not observed (such as acetyl fentanyl and FIBF). The use of targeted methods requires minimal additional cost and effort beyond the purchase of consumables and method validation; however, depending on the class of compounds of interest, systems with different stationary phases may be required, which could be problematic for laboratories with only one or a few instruments. Another interesting possibility, which was not examined here, is the use of dual-injection methods that would allow for analysis of a sample by GC-FID and GC-MS simultaneously, on two separate stationary phases. Combining two different retention times and mass spectral data may provide additional instances of compound discrimination over any of the abovementioned approaches. This study highlights some of the strengths and limitations of two specific analytical workflows. Though there are limitations in the experimental workflow, it does highlight some reasons why laboratories may want to consider changes to their protocols. An ideal workflow would certainly look different across laboratories and would be dependent on factors such as: caseload, personnel, types of cases frequently examined, jurisdictional requirements, and access to instrumentation. While it may not be practical to measure all gains and drawbacks prior to implementing changes to analytical protocols, the ability to test these changes, on a small scale, may prove consequential and may limit instances where new techniques are procured but never implemented into casework. Additional studies investigating different analytical workflows are still ongoing and are the focus of current research. ## Disclaimers Certain commercial products are identified in order to adequately specify the procedure; this does not imply endorsement or recommendation by NIST, nor does it imply that such products are necessarily the best available for the purpose. Certain commercial products are identified in order to adequately specify the procedure; this does not imply endorsement or recommendation by Maryland State Police, nor does it imply that such products are necessarily the best available for the purpose. A portion of this work was supported by Award No. 2018-DU-BX-0165, awarded by the National Institute of Justice, Office of Justice Programs, U.S. Department of Justice. The opinions, findings, and conclusions or recommendations expressed in this publication/program/exhibition are those of the author(s) and do not necessarily reflect those of the Department of Justice. Supplemental Table 2. Reference material sets required to be run for GC-FID verification. Only compounds that required multiple reference materials to be run are listed. The number of reference materials required is listed in parenthesis.

chemsum

{"title": "Comparing Two Seized Drug Workflows for the Analysis of Synthetic Cannabinoids, Cathinones, and Opioids", "journal": "ChemRxiv"}

mechanical_reshaping_of_inorganic_nanostructures_with_weak_nanoscale_forces

## Abstract: Inorganic nanomaterials are often depicted as rigid structures whose shape is permanent. However, forces that are ordinarily considered weak can exert sufficient stress at the nanoscale to drive mechanical deformation. Here, we leverage van der Waals (VdW) interactions to mechanically reshape inorganic nanostructures from planar to curvilinear. Modified plate deformation theory shows that high aspect ratio 2D particles can be plastically deformed via VdW forces. Informed by this finding, silver nanoplates were deformed over spherical iron oxide template particles, resulting in distinctive bend contour patterns in bright field (BF) transmission electron microscopy (TEM) images. High resolution (HR) TEM images of deformed areas reveal the presence of highly strained bonds in the material. Finally, we show the distance between two nearby template particles allows for the engineering of several distinct curvilinear morphologies. This work challenges the traditional view of nanoparticles as static objects and introduces methods for post-synthetic mechanical shape control. ## MAIN TEXT Nanoscience is predicated on the idea that properties are dictated by nanoscale structure in the form of particle size and shape. 1 In the case of inorganic systems, structure control is most often exerted either during the synthesis to generate a desired particle morphology, or post-synthesis to site-specifically remove/deposit material or assemble building blocks into superstructures. Absent among these strategies is the possibility to physically re-shape or re-form inorganic particles via mechanical forces rather than chemical manipulation. Previous reports have investigated the mechanical properties of nanomaterials through nanoindentation and other in situ methods. However, these approaches are low-throughput, single-particle techniques that are often focused more on measuring mechanical properties rather than exercising structural control. Flexibility has been observed in certain nanostructures, but this is often a consequence of random sample preparation processes. The mechanisms driving deformation, their size dependence, and the ability to create new morphologies via flexibility remain unclear. Here, we show that ubiquitous van der Waals (VdW) interactions, which are often considered weak compared to most nanoscale forces, can be leveraged to mechanically deform inorganic nanostructures as a means of post-synthetic shape control. To explore the feasibility of this method, a mathematical model was developed based on continuum mechanics theories for plate deformation. Using the conclusions from this analysis, we show the ability to control the shape of high aspect ratio silver nanoplates by deforming them over small iron oxide template nanospheres. The local deformation caused by a single template particle can furthermore be used as a structural motif to create several unique curvilinear structures. This work challenges the conventional notion that nanoparticles are rigid objects and introduces a new class of curvilinear nanostructures. To understand the forces that might result in nanoparticle deformation, we assumed that typically weak VdW interactions could be leveraged in a situation in which plate-shaped particles interacted with a surface, since this geometry maximizes the VdW contact area compared to other particle shapes. Additionally, we chose to work with noble metal nanoparticles as they are known to be particularly ductile and can readily be synthesized into 2D morphologies. 20, To probe the response to a mechanical stressor under these conditions, Kirchhoff-Love plate theory was employed, which models the elastic deformation and stress behavior of two-dimensional structures. In this formalism, an equilibrium state of mechanical stress is described by a fourthorder partial differential equation, to which there are few tractable solutions. One of the simpler analytical solutions assumes the axially symmetric deformation of a circular disc under a point load (Fig. 1a, inset). Modifying this solution to account for plastic deformation allowed us to compare the relative strength of VdW interactions and mechanical strain energy experienced by nanoplates (see Supporting Information). Given some reasonable assumptions, this analysis demonstrates that plates with a thickness of <10 nm and an aspect ratio of ~100 represent a transition point, below which VdW interactions are capable of causing a significant particle shape transformation (Fig. 1a, see Supporting Information). Importantly, these findings hold for several other common inorganic nanomaterial systems (e.g., SiO2, CdSe) and thus indicate the generalizability of VdW-driven mechanical reshaping (Fig. S12, S13). ## Figure 1. Deformation of thin, silver nanoplates over spherical template particles. (a) Thickness-dependence of the VdW and strain energies as calculated by modified Kirchhoff-Love plate theory. Inset shows the geometry of the strain energy calculation, where F is the concentrated force and a is the disc radius. Schematics of the (b) top-down and (c) cross sectional view for a nanoplate deformed over a spherical template particle. (d) Representative TEM image of a silver nanoplate deformed over one template nanoparticle, (e) a closer look at the unique bend contour that is observed and f-i) several examples of the bend contours that were seen in every instance of a deformed structure. Importantly, the mathematical solution describing plate deformation used above can be translated to and investigated in an experimental context. High aspect ratio silver nanoplates of ~8 nm in thickness were synthesized and deformed over spherical iron oxide template particles to mimic a concentrated, axially-symmetric load (Fig. 1a inset, 1b-c). 24,25 The resulting regions of mechanical strain are evidenced by six-lobed deformation patterns in Bright Field (BF) TEM data (Fig. 1d-e). The unusual pattern of contrast, known as a bend contour, was seen in every instance in which a nanoplate was conformally draped over a spherical template (Fig. 1f-i). Bend contours are a phenomenon that occur when local strain causes nearby crystallographic planes to change their orientation and diffraction condition, resulting in variations in contrast. 20, Although bend contours are well-known features of thin TEM samples, they most often extend over large distances and represent gradual changes in the orientation of the material's lattice. The highly symmetric and punctate nature of the bend contours observed in our samples is unusual and points to a localized stress gradient surrounding the spherical particle template. The diameter of these bend contours extended about an order of magnitude larger than the size of the template itself (103 + 11 nm, n=228), validating the assumption of a point load in the plate theory model (Fig. 1a). To confirm whether the 6-fold symmetry of the observed bend contours is related only to an electron diffraction effect and not a real-space morphological feature, we performed selected area electron diffraction (SAED) and dark-field (DF) TEM analysis. The SAED of a deformed nanoplate exhibits a set of six spots closest to the transmitted beam that represent the 1/3{422} forbidden reflection which is a known feature of structures with internal twinning (Fig. 2a-b). 29 The brighter set of six spots represents the first order diffraction from {220} planes that reveal distinct deformation lobe pairs when selected for in DF TEM imaging (Fig. 2c-h). Therefore, the bend contours that appear in BF TEM images are the convolution of symmetric zone axis (Fig. 2c-h). Atomic force microscopy (AFM) topographical maps show smooth, axiallysymmetric features around template-based deformed regions and are in agreement with bend contour sizes measured by TEM (Fig. 2i, S3). This confirms that the geometry of the proposed model based on plate theory is appropriate for understanding nanoscale shape control. To further validate these findings, experimental and theoretical results were compared against elastic finite element simulations (COMSOL Multiphysics) using a geometry identical to the Kirchhoff-Love model (Fig. 3). The theory and the simulations utilize some of the same input parameters (e.g., boundary conditions, mechanical constants), but employ different loading conditions and solution methods (see Supporting Information). Experimental height profiles of several deformed nanoplates gathered via AFM measurements (black dots, Fig. 3a), show excellent agreement with the displacement fields generated from both the analytical theory and the finite element simulations (Fig. 3a). While the Kirchhoff-Love theory utilizes a point load to achieve deformation, the simulations explicitly employ the experimental geometry of a small sphere deforming a nanoplate. The agreement between the experimental, simulation-based, and theoretical deflection fields, particularly surrounding the center of the deformed region (x = 0, Fig. 3a), suggests that the approximation of a point load in the theory is reasonable. Quantification of the contact radius between the template particle and nanoplate was determined from simulations to be ~5 nm, an order of magnitude less than the radius of the deformed area itself (see Supporting Information); this finding further supports the assumption of a point load. Importantly, if instead of considering only the axially-symmetric deformed region, simulations are performed with the entire triangular plate geometry, displacement fields are found to extend over considerably larger length scales (i.e., 200-300 nm) and no longer agree with experiment (green line, Fig. 3a, S18-S21). Since sedimentary forces are negligibly small for particles of nanometer dimensions, this result suggests that only attractive VdW forces can explain the local deformation observed in these structures. The simulation results also allow us to generate three-dimensional plots of the internal stresses experienced for plates with a given displacement (Fig. 3b). This information can be used to further understand the structural consequences of VdW-driven nanoparticle shape control by mapping regions for which the yield condition has been surpassed and permanent (plastic) deformation has occurred. Although the bulk yield stress of Ag (54 MPa) is surpassed across the entire volume of the plate, it is well known that metal nanostructures often have higher Young's modulus and yield stress values than their bulk counterparts. Quantitative measurements of nanoparticle mechanical constants vary considerably and likely depend sensitively on crystal orientation, dimension, and internal defect structure. Nonetheless, we performed multiple simulations using both bulk and elevated Young's modulus values, appropriate for silver. In all cases, the resulting stresses well exceed even the elevated yield stress values (~1 GPa) expected for nanometer-scale silver particles (Fig. 3b, see Supporting Information). This numerical result suggests that plastic deformation is widespread across the volume of the silver nanoplates displaced by template spheres. This conclusion is corroborated by the fact that if plastic deformation is ignored in the Kirchhoff-Love theory, the energy required to achieve the observed displacement far exceeds what is available to the system in the form of VdW or other attractive interactions. Only by modifying the theory to account for the possibility of plastic deformation do we observe agreement between theory and experiment (see Supporting Information). The curvilinear morphology created by the deformation of nanoplates tilts crystallographic planes and strains the crystal lattice, causing atomic-level distortions. To further understand the role of bond strain and plastic deformation in silver nanoplates, we performed high resolution TEM imaging of regions surrounding the spherical template nanoparticles (Fig. 4). Fast-Fourier Transform (FFT) of these images showed a set of six spots that appear more diffuse in the radial direction compared to analogous spots in the experimental SAED pattern (Fig. 4b). This reflects the presence of distortion in the atomic Ag lattice, spanning a range of different values. To quantify this, three different rings (red, yellow, cyan) were placed at different radial positions over the diffuse spots in the FFT to denote different degrees of lattice distortion, expressed as a percentage deviation from the unstrained lattice spacing (Fig. 4b and inset). This measurement reveals a deviation of 0-5% or more over the vast majority of the deformed region being imaged. This indicates a degree of lattice distortion significantly above what is ordinarily considered the limit of elastic bond strain, further confirming the necessity of including plastic deformation in the model for nanoplate mechanics. Additionally, atomically resolved images show lattice planes and atom positions that are severely distorted with respect to a perfect crystal, indicating numerous broken bonds, defects, and plastic mechanical behavior (Fig. 4c). It is important to note that there exist regions of elastically-strained metal bonds throughout the structure that are consistent with what has been observed to transform a normally inactive noble metal surface to one that can catalyze chemical reactions. This suggests that such curvilinear nanostructures might have a high density of active sites for heterogeneous catalysis. In traditional nanoscale systems, there are canonical structures from which more complex architectures can be built (e.g., spheres assembled into a superlattice or rods lithographically fabricated into metamaterial arrays). 7,42 Similarly, we imagined the morphology associated with a single template particle might serve as a basic structural motif for building more complex curvilinear structures. In order to achieve this, we have investigated the topographies that result when two template particles are near one another and deformed regions overlap (Fig. 5). If the parameter d is defined as the spacing between nearby template spheres, d = 16-31 nm generates a single bend contour that appears slightly larger than one associated with a single template particle (Fig. 5a). Two templates that are separate but closely spaced (d = 37-65 nm) show a distorted sixlobed pattern with a region bridging the two particles (Fig. 5b). Interestingly, when d increases to ~70-93 nm, a saddle point is observed, consisting of areas of high lattice compression between particles and lattice tension over the template peaks (Fig. 5c). Lastly, templates that are greater than ~94 nm apart display bend contours that are completely decoupled from one another (Fig. 5d). This method relies on the mutual mechanical relaxation of neighboring deformation fields and opens up the possibility for complex curvilinear architectures based on substrate topography rather than lithographic patterning. A colormap was applied to BF TEM images to enhance differences in contrast for the purpose of analysis (Fig. S5). In this work we report a simple method for post-synthetic nanoparticle shape modification via mechanical deformation rather than chemical manipulation. Calculations using Kirchhoff-Love plate theory modified to account for plastic deformation create a framework from which to understand the interplay of forces that facilitate this new type of morphological control. Using this in conjunction with simulations and experimental findings, we demonstrate that weak VdW forces can indeed generate enough energy to drive mechanical strain and thereby create a new class of curvilinear structures based on substrate topography. Since these objects would be difficult to generate lithographically, they are expected to result in previously inaccessible electromagnetic modes relevant to the nanooptics community. 43,44 Furthermore, the gradient of strained bonds in these materials has implications for their performance or study in catalytic systems. 45 Overall, this work demonstrates that inorganic nanoparticles may be thought of as being capable of dynamic structural changes, actuated by simple and ubiquitous nanoscale forces. ## ASSOCIATED CONTENT Supporting information is available free of charge at: Experimental details regarding materials and methods, mathematical derivation and calculations, and characterization data. ## Corresponding Author *Corresponding author. Email:[email protected]

chemsum

{"title": "Mechanical Reshaping of Inorganic Nanostructures with Weak Nanoscale Forces", "journal": "ChemRxiv"}

switchable_supracolloidal_3d_dna_origami_nanotubes_mediated_through_fuel/antifuel_reactions

## Abstract: 3D DNA origami provide access to the de novo design of monodisperse and functional bio(organic) nanoparticles, and complement structural protein engineering and inorganic and organic nanoparticle synthesis approaches for the design of self-assembling colloidal systems. We show small 3D DNA origami nanoparticles, which polymerize and depolymerize reversibly to nanotubes of micrometer lengths by applying fuel/antifuel switches. 3D DNA nanocylinders are engineered as basic building block with different numbers of overhang strands at the open sides to allow for their assembly via fuel strands that bridge both overhangs, resulting in the supracolloidal polymerization. The influence of the multivalent interaction patterns and the length of the bridging fuel strand on efficient polymerization and nanotube length distribution is investigated. The polymerized multivalent nanotubes disassemble through toehold-2 mediated rehybridization by adding equimolar amounts of antifuel strands. Finally, Förster Resonance Energy Transfer yields in situ insights into the kinetics and reversibility of the nanotube polymerization and depolymerization. ## Introduction Nature and man-made technologies display abundant examples for hierarchically self-assembled structures and functional materials, which react to external stimuli and reconfigure on demand. 1,2 Particularly intriguing is for instance the cytoskeleton, which employs multiple assemblies for transport, cell movement and cell reorganization: actin filaments, intermediate filaments and microtubules. 3,4 In nature, many of the sophisticated structures -in particular filaments and nanotubes -are built up by monodisperse proteins that organize in a highly specific manner. Using protein engineering concepts, there has been relevant progress to obtain protein-based nanotubes or filaments ex vivo. Even though structural engineering of such proteins has advanced considerably, it remains a challenge to de novo design protein nanoparticle building blocks capable of fibrillar assembly and with elaborate control of switching interactions. In contrast, tailor-made, synthetic building blocks with defined dimensions and controlled interaction patterns could provide an alternative. This way, rational design strategies can be applied for de novo design of self-assembling nanoparticle systems and new types of switching mechanisms can be implemented. One particular challenge is to go beyond simple fibrillar assemblies, that are for instance attainable by block copolymer systems 17 or gold nanorods 18 , and find pathways for the rational design of filamentous nanotube structures that can contain a spatially segregated compartment in the interior. DNA is a promising material to design building blocks for hierarchical assembly due to its precise programmability. For instance, nanotubes can be self-assembled with DNA tiles composed of a few strands. Franco and coworkers demonstrated reversible growth by using pH 23 and toehold-mediated strand displacement, 24 , using a sequence overhang to allow strand displacement by an invading DNA strand, 25 a technique which becomes of growing importance in DNA strand displacement cascades and DNA computing. While such strategies deliver nanotubes, they do not proceed via an intermediate defined nanoparticle/colloid level -similar to engineered proteins -which could provide advantages for e.g. functionalization with enzymes or inorganic nanoparticles. A potential strategy towards precision design of supracolloidal self-assembling systems is the use of 3D DNA origami, where a long DNA scaffold is folded into a pre-designed and distinct nanoparticle shape by staple strands. 30 DNA origami have been successfully employed as building blocks for hierarchical self-assembly, yielding finite-size superstructures 31,32 as well as periodic assemblies like 2D lattices or fibrils. 15, Hollow 3D DNA origami were used to harbor enzymes for cascade reactions and enhanced catalytic activity could be shown by dimerization of them. 36 We previously demonstrated supracolloidal fibrils of solid divalent 3D DNA origami cuboids and showed how classical dsDNA hybridization as well as non-DNA host/guest interactions can be used for their organization, and how multivalency and cooperativity effects can be unraveled using such monodisperse building blocks. 34,37 Saccà and coworkers showed 3D DNA origami fibrils formed by base stacking and reconfigured their stiffness using DNA hybridization. 38 However, reports on DNA origami-based nanotubes are still scarce, and triggers giving additional external control for reversible growth have not been employed for DNA-origami based fibrils so far. 43 Growing interest in DNA strand displacement has shown the extreme potential of using DNA itself as trigger. Toehold-mediated strand displacement uses the thermodynamic gain offered by the DNA toehold to Isolated nanocylinders and their polymerized nanotubes would be of general interest for controlled drug delivery, templated material growth, as membrane channels or even as artificial filaments for biomaterials. 19,22,39,44 Here, we present a first approach towards switchable supracolloidal nanotube assemblies based on distinct 3D DNA nanocylinders (3D-DNA-NC), that are monodisperse in their size and cavity. We report how external fuel strands bridging the ssDNA overhangs emanating from the two faces of these 3D-DNA-NC guide the supracolloidal self-assembly as a function of overhang connector density, salt content and hybridization length. Building on this, we implement fuel/antifuel switching mechanisms using a strand displacement reaction. Moreover, we use Förster Resonance Energy Transfer (FRET) of appropriately functionalized interaction patterns to in situ read out details of the assembly/disassembly as a function of changes in the interaction strength. ## Design of 3D DNA Origami Building Blocks Our building block for nanotube formation consists of a 3D DNA origami hollow nanocylinder, that is abbreviated as 3D-DNA-NC. The DNA strands exiting at both open sides are in general passivated with ssDNA overhangs of 15 thymine nucleobases (nb) to prevent unspecific interactions. However, up to 24 ssDNA strands with a specific sequence protrude from each end, termed "connector overhangs", shown in blue in Figure 1. These connectors are not complimentary, but need an additional bridging strand (fuel strand A*= A1*A2*, shown in black) to hybridize both connector ends A1 and A2 (Figure 1b). This allows to trigger the supracolloidal polymerization and nanotube formation. The bridging strand can be modified with a toehold sequence B (Figure 1c). This introduces the possibility to add an external antifuel trigger strand, B*A, to remove the bridging strand, BA*, via toehold-mediated strand displacement, allowing for a depolymerization of the nanotubes to single colloidal 3D-DNA-NC. Moreover, up to six connector strands per side can be end-modified with fluorophores Alexa Fluor 568 (AF568) and Alexa Fluor 647 (AF647) to enable FRET measurements (Figure 1d). The sequences of all DNA strands used are in Figure S1, Table S1 to S10 and the positions of all connector strands are shown in Figure S2. The 3D-DNA-NC was folded from a M13mp18 scaffold in the presence of a 5x excess of staple strands using a temperature ramp. Transmission electron microscopy (TEM) confirms the correct folding into the 3D-DNA-NC with a length of 30 nm, a diameter of 25 nm and an inner cavity diameter of 15 nm (wall thickness = 5 nm; Figure 1e). Purification by spin filtration was used to remove all excess staple strands. Lanes 1 to 3 of the agarose gel electrophoresis (AGE, Figure 1f) indeed only display one sharp fluorescent band for the 3D-DNA-NC with different connector strand density and modified with AF568 and AF647. Comparison with reference lane 4 confirms that no free fluorophore-labeled connector strands remain. Some 3D-DNA-NC dimers are visible in the AGE. We however attribute their observation to non-specific interactions in the AGE, as TEM displays well separated objects due to the T15 passivation. ## Supracolloidal Nanotube Polymerization The driving force for the self-assembly into supracolloidal nanotubes should in principal depend on the length of the fuel strand (overlap) and on the number of connectors (i.e. the multivalency) at the patches. Additionally, different procedures for assembly can be considered. Indeed, we first investigated two different procedures for the growth into the nanotube structures: (1) in situ fibrillation and (2) post-folding fibrillation. For both we use a fixed connector density of 16 with a bridging fuel strand of 22 nb, hence with a hybridization overlap of 11 nb to both sides (Tm of 38 °C). Firstly, for the in situ fibrillation (Figure 2a), the bridging fuel strand is directly added to the origami folding mixture with a 1:1 equivalence to the connector strands, which again are in 5x excess to the scaffold. Even though this means fuel strands are in excess with respect to the fully formed origami (20 nM), TEM images (Figure 2b) prepared at room temperature show that nanotubes of up to 1 µm (~30 origami) and a number average degree of polymerization of = 5.7 form. This is due to the multivalent design, where cooperative binding and entropic effects ensure that nanotube polymerization is favored over passivation of origami ends by dangling non-bridging fuel strands. 37 After removal of excess staple and fuel strands by spin filtration and further incubation at 37 °C for 2 days the nanotubes grow to over 2 µm (~60 origami) and = 8.7. Further incubation allows for re-shuffling of fuel strands due to the proximity of the incubation temperature to the Tm of 38 °C. This triggers some dynamic self-correction mechanisms and further nanotube growth as the fuel strand is able to dehybridize and rehybridize easily at this temperature, leading to a thermodynamically preferred polymerization. Secondly, in post-folding fibrillation, the bridging fuel strands are added after the 3D-DNA-NC are folded and purified (Figure 2c). After incubation at 20 nM and 37 °C for 2 days to ensure completed polymerization, the nanotube lengths are generally a bit shorter and reach up to 700 nm (~20 origami) with = 4.9. Figure 2e compares the statistical TEM image analysis of the nanotube length distribution for both procedures. In situ fibrillation leads to longer nanotubes, most likely due to the higher dynamics in the system during the annealing procedure as nanotube assembly and building block formation takes place simultaneously. ## Variation of the Connector Strand Density and Mg 2+ Concentration Next, we analyze the influence of changing the connector density and the salt concentration on the length distribution of the growing nanotubes for the in situ fibrillation method. All evaluations were done after purification to remove excess strands and incubation for 2 days at 37 °C. Interestingly, when reducing the connector density from 16 (above Figure 2) to only 8, nanotubes hardly form and mostly dimers are visible (5 mM Mg 2+ ; Figure 3a, c). An increase to 24 connectors leads to a slight increase in nanotube length with = 8.8 (Figure 3e), compared to the previously used 16 connectors. Hence, increasing the multivalency by increasing the connector density intensifies the binding strength between the origami. The cooperativity arises when after the first binding event the other connectors are brought into vicinity, favoring further hybridization with bridging fuel strands. An increase of the Mg 2+ concentration after purification supports nanotube growth by shielding the negative charge of the 3D-DNA-NC (Figure 3b, d). By elevating the Mg 2+ concentration from 5 mM to 20 mM the increases considerably from 8.7 to 13.3 at 16 connectors (Figure 3f). A particular change in the distribution occurs, as monomers, dimers and short oligomers are less abundant. Since the electrostatic repulsion of the 3D-DNA-NCs is reduced at higher ionic strength, the overall binding affinity between the building blocks increases. A similar trend is consistently observed for all connector strand densities, yet for the 8 connectors, the increase in nanotube growth levels off at ca. 10 mM Mg 2+ . This may relate to the general challenge in imaging such fibrils, which is that rupture of nanotubes during droplet deposition cannot be fully excluded and because largest fibrils tend to aggregate (and hence cannot be considered in the statistical evaluation). Therefore, some even longer DNA nanotubes cannot be incorporated into the statistics. Shear-induced rupture during sample preparation is more likely for this low connector density (8) with a mechanically weaker connection and may contribute to an apparent restriction of the nanotube length. TEM images and statistical distributions of the nanotube length for 8 and 24 connectors at higher Mg 2+ concentrations are shown in Figure S3. ## Influence of Hybridization Length of the Bridging Fuel Strand Moreover, the choice of the fuel length is decisive for nanotube growth with respect to the system temperature. We tested this effect for overlap lengths (hybridization lengths) of 5, 8, 11 and 13 nb on each side of the bridging fuel strands, which leads to Tms of ~ 0, 22, 38 and 50 °C as measured by UV-Vis. When performing the in situ fibrillation at a connector density of 16, the shortest bridging strand with 10 nb (5 nb on each side) does not lead to any assembly during incubation at 37 °C (Figure 4a, b). This demonstrates that the multivalency effects are not pronounced enough and lead to a too low binding affinity. This behavior is different to DNA mediated colloid assembly, where large colloids can be efficiently linked using overhangs as short as 4 nb due to strong multivalency effects at comparably flat and large surfaces in such larger systems. Short nanotubes form for a hybridization length of 8 nb at each overhang (total fuel strand length 16 nb) with a Tm of 22 °C. The longest nanotubes with a of 8.7 are observed when the Tm matches the incubation temperature of 37 °C, which is the case for 11 nb per side with a Tm of 38 °C. Interestingly, a further increase of the hybridization length to 13 nb with a Tm of 50 °C leads to a significant decrease in nanotube length with dropping down to 4.2 (Figure 4c). This behavior can be explained by the loss of dynamics for the fuel strand exchange at a Tm substantially higher than the incubation temperature, where rehybridization of fuel strands and corrections of oversaturated origami faces are less efficient. Additionally, TEM indicates a higher amount of ill-formed 3D-DNA-NC for such samples, which is likely due to the fact that the early hybridization with the bridging fuel strands during the folding impedes proper 3D-DNA-NC folding. Hence, we conclude that the inter-origami recognition forces need to be strong enough to ensure stable nanotubes at room temperature, but weak enough to allow reversible binding for self-correction mechanisms. 1 Due to the multivalent design using 16 connectors, small changes in the length of the bridging fuel strand drastically change the interaction strength on the colloidal level (multivalency) and, therefore, the overall behavior of the system as a whole. 51 ## Reversible Switching of Nanotubes Building on this understanding, we will next turn to realizing a switching of the 3D-DNA-NC nanotubes by adding a 15 nb toehold to the original fuel strand containing 11 nb on both sides (Figure 5a). We now use the post-folding fibrillation method and start from individual 3D-DNA-NC (20 nM) and first polymerize them at 37 °C by addition of an equimolar quantity of fuel strand (320 nM) for the 16 connector strands at the 3D-DNA-NC (2 days). Afterwards, the addition of an antifuel strand leads to toeholdmediated strand displacement that we hypothesized to be strong enough for DNA nanotube breakage. Indeed, once the antifuel strand is added (1 eq, 1 h incubation at 37 °C), TEM clearly depicts breakage into individual units. This whole process is highly reversible and further addition of fuel repolymerizes the nanotubes, as confirmed by TEM images shown for three consecutive switches in Figure 5b. Although waste in the form of stable duplexes accumulates with each switch, the statistical distribution of nanotubes shows a very similar length distribution for each cycle. Hence, the switch is highly reversible. This strategy underscores that toehold-mediated strand displacement reactions can be applied in highly multivalent 3D DNA fibrillating systems with strong confinement of the interacting strands at the two sides. In the previous approaches trying to break multivalent 3D DNA origami superstructures a heavy excess of binding partners (at least 10 eq) was needed to break such fibrillar assemblies, because the multivalent binding gain needed to be overcome. 34,37 Here the multivalency can be overcome by the gain in free energy provided by the toehold-mediated hybridization of the full fuel/antifuel strand pair involving the additional toehold area. 25 The susceptibility of the switch to operate with equimolar quantities limits waste accumulation, which may provide higher levels of robustness for reversible switching and reduces crosstalk in more complex systems. Our switching of the nanotubes therefore extends the toeholdmediated fuel/antifuel switching mechanism from previous switching of DNA tile assembly 24,29 and 2D DNA origami 52 to 3D DNA origami filamentous superstructures. ## In Situ Analysis of Reversible Nanotube Assembly by FRET Next, we turn to the particular challenge of developing a strategy to measure this switching mechanism with an ensemble average in situ technique, which to the best of our knowledge has not been realized for such periodic 3D DNA origami assemblies. To this end, we modified six of the connector strands on the respective sides of the 3D-DNA-NC with fluorophores amenable to FRET (Figure 6a). One side bears AF568, while the other side is modified with AF647. Upon nanotube assembly these fluorophores are brought into close vicinity, allowing a FRET from the donor dye AF568 to the acceptor dye AF647. The whole fluorescence spectrum is shown in Figure S4. We first focus on a 3D-DNA-NC with 16 connector strands (Figure 6b). Indeed, the FRET ratio as measured by the ratio of the two emission maxima (IAF647/IAF568 = I670nm/I590nm) increases as soon as 1 eq fuel is added to the fluorophore-modified 3D-DNA-NCs (Figure 6b). The FRET ratio reaches a plateau after ca. 2 h, which is indicative of the majority of the assembly being completed in this time frame. A negative control without any fuel does not show any FRET, as expected. FRET measurements allow to quickly access the in situ behavior at different hybridization lengths of the bridging fuel strand. For post-folding fibrillation at 37 °C, the FRET ratio is highest upon addition of 1 eq fuel with a hybridization length of 13 nb, where the bond is stable at this elevated temperature (Tm = 50 °C, Figure 6b). On the contrary, for 11 nb the bridging fuel is partly hybridized (Tm = 38 °C), giving a reduced FRET. The bridging fuel strand with only 8 nb is almost fully dehybridized at this temperature (Tm = 22 °C), and, consequently, shows no FRET increase at all. This FRET behavior is confirmed by statistical TEM image analysis (Figure 6c), in which an increase of nanotube lengths is achieved by using longer hybridization lengths of the fuel strand. This behavior is slightly different to the in situ assembly method (Figure 4), where the longest fuel strand with a hybridization length of 13 nb yields considerably shorter nanotubes compared to 11 nb due to some crosstalk in the folding process. Here we use pre-folded 3D-DNA-NC and post-folding assembly, which prevents this unwanted crosstalk. Interestingly, despite the large size of the origami units, polymerization proceeds quite fast with the maximum FRET ratio reached in only 0.5 h. The slope of the time-resolved FRET ratio curves correlates with the polymerization rate. By evaluating the initial slope of the FRET increase, it can be concluded that polymerization rate qualitatively increases with the hybridization length and connector density (Figure S5) as the inter-origami binding forces are strengthened. Most importantly, FRET enables to measure the reversible (dis)assembly of nanotubes in situ. We investigated this behavior for the 22 nb fuel strand (11 nb per overhang) with a toehold in conjunction with 3D-DNA-NC bearing different connectors (8, 16 and 24; Figure 6d). Since the post-folding fibrillation at 37 °C does not lead to strong assembly for 8 connectors due to reduced inter-origami interaction (Figure 3), we investigated the reversible switching of nanotubes at 25 °C (for switching at 37 °C, see Figure S6). At this temperature, an increase in FRET ratio can be observed for all connector densities, and a scaling of the FRET ratio with the connector density occurs. This in turn correlates very well with the previously observed increase in nanotube lengths for higher connector densities in the in situ fibrillation (Figure 3). After 2 h of incubation, 1 eq of antifuel was added and the FRET ratio decreases as the nanotubes disassemble. Using this concept, Figure 6d displays a successful and repeated switching for three times. Some shifts in the respective FRET ratios after multiple switching appear, but we suggest that this may be caused by scattering effects as the volume increases with each fuel/antifuel addition and some photobleaching effects. Nonetheless, these shifts are minor thanks to a stable switching of the system and low standard deviations confirm its reproducibility. ## Conclusion In summary, we demonstrated the regulation of the supracolloidal polymerization of 3D DNA origamibased nanotubes using fuel/antifuel strand principles and exploiting toehold-mediated strand displacement reactions. We first explored in detail how multivalency and fuel length overlap provide sufficient driving force for the polymerization and how increased salinity and the correct assembly protocols can assist the formation of long nanotubes. We found that the energetic gain by addition of a short toehold to the bridging fuel strand provides sufficient thermodynamic driving force for disassembly using equimolar amounts of antifuel even for multivalent assembled systems. Additionally, we introduced in situ FRET measurements to monitor supracolloidal self-assembly of 3D DNA origami structures and could give first insights into the kinetics and dynamic behavior of the nanotube polymerization. We believe this work is a promising approach to merge the fields of colloidal self-assembly and DNA nanotechnology while advancing the implementation of biological self-assembly principles into the supracolloidal world. Our approach is versatile and the antifuel approach could be extended to other switches, such as the introduction of a DNA catalytic circuit 29 , an enzymatic reaction network 53 or application of sensors using for example pH 23 . We believe that the FRET approach will allow to study the self-assembly trajectories and energy landscapes of such systems in greater detail in future, as it grants higher temporal resolution and presents a non-invasive form of monitoring the system compared to classical ex situ imaging techniques. ## Experimental Materials. M13mp18 scaffold and folding buffer was purchased from tilibit, DNA strands were purchased from IDT and IBA Lifesciences. Agarose gel was received from AppliChem. MgCl2 (1 M) and NaCl (5 M) were ordered from Fisher Scientific. Boric acid, hexadecane, magnesium acetate and TRIZMA were obtained from Sigma Aldrich. EDTA was purchased from Carl Roth. Carbon film 300 mesh copper grids and uranyl acetate (>98%) were bought at EMS. Devices. TEM images were taken with a FEI L120 operating at 120 kV. Agarose gel electrophoresis was executed in a water-cooled CBS Scientific HSU-020 gel electrophoresis chamber using an Enduro 300 V power source. Gel imaging was done with an INTAS ECL Chemostar. DNA origami were folded in a Biometra TPersonal Thermocycler. Nanotubes were incubated in an Eppendorf ThermoMixer C. UV-Vis measurements were conducted on an AnalytikJena ScanDrop 250 using a Tray cell cuvette from Hellma with a path length of 1 mm or a Hellma microcuvette with a path length of 3 mm. Fluorescence spectroscopy was done with the Tecan Spark plate reader in top mode. Folding of 3D-DNA-NC. The 3D-DNA-NC were designed with the program cadnano 54 and their design confirmed with the software cando. 55,56 Three master mixes of staple strands were prepared. Master mix Purification of 3D-DNA-NC. The folded 3D-DNA-NC mixtures were purified by spin filtration with Amicon 100 kDa spin filters at 10,000 g and 15 °C for 5 min. The samples were washed 6x with FoB5 buffer (5 mM TRIS, 1mM EDTA, 5 mM NaCl, 5 mM MgCl2, pH 7.2) 30 and recovered by turning them upside down into a fresh tube and centrifuging at 5000 g for 3 min. Polymerization of 3D-DNA-NC. In situ fibrillation: Fuel strands were added to the origami folding mixture (800 nM for 8 connectors, 1600 nM for 16 connectors, 2400 nM for 24 connectors) and the origami were folded and purified as described above. After purification, the Mg 2+ concentration was increased if needed and samples were incubated for another 2 days at 37 °C at 300 rpm in a thermoshaker. Post-folding fibrillation: 3D-DNA-NCs were purified by spin filtration and the Mg 2+ concentration was adjusted. The concentration was evaluated by UV-Vis at 260 nm and the fuel strand was added in a 1:1 ratio, in respect to the ssDNA connector strands. The samples were incubated for 2 days at 37 °C and 300 rpm in a thermoshaker unless otherwise stated. Reversible switching of 3D-DNA-NC nanotubes. 3D-DNA-NCs were purified and diluted to 20 nM. 1 eq of 22 nb fuel with toehold was added and the mixture was incubated at 37 °C, 300 rpm for 2 days. For depolymerization, 1 eq of antifuel was added and the sample was incubated for 1h. Next, 2 eq of fuel was added to assure sufficient quantity and the sample was again incubated for 2 days. For the next depolymerization, 2 eq of antifuel was added, followed by incubation for 1 h. For repolymerization, 3 eq of fuel was added, followed by incubation for 2 days. For imaging, nanotubes containing waste of fuel/antifuel from previous switches were washed twice with 200 µL Tris-buffer to improve imaging. TEM sample preparation. 3 µL of sample were incubated for 60 s on plasma-cleaned copper grid, then blotted away using filter paper. 3 µL of milliQ water was dropped on the grid and blotted away immediately afterwards. For negative staining, 3 µL of 1 wt% uranyl acetate solution was incubated on the grid for 20 s before being blotted away. Agarose gel electrophoresis. Gels were prepared with 1.5 wt% agarose in TBE buffer (22.25 mM Tris base, 22.25 mM boric acid, 0.5 mM EDTA, 6 mM magnesium acetate) and cast without stain. Gels were run at 3 V/cm in a cooled chamber set to 15 °C for 2.5 h. A fluorescent DNA ladder was used and the gels were imaged without staining using the fluorescence of the fluorescent connector strands. FRET measurements. For fluorescence intensity measurements, a black 384 well plate from Costar Corning was used. Each well contained 20 µL solution and 10 nM origami. Evaporation was reduced by adding 4 µL of hexadecane on top. The excitation wavelength was set to 495 nm using an excitation filter. Emission wavelengths were measured using filters at 590 nm and 670 nm. Well plates were pre-incubated for 30 min in the plate reader before the fuel was added. If run at 37 °C, the plate was kept on a thermoshaker at 37 °C during pipetting to prevent cool-down. Each measurement was done at least in duplicate and the average and standard error calculated. Quantification of TEM images. Nanotubes were counted using ImageJ. For each sample, an average of 300 species was counted. Measurement of melting curves for fuel. One connector strand was mixed with the fuel in equimolar amounts in FoB5 buffer (480 nM). A UV-Vis spectrum was measured every 180 s while the temperature was cooled down to 2 °C, then heated up to 90 °C and cooled back down over a span of 255 min. An average of at least three separate measurements was used for each fuel strand. The Tm of the 10 nb fuel is too low to be measured and was therefore calculated with the OligoAnalyzer from IDT.

chemsum

{"title": "Switchable Supracolloidal 3D DNA Origami Nanotubes Mediated through Fuel/Antifuel Reactions", "journal": "ChemRxiv"}

targeting_bacillus_anthracis_toxicity_with_a_genetically_selected_inhibitor_of_the_pa/cmg2_protein-p

## Abstract: The protein-protein interaction between the human CMG2 receptor and the Bacillus anthracis protective antigen (PA) is essential for the transport of anthrax lethal and edema toxins into human cells. We used a genetically encoded high throughput screening platform to screen a SICLOPPS library of 3.2 million cyclic hexapeptides for inhibitors of this protein-protein interaction. Unusually, the top 3 hits all contained stop codons in the randomized region of the library, resulting in linear rather than cyclic peptides. These peptides disrupted the targeted interaction in vitro; two act by binding to CMG2 while one binds PA. The efficacy of the most potent CMG2-binding inhibitor was improved through the incorporation of non-natural phenylalanine analogues. Cell based assays demonstrated that the optimized inhibitor protects macrophages from the toxicity of lethal factor.Anthrax is caused by Bacillus anthracis (B. anthracis), a spore-forming encapsulated Gram positive bacterium 1, 2 . The disease is classified depending on the route of exposure; the most common form in humans is cutaneous anthrax, associated with skin lesions and is manageable with antibiotics. Gastrointestinal anthrax causes a much more serious systemic disease but primarily affects livestock that have ingested the bacterial spores. The third form of the disease is pulmonary anthrax, which results from inhalation of airborne spores and is potentially fatal. Pulmonary anthrax is asymptomatic for several weeks as lung macrophages and dendritic cells engulf and kill most inhaled spores 3 . A fraction of the spores survive within the alveolar macrophages and are transported to tracheobronchial and mediastinal lymph nodes, where they germinate, giving rise to mild non-specific symptoms (fever, aches, cough). The disease progresses rapidly from these flu-like symptoms as bacteria reach high levels in the circulation, causing fulminant disease characterised by respiratory impairment, shock and widespread haemorrhage. Antibiotics are without therapeutic benefit from this point onwards due to the accumulation of the bacterial toxins, and death usually occurs within 24 hours 4 .The basis for anthrax virulence is well understood at the molecular level. The genes encoding the secreted binary toxins of B. anthracis, named lethal toxin (LT) and edema toxin (ET), reside on a self-replicating 184-kb plasmid termed pXO1. Anthrax toxin consists of three distinct proteins named protective antigen (PA), lethal factor (LF), and edema factor (EF) 5 . PA is an 83 kDa protein that binds to one of two cell surface receptors, then undergoes furin protease-mediated cleavage to yield a 63 kDa fragment. This cleavage is essential for toxin action and PA harbouring mutations in the furin cleavage site is completely non-toxic and devoid of pathogenic effects in vivo 6 . EF is an 89 kDa calcium and calmodulin-dependent adenylate cyclase that causes a dramatic increase in cytoplasmic cAMP levels, impairing neutrophil function and affecting water homeostasis, leading to edema. LF is a 90 kDa zinc-dependent metalloproteinase that specifically cleaves and inactivates mitogen activated protein kinase kinases (MAPKK), which blocks several signal transduction pathways, leading to apoptosis and lysis within a few hours. The furin-cleaved PA binds one of its two target cell receptors; tumour endothelial marker 8 (TEM8), or capillary morphogenesis gene 2 (CMG2). Once bound to the cell, PA assembles into a ring shaped heptamer that forms membrane spanning pores 7 , acting as a protein translocator, escorting three molecules of LF or EF from the extracellular environment into the cytoplasm 8 . Association of PA monomers occurs spontaneously, and LF and EF can only bind to the oligomeric forms of PA 9,10 . A series of elegant experiments with mice lacking TEM8, CMG2 or both, have demonstrated that the main receptor for anthrax toxicity is CMG2 11 . This may be due to the higher affinity of PA for CMG2 (170 pM) versus TEM8 (1.1 µM) 12 , or the fact that CMG2 is preferentially expressed in cells important for infection and/or toxin-induced death 13 . Previous attempts at targeting anthrax toxicity have included inhibition of proteolytic activation of PA 14 , inhibiting LF enzymatic activity , and inhibiting EF enzymatic activity 18 . In addition to this, cisplatin was identified in a high-throughput screen as binding CMG2 19 , and phage display has been used to identify a 12-residue peptide (AWPLSQLDHSYN) that binds to CMG2 and TEM8, with multiple copies of this peptide used to assemble polyvalent liposomes that inhibited anthrax toxicity 20 . Here we describe the identification and in vitro validation of a linear pentapeptide that binds CMG2 and inhibits the protein-protein interaction (PPI) with PA. ## Results Construction of a PA/CMG2 RTHS and SICLOPPS screening. We employed a genetically encoded, high-throughput screening platform that combines a bacterial reverse two-hybrid system (RTHS) 21,22 to screen a library of 3.2 million cyclic peptides generated by split-intein circular ligation of peptides and proteins (SICLOPPS) . The RTHS links the survival and growth of engineered E. coli to the interaction of the targeted PPI via three reporter genes (Fig. 1A). PA is composed of four distinct domains, termed domain I-IV (Supplemental Figure 1A) 26,27 ; three isopropyl β-D-1-thiogalactopyranoside (IPTG) induced plasmids each encoding full length PA, domain II to domain IV of PA 259-735 , or domain III and IV of PA 488-735 as an N-terminal fusion with the 434 repressor, and the extracellular portion of CMG2 38-218 as an N-terminal fusion with a chimeric P22 repressor were constructed. These plasmids were integrated onto the chromosome of the E. coli heterodimeric RTHS strain as previously detailed 28 . Association of PA with CMG2 will enable the formation of a functional functional 434/P22 repressor that binds to operator sites engineered onto the chromosome of E. coli, preventing expression of 3 reporter genes (HIS3, the yeast auxotroph of the HISB histidine biosynthesis gene which has been deleted form the reporter strain; KanR, encoding kanamycin resistance; and LacZ, encoding β-galactosidase). Thus interaction of the targeted proteins will lead to cell death on selective media. The resulting PA/CMG2 RTHS were assessed for functional repression upon addition of IPTG (causing expression and interaction of PA/CMG2) by drop-spotting. Only the domain III-IV PA 488-735 RTHS showed a reduction in growth in response to IPTG by drop-spotting (Supplemental Figure 1B). This RTHS was further characterised by o-nitrophenyl-β-galactoside (ONPG) assay and additional drop-spotting. An IPTG-dependent reduction in β-galactosidase activity was observed, indicating the formation of a functional repressor with 25 µM IPTG (Fig. 1B). The Figure 1. PA/CMG2 reverse two-hybrid system. (A) PA 488-735 is expressed as a fusion with the 434 bacteriophage DNA binding protein and CMG2 38-218 is expressed as a fusion with a chimeric P22 DNA binding protein. These proteins associate to form a functional repressor that prevents transcription of the 3 reporter genes (HIS3, KanR and LacZ) downstream of the operator sites, leading to cell death on selective media. In the presence of an inhibitor of the PA/CMG2 interaction from the SICLOPPS library, the repressor complex is disrupted, enabling expression of the reporter genes and survival of the host on selective media. (B) ONPG assay of the PA/CMG2 RTHS shows a loss of lacZ expression in response to increased doses of IPTG, with no such effect in the blank strain expression the repressor domains alone. Data represented as mean ± SEM. (C) drop spotting 10-fold serial dilutions (2.5 µL of 10 n cells/mL) of the PA/CMG2 RTHS with a potential SCILOPPS inhibitor. In the absence of IPTG and arabinose, full growth is observed, whereas in the presence of 50 µM IPTG growth of the RTHS is repressed by ~4 spots. In the presence of 6.5 µM arabinose (inducing SICLOPPS) and 50 µM IPTG growth of the RTHS is restored, likely via disruption of the PA/CMG2 PPI. addition of 25 µM IPTG was sufficient to reduce the survival and growth of the PA/CMG2 RTHS on selective media lacking histidine and containing kanamycin (Fig. 1C, top row versus second row), confirming the formation of a functional repressor. An arabinose-induced SICLOPPS library encoding cyclic hexa-peptides with a cysteine in position 1 as required for intein splicing (Supplemental Figure 2A), followed by five random amino acids (CX 5 ) was constructed as previously detailed 24,29 and transformed into the PA/CMG2 RTHS. Peptides disrupting the PA/CMG2 PPI would also disrupt the 434/P22 repressor and enable expression of the reporter genes and host survival and growth on selective media (Fig. 1A). The transformation efficiency of the CX 5 library into electro competent PA/ CMG2 RTHS cells was measured as 3 × 10 7 , ensuring ten-fold coverage of each member of the library in our screen. 480 colonies survived and grew on selective media supplemented with IPTG and arabinose. These colonies were isolated and assessed for retention of phenotype by drop spotting (Fig. 1C); IPTG-dependent formation of the functional repressor was assessed by drop-spotting onto selective media with and without IPTG, and the ability of the SICLOPPS-derived cyclic peptide to disrupt the PA/CMG2 PPI was assessed by drop-spotting onto selective media containing both IPTG and arabinose (Fig. 1C). The relative potency of each cyclic peptide may be indirectly assessed through the number of spots of growth on the IPTG + arabinose plate, with more potent inhibitors enabling further growth. The 27 cyclic peptides that restored the growth of the PA/CMG2 RTHS by two or more spots than the IPTG alone plate (~100-fold improvement in survival) were taken forward for secondary screening. The SICLOPPS plasmid from each of these 27 colonies was isolated and re-transformed into the PA/ CMG2 RTHS for re-confirmation of phenotype. These plasmids were also transformed into another RTHS monitoring for the p6/UEV PPI 30 ; this RTHS is identical to the PA/CMG2 RTHS except for the targeted PPI. Cyclic peptides that enable survival by targeting components of the RTHS other than the PA/CMG2 PPI (e.g. inhibiting the interaction of the repressor domains with DNA) would also be active in the p6/UEV RTHS. Any isolated SICLOPPS plasmids that also enabled survival and growth of the p6/UEV RTHS were therefore discarded. The remaining nine SICLOPPS plasmids were ranked for activity by drop spotting, and sequenced to reveal the identity of the cyclic peptide encoded (Supplemental Table 1). Surprisingly, 7 of the 9 sequences, including all of the top ranking hits, contained a stop codon. This produces a truncated SICLOPPS protein that displays a short peptide aptamer from the C-intein (Supplemental Figure 2B and C), instead of a cyclic peptide. SICLOPPS libraries are constructed with a degenerate oligonucleotide that uses an NNS codon set (N = any base, S = C or G) for each of the 5 random amino acid positions, resulting in only the TAG stop codons being present in the screened library. It should be noted that this is highly unusual; there are no previous reports of this occurrence, and we very rarely isolate sequences containing a stop codon in SICLOPPS screens 31,32 . Interestingly, 2 of the top 9 hits were linear heptamers, generated by deletion of the first T in the SICLOPPS N-intein; this causes a frameshift, changing TGC TTA AGT (the sequence following the last randomized amino acid, encoding C, L, and S) to GCT TAA GT (encoding A and Stop). The observed prevalence of stop codons in the most potent hits from our SICLOPPS library strongly suggests that the cyclic hexapeptide scaffold is not optimal for disrupting the PA/CMG2 PPI, and/ or the optimal pocket for disrupting this PPI does not accept residues displayed by this scaffold. The stop codon results in only linear aptamers being presented, either through incorporation of an amber stop codon, or selective pressure leading to a point deletion, which also results in a stop codon. It is also worth noting that beyond the prevalence of stop codons, the only other consensus in the isolated sequences is between 2 of the 3 most potent hits, which differ by only 1 amino acid (CLRFT and CLRPT). There is little consensus in the sequence or any motif(s) present amongst the other, less potent hits. In vitro quantification of the PA/CMG2 PPI inhibitors. The 3 top ranking compounds isolated from our screen were synthesized by Fmoc solid-phase peptide synthesis and assessed for the ability to disrupt the PA/ CMG2 PPI in vitro. We developed a sandwich ELISA to monitor the interaction between His 6 -PA 488-735 (domains III and IV) and GST-CMG2 38-218 and used this assay to assess the activity of our top 3 inhibitors. The most potent compound was CMNHFPA with an IC 50 of 49.8 ± 2.7 µM, followed by CLRFT with an IC 50 of 77.1 ± 9.5 µM and CLRPT with IC 50 of 153.2 ± 2.9 µM (Fig. 2A,B and C). Given the relatively weak activity of CLRPT, it was not carried forward for further assessment. We repeated the above ELISA using domain IV of PA (His 6 -PA 596-735 ) and GST-CMG2 38-218 ; CLRFT showed a similar level of activity as before with an IC 50 of 71.3 ± 6.5 µM, whereas CMNHFPA lost all activity (Fig. 2D). Given that CMNHFPA is inactive in the absence of domain III of PA, one may hypothesise that this cyclic peptide functions by binding to domain III of PA; however, structural data indicate that domain III of PA is not in direct contact with CMG2 (Supplemental Fig. 1A) 26 . Considering these two points together, one explanation may be that CMNHFPA inhibits the of the PA/CMG2 PPI by binding to an allosteric site on domain III of PA. We next synthesized scrambled analogues of our top 2 inhibitors as negative controls, to assess the sequence dependence of activity. FCRTL (scramble of CLRFT) was found to be inactive in the PA/CMG2 ELISA, whereas HPCNAMF (scramble of CMNHFPA) inhibited the PA/CMG2 PPI with an IC 50 of 152.7 ± 9.3 µM, a 3-fold loss of activity over the selected peptide. Given the retention of some activity of the scramble peptide, we further assessed the sequence specificity of CMNHFPA by replacing phenylalanine with alanine; the resulting molecule (CMNHAPA) disrupted the PA/CMG2 PPI with an IC 50 of 522.2 ± 47.8 µM, a 10-fold loss of activity from the parent molecule. The retention of activity in these control molecules may result from part of the active motif of the parent molecule being retained in the scramble molecule (or reconstituted through folding of the peptide); alternatively, the parent molecule may be a false positive. The protein target of CLRFT was identified, and the binding affinity quantified, using microscale thermophoresis (MST). CLRFT bound CMG2 with a K d of 30.2 ± 1.2 µM (Fig. 2F), while no binding was measured to PA (Fig. 2G). Our ELISA data indicated that CMNHFPA bound to PA (Fig. 2A and D), and we measured a K d of 38.2 ± 4.3 µM (Fig. 2F) for this interaction by MST. Although both of the 2 most potent PA/CMG2 inhibitors identified from the CX 5 library were linear, the in vitro data shows that one acts by binding PA, while the other acts by binding CMG2. CMNHFPA is more potent inhibitor of the PA/CMG2 PPI than CLRFT (Fig. 2A and B), but both peptides bind their respective targets with similar affinity (Fig. 2F and H). Although both these molecules could form the starting point for hit optimization, the binding of CLRFT to CMG2 (rather than PA) may be seen as an advantage, especially with respect to the reduced potential for resistance through mutation. Improving the affinity of CLRFT for CMG2 through non-natural analogues. We synthesized several analogues of CLRFT containing non-natural phenylalanine derivatives, with the aim of probing binding efficacy and improving the potency of this molecule. Phenylalanine was chosen as the residue due to the large number of commercially available non-natural analogues, as well as the loss of activity observed when this amino acid was replaced with a proline (in the third most potent hit identified, CLRPT, Fig. 2A and C), although it should be noted that this loss of activity may be due to the effect of proline on peptide backbone conformation. The analogues were synthesized and their binding affinity for CMG2 measured by MST (Fig. 3). We initially probed the effect of stereochemistry of this residue by incorporating D-phenylalanine in this position, however, we saw little effect on binding (K d = 31.0 ± 2.9 μM, Fig. 3A). We next probed the length of the binding cavity; a 2.5-fold reduction in K d was observed when using homophenylalanine (Fig. 3B), and a 2-fold reduction in K d was observed when using phenylglycine (Fig. 3C). In line with this data, complete loss of binding was observed when 4-benzoyl-phenylalanine was used (Fig. 3D). We next probed the electronic requirements of the binding pocket by using a variety of electron donating and withdrawing substituents (Fig. 3E-K, however, we observed little correlation between this and binding affinity. For example, using tyrosine caused a 3-fold reduction in the binding affinity (K d = 91.9 ± 9.5 μM, Fig. 3E), while using 4-nitrophenylalanine had little effect (K d = 36.2 ± 5.5 μM, Fig. 3F, yet the weaker electron-withdrawing 4-cyanophenylalanine reduced the binding affinity by 2-fold (K d = 61.4 ± 8.0 μM, Fig. 3G). Of the compounds synthesized, only the 4-chlorophenylalanine derivative (Fig. 3L)showed an improvement in the binding, with a 2-fold increase in its affinity for CMG2 (K d = 14.0 ± 3.2 μM, Fig. 3I). ## Assessing the activity of CLR(4-Cl-F)T in cells. We next sought to assess the activity of CLR(4-Cl-F) T (Fig. 3L), our most potent PA/CMG2 inhibitor, in cells. We initially determined binding of this molecule to its extracellular target by using a fluorescent derivative and its scrambled analogue (4-Cl-F)CRTL, generated by tagging cysteine with fluorescein-5-maleimide. The resulting molecules were incubated with BHK-21 cells, and binding to these cells was assessed by fluorescence-activated cell sorting. The data demonstrated that CLR(4-Cl-F) T binds to BHK-21 cells, with similar amount of fluorescence observed using 5 µM or 50 µM of this molecule (Fig. 4A), while 5 µM or 50 µM of the scrambled analogue showed weaker binding (Fig. 4A). Fluorescent microscopy was used to probe the cellular localization of these molecules. CLR(4-Cl-F)T was observed in the membrane of BHK-21 cells, while no binding was observed with the scrambled control at the same dose (Fig. 4B). The ability of CLR(4-Cl-F)T to inhibit CMG2 for LT activity was probed using a toxin neutralization assay. J774 cells were treated with LT in the presence of increasing doses of CLR(4-Cl-F)T or its scrambled analogue, and the number of live cells determined after 18 hours. We observed a significant effect on cell viability from 50 µM of CLR(4-Cl-F)T, with the number of live cells equivalent to those not treated with LT (Fig. 4C). There was no such protection from LT for cells treated with 100 µM of the scrambled control (Fig. 4C). ## Discussion A library of 3.2 million SICLOPPS peptides was screened for inhibitors of the PA/CMGS PPI. The 3 most potent inhibitors were found to contain a stop codon in the randomized region of the library, leading to the production of linear, rather than cyclic peptides. While the NNS codon used for the randomized region of SICLOPPS libraries eliminates 2 of the 3 stop codons, translation termination may still occur via a TAG codon in any of the 5 randomized positions. This is an unusual occurrence; we have not previously selected SICLOPPS hits containing stop codons, and this has not been reported by others 31 . Our findings suggest that members of the cyclic hexa-peptide library do not present their amino acid side chains in an orientation that enables binding to the pockets in the two targeted proteins, forcing the system to select linear peptides. Supporting our hypothesis of selective pressure for linear peptides are the hit peptides that contain a stop codon generated via a point mutation (resulting in a frame shift to give a stop codon). We are currently working to obtain structural information on the selected peptide/protein complexes, which will provide insight into the binding of each of these peptides and the reason for the prevalence of stop codons in their randomized region. Given the high binding affinity of the CMG2/PA interaction (K d of 170 pM) 12 , the identified peptides are unlikely to dislodge PA bound to CMG2 by competing for the same binding interface. Given their K d values, our inhibitors are much more likely to act by binding to an allosteric site on their target protein, and indirectly inhibiting the PPI. The peptides reported above are not the first peptidic inhibitors of the PA/CMG2 PPI; phage display has been previously used to identify linear 12-mer peptides that bind to CMG2 20 . We are unable to compare the affinity of our hits with those previously reported, as the in vitro binding affinity of the previously reported peptides for their target proteins was not reported by the authors. However, there is no homology in the sequence of the previously reported peptides, and those reported here. This suggests that the peptides are binding to different regions for the target proteins, likely a result of the different methods used to identify the hits. Phage display selects for the most potent binding sequence, whereas our RTHS is a functional assay, seeking to identify the sequence that most effectively disrupts the interaction between the two given proteins. In addition, the linear pentamer reported here is substantially smaller than the previously reported 12-mers, and likely to be more readily translated to small molecule inhibitors of the PA/CMG2 PPI. The similarity in the sequence of 2 of the top 3 hits, and the loss of potency caused by the change of phenylalanine to proline indicated the key role played by phenylalanine in binding to CMG2. A modest library of derivatives was therefore synthesized with non-natural phenylalanine analogues in order to improve binding affinity. The most potent analogue contained para-chlorophenylalanine, which bound CMG2 with a K d of 14.0 ± 3.2 μM. This molecule was shown to be active in cells, protecting macrophages from lethal toxin at a dose of 50 µM. While this demonstrates the therapeutic potential of compounds derived from the molecules reported here, additional SAR studies, such as alanine scanning of the lead molecule are required for the design more potent inhibitors, as well as their derivatization into small molecule/non-peptidic compounds. Nonetheless, our screen has provided two sets of scaffolds that may be further developed as potential inhibitors of anthrax toxin entry into cells. While we have chosen to focus on development of the CMG2-binding compound identified here, similar optimization of the PA-binding molecule is also possible. Indeed, a possible strategy for treating anthrax infections may be with a cocktail of derivatives of both sets of molecules, blocking the PA/CMG2 interaction via both the human receptor, and the bacterial protein. ## Methods Construction of the PA/CMG2 RTHS. The RTHS used in this study was constructed as previously detailed for other RTHS 28 . Briefly, CMG2 38-218 was cloned into the first multiple cloning site of pTHCP14 21 via the XhoI and KpnI restriction endonuclease sites, while PA 488-735 was cloned into the second multiple cloning site of this plasmid via the SalI and SacI restriction endonuclease sites. Formation of a functional repressor upon induction of the P22-CMG2 38-218 and 434-PA 488-735 fusion proteins was assessed by drop spotting and ONPG assays as previously detailed 28 , with the data shown in Fig. 1). ## SICLOPPS library construction. A SICLOPPS library encoding CXXXXX (X = any amino acid) was constructed as previously detailed 24 . Briefly, The C-terminal intein from pARCBD 23 was amplified by PCR using the C + 5 forward primer, SICLOPPS reverse primer, and GoTaq (Promega), resulting in the incorporation of a region encoding the CXXXXX random sequence via the forward primer. The PCR product was purified and used as the template for a subsequent PCR reaction using SICLOPPS zipper primer, and SICLOPPS reverse primer (annealing temperature 65 °C and extension time 1 minute 15 seconds). The resulting PCR product and pARCBD plasmid were restriction digested with BglI and HindIII restriction endonucleases. The digested vector was gel purified to isolate the 3376 bp fragment corresponding to the plasmid backbone, and ligated with the restriction digested PCR product (1:3 insert to vector ratio) overnight at 4 °C. Salts were removed from the ligation mixture by dialysis on a nitrocellulose filter (13 mm, 0.025 μm, Millipore), for transformation into electrocompetent cells. ## SICLOPPS screening. The library ligation mixture was transformed into electrocompetent PA/CMG2 RTHS E. coli cells using standard protocols. The transformation mixture was recovered at 37 °C for 1 hour, 2 µL was removed for calculation of transformation efficiency by plating 10-fold serial dilutions of the recovery mixture on LB-agar media containing 30 µg/mL chloramphenicol and counting the number of surviving colonies at the highest dilution. The remaining 998 µL of the recovery mixture was plated onto M9 media-agar plates supplemented with 50 µg/ml ampicillin, 25 µg/mL spectinomycin, 30 µg/mL chloramphenicol, 50 µg/ml kanamycin, 5.0 mM 3-AT, 50 µM IPTG and 6.5 µM arabinose and incubated for 48-72 hours at 37 °C until individual colonies were visible. The 480 surviving colonies were picked and grown overnight in LB supplemented with 30 µg/mL chloramphenicol and drop-spotted onto minimal media plates as above with and without 50 µM IPTG and with and without 6.5 µM arabinose to check for retention of phenotype and rank activity. The SICLOPPS plasmids from the 27 strains that retained their ability to enable survival on +IPTG/+ arabinose plates were isolated and transformed back into the PA/CMG2 RTHS, as well as the p6/UEV RTHS 30 ; both RTHS are identical, except for the interacting protein pair. The resulting recovery mixtures were used to drop spot onto the same minimal media plates as above. Sequences that were active in both RTHS were discarded as non-specific (e.g. targeting a component of the RTHS other than the PPI). The SICLOPPS plasmids from the 9 strains showing selective inhibition of PA/CMG2 were sequenced to reveal the identity of the peptide inhibitors. Peptide synthesis. Peptides were synthesised by Fmoc solid-phase peptide synthesis on a 0.1 mmol scale using Wang resin preloaded with the first amino acid residue. Subsequent steps were performed at room temperature in a sintered funnel with agitation from a stream of argon. The amino acid coupling solution contained an Fmoc-protected amino acid (3 eq.), HOBt (3 eq.) and DIC (3 eq.) and was agitated with the resin in DMF for 1 h. The resin was washed with DMF, DCM and Et 2 O (20 mL of each) and successful coupling was checked using the Kaiser test, and the coupling step repeated if necessary. Fmoc deprotection was carried out by agitating the resin with 20% piperidine in DMF for 20 mins. The resin was washed as before, and successful deprotection checked using the Kaiser test prior to moving on. Upon deprotection of the final residue, the peptide chain was cleaved from the resin with 2 mL of TFA/TIS/H 2 O (95:2.5:2.5) for 2.5 h. The mixture was filtered through a sinter funnel, and the filtrate concentrated in vacuo. Peptides were precipitated from the remaining solution with cold Et 2 O added dropwise until a white precipitate formed. The solid was isolated, dried and dissolved in a H 2 O:MeCN mixture (1:1) prior to purification by preparative reverse-phase HPLC. All HPLC was performed on a Waters 1525 HPLC system using linear gradients of solvents A (0.1% TFA/ H 2 O) and B (0.1% TFA/MeCN). Peptides were purified by preparative HPLC with a Waters XSelect CSH C18 column (5.0 µm particle size, 19 × 250 mm), using a gradient from 95:5 to 50:50 A:B over 25 mins at 17 mL/min flow rate. Analytical HPLC was performed using a Waters Atlantis T3 C18 column (5.0 µm particle size, 4.6 × 100 mm) with the following method: 0-10 min: 95:5; 20-30 min: 40:60; 30-35 min: 95:5 A:B; at 1 mL/min flow rate. Please see supplemental data for the analytical spectra of each peptide. Sandwich ELISA. Glutathione S-transferase (GST)-CMG2 38-218 and His 6 -PA 488-735 were expressed and purified as previously detailed 12 . His 6 -PA 488-735 (1,000 ng) was incubated in Ni 2+ -coated 96-well plates (Pierce) for 1 hour. The wells were washed with 3 × 200 µL of PBS with 0.05% Tween-20. GST-tagged CMG2 38-218 (1,000 ng), incubated with various concentrations of inhibitor and 1 mM MgCl 2 , was added to each well and incubated for 1 hour. The wells were washed as before. Anti-GST (1 in 1000, MA4-004, Neomarkers) was added and incubated for 1 hour, after which the wells were washed as before. Anti-mouse-HRP (1 in 6000, NA931, GE Healthcare) was added and incubated for 1 hour, and the wells washed as before. 100 μL of 3,3′,5,5′-tetramethylbenzide (TMB)-Ultra ELISA solution (Fisher) was added to each well and incubated for 20 minutes. The signal was quenched with 1 M H 2 SO 4 and the plate analysed at 450 nm. The procedure for the CMG2 38-218 and PA 596-735 ELISA was as above, except for the use of truncated PA. ## Microscale thermophoresis. MST experiments were on a Monolith NT.115 system (NanoTemper Technologies) using 100% LED and 40% IR-laser power. Laser on and off times were set at 30 seconds and 5 seconds, respectively. His 6 -CMG2 38-218 and His 6 -PA 488-735 were overexpressed, purified and labelled with NT647 (NanoTemper Technologies) and used at a final concentration of 80 nM. The inhibitors were dissolved in MST-optimised buffer. Samples were filled into hydrophilic capillaries (NanoTemper Technologies) for measurement. FACS analysis. Fluorescein-5-maleimide labeled CLR(4-Cl-F)T, and a scramble control were synthesized by combining fluorescein-5-maleimide with CLR(4-Cl-F)T or (4-Cl-F)CRTL in DMF. The resulting labeled peptides (at the indicated concentrations) were added to BHK-21 cells and incubated for 30 minutes. Cells were washed 3 times with DMEM plus 1% BSA (w/v). Fluorescence was measured by flow cytometry (BD LSRFortessa), with an excitation laser of 488 nm, and an emission bandpass filter of 530/30 nm. Fluorescence microscopy. The above fluorescein-labeled inhibitors (at the indicated concentrations) were added to BHK-21 cells and incubated for 30 minutes. Cells were washed 3 times with DMEM plus 1% BSA (w/v). Fluorescent micrographs were recorded on a confocal microscope (Zeiss). Excitation 488 nm, emission, 490LP filter. Toxin Neutralization Assay. J774 cells were plated at 2 × 10 5 cells/ml (100 µL/well) in 10%DMEM and allowed to adhere for at least 1 hour at 37 °C and 5% CO 2 . PA (1.5 mL at 0.1 mg/mL) was mixed with LF (1.5 mL at 0.1 mg/mL) to generate LT. J774 plates were removed from the incubator, centrifuged and medium was removed. Solutions of LT (50 µL) plus various concentrations of the inhibitor, or scrambled control (20 µL, in PBS), were added to the cells and incubated overnight at 37 °C with 5% CO 2 . The next day, cell supernatants were removed and cell number determined.

chemsum

{"title": "Targeting Bacillus anthracis toxicity with a genetically selected inhibitor of the PA/CMG2 protein-protein interaction", "journal": "Scientific Reports - Nature"}

magnesium-alloy_rods_reinforced_bioglass_bone_cement_composite_scaffolds_with_cortical_bone-matching

## Abstract: Various therapeutic platforms have been developed for repairing bone defects. However, scaffolds possess both cortical bone-matching mechanical properties and excellent osteoconductivity for load-bearing bone defects repair is still challenging in the clinic. In this study, inspired by the structure of the ferroconcrete, a high-strength bifunctional scaffold has been developed by combining surface-modified magnesium alloy as the internal load-bearing skeleton and bioglass-magnesium phosphate bone cement as the osteoconductive matrix. The scaffold combines the high mechanical strength and controllable biodegradability of surface-modified magnesium alloy with the excellent biocompatibility and osteoconductivity of bioglass-magnesium phosphate bone cement, thus providing support for load-bearing bone defects and subsequently bone regeneration. The scaffolds generate hydroxyapatite (HA) during the degrading in simulated body fluid (SBF), with the strength of the scaffold decreasing from 180 to 100 MPa in 6 weeks, which is still sufficient for load-bearing bone. Moreover, the scaffolds showed excellent osteoconductivity in vitro and in vivo. In a New Zealand White Rabbit radius defect model, the scaffolds degrade gradually and are replaced by highly matured new bone tissues, as assessed by image-based analyses (X-ray and Micro-CT) and histological analyses. The bone formation-related proteins such as BMP2, COL1a1 and OCN, all showed increased expression.Large bone defects can result from a wide variety of causes, such as osteonecrosis, trauma, and cancer metastasis 1 . Although bone tissue has a remarkable ability to regenerate and heal itself, large bone defects which exceed the critical size cannot be fully and steadily repaired by themselves. Therefore, it's necessary to graft autologous bone or artificial bone substitutes for treating the defects 2,3 . Although autologous bone grafting represents an effective approach for bone defects repairing, donor site morbidity and source-limitation have hampered its application in large bone defects. In contrast, artificial bone scaffolds have several distinct advantages such as abundant supply 4,5 .Many bone substitutes based on single or composite materials have been fabricated for repairing large bone defects [6][7][8][9] . Metals are one of the desirable and wildly used biomaterials for load-bearing implants, attributing ## Results Characterization of the scaffolds. In order to improve the mechanical and osteoconductive properties of scaffolds for load-bearing bone defects repair, a high-strength bifunctional scaffold of surface-modified magnesium alloy reinforced bioglass-magnesium phosphate bone cement was constructed inspired from the structure of steel reinforced concrete architectures. As depicted in Fig. 1, PCL modified Magnesium (Mg) alloy (as shown in Supplementary Fig. S1) is similar with the inner steel of the reinforced concrete providing excellent mechanical properties. While the bioglass-magnesium phosphate bone cement is similar with the outer concrete providing the scaffold excellent osteoconductivity for load-bearing bone defects repairing. The surface microtopography of BGC were characterized by scanning electron microscopy (SEM), indicating that the BG particles were tightly bond with the magnesium phosphate cement (Fig. 2A,B). Columnar-like crystals were found in the sample represents magnesium phosphate which was formed via the reaction shown below: Due to the mineral-interaction between BGC particles and phosphates in the mixture, BG particles were uniformly dispersed in the magnesium phosphate cement matrix. Energy-dispersive X-ray spectroscopy (EDS) shows the distributions of Mg, P, Ca, Si, and B, indicating the homogeneous reaction of BG with MgP cement matrix as well (Fig. 2C). The crystal structure of BGC scaffolds was further characterized by X-ray diffractometry (XRD). As displayed in Fig. 2D, the BGC shows sharp characteristic peaks at 2θ = 43.2°, 62.7° and 37.1° that are attributed to MgO in consistency with JCPDS.75-1525, while peaks at 2θ = 27.6°, 32.9° and 29.9° are attributed to Mg 3 (PO 4 ) 2 in consistency with JCPDS.48-1167. Degradation behavior and mechanical properties of the scaffolds. In order to investigate the degradation behaviors of different scaffolds, scaffolds of Mg, BGC and BGC-Mg were immersed in SBF at 37 °C for different periods, and the changes in pH, mass, compressive strength and elastic modulus were measured as shown in Fig. 3A-D. Results show that there are significant weight loss for all the scaffolds in the first week, especially for the Mg scaffold. The weight loss of BGC-Mg scaffold is less than that of BGC scaffold. However, after 4 weeks, Mg scaffolds degrade quickly, leading to the quick degradation of the BGC-Mg (Fig. 3A). The pH of the scaffolds immersed solutions were recorded as shown in Fig. 3B. The pH of all samples increased gradually and then reached a dynamic equilibrium in 4 weeks. However, there was a second increase of the pH of BGC-Mg scaffold immersed solution after 4 weeks. The compressive strengths of scaffolds were measured as shown in Fig. 3C. The compressive strength of BGC scaffold was approximately 16.0 MPa, while that of the BGC-Mg composite scaffold was 180.0 MPa, closing to that of cortical bone as100-200MPa 24 . Moreover, the elastic modulus of the composite scaffold was 42.5GPa, which is about twice of 15-25GPa of cortical bone 15 (Fig. 3D). The compressive strength of BGC-Mg composite scaffold was almost completely retained at 180.0 MPa at beginning 4 weeks, and then decreased to 100 MPa in 6th week, which is still much higher than that of BGC scaffold and can match the normal cortical bone. The variation of Ca, Mg, Si, B ions concentrations in soak solution were recorded as well (shown in Supplementary Fig. S2). Both BGC scaffold and BGC-Mg scaffold show gradually increase of Mg, Si and B ions, indicating the degradation of scaffolds. The decrease of Ca ions are attributed to the formation of hydroxyapatite, which is confirmed by the XRD of scaffolds after SBF soaking (Fig. 3E,F). The XRD spectrum of Mg scaffold shows sharp characteristic peaks at 2θ = 38.3°, which can be attributed to the reflections of Mg(OH) 2 in consistence with JCPDS No.86-0441 (Fig. 3G). The surface morphology of all the scaffolds were observed by SEM before and after soaking in SBF for 8 weeks (Fig. 3H). There were large amount of nanoparticles on the surface of BGC scaffold and BGC-Mg scaffold, Biocompatibility of the scaffolds. The effects of scaffolds on cell growth were checked using CCK-8 and live-dead cells staining 9 (Fig. 4). Cell viability assays were firstly carried out using extracts of scaffolds at different concentrations. Compared to the control group (rBMSCs cultured in normal medium), both BGC group and BGC-Mg group showed a slightly decrease of cells viability in high concentrations of extracts (100%), while other conditions no obvious influence on cell growth was found (Fig. 4A,B). However, extracts of Mg group revealed obvious cell growth inhibition at all concentration. The higher concentration of Mg extract, the higher of cytotoxicity was (Fig. 4C). Then the cell viabilities were further assayed by live-dead cells staining using Calcein-AM and PI staining after 1, 3, 7 day of cell culture, where green fluorescence indicated live cells, red fluorescence indicated dead cells (Fig. 4D). No remarkable difference in the number of live cells was found between BGC and control group, which is in correspondence with the results of CCK-8 assay. Furthermore, cell adhesion assay was carried out by seeding rBMSCs onto BGC scaffolds and observed by SEM after co-culturing for 7 d (Fig. 4E). Cells attached tightly on scaffold surface and showed well-flattened and expanded with no significant growth retardation, indicating the BGC shows well biocompatibility with rBMSCs. ## Osteogenic differentiation of rBMSCs in vitro. The differentiation of rBMSCs cultured with the scaffolds was assessed in terms of alizarin red S staining and alkaline phosphate (ALP) activity (Fig. 5) 7 . The results of alizarin red S staining showed that BGC increased mineral deposition of rBMSCs indicating the osteogenic effect of BGC. The ALP activity of rBMSCs with the BGC was much higher than that of the control, indicating the significant increased osteogenic differentiation of the cells. ## X-ray and Micro-CT analyses of bone regeneration by scaffolds in vivo. We adopt Radius bone defect of New Zealand white rabbit as the animal experimental model. The defects were implanted with BGC scaffolds, BGC-Mg scaffolds and Mg scaffolds respectively, while defects of blank group were kept empty as control. No signs of infection were observed. We used X-ray to radiograph the rabbits in order to evaluate the degree of scaffolds degradation and bone formation after 4 and 8 weeks of implantation (Fig. 6). In the BGC group, an www.nature.com/scientificreports/ obvious calcified area was observed around the scaffold after implanting 4 weeks, but the calcified density of bone was lower than that of normal bone tissues. While the bone defect region was filled with bone tissues and completely connected with the host bone margin after 8 weeks. In the BGC-Mg group, Mg alloy was still visible in 4 weeks, indicating well protection of Mg by the PCL coating. But after 8 weeks, Mg alloy disappeared and was replaced by new bone. However, in the Mg alloy group, the Mg alloy scaffolds without PCL coating degraded rapidly in 4 weeks. It is similar with the blank group, the bone defect remained vacant and could not repair itself. The bone regeneration ability was also studied by microscopic computed tomography (micro-CT) after implantation for 8 weeks (Fig. 7A). Both the BGC and BGC-Mg scaffolds group showed the defect region were almost completely occupied by high density new bone. While minimal new bone formation was observed in the Mg group. There was no evidence of bone defect repair for the blank group. Quantitative analyses of fundamental parameters based on the histomorphometric micro-CT analysis were presented, such as bone volume (Fig. 7B), bone mineral density (Fig. 7C), porosity (Fig. 7D), and parameters of bone trabecular (Figure S3 ## Histological analyses of bone regeneration by scaffolds. Masson's trichrome (MT) and H&E stain- ing were used to evaluate the bone regeneration quality of all groups 8 (Fig. 8). In H&E-staining, quantities of new bone tissues were clearly observed in the BGC and BGC-Mg group compared to the Mg and blank group, consistent with previous radiographic results. The margins of defect on the BGC and BGC-Mg scaffolds were connected to the host bone for further new bone formation. In the high-resolution images of the MT-staining, a well-arrayed lamellae of bone matrix with quantities of osteoid seams and blood vessels were displayed in the BGC and BGC-Mg group, whereas few newly formed woven bone was found in the Mg and blank group. These results suggested that the BGC-Mg possessed a remarkable osteopromotive ability to facilitate not only quantities of new bone formation but also a high grade of bone maturation in bone defect sites. To further confirm bone regeneration, immunohistochemistry of bone formation-related proteins were performed (Fig. 9). The level of BMP2 were observed around the newly formed bone of the BGC and BGC-Mg group, implying that the BGC may induce the expression of BMP2, and thus to stimulate osteoblasts to form new bone. Meanwhile, higher level of expression of the COL1a1 and OCN were observed on the BGC and BGC-Mg scaffolds groups (Fig. 9A, blue arrow heads). As shown in Fig. 9B-D, integrated optical density (IOD) values of the BGC and BGC-Mg group were much higher than that of Mg and control group. These results indicated that the BGC and BGC-Mg scaffolds possessed excellent bone formation ability and high efficiency of bone regeneration. www.nature.com/scientificreports/ ## Discussion A variety of synthetic bone scaffolds have been developed in the past decades 6,28,29 . However, lack of mechanical property or ostoeconductivity has hampered their wide application 17,25 . In this study, a novel scaffold with cortical bone-matching mechanical properties and ostoeconductivity has been designed inspired by the structure of steel reinforced concrete architectures. In this scaffold, PCL coated magnesium alloy rod resembles the inner steel of reinforced concrete providing excellent mechanical properties, while the bioglass-magnesium phosphate bone cement matrix provides excellent ostoeconductivity. Compressive strength of this scaffold was 180.0 MPa, which is close to that of cortical bone which has range of 100-200 MPa. The similar compressive strengths could avoid the stress shielding between implants and host bone, thus promoting endochondral and intramembranous bone formation 30 . During the degradation of the scaffolds in SBF, hydroxyapatite formed on the BGC surface, suggesting favorable bioactivity of the scaffolds 26,31 . BMSCs were cultured on the scaffolds and showed proliferation on the scaffolds. Cells viability assayed by CCK-8 and live-dead cells staining showed that the cells viability on the scaffolds was similar to the control group (Cells in the control groups were cultured in normal medium) in 7 days, demonstrating biocompatibility of the scaffolds. Enhanced calcium mineral deposition was detected on the scaffolds by Alizarin red S staining. A significant increase of ALP activity of scaffolds was observed compared to the control group, suggesting ostoeconductivity of the scaffolds. www.nature.com/scientificreports/ Bone regeneration of the scaffolds in vivo was carried out using a rabbit radius bone defect model. After 8 weeks of implantation, the defect was significantly filled with newly formed bone both in the BGC and BGC-Mg group, as illustrated in the X-ray and micro-CT images. BGC partially degraded in 4 weeks, and finally was replaced by newly formed bone in 8 weeks, suggesting that BGC and BGC-Mg scaffolds steadily degraded during the bone regeneration process. The ions released by scaffolds may have stimulated the bone formation 8 . The ICP results indicated that Mg, Si, B ions were released from the scaffolds, but Ca ions were deposited on the scaffolds. These results suggest that enhanced bone formation was mediated by bioactive ions from the degradation of BGC and magnesium alloy 14,15,32,33 . Histological results showed that there was a larger amount of bone formation in the BGC and BGC-Mg group whilst minimum new bone was found in the blank group, which was in agreement with the radiographic results. Masson's trichrome staining revealed that the newly formed bone by BGC-Mg scaffold was mostly lamellae bone, and the bone mineral density was higher than the other groups (equivalent to 93% of cortical bone). The edges of scaffolds were connected to the host bone for further new bone formation. It is worth noting that an active bone remodeling process was observed in the BGC and BGC-Mg groups. It consists of active hyperplasia of osteonal basic multicellular units, abundant blood vessels, osteoblasts and frequently coupled with osteoclasts. Immunohistochemistry results showed that there were obvious up-regulating expression of osteoblast differentiation marker proteins in the BGC and BGC-Mg group. These results indicated that BGC-Mg scaffold features remarkable in vivo osteogenic capability, promoting new bone formation and subsequent bone maturation within the defected area. In the aspect of clinical translation, firstly, there have been many studies on the effect of scaffolds porosity, mechanical properties, and degradation on bone defect repair, but no agreement concerning the optimal values, which is very important for future clinical applications. Besides, bone defects are diverse, so personalized therapy becomes popular. The areas and shapes of bone defects in patient are different in clinical practice, but the design of magnesium-alloy rods reinforced bioglass bone cement composite scaffolds hardly meets the requirements of each patient. Fortunately, depending on the computer and 3D printing technology, scaffolds accommodate to different locations, forms, and mechanical requirements may be expected to solve this problem. Finally, the current production technology of magnesium-alloy rods reinforced bioglass bone cement composite scaffolds is still facing many limits, such as small production scale and low efficiency. These problems limited the clinical application of magnesium-alloy rods reinforced bioglass bone cement composite scaffolds and increasing the www.nature.com/scientificreports/ economic burden on patients. Therefore, it is urgent to simplify the productive process and enhance the output and quality of scaffolds. ## Conclusions In conclusion, inspired by the structure of the reinforced concrete, we have designed and developed a highstrength scaffold with surface-coated magnesium alloy rod as the load-bearing skeleton and bioglass-magnesium phosphate cement as the osteoconductive matrix. This scaffold possesses cortical bone-matching mechanical properties and excellent osteoconductivity. The strength of the scaffold decreases slowly during biodegradation, while new bone formation matched the degradation of the scaffold. The ions released from the BGC and magnesium alloy may have promoted osteoblast differentiation and up-regulate osteogenic genes and proteins expression, resulting in new bone formation and subsequent bone maturation. This high-strength scaffold has potential in accelerating bone tissue growth in load-bearing cases in the clinic. www.nature.com/scientificreports/ (CAS 7778-77-0, Aladdin, Shanghai, CHINA) powder were mixed with 3 g of deionized water, then the mixture was poured into a 3D printed mold and pulled out from the mold after 10 min aging, resulting to the BGC 32,34 . ## Preparation of Borosilicate bioglass (BG) and bioglass-magnesium phosphate bone cement (BGC). Preparation and surface modification of magnesium alloy rods and BGC-Mg matrix composite scaffolds. Magnesium alloy rods (diameter = 2 mm, length = 15 mm) were prepared through vacuum melting method in which the proportion of Mg, Zinc, and Ca is 68wt%, 28wt%, 4wt%. For the surface modification of magnesium alloy rods, Polycaprolactone (PCL, molecular weight = 80,000, CAS 24980-41-4, Macklin, Shanghai, CHINA) was firstly added into dichloromethane (CAS 75-09-2, Aladdin, Shanghai, CHINA) with the mass ratio of PCL to dichloromethane is 1:25, then the mixture were heated to 50℃ at a speed of 3℃/min and were stirred till PCL were absolutely dissolved, then the magnesium alloy rods were dipped into the PCL solution and kept for 10 secs before removing from the solution. After holding in air at room temperature for 1 min, the PCL coated magnesium alloy rods were immersed into ethanol for 5 min to extract the remained dichloromethane 35,36 . The above surface modification process was performed once. To prepare the composite scaffolds, 1.25 g of Bioglass powder, 2.03 g of calcinated MgO and 1.72 g of KH 2 PO 4 powder were mixed with 3 g of deionized water, then the mixture was poured into a prepared molds with the PCL modified magnesium alloy rods in the center (as shown in Supplementary Fig. S1), then the composite scaffold was removed from the mold after 10 min of cement solidification. Biodegradation and bioactivity of the scaffolds. The ability of forming Hydroxyapatite (HA) onto the scaffold was measured by immersing in the simulated body fluid (SBF, PHYGENE, Hercynian, Qinghai Province, CHINA), which is a crucial method assessing the in vitro bioactivity of materials 33,37 . BGC scaffold, BGC-Mg scaffold and pure Mg scaffold were immersed in SBF at 37 °C for 1, 2, 3, 4, 5, 6, 7, and 8 weeks, then rinsed thoroughly in acetone (CAS 5000-48-6, Macklin, Shanghai, CHINA) and dried in room temperature for 2d. Weight loss of the scaffolds and pH variation of the fluid were recorded, and the element content of Ca, Mg, Si, B of the after-immersing SBF were measured by inductively coupled plasma atomic emission spectroscopy (ICP-AES). We tested the mechanical properties of the scaffolds according to ISO 6004:2002. The specimen geometry is a cylinder which length is 50 mm and diameter is 10 mm for compressive strength, and a cylinder which length is 10 mm and diameter is 10 mm for elastic modulus. Cytocompatibility of the scaffolds. The Cytocompatibility of scaffolds were assessed by cck-8 assays, live-dead cells staining and cells adhesion of scaffolds 38 . Firstly, scaffolds were soaked into cell culture medium for 24 h as a ratio of 3cm 2 /ml according to ISO10993-12:2007, then above mixture was collected as extract concentration and diluted by medium to different concentration (100%, 50%, 25%, 12.5%, 6.25%, 3.125%). The rBMSCs were seeded in 96 well plate (6 × 10 3 /well) and then cultured in different concentrations of the extracts. After 1, 3, and 7 days of culture, cell counting kit-8 (CCK-8, Abcam, Shanghai, CHINA) was added to each well, incubated for 2 h at 37 °C, and cellular metabolic activity was measured by optical density at 450 nm using a microplate reader. For the live-dead cells staining, cells were seeded in 24 well plate (2 × 10 4 /well), then the growth medium in the wells were replaced by extract liquid of the scaffolds(50% concentration), after 1, 3, and 7 days of culture, Live-Dead Cell Staining Kit (Calcein-AM and PI, Abcam, Shanghai, CHINA) was added to each well, incubated for 30 min at 37 °C, then the live cells (green) and dead cells (red) were observed with fluorescence excitation of 490 nm and 535 nm by Fluorescence microscope. For cells adhesion, scaffolds were put in the 24 well plate after sterilization, cells were seeded in 24 well plate with scaffolds (4 × 10 4 /well). After 12 h of co-culture, cells on scaffolds were observed by scanning electron microscope. In vitro osteogenic differentiation of scaffolds. The in vitro osteogenic differentiation of scaffolds were assessed by ALP staining and alizarin red S staining. Briefly, rBMSCs were seeded at a density of 2 × 10 4 cells per well in a 24 well plate for ALP staining, while a density of 1 × 10 5 cells per well in a 6 well plate for alizarin red S staining 39 . Then scaffolds were placed into wells inoculated with cells for stabilizing overnight, the culture www.nature.com/scientificreports/ medium (Gibco, Thermo Fisher Scientific Inc. Grand Island, NY,USA) was changed to the osteogenic medium (OSM, comprised of 10 nM of dexamethasone (Dex), 50 mg/mL of ascorbic acid (AA), and 10 mM of b-glycerophosphate (b-gp) in growth medium, Biological Industries, Kibbutz Beit Haemek, Israel). Mineralization was detected by alizarin red S staining after 21 days of culture, cell differentiation was studied by ALP staining after 7 days of culture. Alizarin red S staining (Sigma-Aldrich, St. Louis, MO, USA) was performed according to the manufacturer's instruction. ALP staining (Sigma-Aldrich, St. Louis, MO, USA) of rBMSCs was performed according to the manufacturer's instruction, the stained cells were photographed using a microscope. In vivo rabbit radius bone defects repair. Animal experiments were carried out on rabbit radius bone defects model. All animal use procedures were according to the NIH guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978) and were approved by the Experimental Animal Ethics Committee of Nanchang University. Twelve New Zealand white male rabbits, 6 months old with 2.5-3 kg of weight, were randomly divided into four groups corresponding to blank, BGC, BGC-Mg and Mg scaffolds. 3 rabbits were used for each group. All the rabbits were anesthetized with chloral hydrate (10%, v/v, 2.5 ml/kg, CAS 302-17-0, Aladdin, Shanghai, CHINA), and a 20 mm longitudinal incision was made along the radius. After the skin and musculature were separated, a 15 mm bone defect was made using a reciprocating saw. The bone defect models were established and divided into above four groups. Experimental groups BGC and BGC-Mg and Mg represents implanting with BGC scaffolds, BGC-Mg scaffolds and Mg scaffolds respectively, while the blank group was kept empty as control. The incisions were closed using resorbable suture, and the rabbits were given three days of intramuscular injection of penicillin (CAS 69-57-8, 61-33-6, Aladdin, Shanghai, CHINA) 10,000 units per day. The rabbits were sacrificed with an overdose of chloral hydrate and tissue harvest after 8 weeks of surgery 40 . ## Image-based analyses (X-ray and Micro-CT) and histomorphometric analyses. To evaluate new bone formation in the bone defect sites, the surgery regions were radiographed using an X-ray instrument at each time point which indicates the dynamic changes of the scaffolds 41 . Radiographs were obtained at a suitable magnification, and the degree of new bone formation was determined by the grey scale from the X-ray imaging system. For micro-CT observation 42 , the radius was scanned using a micro-CT imaging system with 60 kV and 300 µA. After micro-CT analysis, the harvested bone specimens were fixed in 10% formalin (CAS 50-00-0, Aladdin, Shanghai, CHINA), dehydrated with a graded ethanol series, defatted with chloroform (CAS 71-55-6, Macklin, Shanghai, CHINA), decalcified using 0.5 M EDTA (pH 8.0, Sigma-Aldrich, St. Louis, MO, USA) for 30 days, and embedded in paraffin blocks sequentially. Vertical sections with a 5 µm thickness were cut from the middle of defect using a microtome, and then stained with H&E (Solarbio, Beijing, CHINA) and Masson's trichrome (Solarbio, Beijing, CHINA) for microscope observation 43 . New bone area was measured using the PhotoShop software (Adobe Systems Inc. USA) and calculated by using the following equation: New bone area (%) = An/Ao × 100%, where An and Ao are the new bone area and original defect area, respectively. For this analysis, eight images were randomly obtained in the same section. For immunohistochemistry, the slides were stained with anti-Bmp2, anti-Col1a1 and anti-OCN antibodies (Thermo Fisher Scientific Inc. Grand Island, NY, USA) 44 . Integrated optical density (IOD) value of the positive area of immunohistochemistry images were measured by Image-pro plus 6.0 (Media Cybernetics, Inc, Rockville, MD, USA). ## Statistical analysis. All experiments were repeated a minimum of three times. Experimental results are presented as the mean ± the standard deviation (SD). Data were analyzed by a two-tailed Student's t-test as appropriate for the data set. Statistical analysis was performed using SPSS 19.0 software (IBM Corporation, USA). Values of p < 0.05 were considered significant, while p < 0.01 were considered very significant.

chemsum

{"title": "Magnesium-alloy rods reinforced bioglass bone cement composite scaffolds with cortical bone-matching mechanical properties and excellent osteoconductivity for load-bearing bone in vivo regeneration", "journal": "Scientific Reports - Nature"}

hydrogen_bonding-induced_oxygen_clusters_and_long-lived_room_temperature_phosphorescence_from_amorph

## Abstract: The study of non-conjugated luminescent polymers (NCLPs) with fluorescence and long-lived room-temperature phosphorescence is of great significance for revealing the essence of NCLPs luminescence, which has gradually attracted the attention of researchers in recent years. Herein, polymethylol (PMO) and poly(3-butene-1,2-diol) (PBD) with polyhydroxyl structures were prepared and their luminescence behaviors were investigated to further reveal the clusteroluminescence (CL) mechanism. Compared with the weak or even non-luminescent behavior of polyvinyl alcohol, PMO and PBD exhibit cyan-blue fluorescence with quantum yields of ca. 12% and green roomtemperature phosphorescence with lifetimes of ca. 89 ms in the solid state. Both fluorescence and phosphorescence exhibit typical excitation-dependent CL behavior. Experimental and theoretical analyses show that the strong hydrogen-bonding interaction of PMO and PBD greatly promotes the formation of oxygen clusters and the through-space n-n interaction of oxygen atoms, enabling fluorescence and phosphorescence emission. The results of this work have important implications for understanding the clusteroluminescence mechanism of NCLPs and provide a new polymer design strategy for the rational design of novel NCLPs materials. ## Introduction Light is an important factor for human survival, health, and development, while fluorescence and phosphorescence are two types of light that humans simulate in nature and play a vital role in optoelectronic devices, chemo-/bioprobes , biological imaging, and other fields . Conventional wisdom holds that chromophores with well-defined large conjugated groups are required to achieve fluorescence or/and phosphorescence emission . However, in recent years, numerous studies have found that many natural and synthetic polymers or small molecules, in the absence of well-defined chromophores or conjugated structures, also exhibit fluorescence or/and even room temperature phosphorescence (RTP), such as polyether , polyester , natural products , poly(maleic anhydride) derivatives , tertiary amine derivatives , poly(hydroxyurethane) , and polysiloxane . The structures of these molecules usually contain heteroatom groups (such as N, O, S, etc.), and their luminescence exhibits concentrationdependent, solid-state fluorescence, and excitation-dependent emission, i.e., typical clusterization-triggered emission (CTE) or clusteroluminescence (CL) properties . The classical through-bond conjugation theory is difficult to explain such non-conjugated luminescence molecules. In this case, CTE or CL has been widely recognized and concerned by researchers since it was proposed [9a, 14] . However, owing to the inclusion of both n and π electrons in the molecular structure, the intrinsic CL mechanism remains obscure, although it has been tentatively uncovered in some previous works [6, 7b, 15] . Therefore, it is urgent to construct a class of typical luminescent model molecules with simple and well-defined structures to further clarify the CL mechanism. Phosphorescence is another aspect and channel to reveal the CL mechanism. But for spinforbidden phosphorescence, the vibration and rotation of molecules and the effects of external conditions such as oxygen and moisture greatly limit the generation of phosphorescence, especially for RTP. To achieve phosphorescence emission, facilitating the singlet-to-triplet intersystem crossing (ISC) to populate the triplet and stabilizing the triplet excitons to inhibit the nonradiative transition pathways are key principles. The phenomenon of RTP has long been synonymous with metallic and inorganic complexes . Nonetheless, over the past few years, purely organic luminophores have gradually been endowed with long-lived RTP through precise molecular design. The main strategies to achieve its RTP are the introduction of heavy atoms (e.g., halogens) , crystallization , and host-guest interactions . For CL, crystallization is an effective and commonly used approach to achieve RTP emission, which can induce intramolecular motion restriction to generate rigid molecular conformations to suppress nonradiative decay [6, 15a, 20] . However, the crystallinity of polymers tends to vary greatly depending on post-processing methods, which affects the emission intensity and lifetime of their RTPs and restricts their specific practical applications. Instead, hydrogen bonding (H-bonding) is a crystallization-like strategy that can be readily constructed in amorphous polymers to achieve conformational rigidification, and many RTP systems also select polymers with multiple hydrogen bonds (e.g., polyvinyl alcohol (PVA)) as a matrix . But developing amorphous RTP NCLPs and revealing their luminescence mechanism remains a challenge. In this work, PMO and PBD with one hydroxyl group on each carbon atom in the backbone and side chain were designed and synthesized, and their luminescence properties were studied in detail to further understand the CL mechanism. The extremely strong H-bonding of PMO and PBD induces the generation of oxygen clusters and through-space n-n interactions of oxygen atoms, which is the source of the strong fluorescence and long-lived RTP. Theoretical calculation analysis shows that the distance between the large number of oxygen atoms is between 2.58−2.83 , which is less than twice the van der Waals radius of oxygen atom (rB: 1.52 ; rP: 1.40 ). The existence of oxygen clusters and through-space n-n interactions are further confirmed. The above results also fully demonstrate that even without crystallization and π electrons, CL can be realized through the action of H-bonding. And if the H-bonding is strong enough, nonradiative decays can also be suppressed to produce RTP. ## Results and Discussion Polyvinyl alcohol (PVA), a well-known polymer with a polyhydroxyl structure, possesses one dissociative -OH group on every two carbon atoms in the backbone. In contrast to PVA, PMO and PBD have one -OH group on each carbon atom in the backbone and side chain (Figure 1). It is of considerable interest in view of strong H-bonding in the study of photophysical properties. To synthesize PMO and PBD, poly(vinylene carbonate) (PVC) and poly(vinylethylene carbonate) (PVEC) were firstly prepared by the radical polymerization of vinylene carbonate and vinylethylene carbonate using AIBN as a radical initiator, respectively (Schemes S1-S2) . The proton nuclear magnetic spectroscopy ( 1 H NMR) and gel permeation chromatography (GPC) data indicated that PVC and PVEC were successfully synthesized, and their number-averaged molecular weights (Mn) and polydispersity indexs (PDI) were 102.8 kg/mol, 1.4 for PVC and 52.1 kg/mol, 1.2 for PVEC, respectively (Figure S1-S4). Then, PVC and PVEC were hydrolyzed in strong alkaline solution to obtain pure white PMO and PBD powers according to literatures (Figure 1 and Schemes S1-S2) . Fourier-transform-infrared (FT-IR) spectra showed that the C=O stretching vibrations of the fivemembered cyclic carbonate of PVC and PVEC at ca. 1800 cm -1 disappeared completely, proving the successful synthesis of PMO and PBD (Figures S5-S6). The glass transition temperatures (Tgs) of PMO and PBD can reach 183.3°C and 113°C, indicating amorphous rather than crystalline states (Figures S7-S8). However, owing to the extremely strong H-bonding, they can't be dissolved in any solvent, which extremely limits the study of optical behaviors in solution. As shown in the structure of Figure 1, there are no other heteroatoms and π electrons in PMO and PBD except oxygen atoms and n and σ electrons. Nonetheless, both PMO and PBD powders exhibited cyan-blue fluorescence and long-lived green RTP with a duration of 2.0 s, which belonged to the typical CL chromophores. To reveal the CL mechanism, PVA showing very weak fluorescence was chosen as a control owing to the similarity in molecule structure. The fluorescence and phosphorescence quantum yields (QYs) of PMO and PBD are 6.83%/5.32% and 6.94%/5.17%, respectively, which are relatively respectable values in NCLPs with RTP, especially for some NCLP systems with only oxygen atoms. Because PMO and PBD have similar optical properties, here the PMO is taken as an example for detailed description. The pure white PMO powder showed distinct excitation-dependent photoluminescence (PL) properties (Figure 2a), similar to many of CL chromophores reported before. The spectrum covered an emission band from 350 to 600 nm, with an emission peak of 438 nm excited by 360 nm (Figure 2a). The fluorescence lifetime measured at the emission peak of 438 nm was 3.95 ns (Figure 2b). Based on the theory of throughbond conjugation, theoretically, there's no fluorescence in PMO because there is no definite conjugation unit in the molecular structure of PMO. Although the presence of oxygen atoms results in n-σ* electronic transitions, the energy gap of the (n, σ*) transition is too high to emit visible light. For example, the energy gap of (n, σ*) transitions of methanol is around 6.7 eV, corresponding to light with a wavelength of 183 nm. Also, the transitions are related to the promotion of an electron from a nonbonding n orbital to σ* antibonding orbital, which are forbidden transitions and therefore are weak intense. Therefore, the fluorescence of PMO does not originate from the (n, σ*) transition of oxygen atom. So, what is the origin of such unusual PL? Tang and Yuan at al. 29] proposed the CTE mechanism and TSI from isolated aromatic rings and heteroatoms with lone-pair electrons to rationally reveal the PL origin of NCLPs. In this case, the only possibility is that the fluorescence originates from the through-space n-n interaction of oxygen. Owing to the overlap of n electrons of oxygen atoms in PMO, new orbitals with lower HOMO-LUMO gaps from oxygen clusters will be generated compared to single oxygen atoms, which can absorb and emit lower-energy (longer-wavelength) light. Furthermore, differences in TSI degree lead to the emergence of different HOMO-LUMO gaps from diverse oxygen clusters, leading to excitationdependent emission characteristics. Meanwhile, green RTP emission with a maximum emission peak at 500 nm and a lifetime of 89.17 ms was observed (Figure 2c-2d), which is comparable to the lifetime of some crystalline small molecules. Similar to the steady-state PL spectrum, the phosphorescence spectrum also shows excitation-dependent emission in the range of 462 to 500 nm at excitation wavelengths from 300 to 360 nm (Figure 2e). This further confirms the existence of diverse oxygen clusters with different conjugation degrees. And the excitation-dependent emission provides an efficient method to realize multicolor fluorescence and RTP emission. For such long-lived RTP emission, polymerization and extremely strong H-bonding play a key role. As reported in our previous work, polymerization is a very efficient method to achieve PL and RTP emission, namely polymerization-induced emission. When the degree of polymerization (DP) of the PMO is 1, 2 or 3, i.e., methanol, ethylene glycol, and glycerol, they emit no PL and RTP as we all known (Figures S9-S11). For erythritol, xylitol, D-mannitol/D-glucitol with DP of 4, 5 and 6, respectively, they are all crystalline. As reported by Yuan and coworkers, crystalline xylitol showed weak blue fluorescence with a QY of 1.5 and an RTP, but not a long phosphorescence lifetime even at a low temperature of 77 K. This suggests that polymerization can induce stronger through-space interaction than crystallization to boost PL and RTP to some extent. Therefore, for amorphous PMO, there must be a critical DP (CDP) to achieve CL. However, owing to the polydispersity of polymers, it is difficult to synthesize monodisperse PMO. So here we can't get the value of CDP experimentally, but it must exist. Another factor that should be emphasized is Hbonding. In fact, polymerization is only a prerequisite for the generation of oxygen clusters and TSI. The H-bonding is the key to fluorescence and RTP, and the H-bonding strength must be strong enough. For example, for PVA with one less hydroxyl group in the building block, the very weak emission signal in the PL spectrum is consistent with what we observed with the naked eye (Figures 1 and 2f). To some extent, the H-bonding strength can be reflected by solubility and Tg. PVA is soluble in hot water and the highest Tg can reach up to 85 o C. Compared to insoluble PMO with a Tg of 183.3°C, the H-bonding strength of PVA is much lower than that of PMO. Therefore, only strong H-bonding can induce the through-space n-n interactions of oxygen atoms and further orbital splitting, showing PL emission. In addition, strong H-bonding promotes conformational rigidification and significantly blocks nonradiative deactivation channels, conferring long-lived RTP emission. Like many traditional chromophores or PL materials without RTP, RTP appears once they diffuse into PVA or other polymers with strong H-bonding. This work provides another avenue to understand the mechanism of PL and RTP. The similar optical properties were observed in PBD with neighboring hydroxyl groups in the side chain (Figure 3), confirming the significance of neighboring hydroxyl groups for fluorescence and RTP. As shown in Figure 3a, it also exhibits excitation-dependent PL emission and emits the same emission peak at 438 nm excited by 360 nm. The RTP peak position and lifetimes of fluorescence and phosphorescence are close to those of PMO (Figure 3b-3d). Therefore, whether the neighboring hydroxyl groups are located in the backbone or side chain has no effect on their luminescent properties. The strong intra-/intermolecular H-bonding interactions of PBD also results in insolubility in most solvents. In other words, when monomers with adjacent hydroxyl groups are polymerized, strong H-bonding can induce physical crosslinking, exhibiting strong intra-/intermolecular interactions. It is further demonstrated the through-space interaction between the oxygen atoms. To further fully confirm that the fluorescence and RTP originate from the through-space n-n interaction of oxygen atoms, optimized conformations of PMO, PBD and PVA based on single polymer chains with fourteen constitutional units were calculated by density functional theory (DFT) at B3LYP/6-31(d, p) level (Figure 4a-4c). Ethylene glycol and 1,2-propanediol, as repeating building blocks of PMO and PBD, were selected as controls and optimized at the same level (Figure 4d-4e). Theoretical calculation analysis shows that the distance between most of the oxygen atoms in PMO and PBD is between 2.58−2.83 (Figure 4a, 4c and Tables S1-S2), which is less than twice the van der Waals radius of the oxygen atom (dO) (rB: 1.52 ; rP: 1.40 ). But for PVA, there are almost no short contacts between oxygen atoms, and most of the oxygen atoms are at a distance greater than dO (Figure 4b and Table S3). Furthermore, for ethylene glycol and 1,2-propanediol, the distance between adjacent hydroxyl groups is about 3.6 , which is much larger than dO. Indeed, no fluorescence was detected in ethylene glycol and 1,2-propanediol (Figures S10 and S12). The importance of polymerization for TSI is well demonstrated, and the above results fully confirm that the fluorescence and RTP of PMO and PBD are ascribed to the through-space n-n interaction between oxygen atoms. In this case, the overlap of electron clouds of oxygen atoms leads to the splitting and coupling of the orbitals and the generation of new molecular orbitals with smaller energy gaps for visible light emission (Figure 4f). The resulting molecular orbitals correspond to the blue visible light of PMO and PBD. Owing to the difference in the distance between the oxygen atoms, the degree of electron cloud overlap and TSI is also different. Thus, it results in the generation of molecular orbitals with different energy gaps and the emergence of excitationdependent PL and RTP emission. That is, the excitation-dependent PL and RTP emission are attributed to diverse oxygen clusters with different conjugated degrees, as detailed schematic diagram is shown in Figure 4f. ## Conclusion In summary, a novel class of amorphous polylols with fluorescence and long-lived RTP properties was prepared. Experimental results and theoretical calculations prove that the through-space n-n interaction of oxygen atoms is the fundamental cause of fluorescence and RTP. Results from controls (ethylene glycol, 1,2-propanediol, and PVA) confirmed that polymerization and H-bonding play key roles in the generation of oxygen clusters and TSI. The difficulty of studying the photophysical behavior of PMO and PBD in solution limits the in-depth understanding of throughspace n-n interactions to a certain extent. Our ongoing efforts are to seek a soluble strong Hbonded NCLP and to develop NCLPs with better optical performance. This work not only provides a new strategy for the design and construction of fluorescence and RTP materials, but also sheds new light on the CL mechanism of NCLPs.

chemsum

{"title": "Hydrogen Bonding-Induced Oxygen Clusters and Long-Lived Room Temperature Phosphorescence from Amorphous Polylols", "journal": "ChemRxiv"}

an_effective_and_versatile_strategy_for_the_synthesis_of_structurally_diverse_heteroarylsilanes_<i>v

## Abstract: A versatile silylation of heteroaryl C-H bonds is accomplished under the catalysis of a well-defined spirocyclic NHC Ir(III) complex (SNIr), generating a variety of heteroarylsilanes. A significant advantage of this catalytic system is that multiple types of intermolecular C-H silylation can be achieved using one catalytic system at a, b, g, or d positions of heteroatoms with excellent regioselectivities. Mechanistic experiments and DFT calculations indicate that the polycyclic ligand of SNIr can form an isolable cyclometalated intermediate, which leaves a phenyl dentate free and provides a hemi-open space for activating substrates. In general, favorable silylations occur at g or d positions of chelating heteroatoms, forming 5-or 6-membered C-Ir-N cyclic intermediates. If such an activation mode is prohibited sterically, silylations would take place at the a or b positions. The mechanistic studies would be helpful for further explaining the reactivity of the SNIr system. Scheme 1 Representative organosilanes and intermolecular C-H silylation. ## Introduction Organosilanes have emerged as an important class of compounds with diverse utilities, 1 serving as versatile organic reagents to mediate many novel organic reactions, 1a,2 functional materials, 3 therapeutic pharmaceuticals, and bioactive chemicals (Scheme 1). 4 Traditionally, silicon-containing molecules have been prepared through the reactions of equivalent organometallic species with electrophilic silicon reagents, 5 which suffer from inferior atom-economy and low functionalgroup tolerance. For several decades, transition-metalcatalyzed intermolecular direct C-H silylation has been developed as a more efficient and attractive strategy. 1c-e Among them, C(sp 2 )-H silylation can generally be promoted with a directing group, which could coordinate with the metal center to form cyclometalated species and thus improve the regioselectivity of reaction. In this feld, a range of directing groups have been developed, 6 which include strongly coordinating pyridines and various azoles, and more weakly coordinating imines, amides, esters, and ketones. In particular, Hou reported an alkoxyldirected Sc-catalyzed silylation of various anisole derivatives. 6a In 2009, a distinctive strategy of using easily-installed and -removed 2-pyrazol-5-ylaniline as a directing group for o-silylation of arylboronic acids has been developed by Suginome. 6b In comparison, undirected C(sp 2 )-H silylation 7 is more challenging due to the loss of interaction between the coordinating group and the catalyst. A breakthrough in undirected silylation was established by Hartwig, 7a which takes advantage of steric effects in controlling regioselectivities. In addition, C(sp 3 )-H bond silylation at the benzylic position 8 of the aromatic ring or next to the heteroatom such as nitrogen 9 or sulfur 10 was also reported, which has expanded the substrate scope and applicability of silylation reaction. Despite those precedent achievements on either directed or undirected C-H silylation reactions, a certain catalytic system could usually be used to activate a specifc type of substrate. Therefore, development of a more general catalytic system for C-H silylation of multiple types of substrates with high regioselectivities for each type of reaction would be in high demand, considering the versatility of this strategy. ## Results and discussion In our previous work, we have developed a well-defned dianionic Ir(III) CCC pincer catalyst (SNIr), 11 which features unique double C(sp 2 )-H bond activation in a polycyclic ligand framework. This unexpected chelation mode reminds us that the central Ir may potentially enable C-H activation upon cleavage of the phenyl Ir-C bond to provide a hemi-open space for substrate activation under certain conditions. Base on this hypothesis, we have developed a versatile strategy for Ir(III)catalyzed C-H silylation of diverse heteroarylsilanes. Herein we present our research results. We started our investigation with 2-phenylpyridine 1a as the model substrate and Et 3 SiH as the silane source to screen the catalysts A-C. A mixture of 1a and A-C (2.5-5 mol%) was frst stirred at 100 C for 6 h. Then a hydrogen acceptor (3 equiv.) and Et 3 SiH (2 equiv.) were added for further reaction. The results are summarized in Table 1. To our delight, the chloride catalyst B could give the highest yield of the desired silylation product 2a (entries 2 vs. 1 and 3), and no reaction was observed in the absence of Ir catalysts or hydrogen acceptors (entries 4 and 5). Further investigation found that tert-butylethylene (tbe) was the most effective hydrogen acceptor (entries 7 vs. 2 and 6). When an increased loading (5 mol%) of B was used in o-xylene solvent, the yield was improved to 85% (entries 9 vs. 7 and 8). Notably, when all reactants and catalysts were added to the reaction simultaneously, the system would become complicated and give a relatively low yield (entry 11). With the optimized conditions in hand, 12 the substrate scope of g silylations with a series of 2-phenylpyridine substrates was frst explored. As shown in Table 2, high yields and regioselectivities were obtained in most cases, while the reaction efficiency could be influenced with the variation of the substitution pattern of substrates. Specifcally, for substituted 2-phenylpyridine (1a-1j), the o-or p-methyl substitution on the benzene ring gave better product yields (83% for 2b, 87% for 2d) compared with the m-substitution (51% for 2c). The substrates with the p-EDG substituted phenyl group could give much higher yields than those with p-EWD substitution (2d and 2g vs. 2e and 2f). A signifcant substituent effect was also observed on different positions of the pyridine ring. For example, 2-methyl substitution afforded a higher yield than 3,4-substitutions (2h vs. 2i and 2j). The scope could be further extended to benzofused substrates (1k-1p), whose reactions could generally afford the desired products in good to high yields (75-92%). Notably, 2-phenylquinoline and 1-phenylisoquinoline could give excellent higher yields (92% for 2l, 90% for 2o). Moreover, for other N-heteroarenes, such as azo-, pyrazolyl-, and iminyl-arenes (1q-1t), they were also amenable in the reaction, affording the corresponding products with high efficiency. In particular, substrates 1s and 1t could mainly give disilylation products in moderate yields along with a trace amount of monosilylation product. Besides, our catalytic system was also well effective toward more inert g C(sp 3 )-H bonds linked to heteroarenes. As a representative example, 8-methylquinoline could afford the gsilylation product 2u in 95% yield. The reactions of 2,6-diethylpyridine (1v) and 2-dimethylaminopyridine (1w) were also feasible, giving products in moderate yields under conditions with elevated temperature. Remarkably, our catalytic system also accommodated the silylation of 1a with other hydrosilanes, such as Ph 3 SiH, Ph 2 MeSiH, or PhMe 2 SiH with good regioselectivities (2x-2z). It is worth nothing that in all cases we were not able to detect other a, b or d silylation products. Subsequent investigation was carried out toward the d-silylation of 2-benzylpyridine 3, 13 and the desired products could be generated in good to high yields in most cases (Table 3). Generally, a higher reaction temperature (120 C) was required than the corresponding g C-H silylation, possibly due to a higher activation energy for the formation of the 6-membered cyclometalated intermediates. Similarly, both electron and steric effects of the benzyl group showed signifcant influence on the reaction outcome. For example, p-EDG substituted substrates gave higher yields than the p-EWG substituted ones in general sense (4d and 4e vs. 4i and 4k). However, for the o-or m-substitutions, both reactions were sluggish and gave poor to moderate yields regardless of either EDG or EWG substituents (4f, 4g and 4j). Delightedly, 2-phenoxypyridine afforded the best result (94% for 4h), probably because of the double activation of the same C-H bond (N to d-C and O to b-C) and electro-donating effect of the ether group. Compared with g C-H silylations of benzo-phenyl pyridines (92% for 2l, 90% for 2o, Table 2), a slow reaction rate and decreased product yields were observed for these d-silylations (74% for 4m, 65% for 4n). Finally, we investigated more universal and practically useful heteroarenes (Table 4), and these silylation reactions showed extremely good regioselectivities and broad substrate scope. For thiophene (5a, 5j and 5k) and furan (5b and 5l) derivatives, silylations generally took place at a positions with good yields, which complemented the normal electrophilic Friedel-Crafts silylation reactions. 1e,14 Further investigation was focused on the derivatives of indole 5c-5i as they have practical utilities in the felds of natural products and drug discovery. 15 In general, silylation always occurred at C-2 positions of indoles except for N-tosyl substituted indole, which directed the silylation to an unusual b-position (6d). 7d The results of a-silylations of indoles indicated that EDG substitutions would give better outcomes than the EWG substitutions (6e-6g vs. 6h and 6i). As for the substituted 2-methyl quinolines and benzo(b)quinoline, b-silylation would occur to afford 6m-6p in moderate to good yields. Moreover, this catalytic mode was also well effective toward the Table 3 Dehydrogenative silylation of d C-H bonds of heteroarenes a a Unless otherwise specifed, reactions were conducted by pretreatment of a solution of 3 (0.5 mmol) and cat. B (5 mol%) in o-xylene (0.5 mL) at 120 C for 6 h, and then tbe (1.5 equiv.) and Et 3 SiH (3 equiv.) were added for further reaction. Computational studies were next conducted to explore the mechanism using 3a (2-BnPy) as a model (Fig. 1). 6f, 16,17 Initially, the cod ligand of cat. To further probe the mechanism, several control experiments were conducted (Fig. 2). First, reactions of 1b or 3d and catalyst B without Et 3 SiH at 120 C for 6 h could generate two brown complexes 1bB and 3dB in 88% and 92% yields, respectively. 1 H NMR, high resolution mass spectroscopy (HRMS) and X-ray analysis confrmed that these complexes contained either a 5-or 6-membered C-Ir-N ring formed from the substrates and catalyst, and both intermediates had a free phenyl group dissociated with Ir. 18 Furthermore, the silylation products 2b and 4d could be generated in 65% and 77% yields, respectively, when 5 mol% 1bB or 3dB was directly used as a catalyst under standard conditions. These results suggested that the iridacycle intermediates might serve as the pre-catalysts during the reaction process. Next, the H/D exchange experiment indicated that the C-H bond activation step might be irreversible (Fig. 2b). The kinetic isotope effect experiment showed a value of 3.1 from two parallel reactions and a KIE of 2.4 from intermolecular competition, which indicated that the C-H bond cleavage process was likely involved in the rate-determining step (Fig. 2c). ## Conclusions In summary, we have developed a general catalyst system based on SNIr for intermolecular C-H silylation of a wide range of substrate types with excellent regioselectivities and good to high yields. In all examples, single silylation products can be obtained in high regioselectivities. Mechanistic experiments and

chemsum

{"title": "An effective and versatile strategy for the synthesis of structurally diverse heteroarylsilanes <i>via</i> Ir(<scp>iii</scp>)-catalyzed C\u2013H silylation", "journal": "Royal Society of Chemistry (RSC)"}

ai-based_atomic_force_microscopy_image_analysis_allows_to_predict_electrochemical_impedance_spectra_

## Abstract: www.nature.com/scientificreports/ (tethered bilayer membranes), and in some cases though not being structural method per se, provides insights into lateral distribution of defects in membranes [8][9][10] . So far, however, there were no attempts to quantitatively relate structural data obtained by AFM and the membrane conductance data measured by EIS, even though experimental capabilities to apply both techniques on the same membrane samples are straightforward. Such comparative measurements would be of great value in studying function of both single and multiple ensembles of membrane damaging protein entities as well as in developing precision biosensors based on tBLMs 11,12 .Recently, significant progress has been made in the development of EIS data analysis of solid supported (tethered) phospholipid membranes [8][9][10]13 . In particular, the theoretical analysis demonstrated that the amount of reconstituted protein pores per surface area can be retrieved from the EIS spectral data. Nevertheless, such theoretical approaches, strictly speaking, should be verified by using data from the independent structural techniques such as AFM.The objective of current study is to explore the possibility to predict the electrochemical impedance spectra from the AFM images of membranes with reconstituted PFTs. The AFM technique allows to detect PFT entities which appear on tBLM surface upon exposure of bilayer to the protein solution. The coordinates of these entities may be measured, and the finite element analysis (FEA) can be applied to model EIS response of such supported membranes. The comparison of predicted and experimental EIS curves obtained from the same sample would allow (1) to independently verify the applicability of FEA approach to theoretically predict EIS spectra developed earlier 9,10 on real, AFM imaged surfaces, (2) to precisely evaluate the physical parameters of supported bilayer membranes, among which the specific resistance of submembrane reservoir separating bilayer from the solid support is of upmost importance. This parameter is strongly correlated with the density of PFT defects in tBLMs 13,14 , therefore, independent verification by AFM can resolve the ambiguities related to such correlation.Typically, only a tiny patch compared to a whole surface area is interrogated by the AFM technique. To establish representative defect densities and their distribution patterns, the sufficiently large areas, in our case, containing hundreds and thousands of defects must by tested. The determination of coordinates of large defect ensembles is a highly time consuming process. To overcome such and similar problems automated algorithms can be applied for AFM image analysis.Typically, the features of different shapes in AFM images are detected via particle or grain analysis based on edge detection. In the majority of cases, a pre-processing takes place to make it easier to measure and observe the features that have been measured 15 . AFM images are always affected by the geometry of a tip and external noise that disturb image features. Although basic image segmentation approaches work well for good-quality image data containing clear and easily distinguishable objects, analysis of noisy, low-resolution or otherwise degraded images requires more sophisticated methods. An important factor is the scarcity of such image data which limits the possibilities of applying machine learning or deep learning methods in a practical way. In some cases researchers still resort to manual work of annotating and quantifying objects of interest in microscopy images 7,16 .Despite the difficulties associated with the automated analysis of AFM images, substantial progress has been recently made in developing practical solutions for certain types of such problems. Meng et al. 17 presented an algorithm based on local adaptive Canny edge detection and circular Hough transform which is suitable for recognizing particles in scanning electron microscope (SEM) or transmission electron microscope (TEM) images. Another study conducted by Venkataraman et al. 18 showed that rotavirus particles in AFM images can be detected by applying a series of image pre-processing, segmentation and morphological operations. Marsh et al. 19 proposed the Hessian blob algorithm for detecting biomolecules in AFM images and showed its superiority against the threshold and watershed image segmentation algorithms. Other recent studies also showed that deep learning techniques can be successfully applied to detect complex-shaped objects in microscopy images. Sotres et al. 20 used the YOLOv3 object detection model and a Siamese neural network to determine the locations of DNA molecules in AFM images and identify the same molecule in different images. Okunev et al. 21 applied a Cascade Mask-RCNN neural network to detect metal nanoparticles in scanning tunneling microscopy (STM) images. In both of these cases the researchers used precision and recall metrics to measure the performance of the proposed models. One more study by Sundstrom et al. 22 involved a supervised learning approach of estimating lengths of DNA molecules in AFM images. A software tool for the automated biomolecule tracing in AFM data (TopoStats) was also recently developed and presented by Beton et al. 23 In this study we investigate the problem of automated detection of membrane bound PFTs in AFM images. Performing this task with adequate accuracy is of practical importance, as the determined coordinates would allow to theoretically calculate EIS spectral features and to compare those features with the experimental EIS data. In addition to applying and testing one of the popular computer vision techniques-convolutional neural network, we present a method for generating synthetic defect sets which resemble detection results of varying accuracy, similar to those obtained by using an actual object detection model. Such datasets are used to perform FEA modeling of EIS spectra and examine the relationship between defect detection accuracy and corresponding variations of EIS spectral features. By doing so we address the question-whether there is some minimal requirement for the precision of the AI based image processing algorithm so that the EIS spectra prediction would fall into acceptable range of uncertainty? MethodsAFM imaging. AFM image data was obtained by measuring three separate tBLM membrane cells. Assembled tethered lipid bilayers were incubated for 30 min with vaginolysin (VLY). Aliquot of a toxin was added to the cell, so that final concentration of VLY was 1 nM . After incubation, cell was washed with 10 mL of phosphate buffer pH7.1 to remove any unbound protein debris, and disassembled under water. AFM imaging was carried out in aqueous environment. More detailed description of experimental settings can be found elsewhere 10 . ## AI-based atomic force microscopy image analysis allows to predict electrochemical impedance spectra of defects in tethered bilayer membranes Tomas Raila 1 , Tadas Penkauskas 2 , Filipas Ambrulevičius 2 , Marija Jankunec 2 , Tadas Meškauskas 1 & Gintaras Valinčius 2* Atomic force microscopy (AFM) image analysis of supported bilayers, such as tethered bilayer membranes (tBLMs) can reveal the nature of the membrane damage by pore-forming proteins and predict the electrochemical impedance spectroscopy (EIS) response of such objects. However, automated analysis involving pore detection in such images is often non-trivial and can require AI-based object detection techniques. The specific object-detection algorithm we used to determine the defect coordinates in real AFM images was a convolutional neural network (CNN). Defect coordinates allow to predict the EIS response of tBLMs populated by the pore-forming toxins using finite element analysis (FEA) modeling. We tested if the accuracy of the CNN algorithm affected the EIS spectral features sensitive to defect densities and other physical parameters of tBLMs. We found that the EIS spectra can be predicted sufficiently well, however, systematic errors of characteristic spectral points were observed and need to be taken into account. Importantly, the comparison of predicted EIS curves with experimental ones allowed to estimate important physical parameters of tBLMs such as the specific resistance of submembrane reservoir. This reservoir separates phospholipid bilayer from the solid support. We found that the specific resistance of the reservoir amounts to 10 4.25±0. 10 • cm which is approximately two orders of a magnitude higher compared to the specific resistance of the buffer bathing tBLMs studied in this work. We hypothesize that such effect may be related in part due to decreased concentration of ionic carriers in the submembrane due to decreased relative dielectric permittivity in this region. Atomic Force Microscopy (AFM) is increasingly used for studying interaction of lipid bilayers with proteins including pore-forming toxins (PFTs) and membrane disrupting peptides . AFM is capable of detecting insertion of proteins, heterogeneous distribution of proteins in membranes 2 in phase separated membranes 3 , formation of rings of PFTs 1 and other structural details important to understand how membrane protein interact with cell membranes. While providing nanoscale-level structural details of reconstituted PFT's and peptides in membranes, AFM does not directly access function of these proteins, neither it can predict the extent of dielectric damage by PFTs and peptide. Such information is important in establishing fundamental relation between structure and function of biological systems. Because of evident reasons the AFM studies of membrane proteins are performed using solid supported phospholipid bilayers 4 . In case the electrical conductance data reflecting functional effects of PFTs or peptides on membranes is sought the tethered bilayer systems are used 5,6 . Also, both techniques, AFM and EIS, are used simultaneously or in parallel to characterize structure and function of PFTs in membranes . The electrochemical impedance spectroscopy (EIS) is a method of choice for detailed studies of electrical effects of PFTs in membranes. The EIS allows accessing the dielectric properties and conductance data of tBLMs For each cell a surface patch of 6 µm × 6 µm was scanned by capturing one 2 µm × 2 µm fragment at a time. Each fragment was imaged with 512 × 512 resolution, thus the overall stitched image consisting of 3 × 3 fragments had 1536 × 1536 resolution. Each image fragment was manually annotated by marking center coordinates (X and Y) of each defect visible in the image. Image fragment sets of each cell were partitioned into training and test subsets by assigning 5 fragments for training and 4 for testing. Test fragments were selected to represent a cohesive 4 µm × 4 µm surface patch at the lower right corner of the fully stitched image. Table 1 shows the total number of annotated defects (N) and average defect density ( N def ) for each AFM image cell and training/test subset. Defect density is expressed as the number of defects per square micrometer. In addition to aforementioned parameters each surface image is also characterized by metric σ which is obtained by computing the Voronoi diagram for a given defect set and calculating the standard deviation of the normalized Voronoi sector areas (multiplied by defect density N def ). This quantity summarizes the degree of defect clustering where higher values correspond to stronger clustering effect (example of defect cluster is highlighted in Fig. 1). Defect clustering has been shown to have significant influence on EIS spectra of tBLM membranes, as presented in earlier research 10 . ## Defect detection accuracy. Although membrane defects are primarily characterized by their center coordinates and defect radius, these attributes can be used to express the defect position in the image as its bounding rectangle. By comparing two sets of bounding rectangles, corresponding to true and predicted defect positions, defect detection accuracy can be quantitatively evaluated. To count the number of correct detections, the bounding rectangle of each true defect position ( B true ) is matched with its closest prediction ( B pred ). The overlap between each such pair of true and predicted bounding rectangles is evaluated by the intersection over union (IoU) metric (1) (also known as Jaccard index), which is expressed as the ratio of bounding rectangle intersection and union areas (Fig. 2): Higher IoU values correspond to a better match between both bounding rectangles. If IoU value is above the chosen threshold (i.e. 0.5), the detection is assumed to be a true positive (TP). Otherwise, if no matching prediction www.nature.com/scientificreports/ exists for a given true position, such detection is counted as a false negative (FN). In the opposite case, when no true bounding rectangle can be matched for a given prediction, a false positive (FP) is assumed. By counting all such cases of correct and incorrect detections, overall defect detection accuracy is summarized by precision and recall metrics 24 : Both precision and recall can also be expressed by the F1 metric: Synthetic defect set generation. In order to assess the relationship between defect detection accuracy and corresponding variations in EIS spectra, a substantial number of defect detection result sets is required. Such detection results should exhibit different precision and recall values distributed in a certain range. However, such specific detection results can be difficult to acquire by applying object detection models trained using real AFM images and annotated true defect positions. We chose an alternative approach of synthetically generating defect coordinate sets which would emulate defect detection results at different accuracy levels. Each synthetic case is generated by starting with the initial set of known true defect coordinates and applying certain modifications (defect addition, removal, coordinate shifting) to acquire a new defect set equivalent to the defects actually being detected by some model with imperfect accuracy. The procedure for generating a series of such synthetic cases from a given true defect set consists of the following steps: 1. Kernel density estimation (KDE) 25 is applied for the set of true defect coordinates. The resulting distribution is used to reduce the chances of defect clustering changing significantly due to new defects being added or existing ones removed. Figure 3 shows an example of a clustered defect set and its corresponding KDE distribution, where warmer colors correspond to the higher values of its probability density function. 2. For each synthetic case: (a) True coordinates ( x (true) and y (true) ) of each existing defect are modified by adding normally-distributed random values: This results in realistically imperfect matches between true and predicted bounding rectangles of the defects. (b) A number n remove of defect coordinate pairs are sampled from the KDE distribution. True defects closest to the sampled coordinates are selected and removed from the initial defect set. This introduces false negatives (FN) into the generated defect set and reduces recall accordingly. (c) A number n add of new coordinate pairs are sampled from the KDE distribution and defects with these coordinates are added into the generated defect set. This represents false positives (FP) and corresponds to lowered precision values. The described algorithm was used to generate the synthetic cases for each of three AFM test images independently. KDE distributions were fitted using the Gaussian kernel and bandwidth parameter set to 400. The standard deviation parameter s of the normal distribution used for defect coordinate shifts was set to 4. Parameters n remove and n add were initially set to 0 and then incremented throughout the generation process by a step quantity corresponding to 3% of true defect count N until the maximum value of N/2 was reached. Table 2 shows the properties (2) Precision = TP TP + FP . (3) of the synthetic defect sets generated by the described procedure. Due to stochastic nature of this algorithm, some variability of clustering effect (expressed in terms of σ ) is still present in the defect sets, as summarized in Fig. 4. ## EIS modeling. Electrochemical impedance (EIS) spectra of each defect distribution are modeled by applying the finite element analysis (FEA) technique. Membrane models were implemented and solved in the same way as described in the previous study 9 . Modeling was performed for each AFM surface from the test set by using the true defect distribution and each of the generated cases, described in "Synthetic defect set generation" and referred to as the predicted set. In order to quantify the discrepancy between the EIS spectra modeled for any given pair of true and predicted defect sets we used the positions of the minima points of the curves (example in Fig. 5) along both frequency and admittance phase axes: www.nature.com/scientificreports/ In order to characterize the relationship between the defect detection accuracy and deviations in the resulting EIS spectra, using F1 metric alone is not enough due to the fact that EIS spectral features are more strongly influenced by the defect size and density than by the specific positions of the defects the membrane surface 9 . For this reason, a predicted defect set might poorly match the true one and thus exhibit a low F1 value, although their corresponding EIS spectra might closely match, as long as the overall properties of defect count and size are similar. To take this effect into account we also use an additional Q N metric which represents the ratio of defect densities (number of defects per square micrometer) from predicted and true defect sets: ## Results and discussion Defect detection with convolutional neural network. To perform the actual defect detection experiments using AFM image data a convolutional neural network (CNN) model was chosen as the current stateof-the-art approach for object detection tasks. Specifically, we used a popular SSD FPN architecture object detector 26 implementing a two-stage object detection approach, where the candidate locations of objects are first identified and then each region is classified separately. Initial model 27 was pre-trained with COCO image dataset 28 to detect objects of 90 different types. In order to adapt it for defect detection in AFM images, the model was re-trained to detect a single type of object (membrane defect) using 15 AFM images described in Table 1 and containing a total of 510 annotated defect instances. Each training image fragment with 512 × 512 resolution was scaled to match the model input of 640 × 640 color (RGB) images. Tensorflow 2.0 framework was used to train and evaluate the model and the training was performed using Nvidia GTX 1080 GPU hardware. The trained model was evaluated with each of 12 test image fragments (Table 1) and the detection results were aggregated to match the layout of 4 stitched fragments per each AFM surface. Bounding boxes of all detected defect instances were equalized to match the width and height of 50 nm, corresponding to defects with circular radius of 25 nm. Defect instances predicted by the model were compared with the true defect positions and the overall model accuracy was evaluated using the precision, recall and F1 metrics for each AFM surface (Table 3). Precision, recall and F1 scores indicate a significant number of inaccurate detections in the test images of all three AFM surfaces. Defect clusters (Fig. 6, left) proved to be difficult to resolve due to poorly visible surface features inside the clusters. However, the model performed fairly well for certain image fragments with no defect clusters present (Fig. 6, right). This is also illustrated by the fact that the test image of AFM surface 3 which indicates the lowest amount of defect clustering in terms of σ (Table 1) also have the highest overall F1 score. ## How much inaccuracies in detection of defects affect the prediction of EIS response of tBLMs? As seen from the previous paragraph, the current AI-based algorithm has limited precision of detection of defects in real AFM pictures. Specifically, as seen from Table 3, both parameter F1, and number of entities Q N are detected with max 75% (F1) and max 96% ( Q N ) precision as judged from the tests on surfaces 1, 2 and 3 (Table 3). It is however, important if inaccuracy in defect recognition can result in significant deviations in predictive power of EIS spectral features. To answer this question we compared the position of characteristic points of EIS spectra obtained via FEA modeling of EIS curves based on coordinates determined by eye ("true coordinates") and EIS curves obtained by applying the AI algorithm. The comparison of the curves are performed by calculating the position of the EIS Bode admittance phase curve minimum in the arg Y vs log f plane. The deviation along the log f axis is measured on a logarithmic scale as f log and the deviation along the arg Y axis is measured on a linear scale as arg Y . Table 3 summarizes the findings. It is obvious that the shift of the position of the phase minima is within the approximate interval 0.1 and -0.027, which translates into the range for relative error in the position of the minimum on a log f scale from 2 to 6%. Even though modern EIS workstations provide much greater measurement precision, given limitations related to the reproducibility of a ( 5) www.nature.com/scientificreports/ specific tBLM experiment such error may be considered as acceptable. The position of the phase minimum on the log f scale is a main parameter from which the defect density can be estimated from the EIS spectra 9,13,14 . So, from this series of tests we may hypothesize that the precision of the prediction of defect density using AI-based algorithm can be increased by recalculating the defect density from the AI-algorithm predicted position of the f min using previously described method 9 . For example, in sample 2, the AI-derived QN is 1.227, i.e, 22.7% more than is located in real AFM images. However, the f log shift is only -0.013, which translates into -3% with respect to a true defect density value. This result is of upmost importance because it suggests that the AI-based AFM image analysis allows to reconstruct EIS spectra with satisfactory precision, while combination of both theoretical analysis techniques, EIS 9 and AI-based AFM image analysis allows to precisely determine defect densities on real tBLM samples. ## Simulation of inaccuracies in detection of defects in tBLMs. In the previous paragraph the evaluation analysis of the AI-based AFM data analysis algorithm was evaluated using images of 3 real samples. To obtain statistically more significant estimate of how the precision of AI-based algorithm may affect the prediction of the EIS spectral features we applied simulation of the inaccuracies in defect coordinate detection. This was done as described in "Synthetic defect set generation" . Starting with true distribution we aimed at generating a large number of defect distributions and determine deviations from true distributions which may arise due to lack of precision of AI-based defect detection algorithm. The simulation data is summarized graphically in Fig. 7. Green points in Fig. 7 plots correspond to the positions of characteristic points of samples 1, 2, and 3, which are included in Table 3. As seen from Fig. 7 deviation of parameter F1 < 1 results in skewed dispersion of both parameters f log and arg Y (see Supplemental Material). Such asymmetry of parameter distribution introduces a systemic shift of f log in AI-derived AFM image data, which for samples 1, 2, and 3 were found to be −0.168, −0.083 and −0.068 respectively (see Supplemental Material) in the F1 values interval from 0.5 to 1.0. The standard deviations of parameter f log are 0.16, 0.13 and 0.14 for samples 1, 2 and 3 correspondingly (F1 interval [0.5,1.0]). Relatively small, though consistent shift of arg Y was also detected. Specifically, the following shifts were observed for samples 1, 2 and 3 respectively: −0.98 deg, −1.68 deg and −0.48 deg in the same F1 interval. The systematic shifts f log decrease rapidly as F1 approaches 1. The f log and its standard deviation for F1 interval from 0.95 to 1.0 are 0.001 and 0.027, −0.002 and 0.031, and −0.016 and 0.027 for samples 1, 2 and 3 respectively. www.nature.com/scientificreports/ Currently, we cannot provide any reasonable explanation for such negative shift. It is obvious that the systemic negative shift may vary in relatively wide intervals causing errors in predictions of EIS spectra features. We may state that the precision of AI-based algorithm reflected in parameter F1 may considerably affect the position of f min so that the relative errors in predicting this parameter may exceed several tens of percent. In our sample surfaces 1, 2 and 3 the values 0.664, 0.611 and 0.742 resulted in (see Supplemental Material Tables S2, S3 and S4, left panes) systemic shifts of f log −0.174, −0.070 and −0.073 respectively. spectra allows one to make estimates of some important physical parameters of tBLMs. Specifically, the specific resistance, ρ , of submembrane layer separating phospholipid bilayer and metal/solution interface (Helmholtz layer) can be estimated. This parameter cannot be independently estimated from the analysis of the EIS response, because it is fully correlated with the defect density N def ## 13 . Independent estimation of N def using AIbased AFM image analysis algorithm allows to resolve the uncertainty. In such exercise the range of defect radius can also be estimated because r def determines the position of the phase minimum of arg Y vs. log f plot of EIS spectra of tBLMs. A series of FEA modeling tasks were performed with each pair of true (established by eye) and predicted defect sets for all three AFM surfaces (test data) separately. Two parameters were varied in each scenario: defect radius r def was adjusted from 1 nm to 13 nm with increments of 2 nm, while the specific conductivity of the submembrane layer ρ sub was adjusted in logarithmic scale from 10 4 to 10 5 • cm with power increments of 0.1, resulting in a total of 77 parameter combinations. Modeled curves of both true and AI-predicted defect sets were matched against the experimental EIS data by minimizing the L1 norm of minimum point coordinates (frequency and admittance phase axes) between a pair of curves. Figure 8 shows the modeled and experimental curves of each surface as well as the specific r def and ρ sub values of the corresponding modeled cases. The mean r def and ρ sub values were found to span interval from 1 to 7 nm and 10 4.0 to 10 4.6 • cm correspondingly. The mean values of the parameters are correspondingly 2.7 ± nm and 10 4.25±0.10 • cm . While r def shows significant standard deviation, which is expected because sensitivity of EIS response to r def is small if relatively modest interval of r def variation is considered 13 . In opposite, ρ sub can be established with considerably better precision, so it is likely that the described AI-based AFM image analysis technique has a good perspective for the use in calibration of tBLMs systems for the precision measurement of defect densities which is of upmost importance in considering tBLMs as quantitative biosensors for the detection of pore-forming toxins. ## Conclusions In this study we investigated the possibilities of automated detection of defects in AFM images of tBLM membranes and possibilities to predict the EIS response of such membranes. By applying the convolutional neural network for the formulated object detection task we demonstrated the potential advantage of this approach in comparison to manual defect annotation, although the results should be considered as preliminary due to the limited amount of image data used and no model tuning. We also attempted to solve the defect detection problem by using TopoStats automated biomolecule tracing tool 23 and compared its accuracy to the performance of the CNN approach (see Supplemental material, Table 5S). The precision of TopoStats proved to be comparable to CNN, while the recall was significantly lower for all AFM images, indicating that a large portion of actual defects were not detected by the tool (illustrative examples presented in Supplemental Material, Fig. 1S). Poor performance of TopoStats can be attributed to the presence of defect clusters in the images. This proves to be a significant obstacle for object detection approaches based on non-AI image processing methods. Using three different samples of tBLMs we found that true and AI-derived sets of defect coordinates though being non-identical produce by FEA modeling similar EIS curves. One of the main EIS spectral features, the predicted position of the phase minimum in Bode plots of admittance was within 2-6% from the true values. Test on larger sample sets, which coordinates were produced synthetically, indicate possibility of a systematic deviations of predicted EIS spectral features. These deviations are sensitive to the AI algorithm's precision parameter F1, and they rapidly decrease as F1 approaches 1. Taken together these findings show that EIS spectra can be predicted sufficiently well however, the systematic errors need to be taken into account. We also showed that automated AI-based algorithm of AFM image analysis allows one to make EIS spectra predictions which can be used to assess important physical parameters of tBLMs such as submembrane specific resistance. Using three different samples of tBLMs we found that the submembrane resistance is 10 4.25±0.10 • cm , a value slightly lower compared to value previously used ( 10 4.5 • cm ). This parameters allows calibration of tBLM biosensors for quantitative detection of activities of pore-forming toxins. In conclusion we provide evidence of applicability of AFM to assess the geometry and density of membrane damaging defects such as pore-forming toxins in tBLMs. This data can be used to theoretically predict EIS response of tBLMs as well as calibrate this response for biosensor applications.

chemsum

{"title": "AI-based atomic force microscopy image analysis allows to predict electrochemical impedance spectra of defects in tethered bilayer membranes", "journal": "Scientific Reports - Nature"}

data-informed_reparameterization_of_modified_rna_and_the_effect_of_explicit_water_models:_applicatio

## Abstract: Pseudouridine is the most abundant post-transcriptional modification in RNA. We have previously shown that the FF99-derived parameters for pseudouridine and some of its naturally occurring derivatives in the AMBER distribution either alone or in combination with the revised 𝛄 torsion parameters (parmbsc0) failed to reproduce their conformational characteristics observed experimentally (Deb I, et al. ## INTRODUCTION Post-transcriptional modifications have been known to be crucial in the regulation of the structure, stability and function of RNA molecules. The MODOMICS database currently lists 172 such modifications 1 . Pseudouridine (Ψ) was the first post-transcriptional modification discovered and is one of the most abundant modifications. Pseudouridine, an isomer of uridine (U), was identified as 5-ribosyluracil and was called the fifth nucleoside . This modified residue contains a C-C base-sugar bond, i.e., in the case of pseudouridine, the uracil base is attached to the sugar by a C1′-C5 bond unlike the C1′-N1 glycosidic linkage found in uridine (Figure 1 (a)). Hence, in contrast to uridine, pseudouridine contains an additional ring nitrogen atom (N1 imino atom) which acts as an additional hydrogen bond donor and is found to be protonated at physiological pH 3,9 . Pseudouridine was reported to be the most commonly observed modification in the stable RNAs, i.e., tRNA, rRNA and snRNA 3 . Further studies involving high-throughput sequencing methods and transcriptome mapping revealed the abundance of pseudouridine as an epigenetic modification, i.e. in mRNA as well as in long noncoding RNA (lncRNA) . Several experimental and theoretical studies suggest the important contribution of pseudouridine to the structure, dynamics and thermal stability of RNA . This modification has been found to reduce the motion of the neighbouring bases, stabilize the C3′-endo conformation and enhance the stability and the stacking propensity in a context-dependent manner 15, . Newby and Greenbaum studied the interaction between Ψ and water in the pre-mRNA branch-site helix and reported that a water-ΨHN1 hydrogen bond contributes to the stabilization of the unique observed architectural features of this helix 18 . In 2016, we reported that the reoptimized set of glycosidic torsion parameters (𝛘IDRP) for pseudouridine developed by us, were sufficient to improve the description of the conformational distribution of the glycosidic torsion space but the description of the sugar pucker distribution for Ψ was still not accurate 24 . In another study in 2020, we checked the transferability of these parameters (𝛘IDRP) to the derivatives of Ψ and observed that the 𝛘IDRP parameters combined with the AMBER FF99-derived parameters 25 and the revised set of 𝛄 torsional parameters predicted the conformational properties of these residues which were in general agreement with the experimental (NMR) data but failed to describe the sugar pucker distributions accurately 26 . In the present study we report a new set of glycosidic torsional parameters (𝛘ND) and a new set of partial atomic charges for pseudouridine (Ψ), 1-methylpseudouridine (m 1 Ψ), 3-methylpseudouridine (m 3 Ψ) and 2′-O-methylpseudouridine (Ψm) (Figure 1). We have compared the results obtained with these parameters with those previously obtained with the FF99 parameters and the FF99 parameters in combination with the 𝛘IDRP parameters and bsc0 𝛄 torsional parameters. In the earlier studies, multiple schemes 27 and/or general schemes 28 were chosen for the quantum mechanical scan and the molecular mechanical energy profiles were fitted with those with the objective that the re-optimized parameters will be able to explore, preferentially, any of the four quadrants (NORTH/syn, NORTH/anti, SOUTH/syn, SOUTH/anti) of the conformational preferences. In the present work, we calculated the quantum mechanical glycosidic torsional energy profiles for five different initial conformations. Then a particular scheme was chosen which outperformed other schemes in reproducing QM profile that was in agreement with the experimentally observed conformational preference. Next, the MM profile was fitted to the chosen QM profile. Additionally, the partial charges were newly generated at the individual modification level before generating the MM profile to incorporate the effect of electrostatic interactions. As a proof of concept, we have chosen pseudouridine and three of its derivatives as a (small) closely related test set that includes molecules with different chemical moieties. For additional validation of our parameter sets, we examined their performance in predicting the conformational and hydration characteristics of the ssRNA trimers and tetramers containing pseudouridine. It has been reported in recent studies that the choice of water model has a significant impact on the predicted RNA structure and dynamics 29,30 . Kührova et al.; based on their study involving the simulation of canonical A-RNA duplexes using explicit water models; i.e. TIP3P 31 , TIP4P/2005 32 , TIP5P 33 and SPC/E 34 , reported that the TIP5P water model was not found to be optimal for simulating RNA systems 29 . Here, we have investigated the impact of the choice of explicit water models on the conformational characteristics and hydration pattern of Ψ, m 1 Ψ, m 3 Ψ, and Ψm. ## Preparation of the initial geometries For the initial geometries of the modified nucleosides Ψ (PSU), m 1 Ψ (1MP), m 3 Ψ (3MP), and Ψm (MRP), we have used the mean values for bonds, angles and dihedral angles corresponding to the ribose sugar following Gelbin et al. (1996) 35 and considered planar geometries for the bases. The three-letter codes of the modified residues are according to Aduri et al. (2007) 25 . These structures were prepared using the molecular structure editor MOLDEN 36 . The geometries of the modified nucleosides were kept either in the C3'-endo/g + conformation or in the C2′-endo/g + conformation and for that the corresponding torsional angles were fixed at definite values. The value of the 𝛄 dihedral angle (O5′-C5′-C4′-C3′) was fixed at 54° (which corresponds to the g + conformation) as observed in the A-form RNA 37 . To compel the nucleoside geometries to stay in the C3'-endo conformation, the values of the 𝜹 (C5′-C4′-C3′-O3′) and O4′-C1′-C2′-C3′ dihedral angles were fixed at 81° and -24°, respectively. To constrain the geometries to the C2'-endo sugar pucker conformation the value of the 𝜹 (C5′-C4′-C3′-O3′) and O4′-C1′-C2′-C3′ dihedral angles were set to 140° and 32° respectively. Five initial geometries, i.e., SC1, SC2, SC3, SC4 and SC5 (Table S1) with constrained values of the H5T-O5′-C5′-C4′ and C1′-C2′-O2′-HO2′ torsional angles were prepared for each of the modified nucleosides, to either promote or restrict the base-sugar hydrogen bonding interactions by maintaining the nucleosides either in C3′-endo or in C2′-endo sugar pucker conformation. The schemes SC1-SC4 were chosen following the values of the torsional angles corresponding to the four schemes chosen in Yildirim et al. 27 and SC5 was chosen based on the syn scheme as mentioned in Deb et al. 24 . SC4 also corresponds to the anti scheme as mentioned in Deb et al. 24 . For the SC4 conformational scheme, the H5T-O5′-C5′-C4′ and C1′-C2′-O2′-HO′2 dihedrals were respectively constrained to 174° and 93° and due to that the O5′-H•••O4 base-sugar hydrogen bonding interaction is restricted and O2′-H•••O4 base-sugar hydrogen bonding interaction is facilitated and hence the geometries corresponding to PSU and its derivatives are compelled towards anti conformation which is not the predominant conformation for these nucleosides. For the SC5 scheme, the values of the H5T-O5′-C5′-C4′ and C1′-C2′-O2′-HO′2 dihedrals were respectively constrained to 60° and -153° to promote the O5′-H•••O4 and restrict the O2′-H•••O4 basesugar hydrogen bonding interactions and hence to force a syn conformation which is predominant for PSU and its derivatives 38 . The SC1 and SC2 conformational schemes were kept in the C2′-endo conformation while SC3-SC5 were kept in the C3′-endo conformation. To prevent any hydrogen bonding interaction between H3T or O2′ and base, so that these interactions cannot affect the glycosidic torsion energy profile, the C4′-C3′-O3′-H3T torsion was fixed at -148° for all the initial geometries. The initial structures corresponding to each of the five conformational schemes are shown in Figure S1. The geometry which corresponds to the SC5 conformational scheme for each of the modified nucleosides (along with the atom names) is shown in Figure S2. ## Quantum mechanical scan All the quantum mechanical calculations were performed using the GAUSSIAN09 software suite 39 . For all the five initial geometries for each of the modified nucleosides, a gas phase PES scan was executed around the glycosidic torsion angle (O4′-C1′-C5-C6) with an increase in its value by 5° resulting in 72 conformations for each nucleoside geometry. Optimization of the structures, during the PES scan, was carried out using the HF/6-31G* level of theory. During the geometry optimization step, the dihedral angles mentioned in Table S1, were kept frozen with the objective of obtaining a smooth QM energy profile. The QM energies (EQM) corresponding to each of the 72 geometry optimized conformations (for each scheme) were calculated using the MP2/6-31G* level of theory. Out of the five quantum mechanical energy (EQM) profiles around 𝛘, we have chosen one particular conformational scheme, i.e. SC5, because the lowest energy minimum for this scheme corresponded to the syn region of the glycosidic torsional space (Figure S3) and experimental (NMR) studies for pseudouridine and its derivatives, under study, reported a preference for the syn conformation . Additionally, the value of energy corresponding to the global minimum of that profile was found to be the least compared to other schemes (Figure S3). ## RESP fitting The new set of partial atomic charges for each of the modified nucleosides was developed corresponding to the lowest energy conformation of the quantum mechanical energy profile of the chosen scheme, i.e. SC5, by RESP 40,41 fitting (Restrained Electrostatic Potential fitting) method using the R.E.D. version III.52 perl program 42 . The partial atomic charges for the atoms of each of the nucleosides are listed in the supporting information (Table S2). ## Molecular mechanical (MM) energy minimization For the calculation of the molecular mechanical (MM) energies (EMM) corresponding to the 72 quantum mechanically (QM) optimized geometries, we have used the AMBER16 software package 43 (Figure 2). During the MM energy minimizations, the dihedral angles (as mentioned in Table S1) were restrained to the values corresponding to the QM optimized geometries by applying a force constant of 1500 Kcal/mol 2 . The starting structures for the MM energy minimization step were the structures equivalent to the QM optimized geometries obtained from the PES scan. The 5′-phosphate group was replaced with a hydrogen (5′-OH) and a hydrogen atom (3′-OH) was added to the 3′ end of the original topology provided by Aduri et al. 25 to create the topologies for all the modified nucleosides used in this study with the parameters corresponding to the 5′-OH and 3′-OH groups taken from the FF99 force field parameter set 44 . During the MM energy minimization, all the glycosidic torsion parameters corresponding to the Aduri et al. 25 parameter set were set to zero for all the modified nucleosides. Minimizations were carried out using the steepest descent method followed by the conjugate gradient method in order to obtain a smooth glycosidic torsional energy profile for each residue. To incorporate the non-bonded interactions during the energy minimization in vacuum, a long range cut-off of 12 was used. MRP residues corresponding to QM calculations (black), MM calculations with the FF99 parameter sets keeping the glycosidic torsion parameters zero (red) and MM calculations with the FF99 parameter sets combined with the newly derived 𝛘 torsional parameters and the newly developed partial atomic charges (FF99_𝛘ND) (green) by fitting the difference between the QM and MM energies. The minimum energies were set to zero for convenience. The ranges 30°-90° and 170°-300° for the 𝛘 torsional angles along the Xaxis, correspond to the syn and anti base orientations respectively. ## Fitting 𝛘 torsion potentials The potential energy due to the glycosidic torsion angle is represented by the difference (ECHI) between the QM energy (EQM) and MM energy (EMM) and is given by the following equation: The 72 values for ECHI obtained from eq. (1) were fitted to the Fourier series as shown in eq. ( 2): ECHI = Where 𝛘 represents the glycosidic torsion angle; i.e. the dihedral around (O4′-C1′-C5-C6) and Vn represents the potential energy barrier around the glycosidic torsion angles (𝛘) and 𝛟n is the phase angle. ## System preparation The starting structures in this study were taken from the original PDB format files for each of the four modified ribonucleoside residues corresponding to their quantum mechanically optimized geometries provided by Aduri et al. 25 , and available in the AMBER 2018 package . These initial structures of these modified ribonucleosides were in a NORTH/anti/g+ conformation. The FF99_𝛘IDRP_bsc0 24 parameter set for Ψ was obtained from Deb et al. 24 , and FF99_𝛘IDRP_bsc0 24 parameter sets for m 1 Ψ, m 3 Ψ, and Ψm residues were obtained from Dutta et al. 26 . The FF99_𝛘ND_bsc0 parameter sets for Ψ, m 1 Ψ, m 3 Ψ, and Ψm residues were prepared by combining our newly derived 𝛘 torsional parameters (𝛘ND) and the revised 𝛄 parameters developed by Pérez et al. 45 (parmbsc0) with the required bond, angle and torsional parameters for each modification from the AMBER provided parameters derived from Aduri et al. parameters 25 . The revised 𝛄 torsional parameters were incorporated by replacing the atom type that described the terms corresponding to the 𝛄 torsion in the default topology files with the torsional terms provided in the revised parmbsc0 force field. The newly developed partial atomic charges for the atoms (except for some atoms as mentioned in the supporting information) of each of the four modified ribonucleosides were introduced replacing the partial atomic charges of these atoms in the preparatory file (prepin) provided by Aduri et al. 25 . We used these revised parameter sets for energy minimization and MD simulation steps. The revised force field parameter sets for Ψ, m 1 Ψ, m 3 Ψ, Ψm (FF99_𝛘ND_bsc0) are given in the supporting information. The modified ribonucleosides Ψ, m 1 Ψ, m 3 Ψ, and Ψm were separately simulated using the FF99_𝛘IDRP_bsc0 and FF99_𝛘ND_bsc0 parameters respectively. Detailed description of the force field parameters used in this study are provided in Table 1. The newly derived glycosidic (𝛘) torsion parameters are listed in Table 2. 25 in combination with revised 𝛄 torsion parameters developed by Pérez et al. 45 (parmbsc0). ## FF99_𝛘IDRP_bsc0 𝛘 and 𝛄 For Ψ, FF99_𝛘IDRP_bsc0 parameters obtained from by Deb et al. 24 and for its three derivatives (m 1 Ψ, m 3 Ψ, and Ψm), FF99_𝛘IDRP_bsc0 parameters 24,25,45 modified by the introduction of required bond, angle and torsional parameters for each modification from the AMBER provided parameters derived from Aduri et al. parameters 25 (obtained from Dutta et al. 26 ). ## FF99_𝛘ND_bsc0 𝛘 and 𝛄 Revised glycosidic torsion parameters (𝛘ND) for Ψ, m 1 Ψ, m 3 Ψ, and Ψm nucleosides and revised 𝛄 torsion parameters developed by Pérez et al. 45 (parmbsc0) in combination with the required bond, angle and torsional parameters for each modification from the AMBER provided parameters derived from Aduri et al. parameters 25 along with the newly developed set of partial atomic charges for each of these modified nucleosides. ## Replica exchange molecular dynamics simulations All replica exchange molecular dynamics (REMD) simulations 46 were performed using the multi-sander approach in AMBER 16 43 in explicit water. To study the effect of the water model on the conformations of these nucleosides, REMD simulations were carried out using the combination of the FF99_𝛘IDRP_bsc0 and FF99_𝛘ND_bsc0 force fields with each of the TIP3P 31 , TIP4P-Ew 47 and SPC/E 34 water models and the hydration patterns for pseudouridine and its three derivatives corresponding to the different force field-water model combinations were analyzed. The modified nucleoside residues Ψ, m 1 Ψ, m 3 Ψ, Ψm were solvated with TIP3P or TIP4P-Ew or SPC/E water molecules in truncated octahedral boxes with a closest distance of 9 between any solute atom and the edge of the box. Energy minimization of the solvated system was carried out in two steps. For the first set of energy minimization which consisted of 500 steps of steepest descent followed by 500 steps of conjugate gradient optimization, the nucleosides were held fixed with the help of a positional restraining force of 500 kcal/mol 2 . The next set of energy minimization was performed without any positional restraining force and consisted of 1000 steps of steepest descent followed by 1500 steps of conjugate gradient optimization. Equilibration of the energy minimized systems was carried out in two steps. In the first step, the systems were heated from 0K to 300K temperature in 20 ps with a 2 fs time step using a constant volume dynamics by the application of a 10 kcal/mol 2 positional restraining force. In the second step of equilibration, whole systems were equilibrated in the absence of any restrain, at 300K temperature for 200 ps with a 2 fs time step using constant pressure dynamics (reference pressure of 1 atm and pressure relaxation time of 2 ps). After the completion of the equilibration steps, the final coordinates obtained were used as the starting coordinates for the REMD simulations. In the REMD equilibration step before the REMD production run, each of the systems was equilibrated at 16 target temperatures that spanned over a range from 300K to 400K (i.e. at T = 300.0 K, 305.8 K, 311.7 K, 317.8 K, 323.9 K, 330.2 K, 336.6 K, 343.1 K, 349.7 K, 356.5 K, 363.4 K, 370.5 K, 377.6 K, 384.9 K, 392.4 K and 400.0 K) and this step was carried out for 1 ns with a 2 fs time step with constant volume dynamics. These equilibrated systems were used for the REMD production runs consisting of 2000 cycles in constant volume. 4000 steps of MDs were performed with a 2 fs time step before the attempted exchange between the neighbouring replicas at the temperatures mentioned above. The REMD production runs generated simulation of 16 ns for each of the replicas, yielding a total simulation of 256 ns in aggregate. For each system-force field and water model combinations, three independent sets of REMD simulations were performed. For propagation of the trajectories, Langevin dynamics (with random velocity scaling with 1 ps -1 collision frequency) was used. The SHAKE algorithm 48 was used to constrain the bonds which involved hydrogen atoms. Particle mesh Ewald (PME) was used for handling the electrostatic interactions. To include nonbonded interactions, a long range cutoff of 8 was used. ## Analysis of conformational ensembles For the analysis of the simulated ensembles we calculated the distribution of sugar pucker conformations, distribution of the syn or anti conformations of the glycosidic torsion angle (𝛘) and the distribution of the 𝛄 torsional angle over different conformational states. The convention followed for the atom names and the dihedral angle nomenclatures was as given in Saenger 38 . The magnitude of the pseudorotation angle was calculated following Altona and Sundaralingam 49 . The pseudorotation angular space was divided into C3′-endo/NORTH (270°≤ P< 90°) and C2′-endo/SOUTH (90° ≤ P< 270°) regions of sugar puckering 50 , which allowed us to directly compare simulated conformational distributions and the equilibrium distributions of the pseudorotation angle (P) as reported in the NMR data. In our analysis, the 𝛘 torsional angle is defined by the atoms O4′-C1′-C5-C4 (for all the modified nucleosides) and was considered to be in the anti conformation if its magnitude was within the angular range of 170°-300° and in the syn conformation if it was within the angular range of 30°-90° 35,51,52 . The values that were beyond these ranges were referred to as others 35,51,52 . For the calculation of the 𝛄 torsional angle, the conformational space with respect to the torsional angle consisting of the atoms O5′-C5′-C4′-C3′ was divided into the conformations referred as g+ (for 60°±30°), g-(for 300°±30°), trans (180°±30°) and others (outside the ranges mentioned for the other conformations). We analysed the hydrogen bonding characteristics, radial distribution function (RDF) for each of the four residues and the distribution of the 𝛉 torsion angle (H2′-C2′-O2′-HO2′) for the Ψ, m 1 Ψ, m 3 Ψ residues. For the calculation of the pseudorotation angle P, the 𝛘, 𝛄, and 𝛉 torsion angles, hydrogen bonds and RDFs, cpptraj tool from Ambertools18 53 was used. RDFs of water oxygen atoms around the HN1 atom was calculated for each of the Ψ, m 3 Ψ and Ψm residues and RDFs of water oxygen atoms around the HN3 atom was calculated for each of the Ψ, m 1 Ψ and Ψm residues. Hydrogen bond formations were taken into account if the distance between the donor and the acceptor atoms was ≤ 3 and the donor-hydrogenacceptor angle was ≥ 135°. The water occupancy maps around the average MD structure (the average MD structures were obtained from 600 frames corresponding to each of the four conformations i.e NORTH/syn, SOUTH/syn, NORTH/anti and SOUTH/anti conformations from a set of 16 ns REMD simulations) of Ψ corresponding to the FF99_𝛘ND_bsc0 and TIP3P force field and water model combination were calculated using the grid routine in cpptraj tool and visualization was done using UCSF-Chimera 54 . ## RESULTS AND DISCUSSION In an earlier study 26 we validated the revised parameter sets for pseudouridine (Ψ) (FF99_𝛘IDRP_bsc0) 24 and checked the transferability of these parameters to the four pseudouridine derivatives i.e. m 1 Ψ, m 3 Ψ, Ψm and m 1 acp 3 Ψ and our observations indicated that the revised parameters for Ψ were transferable to the Ψ derivatives. In the present study we reoptimized the parameters for the glycosidic torsion angle individually for Ψ and its three derivatives m 1 Ψ, m 3 Ψ and Ψm and developed new sets of partial atomic charges for each of these residues and compared the conformational ensembles. The REMD simulations were carried out using the combination of the force fields i.e. FF99_𝛘IDRP_bsc0 and FF99_𝛘ND_bsc0 with the TIP3P, TIP4P-Ew and SPC/E water models. The results are written and discussed below. ## Pseudorotation angle (P) With the AMBER FF99 parameter sets, the distribution of the pseudorotation angle was observed to have a smaller population of the NORTH sugar pucker conformation compared to the experimentally observed population for each of the modified residues except for Ψ 26 (Table S3, Figure 3). Inclusion of the revised 𝛄 torsion parameters (parmbsc0) with the AMBER FF99 parameter sets resulted in an improvement in the propensity of the NORTH sugar pucker conformation for all the Ψ-derivatives. But with the FF99_bsc0 parameters, the propensity of the NORTH sugar pucker conformation for Ψ was significantly lower than the experimentally observed value 26 . For Ψ, the FF99_𝛘ND_bsc0 force field in combination with the TIP3P and the SPC/E models generated a population of the NORTH sugar pucker conformation which were in general much closer to the experimentally observed population than those generated by the FF99_𝛘IDRP_bsc0 force field in combination with each of the water models in this study. But FF99_𝛘ND_bsc0 in combination with the TIP4P-Ew water model generated a much greater population of the NORTH conformers of Ψ than the other force field and water model combinations and also the experimentally observed population. However, FF99_𝛘IDRP_bsc0 + TIP4P-Ew reproduced the experimental value of the NORTH population for m 1 Ψ better than all the other force field-water model combinations. In the case of m 3 Ψ, it was observed that, the ## Glycosidic torsion angle (𝛘) For each of the modified nucleosides under this study, experimental (NMR) studies reported preference for the syn conformation . The FF99 and FF99_bsc0 parameters, for each of the modified residues predicted an excess population of anti conformers (>90%) 26,58 . Earlier, we reported that FF99_𝛘IDRP_bsc0 + TIP3P shifted the equilibrium towards the syn conformation. The FF99_𝛘IDRP_bsc0 parameter sets in combination with each of the TIP4P-Ew and SPC/E water models also generated a much greater population of syn conformation in good agreement with the NMR data than that obtained with the FF99 parameter sets (Table S4, Figure 4). With the revised parameter sets FF99_𝛘ND_bsc0 in combination with each of the three water models, the modified residues adopted a much greater population of the syn conformation than what was predicted by the default AMBER parameters. But for each modified nucleoside, the population of syn conformers predicted by the FF99_𝛘ND_bsc0 parameters were lower than what was predicted by the FF99_𝛘IDRP_bsc0 parameters for each of the water models under this study. ## Gamma torsion angle (𝛄) In our earlier studies, we reported that, with the FF99 parameter sets, the g+ population was much lower than the experimentally observed population for pseudouridine and its derivatives 26,58 . In the present study, it was observed that all the force field and water model combinations predicted the g+ population greater than what was predicted with the FF99 parameter sets, but also than the experimentally observed population (Table S5, Figure 5). As was reported earlier 26 , in the present study also we observed that the inclusion of the revised 𝛄 torsion parameters developed by Pérez et al. 45 (parmbsc0) shifted the equilibrium almost exclusively towards the g+ conformation (∼90%). ## Correlation of the pseudorotation equilibrium with the glycosidic torsion angle (𝛘) The two-dimensional scatter correlation plots of pseudorotation angle (P) vs glycosidic torsion angle (𝛘) revealed that for all the ribonucleosides in this study, with FF99_𝛘IDRP_bsc0 + TIP3P there was a significantly large population of the SOUTH/syn conformations (Figures S4-6). With FF99_𝛘IDRP_bsc0 + TIP4P-Ew,, there were almost equal populations of SOUTH/syn and NORTH/syn conformations for all the four modified nucleosides, but the population of the SOUTH/syn conformers was a little higher in each case. The FF99_𝛘IDRP_bsc0 + SPC/E force field-water model combination also predicted a higher population of SOUTH/syn conformers than the others. In general, with the FF99_𝛘ND_bsc0 force field in combination with the TIP3P and the SPC/E water models, almost equal populations of the SOUTH/syn and NORTH/anti conformers were observed for each of the modified residues. The combination FF99_𝛘ND_bsc0 + TIP4P-Ew predicted a large population of NORTH/anti conformers for Ψ and m 1 Ψ nucleosides. But for m 3 Ψ, this force field-water model combination predicted almost equal populations of the SOUTH/syn and NORTH/anti conformers. With FF99_𝛘ND_bsc0 + TIP4P-Ew, Ψm preferentially adopted the SOUTH/syn conformation. ## Correlation of the pseudorotation equilibrium with the gamma torsion angle (𝛄) From the two-dimensional correlation maps (two-dimensional scatter plots), it was observed that the FF99_𝛘IDRP_bsc0 force field in combination with each of the three water models in the present study, predicted a greater population of the SOUTH/g+ conformers followed by that of the NORTH/g+ conformers for each of the modified nucleosides (Figures S7-9). In general, with the FF99_𝛘ND_bsc0 parameter sets in combination with each of the three water models, we observed that there were almost equal populations of the NORTH/g+ and SOUTH/g+ conformers for all the residues. With FF99_𝛘ND_bsc0+TIP4P-Ew, Ψ preferentially adopted the NORTH/g+ conformation while Ψm preferentially adopted the SOUTH/g+ conformation. The populations of the g-and trans conformers were extremely low due to the inclusion of the 𝛄 torsion parameters developed by Pérez et al. 45 (parmbsc0) as was observed in our earlier study 26 . ## Hydrogen bonding The hydrogen bonds except O5′-H5T---O4 (Figure 6) and O2′-HO2′---O4 hydrogen bonds were observed to be negligible (Tables 3-4). With each of the force field-water model combinations, for all the modified residues (not applicable to Ψm), it was observed that the number of conformers with O2′-HO2′---O4 hydrogen bonding interaction were very small and much lesser than that of the O5′-H5T---O4 hydrogen bonding interaction. ## Radial distribution function From the RDF plot of water oxygen atoms with respect to the HN1 atom of Ψ, it was observed that the FF99_𝛘IDRP_bsc0 force field in combination with each of the water models predicted the formation of a well-defined first hydration shell between 1.5 to 2.5 having a maximum at ~2 (Figures S10-12). This observation was consistent with that of the recent report by Deb et al. 21 . The FF99_𝛘ND_bsc0 force field in combination with each of the water models also predicted the formation of a well-defined first hydration shell between 1.5 to 2.5 having a maximum at ~2 around the Ψ-HN1 atom. For the HN1 atoms of m 3 Ψ and Ψm also, all the force field and water model combinations predicted the formation of a welldefined first hydration shell between 1.5 to 2.5 having a maximum at ~2 . For the Ψ and m 3 Ψ residues, the concentration of the water molecules around the HN1 atom was observed to be slightly higher with the FF99_𝛘ND_bsc0 force field than what was observed with the FF99_𝛘ND_bsc0 force field for each of the water models while for Ψm the concentration was observed to be slightly lower with the FF99_𝛘ND_bsc0 force field than what was observed with the FF99_𝛘ND_bsc0 force field for each of the water models. From the RDF plots of water oxygen atoms with respect to the HN3 atoms of Ψ, m 1 Ψ and Ψm nucleosides, with each of the force field and water model combinations, the formation of a well-defined first hydration shell was observed between 1.5 to 2.5 having a maximum at ~2 . For Ψ and m 3 Ψ, the concentration of the water molecules around the HN3 atom was observed to be similar with each of the force field and water model combinations. Interestingly, for Ψm, the FF99_𝛘ND_bsc0 force field parameters predicted a higher concentration of water molecules around the HN3 atom than what was predicted by FF99_𝛘IDRP_bsc0 in combination with each of the water models. The hydration pattern around pseudouridine (Ψ) corresponding to the FF99_𝛘ND_bsc0 and TIP3P force field and water model combination is shown in Figure 7. ## Orientation of the 2'-hydroxyl group of Ψ, m 1 Ψ and m 3 Ψ nucleosides The orientation of the 2′-hydroxyl groups of RNA has been reported to have a significant contribution to the stability of the A-form RNA helices 59 and also in RNA-protein interactions 60 . The A-RNA duplex has been suggested to be stabilized by a network consisting of water-mediated hydrogen bonds mediated by the 2′ hydroxyl groups and also the extensive individual hydration of the 2′ hydroxyl groups 61,62 . Kührova et al. (2014) reported that the choice of water model has significant effect on the orientation of the 2′-OH atom of nucleotides and hence also on the entire RNA structure 29 . The 𝛉 torsion angle (H2′-C2′-O2′-HO2′) populates three regions, the O3′ domain (value of 𝛉 between 50-140°), the O4′ domain (value of 𝛉 between 175-230°) and the base domain (value of 𝛉 between 270-345°), for C3′-endo sugar pucker conformation 29,63 . It has been reported that the 2'-OH group when oriented towards the base domain can act as a hydrogen bond donor to a water molecule and when it is oriented towards the O3′ domain it can accept a hydrogen bond from the same water molecule 59,61 . NMR studies at low temperatures suggested that the 2′-OH group can be oriented either towards the O3′ domain or towards the base domain and the predominant orientation of the 2′-OH group is reported to be towards the O3′ domain 64 . In the present study, we checked the effect of the combinations of the FF99_𝛘IDRP_bsc0 and FF99_𝛘ND_bsc0 force fields with the three different water models TIP3P, TIP4P-Ew and SPC/E, on the orientation of the 2′-OH atom corresponding to the Ψ, m 1 Ψ, and m 3 Ψ residues (Figure 8). The distribution of the 𝛉 torsion angle (H2′-C2′-O2′-HO2′) angle was similar for each of the three water models for each modified residue. But the distribution differed between the two force fields. The O3′-domain was predominantly sampled (followed by the base-domain) by all the force field-water model combinations in agreement with the experimental and theoretical studies 29,64 . The population of the conformers with the 2′-OH atom oriented towards the O4′-domain were significantly lower than the population of the conformers with the 2′-OH atom oriented towards the other two domains. While a prominent peak was observed at the O4′-domain with the FF99_𝛘IDRP_bsc0 force field in combination with each of the three water models, the FF99_𝛘ND_bsc0 force field did not predict the same. ## CONCLUSIONS In the present study we derived a revised set of of partial atomic charges and glycosidic torsional parameters (𝛘ND) for the nucleosides Ψ, m 1 Ψ, m 3 Ψ, and Ψm following a data-informed approach. At the individual nucleoside level, the partial atomic charges and glycosidic torsional parameters (𝛘ND) were calculated by applying RESP fitting method to the lowest energy conformation of the quantum mechanical energy profile of a chosen conformational scheme and fitting the molecular mechanics energy profile to that scheme-specific quantum mechanical energy profile, respectively. The choice of a particular conformational scheme was dictated by the NMR results that reported a preference for the syn conformation for pseudouridine and its derivatives under study and thereafter looking for the scheme that had the lowest energy value for the syn conformation. The consequences of the application of the revised set of glycosidic torsional parameters (𝛘ND) in combination with the revised 𝛄 torsion parameters (parmbsc0) developed by Pérez et al. 45 and the AMBER FF99-derived parameters 25 for these modified nucleosides were analysed using replica exchange molecular dynamics simulations. The newly derived parameters were validated by comparing the simulated conformational preferences with the available experimental (NMR) data as well as with the observations in Dutta et al. 26 . REMD simulations were carried out using the FF99_𝛘IDRP_bsc0 24 and FF99_𝛘ND_bsc0 force fields in combination with each of the TIP3P, TIP4P-Ew and SPC/E water models. Three independent REMD simulations (each of 16 ns) were carried out in 16 temperature windows ranging from 300 to 400 K, resulting in 768 ns of simulation time in total. It was observed that there were significant differences in the description of the conformational properties of each of the modified nucleosides by different combinations of force fields and water models. The revised force field parameter sets (FF99_𝛘ND_bsc0) with the TIP3P water model was able to closely reproduce the experimentally observed sugar pucker preferences for each of the modified nucleosides in this study. The accuracy of the prediction of the population of the C3'-endo/NORTH conformers might be important for accurate reproduction of the C3'-endo/NORTH pucker conformation associated with the A-form RNA structures. In general, the newly developed force field parameters (FF99_𝛘ND_bsc0) in combination with each of the water models under this study shifted the distribution of the base orientation for each of the modified nucleosides towards the syn conformation in contrast to the excess of anti conformations predicted by the AMBER FF99 and AMBER FF99_bsc0 parameters 25,45 . But the population of the syn conformers predicted by the FF99_𝛘ND_bsc0 force field was observed to be less than that predicted by the FF99_𝛘IDRP_bsc0 force field parameters. The choice of water model was not found to influence the description of the base orientation to a significant extent for the FF99_𝛘IDRP_bsc0 force field parameters. However, the FF99_𝛘ND_bsc0 force field in combination with the TIP4P-Ew water model resulted in a somewhat smaller population of the syn conformers in the case of Ψ and m 1 Ψ nucleosides and a significantly greater population of the syn conformers for Ψm than what were observed with the other two water models. In earlier studies from our group 24,26 , we reported that, at the single nucleoside level, the inclusion of the revised 𝛄 torsion parameters (parmbsc0) developed by Pérez et al. 45 along with the FF99_𝛘IDRP parameter sets did not reproduce the experimentally observed population of the g+ conformers, but predicted a much larger g+ population for pseudouridine and its derivatives. We also noted that the large population of g+ conformers observed with the FF99_𝛘IDRP_bsc0 parameters might be necessary to maintain the g+ conformation of a nucleotide as is observed in the standard A-form of RNA 37 . In the present study, we observed that the newly derived FF99_𝛘ND_bsc0 parameter sets also predicted a large population of the g+ conformation for each of the modified residues. The populations of g+ conformers for all the nucleosides under this study, predicted by each of the force field and water model combinations were similar and were much larger than that predicted with the FF99 parameters. The observations from the calculations of the number of O5′-H5T---O4 and O2′-HO2′---O4 hydrogen bonding interactions for each of the modified nucleosides in this study, suggested that O5′-H5T---O4 hydrogen bonding interaction contributes to the stabilization of the syn base orientation 38 while the O2′-HO2′---O4 hydrogen bonding interaction may facilitate the anti base orientation. The differences in the hydration pattern of the modified nucleosides were better revealed by the radial distribution function calculations. All the force field-water model combinations predicted similar distances of the first hydration shell corresponding to the water molecules around the HN1 atoms of Ψ, m 3 Ψ, and Ψm residues and the HN3 atoms of Ψ, m 1 Ψ and Ψm residues. In general, FF99_𝛘ND_bsc0 parameter sets predicted greater numbers of water molecules around the HN1 atoms of Ψ and m 3 Ψ nucleosides but lesser number of water molecules around the HN1 atoms of Ψm than what were predicted by the FF99_𝛘IDRP_bsc0 ribonucleosides. Table S3. Propensity (in %) for NORTH sugar puckering of Ψ, m 1 Ψ, m 3 Ψ, and Ψm ribonucleosides. Table S4. Fraction (in %) of base orientation states for Ψ, m 1 Ψ, m 3 Ψ, and Ψm ribonucleosides. Table S5. Fraction (in %) of 𝛄 conformational states for Ψ, m 1 Ψ, m 3 Ψ, and Ψm ribonucleosides. RDFs of water oxygen atoms around the HN3 atom of the Ψ, m 1 Ψ, and Ψm residues corresponding to the different force field and water model combinations for the three independent sets of 16 ns REMD simulations respectively.

chemsum

{"title": "Data-informed reparameterization of modified RNA and the effect of explicit water models: Application to pseudouridine and derivatives", "journal": "ChemRxiv"}

one_stone,_three_birds:_one_aiegen_with_three_colors_for_fast_differentiation_of_three_pathogens

## Abstract: Visually identifying pathogens favors rapid diagnosis at the point-of-care testing level. Here, we developed a microenvironment-sensitive aggregation-induced emission luminogen (AIEgen), namely IQ-Cm, for achieving fast discrimination of Gram-negative bacteria, Gram-positive bacteria and fungi by the nakedeye. With a twisted donor-acceptor and multi-rotor structure, IQ-Cm shows twisted intramolecular charge transfer (TICT) and AIE properties with sensitive fluorescence color response to the microenvironment of pathogens. Driven by the intrinsic structural differences of pathogens, IQ-Cm with a cationic isoquinolinium moiety and a membrane-active coumarin unit as the targeting and interacting groups selectively locates in different sites of three pathogens and gives three naked-eye discernible emission colors. Gram-negative bacteria are weak pink, Gram-positive bacteria are orange-red and fungi are bright yellow. Therefore, based on their distinctive fluorescence response, IQ-Cm can directly discriminate the three pathogens at the cell level under a fluorescence microscope. Furthermore, we demonstrated the feasibility of IQ-Cm as a visual probe for fast diagnosis of urinary tract infections, timely monitoring of hospital-acquired infection processes and fast detection of molds in the food field. This simple visualization strategy based on one single AIEgen provides a promising platform for rapid pathogen detection and point-of-care diagnosis. ## Introduction Pathogenic bacteria and fungi are everywhere and pose severe threats to human health and safety. Their infections cause many severe diseases, such as urinary tract infections (UTIs), sepsis and pneumonia. 4,5 The fast identifcation of pathogen type is the frst and most critical step to reduce the abuse of antibiotics and ensure effective treatment. 2,6 The gold standard of diagnosis, pathogen culturing, generally takes several days and results in delayed reports. 3,7 The Gram-staining method can overcome such time limitations by enabling direct observation of the colors of pathogens after staining, but its accuracy rate is low (about 40-60%) due to its complicated multi-step procedure and the low sensitivity of colorimetry. Also, the method can't effectively discriminate between Gram-positive (G+) bacteria and fungi because they both present blue/purple color. Without timely and reliable pathogen information, inadequate antimicrobial therapy could be performed. 11 For instance, in clinics, empirical or broad-spectrum antibiotic therapy is often employed for frst-hand UTI treatment. 12,13 This not only leads to compromised treatment and high chances of hospitalacquired infection, but also signifcantly promotes the emergence of drug-resistant pathogens. 14 Therefore, a simple and reliable visualization strategy is urgently needed for fast discrimination of pathogens. 3,6,15,16 In particular, achieving direct naked-eye visual identifcation of pathogens will be very benefcial for rapid diagnosis at the point-of-care testing level. 17 Fluorescence is a promising visual tool for rapid and reliable identifcation of pathogens because it exhibits more than a one thousand times improvement in sensitivity than colorimetry. 22 Gram-negative (G) bacteria, G+ bacteria and fungi have different surface structures and chemical components (Scheme 1a), 6,23,24 which enable us to visually discriminate them using fluorescence probes. G+ bacteria and fungi only have a cytoplasmic membrane covered by a loose and poriferous cell wall. In contrast, G bacteria possess an additional outer membrane, which performs the barrier function. 23,24 Meanwhile, different from bacteria, fungi are eukaryotic organisms and contain multiple organelles in their cell protoplasm. 25 The difference in surface structures and chemical components of the three types of pathogens allows fluorescence probes to penetrate their cell membrane and thus localizes them in different microenvironments. A lot of fluorophores with donor (D)-acceptor (A) structures are microenvironment-sensitive and show emission color change in response to microenvironmental variation based on the twisted intramolecular charge transfer (TICT) effect. However, with rigid and planar molecular structures, traditional fluorescence probes generally show strong fluorescence background, poor photostability and the aggregation-caused quenching (ACQ) effect, greatly compromising their advantages in sensitivity. 29,30 Thus, it is difficult to grasp the tiny difference among the three types of pathogens and achieve visual discrimination of them based on traditional fluorophores. Diametrically opposed to traditional fluorophores, luminogens with aggregation-induced emission characteristics (AIEgens) provide a good solution for pathogen identifcation. With rotor-stator structures, they generally exhibit weak emission in solution but become highly emissive when the intramolecular motions of rotors are restricted by the surroundings. 31,32 To date, AIEgens have enjoyed great successes in bioanalyte sensing with the merits of low background, high sensitivity and good photobleaching resistance. The multirotor structures of AIEgens also endow them with high sensitivity to the surrounding microenvironment. In particular, when bearing twisted D-A structures, AIEgens can adapt different molecular confgurations and show diverse fluorescence color responses to the microenvironments based on the TICT effect. 27,40 Moreover, the visualization of these fluorescence response colors is guaranteed because the nonradiative relaxation of the TICT state can be effectively suppressed by the AIE properties. 41,42 Thus, rationally integrating the merits of AIE and the TICT effect into one fluorophore is very promising for rapid and visual identifcation of the three pathogens. In this work, we designed and prepared a new cationic AIEactive molecule with a twisted and extended donor-pacceptor (D-p-A) structure, named IQ-Cm, for visual identifcation of pathogen types. Structurally, IQ-Cm consists of three parts: a diphenyl isoquinolinium (IQ) unit, a coumarin-derived (Cm) moiety and a phenyl linker (Scheme 1b). The IQ moiety has a highly twisted molecular structure and was introduced as an AIE-active group. 43 Also, its intrinsic cationic structure allows IQ to act as a strong electron acceptor and a targeting group for the negative pathogen surface. Since many coumarin derivatives are membrane-active, 44 coumarin was introduced to help IQ-Cm effectively interact with the pathogen membrane. A diethylamino group was attached on the coumarin to serve as a strong electron donor. Then, a rotatable aromatic phenyl was employed as the linker between IQ and Cm to generate an extended and twisted D-p-A structure, 42 which endows IQ-Cm with prominent AIE and TICT properties. Driven by the intrinsic structural differences in the outer envelopes and cytoplasm components of the three pathogens, IQ-Cm selectively locates in different sites in them, senses the diverse surrounding microenvironment, and thus successfully transforms pathogen information into distinctive fluorescence colors at the cellular level (Scheme 1c), achieving fast discrimination of them by the naked-eye. Furthermore, we also demonstrated the potential of IQ-Cm in fast pathogen diagnosis in practice, such as fast UTI diagnosis, visual monitoring of hospital-acquired infections and naked-eye detection of molds. ## Synthesis and photophysical properties of IQ-Cm The synthesis of IQ-Cm was readily achieved through two sequential steps of Suzuki coupling and a one-pot multiple component reaction with a high yield of 84% (Scheme 2). The detailed synthesis procedures are described in the ESI. † The chemical structure of IQ-Cm was completely characterized by 1 H NMR, 13 C NMR, and HRMS (Fig. S1-S3 †) and confrmed by X-ray single crystal analysis (Scheme 2 and Table S1 †). The single crystal structure demonstrates that IQ-Cm adopts a twisted 3D conformation with large torsional angles for the aromatic groups, i.e. 72.4-77.8 torsion of phenyl groups from the isoquinolinium core and 23.5-31.8 torsion of coumarin and the isoquinolinium moiety from the phenyl linker (Scheme 2). These rotatable aromatic units would effectively prevent the detrimental p-p stacking, overcoming the ACQ effect. As demonstrated, the distance between the nearest two coumarin or two isoquinolinium planes is about 11 A (Fig. S4 †), much larger than that of prominent p-p interaction (3.3-3.8 A). 45 Multiple intermolecular hydrogen bonds C-H/F were found in the crystal packing (Fig. S4 †), which restrict the motion of aromatic rotors. As a result, the IQ-Cm crystal gives intensive orange-red emission at 620 nm with a fluorescence quantum yield (F F ) of 5.7%. To better understand the molecular characteristics, the electron cloud on the frontier molecular orbitals of IQ-Cm was calculated by density functional theory. It was found that the electron clouds of the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) of IQ-Cm are almost completely separated (Fig. 1a). Its HOMO is mainly contributed by the electron-donating N,N-diethylaminocoumarin unit, while the LUMO is mainly localized on the electron-withdrawing isoquinolinium part. This massive shift of the electron cloud means the occurrence of the obvious TICT upon excitation. 43 This feature endows IQ-Cm with a prominent sensitivity to microenvironmental polarity variation. As shown in Fig. 1b, under UV light irradiation, IQ-Cm presents a remarkable solvatochromism effect. When changing the solvent from dioxane to water, the emission color of the IQ-Cm solution changes from blue (469 nm) to red (625 nm). In contrast, the absorption of IQ-Cm shows little dependence on the solvent polarity, only varying from 399 nm to 437 nm with an extinction coefficient of about 37 000 M 1 cm 1 in DMSO (Fig. S5 †). This obvious change in the emission color of IQ-Cm in response to the environmental polarity greatly favors it for visually identifying pathogens. In addition, IQ-Cm also exhibits AIE properties due to its highly twisted confguration. The F F of IQ-Cm in the solid state (14.6%) is about 18 fold greater than that in DMSO solution (0.8%), showing the typical AIE characteristics. To further illustrate the AIE properties of IQ-Cm, it was studied in DMSO/ water mixtures with different water fractions (f w ). As shown in Fig. 1c and d, when increasing the water content from 0 to 80%, the emission intensity of IQ-Cm at 501 nm gradually decreases and the emission maximum slightly redshifts (Fig. 1c, inset), due to the more polar environment. On further increasing the water content from 80% to 98%, a large red-shifted emission at 643 nm appears and increases (Fig. 1c and d), showing an AIE phenomenon because of the formation of aggregates. As confrmed, with the increase of water fractions above 80%, the scattering intensity of the IQ-Cm solution abruptly increases, indicating the occurrence of aggregation (Fig. S6a †). The DLS result and TEM image show that IQ-Cm forms rod aggregates of about 1 mm (Fig. S6b and c †). This effectively restricts the motion of the aromatic rotors of IQ-Cm and activates its AIE process. This AIE effect dominates over the TICT effect and resists the emission drop caused by nonradiative relaxation of the TICT state, giving the boosted red-shifted emission of the TICT state. 41,42 As confrmed in Fig. S6d, † with the increase in the solvent viscosity, TICT emission at 600 nm appears and gradually increases despite enhanced ICT emission at 500 nm. This further indicates that the restriction of the motion of the aromatic rotors of IQ-Cm can inhibit the non-radiative pathways of the ICT and TICT states and enhance their fluorescence. Additionally, because IQ-Cm aggregates are in the amorphous state (Fig. S6e †), their emission is vulnerable to the surrounding solvent polarity, resulting in the continued red-shift of the emission maximum after aggregation at water fractions above 80% (Fig. 1c, inset). 42 Furthermore, due to the formed loose and amorphous structure, IQ-Cm in the aggregated state still shows weak emission (Fig. 1c), which is conducive for low background. Also, IQ-Cm has a good photostability and shows almost no signal loss at a concentration of 10 mM after continuous irradiation for 60 scans and only about 10% signal loss when the concentration decreases to 1 mM (Fig. S7 †), which is comparable to that of the commercial dyes propidium iodide (PI) and MitoTracker Green. These desired properties of IQ-Cm are highly suitable for the visual identifcation of pathogens as discussed in the following. Visual identifcation of pathogens using IQ-Cm by the nakedeye E. coli (G bacteria), S. aureus (G+ bacteria) and C. albicans (fungi), the three most common pathogens in clinical environments, were chosen as representatives for demonstration. To enable naked-eye identifcation, 10 mM IQ-Cm in PBS solution was used as the working solution. Unlike in the case of water, where rod aggregates are formed (Fig. S6c †), IQ-Cm forms more loose network structures above its critical aggregation concentration of $4 mM in PBS (Fig. S8 †). As a result, IQ-Cm shows a weaker fluorescence background in PBS, which is favorable for the high sensitivity and accuracy of pathogen identifcation. As shown in Fig. 2a, under a UV lamp, the fluorescence of IQ-Cm in PBS solution is negligible. After incubation with the three pathogens, the fluorescence emission of IQ-Cm is obviously enhanced with three distinguishable emission colors (Fig. 2a). E. coli shows weak pink fluorescence, S. aureus presents brighter orange-red fluorescence while C. albicans gives the strongest yellow emission. The corresponding fluorescence spectra and those of the pathogens themselves in PBS solution were also recorded (Fig. 2b). The three pathogens show weak auto fluorescence and cause a variation of emission intensity of IQ-Cm following the order of C. albicans > S. aureus > E. coli. A large blue-shift from 650 nm to 575 nm is induced by C. albicans and a smaller blue-shift to 610 nm is caused by S. aureus. The addition of E. coli causes a blue-shift of the IQ-Cm emission with two peaks centered at about 535 and 610 nm. In order to further prove the feasibility of this naked-eye identifcation method, more pathogens were chosen to be treated with IQ-Cm. Similar fluorescence responses were observed for pathogens of the same kind (Fig. 2c and S9a †), i.e., weak pink for G bacteria, orange-red for G+ bacteria and strong yellow for fungi. The visual identifcation sensitivity of the three pathogens using IQ-Cm is about 10 8 , 10 8 , and 10 7 CFU mL 1 for G bacteria, G+ bacteria and fungi, respectively (Fig. S9b †). These results explicitly demonstrate that IQ-Cm is highly suitable for naked-eye discrimination and identifcation of G bacteria, G+ bacteria and fungi by giving three distinct fluorescence colors. ## Mechanism of visual identication of pathogens To gain insight into the fluorescence color response of IQ-Cm to the three pathogens, the fluorescence imaging technique was employed to directly visualize the interaction of IQ-Cm with the three pathogens at the cellular level. As shown in Fig. 3a, detectable weak fluorescence with low labeling efficiency is observed for E. coli, while S. aureus and C. albicans present bright fluorescence with high labeling efficiency, confrming the different binding affinities of IQ-Cm to the three pathogens. Similar imaging results were also observed for other pathogens of the same kind (Fig. S10 †). The three pathogens labeled at the cell level show different emission colors, i.e., green and orange for E. coli, orange for S. aureus and yellow for C. albicans. Their in situ fluorescence spectra were measured and they further confrmed these different emission colors (Fig. 3b and S11 †). As shown, when IQ-Cm mainly stains the cell membrane of E. coli, the main emission peak is at 530 nm with green emission (Fig. 3a, b, S11a and d †). However, with more IQ-Cm entering the cytoplasm of E. coli, the main emission peak is red-shifted to 600 nm with orange emission. Different from E. coli, IQ-Cm mainly locates in the cytoplasm of S. aureus and C. albicans (Fig. 3a, S11b and c †) and gives an orange emission at 600 nm for S. aureus and a yellow emission at 585 nm for C. albicans (Fig. 3b and S11d †). These in situ spectra on the cell level from the fluorescence microscope are almost consistent with those of their bulk solutions (Fig. 2b). The above imaging results and in situ spectra reveal that IQ-Cm selectively interacts with the three pathogens and locates in different sites, which leads to its discernible emission colors in the three pathogens. Furthermore, to obtain more information on IQ-Cm in the three pathogens, fluorescence lifetime imaging was performed (Fig. 3c), based on the fact that the fluorescence lifetime of a fluorophore relies more on its local environment but less on other variables such as the excitation intensity and the local fluorophore concentration. 46 Two populations with distinct lifetimes of about 1.45 and 1.97 ns were observed for labeled E. coli (Fig. 3d), corresponding to IQ-Cm in the cytoplasm of E. coli and IQ-Cm in the cell membrane of E. coli, respectively (Fig. S12 †). In contrast, IQ-Cm in S. aureus shows one lifetime of about 1.42 ns. In the case of C. albicans, the lifetime of IQ-Cm is widely distributed but mainly at about 1.34 ns. The distinct lifetime means that IQ-Cm experiences different microenvironments in the three pathogens. In response to the difference in the local environment, IQ-Cm adopts different twisted molecular confgurations in the three pathogens, giving different emission colors based on its TICT effect discussed above. These results fully demonstrate that IQ-Cm shows different interactions with the three pathogens and selectively lies in different microenvironments. Next, we further explored the mechanism behind the diversity of interactions and color responses of IQ-Cm to the three pathogens. To understand the lower labeling efficiency of IQ-Cm to G bacteria than to the other two pathogens, we frst investigated their cell envelope structure. As shown in Scheme 1a, compared with G+ bacteria and fungi, G bacteria possess an additional outer membrane, which exhibits the barrier function. 23,24 Therefore, IQ-Cm is effectively prevented from accessing the cytoplasmic membrane of live G bacteria. In contrast, lacking the protection of an outer membrane, IQ-Cm can readily penetrate the cell membrane and enter the inside of G+ bacteria and fungi driven by its cationic (IQ) and membrane- active (Cm) groups (Fig. 3a, S11b and c †). As demonstrated by the zeta potential results (Fig. 4a), after adding IQ-Cm, the surface potentials of S. aureus and C. albicans do not obviously change while that of E. coli becomes more positive. This indicates that IQ-Cm primarily enters the inside of S. aureus and C. albicans but attaches to the surface of E. coli via electrostatic interactions. The cationic IQ-Cm compromises the negative potential of the surface of E. coli. 6 After entering S. aureus and C. albicans, the intramolecular motions of the aromatic rotors of IQ-Cm are restricted effectively by the internal environment, which turns on its emission based on the working mechanism of AIEgens-restriction of intramolecular motion (RIM). 31 As a result, IQ-Cm shows high labeling efficiency for S. aureus and C. albicans, which accounts for the strong emission of their bulk suspension. But the aromatic rotors of IQ-Cm on the surface of E. coli undergo motion with little restriction, making it almost nonemissive. Thus, only a few E. coli with a compromised outer membrane or destroyed cell membrane (dead E. coli) allow IQ-Cm to insert or penetrate their cell membrane, which contributes to the low labeling efficiency by IQ-Cm and the weak emission of the IQ-Cm/E. coli bulk suspension. To verify our claim, co-staining experiments were performed for E. coli using IQ-Cm and propidium iodide (PI, a specifc probe for dead microbes with a destroyed cell membrane along with red emission 47 ). As shown in Fig. 4b, the red emission of PI is observed for orange E. coli but not for green ones. Based on the fact that PI selectively enters the cell protoplasm of dead microbes with destroyed cell membranes, 47 the co-staining results reveal that dead E. coli are labeled with IQ-Cm, which gives orange emission. Meanwhile, the antibacterial results show that IQ-Cm has about 10% killing activity against E. coli, indicating the existence of a few dead E. coli (Fig. 4c). To further prove this, E. coli were killed by medical alcohol to destroy their cell membranes. Evidently, for E. coli treated with medical alcohol, IQ-Cm shows high staining efficiency and enters their cell protoplasm, giving orange emission (Fig. S13 †). These results fully confrm that IQ-Cm destroys the cell membrane of a few E. coli and thus is allowed to enter their cell protoplasm. Unlike PI that specifcally binds with the nucleic acid in the cell protoplasm, 47 IQ-Cm possibly just locates in the cell protoplasm, as the addition of DNA and RNA can obviously enhance the emission of PI rather than IQ-Cm (Fig. S14 †). It has been reported that the cell cytoplasm of bacteria contains a large amount water ($80%) 48 and presents glass-like properties. 49 Hence, in response to such a largely polar and rigid microenvironment, IQ-Cm gives a red-shifted emission of orange color due to the TICT and AIE effects. On the other hand, red emission was not observed for the E. coli with green fluorescence (Fig. 4b), which suggests that the outer membrane of these E. coli was possibly destroyed but the cytoplasmic membrane was intact. To verify this, we destroyed the outer membrane of E. coli and kept their cytoplasmic membrane intact by adding ethylenediaminetetraacetate disodium (EDTA) to remove Ca 2+ or/and Mg 2+ which control the integrity of the outer membrane. 50 As shown in Fig. S15, † IQ-Cm exhibits high staining efficiency for treated E. coli, where E. coli with the hollow green emission is observed. This indicates that IQ-Cm primarily locates at the cell membrane of the E. coli with the compromised outer membrane but intact cytoplasmic membrane. As the cell membrane mainly consists of various lipids with a low surrounding polarity, 51 a blue-shifted green emission of IQ-Cm is exhibited due to the TICT effect. Taken together, with the barrier of the outer membrane, IQ-Cm primarily targets the negative surface of E. coli, and thus a major of E. coli are almost nonemissive. Only a small portion of E. coli with a compromised outer membrane or dead E. coli are lit up by IQ-Cm, where IQ-Cm molecules mainly insert into the cell membrane of E. coli with compromised outer membranes and enter the cell protoplasm of dead E. coli. In response to the difference in these two microenvironments of E. coli, IQ-Cm gives green and orange emission due to the TICT effect. These above factors contribute to two fluorescence colors with low labeling efficiency under fluorescence microscopy and weak pink fluorescence color observed by the naked-eye. Besides, the co-staining experiments with IQ-Cm and PI were also performed for the G+ S. aureus and fungal C. albicans. Clearly, both the orange signal from IQ-Cm and red signal from PI were observed for most of the G+ S. aureus (Fig. 4d). This means that IQ-Cm shows strong interaction with S. aureus and completely kills them, as proved by the nearly 100% killing efficiency of IQ-Cm to S. aureus (Fig. 4c). Similarly to dead E. coli, in the largely polar and glass-like microenvironment of the cell cytoplasm, IQ-Cm gives a red-shifted emission of orange color in S. aureus based on the TICT and AIE effects. Meanwhile, fungal C. albicans presents a different scenario. As shown in Fig. 4e, low staining efficiency from PI was observed, suggesting that IQ-Cm shows low killing activity against C. albicans (Fig. 4c). Both dead and live C. albicans give a similar yellow emission as there is no barrier of the additional outer membrane and IQ-Cm can easily enter the cytoplasm of fungi regardless of the viability. Moreover, fungi are eukaryotic organisms and contain multiple organelles such as mitochondria, the endoplasmic reticulum and the Golgi apparatus in their cell protoplasm. 25 Inspired by the fact that cationic AIEgens possess good specifcity to the mitochondria of eukaryotic cells, 43,52 we hypothesize that the cationic IQ-Cm could also target and accumulate in the mitochondria of fungi. To confrm this, colocalization of IQ-Cm and MitoTracker Green (a commercial probe for mitochondrial imaging of yeast) was performed. As shown in Fig. 4f and S16, † the yellow emission from IQ-Cm and the green emission from MitoTracker Green are almost completely overlapping with a high Pearson's correlation coefficient of 0.81, confrming that IQ-Cm mainly locates in the mitochondria of fungi. Compared with the cell membrane, the mitochondrial membrane contains a greater membrane protein content, 53 which gives rise to a more polar environment than that of the cell lipid membrane but still less than that of the cell protoplasm with a large amount water. Thus, an intermediate yellow emission was observed. Above all, IQ-Cm shows diverse interaction with the three pathogens and selectively locates in different sites. Accordingly, in the three pathogens, IQ-Cm experiences different surrounding microenvironments and fnally gives three discernible fluorescence colors, achieving visual discrimination (Scheme 1c). ## Fast diagnosis of urinary tract infections (UTIs) The high efficiency of IQ-Cm for visual pathogen identifcation inspires us to employ it for clinical diagnosis. UTIs are one of the most common pathogen infections of humans 5 and is thus chosen as a representative example. In clinics, urine culture is recommended as the gold standard for UTI diagnosis, but it generally takes several days. 54 Based on the above results, IQ-Cm shows high potential for fast diagnosis of UTIs. To validate this, UTI models were built by adding E. coli, S. aureus and C. albicans into normal urine to mimic clinical G bacterial, G+ bacterial and fungal infections. Firstly, these UTI model samples were visually identifed by the naked eye. As shown in Fig. 5a, 10 mL infected urine samples were transferred to 10 mL culture medium and then grown for about 5-8 h. This culture step can effectively reduce the interference of the complex components in a patient's urine. The collected pathogens were incubated with IQ-Cm for 10 min and then directly observed under a UV lamp. The sample with weak pink color was iden-tifed as G bacterial infection, orange-red color as G+ bacterial infection and bright yellow color as fungal infection. These identifed results were consistent with the original added pathogen type, conceptually demonstrating the feasibility of IQ-Cm for UTI diagnosis. Although microbial culturing is required, this naked-eye visual identifcation method is simple and only takes a few hours, which is much faster than the several days of traditional urine culture. These fabricated UTI model samples were also visually identifed using a fluorescence microscope. After simply centrifuging the urine samples and resuspending the collected pathogens in PBS, IQ-Cm was added for 10 min, and then the samples were observed under a fluorescence microscope. Based on the distinctive fluorescence response of the three pathogens to IQ-Cm at the cellular level, correct identifcation results were obtained within 30 min (Fig. 5b). Another noteworthy issue is that hospitalized patients often receive hospital-acquired infections due to their compromised immunity caused by the use of broad-spectrum antibiotics or immunosuppressive agents. As a worse case, the initial bacterial infection of patients may evolve into a fungal infection when post-operative antibiotics are inappropriately used. 58 Based on the discernible fluorescence response of IQ-Cm to bacteria and fungi, this hospital-acquired infection process can be easily monitored using IQ-Cm, which is very conducive for clinical decisions. To simulate the occurrence and evolution of opportunistic fungal UTIs from an initial bacterial infection in a hospital, UTI models were built by adding different number ratios of bacteria (S. aureus/E. coli) and fungi (C. albicans) into normal urine. As shown in Fig. 5c, under a fluorescence microscope, the emergence of a very small amount of fungal species in bacterial communities could be noticeably observed, due to their intensely yellow emission. As the fungal numbers gradually increase and exceed that of bacteria, i.e., the number ratios of bacteria and fungi from 100 : 0, 100 : 1, and 100 : 10 to 0 : 100, the species with bright yellow emission become greater in number and fnally become dominant. This means that the cause of infection has been completely changed, and thus the antimicrobial formula should be adjusted accordingly. For a more complicated situation, where the initial infection was caused by two kinds of bacteria, the emergence of fungal species can also be easily monitored (Fig. 5c). These results fully demonstrate the high potential of IQ-Cm for fast clinical diagnosis and timely monitoring of hospital-acquired infection. ## Visual detection of mold The feasibility of IQ-Cm in detecting molds was also explored. Molds are a type of fungus and can be seen everywhere in our life. 59 Unavoidably, tiny spores of molds floating in the air can fall onto food or food processing equipment and grow into molds, which greatly threaten human health. 60 Thus, the rapid detection of molds is very important. The bright yellow emission of fungi labeled with IQ-Cm greatly facilitates the detection of molds. Moreover, the mold number can also be roughly quantifed by the naked-eye under a UV lamp. To demonstrate this, an emission color-fungal amount relationship was frst established (Fig. 6a). It was found that the limit of naked-eye detection of IQ-Cm for fungi is about 10 6 CFU mL 1 . This means that when the fluorescence emission can be observed by the naked-eye, the fungal number is beyond 10 6 CFU mL 1 . Above the detection limit, the emission color changes from light coral and orange to yellow, corresponding to the change in fungal amount from 10 6 and 5 10 6 to 10 7 CFU mL 1 , respectively. This emission color-fungal amount relationship was also verifed by PL spectroscopy (Fig. S18 †). Based on the established emission color-fungal amount relationship, the mold amount grown on food can be easily and visually determined by the naked-eye. To demonstrate this, four classes of representative foods involving preserved fruit (persimmon), vegetable (tomato), fruit (orange) and wheaten food (bread) were taken as the test samples. In appearance, the persimmon did not look moldy, the stem of the tomato was rotten, and the orange and bread were obviously moldy. By nakedeye detection and fluorescence microscope imaging, all four samples were detected to have mold growth (Fig. 6b-e). But the emission colors for the four samples were obviously different from each other. According to the emission color-fungal amount relationship, it can be concluded that the mold number grown on the chosen four samples follows the order of persimmon > tomato z orange > bread (specifcally, $10 7 CFU mL 1 for persimmon, 10 6 to 10 7 CFU mL 1 for tomato and orange, and $10 6 CFU mL 1 for bread). Interestingly, this result does not agree with the observed apparent phenomena. For the obviously moldy orange and bread, the detected mold amount using IQ-Cm is less. This is reasonable because the observed massive "molds" are mainly mycotoxins produced by molds. 59 But there is still a small number of molds hiding among them, as detected using IQ-Cm (Fig. 6d and e). Conversely, the persimmon and tomato with the rotten stem removed seem to have no mold, but a large number of molds are detected using IQ-Cm (Fig. 6b and c). These results suggest that the seemingly benign food possibly hides a large number of molds, just like the situation of the persimmon. In this case, IQ-Cm can rapidly "see" these molds, which is essential for monitoring food quality. ## Conclusions In conclusion, we have rationally designed a simple AIEgen with a twisted D-p-A structure, which serves as a microenvironmentsensitive probe for rapid visual discrimination of G bacteria, G+ bacteria and fungi by giving discernible emission colors. IQ-Cm successfully identifes the subtle differences between the three pathogens by primarily locating in different sites, i.e. cell envelop and cell cytoplasm of G bacteria, cell cytoplasm of G+ bacteria and mitochondria of fungi. In these three cases, IQ-Cm experiences diverse surrounding environments and thus effectively transforms the pathogen information into distinctive fluorescence colors due to its AIE properties and TICT effect. At the naked-eye level, G bacteria are weak pink, G+ bacteria are orange-red and fungi are bright yellow, aiding direct and fast discrimination of them. With a fluorescence microscope, the visual discrimination of the three pathogens is realized at the cell level on the basis of their specifc fluorescence response. More importantly, IQ-Cm shows high potential for fast diagnosis of UTIs, which can reduce the diagnosis time to a few hours by direct naked-eye detection or less than 30 minutes using a fluorescence microscope. Also, based on the distinct fluorescence color response of IQ-Cm to bacteria and fungi, the frequent hospital-acquired infection evolution from an initial bacterial infection to a fungal infection can be timely and visually monitored using IQ-Cm. This is very essential for guiding clinical decisions. Furthermore, thanks to the bright yellow emission of labeled fungi, IQ-Cm can be used for fast detection of molds in the food feld. Moreover, the mold number can also be roughly quantifed according to the established emission color-fungal amount relationship. Therefore, our studies provide a fast and simple platform for pathogen identifcation at the point-of-care level, which exhibits very high potential in offering timely and reliable pathogen information for making clinical treatment decisions, monitoring the trends of infectious diseases and supervising food safety. ## Materials and methods All the chemicals and organic solvents were purchased from J&K, TCI and Sigma-Aldrich Company and used as received. Two Gram-negative (G) bacteria, E. coli (ATCC25922) and P. aeruginosa (JCm5962), three Gram-positive (G+) bacteria, S. aureus (ATCC6538), E. faecalis (JCm5803) and B. subtilis (DSM2109), and two fungi, C. albicans (ATCC10231) and S. cerevisiae (P11), were obtained from the China General Microbiological Culture Collection Center and Beijing Bio-Med Technology Development Co., Ltd. Phosphate buffered saline (1 PBS, pH 7.4) was used throughout the identifcation work of the pathogens. NMR spectra were measured using a Bruker ARX 400 NMR spectrometer. High-resolution mass spectrometry (HRMS) measurements were performed in MALDI-TOF mode on a Finnegan MAT TSQ 7000 Mass Spectrometer. UV-vis absorption and photoluminescence (PL) spectra were recorded on a Milton Ray Spectronic 3000 array spectrophotometer and a Perki-nElmer LS 55 spectrometer, respectively. The absolute fluorescence quantum yield was measured with a Hamamatsu quantum yield spectrometer C11347. The size distribution and zeta potential results were recorded on a ZetaPALS Brochure. Fluorescence images and laser confocal scanning microscope images were collected on a fluorescence microscope (Upright Biological Microscope Ni-U) and confocal laser scanning microscopy (Zeiss LSM800 and Leica SP8), respectively. Fluorescence lifetime imaging was performed using an OLYMPUS IX73 microscope system. The morphology and transmission electron diffraction (TED) pattern of IQ-Cm aggregates were observed and collected by transmission electron microscopy (TEM, JEM 2010). Theoretical calculations were carried out with Gaussian 09 software at the B3LYP/6-31G** level. Naked-eye identifcation of pathogens IQ-Cm was added into a pathogen PBS suspension with a fnal concentration of 10 mM (G bacteria (OD 600 ¼ 1.0), G+ bacteria (OD 600 ¼ 1.0) or fungi (OD 600 ¼ 2.0)). The mixtures were incubated for about 10 min at room temperature, and then observed under 365 nm UV irradiation. ## Pathogen staining and imaging After incubating pathogens with IQ-Cm under the same conditions as described in the experiments of naked-eye iden-tifcation of pathogens, the mixtures were concentrated about 10 times by centrifugation (7100 rpm for 2 min). 2 mL of concentrated pathogen suspension was transferred to a clean glass slide, slightly covered by a coverslip, left for about 2 min for immobilization and then imaged. Imaging conditions of the fluorescence microscope: 100 objective lens, excitation flter ¼ 460-490 nm, dichroic mirror ¼ 505 nm, emission flter ¼ 515 nm long pass. For CLSM imaging using a Leica SP8: 100 objective lens, excitation flter: 488 nm, emission flter: 500-750 nm. Fluorescence lifetime imaging under an OLYMPUS IX73 microscope system: l ex ¼ 450 nm with fs laser pulses and l em ¼ 500-700 nm detected using a hybrid photomultiplier detector (PMA Hybrid 40, PicoQuant). Co-staining experiment: by following the above operation steps, three pathogens were incubated with 10 mM IQ-Cm and 5 mg mL 1 propidium iodide (PI) in PBS solution for 10 min and then imaged. For colocalization with the commercial mitochondrial dye MitoTracker Green, C. albicans was incubated with 1 mM IQ-Cm and 100 nM MitoTracker Green for 10 min. Imaging conditions: in the case of co-staining with PI, the images were collected using a fluorescence microscope with excitation flter ¼ 460-490 nm, dichroic mirror ¼ 505 nm, emission flter ¼ 515 nm long pass for IQ-Cm and with excitation flter ¼ 510-550 nm, dichroic mirror ¼ 570 nm, emission flter ¼ 590 nm long pass for PI. For colocalization with Mito-Tracker Green in C. albicans, the images were obtained using a Zeiss 800 confocal microscope with excitation wavelength l ex ¼ 405 nm and emission wavelength l em ¼ 600-620 nm for IQ-Cm and l ex ¼ 488 nm and l em ¼ 500-520 nm for MitoTracker Green. ## Conflicts of interest There are no conflicts to declare.

chemsum

{"title": "One stone, three birds: one AIEgen with three colors for fast differentiation of three pathogens", "journal": "Royal Society of Chemistry (RSC)"}

facile_route_to_conformal_hydrotalcite_coatings_over_complex_architectures:_a_hierarchically_ordered

## Abstract: An alkali-and nitrate-free hydrotalcite coating has been grafted onto the surface of a hierarchically ordered macroporous-mesoporous SBA-15 template via stepwise growth of conformal alumina adlayers and their subsequent reaction with magnesium methoxide. The resulting low dimensional hydrotalcite crystallites exhibit excellent per site activity for the base catalysed transesterification of glyceryl triolein with methanol for FAME production. † Electronic supplementary information (ESI) available: Full material synthesis and characterisation. See Rising global energy demand over the next 25 years, notably among emergent economies, 1 is driving the quest for sustainable routes to low cost, liquid transportation fuels from biomass feedstocks. 2 Around 9% of transportation energy needs are predicted to be met via liquid biofuels by 2030. 3 The past decade has seen much criticism of first-generation biofuels derived from edible plant materials which are attributed to significant land use changes and deforestation in South East Asia. 4 In order for advanced bio-fuels to be considered truly sustainable, they must be sourced from non-edible crop components, forestry waste, alternative non-food plants such as switchgrass, Miscanthus or Jatropha curcas 5 which require minimal cultivation and do not compete with traditional arable land or drive deforestation, algal sources or the lignocellulosic components of municipal waste such as packaging materials. Although there is burgeoning interest in extracting bio-oils from aquatic biomass, which can yield 80-180 times the annual volume of oil per hectare than plants, 6 process scale-up and the availability of nutrient resources remains challenging. 7 The biorefinery concept affords biomass the simplest and most popular approach to drop-in transportation fuels, 8 based upon carbohydrate pyrolysis and subsequent hydrodeoxygenation (HDO) 9 of the resulting bio-oils or their gasification and subsequent Fischer-Tropsch processing 10 to gasoline and diesel, 11 or lipid transesterification to biodiesel. 12 Catalytic depolymerisation of lignin may also unlock opportunities for the co-production of phenolics and related aromatic compounds via biorefineries for fine chemical and pharmaceutical applications 13 improving their cost-effectiveness. Biodiesel is a clean burning and biodegradable fuel 14 which remains popular for meeting transportation energy requirements in Europe, 15 Asia, 16 the Americas 17 and Africa. 18 Commercial biodiesel is produced almost entirely via the liquid base catalysed transesterification of C 14 -C 20 triacylglyceride (TAG) components of lipids with C 1 -C 2 alcohols 19 into fatty acid methyl esters (FAMEs) which constitute biodiesel. Higher alcohols have also been exploited 20 as they offer a less corrosive FAME with improved physical characteristics. 21 Isolation of the desired biodiesel product from homogeneous base catalysts (and unreacted mono-and di-alkyl glycerides and glycerol by-product) is necessary to circumvent saponification and emulsification side reactions and produce a high quality biofuel. 6 Heterogeneous, solid acid and base catalysts offer facile FAME separation, eliminating the requirement for quenching steps and permitting continuous biodiesel production, 25 and a purer glycerol by-product stream for use as a commodity chemical in the food and cosmetics industry. Among solid base catalysts, hydrotalcites, alkaline earth oxides and alkali-doped mesoporous silicas 34 are good potential candidates for biodiesel formation under mild conditions. Hydrotalcites (of general formula [M(II ) are a subset of microporous layered double hydroxides, 35 conventionally synthesised via co-precipitation from nitrates 36 in the presence of alkalis which are problematic due to NOx emissions 37 and in situ alkali leaching and consequent FAME contamination. We recently reported an alkali/nitrate-free route to tunable Mg-Al hydro-talcite coatings via the direct reaction of Mg(OCH 3 ) 2 with a conventional, bulk alumina support. 41 While the resulting materials exhibited excellent turnover frequencies (TOFs) towards TAG transesterification, they suffer from a number of important drawbacks, namely: poor specific activity per unit mass towards bulky C 18 substrates (0.042 mmol min −1 g −1 ), low surface areas, and restricted (and disordered) pore architectures available through the use of pure alumina templates. In contrast, hydrothermally stable silica frameworks can be readily synthesised, offering diverse pore interconnectivities and bi-or tri-modal pore networks. Here we extend our previous methodology to create crystalline, catalytically active hydrotalcite coatings via a versatile two-step methodology, permitting (i) the first genesis of an ultrathin alumina adlayer over a complex (hierarchically ordered) template, and (ii) facilitating its subsequent reaction with Mg(OCH 3 ) 2 to form a stoichiometric HT/ MM-SBA-15 hydrotalcite catalyst. This novel methodology opens the way to a new class of solid bases built upon the tunable interconnectivity and porosity afforded by underlying silica architectures. The resulting nanocomposite combines the high surface area and excellent mass-transport characteristics of the parent silica, and solid basicity and transesterification performance of a pure hydrotalcite. ## Catalyst synthesis Synthesis of macroporous-mesoporous SBA-15 (MM-SBA-15) An hierarchical macroporous-mesoporous SBA-15 silica was prepared following the method of Dhainaut et al. 22 Briefly polystyrene beads synthesised via the emulsion polymerisation approach of Vaudreuil and co-workers 45 were added to an acidified, aqueous solution of Pluronic® P123 surfactant prior to the addition of tetramethoxysilane. The resulting gel was hydrothermally aged without agitation, and the solid obtained filtered, washed and dried at room temperature before calcination at 550 °C for 6 h in air. ## Alumina grafting onto MM-SBA-15 (Al-MM-SBA-15) The Al-MM-SBA-15 hierarchical framework employed the method of Landau and co-workers developed for MCM-41. 46 Aluminium-tri-sec-butoxide (14.5 g) was dissolved in anhydrous toluene (100 cm 3 ) at 85 °C with stirring. Triethylamine (2.1 cm 3 ) was added to this solution, followed by dried MM-SBA-15 (1 g). After 6 h stirring at 85 °C the solution was filtered under vacuum (∼0.1 bar), with the recovered solid washed three times in toluene (100 cm 3 ). The alumina surface was then hydrolysed in ethanol (318 cm 3 ) containing water (1.6 cm 3 ) for 24 h at 25 °C with stirring, and the resulting solid recovered by vacuum filtration and washed with ethanol (300 cm 3 ) before drying at 80 °C in a vacuum oven overnight. A three-step calcination sequence was utilised to form an alumina monolayer: the material was first heated to 250 °C for 1 h, then 400 °C for 1 h and finally 500 °C for 4 h (each ramp rate 1 °C min −1 ). Consecutive grafting cycles were undertaken employing an identical protocol in order to progressively build-up alumina monolayers over the silica surface, adjusting the quantities to maintain the initial Al:Si stoichiometry. ## Synthesis of hydrotalcite-coated MM-SBA-15 (HT/MM-SBA-15) Magnesium methoxide solution (8-10 wt% in methanol) was added to Al-MM-SBA-15 (400 mg, dried for 1 h at 80 °C), at the minimum quantity to form a homogeneous paste on mixing. After stirring for 15 min, the mixture was dried under vacuum at 80 °C for 1 h to remove excess methanol. The surface Mg : Al atomic ratio was tuned by adjusting the volume of magnesium methoxide (10.8 cm 3 for the MM-SBA-15). The resulting material was calcined at 450 °C for 15 h under 20 cm 3 min −1 O 2 (ramp rate 1 °C min −1 ). After cooling to room temperature under N 2 (20 cm 3 min −1 ), the powder was added to distilled water (50 cm 3 for every 300 mg of powder) in a 100 cm 3 roundbottomed pressure vessel and heated to 125 °C with stirring for 21 h. After cooling to room temperature, the final HT/ MM-SBA-15 sample was filtered, washed with deionised water and dried in a vacuum oven overnight at 80 °C, before storage in a desiccator. This synthesis proved successful on the multigram scale. A conventional hydrotalcite reference material was prepared via our alkali-free, co-precipitation method from Mg- 26 with the Mg: Al atomic ratio tuned to match that of the MM-SBA-15. ## Materials characterisation Nitrogen porosimetry was undertaken on a Quantachrome Nova 1200 porosimeter. Multi-point BET surface areas were calculated over the relative pressure range 0.01-0.3. Pore diameters and volumes were calculated applying either the t-plot or BJH methods to the desorption isotherm. Powder XRD patterns were recorded on a PANalytical X'pertPro diffractometer fitted with an X'celerator detector and Cu Kα source; the Scherrer equation was used to calculate HT crystallite sizes. XPS was performed on a Kratos Axis HSi X-ray photoelectron spectrometer fitted with a charge neutraliser and magnetic focusing lens employing Al K α monochromated radiation (1486.7 eV). Spectral fitting was performed using CasaXPS version 2.3.15. Base site densities were measured via CO 2 pulse chemisorption and subsequent temperature programmed desorption (TPD) on a Quantachrome ChemBET 3000 system coupled to an MKS Minilab QMS. SEM analysis was carried out on a Carl Zeiss EVO SEM fitted with an Oxford Instruments energy dispersive X-ray (EDX) analyser employing Oxford Instruments Inca Software. TGA was performed using a Stanton Redcroft STA780 thermal analyser. ## Transesterification The HT/MM-SBA-15 and conventional HT materials were tested as catalysts in the transesterification of triolein to form methyl trioleate (FAME) using a Radleys Starfish parallel reactor. Briefly, 50 mg of catalyst was added to 10 mmol of triolein using a 30 : 14 : 1 methanol : butanol : oil ratio; butanol was added as a co-solvent to help solubilise the triglyceride. In light of the significant differences in HT content between our conventional and SBA-15 coated materials, a common total mass of catalyst (rather than mass of hydrotalcite) was employed to ensure identical mixing characteristics within the reaction vessel. Reactions were carried out at 90 °C in a modified ACE™ 50 cm 3 round bottom pressure flask, with aliquots removed periodically from the reaction mixture for analysis on a Varian 450 GC with 8400 autosampler ( programmable oncolumn injection onto a Phenomenex ZB-1HT column (15 m × 0.53 mm × 0.15 μm film thickness). Initial rates were calculated from the linear portion of the reaction profile during the first 60 min of the reaction. Turnover frequencies (TOFs) were determined by normalising rates to the total base site density from CO 2 chemisorption. ## Results and discussion Characterisation of Al-MM-SBA-15 Alumina grafted silica (Al-MM-SBA-15) was first prepared as support for subsequent conversion to a high area, hierarchically ordered hydrotalcite coating. The alumina grafting process was repeated four times to obtain a uniform multilayer interface, with textural properties characterised after each grafting in order to examine the evolution of the aluminasilica interface. Low angle XRD and TEM of the parent MM-SBA-15 and the sequentially alumina grafted variants (Fig. S1-2 †) confirmed the presence of ordered mesopores indicative of SBA-15. 47 Characteristic (100), ( 110) and (200) reflections were observed for all materials, indicative of the p6mm space group expected for hexagonally arranged mesoporous channels. 48 Macropore incorporation shifted these reflections to higher angle relative to conventional mesoporous SBA-15, associated with a small contraction in the mesopore lattice parameter. 22 This contraction is attributed to curvature of the mesopore channels as they coalesce around the polystyrene bead template due to strong electrostatic interactions between the beads, block co-polymer and silica precursor. Long range, hexagonally ordered mesopores remained present for Al-MM-SBA-15 even following four consecutive grafting cycles. Nitrogen porosimetry of the parent MM-SBA-15 and alumina grafted analogues confirmed that the mesoporosity intrinsic to the SBA-15 framework is maintained after each grafting cycle (Fig. S3a-b †). However, the BET surface area (and interconnecting micropore area from t-plot analysis), mean mesopore diameter, and total mesopore and micropore volumes decreased progressively with each grafting cycle (Table 1), consistent with an increasing thickness of conformal alumina overlayer uniformly distributed throughout the pore network. The surface of Al-MM-SBA-15 was subsequently investigated by XPS. Successful alumina grafting was confirmed by the presence of surface Al, with the Al : Si atomic ratio increasing monotonically with each cycle, reaching 22.3 wt% Al after four cycles. Fig. 1 compares the Al and Si 2p chemical environments for the parent and alumina grafted mesoporous silicas. The pure MM-SBA-15 exhibited a single Si 2p spin-orbit split doublet centred with Si 2p 3/2 component at 103.1 eV binding energy associated with pure silica. Alumina grafting significantly attenuated the substrate signal consistent with a conformal (Frank-van der Merwe) growth mode, rather than the formation of 3-dimensional alumina islands. This attenuation was accompanied by the emergence of a second low binding energy doublet at 102.2 eV for Al-MM-SBA-15, which can only be associated with a new, interfacial silicon species. This hypothesis is supported by the observation of two distinct Al 2p spin-orbit doublets, at 73.8 eV and 74.7 eV. The former is consistent with pure alumina, and its intensity increases monotonically with grafting cycle relative to the high binding energy state, precisely as expected if the latter was associated with an interfacial alumina species. The opposing binding energy shifts for the interfacial Al and Si species are similar to those previously observed for alumina grafted SBA-15, 49 and can be understood in terms of their different Pauling electronegativities and associated induced dipoles mediated via the Al-O-Si bridges which increase and decrease the local initial state charge on interfacial Si and Al atoms respectively. An estimate of the alumina film thickness may be obtained by comparing the experimentally determined Al surface density (derived from porosimetry and XPS) with that for a crystalline alumina phase such as α-Al 2 O 3 , which exhibits a rhombohedral (4.75 × 4.75 ) surface unit mesh containing three Al atoms within the (006) plane as shown in Scheme 1. 50 With a total surface area of 473 m 2 g −1 , a single α-Al 2 O 3 monolayer covering the entire silica pore network would contain 0.0009 mol Al, equating to an Al loading of 24.3 wt%. This is close to the observed value of 22.3 wt%, and indicates that an alumina film approximately 0.7 monolayers thick (∼0.17 nm) is formed following four grafting cycles, which would constrict the mesopores by 0.34 nm relative to the parent MM-SBA-15, in excellent agreement with the observed pore diameter decrease of 0.3 nm seen in Table 1. ## Characterisation of HT/MM-SBA-15 Powder XRD diffraction patterns for the methoxide functionalised Al-MM-SBA-15 material prepared via four alumina grafting cycles (HT/MM-SBA-15), and a reference bulk HT sample prepared by conventional alkali-free co-precipitation (ConvHT) are shown in Fig. 2. The HT/MM-SBA-15 sample shows a diffraction pattern characteristic of a pure HT phase, very similar to that observed for the ConvHT standard, but with broader reflections indicative of significantly smaller crystallite sizes (as anticipated in light of the highly dispersed alumina substrate, which by inference appears to undergo little restructuring during the crystallisation process) and turbostratic disorder. 51 There was no evidence for brucite 52 and only a single weak reflection likely associated with trace MgO. This confirms the successful synthesis of a hydrotalcite phase through direct reaction of a pre-formed, ultrathin alumina film and magnesium methoxide from solution. Crystallite sizes determined using the Scherrer equation, interlayer spacings, lattice parameters and Mg : Al ratios determined using Vegard's law (Fig. S4 †) are reported in Table 2. The composition, lattice parameter and interlayer spacings of the HT/MM-SBA-15 material were almost identical to that of the ConvHT, confirming that the hydrotalcite phase formed at the surface of the hierarchical silica support was essentially indistinguishable from that of obtained by traditional synthetic methods, but with a surface area around five times higher. However, the significant difference in microporous crystallites size is expected to hinder accessibility of reactants to active sites within the ConvHT interlayers relative to the HT/ MM-SBA-15 sample whose dimensions suggest a hydrotalcite bilayer wherein a far greater proportion of base sites reside on exposed surfaces. Textural properties of the HT/MM-SBA-15 material are compared with those of the Al-MM-SBA-15 precursor in Fig. 3. Nitrogen porosimetry evidences retention of mesopore and macropore character within the adsorption/desorption isotherms following HT crystallisation, although their demarcation is not as clear as for Al-MM-SBA-15 (Fig. S3 †), while Table S1 † shows virtually no change in either the mesopore volume or mean mesopore diameter upon reaction of the alumina adlayer with Mg(OCH 3 ) 2 . This suggests that either extremely thin HT crystallites are formed throughout the bimodal pore network (consistent with XRD), or that hydrotalcite formation is confined to the macropores. The latter would be expected to hinder accessibility of the mesopores (for which macropores serve as the principal conduits), and hence reduce both the mesopore volume and total surface area, in contrast to the observed values reported in Table S1. † SEM of the HT/MM-SBA-15 (Fig. 4) confirms the macropore network present within the parent MM-SBA-15 support is retained throughout the material after hydrothermal treatment, a measure of the excellent stability of silica frameworks towards high temperature water, and conditions that a comparable pure hierarchical alumina structure would be unlikely to survive. TEM shows macropores are decorated with high aspect ratio hydrotalcite nanocrystallites. Thermogravimetric analysis confirms the excellent thermal stability of the HT/MMSBA-15 (Fig. S5 †), with only a small 10% weight loss between 70 and 220 °C, associated with the desorption of physisorbed water from the HT surface and water from within the interlayers, 53 and a 5% loss between 250 and 350 °C attributed to hydroxide anions in the brucite-like layers. 54 EDX elemental analysis of the HT/MM-SBA-15 yields an overall Mg : Al atomic ratio of 2.2 : 1, in good agreement with that derived from Vegard's law in Table 2, and a total Mg content of 19.8 wt% i.e. a quarter that of a bulk hydrotalcite of comparable Mg : Al ratio, 26 consistent with the formation of hydrotalcite nanocrystals approximately 1 nm thick relative to silica walls around 4-5 nm thick in the MM-SBA-15 support. 49 Surface base properties of the HT/MM-SBA-15 and ConvHT bulk reference materials were assessed by temperature-programmed desorption of CO 2 -saturated samples, presented in layers of the bulk hydrotalcite structure may be inaccessible to sterically-demanding substrates. These results confirm that high aspect ratio hydrotalcite crystallites formed over the hierarchical silica support possess similar intrinsic basicity to conventional co-precipitated analogues. Surface analysis of HT/MM-SBA-15 yielded a Mg : Al atomic ratio of 2.21 and Mg content of 16.7 wt%, both very similar to values determined by EDX, evidencing uniform incorporation of Mg into the alumina film throughout the pore network of the Al-MM-SBA-15 precursor. Si 2p XP spectra shown in Fig. 5a reveal that hydrotalcite formation was accompanied by attenuation of the interfacial alumina species, and concomitant appearance of a new low binding energy chemical environment at 101.5 eV. The latter suggests that interfacial silicon atoms are now bound (through oxygen bridges) to a less polarising adlayer relative to alumina, consistent with the exchange of Al 3+ for Mg 2+ cations. Fig. 5b shows analogous changes in the Al chemical environment, with attenuation of the pure (and interfacial) alumina adlayer, and emergence of a high energy Al state ∼74 eV, consistent with the introduction of Mg 2+ cations into the grafted alumina film during hydrotalcite formation. The corresponding Mg 2s XP spectrum of HT/ MM-SBA-15 presents a single chemical environment around 88.5 eV. In summary, over 75% of the MM-SBA-15 silica surface is contacted with a hydrotalcite phase, and a similar proportion of the initially grafted alumina adlayer in Al-MM-SBA-15 is converted into hydrotalcite. ## Transesterification of triglycerides In order to establish the catalytic efficacy of the HT film encapsulating the hierarchical silica template, the HT/ MM-SBA-15 material was screened in the transesterification of glyceryl triolein, a bulky C 18 triglyceride that is a major component on oilseed feedstocks, with methanol for FAME (biodiesel) production. The resulting reaction profiles for HT/ MM-SBA-15 and the co-precipitated ConvHT analogue are shown in Fig. 6. Transesterification proceeded rapidly over both catalysts during the first hour of reaction before slowing dramatically, to give limiting conversions of 34% and 64% for HT/MM-SBA-15 and ConvHT respectively. While the absolute FAME productivity of the bulk hydrotalcite is clearly superior, it is important to recall that the HT/MM-SBA-15 only contains a thin hydrotalcite coating and the majority of this catalyst is composed of inert silica. A fairer comparison of the relative catalytic performance is obtained from their initial rates of triolein conversion and turnover frequencies (TOFs) normalised per base site utilising the CO 2 TPD measurements. This reveals a common initial rate of 1 mmol g catalyst −1 min −1 , however one must recall that the HT/MM-SBA-15 material only contains one quarter of the amount of hydrotalcite present within the bulk ConvHT material, hence the rate normalised per mass of hydrotalcite is four times higher for HT/ MM-SBA-15 catalyst. Since the base site density of the coated hydrotalcite is also ∼34% lower than that of its bulk counterpart, the rate enhancement per base site of the coated material is higher still, translating to TOFs of 7.6 min −1 for the bulk ConvHT standard versus 66 min −1 for HT/MM-SBA-15. Hydrotalcites prepared via conventional co-precipitation are among the most widely-used catalysts for triglyceride transesterifica- tion to FAME, hence the nine-fold rate enhancement observed for our HT/MM-SBA-15 material provides a striking benchmark of its exceptional performance. While the magnitude of this enhancement does fall at longer reaction times, likely due to partial deactivation of the coating, the HT/MM-SBA-15 remains three times as active per base site as the bulk hydrotalcite, even after 1400 min reaction. Since the intrinsic base strength of active sites within the conventional and hierarchical hydrotalcite catalysts is the same (common CO 2 desorption temperatures, Fig. S6 †), we attribute this nine-fold rate enhancement of HT/MM-SBA-15 to superior mass-transport characteristics of the macroporousmesoporous architecture. Indeed, the magnitude of the HT/ MM-SBA-15 enhancement with respect to the ConvHT standard is comparable to that previously reported for a macroporous pure HT material, 28 but affords a far more flexible and hydrothermally stable framework than the latter synthesis. ## Conclusions Sequential, wet-chemical surface modification of nanostructured silicas with Al and Mg precursors offers a versatile route to the preparation of high area, tailored solid base hydrotalcite catalysts. Stepwise grafting and thermal processing of aluminium-tri-sec-butoxide results in a uniform alumina monolayer throughout the bimodal macropore-mesopore network. Subsequent reaction with Mg(OCH 3 ) 2 affords stoichiometric incorporation of aluminium from the alumina adlayer into ∼1 nm Mg 2 Al hydrotalcite crystallites, which possess identical basicity as a co-precipitated, bulk hydrotalcite. In contrast to bulk (monomodal) alumina templates, the development of a silica based methodology results in HT/MM-SBA-15 catalyst exhibits similar specific mass activity in the transesterification of glyceryl triolein with methanol as a bulk hydrotalcite, despite containing only a small fraction of the number of active sites, indicating far greater active site accessibility to the bulky TAG reactant. The latter conclusion is supported by a nine-fold enhancement in the TOF per base site for the hierarchical hydrotalcite, indicating the majority of base sites in HT/MM-SBA-15 reside at the external surface of nanoscale crystallites within the meso-and macropores, rather than within the microporous interlayers of conventional hydrotalcites. Our methodology is readily extendable to diverse silica architectures and other metal oxides, opening up opportunities for the facile introduction of hydrotalcite solid basicity into complex two-or three-dimensional materials, e.g. membranes and monoliths, for catalysis and sorption applications.

chemsum

{"title": "Facile route to conformal hydrotalcite coatings over complex architectures: a hierarchically ordered nanoporous base catalyst for FAME production", "journal": "Royal Society of Chemistry (RSC)"}

agents_for_sequential_learning_using_multiple-fidelity_data

## Abstract: Sequential learning for materials discovery is a paradigm where a computational agent solicits new data to simultaneously update a model in service of exploration (finding the largest number of materials that meet some criteria) or exploitation (finding materials with an ideal figure of merit). In real-world discovery campaigns, new data acquisition may be costly and an optimal strategy may involve using and acquiring data with different levels of fidelity, such as first-principles calculation to supplement an experiment. In this work, we introduce agents which can operate on multiple data fidelities, and benchmark their performance on an emulated discovery campaign to find materials with desired band gap values. The fidelities of data come from the results of DFT calculations as low fidelity and experimental results as high fidelity. We demonstrate performance gains of agents which incorporate multi-fidelity data in two contexts: either using a large body of low fidelity data as a prior knowledge base or acquiring low fidelity data in-tandem with experimental data. This advance provides a tool that enables materials scientists to test various acquisition and model hyperparameters to maximize the discovery rate of their own multi-fidelity sequential learning campaigns for materials discovery. This may also serve as a reference point for those who are interested in practical strategies that can be used when multiple data sources are available for active or sequential learning campaigns.A central concern of the materials discovery and optimization process is a simple, practical question: given limited researcher time and resources, what is the next experiment that should be performed? The urgent need for new energy technologies to mitigate fossil fuel use makes this question especially relevant. Widespread adoption of novel fuel cell catalysts, batteries, thermoelectrics, and other energy technologies requires optimization on many different fronts: materials discovery campaigns may target compounds with improved cost, safety, stability, efficacy, or some combination of these and other goals. The use of artificial intelligence tools to accelerate the discovery and optimization process, hand-in-hand with developments in high-throughput experimentation and analysis, may help us to meet timely goals for decarbonization of the global energy economy.This work is a step towards bridging three relatively recent advances in the materials science research community, which are still realizing their individual and combined potential: (1) the advent of large-scale and freely available databases of computational simulations 1-4 , particularly from density functional theory (DFT) 5,6 , (2) the mainstream accessibility of machine learning tools 7 , and (3) development of high-throughput experimentation hardware and software [8][9][10][11] . DFT has shown its applicability in complementing and even guiding the experimental discovery of materials [12][13][14] . Machine learning exploits the widespread availability of DFT results to allow accurate and interpretable surrogate models to estimate a desired materials property before either experiment or simulation 12,[15][16][17][18][19][20][21][22][23] . By increasing the efficiency of theoretical property prediction, the combination of large-scale DFT and machine learning makes it easier for researchers to obtain theoretical predictions for a wider variety of materials, which can then guide the high-throughput experimental process. The paradigm of sequential (or active) learning (henceforth SL), in which a model solicits new training data and updates its performance in response to this data, is useful both to computational [24][25][26][27][28] and experimental high throughput studies for both optimization and analysis 29 . Some examples of sequential learning include: systems that learn how to perform only the most valuable or relevant DFT simulations using previous iterations 25,30,31 , improve force fields more rapidly for molecular dynamics simulations 24,32 , and synthesize carbon nanotubes at new conditions that promote higher yields and higher qualities of product 33 . The sequential learning paradigm thus can provide a conceptual link between materials optimization and discovery workflows across computational and experimental methodologies.This work also considers a complication in the reality of the scientific discovery process, where there are many sources of data with different costs to obtain them. We use "multi-fidelity" to describe these diverse data Dataset collection and representation. The band gap dataset was collected from two sources: (1) experimentally reported band gaps of inorganic semiconductors aggregated by Zhuo et al. 34 and disseminated via the Matminer 50 package, and (2) GGA-level DFT-computed band gaps generated and disseminated via the Materials Project database 1,51 . We first pulled the experimentally reported compositions and their corresponding band gaps. For each experimental composition, we attempted to obtain the band gap corresponding to the most phase-stable (i.e. lowest computed energy per atom) crystal structure from the Materials Project. Here, the DFTcomputed band gaps using the Perdew-Burke-Ernzerhof (PBE) functional 52 were considered low fidelity data, as GGA has well-known systematic errors that underestimate experimentally measured band gaps by ∼ 0.9 eV 53 . Overall, 3960 unique compositions had both experimental and theory data. Out of the 3960 compositions, 375 contained multiple experimental band gap measurements. For each composition with multiple experimental measurements, the respective minimum band gap value was used. Figure 1 describes the dataset by outlining the element occurrence, which shows abundant oxides, sulfides, and selenides, as well as copper and lithiumcontaining compositions. We processed the collected data by using a fixed-length vector to encode both the compositions of each material and the level of fidelity. More specifically, stoichiometric compositions were featurized with the matminer ElementProperty featurizer 50 . This featurizer offers flexibility to compare experimental and theoretical data when experimental structure information is not available. The levels of fidelity were represented with one-hot encoding, where a binary variable was added for each fidelity level (i.e. for experimental data, a "1" was placed under the "experiment" feature and a "0" under the "theory" feature and vice versa). To improve numerics, the final overall features were scaled such that their distribution had a mean of 0 and a standard deviation of 1. on the recently introduced system for Computational Autonomy for Materials Discovery (CAMD) 31 . CAMD is a framework that abstracts decision-making in sequential learning studies into "agents". Agents perform tasks like training and applying machine learning models or choosing which experiment should be done next based on user-specified criteria. CAMD is open-source and users can add new agents according to their needs (such an agent is one of the contributions of this manuscript). The CAMD framework enables convenient design and testing of acquisition strategies from candidate data points in SL-based optimization. Figure 2 outlines the CAMD framework and highlights the newly constructed multi-fidelity acquisition feature. In a given series of iterations, termed a campaign, the (multi-fidelity) seed data and candidate data (search space) go into an agent. A preprocessing step in the agent featurizes each data point in the seed data and candidate data using the point's composition and fidelity as described in the previous section. The featurized seed data is used to train a machine learning model, which makes predictions on the candidate data for the target property. Using the predictions, the agent then selects candidates at different fidelities. In the CAMD framework, candidates selected by the agent are sent to an experiment API, which collects the experimental data corresponding to the candidate and augments the dataset, allowing candidate data to be moved into the seed data for new active learning iterations. For the sake of active learning simulation, the CAMD experiment is an "after-the-fact" (ATF) API that emulates DFT simulation and experimental measurement, respectively, and returns the results from the known dataset which the agent and CAMD campaign are not aware of prior to the acquisition. This after-the-fact protocol reflects the scope of our study: to benchmark multi-fidelity agent performance in how efficiently they explore a known dataset, demonstrating the gains of multi-fidelity agents with various exploration strategies. The ATF experiment API can be exchanged for one that collects data from and monitors a real experiment, performing new experiments or DFT simulations with the agent that has been designed using ATF simulations 31 . Another CAMD object, the analyzer, monitors the campaign results and provides an analysis of the experiments in the context of the previously collected data (i.e. the seed data) and the progress of the campaign. In our case, the analyzer monitors and reports the cumulative number of materials suitable for solar photoabsorption. Upon the completion of the agent selection, experimental acquisition, and analysis phases of a campaign iteration, newly obtained experimental results are appended to the seed data and removed from the candidate data, and campaign begins in a new iteration with agent selection. Agent design for materials discovery. Designing the agent for a multi-fidelity sequential learning procedure required two steps: (1) selecting appropriate machine learning models and (2) generalizing a CAMDcompatible 31 data acquisition decision-making process to allow for multiple levels of data fidelity. For model selection, we implemented and compared several well-known regression methods, including support vector regression (SVR), k-nearest neighbors (KNN), random forest regression (RFR), and Gaussian process regression (GPR). For each model, we optimized hyperparameters and did comparative performance analysis (detailed results can be found in Supplementary document S1). Based on the results, support vector regression, random forest regression, and Gaussian process regression had qualitatively similar performances and were used for www.nature.com/scientificreports/ framework construction and demonstration. Our implementation is sufficiently general to allow users to choose any scikit-learn-compatible ML model and their choice of hyperparameters. A primary design concern in developing a multi-fidelity agent is mathematically framing the problem of when to draw from low-cost, low-fidelity data vs. high-cost, high-fidelity data. To this end, we designed two agents, an epsilon-greedy multi-fidelity agent (henceforth ǫ-greedy-MF) and a Gaussian process lower confidence bound 54 derived multi-fidelity agent (GPR LCB -MF). The latter exploits the fact that Gaussian Process regression allows for a principled uncertainty estimate "out-of-the-box", whereas the former works for regression algorithms lacking this feature. The salient features of the ǫ-greedy agent are that it takes as input a budget of high-fidelity datapoints n which controls the balance between low-fidelity and high-fidelity data. The agent will only call for high-fidelity measurements in domains that have been previously covered by low-fidelity data (see details in Algorithm S1). The ǫ-greedy-MF agent works using any supervised machine learning regressor from scikit-learn 7 as input. Meanwhile, the GPR LCB -MF agent operates under a total acquisition budget and calls for low-or high-fidelity data in a more sophisticated way. It acquires candidates factoring in Gaussian process regression predicted uncertainties in the LCB setting and hallucination of information gain from low fidelity acquisitions analogous to work of Desautels et al. in batch mode LCB 55 (see full details in Algorithm S2). Hallucination works as such: for a high fidelity candidate, the GPR LCB -MF agent adds the lower fidelity predicted posterior mean into the seed data. As a consequence, the higher fidelity candidate prediction gets updated. Essentially, hallucination refers to the ability of the agent to predict ahead of time how low-fidelity data will impact the uncertainty estimate of the model. Hallucination allows the agent to use low-fidelity candidates to explore potentially promising parts of the domain, while using high-fidelity candidates to exploit promising regions of parameter space, offloading exploratory (higher risk) acquisitions first to lower-fidelity computations. In our formulation, three hyperparameters that must be empirically optimized govern the tradeoff between data fidelities: α , β , and γ . α is the uncertainty multiplier in GPR LCB as shown below: where ŷi is the posterior mean and σ i is the uncertainty given a candidate i. α here sets the weight of uncertainty in the LCB setting. Next, β is a threshold for uncertainty. For a given observation, if its σ i is less than β , the observation is considered to have low uncertainty. A small β makes the agent "risk-averse" around high-fidelity measurements in unexplored regions of space. In the small β regime, unless the uncertainty on a given prediction is very low, it will acquire lower fidelity data first. Inversely, if β is large, the agent is tolerant to high uncertainty for experiments and will more readily add experimental data. In practical applications, β could be set with respect The agent passes the instruction to an "experiment" (a generic term for some kind of data generation, and could alternately be a theoretical calculation). (b) Our work is distinguished by making available two different sources of data with differing degrees of fidelity (here, we use experimental and theoretical data). The agent makes predictions using both sources of data and makes the decision to select new data from one pool or the other. www.nature.com/scientificreports/ to the cost of acquiring high-fidelity data. Lastly, γ is a threshold for the influence of hallucination (denote r ) as shown below: Here, r i is the ranking of an observation ŷi ,LCB in the candidate space based on its distance to the target value. r * i is the new ranking of the observation after hallucination.The agents acquire a high fidelity candidate if �r ≤ γ . If γ is 0, then the prospect of the lower fidelity data has to increase the chances of the experiment being successful. Otherwise, lower fidelity data will be acquired first. Because in this case, r * i has to be a smaller value than r i (i.e. a better ranking). If γ is very large, then the agent does not care about how much low fidelity data affects the potential experiment. Because in this case, r * i can be any value, including a value that is higher than r i (i.e. a worse ranking). The overall influence of these hyperparameters is summed up in a broad overview way in Fig. 3. We simulated various scenarios of these three hyperparameters to optimize the agents. The details and results are in Supplementary document section S3. The Gaussian processes were implemented using the GPy 56 package. Details of agents are also made explicit in the code available via the open-source CAMD repository at https:// github. com/ TRI-AMDD/ CAMD. When a data point x is called for as an experimental candidate, the above flowchart describes the decision-making process for which data source to use. After a data point x is selected for measurement, two conditions are checked: (1) if the corresponding (e.g. with the same formula) low-fidelity measurement has already been made or (2) if the uncertainty associated with the high fidelity measurement is low enough (below a threshold β ). If either is true, the high-fidelity measurement is taken. If neither are true, then the agent must consider the trade-off between low-fidelity and high-fidelity data. The agent thus considers how a low fidelity data point would affect the current ordering of the predicted figure of merit associated with all candidates. If it would alter the ranking by more than γ , the low-fidelity measurement is taken. If not, the high-fidelity measurement is taken. Note that β and γ are user-defined hyperparameters explained in detail in Section S2. Vol:.( 1234567890) 49 . These metrics are ALM, acceleration factor (AF), and enhancement factor (EF), defined as follows and explicated further below: where x and y are agents, N exp is the number of experiments performed (i.e. in our case, the high fidelity data acquired). The function N exp conditioned on ALM (i.e. N exp (x|ALM) and N exp (y|ALM)) refers to the of experiments in the sequential learning campaigns that attained an ALM. Because our emulated discovery campaigns are trying to find materials with a visible-spectrum band-gap, our discovery process can be scored in a binary way: any new data point's band gap is either inside or outside of the target range. Thus, we can compute the fraction of ideal materials which were correctly identified at an iteration given a sequential learning run and so ALM lies within . This metric is defined for a single sequential learning campaign and is most useful for after-the-fact workflows, as the denominator requires some knowledge of the total number of materials which lie within the target range (For a 'real-world' case where the materials are not known ahead of time, the final number of target materials discovered by the campaign can be used in scoring sequential learning, as ALM is a 'time-dependent' property that can change at each iteration step. Also, note that this study focuses on materials that are scored in a binary way as having the band gap property within a target range. For cases where a quantity is optimized around some target, this could be defined using the distance of the best-known material thus far to the current best-known global maximum/minimum target property). Next, acceleration factor and enhancement factor are metrics that compare two sequential learning runs to one another using the ALM. The acceleration factor is the reduction of required budget (e.g. in time, iterations, or some other consumed resource) between an agent and a benchmark case (e.g. random selection, an alternate model, single-fidelity, or manual human selection) to reach a particular fraction of ideal candidates (AF = N budget,benchmark -N budget,agent ). In other words, given ALM vs. N exp , the acceleration factor is the "horizontal-line" distance between two models at an ALM at different "times". A positive value of acceleration factor between a multi-fidelity campaign and a single-fidelity campaign means the former outperformed the latter because it reduced the required budget needed to achieve a certain amount of discovery. Similarly, the enhancement factor is the "vertical-line" distance between two campaigns' ALM score at a given "time", which shows the performance enhancement at the same consumed experiment budget. More specifically, at the same number of iterations, amount of elapsed time, or some other metric of expended resources, enhancement factor quantifies the improvement of materials discovery by a given sequential learning method versus a benchmark method (EF = N discovery,agent N discovery,benchmark ). In the case of comparing a multifidelity campaign to a single-fidelity campaign, when the enhancement factor is greater than one, it indicates that the multi-fidelity campaign outperforms its corresponding single-fidelity campaign at a given budget. ## Sequential learning objective. For multi-fidelity sequential learning campaign simulations and subsequent performance evaluations of the agents, we attempted to model a discovery campaign for photoabsorbers by targeting materials with experimentally measured band gap ⊆ [1.6, 2.0] eV 57 , i.e. those with reasonable solar photoabsorption, were considered ideal and set as the targets. 207 of our 3960 candidate experimental materials are considered ideal based on the target band gap window defined above. In other words, only about one in twenty or 5% of the candidate materials lie within the target window of the discovery campaign. ## Results Figure 4 highlights the campaigns that we performed to benchmark the sequential learning models. In "Boundary cases: all or no DFT data available" (corresponds to campaign A), we demonstrate the performance gains which come from an agent with full a priori knowledge of DFT calculations soliciting experimental data versus an agent which never uses DFT data exploring the same space of experiments. Next, in "In-tandem acquisitions: both DFT and experiment data are acquired" (corresponds to campaign B), we compare the performance of agents seeded with first 500 experimentally discovered compositions in a multi-fidelity versus single-fidelity context, where either both DFT and experimental data are solicited in-tandem (with some DFT data supplied a priori) or exclusively experimental data seeded and solicited. In both cases, we find that the performance of multi-fidelity agents are improved by the inclusion of low-fidelity DFT data. Boundary cases: all or no DFT data available. We first tested the acquisition performance of multifidelity agents in the limiting case where the full suite of DFT calculations was considered as a priori knowledge. The objective here was to determine how an automated experimental sequential learning procedure would be enhanced by a priori knowledge of a large theoretical dataset. This type of acquisition is for a use case where low-fidelity experimental data is much cheaper to acquire than high-fidelity data that full domain coverage is available at the outset of a high-fidelity experimental campaign. Because no new low-fidelity data is solicited, gains in campaign performance are entirely due to the transfer of knowledge from the large, low-fidelity dataset in making predictions and subsequent acquisitions under the high-fidelity, expensive setting. We performed after-the-fact discovery runs with three agents: ǫ-greedy agents that used support vector regression and random forest regression, and a GPR LCB agent. As mentioned previously, ǫ-greedy agents works for regression models lack principled uncertainty estimate, and GPR agent acquire candidates based on both the predicted posterior mean and uncertainty from Gaussian process regression. For each agent, we considered two cases: (1) no low-fidelity seed data at any point in the campaign and (2) all available DFT data as seed data at the outset. Note that both (1) and ( 2) are thus only acquiring high-fidelity data, and this set of six campaigns benchmarks in the most extreme case if and how much a priori low fidelity knowledge can assist in the discovery campaign. For convenience, we designate SVR-SF boundary , RFR-SF boundary , and GPR LCB -SF boundary , SVR-MF boundary , RFR-MF boundary , and GPR LCB -MF boundary (SF denotes single-fidelity, MF denotes multi-fidelity). We gave all the agents a budget of 20 experiment requests in each iteration and simulated each campaign for 100 iterations. In addition, several campaigns have additional stochasticity that requires some thought. More specifically, single-fidelity campaigns with no seed data (i.e. SVR-SF boundary , RFR-SF boundary , GPR LCB -SF boundary ) create initial seeds data randomly, and random forests also have randomness during the bootstrapping of the samples used in building trees. Even though this stochasticity does not change the candidate acquisition strategy of the agents, it could result in varied campaign performance depending on the inputted random seeds. To account for this, we performed ten trials of campaigns that used those four agents (i.e. all three single-fidelity agents and RFR-SF boundary ). This helps us look at the overall campaign performance of those agents more objectively because we have better information about the "average" and "variance" in the performance. Lastly, we bound the performance of our agents above and below by two limiting cases: (1) a perfect agent, where every acquisition is an ideal candidate and the full target space is explored in exactly 202 steps and (2) a naive agent that chooses the next data point from the candidate space at random. Figure 5 shows the results of the simulated discovery campaigns. Where the fraction of the target materials found is plotted against the number of experiments (i.e. high fidelity candidate acquired). The shaded region is the standard deviation of materials found for campaigns with multiple trials. For our initial benchmark, we primarily compare the performance between models. In the single-fidelity case with no access to low-fidelity DFT data, looking at the average target materials found (colored dash lines) in each campaign, random forests agent outperformed support vector regression and Gaussian process regression agents until ∼ 850 experiment requests, at which point close to 60% of the ideal candidates had been discovered. Support vector regression agent started outperforming the other two from ∼ 850 experiment requests. In the multi-fidelity case where all lowfidelity (DFT) data was made available (colored solid lines), all agents performed similarly (with random forests slightly ahead) until ∼550 experiment requests. Afterward, the support vector regression and Gaussian process regression agent outperformed the rest until the end. More importantly, we observed that multi-fidelity agents outperformed their single-fidelity counterparts, demonstrating that these regression algorithms can transfer the knowledge available from the lower-fidelity dataset in making predictions for the high-fidelity target. All of our sequential learning agents consistently outperformed random acquisitions. To compare the performance of single and multi-fidelity agents in more detail, we tabulated acceleration factors at 50% and 80% of the total discovery of target candidates in Table 1. To achieve the discovery of 50% of the candidates designed as ideal, multi-fidelity agents reduce the experiments requested by 160, 80, and 180 for support vector regression, random forests, and Gaussian process regression, respectively. At 80% discovery, the acceleration factors are 160, 60, and 220 for support vector regression, random forests, and Gaussian process regression agents, respectively. The enhancement factors shown in Fig. 6 provided a clearer picture of the comparative performance throughout the campaign. We observe that support vector regression multi-fidelity The y-axis corresponds to the fraction of ideal materials discovered from the search space. SVR, RF, and GPR LCB correspond to agents using support vector regression, random forests, and Gaussian process regression lower confidence bound, respectively. The shaded colored regions are the standard deviation of materials found for campaigns with multiple trials. The random acquisition and ideal acquisition baselines are also labeled in the figure, representing the lower and upper bounds of agent performance. Table 1. Acceleration factor (AF) of multi-fidelity agents in simple acquisitions. The AFs are the reduction in number of experiments performed by multi-fidelity agents to achieve a certain amount of discoveries. For each row, we highlighted the agents used, the experiments performed by single-fidelity agents to achieve 50% and 80% discovery, and the acceleration factor of the multi-fidelity agents at those discoveries. www.nature.com/scientificreports/ agents briefly underperformed their single-fidelity counterparts in the early stages of campaigns (until ∼ 100 experiments). After this point, SVR-MF boundary outperformed SVR-SF boundary by a notable margin to achieve enhancement of a factor of ∼ 1.2 to 1.4 until ∼ 1000 experiments. This factor diminished slowly as candidates were exhausted for the remainder of the campaign. GPR LCB -MF boundary and RFR-MF boundary consistently outperformed their single-fidelity counterpart, with GPR LCB -MF boundary having larger enhancement factors. We also notice a similar diminishing trend of their enhancement factors as candidates were exhausted. In summary, all multi-fidelity agents outperformed their single-fidelity counterparts at all points in the process until most target candidates have been acquired. Between the three agents used, support vector regression and Gaussian process regression agents benefited more from a priori data based on the metrics computed. In-tandem acquisitions: both DFT and experiment data are acquired. Having investigated two boundary scenarios in the previous section, with all-or-no low fidelity data, we now turn to our next main question: when and how should we decide to acquire low-fidelity data to support and minimize the number of high-fidelity measurements during a sequential, closed-loop data acquisition procedure? To answer this, we simulated another set of campaigns benchmarking single-fidelity versus multi-fidelity. First, to mimic a more true-to-life discovery process, we split the compositions into seed data and candidate data based on their year of discovery according to the ICSD 58 timeline of their first publication 59 (Fig. 4). In other words, this rationale for selecting the seed data makes the initial data used for the runs and the successive choice of data by the models entirely deterministic. For single-fidelity campaigns, the data of the first 500 experimentally discovered compositions, up to the discovery year of 1965, were included in the seed data, the remaining (3460 compositions) were included in the candidate data. For multi-fidelity campaigns, the data split was identical, with the addition of corresponding DFT data in each set. Next, we set up the campaigns with a ǫ-greedy agent that used support vectors and a Gaussian processes regression agent (since these two agents had better gains in "Boundary cases: all or no DFT data available"). Therefore, a total of four campaigns were set up: SVR-SF tandem , GPR LCB -SF tandem , SVR-MF tandem , and GPR LCB -MF tandem (SF denotes single-fidelity, MF denotes multi-fidelity). As before, we also included the two limiting cases of (1) random acquisition and (2) 'perfect' acquisition. For the acquisition budget, both SVR-SF tandem , GPR LCB -SF tandem , along with the two limiting cases, had a budget of 5 experiment requests. SVR-MF tandem had a fix-ratio budget of 5 experiments and 5 DFT. GPR LCB -MF tandem had a budget of 5 acquisitions, each acquisition can be either experiments or DFT, depending on the uncertainties and hallucination of information gained from DFT. Based on optimization results in Supplementary document section S3, α=0.08, β = 5, and γ=10 were used for GPR LCB -MF tandem to compare against the other sequential learning campaigns. All campaigns were run until 2000 experiments have been acquired, unless it is stopped due to no discovery after 30 iterations (a setting in the campaign hyperparameter). Figure 7 shows the qualitative results of the simulated campaigns using in-tandem acquisition. Here, SVR-MF tandem agent outperformed its single-fidelity counterpart early in the campaigns (when N experiments reached ∼ 100). It then stayed ahead until ∼ 1200 experiments were acquired, at which point 90% of the ideal materials had been discovered. GPR LCB -MF tandem agent also outperformed its single-fidelity counterpart until 90% of the ideal materials have been discovered (at ∼ 1200 experiments). Compared among all four agents, SVR-MF tandem agent's performance was the best. Furthermore, GPR LCB -MF tandem agent's performance was similar to that of SVR-SF tandem 's. www.nature.com/scientificreports/ The acceleration factor (Table 2) of multi-fidelity acquisitions at 50% discovery were 175 and 85 for intandem support vector machines and Gaussian processes respectively. At 80% discovery, they were 250 and 159, respectively. The enhancement factors (Fig. 8) of in-tandem multi-fidelity support vector regression is very noisy at first (until N experiments reached ∼ 150), which agrees with Fig. 7. Then they stayed above 1 until N experiments reached ∼ 1250. The enhancement of GPR LCB -MF tandem cannot be calculated at first because its single-fidelity counterpart did not have any discovery. After N experiments reached ∼ 100 and its single-fidelity counterpart made some discoveries, the enhancement factors were high but decreased as the acquisition continued and converged to 1 at ∼ 1250 experiments. ## Conclusion In this work, we develop, implement, and benchmark sequential learning agents that allow for the differentiation of data points of different fidelities. Using our implementation in the CAMD sequential learning framework, we simulated a materials discovery process on previously existing experimental and theoretical electronic band-gap data to inform the selection of these models and suggest hyperparameters that could be used to accompany a 'real-life' data acquisition campaign. We found that when all low-fidelity data were provided as a priori knowledge, all multi-fidelity agents outperformed their single-fidelity counterparts and sustained a materials discovery acceleration of 20-60% early on in the campaigns. As the number of experiments acquired in the seed data increased, we saw a decline in additional gain for those multi-fidelity agents. When acquiring low highfidelity data in-tandem with support vector regression and Gaussian process regression multi-fidelity agents, both of them still outperformed their single-fidelity counterparts, suggesting strategic acquisitions of lower fidelity data provides a transfer of knowledge and augment higher fidelity target material discovery. We note that, Gaussian process regression multi-fidelity agent here barely outperformed support vector regression single fidelity agent with the settings we provided, which suggests further investigations of the agent. In summary, we observed a clear trend of multi-fidelity sequential learning agents outperforming those which may only sample at a single-fidelity. The results demonstrate that for studies where low-fidelity data is extremely cheap relative to high-fidelity data, the of separately labeled data either "up-front" or acquired in-tandem with high-fidelity experiments can increase the rate at which valuable experiments are performed. However, the relative performance of multi-fidelity acquisition is sensitive to the dataset size, ML model selection, and acquisition strategy. Furthermore, the multi-fidelity agents can be extended to have data inputs beyond two fidelities. As mentioned in "Dataset collection and representation", since the level of fidelity is represented with one-hot encoding, additional fidelity can be passed as an additional column in the feature. Subsequently, acquisition strategies are easily adaptable for multiple levels of fidelity for both of our proposed Table 2. Acceleration factor of multi-fidelity agents in in-tandem acquisitions. For each row, we highlighted the agents used, the experiments performed by single-fidelity agents to achieve 50% and 80% discovery, and the acceleration factor (AF) of the multi-fidelity agents. The AF's are the reduction in number of experiments performed. algorithms by replicating the logic in a nested fashion. For example, for three levels of fidelity, one may acquire the lowest level of fidelity in order to reduce the uncertainty on a median level of fidelity, and acquire a median level of fidelity to reduce the uncertainty of the highest level of fidelity until the experimental budget threshold for new experiments is reached. Given these dependencies, our framework offers a critical capability that frames automated discovery process itself as an object of study. Our study on multi-fidelity sequential learning campaigns lays a foundation for future research in which both simulations and experiments can be conducted with strategies optimized for their relative cost and accuracy.

chemsum

{"title": "Agents for sequential learning using multiple-fidelity data", "journal": "Scientific Reports - Nature"}

dehydrogenation_of_formic_acid_by_ir–bismetamorphos_complexes:_experimental_and_computational_insigh

## Abstract: The synthesis and tautomeric nature of three xanthene-based bisMETAMORPhos ligands (La-Lc) is reported. Coordination of these bis(sulfonamidophosphines) to Ir(acac)(cod) initially leads to the formation of Ir I (L H ) species (1a), which convert via formal oxidative addition of the ligand to Ir III (L) monohydride complexes 2a-c. The rate for this step strongly depends on the ligand employed. Ir III complexes 2a-c were applied in the base-free dehydrogenation of formic acid, reaching turnover frequencies of 3090, 877 and 1791 h À1 , respectively. The dual role of the ligand in the mechanism of the dehydrogenation reaction was studied by 1 H and 31 P NMR spectroscopy and DFT calculations. Besides functioning as an internal base, bisMETAMORPhos also assists in the pre-assembly of formic acid within the Ir-coordination sphere and aids in stabilizing the rate-determining transition state through hydrogenbonding. ## Introduction Enzyme active sites are a major source of inspiration for scientists in the feld of synthetic chemistry and homogeneous catalysis, because of the high activities and selectivities achieved in chemical transformations and the conceptual strategies employed by these systems. 1 For instance, weak but highly directional hydrogen bonding stands out as a key element used by enzymes to selectively pre-assemble and pre-activate substrates and stabilize transition states. Inspired by this, functional ligands decorated with H-bond donor and/or acceptor groups have been used to construct supramolecular ligand structures, enabling modular ligand families 2 to be utilized in transition metal catalysis and also for substrate preorganization and pre-activation via specifc ligand-substrate interactions. 3 In our quest for novel systems able to undergo hydrogenbonding interactions to steer reactivity we have developed sulfonamidophosphine (METAMORPhos) ligands. These ligands are based on a PNSO 2 scaffold that displays NH-P/N]PH tautomerism (see Fig. 1). They have been employed for mono-and bimetallic Rh-catalyzed hydrogenation, Ru-based heterolytic H 2 cleavage and [2 + 2 + 2] cycloaddition reactions. 4 Formate dehydrogenase metalloenzymes have successfully been employed for the reduction of CO 2 and oxidation of formate. Although there is no complete consensus concerning the mechanism of either the reduction or the oxidation, hydrogen-bonding and proton-shuttling are suggested to play a crucial role in the way in which these enzymes operate. 5 We recently described our initial results with an Ir-catalyst bearing a bisMETAMORPhos ligand in the base-free catalytic dehydrogenation of formic acid, a reaction that has attracted much recent interest in the context of hydrogen storage/release systems. 4f Typically, formic acid (HCOOH) dehydrogenation catalysts require the addition of sub-stoichiometric amounts of base (e.g. 5 : 2 ratio of HCOOH : NEt 3 ). However, this signifcantly reduces the overall hydrogen weight percentage of the reaction mixture. 6 One promising strategy to circumvent the use of exogenous base is to employ catalyst systems bearing cooperative ligands to access metal-ligand bifunctional pathways for substrate activation. 7 Hydrogen-bonding interactions between ligand and substrate as (additional) tools to enhance reactivity have previously been proposed both in the hydrogenation of CO 2 and in the aluminium catalysed dehydrogenation of HCOOH. 8 These interactions were also suggested to play a role in the formation of half-sandwich ruthenium and rhodium hydrides from formic acid. 9 Several studies on the mechanism of HCOOH dehydrogenation have compared conventional bhydride elimination with direct hydride-transfer (Fig. 2A and B). 10 Given the potential H-bonding abilities of METAMORPhos ligands and the protic nature of formic acid, we wondered if biomimetic non-covalent interactions between the ligand backbone and formic acid could play a role in this catalytic system. Herein we show that the proton-responsive ligand not only acts as an internal base (Fig. 2C), but that its hydrogenbonding abilities steer substrate pre-assembly and stabilize catalytic transition states. We will present the synthesis of three bisMETAMORPhos ligands as well as their coordination to iridium, compare the activity of these systems in HCOOH dehydrogenation and explain the dual role of the ligand framework during catalysis by means of both experimental and computational results. ## Synthesis of ligands and complexes METAMORPhos ligands are prepared by a simple condensation reaction between a sulfonamide of choice and a chlorophosphine. They show high stability to both oxidation (at phosphorus) and hydrolysis (of the P-N bond). We attribute this stability to their prototropic character, resulting in an equilibrium between the P III and P V oxidation states that is influenced by R 1 and R 2 . 4e In order to gain insight into the effect of ligand modifcation on the coordination and degree of tuning in HCOOH dehydrogenation catalysis, we prepared bisMETA-MORPhos ligands La-Lc via a three-step synthetic protocol (Scheme 1) that results in selective formation of only the respective meso-isomers, see ESI. †. All three ligands La-Lc display P III /P V tautomerism, but in different ratios for the three possible forms. For ligand La only the P III -P III and P III -P V tautomers were observed in CD 2 Cl 2 in a ratio of 1 : 0.4, as determined by 31 P NMR spectroscopy. For both ligands Lb and Lc all three possible tautomers were observed, with ratios (P III -P III : P III -P V : P V -P V ) of 1 : 1.8 : 0.2 and 1 : 1.9 : 0.1 for Lb and Lc, respectively. These data show that the overall P III /P V ratio is not only determined by the acidity of the N-H bond and basicity of the phosphorus atom but is also greatly influenced by the steric bulk of the substituent on the sulfon group. The P V tautomer provides stability towards oxidation and hydrolysis, even to the extent that La-Lc can be conveniently purifed by column chromatography. Upon coordination to a metal center, the tautomeric behaviour of these ligands is lost. The addition of La-Lc to Ir I (acac)(cod) generated complexes [Ir I (L H )] 1a-c via a single proton-transfer from the ligand to acetylacetonate and displacement of cyclooctadiene. These species show symmetric 1 H and 31 P NMR spectra, irrespective of the specifc ligand substitution pattern, which likely originates from highly fluxional behaviour between the protonated and deprotonated ligand arms. Formal oxidative addition of the remaining ligand -NH group in 1a-1c generates the corresponding Ir III (H)(L) complexes 2a-c (Scheme 2), see also ESI. † The rate for this overall proton-transfer step varies signifcantly for ligands 1a-c. Ir III complex 2a (with La) was obtained quantitatively after 30 hours at room temperature, but no formation of complex 2b was observed under the same conditions. This species could only be obtained after heating the mixture for 40 hours at 70 C. In contrast, the conversion of 1c into 2c was signifcantly faster than the conversion of 1a into 2a, and full conversion was already observed after 16 hours at room Scheme 1 The synthesis of bisMETAMORPhos ligands La-Lc and their three tautomeric forms (P III -P III , P III -P V and P V -P V ). Scheme 2 Synthesis of complexes 1a-c and 2a-c from Ir I (acac)(cod) and La-c. temperature. We previously reported the molecular structure of 2a to be dimeric in the solid state (2a 2 ), with the 'vacant' axial site coordinated by an oxygen from the sulfonamide of a second equivalent of 2a (Fig. 3a). 4f The molecular structure determination for the more bulky analogue 2c revealed a slightly distorted octahedral mononuclear Ir III hydride complex, with axial coordination of a water molecule trans to the hydride to complete the octahedral coordination environment of Ir III (see Fig. 3b). The steric hindrance of the isopropyl groups in complex 2c seems to effectively prevent the formation of dinuclear complexes, as was also found by modelling studies. This fnding also supports the previously proposed mononuclear confguration in solution, based on diffusion NMR data. 4f The N-S bond lengths of 1.554(2) are in agreement with deprotonated sulfonamide fragments. 4f The Ir-O water bond length was found to be 2.2427(18) , which is in accordance with trans-Ir III (H)(OH 2 ) complexes described in literature. 11 ## Dehydrogenation studies with complexes 2a-c The catalytic dehydrogenation of HCOOH was investigated with complexes 2a-c. ‡ Catalysis was performed in toluene at 85 C in the absence of base to generate dehydrogenation curves that are shown in Fig. 4, see also ESI. † The turnover frequencies (TOFs) of 3090 (2a), 877 (2b) and 1791 h 1 (2c) reveal a correlation with the electronic nature of the sulfonamide organic side-group, i.e. high activity is obtained with an electron-donating group in the para-position (2a, n-butyl), whereas an electron-withdrawing group in the para-position (2b, CF 3 ) results in much lower activity (see Computational section for explanation). Also with sterically encumbered complex 2c a lower activity was obtained than with 2a. Variable temperature (VT) NMR spectroscopy was performed to obtain mechanistic insight and detect relevant intermediates. § The addition of an equimolar amount of HCOOH to complex 2a at 223 K led to a signifcant downfeld shift (1.7 ppm) of one of the phosphorus signals (see Fig. 5, top). This might indicate (partial) protonation of one of the ligand arms. Upon increasing the temperature to 298 K the original 31 P NMR spectrum for species 2a is instantaneously restored with no observation of any intermediates.{ Addition of one equivalent of HCOOH to 2a results in an upfeld shift in the 1 H NMR spectrum for the formate proton of 0.13 ppm at 223 K (Fig. 5, right) compared to free HCOOH, which is an indication of HCOOH coordination to the axial vacant site (Fig. 5, bottom). Increasing the temperature leads to decreasing HCOOH signals together with the formation of H 2 . ## Computational investigation into b-hydride elimination The C-H bond cleavage in the dehydrogenation of HCOOH, typically the rate determining step in the catalytic cycle, can either occur via b-hydride elimination or via (ligand-assisted) direct hydride-transfer of the formate hydrogen (HCOOH) atom (see Fig. 2A-C). Both possible mechanisms were computationally investigated using DFT in order to shed light on the potential role of the proton-responsive bisMETAMORPhos ligand. The obtained energy profles for b-hydride elimination towards an equatorial and an axial coordination site of the catalyst are shown in Fig. 6 (only the relevant part of the computed structures is shown).k The frst step in the energy profle towards equatorial b-hydride elimination (red profle) is complete proton-transfer of HCOOH to the cooperative ligand, resulting in the formation of k 1 -formate complex 3I, which is endergonic by 11.3 kcal mol 1 . In this structure the formate C-H bond is pre-organized for b-hydride elimination via high barrier transition state 3II (DG ‡ ¼ 32.2 kcal mol 1 ).** This produces cis-dihydride structure 3III, which is an ). No transition state was found for protonation of the hydride via an O-H group. Transition state 3IV is potentially stabilized by axial coordination of the ligand via the sulfon group, which is in proximity to the iridium (2.42 ). This stabilization cannot occur upon protonation via the O-H moiety, which potentially explains why no transition state could be found. The reductive elimination of H 2 forms Ir I structure 1 (similar to the initially formed complexes 1a-c that lead to the formation of 2a-c) via transition state 3IV 0 , which is 4.6 kcal mol 1 lower in energy (blue energy profle, 11.1 kcal mol 1 ) than 3IV. After release of H 2 the formed structure 1 is 6.4 kcal mol 1 higher in energy than the starting structure 2. This is in agreement with experimental observations, as iridium(I) complexes 1a-c eventually transform to the thermodynamically more stable iridium(III)-hydride complexes 2a-c. The above described pathway to cis-dihydride species 3III via transition state 3II (Fig. 6, red line) seems unlikely to occur, as the activation barrier obtained for C-H cleavage is rather high (32.2 kcal mol 1 ). The b-hydride elimination towards the axial position was found to be energetically more favorable (Fig. 6, black energy profle). Deprotonation of HCOOH by the ligand and formation of k 1 -formate structure 4I is endergonic by 4.1 kcal mol 1 . The protonated ligand showed a N-H/O hydrogen bonding interaction between the ligand and the coordinated formate. b-Hydride elimination from 4I via transition state 4II to yield 4III has an activation barrier of 26.3 kcal mol 1 . The trans-dihydride structure 4III is slightly endergonic by 0.1 kcal mol 1 . Protonation of the Ir-H bond by a ligand N-H group to release H 2 from 4III via transition state 4IV has a high barrier of 24.0 kcal mol 1 . This is most likely related to the signifcant structural reorganization needed to bring the N-H proton in proximity to the hydride. Under catalytic conditions an excess of HCOOH is present, so we decided to investigate whether an additional equivalent of HCOOH could mediate the proton transfer from the ligand to the Ir-H. Indeed, a transition state was obtained wherein protonation of the Ir-H with HCOOH occurred simultaneously with reprotonation of HCOOH by the ligand N-H (Fig. 6, green profle). This transition state (4IV 0 ) turned out to be 13.7 kcal mol 1 lower in energy (10.3 kcal mol 1 ) compared to transition state 4IV. Similar second-sphere interactions were previously proposed in the heterolysis of H 2 assisted by exogenous water. 12 In our case, HCOOH provides a perfect geometrical ft between Ir-H and the N-H moiety of the ligand for second-sphere assisted proton transfer to occur. ## Computational investigation into direct hydride-transfer An alternative mechanism for the dehydrogenation of formic acid could involve a direct hydride-transfer of the formate hydrogen (HCOOH) to the Ir-center, subsequent to, or in concert with, proton transfer of the acidic HCOOH proton to the ligand scaffold. In this mechanism a single metal coordination site is sufficient for effective turnover. To test the validity of such a pathway, we computed different HCOOH-2 adducts. Adducts with either axial or equatorial coordination were all found to exhibit stabilizing hydrogen-bonding interactions with either the nitrogen atom or the coordinated oxygen atom in the ligand scaffold, resulting in structures 5I-8I (see Fig. 7). Axial coordination of HCOOH (5I; interaction with O) is the only structure found to be exergonic, by 3.07 kcal mol 1 , compared to the free complex plus HCOOH. Formation of structure 6I, with an N-H interaction, is endergonic by 3.82 kcal mol 1 . Equatorial coordination of HCOOH, leading to structures 7I or 8I, is associated with a signifcant energy penalty of 11.8 (7I) or 13.8 (8I) kcal mol 1 compared to formation of the axial adducts. The dehydrogenation of HCOOH via a direct hydride-transfer pathway was frst investigated using the axial HCOOH adducts 5I and 6I. Starting from the energetically most stable structure 5I (see black energy profle in Fig. 8), an endergonic rearrangement to 5II was found (+11.8 kcal mol 1 ), which orients the formate hydrogen in a favorable position for direct hydride-transfer to the metal. In transition state 5III (DG ‡ ¼ 26.8 kcal mol 1 and DG ‡ ¼ 29.9 kcal mol 1 with respect to 5I) HCOOH is fully deprotonated by the ligand. This enables facile expulsion of CO 2 leading to a 6-membered Ir/H/C/O/H/O transition state. After release of CO 2 , dihydride complex 5IV is formed in an overall endergonic process (+16.9 kcal mol 1 ), whereafter protonation of the iridium-hydride via transition state 5V (+17.4 kcal mol 1 ) releases H 2 . The HCOOH dehydrogenation pathway was also investigated starting from structure 6I (red profle in Fig. 8). The rearrangement from 6I to 6II (similar to the rotation of 5I to 5II) is endergonic by 12.8 kcal mol 1 . However, transition state 6III was found to be signifcantly lower in energy (DG ‡ ¼ 20.2 kcal mol 1 ) compared to 5III (DG ‡ ¼ 26.8 kcal mol 1 ), leading to formation of structure 4III via an unusual 8-membered Ir/H/C/O/H/N/S/O transition state, see Fig. 6. Structures 6I-III are all stabilized by hydrogen bonding interactions between HCOOH/CO 2 and the ligand pre-assembling HCOOH and stabilizing the transition state. The same role of the ligand was found in the energy profle starting from structure 5I. The Ir-H and N-H bonds in transition state 6III are elongated compared to those found in 4III (Ir-H: 1.76 vs. 1.67 ; N-H: 1.04 vs. 1.02 ), which indicates a late transition state (Fig. 9). A similar interaction was proposed by Hazari et al. for CO 2 insertion of an Ir-H stabilized by hydrogen bonding between a ligand-based N-H group and CO 2 . 13 Calculations performed on structures lacking these Hbonding interactions (by pointing the N-H fragment outwards) yielded unstable structures and no transition states could be found. After CO 2 release from 6III, structure 4III is formed, which releases H 2 with the assistance of another molecule of HCOOH (structure 4IV 0 ), regenerating the catalyst as described above (Fig. 6, green profle). Direct hydride-transfer was also investigated starting from structures 7I and 8I, but these energy profles were found to be signifcantly higher than for 5I and 6I, see ESI. † These calculations suggest that HCOOH dehydrogenation using bisMETAMORPhos-derived complexes 2a-c follows an outer-sphere direct hydride-transfer mechanism at the axial vacant site of these monohydride species. Starting from formic acid adduct 5I, the energetically most favored pathway requires rearrangement of 5I to 6I. This is likely a facile process due to The electronic effect was also investigated theoretically by comparing p-CF 3 with p-CH 3 substituents. Indeed, a higher activation barrier was found for p-CF 3 (24 kcal mol 1 ) compared to p-CH 3 (22.5 kcal mol 1 ), see ESI † for energy profles. We propose the following overall dual role for the bisMETAMOR-Phos ligand in the mechanism of formic acid dehydrogenation (Scheme 3). Species 2 coordinates HCOOH at the vacant axial site, aided by hydrogen-bonding with the coordinated sulfonoxygen atom to give structure 5I. Reorientation of the formate group gives a pre-activated HCOOH unit that participates in hydrogen-bonding with the deprotonated nitrogen of the ligand arm (6II). Release of CO 2 is achieved via transition state 6III (rate determining step) wherein the ligand deprotonates HCOOH and stabilizes the direct hydride transfer by a hydrogen bonding interaction. This gives trans-dihydride 4III that releases H 2 aided by an additional equivalent of HCOOH, which protonates the Ir-H and in turn is reprotonated by the ligand, thereby regenerating starting complex 2. The reversibility of this catalytic system is currently under investigation. ## Conclusions We report several bisMETAMORPhos ligands (La-Lc) based on the xanthene backbone bearing two sulfonamide-phosphine units. The prototropic nature of these PN(H)SO 2 R fragments results in different ratios for the P III and P V tautomers, depending on the electronic and steric nature of the sulfonamide substituents. Formation of Ir I complexes 1a-c was achieved by coordination of La-Lc to Ir(acac)(cod). Subsequent formal oxidative addition of the remaining N-H group resulted in the formation of the corresponding Ir III -monohydride complexes Ir III (H)(L) (2a-c). These three complexes are active catalysts for the base-free dehydrogenation of formic acid, with TOFs of 3090, 877 and 1791 h 1 for 2a-c, respectively. These data reflect the influence of subtle electronic and steric changes in the ligand architecture. The role of the ligand during catalysis was investigated by variable temperature 1 H and 31 P NMR spectroscopic measurements and DFT calculations. Variable temperature NMR data point to the formation of a 2a-HCOOH adduct. DFT calculations indicate that the hydrogen-bonding abilities of the ligand play an important role in the mechanism, resulting in an uncommon direct hydride-transfer mechanism instead of the more commonly proposed b-hydride elimination for HCOOH dehydrogenation. This was also found to be the rate-determining step (23.3 kcal mol 1 ), which confrms experimental observations using DCOOH and HCOOD. 4f A similar mechanism has been proposed for Al-and Fe-based systems reported by Berben and Milstein, respectively. 6c,8c It was found that the combined hydrogen-bonding and protonresponsive properties of the bisMETAMORPhos ligands are essential for the reactivity of complexes 2a-c. These interactions facilitate the pre-assembly of the HCOOH substrate and the stabilization of catalytically relevant intermediates and transition states, allowing an otherwise inaccessible reaction pathway. We thus show that a single coordination site is effective for the dehydrogenation reaction to occur when using a functional ligand scaffold. The proton-responsive and hydrogen-bonding features of ligands (La-Lc) are currently being explored for other reactions. The mechanistic fndings presented herein potentially play a role in other HCOOH dehydrogenative catalysts bearing hydrogen-bonding functionalities in their ligand systems. coordination for reactions in solution is overestimated. Therefore, for reactions in solution, the Gibbs free energies for all steps involving a change in the number of species should be corrected. Several methods have been proposed for the correction of gas phase to solution phase data. The minimal correction term is a correction for the condensed phase (CP) reference volume (1 L mol 1 ) compared to the gas phase (GP) reference volume (24.5 L mol 1 ). This leads to an entropy correction term (SCP ¼ SGP + R ln{1/24.5}) for all species, lowering the relative free energies (298 K) of all associative steps by 2.5 kcal mol 1 . 3k,15 According to some authors, this correction term is too small, and larger correction terms up to 6.0 kcal mol 1 have been suggested. 16 Which correction term is best remains somewhat debatable.

chemsum

{"title": "Dehydrogenation of formic acid by Ir\u2013bisMETAMORPhos complexes: experimental and computational insight into the role of a cooperative ligand", "journal": "Royal Society of Chemistry (RSC)"}

ion-shift_reagent_binding_energy_and_the_shift-mass_correlation_in_ion_mobility_spectrometry

## Abstract: Ion mobility spectrometry is widely used for the detection of illegal substances and explosives in airports, ports, custom, some stations and many other important places. This task is usually complicated by false positives caused by overlapping the target peaks with that of interferents, commonly associated with samples of interest. Shift reagents (SR) are species that selectively change ion mobilities through adduction with analyte ions when they are introduced in IMS instruments. This characteristic can be used to discriminate false positives because the interferents and illegal substances respond differently to SR depending on the structure and size of analytes and their interaction energy with SR. This study demonstrates that ion mobility shifts upon introduction of SR depend, not only on the ion masses, but on the interaction energies of the ion:SR adducts. In this study, we introduced five different SRs using ESI-IMS-MS to study the effect of the interaction energy and size on mobility shifts. The mobility shifts showed a decreasing trend as the molecular weight increased for the series of compounds ethanolamine, valinol, serine, threonine, phenylalanine, tyrosine, tributylamine, tryptophan, desipramine, and tribenzylamine. It was proved that the decreasing trend was partially due to the inverse relation between the mobility and drift time and hence, the shift in drift time better reflects the pure effect of SR on the mobility of analytes. Yet the drift time shift exhibited a mild decrease with the mass of ions. Valinol pulled out from this trend because it had a low binding energy interaction with all the SR and, consequently, its clusters were short-lived. This short lifetime produced fewer collisions against the buffer gas and a drift time shorter compared to those of ions of similar molecular weight. Analyte ion:SR interactions were calculated using Gaussian. IMS with the introduction of SR could be the choice for the free-interferents detection of illegal drugs, explosives, and biological and warfare agents. The suppression of false positives could facilitate the transit of passengers and cargos, rise the confiscation of illicit substances, and save money and distresses due to needless delays. ## INTRODUCTION Ion mobility spectrometry is an analytical separation technique introduced in 1970 by Francis Karasek (Cohen and Karasek 1970). IMS separates ions according to their size to charge ratio under the acceleration of an electric field, while collisions with the buffer gas decelerate the ions (Mason and McDaniel 1988). The collision cross-section, CCS, of the ions is proportional to the time the ions spend to reach the detector. IMS is the instrumental technique most used to detect prohibited substances such as explosives, chemical and biological warfare agents, and drugs in airports, ports, customs (Eiceman and Stone 2004), and prisons (Vautz et al. 2009). False positives caused by interfering ions that overlap with the searched ions are annoying incidents for transporters and passengers. The introduction of shift reagents (SR) in the buffer gas of drifttube ion mobility spectrometers has been used to selectively change ion mobilities and separate analytes that overlap in IMS by adduction with analyte ions. Hence, using SR may decrease the number of false positives by selectively changing ion mobilities and would save time to customs officials, transportation personnel, and passengers. Shift reagents can be considered as a deliberate impurity added to the buffer gas that may react with the ions travelling to lengthen their drift time. Eiceman et al. (1993) eliminated the ammonia interference using ketones as SR to form clusters of hydrazine and monomethylhydrazine; after clustering with ketones, the drift times of these species changed selectively, hence, separating them. Bollan et al. (2007) introduced ketones into the buffer gas to form complexes with hydrazines avoiding ammonia interference. In 2014, overlapping picoline isomers were separated using 2-butanol due to the formation of different nanocluster product ions with different cross-section areas. These cross-sections depended on the position of the methyl group on the pyridine ring (Ghaemi and Alizadeh 2014). The effect of the introduction of polar SR in the buffer gas has been studied and electrostatic attraction, energetically possible conformations, hydrogenbonding, and steric repulsion were proposed as the origin of mobility shifts (Levin et al. 2007). Roscioli et al. (2014) showed that analogous proton affinities of the SR were significant to obtain substantial mobility shifts. The main origin of mobility shifts after the introduction of SR in IMS is the cluster binding energy and the steric hindrance on the charge site instead of size. This was demonstrated by Campbell et al (2014). Tsai et al. (2016) added SR to produce large clusters of ammonium nitrate and urea nitrate, commonly used as improvised explosive devices, to overcome challenges for the detection of these species such as their low mass. Butcher et al. (2019) added solution additives and gas-phase SR to heme proteins and found changes in the mobility profiles depending on the size of the SR used (methanol, acetone, or acetonitrile). They attributed the changes in mobility to clustering and considered that these experiments open new avenues for the manipulation and interrogation of biomolecules in the gas phase. Similar results were obtained for methanol, acetonitrile, and acetone as SR in the analysis of growth hormone-releasing hormone using trapped ion mobility-mass spectrometry (Fouque et al. 2019). Parchami et al. (2017a) eliminated peak overlap in the analysis of biogenic amines (histamine, putrescine, cadaverine, and tyramine) in canned fish samples by using 8-crown-6 as SR in the carrier gas. We have used SR to analyze valinol and other analytes introducing several SR (Fernandez-Maestre 2018). The formation of larger and slower SR-ion adducts than its original size and speed, decreases the mobility of ions upon introduction of SR in the drift gas in IMS. Several parameters are used to explain these changes in mobility: proton affinity and size, inductive effects, SR-ion binding energy, ion charge delocalization, steric hindrance, and number of adduction sites (Rawat et al. 2015). Inductive effects strengthen or weaken ion:SR interactions and the steric hindrance on the charge produced by bulky substituents shields the ion charge from the adduction of SR molecules and delocalizes the charge weakening the ion:SR interactions. Because IMS separates ions based on the size to charge ratio, and the clustering of ions to SR yields larger-sized adducts, it increases the collisions against the molecules of the buffer gas, and resulting in a greater mobility shift. Therefore, larger mobility shifts are obtained with bulkier SR. However, the larger the SR the smaller the number of SR molecules clustering with a certain ion. Proton affinities, intramolecular hydrogen bonds, and steric and inductive effects, are considered when computing the interaction energies of analyte-SR and, consequently, the most used factors to explain mobility changes induced by SR are the number of adduction sites, interaction energies, and SR size (Fernandez-Maestre 2018). Oberreit et al (2015) applied differential mobility-mass spectrometry to observe the sorption of 1-13 vapor molecules onto iodide cluster ions in air at 300 K. Measured CCS shifts were compared to predictions based on the Kelvin-Thomson-Raoult (KTR) and Langmuir adsorption models. They found that the models fit very well to measurements, but the earliest stages of vapor uptake were not well described by the KTR model. The introduction of SR in the buffer gas in IMS has been reviewed (Puton et al. 2008;Waraksa et al. 2016). The mobility shifts of valinol after the introduction of several buffer gas SR have been studied. The following percent changes in mobilities have been obtained:-5.1% (trifluoromethyl benzyl alcohol), -18% (methyl-2chloro propionate), -7.1% (water) -28% (ethyl lactate), -9.8% (2-butanol), and -21% (nitrobenzene) after introducing 2.3, 1.0, 879, 1.7, 6.8, and 1.0 mmol m -3 of the SR in the buffer gas, respectively (Fernandez-Maestre et al. 2010a, 2010b). In the same conditions, serine, a compound with a similar mass and structure showed larger mobility shifts than valinol. As a result, valinol moved away from the mobility shift-mass correlation lines. In this study, we used IMS and theoretical calculations to elucidate the origin of the behavior of valinol in the mobility shift-mass correlations after SR introduction. These mobility shift-mass correlations and the explanation of the departure of some ions from these correlations have not been reported before. This is important to explain this chemical behavior to take a step ahead in the study and design of SR for the IMS detection of illegal substances. ## Instrument The methodology has been modified from that described elsewhere and only a summary is given here (Wittmer et al. 1994). An electrospray-ionization (ESI) atmospheric-pressure ion mobility spectrometer coupled to a quadrupole mass spectrometer was used in these experiments (Figure S1). Routine operating parameters for this instrument were: sample flow, 3 µl min -1 ; ESI voltage, 15.6 kV; voltage at the first ring, 12.1 kV; voltage at the gate, 10.80 ± 0.01 kV; gate closure potential, ±40 V; gate pulse width, 0.1 ms; scan time, 35 ms; number of averages per spectrum, 100-1000; pressure, 688-711 Torr (in Pullman, WA, USA); nitrogen flow, 0.93 liter min -1 ; drift tube temperature, 100-250 ± 2 ºC. The ABB Extrel mass spectrometer (Pittsburgh, PA, USA, 0-4000 amu) was operated in three modes. worked in single ion monitoring ion mobility spectrometry (SIM-IMS), radiofrequency-only ion mobility spectrometry (IMS), and mass spectrometry (MS) modes. In SIM-IMS mode, only the ion mobility spectrum of ions of a given mass to charge ratio or a range of masses is obtained. In IMS mode, the total ion mobility spectrum is obtained; and in MS mode, mass spectra are obtained. The ion mobility spectrometer was made at Washington State University, WSU, (WA, USA) and experiments were performed there. It comprised an electrospray ionization source, a 25-cm drift tube coupled, and a quadrupole mass spectrometer detector. The reaction region length was 7.5 cm. A Bradbury-Nielsen ion gate separated the desolvation and drift regions; a countercurrent of dry, preheated N 2 buffer gas was introduced through the end of the drift tube. ## Reagents Desipramine, ethanolamine, methionine, phenylalanine, serine, threonine, tributylamine, tribenzylamine, tryptophan, tyrosine, and valinol were used as analytes; 2,4-lutidine and 2,6-di-tert-butyl pyridine (DTBP) as chemical standards; and 2-butanol, ethyl lactate, methyl-2-chloro propionate, trifluoromethyl benzyl alcohol, and water as SR. These reagents plus methanol, water, and acetic acid (ACS reagent grade, ≥97 or 98% purity) were purchased from Sigma Aldrich Chemical Co. (Milwaukee, WI, USA). The structures of compounds used in this study are shown in Figure S2. These analytes were selected because their different molecular weights and structures were required to compare the effects of size, interaction energy, and steric hindrance on the mobilities of these molecules with those of valinol with the introduction of SR into the buffer gas. The chemical standards, DTBP and 2,4-lutidine, were selected because they are commonly used as chemical standards (Eiceman et al. 2002). ## Buffer gas N 2 was used as the buffer gas. The humidity of the buffer gas was an average of 10 ppmv. measured with a GE Moisture Image Series 1 Instrument (Billerica, MA, USA), (Fernandez-Maestre et al. 2010a). The drift tube was heated at 150°C using 5-and 10-cm 3/8" firerod cartridge heaters (Watlow, Anaheim CA, USA). We waited until the drift tube was saturated with the SR to start drift time measurements so that the clusters were formed in the ion source. Saturation was considered to be reached when the mobilities of the analytes stabilized over time. The concentration of SR was set using the flow rate of liquid SR, the experimental conditions, and the equation of state. ## Solutions preparation and injection Solutions were prepared at 50-µM (analytes) or 10-µM (chemical standards) concentrations in ESI solution (47.5 % methanol: 47.5% water: 5% acetic acid). Liquid samples or blank solutions (ESI solution) were electrosprayed continuously into the drift tube. Liquid SR were introduced as vapors into the buffer gas line before the buffer gas heater through a heated cross-junction. ## Computational-Theoretical Studies The Gaussian 09 program (Revision D.01) (Frisch et al. 2009) was used for the theoretical-computational calculations at 150°C using the X3LYP-GD3/6-311++(d,p) functional, which includes the Grimme scatter correction. Also, a new, larger, ultrafine grid was used, useful when high precision is desired. Geometry optimization was performed for the analytes, the SR, their protonated species, and their adducts. The structures of the molecules were then used as starting points to determine the geometries and energies. The interaction energies (IE) were calculated using the equation IE = E adduct -(E SR + E analyte ion ). The proton affinities were extracted from the calculations performed on the protonated analytes (Foresman and Frisch 1996). Gibbs free energies (ΔG), enthalpies (ΔH) and entropies, reported as TΔS, were calculated for the complex formation reactions studied. ## Identification of peaks in the spectra The identification of analytes was performed by comparing their m/z signal in mass spectrometry to the molecular weight of their protonated molecules or clusters. Also, analyte peaks and their clusters in the IMS spectrum were analyzed by SIM-IMS for identification. Additionally, the reduced mobilities of the protonated analytes were compared with values from the literature. ## Instrument calibration Under given conditions, the product of the reduced mobility of an ion, K 0 , times its drift time is constant. This lets the reduced mobility to be calculated from that of a calibrant, K 0,c , the calibrant drift time, c t , and the analyte drift time at the same conditions, d t : 2,6-di-tert-butylpyridine was used as a calibrant. ## Experiments For every analyte ion and shift reagent we tried five SR concentrations (for a total 210 experiments) but only 48 at the highest concentrations were reported because the others showed a similar trend. ## RESULTS AND DISCUSSIONS The number of reduced mobility measurements was >3. Reproducibilities of 0.3 to 0.6% were obtained for reduced mobilities. The raw database can be found in Fernandez-Maestre (2017). The mass spectrometer had a low resolution, but this was not a constraint because the peaks of interest were easily differentiated. Figure 2 shows the IMS spectrum of the ESI solvent before introducing any SR in the buffer gas. Both the IMS and mass spectra show no contamination: only water peaks are seen in the mass spectrum and only peaks from the components of the ESI solvent are seen in the IMS spectrum; all other peaks are small. The elimination of contamination is important to prevent the mobility shifts due to clustering of the analytes to contaminants. The peak at 14.80 may be due to clusters of ammonium contaminating ions with water because the ammonium peak always appears before the hydronium peak in IMS (Bahrami & Tabrizchi, 2012). The difference between ammonium and water clusters with the same n number is only one m/z unit. For example, water clusters appear at m/z 37, 55, 73, 91, 109 while the ammonium clusters appear at m/z 38, 56, 74, 92, 110. Because the mass spectrometer was low resolution, the two sets of clusters were not separated in the mass spectrum. All hydronium water clusters in the IMS spectrum merged into one single mobility peak at 17.08 ms, with a weighted average of the ion mobilities of all water ions. Due to the following equilibria, they quickly interconvert into each other many times during their travel through the drift tube, (H 2 O) a H + ↔ (H 2 O) a-y H + + yH 2 O This is also true for the peak at 14.80 ms that is the result of all ammonium water clusters. ## Spectra of the buffer gas 3.2 Spectra of valinol in pure N 2 buffer gas or when the buffer gas was doped with 2-butanol Fig. 3 Mass spectra of a 100-μM solution of valinol in nitrogen-only buffer gas (a) and when 0.68 mmol m -3 of 2-butanol (b) was introduced into the buffer gas. Protonated valinol ion (VH + ), hydrated valinol cluster (VWH + ), doubly hydrated valinol cluster (VW 2 H + ), 2-butanol:valinol cluster (BVH + ), hydrated 2butanol:valinol cluster (BVWH + ), 2-butanol trimer (B 3 H + ), and cluster of valinol with two 2-butanol molecules (B 2 VH + ) are seen Figure 3 shows the mass spectra of valinol solutions in nitrogen-only buffer gas (a) and when 0.68 mmol m -3 (5.0 µL/min flow rate) of 2-butanol (b) was introduced into the buffer gas by the end of the drift tube together with the buffer gas. The protonated valinol ion (VH + ) and its clusters with water (the hydrated valinol cluster, VWH + , and the doubly hydrated valinol cluster, VW 2 H + ) are the dominant peaks without introducing 2butanol in the buffer gas. The water clusters seen in Figure 2 disappeared from the mass spectra by adduction with 2-butanol or the charge was stripped by valinol and 2-butanol due to the lower proton affinity of water. However, when 2-butanol was present in the buffer gas, the intensity of valinol ion decreased due to the displacement of the equilibria to the formation of clusters of valinol with 2-butanol, such as BVWH + , BVH + , B 2 VH + , and others of minor intensity. The peak intensity of the clusters was higher than that of protonated valinol even at a relatively low 2-butanol concentration (0.68 mmol m -3 ) indicating that 2-butanol effectively adducted to valinol without major interferences from steric hindrance and else. Fig. 4 Mobility spectra of a 100-μM solution of valinol in N 2 -only buffer gas (a) and when 0.68 mmol m -3 of 2-butanol was introduced in the buffer gas. There was a 0.80 ms mobility shift when 2-butanol was present in the buffer gas Figure 4a shows the ion mobility spectra of valinol in N 2 -only buffer gas; the valinol peak is observed at 19.88 ms. When 0.68 mmol m -3 of 2-butanol was introduced in the buffer gas the valinol peak displaced to 20.68 ms. This 2-butanol concentration corresponded to a flow rate of 5 µL/hr of 2-butanol vaporized into the buffer gas. The drift time of valinol increased due to the occurrence of a hydrogen bond between the oxygen atom in 2-butanol and the charge on the amine group in valinol that stabilized the charge by sharing it with the hydroxyl group in 2-butanol. The valinol peak shifted from 19.88 to 20.68 ms, a 0.80 ms mobility shift, according to a series of chain equilibria between the protonated valinol (VH + ) and 2-butanol (B). In fact, all ion-molecule reactions that may occur in the drift tube under influence of water (W) and a shift reagent (SR) can be generally presented in Fig. 5. Fig. 5 Possible ion molecule reactions and equilibria that may occur in the drift region, when sample (M) exists in the ionization chamber, and a shift reagent (SR) and water (W) in both the ionization and the drift region. In the absence of sample and SR, only reactant ions of water clusters are formed in the ionization region, as shown in the top first column (blue section). When the sample is added to the ionization region, these ions convert into the product ions shown in the yellow section. This is the case for the peak at 19.88 in Fig. 4 corresponding to all hydrated protonated valinol (VW n H + ). If the shift reagent is added to the drift gas without sample, the green and blue sections show the nature of ions involved in making an ion mobility peak. If sample is added to the ionization region and at the same time SR is added to the buffer gas, the yellow and pink sections show ion-molecule reactions that ultimately generate the peak. This is the case for the peak at 20.68 that includes all protonated valinol clustered with water or 2-butanol (B m VW n H + ) coalesced into a single mobility peak. Ammonium and higher order clusters of sample, M n H + are not considered in these reactions. It is assumed that sample is only added into the ionization region where the hydronium ions are produced, while water (W) and the shift reagent (SR) are present in both the reaction and drift regions. Ammonium ion and higher order clusters of sample, M n H + are not considered in Fig. 5. The system is then described by considering several series of complex reactions or a multi-equilibria system that can proceed in two dimensions, adding a water or a shift reagent molecule to the core ion. This reaction map shows the species that may be observed at different conditions. In general, the peaks of four species may appear on the ion mobility spectrum. Water cannot be totally removed from the drift gas. Hence, in the absence of any sample and SR, (blue section) only water clusters of hydronium ion, W n H 3 O + , as the reactant ion peak, are observed. This is the case for Fig. 1 and 2 where the hydronium reactant ion peak is observed at 17.08 ms. Above the dashed line no sample is present. If sample is added to the ionization region with no SR, (yellow section) only the first column below the dashed line describes the reactions happening in the drift region. Then, a mixture of water clusters of protonated sample (MH + W n ) makes the product ion peak (PIP). This is the case for Fig. 4 a where a peak at 19.88 ms corresponding to water clusters of protonated valinol is observed. If the SR is added to drift region, without any sample, then the reactions proceed towards the right direction above the dashed line and occupy both the blue and the green section. In fact, a vector of W n H + converts into a matrix. Then, the result is a mixture of SR m W n H + ions with n = 1,2,3.. and m = 0,1,2,... Although this is not the case in our experiment, the peak at 223.4 amu in the mass spectrum presented in Fig. 3-b, corresponding to B 3 H + is an example. When the sample is added to the ionization region and the SR is dopped into the drift gas, both water and SR play a role in the ion-molecule reactions and shift the distribution towards heavier SR m MW n H + clusters. In fact, the protonated sample is siege by W and SR molecules. They quickly and frequently take off and on, so that the mobility gets smaller than the original protonated sample. In another word, the final mobility is a weighted average of all cluster ions. This cause the product ion peak shift towards longer drift times as demonstrated in Fig. 4, where the valinol peak shifted from 19.88 to 20.68 ms. This peak corresponds to all ions bellow the dashed line (the yellow and peak sections). Nevertheless, if a valinol molecule is separated from the cluster ion, it jumps to the zone above the dashed line and the remaining ion is in the form of SR m W n H, such as B 3 H + in Fig. 3-b. This reaction. However, is one way since no valinol molecule is available in the drift region. Such ions may appear as a tail between the SR peak and the shifted PIP because they travelled partially as SR or PI. Obviously, the tail will not be observed if the two peaks are not separated well. Another possibility for observing such ion on the mass spectrometer could be the remaining unreacted BH + , formed in the ionization region, that uptake more B molecules on their way while traveling through a bath of B molecules. Examination of the ion mobility spectrum presented in Fig. 4-b show neither tail nor double peak. Hence, the SR peak appears the same drift time as the shifted PIP for valinol with 2-buthanol shift reagent. In summary, all ions can be described either by one dimensional vectors, like the blue or yellow sections of the map, or two-dimensional matrices as described in the green and the pink sections. Adding the sample changes the vector W n H + to a new vector of MW n H + while adding the SR provides an extra dimension for expanding the vector to a matrix of SR m MW n H + . The same scenario can be imagined if ammonium ions are initially considered as the reactant ions. A separate matrix may also form from the dimer ion M 2 H + if the sample concentration is too high. Then a separated peak of the complex mixture of the dimer ion of SR m M 2 W n H + , appears at higher drift time. In the case of valinol as sample and 2-buthanol as shift reagent only few adducts, denoted by oval in Fig. 5, including BVH + and B 2 VH + where observed. Other adducts with a larger number of 2-butanol molecules are less abundant and cannot be seen in the mass spectra but they have been reported (Bollan et al. 2007;Fernandez-Maestre et al. 2010b). The electrostatic surface potential map for one of the SR used in this study, trifluoromethyl benzyl alcohol (F), with methionine, F-MetH + , is shown in Figure S3 (Supplementary Information). This map demonstrates that the nucleophilic regions on the SR disappear after clustering with methionine. The electrophilic regions remain on the cluster for additional adduction. ## Mobility shifts after the formation of SR-ion adducts Fig. 6 a) Mobility and b) drift time shifts of ethanolamine (Et), valinol (V), serine (S), threonine (Thr), phenylalanine (P), tyrosine (Tyr), tributylamine (Tb), tryptophan (Try), desipramine (D), and tribenzylamine (Tz), when different SR were introduced into the buffer gas at 150 °C. The concentrations of SR in the buffer gas were 2.3, 1.0, 879, 1.7, and 6.8 mmol m-3, for F, M, W, L, and B, respectively. R 2 regression coefficients show the mobility shift-mass correlations. Table 1 shows the numeric values of these mobility shifts and SR concentration. B: 2-butanol, F: trifluoromethyl benzyl alcohol, L: ethyl lactate, M: methyl-2-chloro propionate, W: water. The analytes in the graphs, from bottom to top, are: M = Et, V, S, Thr, P, Tyr, Tb, Try, Tz; F = Et, V, S, Thr, P, Tyr, Try; B = Et, V, S, Thr, P, Tyr, Try; W = Et, V, S, Thr, Met, P, Tyr, Try, Tz; L = Et, V, S, D. Figure 6 shows the shifts in mobility and drift time of selected ions, ethanolamine, valinol, serine, threonine, phenylalanine, tyrosine, tributylamine, tryptophan, desipramine, and tribenzylamine, when different SR were introduced into the buffer gas at different concentrations. Table 1 shows these mobility shift values when the SR concentration was increased from 0.0 mmol m -3 to the specific concentration shown in this table at 150 °C. The onsets show the behavior of the analyte versus mass mobility and drift time. It can be seen that the drift time linearly depends on the mass, while the mobility does not. This is due to the fact that drift time depends on the size of the ion. For a homologous series, the size is determined by the number of atoms and it is directly related to mass. The mobility itself is inversely proportional to drift time. Therefore, mobility is not expected to be a linear function of mass. The inverse relation between the mobility and drift time also affects the graphs of the shift versus mass. The plots in Fig 6 show that, both shifts, in mobility and in drift time, have similar decreasing trends with mass, but higher slopes for K are observed. The steeper plots for K may be explained by considering Eq. 2. Based on the plot shown in the onset of Fig. 6-b, the drift time t may be substituted with mass m. Hence, we have, Where b is the intercept of the drift time-mass plot. Eq. 3 shows the relationship between the mobility shift and the drift time shift. Assuming a decreasing t with mass, it is evident that K decreases more rapidly than t, since the squared m exists in the denominator of the right hand side of Eq. 3. This means that, even if the effect of a specific SR on drift times of ions with different masses are similar, or in another word, if the peak displacement, t, is constant, K still decreases with mass. This may mask the effect of SR on a series of ions with different masses. Hence, unlike traditional way, we focus on t rather than K. In fact, t purely reflects the changes in size of an ion due to the presence of SR. All the plots in Fig. 6-a show a general negative slope. This means that the largest drift time shifts were obtained by the compounds of lower mass or smaller size, in this case, ethanolamine (average Δt 5.01 ms) and serine (Δt 4.9 ms) and the smallest shift by the largest mass compound, tribenzylamine (Δt 1.11 ms). This is because the larger the mass or size of an ion, the less its size is affected by the adduction of molecules. Average Δt showed correlation with the ion masses with a 0.70 R 2 regression coefficient. In a homologous series, the mass determines the position of an ion in the mobility or time shift-mass correlation plots when a SR has been injected into the buffer gas in IMS (Figure 5). However, this correlation is affected by the presence of intramolecular bonds and by the interaction energy with the SRs (Fernandez-Maestre 2018). It is clear in Fig. 6 that, methionine departed from these correlations because it is less influenced by the introduction of a SR due to the formation of an intramolecular bond that hinders the adduction with the SR, because the positive charge of the ions is delocalized, and also due to the steric hindrance on the ion´s charge (Nieckarz et al. 2008;Fernandez-Maestre et al. 2012). Karpas (1989) showed that protonated α,ω-diamines have their charge delocalized due to an intramolecular hydrogen bond. This led to more condensed structures than those of the protonated primary n-amines increasing the diamines' mobility more than that of n-amines. Also, due to the formation of intramolecular hydrogen bonds, it was found that α,ω-diamines had a lesser interaction with 18C6 than n-amines (Parchami et al. 2017b). In the graph for water, it is hard to demonstrate a departure from linearity for methionine because its differences with the ion mobilities of tyrosine and phenylalanine were not statistically significant maybe because the experiments were performed at very high flow rates, 1250 µL/hr. At these high flow rates, the content of the 0.25 ml syringe was emptied in only 12 minutes, imposing limited reproducibility to the experimental conditions because the time to reach a homogeneous saturation of water in the drift gas was short. Table 1. Mobility shifts, ΔK 0 , and drift time shifts Δt, for the selected ions when different SR were introduced into the buffer gas at 2.3, 1.0, 879, 1.7, and 6.8 mmol m -3 concentrations for F, M, W, L, and B, respectively. ΔK 0 values were calculated as the mobility difference in nitrogen-only buffer gas and SR-doped buffer gas. Average ΔK 0 showed correlation with the ion masses with a 0.73 R 2 regression coefficient ( Fernandez-Maestre et al. 2010b, 2012). S1. ## The interaction energy of SR-ion adducts The departure from linearity of the other compound, valinol, is more evident in Figure 6 (solid arrows) where the shift-mass lines suffer a strong turn from valinol to serine to retake linearity after this amino acid. Valinol is a small molecule with little steric hindrance and the explanation for its departure from linearity is the interaction energy (Table 2). The lifetimes of the clusters depend on the interaction energies: the higher the interaction energy the larger the lifetime. A larger adduct lifetime increases the average size of the ion because it travels the drift tube a long time as a large adduct. A larger size increases the ions´ collisions with the buffer gas, decreasing the ion mobilities. The strongest interaction energy of the ion:SR adducts originates from the hydrogen bonds formed between the positive charge of the ion and electron-rich groups on the SR. For valinol, the ion interaction energies with any of the SR used were lower than those for serine. This implied that valinol had a shorter adduct lifetime, a smaller average size, and fewer collisions with the buffer gas. Therefore, the mobility of valinol was less affected than those of the other ions and its mobility shifts were also smaller than those of the other ions (Figure 5). The origin of the higher interaction energy of serine with any SR, when compared to valinol, resides in the fact that serine is an amino acid and has one additional OH and carboxylic groups. These groups yield a stronger interaction energy of serine with most SR (Table 2). The effects of intramolecular hydrogen bonding, interaction energy, and proton affinity on ion mobility after the injection of SR have been reviewed (Fernandez-Maestre 2018;Waraksa et al. 2016). Table 2 shows the interaction energies for the compounds and adducts investigated, and Table S3 their interaction energies, Gibbs energies, enthalpies and entropies. The Gibbs energy describes the condition of equilibrium and spontaneity in a process. All Gibbs energies were negative indicating that the complexation processes were exergonic and spontaneous for the formation of clusters in all species corroborating the interaction energies calculations. Table 2. Interaction energies (IE) in kcal/mol for the ions and adducts investigated. Calculations were made using Gaussian 09 (Revision D.01) at 150°C and the X3LYP-GD3/6-311++(d,p) functional. The complete data set with energies in Hartree and kJ/mol is found in Table S2. A compound, 2-methyl-3-pentanamine, was compared to valinol, of similar structure, concerning the interaction energy with the SR (Table 2). The difference of 2-methyl-3-pentanamine with valinol is that the OH group in valinol is replaced by a methyl group (Figure S2). It would be expected that the presence of an OH group in valinol would produce a stronger bond with all SRs than 2-methyl-3-pentanamine based on the comparison made above between valinol and serine. However, in all cases, the opposite result was obtained (Table 2). The reason for this result is the presence of an intramolecular bond in valinol with a -9.2 kcal/mol energy between the positive charge on the nitrogen and the free electrons of the oxygen (Figure S5). This bond hinders the interaction of serine with 2-butanol, as has been demonstrated with atenolol, (Fernandez-Maestre 2018) by dispersing the positive charge on the nitrogen over a larger number of atoms. This bond is not present in 2-methyl-3-pentanamine because of the absence of the hydroxyl group. Figure S6 shows the potential energy surface maps when valinol protonation is carried out. Upon protonation of valinol, the intramolecular bond is formed. If we force the anti-position between the amino group and the hydroxyl group when protonation is carried out, we can observe a ~12 kcal/mol difference in the stabilization energy of the molecule, indicating that the structure with the higher stabilization energy, forming the intramolecular bond, is the most favored. When valinol was excluded from the graphs in Figure 6-b, the R 2 correlation coefficients increased from 0.58, 0.70, 0.76, 0.90, and 0.91 to 0.68, 0.89, 0.93, 0.90, and 0.93, for M, F, B, W, and L shift reagents, respectively, indicating the extent of the departure of valinol from linearity and the effect of the low cluster interaction energy. ## Average shifts and interaction energies Fig. 6 shows that the shift is a function of mass or size with negative slope. However, the slope differs for various SRs. Water gives the smallest shift while ethyl lactate shows the largest shift. The smallest shifts are produced by W, B and F for all analytes and the largest ones are produced by L and M. This is perhaps due to the presence of C=O bond in L and M that generates higher proton affinity than other SRs. Also, these SRs have three interaction sites compared to one site on the other SR. The effect of SR on the shifts can be more accurately discovered if the interaction energy between the SR and the ion is considered. Fig. S7 shows a large correlation (R 2 > 0.98) between the average interaction energies and the average shifts for the selected SRs and analytes. In fact, the shift depends not only to the individual analyte, but also to the tendency of the SR for attachment. ## CONCLUSIONS We observed time and mobility shift-mass correlations for protonated desipramine, ethanolamine, serine, methionine, phenylalanine, threonine, tribenzylamine, tributylamine, tryptophan, tyrosine, and valinol when different SR were introduced into the buffer gas at 150 °C. This correlation is due to the fact that the lighter or smaller ions are more affected by adduction of the SR than the heavier or larger ions. However, the correlation had exceptions in our experiments: valinol, due to the low binding energy with the SR, and methionine, due to the formation of an intramolecular hydrogen bond. Furthermore, it was shown that the average shifts perfectly correlate to the average interaction energies. This shows that the shift is determined by the initial size of the ion as well as the SR-ion interaction. The understanding of this behavior is important in IMS to predict the adequate SR for a given interferent in the detection of illegal substances in airports, ports, and customs. These interferents cause false positives that hinder the transit of passengers and cargo due to unnecessary deeper inspections. SRs can be used to rule out the presence of false positives by facilitating transport and trade. Fig. S6 The potential energy surface map shows that when valinol protonation takes place an intramolecular bond of 9.22 kcal/mol is formed. Upon protonation of valinol, we can notice the change of the electrophilic zone of oxygen and nitrogen from reddish-yellow to dark blue denoting a nucleophilic zone that includes the intramolecular bond. If we force the anti position between the amino and hydroxyl group when protonation takes place, we can observe a small difference in the stabilization energy of the molecule of approximately 0.02 Hartree indicating that the structure with the highest stabilization energy and forming the intramolecular bond is the most favored. PA: proton affinity. IHB: intramolecular hydrogen bond. Average Interaction Energy (kcal/mol) - 32 -30 -28 -26 -24 -22 -20 -18 -16 -14 Average Drift Time Shift (ms) 2 and 3. Table S1. Mobility shifts, %ΔK 0 , for selected ions when different SR were introduced into the buffer gas at different concentrations in mmol m -3 . Ions here were not considered in Table 1 because they form intramolecular bonds or present steric hindrance on the positive charge that deter SR adduction. ΔK0 values were calculated as the percent difference mobilities in nitrogen-only buffer gas and SR-doped buffer gas. The concentrations of SR in the buffer gas were 2.3, 1.0, 879, 1.7, 6.8, and 1.0 mmol m-3, for F, M, W, L, B, and N, respectively (Fernandez-Maestre et al. 2010a, 2010b). -9.2 * The water in the ammonia solution, 106 mmol/m 3 of water, also affects the mobility. A: 2-methyl-3pentanamine, B: 2-butanol, F: trifluoromethyl benzyl alcohol, L: ethyl lactate, M: methyl-2-chloro propionate, N: nitrobenzene, n: ammonia, W: water. (Fernandez-Maestre 2018).

chemsum

{"title": "Ion-shift reagent binding energy and the shift-mass correlation in ion mobility spectrometry", "journal": "ChemRxiv"}

progressive_polytypism_and_bandgap_tuning_in_azetidinium_lead_halide_perovskites

## Abstract: Mixed halide azetidinium lead perovskites AzPbBr3-xXx (X = Cl or I) were obtained by mechanosynthesis. With varying halide composition from Clto Brto I -; the chloride and bromide analogs both form in the hexagonal 6H polytype while the iodide adopts the 9R polytype. An intermediate 4H polytype is observed for mixed Br/I compositions. Overall the structure progresses from 6H to 4H to 9R perovskite polytype with varying halide composition. Rietveld refinement of the powder X-ray diffraction patterns revealed a linear variation in unit cell volume as a function of the average radius of the anion, which is not only observed within the solid solution of each polytype (according to Vegard's law) but extends uniformly across all three polytypes. This is correlated with a progressive (linear) tuning of the bandgap from 3.41 to 2.00 eV.Regardless of halide, the family of azetidinium halide perovskite polytypes are highly stable, with no discernible change in properties over more than 6 months under ambient conditions. ## Introduction Hybrid organic inorganic halide perovskites have created much excitement as promising materials for solar cells, light-emitting diodes, 4,5 and photodetectors. 6,7 These perovskite materials share a general formula AMX3, where the A-site cations occupy the interspace between MX6 octahedra (M being a heavy group 16 element). The compositional variations among A, M and X result in a diverse range of structures with distinct chemical, physical and optoelectronic properties, including band structure, 8 primarily due to variations in M-X bonding interactions and connectivity of octahedra. The most common polytype of these perovskites is the (pseudo-) cubic perovskite formed from a cubic (c-) close-packed stacking sequence of AX3 layers and, as a result, cornersharing MX6 octahedra. In Ramsdell notation 9,10 perovskites formed from entirely cubic closepacked AX3 layers are indicated by the label 3C, representing the three close packing layers of the aristotype cell and C for the cubic lattice type. For lead halide perovskites, such 3C polytypes are favored for tolerance factors, t ≤ 1, which arise for relatively small A-site cations such as Cs + or methylammonium. For larger A-site cations, and t > 1, hexagonal polytypes are obtained. 11 These polytypes contain sequences of both cubic (c-) and hexagonal (h-) close-packed AX3 layers, or only hexagonal (h-) packing. The resulting polytypes are also readily described using Ramsdell notation such as 2H (hh…), 4H (hchc...), 6H (hcchcc…) etc, where, again, the numerical value describes the number of packing layers in the unit cell and H describes the lattice type, which is hexagonal in this instance (although rhombohedral variants such as 9R also exist). These sequences generate a number of possible combinations of corner-sharing and face-sharing octahedra. In general, the bandgap of perovskite materials can be tuned by modifying the ratio of corner-sharing to face-sharing 12,13 octahedra as the nature of octahedral connectivity affects the M-X orbital interactions that determine the energies of the valence and conduction bands. Varying the halide composition is a common strategy for tuning the bandgap in hybrid halide perovskites. Yuiga et al. 14 showed that the bandgap of 3C perovskite MAPbBrxI3-x (MA = methylammonium) varied quadratically from 1.65 to 2.38 eV with increasing Br content accompanied by a symmetry change from tetragonal to cubic. Gratia et al. 16 reported a crystallization process with a progressive structural change from 2H, 4H and 6H to 3C depending on x in (FAPbI3)x(MAPbBr3)1−x (FA = formamidinium); however, DMSO solvent molecules were found to be present in the crystal lattice and it is unclear how their presence may affect the 3 formation of the polytypes and their resulting band structure. Other benefits of mixed halide perovskites include improving solar cell power conversion efficiency 17,18 and the stability of the perovskite materials. 19,20 For example, Jeon et al. reported that incorporating 15% Br in FAPbI3 lead to optimum power conversion efficiency of solar cells . 17 Furthermore, Jun et al. reported that mixing 15-20% Br in MAPbI3 resulted in solar cells that could keep 95% efficiency for more than 15 days after exposure to humidity, while the efficiency of MAPbI3 cells dropped below 50% after the exposure. 19 One of the most commonly studied organic cations used at the A-site of this class of perovskites is methylammonium (MA + ). MA-containing perovskite materials are often used as a reference point for studies of the optoelectronic properties of hybrid perovskites. However, poor resistance to moisture remains an obstacle to the commercialization of MA-containing perovskites, especially for MAPbI3, which decomposes to PbI2 in the presence of water. 21,22 Other cations have been investigated to address the poor stability, such as formamidinium, 23,24 Cs + , 25,26 azetidinium 27 and guanidinium 28 , amongst others. Azetidinium (Az + ) is a four-membered ring ammonium (CH2)3NH3 + , which is calculated to be a possible candidate for organic-inorganic hybrid perovskite with a tolerance factor ranging from 0.98 to 1.02 (from AzPbI3 to AzPbCl3 perovskite). The preparation of both AzPbBr3 12 and AzPbI3 29 from solution have been reported, where 6H and 9R perovskites were obtained, respectively. Our previous study on AzPbBr3 12 showed that the material remains stable in ambient air for over 6 months. AzPbI3 crystals were able to partially maintain the 9R crystalline state after being submerged in distilled water for 50 days 29 and an AzPbI3 thin film was reported to withstand exposure to moisture without decomposing 27 , although the exposure time was only for a few seconds. In the current study, a family of azetidinium mixed halide perovskites, AzPbBr3-xXx (X = Cl or I) were prepared by mechanosynthesis and their structures and optical absorption analyzed by both powder and single-crystal X-ray diffraction and absorption spectra, respectively. Besides the 6H polytype reported previously for AzPbBr3, 12 and 9R polytype reported for AzPbI3, 29 the chloride analogue is shown to also adopt the 6H structure and an intermediate hexagonal 4H polytype is identified for mixed Br-I compositions. Overall, the structure progresses from 6H to 4H to 9R perovskite polytypes with varying halide composition with varying degrees of solid solution 4 formation within each structure type. The structural progression corresponds to a change in ratio of corner-sharing to face-sharing octahedra (Supporting Information, Table S1). Despite this variation in octahedral connectivity, the unit cell volume (normalized per formula unit) as a function of anion average radius varies linearly not only within each solid solution (in accordance with Vegard's law), but also across the entire polytype range. A tuneable bandgap is achieved ranging from 2.00 to 3.41 eV, which varies linearly as a function of average anion radius and the variation factor is similar to the reported factor of APbBr3-xXx (A = MA, or FA, X = Cl or I). The azetidinium halide perovskite polytypes remain highly stable for at least 6 months when stored in the ambient air. ## Synthesis PbBr2 (98%), PbI2 (98%) and hydroiodic acid in water (57%) were purchased from Alfa Aesar. Hydrobromic acid in water (48%) and AzCl (95%) were purchased from Fluorochem. All other reagents and solvents were obtained from commercial sources and used as received. For preparation of azetidinium iodide (AzI), potassium hydroxide (1.30 g, 23 mmol, 1.5 equiv.) was dissolved in 3 mL DI water and mixed with azetidinium chloride (1.45 g, 15 mmol, 1 equiv.) under stirring for 30 min. The intermediate azetidine was extracted at 80 ℃ under reduced pressure and condensed with liquid nitrogen. Hydroiodic acid (3 mL, 23 mmol, 1.5 equiv.) was then added into the condensed azetidine solution and stirred for 30 min at room temperature. The solvent was then removed under reduced pressure at 80 ℃. The crude products were dissolved in 3 mL EtOH and the product recrystallized from diethyl ether. The recovered solid was dried under vacuum for 24 h before use. White needle-like crystals were obtained. The NMR of AzI is shown in Figure S1. Yield: 86%. Mp.:137-138 °C 1 H NMR (500 MHz, DMSO-d6) δ (ppm) 8.42 (s, 2H), 3.98 -3.89 (m, 4H), 2.37 (p, J = 8.3 Hz, 2H). 13 AzPbCl3 samples were prepared by dissolving AzCl and PbCl2 (1:1) in DMSO (2 mL, 0.4 M) at room temperature and in air. After stirring for 1 h clear solutions were obtained. DCM (8 mL) was added slowly into the solution and the vial was shaken for 1 min and then left to stand for 10 min before vacuum filtration. The resulting powders were washed with 10 mL DCM twice and dried under vacuum for 24 h. The samples were white powders. Single crystals of AzPbCl3 were obtained by slow diffusion of antisolvent DCM into the same concentration perovskite/DMSO solution in a sealed vial. White needle-like crystals were obtained. Preparation of both AzPbClxBr3-x and AzPbIxBr3-x solid solutions with 0 ≤ x ≤ 3 was carried out by mechanosynthesis. Appropriate molar ratios of Az/halide source (AzPbCl3, AzBr or AzI) and lead/halide source (PbCl2, PbBr2 or PbI2) were ground together in a Fritsch Pulverisette planetary ball mill at 600 rpm for 1 hour using 60 cm 3 Teflon pots and high-wear-resistant zirconia media (nine zirconia grinding media 10 mm diameter spheres). Single crystals of AzPbCl3, AzPbBr1.5I1.5 and AzPbI3 for comparison with mechanosynthesized samples were prepared by slow diffusion of antisolvent DCM/acetonitrile/acetonitrile into DMSO, DMF/DSMO (4:1) and DMF/γ-butyrolactone (1:1) solution, respectively. AzPbCl3 appears as white needle-like crystals while AzPbBr1.5I1.5 and AzPbI3 crystals are bright yellow and dark red, respectively. During crystallization of AzPbBr1.5I1.5, there was evidence of formation of crystals of other compounds; in one case indexing of the data suggested the presence of AzPbI3. This suggests that the mixed halide is not favored against the single-halide forms of the complex during recrystallization and vice versa. Given some of the data-issues encountered (vide infra), it is possible that the selected crystals of AzPbBr1.5I1.5 may have contained domains or crystallites of AzPbBr3 or AzPbI3. Single crystal samples prepared by precipitation synthesis and powder samples prepared by either precipitation or mechanosynthesis routes were characterized by single crystal and powder X-ray diffraction (SCXRD and PXRD, respectively). SCXRD data were collected at either at 293, 173, or 93 K using a Rigaku FR-X Ultrahigh Brilliance Microfocus RA generator/confocal optics with XtaLAB P200 diffractometer [Mo Kα radiation (λ = 0.71075 )]. Intensity data were collected using ω-steps accumulating area detector images spanning at least a hemisphere of reciprocal space. Details of structure solution and refinement are provided in the Supporting Information. PXRD was carried out either using a PANalytical Empyrean diffractometer with Cu Kα1 (λ = 1.5406 ). Rietveld refinements of PXRD data using GSAS 33 were used to confirm phase formation and for determination of lattice parameters. Optical properties were determined from solid-state absorption spectra recorded using a JASCO-V650 double beam spectrophotometer and bandgaps were calculated using the 'Band-Gap Calculation' program of the spectrophotometer which applies the Tauc method. Sample morphologies were investigated using a Jeol JSM-5600 Scanning Electron Microscope with an accelerating voltage set at 5 kV. 1 H and 13 C Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Advance spectrometer (500 MHz for 1 H, 126 MHz for 13 C). 1 H and 13 C NMR spectra were referenced to residual solvent peaks with respect to TMS (δ = 0 ppm). ## Results and Discussion Commercially available AzCl was found to be too impure (< 90%, discussed in Supporting Information and shown in Figure S2) to use in the mechanosynthesis, so AzPbCl3 prepared by precipitation synthesis, with purity confirmed by powder X-ray diffraction (PXRD), was used as the Az/Cl source. Attempts to prepare AzPbCl3 by mechanosynthesis using commercial AzCl required a stoichiometric excess of AzCl to converge to the desired product. While PXRD showed successful preparation of single-phase AzPbCl3 using this excess (Figure S3), the presence of small amounts of AzCl, which may not be detectable by PXRD, cannot be excluded. Thus, the following analysis of AzPbCl3 was based only on precipitation-synthesized samples. The color progression (Figure 1a) of the as-synthesized AzPbClxBr3-x is subtle and ranges from white (AzPbCl3) to pale yellow (AzPbBr3); the colors of the AzPbBrxI3-x series, by contrast, show a clear and systematic change from pale yellow (AzPbBr3) to red orange (AzPbI3). The PXRD of the mixed halide perovskites are shown in Figures 1b and 1c. The PXRD of AzPbCl3 shows the same 6H hexagonal structure as AzPbBr3, 12 Single crystal X-ray diffraction (SCXRD) at ambient temperature and 173 K confirms the 6H polytype (Figure 2 show additional broad features in the base of the main peaks and which are especially evident around 12 -14°. These features match well with the reported features from bimodal CdS particles 34 and indicate the presence of multiple subpopulations of different sizes of 6H perovskite particles. In our previous study, the PXRD of precipitation-synthesized AzPbBr3 did not show such features. 12 Scanning electron microscopy (SEM) of both mechano-and precipitation synthesized AzPbBr3 (Figure S4) indicate the presence of a large proportion of relatively smaller particles in the mechanosynthesized AzPbBr3, explaining the broad base of the PXRD peaks in Figure 1b. AzPbI3 has been reported previously as a 9R polytype 29 and the Rietveld refinement (Figure S5) of the PXRD confirms that the 9R AzPbI3 perovskite can also be obtained easily by mechanosynthesis compared with the reported two-step recrystallisation method in solution. 29 This is also confirmed by the SCXRD structure, although, as has been the case in previous attempts to determine the structure of AzPbI3 by SCXRD, 29 the apparent crystal quality prevented the confirmation of the Az + cation sites. This was observed for data collected at both ambient temperature and 173 K. In the case of this structure, both resulted in a lattice parameter, a, smaller than that determined by Rietveld refinement, the ambient temperature structure being closer to that seen in the refined PXRD data, however, both SCXRD structures had a c lattice parameter larger than that determined by Rietveld refinement [SCXRD (293 K): a = 9.0835( 5 PXRD data of AzPbBrxI3-x (x ≤ 2) seem to indicate a structure that was neither 6H, 9R nor a twophase mixture of these two polytypes; however, the hypothesis was that AzPbBrxI3-x (x ≤ 2) is still some form of perovskite (or a mixture of perovskite polytypes). Analysis of the PXRD of AzPbBr1.5I1.5, in particular, the d-spacing of the two major peaks at 11.41° and 12.86°, reveals the intermediate structure to be the 4H polytype with P63/mmc space group (Figure 2). The 4H perovskite structure has an (hc)2 stacking sequence in Jagodzinski notation, resulting in alternating face-sharing and corner-sharing octahedra. The goodness-of-fit parameters from the Rietveld refinement of AzPbBr1.5I1.5 to an adapted 4H model (Figure S6) indicate a good fit: c 2 = 3.509, wRp = 7.5%. SCXRD of the AzPbBr1.5I1.5 suggested that in single crystals prepared by precipitation a mixture of phases exist, potentially including the AzPbX3 single-halide materials; no evidence of mixed phase was evident in the same compositions prepared by mechanosynthesis, again highlighting the need for caution for samples prepared using the kinetically-controlled precipitation route compared to thermodynamically-controlled mechanosynthesis. 35 However, it did prove possible to isolate and structurally characterize AzPbBr1.5I1.5 by SCXRD. As was the case with the iodide compound, crystal quality precluded modelling of the Az + sites, both for ambient temperature data, and for that collected at 173 K. Both structures showed lattice parameters smaller than that determined by Rietveld refinement (Table S2), the ambient To study the solid solutions within, and transition between, these polytypes, the lattice parameters of each mechanosynthesized composition were determined by Rietveld refinement of PXRD data. The lattice parameters of the single halide perovskites AzPbX3 (X = Cl, Br, I) and also 4H-AzPbBr1.5I1.5 are shown in Table 1. The average interlayer distance along the c-axis (𝑐̅ ) and lattice parameter a increase with the transition sequence from 6H to 4H to 9R. The cell volume (normalized to the number of formula units per unit cell) of those polytypes varies linearly as a function of average anion radius, Figure 3a (the average anion radius was calculated using rI = 220 pm, rBr = 196 pm and rCl = 181 pm according to Shannon 36 ). While this linear variation within each polytype solid solution is expected in accordance with Vegard's law, it is interesting to note that the linear relationship extends continuously across all three polytypes. Presumably this reflects the AX3 close packing volume; however, it suggests that the polytype adopted is largely driven by the degree of Pb-Pb interactions, which is emphasized in face-sharing (h) layers. The substitution of increasingly large, and less electronegative, halide anions result in an expansion of MX6 octahedra, which decreases the electrostatic energy (Madelung energy) of the ionic crystals and allows for more face sharing octahedral layers and Pb-Pb proximity. * The average interlayer distance along c-axis. In addition to the 4H, 6H and 9R single phase solid solutions, intermediate two-phase regions of 6H-4H and 4H-9R were also identified by PXRD, as shown in Figure 3a. For the 6H-4H two phase-region, the peaks of both phases could be readily identified, but the boundary of the 4H-9R two-phase region was difficult to ascertain due to the overlap of the major peaks (Figure 1b). Attempts at two-phase refinement of PXRD data of both two-phase regions were unsuccessful due to the overall breadth of peaks, overlap of major peaks and relatively low intensities of non-11 overlapping peaks. As a result, no lattice parameters are provided for the 6H-4H two-phase region. For the 4H-9R two-phase region, the data for compositions AzPbBrI2 and AzPbBr0.6I2.4 which appear close to the phase boundaries were refined as single-phase 4H and 9R, respectively, as approximations, and the resulting lattice parameters matched quite well with the linear fit as a function of average anion radius. As a general comparison, the cell volumes as a function of average anion radius for 3C FAPbX3 and MAPbX3 mixed halide perovskites are shown in Figure S8a and display similar linear behavior, but this unlike the AzPbX3 compositions of the current study all MA-and FA-compositions adopt a single (3C) polytype. The optical properties of the different phases were studied by absorption spectroscopy (Figure 4). The absorption onsets are systematically red-shifted with increasing average anion size (from Cl to I). The absorption onset of AzPbBr3-xXx (X = Cl or I, 0 ≤ x ≤ 3) samples show a red shift from ca. 360 nm (3.44 eV, AzPbCl3), to ca. 450 nm (2.76 eV, AzPbBr3), to ca. 615 nm (2.02 eV, AzPbI3). The background absorption of intermediate compositions in AzPbClxBr3-x samples lies above the normalized zero baseline, especially for x = 2.5. This might result from a small number of Br-rich crystallites on the sample surface, the amount of which is too small to be detected in PXRD. The absorption of AzPbI3 bore close resemblance to the reported spectrum, 29 where three well-defined transitions could be detected; the peak maxima of the three well-defined transitions are at 551, 506, 470 nm while the reported transitions peak at 554, 503, 462 nm. all samples were prepared by mechanosynthesis. The bandgap as a function of halide composition for the mixed halide perovskites is shown in Figure 3b. The bandgap of AzPbCl3 and AzPbI3 were calculated to be 3.41 ± 0.01 eV and 2.00 ± 0.02 eV, respectively. The latter is in good agreement with the previously reported value of 1.97 eV. 29 The bandgap varies linearly as a function of average anion radius, despite the change of halide composition and octahedral connectivity. As discussed in our previous study, the varying ratio of corner-sharing to face-sharing octahedral connectivity changes the efficiency of Pb-X orbital overlap; in conjunction with the change in Pb-X bond length, average bond angles and covalency which give rise to the bandgap variation. 12 Comparison of the behavior of the Az-based perovskites with corresponding MA-, and FA-based mixed halide perovskites shows that the lattice parameter progression as a function of halide composition is linear in all cases; however, the reported relation of bandgap versus halide composition is not consistent across these studies. Some studies reported a nonlinear relation, which is described as a bowing effect, 19,30,37 while other studies document a linear progression 38,39 as observed here. Bandgap "bowing" is often fitted to a second order polynomial, with a bowing parameter b as the binominal coefficient of the fitting equation. The bowing parameters of MAPbBr3-xXx (X = Cl or I) are relatively small (7 ´ 10 -4 to 0.33) 19,30 compared to the bowing parameters (0.5 to 1.33) found for other mixed metal perovskite systems. Our study illustrates a good example of a linear variation between bandgap and halide composition, and it is as of yet unclear why both linear and non-linear relationship were reported for other mixed halide perovskites with same organic cation and metal. However, this may related to anion segregation when prepared using kinetically-controlled precipitation routes. 35,44 ## Summary and Conclusions Following on from studies on azetidinium lead bromide, mixed halide compositions, AzPbBr3-xXx (X = Cl or I), were successfully synthesized using a mechanosynthetic grinding method. The single-phase single halide materials AzPbX3 (X = Cl, Br or I) were shown to be stable in air for > six months. In addition to the 6H polytype reported previously for AzPbBr3, 12 and 9R polytype reported for AzPbI3, 29 AzPbCl3 was also shown to form in the 6H polytype and an additional 4H polytype was found for AzPbBr3-xIx (ca. 0 < x ≤ 2) compositions. With varying halide composition, the structure progresses from 6H to 4H to 9R perovskite polytype. A complete (continuous) solid solution is formed for compositions with the 6H structure and partial solid solutions form between the 6H and 4H and 4H and 9R polytypes. A linear variation in unit cell volume (scaled per formula unit) as a function of anion average radius is observed not only within the solid solution of each polytype (according to Vegard's law) but continuously across all three polytypes, which, to the best of our knowledge, is the first time that Vegard's law-type behavior has been observed across several polytypes. This linear relationship extending across all compositions is accompanied by a linearly tuneable bandgap ranging from 2.00 to 3.41 eV as a function of average anion radius without any observations of a "bowing effect". The linear variation of bandgap across all AzPbX3 compositions (and polytypes) is comparable to that observed in APbBr3-xXx (A = MA, or FA, X = Cl or I) but that all adopt a single (3C) polytype. Associated content ## Supporting Information The Supporting Information contains additional experimental information including: details of 1 H NMR analysis, PXRD analysis, SXRD analysis, SEM, examples of Rietveld refinement, absorption spectra and bandgap analysis. The research data supporting this publication can be accessed at []. ## Author information Corresponding authors: [email protected] [email protected]

chemsum

{"title": "Progressive Polytypism and Bandgap Tuning in Azetidinium Lead Halide Perovskites", "journal": "ChemRxiv"}

tunable_circularly_polarized_luminescence_from_molecular_assemblies_of_chiral_aiegens

## Abstract: Circularly polarized luminescence (CPL) is important to chiral photonic technologies. In the molecular systems, besides their intrinsic chemical structures, the architectures of molecular assemblies at the mesoscopic scale also account for the final macroscopic CPL properties. Herein, tunable CPL responses can be induced through architectural regulation of these molecular assemblies in suspension and solid states. A liquid crystalline assembled system of DPCE-ECh exhibiting smectic C* phase with a high dissymmetry factor (gCD = -0.20 and glum = +0.38) is reported. The intense and apparent CD and CPL of the film stem from the intrinsic helical structure of the molecular assembles with weak contribution of Bragg reflection, where the helical axis is perpendicular to the optical axis and is parallel to the direction of the glass substrate. To the best of our knowledge, this large glum factor is very rare for organic compounds even in the assembled state formed by annealing at smectic liquid crystalline temperature. Interestingly, strong CPL signal with glum value of +0.18 is still recorded when DPCE-ECh is annealed at chiral isotropic liquid (Iso*) state. On the other hand, DPCE-ACh can form two coexistence phases of chiral hexagonal and smectic liquid-crystalline phases due to intermolecular hydrogen bonding. The non-periodic molecular orientations of DPCE-ACh break itself helical structure to give a weak negtive CPL signal in 10 -3 order. This work thus provides a new insight for developing efficient chiroptical materials in the aggregate state and profound implications in highperformance CPL-based device. ## Introduction Development of circularly polarized luminescent (CPL) materials has gained increasing interest owing to their potential applications in stereoscopic optical information storage and processing, optical recognition sensor, quantum computing, and circularly polarized electroluminescence for 3D displays. The CPL response of a molecular system is quantified by the dissymmetry factor (glum), where glum = 2(IL-IR)/(IL+IR) and IL and IR denote the emission intensitiy of left-and right-CPL, respectively. The common strategy to achieve CPL is to synthesis molecules with a specific chiral configuration. However, the CPL response of synthetic advanced materials not only relies on chiral functions on the molecular level, but also depends on the mesoscopic architectures of the molecular assemblies. Through a self-assembly approach, nanostructured chiral materials are able to transfer and amplify the molecular functions to an amplified CPL property at a specific length scale. Therefore, investigation on the relationship between hierarchical structure of molecular assembles and their corresponding CPL properties is still an important issue to achieve efficient CPL materials. Normally, the luminescence normalized dissymmetry factor of organic system ranges between 10 −4 to 10 −2 . [3,5, In rare cases, extremely high g-values exceeding 0.2 or even up to 1 have been reported for polyfluorene thin films or cholesteric organic system . In polyfluorene system, the circular polarization is largely determined by the anisotropy of the cholesteric dielectric medium. The glum value is thickness dependent and strong CPL effect originates from the selective CP reflection due to the long-range cholesteric ordering (Bragg reflection). The helical axis of this system is perpendicular to the direction of the substrate. In cholesteric films, hierarchical chiral mesoscopic structures were found in this system. Strong CPL response can be arisen from the sum of two main contributions, including the inherent chiral supramolecular structure and birefringence pattern (Bragg reflection). However, these doped cholesteric systems often suffer the problems of incompatibility and instability. Thus the pursuance of strong chiroptical signal from pure organic compounds remains challenging. Akagi's group reports a glum of +0.29 in chiral bithiophene-phenylene copolymer film annealed in chiral nematic state. They also reports a high glum of -0.23 in chiral disubstituted polyacetylene without no chiral dopant. Recently, chiral molecular assemblies with aggregation-induced emission (AIE) effect become the focus of attention . Benefiting from the enhanced emission intensity upon aggregation of AIEgens, efficient CPL response can be generated in solid state to realize their applications in devices. Although the significant progresses have been achieved to access efficient glum value increasement, the approaches to controlling the mesoscopic structure and the ensuing CPL properties are still limited. Therefore, the CPL properties of chiral luminogens in condensed matter state might have profound implications for the high performance CPL-based device at the macroscopic scale. Herein, two rod-like aggregation-induced emission luminogens with a rigid core containing ester or amide linkage and a cholesterol moiety at one end and long aliphatic chains at the other end, namely DPCE-ECh and DPCE-ACh, are presented and illustrated in Figure 1A. In solid state, DPCE-ECh self-assembles into supramolecular liquid-crystalline smectic C* (SC*) phase and shows an impressive high positive CPL response with glum of +0.380±0.011 and gCD of -0.20. The intense and apparent CD and CPL of the film stem from the intrinsic helical structure of the molecular assembles with small contribution of Bragg reflection, where the helical axis is parallel to the direction of the glass substrate. To the best of our knowledge, this large glum factor is very rare for organic compounds even in the assembled state formed by annealing at smectic liquid crystalline temperature. On the other hand, DPCE-ACh can form two coexistence phases of hexagonal and smectic liquidcrystalline phases with a weak negative CPL response. The glum falls in the range of -0.61 × 10 -3 to -5.96 × 10 -3 . Such non-periodic molecular orientations give a weak CPL signal in 10 -3 order. The large different |glum| is attributed to the amplified artifact induced by the birefringent domains of the thick film. ## Synthesis and characterization The synthetic procedures of DPCE-ECh and DPCE-ACh are outlined in Scheme S1. Their structures were confirmed by NMR and high resolution mass spectroscopies (Figure S1-S15). Thermogravimetric analysis (TGA) revealed that two compounds have high decomposition temperatures (Td) which could up to 300 o C (Figure ## Please do not adjust margins Please do not adjust margins S16), suggesting that they are thermally stable. In dilute THF solution, DPCE-ECh and DPCE-ACh show adsorption band centered at 364 and 360 nm, respectively (Figure S17A). DPCE-ECh and DPCE-ACh exhibit a similar fluorescence spectrum with a peak maximum centered at 430 nm (Figure 17B). As shown in Table S1, two compounds are weekly emissive in THF solutions with quantum yield ΦF, soln. of 0.005 and 0.003 and emissive in the solid powders with ΦF, solid of 0.12 and 0.114. Their αAIE (ΦF, solid/ΦF, soln.) values were calculated to be 24 and 38, suggesting a typical AIE feature of these compounds. ## Chiroptical in solution and suspension The weakly-emissive THF solutions of DPCE-ECh and DPCE-ACh become progressively emissive upon addition of water (Figure 2A, S18-19), demonstrating an AIE phenomenon. Such chiral AIEgens with cholesterol moieties are promising candidates for chiral induction, which might be capable to take supramolecular helicity with assistance of the long alkoxy chains. The chiroptical properties of the aggregates generated in THF/water mixtures with different H2O fractions (fw) were then investigated (Figure 2B-E, Figure S20-35). The aggregates of DPCE-ECh with a ester linkage are CD silent regardless of water fraction variation (Figure S20-28). In contrast, at fw ≥ 40%, aggregates of DPCE-ACh with an amide linkage exhibit obvious CD signals with negative and positive Cotton effects at wavelengths between 300 nm and 400 nm (Figure 2B). It is noted that at fw = 60%, an induced positive split-type Cotton effect consisting of a positive Cotton effect at 375 nm and a negative Cotton effect at 338 nm was observed in DPCE-ACh aggregates, suggesting the formation of organized helical superstructures in solution . The maximum absorption anisotropy factor (gCD) reaches 2.78 × 10 -3 at 375 nm (Figure 2C). In addition, the UV-Vis absorption intensities at long wavelength region began to increase substantially when fw = 60% (Figure S25), this could be attributed to the production of large aggregates with strong light scattering. The above CD and UV data further proved the large-size helical aggregates formation with the addition H2O into THF solvent (fw = 60%). The above CD and UV data reveal the formation of large-sized helical aggregates in THF/H2O mixture with fw of 60%. However, the continuous increment of fw to 90% leads to a dramatic decrease in gCD by an order of magnitude (+2.3 × 10 -4 at 375 nm, Figure 2C), suggesting the dissociation of the chiral helical aggregates. Analog to CD spectroscopy, CPL reflects the chiroptical properties of the luminescent materials upon excitation. Consistent with those of CD results, the isolated species in THF solution and aggregates in THF/H2O mixture of DPCE-ECh are all CPL silent (Figure S29-35). However, the DPCE-ACh aggregates suspended in THF/H2O mixture show positive CPL signal at fw = 40% with glum of 2.0 ×10 -4 (Figure 2D). The detailed glum values of DPCE-ACh aggregates in THF/H2O mixture are depicted in Figure 2E. The maximum glum reaches ~6.0 ×10 -4 at fw between 50% and 70% (Figure 2E). This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins Please do not adjust margins To better understand the origins of the chiroptical properties of DPCE-ECh and DPCE-ACh aggregates, scanning electron microscopy (SEM) was employed to study their assembled structures in THF/H2O mixtures with fw varying from 40%-90% (Figure S36-40). For DPCE-ECh, its aggregates keep spherical structure regardless of the water fraction variation (Figure S36). Such a symmetrical morphology leads to a silent CPL response. For DPCE-ACh, its aggregates show an obvious morphological evolution from intertwined network (fw = 40%, Figure S37) to left-handed helical nanofibers (fw = 60%, Figure 3A,B). Benefited from the helical fibrous morphology, DPCE-ACh aggregates show higher dissymmetry factors compared than those of the spherical aggregates. On the other hand, M-helices and Psupra-helices are found in DPCE-ACh suspension (Figure 3A, B). According Michael D. Barnes' report , they thought the measured g value in disperse phase represents a weighted average of all possible orientations and interaction with the host. As for DPCE-ACh in suspension, the |glum| value detected in this system can be attributed to cancellation effects in ensemble measurements of a randomly oriented (M-helices and P-suprahelices) bulk sample. As seen in Figure 3A, M-helices sturctrues accounts for most of the proportio, thus leading to positive CPL signal. Careful examination of the chemical structures of the AIEgens may account that intermolecular hydrogen bonding between the amide linkages and the chiral nature of cholesterol moiety of DPCE-ACh serve as the external driving forces for forming the helical self-assembled structure to generate CD and CPL signals. To gain further insight into the dynamic nature of hydrogen bonding within the induced helical fibrous structure , temperature-dependent CD spectra are monitored on the DPCE-ACh-based aggregates (fw = 60%, Figure 3C,D, Figure S41). At the low temperature of 5 o C, the CD spectrum of DPCE-ACh aggregates shows an obvious positive Cotton effect with absorption peak at 375 nm and gives a corresponding gCD value of + 2.58 × 10 -3 . The peak at 375 nm is ascribed to the achiral aromatic rigid core of DPCE-ACh, which is induced by the molecular helical arrangement. It is found that the CD signals of helical aggregates were sensitive to the temperature. The CD signals gradually decrease and completely disappear upon heating to 49 o C (Figure 3C). Such temperature-dependent gabs factor variations are summarized and plotted in Figure 3D. It is noted that the gCD of 375 nm was only + 2.5 × 10 -4 at the high temperature of 49 o C, indicating that the hydrogen bonding became very weak and thus lead to the dissociation of helical assembled structure of DPCE-ACh. ## Chiroptical in condensed phase The rod-like molecular structures of two AIEgens with a cholesterol moiety and flexible tails at two ends, makes them promising to form liquid-crystalline phase in a chiral fashion. DPCE-Please do not adjust margins Please do not adjust margins ACh was first explored considering its capability to form helical fibers mentioned above. The phase transition temperatures of DPCE-ACh in solid film are shown graphically in Figure 4A (top, Figure S42). Upon cooling the isotropic liquid of DPCE-ACh to 210 o C, a smectic liquid-crystalline phase with a fan-shaped texture followed by a columnar liquid-crystalline phase with a mosaic texture are observed (Figure 4A, bottom; Figure S43). The molecular orientations in the liquid-crystalline phases are revealed by 1D wide angle X-ray diffraction (1D WAXD, Figure S44-46). The 1D WAXD pattern at 210 o C shows a sharp peak at 2θ = 2.97° and a high-order diffraction peak at 2θ = 5.66°. These two diffraction peaks are associated with a smectic phase structrue . with a layer thickness of 3.05 nm (Figure 4B 110) and ( 200) planes of the hexagonal columnar liquid crystals (Figure 4B, S46). Analysis of small-angle X-ray scattering (SAXS) confirms the lamellar and columnar organization of DPCE-ACh with a lamellar thickness of 3.05 nm and a columnar diameter of 4.33 nm, respectively (Figure S46). Electron density reconstruction was caculated according to the method of previous published paper. The electron density map (Figure 4C) of the phase based on the XRD result shows that the high electron density (red) is concentrated at the center of columns and low-density areas (green and blue) are located at the column periphery associated with the alkyl chains and the cholesterol side chains, respectively. On the other hand, as DPCE-ACh is hexagonal columnar mesophase, the average number (n) of molecules per slice of the column could be obtained by the following formula. n = (ɑ 2 )(√3/2)(hρNA/M) where the notation "ɑ" is the hexagonal lattice parameter, NA is Avogadro's number, M is the molecular mass of the compound and the density (ρ) of these samples is set as 1 g/cm 3 . After calculation, the number of molecules (n) in one disk is approximately 2 for DPCE-ACh. Thus, the possible molecular stacking mode for the hexagonal columns is suggested as Figure 4D, in which a slice is composed of two molecules based on the hydrogen-bonding action between N-H and C=O groups. These results certainly support that the hydrogen bond plays the crucial role for inducing the columnar mesophase of the asymmetrical diphenylacrylonitrile derivatives. As illustrated in Figure 4E, the film exhibits a negative signal at 415 nm with a gCD of -(1.92 ± 0.063) × 10 -3 after annealing at 180 o C. Moderate profile change in the CD spectrum was obtained by rotating the sample at different angles around the optical axis (Figure 4F, S47-49), suggestting that the LDLB effect (birefringent phenomenon) contributes the final CD. Because DPCE-ACh forms both the coexistence of hexagonal and smectic phases, the nonperiodic molecular orientations break itself helical structure to give a weak CD signal in 10 -3 order. On the other hand, we also investigated the CPL spectra at different angles in both sides in a 7 μm of liquid crystal cell. However, a large difference in |glum| was observed at different angles in both sides and the |glum| falls in the range from -0.61 × 10 -3 to -5.96 × 10 -3 (Figure 4F, S50-51). This phenomenon is attributed to the amplified artifact (Bragg reflection) induced by the birefringent domains of the thick film (7 μm). This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins Please do not adjust margins The ester linkage of DPCE-ECh offers only weak the intermolecular interaction and the relationship between the molecular orientations and the chiroptical properties are also investigated. The DSC trace of DPCE-ECh recorded during the first cooling cycle shows three exothermic transitions at around 121, 86 and 30 o C upon cooling from 180 o C (Figure 5A). The POM image shows oily-streak textures that are typical of the sematic phase in the liquid crystalline state at 85 o C (Figure S52). 1D WAXD measurements were then carried out to monitor the structural evolution. It is found that a sharp peak at 2θ = 2.35 o appears at 80 o C, indicating the formation of an ordered structure In addition, a high-order diffraction peak at 2θ = 4.78 o is also observed. The ratio of the scattering vectors of the two peaks is approximately 1:2, indicating the formation of a smectic structure (Figure 5B, S53, 54). Such an ordered structure was retained at the temperature range of 86-33 o C. The transition recorded by DSC at 86 o C corresponds to the clearing point (Figure 5A). In the temperature range of 86-121 o C (DSC), a broad and weak peak compared with that of smectic phase was observed (Figure S55) and we identified it as isotropic liquid phase. To further prove the smectic structure of DPCE-ECh, 2D SAXS and wide-angle X-ray scattering (WAXS) were carried out (Figure 5C,D). The oriented sample for the measurements was prepared by mechanically shearing the melted film at 85 o C. As shown in the illustration in Figure S56, the point-focused X-ray beam was aligned perpendicular to the shear direction. It is noted that the diffuse peaks at smaller (Figure 5C) and larger angles (Figure 5D, marked with dash line) are not orthogonal to each other. This suggests that the director makes a tilt angle with respect to the smectic layer and the angle rotates from layer to layer to form a smectic C phase. CD experiments were conducted to analyse the chiroptical activities of DPCE-ECh solid film on glass slides. No matter rotating or flipping samples, strong and consitent CD signals were obtained (Figure 5E, S57). It implies that the long helical molecular stacking axis was perpendicular rather than parallel to the optical axis (Figure S58A). In addition, the molecular orientation in-plane was supposed to be aligned randomly. In this sense, the LDLB effect in such solid film could be neglected and genuine chiroptical signals from chiral supramolecular structure were resulted. A gCD value of -0.20 at 404 nm was achieved (Figure 5F). Similarly, we also faricated a film in a 7 μm thick liquid crystal cell for CPL measurement. CPL spectra were also obtained by rotating the sample at different angles in both sides (Figure 5G). Strong CPL responses with positive signals were observed in this annealled film with a maximum glum value (average, 70 o C) of +0.380±0.011. However, different glum ranging from 0.342 to 0.438 were observed with vaired angles and sides. The strong CPL response is arisen from the chiral supramolecular structure in which the helical axis is perpendicular to the optical axis and is parallel to the direction of the glass substrate. The large difference in glum (about 0.1) at different angles in both sides is attributed to the birefringence pattern. Because the film thickness is 7 μm for CPL detection which is much thicker than film for CD detection (50 nm), the artifact induced by the birefringent domains is amplified in such a thick film. To the best of our knowledge, such large glum value (+0.380±0.011) with weak contribution of Bragg reflection is very rare for organic compounds. With increasing the annealling temperature (>70 o C), the dissymmetry factor (gCD and glum) of the thin film decreases (Figure 5F,H), indicating the dissociation of smectic C phase. Interestingly, when the isotropic liquid of DPCE-ECh was annealed at temperature range of 90-120 o C followed by CPL measurent, strong CPL signal with gCD value of -0.11 and glum value of +0.18 was still recorded (Figure S59-71). The CPL response of the isotropoic state of DPCE-ECh indicates that a twisted orgainzation is still retained in aggregates and such twisting is still sufficient for CPL induction. Thus, we identify this chiral isotropic state as chiral isotropic liquid (Iso*) in Figure S55 (inset), which was recently discovered new phase. Normally, the measured g value in a disperse phase represents a weighted average of all possible orientations. In THF solution, DPCE-ECh and DPCE-ACh (10 -5 mol L -1 ) are soluble and dispersed isolatedly in this solution. Therefore, the measured g value in dilute solution comes from single molecule itself. For single molecule, chiral function is mainly focused on cholesterol unit and luminescent function is mainly focused on diphenylacrylonitrile unit. Hence, no CPL signal is observed in these single molecules. On the other hand, the aggregates of DPCE-ECh keep spherical structure regardless of the water fraction variation. Such a symmetrical morphology leads to a silent CPL response. Meanwhile, in the solid state, combining the X-ray results and chiroptical activity of DPCE-ECh, a semctic C* phase was identified finally. Moreover, SEM textures of the fracture plane of DPCE-ECh with layered and arched structures further support our hypothesis (Figure S72). Such the smectic C* state leads to a giant CPL response. For DPCE-ACh, positive CPL signal is observed due to M-helical nanofibers formation in suspension. Negative CPL signal is observed due to the complex liquid crystalline (H + S) orientations in solid state. These findings demonstrate that the CPL response (intensity and orientation) of synthetic advanced materials not only relies on chiral functions on the molecular level, but also depends on the mesoscopic architectures of the molecular assemblies. ## Please do not adjust margins Please do not adjust margins ## Conclusions In summary, two AIEgens with rigid cores containing different linkages are developed. These chiral AIEgens show silent CPL response when existed as species in THF solution. In contrast, tunable CPL response is achieved through regulating their aggregated structures in solution and solid states. Driven by the intermolecular hydrogen bonding in DPCE-ACh, opposite CPL responses with glum in 10 -3 order are obtained from M-helical nanofibrous structure and complex liquid crystalline (H + S) orientations. Meanwhile, DPCE-ECh exhibits a liquid crystalline assembled system (smectic C*) with a high dissymmetry factor (gCD = -0.20 and glum = +0.38). The intense and apparent CD and CPL of the film stems from the intrinsic helical structure of the molecular assembles with weak contribution of Bragg reflection, where the lone helical molecular stacking axis is perpendicular to the optical axis and is parallel to the direction of glass substrate. To the best of our knowledge, this large glum factor is very rare for organic compounds even in the assembled state formed by annealing at smectic liquid crystalline temperature. This path opens new capabilities for structural control of molecular assemblies to generate versatile CPL responses that are inaccessible from isolated AIEgen alone. These findings demonstrate that the CPL response (intensity and orientation) of synthetic advanced materials not only relies on chiral functions on the molecular level, but also depends on the mesoscopic architectures of the molecular assemblies. We hope that the present strategy for constructing CPL-active materials in the condensed matter states will open numerous opportunities for applications in photonic devices. ## Experimental Section Chemicals and Methods. All chemicals were purchased from Sigma-Aldrich, J&K Chemical Co. and used as received without further purification unless otherwise specified. Anhydrous THF and CH3CN were used for fluorescence property investigation. Deionized water was used throughout this study. Pre-coated glass plates were used for TLC analysis. Column chromatography was carried out by using silica gel (200-300 mesh) as adsorbent. 1 H and 13 C NMR spectra were measured on a Bruker ARX 400 NMR spectrometer and reported as parts per million (ppm) from the internal standard TMS. High-resolution mass spectra (HR-MS) were obtained on a Finnigan MAT TSQ 7000 Mass Spectrometer System operated in a MALDI-TOF mode. Thermogravimetric analysis (TGA) was performed on a TA TGA Q5000 under nitrogen at a heating rate of 10 °C min −1 . Differential scanning calorimetry (DSC) analysis was performed on a TA Instruments DSC Q1000 at a heating rate of 5 °C min −1 . The sample size was about 2 mg and encapsulated in hermetically sealed aluminum pans, and the pan weights were kept constant. The temperature and heat flow were calibrated using standard materials such as indium and benzoic acid. Polarized optical microscopy (POM) was carried out to observe the liquid crystalline textures of the samples on a Leitz Laborlux 12 microscope with a Leitz 350 hot stage. The morphological structures of the aggregates were investigated by a HITACHI-SU8010 scanning electron microscope (SEM) at accelerating voltages of 200 and 5 kV. Stock solutions of DPCE-ECh and DPCE-ACh in THF (10 -3 mol L -1 ) were prepared. A certain volume (30 μL) of such stock solutions was transferred to small glass vials (5 mL). After addition of appropriate amounts of THF, This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins Please do not adjust margins distilled water was added dropwise under vigorous stirring to afford 5 × 10 -5 mol L -1 of DPCE-ECh and DPCE-ACh solutions. The mixtures were dropped on silicon wafers, the solvents were removed under reduced pressure at room temperature, and the SEM images of the aggregates on silicon wafers were taken. To identify the liquid crystalline structure of DPCE-ECh and DPCE-ACh, 1D XRD experiments were performed on a Philips X'Pert Pro diffractometer equipped with a 3 kW ceramic tube as the X-ray source (Cu Kα), an X'celerator detector, and a temperature control unit of Paar Physica TCU 100. The sample stage was set horizontally. The diffraction peak positions of the 1D XRD were calibrated with silicon powder for wide-angle region and silver behenate for smallangle region, respectively. The data was collected by a Mar165 detector and calibrated by CeO2 powder. The sample temperature was controlled by a Linkman THMSE600 hot stage. The heating and cooling rates in the experiments were 5 °C /min. The data were collected using an exposure time of 120 s. The 2D SAXS and WAXS data of DPCE-ECh were collected on Xeuss 2.0 (Xenocs, France), and the measurement details are listed in Table S2. Absorption spectra were measured on a Milton Roy Spectronic 3000 Array spectrophotometer. Steady-state photoluminescence (PL) spectra were measured on a Perkin-Elmer spectrofluorometer LS 55. The lifetime and the absolute luminescence quantum yield were measured on a Edinburgh FLSP 920 fluorescence spectrophotometer equipped with an integrating sphere (0.1 nm step size, 0.3 second integration time, 5 repeats). Circular dichroism (CD) spectra were recorded with a Chirascan spectrometer (Applied Photophysics, England). Circularly polarized photoluminescence (CPPL) spectra of the films and solution were recorded at 50 nm min -1 scan speed with a commercialized instrument JASCO CPL-300 at room temperature with the resolution of 15 nm. The film samples for CD and CPL measurement were prepared by drop-casting on the quartz substrate from the CHCl3 solution (5 mg/mL) of DPCE-ECh and DPCE-ACh, subsequently by volatilization of CHCl3 solvent at room temperature. Samples were subsequently thermally annealed for 45 min at the indicated temperatures. Preparation took place under inert atmosphere in a nitrogen filled glove box. To freeze temporarily the phase of the DPCE-ECh and DPCE-ACh, the film sample was quenched from the indicated temperatures to liquid nitrogen. The CD and CPL response of the quenched sample was recorded over the same time interval (per 3 min) at room temperature. The magnitude of circular polarisation in the excited state is defined as glum = 2 (IL -IR)/(IL + IR), where IL and IR indicate the output signals for left and right circularly polarized luminescence, respectively. Experimentally, the value of glum is defined as ΔI/I = [ellipticity/(32980/ln10)] / (unpolarized PL intensity) at the CPL extremum . Electron density reconstruction was caculated according to the method of previous published paper . The diffraction peaks were indexed on the basis of their peak positions, and the lattice parameters and the space groups were subsequently determined. Once the diffraction intensities are measured and the corresponding space group determined, three dimensional (3D) electron density maps can be reconstructed, on the basis of the general formula E(xyz) = Σhkl F(hkl) exp[i2π(hx+ky+lz)] (Eqn. 1) Here F(hkl) is the structure factor of a diffraction peak with index (hkl). It is normally a complex number and the experimentally observed diffraction intensity I(hkl) = KF(hkl)F*(hkl) = K|F(hkl)| 2 (Eqn. 2) Here K is a constant related to the sample volume, incident beam intensity, etc. In this paper we are only interested in the relative electron densities, hence this constant is simply taken to be 1 (Eqn. 4) As the observed diffraction intensity I(hkl) is only related to the amplitude of the structure factor |F(hkl)|, the information about the phase of F(hkl), ϕhkl, cannot be determined directly from experiment. However, the problem is much simplified when the structure of the ordered phase is centrosymmetric, and hence the structure factor F(hkl) is always real and ϕhkl is either 0 or π. This makes it possible for a trial-and-error approach, where candidate electron density maps are reconstructed for all possible phase combinations, and the "correct" phase combination is then selected on the merit of the maps, helped by prior physical and chemical knowledge of the system. This is especially useful for the study of nanostructures, where normally only a limited number of diffraction peaks are observed. ## Synthesis of 3-(3,4-bis(dodecyloxy)phenyl)-2-(4hydroxyphenyl)acrylonitrile (2). A mixture of 4hydroxyphenylacetonitrile (1.62 g, 12.0 mmol), compound 1 (5.7 g, 12.0 mmol) and NaOH (0.96 g, 24.0 mmol) in 60 mL of EtOH and 30 mL THF mixture solution was refluxed for 24 h. After the cooling to room temperature, 24 mL of HCl solution (1 M) was poured into the reaction mixture, then the solvent of C2H5OH was removed by a rotary evaporator. And the water (60 mL) was added. The mixture was extracted with ethyl acetate (3 × 50 mL). The combined organic layers were dried with anhydrous Na2SO4 and evaporated under reduced pressure to obtain the crude product. The residue was purified by silica-gel column chromatography using hexane/ethyl acetate (15:1) as an eluent. Compound 2 was obtained as a light brown powder with 35% yield. (2.48 g, 4.2 mmol). 1 (2.45 g, 17.75 mmol) was stirred and refluxed in 60 mL MeCN and 30 mL THF for 12 h at 90 °C. After cooling the room temperature, the solvent was removed by a rotary evaporator. And then the water (60 mL) was added. The mixture was extracted with CH2Cl2 (3 × 40 mL). The combined organic layers were dried with anhydrous Na2SO4 and evaporated under reduced pressure to obtain the crude product. The residue was purified by silica-gel column chromatography (hexane/ethyl acetate =20:1) to yield 2.01 g (84%) of the product as a yellow powder after removal of the solvent. 1 H NMR (400 MHz, CDCl3) δ (ppm): 7.63 (s, 1H), 7.59 (d, J = 9.2 Hz, 2H), 7.34-7.32 (m, 2H), 6.97 (d, J = 8.8 Hz, 2H), 6.91 (d, J = 8.4 Hz, 1H), 4.67 (s, 2H), 4.30 (q, J = 6.8 Hz, 2H), 4.10-4.05 (m, 4H), 1.89-1.82 (m, 4H), 1.59-1.45 (m, 4H), 1.39-1.27 (m, 35H), 0.89 (t, J = 6.0 Hz, 6H); 13 C NMR (100 MHz, CDCl3) δ (ppm): 168.0, 157. 6, 150.5, 148.4, 140.3, 127.9, 126.5, 126.1, 123.3, 118.1, 114.5, 112.4, 112.2, 107.1, 68.6, 68.4, 64.8, 60.9, 31.3, 29.1, 29.06, 29.01, 28.82, 28.79, 28.77, 28.55, 28.49, 25.44, 25.38, 22.1, 13.56, 13.52. MALDI-TOF-MS (C43H65NO5) Calcd.for m/z = 675.9950, found: m/z = 675.4874 (M + ). ## Table of Contents Tunable CPL response is achieved through regulating their aggregated structures in solution and solid states. Driven by the intermolecular hydrogen bonding in DPCE-ACh, opposite CPL responses with glum in 10 -3 order are obtained from M-helical nanofibrous structure and complex liquid crystalline (H + S) orientations. Meanwhile, DPCE-ECh exhibits a liquid crystalline assembled system (smectic C*) with a high dissymmetry factor (gCD = -0.20 and glum = +0.38). The intense and apparent CD and CPL of the film stems from the intrinsic helical structure of the molecular assembles with weak contribution of Bragg reflection, where the lone helical molecular stacking axis is perpendicular to the optical axis and is parallel to the direction of glass substrate.

chemsum

{"title": "Tunable Circularly Polarized Luminescence from Molecular Assemblies of Chiral AIEgens", "journal": "ChemRxiv"}

noncollinear_relativistic_dft+u_calculations_of_actinide_dioxide_surfaces

## Abstract: A noncollinear relativistic PBEsol+U study of the low-index actinide dioxides (AnO 2 , An = U, Np, Pu) surfaces has been conducted. The surface properties of the AnO 2 have been investigated and the importance of the reorientation of magnetic vectors relative to the plane of the surface is highlighted. In collinear nonrelativistic surface models, the orientation of the magnetic moments is often ignored; however, the use of noncollinear relativistic methods is key to the design of reliable computational models. The ionic relaxation of each surface is shown to be confined to the first three monolayers and we have explored the configurations of the terminal oxygen ions on the reconstructed (001) surface. The reconstructed (001) surfaces are ordered as (001)αβ < (001)α < (001)β in terms of energetics. Electrostatic potential isosurface and scanning tunneling microscopy images have also been calculated. By considering the energetics of the low-index AnO 2 surfaces, an octahedral Wulff crystal morphology has been calculated. ## Introduction The surface chemistry of the actinide dioxides (AnO 2 , An = U, Np, Pu) is key to understanding corrosion mechanisms, 1-8 which impacts the design of long-term storage facilities and the industrial reprocessing of nuclear fuels. An oxide layer is inexorably formed on actinide metal surface, which affects the chemistry of the underlying actinide metal. 1-5, 7, 16 The rapid onset of corrosion has resulted in thermal excursions, failure of containment vessels, and the resulting dispersal of nuclear materials. To reduce the risk of nuclear proliferation and assist in nuclear decommissioning, the controlled oxidation of actinide metals offers a means of converting classified nuclear material to simple ingots. 7 In terms of fuel fabrication, the surface energetics of the AnO 2 impact on fuel sintering and particle morphology. 17 As a result of their inhomogeneous and radioactive nature, few AnO 2 experimental surface studies have been completed. 9,13, . To circumvent experimental issues, computational methods offer an attractive alternative and complementary mode of study. However, a computational investigation of heavy-fermion systems is extremely challenging. To investigate the complex electronic structure by computational methods, we must consider exchange-correlation influences, relativistic contributions, and noncollinear magnetic behaviour. Only a limited number of studies have considered relativistic contributions (spinorbit interaction, SOI), which is, however, important in the treatment of actinide systems. In addition, the actinides often have complex (noncollinear) magnetic structures, and thus far no investigation of AnO 2 surfaces has incorporated noncollinear magnetic behavior into the models. The actinides are highly-correlated f-electron systems for which conventional DFT methods calculate an incorrect electronic structure. To model highly-correlated materials correctly, a number of methods have been developed: the self-interaction correction (SIC) method, 28 modified density functional theory (DFT+U), dynamic mean field theory (DMFT), 34 and hybrid density functionals. . As a computationally tractable method, DFT+U offers a means of study in which the electronic structure can be computed. In the Liechtenstein DFT+U formulism, where independent Coulomb (U) and exchange (J) terms treat the on-site Coulomb repulsion of the An f-electrons. The values are derived from higher level ab-initio methods or obtained through semi-empirical analysis. 25 In this study, the low-index AnO 2 (An = U, Np, Pu) surfaces have been investigated by computational methods. The electronic structures of the AnO 2 are heavily influenced by changes in magnetic order and the importance of magnetic vector reorientation is underlined. The effect of transverse 3k AFM behavior on the properties of the UO 2 surface is unknown, whereas investigations on NpO 2 and PuO 2 surfaces are even less common. 9,11 Surface energetics, the degree of ionic relaxation, electrostatic isosurfaces, scanning tunneling microscopy (STM) images and crystal morphologies have been calculated and the impact of oxygen ion reconstruction on the inherently unstable (001) surface is also considered. ©British Crown Owned Copyright 2018/AWE This manuscript has been authored by UT-Battelle, LLC, under contract DE-AC05-00OR22725 with the US Department of Energy (DOE). The US government retains and the publisher, by accepting the article for publication, acknowledges that the US government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this manuscript, or allow others to do so, for US government purposes. DOE will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan (http://energy.gov/downloads/doe-public-access-plan). ## Magnetic Structure The magnetic structure of the AnO 2 is highly complicated. A discontinuous first-order magnetic phase transition (T N = 30.8 K) 38 in UO 2 has been established by heat capacity, magnetic susceptibility 41 and neutron diffraction measurements. A transverse 3k antiferromagnetic (AFM) ground-state has been identified (Figure 1). 25, The ground-state corresponds to an internal Pa 3 (No. 205) crystallographic distortion synonymous with magnetic order (the displacement of the O 2ions is 0.014 ). Figure 1: The longitudinal 3k AFM and transverse 3k AFM phases for the AnO 2 crystal structure. The magnetic structure of NpO 2 remains unresolved. In the absence of interactions that break time-reversal symmetry conditions, the Np 4+ ion (a Kramers ion, one with an uneven number of valence electrons) should order magnetically at low-temperature. 49 A first-order paramagnetic (PM)-AFM phase transition (T = 25.4 K) has been inferred by: magnetic susceptibility 50 and specific heat capacity measurements. In-spite of an exhaustive search, a measurable local magnetic moment has not been identified by: low-temperature Mossbauer (T = 1.5 K), 49 neutron diffraction (12 K < T < 30K), 53 and muon spin rotation (0.3 K < T < 25.4 K) measurements. In terms of the crystal structure, no evidence has been found of an external distortion, which would indicate noncollinear 3k AFM order. 52 An internal O 2ion distortion (indicative of transverse 3k AFM behaviour with Pa 3 (No. 205) crystal symmetry) can be inferred from: the small broadening of Mossbauer spectroscopic lines, 49 and inelastic neutron scattering (INS) (5 K < T < 25 K) measurements. An internal O 2ion distortion of 0.02 has been calculated, which is, however, below the experimental resolution. 52,55,58 In contrast, a longitudinal 3k AFM ground-state has been indicated by: resonant X-ray scattering 59 (10 K < T < 17 K) and 17 O NMR measurements (T = 17 K). 60 The transverse 3k AFM state, relative to the longitudinal 3k AFM state, is 0.002 eV•formula unit -1 lower in energy (as calculated by HSE06 incorporating SOI). 26 An experimental singlet Γ1 diamagnetic (DM) PuO 2 ground-state has been inferred from: magnetic susceptibility (T = 4 K), inelastic neutron scattering (T > 30 K), and nuclear magnetic resonance (T > 4 K) measurements. However, a number of inconsistencies have been identified, and an ordered magnetic ground-state can be assumed. In contrast to experimental measurements, a longitudinal 3k AFM ground-state has been calculated. 18, 22-24, 26, 37, 61-71 It is thought that PuO 2 could be a small-moment insulator (similar to NpO 2 ) for which DFT overestimates the magnetic moments. 26 In this study, transverse 3k AFM order (UO 2 , NpO 2 ) and longitudinal 3k AFM order (PuO 2 ) have been used to describe the crystals. To model noncollinear magnetic behavior, it is imperative that relativistic effects are considered. A significant number of studies ignore the SOI (important in heavy-fermion systems) to reduce the computational cost. 11, A limited number of studies on UO 2 9 and PuO 2 23 consider relativistic contributions to the total energy. The importance of SOI on modeling UO 2 by DFT initially seemed to be inconsequential. 9 In a nonrelativistic treatment of other actinide systems, the study has often been cited to justify the absence of SOI. 9,23,72 The importance of SOI on the PuO 2 (111) surface energies has now been highlighted by hybrid DFT, 23 but all studies have limited themselves to a discussion of collinear 1k AFM order. A major limitation of scalar calculations is the inability to orient the magnetic moments relative to the direction of the surface. In this manner, the magnetic moments are directed orthogonal to the surface plane, which leads to notable inconsistencies within the electronic structure. If not corrected, the orientation of the magnetic field is also directed orthogonal to the surface plane, because the principal axis differs between the surfaces. Consequently, the electronic, magnetic, and crystal structures differ between the bulk crystal structure and individual surfaces. If the magnetic vectors are not reoriented, the energetics and structural relaxations derived by this approach are incomplete. This is particularly concerning when calculating the surface energy, which is derived from the bulk structure and it is therefore important that the magnetic vectors relative to the surface are carefully reoriented. In past studies where this essential transformation has been omitted, the energies of the bulk and surface are therefore often incomparable, which introduces a significant error when calculating the energy of the surface. In this study, the magnetic vectors are reoriented relative to the surface plane, which ensures that we preserve the noncolinear 3k AFM structure. In addition, the reduction of cubic symmetry associated with collinear 1k AFM states (used in past calculations) is avoided. Figure 2: The surface magnetism of a two-dimensional material. The direction of the magnetic moments for the respective surfaces are shown for the first two layers of the bulk crystal structure. The highlighted (01) (green) and ( 11) (blue) surfaces correctly emulate the magnetic structure in the bulk crystal. In contrast, the (11) (red) surface illustrates an incorrect depiction where the magnetic moments are aligned orthogonal to the surface. The magnetic structure is commonly defined by the principal axis. The principal axis of the AnO 2 (111), (011) surface differs from that of the bulk crystal and the final magnetic, electronic and crystal structures are therefore inequivalent. However, this is not the case for the AnO 2 (001) surface which shares the same axes. To illustrate in a two-dimensional material, we consider the first two layers of a collinear 1k AFM material (Figure 2). The (01) surface and the crystal share the same principal axis and the magnetic structures are therefore directly related. In the (11) surface, the principal axis differs from that of the crystal, which results in an unrelated magnetic and electronic structure. It is therefore critical to orient the magnetic vectors to emulate the initial crystal structure. ## Computational Methodology 2.1 Calculation Details All calculations have employed the Vienna Ab-initio Simulation Package (VASP) 28,34,75 using a plane wave basis set, relativistic effective core potentials (ECPs), and the frozen-core projector-augmented wave (PAW) method. The cut-off energy of the plane wave basis set is 500 eV. The uranium (6s 2 , 7s 2 , 6p 6 , 6d 2 5f 2 ), neptunium (6s 2 , 7s 2 , 6p 6 , 6d 2 5f 3 ), plutonium (6s 2 , 7s 2 , 6p 6 , 6d 2 5f 4 ) and oxygen (2s 2 , 2p 4 ) valence electrons are implicitly considered. The integration over the Brillouin zone was performed using the Blöchl tetrahedron method. 76 The influence of the SOI 72 and noncollinear magnetic wave-vectors are considered. The on-site Coulomb repulsion of the An 5f electrons is treated by the Liechtenstein et al. DFT+U formulism. 32 In the Liechtenstein et al. formulism, the Coulomb (U) and exchange (J) modifiers are treated as independent variables. 32 The Coulomb modifier for each ion is written in the parentheses: uranium (U = 3.35 eV), neptunium (U = 4.25 eV) and plutonium (U = 6.00 eV). In the past, the influence of J on noncollinear magnetic materials has been investigated. 77 The introduction of J increased the anisotropic nature of the fstates, 78 and it is therefore not considered in this study. The selected conditions offer an accurate representation of the electronic structure. The integration of the Brillouin zone is performed with a Γ-centered k-point grid. 79 The exchange-correlation energy is evaluated by the revised Perdew-Burke-Ernzerhof for solids (PBEsol) functional. 80 The iteration threshold for electronic and ionic convergence is set at 1x10 -5 eV and 1x10 -2 eV -1 , respectively. As the crystal and electronic structures of AnO 2 are highly dependent on the magnetic state, it is imperative to correctly reorientate the magnetic vectors with respect to the surface plane. Ionic relaxation is a common mechanism by which the surface energy is minimized with respect to the unrelaxed surface. The surface energy (γ) is a measure of the surface stability and is defined by: The number of formula units (N), the total energy of the surface slab (E tot (N)) and the total energy per formula unit ( E AnO 2 ) are defined in the parentheses. In our calculations, all ions are relaxed while the dimensions of the unit cell are fixed. The conjugate gradient method is employed in the relaxation of the ions. Images are visualized by the Crystal Maker 81 and VESTA codes. 82 The density of states have been illustrated by the SUMO code, a commandline plotting tool for ab-initio calculations. ## Low-Index Surface Models The low-index AnO 2 (111), ( 011), (001) surfaces are generated by the METADISE code (Figure 3) 83 from the ionically relaxed bulk material. The nonpolar (111) surfaces are comprised of repeat O-An-O unit layers. In the (111) surface, the individual monolayers are charged, but the surface is characterized by the absence of a dipole moment perpendicular to the surface plane. In contrast, the (011) surface is comprised of nonpolar and charge neutral planes. The polar (001) surface (formed of dipolar An-O layers) is inherently unstable 17, as the electrostatic energy diverges (caused by the formation of an electric dipole) with increasing number of monolayers. In nature, the surface undergoes a reconstruction to prevent the formation of an electrostatic dipole. The reconstruction is influenced by environmental conditions. 85,88 In this study, the dipolar perpendicular to the surface is removed by transposing half of the charged oxygen anions from one surface to the other (Figure 4), which involves the formation of an oxygen-terminated surface with half-filled oxygen vacancies. The result is a non-polar reconstructed (001)r surface, which in a (1•1) unit cell can be either the (001)α or (001)β reconstruction. Although numerous configurations are possible in a (1•2) unit cell, the (001)αβ reconstruction offers a hybridization between the two (1•1) reconstructions and in this study we have calculated the relative stabilities of these three surface configurations. The surface energy is converged with respect to the k-point grid to under 0.05 J m -2 (Figure 5). The (111) surface is calculated from a 5•5•1 Γ-centered k-point grid recommended for hexagonal structures, whereas the (011) and (001) surfaces are calculated from a 4•4•1 Γcentered k-point grid. 84 To minimize potential aliasing errors, the initial bulk structure (from which the surfaces are derived) is calculated with both a 4•4•4 and a 5•5•5 Γ-centered k-point grid for direct comparison with the surface in the surface energy calculations. Finally, the (001)αβ surface is calculated form a 4•2•1 Γ-centered k-point. ## The HIVE Code In the scanning tunnelling microscopy (STM) HIVE code, 89-90 the Tersoff-Hamann model is considered, where the tunnelling-current is equivalent to the local density of states. 91 A point source at a constant height of 2.5 and a Fermi energy sample bias of -2.50 eV is used. Topographies calculated by HIVE include: copper, 92 germanium, 89-90 gold, 93 iron oxide, 94 thorium dioxide. 95 ## Wulff Reconstruction According to the Gibbs thermodynamic principle, the equilibrium crystal morphology is influenced by the total surface energy of the medium interface. An equilibrium crystal morphology that minimises ∆ G i has been calculated as follows (Equation 3): The terms in the parentheses describe the total crystal-medium interface free energy ( ∆ G i ), the surface Gibbs free energy ( γ j ) and the surface area ( A j ). ## Model Constraints 3.1.1 Surface Energetics As a function of the number of formula units used, the energy of the low-index AnO 2 surfaces has been calculated (Additional Information, Figure A1). The ions are fully relaxed while keeping the relative dimensions of the unit cell fixed. In this study, the surface energy is converged to within 0.01 J•m -2 when 12 or more formula units are used. The surface energy increases across the series as (111) < (011) < (001)α < (001)β (typical of fluorite-structured materials) (Table 1). 86,95 The energy difference between the (001)α and (001)β terminations are relatively small in UO 2 (0.08 J m -2 ) and NpO 2 (0.06 J m -2 ), compared to PuO 2 (0.19 J m -2 ) . If one uses a (1•1) unit cell model, the (001)α surface relative to the (001)β surface is energetically favourable, which is confirmed independently by an interatomic potential-based investigation on UO 2 . 88 Compared with past DFT-based methods, the calculated surface energies are considerably greater for each surface. 9, 11, 17-18, 23, 74, 96 although interatomic potential models 88 and relativistic hybrid calculations 15 of UO 2 have resulted in even higher surface energies. In addition, interatomic potential models of UO 2 have calculated lower-energy (001) surface reconstructions, which are formed using a larger unit cell. 88,97 In the reconstruction of the (001) surface in our (1•1) unit cell, only the (001)α and (001)β configurations can be generated, whereas the surface energy of the (001)αβ configuration from a (1•2) unit cell (calculated using 28 formula units) relative to the (001)α and (001)β configurations, is considerably lower in energy (Table 1). This implies a limitation of the DFT (1•1) unit cell model and it is clearly possible that other configurations, in even larger cells, could be more stable. However, increasing the size of the cell increases the computational cost of the system significantly, and a systematic fully relativistic DFT study of bigger simulations cells is currently computationally intractable. ## Ionic Relaxation The low-index AnO 2 surfaces are characterized by the changes in the interlayer spacings (Figure 6-7), which enables a quantitative analysis of the structural relaxation between layers. The interlayer relaxation (Δd interlayer ) is calculated by: where (d i,i+1 ) relaxed is the average interlayer separation of ions in the relaxed surface and d unrelaxed is the average interlayer separation of ions in the unrelaxed surface. The interlayer relaxation is reminiscent of studies on the isostructural CeO 2 material with similar results found for the (111) and (011) surfaces. 98 In the context of An-An relaxation, the (111) surface is marginally distorted. The major difference is confined to the oxygen separation in the second interlayer space. The (011) surface undergoes the greatest overall interlayer relaxation, with the first surface layer experiencing a marked contraction, where the first An layer contracts significantly more than the first O layer. The contraction of the first layer is countered by a slight expansion of An ions in the second layer, but the bulk structure is regained by the fifth layer. The terminal O ions in the (001)α and (001)β surface undergo a significant contraction, although the remainder of the structure is relatively unaffected. In general, the interlayer relaxation is confined to the first 5 , indicating that for investigations of surface reactivity, a slab of minimally 10 thick should be used. Our results are similar to those found in studies of CeO 2 and ThO 2 . 95 In the context of interlayer O-O relaxation, the distortion of the surface is primarily confined to the first three to four monolayers and the degree of ionic relaxation is generally identical in the AnO 2 surfaces, with the exception of the PuO 2 (001)β surface. In the PuO 2 (001)β surface, the relaxation of the oxygen ions is significantly less relative to the UO 2 and NpO 2 (001)β surfaces. Thus, of the (001)r surfaces, the UO 2 and NpO 2 (001)β surfaces undergo the greatest surface relaxation, whereas in PuO 2 , the (001)α surface undergoes the greatest surface relaxation, which is a result of magnetic order and the relaxation in the xy-plane. No significant structural distortion in the xy-plane occurs in the AnO 2 (111), ( 011) or (001)α surfaces, possibly as a result of preserving the Pa 3 (No. 205) or Fm 3 m (No. 225) cubic symmetry from the use of noncollinear 3k AFM order. In contrast, the oxygen ions in the UO 2 and NpO 2 (001)β configuration are shifted from their initial positions by the use of transverse 3k AFM ordering (Figure 8). This distortion is not observed in the corresponding PuO 2 surface in which the ions are relatively fixed, although there is a minor distortion of the surface plutonium ions, potentially as a consequence of using either transverse 3k AFM or longitudinal 3k AFM behavior. By comparison, the oxygen ions in the (001)αβ configuration are relatively static and, instead, the actinide ion is partially shifted toward the terminal oxygen ions. ## Surface Properties 3.2.1 Electronic Structure The electronic structure of the AnO 2 surfaces has been calculated (Figure 9). The covalent nature of the AnO 2 materials (a consequence of An (f) and O (p) mixing) is seen to increase along the series. The Mott-Hubbard insulating nature of UO 2 is characterized by transitions primarily occurring across the An f-bands. Compared to relativistic hybrid DFT calculations of UO 2 , the calculated band gaps for the low-index surfaces are considerably greater. 15 The charge-transfer insulating nature of PuO 2 is characterized by transitions primarily between the valence Pu f-band and conduction O p-band. In NpO 2 , both Mott insulating and chargetransfer characteristics are shown in the surface. In general, the electronic structure is only partially perturbed between surfaces. In addition, the electron affinity and ionization potential of the AnO 2 surfaces has been calculated (Table 2). This information fills a significant gap in the literature where X-ray photoelectron spectroscopy (XPS) and Kelvin probe microscopy studies have yet to be performed. The electron affinity and the ionization potential increases along the (011) < (111) < (001)β < (001)α series. Of the AnO 2 (An = U, Np, Pu) materials, UO 2 is the least reactive, whereas PuO 2 is the most reactive. ## Magnetic Deviation The magnetic structure of the low-index AnO 2 surfaces has been investigated. A complete analysis of the actinide ions can be found in the Additional Information. The localized magnetic normalized root-mean-square deviation (nRMSD) of the first three monolayers has been calculated for each surface (Figure 10). As the monolayer surface depth increases, the magnetic distortion decreases. The total magnetic moment of the U (1.37 µ B •ion -1 ), Np (2.70 µ B •ion -1 ), and Pu (3.80 µ B •ion -1 ) ions remains constant. 011), (001)α surfaces for the first three monolayers. The initial magnetic vector (silver), relaxed magnetic vector (green), actinide (blue) and oxygen (red) are shown. The localized magnetic deviation in NpO 2 for identical surfaces is relatively high. A number of competing low-temperature (T < 25.4 K) magnetic states could cause the distortion. 26 For instance, the transverse 3k AFM state, relative to the FM (111) ground-state, is 0.002 eV per formula unit higher in energy; however, no experimental evidence of a FM (111) ground-sate, which results in a R 3 m (No. 166) crystallographic distortion, exists. 26 In addition, the localized magnetic deviation of the (001)α series can be ascribed to the surface instability. In the first three monolayers of the (001)α surface, a FM and an AFM domain are formed. The lowest RMSD is found for the PuO 2 (011) surface. ## Scanning Tunneling Microscopy The surface energies of UO 2 are extremely sensitive to stoichiometry, defect chemistry, and environmental conditions. Low-energy electron diffraction (LEED) measurements of the UO 2 (111) surface have identified over 16 individual patterns. 102 . To assist experimental analysis, low-index AnO 2 STM images have been calculated (Figure 11) and the resulting images are analogues of experimental STM studies of AnO 2 surfaces. 85, However, in an STM experiment, ionic positions are influenced by perturbations of the electric field caused by the probe and the calculated resolution is therefore considerably greater compared to that of an experimental study. The terminal O 2ions are observed in white, whereas the An 4+ ions area considerably darker. The individual AnO 2 (An = U, Np, Pu) (111), ( 011) and (001)α surfaces patterns are indistinct. In the (111) surface, the O 2ions result in a hexagonal structure, whereas in the (011) surface, a series of darker channels is observed in one direction. In the (001)α surface, the alignment of the O 2ions results in a diamond pattern. As a means of differentiating between compounds, the (001)β surface is influenced by the magnetic state. In the transverse 3k AFM state for UO 2 and NpO 2 , the O 2channels oscillate continuously, whereas in the longitudinal 3k AFM state for PuO 2 , the O 2channels are perfectly linear. In other words, the structures can be differentiated by the transverse 3k AFM state of UO 2 and NpO 2 or by the longitudinal 3k AFM state of PuO 2 which is useful information for comparison with future experimental patterns to deduce the magnetic states. ## Electrostatic Potential Isosurface The electrostatic potential isosurface for the low-index AnO 2 surfaces has been calculated using the PBEsol+U functional (Figure 12 ## Crystal Morphology Low-voltage scanning electron microscopy (SEM) of UO 2 has shown a truncated octahedral Wulff crystal morphology, 105 which to our knowledge is the only experimental study concerning the morphology. The truncated octahedral Wulff crystal morphology of UO 2 is inconsistent with studies of other CaF 2 -type crystal structures and may be the result of environmental influences and the method of sample preparation. The crystals were formed under high pressure (400 MPa) and temperature (1700 °C). A truncated octahedral morphology for a fluorite-structured material has not as yet resulted from any computational approach. In this study, an octahedral Wulff crystal morphology has been calculated (Figure 13) from the surface energies of the low-index (111), (011) and (001)αβ surfaces only. As a result of their relative instabilities, the (001)α and (001)β surface are omitted. Indeed, other high-index surfaces are considerably greater in energy, and their influence on the Wulff crystal morphology is assumed to be negligible. In terms of computational theory, calculations have shown that the crystal structure is influenced by the magnetic state. In theory, the low-temperature octahedral Wulff crystal morphology is linked to the noncollinear 3k AFM state, whereas the high-temperature truncated octahedral Wulff crystal morphology is linked to the PM state. In contrast, the octahedral Wulff crystal morphology of the AnO 2 materials is consistent with fluorite-based materials. The octahedral morphology in the present study is consistent with that calculated by interatomic potentials 88 and with previously reported morphologies for PuO 2 22 and ThO 2 95 calculated by DFT. The (111) surface dominates the morphological features of the particle. Interatomic potential models of the UO 2 (001) surface have indicated surface configurations of lower energy in a (2x2) unit cell, however this energy is not sufficiently low enough to result in a truncated octahedron. 88 In the calculation of (001) surface energetics, the major limitation is the size of the unit cell and there is therefore a possibility that larger cells may result in a configuration of sufficiently low energy to result in a truncated octahedron. In this study, we have used a (1x1) unit cell with either the (001)α or (001)β configuration, although additional configurations are possible in larger supercells. In theory, one of these surfaces may possess sufficiently low energy to affect the morphology. However, a systematic investigation of the (2x2) surface is computationally unfeasible, because of the large number of compute-intensive configurations that must be explored. In another scenario, the experimental sensitivity of UO 2 resulted in a crystal morphology influenced by environmental conditions. It is known that the interaction of oxygen with the AnO 2 surfaces influences the composition range of the solid and the formation of superficial structures. 102 In the past, DFT+U studies have indicated that the truncated crystal morphology is the result of oxygen-rich conditions at 300 K. 106 In addition, interatomic potentials indicate that the AnO 2 (001) surface energy is reduced by hydroxylation, 12,17 which also results in a truncated octahedron. Other models which use interatomic potentials have obtained an octahedral morphology at thermodynamic equilibrium. However, these studies concluded that the truncated morphology is the result of kinetic limitations. 107 Finally, numerous experimental investigations have shown that the surface energies are temperaturedependent. 99,108 4 Conclusions PBEsol+U has been used to investigate AnO 2 surfaces. In the past, collinear 1k AFM states have been used to model surface structures, but these models predominately use scalar approximations of the crystal electric field which causes an inability to reorient the magnetic vectors relative to the plane of the surface. Therefore, the magnetic structures differ across surface indices. This study considers non-collinear 3k AFM behavior and SOI contributions to the surface energetics of the low-index AnO 2 (111), ( 011) and (011) surfaces. The magnetic field is carefully re-oriented relative to the plane of the surface for a complete description of the magnetic surface structure. Localized magnetic distortions have been identified. The interlayer relaxation of the (111), ( 011) and (001)α surfaces is confined to the first 5 . In contrast to past DFT investigations, our surface energies are considerably higher, 11,74 which illustrates the important contribution of the SOI 72 to the calculated surface energetics. Our surface energies suggest that the chemical reactivity of the surface has previously been underestimated. The surface stability increases across the (001)β < (001)α < (011) < (111) series, which is typical of CaF 2 -type structures. From our Wulff reconstruction, the octahedral crystal morphology is completely dominated by (111) facets. As stated, this is consistent with previous calculations of fluorite-type structures. Thus, we have developed a computationally tractable method to model the low-index AnO 2 surfaces with improved energetics, which may serve as the basis for future studies 5 Acknowledgements

chemsum

{"title": "Noncollinear Relativistic DFT+U Calculations of Actinide Dioxide Surfaces", "journal": "ChemRxiv"}

directed,_nickel-catalyzed_umpolung_1,2-carboamination_of_alkenyl_carbonyl_compounds

## Abstract: We report a regioselective, nickel-catalyzed syn-1,2carboamination of non-conjugated alkenyl carbonyl compounds with O-benzoyl hydroxylamine (N-O) electrophiles and aryl/alkylzinc nucleophiles to afford β-and γ-amino acid derivatives. This method enables preparation of products containing structurally diverse tertiary amine motifs, including heterocycles, and can also be used to form quaternary carbon centers. The reaction takes advantage of a tethered 8-aminoquinoline directing group to control the regiochemical outcome and suppress two-component coupling between the N-O electrophile and organozinc nucleophile. Nitrogen-containing small-molecules comprise a significant portion of all known medicines. Thus, novel methods for the formation of carbon-nitrogen (C-N) bonds have been actively pursued. 1,2-Carboamination represents an appealing strategy for converting readily available alkene starting materials into valuable structurally complex amine products in an expedient manner (Scheme 1). This transformation can be carried out using different modes of reactivity, including a classical-polarity approach in which the nitrogen-based reactant functions as a nucleophile (i.e., R2NH) and an umpolung approach where the nitrogen-based reactant is an electrophile (i.e., R2NX, X = halide or pseudohalide), as depicted in Scheme 1a. Catalytic intramolecular (two-component) alkene carboamination involving both polarity types has been extensively studied. Intermolecular (three-component) variants, on the other hand, remain comparatively unexplored and have typically been limited to conjugated alkenes (e.g., styrenes or acrylates). In terms of precedents involving non-conjugated, unstrained alkenes, Liu and coworkers have reported palladium-catalyzed carbonylative 1,2-carboamination using 2oxazolidone or phthalimide nucleophiles to afford terminal βamino acids. [5a] Later, this group reported a similar net transformation involving an azide-containing hypervalent iodine reagent. [5b] These two reports rely on rapid migratory insertion of CO to outcompete side reactions, such as β-H elimination. Our group has reported a palladium-catalyzed directed 1,2carboamination of unactivated alkenes via a classical polarity approach (Scheme 1d). In particular, we demonstrated regioselective anti-addition of imides, amides, sulfonamides, and various azaheterocycles with aryl iodides across alkenes. These contributions notwithstanding, 1,2-carboamination of nonconjugated alkenes employing aliphatic amines and alkyl carbon coupling partners remain unexplored. The goal of the present study was to address this knowledge gap through the development of a three-component umpolung carboamination of a non-conjugated alkene using a substrate directivity strategy. Electrophilic aminating reagents have a rich history in enabling C-N bond formation. During the past few years, examples of umpolung carboamination of alkenes and allenes have been described. [4a, 4c] For example, building on seminal reports by Narasaka, Bower and coworkers described an intramolecular umpolung carboamination of 𝛾,δ-unsaturated oxime esters with arylboronic ester coupling partners. The Zhu group later described analogous reactivity with 1,3,4-oxadiazole C-H nucleophiles (Scheme 1b). Regarding intermolecular examples, in 2013 the Zhang group described an umpolung radical-based copper-catalyzed aminocyanation of styrenes employing N-F reagents (Scheme 1c). [4a] Last year Liu and coworkers published an enantioselective aminoarylation of styrenes also catalyzed by copper using N-F reagents as electrophiles. [4c] In contrast, analogous transformations involving the use of electrophilic aminating reagents in nickel-catalysis have been less extensively studied. [7g, 11] Two-component nickel-catalyzed C-N cross-couplings between organometallic nucleophiles and N-O electrophiles have been described by the Johnson, [11a] Jarvo, [11b] and Knochel groups. [11c] To the best of our knowledge, only a single example of nickel-catalyzed alkene carboamination has been reported to date, [8g] an intramolecular system developed by Selander and coworkers in 2017 (Scheme 1b). [7g, 11d] Realization of an intermolecular nickel-catalyzed carboamination [N]-H = imides, amides, sulfonamides, hydroxamic acid derivatives, and azaheterocycles Table 1. Selected Optimization of Reaction Conditions. [a] [a] Reaction conditions: 1a (0.1 mmol), 2a (1.0-1.2 equiv), Me2Zn (1.0 M in heptane), 60 °C, 18-24 h. 1 H NMR yields reported with CH2Br2 as internal standard. process would present the opportunity to rapidly generate medicinally motifs with dense functionality. We recently described substrate-directed nickel-catalyzed three-component conjunctive cross-coupling reactions that append differentiated alkyl/aryl fragments to β,𝛾-and 𝛾,δunsaturated carbonyl compounds using aryl/alkyl halides and aryl/alkyl zinc reagents. The regioselectivity of these reactions is controlled by a tethered 8-aminoquinoline (8-AQ) directing group that stabilizes 5-or 6-membered metallacycles, thereby suppressing undesired side reactions, such as β-hydride elimination or two-component cross-coupling. Given these results we wondered if it would be possible to employ Obenzoylhydroxylamines as electrophiles in lieu of aryl/alkyl halides to synthesize β-and 𝛾-amino acid derivatives under nickel catalysis. We surmised that this approach would complement our previous palladium(II)-catalyzed method (Scheme 1d) in several respects. Namely it would be synselective, proceed with the opposite sense regioselectivity, enable use of alkyl coupling partners, and potentially compatible with alkenes distal from the AQ group (Scheme 1e). To test this idea, we elected to use alkene 1a as the pilot substrate given its unique effectiveness in earlier work and 2a as the electrophilic nitrogen source based on its success in various other catalytic methods. These starting materials were combined with commercially available dimethylzinc solution in the presence of catalytic nickel. With 20 mol % Ni(cod)2 we observed formation of product 3a (Table 1) in 71% yield (Table 1, Entry 1). DMF, toluene, acetonitrile, and dioxane were also tested under conditions otherwise identical to those in entry 1. The reaction proceeded in toluene and dioxane, though yields were attenuated compared to in THF. Considering conditions from our previous work [13b] we attempted to drive the reaction to completion by using excess Me2Zn, but in this case we found significantly diminished yields when more than one equivalent was used. We also found that the reaction was higher yielding at lower concentrations, with the optimal concentration being 0.075 M 1a in THF. Lower catalyst loadings of 10-15 mol % gave comparable yields, though decreasing the catalyst loading further (5 mol %) led to slightly diminished yield. Increasing the amount of 2a to 1.2 equiv provided 3a in 84% 1 H NMR yield (79% isolated). We also found that the reaction performs comparably well using several bench-stable Ni(II) salts, enabling a glove-box free protocol. The product 3a was isolated in 77% yield using NiCl2 as the precatalyst. Having optimized the reaction conditions, we proceeded to explore the O-benzoylhydroxylamine electrophile scope (Table 2). We found that several hetereocyclic motifs (3b-3g) frequently found in bioactive compounds were well-tolerated, including thiomorpholine, tert-butyoxycarbonyl-protected piperazine, 2-(piperazin-1-yl)pyrimidine, 4,5,6,7tetrahydrothieno[3,2-c]pyridine, piperidine, and pyrrolidine. An array of N-O reagents derived from acyclic amines (2h-2l), including N-methyl-N-benzylamine, diethylamine, dibenzylamine and diallylamine also reacted under optimized conditions. Sterically hindered and especially reactive N-O reagents could not be used as coupling partners in this reaction (see SI). The product 3l was obtained in a similar yield using NiCl2, highlighting its efficacy as a substitute for Ni(cod)2. The reaction was also compatible with a variety of diorganozinc and organozinc halide nucleophiles, though some reactions were found to proceed in diminished yields. In some cases this could be overcome by slow addition of the organozinc nucleophile, demonstrated in the synthesis of 3m, which was isolated in 87% yield. Several other primary alkylzinc nucleophiles were compatible, including propyl (3n), ethyl propionate (3o) and benzyl (3p), providing the corresponding products in moderate yields. Secondary alkylzinc nucleophiles such as cyclobutyl (3q) and cyclohexyl (3r) could also be employed and provided moderate yields. Tertiary carbon nucleophiles were unsuccessful under the optimized reaction conditions (see SI). We observed that monoalkylzinc halides were generally lower yielding that dialkylzinc reagents, likely due to their well-known attenuated nucleophilicity. We hypothesize that the need for excess alkylzinc halide (four times more than in the case of dialkylzinc reagents) is due to competitive reduction of the electrophile 2a before it is able to react in the desired pathway, leading to decreased yields. In the case of secondary nucleophiles, we believe competing β-hydride elimination pathways generates reducing species in solution that facilitate electrophile decomposition. We have also isolated aminoarylated product 3s in 27% yield. We also explored the scope of alkene substrates (Table 3) and found that the reaction was compatible with a variety of substituted alkenes. The relative stereochemistry of 4a was determined by X-ray crystallography, establishing that the reaction proceeded in a syn-selective manner. The stereochemistry of other products derived from internal 1,2disubstituted alkenes were assigned by analogy. 4a was also obtained using NiCl2 as the precatalyst and was obtained with similar yield. We also found that a phthalimide-protected amine could be tolerated under the reaction conditions to afford carboaminated product 4c in moderate yield. Given the success of setting quaternary carbon centers in our previously published dialkylation reaction, [13b] we wondered whether this carboamination reaction could also function in sterically congested environments. We were pleased to find tri-and 1,1disubstituted alkenes could be used to synthesize compounds 4d and 4e, respectively, in good yields, demonstrating the ability of this method to forge quaternary carbons centers at either the β and 𝛾 position. The reaction also proceeded in moderate yields with α-methyl substituted alkenyl carbonyl compounds (4f), though we found the benzyl-substituted analogue to proceed in significantly reduced yields (<10% isolated). We also found the reaction could be extended to 𝛾,δ-unsaturated substrates to afford products 4g-4i and 2-vinylbenzamide-derived product 4j. We next performed the reaction on gram scale to demonstrate its synthetic utility. On 5-mmol scale, we were able to isolate 1.30 g of 3a in 83% yield (Scheme 2). We also validated two methods to remove the AQ directing group. Hydrolysis of 3a afforded β-amino acid 3a' in 76% yield. Using a method published by Ohshima and coworkers, methanolysis of 3a afforded ester 3a'' in 79% yield. Furthermore, we found that a stereocenter at the carbon α to the carbonyl did not racemize under the reaction conditions (see SI for details). Regarding the reaction mechanism, we surmised that two plausible redox manifolds could be operative, namely Ni(0)/Ni(II) or Ni(I)/Ni(III) catalysis (depicted in general form as Ni(n)/N(n+2)). Moreover, the reaction could proceed via two different orders of events (Fig. 3a). In Pathway A, substratebound nickel complex 6c would first undergo oxidative addition with 2 to form intermediate 6a. Transmetalation followed by insertion to the alkene would form 6b, which could reductively eliminate to form products 3-4 and regenerate the active catalyst. In the second potential mechanism, Pathway B, intermediate 6c would first react via transmetalation, after which migratory insertion would lead to intermediate 6d. This species could then oxidatively add to 2 to give nickel intermediate 6e. Reductive elimination would form the key C(sp 3 )-N bond and regenerate the catalytically active low-valent nickel species. A third mechanistic scenario (see SI) in which C-N bond formation precedes transmetalation and C-C reductive elimination cannot be conclusively ruled out at this stage, though we consider it to be less likely because it would involve formation of larger nickelacycles in preference to smaller nickelacycles with both classes of substrates (6 versus 5 with products 3, and 7 versus 6 with products 4). In an effort to disambiguate between these possibilities, we prepared radical clock electrophile 2m (Fig. 3b). Based on literature precedents, the corresponding aminyl radical-which would be formed if SET oxidative addition were operative [11d] - was expected to cyclize with a first-order rate constant of approximately 10 4 s -1 . When this electrophile was subjected to standard reaction conditions, only non-cyclized product 3t was formed in 40% yield. No evidence of cyclization was observed by 1 H NMR of the crude reaction mixture. This result is consistent with a two-electron oxidation addition pathway or alternatively with an SET oxidation pathway involving a radical recombination step with a rate constant >10 4 s -1 . The effect of radical inhibitors was next studied (Fig. 3c). The reaction was not inhibited by the addition of BHT (1 equiv). On the other hand, addition of TEMPO (1 equiv) dramatically suppressed product formation, leading to unreacted starting materials, as well as TEMPO-H and TEMPO-Me adducts, as monitored by 1 H NMR and LC-MS. This result suggests that a Ni(I)/(III) cycle involving a Ni(I)-Me intermediate and SET events may be operative; however, a more detailed mechanistic study is needed before firm conclusions can be drawn. In summary, we have developed an intramolecular umpolung carboamination of non-conjugated alkenes that affords a variety of β-and 𝛾-amino acid and ester derivatives. The reaction is enabled by a removable 8-aminoquinoline tethered directing group, which facilitates formation of stabilized 5-or 6-membered nickelacycles, suppresses β-hydride elimination and two-component coupling, and determines the regiochemical outcome. The reaction tolerates a range of alkenes with various substitution patterns and proceeds in the presence of several synthetically important functional groups.

chemsum

{"title": "Directed, Nickel-Catalyzed Umpolung 1,2-Carboamination of Alkenyl Carbonyl Compounds", "journal": "ChemRxiv"}

the_competing_effects_of_microbially_derived_polymeric_and_low_molecular-weight_substances_on_the_di

## Abstract: To understand the competing effects of the components in extracellular substances (ES), polymeric substances (PS) and low-molecular-weight small substances (SS) <1 kDa derived from microorganisms, on the colloidal stability of cerium dioxide nanoparticles (CeNPs), we investigated their adsorption to sparingly soluble CeNPs at room temperature at pH 6.0. The ES was extracted from the fungus S. cerevisiae. The polypeptides and phosphates in all components preferentially adsorbed onto the CeNPs. The zeta potentials of ES + CeNPs, PS + CeNPs, and SS + CeNPs overlapped on the plot of PS itself, indicating the surface charge of the polymeric substances controls the zeta potentials. The sizes of the CeNP aggregates, 100-1300 nm, were constrained by the zeta potentials. The steric barrier derived from the polymers, even in SS, enhanced the CeNP dispersibility at pH 1.5-10. Consequently, the PS and SS had similar effects on modifying the CeNP surfaces. The adsorption of ES, which contains PS + SS, can suppress the aggregation of CeNPs over a wider pH range than that for PS only. The present study addresses the non-negligible effects of small-sized molecules derived from microbial activity on the migration of CeNP in aquatic environments, especially where bacterial consortia prevail.The cerium dioxide (CeO 2 , F m m3 ) nanoparticle (CeNP) is a nanomaterial that is finding a wide variety of applications to a vast number of products involving fuel additives 1 , fuel cell components 2 , biomedical applications 3,4 , combustion accelerators and abrasives 5,6 , and specialized polishing agents 7 . With all of these applications, it is inevitable that CeNPs will be found in the environment. Unfortunately, in vitro and in vivo experiments with CeNPs have shown that this material can cause chronic toxicity to aquatic organisms 8 , cell death to E. coli 9 , increase of reactive oxygen species levels relevant to human lung cells 10 , and decrease of glutathione levels in cultured human lung epithelial cells 11 . Due to their small size, ~10 nm, the inhaled CeNPs can penetrate into the deep respiratory system 12 and potentially cause adverse health effects despite the existing study reported that CeNPs have low human toxicity 13 . Thus, the distribution and migration behavior of CeNP, as well as other engineered nanoparticles in the ambient environment, is a central issue that requires careful monitoring and modeling 14 . The mobility of CeNP follows the general rules for colloid transport in surface and subsurface environments [15][16][17][18][19] . Colloid transport can be controlled by several processes: sedimentation, filtering effects, hydrodynamic chromatographic effects, and capillary effects. All of these processes are largely dependent on the aggregation processes of CeNPs in natural aquifers 17,20 .The aggregation of colloids is mainly constrained by several factors, including solution pH, electrolyte concentrations [21][22][23] , adsorbed ions 23,24 , and adsorbed organic matter 23,25 . On the other hand, natural and engineered nanoparticles, including CeNPs, can encounter microbial consortia in the subsurface environment 26 due to the ubiquitous occurrence of microorganisms 27,28 . During the interaction between microorganisms and nanoparticles, the extracellular substances (ES) that are released by the microorganisms 29,30 , as essential constituents to form biofilms 31 , adsorb onto the nanoparticle's surface and occasionally lead to particle dissolution 32 , promoting electron transfer 33 , and changing the dispersibility of the nanoparticles in solution 29 . The polymer substances (PS) included in the ES category are generally composed of 40-95% polysaccharides, <1-60% protein, <1-10% nucleic acids, and <1-40% lipids 34,35 . Adsorption of the PS onto the nanoparticles changes the zeta (ζ) potential of aggregates and promotes the dispersibility of particles, increasing the critical aggregation concentration 29, . Adeleye et al. 36 reported that extracellular PS adsorbed onto Cu and CuO nanoparticles can change the ζ potential from positive to negative at pH 4 and narrowed the ζ potential range. Miao et al. 39 also reported the enhanced stability of CuO nanoparticles after adsorption of extracellular PS and polysaccharides due to the electrostatic repulsion and formation of a steric barrier. Adeleye and Keller 37 carried out adsorption experiments of extracellular PS onto TiO 2 nanoparticles. This resulted in a reversal of the surface charge and enhanced particle stabilization. Lin et al. 38 performed adsorption experiments for extracellular PS onto TiO 2 nanoparticles and found that both electric repulsion and steric hindrance were mechanisms of stabilization with increasing mass of the adsorbed extracellular PS. Our previous study 29 revealed enhanced stabilization of CeNPs through steric hindrance and the critical aggregation concentration of NaCl increased from 10 mM to 250 mM when ES was adsorbed onto CeNPs. Despite the fact that effects of extracellular PS have been explored, the previous studies have focused on the polymers only, excluding the effect of small molecules in the ES. Thus, there is limited knowledge on the total competing effects of ES components including small substances. The aim of the present study is to understand the properties of the PS in microbially derived ES, PS, and SS. Secondarily, we aim to evaluate their competing effects on the adsorption processes onto CeNPs, changes in the CeNP surface properties, and the aggregation and sedimentation of CeNPs at various pHs. ## Materials and Methods CeO 2 nanoparticles (CeNPs). Synthetic CeNPs were commercial products purchased from Strem Chemicals, Inc., Newburyport, MA, USA, (part# 58-1400, ~7 nm). The CeNPs had spherical shape and the average diameter was ~7 nm. The surface area was determined to be 70.2 m 2 g −1 using a BET single point analysis, which was smaller than that calculated assuming fully dispersed spherical nanoparticles having <10 nm in size. This indicates that the CeNPs already aggregated prior to use in the present experiments. For a detailed description of these CeNPs, see references 29,30 . ## Preparation of the extracellular substances (ES), the extracellular polymeric substances (PS) and the extracellular small substances (SS). In the present study, Saccharomyces cerevisiae (X-2180) was used as a representative microorganism. First, S. cerevisiae was harvested in 200 mL of sterilized YPD medium, which was composed of 10 g L −1 yeast extract, 20 g L −1 peptone, and 20 g L −1 dextrose. The yeast was incubated for 20 h on a rotary shaker at 120 revolutions per minute (rpm) at 25 ± 1 °C. The suspension of the yeast cells were centrifuged for 10 min at 3000 rpm to be separated. The separated cells were washed three times with 1 mmol L −1 NaCl solution. The yeast cells were put in a polypropylene tube filled with 50 mL of 1 mmol L −1 NaCl. In all solutions the cell density was adjusted to 2.0 ± 0.1 dry g L −1 . The pH of the solutions was initially adjusted to 3.0 ± 0.1 with 1.0 mol L −1 HNO 3 solution. In our previous study 29 , a high concentration of organic matter was extracted at this pH and the composition was similar to the organic matter extracted at higher pHs. A pH meter (TOA tpx-999i; PCE108CW-SR) equipped with an electrode was used to measure pH. After extracting ES for 72 h, the suspension was filtered through a polytetrafluoroethylene (PTFE) membrane filter (Advantec) with 0.20 μm pore size to remove the yeast cells. The filtrate was named as the ES solution. This ES solution contained both polymers and low-molecular-weight species. A portion of ES was dialyzed for 72 hours at 4 °C using a 1000 MWCO Spectra/Por ® 7 (Spectrum) cellulose dialytic membrane. The volume ratio of the ES to ultrapure water was set to 1:3. The water outside the dialytic membrane was exchanged with ultrapure water every 24 hours. This outside solution after the first 24 hours of dialysis was labeled the "extracellular small substances (SS)" solution. The conductivity after 72 hours was measured to be ~0.0 μS. The solution that remained in the membrane tube was labeled PS. The ES, PS, and SS solutions were preserved at 4 °C in a refrigerator and the solutions were adjusted back to room temperature prior to use in experiments. The morphology of the ES was observed by scanning probe microscopy (SPM, DimensionIcon, Bruker AXS, Billerica, USA). The observations were performed under ambient atmospheres using a ScanAsyst probe (ScanAsyst-Air). The specimen for SPM was prepared by dropping the ES solution onto the cleaved pristine surface of biotite and air-dried. Then, the specimen was rinsed with ultrapure water three times and air-dried again. The ES contained various kinds of polymers, organic matter, and inorganic ions, such as H 3 PO 4 . The total phosphate concentrations in the ES, PS, and SS were determined using inductively coupled plasma atomic emission spectrometry (ICP-AES; Agilent 7500c). The detection limit of P was 15 ppb. The concentrations of dissolved organic carbon (DOC) were determined by using a total organic carbon analyzer (TOC; TOC-VE, Shimadzu). The detection limit was 50 μg L −1 and the error was <2%. To further characterize the ES, the dried ES, PS, and SS were analyzed using an attenuated total reflectance Fourier transform infrared spectrometer (ATR-FTIR; Jasco, FT/IR-620) equipped with a deuterated L-alanine triglycine sulfate (DLATGS) detector, a single bounce attenuated total reflectance attachment, and a ZnSe crystal. Thirty-two spectra were obtained with a spectral resolution of 4 cm −1 and averaged. To prepare the dried samples, the pH of the ES solutions was first adjusted to 6.0 ± 0.1 with 1.0 mol L −1 NaOH solution. The ES, PS, and SS solutions were lyophilized and preserved at −10 °C until the measurement. In addition to the FTIR analysis, elemental analysis was completed on the lyophilized ES, PS, and SS to determine the concentrations of C, N, and H. Adsorption of ES, PS, and SS onto the CeNPs. The 5000 mg L −1 CeNPs stock suspension was prepared and ultra-sonicated for 10 min. Five different solutions were prepared in the present experiment: (i) 1 mM NaCl (control); (ii) 1 mM NaCl + 160 μM H 3 PO 4 (160 μM P), of which the P concentration was adjusted to that of the ES solution in the previous study 29 ; (iii) ES solution containing1 mM NaCl solution (conditions during the extraction procedure); (iv) PS + 1 mM NaCl, to adjust the ionic strength to be similar to the other solutions; and (v) SS + 1 mM NaCl. The pH of these suspensions was adjusted to 6.0 ± 0.1 with NaOH. Each of these five solutions were mixed with an aliquot of CeNPs stock solution, in which the concentration of CeNPs was set to 100 mg L −1 so that multiple analytical techniques could be employed. In this study, we did not adjust the C content prior to the adsorption experiments, because the C content does not reflect the actual concentration of specific organic molecules. All ES, PS, and SS contain organic matter with various molecular weights. Thus, it is difficult to quantify the actual concentrations of the non-specified molecules. Rather, the CeNP surfaces were saturated with the organic matters that have concentrations as prepared in the experiments. High-Angle Annular Dark-Field Scanning TEM (HAADF-STEM) and energy-dispersive x-ray spectroscopy (EDX) were completed using a scanning transmission electron microscope (STEM, JEOL, JEM-ARM200CF and JEM-ARM200F, Akishima, Japan). The TEM specimens were prepared by desalinating three times with ultrapure water and dropping the suspension sample on a 300 mesh Cu with Ge or holey carbon supporting membrane followed by air-drying. For the ATR-FTIR analyses, ES, PS, or SS were adsorbed onto the CeNPs at pH 6.0. These suspensions were statically reacted for 24 h. The duration of 24 hours is enough to achieve the apparent equilibrium in this experiment according to our previous study 29 . After the adsorption, the CeNPs associated with organic matter were separated using a 0.025 μm nitrocellulose membrane filter and lyophilized. The analytical procedure of ATR-FTIR for ES, PS, or SS adsorbed to CeNPs was the same as the one described in the previous section. A Zeta Sizer Nano ZEN (Malvern Instruments Inc) was used to measure the ζ potential and average hydrodynamic diameter for the CeNPs suspensions in 1 mM NaCl solution with a capillary cell. The starting pH was set to 6.0 and the pH was shifted to the targeted value using NaOH or HNO 3 . ## Results and Discussion Characterization of ES, PS, and SS. The composition of the ES extracted in 1.0 mmol L −1 NaCl after 72 hours of incubation is summarized in Table 1. The ES contains ~172 mg L −1 organic carbon, ~0.44 mmol L −1 K + , and ~0.59 mmol L −1 total P. The compositions of PS and SS are also given in Table 1. The ES used in the present study contained 2-4 times higher concentrations of organic and inorganic species than the ES characterized in our previous study 29 . The amounts of dissolved organic species in the solutions before and after the adsorption experiments are given in Table S1. Figure 1 shows ATR-FTIR spectra (900 to 1800 cm −1 ) of the lyophilized samples before the adsorption experiments: ES (a), SS (b), and PS (c). The peak assignments, with references, are summarized in Table 2. In line (a), the absorption band at ~1397 cm −1 is assigned to symmetric stretching of COO − groups (ν s C-O) that are included in proteins and polypeptides, and carboxylated polysaccharides . The band at ~1604 cm −1 is assigned to the stretching vibration of C=O groups derived from amide I bonds, which represent amides associated with proteins and polypeptides. The band at ~1518 cm −1 is assigned to the stretching vibration of C-N groups and deformation vibration of N-H groups included in amide II bonds, which correspond to -CO-NH-of proteins and polypeptides . Thus, the two bands at 1604 and 1513 cm −1 suggest that the ES contains proteins and polypeptides. The band at ~1119 cm −1 can be assigned to ring vibrations of C-O-C bonds included in polysaccharides and the stretching vibrations of P=O bonds in proton-dissociated orthophosphate. The band at ~1343 cm −1 is assigned to the carbon backbone coupled with C-O and P-O stretching 44 . Further, the band at ~1050 cm −1 is assigned to symmetric stretching vibrations of P=O derived from phosphoryl groups 24,40,45 . It is difficult to separate the phosphate bands and the polysaccharide vibration bands due to their overlaps; however, the ES released from S. cerevisiae typically contains both polysaccharides and phosphoryl species, which are also included in the ES released from Bacillus subtilis 40,46 and Pseudomonas aeruginosa 45,47 . The FTIR spectrum of SS (b), molecules smaller than 1 kDa, is similar to that of the ES before dialysis. Although the peak position of the PS spectrum is similar to that of the ES spectrum, there is a slight difference in the relative intensity between the peaks. The relative intensity of the peaks derived from phosphate (1044 cm −1 ) and carboxyl (1399 cm −1 ) groups was weaker than that of proteins (1521 cm −1 and 1635 cm −1 ) in the PS fraction. These results indicate that SS contains almost the same compounds as ES, such as inorganic phosphate, amino acids, polysaccharides, and polypeptide, whereas PS contains mainly polysaccharides, proteins and polypeptides of larger molecular sizes >1 kDa. ## Morphology of polymeric substances within the ES. The topological and phase contrast AFM images were obtained for the freshly cleaved biotite (Fig. 2a), the freshly cleaved biotite without the ES after desalination (Fig. 2b), and the ES adsorbed onto the freshly cleaved biotite after desalination (Fig. 2c), which showed the presence of nanoparticles 20-30 nm in diameter and ~3 nm in height (Fig. 2c). The same mode images without ES after desalination did not show any nanoparticles on the surface of cleaved biotite (Fig. 2b). Thus, the particles detected in the former images (Fig. 2c) are polymeric substances of the ES. The shallow height of the nanoparticles indicates a flattened shape after substrate adhesion; thus, the true particle size in solution is likely smaller than 20 nm. STEM of the ES + CeNPs, PS + CeNPs, and SS + CeNPs. Figure 3 shows that HAADF-STEM image and the EDX elemental maps of the samples. ES + CeNPs, PS + CeNPs, and SS + CeNPs exhibit CeNP aggregation ranging from 100 to 500 nm, on which C, N, and P are distributed uniformly, indicating that the polymeric substances of the ES adsorbed onto the CeNP surface. Note that the C map contains interference from the holey carbon supporting mesh. In the PS + CeNPs specimen, the EDX spectrum reveals a clear peak of the S K-line in an aggregate, derived from thiol groups, which is likely attributed to the presence of amino acids such as cysteine and methionine. The P/Ce molar ratio on the aggregates in ES + CeNPs, PS + CeNPs, and SS + CeNPs varies between 0.01 to 0.08, indicating that the adsorption of ES, PS, and SS to CeNP is not homogeneous (Fig. 4). Representative EDX spectra for ES + CeNPs, PS + CeNPs, and SS + CeNPs can be found in Fig. S1. FTIR of ES, PS, and SS adsorbed to CeNPs. ATR-FTIR difference spectra of the experimental samples are shown in Fig. 1: ES + CeNPs (d), SS + CeNPs (e), PS + CeNPs (f), PS + 160 μM P + CeNPs (g), and 160 μM P + CeNPs (h). The CeNPs spectrum was subtracted from the raw spectra to display only the spectra of the adsorbed species. All of the spectra after the adsorption treatment appear similar. After adsorption, the bands for phosphate were broadened, indicating the formation of inner-sphere complexes on the CeNPs, as previously 44 . reported 29 , and proteins and polypeptides adsorbed preferentially onto the CeNP surfaces. Interactions between the extracellular PS and metal oxides generally occur via amide, hydroxyl, and carboxylic groups on the PS amino acids in addition to the phosphate groups from phospholipids or nucleic acids 37 . The amide I peak, derived from proteins and polypeptides, shifted toward higher frequencies and the peak derived from the carboxyl group became minimized after adsorption to the CeNPs, regardless of dialysis treatment. The predominant adsorption of proteins from the bacterial extracellular PS to metal oxides has also been reported in several previous studies 40,48 by forming inner-sphere complexes 45 . Proteins can adsorb onto hydrophilic surfaces and the shift in the peak position typically occurs due to protein conformational changes after adsorption . Although the chemical compositions between ES, PS, and SS were different, the FTIR spectra after adsorption to CeNPs appear identical, strongly suggesting that the molecules adsorbing onto the CeNP surfaces possess similar functional groups, despite the molecular size differences in the ES, PS, and SS constituents. ## The effects of ES, PS, and SS on the surface electric potential (ζ potential). Figure 5a shows the pH dependence of ζ potential for: CeNPs (control), ES + CeNPs, 160 μM P + CeNPs, and PS only. The point of zero charge (PZC) of CeNPs (control) was determined to be 6.9, which was within the range of the reported PZC values from several literature sources; 6.5-8.0 21,53 . The ζ potentials of the CeNPs in 160 μM P were −40 to −50 mV at pH > 5.0, and the isoelectric point (iep) was determined to be ~1.6. In the pH 6 solution, the ζ potential decreased as H 2 PO 4 − and HPO 4 2− adsorbed to the CeNPs. In the ES + CeNPs, the ζ potentials were plotted between the control and 160 μM P values. The P concentration (592 μM) in the present ES was determined to be higher than that measured in our previous study 29 , but within the same order of magnitude. As reported in 29 , phosphate in the ES also adsorbed to CeNPs, although the ζ potential was not affected by the adsorbed phosphate. Furthermore, the ζ potentials of PS were plotted almost identical to the plots of ES + CeNPs. The ζ potentials of ES (Fig. S2) were plotted deviated from PS and ES + CeNPs, indicating that the ES compounds adsorbed onto the CeNP is similar to PS rather than the total ES compounds. It was impossible to measure the ζ potential of SS due to their small sizes. The electrophoretic mobility distribution for the ES + CeNPs was similar to that for PS at pH 2.9-10.0 (Fig. S3). In the diagram for PS at pH 9.99 (Fig. S3), the single peak split into multiple peaks at high pH, most likely because there were several aggregates with different functional groups on the CeNP surface. In the case of ES + CeNPs, the peaks were located at the same mobility value as the case of PS, which may indicate that the large molecules with similar specific functional groups preferentially adsorbed onto the CeNP surfaces. The effect of phosphate adsorption did not appear in the ζ potential in the ES solution because the preferential adsorption of macromolecules, such as proteins and polypeptides, on the outermost surface hinders the effects of orthophosphate. The ζ potentials of three additional systems (PS + CeNPs, PS + 160 μM P + CeNPs, and SS + CeNPs) were also plotted in Fig. 6a, confirming that the presence of inorganic phosphate in the ES did not influence the ζ potential of CeNP in any system where PS was present; that is, the ζ potential of CeNPs reacted with ES was governed by the polymers in the ES rather than the small charged molecules, such as phosphate. In addition, the ζ potential of SS + CeNPs, which contained inorganic phosphate, also exhibit the same trend as that of ES + CeNPs. There are two factors that caused the similarity. One factor is the steric barrier created by the organic matter, even by molecules size smaller than 1 kDa, because the FTIR spectra of ES, PS, and SS revealed that the functional groups of the compounds adsorbed on the CeNPs were nearly identical (Fig. 1). The other factor is the decreased concentration of SS due to dialysis. The SS solution was diluted to approximately one quarter, implying the possibility of decreased amounts of adsorbed inorganic phosphate in the ES. Indeed, the adsorption experiment of inorganic phosphate using various concentrations of phosphate revealed that the ζ potential increases gradually as the P concentration decreases, and the pH dependence at the P concentration of 1.6 µM, which is two orders of magnitude less than the P concentration in ES, became identical to that of the control (Fig. 7a). In addition, the average hydrodynamic diameter also changed concurrently; when P concentration decreased from 160 μM to 16 μM, the ζ potential shifted to a positive value, the iep shifted from ~1.6 to ~4.3, and the pH, at which the average hydrodynamic diameter becomes the maximum, shifted from ~1.9 to ~5.0. The pH dependence of the average hydrodynamic dimeter of the SS + CeNPs appeared similar to that of P + CeNPs (16 µM) (Fig. 7b). When the CeNPs were exposed to 107 µM P, the same P concentration in SS, the iep shifted from ~1.6 to ~2.0, though the pH dependence of the ζ potential was almost identical to the 160 µM P case. Thus, the pH dependence of the ζ potentials for SS + CeNPs and ES + CeNPs can be ascribed to the similar characteristics of the polymeric substances, even when of different molecular sizes. The effects of ES, PS, and SS on the size of the aggregates. Figure 5b shows the average hydrodynamic diameter of CeNPs in three conditions: (i) 1.0 mM NaCl; (ii) 1.0 mM NaCl + 160 μM H 3 PO 4 ; (iii) ES + 1 mM NaCl, over pH 1-11. The size increases at near the iep under all conditions. This indicates that the electrostatic repulsive force resulted from the outermost charge of the particle constrains the aggregation behavior of CeNPs. When the pH was less than 3, the ζ potentials of the ES + CeNPs and the 160 μM P + CeNPs cases were both close to zero, meaning that the electrostatic repulsive force was not effective under low pH conditions (Fig. 5a). However, the size of ES + CeNPs was less than half of that of 160 μM P + CeNPs, indicating that the steric barrier formed by the polymeric substances effectively suppressed the aggregation of CeNPs. Indeed, as described above, the FTIR results indicated a preferential adsorption of proteins onto the CeNP surfaces, causing steric hindrance 39 . On the other hand, between pH 3 and 4, the average particle size of ES + CeNPs was larger than that of the 160 μM P + CeNPs and control cases. Under this pH condition, the ζ potential of ES + CeNPs was close to zero, while that of 160 μM P + CeNPs was as low as −30 mV. This indicates that the aggregation behavior of CeNPs was constrained by both electrostatic and steric repulsion, and the effects of electrostatic repulsion were greater than that of the steric barrier. Figure 6b shows the pH dependence of the average hydrodynamic diameter of ES+ CeNPs, SS + CeNPs, PS + CeNPs, and PS + 160 μM P + CeNPs. The average hydrodynamic diameter for SS + CeNPs exhibited a different trend from that of 160 μM P + CeNPs or ES + CeNPs, most likely because the electrostatic repulsive forces in SS + CeNPs were less than that of the 160 μM P + CeNPs case, due to the lower P concentration in SS, and the steric barrier derived from the molecules <1 kDa in SS was weaker than that in the ES + CeNPs case. In the PS + CeNPs case, the average hydrodynamic diameter increased around the iep of PS itself, whereas that in the PS + 160 μM P + CeNPs case showed only a slight increase around pH ~3. The difference might be attributed to the presence of P adsorbed onto CeNPs rather than PS adsorption on to the CeNPs. CeNP aggregation should Figure 3. HAADF-STEM image with the elemental maps of the CeNP specimen after contact with ES, SS, or PS at pH 6.0 in 1.0 mM NaCl for 24 hours followed by desalination washing with ultrapure water thrice. The suspension of ES + CeNPs was dispersed on the Ge-mesh without using C, while the samples of PS + CeNPs and SS + CeNPs were prepared on holey carbon mesh with a Cu supporting grid. have been promoted at between pH 3 to 5, near the iep of PS (~3.5), because the ζ potential is determined by the largest molecules, regardless of the presence of phosphate. However, aggregation appeared to be suppressed by the inorganic phosphate adsorbed onto CeNPs, likely because the repulsive forces derived from the adsorbed inorganic phosphate became predominant when the distance between CeNP surfaces become sufficiently small. At pH <3, which is close to iep (pH ~1.6) of 160 μM P + CeNPs, both electrostatic repulsive forces and steric barriers from the adsorbed PS reduced aggregation. Although the same trend can be seen in ES + CeNPs, aggregation in PS + 160 μM P + CeNPs was suppressed more profoundly than that in ES + CeNPs. The ES contains free cations and small organic molecules, including amino acids with carboxyl groups. Thus, in the ES + CeNPs system, the electric repulsive forces of inorganic phosphate can be neutralized 54 or other organic matter having hydrophobic adsorption to the CeNP surfaces may lead to a higher affinity between the particles. Indeed, Mosley and Hunter 55 reported that adsorption of organic matter suppresses aggregation through the formation of steric barriers, whereas it can also prevent dissociation of colloid aggregate once they formed aggregates. Because of such effects owing to the presence of other small organic matters in ES, the ES + CeNPs case was found to be slightly less aggregated than that of the PS + 160 μM P + CeNPs case. Figure S4 shows the results of additional experiments measuring the sedimentation rates of CeNPs at pHs of 2.0, 3.0, 3.5, 6.0, 7.0, and 10.0 under three conditions: CeNPs, 160 μM P + CeNPs, and ES + CeNPs. These rates were calculated based on the turbidity time course monitored using UV-Vis spectroscopy. The sedimentation rate of CsNPs was the fastest when the solution pH is close to the iep: 160 μM P + CeNPs at pH 2, ES + CeNPs at pH 3.5, and CeNP at pH 7.0. The sedimentation rate of CeNPs greatly depends on the solution pH, which is consistent with the DLS analysis. Comparison with the other macromolecules. Figure 8 summarizes the surface properties and sizes of CeNPs aggregates under the present experimental conditions. Because the ES is a part of natural organic matter (NOM), and vice versa, it is useful to compare the present results with the well-known effects of NOM such as humic substances (HS) on the aggregation behavior of various engineered nanoparticles 19,23,25, . The presence of NOM typically stabilizes the colloids and reduces aggregation by coating the nanoparticle surfaces: forming a steric barrier and modifying the surface charges 62 . Our results on the role of microbial ES appear to be similar to that of NOMs previously reported 63 . The similarity between the effects of exudate and NOM on the aggregation behavior of TiO 2 nanoparticles has already been recognized 64 . However, comparing the elemental analysis and FTIR data of ES (Table 1) with those of fulvic acid (FA) and humic acid (HA) 65 , the ES contains higher concentrations of nitrogen, reflecting a higher protein and polypeptides content, whereas HS consist of carbohydrates, alginate, amino acids, lignin, and pectin. The clear difference between ES and NOM is the preferential adsorption of protein and polypeptides to the CeNP surfaces. In general, the molecular weights of the differently sized components in HS, FA and HA, range from a few hundred to thousands and from thousands to several millions of Da, respectively 65 . Several studies have reported that large molecular size of HA can reduce aggregation more efficiently than FA 66,67 by forming thick polymer layers. In the case of NOM, it has been reported that adsorbed layer thickness, aromaticity, and molecular weight, are all correlated with aggregation behavior 26,63 . It is also noted that typical surfactants with low-molecular weights can desorb or be displaced by larger molecules, such as NOMs 63 ; however, such phenomena were not observed in the present experiments. This can be ascribed to the fact that the functional groups of the organic substances that adsorbed onto the CeNP surfaces were similar in SS and PS. This is reasonable because proteins and polypeptides, which preferentially adsorbed to the CeNP surfaces in the present experiment, generally adsorb by forming chemical bonds 37,45 . Furthermore, in case of NOMs, large fibrillary polymers can form bridges between nanoparticles, and this occasionally promotes aggregation 68 . Such an enhanced aggregation mechanism did not occur in our PS case because the conformation of the proteins and polypeptides present, as revealed in the AFM images (Fig. 2), were approximately spherical. Rather, the PS further reduced aggregation compared to the SS case (Fig. 8). ## Conclusion Adsorption experiments using ES (PS + SS), PS (>1 kDa), and SS (<1 kDa) to CeNPs were conducted to understand the effect of each fractionated component on the modification of the surface and dispersibility of the CeNPs at pHs ranging from 1.5 to 10. Microscopic and spectroscopic characterization of these components, with and without CeNPs, revealed that the three fractions were composed of organic matter that contained similar functional groups, despite the size difference of the molecules within each fraction because polypeptides and amino acids were present in the SS fraction. The preferential adsorption of proteins and/or polypeptides, and inorganic phosphates were observed in all fractions. The polymeric substances in the ES case formed aggregates as large as a few tens of nm and the chemical composition showed heterogeneous distributions on the CeNP surfaces from the adsorption of multiple components. The ζ potential of CeNP with ES, PS, and SS exhibited the same pH dependence, suggesting that the polymeric substances, even smaller-sized molecules, modified the surface charge of the CeNPs with apparent similarity to the PS case. Although the ζ potentials were governed by the polymeric substances in the ES, other small polymers with different ieps can also adsorb to the CeNP surfaces and reduce nanoparticle aggregation under conditions where the ζ potential is nearly zero. Thus, the suppression effects on the aggregation by ES adsorption onto CeNPs can be expressed under wider pH conditions than those derived from PS adsorption only. There is a wide range of amounts and chemical variations in small and large polymeric substances, as well as geochemical parameters, in natural surface and subsurface environments. Hence, the results of the present study highlight the non-negligible impact of microbially derived polymeric substances, of various molecular sizes, on the migration of CeNP in the environment. The dynamics of adsorption, aggregation, and transformation of various organic matter-nanoparticle couples in realistic environments still remains to be explored as previously pointed out 69 . The quantitative data obtained in the present study can be useful for understanding the role of small-sized molecules derived from microbial activity on the migration of CeNPs in aquatic environments, especially where bacterial consortia prevail.

chemsum

{"title": "The competing effects of microbially derived polymeric and low molecular-weight substances on the dispersibility of CeO2 nanoparticles", "journal": "Scientific Reports - Nature"}

a_9-connected_zirconium-based_metal-organic_framework_for_ammonia_capture

## Abstract: Construction of multifunctional metal-organic frameworks (MOFs) with asymmetric connectivity have the potential to expand the scope of their utilization. Herein, we report a robust 9-connected microporous Zr-based MOF, NU-300, assembled from asymmetric tri-carboxylate ligands and Zr6 nodes. As indicated by single-crystal X-ray diffraction analysis, there exist uncoordinated carboxylate groups in the structure of NU-300 that can participate in ammonia (NH3) sorption through acid-base interactions which yield high uptake of NH3 at low pressure regions (<0.01 bar). In situ infrared (IR) spectroscopy shows the interactions between Brønsted acidic sites and NH3, which 2 suggests that NU-300 can be used as a sorbent for NH3 capture at low pressures. ## Introduction Metal-organic frameworks (MOFs) are a class of porous crystalline materials assembled by metal nodes and organic ligands. 1,2 Because of their high porosity, 3 versatile pore structures, 4 and tunable chemical functionalities, MOFs can be precisely designed at the molecular level for targeted applications including, but not limited to, gas storage and separation, catalysis , chemical sensing, 20,21 and more. 22,23 Particularly, zirconium-based MOFs (Zr-MOFs) have attracted extensive attention in recent years due to their high thermal and chemical robustness, as well as topological diversity. 24,25 Many reported Zr-MOFs contain 12-, 10-, 8-, 6-or 4connected Zr6 nodes with di-, tri-, and tetra-carboxylate ligands, showcasing different network topologies. 26 In designing stable zirconium MOFs, in general, highly symmetric di-, tri-, and tetra-carboxylate ligands are used which results in high symmetry MOFs when combined with symmetric Zr6 nodes. 24,25 In the case of asymmetric ligands, although copper and rare-earth (RE) based MOFs have been constructed with non-planar tri-carboxylate ligands, 27,28 however, Zr-based MOFs have rarely been explored. 29 Asymmetric ligands can introduce new types of coordination environments and potentially unlock new topological structures for novel MOF materials. 27 Thus, the construction of Zr-MOFs with asymmetric ligands remains under explored for understanding the relationship between the different connected Zr6 nodes and ligands within their unique structures, as well as exploiting the potential chemical properties related to the asymmetric ligands. ## 3 The exceptional stability of Zr-MOFs renders them as promising candidates for the capture of ammonia (NH3). Considering the associated corrosiveness and toxicity, the capture of NH3 at extremely low concentrations using porous materials under ambient conditions in industrial settings is of great importance to comply with limits of short-term exposure (35 ppm) and long-term exposure (25 ppm) set by the Occupational Safety and Health Administration (OSHA). 31 Several MOFs including HKUST-1, 33 MOF-74, 34 M(isonicotinic acid)2 (M = Zn, Co, Cu, Cd), 35 MFM-300(Al), 36 M2Cl2BBTA (M = Co, Mn), 37 M2Cl2(BTDD) (M = Mn, Co, Ni and Cu), 38 and UiO-66 series 30,32 have been tested for NH3 uptake. However, the majority of MOFs studied for NH3 uptake showed structural degradation upon exposure or significant loss of uptake after consecutive cycles. So far, only a limited number of MOFs are reported to exhibit good reversible NH3 sorption over multiple cycles, e.g. MFM-300(Al), Co2Cl2BBTA and M2Cl2(BTDD). The development of robust MOFs with reversible NH3 sorption that can withstand multiple cycles remains challenging. With the aforementioned challenges in mind, herein, we report a robust and functional 9-connected microporous Zr-MOF (NU-300) assembled from an asymmetric tri-carboxylate ligand and novel Zr6 node with an unusual linker connectivity. Notably, NU-300 has a high ammonia uptake at low pressures by exploiting Brønsted acidic sites on both the ligand and the node, which can be used an adsorbent for NH3 capture at low concentration. ## Result and Discussion The solvothermal reactions of ZrCl4 and 3,5-di(4'-carboxylphenyl)benzoic acid 4 (H3L) in N,N-dimethylformamide (DMF) with formic acid as a modulator, yielded colorless rhombic-shaped crystals of NU-300 (NU stands for Northwestern University). Single crystal X-ray diffraction revealed that NU-300 crystallizes in the orthorhombic Imma space group. The asymmetric unit of NU-300 contains four Zr 4+ atoms, each uniquely eight-coordinated. As shown in Figure 1a, Zr1 is coordinated by four distinct oxygen atoms from different carboxylates of four H3L ligands and four μ3-O entities. Zr2 is coordinated by two distinct oxygen atoms from carboxylates of two H3L ligands, one oxygen atom from formic acid, one oxygen atoms from DMF and four μ3-O entities. Zr3 is coordinated by three oxygen atoms from various carboxylates of three H3L ligands, one oxygen from DMF and four μ3-O entities. Finally, Zr4 is coordinated by three oxygen atoms from various carboxylates of three H3L ligands, one oxygen from terminal OH/H2O and four μ3-O entities. Two Zr1, two Zr2, one Zr3 and one Zr4 atoms are connected together by eight μ3-O atoms to form the Zr6O8 cluster (Figure S2). This cluster differs from previously reported Zr6 nodes that contain only one or two crystallographically independent Zr 4+ atoms. 26,29, Moreover, the H3L ligand adopts two types of coordination modes. In mode I, two carboxylate groups of H3L adopt a bridging bis-monodentate mode while one is monodentate (Figure 1b). In mode II, two carboxylate groups of H3L adopt monodentate and bridging bis-monodentate modes, respectively, while one carboxylate group remains uncoordinated (Figure 1c) and points to the channel along the a-axis in the 3D structure of NU-300 (Figure 1d). The topological analysis indicates that NU-5 tritopic linkers, since monodentate carboxylic acids are not included in the connectivity counting. Thus, the 3D framework of NU-300 can be simplified as a (3, 3, 3, 9)connected network with a point symbol of (4.6 2 ) (4 2 .6)2 (4 8 .6 20 .8 8 ) (Figure 1e), which is a new topology. The 9-connected Zr6 nodes of NU-300 are different from a previously reported 9-connect node, wherein the carboxylate ligands bridge adjacent Zr atoms in the node. ## 6 The phase purity of bulk NU-300 is confirmed by comparison of simulated and experimental PXRD patterns (Figure 2a). Thermogravimetric analysis (TGA) reveals that the framework of NU-300 starts to decompose at around 400 °C in air (Figure S3), demonstrating the high thermal stability of NU-300. The permanent porosity of NU-300 is confirmed by N2 adsorption measurements at 77 K (Figure 2b). NU-300 exhibits a type I isotherm, indicative of the microporous character of the material. The Brunauer-Emmett-Teller (BET) area and total pore volume for NU-300 are calculated to be 1470 m 2 /g and 0.58 cm 3 /g, respectively, while the pore size distribution based on DFT modeling indicates micropores of ~11 (Figure 2b). The chemical stability of NU-300 is then investigated by soaking NU-300 in 100 °C H2O, 0.01 M aqueous HCl (pH=2) and 0.001 M aqueous NaOH (pH=11) solutions for 24 h. As illustrated by PXRD patterns (Figure 3a), the crystallinity of the NU-300 is retained after these treatments. To further confirm the chemical stability of NU-300, N2 sorption measurements are also conducted after these treatments (Figure 3b). The N2 isotherms of NU-300 in hot water and acidic conditions are almost identical to that of 8 pristine NU-300, confirming its structural integrity and permanent porosity after exposure to boiling water and dilute acid. However, a decrease is observed in surface area and pore volume after base treatment. In light of the presence of free carboxylate groups (-COOH) within the framework, NH3 sorption tests were conducted on NU-300 to investigate potential guest-host 9 interaction. The first run adsorption-desorption isotherm shows an adsorbed NH3 amount of 8.28 mmol/g at 298 K and 1.0 bar (Figure 4a). At pressures less than 0.01 bar (Figure 4b), NH3 molecules preferentially adsorbed to the Zr6 nodes and Brønsted acidic sites of the free -COOH groups in NU-300, exhibiting the steep NH3 isotherm. After regeneration at room temperature under vacuum, an NH3 uptake of 3.30 mmol/g remained in NU-300, likely due to the chemisorption process by the formation of strong interactions between the uncoordinated -COOH groups and NH3 molecules, apart from the acidic -OH groups on Zr6 nodes (Figure S5). This indicates that Brønsted acid sites, particularly the free -COOH groups, aid NU-300 in NH3 uptake at low pressures, allowing NU-300 to reach approximately 4 mmol/g uptake by 0.10 bar and 1.5 mmol/g by 0.01 bar, and the latter can be recycled for at least three times (Figure 4b). There was a loss in NH3 uptake capacity at 1 bar between the first and second cycles while the third cycle of NH3 sorption was nearly identical (5.71 and 5.41 mmol/g at 1.0 bar), suggesting that the loss in capacity occurs primarily in the initial sorption cycle. We then turned to IR spectroscopy to further assess how the carboxylic acid sites in NU-300 interact with NH3 molecules during the adsorption and desorption processes. As observed in Figure 5, with NH3 exposure for 60 min on NU-300, two characteristic NH3 bands are observed, indicating NH3 interactions with NU-300: the degenerate and symmetric deformation of NH3 at 1625 and 1360 cm -1 , respectively. 43 The location of C=O stretching vibration of the free -COOH groups at 1730 cm -1 (Figure S7) decreases 11 to 1710 cm -1 upon NH3 exposure, possibly due to deprotonation and subsequent resonance that weaken the C=O bond strength. 44 The appearance of overlapping bands between 3300 and 3700 cm -1 supports the deprotonation of -COOH by NH3. 45 However, none of these bands between 3300 and 3700 cm -1 disappear upon Ar purge. NH3 exposure also results in a new band at 1480 cm -1 , which could be assigned to the vibration of N-H in NH4 + . 43 These observations indicate that NH3 molecules were protonated in acid-base reaction with Brønsted acidic sites. IR spectra showed that even after Ar purge residual adsorbed ammonia bands were still present which rationalized the loss in uptake between the first and second cycles. ## Conclusions In summary, we have designed a robust 9-connected Zr-based MOF, NU-300 using an asymmetric tri-carboxylate ligand. The presence of free -COOH groups on the ligands provides NU-300 with relevant functional properties for the chemisorption of NH3 molecules at low partial pressures. For the first cycle adsorption, the adsorbed uptake of NH3 was 8.28 mmol/g at 298 K and 1.0 bar. On the other hand, the second and third

chemsum

{"title": "A 9-Connected Zirconium-Based Metal-Organic Framework for Ammonia Capture", "journal": "ChemRxiv"}

high-performance_photoacoustic_probe_for_biopsy-free_assessment_of_copper_status_in_murine_models_of

## Abstract: The development of high-performance photoacoustic (PA) probes that can monitor disease biomarkers in deep-tissue has the potential to replace invasive medical procedures such as a biopsy. However, such probes must be highly optimized for in vivo performance and exhibit an exceptional safety profile. In this study, we have developed PACu-1, the first PA probe designed for biopsy-free assessment (BFA) of hepatic Cu via photoacoustic imaging. PACu-1 features a Cu(I)-responsive trigger appended to an aza-BODIPY dye platform that has been optimized for ratiometric sensing. Owing to its excellent performance, we were able to detect basal levels of Cu in healthy wildtype mice, as well as elevated Cu in a Wilson's disease model and in a liver metastasis model. To showcase the potential impact of PACu-1 for BFA, we conducted a blind study where we were able to successfully identify a Wilson's disease animal from a group of healthy control mice with greater than 99.7% confidence. Significance StatementThe ability to non-invasively detect and track disease biomarkers via photoacoustic imaging can potentially serve as a substitute for invasive medical procedures such as a liver biopsy. While achieving this goal can have a profound impact on disease management, it is an immense challenge that requires novel chemical tools that are sensitive, selective, and safe. Here we report an acoustogenic probe designed for Cu(I), which becomes dysregulated in many disease states. In addition to demonstrating in vivo efficacy in multiple models, we designed a blind study to assess its utility for biopsy-free assessment of hepatic copper levels in Wilson's disease. This work sets the stage for future studies to evaluate the performance of acoustogenic probe designs for biomedical applications. ## Figures 1 to 6 Introduction Photoacoustic (PA) imaging is a light-in, sound-out technique that has emerged as a promising biomedical approach for the non-invasive assessment of various ailments in humans, ranging from arthritis to cancer. 1,2 Excitation of an endogenous pigment such as hemoglobin in blood or melanin in tissue can provide contrast since relaxation via non-radiative decay can trigger thermoelastic expansion of the surrounding tissue. Repeatedly irradiating a region of interest with a pulsed laser can result in pressure waves that can be readily detected by ultrasound transducers. Since ultrasound at clinically relevant frequencies can travel through the body with minimal perturbation, it is possible to accurately pinpoint the source of the signal to afford high resolution images at centimeter imaging depths. 3 Beyond label-free applications, the utility of PA imaging for disease detection has been augmented by the recent development of acoustogenic probes (activatable PA probes) that give an off-on signal enhancement or ratiometric readout. 4,5 Notable examples include those that can visualize dysregulated enzymatic activities, properties of the disease tissue microenvironment, as well as small molecule-and metal ion-based disease biomarkers. However, replacing an invasive medical procedure such as a liver biopsy with an acoustogenic probe is an immense challenge since the in vivo performance and safety profile of such a chemical tool must be exceptional. Thus, in spite of undesirable shortcomings such as the potential to develop severe infections, false negatives due to collection of nondiseased tissue, and the inability to directly monitor disease progression in real-time, 19 liver biopsies are still commonly employed to assess biomarkers in conditions such as Wilson's disease (WD) 20 and cancer. 21 It is noteworthy that elevated levels of hepatic copper (Cu) is a common biomarker shared by these conditions. In WD, Cu accumulates in the liver due to a genetic mutation in the Cu-exporter, ATP7B, and this can lead to chronic liver damage which can become fatal if not treated. 22,23 In the context of cancer, Cu is elevated in many solid tumors including breast 24,25 and lung 26,27 cancers which generally metastasize to the liver. Since Cu can promote angiogenesis and drive tumor progression, BFA of Cu in metastatic lesions is critical. While several probes have been developed for in vivo imaging of Cu, these early examples were designed for fluorescent 28 and bioluminescent 29 methods which are more suitable for shallow imaging depths (mm range) owing to scattering and attenuation of light. More recently, our group 13,30 and others 31 have developed Cu probes for PA imaging to achieve greater tissue penetration and higher resolution. However, these probes are designed to target Cu(II), whereas intracellular Cu exists predominantly in the +1-form owing to a highly reducing environment of the cell. 32 To overcome this challenge, we present the development of PACu-1, the first acoustogenic probe for Cu(I) and its application in BFA of hepatic Cu in a WD model and a liver metastasis model. Moreover, we designed an unbiased BFA blind study to identify a Wilson's disease mouse from a group of healthy wildtype controls using PACu-1. ## Design and Characterization of PACu-1 To target the +1-oxidation state of Cu, we installed a Cu(I)-responsive tris[(2-pyridyl)methyl]amine (TPA) 33 trigger onto an optimized aza-BODIPY dye platform to yield PACu-1 which features ratiometric imaging capabilities. Specifically, we hypothesized capping of the 2,6-dichlorophenol moiety will result in a blue-shift of the wavelength of maximum absorbance (λmax) relative to the uncapped probe. However, the binding of Cu(I) to TPA will induce an oxidative cleavage event of the pendant ether linkage to release the latent dye (Figure 1a). Subsequently, selective irradiation of each form (probe and product) at their corresponding λmax will yield two signals, from which a ratio can be determined. This probe design feature is important for BFA, especially in the liver since we anticipate there to be significant background interference from blood. In addition, we selected the aza-BODIPY platform to develop PACu-1 due to their large extinction coefficients (10 4 to 10 5 M -1 cm -1 ) in the near infrared range and low fluorescence quantum yields since both of properties translate to a stronger PA signal. 30 Lastly, we have determined empirically that many of the aza-BODIPY-based probes we have developed intrinsically localizes to the liver owing to its relatively high hydrophobic properties. After synthesizing PACu-1 (Scheme S1), we evaluated its in vitro response to 20 equivalents of Cu(I) (introduced as [Cu(CH₃CN)₄]PF₆). After 1 h incubation at room temperature, we observed a large spectral shift of 91 nm from 678 nm (probe, Ɛ = 5.3 × 10 4 M -1 cm -1 ) to 767 nm (product, Ɛ = 3.7 × 10 4 M -1 cm -1 ) (Figure 1b). Given that the extinction coefficient of a molecule is a reliable proxy for its PA output, we estimate the ratiometric turn-on will be ~10.4-fold (defined as (770/680Final)/(770/680Initial)). Importantly, irradiation at 680 nm will predominately excite PACu-1, whereas light at 770 nm will only generate a signal that corresponds to the turned over product (Figure 1c-d). Moreover, we were able to observe a dose-dependent response to Cu (LOD = 0.2 µM) (Figure 1e). We were also able to show that PACu-1 can function in the presence of glutathione (GSH), an abundant biological thiol, that can compete with PACu-1 to bind Cu(I) (Figure 1f). Indeed, GSH is present at high levels in the liver and most solid tumors (up to 10 mM). 34,35 Lastly, PACu-1 was shown to exhibit excellent selectivity for Cu(I) against a panel of monovalent and divalent metal ions (Figure 1g). This finding is significant because in addition to Cu(I), the TPA trigger has been tuned to sense other metal ions and thus, may exhibit off-target reactivity. 36,37 ## Metabolic Stability, Biodistribution, and Safety Profile of PACu-1 Because our objective is to employ PACu-1 for BFA of hepatic Cu via PA imaging, it is critical to 1) demonstrate that it is not metabolized in the liver to give false positives and 2) show that it is biocompatible with an excellent safety profile. To this end, we treated PACu-1 with rat liver microsomes rich in metabolic enzymes (e.g., CYP450s). After an incubation period of 1 h, we did not observe any change in the absorbance spectra indicating there would be minimal off-target activation of PACu-1 that can lead to false positive results (Figure S1). We corroborated these results with mass spectroscopy analysis that showed the latent aza-BODIPY was not being released. Next, we performed MTT assays to assess the cytotoxicity of the probe in mammalian cell lines. For instance, HEK293 cells incubated with up to 25 µM of PACu-1 for 24 h were shown to have no significant loss of viability (Figure 2a). Next, we sought to determine the biodistribution of PACu-1 after systemic administration in BALB/c mice via ex vivo PA imaging analysis of the vital organs. Our data indicates that PACu-1 predominantly localizes to the liver and does not accumulate in the heart, kidneys, or spleen (Figure S2). Before PACu-1 could be considered further as a chemical tool for BFA applications, we examined its in vivo safety profile. First, we performed H&E staining on liver samples obtained from mice treated with either a vehicle control or PACu-1. Our results show that the nuclear staining patterns were identical, suggesting that PACu-1 is non-toxic (Figure 2b). Second, we conducted a comprehensive liver function test to measure the levels of albumin, alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate transaminase (AST), bilirubin, blood urea nitrogen (BUN), cholesterol, and glucose in serum. We did not observe any statistical difference between vehicle-and PACu-1-treated animals which further demonstrates that PACu-1 is safe and thus, is ideal for BFA applications (Figure 2c). ## Imaging Exogenous Cu(I) in BALB/c Mice To determine whether PACu-1 can be employed to detect elevated hepatic Cu(I) in live animals, we treated BALB/c mice with CuCl2 via intraperitoneal administration 2 h prior to the introduction of PACu-1. Of note, Cu(II) is rapidly reduced to Cu(I) upon uptake into cells. PA imaging revealed that the PA770/680 ratio was 1.48 ± 0.23 for the Cu-treated animals, whereas the corresponding ratio for the vehicle control was 0.94 ± 0.13 (Figure 3, red and blue, respectively). To confirm that these results were due to the detection of Cu(I), we administered ammonium tetrathiomolybdate (TM), a high affinity FDA-approved Cu chelator drug (Kd = ~10 -20 ), 38 prior to treatment with PACu-1. As anticipated, we did not observe any activation (0.94 ± 0.13) when TM was present since it can outcompete PACu-1 for binding to Cu(I) (Figure 3, yellow). To further validate this finding, we administered Ctrl-PACu-1, a non-responsive control probe that features an attenuated Cu(I) binding trigger (Scheme S2), to a fourth group of animals. Interestingly, PA imaging demonstrated that the ratio was also lower than both the vehicle group and the TM group (0.82 ± 0.12) (Figure 3, green). The lower ratio suggests that PACu-1 can detect basal levels of Cu that are present in the liver. Finally, we performed ICP-MS analysis on liver samples obtained from mice treated with CuCl2. Compared to animals that received a vehicle control, the concentration of hepatic Cu was twice as high (Figure 3c). ## PA Imaging of Hepatic Cu(I) in Wilson's Disease Cu accumulation in the liver is a pathological hallmark of WD which is typically assessed clinically via liver biopsies. 39 Using an established ATP7B genetic knockout model of WD developed by Lutsenko and co-workers (JAX stock #032624), we measured the levels of hepatic Cu in wildtype mice and WD mice using ICP-MS analysis after obtaining biopsied tissue. On average, we found that the Cu levels in WD mice were 17.5-fold greater than wildtype mice (Figure 4a). Likewise, when we employed PACu-1 and PA imaging for BFA of Cu, we found that the PA770/680 ratio was significantly higher in WD mice (1.24 ± 0.16) relative to wildtype mice (0.80 ± 0.11) ( Figure 4b-d). It is critical to note that while ICP-MS analysis reports on total Cu levels, PACu-1 can only access the labile pool which is defined as Cu weakly associated with intracellular chelators such as GSH. To confirm the in vivo imaging results, we harvested the heart, kidneys, liver, and spleen from WD and wildtype mice treated with PACu-1 to perform ex vivo PA imaging. This experiment was performed to demonstrate that the PA signal intensity is higher in the liver of WD mice owing to activation of PACu-1 (Figure S3). ## BFA of Hepatic Cu(I) in Wilson's Disease via a Blind Study To evaluate the potential efficacy of PACu-1 for BFA of hepatic Cu(I) in WD, it is critical to perform a rigorous study that is free of potential bias. To this end, we designed a blind experiment where one investigator randomly selected mice belonging to either the WD or wildtype groups (eight total) for the study (Figure 5a). Each of the animals were then tagged, and their identities were concealed until the completion of the study. A second investigator then administered PACu-1 and employed PA imaging to identify the WD mice. There was no physical indicator that would allow us to distinguish the mice based on appearance. Prior to BFA, a reliable diagnostic threshold was determined in wildtype mice, which is defined as the PA770/680 ratio (0.82 ± 0.10) (vide infra). With this in mind, we identified seven animals with a PA770/680 ratio (0.63, 0.72, 0.82, 0.86, 0.87, 0.93, 0.94) within two standard deviations of the diagnostic threshold which were assigned to Group 1 (wildtype mice) (Figure 5b-d). In contrast, only one of the animals had a PA770/680 ratio (1.16) greater than three standard deviations of the diagnostic threshold and was correspondingly assigned to Group 2 (WD mouse). When the identity of the eight animals were revealed at the end of the study, we were able to correctly identify the WD mouse with greater than 99.7% confidence. ## PA Imaging of Cu(I) in a Liver Metastasis Model Finally, we turned our attention to a second model to further showcase the potential clinical impact of PACu-1. Elevated Cu in cancer of the bone, breast, gastrointestinal tract, and lungs has been associated with aggressive phenotypes and poorer prognosis. 25 There are ongoing efforts to employ Cu chelation therapy to reduce the copper status in primary tumors, as well as in metastatic lesions to treat cancer. 40,41 Since the liver is one of the most common sites of metastasis in the body, BFA of Cu(I) levels would facilitate real-time monitoring during tumor progression and treatment with a chelator. Nu/J mice were either implanted with A549 cells in the liver or received sham surgeries. After four weeks, PACu-1 was administered for PA imaging. We elected to use a PA instrument (MSOT inVision, iThera Medical) capable of whole-body crosssectional imaging for this study because a built-in feature would allow us to readily perform spectral unmixing to distinguish the signal from PACu-1 and blood. Compared to the animals that received sham surgeries (1.06 ± 0.28), the PA fold turn-on (defined as PAFinal/PAInital) of tumorbearing mice was 2.31 ± 0.78 (Figure 6a-c). This indicates that in addition to being able to sense hepatic Cu(I) in WD, PACu-1 can also detect elevated Cu in a lung cancer liver metastases model. ## Discussion One of the major goals of molecular imaging research is to develop high-performance chemical tools that can non-invasively detect and monitor disease biomarkers in a deep-tissue context. PA imaging is ideal for this application because it involves the conversion of safe near infrared light to non-toxic ultrasound waves. Since sound at clinically relevant frequencies can readily pass through the body, it is possible to obtain high resolution images beyond 10 cm in depth. 42 Despite the emergence of various acoustogenic probes for analyte sensing, none have been explored to date for BFA of disease biomarkers of the liver. Thus, our goal is to develop PA probes that can potentially replace or complement invasive biopsies currently in use to provide real-time monitoring capabilities. In this study, we chose to target Cu because while it is an essential metal ion required by all living organisms, aberrant levels are linked to genetic disorders such as WD, as well as most solid cancer types. Our group has previously developed several PA probes for Cu(II), 13,30 however we found that they were not stable when incubated with RLMs. Likewise, after synthesizing RPS1, a PA probe designed to image Cu(II) in a murine Alzheimer's disease model, 31 we discovered that it could not detect exogenous copper in the liver (Figure S4). These results are not surprising since each of these examples were designed to respond to Cu in its +2-oxidation state. PACu-1 on the other hand, is highly selective for Cu(I), affords a robust PA signal enhancement when irradiated at 770 nm, is compatible with ratiometric sensing, intrinsically targets the liver, and most importantly, exhibits an exceptional safety profile. It is worth noting that one of the major differences between BFA using PACu-1 and traditional biopsies is that our probe is designed to detect the labile Cu pool (Cu associated with GSH), whereas the latter technique reports on the total Cu content in the sample. Despite this difference, we can still reliably distinguish WD mice from wildtype controls as shown in our blind study. In addition to detecting Cu in WD, we also demonstrate PACu-1 can be used to detect elevated Cu in a liver metastasis model. We envision PACu-1 can be used to aid in the development of new Cu chelators or in conjunction with existing Cu binding drugs to monitor changes in real-time. As previously mentioned, we employed two different PA instruments for the WD and cancer studies. This indicates that PACu-1 will be compatible with a range of imaging systems including new hand-held scanners, 43,44 wearable devices, 45,46 and endoscopic setups. 47,48 Lastly, we envision this work will inspire the development of other PA probes for BFA applications. ## Materials and Methods In vitro selectivity assay. The initial absorbance (400-800 nm) of PACu-1 (5 µM, 1:1 DMF:HEPES, pH 7.4) was measured before the addition of a panel of metal ions (100 µM). These initial measurements were used to determine the initial ratio770/680 via UV-vis spectroscopy. After addition, the cuvette was sealed and incubated for 1 h. Final measurements were recorded, and the ratiometric fold turn-on was calculated by dividing the final ratio with the initial ratio. All metal solutions were prepared in water from their chloride salt, except for Ag2CO3. Cs2CO3, and tetrakis(acetonitrile)copper(I) hexafluorophosphate. Biopsy assessment of hepatic Cu via ICP-MS. BALB/c mice were anesthetized using isoflurane (1.5 -2.0%). The mice were then intraperitoneally injected with a solution of CuCl 2 (5 mg/kg) or vehicle (sterilized saline). After 2 hours, the mice were euthanized, then the liver was excised and weighed for ICP-MS analysis. The 2-hour incubation time was used to reduce Cu in vivo. To determine the Cu concentration in WT (B6129SF2/J) and WD (B6;129S1-Atp7b tm1Tcg /LtsnkJ) mice, the livers were similarly prepared as the BALB/c mice for ICP-MS analysis, except no intraperitoneal injections were performed. Ex vivo biodistribution of PACu-1 via PA imaging. BALB/c mice were anesthetized using isoflurane (1.5 -2.0%) and retro-orbitally injected with either a solution of PACu-1 (50 μM) or vehicle (10% DMSO in sterilized saline, 50 µL)). After 1 hour, the mice were euthanized, and the liver, spleen, heart, and kidneys were excised. Photoacoustic imaging of the organs was performed at 680 and 770 nm using continuous mode with a 6 second rotation time (Nexus 128+, Endra Life Sciences) The ratio of the PA signals in PACu-1 treated mice obtained upon excitation at 680 nm and 770 nm were normalized to the ratio of the PA signals in vehicle treated mice. ## Determination of the diagnostic threshold. A group of 10 wildtype mice (B6129SF2/J), which are direct controls of the WD mice (B6;129S1-Atp7b tm1Tcg /LtsnkJ), were used to determine the diagnostic threshold for hepatic Cu in Wilson's disease via PA imaging. After the mice were anesthetized using isoflurane (1.5 -2.0%), their abdomens were shaved, and they were positioned in the PA tomographer to facilitate direct imaging of the abdomen. After an image was acquired, an ROI was drawn around the liver to determine the signal intensity. The ratio of the PA signals obtained upon excitation at 680 nm and 770 nm in the ROI provided the initial PA770/680 ratio. The mice were then treated with a 50 μM solution of PACu-1 in saline containing 10% DMSO (50 μL) via retro-orbital injection. The mice were returned to their cages for 60 minutes while PACu-1 was allowed to react with the hepatic Cu. The mice were anesthetized and their livers were imaged as described previously to obtain the final PA770/680 ratio. The diagnostic threshold value (mean ± 2×SD) was determined by dividing the final PA770/680 ratio with the initial PA770/680 ratio. Identification of WD via PA imaging in a blind study. A group of eight mice consisting of one WD animal (B6;129S1-Atp7b tm1Tcg /LtsnkJ) and seven wildtype animals (B6129SF2/J mice) was tagged and randomized by the first researcher. Their identity and the total number of WD mice present was concealed until the end of the study. Importantly, these mice had no distinguishing physical features that would allow us to identify them based on appearance. PA imaging of hepatic Cu using PACu-1 was then performed by a second researcher to determine the PA ratiometric fold turn-on for each animal. Mice with a PA770/680 ratio value greater than 1.02 was assigned to Group 1 (WD) and mice with a PA770/680 ratio value between 0.62 to 1.02 was assigned to Group 2 (wildtype). After PA imaging was performed on all animals, the assignment and identity were revealed to and validated by the corresponding author. Statistical analyses. Statistical analyses were performed in Microsoft Excel. Sample sizes in all experiments were sufficiently powered to detect at least a p value < 0.05, which was significant. All data are expressed as mean ± SD. Multiple group analysis was performed using the Kruskal-Wallis Test. All other in vivo imaging data was analyzed by performing the Student's t-test (α = 0.05). *p > 0.05; **p > 0.01.

chemsum

{"title": "High-performance photoacoustic probe for biopsy-free assessment of copper status in murine models of Wilson's disease and liver metastasis", "journal": "ChemRxiv"}

enhanced_co_evolution_for_photocatalytic_conversion_of_co2_by_h2o_over_ca_modified_ga2o3

## Abstract: Artificial photosynthesis is a desirable critical technology for the conversion of CO 2 and H 2 O, which are abundant raw materials, into fuels and chemical feedstocks. Similar to plant photosynthesis, artificial photosynthesis can produce CO, CH 3 OH, CH 4 , and preferably higher hydrocarbons from CO 2 using H 2 O as an electron donor and solar light. At present, only insufficient amounts of CO 2 -reduction products such as CO, CH 3 OH, and CH 4 have been obtained using such a photocatalytic and photoelectrochemical conversion process. Here, we demonstrate that photocatalytic CO 2 conversion with a Ag@Cr-decorated mixture of CaGa 4 O 7 -loaded Ga 2 O 3 and the CaO photocatalyst leads to a satisfactory CO formation rate (>835 µmol h −1 ) and excellent selectivity toward CO evolution (95%), with O 2 as the stoichiometric oxidation product of H 2 O. Our photocatalytic system can convert CO 2 gas into CO at >1% CO 2 conversion (>11531 ppm CO) at ambient temperatures and pressures. ## C arbon dioxide (CO 2 ) concentrations in the atmosphere have increased drastically over the past few centuries owing to the combustion of carbon-rich fossil fuels such as coal, oil, and natural gas. As a major anthropogenic greenhouse gas, these ever-increasing CO 2 emissions are detrimental to the environment and will affect both ecosystems and the global climate 1 . Therefore, there is a critical requirement of mitigating CO 2 emissions to achieve sustainable development. Since the pioneering work on the photocatalytic conversion of CO 2 into formic acid (HCOOH) and methyl alcohol (CH 3 OH) over semiconductors reported by Halmann and Inoue et al. 2,3 , the photocatalytic conversion of CO 2 into other valuable feedstocks at ambient temperatures and pressures has attracted considerable attention from the scientific community as a feasible strategy for CO 2 storage and conversion . In general, the photocatalytic conversion of CO 2 over an excited semiconductor-based catalyst involves three main steps. First, CO 2 molecules are adsorbed on the photocatalyst surface . Second, the photogenerated electrons react with the adsorbed CO 2 species and protons (H + ) to yield products such as carbon monoxide (CO), methane (CH 4 ), CH 3 OH, and HCOOH. Among these possible reduction products, CO is one of the most useful because it is widely combined with H 2 to provide synthetic gas for use in many chemical processes, such as methanol synthesis 12,13 and the industrial Fischer-Tropsch process that produce various chemicals and synthetic fuels 14,15 . Third, the products are desorbed from the photocatalyst surface. However, as the H/H 2 redox potential (−0.41 V vs. NHE at pH 7) is more positive than that for CO 2 /CO (−0.52 V vs. NHE at pH 7), the generation of H 2 from H + is preferable for the photocatalytic conversion of CO 2 into CO, where H 2 O acts as the electron donor . Moreover, because of the high thermodynamic stability of the linear CO 2 molecule, the fixation and activation of CO 2 are also immense challenges in the photocatalytic conversion of CO 2 by H 2 O 4,19 . Thus, although various heterogeneous photocatalysts have been reported for the photocatalytic conversion of CO 2 into CO with H 2 O as the electron donor , the photocatalytic activity for CO evolution remains limited to a few micromoles, while the photocatalytic conversion rate of CO 2 into CO is <0.15%. Based on the processes involved in the photocatalytic conversion of CO 2 described previously, we deduce that the photocatalytic activity of the photocatalyst for CO 2 conversion can be improved by increasing CO 2 adsorption, charge separation, and product desorption. Due to the fact that CO 2 acts as a Lewis acid that bonds easily with Lewis bases 25 , many studies have focused on improving CO 2 adsorption by modifying the photocatalyst surface with a CO 2 adsorbent, such as NaOH 26 , amino groups 27 , and rare earth species 28 , to increase the photocatalytic activity and selectivity for CO 2 conversion by H 2 O. Our group reported that modifying the photocatalyst surface with alkaline earth metals (e.g., Ca, Sr, and Ba) enhanced the conversion of CO 2 and the selectivity toward CO evolution 29 . Moreover, we found that a Ag@Cr core/shell cocatalyst suppresses the backward reaction from CO and O 2 to CO 2 , and enhances the adsorption of CO 2 , resulting in a highly selective photocatalytic CO 2 conversion 30,31 . In this study, we exploited the above techniques and successfully fabricated a Ag@Cr-decorated mixture of CaGa 4 O 7 -loaded Ga 2 O 3 and CaO photocatalyst, which exhibits a high CO formation rate (>835 µmol h −1 ) per 0.5 g of catalyst, in addition to high selectivity toward CO evolution (>95%) with the stoichiometric production of O 2 as the oxidation product of H 2 O during the photocatalytic conversion of CO 2 by H 2 O. Approximately 1.2% of the CO 2 in the gas phase was transformed into CO (11531 ppm) as a product. The results reported in this study represent almost an order of magnitude higher than most previously published results, as summarized in Supplementary Table 1. ## Results and discussion Photocatalytic reduction of CO 2 by H 2 O. Table 1 shows the formation rates of CO, H 2 , and O 2 , selectivity toward CO evolution, and the balance between consumed electrons and holes over the bare Ga 2 O 3 , Ag-modified Ga 2 O 3 (Ag/Ga 2 O 3 ), Ag@Crmodified Ga 2 O 3 (Ag@Cr/Ga 2 O 3 ), and Ag@Cr-modified Caloaded Ga 2 O 3 (Ag@Cr/Ga 2 O 3 _Ca) photocatalysts during the photocatalytic conversion of CO 2 by H 2 O. No liquid products were detected in the reaction solutions in these photocatalytic systems, and H 2 , O 2 , and CO were detected as gaseous products. As no reduction products other than H 2 and CO were generated, the selectivity toward CO evolution and the balance between the consumed electrons and holes were calculated as follows: where R CO and R H2 represent the formation rates of CO and H 2 , respectively. If H 2 O acts as an electron donor, the value of e − /h + should be equal to 1. We obtained stoichiometric amounts of H 2 and CO as reduction products in addition to O 2 as the oxidation product, indicating that H 2 O serves as the electron donor. Bare Ga 2 O 3 exhibited a particularly low selectivity toward CO evolution (4%) as the electrons generated by charge transfer were not consumed in the reduction of CO 2 , but rather in the production of H 2 from H + . Modifying Ga 2 O 3 with a Ag cocatalyst enhanced the selectivity toward CO evolution (29%); however, this was not sufficient to obtain a selectivity >50%. In contrast, we succeeded in the selective photocatalytic conversion of CO 2 by H 2 O over Ag@Cr/Ga 2 O 3 . A relatively high CO formation rate (499.6 µmol h −1 ) was achieved with 77% selectivity toward CO evolution. The photocatalytic reaction for the conversion of CO 2 by H 2 O over Ag@Cr/Ga 2 O 3 and Ag@Cr/Ga 2 O 3 _Ca was carried out for at least four times, and errors in the product formation rates (H 2 , O 2 , and CO) were smaller than 5%. Controlling both, the bulk and surface of the photocatalysts, is highly important for achieving a considerably high CO formation rate and selectivity toward CO evolution. We found that the amount of Ca species significantly affected the H 2 and CO formation rates (for the product formation rates and selectivity over various Ag@Cr/Ga 2 O 3 _Ca photocatalysts see Supplementary Fig. 1). The formation rate of CO increased first and then decreased as the Ca content increased (Supplementary Fig. 1a-g). In contrast, the formation rate of H 2 over the Ag-Cr/ Ga 2 O 3 _Ca_x samples increased monotonically with increasing amount of Ca species. The Ag-Cr/CaGa 4 O 7 photocatalyst was only active for H 2 evolution derived from water splitting (Supplementary Fig. 1h). The Ag@Cr/Ga 2 O 3 _Ca photocatalyst exhibited the highest CO formation rate (794.2 µmol h −1 ), and the selectivity toward CO evolution was approximately 82%. Additionally, CO production from the photocatalytic conversion of CO 2 after photoirradiation for 15 h over Ag@Cr/Ga 2 O 3 _Ca was more stable than that over Ag@Cr/Ga 2 O 3 (for the product formation rates for 15 h see Supplementary Fig. 2), which indicates that the presence of Ca species is not only beneficial for improving the photocatalytic activity and selectivity, but also for improving stability during the photocatalytic conversion of CO 2 to CO. Various control experiments were carried out to confirm the source of CO during the photocatalytic conversion of CO 2 by H 2 O, the results of which are shown in Supplementary Fig. 3. We did not detect any appreciable amounts of products under dark conditions or in the absence of a photocatalyst. In addition, H 2 was the main product formed when Ar gas was used instead of CO 2 or in the absence of NaHCO 3 . The control experiments confirmed that the evolved CO originated from the CO 2 gas introduced into the samples and not from carbon contaminants. Photocatalyst characterization. The actual amounts of the Ca species loaded into Ga 2 O 3 at different CaCl 2 concentrations were measured using inductively coupled plasma optical emission spectrometry (ICP-OES) (Supplementary Table 2). We found that almost all the Ca species were loaded into the Ga 2 O 3 photocatalyst when the CaCl 2 concentration was <0.001 mol L −1 . However, not all the Ca species could be loaded into Ga 2 O 3 at higher CaCl 2 concentrations. Note that even when no CaCl 2 was added during the preparation of Ga 2 O 3 , trace amounts of Ca were detected in Ga 2 O 3 , which is likely due to Ca impurities present in the experimental vessels or precursor reagents. Hereinafter, we refer to the Ca-loaded Ga 2 O 3 photocatalysts as Ga 2 O 3 _Ca_x (x = 0.32, 0.62, 1.1, 1.6, 2.1, 3.3 mol%) based on the Ca/Ga molar ratio determined by ICP-OES. Figure 1a shows the X-ray diffraction (XRD) patterns of the bare Ga 2 O 3 , Ga 2 O 3 _Ca_x, and CaGa 4 O 7 photocatalysts. As indicated, gradual changes in the diffraction peaks assigned to the (020), (311), (400), (002), and (330) facets of CaGa 4 O 7 (JSPDS 01-071-1613) were observed as the amount of Ca species was increased. In general, a high Ca loading is favorable for the formation of CaGa 4 O 7 . We observed no distinct shifts in the diffraction peaks for the Ga 2 O 3 _Ca_x samples compared with those of bare Ga 2 O 3 . As the ionic radius of Ca 2+ (0.099 nm) 32 is larger than that of Ga 3+ (0.062 nm) 33 , the unshifted XRD peaks imply that Ca 2+ does not act as a dopant in the bulk Ga 2 O 3 lattice. However, there was a clear increase in the peak intensity at 2θ = 30.1°and an apparent decrease in that at 2θ = 30.5°with increasing amount of Ca species (Fig. 1b), which are possibly ascribed to the formation of CaGa 4 O 7 species on Ga 2 O 3 . The increased intensity of the Ca 2p X-ray photoelectron spectroscopy (XPS) peak (Fig. 1c) also indicates that the amount of Ca species on the Ga 2 O 3 surface increased with increasing Ca levels. In addition, the XPS peak locations in the Ca 2p spectra of the Ga 2 O 3 _Ca_x photocatalysts are similar to those of CaGa 4 O 7 , but different from those of CaO. The Ca 2p XPS profiles suggest that a thin CaGa 4 O 7 layer forms on the Ga 2 O 3 surface and that the amount of CaGa 4 O 7 increases as the amount of Ca is increased. We further confirmed the morphological changes in the Ga 2 O 3 _Ca sample by field-emission scanning electron microscopy (SEM), as shown in Fig. 1d. Both ends of the Ga 2 O 3 nanoparticles gradually sharpened and their surfaces became smoother as the amount of Ca species increased, especially when the Ca amount was higher than 1.1 mol%. This smoothing of the Ga 2 O 3 surfaces with increasing Ca/Ga molar ratio resulted in a decrease in the Brunauer-Emmett-Teller (BET) specific surface area of Ga 2 O 3 _Ca_x (Supplementary Fig. 4), which is attributable to the modification of CaGa 4 O 7 , as we confirmed from the XRD patterns and the XPS results that a CaGa 4 O 7 layer was formed on the Ga 2 O 3 surface. The close linkage between CaGa 4 O 7 and Ga 2 O 3 on the Ga 2 O 3 surface was confirmed by field-emission transmission electron microscopy (TEM) and high-resolution TEM (HRTEM) (Fig. 2). The marked lattice spacings (0.296 and 0.255 nm) in Fig. 2b correspond to the (130) and (111) planes of CaGa 4 O 7 and Ga 2 O 3 , respectively. The core-shell-structured Ag@Cr cocatalyst was successfully loaded onto the Ga 2 O 3 _Ca surface using the photodeposition method (Fig. 2c, d), as reported previously by us 31 . Role of the Ca species. Figure 3 shows the Fourier transform infrared (FTIR) spectra of the CO 2 -adsorbed samples after introducing CO 2 at ~0.2 Torr. When CO 2 was introduced into the Ga 2 O 3 sample, three absorbance peaks were observed at 1634, 1432, and 1225 cm -1 , which can be ascribed to asymmetric CO 3 stretching vibrations [ν as (CO 3 )], symmetric CO 3 stretching vibrations [ν s (CO 3 )] of monodentate bicarbonate species (m-HCO 3 -Ga), and OH deformation vibrations [δ(OH)], respectively . The absorbance peaks at 1699 and 1636 cm -1 for the CO 2 -adsorbed CaO sample can be attributed to bridging carbonate stretching and asymmetric CO 3 stretching vibrations [ν as (CO 3 )] of the bicarbonate species, respectively. The broad structureless absorbance peaks between 1480 and 1318 cm -1 can be attributed to the symmetric and asymmetric CO 3 stretching of unidentate carbonate, as well as the symmetric CO 3 stretching [ν s (CO 3 )] of bicarbonate . When the Ga 2 O 3 surface was modified with a small amount of Ca species, absorbance peaks attributable to CO 2 adsorption by both Ga 2 O 3 and CaO were observed after CO 2 was introduced into the Ga 2 O 3 _Ca_1.1 sample. However, when the Ga 2 O 3 surface was modified with large amounts of Ca species, the absorbance peaks attributed to CO 2 adsorption on Ga 2 O 3 had low intensity and mainly corresponded to the broad peaks derived from the adsorption of CO 2 on CaGa 4 O 7 . Supplementary Fig. 5 shows the FTIR spectra of CO 2 -adsorbed Ga 2 O 3 , Ga 2 O 3 _Ca_1.1, Ga 2 O 3 _Ca_3.3, and CaGa 4 O 7 samples after introducing the same amount of CO 2 at various pressures in the 0.1-40.0 Torr range. CO 2 was adsorbed significantly more on the Ga 2 O 3 _Ca_1.1 surface than on the Ga 2 O 3 surface due to its adsorption at both Ga and Ca sites. However, the CaGa 4 O 7 surface was not conducive to CO 2 adsorption; therefore, CO 2 adsorbed less onto the Ga 2 O 3 _Ca_3.3 surface than the Ga 2 O 3 _Ca_1.1 surface. Figure 4 shows the FTIR spectra of the adsorbed CO 2 species on Ga 2 O 3 _Ca_1.1 after different durations of photoirradiation. As the photoirradiation time increased from 0 to 106 h, the bands at 1225 [δ(OH)-Ga] and 1408 cm -1 [ν s (CO 3 )-Ca] decreased and vanished after 104 h. At the same time, new bands gradually appeared at 1581, 1388, and 1353 cm -1 (asymmetric CO 2 stretching [ν as (CO 2 )], CH deformation [δ(CH)], and symmetric CO 2 stretching [ν s (CO 2 )] assigned to formate species (HCOO-Ga/Ca), respectively) . As the photoirradiation continued, the formate species were consumed and gaseous CO (fundamental vibration band at 2143 cm −1 ) 42 was formed simultaneously. This result indicates that the bicarbonate species is the intermediate during the photocatalytic conversion of CO 2 , and the formates transform into CO with photoirradiation, which is consistent with our previous results 43,44 . It is worth mentioning that in addition to the presence of intermediate species on the Ga 2 O 3 surface ([δ(OH)-Ga]), the modification by Ca species further increased the amount of intermediate on Ga 2 O 3 _Ca_1.1. As the photocatalytic conversion of H + into H 2 and the conversion of CO 2 into CO are two competing processes in an aqueous solution, the high adsorption of CO 2 at the base site leads to high photocatalytic activity and selectivity toward CO evolution during the photocatalytic conversion of CO 2 by H 2 O. In order to demonstrate that the presence of CaO on the Ga 2 O 3 surface enhances the photocatalytic activity and selectivity during the photocatalytic conversion of CO 2 into CO, we investigated the photocatalytic performance during the conversion of CO 2 by H 2 O over various Ag@Cr/CaO/Ga 2 O 3 photocatalysts, the results of which are shown in Fig. 5. We found that the Ag@Cr/ Ga 2 O 3 _Ca_1.1 photocatalyst (with a low amount of CaO generated on the Ga 2 O 3 surface) significantly enhanced the rate of CO formation during the photocatalytic conversion of CO 2 by H 2 O compared with bare Ag@Cr/Ga 2 O 3 (Fig. 5a, b). However, no significant change in the rate of CO formation and selectivity toward CO evolution was observed for the sample labeled "Ag@Cr/(1.1 mol%CaO/Ga 2 O 3 )" (in which 1.1 mol% CaO was physically loaded onto Ga 2 O 3 by grinding before loading Ag@Cr cocatalyst onto the CaO/Ga 2 O 3 surface) as compared to bare Ga 2 O 3 (Fig. 5c). Because uncalcined CaO-loaded Ga 2 O 3 easily dissolves in H 2 O, we increased the CaO loading on the Ga 2 O 3 surface to 30 mol% using the same grinding method (labeled "Ag@Cr/(30 mol%CaO/Ga 2 O 3 )"), which resulted in an increased rate of CO formation and a decrease in H 2 formation (Fig. 5d). However, no improvement in photocatalytic activity and selectivity was observed when 30 mol% CaO was mixed with the prepared Ag@Cr/Ga 2 O 3 and ground together (Fig. 5e) or when they were directly mixed in the reaction solution (Fig. 5f). These results clearly reveal that the addition of CaO on the Ga 2 O 3 surface enhances the rate of CO formation and suppresses that of H 2 during the photocatalytic conversion of CO 2 by H 2 O. In addition, the tight junction between Ga 2 O 3 , CaO, and the Ag@Cr cocatalyst is crucial for the superior photocatalytic activity and selectivity of the photocatalyst for the conversion of CO 2 into CO. In our previous work, we confirmed that Ag acts as an active site while the Cr(OH) 3 •H 2 O layer exterior to the Ag core increases CO 2 adsorption 30,31 . Hence, the Ag@Cr cocatalyst should be loaded at the CaO/Ga 2 O 3 interface in order to facilitate contact between the CaO-adsorbed CO 2 species and the Ag active sites. Notably, although CaGa 4 O 7 exhibited high selectivity toward H 2 evolution, the H 2 formation rate for CaGa 4 O 7 was significantly lower than that for Ga 2 O 3 _Ca_3.3 (for the product formation rates over Ag-Cr/Ga 2 O 3 _Ca_3.3 see Supplementary Fig. 6). This indicates that the presence of CaGa 4 O 7 on the Ga 2 O 3 surface enhances the overall photocatalytic efficiency during the photocatalytic reaction, including CO 2 conversion and water splitting. The Mott-Schottky plot (Supplementary Fig. 7) and the absorption spectra converted from the diffuse reflectance spectra using the Kubelka-Munk equation (Supplementary Fig We expect that by exploiting the high CO 2 adsorption of CaO and the high photocatalytic efficiency of CaGa 4 O 7 /Ga 2 O 3 , we can further improve the photocatalytic activity and selectivity of the photocatalyst to maximize the conversion of CO 2 into CO by H 2 O. Figure 6a shows the formation rates of H 2 , O 2 , and CO during the photocatalytic conversion of CO 2 by H 2 O for the Ga 2 O 3 _Ca_3.3 photocatalyst physically mixed with 30 mol% of CaO and Ag@Cr as the cocatalyst. As indicated, a high formation rate of CO (>835 µmol h -1 ) was achieved, in addition to an excellent selectivity toward CO evolution (>95%), with a stoichiometric amount of evolved O 2 . Both 12 CO and 13 CO were detected using quadrupole mass spectrometry (MS), and the peaks at m/z = 28 and m/z = 29 were located at the same positions as those detected by gas chromatography (GC) during the photocatalytic conversion of 13 CO 2 (for the isotopic lead experiments see Supplementary Fig. 10). Indeed, our results indicate that the detected 12 CO was produced from the reduction of 12 CO 2 derived from the NaHCO 3 additive in the solution 43 . As shown in Fig. 6b, with the consumption of 12 CO 2 derived from NaHCO 3 , the amount of generated 12 CO gradually decreased, while the 13 CO content increased under continuous bubbling of 13 CO 2 . The total amounts of 13 CO and 12 CO detected by MS were consistent with the amount of CO detected by GC (Fig. 6c), which indicates that the CO was generated as the reduction product of either CO 2 introduced in the gas phase or from NaHCO 3 , rather than from any organic contaminants on the photocatalyst surface. The converted concentration of CO based on the CO formation rate was found to be 11,531 ppm, indicating that ~1.2% of CO 2 in the gas phase was transformed into CO (see Supplementary Information for the calculation details. The actual amounts of CO detected are shown in Supplementary Movie 1). In our previous work, we had found that basic oxides and hydroxides such as Cr(OH) 3 31 , SrO 44 , and rare earth (RE) hydrates and oxides 28 function as good CO 2 storage materials by generating the corresponding (hydroxy)carbonate compounds (e.g., Cr(OH) x (CO 3 ) y and RE 2 (OH) 2(3−x) (CO 3 ) x ), and they improve the photocatalytic activity and selectivity toward CO evolution. Now, we propose a possible mechanism for the photocatalytic conversion of CO 2 by H 2 O over Ag@Cr/CaO/ CaGa 2 O 7 /Ga 2 O 3 , as shown in Fig. 7. During the photocatalytic conversion of CO 2 in an aqueous solution of NaHCO 3 , the Cr (OH) 3 •H 2 O and CaO species that are in close contact with Ag particles easily form (hydroxy)carbonate species (named M (OH) x (CO 3 ) y , M=Cr or Ca) 31 , which greatly increase the concentration of CO 2 -related species around the Ag active sites, thereby improving selectivity for the photocatalytic conversion of CO 2 into CO instead of water splitting. On the other hand, the Ga 2 O 3 /CaGa 4 O 7 heterojunction improves the efficiency for spatial separation of the photogenerated carriers, which also increases the photocatalytic activity for the conversion of CO 2 into CO. Moreover, while the Cr(OH) 3 •xH 2 O shell outside the Ag particle can be oxidized to Cr 6+ and dissolve into the solution during the photocatalytic conversion of CO 2 46 , the presence of CaO around the Ag active site compensates for the reduced activity from the dissolution of Cr species. As a result, Ag@Cr/ Ga 2 O 3 _Ca is photocatalytically much more stable than Ag@Cr/ Ga 2 O 3 . Herein, we reported the photocatalytic conversion of CO 2 using a Ag@Cr/CaO/CaGa 4 O 7 /Ga 2 O 3 photocatalyst, in which a satisfactory CO formation rate (>835 µmol h −1 ) and an excellent selectivity toward CO evolution (95%) were achieved with the stoichiometric production of O 2 as the oxidation product of H 2 O. Through the use of various characterization techniques, we found that the CaO and CaGa 4 O 7 formed on the Ga 2 O 3 surface improved the adsorption of CO 2 at basic sites in addition to enhancing the total photocatalytic efficiency. In addition, the physical mixing of CaGa 4 O 7 /Ga 2 O 3 with CaO was a particularly simple and convenient technique for exploiting the high CO 2 adsorption ability of CaO and the high photocatalytic efficiency of CaGa 4 O 7 /Ga 2 O 3 . These results are of particular interest, considering that previously, only insufficient amounts of CO 2 reduction products were produced during artificial photosynthesis. ## Methods Ca-modified Ga 2 O 3 (Ga 2 O 3 _Ca) was prepared using the ammonia precipitation method reported by Sakata et al. 47 . In this method, Ga(NO 3 ) 3 •nH 2 O (12.6 g) was dissolved in 200 mL of deionized water or CaCl 2 solution in ultrapure water at various concentrations. Hydroxylation was carried out by dripping an ammonium hydroxide solution until the pH level reached 9.1. The obtained hydroxides were centrifuged and dried overnight. The Ga 2 O 3 _Ca sample was obtained by calcining the precursor at 1273 K for 10 h. Ag@Cr/Ga 2 O 3 _Ca was synthesized using the photodeposition method reported in our previous work 30 . In this method, the asprepared Ga 2 O 3 _Ca powder (1.0 g) was dispersed in ultrapure water (1.0 L) containing the necessary amounts of silver nitrate (AgNO 3 ) and chromium (III) nitrate (Cr(NO 3 ) 3 ). The suspension was purged with Ar gas and irradiated under a 400 W high-pressure Hg lamp with Ar gas flowing for 1.0 h, followed by filtration and drying at room temperature (~298 K). The Ag/Ga and Cr/Ga molar ratios were both 1.0 mol%. Characterization. The as-prepared Ga 2 O 3 _Ca samples were characterized using the following techniques: XRD (Model: Multiflex, Rigaku Corporation, Japan) with Cu Kα radiation (λ = 0.154 nm); XPS (Model: ESCA 3400, Shimadzu Corporation, Japan) with Mg Kα radiation; SEM (Model: SU-8220, Hitachi High-Technologies Corporation, Japan); TEM (Model: JEM-2100F, JEOL Ltd, Japan); and UV-Visible spectroscopy (V-650, JASCO) with an integrated sphere accessory. The BET surface areas of the photocatalyst samples were determined from their N 2 -adsorption isotherms at 77 K using a volumetric gas-adsorption measuring instrument (Model: BELSORP-miniII, MicrotracBEL Corp. (formerly BEL Japan, Inc.), Japan). Prior to these measurements, each sample was evacuated at 473 K for 1 h using a sample pretreatment system (Model: BELPREP-vacII, MicrotracBEL Corp. (formerly BEL Japan, Inc.), Japan). ICP-OES (Model: iCAP7400, Thermo Fisher Scientific, USA) was used to determine the actual amounts of Ca modified on the Ga 2 O 3 surface. The FTIR spectra of the adsorbed carbon species were recorded using an FTIR spectrometer (Model: FT/IR-4700, JASCO International Co., Ltd., Japan) equipped with a mercury-cadmium-tellurium (MCT) detector and cooled with liquid N 2 in the transmission mode at 303 K. Each sample (~30 mg) was pressed into a wafer (diameter: 10 mm) and introduced into the instrument in a cylindrical glass cell with calcium fluoride (CaF 2 ) windows. The wafer was evacuated at 673 K for 30 min before being examined, followed by treatment with O 2 at ~40 Torr for 30 min, after which the wafer was evacuated for 30 min and cooled to 303 K. The data for each FTIR spectrum were obtained from 128 scans with a resolution of 4 cm −1 . The energy gap of the band structure and flat band potential of the Ga 2 O 3 _Ca samples were determined using the Davis-Mott and Mott-Schottky equations, respectively; the experimental details are provided in the Supplementary Information. Photocatalytic reaction. The photocatalytic reduction of CO 2 was carried out using a flow system with an inner irradiation-type reaction vessel. The synthesized photocatalyst (0.5 g) was dispersed in ultrapure water (1.0 L) containing 0.1 M sodium bicarbonate (NaHCO 3 ). The CO 2 was bubbled into the solution at a flow rate of 30 mL min −1 . The suspension was illuminated using a 400 W high-pressure Hg lamp with a quartz filter, and the assembly was connected to a water-cooling system. The amounts of evolved H 2 and O 2 were detected using a gas chromatography system fitted with a thermal conductivity detector (TCD-GC, Model: GC-8A, Shimadzu Corporation, Japan) and a 5A molecular sieve (MS 5A) column, and Ar was used as the carrier gas. The amount of evolved CO was analyzed using a gas chromatography system fitted with a flame ionization detector (FID-GC, Model: GC-8A, Shimadzu Corporation, Japan), a methanizer, and a ShinCarbon ST column, and N 2 was used as the carrier gas. High-performance liquid chromatography (Model: LC-4000, JASCO, USA) was used to detect the presence of liquid products. In the isotope experiment, 12 CO 2 was replaced by 13 CO 2 . The formation rates of H 2 , O 2 , 12 CO, and 13 CO under photoirradiation were detected using a quadrupole mass spectrometer (BELMASS, Microtrac BEL) combined with a TCD-GC detector.

chemsum

{"title": "Enhanced CO evolution for photocatalytic conversion of CO2 by H2O over Ca modified Ga2O3", "journal": "Nature Communications Chemistry"}

approaches_for_enhancing_the_analysis_of_chemical_space_for_drug_discovery

## Abstract: Chemical space is a powerful, general, and practical conceptual framework in drug discovery and other areas in chemistry that addresses the diversity of molecules and it has various applications. Moreover, chemical space is a cornerstone of chemoinformatics as a scientific discipline. In response to the increase in the set of chemical compounds in databases, generators of chemical structures, and tools to calculate molecular descriptors, novel approaches to generate visual representations of chemical space in low dimensions are emerging and evolving. Such approaches include a wide range of commercial and free applications, software, and open-source methods. Herein, the current state of chemical space in drug design and discovery is reviewed. The topics discussed herein include advances for efficient navigation in chemical space, the use of this concept in assessing the diversity of different data sets, exploring structure-property/activity relationships for one or multiple endpoints, and compound library design. Recent advances in methodologies for generating visual representations of chemical space have been highlighted, thereby emphasizing open-source methods. It is concluded that quantitative and qualitative generation and analysis of chemical space require novel approaches for handling the increasing number of molecules and their information available in chemical databases (including emerging ultra-large libraries). In addition, it is of utmost importance to note that chemical space is a conceptual framework that goes beyond visual representation in low dimensions. However, the graphical representation of chemical space has several practical applications in drug discovery and beyond. ## Introduction Chemical space occasionally referred to in the literature as the "chemical universe" is a concept that has become significant in chemoinformatics as an independent theoretical discipline . Chemical space refers to all possible molecules and multi-dimensional conceptual spaces representing their structural and functional properties. In other words, chemical space is a contraction of the "chemical descriptor vector space" defined by the numerical vector D encoding molecular structure and/or property aspects as elements of the descriptor vector D. Therefore, and in contrast to cosmic space, chemical space is not a physical space and is not unique, because anyone is free to customize its vector space based on structural and functional properties. Indeed, structural and functional representation is arguably the most relevant feature in virtually all chemoinformatics or computational studies . Applications of chemical space concept have progressed from drug discovery to other areas in chemistry, including organic synthesis, food chemistry, and material sciences, to name a few examples reviewed in the literature . A key distinction between the different types of the systematic representations of chemical spaces in compound datasets lies in the type of properties or descriptors that are used to represent the compounds of interest. For instance, the nature of the descriptors used to represent small organic molecules is typically different from that describing chemicals with applications in material sciences. In some instances, the qualitative concept of chemical space is actively used to guide drug discovery projects; however, developing a consistent method to visually represent chemical space remains elusive because of the challenge in generating a consistent manner of representing chemical structures. A typical method employed in this area includes analyzing the chemical space of metalcontaining compounds . Initially, in drug discovery, chemical space concept proved useful to understand and generate knowledge of the pharmacokinetic properties and molecular diversity of biologically relevant compounds . As the number of chemical compounds and their information in databases increased, more sophisticated molecular descriptors and visualization techniques were developed to expand their applications. For instance, explorations of chemical space have considerably improved our comprehension of biology and led to the development of several tools for investigating structure-property and structure-activity relationships (SPR, SAR, and SP(A)R) . In addition, this concept has raised interesting questions regarding the estimated size of chemical space, and has motivated several research groups to enumerate large libraries of virtual compounds . Recently, the availability of software libraries and the rise of artificial intelligence (AI) have led to the emergence of several tools that integrate machine learning (ML) methods as versatile tools to design, generate, and visualize the chemical space of small molecules . Most chemoinformatics tools use two discrete procedures to represent chemical space: (i) calculation of molecular descriptors and (ii) projection from descriptor space into a two-dimensional (2D) plane or three-dimensional (3D) volume using one of the several known techniques . The descriptors can be selected from the structure (constitution, configuration, and conformation) or properties (physical, chemical, and biological) of the molecules present. The types of descriptors guide the interpretations and predictions that can be made . Therefore, descriptors based on physicochemical properties have been widely used to encode absorption, distribution, metabolism, and excretion properties that play an important role in determining the characteristics of therapeutic agents, such as absorption, solubility, and permeability through the membrane . Other commonly used molecular representations are fingerprint-based descriptors in which the Molecular Access System (MACCS) Keys and Extended Connectivity Fingerprints (ECFPs) are among the most widely used methods to assess the structural diversity of small organic molecules. To improve the visual representation of chemical space and expand its application to larger compounds such as peptides, oligonucleotides, and complex carbohydrates, Capecchi et al. recently proposed the MAP4 (MinHashed Atom-Pair fingerprint up to four bonds) molecular fingerprint that, in principle, can encode compounds of virtually any size . MAP4 combines substructure and atom-pair concepts to capture global and specific characteristics of the molecular size and shape, which are captured by the bond distance information encoded into the MAP4. To generate graphical representations of chemical space, coordinate- and cell-based approaches have been developed. Recently, molecular networks have been recommended for addressing the dimensionality problem . Because it is complicated to visualize multidimensional spaces, coordinate-based approaches usually rely on dimensionality reduction techniques to transform high-dimensional data into two or three dimensions. Over the past two decades, several research groups have implemented different dimensionality reduction techniques to analyze chemical space. Such advances were extensively reviewed in a previous study . The most common techniques include principal component analysis (PCA), t-distributed stochastic neighbor embedding (t-SNE) , and selforganizing map (SOM) . Previous studies have discussed the exploration of SPR in the context of chemical spaces . The objective of this manuscript is to review recent advances in methodologies for generating lowdimensional visual representations of chemical spaces. We emphasize on freely available and opensource methods. Despite the concept of chemical space having broad applicability in several areas of chemistry, including in organic and inorganic molecules (for instance, metallodrugs used in drug discovery ), this review focuses on the development and applications of chemical space to small organic compounds. It is expected that some of these methods can be extended or adapted to explore chemical space of other types of compounds. Using an analogy with the concept of a multiverse in cosmology, regions in the universe detached from one another exhibit distinct properties , and the systematic description of different types of chemical compounds with varying properties (metalcontaining molecules, larger chemical compounds relevant in polymers, material science, and biochemistry) can be increased to chemical multiverses. ## State-of-the-art applications of chemical space The concept of chemical space has several practical applications. In this study, we organized the applications into four categories: selection of molecules from existing compound libraries, analysis of molecular diversity, SP(A)R, and library design (i.e., to assist the expansion of the chemical libraries). ## Navigation of chemical space: selection of compounds from existing libraries The identification of biologically relevant starting points within a vast chemical space is a particularly relevant task in designing compound collections and selecting compounds from existing libraries for computational and/or experimental screening. Although it is not an easy task, it is possible to utilize the fact that the physicochemical and biological properties of molecules are associated with their molecular structures. This is known as "chemical similarity principle," which states that if two molecules share similar structures, then they will likely have similar bioactivities. Thus, the distribution of the compounds in chemical space guides the search for compounds with a specific set of properties. The choice of descriptors to define chemical space is crucial, however, it is not unique; different from cosmic space, chemical space is not invariant. Therefore, molecular representation is the cornerstone of chemical space (and basically any other computational approach). In this context, different cartographic methods have been proposed to efficiently navigate chemical spaces once a set of descriptors has been selected . Most navigation methods involve positioning a reference query molecule and scanning a large database to identify the adjacent molecules, which are molecules with properties significantly similar to those of the reference structures. Notably, the adjacent molecules to the reference compound can be identified using the full set of descriptors that define chemical space, and this can be performed independently of the visualization method to project the fulldimensional space into a 2D/3D graph. ChemGPS-NP was one of the first chemographic models used to comprehensively describe chemical space of natural products (NPs) using physicochemical properties and has proven to be useful in various applications . ChemGPS-NP is a PCA-based model of physicochemical properties, defined by a training set of carefully selected compounds that act as "satellites" or reference structures with extreme properties. ChemGPS-NP projects or "positions" new molecules into the chemical space by comparing their physicochemical properties with those of the reference structures. Although PCA-based mapping is fast and easy to compute, it omits nonlinear interactions and some map regions are overloaded with data. Some non-linear algorithms that have been implemented for chemical space visualization are t-SNE , and more recently, uniform manifold approximation and projection (UMAP) . These types of algorithms effectively visualize clusters or groups of data points and their relative proximities. Another frequently used method is SOM , a grid-based method that has been used to support lead discovery efforts and target prediction. Examples of the latter include SOM-based prediction of drug equivalence relationships and target inference generator . Generative topographic mapping (GTM) represents a probabilistic alternative to SOMs . This approach has been applied to visualize, analyze and model large collections of data sets for drug design and was also successfully used for large-scale SAR scanning . As discussed in the Introduction section, within non-coordinate-based approaches, chemical space networks (CSNs) were proposed by Bajorath et al. to address the problem of dimensionality . CSNs transform a multidimensional chemical space into a graph with the nodes representing chemical compounds and edges connecting compounds within a specific similarity boundary. These graphs provide immediate visualization that can be easily interpreted. CSNs can also be adequately characterized and compared using generally applicable statistical measures from network science. However, visualization becomes increasingly difficult as the number of compounds increases. Therefore, this method is not directly designed for diversity analysis. Recently, networks have been used as the basis for developing chemical library networks (CLNs) that can be used to explore the diversity of large and ultralarge molecular libraries. In general, representations of a tree-like nature, such as Tree MAP (TMAP), are more suitable for analyzing and interpreting large datasets . To navigate through chemical and biological spaces more intuitively, several researchers have developed methods that seek to improve the interpretation by representing molecules beyond individual data points. An example is the scaffold tree approach that graphically represents chemical space as a tree, where the leaves represent individual chemical compounds and the intermediate nodes represent scaffolds and sub-scaffolds . These representations allow a more consistent scaffold analysis in an SAR/SPR context and facilitate the identification of analog collections . To facilitate the visualization of large analog series Constellation plots have been proposed (see section 2.3) . ## Molecular diversity In drug design, the concept of chemical similarity (or chemical diversity) has been addressed using different approaches, and its applications are mainly found in ligand-based design, for instance, in identifying bioactive compounds when some active compounds are known. Similarly, chemical similarity/diversity analysis provides useful information for projects that seek to prioritize the selection of potentially active compounds for experimental evaluation. Another application is the profiling and selection of compound collections with chemically diverse structures to increase the probability of identifying new scaffolds that can lead to specific biological targets . Similarity and diversity analyses have also been integrated into de novo design strategies to evaluate the structural and molecular novelty of chemical libraries, which play an important role in fairly comparing generative approaches . Several studies reported thus far focus on the use of chemical space as an approach to assess the diversity of different datasets and explore the relationships between compound collections, from which valuable conclusions or interpretations have been obtained. For instance, the chemical space of natural compounds has been compared with other collections of compounds such as drugs approved for clinical use, synthetic molecules, and food chemicals . In general, NPs are characterized by covering a region of chemical space more extensively than synthetic compounds and approved drugs, and they also populate areas in the chemical space that are generally not synthetically accessible . The structural uniqueness and complexity of NPs have encouraged the continued use of these compounds to identify bioactive compounds for further development, optimization, or inspire the synthesis of compounds with unique scaffolds . Recent representative molecular diversity studies include the analysis of novel libraries, such as compounds applied in the food industry , peptides , focused libraries , de novo virtual libraries , and commercially available fragments libraries for medicinal chemistry . The results are summarized in Table 1. For these analyses, new molecular representations and visualization techniques were implemented. For instance, the chemical space of food compounds stored in FooDB was analyzed using ChemMaps, an approach based on reference or "satellite" compounds, that is, molecules whose distance (or similarity) to all other molecules in the chemical space yield sufficient information to produce a visual representation of the space . In principle, it is possible to generate a 3D visual representation of chemical space using satellite structures. TMAP was used in the global analysis of the peptide chemical space, whereas MAP4 was employed as the molecular representation of peptides . A similar approach was used to visualize the chemical space of NPs in the public domain . To assist the processes of decision-making and selecting compound libraries for further virtual screening or compound acquisition for high-or medium-throughput screening for epigenetic drug discovery, Flores-Padilla et al. reported a comprehensive analysis of 11 commercial libraries of varying sizes focused on epigenetic targets (with 53,443 compounds in total) . Analysis of the chemical diversity and coverage of chemical space was conducted with Constellation plots based on the chemical core scaffolds and CLNs . The latter is based on structural fingerprints and facilitates the visual representation of the chemical space of compound datasets with a significant number (millions) of compounds in an efficient manner. The analysis highlighted a commercial library with an extensive coverage of chemical space (despite low intra-molecular diversity) and identified compound collections that cover unique regions of the chemical space not populated by other epigenetic-focused libraries. As previously discussed, diversity analysis of chemical space can be used to evaluate and compare different generative approaches. For instance, Arús-Pous et al. used PCA plots of molecular quantum number (MQN) fingerprints to assess the quality of the training process in generative models . In that study, MQN PCA plots allowed the following up and improvement of the comprehension of the varying architectures of molecular generative models. Another recent and representative example of the use of chemical space to analyze diversity was performed with more than 400,000 purchasable building blocks (PBBs) provided by eMolecules (Zabolotna et al. 2021). Visualization of the chemical space of these PBBs using GTM allowed the identification of the most represented and underrepresented classes of PBBs. The results can be focused to improve PBB libraries in a way that allows efficient synthesis in a relevant medicinal chemistry space. ## Structure-property (activity) relationships As mentioned in the Introduction section, one of the major practical applications of visual representation of chemical space in drug discovery is SAR analysis where the concept of chemical space provides a solid and consistent framework for representing the structural data. When activity data are added (e.g., mapped) into a visual representation of chemical space, it is possible to navigate through the chemical space and exploring (qualitatively or quantitatively) variations in activity upon changes in chemical structures. The massive amount of data stored in chemical databases, including incomplete chemogenomic data or activity data obtained at single concentrations, makes visualization SAR difficult; however, is can be aided by the power of visualization tools. Previous studies highlighted advances in methodologies that explore SAR of compound data sets and screening collections . Recent developments in analyzing SP(A)R include constellation plots. Briefly, constellation plots are 2D graphs that combine the clustering of compound datasets based on chemical scaffolds (in particular, analog series) and the distributions or mutual relationships of analog series based on fingerprint representations. Recently constellation plots were used to analyze the SAR of a large dataset of small molecules tested in a panel of cell lines using high-throughput screening. The authors identified a consistent cell-selective analog series of chemical compounds and proposed statistics to quantify cell promiscuity and consistency . In a separate and recent analysis, constellation plots were used to uncover a promising analog series of inhibitors of tubulin-microtubules. In that study , the authors analyzed the SAR of a curated dataset of 851 compounds with anticancer activity targeting tubulin-microtubules. In particular, the constellation plots identified at least six analog series of compounds with high average activity (known as "bright regions" in chemical space). The plot also indicates an analog series with predominantly inactive molecules ("dark regions" in chemical space). In recent developments, constellation plots have been implemented in DataWarrior such that the user can explore the chemical space interactively. Another recent example of the application of chemical space to SP(A)R analysis lies at the interface of drug discovery and food chemistry . Bayer et al. explored the associations between the chemical structures of 133 compounds with known biological activities and extra-oral bitter taste receptors, which belong to the superfamily of G-protein-coupled receptors. As part of the analysis, the authors represented the chemical space of the compounds using t-SNE as a visualization tool; the compounds were represented using MACCS key fingerprints. It was observed that the visual representation of chemical space grouped chemical compounds with similar functional groups, even though the compounds can belong to different classes (depending on the type of receptors they are related to). ## Compound library design Over the last few decades, medicinal chemistry has made major breakthroughs in increasing the accessible chemical space, which is estimated to contain approximately 10 63 molecules . In this context, having access to more regions of the chemical space can, in principle, augment the probability of finding something "interesting" and valuable. Thus, algorithms and methods to augment and search these spaces can focus on the generation of new molecules to compounds with desirable properties for drug design or discovery projects. In this regard, it remains to determine the medicinally relevant chemical space as the number of therapeutic targets is evolving . A related challenge is to establish the intersection of chemical space with the biological space. These questions are being addressed by computational chemogenomics and have been noted as one of the major challenges in computer-aided drug design . Computational approaches to facilitate the design of functional molecules include the development of de novo algorithms that explore chemical spaces to generate new compounds. For instance, the de novo design algorithm for exploring chemical space scans the space and generates structures in a specific area on a user-selected pane . Similarly, Capecchi et al. developed the peptide design genetic algorithm (PDGA), a computational tool that generates highly-similarity analogs of bioactive peptides with various peptide chain topologies in a chemical space defined by the macromolecule extended atom pair fingerprint . Recently, Aspuru et al. proposed the superfast traversal, optimization, novelty, exploration, and discovery (STONED) algorithm to perform exploration and interpolation in chemical space to obtain novel molecules . STONED uses self-referencing embedded strings , a molecular representation that is more suitable for ML. This algorithm reduces the long training times, large datasets, and handcrafted rules. In general, deep generative models can operate over large spaces of molecular structures and embed the chemical properties of these structures into a vector space. These models can generate new and previously unidentified chemical compounds by decoding from this 'latent' space of chemical structures. Recent reviews of the de novo design have examined progress in generative model architecture and evaluated their efficiency with reference to experimentally validated test cases in the literature . ## Meta-analysis of applications of chemical space To understand the evolution of the concept of chemical space and its applications, a meta-analysis of the literature has been performed using the search terms "chemical space" and "drug design" in PubMed (https://pubmed.ncbi.nlm.nih.gov/). In total, the search yielded 1538 articles (November 2021) that were analyzed using VOSviewer . The results of the meta-analysis indicated that the main concurrent terms associated with the keywords used were SAR analysis and small-molecule library design (Figure 1a). Visualization of chemical space has been used frequently to support the analysis of antineoplastic agents (76 articles), protein kinase inhibitors (60), antibacterials (51), antimalarials (28), and antiviral compounds (21). Similarly, a notable number of articles related to the concept of chemical space are associated with drug repurposing (20). In particular, using network-based representations to predict drug-target interactions and more complex interactions, including drug-disease, protein-disease, and drug-side effect associations, to name a few . According to the author's keywords (Figure 1b), the most recent articles (see color scale) are focused on ML methods such as deep learning. It is also highlighted that the concept of chemical space has had recent applications in Alzheimer's disease and in emerging diseases such as COVID-19. Particularly for COVID-19, chemical space visualization proved to be a fast way to analyze and describe the huge chemical space of known antiviral compounds . For instance, GTM is one of the methods used to represent the chemical space of compounds obtained from medicinal chemistry efforts against coronaviruses (CoVs) . In particular, GTMs helped highlight the structural relationship between antivirals of different categories, predict their polypharmacological profiles, and emphasize frequently encountered chemotypes. Similarly, chemical space concept was very helpful in finding attractive compounds for repositioning and guiding the identification of potent and selective scaffolds with anti-COVID activity . Advances in AI and availability of software libraries have resulted in ML methods, such as deep learning and versatile tools for exploring chemical space for drug discovery applications . Table 2 summarizes the novel approaches using ML methods, some of which have been mentioned previously. Recent advances have focused in identifying molecules with desirable properties in large chemical spaces. To this end, genetic algorithms (GAs) , methods using variational autoencoders (VAEs) , recurrent neural networks (RNNs) , and generative antagonistic networks (GANs) have been developed. In several instances, these algorithms are associated with the generation of new molecules and have exhibited the ability to traverse chemical space more effectively, reaching optimal chemical solutions while considering fewer molecules than allowed by the brute-force screening of large chemical libraries. Similarly, several evolutionary and RNN selection mechanisms have proven successful in multi-objective optimization problems . Emerging approaches in chemical enumeration incorporate chemical reactions into ML-based generation to design novel compounds in a synthetically accessible chemical space . As mentioned, similarity-based compound networks such as CSNs allow the visualization of SAR patterns. To increase the number of practical applications of network-based chemical space representations and decrease biases in ML, it is necessary to incorporate amounts of data from chemical interactomes. In this regard, it is also necessary to improve network visualization to obtain reasonable representations of networks containing thousands of nodes. Addressing these difficulties will be useful in SAR analysis and drug repurposing. Another application of the field of neural networks has been to solve address problems related to big data and visual representation of datasets with a large number of compounds . It is anticipated that more researchers will integrate ML methods to speed up chemical space analysis and realize more efficient outcomes. • Chemical library networks (CLN) Structure-property/activity relationships • Chemical space networks (CSNs) • Constellation plots Design novel compound libraries • Chemical reactions in ML-based generation • Multi-objective optimization algorithms ## Novel implementations for visualization The interactive visualization of 2D and 3D representations of chemical spaces, in particular of large and ultra-large data sets, has been an active area of investigation. The interactive visualization of chemical space was performed using an open-source code and is freely available on websites. Web servers available in the public domain for enabling interactive visualization of chemical spaces have been reviewed recently . This review includes classical and early developments such as Chem-GPS (vide supra), a significant set of public tools developed by Reymond et al. such as Ferun, and PDB Explorer. In the past few months, progress has been made in the interactive analysis of chemical space. A notable example is the "magic rings" developed by Ertl: a freely available web page with an interactive clustering of rings and Bemis-Murcko scaffolds present in compounds in ChEMBL (28 release) with a biological activity value of 10 microM . The interactive clustering available at https://bit.ly/magicrings enables users to quickly identify the main substructures of the major target classes of relevance in drug discovery. Another recent development is the NP navigator , which further bridges the application of cheminformatics in NP research . The NP navigator, publicly available at https://infochm.chimie.unistra.fr/npnav/chematlas_userspace/, is an implementation of the visualization algorithm GTM maps that explores interactively the chemical space of COCONUT (Collection of Open Natural Products database) (currently the largest collection of NPs in the public domain), bioactive molecules in ChEMBL, and purchasable compounds from the ZINC database . Interactive navigation can be used to explore chemical compounds based on different representations such as physicochemical properties, scaffold distribution, commercial availability, and biological activity. In a recent study, Chávez-Hernández et al. implemented an interactive visualization of the chemical space of a newly generated library of HIV-1 viral protease inhibitors assembled from NP fragments. Visual representation of the chemical space was based on TMAPs and molecular fingerprints. The interactive representation of the chemical space enables the user to navigate through a synthetic compound library of pseudo-NPs designed de novo. ## Expert opinion Chemical space is a core concept in chemoinformatics with several practical applications in drug discovery and other areas in chemistry. Typically, chemical space is used for selecting specific sets of compounds for further computational or experimental screening, diversity, and SP(A)R analysis, and to guide the design of novel molecules. The latter application is intended such that the newly generated compounds are at the intersection of the biologically relevant chemical space. In any application, compound representation is a key variable in qualitative or quantitative chemical space analysis (including visual representation); it has to be in line with the objective of the study as it will guide the interpretation of the analysis. Currently, ML methodologies continue to open new possibilities for generating hundreds and thousands of new molecules from an exhaustive search in chemical space. To perform the search in the chemical space faster and more efficiently, in particular for large data sets, the visualization methods should scale well with the number of molecules ("haystack size"); find the most relevant compounds (e.g., find the "needle," irrespective of the size of the haystack); and be affordable to run on standard hardware. In recent years, with a significant an increasing number of molecules to be analyzed, novel methods to generate visual representations of chemical space have been developed. While interpreting such visualizations, one should consider that they are approximations and that the "true" chemical space is defined by the complete set of descriptors used. Because it is challenging to select the appropriate method according to the expected qualities of the visualization, it is advisable to complement the visual (e.g., qualitative) analysis of chemical space with a quantitative analysis considering the entire multidimensional space. In this regard, it is advisable to consider consensus approaches: multiple representations of chemical space (at least more than one), because each visualization will capture part of the "true" chemical space. As part of the progress in method development, there have been notable developments in the implementation of freely available online resources. In this manner, the user can interactively explore the chemical space of compound datasets. There are still challenges in exploring the chemical space for drug discovery, such as developing consistent representations of metal-containing compounds. Other challenges include consistently representing the chemical space of non-traditional small-and medium-sized biologically relevant compounds such as peptides, macrocycles, and metal-containing clinical candidates.

chemsum

{"title": "Approaches for enhancing the analysis of chemical space for drug discovery", "journal": "ChemRxiv"}

functional_role_of_an_unusual_tyrosine_residue_in_the_electron_transfer_chain_of_a_prokaryotic_(6–4)

## Abstract: Cryptochromes and photolyases form a flavoprotein family in which the FAD chromophore undergoes light induced changes of its redox state. During this process, termed photoreduction, electrons flow from the surface via conserved amino acid residues to FAD. The bacterial (6-4) photolyase PhrB belongs to a phylogenetically ancient group. Photoreduction of PhrB differs from the typical pattern because the amino acid of the electron cascade next to FAD is a tyrosine (Tyr391), whereas photolyases and cryptochromes of other groups have a tryptophan as direct electron donor of FAD. Mutagenesis studies have identified Trp342 and Trp390 as essential for charge transfer. Trp342 is located at the periphery of PhrB while Trp390 connects Trp342 and Tyr391. The role of Tyr391, which lies between Trp390 and FAD, is however unclear as its replacement by phenylalanine did not block photoreduction. Experiments reported here, which replace Tyr391 by Ala, show that photoreduction is blocked, underlining the relevance of Tyr/Phe at position 391 and indicating that charge transfer occurs via the triad 391-390-342. This raises the question, why PhrB positions a tyrosine at this location, having a less favourable ionisation potential than tryptophan, which occurs at this position in many proteins of the photolyase/ cryptochrome family. Tunnelling matrix calculations show that tyrosine or phenylalanine can be involved in a productive bridged electron transfer between FAD and Trp390, in line with experimental findings. Since replacement of Tyr391 by Trp resulted in loss of FAD and DMRL chromophores, electron transfer cannot be studied experimentally in this mutant, but calculations on a mutant model suggest that Trp might participate in the electron transfer cascade. Charge transfer simulations reveal an unusual stabilization of the positive charge on site 391 compared to other photolyases or cryptochromes. Water molecules near Tyr391 offer a polar environment which stabilizes the positive charge on this site, thereby lowering the energetic barrier intrinsic to tyrosine. This opens a second charge transfer channel in addition to tunnelling through the tyrosine barrier, based on hopping and therefore transient oxidation of Tyr391, which enables a fast charge transfer similar to proteins utilizing a tryptophan-triad. Our results suggest that evolution of the first site of the redox chain has just been possible by tuning the protein structure and environment to manage a downhill hole transfer process from FAD to solvent. ## Introduction Cryptochromes and photolyases are homologous proteins with a central flavin adenine dinucleotide (FAD) chromophore that fulfl different biological functions, which are most often triggered by light. During a process termed photoreduction, oxidised FAD of cryptochromes or photolyases takes up one or two electrons to convert to the semiquinone or the fully reduced state, respectively. In cryptochromes, which often function as photoreceptor proteins, FAD adopts the oxidised form in darkness. In these proteins the photoreduction is regarded as the frst step of a signal transduction cascade. 1 Cryptochrome of migrating birds functions as molecular compass, due to the radical pair formed in the semiquinone state. In photolyases, which are light triggered enzymes that repair UV-damaged DNA, the chromophore assumes the fully reduced form, FADH , in vivo but converts to the semireduced or oxidised form under aerobic conditions in vitro. Photoreduction is important to ensure a high level of reduced FADH , which is required for DNA repair. Because FAD is embedded in the centre of the protein, a direct transfer of electrons from solution is not possible. Crystal structures and site directed mutagenesis identifed amino acids that constitute an electron transfer (ET) cascade. Most often, replacement of relevant Trp or Tyr by Phe results in a slower or blocked photoreduction. The interpretation of mutant results can however be hampered by the possibility of parallel pathways. Three Trp residues at position 306, 382 and 359 of E. coli photolyase constitute the frst identifed ET cascade of the cryptochrome-photolyase family (PDB 1DNP). 8 These Trp residues are conserved in all other members of class I CPD photolyases, to which E. coli photolyase belongs, in class III CPD photolyases, CRY-DASH proteins, plant cryptochromes, animal cryptochromes and eukaryotic 6-4 photolyases, but not in class II CPD photolyases 9 or FeS-BCP proteins (see Fig. 1 for a phylogenetic tree of photolyases and cryptochromes). In class I and class III CPD photolyases, additional Trp residues have been shown to be involved in photoreduction. For example, in the class III CPD photolyase PhrA, the Trp residue of the classical triad that is closest to FAD is linked to a second, less conserved electron pathway comprising two Trp residues. 5 The class II CPD photolyases have another Trp triad, which is conserved among this group. 10 Trp side chains are chemically ideally suited for ET processes due to their aromatic cycles, low redox potential (around 0.6 V at pH 7) 12 and stable radical state for the deprotonated form. However, in several CPD class I and II photolyases, Tyr residues are also involved in ET. In the class I CPD photolyase from Anacystis nidulans, a Tyr radical is formed within 50 ms after FAD excitation, as detected by ultrafast spectroscopy. 13 In Methanosarcina mazei CPD class II photolyase, a Tyr residue is required for full photoreduction. In the Xenopus laevis (6-4) photolyase the involvement of a Tyr residue in photoreduction was shown by electron paramagnetic resonance. 14 The conservation of Tyr or Trp for electron transfer shows that this selection is not a random process. Factors such as the chemical environment (other nearby amino acids or water) certainly play a signifcant role. Electron transferring Tyr residues are usually located close to the protein surface, surrounded by water and/or located close to deprotonating amino acids. 15,16 Electron transferring Trp residues can occur at the periphery or in the centre of a protein. The group of FeS-BCP proteins is a phylogenetically distinct group of (6-4) photolyases (Fig. 1) with unique properties such as an Fe-S cluster and a 6,7-dimethyl-8-ribityllumazine (DMRL) antenna chromophore. 17 The ET in FeS-BCP proteins differs from all other cryptochrome and photolyase groups in several ways. In the FeS-PCB members PhrB from Agrobacterium fabrum (PDB 4DJA) 17 and CryB from Rhodobacter sphaeroides (PDB 3ZXS) 18 photoreduction proceeds via Trp390 and Trp342 (PhrB numbering), as shown by site directed mutagenesis. These residues are highly conserved in FeS-BCP members (see ESI Fig. S1 †). In both proteins, the Tyr391 side chain is directly located between Trp390 and FAD (see also Fig. 2) suggesting that this Tyr must be part of the ET chain. However, when the Tyr was replaced by Phe, the photoreduction rate of PhrB was not affected, 19 and only slightly affected in CryB. 18 In about 30% of FeS-BCP proteins, a Phe is placed at this position (see Fig. S1 †). These observations raise several questions about Tyr391: is this residue involved in photoreduction as the frst electron donor of FAD, as proposed by its spatial position? if yes, how can the electrons be transmitted via this Tyr residue and why is it a Tyr residue, whereas in other groups of photolyases and cryptochromes a Trp residue serves as electron donor for FAD? -Phe is usually used to interrupt electron chains. Why does the replacement of Tyr391 by Phe not interrupt the electron chain? FAD photoreduction involving a Trp triad has been widely studied by computational approaches in Escherichia coli photolyase, Arabidopsis thaliana cryptochrome, Synechocystis sp. CRY-DASH protein 30 or Xenopus laevis (6-4) photolyase. 31 These studies highlighted the role of Trp in the classical triad or of additional residues 30,31 at an atomistic level, as well as the crucial role of the environment 24,25,29 or the quantum effects in these ultrafast charge transfers. 26 In our group, we have established a quantum mechanics/classical mechanics (QM/MM) scheme based on fragment orbital tight-binding density functional theory (FODFTB) coupled to MM molecular dynamics (MD). 32 Our approach allows direct simulations of the charge propagation along a Trp triad. Previously we established our method by successful reproductions of ET rate constants in E. coli photolyase 23 and Arabidopsis cryptochrome 24 and also highlighted the role of environment in the downhill charge transfer process. Applying these techniques, we are able to shed light onto the molecular evolution of ET pathways in different proteins belonging to the cryptochrome-photolyase family. In the present work, we investigate the role of Tyr391 in PhrB by experimental and QM/MM approaches, comparing PhrB wild type (WT) with its Y391F, Y391W and Y391A mutants. We regard that the analysis of the Tyr to Trp replacement is important for an understanding of the evolution of photolyases and cryptochromes, which most often have a Trp triad as electron transmission. Due to the difference in ionization potentials, it is expected that a Tyr residue slows down or blocks charge transfer, and that the replacement of Tyr by Trp would result in increased ET rates. However, all forward and backward charge transfers along the triad and subsequent possible charge recombination with FAD have to be considered. The comparison between PhrB and other members of the cryptochromephotolyase family shed light on protein fne-tuning, enhancement of FAD photoreduction rates and avoidance of charge recombination. ## Experimental methods Site directed mutagenesis, protein expression and purifcation. A PhrB E. coli expression vector based on pET21b was used for recombinant expression of PhrB in ER2566 cells. The vectors for WT and the Y391F mutant are described in earlier publications. 19,33 To obtain the Y391A and Y391W mutants, site directed mutagenesis was performed according to the Quik Change mutagenesis kit (Agilent) using a pair of complementary primers (Table S1 †) with the desired mutation in the middle for initial polymerase reactions. Mutagenesis success and correctness of the sequences were confrmed by DNA sequencing. Expression and purifcation followed the procedure described in ref. 11 for WT and mutants. In brief, E. coli cells from agar plates were used for the inoculation of 3 l LB containing ampicillin. Following specifc induction of recombinant expression with IPTG and subsequent incubation over night at 28 C, all purifcation steps were carried out at 4 C. Cells were harvested by centrifugation, suspended in 50 ml extraction buffer (50 mM Tris/HCl, 5 mM EDTA, 300 mM NaCl, 10% glycerol, pH 7.8) and extracted with a French Press (America Instrument Company) at 1000 bar. Following centrifugation and precipitation of soluble protein by ammonium sulfate (93% saturation), the protein pellet was suspended in EDTA free buffer. Soluble protein was purifed by Ni affinity chromatography followed by size exclusion chromatography. The fnal buffer was 50 mM Tris/HCl, 5 mM EDTA, 300 mM NaCl, 10% glycerol, pH 7.8. Photoreduction measurements by UV/vis spectroscopy. PhrB WT and mutant proteins were diluted to a fnal concentration of ca. 10 mM. The samples were incubated at 4 C in darkness in saturated oxygen solution. During this treatment, reduced FADH is converted to oxidised FAD, although spectral analyses revealed that the fraction of oxidised FAD differed among the different proteins. Thereafter, 10 mM 1,4-dithiotreitol were added to the protein solution. UV/vis spectra were recorded using a Jasco V550 photometer with temperature control adjusted to 10 C. After the frst recording, the sample was illuminated with blue light emitting diodes (l max ¼ 470 nm) with a light intensity of 55 mmol m 2 s 1 at the position of the cuvette. Subsequent spectra were recorded at a series of time points as given in the results section. For data evaluation, complete spectra, 450 nm or 580 absorbance values, which stand for FAD in the oxidised or semireduced forms respectively, were presented. Cofactor detection and repair assay. For detection of FAD and DMRL, 85 mM protein was denatured by 95 C incubation for 5 min. The insoluble protein and the soluble chromophores were separated by 15 000 g 10 min centrifugation and 10 mL supernatant were analysed by HPLC (Agilent system with a Gemini C18 column (50 4.60 mm, 110 , Phenomenex)). The HPLC buffer conditions were: 5% acetonitrile (ACN) in 0.1% formic acid for 0-5 min; 5-75% ACN in 0.1% formic acid for 5-25 min. The flow rate was set to 0.75 ml min 1 and the column temperature to 25 C. Elution was monitored at 260 nm and 400 nm. The photorepair reaction mixture contained 5 mM of the purifed (6-4) photoproduct of t-repair_1 (Table S1 †) and 8.5 mM protein in repair buffer (50 mM Tris-HCl, pH 7.0, 1 mM EDTA, 100 mM NaCl, 5 mM MnCl 2 , 5% (w/v) glycerol, 14 mM 1,4dithiothreitol). Aliquots were irradiated with 400 nm light emitting diodes (250 mmol m 2 s 1 ) for 3 min. Thereafter, the reactions were stopped by heating to 95 C for 10 min. Samples were centrifuged at 15 000 g for 10 min and the supernatants analysed by HPLC (same column and system as above). The buffer conditions were: 7% acetonitrile (ACN) in 0.1 M triethylamine acetate (TEAA) (pH 7.0) for 0-5 min; 7-10% ACN in 0.1 M TEAA (pH 7.0) for 5-35 min. The flow rate was set to 0.75 ml min 1 and the column temperature to 25 C. ## Computational methods Model structures and molecular dynamics simulations. The structural model of PhrB WT has been derived from the X-ray crystal structure of Zhang et al. (PDB ID 4DJA). 17 The Y391F, Y391A and Y391W mutants have not been crystallized. For the setup of the model structures, we suppose that the mutation of Tyr391 does not affect the structure of the remainder of the protein. Starting from the PhrB-WT model structure, we replaced the aromatic cycle of Tyr391 by a phenyl, a methyl or an indol ring. Two conformations of the indol ring are allowed by the protein structure, but steric hindrance prevents rotation from one to the other (see Fig. 5). In the frst conformation, the Trp side chain can orient toward FAD being in a closer contact than in the second conformation. The conformations are termed Y391Wp (for proximal) and Y391Wd (for distal) in the following. All mentioned simulations were performed with the GRO-MACS 5.0.4 package 34,35 using the AMBER-SB99-ILDN force feld. 36,37 The force feld parameters for neutral (oxidised) FAD and negatively charged cofactor FADc were taken from ribo-flavin and adenosine diphosphate (ADP) models developed in previous studies. 23,24 The GAFF parameters 38,39 were used for the DMRL antenna chromophore. The DMRL atomic charges were calculated by restrained ftting on the electrostatic potential (RESP) 40,41 at HF/6-31G* (ref. 42 and 43) level with Gaussian 09 package. 44 Bonded parameters of the cubic FeS-cluster were taken from ref. 45 and the charges were taken from ref. 46. The loop region from residues 180 to 182, which might impact the DNA binding abilities, 17 was not structurally resolved in the X-ray structure of WT PhrB. It was reconstructed using the MODELLER program. 47 WT and mutated proteins (Y391F, Y391A and Y391W) were solvated in a 106.24 3 cubic box flled by TIP3P water molecules. 48 Twelve sodium ions were added to create a neutral system. Equilibration of the solvated proteins (WT and mutants) starts with a minimization step, followed by 100 ps MD in the NVT ensemble and 100 ps in the NPT ensemble. 100 ns of production NPT MD simulations were performed afterwards. Nose-Hoover thermostat 49 was used to keep a constant temperature at 300 K and Parinello-Rahman barostat 50 to keep the pressure at 1 atm. Covalent hydrogen bonds were fxed on a constant length by the use of the LINCS algorithm. 51 The time step for the MD simulations was 2 fs. Site energy and electronic coupling calculations. To treat the charge transfer processes, a quantum mechanical treatment of the active site has to be included via a so called combined Quantum Mechanics/Molecular Mechanics (QM/MM) scheme. The structural part of interest for charge transfer, which is treated at QM level, contains the side chains of amino acids involved in the called triad (A, B and C, see Fig. 2) and the isoalloxazine ring of FAD. The remaining atoms are treated classically using force felds (MM) and affect the QM zone by electrostatic interactions. Hydrogen link atoms 52 are inserted at the QM/MM boundary, namely in the C a -C b bond of A, B and C side chains or in the C1-C2 bond of the FAD D-ribitol tail. To compute the electronic properties along the classical MD simulations, we use the semi-empirical Tight-Binding Density Functional theory (DFTB) method, 53 which is derived from density functional theory (DFT) but roughly 2-3 orders of magnitude faster than standard GGA-DFT methods with medium sized basis sets. Running a fragmentation of the QM region into several functional parts speeds up the calculation signifcantly and allows to systematically correct for errors well known in DFT-GGA, like self-interaction error (for a detailed discussion see ref. 54 and 55). Each fragment is represented by only one frontier orbital and the chosen i-th fragment orbital (FO) of the fragment m is expressed in an atomic basis set c m and determined by FO coefficients c m i : 32,56 This FO-DFTB approach has been extensively evaluated and tested in previous publications 32,54,57 and has been so far successfully applied to describe the charge transfer in photolyase, 23 cryptochrome 24 and DNA 58,59 where the HOMO's of the fragments were used to calculate the evolution of electronic couplings and site energies. The Hamiltonian H mn matrix is built out of the FO coefficients and the Hamiltonian H mn in the atomic-orbital-like basis function of the fragments: The diagonal elements of the Hamiltonian matrix correspond to the site energies 3 m : The HOMO energies for different molecules show nonsystematic errors, which can be corrected adding a constant energy shift depending on the chemical identity of the fragment. This correction was evaluated in ref. 58 for the relative energies of Trp and Tyr and expanded in this work to Phe and FAD relative to Trp (see Table S2 †). The energy gap between two sites can be related to the driving force of a charge transfer as described in the Marcus theory. The off-diagonal elements of the Hamiltonian correspond to the direct electronic coupling H DA between two sites. The FO-DFTB electronic coupling calculations were also validated in comparison with higher theoretical level results on set of organic stacked molecules. 56,60 These matrix elements (H DA ) can be used to compute two different charge transfer regimes. When large energy barriers occur along the charge transfer pathway, tunnelling matrix elements (T DA ) can be computed ('super-exchange tunnelling'), for small barriers, a 'direct' hopping-type mechanism may appear, which we treat using a QM/MM non-adiabatic MD technique, also based on the same computed matrix elements H DA . Super-exchange tunnelling. In case of super-exchange tunnelling mechanism for a bridged charge transfer, the n fragments of the bridge B must be included in the electronic coupling T DA calculation. The system is thus divided into the donor/acceptor (D/A) and bridge subspace and an effective Hamiltonian is calculated in which off-diagonal elements correspond to T DA (for detailed discussions see ref. 54 and 61): where b DA describes the direct electronic interaction between D and A, b Di and b jA the electronic interactions between D or A and the i or j-th fragment of the bridge. The tunnelling energy 3 tun corresponds to the average of the eigenvalues of the effective Hamiltonian and G ij is an element of the Green's function matrix, describing the probabilities for the electron to tunnel through the i-j space of the bridge. Direct electron transfer. We established in our previous work a multi-scale method to describe charge transfer reactions in cryptochromes and photolyases. 23,24 This non-adiabatic charge propagation scheme allows simulations where the charge transfer occurs on the same timescale as environment response charge state. Nonadiabatic QM/MM MD simulations directly calculate the population of the charge which is located at a specifc site of the QM zone. 62 This method doesn't force a specifc charge transfer process; however it can describe a hopping or even a band-like conduction. 32,57 The wave-function associated with the transferred charge and atomic coordinates are simultaneously propagated solving time-dependent Schrödinger equation and classical Newton equations at every MD step, respectively. The excess charge corresponds to a second order perturbation of the neutral system Hamiltonian while environment interacts with the QM part as point charges. To take into account charge transfer, the partial charge of the QM part is calculated at every MD step to model the interaction of the moving charge with the charge distribution of the environment. More detailed description can be found in ref. 32, 57 and 62. As for the electronic coupling calculations, charge propagation simulations were performed using an in-house GROMACS 4.6 version. 63 The charge transfer between the triad and the isoalloxazine ring is initiated by the excitation of the FAD cofactor. The frst transfer continuing the excitation was studied in some photolyases to happen within one picosecond. 64 These two ultra-fast events were excluded in the previous studies 23,24 to reduce complexity and to focus on charge transfer along the Trp triad. To keep consistency, the same exclusion was also applied here and the QM part consists in A, B and C. Charge propagation simulations start with an electronic state where FADc is a radical anion and the frst site A a radical cation. The hole on the frst site is then propagated along the triad and moves in the reverse direction compared to the electron. These simulations were performed on PhrB WT and the Y391W mutant. Charge occupation of each site, varying from 0 (neutral state) to 1 (fully oxidized state) is followed during 1 ns QM/MM MD simulations. We randomly chose 20 to 25 starting structures from classical MD trajectories to guarantee well equilibrated systems while sampling different initial conditions. A kinetic model 23,24 was used to ft the average occupations (from the individual simulations) of each site and thus determine the different reaction rate constants. ## Photoreduction of PhrB and mutants For photoreduction studies, we generated the Y391F, Y391W and Y391A mutants of PhrB. All mutants and WT PhrB were expressed in E. coli and purifed by Ni chromatography and size exclusion chromatography. Whereas protein yields of Y391F and Y391A are comparable to those of WT, the yield of Y391W is ca. 10 times lower. Absorbance spectra of Y391A and Y391F in the oxidised FAD state are comparable with WT (Fig. 3), although detailed analyses reveal different chromophore to protein ratios and/or different fractions of reduced FAD at starting time. The absorbance of the Y391W in the blue spectral range is very weak (Fig. 3). Chromophore analyses show that this mutant contains only (1.3 AE 0.1)% FAD and (1.8 AE 0.2)% DMRL as compared to WT. These values are (94 AE 1)% and (87 AE 3)% for FAD and DMRL of Y391F, respectively, and (53 AE 2)% and (53 AE 2)% for FAD and DMRL of Y391A, respectively. We propose that the replacement of Tyr 391, which is located close to FAD, by bulky Trp in Y391W results in opening of the FAD pocket and loss of FAD binding capacity. The DMRL pocket is formed by amino acids of the N-terminus and more distant from the mutation. The loss of DMRL results therefore probably from FAD depletion. The partial loss of both chromophores to equal percentages in the Y391A mutant supports this idea. The 410 nm peak in the spectrum of the Y391W mutant is assigned to the iron sulphur cluster. During blue light irradiation, the spectra of WT PhrB and the Y391F mutant change in a characteristic manner. The transient increase at 580 nm, the maximum of the protonated FAD semiquinone, and the loss of absorbance at 450 nm, characteristic for the loss of oxidised FAD (Fig. 4), are comparable to data published earlier. 33 Here, the relative A 450 nm decrease at t ¼ 90 min in the Y391F mutant appears smaller than in WT. This can be due to slower photoreduction or smaller fraction of oxidised vs. total FAD in the mutant. Both decay curves can be ftted with monoexponential decay functions which yielded time constants of 36 AE 1 min and 32 AE 1 min for WT and Y391F, respectively. Thus, the rate of overall photoreduction is not affected by the Tyr to Phe replacement, but the oxidation state of Y391F was incomplete at the start of the photoreduction experiments. Formation and decay of the semiquinone intermediate absorbing at 580 nm is slower in Y391F (rise and decay times of 6 AE 0.3 min, 110 AE 30 min for WT and 7 AE 0.3 min, 200 AE 110 min for Y391F, respectively). This results shows that the role of Tyr or Phe differs in the frst and second electron transfer. In summary, the present and published data 19 show clearly that the replacement of Tyr by Phe into the proposed electron path does not block photoreduction. We do not observe any light induced absorbance changes in the Y391A mutant (Fig. 3). This result suggests that position 391 is critical for photoreduction, as proposed above. DNA repair in the presence of Mn 2+ (ref. 11) is complete after 5 min for WT PhrB and Y391F mutant, whereas no repair activity is observed for Y391A and Y391W mutants under these conditions. When the repair time is prolonged to 120 min, Y391W repairs about (8.7 AE 0.7) % of damaged DNA, whereas with Y391A still no repair is observed. ## Molecular dynamics simulations We performed 100 ns classical MD simulations of PhrB WT and the Y391F, Y391A and Y391W mutant, based on the crystal structure of PhrB. In these simulations, no major conformational change of the overall protein structures is observed. The residues A, B and C (see Fig. 2) involved in the triad occupy similar positions in WT and the Y391F or Y391A mutants. In the model structure of the Y391A mutant, water molecules fll the space let by the replacement of Tyr to Ala (see Fig. S2 †). In WT, Y391F and Y391A, a water molecule, present in the crystallographic structure, interacts with isoalloxazine O4 and A backbone (see Table S3 and Fig. S3 †). The experimental fndings for the Y391W mutant, which has lost both chromophores, suggests a more drastic impact on the protein structure which would require simulation protocols dedicated to protein folding and FAD docking. Our simulations, which follow the dynamics of Y391W on relatively short time-scales, however, allow us to investigate the theoretical role of an amino acid independent on large structural changes that might occur on much longer time-scales. Such simulations help us to compare the relationship between environment and the frst site in different members of the cryptochrome-photolyase family. In the end, we have to combine experimental and theoretical results to obtain the maximum information for the role of each amino acid. The simulations for the Y391W mutant revealed two conformations of the Trp391 side chain (A) (see Fig. 5), denominated as Y391Wd and Y391Wp. In Y391Wd, A stays continuously close to the second Trp side chain B whereas in Y391Wp, A stays most of the time close and parallel to FAD isoalloxazine ring, but moves closer to B during a few nanoseconds (see Fig. 5, S4 and S5 †). As shown in Table 1, the distances between all neighbouring sites (FAD-A, A-B, B-C) in WT and mutant structures are between 5 and 8 . These are typical nearest neighbour distances reported in our MD simulations of Arabidopsis cryptochrome and E. coli photolyase which allow for sufficiently large electronic couplings in order to enable fast charge transfer. The larger distances between FAD and the second neighbour between 12 and 13 (FAD-B) suggest that a direct electron transfer from B to FAD is unlikely because the electronic coupling decreases exponentially with distance in absence of charge transfer bridge. We also report the distance between FAD or B and another tyrosine, Tyr395 (see also Fig. 6), which is close to the charge transfer chain. Mutations have no impact on the position of Tyr395; it stays at around 8 from FAD and slightly less than 7 from B. ## Site energy and electronic coupling Upon excitation of FAD by light or by energy transfer from the excited antenna chromophore, this site is reduced to the negatively charged FADc species by an ET from the triad, which then in turn becomes positively charged. In a frst step, we investigate the electronic structure of the neutral FAD-triad system by computing site energies and electronic couplings. In a second step, we compute the changes in the electronic structure due to the charge transfer. In previous work, we have studied Arabidopsis cryptochrome and E. coli photolyase in a similar way. 23,24 Both proteins show a highly exergonic and fast charge transfer on a picosecond time scale. To investigate the effect of the mutation at site A, we compare the relevant parameters for charge transfer in the present study with these two reference systems. The charge transfer parameters have been evaluated for oxidised FAD and a neutral triad along the 100 ns classical MD simulations on the neutral state of the protein using the FO-DFTB/MM scheme as discussed above. The orbital energies of A, B and C are called site energies and are a direct measure of the (relative) ionization potentials (IP) for the residue in the protein. The electronic couplings between the different partners are a measure for the charge transfer probability, i.e. they can be directly related to the prefactors in Marcus theory. 65 Average values of energies and couplings are reported in Fig. 7, the error bars indicate the associated standard deviation. According to these data, site energies of Trp390 and Trp342 (B and C) are similar, independent of the chemical nature of A. The electronic coupling values associated with the charge transfer between these Trp's are about 4.1-4.2 meV for WT and the Y391F mutant, 5.9 meV for the Y391A mutant and between 7.0 and 9.0 meV for the two rotamers of the Y391W mutant. These values are comparable with those found in Arabidopsis cryptochrome and E. coli photolyase. The small increase of electronic coupling from WT to Y391W corresponds to a small decrease in B-C distance in the mutant (Table 1). For reference, we also show the location of the HOMO level of neutral FAD, which is the electron acceptor after excitation of one of its electrons. The site energy of A as an aromatic residue (Trp, Tyr or Phe) inside the protein follows the same order as the HOMO energies in gas phase (Table 2, HOMO z IP): HOMO(Trp) > HOMO (Tyr) > HOMO(Phe). In the protein, A has a value of about 5.8 eV for Trp, which is substantially decreased to 6.3 eV for Tyr and to around 7 eV for Phe. In the Y391W mutant, the energy of A is similar to energies of B or C, leading to a small energy gap between the charge transfer partners (within about 0.3 eV). Electronic couplings are twice as large as in PhrB WT or Y391F. These results support the possibility of a charge migration involving a Trp triad in the Y391W mutant. The distal conformation Y391Wd facilitates B / A charge transfer by decreasing the A-B energy gap by 0.08 eV and the average A-B distance by 0.64 compared with Y391Wp (Table 1 and Fig. 7). On the contrary, in the proximal conformation, the aromatic ring of A and the isoalloxazine ring are in a close position which must enhance A / FAD charge transfer. As expected, the lower site energies of Tyr in PhrB WT and Phe in Y391F may present a barrier for hole transfer from FAD, as clearly seen in Fig. 7. While our previous studies on Arabidopsis cryptochrome or E. coli photolyase showed a hopping type mechanism along the Trp-triad, one may expect in WT and Y391F a tunnelling mechanism to be in operation, which would lead to less efficient charge transfer. We calculated the electronic coupling between FAD and B which involves A as a bridge for the WT and Y391F by a previously published method. 54 Our calculation shows a coupling of 0.06 and 0.02 meV for WT and Y391F which is ten-fold more than the direct (assuming no bridge residue) coupling between FAD and B (see ESI Table S4 †). Electron tunnelling through the A side chain seems possible, as also indicated by the experiments on Y391F. Tunnelling involving a Phe and protein backbone has been also described for the E. coli photolyase. 67 Therefore, in the case of Y391F, where a barrier of more than 1 eV is apparent, we defnitely have to consider tunnelling, for the smaller barrier resulting from the presence of Tyr391 in WT this is not necessarily the case. This has been discussed for charge transfer in DNA, 68 where small charge transfer barriers up to 0.4 eV may easily be overcome, especially for short bridges due to molecular fluctuations. The hole transfer between FAD and A falls in this range, allowing a direct hopping mechanism (Fig. 7). Electron hopping via Tyr would involve a transiently positively charged side chain, which must be followed by deprotonation. However, a nearby proton acceptor is missing in the PhrB structure, ruling out the deprotonation mechanism of Tyr391. Oxidised Tyr cannot be stabilised while the electronic coupling between A and B is strong (see Fig. 7). If FAD / A transfer occurs, the following hole transfer between A and B must be very fast. Other charge transfer pathways can also be considered from the crystallographic structure. A charge transfer chain via Tyr399 and Tyr40 has been suggested in our previous experimental study. 19 Spectral changes related to FAD reduction in Y399F mutant are slightly slower compared to WT. However, the absence of FAD reduction in both W390F and W342 mutants clearly indicates that Trp390 and Trp342 are essential in the charge transfer process, which cannot be compensated by a transport via Tyr399 and Tyr40. Consequently, the Tyr399-Tyr40 charge transfer pathway was rejected. Another possibility could be a transfer between FAD and Trp390 via Tyr395 (see Fig. 6). This residue could substitute site A (Tyr391), since it has similar distances to FAD and site B, as shown in Table 1. Interestingly, theses distances do not change upon mutation at site A, i.e. the FAD binding pocket and the connection via Tyr391 is very similar in all variants. According to the computed electronic couplings and site energies (Table S5 †), this pathway seems to be possible as well. However, our photoreduction studies for the Y391A mutant clearly indicate the absence of any charge transfer, i.e. the pathway does not seem to be a possible alternative. Although it has been shown that our scheme for the calculation of charge transfer couplings is very reliable for all couplings between the oxidisable residues, 60 the couplings between FAD and site A respective Tyr391 are very small, i.e. a small change in geometry could lead to a different result. For example, the PhrB structure in solution (experimental photoreduction measurements) could differ from the crystal structure used for calculations. Further, the couplings are computed for neutral FAD in the ground state, whereas the active state is an excited state, which may have some impact on the value of the coupling. As a consequence, within the accuracy of the calculations reported here, the rather small value reported for the FAD-Tyr395 coupling (Table S5 †) could as well turn out to vanish. Therefore, we believe that under the conditions the experiments are performed, the pathway via Tyr395 seems to be impeded. However, since an analogous alternative pathway has been reported for the PhrB homologue CryB, 18 it could be interesting to investigate under which conditions this pathway could be activated in PhrB. This, however, would require further investigations which are beyond the scope of the present work. For the main focus of this work, it is very interesting to note that for this alternative pathway a tyrosine is the primary electron donor of FAD, i.e. highlighting the role of tyrosine in the charge transfer cascade, which is the main emphasise of this work. To estimate the effect of a water molecule bridging the FAD and the backbone of site A (see ESI †), we also applied the pathways model from Beratan and co-workers. 69 The couplings for these pathways are very small, therefore we did not consider these pathways further (see Table S4 and Fig. S6 †). We now discuss the energetics and couplings for the case, where FAD and one of the residues of the triad are charged. The energetic landscape is changed drastically when a charged FAD and a charged A are considered (Fig. 8). As discussed recently, the fast charge transfer in cryptochromes and photolyases can be explained by a steep downhill energetics, which results from the interaction of the charge with the protein and solvent environment. 23,24 The frst ET results in negatively charged FADc and a positively charged side chain on one site of the triad. Charge separation has a sizable effect on the site energies. The positive charge on A, B or C leads to a strong polarization of the environment. This polarized environment in turn leads to a stabilization of the charge at the respective site. In Marcus theory, this effect is called outer-sphere reorganization energy; in our simulations it manifests itself by the lowering of the site energies with regard to the neutral states. For each charge state on A, B and C, we compute the site energies along 1 ns MD simulations containing the charge on the respective site during the MD simulations, as shown in Fig. 8. We have discussed this effect in some details in our previous work, showing that the solvent has a distinct impact, in particular on those sites which are more solvent exposed, 24 like site C which is located on the protein surface. In previously studied Arabidopsis cryptochrome or E. coli photolyase, the positive charge stabilization follows a downhill scheme, with an increasing energy gap between neutral and charge residue from A to C and where neighbouring sites have an energy difference of about 0.5 eV (Fig. 8). This leads to the fast charge transfer in a picosecond regime. In PhrB, a similar stabilization on C occurs, this results from the solvent exposure. However, surprisingly also site A is massively stabilized, for Tyr even more than for both Trp rotamers. In the latter, A and B site energies are similar and the charge transfer from B to A no longer follows the downhill scheme described for the WT, E. coli photolyase or Arabisopsis cryptochrome. In total, the energy difference between site C and A in Y391W is only half of the value compared to the other two systems (Fig. 8), which has a drastic effect on the charge transfer equilibrium. There is an obvious energetic difference for the site A Trp in Y391W, E. coli photolyase and Arabidopsis cryptochrome. In Y391W, A is nearly isoenergetic to B, while in the other proteins, positively charged B is more stable than A by about 0.4 eV. In our previous work, we have analysed structural reasons for this energy gap between A and B. In the E. coli photolyase or Arabidopsis cryptochrome, A is buried in a pocket with more than 5 distance from any water molecule, as documented by calculation of water distribution functions along MD simulations (Fig. 9). On the contrary, water molecules can easily move toward A in PhrB: the radial distribution function of water around Tyr391 presents a peak around 5 , while the frst peak around B is observed at 7 (Fig. 9). Moreover, a stable hydrogen bond network, involving Thr373, a water molecule, Tyr385, Thr358 and the backbone of Val354 (Fig. 6) can also participate in Ac + stabilization in our simulation. In E. coli photolyase and Arabidopsis cryptochrome, sites B and C are stabilised by solvent interactions, leading to a larger solvent reorganization energy. This explains the downhill energetics as shown in Fig. 8. On the contrary, water molecules close to the FAD-A complex in PhrB help to stabilize the charge-separated RP-A (FADc -Ac + ) state, and compensate the unfavourable intrinsic IP of Tyr. Likewise, this could impact the charge transfer efficiency in the Y391W mutant by increasing the probability to localize the charge on the frst member of the triad. ## Charge transfer simulations To study the implication of the energetic landscape on the charge transfer dynamics, we performed unbiased simulations of the charge transfer through the residues A, B and C in WT protein and Y391W rotamers. Y391F and Y391A are not considered in this part as Phe and Ala cannot be oxidised. These simulations indicate the formation and lifetime of each radical pair state: RP-A (FADc -Ac + ), RP-B (FADc -Bc + ), RP-C (FADc -Cc + ), summarized in Fig. 10. We present the averages over 25 simulations for WT and 20 simulations for the two Y391W mutants; more details and charge transfers movies are given in ESI. † The charge is considered to be on a specifc site when the occupation of the site is larger than 50%. The time dependence of the average site occupations have been ftted using a kinetic model previously described 23,24 which allows to obtain rate constants (Table 3) corresponding to the following steps: In WT, the population of the frst radical state RP-A drops within a few ps and no back transfer is observed. The second state, RP-B, is transiently occupied by 60% of the total positive charge before decay and formation of the third state RP-C. After 25 ps simulation time, the charge distribution remains stable, with roughly 85% of the charge on the third state, while 15% remains on the second state. Due to the averaging over several simulations, these numbers show a statistical distribution rather than a charge delocalization over different sites. The positive charge is therefore well stabilized on the solvent accessible Trp342. No backward charge transfers from B to A occur during 1 ns of simulation time. The very fast frst charge transfer from Tyr391 to Trp390 shows a signifcantly larger transfer rate than that calculated for plant cryptochrome. 1 PhrB k 23 and k 32 are consistent with values from Arabidopsis cryptochrome and E. coli photolyase, with a backward transfer 10 fold smaller than the forward one. The Y391W mutants show a different behaviour. On average, the hole remains on RP-A during the frst 50 ps in Y391Wp and during the frst 21 ps in Y391Wd, respectively. The fnal stabilization of 70-80% of the charge on site C occurs after 70 ps for Y391Wp and 33 ps for Y391Wd. For the A-B transfer, the forward and backward rate constants are very similar in each rotamer simulation, as shown in Table 3. In Y391Wd, the k 21 rate is also close to k 23 . For the transfer from B to C, the forward rate is 10-fold higher than the backward rate. All rate constants are in the same order of magnitude as those of Arabidopsis cryptochrome or E. coli photolyase. The main difference between the Y391W mutant and Arabidopsis cryptochrome is the strong back transfer rate from B to A (k 21 in Table 3). Indeed, during all charge transfer simulations of our different cryptochromes and photolyases proteins, we observe several backward transfers to A. No backward transfer is present in PhrB WT simulations, as the frst residue is a Tyr. We compare the number and the stability of backward transfers in Table 4 for different systems: Y391Wp/d and E. coli photolyase. In Y391W, we observe about 30 crossings in which the positive charge moves back to A and forms a stable RP-A for at least 500 fs which is ten-fold more than in E. coli photolyase. The difference between the two conformations of Y391W is related to the A-B distance: in Y391Wd, A is closer to B, which facilitates backward and forward charge transfer between them. On the contrary, in Y391Wp, A is closer to negatively charged FAD which contributes to stabilize the positive charge on A. Furthermore, a charge recombination on the isoalloxazine ring becomes more likely. Nevertheless, in 100 ns MD simulation of Y391Wp, motion of A to a distal conformation is observed and can also contribute to an enhanced charge transfer between the two Trp residues. In both conformations, the charge is more often back transferred to A, but stays less time for one transfer than in E. coli photolyase. ## Discussion and conclusion In most members of the cryptochrome-photolyase family, the central ET pathway contains a triad of Trp residues, from the surface of the protein to the FAD chromophore. The mechanism commonly accepted for the FAD photoreduction consists of three successive hole transfer steps: FAD* / A, A / B and B / C. 31 Like other members of the cryptochrome-photolyase family, PhrB is able to reduce FAD upon light absorption via a long range ET involving aromatic residues. Site directed mutagenesis experiments have shown that two Trp, Trp390 and Trp342, are essential for the reduction process. The distance between the isoalloxazine ring and Trp390 is roughly 12-13 (Table 1) and thus too far from FAD for direct ET. There is no other Trp in the structure that can complete the triad, which raises the question of the role of the closest residue to FAD involved in charge transfer. The presence of Tyr391 is intriguing: it is situated between FAD and Trp390 in a suitable place to take part in the ET, but its oxidation appearsat frst sightnot required for FAD reduction: (i) mutation of Tyr391 to redox inert Phe residue neither blocks FAD photoreduction nor DNA repair; (ii) in 464 PhrB homologs, this residue is either Tyr or Phe (Fig. S1 †). However, experimental mutation of Tyr391 to Ala blocks FAD photoreduction and DNA repair, underlining its relevance in electron transfer process. This observation rules out other alternative pathways e.g. through Tyr395 or water molecules. In both WT and Y391F mutant we fnd an energy barrier at site A. Therefore, we considered a super-exchange tunnelling process for both residues, which is a viable hypothesis to explain the experimental charge transfer between FAD and Trp390 in Y391F mutant where a large energy barrier occurs, but the computed couplings are sufficient to allow charge transfer according to this mechanism. 70 We observe similar electronic couplings between FAD and B when a Tyr or Phe aromatic cycle is included as a bridge. One can notice that in WT and Y391F, the Tyr391 or Phe391 aromatic cycle is parallel to the isoalloxazine ring of FAD and in a suitable conformation for pp orbitals interaction. Hole transfer via tyrosines is usually not favourable due to the high ionisation potential, which is 0.6 eV larger than for tryptophans (Table 2). The oxidised state of tyrosines can be stabilized by proton transfer, but there is no proton acceptor in the neighbourhood of Tyr391. Therefore, an efficient hopping mechanism, where Tyr391 is transiently oxidized, seems improbable at frst sight. The energy of the Tyr391 HOMO, however, is signifcantly reduced in PhrB. The Tyr391 oxidized state is stabilized by the protein environment instead, in the immediate environment a hydrogen-bond network (Fig. 6) can attract the proton from Tyr391 and allow the O-H bond elongation to compensate an electronic density decrease on the cycle. Therefore, the charge transfer from FAD to B via a transiently oxidized tyrosine seems to be possible. In addition to tunnelling, a much more efficient hopping regime seems to be enabled in PhrB due to the specifc protein environment of site A. Mutation of Tyr391 to Phe disables this efficient pathway, but does not impede tunnelling as shown by both, experimental and theoretical results. Mutation to Ala, however, blocks the pathway, as shown in experiments and calculations, and thereby blocks FAD photoreduction due to vanishing electronic couplings. Most photolyases and cryptochromes carry a Trp triad. In these cases, the strong exothermicity of the charge transfer results from stabilization of the positive charge by the solvent, as discussed in detail in our previous work. The question arises therefore, why PhrB makes use of a Tyr at site A instead of a Trp residue. Electron transfer can be described by an energy landscape as shown schematically in Fig. 11. It is clear that a Tyr substitution would introduce signifcant barriers into the charge transfer pathway when it would be placed into the photoreduction pathway of e.g. E. coli photolyase. The barrier, when estimated using the gas-phase ionisation potentials (Table 2) is in the order of 0.6 eV, which surely would block an efficient hopping type charge transfer, as found for proteins with a Trp-triad. In PhrB, however, both Tyr and Trp at site A show a stronger stabilization, due to interactions with water and the protein environment, when compared to other members of the cryptochrome-photolyase family which we have already simulated. 23,24 Since site A in PhrB is very close to water molecules in the binding pocket, the intrinsic IP of Tyr and Trp are substantially Table 4 Average number n of backward transfer from B to A and average total time s of occupied A. The charge is considered to be on A when the occupation of the site is larger than 50%. A transfer is counted in s when the charge stays more than 500 fs on site A. stabilized by this polar environment. This stabilization results in a downhill hole transfer from FAD to C in PhrB WT but in similar energies for site A and B in Y391W mutant. The calculations suggest a reason for the presence of Tyr instead of Trp in PhrB. The forward transfer is less efficient for Tyr compared to Trp due to the slightly higher IP, however, the back-transfer seems to be too efficient in Y391W mutant, which may lead to unproductive cycles. The Y391W mutation, because of the higher intrinsic IP of Trp, disrupts the downhill energetics and allows charge recombination of FAD. The protein may therefore trade a slightly less efficient forward transfer for blocking backtransfer when using a tyrosine at site A. Absorbance spectra showed that the Y391W mutant has lost both FAD and the antenna chromophore DMRL almost completely. The loss of FAD could be due to a modifcation of the chromophore pocket by Trp, which is larger than Tyr. The small percentage of DNA repair by the Y391W mutant suggests that a small fraction is still able to bind FAD. Quantitative comparisons between photoreduction of Y391W and WT are not possible experimentally. However, our simulations of the Y391W mutant (where loss of FAD was not considered) provide valuable insight of the importance of the Tyr residue in comparison with previously studied cryptochromes and photolyases. 23,24 The radical pair state of RP-A is stabilized in our Y391W simulations due to strong electronic couplings and small energy gaps between Trp390 and Trp391 and charge is transferred to Trp342 within 33-70 ps (Fig. 10). Although rate constants for the hopping mechanisms in the Y391W mutant of PhrB are comparable with the Trp triads in E. coli photolyase and Arabidopsis cryptochrome, 23,24 backward charge transfers to A occurs more frequently in the PhrB mutant than in these two proteins. Such back transfer increases the risk of charge recombination on FAD and hence a more likely inefficient charge transfer mechanism. If a Trp residue corresponded to the frst site and if the FAD binding were efficient, the environment would stabilize the RP-A state in PhrB obviously more than in Trp triads of other members of the cryptochromephotolyase family. Taken together, our experimental and theoretical results indicate the following: the protein environment is quite different in PhrB from other groups of photolyases and cryptochromes. Residues bigger than Tyr at position A result in loss of FAD binding. Solvent can come closer to A, stabilizing the FADc -Ac + state due to reorganization energy. The presence of a Tyr residue instead of a Trp at this site preserves the structure, the energetics, and therefore the function of PhrB. ## Conflicts of interest There are no conflicts to declare.

chemsum

{"title": "Functional role of an unusual tyrosine residue in the electron transfer chain of a prokaryotic (6\u20134) photolyase", "journal": "Royal Society of Chemistry (RSC)"}

potential_sars-cov-2_nonstructural_protein_15_(nsp15)_inhibitors:_repurposing_fda-approved_drugs

## Abstract: Purpose: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused millions of deaths worldwide, pushing the urgent need for an efficient treatment. Nonstructural protein 15 (NSP15) is a promising target due to its importance for SARS-CoV-2's evasion of the host's innate immune response.Methods: Using the crystal structure of SARS-CoV-2 NSP15 endoribonuclease, we developed a pharmacophore model of the functional centers in the NSP15 inhibitor's binding pocket. With this model, we conducted data mining of the conformational database of FDA-approved drugs.The conformations of these compounds underwent 3D fingerprint similarity clustering, and possible conformers were docked to the NSP15 binding pocket. We also simulated docking of random compounds to the NSP15 binding pocket for comparison.Results: This search identified 170 compounds as potential inhibitors of SARS-CoV-2 NSP15.The mean free energy of docking for the group of potential inhibitors were significantly lower than for the group of random compounds. Twenty-one of the compounds identified as potential NSP15 inhibitors were antiviral compounds used in the inhibition of a range of viruses, including MERS, SARS-CoV, and even SARS-CoV-2. Eight of the selected antiviral compounds in cluster A are pyrimidine analogues, six of which are currently used in a clinical setting. Four tyrosine kinase inhibitors were identified with potential SARS-CoV-2 inhibition, which is consistent with previous studies showing some kinase inhibitors acting as antiviral drugs. Conclusions:We recommended testing of these 21 selected antiviral compounds for the treatment of COVID-19. ## INTRODUCTION Coronavirus disease-2019 (COVID-19) is a respiratory disease caused by SARS-CoV-2. As of August 1st, 2021, SARS-CoV-2 has cumulatively infected over 198 million people and killed over 4 million individuals in almost 200 countries and regions (https://coronavirus.jhu.edu). The serious threats to global public health and the economy presented by SARS-CoV-2 create an urgent need to identify novel tools to provide new pharmacologic leads that can improve survival for those already infected. SARS-CoV-2 is a positive-sense, single-stranded, RNA betacoronavirus with a genome size of approximately 30kb. The genomic RNA contains a 5'-cap structure and a 3'-poly(A) tail. During infection, the genome is translated to generate viral polyproteins and transcribed to generate negative-sense RNA and subgenomic RNAs. The SARS-CoV-2 genome contains 14 open reading frames (ORFs) that encode 29 proteins, including nonstructural proteins (NSPs), structural proteins, and accessory proteins. The two main units, ORF1a and ORF1b are located at 5'-terminus and produce 16 NSPs through proteolytic cleavage by two viral proteases: the 3Clike protease (3CL pro ) and the papain-like protease (PL pro ). NSPs are essential for RNA transcription, replication, translation, and suppressing the host antiviral response . Targeting viral proteins to disrupt replication is an important approach in developing a therapeutic treatment against SARS-CoV-2 infection. Ideally, one can target highly conserved viral proteins that are unlikely to acquire resistance as the outbreak progresses. Recent studies report SARS-CoV-2 genomic variations in over 10% of isolated sequences, with the most frequent mutations being P323L in NSP12 and D641G in the spike protein 4,5 . In contrast, NSP15, an RNA uridylate-specific endoribonuclease (with a C-terminal region homologous to EndoU enzymes), is highly conserved, making it an attractive target for drug development. NSP15-like endoribonucleases are found in all coronavirus family members, suggesting its endonuclease function is critical for the viral life cycle. The amino-acid sequence alignment of NSP15 from SARS-CoV and SARS-CoV-2 showed 88% sequence identity and 95% sequence similarity 6 . NSP15 recognizes uracil and cleaves single stranded RNA through an Mn 2+ requiring transesterification reaction 7 . Recent studies indicate that NSP15 is not required for viral RNA synthesis; rather, NSP15 suppresses the host protective immune response through evasion of host dsRNA sensors 8 . Most recently, NSP15 was reported to participate in viral RNA processing by degrading viral polyuridine sequences. This may prevent the host immune sensing system from detecting viral RNA via cell pathogen-recognition receptors, which subsequently inhibits both direct and indirect antiviral effects 9 . These mechanisms are important for normal coronavirus infection of host cells. In the absence of NSP15 activity, viral replication is slowed significantly, and therefore NSP15 remains an attractive target for addressing SARS-CoV-2 infection 10 . NSP15 is only active as a hexamer, which is formed as a dimer of trimers. The NSP15 monomer contains three domains: a N-terminal domain responsible for oligomerization, a middle domain, and a C-terminal domain, which contains the catalytic domain 11 . Binding sites of each of the catalytic domains are accessible despite hexamerization. A recent publication showed the first two crystal structures of SARS-CoV-2 NSP15 with 1.90 and 2.20 resolution 6 . In the Cterminal catalytic domain of SARS-CoV-2 NSP15, the active site carries six key residues: His235, His250, Lys290, Thr341, Tyr343, and Ser294. Among of these residues, His235, His250, and Lys290 are suggested to constitute the catalytic triad for its nuclease activity. His250 acts as a general base to activate the 2'-OH of the ribose while His235 functions as a general acid to donate a proton to the leaving 5'-OH the ribose 6,11 . Ser294 together with Tyr343 determine uridine specificity. Ser294 is a key residue to recognize uracil and is assumed to interact with the carbonyl oxygen atom O2 of uracil, while Tyr343 orients the ribose of uridine for cleavage by van-der-Waals interactions 11 . In the crystal structure of the NSP15 citrate-bound form, the citrate ion forms hydrogen bonds with active site residues including His235, His250, Lys290, and Thr341 6 . In the crystal structure of NSP15 complexed with uridine-5'-monophosphate (5'-UMP), 5'-UMP was found to interact with all six active site residues. The uridine base of 5'-UMP interacts with Tyr343 through van der Waals and forms hydrogen bonds with the nitrogen atom of Ser294, Lys290, and His250 12 . This structural information is important for exploring binding of uridine analogues as a potential SARS-CoV-2 NSP15 inhibitors. Tipiracil, an uracil derivative, is a thymidine phosphorylase inhibitor. It is an FDA-approved drug used with trifluridine to treat metastatic colorectal and gastric cancer. Previously, tipiracil has been reported to form hydrogen bonds with SARS-CoV-2 NSP15 active site residues Ser 294, Lys345, and His250 12 . Tipiracil suppresses RNA nuclease activity of NSP15 and modestly inhibits SARS-CoV-2 virus replication in vitro without affecting viability of host cells most likely through competitive inhibition 12 . Moreover, recent in-silico-based approaches have identified other potential NSP15 inhibitors that await further structural and biochemical validation 13,14 . The current COVID-19 pandemic brought attention to the repurposing of existing drugs and the rapid identification of candidate compounds. In this study, we use structure-based pharmacophore model and molecular docking to identify potential inhibitors of NSP15 by screening FDA approved drug database. ## MATERIALS AND METHODS The crystal structure of SARS-CoV-2 NSP15 endoribonuclease (PDB ID: 6WXC) in complex with the ligand tipiracil (5-chloro-6-(1-(2-iminopyrrolidinyl)methyl)uracil) was downloaded from the RCSB protein data bank. Using Molecular Operating Environment (MOE; CCG, Montreal, Canada), we analyzed the key binding site residues that are responsible for interaction between the NSP15 and tipiracil and employed a structure-based approach to construct our pharmacophore model of NSP15. The default forcefield is Amber 10: EHT with R Field solvation. Our pharmacophore model was created with seven features and excluded volume R= 1.6 . It had 1 donor, 3 acceptors, 1 cationic atom&donor, and 2 hydrophobic centroids. Based on this developed pharmacophore, we conducted a pharmacophore search on our conformational database of 2356 FDA approved drugs. Pharmacophore partial match was used for a 5 of 7 features search. For multi-conformational docking of the selected compounds, we prepared the NSP15 structure with Protonate 3D application, isolated the ligand and pocket, visualized the space available for docked ligands, defined the binding pocket based on the known key residues for its nuclease activity and uridine specificity, and generated ligand conformations using the bond rotation method. The compounds were docked into the pocket using the Triangle Matcher Method and London dG scoring for placement; and the Induced Fit Method and GBVI/WSA dG scoring for refinement. Poses were ranked by GBVI/WSA binding free energy calculation in the S field. The 56 random control compounds were selected from the FDA drug database. To further analyze ligand interactions for some of the above models, the structures were divided into ligand and protein pdb files. The separate structures were protonated: the protein with VMD (Visual Molecular Dynamics, v1.9.4) and the ligand with Avogadro v1.2.0. VMD was used to generate a psf (NAMD protein structure file) file for the protein and the Ligand Reader and Modeler from charmm-gui.org, was used to generate the psf and prm files for the ligand. VMD was then used with the CHARMM36 forcefield to re-combine the ligand and protein, thus solvating the structure and generating the required psf and pdb files 15,16,17 . NAMD v2.14 was used to run 100 steps of minimization followed by 100 ns of dynamics with 2fs/step (50,000,000 iterations). The simulation conditions were rigid bonds involving hydrogen (rigidbonds set to "all"), a splitting distance of 12A between the short range and PME long range potential, Langevin dynamics at 310K with hydrogen atoms excluded (Langevin hydrogen set to "off"), and Periodic Boundary Conditions 15,16,17 . ## Pharmacophore model creation and search of drugs database A recent publication of the crystal structure of SARS-CoV-2 NSP15 endoribonuclease in complex with the ligand tipiracil provides detailed information regarding key residues responsible for the catalytic activity of NSP15 and its interactions with the potential ligands 6 . Based on the binding information for these key residues, we generated a pharmacophore model with potential functional centers that bind to the residues in the pocket (Figure 1A). The pharmacophore search with a partial match 5 of 7 centers identified 803 compounds. We selected 170 compounds from the search based on the number of H-bonds and hydrophobic interactions in the best docking pose. Minimum three H-bonds and two hydrophobic interactions was the criteria for selection. Then we clustered the selected compounds using the Similarity Clustering of the MOE Database Viewer with a fingerprint GpiDAPH3 and similarity-overlap parameter SO = 45%. The search identified three major hit clusters, containing ten or more compounds, along with several clusters containing less than ten compounds (from nine to two) and 36 single clusters with just one compound (Table 1). The two largest clusters (A and B) contain 16 and 35 compounds respectively, clusters C, D, E, F, G and H contain 11, 9, 7, 7, 5, and 5 compounds respectively; clusters I, J and K contain 4 compounds each, clusters L to V contain 2 to 3 compounds each, and there are 36 not clustered single compounds (Table 1). 1 and 2). ## Computational docking For docking the selected compounds, we used the crystal structure of SARS-CoV-2 NSP15 endoribonuclease (PDB ID: 6WXC), which was imported into MOE. After the structure preparation and the model's binding pocket was defined, based on known key residues for its nuclease activity and uridine specificity, ligand conformations were generated using the bond rotation method. These were then docked into the site with the Triangle Matcher method and ranked with the London dG scoring function. The retain option specifies the number of poses (30) to pass to the refinement, which is for energy minimization in the pocket, before rescoring with the Induced Fit method and GBVI/WSA dG scoring function. To validate docking, 56 random control compounds were selected from the FDA drug database, using a random number generator without repetitions. The values of docking free energies of the selected and random compounds are shown in Figure 3. The means of the selected and random compounds are −6.50 kcal/mol and −5.79 kcal/mol, respectively. Furthermore, the p value of one tail for selected vs random compounds is 1.31 E-06. Energies of interaction with the NSP15 active site are shown in Table 2 and Table S1. Figure 3. Free energies of docking interaction of selected and random compounds with SARS-CoV-2 NSP15. The means of the selected and random compounds are −6.50 and −5.79 kcal/mol, respectively. The p value of one tail is 1.31E-06. Table 2. List of selected compounds sorted by their energies of interaction with SARS-CoV-2 NSP15 in the docked positions. All compounds shown have an energy less than -7. DFE: Docking free energy. ## Molecular dynamics simulations We selected the top three compounds in docking energies to further analyze stability of ligand interactions: cefmenoxime, cefotiam, and ceforanide. The final configuration of the compoundprotein complexes resulting in this MD simulations are shown in Figure 4 and Table 3. Cefmenoxime (Figure 4A and 4D) had 6 major ligand interactions with NSP15, the shortest distance of which was 2.73 with the residue Lys290. Cefotiam (Figure 4B and 4E) had 4 major ligand interactions with NSP15, the shortest distance of which was 2.693 with the residue Leu246. Finally, ceforanide (Figure 4C and 4F) had 2 major ligand interactions with NSP15, the shortest distance of which was 2.70 with the residue Lys290. Figure 5 shows the measures though MD distances between the NZ atom of LYZ290 of protein with the geometric center of these compounds. One can see that these distances pretty stable during MD simulations. 4). Table 4. List of selected compounds with known antiviral activity. According to the DrugVirus.info database 18 , 13 of the antiviral compounds selected by the pharmacophore-based search showed activity against a total of 40 viruses in cell-culture, animal, and clinic models (Figure 6). The other eight antiviral compounds were not in the database. A previous study did not identify any of these compounds as potential NSP15 inhibitors, and their top selected drugs did not show antiviral activity 14 . Differences in methodology may explain these discrepancies of results. Specifically, Chandra and co-authors used NSP15 PDB ID 6W01 structure with a citrate ion 14 ; we used crystal structure of NSP15 in complex with tipiracil that binds to NSP15 uracil site. We assume that the pharmacophore model generated on this protein structure includes the key features responsible for ligand interaction with residues in NSP15 active site. We did notice that tipiracil, the positive control, did not have a low free energy. However, an in-vitro study confirmed that tipiracil can inhibit uracil binding to the NSP15 active site presumably through competitive inhibition and modestly suppress SARS-CoV-2 viral replication in cellular assays 12 . Cluster A includes six pyrimidine analogues that are currently used as viral inhibitors: HIV reverse transcriptase inhibitors-zidovudine and stavudine, HBV DNA polymerase inhibitor-telbivudine, and HSV DNA polymerase inhibitors-brivudine, edoxudine, and trifluridine (Table 4 and Figure 6). The other two drugs in cluster A, tipiracil 12 and floxuridine 19 , are anticancer drugs that have antiviral properties. All these pyrimidine analogues are polymerase inhibitors, which is a major class of antiviral drugs. These results support using NSP15's pharmacophore features to identify potential antiviral compounds containing a pyrimidine-like scaffold and further development of nucleotide-like drugs with higher affinity for the active site of NSP15. Recent studies demonstrated that tyrosine kinase inhibitors have antiviral potential through inhibition of key kinases required for viral entry and reproduction 20,21 . Thus, repurposing receptor tyrosine kinase inhibitors is an effective strategy in the fight against COVID-19 22 . Our pharmacophore model successfully identified four tyrosine kinase inhibitors with antiviral activity in cluster G, the binding affinities of which are high. Dasatinib, an approved drug for chronic myelogenous leukemia (CML), has activity against both MERS-CoV and SARS-CoV in vitro and possible protection against SARS-CoV-2 infection 23,24 . EGFR inhibitor gefitinib has demonstrated in vitro activity against HCV, BKV, CMV, and VACV (Figure 6). Lapatinib was just recently found to potently inhibit SARS-CoV-2 replication at clinical doses, strongly supporting our screening result 25 . Promising antiviral drugs from cluster U includes HIV proteinase inhibitor amprenavir. Specifically, amprenavir has a free energy of −7.29 kcal/mol and modestly inhibits replication of SARS-CoV-2 in vitro 26 . Outside of clusters A, G, and U, other antiviral drugs include influenza neuraminidase inhibitors peramivir and oseltamivir, HIV non-nucleoside reverse transcriptase inhibitor doravirine, and HCV NS5B polymerase inhibitor sofosbuvir, which displayed activity against SARS-CoV-2 27 . Interesting to note that some of randomly selected FDA-approved drugs had free energies below −7.00 kcal/mol, namely gadoxetate (−8.31 kcal/mol), iohexol (−7.45 kcal/mol), and chlortetracycline (−7.11 kcal/mol) (Table S1). These compounds also can be potential inhibitors of NSP15. ## CONCLUSION Given the severity of the COVID-19 pandemic, we need a fast way of finding treatment. Identification of FDA-approved drugs to inhibit SARS-CoV-2 could lead to advances in this field. Though this study is limited due to only using computer-based screening, the implications of the 170 compounds is a key step in finally finding a treatment. Twenty-one of these drugs have known antiviral properties, some of which have demonstrated inhibition of SARS-CoV-2 in vitro. We recommended testing of selected compounds for the treatment of COVID-19, especially those in clusters A, G, and U. ## ACKOWLEDGEMENTS We would like to thank the people of San Diego Supercomputer Center and CCG (Montreal, Canada).

chemsum

{"title": "Potential SARS-CoV-2 Nonstructural Protein 15 (NSP15) Inhibitors: Repurposing FDA-Approved Drugs", "journal": "ChemRxiv"}

ap-net:_an_atomic-pairwise_neural_network_for_smooth_and_transferable_interaction_potentials

## Abstract: Intermolecular interactions are critical to many chemical phenomena, but their accurate computation using ab initio methods is often limited by computational cost. The recent emergence of machine learning (ML) potentials may be a promising alternative. Useful ML models should not only estimate accurate interaction energies, but also predict smooth and asymptotically correct potential energy surfaces. However, existing ML models are not guaranteed to obey these constraints. Indeed, systemic deficiencies are apparent in the predictions of our previous hydrogen-bond model as well as the popular ANI-1X model, which we attribute to the use of an atomic energy partition. As a solution, we propose an alternative atomic-pairwise framework specifically for intermolecular ML potentials, and we introduce AP-Net-a neural network model for interaction energies. The AP-Net model is developed using this physically motivated atomic-pairwise paradigm and also exploits the interpretability of symmetry adapted perturbation theory (SAPT). We show that in contrast to other models, AP-Net produces smooth, physically meaningful intermolecular potentials exhibiting correct asymptotic behavior. Initially trained on only a limited number of mostly hydrogen-bonded dimers, AP-Net makes accurate predictions across the chemically diverse S66x8 dataset, demonstrating significant transferability. On a test set including experimental hydrogen-bonded dimers, AP-Net predicts total interaction energies with a mean absolute error of 0.37 kcal mol −1 , reducing errors by a factor of 2-5 across SAPT components from previous neural network potentials. The pairwise interaction energies of the model are physically interpretable, and an investigation of predicted electrostatic energies suggests that the model 'learns' the physics of hydrogen-bonded interactions. ## I. INTRODUCTION Recent advances in the field of machine learning (ML) offer an exciting new perspective on the perpetual costaccuracy trade-off of quantum chemistry. 1,2 Models like neural networks (NNs), which are flexible universal function approximators, can be used to predict a variety of chemical properties. For a target molecular system, a parameterized (or trained) model can estimate a property in a small fraction of the time needed to compute it with quantum methods. These models come with some caveats, however. The accuracy of a prediction is highly dependent on the amount of data used to train the model and the similarity of the training data to the molecule to be predicted on. Also, it is nontrivial to design and train an ML model. Specific choices made in the architecture of a neural network, the choice of features, hyperparameters, etc. can have a large impact on the model's accuracy and transferability. Over the past few years, ML models have been developed for seemingly every chemical property that can be computed with an ab initio method. The chemistry community has investigated electrostatic multipoles 3 , rate constants 4 , chemical shifts 5 , etc. However, a significant amount of attention has been dedicated to ML potentials (i.e. energy prediction). These ML potentials possess one commonality, which is that their development and application have emphasized covalently bound systems and accurate total energy predictions. Less attention has been paid to noncovalent interactions (NCIs) and interaction energies, which are of fundamental importance to drug binding, liquid structure, biomolecular structure, molecular crystals, etc. In many of these applications, obtaining accurate interaction energies is a more important goal than total energies. This is not to say that existing ML potentials totally neglect NCIs. The ANI-1X model, for example, was trained using a dataset containing many molecular dimers. 14 Also, accurate total energy predictions can be used to obtain accurate intermolecular energy predictions. For a dimer, the interaction energy (∆E int ) is defined as: where E AB , E A , and E B are total energies of the dimer and two monomers. For any ML potential, dimer interaction energies can be evaluated with this so-called 'supermolecular' approach. One challenge for NCIs is that to obtain accurate interaction energy, one needs good cancellation of errors in all three component energies, E AB , E A , and E B . Alternatively, more accurate models of interaction energies might estimate ∆E int directly. Creating such an intermolecular ML potential poses some unique challenges, as many standard representations of molecular systems for ML are not necessarily applicable, and special care must be taken in generating useful training data. These and other concerns are discussed in our recent pilot study of hydrogen-bonding interactions, which to our knowledge is the first purely ML intermolecular potential designed to work on an entire class of chemical systems. 19 In that work, neural networks were trained to predict symmetry adapted perturbation theory (SAPT) interaction energies, resulting in a model which we refer to here as SAPT-ML. The SAPT-ML intermolecular potential was designed in the same spirit as many of the popular total potentials, including the use of an atomic energy partition, discussed in more detail in Section II B. On a dataset of crystallographic and artificially constructed hydrogen bonding dimers, SAPT-ML predicted interaction energies with a mean absolute error of 1.2 kcal mol −1 , approaching quantitative accuracy, while using a relatively small training dataset for deep learning tasks. The primary evaluation metric for any potential, SAPT-ML included, is generally a summary of the error distribution such as the mean absolute error, max absolute error, etc. preferably computed for a large and representative test dataset. A useful ML potential will make predictions with small errors, but these summary statistics alone are not sufficient criteria for usefulness. Of arguably equal importance is the smoothness of the predicted potential energy surface (PES). Jagged PESs yield inaccurate forces, which is a particularly problematic concern for molecular dynamics simulations, a target application of ML potentials. Smoothness is also a requirement for geometry minimization and transition state searches. For an intermolecular PES, an ML potential should adhere to additional asymptotic constraints. The predicted interaction energy should be approximately zero at large separations and strongly repulsive at near separations. The predicted PES should not only be locally smooth, but also exhibit minima with approximately the same energies and locations as the true PES. The importance of smoothness for ML potentials has been acknowledged by ML approaches that use energy gradients in the training procedure, 20 but to our knowledge global PES smoothness has not been studied in detail, particularly in the context of intermolecular interactions. In the pilot hydrogen-bonding study, we did not explicitly examine smoothness nor asymptotic convergence of the SAPT-ML model. Here, we re-examine our SAPT-ML potential and also investigate the popular ANI-1X potential. We find that both models produce intermolecular PESs with unphysical irregularities. This appears to be a fundamental weakness of atomic neural network potentials, which predict an atomic partition of the target property. To overcome this deficiency we propose an alternative atomic-pairwise interaction paradigm, and we introduce AP-Net, a corresponding atomic-pairwise neural network intermolecular potential. To illustrate the advantage of this new approach, we test AP-Net on the same hydrogen-bonding task as SAPT-ML and report up to a 5-fold reduction in errors across the SAPT interaction energy components. The AP-Net model, trained only on a modest number of mostly hydrogen-bonded dimers, is also tested on the chemically diverse S66x8 benchmark dataset. 21 This atomic-pairwise models exhibits surprising transferability, making reasonable estimates of intermolecular PESs dominated by π−π stacking and dispersion interactions for systems where ANI-1X generates incorrect potentials. Overall, we observe that AP-Net uniquely displays correct asymptotic behavior and makes smooth predictions along intermolecular coordinates, both necessary aspects of intermolecular potentials. Lastly, we examine the individual atomicpair energies predicted by AP-Net and find good agreement with chemical intuition. This suggests that AP-Net predicts interaction energies by 'learning' some physically meaningful chemical representation. ## II. METHODOLOGY A. Symmetry Adapted Perturbation Theory In order to train an intermolecular ML potential, reference interaction energies (or labels) are needed from some quantum chemistry calculation. In principle, any wavefunction or density functional theory (DFT) method could be used in conjunction with Equation 1 to obtain supermolecular reference interaction energies. An alternative theoretical approach for computing the interaction energy is symmetry adapted perturbation theory (SAPT). In its wavefunction-based formulation, SAPT accounts for the interaction between two monomer Hartree-Fock wavefunctions through a triple perturbation series in monomer A correlation, monomer B correlation, and intermonomer interaction. SAPT has a few advantages over the supermolecular approach. Firstly, SAPT is formally correct in the limit of the perturbation series, and it recovers the full configuration interaction solution to the time-independent Schrödinger equation. However, a major appeal of SAPT is the accuracy of early truncations of the series. One of the most popular and economical SAPT methods, 0 th order intramonomer correlation and 2 nd order intermonomer interaction, is commonly referred to as SAPT0. Combined with an appropriate jun-cc-pVDZ basis set, SAPT0 has been shown to be surprisingly accurate, in part due to fortuitous error cancellation. 25 At only O(N 5 ) in cost, SAPT0 is is affordable enough to probe interactions in systems with hundreds of atoms, large enough to be of biological interest. 26 The most useful feature of SAPT for an ML potential, however, is the resulting physically meaningful decomposition of the interaction energy. The individual terms of the perturbation series correspond to standard interpretations of intermolecular interactions: interactions between permanent charge distributions of opposite monomers (electrostatics, E elst ), induction or polarization of a charge distribution on one monomer by a charge distribution on the other (induction, E ind ), simultaneous correlation between charge distributions on opposite monomers (dispersion, E disp ), and finally fermionic Pauli exchange between electrons on opposite monomers (exchange, E exch ): These components are often exploited in modern force field development, where separate physically-motivated functional forms are developed and parameterized for each term. An analogous approach can be taken in an intermolecular ML potential by structuring a model to predict these individual components, therefore allowing the model to exploit the interpretability of SAPT. Detailed equations for the exact specification of individual SAPT0 components and their efficient implementation through density-fitting techniques are presented in Refs 35 and 36. ## B. Pairwise Energy Partition Nearly every published ML potential follows the same general formulation. For a given molecular system, there exist many arbitrary partitions of the total energy (E) into atomic energies (E i ): It is assumed that a partition exists such that E i is a learnable and transferable function of the local environment around atom i. Regression models such as neural networks are used to predict E i for each atom. No ab initio E i labels exist, as atomic partitions of the energy are not generally computable with quantum mechanical methods. Instead, the sum of predicted E i 's is constrained to match E, which in most ML frameworks is easily specified. Thus, the predicted total energy is estimated through a learned atomic energy partition. The various ML approaches primarily differ in how the regression model is parameterized to predict the partitioned energy E i of each atom i. The atomic nature of this type of potential has an important consequence. Because the regression is performed at the atomic level, models are transferable to many different molecules, provided that the local environments of atoms in the molecules are similar. For example, an atomic potential trained on conformations of butane and hexane should provide reasonable estimations for the PES of a pentane molecule. The transferability of atomistic models of this kind can be contrasted with traditional system-specific potentials, which must be created and parameterized separately for each molecular system of interest. A weakness of atomic ML potentials is their poor performance in capturing long-range interactions. 10,13 This is because the enforced locality of the atomic partition is at odds with the long-range nature of NCIs. Most ML potentials limit an atom's environment to include neighboring atoms within some distance cutoff that can be as short as 5 . This is insufficient in capturing small but chemically important long-range electrostatics, and to a lesser degree, van der Waals effects. If one chose to use spatially large atomic environments, the transferability and computational cost of the resulting ML model would suffer. To address this issue, some machine-learning potentials simply add a classical force-field model (like Grimme's D3 dispersion correction) to their predictions in order to describe distant interactions 9,13 . Other models are parameterized to predict atomic charges, which are used to evaluate a separate long-range electrostatic energy. 17 In an attempt to explore the modeling of NCIs with flexible neural network models, we recently adapted the atomic partitioning approach to the explicit prediction of dimer interaction energies. 19 The resulting model, SAPT-ML, was trained to predict the four SAPT0 components for a set of hydrogen-bonded dimers. A consequential design choice of the SAPT-ML model was that the interaction energy could also be partitioned into individual atomic contributions, each of which is a learnable function of an atom's local environment: where a and b index the atoms of monomers A and B respectively. Eq. 4 is a straightforward intermolecular adaptation of Eq. 3, the standard formulation of ML potentials. The current work is motivated by the observation that NCIs between molecules are well understood to be approximately a sum of interactions between pairs of atoms. Atomic-pairwise additivity corresponds to a different partition of the interaction energy: This paradigm is fundamental to NCIs and dates at least as far back as the original Lennard-Jones potential. 37 Pairwise additivity is still the basis of the many popular classical force fields like AMBER, CHARMM, and OPLS. Advanced polarizable force fields such as AMOEBA add small, self-consistent corrections on top of a pairwise additive model. 41 The D3 dispersion correction of Grimme and coworkers captures the purely quantum mechanical phenomenon of dispersion with yet another pairwise model. 42 Our group has even developed postprocessing methods for partitioning a calculated SAPT0 interaction energy into both atom and functional group pairs. 43,44 In the design of machine learning models, incorporating prior knowledge about the nature of the function to be approximated is a fundamental priority. For example, major advances in the field of computer vision are a result of encoding locality and shift invariance in neural networks (through so-called "convolutional" layers). 45,46 Given the overwhelming amount of chemical intuition and empirical evidence supporting the pairwise additive nature of NCIs, it seems imperative to incorporate this information into the model. This insight is the motivation behind AP-Net, an atomic pairwise neural network model for interaction energies, developed in this work. Functionally, AP-Net is similar to SAPT-ML in that both models use a geometric description of a molecular dimer to predict an interaction energy. The important difference lies in the atomic-pairwise nature of AP-Net's architecture. ## C. Features Regression problems require a careful choice of variables (or features/descriptors) from which a mapping is approximated (or learned) to some desired property (or label). The selection of features can greatly affect both the accuracy of the final model and its ability to generalize well to unseen data. These concerns are particularly relevant for chemical systems, since standard feature engineering techniques don't immediately apply to the unique graph-like structure of molecular geometries. Thus, the representation of chemical data is an essential component of machine learning potentials. Some aspects of useful features can be reasoned about a priori. For one, features should obey the same invariances as the predicted property. For potentials, this means that the features should be invariant to relabeling of identical atoms, molecular translation, and molecular rotation. While many features are used across various atomic potentials, the atomic-pairwise model described in this work necessitates the development of features that can describe a pair of atoms. To begin with, the atomic pair paradigm has an immediate set of sensible and descriptive features not explicitly available in the original atomic paradigm: Z a , Z b , and r ab . The existence of simple, qualitatively correct, nonbonded force fields based on these variables suggests that they can be used to account for a large fraction of interaction energies, and we use them as inputs to AP-Net. However, these three variables alone are not sufficient to describe all of NCIs, as they don't contain information about individual atomic environments or their orientations with respect to the other monomers. Thus, additional descriptors are necessary. For this purpose, the popular atom-centered symmetry function (ACSF) 7 is reviewed and the new atom-pair symmetry function (APSF) is introduced. ## Atom-Centered Symmetry Functions (ACSFs) The prediction of pseudo-atomic properties (like charge) and atomic partitions of molecular properties (like energy) are common applications of machine learning to chemistry. As such, much effort has been put into the design of features that encode local atomic environ-ments to use in ML models. In contrast, this work seeks to predict a partitioning of the interaction energy over pairs of atoms, not individual atoms. Nevertheless, a reasonable starting point for a feature to describe a pair of atoms is simply the concatenation of individual atomic features. A well-established descriptor for this purpose is the radial atom centered symmetry function (ACSF) of Behler and Parinello. 7 A radial ACSF of atom i (G rad i ) describes the atom's local environment in terms of the radial distribution of neighboring nuclei: G rad i encodes the radial density of atom i's neighbors. The δ ZZj term filters only neighboring nuclei with a particular atomic number. The parameters µ and η define the radius and width of a spherical Gaussian shell upon which the neighbor density is projected onto. Lastly, the cutoff function f c ensures locality by removing contributions of neighbors outside of a chosen cutoff radius r c . In practice, many radial ACSFs are used to describe the complete radial environment around atom i (i.e. a collection of G rad i with varying µ, η, and Z). We denote such a collection or vector of ACSFs for a single atom with different {(µ, η, Z)} as G rad i . Figure 1 depicts individual elements of G rad i for an example system. In the dimer picture, we further indicate that the ACSF vector is formed from only intramonomer neighboring nuclei as G rad a or G rad b . It is important to note that, while we chose radial ACSFs to describe the intramolecular environment for their speed and simplicity, any other feature could be substituted or added, including the more expensive angular ACSFs. In particular, the increasingly popular message-passing framework 10,12,17,47 could be adapted to the dimer picture and used here. ## Atom Pair Symmetry Functions (APSFs) The monomer ACSFs G rad a or G rad b as defined above encode an atom's local environment within its respective monomer, containing no information about the identities of, distances from, or orientations to atoms in the other monomer. However, when these monomer ACSFs are combined with Z a , Z b , and r ab , we can achieve a nearly complete description of a pair of atoms in opposite monomers, lacking only information about intermonomer orientation. A model developed to use only these features would therefore result in an isotropic description of atom-atom interactions. This can be a poor approximation, since the electron density around an atom may be anisotropic (e.g. higher along a bond axis or in the direction of an electron lone pair). ). Each µ is associated with a direction from the hydrogen, drawn as a blue ray. The dependence of this direction on the HO intermolecular axis is indicated in red. Here, we introduce angular atom-pair symmetry functions (APSFs) as a way to account for orientation of atomic environments within the atom-pair paradigm. The angular APSF of an atom a in monomer A with respect to an atom b in monomer B is defined as: where index a runs only over atoms in the same monomer as atom a. The angular APSF closely resembles the original radial ACSF, and the two features are compared in Figure 1. In the APSF, atom a is still described by the spatial distribution of its neighboring nuclei within monomer A. The terms f c (r aa ) and δ ZZ a serve analogous roles as in ACSFs, enforcing spatial locality and filtering out nuclei by atomic number, respectively. The essential difference of this descriptor is the shape of Gaussian that the neighbor density is projected onto, which is now cone-like instead of spherical. The apex of the cone is at nucleus a and the cone is aligned with the ab axis. µ determines the apex angle of a cone (instead of the radius of a sphere), and η is still the Gaussian width. Different values of µ define cones with different angles. The value cos θ a ab is determined by the alignment of the vector r aa with the ab axis. The range of reasonable µ values is the same range as the cosine function, [−1, 1]. If an ACSF is understood to encode an atom's neighbor density as a function of distance, the APSF can be thought of as encoding an atom's neighbor density as a function of angle or orientation, where an atom in a different monomer is the third point in the angle. The vector notation used for the ACSFs is also used for APSFs. Note that G ang a(b) is not equal to G ang b(a) . The former describes the environment of atom a in monomer A with respect to the orientation of atom b in monomer B while the latter describes the environment of b with re-spect to the orientation of a. The intent behind the APSF descriptor is to explicitly decouple intermonomer orientation from intermonomer distance, which is already captured by using r ab as a feature. Without the APSF, AP-Net could not be able to account for atomic anisotropy. It would be possible to use additional hyperparameters in the APSF, for example by including an additional spherical Gaussian shell to project r aa onto with its own µ and η, much like an ACSF. However, minimizing the number of necessary features is a worthwhile pursuit if AP-Net is to be used in applications such as high-throughput screening or molecular dynamics simulations. ## D. Network Architecture, Training, & Implementation A common practice in the design of neural network potentials under the atomic partition is to parameterize a separate neural network for each element (atom type): This splitting out of networks has been seemingly necessary to achieve good performance, but is undesirable for a number of reasons. For one, it results in unwieldy implementations that scale in size with the number of treated atom types. The use of separate atomic networks is also not very data-efficient, since generalization across atom types by definition cannot be learned by independent networks. Data-efficiency is particularly important when some atom types are much less common in a dataset. In accordance with recent work on shared-weight models, 48 the proposed AP-Net architecture avoids this problem. For each SAPT component energy (E comp ), a single neural network is trained to predict atomic pair partitions (E ab,comp ) of the component energy: where the notation (•, •) represents concatenation. The vector Z i is a concatenation of the atom's atomic number and a binary variable (or one-hot) encoding of the atomic number. (For a model that accommodates N atom types, Z i has length N +1, and the last N elements are all zeros except for a one in the position corresponding to the current atom type.) The network is also symmetrized with respect to a and b by averaging the output over the two possible orders, ensuring that predictions are independent of the order of the monomers. An important final adjustment to the internal neural network architecture of AP-Net is made to encourage correct asymptotic behavior. Rather than using a raw neural network output as a prediction for E ab,comp , the output of the last network layer is scaled by r −1 ab for every component. This normalization guides predictions to have small magnitude at large interatomic distance. Thus, r ab is both an input to the model and an explicit part of the functional form. AP-Net was developed with version 2.1.0 of the Ten-sorFlow library. 49 The model was constructed to handle the six atom types present in the datasets described in Section II E (H, C, N, O, F, and S). For the ACSF feature, η is fixed at 100.0 and µ varies from 0.8 to 5.0 in increments of 0.1 . For the APSF feature, η is fixed at 25.0 and µ varies from −1.0 to 1.0 in increments of 0.1. The cutoff radius, r c is fixed at 8.0 . Each network consists of three dense layers of 128 nodes and a final output layer of a single node. As discussed previously, the output layer is scaled by r −1 ab to form E ab,comp . Additionally, both ACSF and APSF vectors are preprocessed with separate dense layers of 100 and 50 nodes, respectively. All layers use the rectified linear activation function, except for the output layer which uses a linear activation function. Each network is trained for 200 epochs using the Adam optimizer with a learning rate of 1.0 × 10 −4 and a batch size of a single molecule. 50 While we previously used a multi-target loss function that balanced the accuracy of individual component energies and total interaction energies, here we chose to train networks separately, prohibiting explicit error cancellation. 19 The training procedure minimizes the mean squared error of the predicted component energy. The network weights resulting in the lowest error on a held-out validation subset of the training data over the 200 epochs are used as in the final model. Eight randomly initialized networks are trained per SAPT component, and AP-Net reports the average prediction of the eight networks. The variance of the ensemble predictions can also be used as an uncertainty metric. ## E. Training and Testing Datasets The hydrogen-bonded dimer dataset previously developed with SAPT-ML is revisited. 19 This dataset features interactions between N-methylacetamide (NMA)a popular model system for peptide bonding and universal hydrogen-bond donor and acceptor-paired with other small hydrogen-bond acceptors and donors. The training data was created by selecting a number of these other molecules, placing them in a favorable hydrogenbonding orientation with NMA, and then procedurally varying the monomer separation and orientation. Intramonomer geometry was also sampled with small, random perturbations from equilibrium so that models trained on the dataset are capable of disentangling the effect of intramolecular geometry on the interaction energy. This was done for 92 small molecules expressing 149 chemically distinguishable donor and acceptor sites hydrogen bonded with NMA in 7784 different configurations. The training data was further supplemented with the 2192 neutral dimers in the sidechain-sidechain interaction (SSI) dataset. 51 The testing data consists of NMA in complexation with donors and acceptors absent from the training set, and was either taken from crystallographic data, 52,53 or, in the case of isoquinolone as an acceptor, generated using the same sampling method as described above for the training data. A subset of these configurations is shown in Figure 2. We will refer to these two datasets of dimers as NMA-training and NMA-testing. Throughout this work, a randomly selected subset of 498 dimers from NMA-training is used for validation as described in Section II D. Experiments are performed to assess AP-Net's ability to describe a large expanse of the intermolecular PES of a single hydrogen-bonded dimer. We examine the H 2 O dimer and the dependence of the interaction energy on intermonomer separation and orientation. We report improvements on the shape, asymptotic behavior, and smoothness of the neural network PES. We also test the transferability of AP-Net to the diverse S66x8 benchmark dataset, which contains 66 small dimers each at 8 different geometries along the radial dissociation curve. 21 The dimers consist of small, closedshell, neutral molecules and span a wide range of interaction types, from hydrogen-bonding to π − π stacking. Of the eight configurations, one is the equilibrium geometry, five are slightly dissociated (×1.05, ×1.10, ×1.25, ×1.50, and ×2.00), and two are slightly compressed (×0.95 and ×0.9). ## A. NMA Dataset We compare the accuracy of AP-Net to the reported accuracy of the original SAPT-ML model. Both models are trained on exactly the same NMA-training dataset, down to the random subset of the data used for validation. The performance of the two models on the NMAtesting dataset is shown in Table I. AP-Net exhibits a significant improvement in the prediction of all four SAPT0 components as well as the total SAPT0 interaction energy. For 8 of the 10 dimers, the AP-Net error is lower for all four energy components and the total SAPT0 energy. One exception is NMA/cyclohexanone, for which only five configurations are present in NMA-testing. With such a small sample, this behavior appears to be an instance of SAPT-ML benefiting from some combination of randomness and fortuitous error cancellation. Also, both models predict this interaction with small errors. The other dimer, 3-methylbutan-2-one features improved predictions for each component, but is narrowly worse at total interaction energies. Although AP-Net's predictions for all four SAPT0 components are an improvement over SAPT-ML, the relative magnitudes of errors between components are largely the same for the two models. Errors in the prediction of E elst remain the highest, followed by E exch , E ind , and finally E disp . The average component error is partially explained by the range of possible energies for each component. Figure 3 shows that for the NMA-testing dataset, E elst and E exch take on a wider range of values (approximately 20 and 30 kcal mol −1 respectively) than E ind and E disp (both approximately 10 kcal mol −1 ), a trend that the average errors roughly mirror. However this is not a full explanation, since, for example E elst errors are actually larger than those of E exch , despite the labels having a smaller range. The existence of relative difficulties in predicting different SAPT components may imply that the physical nature of these interaction types governs how easily they can be modeled. This would explain why AP-Net does a particularly good job at predicting E disp , with an weighted total MAE of only 0.03 kcal mol −1 , an approximately 10-fold improvement over SAPT-ML: dispersion is often well modeled by an atomicpairwise functional form. 42 The use of an individual neu-ral network per component allows the model to separately learn the different physics governing each of them, a unique advantage of training to reproduce SAPT components rather than the total interaction energy. Further tailoring of these individual networks to match known physics could yield additional improvements. Another similarity between AP-Net and SAPT-ML is the relative difficulty of predicting interactions of different monomers in the NMA-testing dataset. The largest errors are still in the prediction of uracil and benzimidazole, and the smallest errors in the prediction of isoquinolone. We note that the experimentally measured NMA/uracil hydrogen bond lengths are shorter than any of the hydrogen bond lengths in the theoretically gen- Figure 4 shows a training saturation curve for AP-Net, illustrating the incremental improvements in accuracy with more training data. The validation error consistently declines for all four SAPT0 components as up to 9000 dimers are used in training. The lack of a plateau is a positive sign, as it suggests that the predictive capability of the relatively simple AP-Net architecture is not yet saturated. Improved performance could be attained simply by adding more training data than is used in this work. The low-data limit is also encouraging. Using only 200 training dimers, AP-Net's predicted total SAPT0 in-teraction energies already reach "chemical accuracy" or 1 kcal mol −1 MAE. The ability to make reasonable predictions with little data illustrates the appropriateness of the atomic-pairwise paradigm. Another interesting detail in the saturation curve is the existence of a crossover between errors of the electrostatic and exchange components. This can probably be attributed to the variable and long-range nature of electrostatic interactions, which makes prediction slightly more difficult even with many training dimers. The electrostatic energy is the only SAPT component that can be both attractive and repulsive, and its r −1 decay is slowest among the four components. This learning curve also further reinforces the observation that the dispersion functional form is well described by a pairwise additive model. The validation MAE for E disp reaches 0.02 kcal mol −1 by 5000 training dimers. This error is much smaller than the errors expected in the SAPT0 dispersion energies themselves, as they are computed using only second-order perturbation theory. 25 Lastly, the parallel behavior of the electrostatic component and total interaction energy errors is particularly striking. Further improvements in the prediction of the SAPT0 interaction energy will necessitate focusing on this component. ## FIG. 4. Saturation curve of AP-Net mean absolute error in the total SAPT0 interaction energy and components. The number of training dimers is varied from 100 to 9000. The mean absolute error is computed on a randomly selected set of 498 validation dimers from the same distribution as the training data. All models were trained using the same procedure and hyperparameters described in Section II D. ## B. H2O Dimer Next, we examine the performance of AP-Net at describing the hydrogen-bonded water dimer. This dimer is absent from the NMA-training dataset, so this experiment is partially a test of AP-Net's ability to generalize to a different intermolecular interaction. More importantly, the H 2 O dimer is of incredible practical relevance and captures the essential, minimal hydrogen bond. We scan the intermolecular dissociation and rotation coordinates of the dimer, illustrated in Figure 5. In generating these coordinates, calculations are performed every 0.01 or 0.5 • so that we can assess not only the asymptotic predictions of the models, but also the smoothness of the ML potential. Predicting smooth intermolecular PESs is a necessary requirement for using AP-Net in molecular dynamics or searches for stable intermolecular configurations. FIG. 6. The total SAPT0 interaction energy of the H2O dimer along an intermonomer radial dissociation coordinate is plotted. Predictions of AP-Net and SAPT-ML models are compared to the true SAPT0 values. The first coordinate probes the dependence of the hydrogen bond on intermonomer separation. Note that the monomer geometries are kept rigid, so any change in AP-Net's prediction along the scanned coordinate must attributed to changes in the features r ab , G ang a(b) , and G ang b(a) ; the other features (Z A , Z B , G rad a , and G rad b ) are constant. The total SAPT0 predictions of AP-Net and SAPT-ML in Figure 6 show that only AP-Net correctly captures the shape of the SAPT0 potential. AP-Net predicts a minimum in the total interaction energy at approximately the correct intermonomer separation, although the strength of the hydrogen bond is underestimated by approximately 1 kcal mol −1 . The most striking difference between SAPT-ML and AP-Net, however, is the smoothness of the intermolecular PES. For a given component, neighboring points on the SAPT-ML potential curve fluctuate as much as an entire kcal mol −1 . We believe this noisiness to be a result of the intermolecular descriptors used by SAPT-ML. The availability of actual distances as a feature in AP-Net's atom-pair paradigm combined with the carefully designed APSF feature is a significant improvement over the intermolecular ACSFs used in SAPT-ML. The second coordinate, shown in Figure 7, is a particularly challenging test of an intermolecular potential, as it isolates the angular dependence of the hydrogen bond. Not only are intramonomer geometries rigid, but also r OH is constant value of 1.95 for the hydrogenoxygen pair participating in the hydrogen bond interaction. Therefore, most of the change in predicted energy over this coordinate must be accounted for by the new APSFs. AP-Net is still able to correctly predict the trend of the total interaction energy through the rotation. The decline in prediction quality at the smallest angle is a result of a strong repulsive clash between the two oxygen atoms, which become unreasonably close if we follow the curve all of the way to 90 • ; this repulsive contact is unlike any in the training dimers. The strong curvature of the potential in Figure 6 is an important result, as it shows that AP-Net is able to account for molecular anisotropy, a necessary aspect of accurate NCI potentials. Predictions are still smooth, especially when compared to SAPT-ML. ## C. S66x8 We have shown AP-Net to be an effective general model for hydrogen-bonded dimers when trained on the NMA-training dataset of similar hydrogen-bonded dimers. However, a useful intermolecular potential must also accurately describe all types of NCIs. This could obviously be accomplished by including additional and diverse training data. Here, we examine transferability from an alternate perspective: How well can the AP-Net model, a model specialized at hydrogen-bonding, describe other kinds of interactions? This type of analysis is necessary for ML potentials, since we cannot always expect a target system to be well represented in the model's training data. Some combination of interpolation and extrapolation within chemical space will always be necessary. AP-Net's transferability is assessed by examining performance on the popular S66x8 benchmark dataset for intermolecular interactions. As a comparison, we also test the ANI-1X ML potential on the same benchmark. ANI-1X is a neural network potential for organic molecules, trained on a dataset of 5.5 million molecular conformations. This dataset includes many noncovalent complexes, making ANI-1X one of the best candidates for a robust intermolecular ML potential. Because AP-Net and ANI-1X are trained to match different levels of theory (SAPT0/jun-cc-pVDZ and ωB97X/6-31G* respectively), it would be unfair to compare each model's predictions to a single approximate reference method. Instead, we show predicted interaction energies for each model alongside the reference interaction energy at the same level of theory used to parameterize that model. Of the 66 different dimers, two pathological but representative cases are shown: the benzene-benzene sandwich dimer in Figure 8 and the cyclopentane-neopentane dimer in Figure 9. Predictions on the remaining 64 dimers can be found in the SI. The intermolecular PESs predicted by AP-Net are at least as accurate and reasonable as ANI-1X's on average. For the benzene dimer, both AP-Net and ANI qualitatively predict the required interaction energy trend-repulsive at close separation and near zero at dissociation, with a slightly attractive minimum somewhere in between. However, ANI-1X predicts a steeper repulsive wall and more separated minimum, potentially as a result of missing the tricky chargepenetration effects known to occur in π − π interactions. AP-Net closely matches the entire potential, even though this dimer is chemically unlike the NMA hydrogen-bond dimers that make up its training data. AP-Net's behavior is unlikely to be the result of a lucky guess, since the predicted potentials of other π − π interactions in S66x8 are similarly correct. Presumably, the AP-Net model learned a representation of aromatic carbons via secondary interactions present in hydrogen-bonded dimers such as NMA/benzene. The cyclopentane-neopentane dimer is an even more extreme comparison, and a good example of the disadvantages of relying solely on error statistics. Although the ANI-1X prediction is fairly accurate in terms of mean absolute error, the shape of the potential is unphysical, containing a spurious minimum and maximum along the coordinate. These artifacts would severely hinder the practical use of this potential. While AP-Net's prediction has only a slightly better MAE than that of ANI-1X, the predicted potential is parallel to the correct potential, and could be reasonably used for molecular dynamics or a rigid monomer geometry optimization. This occurs despite the fact that the AP-Net model was trained on a much smaller and more chemically homogeneous dataset than ANI-1X. The physical appropriateness of the atomic-pairwise representation is uniquely responsible for the dramatic generalization ability of AP-Net. It should also be emphasized that this type of purely dispersion-bound interaction was not well represented in the NMA-training dataset. ## D. Pairwise Partition Analysis So far, the atomic-pairwise energy predictions of AP-Net are not individually used. They are summed to produce a predicted dimer interaction energy, which is then compared with the ab initio interaction energy. However, the existence of pairwise energy predictions is a unique feature of the atomic-pairwise paradigm, and analyzing these energies can provide insight into the AP-Net model and its representation of chemical systems. For example, AP-Net's pairwise partition of the interaction energy could be compared with empirical force-field models. Pairwise energy predictions of the H 2 O dimer along the radial dissociation coordinate from Section III B are shown in Figure 10. As discussed earlier, the predicted SAPT0 interaction energy of this dimer as well as the four SAPT0 components correctly decrease in magnitude along the coordinate. Here, we see that the individual atompair predictions are similarly distance-dependent. This distance-dependence is a good validation of the AP-Net model, since it matches our intuitive understanding of interactions-any atom-pair interaction should grow weaker as the atoms become farther apart. AP-Net's predictions also qualitatively match the known physics of intermolecular interactions. Electrostatics, the longestrange SAPT component, is correctly predicted to decay slowest of the four components. The pairwise exchange, induction, and dispersion energies have the correct sign. These components are by definition only repulsive or attractive, and we would expect the pairwise energies to match this. It is also interesting to note that the signs of individual electrostatic energy predictions. Interactions between hydrogen and oxygen are attractive, while interactions between two atoms of the same type are repulsive. These predictions reflect the partial-charge picture used in virtually every force-field, even though AP-Net is not trained to predict anything related to electron density. This phenomenon is a natural consequence of the pairwise framework and is an exciting result, since it suggests that AP-Net contains some fundamental representation of the actual physics occurring in intermolecular interactions. This analysis of pairwise energies shows that AP-Net's predictions are uniquely physically interpretable, and it works towards countering the longstanding "blackbox" criticism of ML models. In Figure ??, the interpretation of pairwise energies is taken a step further by comparing AP-Net's predictions to those of an ab initio force field developed by Van Vleet et al. 32 . The force field uses atomic dispersion coefficients (C 6 , C 8 , C 10 , C 12 ) computed from H 2 O monomer frequency-dependent polarizability tensors. Because this force field is fit to DFT-SAPT while AP-Net is fit to SAPT0, complete agreement between the two predictions is not expected. Still, the total dimer dispersion energy predicted by AP-Net and the force field are exceedingly close across the entire dissociation coordinate. Although the two predictions agree on the value of E disp , they differ significantly in the atomic-pairwise partition of E disp . AP-Net attributes most of the interaction energy to the close oxygen-hydrogen pair, while the force field assigns an approximately equal split between the close oxygenhydrogen pair and the oxygen-oxygen pair. The two partitionings are not completely irreconcilable, as they both yield the same ordering of pairwise interactions by magnitude. The qualitative agreement between AP-Net and the force field further validates the physical grounding of the AP-Net model. The close correspondence of AP-Net to an ab initio force field is something very few (if any) ML models can claim. AP-Net's predictions could be used to extract a different set of dispersion coefficients. This comparison also illustrates an important lesson in using any sort of energy partition, which is that there is not a singular 'correct' partition. ## IV. CONCLUSIONS The development of ML potentials is a rapidly evolving endeavor, as evidenced by increasingly technical model architectures, more exhaustively constructed datasets, and lower reported errors on common benchmark tasks. As the field progresses, assessing and developing ML potentials for practical use will become even more important. One such practical application is the quantitative description of of NCIs, where estimated PESs must be smooth and physically reasonable. Although our previously developed SAPT-ML intermolecular potential obtained average errors near chemical accuracy, an investigation of the model's predictions revealed serious shortcomings in the estimated PESs. In order to address these concerns, here we propose a different formulation of ML potentials specific to the intermolecular case. Instead of the usual atomic partition of energy central to nearly all ML models, the new formulation substitutes an atomic-pairwise partition of interaction energies. Although the energy partition used in any ML potential is arbitrary, an atomic-pairwise partition is decidedly more physically motivated, and therefore it should improve the accuracy and generalizability of any ML potential that make use of it. To test this claim we introduce AP-Net, an atomic-pairwise neural network model for the prediction of interaction energies. New ML descriptors that efficiently represent of a pair of atoms are also developed for AP-Net, including an APSF feature that captures the orientation dependence of monomers, a key aspect of NCIs. AP-Net is applied to the SAPT0 hydrogen-bonding task developed with the atomic SAPT-ML model, and we find that the new atomic-pairwise model yields dramatic improvements in both the accuracy of single-point energies and the smoothness of predicted potentials. On an experimental NMA hydrogen-bond test dataset, AP-Net predicts a smooth and relatively accurate intermolecular PES for the H 2 O dimer, correctly describing both the radial and angular dependence of a hydrogen-bond, after being trained on a dataset entirely absent of this dimer. This hydrogen-bond specialized AP-Net model also shows a surprising ability to generalize across chemicalspace, achieving a mean absolute error of 1.1 kcal mol −1 on the entire S66x8 noncovalent interaction benchmark. This level of accuracy surpasses that of the general universal neural network potential ANI-1X. AP-Net predicts more physically reasonable potentials than the aforementioned ML potential on this benchmark, free of spurious optima, while using fewer and less diverse training data. The ability of AP-Net to make reasonable predictions for disparate interaction types, i.e. extrapolation across chemical space, is an incredibly important characteristic of an ML potential. This behavior is a direct consequence of the atomic-pairwise framework, which provides the model with a physically motivated inductive bias. Put another way, AP-Net regresses over many atomatom interactions, while an atomic model like SAPT-ML regresses over fewer atom-monomer interactions. The atom-atom 'chemical-space' is much smaller than the atom-monomer 'chemical-space', making the regression problem simpler and the predictions more accurate under an atomic-pairwise energy partition. Future work related to AP-Net will focus on using a larger, more chemically diverse dataset that samples a greater expanse of the intermolecular PES. Curating more accurate interaction energies than the SAPT0 labels used here is also pertinent, given that AP-Net's errors with respect to SAPT0 are on average smaller than the errors in the ab initio SAPT0 calculation. Lastly, we note that the general atomic-pairwise framework advocated for in this work could be easily adapted to work with other features, model architectures, and learning tasks. ## SUPPLEMENTARY MATERIAL The supplementary material contains additional analysis of AP-Net's predictions on the NMA-testing dataset. Predictions on all 66 radial coordinates in the S66x8 benchmark are also included. ## DATA AVAILABILITY Code to create an AP-Net model, all datasets used in this work, and the trained AP-Net model used in this work will be included with the final manuscript.

chemsum

{"title": "AP-Net: An atomic-pairwise neural network for smooth and transferable interaction potentials", "journal": "ChemRxiv"}

fundamental_analysis_of_piezocatalysis_process_on_the_surfaces_of_strained_piezoelectric_materials

## Abstract: Recently, the strain state of a piezoelectric electrode has been found to impact the electrochemical activity taking place between the piezoelectric material and its solution environment. This effect, dubbed piezocatalysis, is prominent in piezoelectric materials because the strain state and electronic state of these materials are strongly coupled. Herein we develop a general theoretical analysis of the piezocatalysis process utilizing well-established piezoelectric, semiconductor, molecular orbital and electrochemistry frameworks. The analysis shows good agreement with experimental results, reproducing the time-dependent voltage drop and H 2 production behaviors of an oscillating piezoelectric Pb(Mg 1/3 Nb 2/3 )O 3 -32PbTiO 3 (PMN-PT) cantilever in deionized water environment. This study provides general guidance for future experiments utilizing different piezoelectric materials, such as ZnO, BaTiO 3 , PbTiO 3 , and PMN-PT. Our analysis indicates a high piezoelectric coupling coefficient and a low electrical conductivity are desired for enabling high electrochemical activity; whereas electrical permittivity must be optimized to balance piezoelectric and capacitive effects. Piezocatalysis is a new approach toward enabling or enhancing electrochemical processes by making use of the strain state of a piezoelectric material 1 . Piezocatalysis is the product of an intimate interaction between the native electronic state of the piezoelectric material, the chemistry of the surrounding medium, and a strain induced piezoelectric potential. The action of mechanically deforming a piezoelectric material induces a perfuse electric field which augments the energetics of both free and bound charges throughout the material 1,2 . The thermodynamic feasibility and kinetics of electrochemical processes occurring at the surface of the piezoelectric material sensitively depend upon the electrochemical potential difference between charges on the piezoelectric's surface and in the surrounding medium 3-6 . Thus piezoelectric potential, which can dramatically affect the difference between these electrochemical potentials, is a new means of modulating the material's electrochemical activity via its strain state.Recently, a piezocatalysis process was demonstrated by our study of a strained ferroelectric Pb(Mg 1/3 Nb 2/3 )O 3 -32PbTiO 3 (PMN-PT) beam in a deionized (DI) water system, from which a strong dependence of hydrogen evolution from the aqueous surroundings on the material's piezoelectric potential was observed 1 . In addition, numerous recent works have confirmed the correlation between electrochemical activity and piezoelectric or ferroelectric polarization in a broader sense. For example, a study conducted using ferroelectric poly(vinylidene fluoride) (PVDF) has demonstrate that in-situ piezopotential can influence lithium battery charging behavior 7 . Electrochemical deposition was found to be selectively activated by the ferroelectric domain polarization [8][9][10][11][12][13][14][15] . However, to date there is no general theoretical analysis of the piezopotential's effect on electrochemical activities, e.g. how one piezoelectric material's activity differs from another; the influence of metallic electrodes as compared to bare piezoelectric surface on reaction output, and how free charge in piezoelectric material systems affects the piezocatalysis process. In this paper, we address the piezocatalysis system in generality to elucidate the details underlying these open questions and illuminate trends for further experimental study.In order to clearly understand the piezocatalysis process, a conventional electrocatalysis process is discussed first, where the application of electrical potential from an external power source is a typical means of driving the electron transfer reactions (Fig. 1a). The applied potential can result in one of the following two processes: (1) lowering electronic energy levels of unoccupied states within the electrode to a magnitude less than that of the highest occupied molecular orbital (HOMO) in solution; (2) raising occupied states within the electrode above the lowest unoccupied molecular orbital (LUMO) in solution. Under the first condition, electrons will leave the HOMOs in solution and transfer to the unoccupied states within the electrode -oxidizing the solution (center panel of Fig. 1a). Under the second condition, electrons will leave the occupied states in the electrode and transfer to the LUMOs in solution -reducing the solution (right panel of Fig. 1a). When the electrode is a non-metallic solid, the eQ HOMO and ew LUMO logic extends to the materials valence (eQ VB ) and conduction band (eQ CB ) edges, respectively. For piezocatalysis, the external power source is replaced by piezoelectric potential which results from the piezoelectric polarization (P PZ ). A perfectly insulating piezoelectric material is the most ideal and simplest case and it will be analyzed first. For such a material, mechanical deformation creates a perfuse electric field inducing a total energy shift across the material, V p max , given by: where T k is an applied stress in the k dimension, d xk are the piezoelectric moduli, e 0 is the electrical permittivity of free space, and e r,x is the relative permittivity in the x dimension and W x is the width of the piezoelectric material in the x dimension. In a one-dimensional case, x and k are equal and the subscripts are dropped. This piezoelectric potential, which changes the energetics of the valence band (VB) and conduction band (CB) across the piezoelectric material, can have a dramatic effect on the material's interaction with its environment (Fig. 1b). In this model it is assumed that stain does not change the magnitude of the band gap. If the eQ VB approaches ew LUMO , it becomes energetically favorable for electrons to leave the VB and enter the LUMO (center panel of Fig. 1b). If the eQ CB approaches eQ HOMO , it becomes energetically favorable for electrons to leave the HOMO and enter the CB (right panel of Fig. 1b). Placing metal electrodes between the piezoelectric and solution, with their continuous density of states about their Femi energies, simplifies this situation: the piezopotential now acts as a bias, lifting and lowering the metal's Fermi energy eQ M (Fig. 1c). Piezoelectric materials, which are the source of bias in the case of piezocatalysis, are capable of achieving extremely high potentials (tens to hundreds of volts) when subjected to moderate to severe strain 16 . Under such circumstances (i.e. where electrode potentials versus standard hydrogen electrode (SHE) exceeding ,3 volts) many chemical species in contact with the piezoelectric will be thermodynamically capable of undergoing reduction or oxidation reactions. In order to deduce the maximum quantity of oxidation or reduction reactions possible for a given deformation, it is necessary to quantify the interaction between charge exchange at the piezoelectric/electrolyte interface and the effect of charge exchange on the piezopotential. A characteristic of piezocatalytic systems is the piezopotential drop that takes place during charge transfer to and from its surfaces (or electrodes). In this way the system acts as a capacitor. The rate of piezopotential drop is dependent upon both the piezoelectric material's properties and the nature of the solution in which it is submerged. In the case where the concentration of reactive species in solution is low, e.g. when pure Milli-Q water is used for the hydrogen reduction reaction, and the power available for driving electron-transfer reactions is sufficiently high, the rate of electrochemical reactions at the electrode is dominated by the diffusion rate of redox species to the electrode. In the case of Milli-Q water, the redox species are protons, hydroxide and impurity species. Mass transfer to the electrode is described by the Nerst-Planck equation, which in its one-dimensional form along the x-axis is written as: where J i (x) is the flux of species i at distance x from the surface, D i is the diffusion coefficient, LC i (x) Lx is the concentration gradient at distance x, F is the Faraday constant, R is the gas constant, T is absolute temperature, LQ(x) Lx is the potential gradient, C i is the concentration of species i, and n(x) is the velocity with which a volume element in solution moves along the axis 17 . The expression describes the contributions of diffusion, migration and convention, respectively, to the flux of species i. Neglecting migration and convective phenomena, the flux of species i is dependent upon diffusion alone. In the diffusion limited regime, the maximum rate of electron transfer from the piezoelectric is equal to the rate of reactant diffusion to the piezoelectric, resulting in a current density j given by: where n i is the number of electrons per reaction event with species i 17 . Applying appropriate boundary conditions to equation ( 3) and taking account of the capacitive nature of the piezoelectric bias source, yields an expression of the piezopotential V p as a function of time (a variant on the Cottrell equation): where c i is the bulk concentration of species i, f i is a parameter taking on a value between 0 and 1 that toggles the kinetics of the electrodes' reactivates with species i, and t is time 18 . Under conditions of low reactant and electrolyte concentrations and high positive electrode potential (ew LUMO .2eV vs SHE), a kinetics parameter (f i ) less than 1 causes a semi-diffusion controlled regime to form where charged reactant species (e.g. protons and metal ions) capactively couple to the electrode's surface, effectively reducing the surface potential before they are electrochemically reduced or oxidized (Fig. 2a). Under the application of a large positive electrode potential, a dilute (e.g. Milli-Q water) system's capacitance is well approximated by the Helmholtz model 17 . The voltage drop expected in time t by both electron transfer reactions and capacitive effects is given by integrating equation (4): where z i is the charge sign (1 for cation, 21 for anion) of species i, w H is the thickness of the Helmholtz layer, and l is the number of different species in solution. The positive potential of the electrode dictates that a positive charge in solution will contribute to a positive capacitive current, lowering the piezopotential. The first term in equation ( 5) describes the piezopotential change as redox reactions proceed, the second term describes the potential change due to capacitive coupling of not yet reacted species at the piezoelectric material's surface. Equation ( 5) governs how the potential on the piezoelectric's wall should drop from the time (t 5 0) of initial mechanical deformation, when the piezopotential can be a value of tens or hundreds of volts, to a time (t 5 t p ) when the potential has been reduced to a reasonable value about the SHE (e.g. 62 V vs SHE). Between time 0 , t , t p , the electrode potential is sufficiently high as to reduce or oxidize species within solution in an effectively nonselective manner. At t . t p , the piezoelectric potential falls within the range of typical eQ HOMO and ew LUMO values of ions in solution resulting in a more selective reduction and oxidation process (Fig. 2b). The processes described above pertain to both surfaces of the piezoelectric material; on one surface oxidation dominates while reduction dominates on the other. The full voltage change, as measured across the piezoelectric material, is a combination of both of these processes happening simultaneously. An electrode potential above (in the case of oxidation current) or below (reduction current) the selective potential window of ,62 V versus SHE here are treated identically. This results in a total voltage reduction across the piezoelectric material of twice the expected magnitude from a single wall (equation ( 5)). As the piezopotential reaches a modest range about 0 V (t 5 t p ), the electrochemical activity of some ions is effectively switched off while others will remain highly active. Those ions which are no longer electrochemically active are still free to participate in capacitive effects (Fig. 2b and second term in equation ( 5)). The voltage decrease across the piezoelectric from t p $ t $ ' is thus due to a separate set of electrochemical and capacitive effects conducted by a subset (i...m) of the total chemical species present (i…l). By applying equation ( 5) to both the selective and unselective regime we can describe the magnitude of piezopotential at all times by the following derived expression: where H(x) is the Heaviside function defined as 0 when x is less than 0, and 1 when x is equal to or greater than 0. e H is the electrical permittivity of the Helmholtz layer. The third and fourth terms in equation ( 6) acts to add continuity between the selective and unselective regimes. Equation ( 6) is a simplification of the activity occurring around the piezoelectric as the piezopotential drops to within the vicinity of eQ HOMO and ew LUMO of species in solution. As the energetic advantage for electron transfer diminishes, the current density j begins to show its exponential dependence upon driving force (eQ HOMO 2 ew LUMO ). The current density exponentially depends upon applied potential about E e : where j 0 is the exchange current density and is equal to: n is the number of electrons transferred in the reaction, k 0 a and k 0 C are the rate constants for the anodic and cathodic current, respectively, C R and C O are the concentrations of the reduced and oxidized species, respectively, a a is the anodic transfer coefficient, E e is the redox potential of a species in solution, and g is the applied bias (i.e. Fermi energy difference between eQ m and E e ) 18 . For the purposes of simplification, the effects of equation ( 7) will be approximated by utilizing two different kinetics constants acting in equation ( 6) for 1 $ i $ m: f 1,i and f 2,i for the nonselective time interval 0 # t # t p and the selective interval t p $ t $ ', respectively. To elucidate the behavior of the system, Fig. 2c is a plot of equation ( 6) as it is applied to the case of a piezoelectric material (PMN-PT) strained in Milli-Q water. We take a value of electrical permittivity e PMN{PT,x 5 8000 and a thickness of PMN-PT w th 5 .23 mm 19 . The value of the Helmhotlz layer thickness (w H ) was fixed to 2.75A ˚with a e H 5 80. Under standard conditions, Milli-Q water has a resistivity value of 18 V?cm due to the contribution of l ''impurities'', including hydroxide and hydronium (c 1,2 5 ,0.100 ppb?mol?cm 23 , D 1,2 9:3 Ã 10 {5 cm 2 sec), organics (c 3 5 1.106 ppb?mol?cm 23 , D 3 ~0:52Ã 10 {5 cm 2 sec), silicates (c 4 5 0.5534 ppb?mol?cm 23 , D 4 ~1:28Ã 10 {5 cm 2 sec), and heavy metal ions (calcium, sodium, chloride, etc; c 5 5 0.1106 ppb?mol?cm 23 , D 5 ~1:2 Ã 10 {5 cm 2 sec) 20 . Based on experimental measurements discussed in the following section, t p is chosen at 0.042 s and f 1i is 0.715 (between 0 §t.t p , 71.5% of all species immediately undergo redox reactions upon their contact with the electrode, 28.5% of species first capacitively couple to the electrode). The physical consequences of the voltage decrease described by equation (6) are: (1) the creation of reaction byproducts in the medium surrounding the piezoelectric material (e.g. hydrogen gas, oxygen gas, chlorine gas, oxidized organics, etc); (2) the buildup of a capacitive layer in solution alongside the piezoelectric material; and (3) a reduction in the electric field present inside the piezoelectric material. These effects will continue to change with time until the potential decrease (equation ( 6)) approaches the total potential originally generated (equation ( 1)). The rate of these effects depend sensitively upon the rate constants f 1i and f 2i , the concentration of reactive species and their diffusion coefficients. The natural driving force for these chemical changes is the piezopotential which, as described by equation ( 1), depends upon the electrical permittivity, mechanical strain, Young's moduli and piezoelectric moduli. This is true regardless of whether the electrochemically active medium is in contact with metallic electrodes or the piezoelectric material's bare surfaces. In the case of bare piezoelectric surfaces, the band gap and positions of ew CB and ew VB relative to eQ HOMO and ew LUMO also determine the nature of what happens during straining. When comparing the piezocatalytic abilities of various piezoelectric materials, it is useful to choose a performance metric from equation ( 6) such as the quantity of a reaction byproduct, e.g. H 2 gas. This value depends sensitively upon the parameters we choose. Previous work reported the production of hydrogen gas via piezocatalysis between a PMN-PT single crystal slab (2 mm 3 10 mm in size) and Milli-Q water 1 . Using recursion we are able to fit equation (6) to the piezopotential verses time profile obtained from a working electrode on a PMN-PT surface under one strain (Fig. 2d). The results yield t p 5 0.042 s, f 1i 5 0.715, and f 2i 5 0.07 under the constraints that after t 5 t p only H 1 reduction and H 2 O oxidation were possible at the electrodes, whilst unreacted H 1 , OH 2 , silicate and heavy metal ions only acted capacitively. These low kinetics values likely resulted from a low concentration of electrolyte. The switch over at t p 5 0.042 s from one reaction regime to the other occurred at a potential (difference) value between the electrodes of approximately 2 V. A gold electrode in equilibrium with pH 7 Milli-Q water is in equilibrium with the H 1 reduction and H 2 O oxidation reactions. This places the Au electrode's potential (Q ElectrodeHOMO ) at 0.41 V vs. SHE (0.615 V below the eQ LUMO of H z and 20.615 above the eQ HOMO of H 2 O). A symmetric voltage shift applied to both Au electrodes of 1 V at t p means an overpotential (Q Electrode HOMO { Q LUMO ) of 20.385 V for the H z reduction reaction and an overpotential (Q Electrode HOMO {Q LUMO ) of 0.385 V for the H 2 O oxidation reaction. These are the potential values taken at which the system switches from a nonselective regime to a selective regime. In order to derive a function of H 2 evolution dependency on strain, the minimum strain S min,1 capable of driving electrochemical reactions for an arbitrary piezoelectric is necessary and determined by augmenting equation ( 1): where Y is the Young's modulus of the material. For metallic Au electrodes, the preceding analysis determined the potential necessary to drive electrochemical H 2 production under the observed conditions to be 0.615 (Q Electrode HOMO {Q LUMO ). In the case of a bare piezoelectric, (Q Electrode HOMO {Q LUMO ) becomes Q VB {Q LUMO ð Þ . S min,2 is denoted as the strain necessary to transition from the selective to nonselective regime, e.g. where (Q Electrode HOMO {Q LUMO ) in equation ( 9) becomes (Q Electrode HOMO {Q LUMO z0:385V). Using the value parameters fit from experiment in conjunction with the values of piezoelectric materials' parameters listed in Table 1, the H 2 generation capacity per straining event (H Metal,Total ) for a multitude of Au-electrode coated piezoelectric materials is depicted in Figure 3a and is given by a manipulation of equation ( 6): H Metal,Total ~H(S{S min,1 )H Metal,SmallS H(S min,2 {S)z H Metal,SmallS is the H 2 production for piezopotentials that fall within the selective regime and is given by: where t 1 is the time required to exhaust the hydrogen production capability for a piezoelectric operating only within the selective regime (S min,1 vSvS min,2 ) and is given by: where e H2O is the electrical permittivity of water. H Metal,LargeS is the H 2 production for piezopotentials that reach the unbiased regime (SwS min,2 ) and is given by: where t 2 (equation ( 14)) is the time it takes for the piezopotential to fall from an arbitrarily large potential (V p max ) to that which brings it into the selective regime (Q LUMO 1 Q Op , where Q Op is the overpotential necessary to transition between reaction regimes, e.g. 0.385 V), and t 3 (equation ( 15)) is the time it takes for that same piezoelectric to reduce its potential from and At large strains (strain . .00025), PZT dominates H 2 gas production per unit strain with a value of 9.1 3 10 14 H 2 ?strain, PMN-PT and BTO have similar production rates of 5.4 3 10 14 H 2 ?strain and 4.5 3 10 14 H 2 ?strain respectively, while ZnO maintains a modest H 2 production rate of 79 3 10 13 H 2 ?strain. From equation ( 13) it can be determined that the H 2 production depends upon the relative weight of f 1,H z to f 2,H z, d xk and e r,x . When f 1,H z is much larger than f 2,H z, ffiffiffi ffi t 2 p dominates the expression where a large ffiffiffi ffi t 2 p value results from a larger V p max combined with a large e r,x . However e r,x is contained within the denominator of the expression determining V p max (equation (1)). Thus, while a large d xk is always valuable to the piezocatalysis process, a balance must be achieved with e r,x . This is why PZT, possessing both large d xk and moderately large e r,x , has the largest H 2 production rate. Physically this competition represents a balancing between the piezoelectric, voltage generating capabilities of the material, and the capacitive nature of the material. The inset in Figure 3a enlarges the small strain region (strain , 0.00004), demonstrating the dynamics of initiation H 2 production. Materials with the smallest e r,x d xk ratio, the most prominent being ZnO, begin H 2 production at strains far smaller than a material like PMN-PT. This is because the thermodynamic accessibility of electrochemical reactions depends upon the relative energetics of donor (eQ HOMO ) and acceptor (ew LUMO ) states and a high ratio of piezoelectric moduli to electrical permittivity strongly couples the strain state of the material to these state energetics (equation ( 9)). The predicted H 2 production capacity is compared to experimental data obtained from an oscillating gold coated PMN-PT cantilever with a peak piezopotential of 20 volts (Fig. 3b) 1 . At 20 Hz, each straining action takes place during 0.025 seconds, which is insufficient for depleting all piezopotential and reaching a thermodynamic equilibrium. Nonetheless, an approximation of the experiment can be constructed by truncating the nonselective regime at 0.025 seconds and applying a window of acceptable values on the kinetics parameter f i from 0.07 to 0.715 (inset of Fig. 3b). Experimental data fall around the lower bound of predicted H 2 evolution rate, exhibiting good agreement. This indicates that there remains much room for experimentally improving the H 2 production rate by means of adding a proton reduction co-catalyst and thus increasing the f i value. Several factors change when comparing a piezoelectric material coated with metal electrodes to a naked, insulating one. In general, a naked and perfectly insulating piezoelectric material does not necessarily possess a continuum of states about the redox potentials in solution. To achieve piezocatalysis (including H 2 production) with such a piezoelectric material, the conduction band and valence band must be moved sufficiently in potential so that their energies coincide with redox potentials under examination. Taking the production of H 2 as our prototypical case, the energetics of top-most valence band electrons (eQ VB ) must be lifted above the (ew LUMO ) of H 2 . The minimum strain, and thus potential, required to achieve this criteria is again given by equation ( 9), except in the case of a bare piezoelectric Q ElectrodeHOMO becomes Q VB . The values of potentials necessary to cause the reduction of H 1 and oxidation of H 2 O for various piezoelectric materials can be found in Table 1. The hydrogen production capacity of a bare piezoelectric material can be calculated by modifying equation ( 10): H Insulator,Total ~H(S min,1 {S)H Insulator,SmallS H(S{S min,2 )z where H Insulator,SmallS is the H 2 production for piezopotentials that fall within the selective regime (S min,1 vSvS min,2 ), H Insulator,LargeS is the H 2 production for piezopotentials capable to reaching the nonselective regime (SwS min,2 ). Applying the values from Table 1 and the same kinetic parameters as in the metal electrode case (f 1i 5 0.715, f 2i 5 0.07, and Q Op 5 20.385 V), Fig. 3c shows the H 2 production rate for various naked piezoelectric materials. Subjected to large strains (strain..001), both naked and Au coated piezoelectric materials perform consonantly. Discrepancies between these two are more prominent under small strain (strain,0.0002), which are a result of the difference in the band structures of individual piezoelectric materials. Under small strain, each piezoelectric's H 2 -production's turn-on strain depends not only upon the e r,x d xk ratio but also upon the value of the individual piezoelectric material's (eQ VB {eQ LUMO ) value. Concerns over the stability of a piezocatalysis system comprising a bare piezoelectric material can be addressed by comparing piezocatalysis with a controlled corrosion process, where reduction reactions take place through the piezoelectric's oxidation and vice versa. The maximum piezoelectric charge density at the surface is on the order of 10 13 charge?cm 2 , two orders of magnitude lower than the average atomic density of a solid-state surface. With oxidation events of this magnitude, it is expected that a piezoelectric capable of withstanding photocatalytic reactions with water is also able to survive the piezocatalysis process. In contrast to the previous analysis, most piezoelectric materials are not perfect insulators, mobile electrical charges inside the piezoelectric material respond to and rearrange according to its internal electric field. In bulk systems, these mobile charges act as extremely effective screening agents of the piezopotential, resulting in an effective piezopotential given by a manipulation of the Gouy-Chapman capacitance model: where k is Boltzmann's constant,, and n 0 is the bulk concentration of free charge 17 . According to equation (17), a highly insulating bulk piezoelectric is required to reach an appreciable potential (Fig. 4a). Taking the values from Table 1 for various piezoelectric materials subjected to a strain of 0.002, the maximum concentration of free charges allowed in order for the piezoelectric materials' surfaces to obtain the H 2 production potential were 5.22 PZT respectively. The H 2 production capacity for these piezoelectric materials subjected to 0.2% strain was determined as a function of free charge by manipulating equation ( 16): where H Highn0 is the expression for H 2 production while the piezopotential is within the selective regime and H Lown0 describes the H 2 production for a piezoelectric that has achieved a potential for accessing the nonselective regime. n max,1 is the maximum number of free charges allowed for a given piezoelectric material under 0.2% strain to reach the threshold potential Q VB {Q LUMO ð Þfor driving H 2 production. n max,2 is the charge concentration necessary to reach the potential threshold between the two reaction regimes In equation (18) the dependence of H 2 production on n 0 comes exclusively from the fact that in the case of a semiconducting piezoelectric material, V p,max [equation ( 13) & ( 14)] becomes V Semi [equation (17)]. The effect of free charge on H 2 production for various piezoelectric materials is demonstrated in Fig. 4b, where the hydrogen production for a strain of 0.2% is shown as a function of mobile charge concentration, n 0 . Behavior of particular note is the dramatic increase in H 2 production that results from decreasing the mobile charge concentration ,5 orders of magnitude below the minimum concentration required to reach the H 2 potential for the piezoelectric materials. Additional mobile charge reduction beyond that initial 5 orders reduction has relatively little effect on H 2 evolution. ## Discussion A number of factors have been shown to augment the efficacy of strain to induce electrochemical reactions. The relative energies of states within the electrode with respect to HOMO and LUMO energies in solution can both dramatically change the rate of chemical reactions and be a determining factor in which chemical reactions are allowed to proceed. These energy state positions depend directly on the magnitude of strain and piezoelectric coefficient while depending inversely upon the electrical permittivity. In the presence of freecharge, generated either by doping, photo or thermal excitations, the piezopotential can be markedly decreased with direct repercussions on reactivity. In order to increase the electrochemical activity of a strained piezoelectric, the value electrical permittivity needs to be optimized. The piezoelectric, semiconductor and molecular orbital frameworks discussed herein have historically and successfully been applied to solid state and electrochemical systems. It is expected that these frameworks can be extended to the piezocatalysis theory. The theoretical work presented here can provide fundamental guidance for designing and understanding the novel piezocatalysis phenomenon from broad electrochemistry and piezoelectric material systems. ## Methods All calculations were performed in Wolfram Mathematica 8. To calculate the total H 2 output from an insulating piezoelectric with metal electrodes, we first calculated the H 2 generated as the piezoelectric potential dropped from the maximum potential induced by strain [equation( 1)] to the potential present at time t P . Then, the H 2 generated after time t p was calculated using another value of the kinetic parameter. Summation of these two H 2 quantities gave the total H 2 output. The first time segment utilized kinetics parameter f 1 5 0.715; while the second segment utilized f 2 5 0.07. To accomplish this we used equation (10), using the variable values found within the paper. Species i 5 1, … m was designated as H 1 ions only, while i 5 m 1 1, … l included all other species in solution. Calculations conducted on bare, insulating piezoelectric materials used the same methodology as those done for metal electrodes-covered insulating piezoelectric materials. The only difference is that the valence band and conduction band energies of the piezoelectric material were used in place of the metal's Fermi energy (equation 16 and Table 1). In the case of the semiconducting piezoelectric, the same procedure was followed the case of the insulating piezoelectric [equation (18)] except that V Semi [equation ( 17)) was used in place of V P max . The experimental data was acquired by using a PMN-PT single crystal slab as the piezoelectric component for piezocatalyzed water splitting (from our previously publication 1 ). The piezoelectric cantilever architecture was constructed and placed within a sealed chamber with access ports for piezoelectric actuation and monitoring, environmental purging, and atmospheric sampling. Straining of the piezoelectric cantilever was achieved by external electronic actuators. The H 2 concentration was determined by an AMETEK Analyzer ta3000F H 2 gas analyzer. The piezoelectric potential was measured by a digital oscilloscope and a potentiostat, respectively. See reference 1 for more details.

chemsum

{"title": "Fundamental Analysis of Piezocatalysis Process on the Surfaces of Strained Piezoelectric Materials", "journal": "Scientific Reports - Nature"}

carbene-based_difluoromethylation_of_bisphenols:_application_to_the_instantaneous_tagging_of_bisphen

## Abstract: The rapid and efficient difluoromethylation of a panel of eleven bisphenols (BPs) for their enhanced detection and identification by Electron-Ionization Gas Chromatography-Mass Spectrometry (EI-GC-MS) is presented. The derivatization employs the inexpensive, environmentally benign agent diethyl (bromodifluoromethyl) phosphonate (DBDFP) as a difluorocarbene-generating species that converts the BPs into bis-difluoromethylated ethers that can be detected and identified by GC-MS means. Key attributes of the protocol include its extreme rapidity (30 seconds) at ambient temperature, high specificity for BPs amidst other alcohol-containing analytes, and its biphasic nature that allows for its convenient adaptation to the analysis of BPs in organic as well as aqueous matrices. The protocol furnishes stable, novel BP ethers armed with a total of four fluorine atoms for their subsequent analysis by EI-GC-MS. Furthermore, each derivatized bisphenol exhibits unique retention times vastly different from their native counterparts leading to their unequivocal identification. The effectiveness and robustness of the developed methodology was applied to the tagging of the most famous member of this family of compounds, bisphenol-A (BPA), when spiked (at 1 μg.g −1 concentration) in the physically and compositionally complex Nebraska EPA standard soil. The method detection limit (MDL) for the bis-difluoromethylated BPA was determined to be 0.01 μg.mL −1 . The bis-difluoromethylated BPA was conveniently detected on the organic layers from the biphasic, derivatized mixtures, highlighting the protocol's practicality and utility in the rapid, qualitative detection of this endocrine disruptor during environmental analysis.Among the most persistent chemicals in the environment and whose emerging negative reputation is starting to garner considerable supporting data are the bisphenols. Bisphenols (BPs) are a class of aromatic hydrocarbons comprising two phenolic units linked together by a carbon atom (or a heteroatom such as sulfur) that may be, in the simplest member of the family, a methylene unit (CH 2 ) or a more elaborate structural motif (e.g. methylphenyl, bis-trifluoromethyl). While the two phenolic units may be linked symmetrically in three different ways, ## Results and Discussion Use of the fluorinated carbon units, mostly as the difluoromethyl (-CF 2 H) and the trifluoromethyl (-CF 3 ) moieties, as isosteric alternatives for the methyl (-CH 3 ) moiety in drug discovery is well documented 24 . Their use over the methyl group stems for the fact that once nitrogens, sulfurs and oxygen atoms present in a drug candidate are modified with this functionality, the net result is the blockade of metabolizing enzymes at that position that results in the increase of a drug's bioavailability and serum half-life. Equally important and within the realm of analytical chemistry, fluorine-bearing substituents play a crucial role as valuable chemical tags for analytes whose conventional detection by GC-MS is problematic as a result of their inherent low ionization potential 25 . Introduction of fluorine atoms into analytes, mostly accomplished as the universally-employed trifluoroacetyl or pentafluorobenzyl groups, act in synergistic and additive fashion to enhance their detection by augmenting their electron-capture ability. Introduction of the trifluoroacetyl moiety is accomplished using trifluoroacetic anhydride (TFAA) while the pentafluorobenzyl tag requires the use of pentafluorobenzyl bromide in the presence of a base 26,27 . As efficient and established as these reactions are in the analytical chemistry toolbox of derivatization methods, the cross-reactivity of the reagents with virtually any nucleophile is unavoidable leading to the fluorination of most, if not all, analytes in a mixture. Even though the areas of complex data analysis, processing and deconvolution have experienced an exponential evolution in the last decade, the avoidance of unnecessary, derivatized side-products that can interfere with the analysis has remained a prime and often welcomed requirement in analytical chemistry. Now, with regards to the difluoromethyl group, clever ways have been devised by chemists to introduce them into molecules, with the majority of these applicable for only modifying acidic hydroxyl groups (i.e. phenols) . Most of these methods involved heating (>100 °C) for prolonged periods of time (often >16 h), until the Zafrani group introduced the stable and environmentally benign agent diethyl (bromodifluoromethyl) phosphonate (DBDFP) that expedited the difluoromethylation of acidic phenols under much milder conditions 31 . Due to its exclusive employment in the fields of medicinal chemistry and drug development, the difluoromethyl tag has experienced extremely limited use in the fields of analytical chemistry and forensic science. Earlier work by our group focused on the use of DBDFP for the derivatization of chlorinated phenols (pK a ~ 7-9) and demonstrated its efficiency, speed, and ability for detection of particularly toxic species by Proton and Fluorine Nuclear Magnetic Resonance ( 1 H and 19 F-NMR) spectroscopy 29 . Building on this work, we turned to expanding the methodology for the derivatization of bisphenols and their subsequent analysis by GC-MS means as shown diagrammatically in Fig. 1. Thus, under the basic conditions used in the protocol, DBDFP breaks down into diethylphosphonic acid and a fleeting bromodifluoromethyl carbanion that undergoes the loss of a bromide ion to generate the highly reactive difluorocarbene species that immediately reacts with the bisphenol to furnish the bis-difluorinated product (Fig. 1). At the outset, we sought to determine the number of equivalents needed to successfully derivatize a given bisphenol without generating too many by-products from the degradation of excess reagent. After determining that 1.5 equivalents per hydroxyl group (thus a total of 3 for all bisphenols in this study) were sufficient for the derivatization of BPA without the creation of many by-products, we performed the derivatization on ten additional, structurally diverse bisphenols (Table 1). Interestingly, we were delighted to find that using large excess of DBDFP did not deleteriously affect the course of the reaction or the subsequent data analysis by providing additional by-products or interferences. Naturally, when encountering real world samples, one makes use of excess derivatization reagents to make sure that all of the analytes have been modified (e.g. BSTFA-mediated silylation), which is the reason why we employ such elevated concentrations when dealing with the soil samples (vide infra). Two interesting points that arise from the GC-MS studies of the reaction are, 1) the derivatization results in fluorinated bisphenol products with shorter retention times relative to the parent bisphenol, thus making them particularly useful in the analysis of late eluting analytes (e.g. B-FL with 38.7 min, SI, Page S16) with the difference in retention times between the derivatized and underivatized bisphenols ranging from 3.6-5.9 min., and 2) the protocol yields products that are m/z + 100 relative to the starting bisphenols, thus providing a strong, diagnostic, molecular ion peak when analyzing the data (Table 1). In all the derivatized bisphenols studied in this work, the molecular ion peak is highly visible and in the case of bisphenol FL it is the base peak in the mass spectrum. In all other cases studied, the base peak was found to correspond to the species generated from the cleavage of one of the C-C bonds at the bridging carbon of the bis-difluoromethylated product (Fig. 2). This mode of ionization can be anticipated as the resulting radical carbocation generated at the bridging carbon is a highly stabilized doubly benzylic species while it is an even more stable trityl species where the remaining R group attached to the bridging carbon is a phenyl group. Thus, starting with bis-difluoromethyl bisphenol G (BP-G(CF 2 H) 2 ), its relatively simple mass spectrum shows the molecular ion peak at m/z = 412 [M] + as well as the spectra's base peak at m/z = 397 [M − CH 3 ] + (Figs. 2A and SI, Page S19). Similarly, in the case of BP-E(CF 2 H) 2 , one can observe its molecular ion peak at m/z = 314 [M] + while the base peak can be clearly noted at m/z = 299 [M − CH 3 ] + (Figs. 2B and SI, Page S13) arising from the loss of the bridging carbon's methyl group. Interestingly, it is the loss of a methyl rather than a proton to yield a more stable tertiary radical carbocation species that provides the base peak in the spectra of BP-E(CF 2 H) 2 , a dominant fragmentation pattern that is also exhibited by the native BPE (SI, Page S12). Other fragments that can be accounted for but yield very low abundance are the ones at m/z = 263 [M − CF 2 ] + and m/z = 247 [M − OCF 2 ] + . For the tetramethylated bisphenol analog, compound BP-C(CF 2 H) 2 , its mass spectrum seems to also offer very few fragment ions that include the molecular ion peak at m/z = 356 [M] + and its base peak at m/z = 341 [M − CH 3 ] + arising from the loss of one of the methyl groups at the bridging carbon (Figs. www.nature.com/scientificreports www.nature.com/scientificreports/ as one of the substituents at the bridging carbon, we begin our discussion with BP-AP(CF 2 H) 2 which provides us with one of the simplest mass spectra in our study (Figs. 2F and SI, Page S7). The spectrum is dominated by the presence of a low molecular ion peak at m/z = 390 [M] + and the base peak at m/z = 375 [M − CH 3 ] + arising from loss of the methyl group at the bridgehead, leaving a stabilized trityl radical cation. In the case of BP-BP(CF 2 H) 2 one can observe the molecular ion peak at m/z = 452 [M] + and the base peak at m/z = 375 [M − Ph] + arising from loss of one of the phenyl rings at the bridgehead for a species that resembles the one obtained with BP-AP(CF 2 H) 2 (vide supra) (Figs. 2G and SI, Page S9). Other peaks of interest that are significant in the spectrum are m/z = 309 [M − C 7 H 5 OF 2 ] + arising from the loss of one of the substituted phenyl groups to generate a trityl radical carbocation but not as strong as the one featured on the base peak. One interesting product that provides a rich mass spectrum as its fluorinated derivative is BP-FL(CF 2 H) 2 . In the spectrum one can see several strong peaks arising from the breakdown of the product in and something quite interesting, the base ion peak is also the molecular ion peak at m/z = 450 [M] + . Other peaks in the spectra include one at m/z = 383 [M − OCF 2 H] + representing the loss of one of the difluoromethoxy groups. The peak at m/z = 307 [M − C 7 H 5 OF 2 ] + represents the loss of one of the substituted phenyl rings from the bridgehead junction to yield a stable trityl radical carbocation akin to the process observed for BP-BP(CF 2 H) 2 (Figs. 2H and SI, Page S17). In similar fashion, the spectrum of BP-Z(CF 2 H) 2 provides us ion fragments that seem to be analogous 1. Gas chromatographic properties for the native and bis-difluoromethylated bisphenols described in this work. The corresponding masses for the more abundant fragment ions along with their relative intensity to the base peak have been in included (in parentheses). In the MS peaks columns, the molecular ion peaks for each compound have been italicized. representing the loss of one of the difluoromethoxy groups. One of the most important bisphenols studied in this work is BPS due to its long time notoriety as a suitable substitute of BPA for manufacturing processes but still remaining a threat to human health as evidenced by numerous studies 32 . During the course of our studies, the native BPS possessed low response during the EI-GC-MS analysis (SI, Page S20) a characteristic that was diametrically obvious in its derivatized counterpart BP-S(CF 2 H) 2 . In the derivatized compound, BP-S(CF As the protocol involves a highly basic solution for the generation of the difluorocarbene species, we hypothesized that the acidic bisphenols would be amenable for selective derivatization over other less acidic alcohol species. Thus, in order to test the selectivity of the protocol for bisphenols over other alcohols, we carried out the difluoromethylation of BPA in the presence of six other alcohols. The alcohols were chosen so as to span a range of structural diversity and reactivity. Thus, incubation of BPA spiked in a mixture of 3-pentanol, 2-methyl-2-pentanol, 2,2-thiodiethanol, pinacolyl alcohol, 3,7-dimethyl-3-octanol and N,N-dimethylaminoethanol followed by treatment of the mixture with excess DBDFP resulted in the sole derivatization of BPA over the other alcohols (Fig. 3). This demonstrates that the protocol is a viable method for the specific labeling of other acidic alcohols (i.e. chlorophenols, resorcinols) under the basic conditions employed for its execution 23 . The observed selectivity lies in the magnitude of the pK a values for phenols that are orders of magnitude lower (i.e. ~7-8) than those exhibited by most alcohols (i.e. ~15-17). Consequently, under the basic conditions (pH ~ 12.8) at which the www.nature.com/scientificreports www.nature.com/scientificreports/ derivatization is conducted, the phenoxide not the alkoxide anion is the predominant species in the mixture and thus the most reactive towards difluoromethylation. Using the Henderson-Hasselbalch equation (Eq. 1) one can deduce that at pH = 12.8, the bisphenols practically exist as their phenoxide ions (with a pK a = 7, 99.9%) while the aliphatic alcohols employed in this study would only marginally exist as their alkoxide counterparts (with a pK a = 16, 0.001%). a In addition, one can invoke the much higher nucleophilicity of a phenoxide anion over that of an alkoxide anion as a reason for the selectivity observed in the process, however, this may not be a relevant contributor to the observed behavior as excess of the DBDFP is employed as eventually that could lead to the derivatization of the other alcohols. In order to exploit the advantage that this protocol offers over other derivatization methods, we decided to test its derivatizing power in a Nebraska soil sample that was spiked with BPA initially at a 10 μg.g −1 concentration and then at a 1 μg.g −1 concentration. The protocol involves the direct conversion of the contaminated soil into a biphasic mixture (KOH/H 2 O//CH 3 CN) followed by the in situ derivatization of BPA with DBDFP. Upon vortexing and subsequently allowing the soil residue to settle to the bottom of the vial along with the aqueous layer, the top acetonitrile layer was then aliquoted, dried and analyzed by GC-MS. Nebraska soil was selected as a matrix due to its composition that encompasses several characteristics not normally present in other soils such as a total organic content (TOC, 1.9%) that provides several organic analytes that can act as interferences. The soil also possesses a large percentage of silt (58%) and clay matter (32%) that are recognized as challenging matrices in the field of soil extraction and analysis due to their inherent reactivity and its fine particulate texture making it difficult for derivatizing agents to access trapped analytes 33 . When carrying out the derivatization on the Nebraska soil, we were delighted to find it was successful at tagging the BPA providing its bis-difluoromethylated ether in enough concentration to be easily detected by GC-MS means (Fig. 4). After this initial assessment at 10 μg.g −1 BPA concentrations, we decided to explore the derivatization at an order of magnitude lower concentration of BPA (1 μg g −1 ) in the same soil. Again, the derivatization of BPA was successful in yielding the bis-difluoromethylated analog in the soil. Interestingly, when dealing with such a low concentration of the analyte, we made use of a selected-ion extraction mode (m/z = 313, strongest ion in the chromatograph) in order to unambiguously identify the fluorinated BPA demonstrating the ability of the protocol to be not only rapid (30 seconds) at tagging the BPA, but successful at detecting this endocrine disrupting compound at such low concentration (Fig. 5). The method detection limit (MDL) for the bis-difluoromethylated BPA in this soil was determined to be 0.01 μg.mL −1 (Supporting Information, Page S4). ## Methods Materials. All chemicals were purchased from commercial suppliers and used as received. Acetonitrile, methylene chloride, Bisphenol E, Bis-(4-hydroxyphenyl)methane (Bisphenol F), Bisphenol BP, Bisphenol G, 4,4′-(9-fluorenylidene)diphenol (Bisphenol FL), 3-pentanol, 2-methyl-2-pentanol, 2,2′-thiodiethanol (thiodiglycol), pinacolyl alcohol (3,3-dimethyl-2-butanol), 3,7-dimethyl-3-octanol, and 2-dimethylaminoethanol were Nebraska (EPA standard 54-135-4) soil was obtained from the soil matrix library at the Forensic Science Center in the Lawrence Livermore National Laboratory. All new bis-difluoromethylated bisphenols were purified using a Biotage Isolera flash column chromatography purification system using SNAP KP-Si gel column cartridges. HRMS analyses were obtained in the Forensic Science Center at the Lawrence Livermore National Laboratory using chemical ionization (CI). Combustion analyses were conducted at Galbraith Laboratories (Knoxville, TN). ## EI-GC-MS experiments. EI-GC-MS analyses were carried out employing a 6890 Agilent GC equipped with a 5975 MS detector featuring a split/splitless injector 23,33 . The column used for our analyses was an Agilent DB-5MS capillary column (dimensions: 30 m × 0.25 mm i.d. × 0.25 µm i.f.). Ultra-high purity helium was employed as the carrier gas at a flow rate of 0.8 mL.min −1 . The injector temperature was set to 250 °C, and the injection volume was 1 µL. The oven temperature program used for the work was the following: 40 °C (held for t = 3 min), increased at a rate of 8 °C.min −1 to 300 °C and then held for t = 3 min. The MS ion source and quadrupole temperatures were set at 230 °C and 150 °C, respectively. The electron ionization energy used was 70 eV. The MS was operated to scan from m/z = 29 to m/z = 600 in t = 0.4 sec. ## Derivatization protocol of individual bisphenols. The initial protocol conditions included longer reaction times and stirring rather than vortexing aimed at the definitive derivatization of the panel of 11 BPs. Thus, the bisphenol (0.04 mmol) was placed in a glass autosampler vial equipped with a small stir bar and treated sequentially via pipette with 0.1 M KOH/H 2 O (800 μL), acetonitrile (CH 3 CN, 800 μL) and diethyl (bromodifluoromethyl) phosphonate (DBDFP, 21.4 μL, 0.12 mmol, 3.0 equiv. to bisphenol). The vial was capped and stirred vigorously at ambient temperature for 5 minutes. After the stirring was finalized, the mixture was allowed to stand to reveal a biphasic mixture and 500 μL of the clear, top layer (acetonitrile) was aliquoted into another glass autosampler vial containing anhydrous sodium sulfate (50 mg). The dried, organic fraction was passed through a syringe PTFE filter disc (0.45 μm) and 20 μL of the filtrate were aliquoted and diluted to 1.5 mL total volume with methylene chloride in an autosampler vial for GC-MS analysis. Assessing the selectivity of the derivatization of bisphenol A in a mixture of aliphatic alcohols. In a 2 mL glass scintillation vial equipped with a stir bar, a 500 μL stock solution of seven alcohols, six of them aliphatic (3-pentanol, 2-methyl-2-pentanol, 2,2-thiodiethanol, pinacolyl alcohol, 3,7-dimethyl-3-octanol and N,N-dimethylaminoethanol) and BPA (each one at a 1000 μg mL −1 concentration) were treated sequentially with 0.1 M KOH/H 2 O (800 μL), acetonitrile (800 μL) and DBDFP (50 μL). The resulting biphasic mixture was vigorously stirred for 5 minutes at ambient temperature. After the stirring was stopped and the biphasic mixture revealed, 20 μL of the top layer (acetonitrile) was aliquoted into another autosampler vial and diluted to 1.5 mL with methylene chloride for GC-MS analysis. The extended period of time (5 min.) in contrast to the established Detection of bisphenol A in Nebraska soil sample at a 10 μg.g −1 concentration. Three sets of Nebraska soil samples (100 mg) in 4 mL vials were spiked with a BPA solution (1 μg mL −1 ) in methylene chloride and mixed, via tumbling, using a rotary evaporator at 40 °C for 15 minutes that after fully drying leads to a 10 μg g −1 BPA-contaminated soil. The contaminated soil was treated with a 0.1 M KOH aqueous solution (800 μL), followed by the sequential addition of acetonitrile (800 μL) and DBDFP (30 μL). The vials were capped, and the resulting biphasic suspensions were each mixed using a vortex for 30 seconds. After this time, 800 μL of the organic, top layer was aliquoted into an autosampler vial and dried with anhydrous sodium sulfate (30 mg). After drying, 100 μL of the organic phase was aliquoted into an autosampler vial equipped with a glass insert for GC-MS analysis. Direct derivatization protocol for bisphenol A-treated Nebraska soil spiked at 1 μg.g −1 concentration. Nebraska soil (100 mg) was placed in a 4 mL vial and spiked with a BPA solution (0.1 μg mL −1 ) in methylene chloride. The suspension was mixed, via tumbling, using a rotary evaporator at 40 °C for 15 minutes that after fully drying leads to a 1 μg g −1 BPA-contaminated soil sample. The soil was treated using a pipette with 0.1 M KOH/H 2 O (1 mL), and then sequentially with acetonitrile (1 mL) and DBDFP (30 μL). The mixture was vortexed for 30 seconds at ambient temperature after which time an aliquot was extracted via pipette (800 μL), dried over anhydrous sodium sulfate (Na 2 SO 4 , 30 mg) and transferred (100 μL) to an autosampler vial equipped with a glass insert for GC-MS analysis. This set of experiments were conducted in triplicates. General procedure for the synthesis of bis-difluoromethylated bisphenols. The bisphenol (0.84 mmol) is dissolved in CH 3 CN (3 mL) and then treated sequentially with DBDFP (500 μL, 2.8 mmol, 3.3 equiv. to bisphenol) followed by 0.1 M KOH/H 2 O (pH 12.8, 3 mL). The resulting mixture was stirred at ambient temperature for 30 minutes. The acetonitrile layer was dried over Na 2 SO 4 , evaporated in vacuo and purified by flash column chromatography (hexanes → 7:3 EtOAc/hexanes) to furnish the bis-difluoromethyl-bisphenol. All characterization data associated for each bis-difluoromethyl-bisphenol used in this study can be found in the SI section of this manuscript. ## Conclusions The methodology described herein represents the first expedient and practical derivatization of BPs via difluoromethylation for their enhanced and unequivocal detection by GC-MS. A remarkable aspect of the protocol is the fact that for the determination of BPs in soil, there are no prior extraction/concentration steps involved, thus greatly speeding up the process by removing the sample preparation step from the analysis. The fast and direct derivatization of BPA in Nebraska soil (at 1 μg.g −1 ) showcases the protocol's practicality and establishes it as a strong derivatization tool in the analytical chemist's toolbox for these types of compounds.

chemsum

{"title": "Carbene-based Difluoromethylation of Bisphenols: Application to the Instantaneous Tagging of Bisphenol A in Spiked Soil for Its Detection and Identification by Electron Ionization Gas Chromatography-Mass Spectrometry", "journal": "Scientific Reports - Nature"}

bioinspired_nonheme_iron_complex_that_triggers_mitochondrial_apoptotic_signalling_pathway_specifical

## Abstract: The activation of dioxygen is the keystone of all forms of aerobic life. Many biological functions rely on the redox versatility of metal ions to perform reductive activation-mediated processes entailing dioxygen and its partially reduced species including superoxide, hydrogen peroxide, and hydroxyl radicals, also known as reactive oxygen species (ROS). In biomimetic chemistry, a number of synthetic approaches have sought to design, synthesize and characterize reactive intermediates such as the metal-superoxo, -peroxo, and -oxo species, which are commonly found as key intermediates in the enzymatic catalytic cycle.However, the use of these designed complexes and their corresponding intermediates as potential candidates for cancer therapeutics has scarcely been endeavored. In this context, a series of biomimetic first-row transition metal complexes bearing a picolylamine-based water-soluble ligand, [M(HN 3 O 2 )] 2+ (M ¼ Mn 2+ , Fe 2+ , Co 2+ , Cu 2+ ; HN 3 O 2 ¼ 2-(2-(bis(pyridin-2-ylmethyl)amino)ethoxy)ethanol) were synthesized and characterized by various spectroscopic methods including X-ray crystallography and their dioxygen and ROS activation reactivity were evaluated in situ and in vitro. It turned out that among these metal complexes, the iron complex, [Fe(HN 3 O 2 )(H 2 O)] 2+ , was capable of activating dioxygen and hydrogen peroxide and produced the ROS species (e.g., hydroxyl radical). Upon the incubation of these complexes with different cancer cells, such as cervical, breast, and colorectal cancer cells (MDA-MB-231, AU565, SK-BR-3, HeLa S3, HT-29, and HCT116 cells), only the iron complex triggered cellular apoptosis specifically for colorectal cancer cells; the other metal complexes show negligible anti-proliferative activity. More importantly, the biomimetic complexes were harmless to normal cells and produced less ROS therein. The use of immunocytochemistry combined with western blot analysis strongly supported that apoptosis occurred via the intrinsic mitochondrial pathway; in the intracellular network, [Fe(HN 3 O 2 )(H 2 O)] 2+ resulted in (i) the activation and/or production of ROS species, (ii) the induction of intracellular impaired redox balance, and (iii) the promotion of the mitochondrial apoptotic signaling pathway in colorectal cancer cells. The results have implications for developing novel biomimetic complexes in cancer treatments and for designing potent candidates with cancer-specific antitumor activity. ## Introduction Cancer is one of the most harmful and serious heterogeneous diseases that represent abnormal cellular energy metabolism and remains one of the major causes of death in most developing and developed countries. 1 Although extensive labours have been devoted to discovering targeted therapies, it is still challenging to overcome poor prognoses and high mortality. Thus, the pursuit of new chemical tools dealing with biomedical functions of metal complexes has garnered tremendous interest due to their broad pharmaceutical properties as potent anticancer agents. For instance, cisplatin, one of the most clinically successful examples of platinum-based anticancer drugs, opened a new era for the development of anticancer transition metal compounds by covering about 50% of all cancer treatments to date. Despite the outstanding applicability of Pt-based drugs, they suffer from low stability under physiological conditions resulting in copious toxic side effects such as necrosis, tissue injury, nausea, vomiting, and neurotoxicity. Ever since the pioneering work of the Au(I)-NHC complex was reported by Bernes-Price and co-workers in 2004, 9 recent developments in the design of non-Pt-based metal Nheterocyclic carbene (NHC) complexes, such as those of Ru, Au, Ir, and Pd, have succeeded in the signifcant improvement of their stability due to the strong donating ability of the NHC ligand. Still, these metals are non-existing elements for the human body and may be the source of unexpected side effects along with the development of drug resistance. Therefore, the use of frst-row transition metals such as Mn, Fe, Co, and Cu would be a credible alternative route for next generation anticancer agents with low general toxicity because they are biorelevant trace elements. In particular, iron, a redox-active essential element involved in oxygen transport in mammals and electron transport in iron-sulfur proteins, would be expected to bypass the cytotoxicity concerns and function both as an electron donor and acceptor in order to disturb the redox homeostasis of the reactive oxygen species (ROS) level in tumors. 25 However, the anticancer activity of frst-row transition metal-based drugs have mostly been examined by introducing or vectorizing metal chelators as potential chemotherapeutics; their in situ mechanism of action and reactivity were scarcely scrutinized due to the intrinsic instability of reactive metal-oxygen intermediates. Recent investigations have highlighted the importance of redox balance and the deregulation of redox signalling in cancer cells due to the raised levels of ROS from multiple intracellular factors such as increased metabolic activity, mitochondrial dysfunction, and peroxisome activity. On the contrary, ROS, which are by-products of normal cellular activity mainly generated in mitochondria and membrane-bound NADHP oxidase, 36,37 are persistently produced in a highly controlled manner in normal cells because the canonical production of ROS is required for the signalling processes of cell division, autophagy, and cellular proliferation. 38,39 Hence, a wealth of recent evidences have underlined ROS as a double-edged sword in cancer cells (e.g., a cancer-stimulating or a cancersuppressing agent). Such dichotomic role in cancer is also relevant to nitric oxide. 40,41 Nevertheless, aiming ROS regulation for the clinical treatment of cancer represents a valuable challenge to advance cancer therapeutic approaches. In this context, we reasoned that the use of biomimetic metal complexes would (i) advance our understanding of the molecular basis of their mechanism of action, (ii) offer an elegant way to control intracellular ROS dysregulation, and (iii) provide a wealth of repertoire of metallodrugs having a broader spectrum of cancer-specifc antitumor activity with a prudent choice of ligands. In the course of our investigation, we employed biomimetic metal complexes having picolylamine-based water-soluble ligand, which accommodates frst-row transition metals such as manganese, iron, cobalt, and copper. Their reactivity toward dioxygen and ROS was examined and then their anticancer activity was directly evaluated upon their incubation with cancer cells. It was found that the iron complex that was capable of activating both dioxygen and ROS triggered an efficient mitochondrial apoptotic signalling pathway of colorectal cancer cells by producing hydroxyl radicals and provoking the intrinsic mitochondrial apoptotic pathway (Scheme 1). ## Synthesis and characterization of rst-row transition metal complexes First-row transition metal complexes bearing a N,N-bis(2picolyl)amine backbone with a pendant ethoxyethanol side chain (HN 3 O 2 ), [M(HN 3 O 2 )] 2+ (M ¼ Mn, Fe, Co, and Cu) were synthesized according to the modifed literature procedure (see ESI †). 42 The X-ray crystal structures of manganese and iron complexes, [Mn(HN 3 O 2 )(Cl) 2 ] and [Fe(HN 3 O 2 )(H 2 O)] 2+ (1), were obtained when recrystallization was carried out under nitrogen atmosphere (Fig. 1a, b and Table S1 †). As depicted in Fig. 1 were stable in both organic and aqueous solution; they did not undergo demetallation, aggregation, and precipitation. Interestingly, only when 1 was recrystallized in a solvent mixture of CH 3 CN : ether (v/v 1 : 2) under aerobic conditions for several days, deep yellow single crystals due the formation of the m-oxobridged diferric complex, [Fe 2 (m-O)(HN 3 O 2 ) 2 ] 4+ (2), was obtained (Fig. 1c and Table S2 †). This suggested that among [M(HN 3 O 2 )] 2+ , only 1 was susceptible to conduct the dioxygen activation reaction depending on the reaction conditions (vide infra). 47,48 From the charge consideration, the iron ions in 2 were both in the +3 oxidation state. Each Fe ion was at the center of a distorted N 3 O 3 octahedron with one bridging oxo ligand forming a {Fe 2 (m-O)} core with a Fe-O-Fe angle of 165.08(6) (Table S2 †). The Fe/Fe distance (3.530(1) ) and the Fe-O distance (1.7799(4) ) were consistent with a m-oxo bridged diiron complex (Table S2 †). ## Reactivity of 1 toward dioxygen and hydrogen peroxide To provide more insight into the reactivity of complex 1, we scrutinized the reactivity of complex 1 toward dioxygen and hydrogen peroxide (H 2 O 2 ). The UV-vis spectrum of isolated 2 clearly exhibited an intense absorption band at 355 nm (3 ¼ 6000 M 1 cm 1 ) in CH 3 CN at 20 C, which differed from that of 1 (Fig. 2a). Interestingly, when 1 was treated with iodosylbenzene (PhIO) or H 2 O 2 , 2 was generated instantaneously (Fig. S4 †). The ESI MS spectrum of 2 clearly exhibited two prominent peaks at m/z of 350.1 and 799.1, whose mass and isotopic distribution patterns are in a good agreement with [Fe 2 (O)(N 3 O 2 ) 2 ] 2+ and [Fe 2 (O)(N 3 O 2 ) 2 (ClO 4 )] + , respectively (Fig. 2, inset and Fig. S5a †). The use of 18 O-labeled PhIO resulted in two mass unit shift of the peak at m/z from 799.1 to 801.1, indicating that the oxygen atom is originated from PhI 18 O (Fig. 2, inset). On the other hand, the addition of 1-benzyl-1,4dihydronicotinamide (BNAH), known as an NADH analog, to an CH 3 CN-solution of 1, also afforded the formation of 2 in the presence of acid (e.g., perchloric acid or hydrochloric acid) under aerobic conditions (Fig. S6 †). 54 The ESI MS spectrum of 2 generated upon the use of 18 O 2 evenly showed a peak at m/z 801.1, demonstrating that the source of the bridging oxygen atom was dioxygen (Fig. S5b †). Therefore, 1 is capable of activating dioxygen using biological ingredients (e.g., proton and cofactors). The electrochemical property of 1 was examined by cyclic voltammetry. Despite an irreversible redox wave of 1, the reduction peak potential (E red ) value of 0.15 V vs. Fc/Fc + in CH 3 CN was found; when an identical experiment was performed in deionized water, the E red value was positively shifted (e.g., 0.08 V vs. Fc/Fc + ) (Fig. S7 †). According to the wellestablished results in the literature, the E red value below $0.1 V vs. Fc/Fc + is defned to be a prerequisite value for dioxygen activation by nonheme iron(II) complexes. 47,55 Hence, 1 activates dioxygen with the help of a cofactor such as the NADH analog and proton under aerobic conditions or activates H 2 O 2 (Fig. S4 and S6 †); this results in the formation of the m-oxobridged dinuclear iron complex. Furthermore, we attempted to detect the generation of ROS species such as hydroxyl radical ($OH) due to 1 using fluorescence probe (e.g., terephthalic acid (TA) assay). 56 Upon incubation of 1 with non-fluorescent TA under aerobic conditions for 24 h, a brilliant fluorescence at 395 nm was detected by fluorescence spectrophotometry (l ex ¼ 310 nm), indicating the hydroxylated product, 2-hydroxyterephthalic acid (TA-OH) was formed upon 24 h incubation (Fig. 2b). Indeed, over 50% generation of TA-OH in the reaction between TA and 1 can be achieved within 1 h under identical reaction conditions (Fig. S8 †). Since TA, 1, and 2 did not show any fluorescence at 310 nm excitation wavelength, the appearance of the fluorescence intensity at 395 nm due to TA-OH solely comes from the reaction between 1 and TA under aerobic conditions. Interestingly, the incubation of other metal complexes or ligands only with TA did not afford such strong fluorescence (Fig. 2b). Therefore, the present result confrmed the efficacy of 1 for the in situ production of $OH under aerobic conditions. By virtue of well-established dioxygen activation mechanism, 47,55, 57 1 could react with dioxygen to form a putative ironsuperoxo species (Scheme 2, pathway a), which might further be converted to a m-peroxo-bridged diferric species (Scheme 2, pathway b). Subsequent homolytic O-O bond cleavage results in the generation of iron-oxo species (Scheme 2, pathway c), followed by a comproportionation reaction with 1 to produce 2 (Scheme 2, pathway d). The addition of H 2 O 2 might facilitate the formation of the iron-oxo species and produce the cytotoxic hydroxyl radical species as seen in the Fenton-like reaction (Scheme 2, pathway e and f). 58 ## Cell viability and intracellular ROS detection Encouraged by the abovementioned dioxygen and ROS activation by the iron complex, we surmised that 1 could promote the deregulation of the intracellular ROS level and could induce cancer cell cycle arrest and cell death. Indeed, it has also been shown that the m-oxo-bridged diiron(III) complexes could exhibit tumor-specifc anticancer activity by generating the toxic hydroxyl radical. The potential anti-proliferative activities of the above-mentioned biomimetic metal complexes against breast cancer cells (e.g., MDA-MB-231, AU565, and SK-BR-3), cervical cancer cell (e.g., HeLa S3), and colorectal cancer cells (e.g., HT-29 and HCT116), were examined by the WST-8 assay monosodium salt), which relies on the mitochondrial activity (Fig. 3 and S9 †). Among biomimetic metal complexes, the iron complex, 1, only exhibits the anticancer activity toward the cancer cells, more specifcally, the colorectal cancer cell lines such as HT-29 and HCT116; 1 is signifcantly effective to HCT116 than HT-29 (Fig. 3a and b). As shown in Fig. S10, † 1 clearly revealed enhanced cytotoxicity against HCT116 cells while other metal complexes did not. The half-maximal inhibitory concentration (IC 50 ) of 1 toward HCT116 cells is determined to be 24.5 mM (Fig. 3c). Such an IC 50 value is greater than that of other reported Pt-based anticancer agents; however, it is still largely below the millimolar range, thereby showing signifcant cytotoxic activity against HCT116 cells. This result led us to, notably, the time-dependent WST-8 viability, which showed that the anticancer activity of 1 was observed with a clear viability decay up to $50% within 24 h while other metal complexes did not exhibit any kinetics of cytotoxicity on the HCT116 cells (Fig. S11 †). To show that 1 is not harmful to normal cells, we incubated 1 with CCD-986Sk cells, which are fbroblast cells for skin, under identical conditions. Very interestingly, all frst-row transition metal complexes including 1 did not show any perceivable cell death (Fig. 3d). Contrary to other abiological metal-based anticancer drugs including Pt, Pd, and Ru that are cytotoxic by nature, these biomimetic metal complexes are signifcantly less toxic in the biological environment. These results strongly advocate that the use of the bio-relevant metal ions accommodating complexes might circumvent the side effects by better dealing with human physiological homeostasis. 65 The qualitative and quantitative detection of intracellularly generated ROS by 1 in HCT116 cells and CCD-986Sk cells were carried out under identical incubation conditions using the cell permeant reagent 2 0 ,7 0 -dichlorofluorescin diacetate (DCFDA) (Fig. 4). Consistent with previously demonstrated results with Scheme 2 Proposed mechanism for the formation of the m-oxobridged diferric complex via the reductive activation of dioxygen or hydrogen peroxide. TA, only 1 effectively generated the ROS in HCT116 cells; the amount of intracellularly generated ROS was $1.8 fold greater than that observed in the negative control (NC) case (Fig. 4a). This result revealed that 1 efficiently penetrated into the HCT116 cells and produced intracellular ROS in the heterogeneous environment. Very importantly, the intracellular production of ROS by 1 in normal cells (i.e., CCD-986Sk) was determined to be $1.2 fold greater (Fig. 4b). These observations allowed us to speculate that the intracellular generation of ROS by 1 may be favored in heterogeneous environment (i.e., cancer cells) than in homogeneous environment (i.e., normal cells). It has been demonstrated that an increase in ROS was associated with cancer cell growth as compared to normal cells and there might exist a certain threshold of ROS concentration that is incompatible with cellular survival. 66 In the present case, we proposed that (i) 1 in HCT116 cells would activate intracellular ROS, (ii) provoke excessive levels of ROS that reaches a certain threshold, and (iii) fnally exert cytotoxic effect. However, the less activation of ROS by 1 in normal cells would barely reach the threshold and the redox homeostasis would be maintained. Therefore, we concluded that once 1 was penetrated into the heterogeneous HCT116 cells, 1 increased the intracellular concentration of ROS in order to prompt the cell death by presumably affecting many regulatory signalling processes closely related to intracellular ROS homeostasis (vide infra). ## Immunocytochemistry, qRT-PCR, and western blot analysis To verify that 1 roused the signalling pathway of cell death in colorectal cancer cells, we frst performed immunofluorescence staining with 4 0 ,6-diamidino-2-phenylindole (DAPI), which is frequently used to visualize the nuclear DNA in both living and fxed cells, in HCT116 cells within 24 h after treatment with 1 (Fig. 5a). The structural changes of the nuclei including nuclear condensation and shrinkage were noticed in HCT116 cells treated with 1; this result suggested the production of the apoptotic nuclei upon treatment with 1 (Fig. 5a, shown with a white arrow). We further examined the area and number of nuclei in HCT116 cells treated with 1 (Fig. 5b). A signifcant decrease in the nuclei area as well as the average nuclei area per image in HCT116 cells incubated with 1 as compared to the control were confrmed. In addition, a decreased number of nuclei also suggested that 1 has a critical role in anticancer activity. In order to understand whether the anticancer effect of 1 influenced the apoptosis of HCT116 cells, we monitored the level of genes and proteins closely related to the apoptotic pathways. We frst checked the expression of the BCL-2 family members using qRT-PCR (Fig. 5c). It is well-documented that the BCL-2 family is considered to be divided into anti-apoptotic and pro-apoptotic members according to their functions. 67 Even though the expression of BCL-2 alpha and beta, known as antiapoptotic members, was slightly attenuated, the expression of BAX and BAK, known as pro-apoptotic members, was increased and prominent in HCT116 cells treated with 1. This clearly suggested that 1 induced apoptotic cell death. Since up-regulated BAX and BAK commonly concerned the release of cytochrome c into the cytosol by increasing the permeability of the mitochondrial membrane, 68 we performed immunofluorescence for COX IV to ascertain whether 1-triggered apoptosis was mediated by the mitochondria. COX IV is an inner membrane mitochondrial marker and their level is increased at the early stage of apoptosis and then decreased after the release of cytochrome c into the cytosol during the apoptotic process. 69 As shown in Fig. 5d, COX IV was remarkably reduced in HCT116 cells treated with 1, whereas its level was abundant in control. These results supported that the apoptosis of HCT116 cells caused by 1 might be accompanied by a decrease in the mitochondrial function. Since it has been wellestablished that the decrease in the level of COX IV affected the activation of caspases, for instance, caspase 9 and 3, 69 we examined the level of the cleaved caspase 9 and 3 after incubating HCT116 cells with 1 for 24 h. Western blot analysis undoubtedly showed that the level of the cleaved caspase 9 and 3 were remarkably increased in HCT116 cells (Fig. 5e). Moreover, an increased level of the cleaved poly(ADP-ribose) polymerase 1 (PARP1), which is a hallmark of apoptosis due to the caspases, 70 was noticeably perceived in HCT116 cells treated with 1 (Fig. 5e). Next, to investigate whether 1-triggered apoptosis was mediated by the extrinsic pathway through FADD (fasassociated protein with death domain), immunofluorescence for E-cadherin, which collaborates with death receptors, was conducted (Fig. S12 and S13 †). 71,72 We observed that there was no signifcant change in the E-cadherin level after treatment with 1. In addition, the expression of BID, which is proteolytically activated by FADD signalling, was not signifcant between HCT116 cells treated with 1 and the controls (Fig. S14 †). These results supported that the apoptosis induced by 1 was presumably mediated by the intrinsic pathway including mitochondrial dysfunction but not by the extrinsic pathway. In addition, we performed the cell viability assay in the presence and absence of Z-VAD-FMK to evaluate the apoptotic effect of 1 on HCT116 cells. Z-VAD-FMK is a cell permeable caspase inhibitor that impedes caspase processing and apoptosis by irreversibly binding to the catalytic site. 73 1 induced cell death only in HCT116 and co-treatment with Z-VAD-FMK partly improved the viability level (Fig. 5f). CCD-986Sk is not affected by 1 and Z-VAD-FMK. These results showed that 1 was closely associated with the apoptosis-dependent pathway. Taken together, we propose that after cellular uptake, the intracellular generation of ROS by 1 prompted mitochondrial dysfunction and resulted in the apoptotic signalling pathway as follows: (i) the mitochondrial release of cytochrome c in cytosol, followed by (ii) the cleavage of caspase 9 and then that of caspase 3 occurred to fnally (iii) stimulate the cleavage of PARP1. Thus, the intrinsic mitochondrial apoptosis pathway was activated to cause HCT116 cell cycle arrest upon treatment with 1. ## In vivo studies Having established that 1 elevated the intracellular ROS concentration, we investigated the anticancer activity of 1 in vivo. To evaluate the anticancer effect of 1 in vivo, six mice models injected with HT29 cells were used; three were used as the vehicle with sterile distilled water and another three were treated with 1 intraperitoneally for 5 days (Fig. 6a). When the mice were treated with 5 mg kg 1 of 1, no adverse side effects were detected. Three cases of 1 resulted in approximately 50% to 80% tumor growth inhibition with respect to the control (Fig. 6b). Furthermore, the injection of 1 at 5 mg kg 1 signifcantly inhibited the tumor growth of the xenograft relative to the control in the subcutaneous colorectal cancer model, indicating that 1 is a potential antitumor agent that operates in vivo (Fig. 6c). These results supported that 1 has the possibility of therapeutic target in vivo. ## Conclusions In conclusion, we successfully synthesized and characterized a series of biomimetic frst-row transition metal complexes with various spectroscopic methods including X-ray crystallography. Among these chemically designed complexes, only iron complex, 1, was capable of activating dioxygen and reactive oxygen species and producing hydroxyl radical species. This illustrated the importance of the choice of the redox active metal ion at the center of the biomimetic complex in order to judiciously attribute their chemical functionality and reactivity. This interesting chemical property of 1 was believed to trigger the cell cycle arrest of the cancer cells, more specifcally colorectal cancer cells, by efficiently producing intracellular ROS in a heterogeneous environment. Strikingly, when normal cells were treated with 1 under identical conditions, cell death did not occur due to the less production of ROS under homogeneous environment. The accumulated evidences presented in the cell viability assays clearly supported the effective intracellular generation of ROS by 1 as compared to other biomimetic metal complexes. More detailed investigation on the signaling pathway of cell cycle arrest revealed that the intrinsic mitochondrial apoptotic pathway was activated by 1 through (i) the release of cytochrome c, (ii) the cleavage of caspase 9 and caspase 3, and (iii) the cleavage of PARP1 located in the nucleus. Finally, we confrmed the anticancer effect of 1 both in vitro and in vivo as the inhibition of tumor growth by 1 in the subcutaneous colorectal cancer model was verifed. Overall, the present strategy (e.g., the use of biomimetic metal complexes) might tremendously reduce the occurrence of off-target effects, thereby proving the possibility of a wide range of biological application and justify further investigation. Moreover, this would be an important asset-or, at least one of the central functions-that the designed biomimetic metal complexes merit more attention in the future direction of the cancer therapeutic research. ## Materials Commercially available chemicals were used without further purifcation unless otherwise indicated. Solvents were dried according to the published procedures and distilled under Ar prior to use. 74 was prepared by a literature method. 75 The nonheme manganese, iron, cobalt, copper(II) complexes were prepared according to the modifed literature methods. 42 ## Instrumentation The UV-vis spectra were recorded on a Hewlett Packard Agilent Cary 8454 UV-visible spectrophotometer equipped with a T2/ sport temperature-controlled cuvette holder. Electrospray ionization mass spectra (ESI MS) were collected on a Thermo Finnigan (San Jose, CA, USA) LTQ™ XL ion trap instrument by infusing the samples directly into the source at 5.0 mL min 1 using a syringe pump. The spray voltage was set at 4. to each well and the absorbance at 450 nm was measured by Synergy™ HTX Multi-Mode Microplate Reader from Bio-Tek (Winooski, VT, USA) and VICTOR Nivo™ Multimode Plate Reader on a PerkinElmer (Waltham, MA, USA). Chemiluminescent signals were induced by chemiluminescent detection reagent from ATTO (Taito, Tokyo, Japan) and detected by Amersham Imager 600 from GE Healthcare (Chicago, IL, USA). The images observed with a LSM 700 laser scanning confocal microscope and Axio Vert. A1-inverted microscope from Zeiss (Oberkochen, Baden-Württemberg, Germany) and analyzed using ImageJ from NIH (Bethesda, Maryland, USA). qRT-PCR was measured by LightCycler 96 Instrument from Roche (Basel, Basel-Stadt, Switzerland). Synthesis and characterization of mononuclear manganese(II), iron(II), cobalt(II), and copper(II) complexes 77 The CCD data were integrated and scaled using the Bruker-SAINT software package. 78 An empirical absorption correction was applied using the SADABS program. 79 The structures were solved by direct methods, and all non-hydrogen atoms were subjected to anisotropic refnement by full-matrix least squares on F 2 using SHELXTL Ver. 6.14. 80 The crystallographic data and selected bond distances and angles are listed in Tables S1 and S2, † respectively. ## Cell culture The human carcinoma-derived cells such as HCT116 and HT- ## Cell viability To determine cell viability, cells were seeded in 96-well plates and allowed to recover for 24 h. Then, the cells were exposed to the metal complexes for 24 h. The metal complexes were dissolved in distilled water and diluted in fresh culture medium (fnal metal complex concentration is 50 mM). The added concentration of Z-VAD-FMK was 25 mM (Selleck Chem. #S7023). After 24 h treatment of the complexes, the cytotoxicity of the metal complexes was determined using the 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-8) assay as previously described. 81 Cellular reactive oxygen species (ROS) assay ROS were detected using 2 0 ,7 0 -dichlorofluorescin diacetate (DCFDA) cellular ROS detection assay kit (Abcam # ab113851) following the manufacturers' protocol. Cells were plated overnight in 96-well plates in their medium with or without metal complex. The fluorescence excitation/emission 485/535 was measured using the Synergy HTX Multi-Mode Microplate Reader (Bio-Tek). ## Statistical analysis Statistical analysis was performed using two-tailed paired t test, one way ANOVA-Dunnett's test, and nonlinear regression analysis in GraphPad Prism 5.01 Software (San Diego, CA, USA). ## Western blot analysis Cells were seeded in a 6-well plate and cultured in a 37 C, 5.0% CO 2 incubator to adhere to the cells. The old culture medium was discarded and replaced with a fresh medium containing 50 mM of the iron complex. Incubation was then continued for 24 h. After treatment was completed, the cells were washed with PBS. The total protein was extracted using an RNA/protein extraction kit (MACHEREY-NAGEL) according to the manufacturer's instructions. Lysates 491A). The horseradish peroxidase-conjugated secondary antibodies in 2.0% skim milk in PBS with 1.0% Tween-20 were incubated at room temperature for 1 h. Signals were induced using the chemiluminescent detection reagent (ATTO) and detected using an Amersham Imager 600 (GE Healthcare). ## Immunocytochemistry Cells were fxed with 4% paraformaldehyde and permeabilized with 0.20% Triton X-100 in phosphate-buffered saline (PBS). Following fxation, cells were incubated at 4 C overnight with anti-COX IV and anti-E-cadherin primary antibody in PBS with 1.0% bovine serum albumin (BSA) and 0.20% Triton X-100. The stained proteins were visualized using Alexa Fluor 488 and Alexa Fluor 594-conjugated secondary antibodies. The nuclei were counterstained with DAPI. The stained cells were observed with a LSM 700 Confocal Laser Scanning Microscope (Zeiss). The nuclei area and number of nuclei were measured by the ImageJ software (NIH). ## Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) The total RNA was extracted using an RNA/Protein extraction kit (MACHEREY-NAGEL). cDNA was synthesized from total RNA (2 mg) using M-MLV Reverse Transcriptase (Promega, M1705). qRT-PCR was performed on a LightCycler 96 System (Roche) using qPCRBIO SyGreen Blue Mix (PCR Biosystems, PB20.15), according to the manufacturer's instructions. The following targets were amplifed using the indicated primer pairs (Table S3 †). ## In vivo experiments All animal experiments were performed according to the guidelines of the Chonnam National University Medical School Research Institutional Animal Care Committee, and all experimental protocols were approved by the committee. Five-sixweek-old Balb/c-nu mice were obtained from OrientBio, Inc. (Seongnam, Korea) and housed in metal cages with free access to water and food. To generate the subcutaneous colon cancer models, HT29 human colon cancer cells were harvested during exponential growth and resuspended in a 1 : 1 mixture of saline and Matrigel (BD Biosciences). Next, 1 10 7 HT29 cells per mouse were injected subcutaneously into the flank of Balb/c-nu mice. Mice were randomly assigned to three groups (N ¼ 3): sterile distilled water (control), 1 (concentration: 1 mg kg 1 ), and 1 (concentration: 5 mg kg 1 ). When the average tumor size reached a volume of approximately 100 mm 3 , 1 was administered via intraperitoneal injections fve times for 5 days. Tumor growth was monitored every 2 to 3 days; mice were monitored for signs of toxicity, and the size of the tumors were measured.

chemsum

{"title": "Bioinspired nonheme iron complex that triggers mitochondrial apoptotic signalling pathway specifically for colorectal cancer cells", "journal": "Royal Society of Chemistry (RSC)"}

nhcs_and_visible_light_co-catalyzed_1,4-sulfonylacylation_of_1,3-enynes_for_tetrasubstituted_allenyl

## Abstract: The modulation of selectivity of highly reactive carbon radical crosscoupling for the construction of C-C bonds represents a challenging task in organic chemistry. N-Heterocyclic carbenes (NHCs) catalyzed radical transformations opened a new avenue for acyl radical cross-coupling chemistry. With this method, highly selective cross-coupling of acyl radical with alkyl radical for efficient construction of C-C bonds were succussfully realized. However, the cross-coupling reaction of acyl radical with vinyl radicals represents an uncharted domain. We herein describe NHCs and photocatalysis co-catalyzed radical 1,4-sulfonylacylation of 1,3-enynes, providing structurally diversified valuable tetrasubstituted allenyl ketones. Mechanistic studies indicated that ketyl radicals are formed from aroyl fluorides via oxidative quenching process of excited photocatalysis, allenyl radicals are generated from chemo specific sulfonyl radical addition to the 1,3-enynes, finally, unprecedented key allenyl and ketyl radical cross-coupling provides tetrasubstituted allenyl ketones. Radical cross-coupling between two carbon radicals emerged as a powerful platform for constructing C-C bonds and received increasing attention. 1 Since the radical-radical coupling reactions proceeded via a diffusion-controlled manner, selectivity modulation is the critical challenge. 1b Via radial addition to unsaturated bond to form C-C bond, acyl radicals have been utilized in preparing diverse carbonyl compounds. 2 However, radical-coupling reaction between acyl and other carbon-centered radicals is rare. N-Heterocyclic carbenes catalysis (NHCs) has emerged as an attractive strategy in synthetic chemistry to access value-added organics via the formation of key Breslow intermediate (BI). 3 Recently, the single-electron-transfer (SET) of BI was found to provide ketyl-type radical species, which opens a new avenue for acyl radical chemistry. As a result, NHCs catalyzed radical-couplings have attracted great attention after the pioneer work of Ohmiya in 2019. 7a Alkyl radical sources such as redox-active esters, 7 Katritzky pyridinium salts, 8 Hantzsch ester, 9 benzylic C-H bonds, 6e alkylborates, 10g olefins 6c,10 as well as cyclopropanes 6f could be used to perform cross-coupling reaction with acyl radicals to form C-C bond (Fig. 1a). However, to the best of our knowledge, radical-coupling reaction between vinyl radical and acyl radical has never been reported. On the other hand, radical 1,4-difunctionalization of 1,3-enynes provides an elegant and versatile strategy for tetrasubstituted allenes from easily available feedstocks. In this regard, in situ generated allene radicals undergo cyanation, 14a-d arylation, 14e-h halogenation, 14i alkynylation, 14j trifluoromethylation, 14k or intramolecular cyclization 14l to afford functionalized allenes. Radical acylation of 1,3enynes may provide straightforward access to value-added allenyl ketone units, which are crucial core in important nature products 15 and synthetic intermediates. 16 Recently, Studer et al. developed acylative difunctionalization of olefins 6c /cyclopropanes 6f and formal alkenyl 6d /benzylic 6e C−H acylation by employing aroyl fluorides as ketyl-type radical precursors. Inspired by those elegant approaches, we speculated that NHCs and visible light co-catalyzed system 6c-6f,9,11-12 enable generation of allenyl radicals and NHCs stabilized ketyl radicals under extremely mild conditions, which may offer an opportunity for radical acylation of 1,3-enynes. As our continuous interests in radical chemistry, 17 we now describe the development of NHCs and photocatalysis cocatalyzed three-component radical 1,4-sulfonylacylation of 1,3-enynes, providing direct access to structurally diversified tetrasubstituted allenyl ketones (Fig. 1b). ## Results and discussion Reaction conditions development. We commenced our investigation by employing 1,3-enyne (1a), benzoyl fluoride (2a), TolSO2Na (3a) as the prototype substrates and PC-1 (1.5 mol %), NHC-1 (15 mol %) as catalysts. Pleasingly, in dichloromethane (DCM) under irradiation with Blue LED at room temperature for 4 h, the expected allenyl ketones 4 was obtained in 10% yield combination with competitive by-product 5 (Fig. 2, entry 1). Ir-based photocatalysis PC-2 and PC-3 improved reactivity and selectivity (entries 2 and 3), while PC-4 and PC-5 were inefficient for this reaction (entries 4 and 5). The employment of other solvents such as CH3CN, PhCF3, or THF provided 4 in relatively lower yields (entries 6-8). The structure of NHCs was crucial for chemo-selectivity control (entries 9-13). The NHC-2 and NHC-3 were unsatisfactory (entries 9 and 10). The N-2,6-diethyl phenyl substituted catalyst NHC-4 afforded 4 with a slightly diminished yield compared to NHC-1 (entry 11). For NHC-5 or NHC-6, decreased yield was observed (entries 12-13). To our delight, yield could be further improved upon running the reaction at lower concentration (entries 14−15), and affording 4 in 80% isolated yield with negligible 5 in 4 mL DCM (entry 15). The desired 1,4-sulfonylacylation product was isolated in 75% yield when the reaction was run at 0.2 mmol scale (entry 16), and these conditions were thus defined as the standard reaction conditions for subsequent investigations. ) (1:1 dr.) yield. Furthermore, the fluorides derived from natural products such as telmisartan and mefenamic acid were successfully converted into 54 and 55 in 85% and 61% yields, respectively. Synthetic applications. Large-scale synthesis and derivatization reactions were performed to showcase synthetic applications (Fig. 4a). Scale-up synthesis of 17 has been achieved at a 2.0 mmol scale, and a comparable yield was obtained (Fig. 4a1). When employing PhLi as a base, the tetrasubstituted allenyl ketones 4 could isomerize to diene product 56 in 78% yield. 4 could undergo reduction of ketone unit with NaBH4. The allenyl ketone 4 could easily be transformed into conjugated viny selenyl ether 58 in 50% yield with excellent Z/E selectivity. When treated with concentrated H2SO4, Nazarov cyclization product 59 was isolated in 86% yield. ## Mechanism investigations. A series of control experiments were performed to unravel the reaction mechanism (Fig. 4b). Light, NHCs, and photoredox catalysis were indispensable for this 1,4-sulfonylacylation reaction (Fig. 4b1). When the radical scavenger 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO) was added, the reaction was suppressed, and TEMPO-trapping product 60 was separated in 55% yield (Fig. 4b2), thus suggesting the formation of ketyl radicals. Furthermore, a trace amount of 4,4'dimethyl-1,1'-biphenyl (62) was isolated under standard conditions, indicating the involvement of a sulfonyl radical. The intermediacy of acyl azoliums has been confirmed by coupling of acyl azolium ion 61 with 1,3-enynes 1a and sodium benzenesulfinate 3a in the absence of NHC (Fig. 4b3). The radical chain process could rule out based on light/dark experiments (Fig. S4, see Supplementary Information). Then Stern-Volmer quenching studies were conducted to clarify the plausible photoredox mechanism (Fig. 4c). 1,3-Enynes 1a and sodium benzenesulfinate 3a do not show a significant luminescence quenching effect to the excited state of the Ir*(III). In contrast, Ir*-complex was effectively quenched by acyl azolium ion 61, pointing to the oxidative quenching process. In summary, we have realized an efficient 1,4-sulfonylacylation of 1,3-enynes by merging photocatalysis with NHCs. This transformation provided a facile and direct entry for tetrasubstituted allenyl ketones under mild conditions with broad functional group tolerance and excellent chemo-and regioselectivity. Mechanistic studies indicated that the key step of the transformation is unprecedented allenyl and ketyl radical cross-coupling, proving a new avenue for NHCs catalyzed radical chemistry. Ketyl radical was formed from aroyl fluorides via the oxidative quenching process of excited photocatalysis. Further extension of this cross-coupling system to other destabilized transient radicals is ongoing in our laboratory. ## Methods General procedure for the synthesis of tetrasubstituted allenyl ketones. Into a nitrogenfilled glove box, a vial (15.0 mL) equipped with a magnetic stir bar was charged with NHC-1 (12.6 mg, 0.03 mmol), Cs2CO3 (130.3 mg, 0.4 mmol), PC-3 (2.7 mg, 0.003 mmol), sulfinate (71.3 mg, 0.4 mmol) and DCM (8.0 mL). Then 1,3-enynes (0.2 mmol) and acyl fluorides (0.4 mmol) were added. The vial was removed from the glove box, and then the reaction mixture was irradiated with Blue LED at room temperature for 4 hours. After the reaction finished that monitored by TLC, the reaction mixture was quenched by water. The mixture was extracted with EtOAc (3 x 5.0 mL). The combined organic phases were dried over anhydrous Na2SO4, and the solvent was evaporated under vacuum. The residue was purified by flash column chromatography (petroleum ether/ethyl acetate = 10 : 1) to get the desired product.

chemsum

{"title": "NHCs and Visible Light Co-catalyzed 1,4-Sulfonylacylation of 1,3-Enynes for Tetrasubstituted Allenyl Ketones", "journal": "ChemRxiv"}

biomimetic_co_oxidation_below_-100_⁰c_by_a_nitrate-containing_metal-free_microporous_system

## Abstract: CO oxidation is of importance both for organic and inorganic systems. Transition and precious metals on various supports can oxidize CO to CO2. Among them, few systems, like Au/TiO2, can perform CO oxidation at the low temperature of -70 ⁰C. Living (an)aerobic organisms perform CO oxidation with nitrate using complex enzymes under ambient temperatures which is an important pathway of their living cycle that enables them to "breathe"/produce energy in the absence of oxygen and leads to the carbonate mineral formation. Herein, we report that CO can be oxidized to CO2 by nitrate at -140 ⁰C in completely inorganic system (zeolite) without metals. The transformation of NOx and CO species in zeolite as well as the origin of this unique activity (catalyzed by Bronsted acid sites) are clarified using spectroscopic and computational approach. CO oxidation is important both for automotive emissions control and in living microorganisms . More specifically, inorganic materials such as transition/noble metals on solid supports are capable of oxidizing CO at elevated temperatures . Among such systems, Au nanoparticles supported on titania, discovered by Haruta, represents a material active for CO oxidation at the lowest known temperature of -70 ⁰C . In anaerobic and aerobic microorganisms, enzymes evolved to oxidize CO to CO2 by nitrate as shown in the pioneering studies of King and co-workers 8]. The energy produced is used to sustain life while emitted CO2 leads to formation of carbonate minerals, for example . Moreover, search for carbonate minerals/CO2 on other planets (Mars) is ongoing to possibly confirm whether anaerobic life was ever present on such planets . We discovered a pathway of CO oxidation by nitrate in a completely inorganic, non-metal crystalline system at -140 ⁰ C, more specifically zeolite SSZ-13. First, we had to clarify the chemistry of NOx species in zeolite under different conditions. In the 1980s, it was first discovered that NO + species can form in Na-zeolites upon interaction with (NO+O2) or NO2 using Raman and infra-red spectroscopy . Later on, K. Hadjiivanov and co-workers' pioneering studies allowed to establish, using FTIR spectroscopy and isotopic methods on NxOy molecules, that NO + can indeed form in ZSM-5 upon interaction with (NO+O2) mixture. NO2 was shown to disproportionate to NO + and NO3in the zeolites in Li/K/Na forms as well . Although it was clear that NO + can be produced in zeolites from NO+O2 mixtures and specific mechanisms were proposed, we investigated its production and chemical properties using spectroscopy and density functional theory calculations. We started by introducing NO2 onto small pore H-SSZ-13 zeolites with Si/Al ratios 12 and 6. SSZ-13 zeolite was chosen because it is the one often encountered in nature , it is a robust, hydrothermally stable framework used extensively in catalytic aftertreatment systems to decrease automotive pollution . Additionally, it is the framework with only one equivalent framework T site which is more straightforward to model than frameworks with broad distribution and location of T-sites. cm -1 (belonging to NO + ) and one with a maximum at ~1650 cm -1 (belonging to free HNO3). This corresponds to disproportionation reaction of NO2 to NO and HNO3: 2NO2 + H-zeolite  Zeolite-NO + + HNO3 Indeed, NO2 is known to be easily dimerizable to N2O4 which, in highly polar solvents (such as ethylacetate, for example) or in the highly polar zeolitic micropores favors disproportionation to , which can react with zeolite protons (acid denoted as H-zeolite) in a scheme depicted above. The band representing NO + is not symmetrical and it corresponds to two NO + stretches at ~2195 and ~2171 cm -1 . Concomitant to the development of these two absorption bands is the gradual intensity decrease of the two OH stretching features at 3612 and 3585 cm -1 (Fig. 2B). Thus, NO + replaces protons near at least two distinct Al atom environments. Concurrently, a new OH stretching band appears at 3667 cm -1 , corresponding the OH stretch of nitric acid. Thus, NO2 reacts with zeolite to form NO + near the framework oxygens (in place of acidic protons) and HNO3. Similar chemistry is observed for H-SSZ-13 with a different Si/Al ratio ~ 6 with slightly different distribution with NO + species formed (see difference spectra in SI Fig. S1, HAADF-STEM images of SSZ-13 crystals are shown in Fig. S2). This is fully consistent with the DFT calculations, which show that the reaction: Zeo/H + + 2×NO2(g) → Zeo/NO + + HNO3(g) is exothermic by -46 kJ/mol. Conceptually, NO + can bind with an Xanion in two fundamentally different ways to form NOX complexes. 1). [N≡O] + complexes with weakly coordinating anions, like BF4 -. In these complexes NO + behaves as a free (or semi-free) cation exhibiting an N-O stretching vibrational frequency of ≥2,300 cm -1 , for example 2340 cm -1 in NO[BF4], 2326 cm -1 in NO[AuF6], and 2298 cm -1 in concentrated sulfuric acid solutions for free [NO] + . 2). NO + forms complexes with X-but a covalent bond between N and X (X is most often a halogen) is preserved: in this case a bent O=N-X molecule forms with an O-N-X angle <180⁰. Complexes like these exhibit N-O vibrational frequencies between 1,950 and 1,800 cm -1 . Furthermore, even for non-halogens the bent nonionic M-N=O moieties with fully covalent N-M bonds and bent Metal-N-O angle ( 120<angle<180) show νNO<1,900 cm -1 in the IR spectra . DFT calculations for free NO + and NOX complexes are in good agreement with the experimental trend (Table S1). However, for the NO + in zeolite we observe νNO in the intermediate region between 2,300 and 1,950 (but closer to the former). This does indeed suggest that NO + and -O-Zeolite interaction has a significant covalent character (i.e., NO + is not a free-floating ion in zeolite). Further confirmation of this is the observation of the same FWHM of NO + band upon warming NO + /SSZ-13 system from 77 to 298 K (the NO + band FWHM does not change, Fig. S3) suggesting that NO + structure is attached to the zeolite and remains stable with temperature change (no shifting or broadening upon significant temperature changes). Unlike the 2133 cm -1 NO + band in ZSM-5 , the NO + bands in SSZ-13 are located at higher wavenumbers (at ~2170 and 2200 cm -1 ) and are characterized by higher thermal stability up to 200 ⁰C under high vacuum (Fig. S4) in contrast to NO + in H/ZSM-5 located at 2133 cm-1 and which starts desorbing above room temperture. Adsorption of excess NO2 on NO + /zeolite with Si/Al ~ 6 (Fig. S5) and 12 (Fig. 1C) leads to the decrease of the intensity of the NO + stretching band in the expense of a new NO band at ~2230 cm -1 (lying higher than the original NO + bands by ~35 and ~58 cm -1 correspondingly). Simultaneously, two new vibrational features appear,: a sharp band at ~1740 cm -1 and a shoulder at ~2080 cm -1 (clearly seen in the difference spectra and spectra upon evacuation, Fig. S5, S8) as the excess NO2 produces an NO + -NO2 complex. The formation of such a complex has been suggested, on the basis of Rietveld refinement of synchrotron XRD data, for NO + interacting with excess NO2 in the supercages of Ba-FAU zeolite . The onset of the formation of this complex coincides with the appearance of the band at 1746 cm -1 . DFT calculations (Fig. S10) show the N-O stretches of NO + -NO2 complex lie at ~2042 and 1722 cm -1 . Considering the systematic shift of calculated DFT NO stretches relative to experimental values by 20-30 cm -1 (due to the non-innocent nature of NO adsorption, unlike in the CO case) this agrees well with ~2080 and ~1746 cm -1 bands for the NO + -NO2 complex. The nature of the 2,230 cm -1 band is discussed below. The chemistry we observe when reacting a mixture of NO+O2 over H-zeolites is somewhat different from the case discussed above for NO2. It was shown in the 1980s that NO+O2 in microporous materials can easily produce NO2 . The thus formed NO2, in turn, can either dimerize to form N2O4 or react with NO to form N2O3. Due to the presence of excess NO in the system the primary reaction is N2O3 formation. The thus formed N2O3 can disproportionate to the ion pair NO + NO2that, in turn, can react with a zeolitic proton. Indeed, when we mix NO with sub-stoichiometric amounts on oxygen, we see the immediate appearance of the IR bands characteristic of both NO + (2174 cm -1 ) and N2O3 (1570 and 1970 cm -1 ) (Fig. S6). The intensities of the IR bands of N2O3 initially grow rapidly, and then, after reaching their maxima, they begin to lose their intensities. The IR feature of adsorbed NO + continuously grows during the entire experiment (Fig. S6) while Bronsted acidic protons are consumed: HNO2 then quickly reacts with H + to form NO + and H2O. The major difference between the two NO + formation processes (NO2 vs. NO+O2) is the production of nitrates in the NO2 only process, and the formation of H2O in the NO+O2 reaction. NO + , formed in either processes, is stable under vacuum at 77 K and only desorbs above 150 ⁰C under high vacuum. Furthermore, the chemistry of NO + confined in zeolite is peculiar. Adsorption of NO2 at room temperature leads to the formation of NO + -NO2 complex as well as a shift of the NO + band to 2230 cm -1 . Evacuation restores the IR signature of the original NO + as the [NO + -NO2] complex decomposes (Figs. S7 and S8). Adsorption of CO at low temperature (77 K) on the NO + /H/SSZ-13 shows the production of an NO + -CO complex evidenced by the blue shift of the NO + vibrational signature to ~2220 cm -1 (vide infra) (Fig. 3A). In contrast, upon the adsorption of NO on the NO + /H/SSZ-13 sample at 77 K the IR band of NO + redshifts to 2013 cm -1 evidencing the selective production of the NO + -NO complex. Thus, this novel chemistry provides a hitherto unknown insight how adsorption of an adsorbate (NO, CO, NO2) changes the properties of the cation with which it interacts. Normally, such cations are metal cations that have no corresponding IR active vibrations, and the information regarding the changes incurred during adsorption is hidden. In our case, however, because NO + itself has an active N-O vibration, we see that adsorption of CO, NO and NO2 shifts its electronic signature, our DFT calculations showed that the interaction between NO + and CO or NO2 is relatively weak with a binding energy of the adsorbed molecule below -20 kJ/mol in absolute value (Table S1). Hence, we conclude that since the concentration of CO or NO2 is high in the zeolite pores, these gas phase molecules slightly shift NO + further from its equilibrium position in the zeolite. This, in turn, weakens the interaction between NO + and zeolite, leading to a shortening in the N-O distance and a blue shift of N-O vibrational frequency to ~2230 cm -1 (in the case of NO2) and 2220 cm -1 (in the case of CO). This provides a unique insight into the interaction of extra-framework cations with adsorbates, not routinely available even from the most sophisticated synchrotron XRD and Rietveld refinement methods. However, in the case of NO, the shift is to a significantly lower frequency. This finding can be rationalized by our DFT results, since in this case a stable Zeo/NO + -NO complex is formed (Fig. S10) in the zeolite with a binding energy of NO to Zeo/NO + of -52 kJ/mol (Table S1). This structure has two frequencies at 2009 and 1911 cm -1 . They can rationalize the experimental bands at 2013 and 1871 cm -1 . In addition, calculated ONNO species in gas phase has frequencies at 1879 and 1727 cm -1 . Thus, they can rationalize the experimental bands at 1870 and 1685 cm -1 . In-situ heating of the NO-CO complex produced from N2O3 reaction with H-SSZ-13 leads to no CO2 formation (Fig. S9). However, when the NO + /SSZ-13 sample prepared by the disproportionation of NO2 (i.e., contained large amounts of nitrates) was heated from -170 to -140 ⁰C in the presence of CO, the immediate formation of CO2 inside the zeolite micropores was observed, evidenced by the appearance of a sharp band at 2345 cm -1 characteristic signature of adsorbed CO2 . CO was oxidized by nitrates: as the intensity of the characteristic vibrational feature of adsorbed CO2 gradually increased, the intensity of the 1645 cm -1 nitrate band simultaneously decreased. These results unambiguously show that CO can be oxidized by nitrates in this zeolite at the very low temperature of -140 ⁰C ( Fig. 3). This biomimetic chemistry by a completely inorganic non-metal system occurs at temperatures previously unseen for such a conversion. Moreover, when we react CO with NO3at room temperature (in the same system), no reaction takes place at all! How can this seemingly puzzling fact be rationalized? It is well-known the catalysis can occur when the reacting molecule is adsorbed/chemisorbed. In the case of CO, at room temperature CO is not adsorbed by the Bronsted acid protons of -Si-OH-Al groups, as evidenced by the lack of CO stretches other than gas-phase CO. However, CO interacts with Bronsted acid protons of the zeolite at lower temperatures forming -H + ---CO complex . IR CO stretching feature in this complex is centered at ~2175 cm -1 . The major consequence of the binding of CO to H + is the polarization of the C-O bond as C (δ+) -O (δ-) because in the H + -CO complex no backdonation from the proton to CO is present, and only electrostatic interaction and formation of a sigma bond takes place with charge transfer from C to H + . This polarization (i.e., formation of C (δ+) -O (δ-) ) makes CO susceptible to nucleophilic attack by NO3to form CO2 and reduced NOx species. DFT calculations further support the proposed route. We considered four mechanisms for CO oxidation by HNO3 in H-CHA. They are presented schematically in Fig. 4, as well as the corresponding energetic diagrams. Shorter names are used for the structures in the energy diagrams with respect to the schemes above, as: IS -Zeo1/CO/HNO3; FS -Zeo1/CO2/HNO2; IC -Zeo2/NO2 + /HCO2 -; ID -Zeo2/HCO2 -/NO2 + , FS2 -Zeo2/CO2/HNO2. Color coding: Si -blue, O -red, Al -green, Nlight blue, C -brown, H -white. All mechanisms start with the adsorption of the reactants, CO and HNO3, via hydrogen bonds to the bridging OH group and basic zeolite oxygen center, respectively. In this initial state (IS) structure (Zeo1/CO/HNO3) the C-O vibrational frequency was calculated to be 2175 cm -1 , in line with the corresponding experimental band. Mechanisms A and B are one-step mechanisms. In the former one the CO molecule is oxidized by the HNO3 via transition state structure TS1 with no direct participation of the zeolite. In mechanism B, the CO molecule is oxidized to CO2 via transition state structure TS2, where zeolite participates actively in the process via movement of the proton from the bridging OH group towards C atom from the CO molecule and via the basic zeolite oxygen, which attracts the nitric acid's proton. TS2 structure seems to correspond to a bifurcation point, since it may be decomposed in two ways (i) directly to CO2 and HNO2 via the electron transfer shown by the arrows in the mechanism B; and (ii) to formation of a complex HCOO -NO2 + (IC) as the zeolite proton interacts with an O center from NO2 + , as shown in mechanism C. From the latter intermediate can be formed the final products (CO2 and HNO2) via the same TS2 structure (Mechanism C). Alternatively, if the intermediate is coordinated to the zeolite (see Zeo2/HCO2 -/NO2 + , ID) CO2 and HNO2 can be formed via TS3, which includes a Htransfer from HCOOto NO2 + (Mechanism D). In all mechanisms considered the CO oxidation was calculated to be exothermic by 187 to 217 kJ/mol, depending on the coordination of the products (Figure 4), thus we investigated only kinetic aspects in depth. Energy diagrams show that the most plausible are Mechanisms B and C with a barrier of the rate limiting steps (which are the same) of 81 kJ/mol. In addition, we calculated the Gibbs free energy barriers of this rate limiting step of both mechanisms at 140 K to be 58 kJ/mol. Since these steps are the first ones, formation of an intermediate is not expected in line with our experimental results. Both mechanisms involve the direct participation of the zeolite via H + transfers and as well as the formation of initial complex of CO to the zeolite H + is crucial for the oxidation process. The calculated energy barriers for the rate limiting steps of mechanisms A and D are notably higher (Figure 4). We also considered CO oxidation on Zeo/NO2 + (Fig. S11), in which NO2 + are the chargecompensating species of the negatively charged zeolite framework, forming kind of nitrate with the O center from the zeolite. The calculated IR frequencies of such Zeo/NO2 + species are 2046 and 1358 cm -1 and move to 2055 and 1353 cm -1 after CO adsorption. However, the barrier for the reaction: Zeo/NO2 + + CO → Zeo/NO + + CO2 is very high, 143 kJ/mol, thus the role of NO2 + species as a CO oxidizing agent can be discarded. In summary, we discovered that CO can be oxidized to CO2 by nitrate in zeolite micropores at temperatures as low as -140 ⁰C. This reaction was previously known to be catalyzed by complex enzyme molecules in living (an)aerobic organisms. However, no noble (or other) metal systems have been known that can perform this biologically important reaction under mild conditions. Remarkably, fully inorganic system with no (noble) metals performs such a reaction at -140 ⁰C. Interaction of CO with Bronsted acid protons in confined nanopore produces -H + -CO complex making the carbon atoms susceptible for nucleophilic attack by a nitrate, revealing a hitherto unknown pathway for CO conversion chemistry in inorganic systems at low temperatures. ## Supplementary Materials: Materials and Methods H-SSZ-13 was synthesized using previously described methods [14,16 in the main text]. More specifically, a Na-form of SSZ-13 with Si/Al ratio ~6 and 10-12 were prepared, and then exchanged three times with 2 M ammonium nitrate solution at 80 ⁰C. The powder was purified by consecutive centrifugation cycles after washing with DI. The wet powder was dried at 100 ⁰ C, and then calcined at 550 ⁰C for 5 hours in the flow of dry air. All the chemicals used were the highest-grade purity available. The in situ static transmission IR experiments were conducted in a home-built cell housed in the sample compartment of a Bruker Vertex 80 spectrometer, equipped with an MCT detector and operated at 4 cm -1 resolution. The powder sample was pressed onto a tungsten mesh which, in turn, was mounted onto a copper heating assembly attached to a ceramic feedthrough. The sample could be resistively heated, and the sample temperature was monitored by a thermocouple spot welded onto the top center of the W grid. The cold finger on the glass bulb containing CO was cooled with liquid nitrogen to eliminate any contamination originating from metal carbonyls, while NO was cleaned with multiple freeze-pump-thaw cycles. Prior to spectrum collection, a background with the activated (annealed, reduced or oxidized) sample in the IR beam was collected. Each spectrum reported is obtained by averaging 64 scans. HAADF-STEM was performed with a FEI Titan 80-300 microscope operated at 300 kV. The instrument is equipped with a CEOS GmbH double-hexapole aberration corrector for the probeforming lens, which allows for imaging with 0.1 nm resolution in scanning transmission electron microscopy mode (STEM). The images were acquired with a high angle annular dark field (HAADF) detector with inner collection angle set to 52 mrad. ## Computational Details and Models Periodic DFT calculations were performed using the Perdew-Burke-Ernzerhof (PBE), exchange-correlation functional with the additional empirical dispersion correction as proposed by Grimme (DFT-D2), as implemented in Vienna ab initio simulation package (VASP). . We also employed PAW pseudopotentials and the valence wave functions were expanded in a plane-wave basis with a cutoff energy of 415 eV. The Brillouin zone was sampled using only the Γ point. We used a monoclinic unit cell of the CHA framework, which consists of 36 T atoms. It was optimized for the pure silicate structure with dimensions: a = b = 13.675 , c = 14.767 ; α = β = 90 o , γ = 120 o . One Si center in the unit cell located in one six-member ring was replaced with Al, as the negative charge around this Al was compensated by an H + cation. During the geometry optimization, atoms were allowed to relax until the force on each atom became less than 5×10 −2 eV/. The vibrational frequencies were calculated using a normal mode analysis where the elements of the Hessian were approximated as finite differences of gradients, displacing each atomic center by 1.5×10 −2 either way along each Cartesian direction. The reported binding energies (BE) of the various adsorbates (CO, CO2, NO, HNO3, HNO2) were calculated as BE = -Ead -Esub + Ead/sub, where Ead is the total energy of the adsorbate in the gas phase (ground state), Esub is the total energy of the pristine zeolite system, where the framework negative charges are compansated by some of the modeled cationic species (H + , NO + , or NO2 + ) considered, and Ead/sub is the total energy of the zeolite, together with the adsorbate in the optimized geometry. With the above definition, negative values of BE imply a favorable interaction. When Gibbs free energies were obtained the enthalpy values were calculated from the total energy values (Eel) corrected for the internal vibrational energy (Ev) and zero point vibrations (ZPE) derived from frequency calculations of the optimized structures: H140 = Eel + Ev + ZPE. The entropy values of the initial and transition states (TS) include only the vibrational degrees of freedom (Sv), since the adsorbates are bound to the zeolite and the rotational and translational degrees of freedom are converted into vibrations . The expressions of all enthalpy and entropy contributions can be found elsewhere . ## Table S1 Binding energy (in kJ/mol) of NO2 and NO to Zeo/NO + structure, vibrational frequencies (in cm -1 ), ν(N-O), as well as selected interatomic distances, R(A-B), in pm.

chemsum

{"title": "Biomimetic CO oxidation below -100 \u2070C by a nitrate-containing metal-free microporous system", "journal": "ChemRxiv"}

j_o_u_r_n_a_l_na_me_local_energy_decomposition_analysis_and_molecular_properties_of_encapsulated_met

## Abstract: Methane has been successfully encapsulated within cages of C 60 fullerene, and it is an appropriate model system to study confinement effects. Its chemistry and physics is also relevant for theoretical model descriptions. Here we provided insights into intermolecular interactions and predicted spectroscopic responses of the CH 4 @C 60 complex and compared with results from other methods and with literature data. Local energy decomposition analysis (LED) within the domain-based local pair natural orbital coupled cluster singles, doubles, and perturbative triples (DLPNO-CCSD(T)) framework was used, and an efficient protocol for studies of endohedral complexes of fullerenes is proposed. This approach allowed us to assess energies in relation to electronic and geometric preparation, electrostatics, exchange, and London dispersion for the CH 4 @C 60 endohedral complex. The calculated stabilization energy of CH 4 inside the C 60 fullerene was −13.5 kcal/mol and its magnitude was significantly larger than the latent heat of evaporation of CH 4 . Evaluation of vibrational frequencies and polarizabilities of the CH 4 @C 60 complex revealed that the infrared (IR) and Raman bands of the endohedral CH 4 were essentially "silent" due to dielectric screening effect of the C 60 , which acted as a molecular Faraday cage. Absorption spectra in the UV-Vis domain and ionization potentials of the C 60 and CH 4 @C 60 were predicted. They were almost identical. The calculated 1 H/ 13 C NMR shifts and spin-spin coupling constants were in very good agreement with experimental data. In addition, reference DLPNO-CCSD(T) interaction energies for complexes with noble gases (Ng@C 60 ; Ng = He, Ne, Ar, Kr) were calculated. The values were compared with those derived from supermolecular MP2/SCS-MP2 calculations and estimates with London-type formulas by Pyykkö and coworkers [Phys. Chem. Chem. Phys., 2010, 12, 6187−6203], and with values derived from DFT-based symmetry-adapted perturbation theory (DFT-SAPT) by Hesselmann & Korona [Phys. Chem. Chem. Phys., 2011, 13, 732−743]. Selected points at the potential energy surface of the endohedral He 2 @C 60 trimer were considered. In contrast to previous theoretical attempts with the DFT/MP2/SCS-MP2/DFT-SAPT methods, our calculations at the DLPNO-CCSD(T) level of theory predicted the He 2 @C 60 trimer to be thermodynamically stable, which is in agreement with experimental observations. ## Introduction Carbon displays a rich chemistry and physics with a variety of molecular allotropes, including common graphite and diamond, but also fullerenes, carbon nanotubes, and graphene. The most investigated fullerene is the C 60 "Buckminsterfullerene" composed of 20 hexagons and 12 pentagons of sp 2 -hybridized carbon atoms fused into a pseudosphere with a ∼7 diameter, as displayed in Figure 1. C 60 occurs in trace amounts on Earth in carbon-rich rocks and soot. 5,6 It has also been observed in the micrometeorite impact residue on the NASA Long Duration Exposure a b Fig. 1 The C 60 fullerene (a) and its endohedral complex CH 4 @C 60 (b). Facility orbiter, 7 which indicates that it either survived impact at nominal encounter velocity of orbital debris (∼11 km/s), 8 or was created in situ in space. Fullerenes isolated from meteorites re-vealed encapsulated atoms of noble gases with a 3 He/ 4 He isotope ratio of clearly extraterrestial origin. 9 Moreover, analyses of the 2019 data collected by the NASA/ESA Hubble Space Telescope confirmed spectral features of the ionized C + 60 species in diffuse interstellar bands making it the largest molecule observed in space and indicating that fullerenes might play an important role in interstellar chemistry. 10,11 The properties and chemistry of C 60 have been studied; for example, the wave-particle duality was experimentally observed for C 60 . 12 Methods for preparation and separation have been established, 13,14 and the possibilities to encapsulate atoms and molecules inside the fullerene cage was recognized soon after its discovery. 15 C 60 endohedral complexes with metal ions, noble gases, H 2 , N 2 , and H 2 O have been prepared by high-energy collisions of ionized fullerene species, harsh conditions of high temperature and pressure, electric arc, or by organic synthesis methods called as molecular surgery. The successful synthesis of the endohedral complex with CH 4 was reported in 2019 by Whitby and coworkers. 20 Methane is the largest, and the first organic molecule encapsulated in the C 60 fullerene, and such a complex denoted as CH 4 @C 60 is the main object of this study. There is no obvious direct route to measure the stabilization energy in fullerene endohedral complexes and obtain insights into the interaction mechanisms. Experimental observations in this respect have been limited to assessing the efficiency/probability of the given complex to be formed, and the main focus has been on spectroscopic and diffraction studies in relation to unusual physical properties of the encapsulated species. 21 Substantial theoretical efforts have directed to studies of C 60 endohedral complexes and associated intermolecular interactions. Pioneering ab initio studies by Jerzy Cioslowski at the Hartree-Fock (HF) level of theory were expanded by studies of Bühl et al. 25,26 , Patchkovskii et al. 27 , Darzynkiewicz et al., 28 and Autschbach et al. 29 among others, where density functional theory (DFT) and secondorder Møller-Plesset perturbation theory (MP2) were used. However, within the supermolecular approach with the interaction energy being the arithmetic relation of related energies (E int = E AB − (E A + E B )), DFT is essentially blind to long-range dispersion. This limitation has typically been addressed by using empirical correction schemes for the dispersion contributions. MP2 is the lowest ab initio method that accounts for "real" dispersion effects but it is unbalanced and its performance for weak, noncovalent interactions are modest and system dependent. 41 Symmetry-adapted perturbation theory (SAPT) with monomers description at the DFT level (DFT-SAPT) developed by Krzysztof Szalewicz and coworkers is an alternative approach to account for dispersion contributions. Within SAPT, the interaction energy is obtained as a sum of physical contributions, free from basis set superposition error (BSSE). 45 Hence, the most reliable stabilization energies for C 60 endohedral complexes so far have been obtained with DFT-SAPT by Hesselmann and Korona. 46,47 In parallel developments, approximate London-type formulas have been derived by Pyykkö and coworkers for the estimation of dispersion interaction in endohedral systems. 48,49 Attractive dispersion interactions between nonpolar species such as C 60 and CH 4 (or noble gases) are purely quantum me-chanical and originate from instantaneous effects of dynamical electron correlation. 50 For systems of chemical interest that can be correctly described by a single reference wave function, the most robust (and still tractable) way of introducing electron correlation is the coupled cluster singles, doubles, and perturbative triples CCSD(T) method. 51 It is the "gold standard" of quantum chemistry. However, a canonical implementation of the CCSD(T) model exhibits a seventh-order scaling with the system size, which results in tremendous computational expenses when considering systems larger than 15−25 atoms. Frank Neese and coworkers have recently developed an efficient implementation of the domain-based local pair natural orbital coupled cluster method (DLPNO-CCSD(T)). Briefly, in the DLPNO-CCSD(T) approach the correlation energy is expressed as a sum of electron pair correlation energies, which enables the distinction between the "weak pairs" with negligible contribution to the total correlation energy and the "strong pairs" that constitute the dominant, desired part. In this way the "weak pairs' can be treated with a computationally more efficient second-order perturbation theory, whereas only the essential "strong pairs" are subjected to an accurate coupled cluster treatment, which greatly reduces the computational complexity. With appropriately selected pairselection thresholds, this model is capable to recover 99.9 % of the correlation energy of its canonical counterpart. It reproduces the CCSD(T) results within a chemical accuracy at substantially reduced computational efforts. 55,56 This approach extends the possibility of obtaining accurate ab initio energies to systems for which only DFT has been applicable so far. 57,58 Moreover, using a local energy decomposition (LED) protocol allows for a physical meaningful decomposition of the interaction energy within the DLPNO-CCSD(T) framework. 50,59,60 In this study, the goal was to providing an accurate interaction energy decomposition for the CH 4 @C 60 complex and the encapsulation energy barrier using the DLPNO-CCSD(T) method. This approach is not biased by the parametrization inherent to the DFT models, including type of exchange-correlation approximation and dispersion correction scheme. The reference interaction energies for endohedral complexes with noble gases were provided and compared with results by Pyykkö et al. and Hesselmann and Korona. ## Methods The counterpoise-corrected interaction energy of molecular fragments X and Y can be expressed as: 59 ## ∆E where the E B A (C) notation denotes energy of fragment A calculated at the energy optimized coordinates of B and using a basis set of system C. The ∆E int term is the "electronic interaction", whereas ∆E geo−prep is the geometric preparation contribution that accounts for the differences between equilibrium molecular ge-ometries of isolated fragments and those in a complex ("deformation energy"). The electronic interaction energy ∆E int is decomposed within the following DLPNO-CCSD(T)-LED decomposition scheme: The interaction energy ∆E int is decomposed into that from the Hartree-Fock level of theory ∆E HF int and the corrections due to inclusion of electron correlation ∆E C int . The latter is decomposed further into the interaction energy contribution at the CCSD level of theory ∆E C−CCSD int and that resulting from the perturbative triple excitations ∆E ## C−(T) int . The Hartree-Fock interaction energy ∆E HF int is decomposed into the electronic preparation contribution ∆E HF el−prep , which correspond to the energy needed to bring the electronic structures of the isolated fragments into the one optimal for the interaction ("energy investment") and into attractive electrostatic E elstat and exchange E exch contributions. The CCSD correlation interaction energy is partitioned further into the genuine London dispersion interaction energy E C disp and the nondispersive correlation contribution ∆E C non−disp . The latter provides (dynamical) corrections to the Hartree-Fock polarization effects, "dynamic charge polarization". We refer the reader to the original articles for a detailed description of the method and implementation. 50,59,60 ## Computational details All calculations were performed with the ORCA code 61,62 using a very tight convergence tolerance of 1 × 10 −9 E h . The evaluation of Coulomb and exchange integrals was accelerated with the RIJCOSX approximation 63 with def2/J Coulomb-fitting basis set 64 and tightened grid (GridX5; further increase was verified to have negligible effect). Geometry optimizations were converged to very tight thresholds (VeryTightOpt setting) using the revised PBE "revPBE" exchange-correlation DFT approximation 65,66 together with atom-pairwise dispersion correction based on tight binding partial charges (D4). 40 The polarization-consistent segmented pcseg-1 basis set, 67 and the increased DFT integration grid (Grid5 NoFinalGrid) were used. The choice of the revPBE model was based on its performance in a recent and thorough benchmark study (best among the computationally efficient gradient-corrected GGA functionals). 68 To confirm the global energy minima at the potential energy surfaces, and to evaluate vibrational IR and Raman spectra, Hessians and dipole polarizabilities were calculated. The transition state search involved many consecutive computations of the Hessians towards the firstorder saddle point. Thereby computational efforts were reduced by using the smaller pcseg-0 basis set for atoms of aryl groups for the open-cage models. Cartesian coordinates of the models are provided in the Electronic Supplementary Information (ESI) † . DLPNO-CCSD(T) calculations were performed with a Foster-Boys localization scheme, a full MP2 guess, and employed correlationconsistent cc-pVXZ (X = D, T, Q, 5) orbital basis sets together with the corresponding cc-pVXZ/C auxiliary basis sets. 72 The chosen PNO truncation thresholds are discussed in the results and discussion section. Computations were performed on a cluster node equipped with two Intel R Xeon R Gold 6126 CPUs (2.6 GHz; 12-core) and 256 GB of RAM. ## The choice of PNO truncation thresholds and basis sets To facilitate accuracy control in a user friendly manner, the authors of the DLPNO-CCSD(T) method have implemented three levels of predefined PNO truncation thresholds. These levels converge towards the method limit at increasing computational cost: LoosePNO, NormalPNO, and TightPNO. 55 The first two offer sufficient accuracy for most applications (<0.5 kcal/mol deviation for the evaluations of total energy with respect to the CCSD(T) reference), 55 but for analysis of weak intermolecular interactions, the TightPNO setting should be used. It ensures that the electron pairs that dominate the interaction are being treated at the coupled cluster level. 59 However, fullerenes are challenging for local coupled cluster methods. The large number of (long-range) π − π interactions in the highly delocalized π-system of fullerenes render calculations with the TightPNO setting and accurate basis sets very demanding. 56,73,74 The DLPNO-CCSD(T) in its current implementation is formally a linear-scaling method when considering the iterative part, but the RI-PNO integral transformations on large systems add substantial prefactors to the total computation times, which in turn limit the feasibility, also due to the memory and disk space requirements. Therefore, based on test calculations for the CH 4 • • • C 6 H 6 dimer (Figure 2), which is expected to exhibit similar nature of noncovalent interactions to those in the CH 4 @C 60 complex, we used a multilevel approach as proposed by Sparta et al. 57 Within the multilevel DLPNO approach the test CH 4 • • • C 6 H 6 system was divided into CH 4 and C 6 H 6 fragments. By this division, the intrafragment electron pairs with their orbitals entirely localized on one molecular fragment could be separated from those that gave rise to intermolecular interaction. 57 In Table 1, dispersion interaction and time used for the DLPNO-CCSD(T)-LED calculations for the CH 4 • • • C 6 H 6 dimer are presented. We monitored the convergence of the energy component for the dispersion interaction (E C disp ) since it depends solely on the treatment of the electron correlation, and therefore is critically sensitive to the truncation thresholds of the PNO and the (in)completeness of the basis set. By using a TightPNO setting for both intrafragment and interfragment pairs, smooth convergence towards the limit of a complete basis set (CBS) was observed (see Fig. 2). The dispersion interaction energy essentially converged at the TightPNO/cc-pVQZ level. Unfortunately, this setup would involve prohibitive computational costs when applied to endohedral fullerene complexes. Therefore, for routine applications we propose a more tractable multilevel DLPNO scheme. In this scheme, the truncation thresholds for the intrafragment PNO Open circles correspond to the calculation with the multilevel scheme proposed in the last row of Table 1. for the guest (in this case CH 4 ) and for a troublesome delocalized π-system (C 6 H 6 , C 60 ) are reduced to the NormalPNO and LoosePNO, respectively, whereas the critical interfragment pairs are subjected to an accurate TightPNO evaluation. Together with the combination of the cc-pVQZ/cc-pVTZ basis sets, the multilevel scheme proposed in the last row in Table 1 offers massive computational savings without compromising accuracy to any significant extent. For a test conducted for the CH 4 • • • C 6 H 6 dimer, this approach recovered >95 % of the dispersion interaction energy when compared to the TightPNO/CBS reference, while being only twice as expensive as the TightPNO/cc-pVDZ calculation. The contribution from weak pairs to the E C disp was less than 4 % throughout the calculations presented in Table 1. Moreover, the related interaction energies compared well with previously reported accurate ab initio calculated energies for the CH 4 • • • C 6 H 6 dimer. When using the proposed multilevel DLPNO-CCSD(T) setup, a total interaction energy ∆E int =−1.32 kcal/mol was calculated, which agreed very well with the CCSD(T)/aug-cc-pVTZ result of −1.39 kcal/mol by Ringer et al. 77 The estimated dispersion contribution of −2.03 kcal/mol at the SAPT2/aug-cc-pVDZ level of theory by Ringer et al. 77 was close to our value of −2.22 kcal/mol. Above indicated that the multilevel DLPNO-CCSD(T) setup tailored for substantially more demanding calculations on endohedral complexes of fullerenes is robust. ## DLPNO-CCSD(T)-LED analysis of the CH 4 @C 60 In Table 2 the corresponding bond lengths of energy optimized geometries of the CH 4 , C 60 , and CH 4 @C 60 endohedral complex are shown. As a consequence of I h symmetry the C 60 fullerene molecular structure is defined by the two distinct C−C distances r 1 and r 2 that originate from the bonds between fused pentagons and hexagons (r 1 ) and the shorter ones between two hexagons (r 2 ). The energy optimized model of C 60 exhibited excellent agreement with experimental bond lengths estimates. For r 1 the deviation was <0.005 , and r 2 coincided with the empirical C−C distance. This indicated that the revPBE-D4/pcseg-1 level of theory was capable to deliver robust models of fullerene systems. For comparison, reported geometries of C 60 optimized at the (ab initio) HF and MP2 levels of theory have revealed considerable deviations of C−C bond lengths, 33,34 whereas previous tests of different DFT approximations have not included dispersion corrections. 33,34 Noteworthy, at the revPBE-D4/pcseg-1 level of theory the geometries of both CH 4 and C 60 remained essentially unchanged upon formation of the CH 4 @C 60 endohedral complex. ## DLPNO-CCSD(T)-LED Repulsion Stabilization Fig. 3 Results of the DLPNO-CCSD(T)-LED/cc-pVQZ(CH 4 )/cc-pVTZ(C 60 ) local energy decomposition analysis for the CH 4 @C 60 endohedral complex, energies are given in kcal/mol. Inset shows total interaction energies (∆E int ) corresponding to the Hartree-Fock, CCSD, and CCSD(T) levels of theory. respond to an energy minimum at coupled cluster level of theory, for which the equilibrium C−H distance was shorter, and closer to experimental estimate. The encapsulation of CH 4 in C 60 is associated with a tiny shortening (<0.001 ) of the C−H bond lengths. Therefore, the total DLPNO-CCSD(T) energy of the CH 4 molecule calculated at molecular geometry corresponding to that in the CH 4 @C 60 complex was lower compared to that for the isolated CH 4 . Although this effect was very small and had no implications on the evaluation of the electronic interaction energy ∆E int in the CH 4 @C 60 complex, it would lead to an unphysical lowering of the "deformation energy", ∆E geo−prep term in Equation 1. Therefore, to provide the most realistic values the evaluation of ∆E geo−prep included only the contribution from the deformation of the C 60 cage. In Figure 3, interaction energy contributions are presented from the DLPNO-CCSD(T)-LED analysis of the CH 4 @C 60 endohedral complex. The total interaction energy for the complex is represented as a sum of seven physical contributions: , and ∆E geo−prep (according to Equations 1 and 2). The large and positive electronic preparation term ∆E HF el−prep = +81.22 kcal/mol is counteracted by attractive contributions due to electrostatics and exchange (E elstat = −39.73 kcal/mol, E exch = −21.00 kcal/mol). However, the summed components of the interaction energy at the Hartree-Fock level (∆E HF el−prep + E elstat + E exch ) resulted in substantially repulsive interaction of ∆E HF int = +20.49 kcal/mol. This value was basically identical to the value calculated by Pyykkö and coworkers at the HF/def2-QZVPP level of theory (the same as the +20.50 kcal/mol). 49 This summation can be regarded as an estimate of the extent of steric repulsion. 48 Noteworthy is that the CH 4 @C 60 is predicted to be unstable also by DFT if empirical dispersion corrections are not used. 30,31 The non-dispersive correction due to electron correlation was small and repulsive (∆E C non−disp = +1.00 kcal/mol). As expected, London dispersion is the dominant intermolecular interaction mechanism. The magnitude of the E C disp term of −29.96 kcal/mol was larger than the substantially repulsive Hartree-Fock interaction and the ∆E C non−disp correction, resulting in an endohedral complex stabilization by −8.47 kcal/mol. Yet, further attractive correction came from the contribution from perturbative triple excitations ∆E C−(T) int that stabilized the complex by an additional estimated contribution of −5.03 kcal/mol. The correction from perturbative triples was important, given that it increased the net binding energy in the complex by nearly 60 % (from −8.47 to −13.50 kcal/mol; see inset in Fig. 3). Therefore, the final electronic interaction energy at the DLPNO-CCSD(T)/cc-pVQZ(CH 4 )/cc-pVTZ(C 60 ) level of theory for the CH 4 @C 60 endohedral complex was −13.50 kcal/mol. The geometry preparation ("deformation energy") term was very small (∆E geo−prep = +0.03 kcal/mol). C 60 is a rigid molecule and encapsulation of the CH 4 guest had a negligible effect on its geometry (see Table 2). Our reference stabilization energy of ∆E = −13.47 kcal/mol was compared with best reported estimates. For the CH 4 @C 60 complex, the most robust results have been reported by Pyykkö and coworkers. 49 In that study interaction energies were obtained with supermolecular MP2 and its spin component scaled counterpart (SCS-MP2). Calculations were performed with the def2-TZVPP and def2-QZVPP basis sets, interaction energies were corrected for basis set superposition error and extrapolated to the complete basis set limit. The obtained MP2 interaction energy of −21.37 kcal/mol was clearly overestimated. The value obtained with SCS-MP2 (−11.97 kcal/mol) was closer to the coupled cluster reference, but underestimated. These calculated interaction energies followed the pattern observed in previously reported benchmark calculations. For the CH 4 • • • C 6 H 6 dimer, MP2 overesti-mates the CCSD(T) interaction energy, as was shown by Ringer et al., 77 and for the endohedral CH 4 @C 60 complex, this overestimation seems to be even more pronounced. The same trend of deviation was observed by Pyykkö and coworkers for dimers composed of atoms of noble gases and benzene (Ng• • • C 6 H 6 ), where MP2 overestimated the CCSD(T) reference interaction energies significantly, whereas SCS-MP2 was generally much closer to coupled cluster results, but consistently underestimated the interaction. 49 Pyykkö and coworkers have also developed London-type formulas to estimate dispersion interaction energies in endohedral systems. 48,49 The input parameters to these formulas such as ionization potentials and polarizabilities can be readily computed at the DFT level. Using the data from the study of Pyykkö and coworkers 49 (Table 17, equations 69+86) the dispersion energy estimate of −17.33 kcal/mol was obtained for the CH 4 @C 60 complex. This energy was much smaller than for the DLPNO-CCSD(T)-LED (−29.96 kcal/mol), and would not overcome the steric repulsion estimate of +20.49 kcal/mol, and the complex would not be estimated to be stabilized in that description. vations that high pressure and elevated temperature conditions (1645 atm, 190 • C for 22 h) are necessary to achieve a high degree of CH 4 insertion. 20 Noteworthy is that the electronic repulsive interaction at the orifice ∆E int = + 9.19 kcal/mol amounted to only less than half of the insertion energy barrier. The remaining geometry preparation term corresponded to the energy needed to deform the open-cage fullerene from its equilibrium geometry to the one optimal for CH 4 insertion (∆E geo−prep = +10.62 kcal/mol). After insertion, the CH 4 molecule is predicted to be stabilized inside the open-fullerene cage by ∆E = −10.16 kcal/mol. ## Spectroscopic properties of the CH 4 @C 60 in IR and UV-Vis Harmonic vibrational frequencies of the endohedral CH 4 @C 60 complex have been computed at the level of GGA and hybrid DFT approximations without using dispersion corrections, 30,31 and at the Hartree-Fock level of theory. 32 Hence, those frequencies were evaluated on structures corresponding to energy minima in situation where London dispersion interactions had not been accounted for. In those studies the intensities in the resulting calculated IR and Raman spectra were not discussed as well. Therefore, we calculated the harmonic vibrational frequencies together with the respective IR absorption coefficients and Raman scattering factors for the CH 4 , C 60 , and the CH 4 @C 60 complex at the revPBE-D4/pcseg-1 level of theory. Related frequencies, absorption coefficients and scattering factors are presented in Table 3. The calculated vibrational frequencies of the C 60 were in very good agreement with experimental data and virtually not changed upon CH 4 encapsulation. This situation was in agreement with the experimental IR spectrum of the H 2 O@C 60 , where the vibrational frequencies of the fullerene cage were the same as those of the free C 60 . 19 The calculated IR absorption coefficients and Raman scattering factors (for the fullerene cage) were predicted to be slightly affected by CH 4 encapsulation and resulted on average in a <5 % loss in spectral intensity. Frequencies of the encapsulated CH 4 on the other hand were blue shifted with respect to the free CH 4 molecule, and were in agreement with previous theoretical predictions. 31,32 Our results suggested that the triple degeneracy of the asymmetric bending and stretching IR modes (1287 and 3107 cm −1 ) of CH 4 was partially removed due to the interaction with the cage. However, what was the most important, for both IR and Raman a substantial loss in spectral intensities for the encapsulated CH 4 was revealed. This intensity loss was in line with experimental IR spectra of the CH 4 @openfullerene complex, where vibrations of the CH 4 could not be observed. 35 In addition, vibrational features of the H 2 O were very weak in the experimental IR spectrum of H 2 O@C 60 , and the potential screening effect of the fullerene cage was indicated. 19,83 Dielectric measurements conducted at low temperature and IR spectra of H 2 O@C 60 collected at liquid helium temperature have revealed that the dipole moment of the encapsulated H 2 O was 0.5±0.1 D, 84,85 which is about 25 % of free the H 2 O molecule (in agreement with theoretical predictions 86 ). A very similar extent of dipole moment reduction has been observed for HF in HF@C 60 as well. 87 Table 3 Harmonic vibrational frequencies (ν; cm −1 ), IR absorption coefficients (A; 10 5 cm/mol) a and Raman scattering factors (S; 4 /amu) a calculated at the revPBE-D4/pcseg-1 level of theory b ; experimental values for the free CH The fullerene cage protects encapsulated species from influence of the external electric field, and acts as a molecular Faraday cage. 88,89 Such a screening effect can be assessed by calculating a difference between the dipole polarizability (α) of an endohedral complex, and the sum of polarizabilities of an isolated guest and the empty fullerene: 90 Negative ∆α corresponds to the polarizability depression that results from the decrease of polarizability of the encapsulated guest. Therefore, the dielectric screening coefficient can be evaluated: 90 To inspect these effects for the CH 4 @C 60 endohedral complex, the dipole polarizabilities of CH 4 , C 60 , and CH 4 @C 60 were calcu-lated at the CAM-B3LYP/Sadlej-pVTZ level of theory. The rangeseparated and Coulomb-attenuating method called as the CAM-B3LYP DFT approximation 91 was shown to deliver accurate polarizabilities, 92 and a balanced description of electronic excited states. 93 The Sadlej-pVTZ basis set was specifically developed for calculations of polarizabilities and other electric molecular properties. The calculated polarizabilities and the respective values obtained from Equations 3 and 4 are shown in Table 4. The values for the polarizabilities for CH 4 and C 60 were in good agreement with experimental data. The polarizability of the CH 4 @C 60 complex was predicted to be almost the same as that of the empty C 60 , which in turn was reflected in a substantial polarizability depression of ∆α=−2.48. The value for the dielectric screening coefficient (c = 0.97) indicated a particularly strong effect for the CH 4 @C 60 endohedral complex. The polarizability of the encapsulated CH 4 molecule was essentially quenched. a From ref. 97,98 The electronic excited states energies and absorption in the UV-Vis domain were calculated at the CAM-B3LYP/cc-pVTZ level of theory with a time-dependent DFT (TD-DFT) within the efficient sTD-DFT implementation of Bannwarth and Grimme. 99 Transition energies, wavelengths, and oscillator strengths for C 60 and CH 4 @C 60 are shown in Table 5. The predicted spectroscopic characteristics of C 60 and CH 4 @C 60 were almost identical in the UV-Vis range. Both entities exhibited the same transition energies. The oscillator strengths were marginally lower for the complex compared to the empty fullerene. These results were in agreement with experimental observations for C 60 and H 2 O@C 60 . They display close to identical UV-Vis spectra, despite revealing a slightly lower absorption for the complex. 19 The data in Table 5 compare favorably with the experimental UV-Vis spectrum of C 60 in the gas phase. 100 The set of three most intense bands at 187/231/300 nm that reveal oscillator strengths of 1.907/1.922/0.384 correspond to the experimentally observed transitions at 205/257/330 nm that exhibit extinction coefficients of 4.8/3.5/0.9 (10 5 L mole −1 cm −1 ). 100 Hence, the pattern of UV-Vis bands correlated well between theoretical predictions and the experimentally observed spectra, however, transition energies were overestimated (too short wavelengths) at the TD-DFT level of theory. Because of the rich chemistry of the ionized fullerene species (C n+ 60 ; n = 1, 2, 3), it is interesting to compare the ionization potentials of the C 60 and its endohedral complex. Ionized fullerenes exhibit a diversity of ionization mechanisms and a variety of reactions with potential implications to chemistry in the interstellar medium. 101 The vertical ionization energies (V IE) for C 60 and CH 4 @C 60 were calculated at the CAM-B3LYP/Sadlej-pVTZ level of theory according to: where E n+ cation and E 0 neutral denote the total energies of the ionized and neutral species, respectively. They were calculated at the equilibrium geometry of the ground state. The calculations for ionized species involved an unrestricted (UDFT) formalism due to higher than singlet multiplicities. The obtained results indicated that the ionization potentials of the CH 4 @C 60 complex were almost identical to those of empty C 60 . The calculated V IE for C 60 agreed very well with experimental data, as can be seen from values in Table 6. ## NMR properties of the CH 4 @C 60 Fullerenes constitute the only known allotrope of carbon that can be dissolved in organic solvents at room temperature. 103 Therefore, high resolution liquid-state NMR spectra of C 60 and its endohedral complexes can be measured in common NMR solvents. 104,105 Whitby and coworkers collected and analyzed 1 H and 13 C NMR spectra of the CH 4 @C 60 complex dissolved in 1,2dichlorobenzene. 20 For prediction of such spectra with quantum chemistry methods, a robust model including subtle interactions of the fullerene cage with solvent molecules is important. Hence, we constructed systems composed of the C 60 and CH 4 @C 60 explicitly solvated by 25 molecules of 1,2-dichlorobenzene, whereas solvent effects at the outer sphere were accounted implicitly by a polarizable continuum model (PCM) assuming a dielectric con-stant ε = 9.93. The initial configuration was obtained with the Packmol software, 106 and the coordinates were energy optimized at the revPBE-D4/pcseg-1(CH 4 , C 60 )/pcseg-0(C 6 H 4 Cl 2 ) level of theory up to the energy change of < 5 × 10 −6 E h ; see Figure 5. The 1 H and 13 C nuclear magnetic shielding tensors 107,108 (σ σ σ ; assessed with the GIAO approach 109 ) and indirect nuclear spinspin coupling constants (J) were calculated at the level of PBE0 DFT approximation. 110 The calculations were performed with segmented pcS-1 and pcJ-1 basis sets, which have been specifically developed to provide fast convergence towards the Kohn-Sham limit for NMR shieldings and spin-spin couplings. 111,112 The chosen setup represents a reasonable compromise for the calculations of 1 H/ 13 C NMR parameters when compared to more accurate methods given the size of the system. 113,114 For solvent molecules, the pcseg-0 basis set was used. The calculations of NMR observables involved all electrons (no frozen core) and very tight grids (GridX8 Grid7). The calculated isotropic 1 H/ 13 C NMR shielding: was converted into isotropic NMR chemical shift δ according to: where δ j and σ j correspond to the chemical shift and shielding of an atom of interest j, whereas σ re f , calc CH 4 (gas) and δ re f , exp CH 4 (gas) represent the calculated shielding and experimental shift of the reference, respectively. The CH 4 molecule was used as a reference, since its proton and carbon chemical shifts measured in the gas phase are available. 115 Shifts of chemically equivalent atoms were averaged. Spin-spin coupling constants were represented as a sum of four physical contributions: the Fermi contact (FC), spin-dipole (SD), paramagnetic spin-orbit (PSO), and diamagnetic spin-orbit (DSO) terms. a From ref. 20,115,116 The calculated 1 H and 13 C NMR chemical shifts as well as the 1 H− 13 C spin-spin couplings presented in Table 7 revealed a very good agreement with experimental data. For protons in the CH 4 molecule, the change in chemical shift (∆δ ) upon encapsulation in C 60 was predicted to be −7.54 ppm, which compared very well to −7.88 ppm observed in an experiment. 20 This change on encapsulation is associated with the NMR shielding inside the fullerene cage, where the locally induced magnetic fields counteract the applied external field of the NMR instrument. The corresponding effect for the 13 C in CH 4 was slightly smaller, as revealed by the calculated and experimental ∆δ values of −5.87 and −4.98 ppm, respectively. For carbon atoms of the fullerene cage, the presence of endohedral CH 4 results in a deshielding of the 13 C NMR signal. Therefore, this effect is opposite to that observed for the 13 CH 4 inside the cage. The calculated 13 C deshielding of the cage of +0.51 ppm was in an excellent agreement with the experimental value of +0.52 ppm. 20 The spin-spin coupling constant 1 J HC in the CH 4 was dominated by the Fermi-contact (FC) mechanism. The calculated coupling strength of 1 J HC = 126.4 Hz for the free CH 4 was close to the experimental value of 125.3 Hz. 116 Encapsulation of CH 4 in C 60 had little effect on the 1 J HC , and the calculated value of 124.1 Hz was very close to the experimentally determined 124.3 Hz. 20 Therefore, our theoretical model predicts correctly the sign and the small magnitude of the ∆J. The small change of the coupling was consistent with negligible deformation of the CH 4 geometry upon encapsulation. Complexes with noble gases Ng@C 60 (Ng = He, Ne, Ar, Kr) The efficient DLPNO-CCSD(T) setup designed for probing intermolecular interactions in the CH 4 @C 60 complex was also used to obtain reference interaction energies for endohedral complexes with atoms of noble gases (Ng@C 60 ; He, Ne, Ar, Kr). Hence, in this section we compared our coupled cluster results to previously reported estimates at lower levels of theory. a Two-point extrapolation to the CBS limit based on the def2-TZVPP/def2-QZVPP basis sets; results from ref. 49 b DFT-SAPT calculations with PBEac functional, aug-cc-pVDZ(He, Ne), aug-cc-pVTZ(Ar, Kr), and TZVP(C) basis sets; results from ref. 47 In Table 8, interaction energies (∆E int ) calculated at the DLPNO-CCSD(T)/cc-pVQZ(Ng)/cc-pVTZ(C 60 ) level of theory are presented together with values from the MP2/SCS-MP2 calculations by Pyykkö and coworkers, 49 and from the DFT-SAPT calculations by Hesselmann and Korona. 47 The MP2 method exhibited the most unbalanced performance. The associated interaction energies for He was too low with this method, for Ne they were quite reasonable (accidentally), but those for Ar and Kr were severely overestimated. These trends resembled those observed for values derived with the MP2 method for the CH 4 @C 60 complex. The spin component scaled variant (SCS-MP2) displayed an improved description. Although interaction energies for He and Ne were too low, the result for Ar was close to the coupled cluster reference, and the overestimation for Kr was not as severe as with the MP2. The situation with the DFT-SAPT results was more complex and somewhat difficult to judge. On the one hand, all interaction energies for these systems obtained with the DFT-SAPT were consistently underestimated when compared to the DLPNO-CCSD(T) values. On the other hand, the performance of the DFT-SAPT was consistent and well balanced across the He, Ne, Ar, and Kr complexes. This complexity became apparent when the results of DLPNO-CCSD(T) were plotted against the DFT-SAPT counterpart in Figure 6a. The correlation among the two approaches was very good, despite the consistently underestimated interaction energies by the latter. For comparison, the correlation with the SCS-MP2 data shown in panel b was much worse and significantly less convincing. In Table 9, estimations of dispersion contributions to the interaction energy for the Ng@C 60 complexes are presented for different methods of calculations. The dispersion contributions (E C disp ) from the DLPNO-CCSD(T)-LED were in good agreements with the dispersion components from the DFT-SAPT for all four considered complexes. Hence, it was concluded that the DFT-SAPT compared favorably to the DLPNO-CCSD(T)-LED for prediction of the dispersion interaction in fullerene complexes. This favorable comparison for DFT-SAPT is illustrated in Figure 7a where results of DLPNO-CCSD(T) and DFT-SAPT are plotted against each other. Therefore, the too low interaction energies obtained with the DFT-SAPT for the complexes with noble gases did not originate from inappropriate description of the dispersive part of the in- Fig. 6 Interaction energies (∆E int ) calculated at the DLPNO-CCSD(T)/cc-pVQZ(Ng)/cc-pVTZ(C 60 ) level of theory plotted against the results from DFT-SAPT (a), 47 and supermolecular SCS-MP2 (b). 49 Grey line corresponds to the ideal correlation y = x, whereas red line to the linear regression fit. teraction, but resulted from deficiencies in the remaining components of the interaction energy. Analysis with DLPNO-CCSD(T)-LED revealed significant contributions from perturbative triples (∆E ## C−(T) int ), and yet, for small guests (He, Ne) for which repulsive interaction at the Hartree-Fock level (∆E HF el−prep + E elstat + E exch ) is small, the non-dispersive corrections due to electron correlation (∆E C non−disp ) were attractive. The dispersion interaction as predicted with London-type formulas by Pyykkö and coworkers were substantially underestimated when compared to the DLPNO-CCSD(T)-LED results, although a linear trend with the latter was revealed; see Table 9 and Figure 7b. This underestimation indicates that the sum of the dipole-dipole and quadrupole-quadrupole terms in the "Pyykkö model 2010" (equations 69+72 from ref. 49 ) was not sufficient, and that the higher order multipole-multipole contributions are necessary to include to obtain better agreement with high-level quantum chemistry methods. 47,49 The He 2 @C 60 trimer The existence of the He 2 @C 60 trimer, where two helium atoms are encapsulated inside the C 60 was discovered with 3 He NMR by Rabinovitz and coworkers. 117 The observed He 2 @C 60 :He@C 60 ratio of 1:200 was 10 times smaller than that for the He 2 @C 70 :He@C 70 (1:20). This reduction suggested that the smaller cavity of C 60 was significantly less suited for the encapsulation of two He atoms as compared to the C 70 fullerene. The stability of the He 2 @C 60 trimer was studied with quantum chemistry methods by Darzynkiewicz & Scuseria, 28 Krapp & Franking, 118 and Hesselmann & Korona. 47 However, all methods applied, including DFT, MP2, SCS-MP2, and DFT-SAPT predicted repulsive interaction in the range from +1.13 to +10.23 kcal/mol, depending on the method and the basis sets used. Hence, theoretical investigations reported so far suggest that the He 2 @C 60 is thermodynamically unstable towards loss of the noble gas atom, in stark contrast to the experimental observation. To gain insight into this challenging system and confront discrepancy between theory and experiment with the DLPNO-CCSD(T) approach, a relaxed potential energy surface scan at the revPBE-D4/pcseg-1 level of theory was performed for the He 2 @C 60 . The He−He distance was sampled with a 0.02 increment, and for each step all other coordinates were subjected to unconstrained optimization. Subsequently, single-point DLPNO-CCSD(T) calculations were performed on the resulting geometries to locate the "true" energy minimum; see Figure 8. At the DLPNO-CCSD(T)/cc-pVQZ(He)/cc-pVTZ(C 60 ) level of theory, the equilibrium He−He distance inside the C 60 was 1.94 . This distance was not only substantially shorter than that of 3.00 calculated for the free He 2 dimer, but it corresponded to a clearly repulsive regime for the latter. comparably smaller. Note that the interaction well for the isolated He 2 dimer was relatively shallow, whereas the relation of potential energy change upon He−He distance variation was very steep for the He 2 @C 60 trimer. For the equilibrium distance r He−He = 1.94 inside C 60 the stabilization energy ∆E int of the He 2 @C 60 trimer: − (E XY Z X (XY Z) evaluated at the DLPNO-CCSD(T)/cc-pVQZ(He 2 )/cc-pVTZ(C 60 ) level of theory was −1.43 kcal/mol. Noteworthy was that the stabilization energy for the He 2 @C 60 trimer at the DLPNO-CCSD(T) level of theory was almost as high as for the complex with a single He atom with the DFT-SAPT. The ab initio calculations predicted the He 2 @C 60 trimer to be stable, which is in agreement with experimental observations. ## Conclusions The reference interaction energies for endohedral complexes of the C 60 fullerene with He, Ne, Ar, Kr, and CH 4 were calculated at the DLPNO-CCSD(T) level of theory and decomposed into physical contributions with the LED scheme. An accurate and efficient multilevel DLPNO-CCSD(T) setup was proposed, which was applicable to routine studies of endohedral complexes of C 60 and larger fullerenes. Calculated molecular properties of the CH 4 @C 60 complex revealed that the IR and Raman bands of the endohedral CH 4 were essentially "silent" due to the dielectric screening effect of the C 60 , which acted as a molecular Faraday cage. Absorption spectra in the UV-Vis and ionization potentials of C 60 and CH 4 @C 60 were predicted to be almost the same. Calculated 1 H/ 13 C NMR shifts and spin-spin coupling constants were in very good agreement with experimental data. Lastly, selected points at the potential energy surface of the endohedral He 2 @C 60 trimer were calculated at the DLPNO-CCSD(T) level of theory. In contrast to previous theoretical studies with DFT, MP2, SCS-MP2 and DFT-SAPT, where all these methods predicted the He 2 @C 60 to be thermodynamically unstable towards the loss of the noble gas atom, our calculations predicted the He 2 @C 60 to be stable, which is in agreement with experimental observations. Therefore, the case of the He 2 @C 60 trimer clearly indicated that the DLPNO-CCSD(T) level of theory is indispensable in studies of weakly interacting systems, and should be used whenever applicable. ## Conflicts of interest There are no conflicts to declare.

chemsum

{"title": "J o u r n a l Na me Local energy decomposition analysis and molecular properties of encapsulated methane in fullerene (CH 4 @C 60 ) \u2020", "journal": "ChemRxiv"}

integration_of_exonuclease_iii-powered_three-dimensional_dna_walker_with_single-molecule_detection_f

## Abstract: Initiator caspases are important components of cellular apoptotic signaling and they can activate effector caspases in extrinsic and intrinsic apoptotic pathways. The simultaneous detection of multiple initiator caspases is essential for apoptosis mechanism studies and disease therapy. Herein, we develop a sensitive nanosensor based on the integration of exonuclease III (Exo III)-powered three-dimensional (3D) DNA walker with single-molecule detection for the simultaneous measurement of initiator caspase-8 and caspase-9. This assay involves two peptide-DNA detection probe-conjugated magnetic beads and two signal probe-conjugated gold nanoparticles (signal probes@AuNPs). The presence of caspase-8 and caspase-9 can induce the cleavage of peptides in two peptide-DNA detection probes, releasing two trigger DNAs from the magnetic beads, respectively. The two trigger DNAs can serve as the walker DNA to walk on the surface of the signal probes@AuNPs powered by Exo III digestion, liberating numerous Cy5 and Texas Red fluorophores which can be quantified by single-molecule detection, with Cy5 indicating caspase-8 and Texas Red indicating caspase-9. Notably, the introduction of the AuNP-based 3D DNA walker greatly reduces the background signal and amplifies the output signals, and the introduction of single-molecule detection further improves the detection sensitivity. This nanosensor is very sensitive with a detection limit of 2.08 Â 10 À6 U mL À1 for caspase-8 and 1.71 Â 10 À6 U mL À1 for caspase-9, and it can be used for the simultaneous screening of caspase inhibitors and the measurement of endogenous caspase activity in various cell lines at the single-cell level. Moreover, this nanosensor can be extended to detect various proteases by simply changing the peptide sequences of the detection probes. ## Introduction Apoptosis is programmed cell death which can result in the disappearance of cells without any inflammatory phenomena. 1 Caspases are a family of cysteinyl aspartate-directed proteases, and they are the central executioners of apoptosis. 2 Once activated by a specifc stimulus (e.g., ultraviolet (UV) radiation, girradiation, heat, DNA damage, viral virulence factors), 3 caspases execute the part-proteolysis of downstream substrates to trigger a cascade of events that culminates in the desired biological response: disruption of cellular membranes, breaking down of cytoplasmic and nuclear skeletons, extruding of cytosol, degradation of chromosomes, and fragmentation of the nucleus. 4 Inappropriate apoptosis and deregulation of caspase activity are implicated in various human diseases, including Alzheimer's disease, 5 ischemic damage, 6 autoimmune diseases, 7 and cancers. 8 The apoptotic-associated caspases are classifed into initiators and effectors by their location in the apoptosis cascade signalling pathways. The initiator caspases (e.g., caspase-8, caspase-9, and caspase-10) are responsible for initiating the apoptosis cascade, and the effector caspases (e.g., caspase-3, caspase-6, and caspase-7) can be activated by initiator caspases to destroy cellular proteins and execute apoptosis. 9 Extrinsic and intrinsic apoptotic pathways are the only two pathways of apoptosis, and caspase-8 and caspase-9 are critical participants in extrinsic and intrinsic apoptosis, respectively. In the extrinsic apoptotic pathway, caspase-8 is specifcally activated by death receptors such as Fas/CD95. 10 In the intrinsic apoptotic pathway, caspase-9 is activated by the accumulation of cytochrome C in the cytosol due to dissipation of the mitochondrial membrane potential. 11 Therefore, the simultaneous measurement of caspase-8 and caspase-9 are of great importance for the study of the apoptosis mechanism and disease therapy. Conventional methods for caspase assays include enzymelinked immune sorbent assay (ELISA), 12 western blot, 13 flow cytometry, 14 and mass spectrometry. 15 They usually involve multi-step and time-consuming processes, 16 and extensive pretreatment of samples. 17 Recently, a variety of new methods have been developed for in vitro and in vivo detection of caspases, including electrochemical, 18 colorimetric 19 and fluorescence measurements. 20,21 Most of these methods rely on either antibodies 18 or peptide substrates. The main limitation of antibody-based assays is the requirement for high quality antibodies. 22 Alternatively, peptide substrates are a more attractive choice for caspase assay with the distinct advantages of accessibility, simplicity, cost-effectiveness, and chemical defnition. 23 In the peptide substrate-based assays, peptide substrates are often labelled with fluorophores and the fluorescence signals are directly measured after caspase cleavage, resulting in limited sensitivity. 24 The development of new methods for sensitive caspase assay is highly desirable. Due to the highly selective and programmable Watson-Crick complementarity, DNA is becoming an increasingly attractive building material for the construction of a series of functional nanodevices (e.g., walker, gear, 28 and tweezer 29 ). In particular, a DNA walker that is able to convert chemical energy to mechanical motion is the most widely studied DNA nanodevice and has been extensively applied for molecular transport, 30 DNA computing, 31 chemical synthesis, 32 and biosensing. 33,34 DNA walkers can be classifed into one-dimensional (1D), twodimensional (2D), and three-dimensional (3D) DNA walkers in terms of dimensionality. The 1D DNA walkers move along 1D tracks such as double-stranded DNA (dsDNA) and single walled carbon nanotubes (SWCNTs). 35,36 2D DNA walkers can move along the 2D surfaces of gold electrodes and DNA tile. 37,38 3D DNA walkers can move along 3D scaffolds on nanoparticles such as gold nanoparticles (AuNPs), 39 magnetic beads (MBs), 40 SiO 2 @CdTe, 41 Au@Fe 3 O 4 , 42 hollow carbon nanospheres (HCS), 43 and DNA origami. 44 Such 3D DNA walkers are usually powered by nicking endonucleases, 45,46 exonuclease III (Exo III), 47 or the DNA catalytic hairpin assembly reaction, 48 and possess large specifc surface area and high DNA loading capability, 49 and they can greatly enhance the local concentration of track DNA for improved walking efficiency and higher processivity, 50 facilitating high signal gain in a given period of time. 51 A rolling circle amplifcation (RCA)-based method has been developed for caspase assay, 52 but it is only suitable for single target detection and is prone to a high background due to nonspecifc amplifcation. In addition, it requires laborious procedures for the preparation of the circular template, and its accuracy is challenged by the inner flter effect and the collisional quenching of ensemble fluorescence measurements. Herein, we develop a simple and sensitive caspase nanosensor with the capability of multiplexed assay based on the integration of an Exo III-powered 3D DNA walker with singlemolecule detection. The Exo III mediates highly efficient signal amplifcation through catalyzing the cyclic digestion of one strand of dsDNA from the 3 0 end, 53 which can be performed under isothermal conditions with the elimination of complex thermal cycling and nonspecifc artifcial amplifcation involving in polymerase chain reaction (PCR). 54 In comparison with other isothermal amplifcation methods such as strand displacement amplifcation (SDA), 55 exponential amplifcation reaction (EXPAR), 56 rolling circle amplifcation, 57 and loop mediated isothermal amplifcation (LAMP), 58 the Exo IIImediated amplifcation takes advantage of a much simpler reaction scheme without the requirement of a specifc recognition sequence for nicking endonuclease, complicated procedures for preparation of the circular template, and multiple primers for completing the amplifcation reaction. Moreover, in comparison with the ensemble measurement, single-molecule detection has the distinct advantages of high sensitivity, simplicity, and low sample consumption. This assay involves two peptide-DNA detection probe-conjugated magnetic beads and two signal probe-conjugated gold nanoparticles (signal probes@AuNPs). The integration of Exo IIImediated amplifcation with the AuNP-based DNA walker facilitates facile and efficient amplifcation of the caspase signal to acquire high sensitivity with reduced background, and the introduction of single-molecule detection further improves the detection sensitivity. The presence of caspase-8 and caspase-9 can induce the cleavage of peptides in two peptide-DNA detection probes, releasing two trigger DNAs from the magnetic beads. The two trigger DNAs can serve as the walker DNA to walk on the surface of signal probes@AuNPs powered by Exo III digestion, liberating numerous Cy5 and Texas Red fluorophores which can be quantifed by single-molecule detection, with Cy5 indicating caspase-8 and Texas Red indicating caspase-9. This nanosensor is very sensitive with a detection limit of 2.08 10 6 U mL 1 for caspase-8 and 1.71 10 6 U mL 1 for caspase-9, and it can be used for simultaneous screening of caspase inhibitors and measurement of endogenous caspase activity in various cell lines at the single-cell level. To the best of our knowledge, the integration of Exo III-powered 3D DNA walker with single-molecule detection for the simultaneous measurement of multiple caspases has not been explored so far. ## Results and discussion The schematic illustration of the nanosensor based on the integration of the Exo III-powered 3D DNA walker with singlemolecule detection for the simultaneous measurement of initiator caspase-8 and caspase-9 is shown in Scheme 1. This assay involves two peptide-DNA detection probe-conjugated magnetic beads and two signal probe-conjugated gold nanoparticles (signal probes@AuNPs). The biotinylated peptide-DNA detection probes (Fig. S1, ESI †) can be self-assembled on the streptavidin-coated MBs through the specifc streptavidinbiotin interaction. The peptide domain of detection probe 1 (Scheme 1, green color) contains a tetrapeptide sequence Ile-Glu-Thr-Asp (IETD) for caspase-8 recognition, and the peptide domain of detection probe 2 (Scheme 1, magenta color) contains a tetrapeptide sequence Leu-Glu-His-Asp (LEHD) for caspase-9 recognition. The DNA domain of detection probe 1 (Scheme 1, yellow color) is complementary to signal probe 1 (Scheme 1, blue color), and the DNA domain of detection probe 2 (Scheme 1, cyan color) is complementary to signal probe 2 (Scheme 1, red color). Signal probes are attached to the surface of AuNPs via a Au-S bond to form the 3D scaffold of the DNA walker which is powered by Exo III. In the presence of caspase-8, it specifcally recognizes the sequence IETD and hydrolyzes the peptide bond adjacent to the carboxylic group of the aspartic acid residue (i.e., the peptide bond between aspartic acid and glycine), releasing the cleaved detection probe 1 from the magnetic beads (Fig. S1A, ESI †). In the presence of caspase-9, it specifcally recognizes the sequence LEHD and hydrolyzes the peptide bond between aspartic acid and glycine, releasing the cleaved detection probe 2 from the magnetic beads (Fig. S1B, ESI †). The intact detection probes are separated by magnetic separation. The trigger DNA in the cleaved detection probes (Scheme 1, yellow and cyan color) can serve as the walker DNA to initiate the recyclable cleavage of signal probes on the surface of the AuNPs, releasing numerous Cy5 and Texas Red fluorophores from the signal probes@AuNPs. Specifcally, the hybridization of trigger DNA 1 of the cleaved detection probe 1 (Scheme 1, yellow color) and signal probe 1 (Scheme 1, blue color) form a dsDNA with 5 0 overhangs, which can be recognized by Exo III. The stepwise catalytic removal of mononucleotides from the 3 0 -terminus of signal probe 1 (Scheme 1, blue color) leads to the liberation of the Cy5 fluorophore from the signal probe 1@AuNPs, while the digestion of trigger DNA 1 (Scheme 1, yellow color) is blocked by the oligo-T tail. As the digestion of signal probe 1 (Scheme 1, blue color) proceeds, the trigger DNA 1 (Scheme 1, yellow color) is partially released and hybridizes with another signal probe 1 (Scheme 1, blue color) nearby. In this way, the trigger DNA 1 (Scheme 1, yellow color) walks along the AuNP surface, and abundant Cy5 fluorophores are liberated from the signal probe 1@AuNPs, resulting in the restoration of Cy5 fluorescence which can be quantifed by single-molecule detection, with Cy5 indicating the presence of caspase-8. Similarly, the hybridization of trigger DNA 2 of the cleaved detection probe 2 (Scheme 1, cyan color) with signal probe 2 (Scheme 1, red color) forms a dsDNA with 5 0 overhangs, which can be recognized by Exo III. The Exo III-mediated stepwise catalytic removal of mononucleotides from the 3 0 -terminus of signal probe 2 (Scheme 1, red color) leads to the liberation of abundant Texas Red fluorophores from the signal probe 2@AuNPs, resulting in the restoration of Texas Red fluorescence which can be used for the quantifcation of caspase-9. Because there are 120 signal probes per AuNP (ESI †), the high density of the scaffold DNA enhances the movement of trigger DNA along the 3D tracks, greatly amplifying the fluorescence signals. In contrast, in the absence of caspase-8 and caspase-9, the detection probes remain intact, and no trigger DNA is released. Consequently, no Exo III-mediated digestion of signal probes occurs, and neither the Cy5 nor Texas Red signal can be detected. This assay mainly relies on the caspase-mediated cleavage of peptide to initiate the recycling liberation of Cy5/Texas Red fluorescent molecules from the signal probes@AuNPs. We used 12% nondenaturing polyacrylamide gel electrophoresis (PAGE) to verify the cleavage of the detection probes by caspase-8 and caspase-9. For the caspase-8 assay, when caspase-8 is absent, only one distinct band is observed (Fig. 1A, lane 2), which is identical to that of the intact detection probe 1, indicating that the cleavage of detection probe 1 cannot occur in the absence of caspase-8. When caspase-8 is present, a new smaller-sized band is observed (Fig. 1A, lane 3), which corresponds to the cleaved peptide-DNA 1 (Fig. 1A, lane 1), indicating the cleavage of detection probe 1 induced by caspase-8. When only caspase-9 is present, only one identical band to that of the intact detection probe 1 (Fig. 1A, lane 2) is observed (Fig. 1A, lane 4), indicating that the cleavage of detection probe 1 cannot occur in the presence of caspase-9. For the caspase-9 assay, when caspase-9 is absent, only one distinct band is observed (Fig. 1B, lane 2), which is identical to that of intact detection probe 2, indicating that the cleavage of detection probe 2 cannot occur in the absence of caspase-9. When only caspase-8 is present, only one distinct band identical to lane 2 (Fig. 1B) is observed (Fig. 1B, lane 3), indicating that the cleavage of detection probe 2 cannot occur in the presence of caspase-8. When caspase-9 is present, a new smaller-sized band is observed (Fig. 1B, lane 4), which corresponds to the cleaved peptide-DNA 2 (Fig. 1B, lane 1), indicating the cleavage of detection probe 2 induced by caspase-9. To verify the feasibility of the proposed nanosensor, we performed fluorescence measurements (Fig. 1C and D). As shown in Fig. 1C, a distinct Cy5 fluorescence signal with a characteristic emission peak of 670 nm is observed in the presence of 0.025 U mL 1 caspase-8 (Fig. 1C, red line). In contrast, in the control group without caspase-8, a low Cy5 signal (Fig. 1C, black line) is detected. As shown in Fig. 1D, a distinct Texas Red fluorescence signal with a characteristic emission peak of 620 nm is observed in the presence of 0.025 U mL 1 caspase-9 (Fig. 1D, green line). In contrast, in the control group without caspase-9, a low Texas Red signal (Fig. 1D, black line) is detected. We further employed single-molecule detection technology to detect the fluorescence signals (Fig. 2). In the absence of caspase-8 and caspase-9, neither Cy5 (Fig. 2A) nor the Texas Red fluorescence signal (Fig. 2E) is observed, indicating that the Exo III-mediated recycling liberation of fluorophores cannot occur in the absence of caspase-8 and caspase-9. In contrast, in the presence of 0.025 U mL 1 caspase-8, distinct Cy5 fluorescence signals are observed (Fig. 2B, red color), but no Texas Red fluorescence signal is detected (Fig. 2F), indicating that caspase-8 can induce the recycling liberation of Cy5 fluorophores from the signal probe 1@AuNPs. In the presence of 0.025 U mL 1 caspase-9, distinct Texas Red fluorescence signals are observed (Fig. 2G, green color), but no Cy5 fluorescence signal is detected (Fig. 2C), indicating that caspase-9 can induce the recycling liberation of Texas Red fluorophores from the signal probe 2@AuNPs. In the presence of both caspase-8 and caspase-9, distinct Cy5 (Fig. 2D, red color) and Texas Red fluorescence signals (Fig. 2H, green color) are simultaneously observed. Notably, both the Cy5 and Texas Red fluorescence spots exhibit single-step photobleaching (Fig. S3, ESI †), suggesting that the observed individual fluorescent spot originates from a single dye molecule. These results clearly demonstrate that the proposed nanosensor can be applied for the simultaneous detection of caspase-8 and caspase-9 at the single-molecule level. Under the optimized experimental conditions (Fig. S4 and S5, ESI †), we evaluated the sensitivity of the proposed nanosensor by measuring the variance of fluorescent counts with the caspase concentration. As shown in Fig. 3A, the Cy5 counts improve with increasing concentration of caspase-8 from 2.50 10 6 to 0.05 U mL 1 . In the logarithmic scale, the Cy5 counts show a linear correlation with the concentration of caspase-8 over the range from 2.50 10 6 to 2.50 10 3 U mL 1 . The regression equation is N ¼ 510.06 + 89.12 log 10 C with a correlation coefficient (R 2 ) of 0.9990 (inset of Fig. 3A), where N is the measured Cy5 count and C is the concentration of caspase-8 (U mL 1 ). By evaluating the average response of the control group plus three times the standard deviation, the limit of detection (LOD) was calculated to be 2.08 10 6 U mL 1 (7.51 pM). The sensitivity of the proposed nanosensor is 96-fold higher than the conjugated polymer-based fluorescence assay (0.2 U mL 1 ), 66 and 439-fold higher than the reported fluorescence assay (3.3 nM). 67 As shown in Fig. 3B, the Texas Red counts improve with increasing concentration of caspase-9 from 2.50 10 6 to 0.05 U mL 1 . In the logarithmic scale, the Texas Red counts show a linear correlation with the concentration of caspase-9 over a range from 2.50 10 6 to 2.50 10 3 U mL 1 . The regression equation is N ¼ 451.37 + 71.46 log 10 C with a correlation coef-fcient (R 2 ) of 0.9997 (inset of Fig. 3B), where N is the measured Texas Red count and C is the concentration of caspase-9 (U mL 1 ). The LOD was calculated to be 1.71 10 6 U mL 1 (92.37 pM). The sensitivity of the proposed nanosensor is 40-fold higher than the up-conversion nanoparticles-based FRET assay (0.068 U mL 1 ), 68 and 650-fold higher than the gold nanoparticle-polydopamine-based electrochemical immunosensor (0.06 mM). 18 The improved sensitivity of the proposed nanosensor can be ascribed to (1) the efficient conversion of the caspase activity signal into the DNA signal through the cleavage of the peptide-DNA detection probe, (2) recyclable liberation of fluorescent molecules from the signal probes@AuNPs induced by the Exo III-driven 3D DNA walker, and (3) the high signal-tonoise ratio of single-molecule detection. To investigate the selectivity of the proposed nanosensor for caspase assay, we used caspase-3, DNA (cytosine-5)methyltransferase 1 (Dnmt1) and human alkyladenine DNA glycosylase (hAAG) as the negative controls. Caspase-3 is a core effector caspase that can be activated by caspase-8 in the extrinsic apoptotic pathway and can be activated by caspase-9 in the intrinsic pathway, but it cannot cleave the peptide substrates of caspase-8 and caspase-9. 16 Dnmt1 can specifcally recognize the hemimethylated sequence 5 0 -CG-3 0 , and catalyze the transfer of a methyl group from S-adenosyl-L-methionine (SAM) to the cytosine in genomic DNA. 69 hAAG can recognize and excise a diverse group of alkylated purine bases and cleave the N-glycosidic bond between the sugar and the damaged base. 70 As shown in Fig. 4, in the presence of caspase-3, Dnmt1 and hAAG, neither Cy5 nor Texas Red fluorescence signal is observed, consistent with the control with only the reaction buffer. In contrast, in the presence of caspase-8, an enhanced Cy5 fluorescence signal is observed, but no Texas Red fluorescence signal is detected. In the presence of caspase-9, an enhanced Texas Red fluorescence signal is detected, but no Cy5 fluorescence signal is detected. Moreover, in the presence of both caspase-8 and caspase-9, both Cy5 and Texas Red fluorescence signals can be simultaneously detected. These results demonstrate that the proposed nanosensor exhibits excellent selectivity toward caspase-8 and caspase-9. To investigate the feasibility of the proposed nanosensor for kinetic analysis, we measured the initial velocity (V) in response to various concentrations of the detection probes. To evaluate the enzyme kinetic parameters of caspase-8, we measured the V in the presence of 0.025 U mL 1 caspase-8 and different concentrations of detection probe 1 for 5 min at 37 C. As shown in Fig. 5A, the initial velocity of caspase-8 enhances with the increasing concentration of detection probe 1. V max is calculated to be 146.55 min 1 and K m is calculated to be 1.39 mM. The K m value is consistent with that obtained by the rolling circle amplifcation (RCA)-based fluorescence assay (1 mM). 52 To evaluate the enzyme kinetic parameters of caspase-9, we measured the V in the presence of 0.025 U mL 1 caspase-9 and different concentrations of detection probe 2 for 5 min at 37 C. As shown in Fig. 5B, the initial velocity of caspase-9 enhances with the increasing concentration of detection probe 2. V max is calculated to be 109.12 min 1 and K m is calculated to be 1.21 mM. The K m value is consistent with that obtained by the multicolor gold-selenium bonding nanoprobe-based fluorescence assay (8.53 mM). 71 These results suggest that the proposed nanosensor can be used to accurately evaluate the kinetic parameters of caspases. Fluoromethylketone (FMK), 72 chloromethylketone (CMK) 73 and difluorophenoxymethyl (OPh) 74 are competitive caspase inhibitors, and they can irreversibly inactivate caspases by forming a covalent thioether adduct with the cysteine of the active site in caspases. We used the caspase-8 inhibitor Z-IETD-FMK, caspase-9 inhibitor Ac-LEHD-CMK and broad-spectrum caspase inhibitor Q-VD-OPh as the model inhibitors to investigate the feasibility of the proposed nanosensor for the caspase inhibition assay. As shown in Fig. 6A, the relative activity of caspase-8 decreases with the increasing concentration of Z-IETD-FMK from 0 to 20 mM. The concentration of inhibitor required to reduce the activity of caspase-8 by 50% (IC 50 ) is determined to be 1.95 mM, consistent with that obtained by the RCA-based fluorescence assay (0.9656 mM). 52 As shown in Fig. 6B, the relative activity of caspase-9 decreases with increasing concentration of Ac-LEHD-CMK from 0 to 300 nM. The IC 50 is determined to be 72.80 nM, consistent with the data from the manufacturer (70 nM). As shown in Fig. 6C and D, the relative activities of caspase-8 and caspase-9 decrease with increasing concentration of Q-VD-OPh, respectively. The IC 50 is determined to be 0.28 mM for caspase-8 and 0.31 mM for caspase-9. These results suggest that the proposed method can be applied for the screening of caspase inhibitors. To demonstrate the feasibility of the proposed method for cellular caspase assays, we simultaneously measured endogenous caspase-8 and caspase-9 activity in a human cervical cancer cell line (HeLa cells), a human breast cancer cell line (MCF-7 cells), and a human acute T-lymphocytic leukemia cell line (Jurkat cells). Staurosporine (STS) is a broad-spectrum inhibitor of protein kinases with the capability of inducing in vitro apoptosis. 75 To avoid the interference from nonspecifc caspases, we employed anti-caspase-8 and anti-caspase-9 antibodies to completely inhibit target caspase-8/9 activity (Fig. S6, ESI †), and calculated the specifc caspase-8/9 signal DC according to eqn (1). where C is the fluorescent count in the absence of antibody, and C a is the fluorescent count in the presence of antibody. We frst verifed its capability of inducing apoptosis. In contrast to the low background signal in the control group without any cell extracts (Fig. 7A and B DC Texas Red in HeLa cells (Fig. 7A and B, green columns), MCF-7 cells (Fig. 7A and B, orange columns) and Jurkat cells (Fig. 7A and B, cyan columns) compared to the cells without STS treatment (Student's t-test, P < 0.001), suggesting that the activation of caspases is involved in STS-induced apoptosis. These results demonstrate that the proposed nanosensor can be used for the sensitive detection of endogenous caspases activity. ## Conclusions In summary, we develop a sensitive nanosensor based on the integration of exonuclease III-powered 3D DNA walker with single-molecule detection for simultaneous measurement of initiator caspase-8 and caspase-9. The presence of caspase-8 and caspase-9 can induce the cleavage of peptides in two peptide-DNA detection probes, releasing two trigger DNAs from the magnetic beads. The two trigger DNAs can serve as the walker DNA to walk on the surface of signal probes@AuNPs powered by Exo III digestion, liberating numerous Cy5 and Texas Red fluorophores which can be quantifed by singlemolecule detection, with Cy5 indicating caspase-8 and Texas Red indicating caspase-9. This nanosensor possesses the following distinct advantages: (1) the introduction of the AuNPbased 3D DNA walker greatly reduces the background signal and amplifes the output signals, and the introduction of singlemolecule detection further improves the detection sensitivity, endowing this nanosensor with higher sensitivity and less assay time than the reported caspase assays (Table S1, ESI †) and facilitating the accurate detection of low-abundant caspase with low sample consumption for biomedical research and disease diagnosis; (2) the amplifcation reaction can be conducted at constant temperature without the requirement of complicated thermal cycling, and the signal can be easily quantifed by single-molecule counting; (3) the use of peptide-DNA detection probe-conjugated magnetic beads and signal probes@AuNPs greatly simplifes the experimental procedures, facilitating the simultaneous detection of multiple caspases. This nanosensor is very sensitive with a detection limit of 2.08 10 6 U mL 1 for caspase-8 and 1.71 10 6 U mL 1 for caspase-9, and it can be used for the simultaneous screening of caspase inhibitors and measurement of endogenous caspase activity in various cell lines at the single-cell level. Moreover, this nanosensor can be extended to detect various proteases by simply changing the peptide sequences of the detection probes. ## Chemicals and materials All oligonucleotides (Table 1) ## Preparation of the detection probe-conjugated MBs The assembly of detection probes onto the MBs was carried out according to the protocol of the manufacturer. 50 mL of the 10 mg mL 1 streptavidin-coated MBs solution was washed three times using 1 PBS. After resuspending with 50 mL of 1 PBS, 10 mL of 10 mM detection probes were added to form the detection probes-MB nanostructure through biotin-streptavidin interaction at room temperature for 30 min. The detection probe-MBs were then washed fve times using 1 PBS to remove the uncoupled probes by magnetic separation, followed by resuspending in 50 mL of 1 PBS. Construction of signal probes@AuNPs AuNPs (10 nm) were functionalized with thiolated signal probes using the freeze-directed methods. 76 The method is reagentless without the involvement of extra salts, acids, and surfactants. Specifcally, 20 mL of 100 mM signal probes were mixed with 1 mL of AuNPs. The mixture was subsequently placed in a laboratory freezer at 20 C for 2 h, followed by thawing at room temperature. At last, the signal probe-coated AuNPs were centrifuged and washed three times with ultrapure water to remove excess signal probes, and resuspended in 40 mL of sterile water, and stored at 4 C. In the signal probes@AuNPs solution, the concentration of DNA was measured using a NanoDrop 2000c Spectrophotometer (Thermo Scientifc, Wilmington, Delaware, USA) and the number of signal probes per AuNP is estimated to be 120 AE 1 for both Cy5-labeled signal probe 1 and Texas Red-labeled signal probe 2 (Fig. S2, ESI †). ## Detection of caspase activities The caspase activity assay includes two steps: (1) caspasemediated cleavage of detection probes, and (2) recycling liberation of fluorophores induced by the Exo III-powered 3D DNA walker. The caspase-mediated cleavage of the detection probe was performed in 20 mL of solution containing 3.5 mL of 1 PBS, 1 mM DTT, 8 mL of detection probe 1-conjugated MBs or/and detection probe 2-conjugated MBs, and 0.5 mL of caspase at different concentrations at 37 C for 1 h. After magnetic separation, the supernatant with cleavage products was collected for the next-step use. The Exo III-powered 3D DNA walker-based recycling liberation of fluorophores was performed in 20 mL of reaction solution containing 1 CutSmart buffer (50 mM potassium acetate, 20 mM tris-acetate, 10 mM magnesium acetate, 100 mg mL 1 BSA, pH 7.9), 0.71 mL of signal probe 1@AuNPs or/and 0.36 mL of signal probe 2@AuNPs, and 0.5 U of Exo III at 37 C for 1 h. ## Ensemble uorescence measurement The 20 mL of reaction products was diluted to a fnal volume of 50 mL with ultrapure water for the measurement of fluorescence emission spectra using a 1 cm path length quartz cuvette on a Hitachi F-7000 fluorescence spectrophotometer (Tokyo, Japan). The excitation wavelength was 645 nm for Cy5 and 560 nm for Texas Red, and the emission spectra were recorded in the wavelength range of 660-750 nm for Cy5 and 600-680 nm for Texas Red with a slit width of 5 nm for both excitation and emission. ## Single-molecule detection and data analysis The reaction products of caspase-8 were diluted 200-fold in the imaging buffer (1 mg mL 1 glucose oxidase, 0.4% (w/v) Dglucose, 0.04% mg mL 1 catalase, 50 mg mL 1 BSA, 67 mM glycine-potassium hydroxide, 1 mg mL 1 Trolox, 2.5 mM magnesium chloride, pH 9.4), and the reaction products of caspase-9 were diluted 500-fold in the imaging buffer. For total internal reflection fluorescence (TIRF) imaging, 10 mL of the sample was directly pipetted onto the coverslips. The Cy5 and Texas Red fluorescent molecules were excited by the sapphire 640 and 561 nm lasers (Coherent, USA), respectively. The resulting photons were collected by an oil immersion objective (CFI Apochromat TIRF 100). The Cy5 and Texas Red fluorescence signals were imaged on an Andor ixon Ultra 897 EMCCD camera (Andor, Belfast, UK) with an exposure time of 500 ms. For data analysis, the ImageJ software was used for counting the Cy5 and Texas Red fluorescent molecules from an imaging region of 600 600 pixels. ## Gel electrophoresis The reaction products were analyzed by 12% nondenaturing polyacrylamide gel electrophoresis (PAGE) in 1 TBE buffer (9 mM Tris-HCl, 9 mM boric acid, 0.2 mM ethylenediaminetetraacetic acid, EDTA, pH 7.9) at a 110 V constant voltage at room temperature for 50 min. The gels were stained by SYBR gold and analyzed by a Bio-Rad ChemiDoc MP Imaging System (Hercules, CA, USA). ## Kinetic analysis To evaluate the enzyme kinetic parameters of caspases, we measured the initial velocity in the presence of 0.025 U mL 1 caspase (caspase-8 or caspase-9) and different concentrations of the detection probes at 37 C for 5 min. The kinetic parameter is ftted to the Michaelis-Menten equation. where V max is the maximum initial velocity, [S] is the concentration of the detection probe, and K m is the Michaelis-Menten constant. ## Inhibition assay To evaluate the effect of inhibitor upon the caspase activity, different concentrations of the inhibitor (Z-IETD-FMK for caspase-8, Ac-LEHD-CMK for caspase-9, and broad-spectrum caspase inhibitor Q-VD-OPh for both caspase-8 and caspase-9) were preincubated with 0.025 U mL 1 caspase (caspase-8 or caspase-9) at room temperature for 15 min, respectively. Then 8 mL of detection probe 1-conjugated MBs or detection probe 2conjugated MBs were added into the mixture, and the reaction volume was adjusted to 20 mL with 1 PBS, followed by the same detection procedure as described above. The relative activity (RA) of caspase was measured according to eqn (3). where N 0 represents the Cy5 counts in the absence of caspase-8 or the Texas Red counts in the absence of caspase-9; N t represents the Cy5 counts in the presence of caspase-8 (0.025 U mL 1 ) or the Texas Red counts in the presence of caspase-9 (0.025 U mL 1 ); and N i represents the Cy5 counts in the presence of caspase-8 (0.025 U mL 1 ) + inhibitor or the Texas Red counts in the presence of caspase-9 (0.025 U mL 1 ) + inhibitor. The IC 50 value was calculated from the curve of RA versus the inhibitor concentration. ## Cell culture and preparation of cell extracts HeLa cells and MCF-7 cells were cultured in Dulbecco's Modifed Eagle's Medium (DMEM, Life Technologies, USA) with 10% FBS (Life Technologies, USA) and 1% penicillin-streptomycin (Gibco, USA). Jurkat cell was cultured in 1640 cell medium (Life Technologies, USA) with 10% FBS (Life Technologies, USA) and 1% penicillin-streptomycin (Gibco, USA). The cells were cultured at 37 C in a humidifed atmosphere containing 5% CO 2 . For real sample analysis, cells in the exponential phase of growth were collected and counted using Countstar BioTech Automated Cell Counter IC1000 (Shanghai, China), washed twice with ice-cold 1 PBS, and centrifuged at 800 rpm for 5 min. To isolate cytoplasmic components from nuclear ones, the cells were treated with a nuclear protein extraction kit (Beyotime Biotechnology, Wuhan, China) and centrifuged at 3400 rpm for 15 min at 4 C. For STSinduced apoptosis analysis, cells were incubated in 5 mL of cell medium containing 0.4 mM STS for 4 h prior to the cell lysis procedure. The protein concentration was measured using a NanoDrop 2000c Spectrophotometer (Thermo Scientifc, Wilmington, Delaware, USA).

chemsum

{"title": "Integration of exonuclease III-powered three-dimensional DNA walker with single-molecule detection for multiple initiator caspases assay", "journal": "Royal Society of Chemistry (RSC)"}

truly_random_degradable_vinyl_copolymers_via_photocontrolled_radical_ring-opening_cascade_copolymeri

## Abstract: Degradable vinyl polymers by radical ring-opening polymerization have become a promising solution to the challenges caused by the widespread use of non-degradable vinyl plastics. However, achieving even distribution of labile functional groups in the backbone of degradable vinyl polymers remains challenging. Herein, we report a photocatalytic approach to truly random degradable vinyl copolymers with tunable main-chain composition via radical ringopening cascade copolymerization (rROCCP). The rROCCP of the macrocyclic allylic sulfone and acrylates or acrylamides mediated by visible light at ambient temperature achieved near-unity reactivity ratios of both comonomers over the entire range of the comonomer compositions and afforded truly random vinyl copolymers with degradable units evenly distributed in the polymer backbone. Experimental and computational evidence revealed an unusual reversible inhibition of chain propagation by in situ generated sulfur dioxide, which was successfully overcome by reducing the solubility of sulfur dioxide in the reaction mixture. This study provided a powerful approach to truly random degradable vinyl copolymers with tunable main-chain labile functionalities and comparable thermal and mechanical properties to traditional non-degradable vinyl polymers. ## Introduction Vinyl polymers have been widely used in an array of applications including packaging, structural materials, synthetic fibers, coating, absorbent, and many others. While the all-carbon backbone makes vinyl polymers highly robust materials, it has also created significant challenges in their degradation, leading to critical environmental issues caused by plastic accumulation in landfills and the ocean. Therefore, significant efforts have been made in recent years to develop innovative synthetic polymers that possess thermal and mechanical properties comparable to the original, nondegradable vinyl polymers and can undergo facile degradation at the end of their life cycle. Among various approaches to degradable vinyl polymers, radical ring-opening polymerization (rROP) is of great interest. Attractive features of rROP include its abilities to incorporate labile functional groups (e.g. esters, thioesters, disulfide, etc.) into the polymer main chain and interface with a plethora of reversible deactivation radical polymerization (RDRP) techniques for the synthesis of polymers with complex and defined macromolecular architectures. 8 Since the advent of rROP, various cyclic monomers have been successfully developed for the synthesis of degradable vinyl (co)polymers. 6 As a representative class of rROP monomer, cyclic ketene acetals (CKAs) have been extensively investigated since 1980s. 9 Despite recent progress made by Dove, Nicolas, and Sumerlin, unfavorable reactivity ratios in the copolymerization of CKA with other vinyl monomers often lead to gradient or tapered compositions of the resultant copolymer. 23 The gradient composition in turn resulted in highly dispersed degradation products and large non-degradable fragments, as part of the copolymer lacked main-chain degradable units. Although new cyclic monomer classes including macrocyclic allylic sulfide (MAS) and dibenzo[c,e]oxepane-5-thione (DOT) (Figure 1A) have demonstrated promising properties, truly random copolymerization of these cyclic monomers with acrylates or acrylamides remains challenging. In 2018, we reported an approach to the radical ring-opening cascade polymerization of allylic sulfone macrocyclic monomers. 31 The radical cascade reaction of macrocyclic allylic sulfone could extrude sulfur dioxide (SO2) and generate a secondary alkyl radical capable of controlled chain propagation. 32 However, copolymerization of the macrocyclic allylic sulfone and acrylates exhibited unfavorable reactivity ratios at high temperatures. Therefore, it is essential to develop a method that provides access to truly random copolymers with tunable compositions and evenly distributed main-chain functional groups. Recent studies suggest that temperature has a strong influence on the reactivity ratios in the radical copolymerization of cyclic and acyclic vinyl comonomers. 6 We reasoned that performing the copolymerization at lower temperatures would provide a key opportunity to modulate the reactivity ratios of vinyl comonomers. Therefore, we turned our attention to light-mediated polymerization techniques, as recent works have demonstrated that they are versatile tools to mediate controlled polymerization following radical, cationic, and metathesis pathways at ambient temperature (Figure 1B). 50 In particular, we envisioned that the photoinduced electron/energy transfer-reversible addition/fragmentation chain transfer (PET-RAFT) polymerization developed by Boyer and coworkers could be employed to mediate radical ring-opening cascade copolymerization (rROCCP) of the macrocyclic allylic sulfone and acrylates or acrylamides (Figure 1C). Unlike the polymerization initiated by azobisisobutyronitrile (AIBN) that required high temperatures (80-100 °C) to maintain a sufficiently high rate of propagation, PET-RAFT can be performed at mild temperatures, thereby enabling favorable comonomer reactivity ratios in copolymerization. To the best of our knowledge, the photocontrolled rROCCP represents the first method that achieved truly random radical copolymerization of cyclic monomers and acrylic monomers over the entire range of comonomer compositions. ## PET-RAFT Polymerization of Macrocyclic Allylic Sulfones Our investigation began by screening various wellestablished photocatalysts to mediate the photocontrolled homopolymerization of allylic sulfone macrocyclic monomer 1 under visible light irradiation (Table S1). We screened an array of photocatalysts, including fac-[Ir(ppy)3], Ru(bpy)3Cl2, ZnTPP, and Eosin Y, and identified fac-[Ir(ppy)3] as a promising photocatalyst for the reaction due to the excellent control over the polymerization when combined with CTA1. At a monomer/CTA ratio of 50:1, our initial attempt of the polymerization of macrocyclic allylic sulfone 1 mediated by fac-[Ir(ppy)3] and CTA1 under 450 nm light irradiation yielded P-1 with Mn (SEC) of 9.8 kg/mol and Ð of 1.11 (Table S2). Further examination of the reaction conditions found that optimal polymerization was achieved when the monomer concentration was at 0.2 M in DMF and the catalyst loading reached 200 ppm (Table S3-S5). Polymerization of 1 at other monomer/CTA ratios of 25:1, 100:1, and 200:1 successfully yielded polymers with predictable Mn and low Ð, demonstrating excellent control over the polymerization (Table S6). Similarly, macrocyclic allylic sulfone 2 with a smaller ring size was also polymerized with good control under the same conditions (Table S7). It is noteworthy that no ring-retaining propagation of both allylic sulfone macrocyclic monomers 1 and 2 has been observed. Following the exploration of reaction conditions, we examined the living characteristics of the polymerization. First, the kinetic analysis revealed that the polymerization of 1 deviated from first-order kinetics in the late stage (Figure S1). This observation was consistent with our previous results when the cascade polymerization of macrocyclic allylic sulfone was thermally initiated. 31,59 Despite the kinetic anomaly, the polymerization of 1 still exhibited a linear increase of Mn with respect to the monomer conversion and remained low Ð throughout the reaction, suggesting that control over the polymerization was well maintained even after the rate decreased in the late stage (Figure 2A). 1 H-NMR analysis of P-1-6k (M n (SEC) = 6.4 kg/mol, Ð = 1.07) confirmed the fidelity of the chain end groups (2.46 and 1.21 ppm for a-chain end and 4.81 and 3.36 ppm for w-chain end, Figure S2), an important indicator of controlled polymerization. Besides, the discrete oligomers of P-1-5k (M n (SEC) = 5.5 kg/mol, Ð = 1.10) observed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry showed masses consistent with the predicted values of these oligomers with intact chain ends (Figure 2B). Furthermore, chain extension of the macroinitiator P-2-4k (M n (SEC) = 3.9 kg/mol, Ð = 1.16) by 1 exhibited a clear shift to the higher molecular weight region on the SEC chromatogram, suggesting the formation of a diblock copolymer P-2-b-P-1 (M n (SEC) = 13.0 kg/mol, Ð = 1.20, Figure 2C). Finally, the reaction exhibited excellent temporal control: chain propagation completely halted when the light was switched "off"; polymerization resumed efficiently after the light was switched back "on" (Figure 2D). Taken together, these results unambiguously supported that the PET-RAFT polymerization of macrocyclic allylic sulfones maintained an excellent control throughout the reaction despite the deviation from first-order kinetics at the late stage. ## Copolymerization of Macrocyclic Allylic Sulfones and Acrylates or Acrylamides Building upon the results of photocontrolled homopolymerization of macrocyclic allylic sulfones, we then investigated copolymerization of 1 and various acrylates or acrylamides (denoted hereafter as comonomer B). First, 1 was copolymerized with methyl acrylate (MA) at the feed composition of 𝑓 𝟏 " = 0.05, where 𝑓 𝟏 " is the molar fraction of 1 in the initial comonomer mixture, yielding copolymer P-1-co-MA with Mn (SEC) of 44.0 kg/mol and Ð of 1.28 (Table 1, entry 1). The propagation of both comonomers demonstrated first-order kinetics throughout the copolymerization (Figure 3A). The molecular weight also increased linearly with respect to the overall monomer conversion, which is defined by Eq 1: where [1(t)] and [B(t)] are the respective instantaneous concentrations of 1 and comonomer B at time t, and [1(0)] and [B(0)] are the respective initial concentrations of 1 and comonomer B (Figure 3B). Importantly, the instantaneous molar fraction of 1 incorporated in the copolymer (denoted hereafter as 𝐹 𝟏 ) remained identical to 𝑓 𝟏 " throughout the copolymerization (Figure S3). Correspondingly, the final copolymer composition, 𝐹 𝟏 (*+,) , when the reaction reached the end point, was also identical to 𝑓 𝟏 " (Table 1, entry 1). These results suggested that the reactivities of the two comonomers are highly similar in chain propagation. To determine the reactivity ratios of the copolymerization, the compositional data of 1 and B throughout the copolymerization was fitted to the Beckingham−Sanoja−Lynd (BSL) integrated model reported by Lynd et al. 60 𝑐𝑜𝑛𝑣. = 1 − 𝑓 𝟏 " , 𝑐𝑜𝑛𝑣. = 1 − 𝑓 𝟏 " , where 𝑟 𝟏 and 𝑟 ) are reactivity ratios of 1 and comonomer B. It is noteworthy that although the BSL model is derived for ideal copolymerization where 𝑟 . × 𝑟 ) = 1, such as ionic or metalcatalyzed copolymerization systems, we reasoned that the copolymerization of the macrocyclic allylic sulfone and acrylic monomers is a close approximation of the ideal copolymerization, because the allylic sulfone motif was designed such that the propagating secondary alkyl radical formed after the radical cascade process is structurally similar to the propagating radical of polyacrylates. 61 Independent fitting of the polymer compositional data to Eq 2 and Eq 3 supported this rationale, as the derived reactivity ratios of the comonomers were 𝑟 𝟏 = 1.07 and 𝑟 ) = 0.94, with 𝑟 𝟏 ´ 𝑟 ) = 1.006 (Figure 3C). These results suggest that the copolymerization is truly random and that it is indeed highly analogous to an ideal copolymerization in which the product of the two reactivity ratios equals 1. The reactivity ratios of 1 and MA in the entire range of monomer feed compositions (𝑓 𝟏 " = 0-1) remained close to unity ( To investigate how degradability was influenced by the composition and distribution of degradable building blocks in copolymers, copolymers were treated with sodium methoxide to cleave the main-chain esters. SEC analysis of the degradation of the copolymer P-(1-co-MA) prepared by the photocontrolled rROCCP ( 𝐹 𝟏 (*+,) = 0.10, Mn (SEC) = 19.2 kg/mol, and Ð =1.27) exhibited a dramatic molecular weight reduction after degradation, resulting in oligomers with Mn (SEC) of 1.3 kg/mol and Ð of 1.33 (Table 2, entry 2 & Figure 4A). In contrast, degradation of the copolymer with a similar overall composition ( 𝐹 𝟏 (*+,) = 0.08, Mn (SEC) = 16.4 kg/mol, and Ð =1.52) generated by the thermallyinitiated copolymerization produced frag ments with higher Mn and Ð (Mn (SEC) = 7.3 kg/mol, Ð = 1.98) (Figure 4A). Furthermore, the degradation of copolymers with different comonomer compositions generated by the photocontrolled rROCCP consistently produced fragments with low Mn and narrow molecular weight distributions (Table 2). These results indicated that while the thermally initiated copolymerization yielded a gradient copolymer that could only be partially degraded, copolymers generated by the photocontrolled rROCCP possessed even and tunable distributions of main-chain degradable functionalities and could be degraded efficiently into low molecular weight fragments. The thermal properties of copolymers were further evaluated by thermogravimetry (TGA) and differential scanning calorimetry (DSC) analyses. P-(1-co-MA) with main-chain degradable functionalities at different copolymer compositions established similar thermal stability comparable to polymethylacrylate with 5% weight loss decomposition temperature (Td) between 363-368 °C (Figure 4B). Furthermore, glass transition temperature (Tg) of P-(1-co-MA) can be fine-tuned by the initial comonomer feed composition in copolymerization, highlighting the potential utility of this method in generating degradable vinyl polymers with tailormade material properties (Figure 4C). ## Understanding the Unusual Kinetic Behavior Our studies have shown that while the PET-RAFT homopolymerization and copolymerization (Figure S3 & S20-26) involving macrocyclic allylic sulfones deviated from the first-order kinetics, the polymerization remained well-controlled. This phenomenon was in stark contrast to traditional controlled polymerization in which deviation of first-order kinetics is usually a sign of loss of control, suggesting an unusual kinetic behavior that warranted further investigation. We suspected that the in situ generated SO2 in the radical cascade polymerization affected the reaction kinetics. To investigate this hypothesis, Density Functional Theory (DFT) calculations were carried out using the M06-2X/6-311++G(d,p)//B3LYP/6-31G(d) method in conjunction with the Solvation Model based on Density (SMD) simulating the effect from DMF to compute a plausible potential energy surface of the cascade process in the polymerization of macrocyclic allylic sulfones (Figure 5B). 65 Our calculation showed that the ascission/SO2 extrusion step (G2 to G3) has a low energy barrier of 5.9 kcal/mol, and that this transformation is exergonic by 2.8 kcal/mol. The low activation energy and relatively small change in Gibbs free energy indicates that this step is likely reversible. The DFT calculations also suggest that G3, with the lowest energy in the whole cascade process, exists at a high enough concentration during steady-state conditions, making it a plausible intermediate for chain propagation (Figure S27). Compared to chain propagation (G3-TS4-G4, with an energy barrier of 20.7 kcal/mol), two alternative reaction pathways of G3 with lower energy barriers are the reversible addition by the CTA (G3-TS5-G5, with an energy barrier of 12.0 kcal/mol) or SO2 (G3-TS3-G2, with an energy barrier of 8.7 kcal/mol). While the former serves as the reversible deactivation of the chain propagation to achieve controlled polymerization, the latter is a reverse reaction of the ascission/SO2 extrusion step and regenerates the sulfonyl radical G2. Because of a high energy barrier of 19.7 kcal/mol and being endergonic by 9.8 kcal/mol, chain propagation of G2 by the monomer (G2-TS6-G6) is prohibited thermodynamically and kinetically. These results indicate that excess SO2 in the reaction could indeed recombine with the propagating alkyl radical to regenerate the sulfonyl radical and inhibit chain propagation. To provide further evidence of the presence and accumulation of sulfonyl radical over the course of reaction, we employed electron paramagnetic resonance (EPR) to monitor the evolution of radical species in the reaction in situ (Figure 5C). In the early stage (initial two hours) of the reaction, the EPR spectrum only consisted of signals corresponding to the alkyl radical a (g0 = 2.004) and the degenerative intermediate b (g0 = 2.009) (Spectrum I, Figure 5C). The g-values of the peaks and patterns of the spectrum are consistent with the radical species generated in the radical polymerization of MA. The EPR spectrum gradually evolved as the polymerization proceeded. In the late stage (after five hours) of the reaction, a new peak c with a gvalue of 2.014 appeared in the EPR spectrum (Spectrum II, Figure 5C), which is consistent with the g-value of the sulfonyl radical reported in literature. 66 Furthermore, simulated EPR spectra (dotted lines) based on the absence and presence of the sulfonyl radical in the reaction perfectly fit the experimental data as shown in Spectrum I and II, respectively, confirming the proposed assignments. Notably, Spectrum II is also consistent with Spectrum III obtained after the exogenous SO2 gas was introduced to the system at the early stage of the reaction (Figure 5C). Collectively, the DFT calculations and EPR analyses are consistent with the observed kinetic results, confirming that G2 (peak c in Figure 5C), G3 (peak a in Figure 5C), and G5 (peak b in Figure 5C) are long-lived radical intermediates in the polymerization of macrocyclic allylic sulfones, and that the concentration of SO2 could have a significant effect on the direction of the reaction. Overcoming the Propagation Inhibition by SO2. Based on DFT calculations, we reason that the propagation inhibition by the in situ generated SO2 may be reversible, given the low energy barrier of the process. This reversibility implies that the extrusion of SO2 and the formation of the alkyl radical are favored at low SO2 concentrations, whereas the recombination of SO2 and the formation of the sulfonyl radical are favored at high SO2 concentrations. Therefore, the propagation inhibition could be alleviated by removing SO2 from the reaction. Indeed, we found that sparging the reaction mixture with argon steadily increased the rate of the PET-RAFT homopolymerization of 1 in the late stage of the reaction at 25 °C (Figure S28). In fact, both the SO2 inhibition and reactivation of chain propagation by argon sparging were reversible and the polymerization could be switched "on"/"off" by alternating the exogenous SO2 and argon introduced into the reaction vessel (Figure S29-S30). Similarly, the propagation inhibition was also alleviated in the copolymerization of 1 and BnA (𝑓 𝟏 " = 0.09) by argon sparging at 25 °C (Figure S31). Additionally, increasing the reaction temperature to 50 °C was also found to improve the rate of the homopolymerization of 1 in the late stage (Figure S32-S33). Combining the argon sparging and the temperature elevation to 50 °C proved to further improve the reaction kinetics of the homopolymerization of 1, allowing it to remain pseudo first-order throughout the reaction (Figure 6A). The rate of copolymerization of 1 and MA was also improved when the reaction temperature was elevated to 50 °C (Figure S34), but a modest deviation of the comonomer reactivity ratios from unity was observed (Figure S35). We reasoned that an alternative strategy to reduce the propagation inhibition by SO2 was to switch the solvent from DMF to dioxane, in which SO2 has lower solubility (Figure S36). Encouragingly, we found that the kinetics of copolymerization of 1 and BnA at 25 °C remained pseudo first-order throughout the reaction when dioxane was used as the solvent (Figure 6B). ## Conclusion A novel approach to the truly random degradable vinyl polymers with tunable main-chain composition via photocontrolled radical ring-opening cascade copolymerization (rROCCP) is presented in this article. Compared to existing rROP systems, the photocontrolled rROCCP enabled the synthesis of truly random degradable vinyl copolymers with evenly distributed, tunable composition of the main-chain labile groups at ambient temperature. Computational and EPR analyses revealed that the reversible inhibition of the chain propagation by in situ generated SO2 caused an unusual kinetic behavior that showed a deviation from first-order kinetics in the late stage of the reaction. Removal of SO2 was found to reverse the inhibition of the chain propagation and improve the reaction kinetics in both the homopolymerization and the copolymerization involving macrocyclic allylic sulfones. Taken together, excellent control and favorable comonomer reactivity ratios make photocontrolled rROCCP a powerful strategy for the preparation of truly random degradable vinyl copolymers with tunable main-chain compositions for a wide range of applications. In addition, the mechanistic insights into the reversible inhibition of chain propagation by SO2 shed light on using chemical cues to control radical chain-growth cascade polymerization systems.

chemsum

{"title": "Truly Random Degradable Vinyl Copolymers via Photocontrolled Radical Ring-Opening Cascade Copolymerization", "journal": "ChemRxiv"}

cost-effective_sol-gel_synthesis_of_porous_cuo_nanoparticle_aggregates_with_tunable_specific_surface

## Abstract: CuO nanoparticles (NPs) are applied in various key technologies, such as catalysis, energy conversion, printable electronics and nanojoining. In this study, an economic, green and easy-scalable sol-gel synthesis method was adopted to produce submicron-sized nanoporous CuO NP aggregates with a specific surface area > 18 m²/g. To this end, a copper-carbonate containing precursor was precipitated from a mixed solution of copper acetate and ammonia carbonate and subsequently calcinated at T ≥ 250 °C. The thus obtained CuO nanopowder is composed of weakly-bounded agglomerates, which are constituted of aggregated CuO NPs with a tunable size in the range of 100-140 nm. The CuO aggregates, in turn, are composed of equi-axed primary crystallites with a tunable crystallite size in the range of 20-40 nm. The size and shape of the primary CuO crystallites, as well as the nanoporosity of their fused CuO aggregates, can be tuned by controlled variation of the degree of supersaturation of the solution via the pH and the carbonate concentration. The synthesized submicron-sized CuO aggregates can be more easily and safely processed in the form of a solution, dispersion or paste than individual NPs, while still offering the same enhanced reactivity due to their nanoporous architecture.Advanced nanotechnologies in the fields of catalysis, energy conversion, storage and sensing devices rely on the accurate control of the shape and the size of materials from the nano-up to the micrometer scale. This requirement has boosted the development of a wide range of synthesis techniques for producing metallic, insulating and semiconducting nanoparticles (NP) of controllable sizes and shapes (cf. reviews 1 and 2 ). Reported studies on engineered and environmental NP-based systems generally focus on the size and shape of the smallest undividable entity (or building block), commonly referred to as the primary particle (or crystallite). However, successive manufacturing and processing steps (or environmental exposure) generally induce aggregation and/or agglomeration of primary NPs into larger entities with sizes of up to several microns. NP aggregates (or secondary particles) consist of strongly bonded or fused primary NPs, which cannot be separated by subsequent handling and processing steps (i.e. the aggregation process is irreversible). NP agglomerates comprise assemblies of more weakly bound primary NPs and NP aggregates (and/or a mixture thereof), which can be separated into their individual constituents by providing sufficient external energy and stabilized through the addition of suitable dispersion agents 3 . For practical applications, the actual properties of the nanomaterial (e.g. specific surface area, chemical reactivity, dispersibility and toxicity) will be governed by the size, shape and density (i.e. nanoporosity) of the NP aggregates and/or agglomerates and not solely by the primary particles 4,5 . For example, the extent to which the internal surface of a loosely clustered (and thus nanoporous) NP aggregate is accessible to interpenetrating gaseous or liquid species will critically influence its dispersibility, chemical reactivity and sintering behavior, which are of key importance for the development of e.g. catalysis, printed electronics and joining technologies 6,7 . Notably, sub-micron sized nanoporous NP aggregates also have the advantage that they can be more easily (i.e. ; in addition of OH − ) and the pH 24,25 . Inorganic or organic additives, such as urea, polyethylene glycol or polyvinylpyrrolidone can be added to steer the precipitation and aggregation of the precursor phase, thus enabling the formation of specific hierarchical CuO nanostructures 22,26 . For example, Cu(NO 3 )-salt solutions in the presence of urea were aged at 90 °C to yield spherically-shaped malachite (CuCO 3 •Cu(OH) 2 ) precursor particles, which after calcination at 700-800 °C transform into micro-sized spherical CuO particles 27 . Reproducible results can only be obtained if key factors such as the pH, type and concentrations of the reactants are well controlled 22 . In the present study, a simple, green and low-cost sol-gel synthesis method based on a copper-carbonate species containing precursor phase, using copper acetate and ammonia carbonate salts without the addition of any additives, was selected and optimized to produce loosely clustered (and thus nanoporous) CuO NP aggregates with an average size in the range of 100-140 nm and a resulting SSA that is significantly higher than typical SSA values in the range of 10-15 m²/g, as reported for commercially available high-purity (i.e. ≥99.99%) CuO nanopowders. Sol-Gel processes are usually used to synthesize nonmetallic inorganic materials from particle dispersions. They are easily upscalable and can be conducted with cheap and non-toxic chemicals 28 . Importantly, the synthesis was performed without organic additives (which contaminate the product phase and complicates the complex forming process of precursor phases). The evolution of the crystal structure, size and shape of the primary crystallites, which develop from the precursor phase during the calcination treatment, were monitored by measuring the peak broadening through high temperature in-situ XRD. The size, shape and cluster density of the resulting CuO aggregates, as constituted of individual clusters of firmly bonded primary CuO crystallites, was investigated by complementary analytical techniques, including Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), Dynamic Light Scattering (DLS) and Brunauer-Emmett-Teller (BET) analysis. Key parameters in the sol-gel synthesis procedure were identified (e.g. the supersaturation level, carbonate concentration and the resulting pH of the mixed solution) and tuned to obtain an enhanced SSA of the synthesized CuO nanopowder for the targeted applications. ## Experimental Material synthesis. Synthetic malachite can be prepared by reacting a solution of copper(II) nitrate with a solution containing the equivalent amount of sodium carbonate at room temperature; i.e. the copper-to-carbonate concentration ratio is fixed at [CO 3 2− ]/[Cu 2+ ] = 1.0 29 . Accordingly, solutions were prepared by mixing stoichiometric amounts of fresh aqueous 15 mM copper acetate (Sigma Aldrich) and 15 mM ammonia carbonate (Alfa Aesar) solutions at room temperature, while continuously stirring the mixed solution 30 . This resulted in a pH of 5.8 for the mixed solution, which remained stable during a stirring duration of 2 h, as monitored using a pH-meter (Metrohm 914 pH/Conductometer). Upon combining the starting solutions at room temperature (further referred to as mixed solution), precipitation of particles (the gel) from the blue-greenish solution was immediately observed. In an acidic environment, copper(II) ions would remain in solution without forming precipitates over the described time period. For more neutral environments of a pH above 5.6 (as is the case for the present study; see above), complexation of Cu 2+ leads to the formation of precipitates. Due to the supersaturation of the solution with respect to the formation of malachite, successive precipitation of the copper(II) complexes and gel formation occurs, and the composition of this precursor phase moves towards the composition of malachite. The chemical reaction to malachite can be written as www.nature.com/scientificreports www.nature.com/scientificreports/ When the pH exceeds 8.5, the copper-carbonate-hydroxide precursor phase complexes would gradually dissolve over time by ammonia leaching (resp. they would not even be formed), eventually leading to the formation of primarily copper(II) tetra ammine complexes 31 , i.e. The stability window for the formation of malachite in the mixed solution is therefore between pH 5.6 and 8.5. To evaluate the effect of the pH of the mixed solution on the size and shape of the copper-containing precursor phase (and thus on the final CuO end product), additional synthesis routes were performed at a fixed pH value of either 5.6 or 7.0 by adjusting the added volume of the ammonia carbonate solution, resulting in [CO 3 2− ]/[Cu 2+ ] ratios of 0.4 and 2.5, respectively. Depending on the regulated pH, either an ammonium carbonate deficiency (i.e. [CO 3 ## 2− ]/[Cu 2+ ] < 1 for pH < 5.83) or surplus [CO 3 ]/[Cu 2+ ] > 1 for pH > 5.83) is established, which affects the supersaturation level of the mixed solution with respect to the nucleation of the targeted malachite precursor phase. Unless stated otherwise, the mixed solution was continuously stirred over the defined duration time of t stirring = 2 h. Afterwards, the gel precipitate was collected by centrifugation, washed with ethanol and distilled water. The freshly washed precipitate was then dried in a muffle furnace in air at a temperature of 60 °C for a fixed duration of t drying = 6 h. The resulting (largely) dehydrated precursor phase was decomposed into CuO by annealing in a muffle furnace in air at 400 °C for a fixed duration of t calcination = 4 h (further referred to as calcination), i.e. A schematic of the above-described synthesis procedure is depicted in Fig. 1. ## Material characterization. The crystallinity of the gel precursor, as well as the phase purity and average primary crystallite size of the synthesized CuO nanopowder, were investigated by X-ray diffraction (XRD) using a PANanalytical X'Pert Pro Multi-Purpose (MPD) X-ray diffractometer. The XRD data were collected in the 2Θ range of 10-100° 2Θ with a step size of 0.026° using Cu-Kα 1-2 radiation (λ average = 0.15418 nm, 45 kV and 40 mA). A time-resolved XRD study of the precursor-to-CuO transformation during calcination was conducted using a PANalytical X'Pert PRO MPD X-ray diffractometer (same measurement parameters) with a gas-tight Anton Paar XRK-900 heating chamber equipped for heating and gas feeding (5850 TR, Brooks instrument) in static air. Thermo-gravimetric analysis (TGA) combined with differential scanning calorimetry (DSC) was conducted using a Netzsch STA449 F3 Jupiter with a low-temperature silver oven. The sample (mass: ~31.6 mg) was handled in an Al sample pan (85 μl) and heated from room temperature to 400 °C at a rate of 5 K/min under flow of synthetic air (60 ml/min). The data were corrected by an empty pan scan measured under identical conditions. The morphology and elemental composition of the calcinated nanopowder was investigated using a Hitachi S-3700N scanning electron microscope (SEM), equipped with an energy-dispersive X-ray detector (EDX) (Ametek EDAX, www.nature.com/scientificreports www.nature.com/scientificreports/ Octane Pro). In addition, high-resolution SEM investigations were conducted using a FEI Nova NanoSEM 230. For the analysis by transmission electron microscopy (TEM; JEOL JEM-2200FS operated at accelerating voltage of 200 kV), the synthesized particles were dispersed in ethanol and transferred onto a gold grid (300 mesh, TED PELLA, INC). ATR-FTIR analysis of the different precursor samples were performed with a Cary 640 FTIR spectrometer (Agilent). The diamond ATR accessory with a type IIa synthetic diamond crystal has a penetration depth of ~2 µm. The spectra were recorded in a frequency range of 4000-600 cm −1 with a spectral resolution of 2 cm −1 . A total of 128 scans were co-added for every spectrum. The background was measured with the same settings against air. The spectrometer was controlled by Agilent Resolutions Pro software 5.2.0. DLS experiments were conducted to determine the size distribution of the CuO NP aggregates. To this end, the synthesized nanopowder was dispersed in a water-based solution with 0.5% sodium dodecylsulfat (SDS) as a detergent and ultra-sonicated (UP200ST with VialTweeter, hielscher) to disrupt the weakly linked agglomerates. The DLS measurements were performed with a Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, UK) equipped with a max 4 mW He-Ne laser (emitting at 633 nm). Each measurement was performed at the non-invasive backscatter angle (NIBS) of 173° at a temperature of 25 °C and was preceded by a 30 s equilibration time. The ultrasonic treatment procedure was optimized to yield a stable minimal particle size after successive DLS measurements of the dispersion (thus ensuring near-complete disruption of the agglomerates). The specific SSA of the synthesized CuO nanopowders after calcination, as well as after ultrasonic treatment and subsequent drying in air, was determined from a 5-point N 2 adsorption BET isotherm, as measured with a Beckman-Coulter SA3100 instrument (Switzerland). Before the BET analysis, the powder samples were dried for 2 h at 180 °C in synthetic air. ## Results and Discussion Transformation of the precursor phase into CuO by calcination. The nature of the freshly-washed blueish-greenish precipitate collected for [CO 3 ## 2− ]/[Cu 2+ ] = 1.0 at pH = 5.83 (step 5 → 6 in Fig. 1) to CuO was first investigated by XRD (Fig. 2). The measurement indicates that the precipitated gel is XRD-amorphous, see Fig. 2a. Only a very broad intensity hump characteristic of an amorphous phase was detected in the 2-theta region of the expected principal (10-2) reflection of crystalline malachite, but no indications of crystalline CuO were found. To confirm the presence of a malachite-like precursor phase, the obtained blue-greenish precipitate was slowly dried to completion over 48 h at room temperature (RT) in air (note: for the standard synthesis route, the precipitate is dried at an elevated temperature for much shorter times, as described above). The XRD pattern after drying indeed matched the crystalline structure of malachite, which crystallizes in a monoclinic space group P21/a1 with lattice parameter a = 9.502 , b = 11.974 , c = 3.24 , alpha = 90.00°, beta = 98.75° and gamma = 90.00°3 2 : see Fig. 2b. These findings suggest that the amorphous precursor phase is chemically and structurally similar to crystalline malachite. Subsequent calcination of the thus-obtained crystalline malachite phase (as obtained by slow drying at RT in air), as well as of the amorphous precursor phase (obtained using the default synthesis route), both lead to the formation of CuO: see Fig. 2c and Eq. ( 3). CuO crystalizes in the monoclinic space group C12/c1 with lattice parameters a = 4.6837(5) , b = 3.4226(5) , c = 5.1288(6) , alpha = 90.000°, beta = 99.54(1)° and gamma = 90.000°3 3 . In a next step, the calcination of the amorphous precursor gel, as obtained from the standard synthesis procedure was studied by in-situ HT-XRD: see Figs 3 and S1. To this end, a drying step was performed first for 12 h at 75 °C (i.e. 15 K higher than the default drying step), after which the temperature was step-wise increased with a rate of 1 °C/min to 250 °C, followed by an isothermal annealing step of 8 h at 250 °C (see Fig. 3a). In parallel, the reflections of the 2Θ region from 20-40°, which contains the principle reflections of crystalline malachite, Cu(OH) 2 , 4) and the Williamson-Hall formula (W-H) (5), i.e. where β is the full width at half maximum of the respective reflection at the Bragg angle 2Θ, K is a numerical factor (here: K = 0.9, which is a good approximation for spherical particles in the absence of detailed shape information ), ε is a factor for the strain induced peak broadening and λ is the incident X-ray wavelength (here Cu-Kα) 37,38 . As evidenced by Fig. 3c, the average size of the CuO nanocrystallites synthesized at pH = 5.8 initially increases rapidly with increasing annealing time at T = 250 °C, reaching an average final size of roughly 17 ± 2 nm (by D-S) and 17.4 ± 0.4 nm (by W-H) within a few hours. Here it is emphasized that the XRD analysis probes the average size of the coherent scattering domains and therefore presents a measure of the smallest CuO crystalline building block only (and not of the CuO NP aggregate size). Notably, the Debye-Scherrer analysis on the basis of the CuO(111) and CuO(11-1) reflections gives similar crystallite sizes, which indicates that roughly equiaxed CuO crystallites are formed (i.e. single crystallites with a platelet or needle morphology are of minor importance). After complete calcination, only reflections that match the reference pattern of CuO remain (see Fig. 2c), which suggests that the amorphous precursor phase is fully transformed into single-phase CuO within several hours of calcination at T ≥ 250 °C, in accordance with ref. 30 . Differential scanning calorimetry (DSC) coupled with thermal gravimetric analysis (TGA) was carried out in synthetic air (60 mL/min) and at a heating rate of 5 K/min to 400 °C as shown in Fig. S2. An exothermic effect was detected at ~190 °C with a subsequent endothermic effect at ~260 °C, associated with a pronounced weight loss. The exothermic effect can be attributed to the crystallization of the amorphous precursor phase, as formed for the default synthesis route at pH = 5.8. The following endothermic effect is caused by the subsequent thermal decomposition of the crystallized precursor phase to CuO. The relative weight loss of 29%, as detected with TGA during the thermal decomposition, is in good agreement with the theoretical value of malachite decomposition: i.e. 1 − (2 × M CuO )/M malachite , where M CuO and M malachite correspond to the molar masses of CuO and malachite, www.nature.com/scientificreports www.nature.com/scientificreports/ respectively. This corroborates the findings from XRD that the thermal decomposition of the precursor gel into CuO (in static air) becomes thermally activated at around 250 °C. A somewhat lower transformation temperature of 180 °C was reported in ref. 39 , which might be due to a slightly different nature of the precipitate phase and/or the much longer annealing time of 48 h. Noteworthy, DSC-TGA shows a clear crystallization peak upon heating whereas no distinct crystalline malachite reflections could be detected during heating by in-situ XRD (see Figs 3 and S1). This can be attributed to the overlap of the crystalline malachite peaks in XRD with the broad peak of the amorphous as-prepared gel (cf. Fig. 2), which hinders unique identification of the malachite phase as long as the amorphous gel is still present. ]/[Cu 2+ ] < 1 for pH < 5.83) or surplus (i.e. [CO 3 ## Effect of pH and [CO 2− ]/[Cu 2+ ] > 1 for pH > 5.83), respectively. To provide a better insight into the precursor composition, the chemical speciation of Cu in aqueous solution, as well as the thermodynamic equilibrium with possible solid precipitate species (i.e. malachite, azurite, copper carbonate, copper hydroxide and copper oxide), were assessed as a function of pH for different carbonate concentrations using the MEDUSA software and the respective equilibrium and solubility constants from the HYDRA database 40 . The predictions were performed for various ammonia carbonate and copper acetate starting concentrations, as used in the experiments. The calculation results are plotted in Fig. 4a-c 4a-c,). Therefore, a second set of calculations was performed, which only considered the species remaining in solution (orange colored bars in Fig. 4a-c). The respective XRD patterns which were experimentally collected for the precipitate gels after 2 h of stirring are plotted for comparison in Fig. 4d-f. For a carbonate deficiency of the mixed solution (Fig. 4a, pH = 5.6), [Cu 2+ •(OAc) − ] + , [Cu 2+ •2(OAc) − ] (with OAc = CH 3 COO) and [Cu 2+ •(HCO 3 ) − ] + are the dominant Cu complexes in solution. Due to the low solubility of malachite, the equilibrium is largely shifted towards the solid malachite phase (compare calculation with and without precipitation species in Fig. 4). According to the thermodynamic equilibrium assessment, Cu can to some extend also precipitate as CuO. Indeed, as predicted both malachite and CuO are being detected by XRD in the (freshly washed) precipitated gel, as collected after the default stirring duration of 2 h (see step 4 → 5 in Fig. 1). The CuO signal appears to be much more dominant than that of malachite in the recorded diffractograms, ]/[Cu 2+ ] concentration ratios and pH's of (a-c) are shown in (d-f) respectively including reference pattern for CuO (red) 33 and malachite (blue) 32 . (2019) 9:11758 | https://doi.org/10.1038/s41598-019-48020-8 www.nature.com/scientificreports www.nature.com/scientificreports/ which would implicate that the formation of malachite is suppressed compared to CuO under carbonate-deficient conditions, in contrasts to the thermodynamic assessment. However, the crystalline reflections of malachite and CuO are superimposed on a very broad intensity hump indicating the presence of an amorphous (malachite-like) precipitate phase. In this case, the summed-up signal intensity from crystalline malachite plus the amorphous malachite-like precursor clearly dominates over that of CuO, in accordance with the model predictions. For the [CO 3 ## 2− ]/[Cu 2+ ] ratio equal to 1.0, which corresponds to a slightly higher pH of 5.8, similar calculation results are obtained, although the CuCO 3 -complexes get slightly more dominant and the equilibrium further shifts to malachite, being by far the most dominant precipitating species (note: the predicted fraction of CuO becomes negligible, Fig. 4b). The freshly washed precipitate collected for the 1:1 ratio mixture is fully XRD-amorphous (Fig. 4e), but transforms into crystalline upon complete drying in air (see Fig. 2b). Finally, the calculations for a carbonate surplus of [CO 3 ]/[Cu 2+ ] = 2.5 (pH = 7.0) predict CuCO 3 as the most dominant solution species and malachite as the only precipitation species; the acetate-containing copper complexes have only a minor contribution and the [Cu 2+ •(HCO 3 ) − ] + contribution is increased (Fig. 4c). In this case, the XRD analysis of the freshly-washed precipitate indeed only shows crystalline malachite, as well as the amorphous copper-carbonate-hydroxide (malachite-like) precursor phase (Fig. 4f). The three different wet precursors with [CO 3 2− ]/[Cu 2+ ] = 0.4, 1.0 and 2.5 were also analyzed by ATR-FT-IR to disclose the differences in their molecular compositions. The recorded ATR-FT-IR spectra are shown Fig. S3 in the Supplementary Information. In all cases, vibration bands at 3311 cm −1 , 2400-1900 cm −1 and 1637 cm −1 , corresponding to vibrations of the H 2 O solvent were identified. The FTIR spectra do not show any characteristic vibration bands for copper acetate 41 or acetate ions 42 , which indicates that acetate is not contributing to the precursor phase and is removed during the washing process. The vibration detected at 880 cm −1 for [CO 3 2− ]/ [Cu 2+ ] = 1.0 and 2.5 corresponds to the non-planar rocking of bonded CO 3 2− , which does not appear for the sample with carbonate deficiency (i.e. [CO 3 ]/[Cu 2+ ] = 0.4) 43 . Also the absorption bands for the symmetric C-O stretching at 1045 cm −1 and 1085 cm −1 appear less intense for the sample with carbonate deficiency. The largest differences in FTIR spectra for the three synthesis conditions are detected in the region of 1300-1530 cm −1 , in which asymmetric C-O stretching and CO 2 stretching vibrations of carbonate species appear 43 . The precursor obtained from the carbonate surplus reaction shows an absorption at ~1510 cm −1 , corresponding to asymmetric C-O stretching in basic conditions of complexated carbonates (fully deprotonated CO 3 2− ) 43 and at ~1400 cm −1 (CO 2 stretching in carbonates). Its small shoulder at 1420 cm −1 coincides with the broad absorption of the [CO 3 2− ]/[Cu 2+ ] = 0.4 sample. In refs 43 and 44 it was shown that the carbonate vibrations in acidic environments and in bicarbonate compounds shift as compared to their carbonato counterparts. The asymmetric C-O stretching vibrations in acidic environments shift towards 1620-1660 cm −1 and are thus covered by the absorption band of the solvent. The CO 2 stretching vibrations in HCO 3 − compounds appear at ~1410 cm −1 and ~1475 cm −1 . Hence the FTIR analysis reflects a dominant contribution of HCO 3 − species (1410 cm −1 and 1475 cm −1 ) and only a minor contribution of CO 3 2− species (visible at 1045 cm −1 ) at a carbonate deficiency, in agreement with the theoretical assessment of the solution species. Accordingly, the CO , as expected for [CO 3 2− ]/[Cu 2+ ] = 2.5 and 0.4, can be identified. The FTIR analysis thus indicates that the amorphous precursor phase, as formed for the default synthesis route at pH = 5.8, is predominantly constituted of copper carbonate and bicarbonate species and can thus be designated as an "amorphous malachite-like precursor phase". In conclusion, the experimental findings in combination with the thermodynamic calculations indicate that the observed amorphous phase is composed of randomly-packed clusters and chains of the predicted copper-complexes with HCO 3 − , CO 3 2− and H 2 O ligands (see Fig. 4). For the stoichiometric ratio of [CO 3 ]/[Cu 2+ ] = 1.0, this amorphous precursor phase is the dominant product phase of the complex forming reaction and preferably convert into crystalline malachite upon slow drying and/or subsequent calcination. For deviations from the ideal ratio (and its corresponding pH-value), the crystallization of malachite from the amorphous precursor phase seems to be accelerated, but also the crystallization of CuO can then be observed. ## Effect of pH, [CO 3 2− ]/[Cu 2+ ] -ratio and precipitation time on the morphology and size of the calcined CuO nanopowder. In order to tailor the crystallite and agglomerate sizes of the CuO nanopowder, the influence of the solution concentrations and resulting pH on the size and shape of the synthesized CuO NP aggregates as function of precipitation time was investigated in more detail. Since the hierarchical structures of the Cu precursor phase are generally largely preserved upon thermal decomposition into CuO by calcination 22 , the effect of the calcination treatment on the synthesized CuO product was not specially considered in the present study. Therefore, all calcinations were performed for 4 h at 400 °C. Calcination of the precipitate collected after different stirring times of 1 min, 1 h, 2 h and 72 h leads to a clear difference in shape and compactness of the primary CuO crystallites and aggregates for the two [CO 3 2− ]/ [Cu 2+ ]-ratios considered, as becomes apparent from the SEM analysis of the CuO nanopowder (Fig. 5). For a very short stirring time of 1 min, the aggregated NPs and agglomerates produced at both pH values are predominantly constituted of large crystallites with needle-and platelet-like morphologies. This anisotropic crystallite shape found at the onset of mixing hints at the presence of an intermediate precursor phase which differs from both the above discussed copper-containing amorphous malachite-like precursor phase and crystalline malachite, which can be found after 2 h of stirring. A likely explanation is the formation of a precipitating phase similar to the amorphous malachite-like precursor phase, but with an increased hydroxide content, which is typical for the formation of needle-and platelet-shaped Cu-hydroxides 30,45 . Indeed, it may be assumed that the complexation of Cu 2+ by (larger and less mobile) carbonate CO 3 2− and bicarbonate HCO 3 − ions, as associated with the nucleation and growth of a malachite precursor phase, is relatively sluggish as compared to complexation of Cu 2+ by OH − . (2019) 9:11758 | https://doi.org/10.1038/s41598-019-48020-8 www.nature.com/scientificreports www.nature.com/scientificreports/ Upon further stirring, this intermediate precursor phase gradually dissolves again, as the more sluggish complexation of Cu 2+ by the larger carbonate CO 3 2− and bicarbonate HCO 3 − ions progresses, leading to the formation of the amorphous malachite-like precursor phase. As follows from the structural analysis of malachite in ref. 24 , malachite itself shows not only selective bonding along crystallographic directions by the interconnection of Cu(OH) 2 building blocks, but also produces strong Cu-O with the carbonate ions in the (001) planes 24 . Assuming a similar local ordering for the amorphous malachite-like precursor, the progressive complexation of Cu 2+ by carbonate CO 3 2− ions 46 then results in the observed change in the crystallite shape of the CuO NPs from anisotropic to equiaxed morphology with increasing stirring time: see Fig. 5. As follows from a comparison of Fig. 5c with 5g, the (agglomerated) CuO aggregates synthesized for stirring times of 2 h appear much less densely clustered for pH = 7.0 as compared to pH = 5.6. For longer stirring times, the degree of NP aggregation increases for both pH-values, hinting at increasing densification of the amorphous precursor phase. Hence, a stirring time of 2 h can be considered as the optimum aging time for production of nano-porous nanopowders. The TEM micrographs of the calcinated CuO nanopowders obtained at pH = 5.6 and 7.0 for the optimum stirring time of 2 h are shown in Fig. 6. Deposition of the nanopowder dispersions on the electron-transparent TEM grid leads to a mixture of CuO aggregates and their agglomerates, which hinders a determination of the true CuO NP aggregate size (which is obtained by DSL in the present study). However, the TEM analysis clearly evidences that the agglomerated aggregates are constituted of clusters of much smaller primary crystallites. As is evident from comparison of Fig. 6c,d, the average size of the primary crystallites increases with increasing pH. The primary crystallite size of 23 ± 6 nm for pH = 5.6, as determined by TEM (see Fig. 6a), complies well with the CuO crystallite sizes of 20 ± 6 nm (from D-S) and 23 ± 9 nm (from W-H), as determined by XRD. Notably similar crystallite sizes were obtained in the in-situ HT XRD study of the calcination process (for pH = 5.8) as discussed above. For pH = 5.6, CuO particles with considerably larger crystallite and aggregate sizes could be observed by TEM in very few occasions. These much larger particles may be attributed to the observed formation of fewer primary CuO crystallites during the synthesis step. Investigation of the obtained particles from pH = 7.0 show a larger average crystallite size of 38 ± 8 nm which also complies reasonably well with the corresponding XRD values of 25 ± 7 nm (for D-S) and 28 ± 13 nm (for W-H). Notably, for the synthesis at pH = 7.0 the shape of the primary crystallites appears smoother and more spherical, and the resulting (agglomerated) CuO aggregates are more loosely packed, corroborating the observations made by SEM: compare Fig. 6a-d. A possible explanation for the observed difference in primary crystalline as well as in aggregate size and morphology between pH = 5.6 and pH = 7.0 is the following. For the pH = 5.6 solution, a [CO 3 2− ]/[Cu 2+ ] = 0.4 ratio induces a deficiency of CO 3 2− ions with respect to Cu 2+ ions (see FTIR analysis). It may be assumed that upon gel formation, i.e. condensation of the Cu-complexions towards the nominal malachite composition, a much denser network of Cu 2+ and CO 3 2− is developed than in case of an equimolar ratio of Cu 2+ and CO 3 2− , i.e. more Cu-carbonate-Cu-bridges are formed. As a consequence the formed gel is much more compact, and calcination of the gel then leads to larger primary CuO crystallites with higher cluster density, i.e. denser aggregate morphology. The observed difference in primary crystalline and aggregate size between pH = 5.6 and pH = 7.0 can be rationalized as follows: upon mixing of the starting solutions, the concentrations of the competing Cu complexation species practically instantaneous reach critical supersaturation, which for the ideal case of a homogeneous mixed solution under thermodynamic equilibrium, would result in the instantaneous homogeneous nucleation of the most stable precursor phase (i.e. crystalline malachite). These initial nuclei can grow upon aging of the ]/[Cu 2+ ] = 2.5 and pH = 7.0 (compare Fig. 4a,c). The CuO aggregates (as obtained after calcination), as synthesized from a carbonate deficient solution at pH = 5.6, are much more compact than the ones synthesized in a carbonate enriched solution at pH = 7.0 (compare Fig. 6d,f, as well as Fig. 5a,c,d). Here we propose the following explanation for the much higher compactness of the CuO aggregates synthesized at pH = 5.6. The precipitating amorphous precursor phase consists of randomly-packed clusters and chains of the predicted copper-complexes with HCO 3 − and CO 3 2− ligands (see Fig. 4), resulting in an amorphous precursor phase. The lower the [CO 3 2− ]/[Cu 2+ ]-ratio, the closer the proximity between neighboring copper cores in the precipitated amorphous precursor gel, which upon calcination result in a more dense CuO aggregates. Finally it is noted that, according to LaMer's-Model, larger primary crystallites are formed at a lower supersaturation (i.e. pH = 5.6), which is not observed in this study: the primary crystallites formed at pH = 5.6 are smaller than those grown at pH = 7.0 (as evidenced by XRD and TEM). In this regard it is emphasized that a variation of the carbonate molarity not only affects the supersaturation, but also the pH of the solution and thereby the stability (i.e. solubility) of the solid precursor phase in the mixed solution as well as its composition (thus affecting its final size resulting from Oswald ripening during continuous stirring). Both the higher carbonate molarity and the higher stability of the malachite phase promote diffusion-limited growth of the primary malachite nuclei at pH = 7.0. Determination of the CuO aggregate size by DLS. DLS was applied to determine the average size of the CuO aggregates, as dispersed in a water-based solution with 0.5% sodium dodecyl sulfate (SDS) following an ultrasound treatment. In this regard, it is emphasized that DLS records the intensity of the scattered light at very high temporal resolution from which the hydrodynamic diameter is calculated, which is the diameter of the particle or aggregate plus any ligands, ions or molecules that are attached to their surface (here: SDS). The ultrasonic Determination of the specific surface area (SSA) of the CuO nanopowder by BET analysis. The specific surface area (SSAs) of the synthesized CuO nanopowders after calcination, as well as after ultrasonic treatment and subsequent drying in air, were determined by BET analysis. The compact CuO aggregates synthesized at pH = 5.6 have an SSA of 16 m²/g, which can be compared with typical SSA values in the range of 10-15 m²/g, as indicated for commercially available high-purity (i.e. ≥99.99%) CuO nanopowders (e.g. US research Nanomaterials © , Plasmachem © , GetNanoMaterials © , Nanografi © and Nanoshel © ). In a next step, the same CuO nanopowder (i.e. synthesized at pH = 5.6) was dispersed in a water-based solution with 0.5% SDS using ultrasound waves and dried in air. Strikingly, a similar SSA of 15.33 m²/g was determined after the ultrasonic treatment and subsequent drying. This indicates that the ultrasonic treatment of the nanopowder dispersion is not affecting the effective surface area. In other words, although the more weakly linked agglomerates will be dispersed during the ultrasonic treatment, this has no distinct effect on the effective available surface area of compact aggregates. The loosely-clustered CuO aggregates synthesized at pH = 7.0 (with an average size of 93 nm; see Fig. 7b) have an SSA of 18.73 m²/g before and 18.92 m²/g after the ultrasonic treatment, which is about 20% larger than the SSA of the compact CuO aggregates (with an average size of 135 nm; see Fig. 7a). The maximum theoretical SSA that can be achieved for spherical nanoparticles is plotted as function of the particle diameter in Fig. 8. The SSA's of the compact and nanoporous CuO aggregates, as synthesized in this study are compared with the indicated SSAs for commercially available CuO nanopowders. It follows that the SSA of ≈19 m²/g, as obtained for the nanoporous CuO NP aggregates formed from pH 7 solution in the present study, is not only significantly higher than for most commercial CuO nanopowders, but also much closer to the theoretical value of ≈25 m²/g for the respective primary crystallite size (by TEM) of 38 ± 8 nm. Strikingly, the SSA for the compact CuO aggregates with a smaller primary crystallite size of 23 ± 6 nm, as synthesized at pH = 5.6, falls far below the respective theoretical SSA. Although a smaller primary crystallite size should result in a higher SSA (see Fig. 8), this was not observed in the present study. It is thus concluded that the BET analysis effectively probes the enhanced nanoporosity of the loosely-clustered CuO aggregates synthesized at pH = 7.0: i.e. the nanoporosity of the CuO aggregates at pH = 7.0 is much better accessible (permeable) to the infiltration by a gas (or liquid) than in case of the denser aggregates for pH = 5.6. These findings underline the importance of tuning the cluster density of the primary crystallites in stable NP aggregates (and not solely the primary crystallite size) for achieving a high SSA and thereby an enhanced chemical reactivity (often the main property aimed at with the use of nanoparticles) and sinterability of the nanopowder when processed in the form of a dispersed solution, paste or nanocomposite. First trials of low temperature joining with the less dense CuO NP aggregates processed as a nanopaste have confirmed that the sintering temperature and time for the bonding process can both be effectively lower by tuning the SSA of the CuO NP aggregates (work in progress). ## Conclusion The cost-effective and green sol-gel synthesis of CuO nanopowders via thermal decomposition of an amorphous malachite-like precursor phase was successfully implemented to tune size, shape and cluster density of the primary crystallites in the CuO NP aggregates. In-situ heating XRD measurements showed that the transformation of the amorphous malachite-like precursor phase into single-phase CuO upon heating in air becomes thermally activated at T ≥ 250 °C. The resulting CuO nanopowder is composed of (agglomerated) CuO aggregates, which are constituted of clusters of much smaller primary CuO crystallites. The size, shape and nanoporosity (i.e. aggregate cluster density) of these primary CuO crystallites can be tuned by controlled adjustment of the pH and the degree of supersaturation of the mixed solution with respect to the nucleation of malachite. To this end, the mixed solution was regulated to a specific pH value of either 5.6 or 7.0 by adjusting the added volume of ammonia carbonate solution, which resulted in an carbonate deficiency (i.e. [CO 3 2− ]/[Cu 2+ ] < 1 for pH < 5.83) or surplus (i.e. [CO 3 ## 2− ]/[Cu 2+ ] > 1 for pH > 5.83), respectively. This resulted in an average CuO aggregate size of 135 nm ± 43 nm at pH = 5.6 and of 93 nm ± 40 nm at pH = 7.0. The loosely clustered (and thus nanoporous) CuO aggregates synthesized at pH = 7.0 have a specific surface area of 18.73 m²/g, which is about 20% larger than the SSA of 15.97 m²/g for the compact CuO aggregates, despite a smaller NP crystallite size of the latter structure. It follows that the cluster density of the primary crystallites in the synthesized CuO NP aggregates can be tailored to enhance the specific surface area of the resulting CuO nanopowder for targeted applications in the field of e.g. catalysis, nanojoining, energy conversion and energy storage. ## Data Availability The datasets generated and analysed during the current study are available from the corresponding author upon reasonable request.

chemsum

{"title": "Cost-effective sol-gel synthesis of porous CuO nanoparticle aggregates with tunable specific surface area", "journal": "Scientific Reports - Nature"}

a_universal_stamping_method_of_graphene_transfer_for_conducting_flexible_and_transparent_polymers

## Abstract: transfer method of chemically vapor deposition graphene is an appealing issue to realize its application as flexible and transparent electrodes. A universal stamping method to transfer as grown graphene from copper onto different flexible and transparent polymers (FTPs) reported here ensures simple, robust, rapid, clean and low-cost. This method relies on coating ethylene vinyl acetate (EVA) onto the as grown graphene, binding EVA coated graphene/Cu with FTPs and delamination by hydrogen bubbling process, which is analogous to the method used by stamping process where ink carries the imprint of the object onto any materials. the fate of the stamping method depends on how strongly the adhesion of EVA coated graphene/Cu with target FTPs. Interestingly, we have found that the thin film of EVA/ graphene/Cu can only bind strongly with the FTPs of less than 25 µm in thickness and lower glass transition temperature value to the EVA while wide range of other FTPs are considered upon surface engineering to enhance the binding strength between FTPs and EVA. What's more, the electrical performance was investigated with a demonstration of triboelectric nanogenerators which confirmed the reliability of graphene transfer onto the FTPs and prospect for the development of flexible and transparent electronics. Since the discovery of mechanically exfoliated graphene 1 , production of high quality chemically vapor deposition (CVD) graphene for the industrial scale has remained challenging . Large domain size graphene grown on Cu may explore the possibilities of realistic application upon transfer onto the dielectric substrates . Graphene on polymer substrates are especially appealing as replacement of Indium tin oxide (ITO) and also one of the essential flexible and transparent electrodes for a wide range of optoelectronics devices such as touch screen displays and solar cells . Polymethyl methacrylate (PMMA) polymer is used to transfer graphene from Cu metal substrate onto the dielectric substrates 13,14 , but only limited to study for the fundamental properties. Till date, as grown CVD graphene was transferred onto many different types of flexible polymers . In majority of transfer case, PMMA and thermal releasing polymers are used as intermediate polymer to transfer graphene onto different polymers which scarifies the Cu substrates and induces cracks 19,22 . Roll to roll transfer of graphene onto polyethylene ptherapthalate (PET) was also achieved via thermal release tape 10 , epoxy resin 15 and ethylene vinyl acetate(EVA) 23 as binding source between graphene and PET. Roll to roll graphene transferred onto the EVA/PET by bubbling method 24 and green transfer 23 is hopeful to achieve the low cost, light weight, flexible and transparent electrodes. To our best of knowledge there is no report found on graphene transfer onto many other polymers, except PET, using EVA as binding agent for the industrial scale. EVA is explored against PMMA mediated graphene transfer for conformal contact with the target substrates 25,26 . Graphene transfer onto different polymers of characteristics properties suits wider range of applications still remains challenging and not many efforts have been made in this direction 27 . Recent advances in flexible electronics expect to soon realize the industrial production of graphene based hybrid transparent electrodes 28 . Our study met the challenge to transfer graphene onto different polymers by a fast and clean process. In this work, we designed a stamping method to transfer as grown graphene on Cu onto different flexible and transparent polymers (FTPs) which is applicable to the large scale graphene requirement as flexible and transparent electrode in modern electronics. This innovative method (Figs 1a and S1) relies on coating EVA onto the as grown graphene on Cu, binding EVA coated graphene/Cu with different polymers and delamination by hydrogen bubbling process, which is analogous to the method used by stamping process where ink carries the imprint of the object onto any materials. EVA has characteristic properties such as excellent transparency, flexibility and adhesivity as a function of temperature, which is already being used as encapsulating layers in solar panels 29 , is the major role in this transfer process. Next generation of wearable/bendable electronics demands the potential supply of flexible and transparent polymers as transparent electrode substrates in the areas of energy conversion, environmental monitoring, healthcare and communication and wireless network 30 . Although either big or small differences in polymer properties such as transmittance, flexibility, oxygen and water permeability, temperature resistance may largely impact on the functional properties desired for the respective applications. We chose thermoplastic transparent and flexible polymers, but not limited to, such as polyethylene terephthalate (PET), polyimide(PI), polycarbonate(PC), polyvinyl chloride(PVC), TOPAS (thermoplastic polymer mr-I T85) and CYTOP (an amorphous fluoropolymer type: CTL-809M, was purchased from AGC Asahi Glass) as target substrates for graphene based conductive film which could open the new avenue either in the modern electronics. The fate of the stamping method depends on how strongly the adhesion of EVA coated graphene/Cu with target FTPs. Interestingly, we have found that the thin film of EVA coated on graphene/Cu can only bind strongly with the FTPs of less than 25 µm in thickness and lower glass transition temperature (Tg) value to the EVA without any pretreatment (unmodified-FTPs) while wide range of other FTPs such as higher or lower Tg to that of EVA with low thermal conductivity in thicker polymer substrates are considered upon surface engineering (surface engineered-FTPs) to enhance the binding strength between FTPs and EVA. What's more, the electrical performance was investigated with triboelectric nanogenerators (TENG) , which confirms that our transfer process is reliable for the different polymers and prospect for the development of flexible and transparent electronics. Graphene transfer by stamping method. After CVD grown graphene on copper, an EVA solution (1-4 wt% dissolved in cyclohexane, Aladdin Industrial Corporation, Shanghai) was coated onto the graphene side of the copper by spin/spray/blade coating process. Prior to coating process, Cu was flattened using glass rod; edges of the graphene/Cu were covered with scotch tape to avoid the coating of EVA onto the backside of the graphene/ Cu. Spin coating is done with two spinning steps; 200 rpm for 30 seconds and 400 rpm for 60 seconds. Two times coating was done to ensure the continuity of the thin film EVA. In spray coating process, EVA solution was kept at hot container to make solution free flow through the nozzle. The spray nozzle movement and pressure were adjusted to 20 mm/s and 0.2 Mpa, respectively. We also demonstrated blade coating process, which is most suitable for the large scale graphene transfer attributed to roll to roll process. Speed of the blade movement was adjusted to 10 mm/s while distance between the blade and sample to be coated was set to 0.5 mm. The temperature of the sample holder both in spray and blade coating were kept at 50 °C to make fast evaporation of the solvent. After complete evaporation of solvent after being kept in dry box for 60 minutes, the resultant EVA/graphene/ Cu was bound onto any of the flexible polymers (cleaned using isopropanol and blow-dried with a nitrogen gun) using hot lamination method to form FTP/EVA/graphene/Cu. Finally, graphene/EVA/FTPs is delaminated from the metal substrate by electrochemical hydrogen bubbling method 21,33 and dried in air. ## Characterization. The morphology was characterized by optical microscopy (Olympus BX51), scanning electron microscopy (ZEISS-Merlin) and atomic force microscopy (Bruker). Quality of the CVD grown graphene transferred onto Si/SiO 2 was evaluated using a Raman spectroscope (Horiba, LabRAM HR Evolution) with a laser excitation wavelength of 532 nm. Monochromatic Al X-ray (Physical Electronics 56000 multitechnique system) was used to analyze any metal residue remained after graphene being transferred onto FTPs. The transmittance measurement was examined using UV-Vis-NIR spectrophotometer (Perkin Elmer, Lambda 750 s, 190-3300 nm). Contact angle measurement was done by contact angle tester (AST VCA Optima XE). Sheet resistance measurement was carried out using four probe system (Guangzhou 4-probe Tech Co. Ltd., RTS-4) with probe spacings of ~1 mm. Fabrication of triboelectric nanogenerator. The final product from this transfer process graphene/EVA/ FTPs film was used as the electrode of TENG. The fabricated device consists of a graphene/EVA/FTP electrode coated with CYTOP film (triboelectric layer) (18 × 20 mm) and a PET substrate coated with ITO films. Both two components were placed in an acrylic glass as a proof mass which can be driven by external vibration. The acrylic mass and transparent substrate were assembled in the elastic holder to make an arched structure with a dimension of 45 × 45 × 10 mm. The CYTOP polymer coated on the bottom plate is charged in a custom-built corona charging setup. The setup consists of a grounded electrode, a metal mesh grid (V g = −2000 V) and a high-voltage probe tip (V H = −6 kV). The device is driven by a mechanical shaker with controlled frequency and amplitude, where an accelerometer is used to monitor the acceleration during the measurement. The shaker is driven by an excitation signal generated from a signal generator (Brüel&Kjaer, LAN-XI 3160) and a power amplifier (Brüel&Kjaer, 2719). ## Results Monolayer graphene is grown on electrochemically polished 25 µm thick copper foils by low-pressure chemical vapor deposition (LPCVD) 2 . Both optical microscopy (OM) (Fig. S2a) and scanning electron microscope (SEM) (Fig. S2b) of graphene transferred onto SiO 2 by PMMA revealed continuous monolayer graphene with bilayer or multilayer graphene domains. Figure S2c shows the Raman spectroscopy of the graphene on SiO 2 confirms the high quality graphene by showing the negligible D peak which is raised commonly due to the PMMA mediated transfer. Figure 1a shows the schematic of stamping method to transfer CVD-grown graphene from Cu substrate to a FTPs comprises three major steps; (i) EVA is coated onto the as grown graphene on Cu by any of the three modes such as spin coating for small sample, spray coating and blade coating for large samples (Fig. S1a). The coated sample represents as ethylene vinyl acetate/graphene/copper (EVA/graphene/Cu) thin film. Figure 1b shows the photograph which distinguishes the graphene on Cu before and after EVA coating, where bright contrast of Cu/graphene becomes dull upon EVA coating which revealed the EVA film formation. A thin close contact of EVA upon blade coating is evidenced with the vertical cross-sectional SEM (Fig. 1c). (ii) Binding the EVA/ graphene/Cu thin film with the target FTP substrate using hot lamination (Fig. S1b) to form FTP/EVA/graphene/ Cu stack as similar to the graphene transferred onto commercially available EVA/PET 23,24 . Hot lamination in our graphene transfer method is reliable for the flexible polymers as coated EVA layer on Cu/graphene unchanged the flexible property. Roller temperature was adjusted according to the Tg of EVA. Higher temperature than Tg of EVA makes the FTPs film deform. We found some interesting mechanisms of binding EVA coated graphene/Cu thin film with the smooth surface morphology FTPs (less than 25 µm) having similar Tg of EVA such as TOPAS, CYTOP, PET. 25 µm thin FTPs with lower Tg undergoes easy deformation at 120 °C, since as grown graphene on Cu has considerable roughness 35 , smoother FTPs adhered tighter with EVA/graphene/Cu due to roughness impression carried from graphene/Cu. But in the case of thicker FTPs of 100 µm such as PET, PC, PVC, we cannot www.nature.com/scientificreports www.nature.com/scientificreports/ found tight adhesion with EVA/graphene/Cu due to insufficient supply of required temperature to attain Tg value for thicker FTPs in lamination process. (iii) Delamination of FTP/EVA/graphene/Cu stacks to transfer graphene onto FTP/EVA from Cu substrate using electrochemical hydrogen bubble method by polarizing Cu/graphene/ FTP at 2 V 24,36,37 (Fig. S1c). Figure 1d shows the photographs of the graphene/EVA/FTP (bottom row) which are comparable to that of EVA/FTP (top row), indicating that transfer process has little effect on the transmittance of target FTP upon graphene transfer. Note that all FTPs are resistant to the NaOH (electrolytic solution) and the method ensures successful graphene transfer without damaging either Cu substrates or EVA layer coated on graphene/Cu. Since FTP/EVA/graphene/Cu stack was used as cathode in the delamination process, H 2 bubble generates between graphene and the Cu substrates which leads to the delamination of graphene onto the EVA/ FTPs. Oxidation of graphene/EVA/FTPs is avoided while Cu undergoes slight oxidation due to the basic electrolytic solution but it favors the growth of high quality graphene for the second time 23 . The graphene/FTPs film is rinsed with deionized water and blow dried with nitrogen to ensure it to be free of chemical residues. Prior to the coating EVA solution onto the graphene on Cu, the method further comprises the step of flattening the CVD-grown graphene on Cu foil (Fig. S3a). Spin coating is popular and commonly used to transfer graphene using PMMA solution because it helps to control the thickness of the thin film formation of PMMA layer 13 . The innovation of our work relies on the thin film deposition of EVA onto as grown graphene on Cu and surface engineering of FTPs, where EVA acts as a binding agent between graphene and target substrates. In the process of coating, EVA solution preparation and optimization process is very important because we are the first using EVA to transfer graphene on to different FTPs. The preparation method is as follows: 1-4 wt.% EVA solution is made by dissolving EVA and stirring in cyclohexane at 75 °C. In fact, we demonstrated the coating method based on the sample size; spin coating is generally suitable for small size graphene since it is easier and makes the process fast. A thin layer of 1% EVA was spin coated twice to ensure a continuous layer, parameters were set similar to the PMMA coating 38 . On the other hand, for large area of graphene samples especially for roll to roll CVD graphene, either spray coating or blade coating process enables the process efficient and easier. Since the diameter of spray nozzle is small, 1% EVA is used in the spray coating method to avoid the blockage of nozzle. It should be noted that spray coating can control the tight contact of EVA with graphene upon adjusting the spray pressure to form the compact thin uniform EVA film. Multi spray coating deposition can also be done to control the thickness, which is attributed to achieve graphene transfer onto surface engineered polymers. To obtain thicker film on large area graphene, 4% EVA was coated by blade coating machine. By adjusting the distance between Cu/ graphene and blade, thickness can be controlled up to 20 µm. Detailed procedure of coating method is described in experimental part. The samples after deposition were kept at room temperature for 60 minutes to evaporate the solvent. By comparing the vertical cross-sectional SEM images of Cu/graphene (Fig. S3d) and EVA coated Cu/graphene (Fig. S3e), the continuity of EVA layer without any disruption was confirmed. Note that there is no reaction found between EVA solution and graphene/Cu metal substrate, though it helps the formation of thin film very fast due to fast evaporation of the solvent. Both blade coating and spray coating can be integrated in the roll-to-roll processing of graphene transfer. Interestingly, we found our EVA mediated stamping method of graphene transfer onto smooth FTPs can only be realized when their thickness and Tg is less than 25 µm and ~140 °C, respectively. Weak binding force between the two smooth surface polymers films were noticed in the fabrication of graphene/metal nanowire transparent electrodes 39 , which signalling that the surface engineering of the target FTPs are deciding factors to clamp thin film EVA to achieve efficient graphene transfer in our stamping method. It is also reported that EVA film shows excellent adhesive bonding to solar glass which are rough in surface 40 . In addition, our method can also extend to FTPs thicker than 25 µm or with higher Tg temperature than EVA after proper surface engineering. The basic prerequisite of the target FTPs substrate should be surface roughness and hydrophobicity. Surface engineering in our method comprises two steps; (i) FTPs are and blasted to make the surface rough with the formation of crest and trough in large scale. (ii) Fill the crest and trough by coating 1% EVA solution using various methods. Generally, graphene transfer is only valid onto smooth surface materials for electronic applications 41 . To transfer graphene onto a wide variety of FTPs having higher Tg value to that of EVA and low thermal conductivity in thicker polymer substrates, surface engineering is inevitable. EVA mediated transfer overcome the challenge to transfer graphene onto rough surface by providing smooth surface basement to graphene upon EVA coating onto the target rough surface polymers. Here, we used but not limited to PI as FTP target substrates to demonstrate graphene transfer onto the smooth surface with a higher Tg value to that of EVA. Graphene transfer from Cu/ graphene/EVA stack onto the PI is unsuccessful, where arrow in the Fig. 2a shows the EVA/graphene detachment from PI substrate. EVA/graphene thin film detachment from ultra-smooth PI substrate is clearly seen in SEM image (square mark of Fig. 2a), which signs that the graphene transfer can only be done upon surface engineering of target polymer substrates of higher Tg value and higher thickness (Fig. 2b). Figure S4a shows the schematic illustration of FTPs surface modification, surface was thoroughly rubbed by sand paper, forming uneven surface with crest and trough and the same was made smooth by filling EVA solution by spin coating method. Crest and trough of rubbed FTPs were clamped tightly by the coated EVA which forms the flat surface, as shown in the vertical cross-sectional image of SEM (square mark of Fig. 2b). Further surface morphology characterizations of surface engineered-FTPs and graphene transfer onto the FTPs were carried out by OM and atomic force microscopy (AFM). OM shows that the crest and trough in FTPs ensures that the entire surface is uneven (Fig. 2c), while it turned to flat uniform surface upon EVA coating (Fig. 2d) and finally graphene transferred onto the EVA coated FTPs remains surface flat which is identified with the graphene grain boundary (arrow in Fig. 2e). Our AFM observations show that the FTPs of higher Tg rubbed with sandpaper bears the uneven surface (Fig. 2f; bottom) with random crest and trough of several micrometers (line section of the surface morphology; Fig. S5a), which helps to clamp the EVA very tight (Fig. 2b). Upon EVA coating, crest and trough were filled to form a thin uniform film (Fig. 2f; middle) to satisfy the surface requirement to transfer graphene, surface roughness was decreased to several nanometers (Fig. S5b). After graphene transferred onto the surface engineered-FTPs, surface roughness was found to be quite increased as compared to the EVA coated FTPs before transfer, which is due to the surface morphology of graphene grown on copper 16 (Fig. 2f; top and Fig. S5c). Contact angle (CA) measurement was carried out to measure to the surface behavior of surface engineered FTPs before and after graphene transfer. In fact, rough surface FTPs shows hydrophilicity due to the rough surface morphology (CA = 75°, Fig. 2c), while EVA coated FTPs before (CA = 105°, Fig. 2c) and after Gr transfer (CA = 97°, Fig. 2e) remains hydrophobic which strongly shows that the graphene transfer upon surface engineered polymer is successful by stamping method. In contrast, topographical AFM images shown in Fig. 2f distinguishes the surface roughness of FTPs rubbed with sand paper (bottom), smoother upon EVA coating (middle) and finally turns to be negligible rough after graphene transfer (top). It is noteworthy that graphene grown on Cu foil by CVD shows high quality (Fig. S2). Surface morphology of the graphene on different polymers by stamping method was characterized by SEM. The graphene grain boundaries (arrow mark in Fig. 3a-f) are observed as the characteristics confirmation of graphene transfer onto the different polymers such as TOPAS, CYTOP, PET, PVC, PC and PI. No voids or cracks are seen from our transfer, which confirms the good contact between the target substrates and the EVA/graphene transferred from the Cu substrates. Surface of graphene/EVA on polymers shows slight wavy morphology due to the surface engineered FTPs substrates and the graphene/Cu. In the hot lamination process, EVA softens at 120 °C and mimics the surface morphology of both the FTPs and Cu, which results in the rough graphene/EVA/FTPs surface compared to that of EVA/FTPs before graphene transfer, as shown in the AFM image of Fig. 2f (See Supplementary Fig. S5c). However, OM image of graphene/EVA/FTP shows the confirmation of graphene transfer in large area, where graphene grains on Cu can be compared with the graphene grains on EVA/FTPs (Fig. S6a). Following coating and binding process, electrochemical bubbling method results the final graphene on FTPs; we found no surface contamination on graphene/EVA/FTPs by XPS and UV-Visible spectra. Figure 3g shows the XPS full spectra of graphene/EVA/FTPs. Two predominant XPS peaks of C 1 s and O 2 s found at 284.5 eV and 530 eV, which are ascribed to the sp2 carbon of graphene and the oxygen in EVA, respectively. The deconvoluted XPS spectra of graphene/EVA/FTPs in inset of Fig. 3g shows no predominant Cu peak in the binding energy between 930 and 960 eV at the detection limit of XPS. This observation indicates that the coating-lamination-bubbling process in the stamping method is efficient to transfer graphene/EVA from Cu onto FTPs. We then evaluated the UV-Visible spectrum of graphene/EVA/FTPs to confirm whether the quality of transparency is affected in the transfer process. Fig. S6b shows the UV-Visible spectrum of graphene transferred onto EVA/FTP showing 97.4% transmittance close to the theoretical value of graphene/Quartz, which confirms that our transfer process is successful with no surface contamination in the entire transfer process; note that the substrate transmittance was subtracted. The transmittance of graphene/EVA on different FTPs such as TOPAS, CYTOP, PET, PC, PVC and PI were evaluated showing that original transmittances of the FTPs were unaffected upon graphene/EVA transfer. The total T% values were found for graphene on different FTPs such as TOPAS, CYTOP, PET, PC, PVC and PI are 96%, 96%, 86%, 84%, 83% and 55%, respectively (Fig. 3h). Conductivity is one of the main concerns of graphene transferred onto FTPs. Sheet resistance of graphene/EVA/FTPs was evaluated with four point probe system. The large area of graphene on different-FTPs was measured with sheet resistance and the result value lies between 1-10 kohm/sq, which is acceptable range of graphene on EVA/PET by green transfer 23 . Because of high sheet resistance of polycrystalline graphene on dielectric substrates, metal nanowires are used to fabricate www.nature.com/scientificreports www.nature.com/scientificreports/ graphene based hybrid transparent electrodes to perform equivalent to that of ITO electrodes 24,42 . Figure S6c shows the schematic illustration for the reason of higher Rs found at graphene/EVA/FTPs. Four probes shown in the Fig. S6c spotted at within the grain size or between the large grain size Rs value is considerable lower than that of four probes spotted at small grains or higher grain boundary region results in higher Rs of graphene/EVA/ FTPs. Either with the improved growth of large area domain size graphene on Cu 43 , metal nanowire networks 44 or chemical doping 45 are strong strategic direction that our transfer method is efficient to resolve the higher Rs value in graphene/FTPs. In contrast, graphene on unmodified-FTPs shows a Rs of 10 Kohm (Fig. 3i) while graphene on surface engineered-FTPs shows higher Rs of about 10-20 K Ohm (Fig. 3j), indicating the surface engineering process affects the electrical properties slightly. The distribution curve of sheet resistance of graphene on different unmodified FTPs is observed over the 3 × 4 cm graphene/EVA/FTPs is found to be 1-10 k ohm/sq, which are measured with the typical probe spacing 1 mm (Fig. S6d). The Cu foil was not damaged by any process involved in stamping method (Fig. 1b), neither of chemical residue remained while EVA coating on graphene/Cu or tore Cu foil in the lamination and bubbling method. Vertical SEM images of Cu after graphene/EVA transferred onto FTPs show that there are no such mechanical damages found on Cu substrates (Fig. S3f). Figure 4a (left) shows the Cu foil after graphene/EVA thin film transferred onto the FTPs showing some part undergoes oxidation which is an advantage for the growth of high quality graphene 46 , while graphene grown upon reused Cu (right of Fig. 4a) turns to be shine which is similar to the Cu used to grow graphene at first time (Fig. 1b; left). The high-quality nature of graphene on reused Cu is evidenced by OM (Fig. 4b), SEM (Fig. 4c) and Raman spectrum (Fig. 4d). The enlarged domain size and less grain boundary may favor the quality improvement of graphene on FTPs, which is significant for economic industrial scale. To confirm the graphene transfer performance on the electrical conductivity and flexibility, we have applied graphene/EVA/FTPs electrodes in the TENG. The fabricated device consists of a FTPs/EVA/graphene electrode coated with CYTOP film (18 × 20 mm2) and a PET substrate coated with ITO films sketched in Fig. 5a. The ITO plays dual roles as electrode and contact surface, while CYTOP plays the role as the other contact surface. Graphene is used as back electrode. Figure 5a (right) shows the photograph of the graphene/EVA/PI revealing the bending capability without causing any damage to graphene/EVA. To fabricate the device, cast acrylic glass was prepared as a proof mass which can be driven by external vibration. Both the acrylic mass and transparent substrate were assembled in the elastic holder to make an arched structure with a dimension of 45 × 45 × 10 mm 3 . The CYTOP polymer coated on the bottom plate is charged in a custom-built corona charging setup (Fig. 5b). The setup consists of a grounded electrode, a metal mesh grid (V g = −2000 V) and a high-voltage probe tip (V H = −5 kV). After charging for 15 min, the surface potential of the electrets layer is mapped in Fig. 5d. The energy harvesting performance of the device is characterized with a shaker setup shown in Fig. S7a. Our device is driven by a mechanical shaker with controlled frequency and amplitude, where an accelerometer is used to monitor the acceleration during the measurement. The shaker is driven by an excitation signal generated from a signal generator (Brüel&Kjaer, LAN-XI 3160) and a power amplifier (Brüel&Kjaer, 2719). Figure 5c shows a typical www.nature.com/scientificreports www.nature.com/scientificreports/ driven vibration with amplitude of 170 m/s 2 at 46 Hz. To find the optimal work frequency, we have explored the relationship between the output power of the TENG and the frequency of the vibration source under different amplitudes, as shown in Fig. S7b. An output power peak can be seen at 46 Hz, which is exactly the same as the resonant frequency of the device. At resonance, the output voltage of TENG based on FTPs/EVA/graphene/CYTOP VS ITO/PET was presented in Fig. 6a with a closed-up view shown in Fig. 6b. The voltage peak reaches 0.23 V. Output current was found with different values for the same area size of graphene on different FTPs shown in Fig. S8. Here we demonstrated graphene transfer onto the unmodified-FTPs, found output voltage was 0.008 V for graphene transferred onto the thin CYTOP, TOPAS and PET (Fig. S8a-c), while graphene on surface engineered FTPs such as thicker PET (Fig. S8d) and higher Tg value polymers such as PI (Fig. S8e) output voltage was found to be increased due to wavy surface morphology of graphene. CVD graphene surface roughness increases the triboelectric effect 47 . Furthermore, it is observed that the electric signal also can be generated by hand driving periodically; the output result of device FTP/EVA/graphene/CYTOP VS ITO/PET was shown in Fig. 6c. And it was proved that the electricity generated by our device is effective when a 10 μF capacitor is charged successfully from 0 V to 3 V in less than 20 min, as Fig. 6d presented. Meanwhile, we also proved the electricity generated by our device is powerful enough to power LEDs, as 6 LEDs can be powered successfully as showed in Fig. 6e,f. This demonstration showed that the binding energy between graphene/EVA and the FTPs are strong enough and as well materials could be used as flexible and transparent electrode in the modern electronics. Even though only five polymer examples are shown, the procedure worked consistently on all other target substrates such as PC and PVC which justifies that our graphene transfer is universal. ## Conclusion We have demonstrated a stamping method to transfer graphene onto different FTPs using EVA as binder only between graphene and target substrates without affecting the Cu substrates which could be used for repeated graphene growth. Surface modification of FTPs to alter the effective surface interaction with the EVA widens the choice for target substrates. Our transfer method is simple and fast, which ensures the clean and efficient transfer without inducing any damage either onto the graphene/substrates or Cu foil. What's more, the electrical output performance is demonstrated with the fabrication of TENG and implied that our transfer method is realistic to scale up the graphene on plastics for industrial-scale. We believe this approach may be further improved by adopting effective strategies like metal nanowire based graphene transparent and flexible electrode reported elsewhere to replace ITO in optoelectronic devices 24 .

chemsum

{"title": "A Universal Stamping Method of Graphene Transfer for Conducting Flexible and Transparent Polymers", "journal": "Scientific Reports - Nature"}

electrophilic_reactivities_of_cyclic_enones_and_α,β-unsaturated_lactones

## Abstract: The reactivities of cyclic enones and a,b-unsaturated lactones were characterized by following the kinetics of their reactions with colored carbon-centered reference nucleophiles in DMSO at 20 C. The experimentally determined second-order rate constants k 2 were analyzed with the Mayr-Patz equation, lg k ¼ s N (N + E), to furnish the electrophilicity descriptors E for the Michael acceptors. Cyclic enones and lactones show different reactivity trends than their acyclic analogs. While cyclization reduces the reactivity of enones slightly, a,b-unsaturated lactones are significantly more reactive Michael acceptorsthan analogously substituted open-chain esters. The observed reactivity trends were rationalized through quantum-chemically calculated Gibbs energy profiles (at the SMD(DMSO)/M06-2X/6-31+G(d,p) level of theory) and distortion interaction analysis for the reactions of the cyclic Michael acceptors with a sulfonium ylide. The electrophilicities of simplified electrophilic fragments reflect the general reactivity pattern of structurally more complex terpene-derived cyclic enones and sesquiterpene lactones, such as parthenolide. ## Introduction Cyclic carbonyl compounds with a,b-unsaturated positions are important motifs within many natural products (Chart 1). Previous studies of their cellular reactivities with endogenous proteins revealed intriguing insights into their target profles. 4,5 The ability of these biomolecules to react as electrophiles with nucleophilic sites furnishes them with a multitude of biological functions, 6,7 e.g. the recently reported inhibition of focal adhesion kinase 1 by parthenolide, 5a,b the cytotoxic activity of dehydroleucodine against human leukemia cells, 8 or the ability of nimbolide to inhibit metastasis. 5c Nature has structurally tailored the reactivity of a,b-unsaturated cyclic carbonyl compounds in different variants. In particular, a-methylene-gbutyrolactones exhibit superior cellular protein binding compared to lactones with endocyclic p-system, likely associated with an elevated reactivity. 4a,9 For the sesquiterpene lactones costunolide and dehydrocostus lactone, 10 a,b-unsaturated d-lactones such as leptomycin, fostriecin or the anguinomycins, as well as for simple fragments, such as tulipalin A, 16 it has been analyzed that their biological activities mainly depend on the ability to alkylate biomacromolecules through Michael additions. Sometimes these Michael additions are coupled with subsequent steps to achieve irreversible covalent enzyme inhibition. 15 On the other hand, in modifed rugulactones the a,b-unsaturated d-lactone unit does not Chart 1 Examples for electrophilic natural products. contribute to the antibacterial effects and bioactivities of rugulactone were instead assigned to the reactivity of the a,b-unsaturated ketone unit. 17 Despite these insights into proteome reactivity, a systematic analysis of the individual electrophilicity of the Michael acceptor moieties in different natural products or their truncated analogs is lacking. Knowledge of the reactivity of such biologically occurring electrophilic fragments would facilitate the identifcation of pharmacophores and is, therefore, of fundamental interest in biochemistry, toxicology, medicinal chemistry, and drug discovery. 9,18 Moreover, Michael acceptors with endo-and exocyclic unsaturation are also a structural motif of signifcant importance for synthetic chemists. 3 In life-sciences, rate constants for the reactions of electrophiles with glutathione (GSH) are frequently used for estimating the reactivity and potential toxicity of various electrophilic compounds. However, the most comprehensive overview of polar organic reactivity is currently given by Mayr and co-workers who used eqn (1) to characterize the reactivities of more than 1200 nucleophiles and over 300 electrophiles in solution phase. 24 lg k 2 (20 C) ¼ s N (N + E) Eqn ( 1) is a linear free energy relationship that allows for the semi-quantitative prediction of second-order rate constants k 2 for the reactions of electrophiles with nucleophiles from three parameters: the electrophilicity parameter E and the solventdependent nucleophilicity parameters N and s N (susceptibility). Recently, we determined the nucleophilic reactivity parameters N and s N of GSH in aqueous solution, which facilitates to interconnect both approaches. Bioassay-derived GSH kinetics can now be used to roughly estimate Mayr electrophilicity parameters E, and vice versa. In this way, Mayr electrophilicities E for more than 70 acyclic Michael acceptors were estimated based on their previously determined kinetics toward GSH. 25 More precise electrophilicities E for a series of structurally simple acyclic Michael acceptors were determined from the kinetics of their reactions with carbon-centered one-bond nucleophiles (reference nucleophiles), that is, mainly with pyridinium and sulfonium ylides. 26,27 We now set out to determine the Mayr electrophilicity parameters E of cyclic enones 1-3 and a,b-unsaturated lactones 4-5 by studying the kinetics of their reactions with the reference nucleophiles 6-7 (Chart 2). We then tested whether the Mayr E parameters obtained for the electrophilic core structures 1-5 are also representative of the reactivity profle of structurally more complex natural products that bear these fragments in their molecular scaffold. Quantum-chemical calculations were used to rationalize the observed reactivity trends which signifcantly differ from those for analogous acyclic (open chain) ketones and esters. ## Product studies The formal 1,3-dipolar cycloadditions (Huisgen reactions) of simple electron-defcient alkenes with pyridinium ylides, generated from N-alkylated pyridinium salts under basic conditions, are well-known to yield tetrahydroindolizines. Subsequent oxidation (e.g. with air or chloranil) efficiently aromatizes the newly formed heterocycles to afford diversely substituted indolizines. In contrast, formation of the analogous tricyclic cyclopenta-, cyclohexa-, or cyclohepta-indolizines has rarely been studied. Only Tamura reported the formation of cyclohexaindolizines in low yield (10%) in a vinylic substitution reaction that used the pyridinium ylide 6c (R ¼ CO 2 Et) and 3chlorocyclohexanone as educts. 32 Direct 1,3-dipolar cycloaddition reactions of pyridinium ylides with cyclic enones or a,bunsaturated lactones have not been reported to the best of our knowledge. We planned to use the pyridinium ylides 6 as colored reference nucleophiles to follow the kinetics of their reactions with cyclic Michael acceptors by photometric methods. Given the lack of knowledge about the outcome of these reactions, we decided to characterize the products of a subset of the electrophile/nucleophile combinations under the conditions of the kinetic experiments, that is, in DMSO at 20 C (Scheme 1). Treatment of a 1 : 1-mixture of the pyridinium salt 6b$HY (HY ¼ HCl, HBr) and sodium carbonate with a DMSO solution of cyclopentenone (1a, 2 equiv.) resulted in a (3+2)-cycloaddition to give a mixture of diastereomeric tetrahydroindolizines. Due to their high sensitivity toward oxidation 33 and to facilitate the product purifcation, we oxidized these initial adducts to the aromatic indolizine 8, which was isolated in 18% yield and characterized by single-crystal X-ray diffraction. We were delighted to fnd that analogous reactions of 6b with cyclohexenone (2a), cycloheptenone (3) as well as with the lactone 5a gave the corresponding indolizines 9b, 10, and 11, respectively, in signifcantly higher yields (72-86% of isolated products). Furthermore, cyclohexaindolizines 9a and 9c were isolated in high yields from the reactions of the ester-and keto-stabilized pyridinium ylides 6a and 6c with cyclohexenone (2a). To diversify the types of reference nucleophiles in our kinetic studies, we also investigated the reactions of the cyclic electrophiles with the sulfonium ylide 7. Treatment of a solution of the sulfonium tetrafluoroborate 7$HBF 4 and a cyclic Michael acceptor in DMSO with potassium tert-butoxide generated the sulfonium ylide 7 which then underwent cyclopropanation reactions with the electrophiles 1a, 2a, 4a, and 5a (Scheme 2). The cyclopropanes 12-15 were obtained as mixtures of diastereomers. Separation of the diastereomers by column chromatography was not always possible. However, purifed diastereomers of 12 and 15 could be crystallized and characterized by single-crystal X-ray crystallography, providing unequivocal evidence for the cyclopropanation reaction. The lactones 4b and 5b reacted with the sulfonium ylide 7 at their exo-methylene groups to give diastereomeric mixtures of 16 (46%) and 17 (69%), respectively. Owing to their sufficiently different polarity these diastereomeric mixtures were separable by column chromatography. One diastereomer of 16 and one of 17 were crystallized and analyzed by single-crystal X-ray diffraction (Scheme 2). As a general trend, the yields of the cyclopropanes 12-17 depended on two factors: (a) the excess and (b) the absolute concentration of the electrophiles. A survey of the reaction conditions showed that highest yields were obtained when the cyclic Michael acceptor was present in excess (up to 10 equiv.) over the pronucleophile 7 and/or at low concentrations (<0.01 M). Experimental protocols with higher concentrations of the Michael acceptors or reduced excess (1.5 equiv.) resulted in complete consumption of the colored ylide 7, too, but the cyclopropanes were only formed as minor products under these conditions. Instead, 7 isomerized in a background reaction to furnish the sulfde 18, 34,35 presumably through a Sommelet-Hauser type of rearrangement (Scheme 3). 36 The sulfde 18 is the starting material for BAY 85-8501, a candidate for the treatment of inflammatory diseases such as acute lung injury. 35 We characterized 18 by single-crystal X-ray diffraction. Solutions with low concentrations of 7 in DMSO isomerized slower (t 1/2 ¼ 30 min at 0 ¼ 1 10 4 M) than solutions with higher concentrations of 7 (t 1/2 ¼ 4 min at 0 ¼ 0.057 M, monitored by time-resolved 1 H NMR spectroscopy). Thus, the isomerization of 7 into 18 partially consumed the nucleophile when 7 was combined with weakly or only moderately reactive electrophiles, whose cyclopropanations proceeded at comparable time scale as the Sommelet-Hauser rearrangement of 7. ## Kinetics The kinetics of the reactions of the colorless electrophiles 1-5 with the pyridinium (6) and sulfonium (7) ylides were determined by following the decay of the UV-vis absorbance of the colored nucleophiles. Reactions at the seconds to minutes timescale were followed by conventional photometry. Stopped-flow photometric methods were employed for faster reactions in the millisecond regime. DMSO was used as the solvent for all Scheme 1 Products for the reactions of the pyridinium ylides 6 (generated by deprotonation of 6$HY) with cyclic Michael acceptors (yields of isolated products after chromatography, see ESI † for details). Insert: Single-crystal X-ray structure of 8. Thermal ellipsoids drawn at a 50% probability level. Scheme 2 Products for the reactions of the sulfonium ylide 7 with the electrophiles 1-5 (yields of isolated products after column chromatography, thermal ellipsoids drawn at 50% probability level, see ESI † for details). [a] 5 equiv., [b] 10 equiv., [c] electrophile-nucleophile combinations, which were uniformly studied at 20 C. Solutions of the ylides 6 and 7 in DMSO were generated by adding stoichiometric amounts of potassium tert-butoxide to the corresponding pyridinium or sulfonium salts. In the next step, these DMSO solutions were mixed with an excess (>10 equiv.) of the electrophiles 1-5. With this ratio of reactants, the concentration of the excess compound can be assumed to remain practically constant during the kinetic measurements, which simplifes the kinetics and makes it possible to determine rate constants k obs under pseudo-frst order conditions. In general, the timedependent change of the nucleophile's absorbance followed a mono-exponential decay. The frst-order rate constants k obs were then determined by a least-squares ftting of the monoexponential decay function A t ¼ A 0 exp(k obs t) + C to the experimental absorbances A t (Fig. 1A). The correlation of k obs with the concentration of the electrophiles 1-5 revealed a linear relationship, the slope of which corresponds to the second-order rate constant k exp 2 (Fig. 1B, Table 1). Isomerization (of 7) and/or decomposition of the colored reference nucleophiles proceed concurrently and impede the kinetic study of slower reactions. Therefore, only the highly reactive nucleophile 6c was available to study the rather unreactive 2-and 3-methylated cyclic enones 1c, 2b, and 2c. Based on the set of experimental second-order rate constants k exp 2 , we calculated the electrophilicity parameters E for compounds 1-5 by applying eqn (1) and the reported Mayr nucleophilicity parameters N and s N of the reference nucleophiles. 24d, 30,37 Electrophilicity of natural products Natural products. Cyclic Michael acceptors are frequent moieties in natural products (NPs), such as sesquiterpene lactones. 13,14 We, therefore, set out to assess whether the electrophilicity parameters determined for the simple cyclic Michael acceptors 1-5 (Chart 2) also hold to estimate the correct order of reactivity for analogous, but more complex, natural products. We made use of the reference nucleophiles 6c and 7 to investigate the kinetics of their reactions with different classes of electrophilic natural products with embodied cyclic enone or exo-methylene lactone units (Chart 3). The monoterpenes carvone (19) and verbenone (20) as well as the sesquiterpene nootkatone (21) were used to test the reactivity of naturally occurring cyclic enones. Four sesquiterpene lactones (parthenolide 22, costunolide 23, dehydroleucodine 24, dehydrocostus lactone 25) were chosen to gain insight into the reactivity of exomethylene lactones. Product studies. 1 H NMR spectroscopic studies of the products of the reactions of the natural products 22-25 with the nucleophile 7 indicated exclusive cyclopropanation at the amethylene lactone fragments. Neither was nucleophilic opening of the epoxide ring in parthenolide ( 22) nor cyclopropanation of the 3-methylcyclopentenone moiety in dehydroleucodine (24) observed. However, the cyclopropanations of 22-25 by 7 do not proceed with noticeable stereoselectivity and mixtures of up to four diastereomers were obtained, e.g. with dehydroleucodine (24). Luckily, the reaction of 7 with parthenolide (22) furnished a mixture of only two major diastereomeric products after separation by preparative thin layer chromatography, and single-crystal X-ray crystallography of 26 (arbitrarily taken from the diastereomeric mixture of crystalline material) corroborated the structural assignment on the fundament of our NMR spectroscopic analysis (Scheme 4). Reactivity studies. Direct kinetic measurements in DMSO at 20 C and the determination of second-order rate constants k 2 in analogy to those for the fragments 1-5 require access to sufficient quantities of the electrophilic reaction partner used in excess over the colored reference nucleophiles. The available quantities were sufficient to follow this strategy for the natural products 19-22, and the kinetics of their reactions were studied toward the pyridinium ylide 6c as reference nucleophile. The Mayr electrophilicities E of 19-22 (Table 2) were estimated by substituting the experimental second-order rate constants k exp 2 and the known N and s N for 6c in eqn (1). Competition experiments were performed to estimate the Mayr electrophilicities E of the exo-methylene lactones 22-25 (¼ natural products, NP). The lactone 4b (E ¼ 19.4) was chosen as the competition partner because it contains the entire core structure of the electrophilic moiety in the natural products. As outlined in Scheme 5, 38 the experiments were performed such that the reference nucleophile 7 (generated in solution from 7$HBF 4 with KOtBu) was completely consumed in reactions with an excess of the two competing electrophiles NP and 4b. Consequently, the product mixture contained the remaining electrophiles NP and 4b as well as both products, the respective cyclopropanated natural product CNP (from NP + 7) and 16 (from 4b + 7). The reaction mixture was analyzed by 1 H NMR spectroscopy to determine the competition constant k according to eqn (3). The competition constants k were then used to estimate the E parameters for the electrophilic NPs 22-25 (Table 2). The reactivity of parthenolide (22) was characterized by both approaches. An electrophilicity E ¼ 19.0 was determined from the direct kinetic measurements with 6c and E ¼ 18.5 resulted from the competition experiment (vs. 4b) with the nucleophile 7. Thus, the E values determined by the two different experimental methods agreed within one order of magnitude, and an averaged E ¼ 18.8 is a realistic semiquantitative estimate for the electrophilicity of parthenolide (22). Substituents remote from the electrophilic p-system have only a minor impact on the observed reactivity. Carvone ( 19) is almost as reactive as 2-methylcyclohexenone (2b) and verbenone (20) has a similar reactivity as 3-methylcyclohexenone (2c). Only a signifcant increase of the steric hindrance in the vicinity to the reaction center, for example in nootkatone (21), causes another slight decrease in electrophilicity in comparison with the model fragment 2c. ## Application of electrophilicity parameters in synthesis The levels of electrophilicity derived from the ranking of 1-5 in the Mayr electrophilicity scale (Fig. 2) facilitate assessing the reaction times and experimental conditions required for successful reactions with C-nucleophiles (see comment box in Fig. 2). 24d Usually, reactions with predicted second-order rate constants of k 2 < 10 5 M 1 s 1 (at 20 C) will need catalytic activation, heating or signifcantly extended reaction times to furnish products. In the subsequent summary, the reaction conditions of reported procedures are compared with predictions based on the Mayr-Patz eqn (1). In accord with the determined electrophilicity, tulipalin A (4b, a-methylene-g-butyrolactone, E ¼ 19.4) was reported to undergo high yielding DBU-catalyzed Michael reactions with the nitromethane-(N/s N ¼ 20.7/0.60 in DMSO) (25 to 20 C, 16 h) 39 and 2-nitropropane-derived carbanions (N/s N ¼ 20.6/0.69 in DMSO) (20 C, 48 h). 40 The Michael adduct from the reaction of 4b with the deprotonated diethyl 2-chloromalonate (N/s N ¼ 18.2/0.74 in DMSO) (in THF, r.t., 6 h, 85% yield) was accompanied by traces of the corresponding 4-oxo-5-oxaspiro heptane, generated via a cyclopropanation reaction. This sequence of nucleophilic attack at 4b with subsequent ring closure was exclusively observed when diethyl 2-bromomalonate was used as the pronucleophile in the analogous reaction with 4b (in THF, r.t., 10 h, 75% yield). 41 Furthermore, the piperidine-catalyzed Michael addition of malononitrile (N/s N ¼ 18.2/0.69 in MeOH) at 4b was reported to be facile at ambient temperature in ethanol. The reaction did not stop at the 1 : 1 stage and furnished the two-fold alkylated malononitrile (piperidine cat, EtOH, 2-3 min, product precipitates, 75% yield). 42 The carbon-carbon bond-formation between 4b and the weakly nucleophilic Meldrum's acid-derived enolate ion (N/ s N ¼ 13.9/0.86 in DMSO) is predicted by eqn (1) to be very slow at 20 C (k eqn (1) 2 ¼ 4 10 7 M 1 s 1 ), and effective product formation required phase transfer catalysis and elevated reaction temperatures (TEBA-Cl in MeCN, 50 C, 10 h, 64% yield). 43 Reactions of dehydrocostus lactone (25, E ¼ 19.0) with the anion of nitromethane (N/s N ¼ 20.7/0.60 in DMSO, 90% yield) were carried out under the same experimental conditions as applied for 4b, the core electrophilic fragment of 25. 39 Given the almost identical electrophilic reactivities, it is unsurprising that reported Michael additions or cyclopropanation reactions of a-methylene-pyranone (5b, E ¼ 19.5) cover the same spectrum of carbon-nucleophiles as for 4b. Carbanions generated by deprotonation of diethyl 2-chloroand 2-bromomalonate (for 2-Br-malonate: in THF, r.t., 10 h, 85% yield) 41 and 2-nitropropane (DBU-catalyzed in MeCN, r.t., 4.5 h, 81% yield) 44 were successfully used to functionalize 5b. Michael additions of nitroethane to cyclopentenone (1a, E ¼ 20.6) and cyclohexenone (2a, E ¼ 22.1) under basic conditions were reported. 45 Enantioselective additions of the anion generated by deprotonation of dimethyl malonate (N/s N ¼ 20.2/ 0.65 in DMSO) to 1a were carried out in the presence of a bifunctional amine-thiourea catalysts (toluene, 50 C, 20 h, 84% yield). 46 Alkylations of the cyclic enones 1a, 2a, and 3 (E ¼ 22.0) at their b-positions were also reported when dimethyl malonate was deprotonated by potassium tert-butoxide (in THF, r.t., 92-95% yield) 47a or when 3 reacted with the slightly less reactive ethyl acetoacetate-derived carbanion (in ethanol, 25 C, 21 h, 52% yield). 47b Furthermore, the cyclic enones 1a and 2a were used as substrates for cyclopropanation reactions with the sulfonium ylide generated from trimethylsulfoxonium iodide (N/s N ¼ 21.3/0.47 in DMSO). 48 Elongated reaction times were needed (DBU, CHCl 3 , r.t., overnight, 82% yield), however, when a bicyclic framework was constructed from cyclopentenone 1a with the less nucleophilic sulfonium ylide derived from ethyl (dimethylsulfonium)acetate bromide (N/s N ¼ 15.9/0.61 in DMSO). 49 With the same sulfonium ylide as the nucleophile, the butenolide 4a (E ¼ 20.7) was reported to produce only a poor yield (22%) of the attempted cyclopropanation product (Cs 2 CO 3 , DMF, r.t., reaction time not given). 50 Conjugate additions of silyl ketene acetals (N/s N ¼ 9.0/0.98 in CH 2 Cl 2 for Me 2 C]C(OMe) OSiMe 3 ) to 4a and 5,6-dihydro-2H-pyran-2-one 5a (E ¼ 21.8) required activation e.g. by Lewis acid catalysts to be productive. 51 The highly reactive phenyl lithium reacted with 2-methyl cyclohexenone (2b) through 1,2-addition at the carbon atom of the carbonyl group. 52 The electron-poor olefn 2b (E ¼ 27.5) underwent conjugate additions, however, with the anion of dimethyl malonate (N/s N ¼ 18.2/0.64 in MeOH) in methanol or ethanol after initial heating and long overall reaction times (MeOH, 16 h (ref. 53) and EtOH, 60 C for 5 h + 20 C, 12 h). 54 The Michael addition of nitroethane (N/s N ¼ 21.5/0.62 in DMSO) to 2b was accomplished by deprotonation of the pronucleophile with N,N,N 0 ,N 0 -tetramethylguanidine and stirring the acetonitrile solution for 3 days at ambient temperature (62% yield). 45 Analogous reactions of deprotonated nitroethane with the even less electrophilic 3-methylcycloalkenones 1c (E ¼ 28.9) and 2c (E ¼ 29.6) were carried out under phase transfer catalysis to avoid too long reaction times (K 2 CO 3 /TEBA-Cl in benzene, r.t. for 4 days, 51% yield from 1c). 45 Accordingly, 2c (E ¼ 29.6) requires a reaction time of 9 days for the Michael addition of the diethyl malonate-derived anion (N/s N ¼ 18.2/0.64 in MeOH) in ethanol at ambient temperature (74-76% yield). 55a In an alternative procedure, the diethyl 2methylmalonate-derived carbanion (N/s N ¼ 21.1/0.68 in DMSO) added to 2c under 15 kbar pressure (DBN, MeCN, 45 C, 36 h) in a yield of 50%. 55b The reaction of 2c with the highly nucleophilic lithiated phenylacetonitrile (N/s N ¼ 29.0/0.58 in DMSO, estimated based on data for 2-phenylpropionitrile) 24d delivers within a few minutes the allyl alcohols via kinetically controlled 1,2-addition (90 C in THF). The 1,2-addition is reversible, however, and extended reaction times or slightly higher temperatures furnish the corresponding ketone via the thermodynamically favored 1,4-attack (THF, 60 C, 120 min, 95%). 56 This survey of reported reactions of C-nucleophiles with the cyclic Michael acceptors characterized in this work shows, that the determined Mayr electrophilicities E for the electrophiles 1-5 and the dehydrocostus lactone (25) are well in accord with practical experience in organic synthesis. Structure reactivity relationships. Embedding the cyclic electrophiles 1-5 and electrophilic natural products 19-25 in the Mayr electrophilicity scale makes it possible to compare their reactivities with those of acyclic Michael acceptors (Fig. 2). The analysis in Fig. 3 reveals that cyclization changes the reactivity of enones and a,b-unsaturated esters in a way that is difficult to predict by intuition. Cyclic enones are by 2-3 E units weaker electrophiles than acyclic b-substituted enones. The opposite trend is observed for lactones: a,b-unsaturated lactones 4-5 are more reactive by 2-3 E units compared to their acyclic counterparts. We performed quantum-chemical calculations to rationalize these antipodal reactivity trends. ## Quantum chemical calculations Energy profles. To gain further insight into the observed reactivity ranking and structural factors that influence the observed reactivity of cyclic Michael acceptors, we calculated the reaction profles for the addition of the sulfonium ylide 7 to the electrophiles 1-5 at the SMD(DMSO)/M06-2X/6-31+G(d,p) level of theory using the Gaussian software package. 57 As depicted in Fig. 4 for the reaction of 7 with cyclopentenone 1a, zwitterionic intermediates IM are generated in the frst step of the reaction mechanism (via TS1). The newly formed C-C bond connects two stereocenters, and the reaction can proceed through a cis-and a trans-attack. As displayed in Table 3, the computational results indicate that the trans-attack is slightly favored over the cis-attack for the cyclic Michael acceptors, except for the 2-and 3-methyl substituted electrophiles 1b, 2b and 2c. In general, however, the computed differences between the cis-and the trans-pathways are small, in accord with the experimental observation that mixtures of diastereomeric products were isolated in moderate yields (Scheme 2). Hence, we refrained from interpreting the stereoselectivity of the cyclopropanation reactions and used the most favorable pathway for our subsequent analyses (if not stated otherwise). In the fnal step, an intramolecular S N 2 reaction eliminates dimethyl sulfde from IM via TS2 to yield the highly exergonic products, namely, dimethyl sulfde and cyclopropanes with cisor trans-confguration. For all entries in Table 3, the relative Gibbs activation energies for TS1 and TS2 indicate that the addition (via TS1) is the rate-determining step in the reactions of 7 with 1a. As shown in Table 3 and graphically in Fig. 5A, the quantumchemically calculated activation barriers DG ‡ (TS1) agree reasonably well (AE11 kJ mol 1 ; mean deviation: AE3.3 kJ mol 1 ) with the experimental DG ‡ determined either by experiment (k exp 2 ) or by utilizing eqn (1) (k eqn (1) 2 ). Accordingly, there is also a reasonable correlation of DG ‡ (TS1) with the electrophilicity parameters E from Table 1 (Fig. 5B). Enone conformation. If compared to analogous acyclic Michael acceptors, the cyclic enones studied in this work experience a signifcantly reduced conformational flexibility. Experimental electrophilicities were so far only determined for (E)-confgured acyclic Michael acceptors. However, relevant information about the reactivity of (Z)-confgured conformers, which is required for the discussion of stereoelectronic effects in cyclic enones, is missing. To get insights into the effects of locked conformations on transition state energetics, we set out to perform quantum-chemical calculations for the reaction of 7 with both (E)-and (Z)-pentenone. As discussed by Bienvenüe on the basis of UV and IR spectroscopic data, (E)-and (Z)-enones exist in both the s-trans and scis form owing to the hindered rotation around the central carbon-carbon s-bond (Fig. 6, top). 58 Experimental data 58 as well as computations (this work) agree that for (E)-pentenone both s-cis and s-trans conformers are of comparable energy. For (Z)-pentenone, however, the calculations indicate a signifcant preference for the s-cis form (cis/trans ¼ 93 : 7). We then computed Gibbs energies for the transition states of the addition of 7 at both (E)-and (Z)-pentenone. We found that the s-cis conformers of (E)-and (Z)-pentenone both react with 7 via lower energy barriers than the respective s-trans conformers. As shown in Fig. 6 (bottom, left), the transition state energy for the s-cis-(E)-pentenone is 8.6 kJ mol 1 lower than that for the strans-(E)-conformer. The difference between the transition states for s-trans-(Z)-and s-cis-(Z)-pentenone amounts to 5.0 kJ mol 1 (Fig. 6, bottom, right). When the most favored transition states for (E)-and (Z)-pentenone are compared, the (E)-isomer of pentenone can be expected to be by approximately one order of magnitude more reactive than the (Z)-confgured isomer (DDG ‡ ¼ 7.6 kJ mol 1 ). Furthermore, the calculations suggest that the experimentally characterized (E)-pentenone reacts via the s-cis transition state (DG ‡ ¼ 69.7 kJ mol 1 ) with nucleophiles (such as 7). Conformationally locked cyclic species, such as 1a or 2a, adopt transition states similar to the unfavorable s-trans pathway for (Z)-pentenone (DG ‡ ¼ 82.3 kJ mol 1 ). Thus, we can roughly estimate that cyclic enones are at minimum by two orders of magnitude less reactive than analogously substituted a,bunsaturated open-chain ketones. The Mayr E values for (E)pentenone (E ¼ 18.8), cyclopentenone (1a, E ¼ 20.6), Ester vs. lactone. Due to the analogous conjugated p-systems of unsaturated ketones and esters, (E/Z)-confgurations and scis/s-trans conformations should influence the reactivity of esters in a similar manner as in ketones. Counterintuitively, (Z)lactones are more electrophilic than their open-chain ester analogs with (E)-confgured CC double bond (cf. Fig. 2), and other stereoelectronic effects seem to dominate their reactivity. In line with the relative reactivity ranking in our work, lactones are well-known to undergo signifcantly faster alkaline hydrolysis than acyclic esters. This fnding was explained by unfavorable orbital interactions in the transition state 59 or through differences in the dipole moments leading to ground state destabilization of (Z)-confgured ester units. 60 More recently, stereoelectronic effects were suggested to explain the higher reactivity of unsaturated lactones. 61 Fig. 4 Gibbs energy profile for the reaction of 7 with cyclopentenone (1a) at the SMD(DMSO)/M06-2X/6-31+G(d,p) level of theory (see ESI, Fig. S1, † for a distortion/interaction analysis). Table 3 Quantum-chemically calculated energy profiles (in kJ mol 1 ) for the addition of the sulfonium ylide 7 to the electrophiles 1-5 at the SMD(DMSO)/M06-2X/6-31+G(d,p) level of theory Trans-pathway Cis-pathway For acyclic esters, the s-(Z) conformation is generally preferred, in which the n / s* interaction donates electron density from the oxygen lone pair into the antiperiplanar antibonding s * CO orbital (Fig. 7). This negative hyperconjugation reduces the electron-defciency of the p-system and, in consequence, electrophilicity. In contrast, the locked s-(E) conformation in lactones impedes such a transfer of electron density and gives rise to an unattenuated electrophilic reactivity of the conjugated p-system (Fig. 7). 61 The oxygen atom of the alkoxy group can affect the reactivity of the p-system only through a minor inductive effect. In line with this interpretation, the quantum-chemically calculated transition state structures for the addition of 7 at the ketone 2a and the lactone 5a are highly similar in geometry and energetics (Table 3, Fig. 8) in agreement with the almost identical experimentally determined second-order rate constants k exp 2 for both reactions (Table 1). The unsaturated lactones 4b and 5b bearing an exo-methylene group are more electrophilic than the lactones 4a and 5a with endocyclic unsaturation. The higher reactivity can be attributed to the favorable interplay of two effects. First, the s-trans geometry is locked in lactones 4b and 5b in both the reactants and the transition states. Additionally, we assumed that the absence of substituents at the site of nucleophilic attack introduces less steric constraints in 4b/5b than in 4a/5a. To assess this hypothesis, a distortion interaction analysis (DIA) 62 was performed, which compared the transition states of the frst step in the reactions of the S-ylide 7 with 2a, 5a, and 5b, respectively (Fig. 8A). While the distortion energy of ylide 7 is identical in the reactions with 2a, 5a and 5b, the distortion energy of the electrophile is signifcantly lower for 5b than for 2a or 5a. It can be expected, that variable demand for geometrical changes at the electrophiles' reactive carbon atom upon C-C bond formation is key for the observed distortion energy difference in the comparison of 5b vs. 5a. We used the distance of the attacked C-atom of the electrophile from the plane defned by the three surrounding atoms in the transition state, as depicted in Fig. 8B, to describe the degree of pyramidalization D in the transition state. In line with Hine's principle of least nuclear motion (PLNM), which predicts 'that those elementary reactions will be favored that involve the least change in atomic position', 63 we observed a higher requirement for pyramidalization in the transition state of the reaction of 7 with 5a (D ¼ 0.227 , distortion energy: +39.7 kJ mol 1 ) than in the analogous transition state for the faster reaction of 7 with 5b (D ¼ 0.174 , distortion energy: +34.3 kJ mol 1 ). Let's now analyze the higher interaction energy in the reaction of 7 with 5b (40.6 kJ mol 1 ) than in the reaction of 7 with 5a (33.1 kJ mol 1 ). It has previously been shown, that interaction energies can be further decomposed by energy decomposition analysis (EDA). 64 In this work, we applied symmetryadapted perturbation theory (SAPT) at the sSAPT0/jun-cc-pVDZ level of theory, which decomposes an interaction into its electrostatic, exchange, induction, and dispersion components. 64 The SAPT analysis was performed in gas-phase with an entirely different theoretical method and, therefore, absolute numbers of the interaction energies differ from the results of our DFT method. Nevertheless, we expected the relative trends to hold. As shown in Fig. 8C, the interaction energy (DE sSAPT0 ) for 5b is generally more negative than for 5a. Depending on the extent of bond formation, this is due to different origins. (1) During the approach to the transition state (located at 2.20 ), it is the stronger electrostatic interaction that favors 5b over 5a. (2) At the transition state and in the further course of the reaction, however, the induction component becomes the decisive factor. In the transition state, the LUMO energy of the distorted 5b is lower (3 LUMO ¼ 0.03527 Hartree) than that of the distorted 5a (3 LUMO ¼ 0.03309 Hartree) while the HOMO energies of 7 are essentially identical (with 5b: 3 HOMO ¼ 0.21875 Hartree; with 5a: 3 HOMO ¼ 0.21867 Hartree). The smaller energetic gap for 7 + 5b (4.99 eV) indicates a more favorable HOMO-LUMO interaction for the couple 7 + 5b than for the combination 7 + 5a (5.05 eV), in accord with the relative DE induction for 5b and 5a in the SAPT analysis. Effects of 2-and 3-methyl substitution. Methyl substituents in the aor b-position strongly influence the reactivity of enones. In a similar but more distinct way than in acyclic systems (Fig. 9A), 25,27 alkyl substitution of the C]C double bond drastically lowers electrophilicity of cyclic enones. A methyl group in the a-position reduces the electrophilicity E of cycloenones by 2 to 6 units (cf. Fig. 2). For substituents placed in the b-position this effect is even more pronounced: the reactivity of b-methyl cycloenones 1c and 2c is reduced by approximately 8 units on the Mayr E scale if compared to the unsubstituted analogs 1a and 2a, respectively. The retarding effect of aand balkyl substituents at the cyclic enones may be caused by steric constraints and/or the electron donating ability of the alkyl group. Again, DIA was used to quantify the effects. To keep the transition-state conformations comparable (Fig. 9B), the trans-TS for the reaction 1b + 7 was evaluated in the DIA instead of the (by 1.6 kJ mol 1 ) preferred cis-TS. As the C-C bond length in the transition state of the reaction 1a + 7 differs from that of the reaction 1b + 7, the entire pathways of the reactions of 7 with 1a, 1b, and 1c, respectively, were analyzed. The positions of the distortion and interaction energy curves of 1c (Fig. 9C) and 1b (Fig. 9D) relative to those of 1a reveal the reasons responsible for the reduced reactivity in both cases. Let us frst discuss the effect of 3-substitution (Fig. 9C): the distortion energy for 1c is signifcantly more positive than that for 1a while the interaction energy is slightly more negative for 1c than for 1a. As the C-C bond lengths in the transition states are similar for the reactions of 7 with the 3-substituted cycloenones and their unsubstituted analogs, these observations can also be assessed in a DIA of the respective transition state geometries of 1a and 1c (and analogously for 2a and 2c). As shown in Fig. 9E, the signifcant decrease of reactivity of b-substituted enones is mostly due to an increase of the distortion energy in both the enone fragments and the ylide 7. As previously discussed for 5a and 5b, pyramidalization D and Hine's PLNM can be utilized to rationalize the higher distortion energies of the 3-methyl substituted cycloenones. The reaction of 1a with 7 requires a minor extent of pyramidalization (D ¼ 0.222 ) than for the much less electrophilic 1c (D ¼ 0.303 ). 65 Moreover, the higher distortion energy of 7 in the reaction with 1c than in that with 1a can be rationalized by comparing the structures of the transition states (Fig. 9B). Different from the transition states for the reactions of 7 with 1a or 1b, the SMe 2 group of 7 is rotated in the transition state of the reaction with 1c to avoid a clash with the methyl group of the electrophile. Also 2-substituted cyclic enones were found to be weaker electrophiles than the unsubstituted analogs, though, for a different reason. The distortion energy curves for 1a/1b (Fig. 9D) are highly similar or, for 2a/2b (not depicted) even indicate a lower distortion component for 2b. Hence, the nucleophilic b-attack is not sterically hindered by the presence of an a-methyl group. However, the reaction path for 1b suffers from a slightly less negative interaction energy than for the analogous reactions of 7 with 1a. 66 Analysis of the involved HOMO/LUMO interactions of the fragments in the transition state resulted in a slightly stronger orbital interaction in the reaction of 7 with 1a (5.14 eV) than in the reaction of 7 with 1b (5.19 eV). Moreover, Hirshfeld atomic charge analysis of the fragments showed that in the transition state the reactive center of 1b (+0.0391) is less positively charged than that of 1a (+0.0472), presumably due to the electron-donating effect of the methyl group in 1b (ESI, Fig. S3 †). ## Rate constants toward glutathione (GSH) Helenalin is a sesquiterpene lactone isolated, e.g., from Arnica montana, which embodies two different electrophilic units. 67 GSH was reported to attack faster, yet reversible, at the cyclopentenone moiety of helenalin than at the a-methylene butyrolactone part. 68 This kinetic preference differs from the ordering of electrophilicities derived from our measurements, which predict higher reactivity for the unsaturated lactone from E ¼ 19.4 for 4b and E ¼ 20.6 for 1a (Fig. 10). We, therefore, set out to evaluate whether the electrophilicity parameters E for the cyclic Michael acceptors, which we determined from their reactions with carbon-centered nucleophiles in DMSO solution, would enable us to also predict their reactivity toward glutathione (GSH) in aqueous solution. The rate constants for the reaction of GSH with the selected electrophiles were measured in aqueous, buffered solution at pH 7.4 by utilizing a modifed bioassay. Typically, eqn (1) allows one to calculate second-order rate constants within a precision of factor <100 for reactions in which one new s-bond is formed. Table 4 shows that eqn (1) estimated the second-order rate constants for the additions of GSH at cyclopentenone (1a), cyclohexenone (2a), the dihydropyranone 5a and the a-methylene-pyranone 5b within a factor of 20. For 2-methyl-cyclopentenone (1b) and the exoand endocyclic lactones 4a and 4b k exp 2 and the calculated k eqn (1) 2 agreed within a factor of 2. It can, thus, be concluded that the general reactivity pattern of cyclic electrophiles toward GSH is represented by their E parameters. In agreement with previous studies by Schmidt on the dual electrophilicity of helenalin toward GSH, 67,68 we determined k GSH (1a) > k GSH (4b). Hence, we have to note that small reactivity differences within one or two orders of magnitude are not unequivocally resolved by the simple three-parameter eqn (1). Changing the experimental method for determining the kinetics, swapping from a C-to an S-centered reference nucleophile as well as the neglect of constraint conformational space in natural products by the fragment approach may twist the relative reactivity order of similarly reactive Michael acceptors. Also the influence of solvents on the reactivity of carbonyl compounds needs further investigation. The relative position of the lactone 5a (E ¼ 21.8) and (E)pent-3-en-2-one (E ¼ 18.8) or (E)-hex-4-en-3-one (E ¼ 18.9) in the electrophilicity scale (Fig. 2) is in accord with the preferential binding of the ketone unit of rugulactone by nucleophilic sites in the course of covalent enzyme inhibition. 17 This illustrates that DE > 2.5 enables a safe prognosis of the reactive site in a natural product with dual electrophilicity. ## Conclusions In summary, sulfonium and pyridinium ylides were utilized as one-bond reference nucleophiles in kinetic experiments to characterize the Mayr electrophilicity parameters E for various cyclic enones and a,b-unsaturated lactones in DMSO at 20 C. By combining the electrophilicity parameters E with tabulated nucleophilicity descriptors N (and s N ) eqn (1) can be used to predict the rate constants for the reactions of 1-5 with various C-nucleophiles, as demonstrated by comparison with reported synthetic protocols. Most valuable, the reactivities of cyclic core fragments of the Michael acceptors 1-5 agree with the observed electrophilicities of natural products (terpenes) of more complex structure and considerably higher molecular weight that contain the same reactive moiety. The distinct different reactivity of cyclic enones and unsaturated lactones compared to their acyclic analogs was analyzed by quantum-chemical calculations, distortion interaction analysis, and by considering stereoelectronic effects. The most important structural effects on the reactivity of a,bunsaturated carbonyl compounds are summarized in Fig. 11. The locked conformations of cyclic Michael acceptors have a signifcant impact on their electrophilic reactivities. If compared to analogous open-chain enones, the electrophilicity of cyclic enones is signifcantly reduced by the fxed (Z)-geometry of the s-trans confgured p-system. Alkyl groups in either aor b-position of the cyclic enones further attenuate the electrophilicity of cyclic enones by positive inductive effects and steric bulk in vicinity or directly at the electrophilic reaction center. Thus, b-alkylated cyclohexenones are among the least electrophilic species characterized so far in Mayr's reactivity scales. 24 In contrast, the rigid cyclic structures of a-methylene-gbutyrolactones facilitate synergistic stereoelectronic effects which favorably combine with a lack of steric hindrance at the reactive site to furnish a privileged class of highly potent electrophiles. In contrast to simple alkyl acrylates of comparable electrophilic reactivity, the cyclic scaffold of sesquiterpene lactones can be loaded with stereochemical information needed for recognition processes and selective reactions in living organisms. It is, therefore, not surprising that plants have chosen a-methylene-g-butyrolactones as most abundant electrophilic fragment in biologically active sesquiterpene lactones. The reactivity parameters determined in this work, together with those of previously characterized acyclic Michael acceptors, now provide an extensive basis for the systematic development of reactions with various classes of nucleophiles. Derivatization of natural products with the studied electron-defcient cyclic core fragments can in future be exploited in a more straightforward manner, thus saving limited natural resources, energy, and human effort. Knowledge of the electrophilic potential of these cyclic Michael acceptors to undergo

chemsum

{"title": "Electrophilic reactivities of cyclic enones and \u03b1,\u03b2-unsaturated lactones", "journal": "Royal Society of Chemistry (RSC)"}

modeling_variance:_a_variance-motivated_approach_to_molecular_prediction

## Abstract: Using machine learning to predict molecular properties is an exciting research area at the interface of computer science, statistics, chemistry, and physics. Thus far, a great deal of work has been done on training various ML models to predict ground state energy, using the so-called 'Coulomb matrix', a global molecular descriptor as inputs. In this variance-motivated study, the variance of a multi-thousand molecular dataset of Coulomb matrices is analyzed; a variance analysis. This paper presents novel statistical methods and models that can aide in prediction and analysis of molecular properties and molecules using the Coulomb matrix. Analysis is performed after a detailed literature review of molecular prediction and Normality of data assessment. It is also hoped that the models introduced in this variance analysis can be generalized to other areas of interest. ## INTRODUCTION Quantum mechanics has been combined with machine learning to predict molecular properties. 1 Various ML models for this purpose have been introduced 1,2,3 ; they generally share one thing in common: models are trained to predict ground state energy. One common input for such an ML model is the Coulomb Matrix (CM), made popular by Dr. Matthias Rupp. CM is a global 3D molecular descriptor. 1D molecular descriptors capture composition, 2D captures the molecular graph, and 3D captures shape. Global descriptors characterize the entire molecule. CM is an alternative to using the Schrodinger equation to determine molecular properties, which is rather cumbersome. Using the Hamiltonian, a component of the Schrodinger equation, and the Schrodinger equation to determine molecular properties is the classical quantum mechanical approach. The Coulomb matrix is a square and symmetric matrix with size equal to the number of atoms in the molecule, squared. It represents electronic interactions of the atoms of a molecule with themselves and each other. Diagonal elements represent a polynomial fit of atomic energies to nuclear charge, hence, they represent the fitted atomic energies of the atoms. Off-diagonal elements represent Coulomb repulsion between atoms. As can be seen in figure 1, the number of elements is the number of atoms, squared, hence there are 9 elements. The diagonal elements represent an atom interacting with itself, which is represented in the CM as a polynomial fit: .5*Zatom 2.4 . Where Z is the atomic number of the atom. Hence element 1,1 (row 1, column 1) has a fitted atomic energy of 36.86. All elements that are not diagonal represent Coulomb repulsion, as stated earlier; the numeric representation is: ZIZJ/|RI-RJ|, where RI-RJ is the distance between atoms I and J. It has been shown computationally 1 that a plot of an ML model that used the Coulomb matrix as inputs, based on thousands of molecules, showed a 1:1 relationship between predicted atomization energies and reference atomization energies. The model was said to be a better predictor of atomization energy than semi-empirical quantum chemistry. The energy estimates used in the ML model are a weighted Gaussian sum. Molecules used in the study were from a Molecular Generated Database (MGD) of nearly 1 billion stable organic molecules. Regression tree algorithms have also been used to predict ground state energies 2 , using CM as input-"features". Using a dataset that contained over 16,000 molecules, ground state energies were computed on trained ML models. The model, boosted regression tree, was shown to have increased accuracy and reduced computational cost. The computational based study has potential applications in molecular discovery and informatics. The actual prediction is pseudo-atomization energy, which is a function of ground state energy and pseudo atomic energy. The absolute value of pseudo-atomization energy for the sample size of 16,242 molecules was shown to have a Normal distribution. Not all seek alternatives to solving the Schrodinger equation for molecular prediction. AFLOW 4 , a high throughput (HT) framework uses ab initio calculations to solve the Schrodinger equation to yield information such as energies and electron densities. AB initio calculations use only restrains and coulomb interactions as inputs. AFLOW is also used to calculate crystal structure properties of alloys, inorganic compounds, and intermetallic compounds. AFLOW is designed to run atop structure energy software (DFT is common). After selection of starting structures from a database, AFLOW adjust lattice parameters, and creates an input file with all parameters necessary for relaxations, and static and bond structure runs. AFLOW computes structure total energies and electronic bond structures. It also can perform Monte Carlo calculations, generate nanoparticle structure files, and identify interstitial sites inside any crystallographic structure (from an input of atomic positions). Whether AFLOW, which uses classical quantum mechanics, or the CM is used, both can perform powerful molecular characterizations. Both however require a detailed and descriptive list of inputs to function. In addition to using the CM in ML to predict atomization energies, it has also been stated that the CM can be used to for rotational spectra interpretation 3 . In the computationally driven study revolving around molecular isomerism, the author used an ETKDG algorithm to generate 1000 random conformations of 309 isomers. The prediction task was to distinguish between a given isomer from all other isomers. The misclassification rate was defined as proportion of incorrect assigned labels (incorrectly identifying an isomer). Various ML models were used: logistic regression, decision tree, random forest, gradient boosted trees, support vector machine, and k-nearest neighbor. Models were trained and cross-validated. The decision tree ML model had the highest misclassification rate and support vector machine had the lowest. The mean largest eigenvalue of CM (averaged over conformers) was said to be inefficient at distinguishing isomers. Now it's time to discuss a blended computational-laboratory study on the using ML to label the degree of peptide reactivity with chemicals that contain allergens 5 . Chemical allergens react with proteins, inducing skin sensitization 5 . The majority of allergens are electrophilic and react with nucleophilic amino acids. The purpose of the study was to determine whether and to what extent reactivity correlates with skin sensitization potential. They evaluated 82 allergen containing chemicals (of different potencies) and non-allergen containing chemicals for their ability to react with amino-acid containing molecules. After a set reaction time, UV detection was used to quantify the depleted amount (reacted amount). The reactivity data and existing data was used to build a classification tree that allowed ranking of reactivity: minimal, low, moderate, high. The classification tree ML model had 89% prediction accuracy (based on cysteine and lysine amino acids). The splitting rule for the tree was based on average peptide depletion exceeding a threshold. Thus far various ML models have been presented that predicted molecular properties. In addition, a non-ML (quantum mechanic) model was presented (AFLOW) that had a much broader spectrum of molecular characterization, though it relied heavily on input files. Predictions for the ML models were usually limited to atomization energy, though several other molecular predictions were presented as well. Isomerism was predicted via comparison of various ML models and the reactivity of chemicals containing allergens was predicted using classifications trees. Except for the last study and AFLOW, all others presented used the CM as input for the ML model. One may wonder how this relates to This study of CM variance. The purpose of This study is to model the variance of the fitted atomic energies for a random sample of molecules from a large molecular database, in an effort to make inference to broader populations of molecules, using current and novel models. The goal also is to use smaller and simpler inputs to predict molecular properties. To analyze and model variance, parametrically, it is necessary to assign a distribution to variances of fitted atomic energies. Of note is the fact that fitted atomic energy variance is not the same as sampling variance. To begin, let's briefly review some literature on characterizing and assessing probability distributions and data. The Normal distribution is a common distribution for modeling data, especially of a large size. Hence, it can model and statistically summarize Normal data. Common methods to assess normality are graphical, usually the data quantiles are plotted against the standard Normal quantiles. Another common test, that is non-graphical is the Shapiro-Wilk test, which is generally only recommended for sample sizes 50 and under. Box-plots can also be used as a simple quantile graphical tool to assess normality. A histogram plot of the data is yet another common graphical method for assessing normality. It is often that analysts will use these graphical tools to deem data that 'roughly' fits as normal or 'approximately Normal'. Let us look at a study that presents an alternative graphical method to assess Normality that was compared with various tests using Monte Carlo testing 6 . The results of the study suggest a potential more evident way to reject un-Normal data. According to the study, in finance literature, a plot of empirical and fitted normal densities on the log scale is regularly preferred as a graphical means to assess normality. The study argues that interpretation of quantile-quantile plots can be compromised; assessing degree of curvature in the plot to designate data as not Normal is largely subjective. They then discuss a graphical alternative, the logdensity (empirical density) plot. The graphical procedure involves plotting empirical density alongside fitted Normal density. The log-scale is used to clearly display the tails of non-Normal data. One weakness of this technique is that it was said to be possibly misleading for small sample sizes (which shouldn't be a surprise). In the study, a 1,000 simulation Monte Carlo test was performed to simulate a p-value for the log density method, which was compared to other Normality assessing tests (Shapiro-Wilk, Anderson-Darling, and Cremer von-Mises). Power was used to compare the various tests. Data from three distributions were used: Cauchy, t(with 6 degrees of freedom), and Gumbel (extreme value). In all but the extreme value distribution, the Monte Carlo log density test had the highest power. The author argues this is due to the sensitivity of the Monte Carlo Log Density to fat-tailed departures from normality, but being weaker for skewed (extreme value) distributions. Another test comparison between quantile-quantile plots and log density plots showed a Normal appearing quantile-quantile plot, but an obvious non-Normal log density plot. One of the biggest drawbacks of the log density method presented in the study is that it is cumbersome to compute empirical density, hence most would likely prefer the more easily implemented quantile-quantile plots for approximating normality. A brief literature review of skewness 7 will conclude the data characterization review. The paper is essentially a review of skewness and argues in favor of incorporating it into statistical education. There are various ways to assess skewness, graphical and quantitative. One quantitative method is in the form of a skewness statistic, which compares the mean to the median. Visual tools to assess skewness include beam-and-fulcrum plots, boxplots, and dotplots; dotplots being deemed the best visual tool to assess skewness. The Fisher-Pearson coefficient of skewness, measures skewness and is the ratio of average distance cubed from the mean to average distance squared from the mean (the denominator is raised to the 3/2 power). Of note is that the statistic presented in the paper consists of sample distance from the mean; the mean being the sample average. The brief review of skewness was necessary, because it is often the case that data exhibits non-Normal deviation in the quantile-quantile plots at the extreme ends (tails), and it is necessary to have a means to measure tail deviation as significant skewness, or not. Now that the literature review has been completed, it is time to discuss the experiments and data analysis. ## COMPUTATIONAL METHODS The QM7 database 8 was used to obtain the CMs. The dataset contains 7165 molecules and is a subset of the GDB-13 database (contains nearly 1 billion stable organic molecules). QM7 contain molecules no larger than 23 atoms, and contains the CMs for each molecule and atomization energies. ML models commonly aim to predict the atomization energies using CMs as input. CMs were stored as row elements in the QM7 database, so it was necessary to regenerate the CMs in matrix form. A random sample of size 500 was taken from the 7165 molecule database, each sample corresponding to a molecule (stored as a CM and atomization energy). Atomic energies (diagonals of CM) were extracted for each CM in the sample. Mean atomic energy and variances were computed for each molecule and stored in vectors. Only molecules with variance of no more than 1000 (units: protons 5.76 ) were considered, reducing the sample size to 477. Unless noted otherwise, all calculations are rounded to the nearest whole number. Unless stated otherwise, variance will refer to sample variance. Unless stated otherwise, atomic energy refers to CM model fitted atomic energy (.5Z 2.4 ) ## RESULTS AND DISCUSSION The density histogram of atomic energy (fitted) variance of the random sample of size 477 molecules is shown in Figure 2. The sample mean for this distribution is 562 protons 5.76 and the corresponding standard error is 118 protons 5.76 . Note that the units here are the same, because the measured quantity is variance. The sample median for this distribution (557 protons 5.76 ) is very near in value to the sample mean, indicating the symmetry of this distribution and providing good justification to further assess normality of data. ## Figure 2. Density histogram of atomic energy variance for the 477 molecule sample Figure 3 shows the density histogram of the mean fitted atomic energies for the random sample of size 477. For this distribution, the sample mean, median, and variance are 20 protons 2.4 , 20 protons 2.4 , and 19 protons 5.76 . Though the mean and median are equal, the distribution is visually not symmetric with high variance relative to the sample mean; they are approximately equal. Both distributions are visibly continuous. Figure 4 shows the density histogram of atomic energy variance with fitted normal density superimposed. From the histogram, the Normal distribution is a good approximation of the sample data. To further assess normality of atomic energy variance of the sample, a quantile-quantile plot was graphed (Figure 5), which showed bulk normality, but normality deviation for lower and higher quantiles. To assess normality further, an algorithm was created that superimposed Normal density over the density of atomic energy variance. The Normal density represented a population of atomic energy variance with mean(atomic energy variance) equal to the estimated mean (562 protons 5.76 ) and standard deviation equal to estimated standard deviation (128 protons 5.76 ); note that the units are the same due to the measured quantity being variance. The size of the population was set equal to the original population size of the QM7 dataset, 7,165 molecules. Each simulation (algorithm run) superimposed randomly generated Normal density with mean variance and standard deviation given by the estimates. The density histogram of a simulation is shown in Figure 6. The results further support that the Normal probability distribution is a good model to summarize and approximate the atomic energy variance sample data. Next, an alternative model to classify data as approximately Normal will be introduced. The model is a combination of a classification tree and statistical hypothesis testing, and will be referred to as TDT (Test-performing Decision Tree). Figure 7 displays the decision tree. The advantage of the test over visual (subjective) methods to assess and 'measure' normality is that TDT performs a hypothesis test for Normality. The test ends in a composite test statistic, which is used to make a decision that the data is Normal or not Normal. The test is also fully automated; user interpretation is not a component of the test. Note that Normal refers to being Normal from an input population (or parent data). The decision tree splits are based on the data proportions of a Normal distribution: 68% of the data falls within 1 standard deviation units of the mean, 95% of the data falls within 2 standard deviation units of the mean, and 99.7% of the data falls within 3 standard deviation units of the mean. The actual acceptance criteria for proportions is relaxed, since the TDT tests for approximate Normality: 58% to 83% of the data must fall within 1 standard deviation unit of the mean, 85% or more of the data must fall within 2 standard deviation units of the mean, and 95% or more of the data must fall within 3 standard deviation units of the mean. The test tallies the counts in each class and computes proportions. The proportions form the composite test statistic. Data is deemed Normal only if the proportions fall within the percentage ranges discussed prior. TDT performs a hypothesis test, the null hypothesis is that the Data is Normal (with mean equal to the population mean and standard deviation equal to the population standard deviation). The alternate (research) hypothesis is that the data is not Normal (with mean and standard deviation equal to that of the population). The model inputs are the sample and the parent sample (or population). To assess the strength of the model at correctly identifying Normal data from a given population as Normal, 10,000 simulations were performed to measure type 1 error (rejecting the null hypothesis when the null hypothesis is true). TDT inputs were a population (size 7,165) of randomly generated realizations from the standard normal distribution and a random sample (size 477) from the population. The sizes of the population and sample correspond to the size of the QM7 dataset and variance random sample used in This study. Three iterations of 10,000 simulations were performed, each having a type 1 error rate of 0. TDT was then used on the atomic variance sample of interest, which classified the data as Normal. Figure 8 shows the R output of the TDT test. test statisticA, p_1sd, is: 0.6771488 .test statisticB, p_2sd, is: 0.9979036 . test statisticC, p_3sd, is: 1 . Accept null. X ~ Normal(mean_population,sd_population) ## Figure 8. Output of Normality classification algorithm, classifying 477 size atomic energy sample as Normal The next part of the analysis is determining if the atomic energy variance can be modeled as a linear function of mean atomic energy. Figure 9 is a scatter plot of the 477 molecule sized sample. ## FIGURE 9. Scatter plot of atomic energy variance against mean atomic energy for the 477 sized random sample The plot shows a clear positive correlation between atomic energy variance and mean atomic energy, however it also shows a fanning pattern and it is not hard to see that a line fit through the data would have residuals of nonconstant variance, a violation of linear regression. To reduce the fanning so that a linear model could be fit to the data, only molecules with a maximum variance of 350 protons 5.76 were considered, shrinking the sample size to 25. Of the size 25 random sample, only 8 data points were unique, in other words multiple molecules in the sample shared the same atomic energy variance and mean atomic energy. This is understandable, since the population (QM7 dataset of 7,165 molecules) contain small organic molecules, isomerism will result in numerous molecules having the same CM diagonals (fitted atomic energies), thus they will have the same mean atomic energies and same atomic energy variance. The linear model that will be discussed next consists of the 8 molecules with unique CM diagonals; isomers are not included. Figure 10 shows the scatter plot, showing a linear relationship between atomic energy variance and mean atomic energy. Figure 11 is the same plot with the best fit regression line shown. Figure 12 shows the R output of the linear model, and figure 13 is a plot of the residuals against mean atomic energy. To summarize the regression results, for a random sample of 8 small organic molecules with maximum atomic energy variance of 350 protons 5.76 , the atomic energy variance can be expressed as a linear function of mean atomic energy. Though the sample consists only of 8 molecules, the results support that for a small number of molecules with low atomic energy variance, the mean atomic energy can be used to predict atomic energy variance, and may potentially be an alternative molecular descriptor (for a small number of molecules). The final portion of the data analysis concludes with variance visualization. Though commonly used to characterize data in statistical analysis, visualization of variance is not regularly used in statistical analysis and data visualization. This portion of the analysis presents a model to visual variance. Atomic energy sample variance is known for each of the 477 molecules in the random sample of interest. Sample variance is the average squared distance from the sample mean; emphasis here is put on the fact that it is a distance. The distance between two points on a line can be calculated as the hypotenuse of a triangle: c=(a 2 +b 2 ) 1/2 , where a is the horizontal segment length between the two points and b is the vertical segment length between the two points. The variance visualization technique involves computing the vertical component of sample variance for a fixed horizontal component (this set represents point B), then plotting a line containing 0,0 (point A) and point B. The length of this line is the sample variance. The variance, represented as a vector, is translated to the origin (all variance vectors use the origin as point A). Figure 14 is a plot of a random sample of size 30 from the atomic energy variance sample (size 477). The mean, median, and standard deviation of this sample is 584 protons 5.76 , 583 protons 5.76 , and 121 protons 5.76 , which are near in value to the values of the parent sample. The horizontal component of the variance was fixed at 1. Figure 14 shows the distribution of variance, visualized as vectors representing atomic energy sample variances. Figure 15 ## Conclusion Variance was modeled for a random sample of molecules from the QM7 database. Modeled variance represented fitted atomic energy variance. A rigorous assessment of normality was performed using simulation and multiple visualization methods, including quantile-quantile plots. An alternate model to assess normality was introduced, the TDT (Test-performing Decision Tree), which computes a test statistic for accepting or rejecting data as Normal based on proportion amounts of data that are within 1,2, and 3 standard deviations of the mean. TDT is a combination of statistical hypothesis testing and machine learning/statistical learning classification trees that uses sample and population data (or parent sample data) as inputs. Three iterations of 10,000 simulations were performed using TDT, each resulting in a type 1 error rate of 0. TDT classified the fitted atomic energy variance sample as Normal. TDT is a fully automated model for assessing normality that is independent of user interpretation. Linear regression was performed on a small random sample and showed that atomic energy variance can be expressed as a linear function of mean atomic energy. This will be further explored to see if mean atomic energy can be used as a molecular descriptor for some molecules, which would greatly reduce the dimensions of molecular datasets like the one in This study. For future analysis, similar groups will be considered from QM7 (example atmospheric or greenhouse gases, greenhouse related gases) and linear regression will be reattempted. The variance-driven analysis concluded with the introduction of a variance visualization nonparametric model, which provided visualization of variance about a fixed location (the origin). It is hoped that the models in this paper will be considered and used to augment statistical analysis, in numerous areas of application.

chemsum

{"title": "MODELING VARIANCE: A VARIANCE-MOTIVATED APPROACH TO MOLECULAR PREDICTION", "journal": "ChemRxiv"}

simultaneous_morphology_manipulation_and_upconversion_luminescence_enhancement_of_β-nayf4:yb3+/er3+_

## Abstract: A strategy has been adopted for simultaneous morphology manipulation and upconversion luminescence enhancement of β-NaYF 4 :Yb 3+ /Er 3+ microcrystals by simply tuning the KF dosage. X-ray power diffraction (XRD), field emission scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS) and photoluminescence spectra (PL) were used to characterize the samples. The influence of molar ratio of KF to Y 3+ on the crystal phase and morphology has been systematically investigated and discussed. It is found that the molar ratio of KF to Y 3+ can strongly control the morphology of the as-synthesized β-NaYF 4 samples because of the different capping effect of F − ions on the different crystal faces. The possible formation mechanism has been proposed on the basis of a series of time-dependent experiments. More importantly, the upconversion luminescence of β-NaYF 4 :Yb 3+ /Er 3+ was greatly enhanced by increasing the molar ratio of KF to RE 3+ (RE = Y, Yb, Er), which is attributed to the distortion of local crystal field symmetry around lanthanide ions through K + ions doping. This synthetic methodology is expected to provide a new strategy for simultaneous morphology control and remarkable upconversion luminescence enhancement of yttrium fluorides, which may be applicable for other rare earth fluorides.In recent years, the synthesis of inorganic nano-/microstructures with controllable morphologies and accurately tunable sizes has attracted much attention not only for fundamental scientific interest but also for their potential applications in the fields of photoelectric device, sensor, catalysis, biological labeling, imaging and drug delivery [1][2][3][4] . It is generally accepted that most of the applications of such materials strongly depend on various parameters, including crystal structure, morphology, size, and dimensionality. Subsequently, simultaneous control over shape, size and phase purity of crystals has been becoming the research focus and one of the challenging issues. Until now, a variety of inorganic crystals, such as oxides, oxyfluorides, fluorides, sulfides, hydrates and other compounds, have been prepared with different shapes and sizes by various methods [5][6][7][8] . However, the precisely architectural manipulation of inorganic functional materials with predictable size, shape and crystal phase is still a challenging and urgent task, owing to the complexity of crystal structures and compositions of materials. To clarify these issues clearly, a deep understanding on the nature of shape evolution and phase transition is still needed. As a result, it is very important for us to establish the relationship between the observed complex phenomena of crystal growth with the underlying fundamental theories and principles, which could be regarded as a reference to controllable synthesis of other inorganic materials. As a significant class of rare earth compounds, rare earth (RE) fluorides have been become a research focus in the material field due to their unique applications in optical communications, three-dimensional displays, solid-state laser, photocatalysis, solar cells, biochemical probes and medical diagnostics . Among them, NaYF 4 has been regarded as one of the most excellent host lattices for performing multicolor upconversion (UC) luminescence of the doped RE ions, due to its low phonon energy, high chemical stability and good optical transparency over a wide wavelength range . As we known, the crystal structure of NaYF 4 exhibits two crystallographic forms, namely, cubic (α -) and hexagonal (β -) phases, depending on the synthesis conditions and methods 18 . Previous studies have indicated that the hexagonal polymorph exhibits considerable enhanced UC emissions compared with the cubic one 14,15 . Consequently, how to obtain pure β -NaYF 4 is crucial in successfully achieving high luminescence performance. Till now, many efforts have been dedicated to exploring excellent routes to the synthesis of hexagonal NaYF 4 with various sizes and shapes, such as nanospheres, nanoplates, nanorods, nanotubes, microrods, microtubes, microshpheres, micro-bipyramids, microplates and microprisms . However, it is still limited on the investigation of the mechanism underlying the shape and phase evolution of NaYF 4 microcrystals. A deep understanding on the dynamic process governing nucleation and growth of the complex fluoride microcrystals is further needed. Compared with other phosphors, such as organic fluorophores and quantum dots, lanthanide ions doped β -NaYF 4 crystals have many advantages, including sharp emission peaks, large anti-Stokes shifts, long-lived excited electronic states and high photostability . But in spite of these advances, improvements are still needed to optimize UC luminescence properties for further potential commercialization. The remarkable challenge for us is how to further enhance the UC intensities of RE ions doped β -NaYF 4 crystals, which has considerable significance to their applications. So far, several attempts have been devoted to improving UC intensity via internal adjustment and external approaches, such as sensitizing mechanisms 26 , the formation of core-shell structure 27 , the introduction of non-lanthanide ions 28,29 and the incorporation of noble metals 30,31 . Among these methods, co-doping with non-lanthanide ions provides an alternative approach to enhance UC luminescence intensity by adjusting the crystal field symmetry. Herein, we demonstrate a facile and effective hydrothermal process to synthesize β -NaYF 4 microcrystals using KF as fluoride source. In our experiments, the KF serves two purposes: (1) to tune morphology of the final products based on different capping effect of F − ions on the different crystal faces; (2) to tailor the local crystal field of host lattice by K + ions doping. By simply tuning the molar ratio of KF to Y 3+ , regular β -NaYF 4 crystals with controllable morphologies can be obtained. In addition, the phase and morphology evolution process as well as the formation mechanism have been systematically investigated and discussed in detail. Meanwhile, significant enhancement of UC luminescence intensity was also observed in β -NaYF 4 :Yb, Er microparticles by simply tuning the KF dosage. To the best of our knowledge, simultaneous morphology control and UC luminescence enhancement for Yb, Er co-doped β -NaYF 4 microcrystals has been reported for the first time, and KF is rarely used as fluoride source. ## Results and Discussion Structures and Morphologies of As-prepared NaYF 4 samples. Here, we mainly focus on the effect of the KF/Y 3+ molar ratios on the phases and morphologies of the final products. In our experiments, the other synthetic parameters were set as reaction temperature 220 °C, reaction time 24 h, Na 3 Cit 1 mmol. Figure 1 shows the XRD patterns of these samples obtained at different molar ratio of KF/Y 3+ as well as standard data of pure hexagonal NaYF 4 phase for comparison. As shown, all crystals exhibit diffraction patterns corresponding to the hexagonal phase of NaYF 4 according to JCPDS No. 28-1192. The crystal structure of the hexagonal phase is determined with lattice parameters of a = 0.596 and c = 0.353 nm, space group P6 3 /mmc 15 . No trace of characteristic peaks is detected for other impurity peaks such as KYF 4 , YF 3 , indicating that the simple hydrothermal method is a feasible route to synthesize pure β -NaYF 4 microcrystals using KF as fluoride source. Moreover, careful observation reveals that the relative diffraction peak intensity of XRD patterns varies with different molar ratio of KF to Y 3+ , implying the morphology evolution of as-prepared β -NaYF 4 crystals. The typical morphology evolution of β -NaYF 4 microcrystals obtained with increasing KF/Y 3+ molar ratio is presented in Fig. 2. As shown, the molar ratio of KF to Y 3+ has a profound influence on the morphologies of the as-synthesized samples. At low molar ratio of KF to Y 3+ (KF/Y 3+ = 16), the SEM images in Fig. 2a reveal that the sample is composed of a large quantity of microrods with uniform size of 11.8 μ m in length and 2.3 μ m in diameter. From the images of a higher magnification (Fig. 2b) and a typical individual microrod (Fig. 2c), we can see that the products are prismatic microrods with smooth and flat surfaces as well as sharp ends. Furthermore, the ends of rods are of hexagonal pyramid structure, as shown in Fig. 2c. At a medium molar ratio of KF to Y 3+ (KF/Y 3+ = 30), the general images of β -NaYF 4 sample are shown in Fig. 2d. It clearly indicates that the as-prepared product consists of a great deal of hexagonal microprisms with prefect uniformity, monodispersity and well-defined crystallographic facets. The mean size of microprism is calculated to be about 2.6 μ m in diameter and 12.1 μ m in length. Further investigation under higher magnification (Fig. 2e,f) indicates that both tops and bottoms of these microprisms exhibit flat planes. At high molar ratio of KF to Y 3+ (KF/Y 3+ = 50), the regular hexagonal prism-shaped β -NaYF 4 with an average size of 11.5 μ m in length and 2.8 μ m in diameter are observed from Fig. 2g. A magnified SEM image (Fig. 2h) reveals that the large-scale, regular and monodisperse prismatic microrods with soomth and flat surfaces are obtained in this experimental condition. Interestingly, the surfaces of top/bottom have very regular concave centers, as depicted in Fig. 2i. It's worth mentioning that the above-mentioned experiments have been repeated three times at least and the same conclusion could be drawn from these experiments. From the above investigations, it can be concluded that the morphology of products can be tuned accordingly with no further change in particle size by changing the molar ratio of KF/Y 3+ . Growth Mechanism. To understand the formation process of β -NaYF 4 microcrystals with different morphologies, reaction samples have been carefully investigated by quenching the reaction at different time intervals. Although we know that the reaction doesn't stop immediately after the autoclave is removed from the heater because of heat transfer, we do believe that the products synthesized at that time represent certain stage in the formation process. Figure 3 shows the XRD patterns of the NaYF 4 samples synthesized with 50:1 KF/Y 3+ at different reaction times as well as standard data of α -NaYF 4 (JCPDS No. 77-2042) and β -NaYF 4 (JCPDS No. 28-1192) phases for comparison. It reveals that the samples exhibit distinctively different XRD patterns at different reaction times. The sample obtained at t = 1 h is pure cubic NaYF 4 (Fig. 3a). A new hexagonal NaYF 4 phase emerges in addition to cubic NaYF 4 phase with the reaction proceeding for 2 h (Fig. 3b). With the further reaction from 2 h to 4 h, the fraction of β -NaYF 4 increases dramatically while the amount of α -NaYF 4 decreases. The result indicates that the phase transformation (α → β ) takes place through a dissolution-renucleation process . When the reaction time increases from 4 h to 24 h, pure β -NaYF 4 can be successfully obtained, as shown in Fig. 3c-f. Based on the above analysis, it can be concluded that the crystal evolves from cubic phase to mixed phase and ultimately to hexagonal phase with the prolonged reaction time. At the meantime, the morphologies of the products are carefully investigated by quenching the reaction at different time intervals. Figure 4 shows the corresponding FE-SEM images of the different intermediate samples at different reaction stages. It clearly reveals that the six samples exhibit dramatically different morphologies in the process of crystal growth. At a short reaction time of 1 h, the α -NaYF 4 sample consists of spherical-like nanoparticles with a mean diameter of 40 nm (Fig. 4a and Fig. S1 in Supplementary Information). But in the present situation, the α -phase of NaYF 4 is unstable and these nanoparticles would serve as seeds for the growth of β -NaYF 4 hexagonal microrods by a dissolution-renucleation process. With further reaction, these unstable α -NaYF 4 nanoparticles convert to β -NaYF 4 microprisms gradually. After 2 h of growth, the regular and well-defined microprisms begin to appear in the intermediate product. Moreover, a large amount of nanoparticles are attached on the surface of hexagonal microprisms, as shown in Fig. 4b. According to the corresponding XRD pattern, the coexisting of two shapes results from the presence of the mixture crystal phase (α + β ). As we known, the cubic NaYF 4 has isotropic unit cell structure, resulting in an isotropic growth of particles. As a result, spherical-like particles are observed. By comparison, hexagonal NaYF 4 has anisotropic unit cell structure, which can induce anisotropic growth along crystallographically reactive directions, leading to the formation of hexagonal-shaped structure 35 . On the basis of above analysis, it can inferred that these crystals with different morphologies should be cubic NaYF 4 (nanoparticles) and hexagonal NaYF 4 (microprisms), respectively. As reaction time extended to 4 h, the α -NaYF 4 nanoparticles disappear completely and only the fairly uniform well-defined β -NaYF 4 microprisms exist. The result also reveals that the phase transition (α → β ) can directly induce obvious change in the morphology of NaYF 4 crystals. As shown in Fig. 4c, the mean length and diameter of rods could be estimated to be 5.2 μ m and 1.0 μ m, respectively. With the further reaction from 8 h to 24 h, there are no further change in morphology, but the size of microprisms increases from 8.8 μ m to 11.6 μ m in length and from 1.9 μ m to 2.8 μ m in diameter (Fig. 4d-f), indicating the longitudinal and transversal growth of NaYF 4 microprisms along with the reaction time. We also investigate the growth process of β -NaYF 4 crystals synthesized with other KF/Y 3+ molar ratios (KF/Y 3+ = 20, 25, and 40). As can be seen from the XRD patterns (Fig. S2-S4 in Supplementary Information), these crystals exhibit very similar phase transformation process to that of these three samples. In addition, the FE-SEM images (Fig. S5-S7 in Supplementary Information) also reveal a similar phase transformation process (α → β ) to these three products. Based on the above results, a possible phase and morphology evolution mechanism is shown in Fig. 5 and described as follows. At the beginning, the citrate anions (Cit 3− ) introduced into the reaction system can form complexes with Y 3+ ions through strong coordination interaction. At the same time, KF dissolves in the aqueous solution to form K + and F − ions. Under the conditions of high temperature and high pressure, the chelating ability of the Y 3+ − Cit 3− complexes would be weakened by slow degrees during hydrothermal process, resulting that the Y 3+ ions could be released gradually. Then Na + , K + and F − ions in this solution react with Y 3+ ions to generate small nuclei. In a very short reaction time, these nuclei quickly aggregate together and grow into cubic-phased K x Na (1−x) YF 4 nanoparticles. However, these grown up α -K x Na (1−x) YF 4 nanoparticles are thermodynamically unstable and evolve inevitably to hexagonal K x Na (1−x) YF 4 seeds through dissolution-renucleation process. Meanwhile, this phase transformation (α → β ) results in dramatic morphology change of the samples, which could be related to different characteristic unit cell structures for varying crystallographic phases. The dissolution-reconstruction process of cubic-phased nanoparticles preferentially happens at the circumferential edges of each prismy microrod along crystallographically reactive direction, resulting in the formation of rod-like β -K x Na (1−x) YF 4 with a well-defined cross-section. With the further reaction, the morphology of products changes from spherical nanoparticles into short hexagonal microrods. As we known, the shape evolution of β -NaYF 4 microcrystals is significantly dependent on external factors such as the molar ratios of KF to Y 3+ and Na 3 Cit to Y 3+ , the pH values in the solution 36 . In this experiment, the subsequent crystal growth of β -K x Na (1−x) YF 4 seeds is significantly affected by the KF/ Y 3+ molar ratio, resulting in different morphologies of hexagonal K x Na (1−x) YF 4 microcrystals. Although the exact mechanism is not very clear at present, the explanation for the change of morphology can be provided as follows. According to the general principle of crystal growth, the growth of crystals is related to the relative growth rate of different crystal facets 33,37,38 . The different growth rate of various crystal planes results in diverse appearance of the crystallite. Generally speaking, crystal facets perpendicular to the fast directions of growth have smaller surface area and show growing faces therefore dominate the morphology of the final crystal. The growth velocity in different crystallographic facets of β -K x Na (1−x) YF 4 crystals could be influenced by the coordination effect between F − and Y 3+ ions. According to the Gibbs-Thompson theory, the relative chemical potential of crystal plane is simply proportional to its surface-atom ratio, determined by the average number of dangling bonds per atom over the entire crystal facet 20 . The capping effect of F − ions could decrease the average number of dangling bonds and further reduce the chemical potential of the crystal plane. Moreover, the different density of Y 3+ on various crystal planes leads to the difference in chemical potential of crystal facets. Consequently, the chemical potential of different crystal facets could be modified, and the relative growth rates could be affected by different molar ratio of KF to Y 3+ , finally leading to different crystal shapes. For β -K x Na (1−x) YF 4 crystals, the density of Y 3+ on the prismatic planes ({10-10} crystal planes) is bigger than that on the top/bottom facets ({0001} crystal planes), resulting in the selective adsorption ability of F − ions on the prismatic facets being bigger than that on the top/bottom planes. Finally, the relative growth rate along is much quicker than that of along 10-10, resulting in the hexagonal microrods with long length and high aspect ratio. The difference in the top ends of hexagonal microrods is relate to the different capping effect of F − on the {10-11}, {-101-1} and {0001} crystal planes, as depicted in Fig. 6. At low F − concentration, the capping effect of F − on the {10-11} and {-101-1} crystal planes is greater than it on the {0001} plane. Consequently, the growth rate of {10-11} and {-101-1} crystal facets is faster than that of {0001} planes, resulting in the formation of sharp ends. At medium F − concentration, the capping effect of F − ions on the {0001} facets is greater and the fast growing faces ({0001} crystal planes) therefore induce to the flat ends. The concave structure is observed at the top/bottom facets by further increasing the F − concentration in the solution. The presence of the concave ends demonstrates that the growth rate of the prismatic side facets ({10-10} planes) is a little faster than that of the top/bottom facets ({0001} planes). ## Upconversion luminescence properties. To investigate the UC luminescence properties of β 4 synthesized with different molar ratios of KF to RE 3+ , Yb 3+ /Er 3+ are selected as co-doped ion pairs to form β -NaYF:Yb 3+ , Er 3+ microcrystals. Figure 7 shows the UC emission spectra of β -NaYF 4 :20%Yb 3+ , 2%Er 3+ samples (20 mg powder is dispersed in 10 mL ethanol under ultrasound treatment) synthesized by different molar ratio of KF to RE 3+ under 980 nm laser diode excitation (power density: 0.2 W/mm 2 ). As can be seen clearly, the six samples show the same emission peaks yet with quite different emission intensity. As shown in Fig. 7a, the characteristic UC emission bands centered at 521 nm, 540 nm and 656 nm can be ascribed to 2 H 11/2 → 4 I 15/2 (green), 4 S 3/2 → 4 I 15/2 (green) and 4 F 9/2 → 4 I 15/2 (red) transitions of Er 3+ , respectively 39 . It is worth noticing that the UC luminescence intensities in both the green and red regions increase notably with the increment of molar ratio of KF to RE 3+ . Figure 7b exhibits the integral intensity of green and red emissions as a function of the KF dosage. By increasing the molar ratio of KF to RE 3+ , the integral intensities of 521 nm, 540 nm and 656 nm emissions are enhanced dramatically. The integrated green (500-600 nm) and red (600-700 nm) emissions in KF6 sample are measured to be about 15 and 12 times as high as that of KF1 sample (Table S1 in Supporting Information). In addition, the similar trend of green and red emissions also suggests the same upconversion pathways for them. To visualize the enhancement of UC emission, the corresponding luminescence photographs of β -NaYF 4 :20%Yb 3+ , 2%Er 3+ crystals synthesized with different KF dosage are provided in Fig. 7c. The green emission of six samples can be seen clearly by naked eyes. Moreover, the emission intensity of KF6 sample is the strongest, which agrees with the results in Fig. 7a,b. To accurately demonstrate enhancement of UC luminescence, the quantum yields of the KF1 and KF6 samples were measured. Importantly, the UC efficiencies of KF1 and KF6 are roughly estimated to be ~0.3% and ~1.6%, respectively. The detailed measuring procedure for quantum yield has been presented in supporting information (Fig. S8). Additionally, the obvious UC enhancement can also observed in Yb 3+ /Tm 3+ co-doped β -NaYF 4 samples (Fig. S9 in Supplementary Information). Notably, the integrated blue emission of the β -NaYF 4 :20%Yb 3+ , 0.5%Tm 3+ sample obtained with KF/RE 3+ molar ratio 50 are enhanced by about 80 times (Table S2 in Supporting information), resulting in the strongest blue emission under the excitation of 980 nm laser diode. The results are quite similar to the Yb 3+ /Er 3+ co-doped β -NaYF 4 samples, revealing the generality of the approach. In order to deeply investigate the relevant UC mechanism in the as-synthesized β -NaYF 4 :20%Yb 3+ , 2%Er 3+ crystals, the excitation power-dependent UC emissions of green and red are calculated accordingly. It is generally known that the output UC emission intensity (I uc ) is proportional to the infrared excitation power (I IR ): I uc ∝ (I IR ) n , where n is the absorbed photon numbers per visible photon emitted, and its values can be acquired from the slope of the fitted line of the plot of log(I uc ) versus log(I IR ) 40,41 . The pump power dependence of the UC emissions in KF6 sample under 980 nm LD excitation is presented in Fig. 7d. As shown, the slopes of the linear fit of log(I uc ) versus log(I IR ) for 521 nm, 540 nm and 656 nm are 1.67, 1.86 and 1.94, respectively. The result indicates that only two-photon process is involved to produce the green and red UC emissions, whereas a saturation effect can be observed at relatively higher excitation power. Based on the above results, the proposed UC mechanism in β -NaYF 4 :Yb 3+ /Er 3+ under 980 nm LD excitation is shown in Fig. 7e and briefly described as follows 42 . Firstly, the electron of Yb 3+ is excited from 2 F 7 to 2 F 5 level in β -NaYF 4 : Yb 3+ /Er 3+ microcrystals under 980 nm LD excitation. An initial energy transferred from Yb 3+ ions in the 2 F 5/2 state to Er 3+ ions populates the 4 I 11/2 level of Er 3+ ions. Then, a second 980 nm photon transferred by the adjacent Yb 3+ ions can populate the 4 F 7/2 level of Er 3+ ions, whose energy lies in the visible region. The Er 3+ ions can relax nonradiatively to the level of 2 H 11/2 , 4 S 3/2 and 4 F 9/2 . Through a two-photon UC process, the dominant green and red emissions are observed by these transitions from the aforementioned states to 4 I 15/2 level. In order to demonstrate the UC enhancement more theoretically, the emission decay curves of 4 S 3/2 → 4 I 15/2 (540 nm) and 4 F 9/2 → 4 I 15/2 (656 nm) transitions in the six samples were measured at the excitation wavelength of 980 nm, as shown in Fig. 8. The effective experimental lifetime is evaluated using eff where I(t) represents the luminescence intensity at time t after the cutoff of the excitation light 43 . It can be seen clearly that the lifetimes of 4 S 3/2 and 4 F 9/2 states in β -NaYF 4 :Yb 3+ /Er 3+ samples are prolonged gradually with the increase of molar ratio of KF to RE 3+ . Furthermore, the variation trend of lifetimes is also consistent with the enhancement of UC luminescence intensity. The average lifetimes of 4 According to the UC mechanism and experimental results, it can be concluded that the energy transfer process between Yb 3+ and Er 3+ hasn't been changed by tuning the KF dosage. Moreover, the nonradiative transition rate should not have obvious effect on the great enhancement of UC luminescence because of the same experimental and excitation conditions for these samples. Therefore, we can ascribe the as-observed much longer lifetimes (τ) to the increase of theoretical lifetimes (τ rad ), namely the modification of lattice parameters. The introduction of K + ions can tailor the local crystal field of the host lattice, and therefore can modify the theoretical lifetimes of Er 3+ ions through slightly changing their wave functions. Enhancement mechanism for UC emissions. What accounts for the obvious enhancement of UC luminescence when tuning the KF dosage in our experiments? Three important factors for UC enhancement should be in consideration: crystal phase, particle size and morphology, local crystal field. First of all we should investigate the crystal phase, particle size and morphology of β -NaYF 4 :Yb 3+ /Er 3+ crystals synthesized with the addition of different KF dosage. The XRD patterns and FE-SEM images of as-prepared β -NaYF 4 :Yb 3+ /Er 3+ samples are presented in Fig. S12 and Fig. S13, respectively. It is evident that all the peaks can be indexed to pure hexagonal-phased NaYF 4 according to JCPDS card (No. 28-1192). No additional peaks can be detected, indicating that the addition of KF has not lead to the formation of other impurity phases. Although the morphology of crystals has changed a lot, the specific surface areas of the six samples show a little distinction from each other (Table S3 in Supplementary Information). Moreover, the luminescent property of phosphors is relate to their specific surface area of the materials. Consequently, it is obvious that the first two factors should not have an obvious effect on marked enhancement of UC luminescence. However, careful observation reveals that the position of the main diffraction peak at 17.1° in the XRD pattern shifts slightly towards small angles as the molar ratio of KF to RE 3+ increases (Fig. 9a). The variation of lattice parameters and unit cell volume with the KF dosage were calculated form the observed XRD data and are presented in Fig. 9b. According to the Bragg's law (nλ = 2dsinθ), the decrease of Bragg angle (θ) indicates that the spacing between the planes in atomic lattice (d) increases, resulting in the expansion of unit cell. Considering that KF is used as fluorine source and r K (1.51-1.57 ) > r Na (1.13-1.53 ), we can easily infer that some K + ions are doped in β -NaYF 4 host lattice and may occupy lattice sites by the substitution of Na + ions. Furthermore, X-ray photoelectron spectroscopy (XPS) was used to determine the successful incorporation of rare earth ions (Yb 3+ , Er 3+ ) and K + ions into the β -NaYF 4 host matrix. As shown in Fig. 9c, the peaks observed at 285.5 and 302.0 eV can be assigned to the binding energy of K2p 1/2 and K2p 3/2 , respectively 45 . The characteristic peaks at 198.8 and 186.5 eV can be assigned to the binding energy of Yb 4d and Er 4d, respectively (Fig. 9d) 46 . The EDS and ICP-AES analyses (Table 2 and Fig. S15 in Supplementary Information) are also used to confirm the successful incorporation of K + , Yb 3+ , Er 3+ ions into β -NaYF 4 matrix. Evidently, with increasing of the KF dosage in the product, K content increases gradually, and the Yb and Er contents keep unchanged. Therefore, it is believed that the introduction of K + ions into the host lattice leads to the UC enhancement. Based on the above results, we can provide the following explanation for this obvious enhancement of UC luminescence. According to the single-crystal X-ray powder diffraction data, the crystal structure of hexagonal NaYF 4 with the P6 3 /mmc has three types of cation sites in a unit cell: one for rare earth ions (site 1a), one for both rare earth and sodium ions (site 1f), and the third for sodium ions (site 2h). Site 1a and 1f both have C 3h symmetry, whereas site 2h has C s symmetry 18,47 . When trivalent lanthanide ions (Yb 3+ , Er 3+ ) are doped into β -NaYF 4 lattice by isomorphic substitution of Y 3+ ions without charge compensation, only one kind of substitutional site with a crystallographic site symmetry of C 3h could be observed in the unit cell. It is well known that the electric-dipole transitions between 4f n configuration (f-f) with the same parity are parity-forbidden for free lanthanide ions according to the quantum selection rules. However, such prohibition can be broken by mixing of opposite-parity configuration, resulting that the electric-dipole transitions can be weakly allowed in crystal lattice. In order to greatly increase the electric diploe transitions probability, an asymmetric crystal field is required. When K + ions are doped in crystal lattice, in view of the bigger ionic radius of K + relative to that of Na + , K + ions only occupy the lattice sites by the substitution of Na + ions in the crystal, corresponding to the situation presented in Fig. 10 48 . As a result, the coordination shell around site 1f originally statistically distributed by Y 3+ and Na + ions could be perturbed seriously. Accordingly, the displacement patterns of various Y/Na coordination shell around each subset of lanthanide ion can be slightly different. According to the microscopic model of disorder, the local site symmetry around the lanthanide ions may descend from C 3h to lower symmetries C s . For rare earth ions embedded in solid materials, lower crystal symmetry caused by tailoring the crystal structure is generally favorable for higher UC emission intensity. Therefore, the introduction of K + ions into β -NaYF 4 crystal lattice would change the crystal field and lower the local crystal field symmetry around lanthanide ions, resulting in the enhancement of UC luminescence intensity 49 . Tailoring the local crystal field of host lattice has become a general explanation for increasing UC emission intensity when introducing non-luminescent ions, such as Li + , Bi 3+ , Sc 3+ , Fe 3+ ions 29,46,50,51 . However, the direct evidence of tailoring local crystal field is still lacking. So, investigation on the local structure and site symmetry of lanthanide ions, for example, by using high-resolution photoluminescence spectroscopy at low temperature (10 K), are further needed to provide such proof. In conclusion, we have described a facile and general strategy for simultaneous morphology manipulation and UC luminescence enhancement of β -NaYF 4 :Yb 3+ /Er 3+ samples using KF as fluoride source. Through the simple manipulation of the KF/Y 3+ molar ratio, regular β -NaYF 4 microcrystals with predictable shapes have been synthesized. A mechanism for how the molar ratio of KF to Y 3+ influences the anisotropic growth and morphology evolution of β -NaYF 4 crystals is proposed. Based on the phase and morphology evolution, the possible formation mechanism for hexagonal NaYF 4 is discussed in detail. Notably, the UC luminescence intensity of β -NaYF 4 :Yb 3+ /Er 3+ sample is significantly enhanced by increasing the KF dosage. It is found that the doping of K + ions into β -NaYF 4 crystal lattice can tailor the local crystal field and lower the local crystal field symmetry around lanthanide ions, which is the main reason for the UC enhancement. This study provides a reference for simultaneous morphology control and UC luminescence enhancement of rare earth fluorides, which will have great potential in fields of sensors, solar cells and photocatalysis. Preparation. β -NaYF 4 :Yb 3+ , Er 3+ microcrystals have been synthesized via a facile hydrothermal method assisted with trisodium citrate. In a typical procedure for the synthesis of β -NaYF 4 : 20%Yb 3+ , 2% Er 3+ , 1.56 mmol YCl 3 .6H 2 O, 0.40 mmol YbCl 3 .6H 2 O, 0.04 mmol ErCl 3 .6H 2 O were firstly dissolved in 10 mL H 2 O with magnetic stirring to form rare earth chloride solution. Then this solution was added into 20 mL aqueous solution containing 2 mmol trisodium citrate (Na 3 Cit) to form the metal-citrate complex. After vigorous stirring for 30 min, 30 mL aqueous solution containing different amounts (32, 40, 50, 60, 80, 100 mmol) of KF.2H 2 O was introduced into the above solution. After additional agitation for 15 min, the as-obtained mixing solution (60 mL) was transferred into a Teflon bottle (100 mL) held in a stainless steel autoclave, sealed, and maintained at 220 °C for 24 h. As the autoclave was cooled to room temperature naturally, the precipitates were separated by centrifugation, washed with deionized water and ethanol in sequence, and then dired in air at 80 °C for 12 h. In addition, different molar ratios (16:1, 20:1, 25:1, 30:1, 40:1, 50:1) of KF to RE 3+ (RE = Y, Yb, Er) and hydrothermal treatment times (0.5 h, 1 h, 3 h, 8 h, 12 h, 24 h) were selected to investigate the effects of these factors on the morphological, structural and luminescent properties of the as-obtained sampleaccs. According to the KF/RE 3+ molar ratio, the resulted samples were denoted as KF1, KF2, KF3, KF4, KF5, and KF6, resspectively. These as-syntheszied products were used to characterize without any further purification. ## Methods Characterization. Powder X-ray diffraction (XRD) measurements were performed on a ARL X' TRA diffractometer at a scanning rate of 10°/min in the 2θ range from 10° to 80° with Cu Kα radiation (λ = 0.15406 nm). SEM micrographs were obtained using a field emission scanning electron transmission microscope (FE-SEM, SU8010, Hitachi). Transmission electron microscopy (TEM) was recorded on a JEM-200CX with a field emission gun operating at 200 kV. Images were acquired digitally on a Gatan multiple CCD camera. The chemical compositions were determined by inductively coupled plasma (ICP) technique using a Perkin-Elmer Optima 3300DV spectrometer. X-ray photoelectron spectroscopy (XPS) spectra were performed with a PHI5000 VersaProbe system, using a monochromatic Al Kα X-ray source. UC emission spectra were recorded with a Jobinyvon FL3-221 fluorescence spectrophotometer equipped with a 980 nm LD (laser diode) Module (K98SA3M-54W, China) as the excitation source. Upconversion decay lifetimes were measured with a customized UV to mid-infrared steady-state and phosphorescence lifetime spectrometer (FSP920-C, Edinburgh) equipped with a digital oscilloscope (TDS3052B, Tektronix) and a tunable mid-band OPO pulse laser as excitation source (410-2400 nm, 10 Hz, pulse width ≤ 5 ns, Vibrant 355II, OPOTEK). All the measurements were performed at room temperature.

chemsum

{"title": "Simultaneous morphology manipulation and upconversion luminescence enhancement of \u03b2-NaYF4:Yb3+/Er3+ microcrystals by simply tuning the KF dosage", "journal": "Scientific Reports - Nature"}

high_electrical_conductivity_and_high_porosity_in_a_guest@mof_material:_evidence_of_tcnq_ordering_wi

## Abstract: The host-guest system TCNQ@Cu 3 BTC 2 (TCNQ ¼ 7,7,8,8-tetracyanoquinodimethane, BTC ¼ 1,3,5benzenetricarboxylate) is a striking example of how semiconductivity can be introduced by guest incorporation in an otherwise insulating parent material. Exhibiting both microporosity and semiconducting behavior such materials offer exciting opportunities as next-generation sensor materials.Here, we apply a solvent-free vapor phase loading under rigorous exclusion of moisture, obtaining a series of the general formula xTCNQ@Cu 3 BTC 2 (0 # x # 1.0). By using powder X-ray diffraction, infrared and X-ray absorption spectroscopy together with scanning electron microscopy and porosimetry, we provide the first structural evidence for a systematic preferential arrangement of TCNQ along the (111) lattice plane and the bridging coordination motif to two neighbouring Cu-paddlewheels, as was predicted by theory. For 1.0TCNQ@Cu 3 BTC 2 we find a specific electrical conductivity of up to 1.5 Â 10 À4 S cm À1 whilst maintaining a high BET surface area of 573.7 m 2 g À1 . These values are unmatched by MOFs with equally high electrical conductivity, making the material attractive for applications such as super capacitors and chemiresistors. Our results represent the crucial missing link needed to firmly establish the structure-property relationship revealed in TCNQ@Cu 3 BTC 2 , thereby creating a sound basis for using this as a design principle for electrically conducting MOFs. ## Introduction The development of electrically (semi-)conductive metalorganic frameworks (MOFs) is of great scientifc and technological interest, offering the opportunity of making electronic devices with permanent microporosity. 1,2 MOFs are supramolecular coordination complexes composed of metal ions or clusters that are linked by polydentate organic ligands to form 2D or 3D frameworks with accessible porosity. 3 By combining both long-range order found in inorganic semiconductors and high chemical tunability found in organics, semiconducting MOFs are unique among the different classes of conducting materials. In principle, the deep understanding of coordination chemistry and crystal engineering enables rational design of MOF systems at a level that is difficult to achieve in other (particularly non-crystalline) materials. 4,5 However, most MOFs are insulators, originating from the rather ionic coordination bonds of metal carboxylates that tend to suppresses charge transfer between the metal node and linker. 6 Typically, design strategies for electrically conductive MOFs focus on the use of organic linkers that form coordination bonds with improved orbital delocalization between metal and ligand to facilitate charge transfer, and on the use of metal ions with high-energy valance electrons such as Cu 2+ and Fe 2+ . 6,7 In 2014, some of us applied a radically new approach to impart electrical conductivity to MOFs by incorporating the redox-active molecule TCNQ (7,7,8,8-tetracyanoquinodimethane) in the pores of Cu 3 BTC 2 (also known as HKUST-1; BTC ¼ 1,3,5-benzenetricarboxylate). 8 Since then, this striking observation has been confrmed by other groups; 9,10 however, the detailed nature of the host-guest complex and the conductivity mechanism are mainly unsolved. It is proposed that TCNQ bridges two Cu dimer units via two geminal nitrile groups, which is strongly supported by both theory and spectroscopic data. 8,11 Initial measurements of the Seebeck coefficient revealed that holes are the majority charge carriers, 12 and an underlying super-exchange mechanism was proposed. 13 Neumann et al. reported calculations supporting this hypothesis, but required the majority carrier to be electrons, which is in apparent conflict with the abovementioned Seebeck measurements. 10 Furthermore, detailed and clear structural evidence for the bridging binding motif has not been obtained, despite intense research efforts. A factor confounding interpretation of prior results is the use of solution-phase techniques for the infltration of TCNQ into the host framework, during which solvent molecules and TCNQ compete for free Cu(II) coordination sites. This likely impedes long-range ordering of TCNQ molecules in the framework and disrupts diffusion pathways for the guest molecule. Moreover, TCNQ is easily reduced (redox potential of +0.2 V vs. Ag/AgCl), 14,15 and in turn can react with the organic solvent and/or water molecules, preventing full control of the oxidation state of TCNQ molecules adsorbed in the Cu 3 BTC 2 framework. In other words, the material obtained via liquid phase infltration lacks rigorous experimental evidence possibly needed for a profound understanding of the conductivity mechanism due to a wider parameter space of compositional and structural characteristics. Is there a single type of TCNQ guest or is there a range of guest species including neutral and charged and how relevant is a homogenous long range ordering vs. short range (nanodomains) or even random and disordered distribution of TCNQ? Is the material phase pure or are their impurities to be considered? What is the role of water and solvent coordinated to Cu centers that are not occupied by TCNQ and do traces of physisorbed water or other protic impurities (e.g. methanol, ethanol) influence the conductivity of the material? Pristine Cu 3 BTC 2 samples (bulk as well as thin flm) are known to exhibit signifcant amounts of Cu(I) defect sites. Do these sites play a role when the material is loaded with redox non-innocent guests such as TCNQ? This more complex scenario of questions prompted us to investigate preparative concepts for loading Cu 3 BTC 2 with TCNQ. Recently, D'Alessandro and co-workers applied a vacuum vapor-phase infltration (VPI) for the incorporation of TCNQ into Cu 3 BTC 2 . 16 In their approach, however, infltrated samples were subsequently washed with solvent to remove excess, uninfltrated TCNQ. Furthermore, samples were treated under ambient conditions, compromising efforts to keep the hostguest system free from solvent and moisture. Our study builds on their important work, applying an optimized VPI method with rigorous exclusion of moisture and solvent contaminations. Following this new protocol, we obtain samples with precise stoichiometry, having the general formula xTCNQ@Cu 3 BTC 2 (0 # x # 1), that are devoid of complicating factors associated with liquid-phase infltration. The materials were characterized by powder X-ray diffraction (PXRD), infrared (IR) and X-ray photoelectron spectroscopy (XPS) and electrical conductivity measurements, with all measurements strictly conducted under inert conditions. Strikingly, we fnd evidence of long-range order for TCNQ@Cu 3 BTC 2 samples with highloadings. Despite the observation that minor amounts of Cu(TCNQ) form as byproduct on the surface of Cu 3 BTC 2 crystals, we fnd a relatively high electrical conductivity of these samples. Moreover, by avoiding the use of solvent and careful sample handling, our VPI method yields high permanent porosity as wellmore than fve times higher than achieved previously using liquid-phase infltration 8demonstrating that high electrical conductivity and large pore volume in MOFs can be compatible. ## Results and discussion The physical mixture of activated, desolvated Cu 3 BTC 2 and TCNQ (ground together in a mortar) was annealed under vacuum in a glass ampule and heated for 3 days at 180 C to yield the host-guest complex. Using the VPI approach, we produced a series of samples of the general formula xTCNQ@Cu 3 BTC 2 (x ¼ n(TCNQ)/n(Cu 3 BTC 2 ) with x ¼ 0.1, 0.2,., 1.0) with precisely defned guest loadings by varying the amount of TCNQ with respect to a fxed amount of Cu 3 BTC 2 . Even though a loading of x ¼ 1.5 would be necessary to saturate all open Cu sites, unreacted, crystalline TCNQ was observed by powder X-ray diffraction (PXRD) and scanning electron microscopy (SEM) for samples with x > 1.0. This is in accordance with the maximum loading capacity of two TCNQ per large pore (one TCNQ per formula unit Cu 3 BTC 2 , x ¼ 1) predicted by theory. 8 The PXRD data of the loaded series of xTCNQ@Cu 3 -BTC 2 (Fig. 1) show that the overall crystal structure of Cu 3 BTC 2 is retained upon TCNQ infltration. The largest change observed is in the intensity of the (111) reflection at 2q ¼ 5.82 , which increases monotonically with the loading amount of TCNQ (highlighted in Fig. 1). Additionally, new reflections appear at higher angles, of which only the weak reflection at 15.77 matches with the principal reflection of a potential byproduct, i.e. Cu(TCNQ) phase I (cf. Fig. S1 †). The increase of the (111) reflection has previously been interpreted as an indicator for chemisorption of guests at the open Cu sites, 17 and is here indicative of the incorporation of TCNQ into Cu 3 BTC 2 . Since the modifed VPI method excludes any intensity changes due to solvent incorporation, this fnding is consistent with the suggested bridging binding mode of TCNQ, as the Cu atoms of the paddlewheel nodes direct the planar TCNQ molecule into the (111) lattice plane (Fig. 2). Nevertheless, there will be disordered TCNQ inside of the pores considering that there are two types of large pores in Cu 3 BTC 2 and only one features open Cu sites pointing to the center of the pore, i.e. are available for coordination of TCNQ. Furthermore, we observed weak reflections with increasing x in xTCNQ@Cu 3 BTC 2 that are not in agreement with the parent face-centered cubic cell of Cu 3 BTC 2 , e.g. at 10.65 , 15.44 and 18.20 . However, they are consistent with a primitive unit cell of parent Cu 3 BTC 2 having similar lattice parameters, therefore pointing to decreased lattice symmetry for the infltrated MOF. For example, a Pawley profle ft with reduced symmetry (Pn 3m) and maintaining the same lattice parameters as the parent Cu 3 BTC 2 can account for all additional reflections (Fig. S4 †). A doubling of the unit cell parameters while keeping the face-centered symmetry could similarly lead to a good profle ft, which might be in better chemical agreement, reflecting the different pores with and without available Cu sites. Determining the origin of these reflections requires additional structural analysis that is complex and extends signifcantly beyond the scope of the present report. However, this fnding is intriguing, pointing to an ordering phenomenon related to TCNQ molecules and a subsequent symmetry reduction of the host-guest system that has not been previously observed. Based on the PXRD measurement, we therefore conclude that our modifed VPI method leads to incorporation of TCNQ into Cu 3 BTC 2 with the potential formation of a supercell. In order to probe the porosity as function of x in xTCNQ@Cu 3 BTC 2 , we performed nitrogen adsorption experiments. As expected, a decrease of the Brunauer-Emmett-Teller (BET) surface area is observed, from 1833.0 m 2 g 1 for pristine Cu 3 BTC 2 to 573.7 m 2 g 1 for 1.0TCNQ@Cu 3 BTC 2 (Fig. 3), about two thirds of the initial BET surface area. In addition, the decrease is linear with TCNQ loading, providing additional evidence for the incorporation of the guest molecule into the framework. Importantly, the decrease cannot be explained by pore blocking or a simple adsorption of TCNQ at the crystal surface. For instance, the BET surface area of a physical mixture of both components for 1.0TCNQ@Cu 3 BTC 2 would still amount to 1370.4 m 2 g 1 (see detailed discussion in ESI †). It is further important to note that our measured surface areas considerably exceed those for TCNQ-loaded Cu 3 BTC 2 samples reported in the literature. For example, the VPI 0.5TCNQ@Cu 3 BTC 2 sample exhibits a surface area of 1145 m 2 g 1 , whereas the surface area of the material with the same loading synthesized via liquid phase infltration is reported as 214 m 2 g 1 , which is likely due to residual adsorbed water or solvent. 8 The microstructure and phase homogeneity of the TCNQloaded samples was further investigated using SEM (Fig. 4). Nanowire-like structures were discovered on the external surface of the TCNQ-loaded MOF crystallites; the amount and dimensions of these increase with the amount of TCNQ employed during the VPI. To determine the chemical nature of the nanowires, scanning Auger electron microscopy was performed. These data show a homogenous distribution of Cu, C, and N (Fig. S13 †). In combination with the characteristic morphology 18 and the reflection at 15.77 in the diffraction pattern, the nanowires were identifed as phase I of Cu(TCNQ). For the formation of Cu(TCNQ) to occur, both TCNQ and Cu(II) must be reduced to TCNQ and Cu(I), presumably involving an oxidation (decarboxylation) of the BTC linker under the conditions of the loading experiment. In the elemental analysis data of the concentration series (Table S2 †), slightly decreased carbon contents compared to the calculated values are found, whereas the nitrogen contents are slightly increased, supporting a decomposition of some BTC molecules. Moreover, Cu 3 BTC 2 is known to contain small quantities of Cu(I) species, which have been related to intrinsic defects and thermal treatment during activation. 19,20 Surface sensitive XPS measurements (Fig. S19 †) reveal a relatively high abundance of Cu(I) species at the surface of Cu 3 BTC 2 crystallites. This Cu(I) concentration is further increased by the inevitable prolonged thermal treatment during the VPI process. The Cu(I) species, in turn, are able to reduce neutral TCNQ (via Cu(I) / Cu(II) + e ) and thus enable the formation of the surface impurity phase Cu(TCNQ). The presence of several TCNQ species (i.e. coordinated, uncoordinated, radical anion) is suggested by the broadening of the N 1s signature in XPS (Fig. S20 †). The formation of the Cu(TCNQ) byproduct could be reduced, but not fully inhibited, by using lower reaction temperatures (Fig. S15-S17 †). However, reduced temperature also compromise the ordering of TCNQ, as evidenced by the absence of the pronounced (111) reflection in PXRD (Fig. S2 †). The loading and coordination of TCNQ to open Cu sites can also be tracked by vibrational spectroscopy. The infrared (IR) spectra of pristine and TCNQ-loaded Cu 3 BTC 2 are shown in Fig. 5. Whereas in pristine Cu 3 BTC 2 a band in the CN vibration region is absent, a CN vibration mode at 2222 cm 1 appears in TCNQ-loaded samples and becomes more pronounced with higher TCNQ loading. Notably, a second vibrational mode at 2200 cm 1 and a shoulder at 2170 cm 1 appear for samples with high TCNQ loading (cf. Fig. S6 †). A red shift of the CN vibration was previously attributed to coordinated TCNQ involving a partial charge transfer. 8,21 Due to the presence of the CuTCNQ byproduct in the samples, we here also assign some contribution to the signal at 2000 cm 1 and the shoulder at 2170 cm 1 to Cu(TCNQ), whose IR signature is known from the literature. 22 The indication of multiple TCNQ species in the IR spectra matches well with the XPS signature in the N 1s regime. Thermogravimetric analysis (TGA) of 1.0TCNQ@Cu 3 BTC 2 (Fig. S7 †) shows a decomposition that proceeds in one step with an onset at 300 C. This temperature is slightly lower than observed for pristine Cu 3 BTC 2 , which starts to decompose at 330 C. The corresponding features in the differential scanning calorimetry (DSC) data are at 349.7 C and 338.3 C for the pristine and the loaded MOF, respectively. The 1.0TCNQ@Cu 3 BTC 2 sample shows an additional smaller peak at 314.2 C, indicating a step-wise decomposition. Electrical transport measurements were performed on pressed pellets using an air-tight two-point-probe setup. I-V curves were recorded from 5 V to 5 V (Fig. S9 †) and the conductivity s was calculated using eqn (1): in which I, V, d and A are the measured electrical current, the applied voltage, the thickness of the pellet and the area of the pellet, respectively. The calculated room-temperature conductivities are plotted against the TCNQ loading (Fig. 6). We observe an exponential increase of the electrical conductivity upon TCNQ loading, starting from immeasurably small values for pristine Cu 3 BTC 2 to 1.5 10 4 S cm 1 for 1.0TCNQ@Cu 3 -BTC 2 . This value is lower than the electrical conductivity reported for thin flm samples infltrated via the liquid phase (0.07 S cm 1 ), 8 pointing at additional influence of grain boundaries and particle sizes as well as at the different measurement techniques used (pressed powder vs. thin flm). The observed exponential increase of the electrical conductivity can be described by classical percolation theory. 24,25 At low TCNQ loadings only few of the copper paddlewheel units are bridged by TCNQ and form localized conducting regions. With increasing TCNQ loading these bridged domains become interconnected and give rise to charge transport through the framework. Given the fact that TCNQ@Cu 3 BTC 2 samples in the literature showed high conductivities but no pronounced (111) reflection, disordered TCNQ might also contribute to the charge transport through the material. The impact of the Cu(TCNQ) byproduct on the electrical transport, however, is intrinsically difficult to assess because Cu(TCNQ) can crystallize in two different phases. Phase II is a poor conductor, whereas phase I is an electrical semiconductor with a room temperature conductivity of 0.25 S cm 1 . 18 Mechanical removal of the nanowires by sonication was unsuccessful and led to a disordering of the TCNQ molecules and a signifcant decrease in the conductivity (Fig. S3, S11 and S18 †). A physical mixture of 1% CuTCNQ/ Cu 3 BTC 2 (estimated amount from SEM images) did not show any conductivity. In combination with previous fndings in the literature for TCNQ@HKUST-1 in which Cu(TCNQ) as an impurity phase was not observed, we ascribe the increasing electrical conductivity as function of TCNQ loading to the formation of our host-guest system. Moreover, 1.0TCNQ@Cu 3 BTC 2 exhibits one of the highest-reported electrical conductivities paired with permanent porosity reported to date, comparing with other conductive MOFs of similar porosities, such as Cd 2 (TTFTB). 6,26 The synthesis of a reference sample of xTCNQ@Cu 3 BTC 2 (x ¼ 0.4 as determined by EA) via liquid phase infltration revealed no indication of TCNQ ordering, but a conductivity in the order of 10 1 S cm 1 , which is signifcantly higher than for samples with comparable TCNQ loading prepared via VPI. However, this sample shows a non-ohmic behavior (deviation from the linear I-V curve) at high potentials that we assign to electrochemical processes of water/ solvent inside the pores (Fig. S21-S24 †). This fnding emphasizes the importance of solvent exclusion during the guest infltration step and highlights the need for in-depth structural and spectroscopic studies of Guest@MOF systems to develop and validate structure-property relationships. ## Conclusions We developed an optimized VPI strategy under thermodynamic control and used it to introduce stoichiometric amounts of TCNQ to obtain a concentration series of xTCNQ@Cu 3 BTC 2 with 0 # x # 1.0 under strict inert conditions. No washing step is needed to remove excessive TCNQ, which is advantageous over the kinetically controlled loading procedure presented in previous studies. 16 High reaction temperatures and long exposure times evidently promote an ordered, periodic arrangement of TCNQ within the (111) crystal lattice plane and the bridging coordination motif of TCNQ and the Cu atoms of two neighbouring paddle-wheel units. To our knowledge this is the frst crystallographic evidence for the integration of TCNQ into the framework of Cu 3 BTC 2 , and further demonstrates that the introduction of a non-innocent guest molecule can be regarded as a new element of MOF property design. Additional evidence supporting the accommodation of TCNQ within the pores of the framework is the systematic decrease in the BET surface area. SEM images of the infltrated samples show the formation of nanowires on the MOF crystal surface during VPI that were identifed as Cu(TCNQ). Their formation can be reduced but not entirely suppressed at lower temperatures. IR data recorded during this work shows two nitrile vibration modes that can be attributed to TCNQ molecules inside the Cu 3 BTC 2 framework and to the TCNQ anion in Cu(TCNQ). The electrical conductivity of the concentration series increases exponentially with the amount of TCNQ used in the VPI. Even though, a quantitative analysis of the contribution of the hostguest complex and the Cu(TCNQ) byproduct to the electrical conductivity of the material is difficult, the obtained material shows high conductivity values accompanied by high permanent porosity. In fact, the BET surface area of 1.0TCNQ@Cu 3 BTC 2 is among the highest for electrically conductive MOFs. 6,26 The combination of these two properties creates a high potential for using this material in sensing or electronic device fabrication where access to the pores provides an essential new function. In this respect, well defned, oriented Cu 3 BTC 2 thin flms deposited and infltrated with TCNQ using solvent-free vapor phase techniques represent the ultimate goal. Pointing to the complexity of this system, further detailed analysis is required, beyond standard characterization techniques, to fully understand the ordering of TCNQ and the conductivity mechanism. ## General information All chemicals were purchased from commercial suppliers (ABCR, Acros Organics, Alfa Aesar, Sigma Aldrich) and used without further purifcation unless otherwise stated. Solvents used for the synthesis and washing steps were reagent grade or higher. ## Vapor phase inltration Cu 3 BTC 2 was synthesized following the literature procedure. 27 The tile crystalline product was continuously washed with ethanol and dichloromethane in a Soxhlet apparatus for one week, respectively, to rigorously remove all reactants and highboiling solvents. The washing solution was replaced by the respective fresh solvent once. Subsequently, the blue powder was desolvated under high vacuum ($10 6 mbar) at 180 C to yield pristine Cu 3 BTC 2 . TCNQ was recrystallized three times from acetonitrile under inert conditions to yield molecular, crystalline TCNQ in high purity. Inside an argon glovebox, 200 mg of Cu 3 BTC 2 and distinct amounts of TCNQ were physically mixed and flled into borosilicate glass tubes to prepare a concentration series of xTCNQ@Cu 3 BTC 2 (x ¼ n(TCNQ)/n(Cu 3 BTC 2 ) with x ¼ 0.1, 0.2,., 1.0). The glass tube was then evacuated ($10 3 mbar) and flame sealed to give a closed system for the VPI process. The sealed ampules were placed inside a convection oven at 180 C for 72 hours allowing the TCNQ to sublime and diffuse into the MOF. After cooling down to room temperature the ampules were transferred into the glovebox and stored for further characterization. ## Powder X-ray diffraction Powder X-ray diffraction (PXRD) data was collected in a 2q range of 5-50 in steps of 0.0016413 (2q) on a PANalytical Empyrean equipped with a Cu X-ray tube operated at 45 kV and 40 mA. The samples were flled into borosilicate capillaries of 0.7 mm diameter and mounted onto a capillary spinner. The radiation was focused onto the sample through a focusing X-ray beam mirror equipped with a 1/8 divergence slit and 0.02 radian soller slits. The diffracted beam was detected by a PIXcel1D detector in receiving slit mode equipped with a 1/8 anti-scatter slit and 0.02 radian soller slits. Thermogravimetric analysis/differential scanning calorimetry TGA/DSC measurements were carried out on a Mettler Toledo TGA/DSC 1 equipped with an auto sampler unit. Aluminum crucibles (100 mL) were flled with 5-10 mg of MOF powder inside the glovebox and tightly capped with an aluminum lit. The crucible was transferred into the TGA chamber and a hole was pinched into the lit while the stream of Ar (40 mL min 1 ) was already on. The temperature was ramped from 30 C to 550 C at a rate of 5 C min 1 . The instrument was calibrated to a blank sample prior to the measurements. ## Porosimetry measurements Porosimetry measurements of pristine and guest-loaded MOF powders were performed using a Micromeritics 3flex to determine their BET surface area. Therefore, approximately 60 mg of a sample was flled into a BET tube and evacuated for 3 h at room temperature prior to the measurement. Nitrogen isotherms were recorded at 77 K. The BET surface area was calculated from data points in the relative pressure range of 0.01 to 0.1. ## Fourier transform infrared spectroscopy Fourier transform infrared spectroscopy (FTIR) of powder samples was done in argon atmosphere on an ALPHA FTIR spectrometer (Bruker) equipped with a Pt attenuated total reflectance (ATR) unit at room temperature in the range of 400-4000 cm 1 with a resolution of 2 cm 1 . 64 scans were recorded per measurement. ## Elemental analysis Elemental analysis (EA) for the elements C, H, N, and S was carried out on a Hekatech EuroEA Elementaranalysator. Cu contents were determined by atom absorption spectroscopy (AAS) after decomposing the sample in a mixture of sulfuric acid and nitric acid in a CEM microwave. ## Scanning electron microscopy Top-view micrographs of MOF powders were conducted on a Merlin high-resolution scanning electron microscope (Carl Zeiss) using an acceleration voltage of 5 kV. The samples were transferred from a glove box into the analysis chamber under argon atmosphere using a transfer vessel (Leica EM VC500). To avoid charging, the sample was coated with 5 nm of Pt prior to the measurement using a Leica EM ACE600. Energy dispersive X-ray spectroscopy (EDX) analysis was conducted using an XMAX EXTREME EDX detector (Oxford Instruments). Measurements were carried out by application of an acceleration voltage of 5 kV and a probing current of 100 pA for SEM and 1000 pA for EDX. ## Auger electron spectroscopy AES measurements were performed at room temperature with a scanning Auger electron spectroscope (JEOL Ltd. JAMP-9500F feld emission scanning Auger microprobe) system. Samples were prepared by spreading powder particles over a gold-coated surface. AES spectra were acquired with a primary beam of 10 keV. The take-off angle of the instrument was 0 . The differential energy spectrum was used to subtract background from the direct Auger spectrum for calculating the band-to-band intensity. The frst differential d(N(E))/d(E) Auger spectra were obtained by numerical derivation of the direct N(E) integrated Auger data displaying an absolute scale with counts/second units by a universal Savitzky-Golay (SG) quadratic differential flter using seven points and used to calculate the band-to-band intensity of Auger electrons and derive the elemental compositions. The differential spectrum is simply the differential of the direct spectrum with respect to energy. The spectra were calibrated with the carbon band at 263.0 eV. For Auger elemental analysis an 8 nm probe diameter was used. Elemental mapping was analysed by AES. Elemental images were acquired with a primary beam of 10 keV. The take-off angle of the instrument was 0 . The coloured images are elemental

chemsum

{"title": "High electrical conductivity and high porosity in a Guest@MOF material: evidence of TCNQ ordering within Cu₃BTC₂ micropores", "journal": "Royal Society of Chemistry (RSC)"}

reconstructing_reactivity_in_dynamic_host-guest_systems_at_atomistic_resolution:_amide_hydrolysis_un

## Abstract: Spatial confinement is widely employed by Nature to attain unique efficiency in controlling chemical reactions. Notable examples are enzymes, which selectively bind reactants and exquisitely regulate their conversion into products. In the attempt to mimic natural catalytic systems, supramolecular metal-organic cages capable of encapsulating guests in their cavity and of controlling/accelerating chemical reactions under confinement are attracting increasing interest. However, the complex nature of these systems, where reactants/products continuously exchange in-and-out the host, makes it often difficult to elucidate the factors controlling the reactivity in dynamic regimes.As a case study, here we focus on a coordination cage that can encapsulate amide guests and enhance their hydrolysis by favoring their mechanical twisting towards reactive molecular configurations under confinement. We designed an advanced multiscale simulation approach that allows us to reconstruct the reactivity in such host-guest systems in dynamic regimes. In this way, we can characterize the amide encapsulation/expulsion in/out the cage cavity (thermodynamics and kinetics), coupling such host-guest dynamic equilibrium with the characteristic hydrolysis reaction constants.All computed kinetic/thermodynamic data are then combined, obtaining a statistical estimation of reaction acceleration in the host-guest system that is found in optimal agreement with the available experimental trends. This shows how, to understand the key factors controlling accelerations/variations in the reaction under confinement, it is necessary to take into account all dynamic processes that occur as intimately entangled in such host-guest systems. This also provides us with a flexible computational framework, useful to build structure-dynamics-property relationships for a variety of reactive host-guest systems. ## Introduction In billion of years of evolution, Nature has evolved systems (or materials) to control chemical reactivity with unique specificity, efficiency and fidelity. 1,2 Enzymes, capable of catalyzing reactions in their substrate-binding cavities, are a notable example. 3,4 In these systems, the reactants are dynamically encapsulated in the enzymes binding site, where the reaction occurs, and the products are then expelled leaving the reaction site free for hosting another reaction. 5 Such a machinery is controlled by a delicate modulation of host-guest interactions, which control key factors, such as, e.g., the residence time of the reactants/products (guest) in the reaction site of the enzymes (host), conformational changes in the guests favoring the reaction, etc., controlling de facto, the reactivity in the system. In the attempt to mimic natural catalytic systems, chemists have designed synthetic cavities capable of encapsulating reactants with high selectivity. In particular, supramolecular coordination cages have been designed, which can host catalytic reactions in their internal cavities. 10, Reactivity, regio-selectivity and enantio-selectivity can be manipulated in such systems, e.g. by engineering the structural and electronic properties of the cage frameworks or by changing the guest structure. 9,16 Noteworthy, mimicking what happens in some proteolytic enzymes, 17 in a recent work it has been shown that the encapsulation of amide guests in a coordination cage may result in a considerable acceleration of amide hydrolysis. In particular, the molecular crowding in the cavity of the cage was found to favor the mechanical twisting of the amides towards reactive (cis isomers) configurations. 10 The rational design of similar synthetic molecular systems requires a detailed comprehension of (i) the molecular and chemical-physical factors that control their reactivity in space and time, and (ii) how to master them in order to produce new classes of catalytic systems. However, in such supramolecular host-guest systems, the reactivity is coupled with a dynamic equilibrium where the guests exchange in/out the cage hosts, which makes them difficult to rationalize. 16 In general, the reactivity in such systems is controlled by several factors, such as the guests binding/unbinding, the solubility of the guests in the outer solution, the character-istic timescale for the reactions, possible entrapment in metastable states, and molecular concentrations in the system. In presence of coordination-cages, a major role in the reactivity is played by the non-covalent cavity-guest interactions. 16 Despite notable efforts, 9,15, , reaching a detailed understanding in terms of the molecular factors that control the reactivity in systems in which the molecular species are in continuous exchange is typically difficult at experimental level. Computer simulations are extremely useful to this end. Quantum mechanical (QM) approaches, 21 or semi-empirical approaches such as Density Functional Tight Binding methods (DF-TB) 22 have been employed to study chemical reactivity and reactive pathways with notable precision. Ab initio molecular dynamics (MD) and metadynamics simulations have been also widely adopted to study chemical reactions of reactants in reactive configuration, in cases where the reactions require crossing free-energy barriers. However, the study of such systems on timescales that allows accounting also for their supramolecular host-guest dynamics (i.e., dynamic exchange of guests in-and-out the host) is not trivial. For this reason, a comprehensive description of such complex dynamic systems has been until now difficult to attain. Recent computational approaches based on enhanced sampling methods, such as, e.g., metadynamics (MetaD), holds great potential in this sense. 16, Recently employed, e.g., to study the isomerization of photochromic switches (azobenzenes) encapsulated in coordination cages, such approaches allowed to demonstrate the tight interplay between isomerization dynamics, molecular crowding, and host-guest exchange dynamics. 16 This approach is versatile and holds a high potential for studying chemical reactions in host-guest systems in dynamic regimes in general. As a case study, here we focus on the neat host-guest system recently reported by the group of Fujita, 10 where the encapsulation of amide guests within a coordination cage was found to considerably enhance the amide hydrolysis. This is an interesting test case not only because one amide functional group (the peptide bond) is the structural foundation of proteins, but also because the hydrolysis of amides is a classic example of a spontaneous reaction hindered by very high kinetic barriers, which can be lowered by physical means. 32 X-ray crystallography and NMR measurements were used in this system to characterize the encapsulation of a number of electron-rich diaryl amides into different octahedral coordination cages, differing in their metal corners, having electron-deficient walls. In particular, here we focus on the host-guest system of Figure 1, for which an acceleration in the amide hydrolysis of ⇠ 14x (compared to the same amide free in solution) has been experimentally reported when amide 2 is co-encapsulated with co-guest cage 3 inside cage 1 (Figure 1b: red vs. blue curves). This has been imputed to the fact that reactive conformations of the amide guest (cis-twisted) are stabilized within the cage host. 10 Such evidence found consistency with single-crystal X-ray diffraction analyses, showing that the amide can twist when encapsulated within the cage cavity. To obtain a submolecular-resolution insight into the behavior of such system in dynamic regime, here we combine all-atom (AA) MD and MetaD simulations that allow us to reconstruct the structural and dynamical features of this host-guest system. Ab initio MetaD simulations 25 allow us to study the chemical reactivity of the amide guest in the conformations that are most favored within the cage cavity. Coupled with the AA-MetaD thermodynamic/kinetic study of the amide (and co-guest) exchange in-and-out the cage, we can formulate a general scheme revealing the key factors controlling the reaction enhancement in this system. We obtain results that are found in excellent agreement with the available experimental trends, as well as a flexible computational framework, which can be used, in principle, to study a variety of dynamically reactive host-guest systems and to draw structure-dynamics-reactivity relationships useful for rational design. ## Results and Discussion Atomistic modeling of the host-guest system As a representative example of a supramolecular host, here we focus on the coordination cage 1 reported in Figure 1a recently employed by Takezawa et al. to host the hydrolysis of encapsulated amide guests. 10 This is an octahedral coordination cage composed of four self-assembled electron-deficient panel ligands (2,4,6-tris(4pyridyl)-1,3,5-triazine) and six metal-based corners (cis-endcapped Pd(ii) complexes). 33 As a first step, starting from the X-ray crystal structure reported in the literature, 10 we built an AA model for cage 1 which was then preliminary minimized and equilibrated in explicit water solvent and in standard (room) conditions of temperature and pressure via a classical MD simulation. In particular, all the AA models used herein have been parametrized based on the General Amber Force Field (GAFF) 34 and all simulations have been run with the GROMACS-2020.2 software 35 patched with Plumed-2.7. 36 (details available in the Computational Methods section in the Supporting Information, SI). We analyzed the equilibrium MD trajectories to study the degree of flexibility of the cage in realistic conditions. Analysis of the evolution along the MD of, e.g., the internal volume of the cage cavity, Root Mean Square Deviation (RMSD) of the atomic positions, and of two variables (d1 and d2) estimating respectively the height and equatorial width of the octahedral cage revealed that the cage structure is rather rigid in experimentally relevant conditions (see FigureS2 in the SI). This is due to the tetrahedral T d symmetry of this cage. Differently from other examples of flexible cage hosts, 16 cage 1 thus shows a large volume (⇠1.2 nm 3 ), open, with a rather persistent hydrophobic cavity that allows encapsulating one or multiple guest molecules in its interior (the electron-acceptor ⇡ planes of the triazine-based ligands of 1 are particularly apt at interacting with electron rich guests). 10,33 As the main guest, here we focus on guest 2, an N-(2,4-Dimethoxyphenyl)thiophene-2-carboxamid, an electron-rich diaryl amide (Figure 1a) that was experimentally shown to produce the considerable reaction acceleration following to encapsulation in cage 1 (Figure 1b). 10 The central bond in amide guest 2 can undergo hydrolysis according to the reaction schematized in Figure 1b (top). Guest 2 is mildly apolar (logP = 2.77). 37 Experimentally-obtained crystal structures show that cage 1 can encapsulate at the same time up to two 2 molecules in its internal cavity. 10 An AA model was developed for guest 2, paying particular attention to the accuracy in the force field parametrization of the central amide bond dihedral, which defines the trans and cis conformers of the amide, how much one is energetically favored respect to the other, and the related transition barrier (see Figure S1 and details in the Computational Methods section in the SI). We built inclusion complexes where guest 2 is encapsulated within cage 1 in different stoichiometries: i.e., 2 ⇢ 1 and 2 2 ⇢ 1, where respectively one or two 2 amide guests are encapsulated inside cage 1. In particular, the AA model for the 2 2 ⇢ 1 complex was built starting form the available experimental crystal structure for this complex, 10 while that for the 2 ⇢ 1 was obtained by deletion of one of the 2 guests. Aromatic amides, like 2, exist mainly in a trans-planar conformation in solution. However, experimental evidences demonstrated that 2 may adopt a cis-twisted conformation within the T d symmetric cavity (forming a pseudo-S 4 symmetric dimer) in cage 1. 10 In particular, X-ray and NMR measurements showed signals corresponding to a cis:trans 1:1 2 dimer in the 2 2 ⇢ 1 complex, and to a cis-twisted conformation in the 2 • 3 ⇢ 1 complex. To the purpose of our investigation, here we decided to model all possible combinations of conformers, mainly aiming to explore the presence of any correlation between confinement, crowding and the rotation of the amide bond. We also parametrized an AA model for coguest 3 (Figure 1a). Starting also in this case from a corresponding experimentally available crystal structure, 10 we use this to built an additional AA model for the 2 • 3 ⇢ 1 complex, a ternary inclusion complex where one 2 guest and one 3 co-guest are simultaneously encapsulated in cage 1. Particularly interesting for this study, the 2 • 3 ⇢ 1 system was observed experimentally to produce the larger hydrolysis acceleration among all explored cases (Figure 1b: ⇠ 14x acceleration compared to the free amide). 10 We then ran classical MD simulations to equilibrate all the considered complexes in explicit water and in standard (room) conditions of temperature and pressure (at least 1 µs for each system -see SI for details). From the equilibrium MD trajectories, we then extracted multiple data indicative of the encapsulation (Figure 2). When in the cage, 2 tends to stay shifted from the geometrical center of the cavity to maximize interactions with the walls, with the 2 trans conformer showing in general a larger shift compared to 2 cis (Figure 2b). This is imputable to the thiophene ring, which tends to partially stick out of the cavity preferring interaction with the solvent (scheme of Figure 2a, top: in cyan). Co-encapsulation with 3 induces a larger decentralization of the amide guests. Noteworthy, the augmented growing in such a case is observed to causes a significant reduction in terms of 2 cis mobility in the cage cavity, as evidenced by the narrower distribution in Figure 2b (solid red curve). This behavior correlates with an augmented number of contacts between 2 and the cage 1 (Figure 2c). In general, the 2 cis guest shows increased contacts than the 2 trans one (augmented guest-cage interaction), while this trends is even increased when co-guest 3 is also present within 1 (higher crowding). We will discuss The Solvent Accessible Surface Area (SASA) of the guests shows that 2 is less exposed to the solvent when this is encapsulated within 1, and even less when this is co-encapsulated together with 3 (Figure 2d). In general, we observe that the 2 cis conformer has a smaller SASA, and is mode compact than 2 trans in all cases. These SASA and contacts data provide information on the different packing of the 2 cis and 2 trans conformers within the cage in increasing crowding conditions. These are consistent with the FESs reconstructed from the histograms extracted from the MD of Figure 2a, where it is evident how 2 cis allows for a tighter packing within the cage (bottom). At the same time, the 2 cis conformer is found less mobile within 1, as it is demonstrated by a narrower FES dark minimum (minimum energy configuration) compared to that of the 2 trans conformer (top). As a fist and generally rate-limiting step, the amide hydrolysis reaction requires the nucleophilic attack of water (or of OH , if the reaction occurs as catalyzed in basic conditions) to the carbonyl carbon. 32,38 While the cavity of cage 1 is markedly apolar, and the encapsulation of the guests in the cavity is essentially driven by hydrophobic effects, some accessibility by the solvent is therefore still required for the reaction to take place. We calculated the number of contacts between the solvent molecules and the carbonyl group of the 2 guest in the various case. In general, we can observe that the amide of 2 trans is slightly more accessible to the solvent compared to that of 2 cis when the guests are free in solution (dotted distributions), and a similar depletion is observed upon mono-guest confinement (dashed). However, the situation is surprisingly switched in the tightly packed 2•3 ⇢ 1 systems (Figure 2e). In highly confined conditions, the amide of 2 cis is found more exposed to the solvent within cage 1 compared to that of 2 trans (red vs. blue solid curves). This suggests that, while on the one hand the cis conformer of 2 is more tightly packed within the cage, the equilibrium configuration of 2 cis in the 2 • 3 ⇢ 1 complex may allow, at the same time, for an increased propensity to react. We explore at a deeper level the difference in terms of reactivity of the trans vs. cis conformers of 2 in the next section. ## Relationship between amide conformations and reactivity The encapsulation of amide 2 within cage 1 was shown to enhance amide hydrolysis in considerable way in mildy basic conditions (experimental results of Figure 1b have been obtained at [NaOH] = 100 mM 10 ). To obtain information on the effect of confinement on the hydrolysis reaction (slow-down, inhibition, acceleration, etc.), here we modeled the hydrolysis reaction when the amide is free in solution vs. when this is encapsulated the the cavity of cage 1. In moderate pH conditions (6 < pH < 13), and in the absence of other catalysts, the hydrolysis of amides occurs via hydroxide attack, forming a tetrahedral intermediate (TI), followed by a second step consisting in the C-N bond rupture. 32 The first step of the reaction is typically considered the rate determining step of the process 38 (although in some cases the subsequent bond rupture can contribute to, and also control, the rate of hydrolysis). 32 For this reason, for the study of the hydrolysis of amide 2, here we focused our investigation only on the formation of the TI via nucleophilic attack by a solvated hydroxide (Figure 3a). Previously used, e.g., to investigate the hydrolysis of formamide in basic conditions, 38 here we relied on ab initio well-tempered metadynamics (WT-MetaD) simulations 39 to study the reactivity of 2 as free in solution vs. confined in the cage cavity. Considered the computational cost of these simulations, and the complexity of our systems, we employed a semi-empirical density-functional tight-binding (DF-TB) method, 40,41 in its self-consistent charge corrected variant SCC-DFTB. 42 Recently shown to provide comparable accuracy to DFT with large basis sets in terms of prediction of barrier heights and reaction energies for organic molecules, 43 SCC-DFTB guarantees satisfactory accuracy in our case at an affordable computational cost. 22 We simplified our models by studying a system with an OH nearby the amide (Figure 3a), and constraining the C-N-C(O)-C dihedral (!) of amide 2 to representative values, in order to simulate attack to different conformers. We compared 2 conformers with ! equal to 0 (cis conformer), ⇡ (trans conformer), and ⇡/4 (a twisted cis The blue profile refers to 2 in trans conformation, the red one refers to the reaction when 2 is cis, while the violet profile refers to the free energy profile of a cis-distorted configuration of 2 with ! = ⇡/4. A relative reactivity score for each amide conformers ( ! ), normalized based on the maximum measured value (i.e., that for !=⇡/4, set to 1), is associated to the simulated conformers of 2 (right secondary y axis). (c) Isomerization of 2 in the cage, co-encapsulated with 3 (left: trans-2, right: cis-2). (d) Free energy profiles for the isomerization of 2 (i) when this is free in solution (dotted curve, cis in pink), (ii) when 2 is encapsulated in 1 (dashed curve, cis in dark pink), (iii) when this is co-encapsulated with another (trans) 2 guest in cage 1 (dot-dashed curve, cis in light red), and (iv) when 2 is encapsulated in 1 together with the co-guest 3 (solid curve, cis state in light red). The data show that an increasing crowding stabilizes more and more the reactive 2 conformations (e.g., cis) in the cage cavity. Right secondary y axis: relative probabilities (P conf ! ) for the different conformations (!) of 2 in the various host-guest systems calculated based on the G values extracted from WT-MetaD simulations. conformer). We employed replica infrequent ab initio WT-MetaD simulations 25 to obtain information on the reaction coefficients (rate of hydroxide attack and TI formation) for the various conformers of 2 (see Methods section in the SI for details). The reaction barrier of ! = ⇡/2 has been also tested, but this conformer was found too unstable to compute meaningful kinetic data. From multiple infrequent WT-MetaD runs activating/biasing the transition (R!TI, and the TI!R processes), we reconstructed the unbiased kinetics for the transition events and could estimate the characteristic transition times, ⌧ of f and ⌧ on . The kinetic constant for hydroxyde release can be calculated as k of f = 1/⌧ of f . The kinetic constant for hydroxyde attack (k on ) can be obtained in similar way from the ⌧ on and accounting for the OH concentration in the system (in our simplified setup, where OH is constrained in close proximity of the amide -perfectly basic conditions -, the theoretical OH concentration to be considered is equal to that of pure water: 55.6 M -see Methods for details). This simulation approach allowed us to obtain an throughout thermodynamic and dynamic characterization of the reaction as a function of the amide conformation (for some relevant discrete ! values). The results of Figure 3b show a strong dependency of reactivity on the ! dihedral. As expected, the lowest reactivity against hydroxyde attack was observed for the trans conformer, while the cis and the twisted cis amide conformers were found as more reactive. In particular, the latter is the only conformer with a K reac = k on /k of f >1 (Figure 3b: R is higher in free energy than TI -see complete data in Table S2). In particular, from the K data, we can obtain relative reactivity scores ( ! ) useful to compare the reactivity between the different amide conformers. The ! scores of Figure 3b (right secondary y axis) clearly show how, compared to the twisted cis amide conformer (!=⇡/4), the cis amide (!=0) is ⇠ 1000 times less reactive, while the reactivity of the trans amide conformer (⇡) is basically negligible (⇠ 10 9 times less reactive than the ⇡/4 conformer). To our purpose, the reactivity of the different conformers of 2 has to be put into context, namely, considering the actual probability for finding such conformers in solution vs. in the different encapsulated systems (i.e., under different crowding conditions). In realistic conditions, amide bonds are usually found in trans configurations with a torsion angle ! close to ⇡, with a sparse population in cis conformation (! ⇠ 0). The degree of steric conflict of the two residues flanking the amide bond is typically larger in cis amides, resulting, for example, in only ⇠ 5 6% occurrence of cis peptide bonds in protein structures. 44,45 To estimate the relative probabilities for finding in different conformers in different conditions, we used WT-MetaD simulations. As a fist step, we optimized the amide ! dihedral force field parameters, in order to obtain a trans-to-cis isomerization free energy profile consistent with the available experimental data (see extended details in the Methods section in the SI). 46 The trans-to-cis isomerization of ! angles consists of a local conformation change that is often compensated by local variations of the backbone angles and of the residues flanking the amide. 44 In order to asses how the free energy profile for the isomerization of ! is affected by the torsion of and , we selected these 3 dihedral angles as our CVs and ran WT-MetaD simulations activating/biasing the trans-to-cis transition of 2 (i.e., the torsion around the amide bond) in different conditions (Figure 3c-d). Preliminary WT-MetaD simulations showed that the free energy profile of the ! isomerization is not particularly influenced by the and torsions. Well-converged WT-MetaD runs allowed us to reconstruct the differences in free energies between the conformers ( G) and estimate the free energy barriers (to this end we used infrequent WT-MetaD simulations, as recrossing WT-MetaD simulations may underestimate the barrier heightssee extended Methods for details). The results in Figure 3d compare four cases where: (i) 2 is free in solution (Figure 3d: dotted curve, cis conformer in pink), (ii) 2 is encapsulated in cage 1 (dashed curve, cis in dark pink), (iii) the isomerizing 2 is co-encapsulated in cage 1 with another trans 2 guest (encapsulated 2 dimer: dot-dashed curve, cis in light red), and (iv) 2 is co-encapsulated in cage 1 with co-guest 3 (dot-dashed curve, cis in light red). The results show that the stability of the conformers of 2 is significantly affected by confinement. The free energy differences between the cis and trans conformers (Figure 3d) indicate that, while trans is always the most stable configuration of the guest, the cis conformer is more and more stabilized as the crowding in the cage cavity increases. The transition barrier also decreases while increasing the crowding. This is captured by the G and the K conf values, as well as by the relative probability profiles P conf ! of Figure 3d. In particular the P conf ! plots the relative probability for different conformers (!) of 2 with respect to the trans conformer in all simulated complexes. We move from a cis:trans ratio of ⇠ 10 7 :1 for one 2 free in solution to ⇠ 10 5 :1 in the mono-encapsulated system (2 ⇢ 1), ⇠ 10 4 :1 when two 2 guests are co-encapsulated in the cage (2 2 ⇢ 1), to ⇠ 10 2 :1 in the 2 • 3 ⇢ 1 system. The 2 dimer encapsulation (2 2 ⇢ 1) system, where one of the two guests is kept fixed in a trans configuration in accordance with experiments, 10 falls in between the 2 ⇢ 1 and 2 • 3 ⇢ 1 cases. The strongly twisted at ! = ⇡/4 remains extremely unlikely in all systems, despite a similar thousand-fold stabilization via confinement. The most crowded case, 2 • 3 ⇢ 1, shows a ⇠ 10000⇥ increase in the probability for finding the more reactive cis conformer respect to the case where 2 is free in solution. This is remarkable, considered that experimentally this case is the one showing the strongest acceleration in the hydrolysis reaction. 10 Table 1: Thermodynamic and kinetic data for 2 isomerization in all simulated complexes. Free energy differences ( G trans!cis ) related to the trans-to-cis isomerization, equilibrium constants for the conformational change (K conf ), the height of the free-energy barriers ( G ‡ trans!cis ) from the trans state, characteristic timescales (t trans!cis and t cis!trans ) are reported. a The second 2 trans guest was kept in trans conformation during the simulation. These results provides a new perspective for interpreting the reactivity ranking obtained in Figure 3b. (i) The most probable conformer in all states, 2 trans , is also the least reactive. (ii) The most reactive twisted conformers (! = ⇡/4 or, e.g., ⇡/2) are, at the same time, highly improbable, even at increased molecular crowding. (iii) The 2 cis conformer, moderately reactive (but sensibly more reactive that the 2 trans one), is unfavored in solution against 2 trans , but it becomes more and more relatively favored as the crowding increases upon confinement, emerging as the prominent reactive species in the cage. The available experimental X-ray structures for these complexes show a 2 cis -2 trans dimer in the 2 2 ⇢ 1 case, and a 2 cis -twisted conformation in the 2 • 3 ⇢ 1 complex. This seems to indicate that in these complexes a 2 cis conformer is more favored than 2 trans . While this may seem to contradict the simulation results discussed above, it is worth noting that all the results collected up to this phase are valid only under the assumption that the encapsulated guests remain always within the cage cavity. Nonetheless, these are host-guest systems, in which the probability for finding the guests within the cage obeys a well-defined supramolecular equilibrium. Estimating the effective probability for finding the guests within the cage requires also studying the dynamics of guest encapsulation/exchange in-and-out the cavity. As it will be demonstrated in the next section, accounting also for the intrinsic supramolecular dynamics of these host-guest systems provides results that are globally in very good agreement with all available experimental evidences. ## Amide encapsulation/expulsion in-and-out the cage cavity In realistic conditions, the encapsulation/expulsion of guests as, e.g., 2 or 3 in/out the cavity of cage 1 may require crossing considerably high free energy barriers, 16,30 which makes them rare events in the timescales accessible via classical atomistic MD simulations. As recently done for other host-guest 16 and dynamic supramolecular systems, 31,47 we thus reconstructed the thermodynamics and kinetics for the processes of encapsulation/expulsion of amide 2 in/out the cavity of cage 1 by means of a well-suited WT-MetaD 48 simulation protocol (complete computational details are available in the Supplementary Information). 16,31,47,49,50 The extracted data are collected in Table 2 and Figure 4. Table 2: Equilibrium and kinetics of the amide encapsulation/expulsion in/out cavity. For each simulated host-guest complex, encapsulation free energies ( G), equilibrium constants K enc , expulsion free energy barriers ( G ‡ of f ), characteristic in-cavity residence times (t of f ), and the associated transition rates (k of f and k on ) estimated from the WT-MetaD simulations are reported. Comparing the encapsulation of the different isomers of 2 in the cavity of cage 1, either alone or when 1 also contains a co-guest (3 or 2 trans ), we could observe that in general the encapsulation of 2 cis is more favored than that of the 2 trans isomer in all studied cases (see Table 2 and Figure 4). The kinetic constants measured for the 2 trans encapsulation/expulsion in/out the cavity indicate that in all complexes the dynamics of the transitions are marginally affected by the presence of other guests in the cage (k of f and k on in the same orders of magnitude). On the other hand, the dynamics (and stability) of the 2 cis -complexes is more impacted by the presence of co-guests in the cage cavity, which are found to stabilize the 2 encapsulation in the cage cavity by ⇠ 2 4 orders of magnitude in the presence of 3 (lower k of f : more improbable/slower 2 expulsion out of cavity). Shown in Table 2, the estimated k of f for the expulsion of the 2 cis guest out from the cage cavity drops from ⇠ 7.1 ⇥ 10 1 s 1 , when only one 2 cis is present in the cavity of 1, to ⇠ 1 s 1 or ⇠ 5.9 ⇥ 10 2 s 1 , when 2 cis is co-encapsulated in the cavity of 1 with a 2 trans or with a 3 co-guest respectively. The k on , on the other hand, is found globally similar in all simulated cases (see Table 2). Altogether, these data suggest that differences in the host-guest equilibrium in such systems in is mainly controlled by the interactions/affinity between the guest (2) and the effective host cavity, thought of as that accessible for the guest considering the cage and the presence of eventual encapsulated co-guests. From the calculated G, we could also estimate the host-guest affinity constants (K enc ) for all the host-guest systems (Table 2). It is worth noting that the K enc for the 2 cis conformers are in general orders of magnitude higher than those of the 2 trans complexes. In particular, this is evident for the 2 • 3 ⇢ 1 complexes. In such a case there is a difference in the encapsulation Ks of ⇠ 5 orders of magnitude (Table 2). This means that the probability for having 2 cis co-encapsulated in the cavity together with 3 is ⇠ 100 0 000⇥ higher than that of finding a co-encapsulated 2 trans . Despite the fact that, in theory, the 2 trans conformer is found ⇠ 100⇥ more favored compared to the 2 cis one in the cage cavity (see Figure 3d: right secondary axis), such a statistical penalty for having a 2 trans conformer effectively encapsulated within the cage cavity -emerging from the host-guest equilibrium -explains why the experimentally obtained X-ray structures of these complexes always show a 2 cis encapsulated guest. Nonetheless, the high K enc values indicate that in all the cases simulated herein the amide guest can be, in good approximation, assumed as always encapsulated in the cage cavity. In fact, from the thermodynamic data we can extrapolate the partition probability The P in indicates the relative probability for having 2 in vs. out the cage cavity. In particular, P in tends to 1 when the K is high and the guest has a high probability of being encapsulated in the system, as it is the case in general for all host-guest complexes explored herein (P in ⇠ 1 in all cases, see also next section). ## Molecular determinants of reactivity in dynamic host-guest systems Based on all the parameters obtained from our simulations, we can define a reaction acceleration index, a, as the ratio between the observed reactivity with or without the presence of the cage in the system -i.e., when the reactant, guest 2, is encapsulated within the cage cavity (K(cage)) or when it is free in solution (K(sol)): In the real system, amide hydrolysis can in principle take place both when 2 is en- capsulated in the cage cavity or when this is out of the cage (with the observed reaction coefficients determined by the probabilities for finding the different reactive conformersplanar or twisted -in the two environments). In general, the reaction acceleration a will thus depend on the likelihood that the hydrolysis of 2 occurs in vs. out of the cage. From our simulations we have seen that the conformational free energy landscape of the amide guest may change upon encapsulation (changing the relative free energy difference between cis and trans conformers). As a consequence, the probability for crossing the rotational barrier around the amide bond also changes. In particular, we could observe that the more reactive 2 cis conformer is more and more stabilized as the crowding increases in the cavity of cage 1 (Figure 3d). The simulations also show that the encapsulation of 2 cis within the cage cavity is considerably more stable than that of 2 trans , showing a higher affinity and retention time (Figure 4 and Table 2). Altogether, this indicates that it is more likely to observe 2 cis rather than 2 trans encapsulated within the cavity of the cage, which is consistent with the fact that the 2 cis conformer is present in the crystal structures obtained experimentally. 10 Such a dynamic complexity is represented in the scheme of Figure 5a. More in detail, the reactivity in the system depends on the propensity of the visited 2 conformers to react, their relative population in the different complexes, their probability of encapsulation (i.e., the relative population ratio between having 2 in the cage vs. in solution at the equilibrium), and the solvent molecules accessibility to the amide (i.e., the solvent is another key reactants) upon encapsulation. Noteworthy, all these parameters that can be extracted from our simulations. In general, we can define a global reaction constant for the case when hydrolysis takes place within the cage cavity, K(cage), as the sum of the reaction constants (K ! (cage)) for all amide conformers (!) visited by the guest reactant 2 in the cage cavity: where ! is the hydrolysis reaction constant associated to the possible amide conformers ! (see Figure 3b), P conf ! (cage) is the relative statistical weight for all different conformers ! in the cage cavity (Figure 3d), P in ! (cage) is the probability for effectively having each specific conformer ! in the reactive environment -in this case, inside the cage cavity (see Table 2) -, and NW ! (cage) is the average number of contacts between solvent molecules (water or OH : key reactants for amide hydrolysis) and the amide's carbonyl (estimated as in Figure 2e). Accordingly, the global reaction constant in the absence of the cage in the system (2 alone in solution), K(sol), can be defined as: where in this case P conf ! (sol) and NW ! (sol) refer respectively to the relative probabilities for 2, when alone in the solvent, to assume the different conformers !, and the corresponding number of amide carbonyl-solvent molecules contacts. In this case, in the absence of the cage in the system, guest 2 is by definition always out of the cage, and P in ! (sol) = 1. Thus, Equation3 simplifies into: Moreover, it is worth noting that, given the high G values in Figure 4c and Table 2, when the cage is present in the system, the guests can be also considered as always encapsulated within the cage cavity, so that in Equation 2 the P in (cage) term tends to ⇠1 (vide supra): Given the high propensity to guest encapsulation (P in (cage) ⇠1), in this specific case the reaction acceleration in the system is found to be little dependent on the guest encap-sulation/expulsion equilibrium. On the other hand, the reactivity turns out to be rather controlled by the fact the guest is more favored to assume reactive conformations inside the cage cavity (compared to the case when this is free in solution). This is in full agreement with the available experimental evidences on these systems. 10 Finally, it is worth noting that while the summations in Equation 5and Equation 4 run in principle over all possible values of ! (have different reactivities -see Figure 3b), the data of Figure 3d clearly show that, due to the intrinsically high isomerization barrier, the relative probability for observing twisted (an extremely reactive) 2 conformers (e.g., ⇡/2, ⇡/4, etc.) is very low. These are distorted, very unstable conformers, with a survival lifetime which tend to 0, and for which the product ! • P conf ! ⇠ 0. The unique conformers with survival life and P conf ! 6 = 0 are 2 cis (! = 0) and 2 trans (! = ⇡). The latter, however, is substantially non-reactive (Figure 3b: ⇡ ⇠ 0), so that also in this case ⇡ • P conf ⇡ ⇠ 0. Based on these observations, in our case the reactivity of the system seems thus to be largely related to (i) how much over-stabilized is the reactive 2 cis conformer and (ii) how accessible is the amide to the co-reactant solvent molecules in such conformation in the cage cavity vs. in solution. Combining these data, we estimate the reaction acceleration a for the various host-guest complexes reported in Figure 5b). We observe that, in this case, the reactivity increases with the crowding in the system. While a ⇠26-fold acceleration is computed for the monoencapsulated case (2 ⇢ 1), a double-encapsulation gives a ⇠64-fold increase for the 2 2 ⇢ 1 system. A dramatic a ⇠ 150 is obtained for the 2 • 3 ⇢ 1 complex. While such estimated a values may differ quantitatively from those obtained from the experiments (this can be expected, given the deviations of such ideal models from realistic systems/conditions), the trends can be still safely compared. Figure 5c shows a remarkable trend between our calculated acceleration data and the experimental ones. This validates our simulation approach. It is worth noting that the mono-encapsulation case (2 ⇢ 1) does not have an experimental counterpart, due to the tendency of 2 to dimerize within the cage. Nonetheless, this extracase (where crowding is lower than in, e.g., 2 2 ⇢ 1 and 2 • 3 ⇢ 1) provides an additional case useful for comparison. In particular, the limited computed acceleration seen in this case supports the evidence that molecular crowding within the cage cavity is a key player for the reactivity in the host-guest system. In order to obtain an insight into the key molecular determinants controlling the reaction acceleration in these host-guest systems, in Figures 5d-g we plot the computed a parameters against some of their key constitutive terms. We have seen that the difference in affinity between cis and trans conformers among the different systems is the main factor determining the final reaction acceleration using Equation1, this being shown by the nearly perfect exponential correlation between a and the relative probability of finding the 2 cis conformer with respect to the 2 trans conformer in solution and in the different encapsulation complexes (Figure5d). The trend suggests that small incremental stabilizing effects on this conformation, e.g. by changing affinity and size of the co-guest, could result in potentially outstanding enhancements of reactivity for guest 2, keeping all other parameters constant. Noteworthy, a quasi-exponential trend is observed between the computed reaction acceleration a and the encapsulation free energies ( G enc ) for 2 cis in all systems (Figure 5e). In these systems, where the reactivity is observed to increase with the crowding inside the cage cavity, the a is also clearly related to the host-guest interaction (namely, to obtain a stable complexation, a strong host-guest affinity is necessary to compensate the crowding penalty associated to the binding). A similar trend can be observed also when looking at the weighted number of contacts between the host and the guest (Figure 5f, evaluated from the distributions of Figure 2c, i.e. a proxy for the host-guest interaction energy). If we consider the interaction between 2 and cage 1 to be consistent among all the investigated systems, we can trace the trend back to the interaction between guest and co-guest (or the absence thereof in the 2 ⇢ 1 case), with 3 showing a greater stabilizing effect for 2 cis within the cage cavity compared to another 2 co-guest. Noteworthy, as revealed by the obtained trends of Figures 5e-f, such favorable affinity can stabilize the reactive conformer of amide 2 to a higher extent, which results in a remarkable increase in the reaction acceleration a. To obtain information on how much of the host-guest interaction is due to solvophobic effects, we calculated the reduction in the Solvent Accessible Surface Area (SASA) of the 2 cis conformer when this is encapsulated in the cavity of the various complexes vs. when this is alone in solution. 16,47,51 While a correlation with the computed reaction acceleration is observed (see Figure 5g), the trend becomes less neat. The trend is respected while moving from the amide in solution to mono-guest (2 ⇢ 1) and double-guest complexes (2 2 ⇢ 1 and . However, the differences in acceleration between the various systems do not correlate in a neat manner with the SASA calculated for the various cases. This reveals that (i) non-specific hydrophobic effects alone are not sufficient to grasp the complexity of these reactive system, and suggesting that (ii), like in most of receptor-ligand complexes in Nature, specific molecular interactions are probably relevant in controlling the host-guest affinity. ## Conclusions Understanding reactivity in dynamic regimes and in systems in which reactants and products are in continuous exchange is a non-trivial task. Here we report a computational approach that allows us to reconstruct the reactivity in dynamic host-guest systems and to study at atomistic detail the key (molecular and dynamic) factors that control it. As a case study, we focus on the hydrolysis of amide guests encapsulated in the cavity of a coordination cage, for which experimental evidence for reaction acceleration has been recently reported. 10 By combining a multi scale modeling scheme with metadynamics simulations, we couple the study of the intrinsic dynamics of the host-guest system with that of the amide hydrolysis reaction. The approach allow us (i) to characterize the barriers to the hydrolysis reaction as a function of the conformation assumed by the amide, (ii) to study the conformations that the amide guest can assume and estimating their relative probabilities in vs. out the In the specific case study used here, we obtain clear evidence that the reaction acceleration is controlled by the crowding effects accompanying the guest encapsulation. We compared four cases, where the amide guest 2 is alone in solution, when it is encapsulated in the cage (2 ⇢ 1), or when it is co-encapsulated together with other co-guests in the cage cavity (2 2 ⇢ 1 and 2 • 3 ⇢ 1), we characterized all of them, and we estimated their hydrolysis acceleration factor. The results show unambiguously that the encapsulation of the amide in the cage cavity tends to stabilize the reactive conformers of the amide guest. This is key in such specific systems, considered the high encapsulation constants obtained for all studied cases (i.e., high probability to find the amide guest within the cage cavity). We clearly observe how, when a co-guest is also co-encapsulated with the amide guest in the cage, the crowding in the cavity augments, and the reactive cis conformer of the amide guest is more and more stabilized. The acceleration scores estimated from our computations are found in remarkable trends with the reaction acceleration observed experimentally. Overall, our computational results are found in optimal agreement with the experimental results by Takezawa et al.. 10 , while it is worth noting that in our approach these emerge bottom-up, from a comprehensive study of the molecular and supramolecular dynamics of the host-guest system and of its key molecular equilibria. This provides a general character to this approach, as confinement-induced reaction acceleration (or deceleration) in such supramolecular (and intrinsically dynamic) host-guest system can only be explained by taking into account of all the dynamic processes that occur within them. The comprehensive picture of hydrolysis, and of how this may be modulated under confinement that we obtain here provides a general high-resolution framework for building structure-property relationships. This approach constitutes a useful general-purpose platform, useful to explore strategies towards the rational design of host-guest systems with a molecular-level control of chemical reactivity. Based on the results that we show herein, this approach offers a useful platform to explore, e.g., the effect of tuning the properties/features of the guest (e.g., hydrophobicity, symmetry, interactions, size, etc.) or of the cage cavity (e.g., structure, flexibility, hydrophobicity, etc.), as recently shown in other host-guest reactive systems. 16 Its versatility and generality may be also useful to explore ways to gain control over the system reactivity by tuning the host-guest dynamics by using, e.g., guests mixing and guest-guest encapsulation competitions, 10 which could allow tuning the residence time and fractions of encapsulated guests within the cage cavity and, consequently, the reaction in the system. In general, all these considerations underline how gaining control over the dynamics of these systems is key to control reactivity within them, and indicate how approaches such as that described herein can offer a relevant support towards the development of new types of reactive supramolecular systems. ## Computational methods and supplementary figures Creation and parametrization of the molecular models All atomistic model systems have been parametrized based on General Amber Force Field (GAFF) 1 , with the exception of the amide ! dihedral angle, which has been reparametrized to improve its accuracy as explained in the following section below. The donor-acceptor (Pd-N) bonds were parametrized using Seminario's method 2 following the Metal Center Parameter Builder (MCPB) protocol 3 as recently done in the atomistic models of other coordination cages. 4 The partial charges for the whole atomistic system (cage and the guests) were calculated using the RESP approach 5 as implemented in CP2K. 6 The quantum mechanical calculations for this purpose were performed using CP2K, with a BLYP-D2 functional 7,8 , paired with the Goedecker-Teter-Hutter pseudopotentials 9 and a triple-⇣ basis set with polarization functions (TZVP). 10 The atomistic parametrization was then carried out using the ANTECHAMBER software. 11 All the atomistic simulations in this work have been conducted in explicit solvent using the GROMACS-2020.2 software 12 patched with Plumed-2.7. 13 All systems were immersed in a simulation box filled with explicit TIP3P water molecules. 14 All simulated systems consist of a cubic box of different side length depending on the system: 37.2 for the amide in solution solvated by 1684 water TIP3P molecules, and ⇠51.0 with ⇠4290 water molecules for the other 3 host-guest systems. Moreover, for those models containing the cage, 12 molecules of NO 3 have been also added to neutralize the total charge present in the systems, consistent with the experimental conditions. 15 Reparametrization of ! dihedral potential terms of amide 2 Peptide bonds in protein structures are mainly found in trans conformation with a torsion angle ! close to ⇡. Due to the small population of the cis conformation 16,17 , the kinetics of isomerization of peptide bond has always been difficult to characterize experimentally and, ## S2 as a consequence, only limited data on the free energy barriers separating the two isomers are available. 18 Being generally derived from quantum mechanical calculations on model compounds, or based on experimental data from thermodynamic and kinetic studies, 19,20 the AMBER force field shows limitations in the estimation of the accurate free energy difference between the cis and trans conformations in peptide bond. Herein, this was evidenced and proven by preliminary metadynamics tests in this sense. As showed in section 3, the description of the cis-trans equilibrium for the amide 2 has a central role in the study of its reactivity, and thereby, our aim is to evaluate the accuracy of the potential terms involved in the process of isomerization of the ! dihedral angle. In the AMBER force field, the torsional potential energy term is expressed as the Pitzer potential 21 , a Fourier series term given by: where V n , n, µ, and are, respectively, the dihedral force constants, periodicity, torsional angle, and phase angle. To reproduce the torsional energy profile of the peptide bond, only the first two terms in this series are relevant: V 1 (n=1, = 0), and V 2 (n=2, = 180); the former describes the cis-trans equilibra, while the latter is responsible for the barriers to rotation about C-N bond. In order to test the accuracy of the dihedral amide parameters, we compared the freeenergy surface (FES) for the torsion of an N-methylacetamide (NMA) amide -the simplest analog of the peptide bond within amides, that here we use as a reference to optimize the amide force field parameters -obtained performing WT-MetaD simulation with the experimental energy profile obtained with NMR studies. 18 In these WT-MetaD simulations, as the collective variable (CV) we chose the ! dihedral angle of the amide, with a bias factor of 30 deposited every 500 steps using Gaussians of initial height of 1. More precisely, these dihedral force field parameters have been modified until a good agreement was reached between our cis-trans isomerization FES and the experimental free energy profile for NMA (see Figure S1), modifying the main torsional parameters involved in ! bond rotation, while all the other non-bonded parameters were left unchanged (Table S1: in our procedure only the V 2 term for the general X-C-N-C torsion, and the V 1 and V 2 0 potential parameters specific of the H-N-C-O torsion have been modified). to keep a 300 K of temperature and 1 bar of pressure in the systems. The electrostatic interactions have been treated using the Particle Mash Ewald (PME). 25 The cutoff for the real part of the summation was 1.0 nm. The cutoff for the van der Waals interactions was set to 1.0 nm. All of the bonds involving hydrogen atoms were constrained using the LINCS algorithm. 26 The leap-frog integrator was used to integrate the equations of motions. All the analyses of Figure2 have been performed with Plumed-2.7. 13 . For the contact analysis a distance cutoff of 0.5 nm has been chosen between the involved groups, paired with a switching function with parameter D 0 =0.35 nm. The Solvent Accessible Surface Area (SASA) was computed with the default probe radius of 0.14 nm. ## Preface on reconstructing dynamics from metadynamics In these atomistic models, transitions such as, e.g., the isomerization of the amide, as well as amide encapsulation/expulsion in/out the cage are typically rare events, which cannot be effectively sampled via classical MD simulation. Recently, it has been demonstrated that the kinetics of rare events can be efficiently reconstructed by means of infrequent WT-MetaD simulations. Provided that the the CV and setup of the WT-MetaD simulations are opportunely chosen, this approach relies on the fact that the real unbiased timescale of an event can be retrieved from the statistics obtained for the biased event, by calculating the transition time distributions. We exploited this approach to calculate the characteristic timescales and the related kinetics of all the key processes studied herein -i.e., the amide hydrolysis rate limiting step, the cis-trans isomerization of the amide, and its encapsulation/expulsion in/out the cage (details on the WT-MetaD simulations setup for each of these studies are provided in the dedicated sub-sections below). In particular, the unbiased transition time (t) can be calculated from the biased transition time (t W T ) extracted from each individual infrequent WT-MetaD run (in which the transition is biased/activated) as: where V (s(R, t)) is the time dependent bias used during the simulation, and the exponential is averaged over the WT-MetaD run. The main idea behind this approach is to infrequently deposit the bias onto the free energy landscape to speed-up the transition and to effectively observe it at atomistic resolution during the run. At the same time, the V (s(R, t)) has to be deposited infrequently during the WT-MetaD, in such a way to prevent/minimize the deposition of bias on the transition barrier. The transition times (t) estimated from multiple infrequent WT-MetaD runs allow building a transition probability distribution, P n 1 , defining the probability to effectively observe at least one transition by time t: where ⌧ is the characteristic timescale expected for unbiased transition. For a rare event, the statistics of transition times fit well with a Poisson distribution. 29,31 The kinetic rate constant (k) associated to such rare events can be then estimated from the characteristic transition timescales (⌧ ) as: This allows to estimate the unbiased kinetics of a biased transition. The associated transition barrier ( G ‡ ), can be then estimated using the Eyring equation: where  is the transmission coefficient (set to 1 in all cases studied herein, based on the fundamental no-recrossing assumption of transition state theory), k B is Boltzmann's S7 constant, and h is Planck's constant. ## Ab initio metadynamics simulations Ab initio metadynamics simulations have been performed using the CP2K package. 6,32 We employed the semi-empirical density-functional tight-binding (DF-TB) method, 33,34 in its self-consistent charge corrected variant SCC-DFTB. 35 Since our reactions include mostly organic molecules in water, we employed the well validated parametrization set miomod:nh. 36 Convergence of the SCF was set at 1.0 6 Ha. All simulated model systems used for the study of the hydrolysis reaction consist of a cubic box of side length 17.0 , containing amide 2 and 160 explicit water molecules, for a total of 510 atoms per simulation cell (Figure S3). Initial minimization and equilibration of this simulation box were performed for the neutral system. Subsequently, the initial configuration for the hydroxide ion was obtained by deleting one proton from the water molecule closest to the amide bond. The Coordination Number (CN) of the oxygen of the OHwith respect to the hydrogens in the system was then constrained to be 1, in order to preserve the geometry of an hydroxide ion, following Crespo et al. 37 The CN was defined using a Fermi function of the following form: (1 [r/r 0 ] NN )/(1 [r/r 0 ] ND ), where r 0 = 1.32 , NN = 16, and ND = 56 with respect to all of the hydrogens in the system. This CN ensures that the geometry of this species is constrained to be that of the hydroxide ion, which prevents complications arising from the identity of the hydroxide changing due to the Grotthuss mechanism, while still allowing the ion to react with amide. Ab initio metadynamics simulations of the hydroxide ion attack of the amide have been conducted on different amide conformers, having different amide ! dihedral values which have been kept as contrained with an harmonic potential during the simulations. In particular, having initially set the constraints, every system (containing different ! amides) has been further thermalized for 3-4 ps. All simulations were run at 333 K in the NVT ensemble using the Canonical Sampling through Velocity Rescaling (CSVR) thermostat. 23 To estimate the characteristic rates of the OH attack/detachment from the carboxyl S8 In particular, we compared four cases, where the ! dihedral of amide 2 was constrained to 0, ⇡, ⇡/4 and ⇡/2. From the binding/unbinding transition timescales extracted from the 30 infrequent MetaD runs, we could reconstruct the characteristic unbiased kinetics for the events and estimate the characteristic unbinding/binding timescales, ⌧ of f and ⌧ on respectively. For all modeled cases, the kinetic constants for hydroxyde ion release from the amide has been calculated as k of f = 1/⌧ of f . The kinetic constant for hydroxyde ion attack (k on ) can be obtained the same way from ⌧ on -the obtained values have been then corrected considering that our simulation setup is consistent with a hypothetical OH concentration equal to the one of pure water (55.6 M), as the OH ion starts already (and remains) in close proximity of the amide. We could then estimate the equilibrium constant for the amide hydrolysis (K -see Table S2) as: In particular, the case with !=⇡/2 demonstrated to be highly reactive and unstable, proving that this conformer of amide 2 is statistically irrelevant in this case. In these ab initio infrequent WT-MetaD simulations we used the distance between O OH and the C=O carbon as the CV. We deposited hills (initial height = 2.0E-3 Ha) every 100 steps, using a bias-factor of 50. A potential wall was imposed to limit CV values larger than 3 . The CV was monitored to determine the transition events, with a cutoff of < 1.4 for ⌧ on and of > 2.8 for ⌧ of f . Table S2 reports the obtained data for the three dihedral angles. Table S2: Reaction equilibrium and kinetic constants. To each system here studied we report the measured residence times (⌧ of f and ⌧ on ), kinetic constants (k of f and k on ), reaction constant (K reac ) and reactivity score ( ! ). ## S10 For the characterization of the barriers and kinetics of the trans-to-cis isomerization, we turned to infrequent WT-MetaD simulations. We ran 51 infrequent WT-MetaD simulations have been ran for each system where the isomerization of the amide has been activated. In particular we, focused on the isomerization along ! and dihedral angles for each of our host-guest systems. This allowed us to compare how the encapsulation, and then the molecular crowding inside the cage cavity, affect the dynamics of isomerization of 2. In these infrequent WT-MetaD runs, the bias was deposited every 5000 steps (10 ps of simulation time) using Gaussians of initial height of 1.2 kcal/mol, of 0.23 rad, with bias factors between 6-16 depending on the system. ## Encapsulations/expulsion metadynamics simulations The isomers of amide 2 show different encapsulation/expulsion kinetics in/out the cage cavity, with different affinities ( G), retention times (⌧ ) and characteristic transition rates (k). As expected, these quantities are influenced also by the presence of a co-guest (3, or another 2) inside the cavity of 1. For evaluation of the kinetics constant of the guests encapsulation/expulsion in/out the cage cavity, we conducted multiple infrequent WT-MetaD simulations in which the encapsulations/expulsion of the amide have been activated. In these WT-MetaD simulations, we used as the CVs the contacts between heavy atoms of guest and host-cage (CV1), the host-to-guest center-to-center distance (CV2), and the standard deviation of the contacts between heavy atoms of the guest and of the host-cage (CV3). We conducted a first explorative WT-MetaD simulation, where the amide was biased to exchange in/out the cage cavity. Qualitatively, similar to what seen also in other hostguest 30 and dynamic supramolecular systems, 39 this simulation showed that the exchange of a guest from the cage cavity into the solvent is most likely a 2-steps process, where (i) the encapsulated guest is first expelled out of the cage cavity and it remains absorbed onto the cage surface (Figure S4a,b: in-to-out cavity transition), and then (ii), from such absorbed state, it jumps in solution. The same holds for the back (encapsulation) process, but in S11 opposite direction. As explained in the main text, for the purposes of this specific work, the exchange step (i) is the key one. In fact, this transition relates the state where 2 is encapsulated within the cage cavity vs. the closest state in free-energy where 2 is out of the cavity of 1 -i.e., the most likely transition (accompanied by the lowest exchange barrier) having consequences in terms of stabilization of the reactive 2 cis conformers seen in Figure 3d of the main paper). Indeed, as the crowding has a direct effect on the guest favored conformers and on their related reactivity, being out of the cage and adsorbed on the surface or in solution has negligible effects in terms of crowding for amide. Reaching a robust convergence in a single WT-MetaD simulation for such a complex multi-step exchange process is prohibitive, and the FES shown in Figure S4a,b has thus just a qualitative/explorative value. As recently done also for other similar systems, 4,40 to characterize the key step (i), we thus opted to reconstruct the guest encapsulation/expulsion exchange profile, the associated G, transition barriers, and characteristic kinetics from multiple infrequent WT-MetaD simulations (see Figure S4c, and Figure 4 in the main paper). In these infrequent WT-MetaD simulations, the bias was deposited every 5000 steps (10 ps of simulation time) using Gaussians of initial height of 0.12 kcal/mol, with bias factor ranging between 10 and 14 (depending on the system) for the expulsion out of the cage cavity, while we used 5 as the bias factor for the WT-MetaD simulations activating the guest encapsulation event. The deposed Gaussians had equal to 4.0, 0.04 nm, 2.0 along CV1, CV2 and CV3 respectively. For each system, the estimation of the kinetic constants (k) for the amide encapsulation/expulsion transitions in/out the cage cavity have been estimated from 50 WT-MetaD simulations. From these, we then could reconstruct all the associated thermodynamic quantities as described in the previous section above (see Equation S2-S5). As demonstrated by the data obtained for all cases, it is clear that in such host-guest systems the amide guest can be in general considered to be always inside the cage cavity (see Figure S4c, Figure 4 and Table 2 in the main paper) -i.e., in Equation 5 in the main paper P in ⇠ 1 in all simulated cases.

chemsum

{"title": "Reconstructing Reactivity in Dynamic Host-Guest Systems at Atomistic Resolution: Amide Hydrolysis Under Confinement in the Cavity of a Coordination Cage", "journal": "ChemRxiv"}

aggregation-free_and_high_stability_core–shell_polymer_nanoparticles_with_high_fullerene_loading_cap

## Abstract: Fullerenes have unique structural and electronic properties that make them attractive candidates for diagnostic, therapeutic, and theranostic applications. However, their poor water solubility remains a limiting factor in realizing their full biomedical potential. Here, we present an approach based on a combination of supramolecular and covalent chemistry to access well-defined fullerene-containing polymer nanoparticles with a core-shell structure. In this approach, solvophobic forces and aromatic interactions first come into play to afford a micellar structure with a poly(ethylene glycol) shell and a corannulene-based fullerene-rich core. Covalent stabilization of the supramolecular assembly then affords core-crosslinked polymer nanoparticles. The shell makes these nanoparticles biocompatible and allows them to be dried to a solid and redispersed in water without inducing interparticle aggregation.The core allows a high content of different fullerene types to be encapsulated. Finally, covalent stabilization endows nanostructures with stability against changing environmental conditions. ## Introduction Fullerenes are a family of carbon cages. They are characterized by high electron affinities, reactive exteriors, and inert interiors. The frst two characteristics enable them to quench reactive oxygen species, which are considered to be mediators of oxidative stress and cause for numerous chronic and acute diseases. 5 The interior space can entrap metal atoms such as gadolinium to assist in magnetic resonance imaging. 6 This approach of confning the active and toxic metal atoms to the carbon cage allows overcoming the stability and toxicity issues associated with the gadolinium chelates typically used for imaging purposes. 7 However, despite great potential, the applications of fullerenes in biomedical sciences remain rather limited due to their poor water solubility. The general approaches to enhance their water solubility include covalent modifcation of the exterior and supramolecular complexation with a variety of hosts (Fig. 1). The former suffers from unknown toxicological profles of new fullerene derivatives and complex synthesis involving regio-and stereoisomers. Although Fig. 1 The most common strategies for preparing water-soluble fullerenes involve supramolecular complexation with a polymer chain or covalent modification of the carbon scaffold (top). The attributes of a fullerene-containing core-shell nanoparticle morphology as explored in this work (bottom). great strides have been made recently with the help of 'click' chemistry, which allows for multi-fullerene molecules to be prepared by multi-step organic synthesis. 18 The latter approach suffers from low fullerene loading capacity ($1 wt%) and reduced in vivo stability of the noncovalent constructs. Interestingly, C 70 is shown to be a better candidate in terms of biological applications. 19 However, studies involving C 70 are scarce due to an even higher restriction on access to water-soluble larger fullerene structures. To address these issues, we designed a covalently stabilized block copolymer micellar system in which (i) host-guest interactions in the micellar core enable a high (8 wt%) fullerene loading capacity (Scheme 1), (ii) the flexibility of the host units and their multiple numbers allow smaller (C 60 ) and larger (C 70 ) guest surfaces to be encapsulated irrespective of their sizes (Fig. 2), (iii) core-crosslinking renders the structures robust against changing environmental conditions, and (iv) a well-defned PEG-based shell provides solvation in water, hemocompatibility, and protection from unfavorable interparticle and protein-particle interactions. A preliminary property study is also carried out, which indicates that C 70 nanoparticles are more active than C 60 nanoparticles towards free radical scavenging in aqueous solutions. This observation further reinforces the idea that larger fullerenes offer superior biorelevant properties. ## Molecular design Corannulene (C 20 H 10 ) can be considered a fragment of fullerene C 60 . 20 However, its tour-de-force synthesis by Barth and Lawton predates the discovery of fullerenes by nearly two decades. 21 Since then, many properties of this fascinating molecule have been studied, including its capacity to host fullerene C 60 through complementarity of the curved p-surfaces. 23 To introduce this motif into a polymer chain, a post-polymerization modifcation strategy was developed (Scheme 2). For this, initially, an atom transfer radical polymerization of glycidyl methacrylate (GMA) monomer through a poly(ethylene glycol) (PEG)-based macroinitiator (PEG-Br) (M n(GPC) ¼ 8600, M w /M n ¼ 1.1) was carried out to access the reactive block copolymer (PEGb-PGMA) scaffold (M n(GPC) ¼ 12 700, M w /M n ¼ 1.2) (ESI Fig. S1 †). Area integration analysis in 1 H NMR spectroscopy indicates that the degree of polymerization for the GMA block was approximately 17 repeating units (ESI Fig. S2 †). The ring opening reaction between the epoxide group of PGMA and mercaptocorannulene then afforded host polymer 1 (M n(GPC) ¼ 17 800, M w /M n ¼ 1.2). 1 H NMR spectroscopy indicated that the postsynthesis modifcation reaction was quantitative; therefore, Scheme 1 Chemical structure of host polymer 1 encoded with corannulenes and its assembly with fullerenes through ball-and-socket interactions between concave and convex aromatic surfaces to furnish micellar nanoparticles. Fig. 2 The hosting polymer block contains many corannulene units attached to the polymer backbone through a linker that can adjust upon receiving the guest molecule. These features allow accommodating different fullerene sizes in the micellar core. approximately 17 corannulene units were incorporated into each diblock copolymer chain. This synthetic strategy was chosen because the ring opening reaction produces a reactive hydroxyl group that could be used for the core-crosslinking reaction once the amphiphilic block copolymer assembles into a micellar nanostructure. The choice of PEG as the shell component was due to its ability to avoid fouling with proteins and to impart water solubility and prolonged blood circulation kinetics to the nanoparticles. 24 Micelle formation and covalent stabilization 25 Above a certain concentration, block copolymers carrying hydrophilic and hydrophobic segments can assemble into a micellar structure in a solvent that selectively solubilizes only one of the polymer blocks. 26 The concentration is referred to as the critical micelle concentration (CMC). Micelles can be produced with different morphologies and functions. However, their supramolecular nature makes them susceptible to changing environmental conditions. For example, a solvent that can solubilize both polymer blocks can dissolve the supramolecular structure. In the context of biological applications, shear forces under flow conditions are also expected to disrupt such noncovalent assemblies. Crosslinking of the core or shell is required to stabilize the assembled structure against any changes in solvent, concentration, or temperature. This path leads to robust nanoparticles. 25, To study micelle formation from polymer 1 under aqueous conditions, the fluorescence emission of corannulene was monitored at 465 nm. As the polymer chains begin to assemble, the emission signal intensity decreases due to the aggregation-induced self-quenching process. 32 These data allows the determination of the CMC and indicate that at concentrations above 12 mg mL 1 , polymer 1 forms micellar structures (Fig. 3). Dynamic light scattering (DLS) studies were undertaken to investigate the micellar sizes. In dimethylformamide (DMF), a good solvent for both polymer blocks, a hydrodynamic diameter of <1 nm indicated individual polymer chains in the solution. In water, a solvent preferential for the poly(ethylene glycol) block, larger structures with an average hydrodynamic diameter of 37-38 nm could be observed, indicating micellar assembly. These micelles could be stabilized through crosslinking of the hydroxyl groups present in the core with adipic acid using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) as the carboxyl activating agent and dimethylaminopyridine (DMAP) as the catalyst for the formation of the ester bonds. Upon crosslinking of the core, the micellar size decreased slightly (31-32 nm). This is most likely due to crosslinking-induced shrinkage of the core. Since, such an intermolecular crosslinking process requires a very low fraction of the reactive sites, and the polymer chains contained the ester groups already (before formation of new ester bonds upon core-crosslinking), typical characterization tools such as NMR and IR spectroscopies were found to be of no help in characterizing the crosslinked structure. However, the success of the crosslinking process could be verifed by dissolution of the micelles in DMFa solvent that is good for both polymer blocks and therefore capable of solubilizing the supramolecular micellar assembly into individual polymer chains (Fig. 3). Upon crosslinking, the micelles could be dispersed in DMF without destroying the nanostructure (Fig. 3). This indicated that the crosslinking process was successful in covalently stabilizing the micellar structure. However, larger particle sizes were observed (82-83 nm), presumably due to swelling of the crosslinked core in DMF. ## Fullerene loading Two different approaches were examined to incorporate fullerenes into the micellar structures (Fig. 4). In the frst approach, preformed micelles were exposed to C 60 /C 70 through sonication in water. Crosslinking was then performed to lock the C 60 /C 70 that entered into the core. Alternatively, polymer and C 60 /C 70 were mixed together in tetrahydrofuran, and then water was added to allow the formation of micelles. Finally, the supramolecular structure was crosslinked. In the second approach, the polymer chains can interact with the fullerenes before formation of the secondary structure. To study the loading capacity arising from these different preparation methods, UV-Vis spectroscopy was used (ESI Fig. S3 †). In water, the molar absorption coefficient (3) of C 60 is 49 000 at 340 nm, and 34 000 for C 70 at 384 nm. 15 This allows a comparison to be made and calculation of the concentration of fullerenes in the crosslinked nanoparticles to be performed. These data indicated that the frst approach resulted in the incorporation of approximately 1.22 wt% fullerenes into the particle core. The second approach, however, was superior and furnished 8.12 and 8.01 wt% C 60 and C 70 , respectively, into the nanoparticles. To investigate the supramolecular complexation between fullerenes and corannulene through interaction of their curved surfaces, 13 C-enriched fullerenes were utilized, and the crosslinked nanoparticles were studied with the help of 13 C NMR spectroscopy (Fig. 5). In the case of C 60 nanoparticles, an upfeld shift of 1.4 ppm (d ¼ 142.8 free C 60 ; d ¼ 141.4 complexed C 60 ), indicative of aromatic shielding effect and consistent with the previously reported supramolecular fullerene complexes, 33 was observed in the 13 C resonance of C 60 . To rule out any unspecifc interactions as the cause for this shift, an external standard tube (of larger diameter than that of the original NMR tube) containing a pure solution of C 60 in deuterated tetrachloroethane was used. These samples showed signals from the uncomplexed fullerene (present as an external standard) and the signal from the C 60 nanoparticles also in deuterated tetrachloroethane. These data confrmed that the shift of 1.4 ppm was due to specifc complexation of C 60 with corannulenes in the nanoparticle core. A broadening of the C 60 signal was also observed, possibly as a result of the restricted rotational freedom of complexed C 60 molecules in the nanoparticle core. Unlike C 60 , which possesses only one type of carbon atom, C 70 possesses 5 different types of carbons. Furthermore, the commercially obtained 13 C-enriched C 70 sample was found to be contaminated with C 60 . Therefore, the 13 C NMR displayed a total of 6 signals from a mixture of fullerenes (ESI Fig. S4 †). The same external standard technique was employed to confrm the signal shift. This study indicated that in the nanoparticles, the signals shifted by approximately 1, 1.2, 1.5, 1.1, and 1.1 ppm upon C 70 complexation. The signals were also relatively broad. These results again indicated that the fullerenes were localized in the particle core. Interestingly, when the two fullerenes are part of the same nanoparticle core, the shift in the C 60 signal is nearly half (0.7 ppm) that when there is no competition from C 70 . This indicates that C 70 is a preferred guest and binds more strongly to the multicorannulene host. In DLS, the Fig. 4 Different approaches for the encapsulation of fullerenes into the host polymer micelle. The top shows exposure of pre-formed micelles to fullerenes, which leads to low fullerene loading capacity. The bottom shows an alternative approach in which the micelles are allowed to form in the presence of fullerenes leading to a higher fullerene loading capacity. nanostructure sizes increase when the core nests the fullerenes (Fig. 6). In transmission electron microscopy (TEM), these cores could be visualized without the need for staining the samples due to their high electron densities (Fig. 7 and ESI Fig. S5 †). Finally, corannulene was replaced with a simple phenyl group in polymer 1 (Scheme 3). This effort was directed at examining the antithesis that fullerene encapsulation in the micellar core required only a hydrophobic atmosphere and simple aromatic groups for stabilization. For this, the ring opening reaction was carried out using thiophenol to give polymer 2 (ESI Fig. S6 †). Polymers 1 and 2 were obtained from the same PEG-b-PGMA scaffold. Hence, they contained identical number of repeating units in the polymer chains (m z 113, n z 17) necessary for comparison. Polymer 2 successfully formed micelles in water (ESI Fig. S7 †). Importantly, however, it failed to show any fullerene uptake in its micellar core (ESI Fig. S8 †). The 13 C NMR and UV-Vis spectroscopies, therefore, suggest that the specifc convex-concave interactions between fullerenes and corannulene are key to encapsulating a large amount of fullerenes in the nanoparticle core. ## Structural integrity of the nanoparticles Typically, aqueous dispersions of fullerene nanoparticles are prone to aggregation in the drying process, thereby losing their structural integrity. In the current molecular design, the particle shells are noncrosslinked and provide a molecularly defned layer of solvation (hydration) and protection to the crosslinked core against changes in concentration. The removal of the solvent and a redispersion therefore do not lead to any adverse effect on the integrity of the nanoparticles. This can be observed by drying the nanoparticulate matter and then redissolving it in water. The hydrodynamic volume before and after this process remains unchanged (ESI Fig. S9-S11 †). This characteristic allows nanoparticles to be stored and transported in a powdered form. The aqueous solution can be prepared when required. Furthermore, once the solution is prepared, no changes are observed in the particle sizes even for months after their dissolution (ESI Fig. S12 †). Encouraged by these results, the thermal stability of the cargo was studied with the help of 13 C NMR spectroscopy (Fig. 8). The goal of these experiments was to heat the nanoparticles and determine whether uncomplexed fullerene could be detected in solution. If so, it would indicate that the guestloaded core is unstable at higher temperatures and releases the guest molecules into the solution. In these experiments, an external standard was not employed to avoid overlap between the free fullerene signals. Initially, the C 60 -containing nanoparticles were heated at 60 C for 24 hours, and no signal belonging to the free fullerene could be observed in the solution. Therefore, the temperature was increased to 100 C, and the sample was heated for 70 hours. In this case also, the complexed fullerene signal remained intact. Finally, nanoparticle stability was evaluated against acidic and basic conditions. For this, nanoparticles were dissolved in PBS (pH ¼ 7.4) and citric acid/Na 2 HPO 4 (pH ¼ 2.5) buffers and monitored with the help of DLS. The nanoparticles did not display any change in their diameter under either conditions (ESI Fig. S13 †). The ability to switch from bulk materials to aqueous solution and withstand demanding thermal/pH conditions relates to the core-shell morphology of the nanostructures and is unprecedented in the fullerene-based water-soluble nanoparticle arena. ## Radical scavenging activity Having access to fullerene nanoparticles, their free radical scavenging activity was assessed with the help of a 2,2 0 -azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay. 34 In this assay, the interaction between an antioxidant and the pregenerated ABTS radical cation was examined with the help of UV-Vis spectroscopy (Fig. 9). Upon successful quenching of the radical cation, a decrease in the absorption intensity of the signals located at 645, 734, and 815 nm could be observed. The amount of antioxidant required to quench this intensity by 50% is defned as the inhibition concentration 50 (IC 50 ). This study produced interesting results. The micelles without C 60 show moderate antioxidant capacity (Fig. 9a), most likely due to (b). Polymer nanoparticles with fullerene C 70 (c). A comparison of the three systems to evaluate the amount needed to quench 50% of the free radicals in the system (d). corannulenes in the micellar core (IC 50 $ 1000 mg mL 1 ). The presence of C 60 enhanced this capacity (IC 50 ¼ 529 mg mL 1 ) (Fig. 9b). In the case of C 70 , the visual color changes could already be appreciated beginning at 100 mg mL 1 (Fig. 9c). In this system, to reach IC 50 , a relatively lower amount of 384 mg mL 1 was required. C 70 -containing nanoparticles, therefore, are the most active system in this regard (Fig. 9d). ## Protein adsorption This aspect was studied with the help of bovine serum albumin (BSA) and the Bradford method to quantify the amount of the adsorbed protein onto the nanoparticles. 35 In this method, binding of the protein to a dye molecule changes its absorbance from red to blue region (ESI Fig. S14 †). If no protein binds, then the solution remains brown. Thus, the amount of complex present in solution is a measure of the protein concentration and can be estimated by use of an absorbance reading. To achieve this, the particles were incubated with BSA for 3 hours to achieve equilibration. After this time, the particles were removed from the solution by centrifugation, and the supernatant was examined by means of UV-Vis spectroscopy (595 nm) to quantify the amount of the 'free' (unadsorbed) protein. In this way, the amount of adsorbed protein was calculated to be 3.5 and 3.8% for nanoparticles containing C 60 and C 70 , respectively. ## Hemolysis As a measure of biocompatibility, a hemolysis assay was performed to assess the lytic activity of nanoparticles using sheep red blood cells (RBCs). In this study, the released hemoglobin was detected by means of UV-Vis spectroscopy at a wavelength of 540 nm and compared to positive and negative controls. The negative control was PBS buffer, in which the RBCs do not disintegrate owing to the isotonic effect and a net zero movement of molecules across the membrane. 36 The positive control was deionized water, in which RBCs disintegrate fully due to a hypotonic effect and movement of pure water into the cells through osmosis, resulting in bursting of the cell walls. In the case of nanoparticles, however, no hemolysis was observed even at a concentration of 1000 mg mL 1 (ESI Fig. S15 †). This suggests that nanoparticles have no toxic effects on mammalian cell walls, which bodes well for their future applicability as diagnostic probes. ## Conclusions In summary, core-crosslinked polymer nanoparticles can be prepared by combining a supramolecular complexation and covalent stabilization strategy. The particles contain a high loading (8%) of smaller (C 60 ) and larger (C 70 ) fullerenes and provide an opportunity to compare their properties. Radical scavenging leads to the conclusion that C 70 particles possess higher antioxidant activity (IC 50 ¼ 384 mg mL 1 ). The particles can be stored in a powdered form in bulk and can be redispersed when required. They are stable even when heated to 100 C for 70 hours. They resist the accumulation of proteins (<4%) and are nonhemolytic even at high concentrations (1000 mg mL 1 ). It is anticipated that replacing normal fullerenes from the present design with endohedral fullerenes containing a contrast agent, such as Gd(III), will lead to nontoxic imaging probes with long circulation times due to their nanometer sizes and a poly(ethylene glycol) shell. Furthermore, the preparation of water-soluble nanoparticles with higher fullerenes 37 (e.g., C 76 and C 84 ) and the study of their biorelevant properties appear to be an enticing research direction. ## Experimental details Micelle formation First approach. Diblock copolymer 1 (3 mg) was frst dissolved in THF (150 mL). After adding this suspension dropwise into deionized water (3 mL) under stirring, the micellar solution was dialyzed (dialysis tube: cutoff 1 kDa) against deionized (DI) water for 2 days to remove the THF. The fnal polymer concentration was about 1 mg mL 1 . Fullerene (1 eq. per corannulene unit) was added into the micellar solution (3 mg mL 1 ) and sonicated for 30 minutes. The solution was then fltered through a 0.8 mm syringe flter (cellulose acetate). Second approach. Diblock copolymer 1 (3 mg) and fullerene (1 eq. per corannulene unit) were frst suspended in THF (150 mL) and stirred for 30 minutes. After adding this suspension dropwise into deionized (DI) water (3 mL) under stirring, the micelle solution was then fltered through a 0.8 mm syringe flter (cellulose acetate). The mixture was dialyzed (dialysis tube: cutoff 1 kDa) against deionized water for 2 days to remove the THF. ## Crosslinking of micelles To a solution of adipic acid (0.15 mg, 1 mmol (0.5 eq. per hydroxyl unit)), EDCI (0.5 mg, 0.2 mmol) and DMAP (0.3 mg, 0.2 mmol) in DI water (50 mL) were added dropwise into the micellar solution (1 mg mL 1 ). The reaction mixture was stirred at room temperature overnight. After this time, the reaction mixture was dialyzed (dialysis tube: cutoff 1 kDa) against DI water for 1 day. The crosslinked micelles were lyophilized to dryness. ## Conflicts of interest There are no conflicts to declare.

chemsum

{"title": "Aggregation-free and high stability core\u2013shell polymer nanoparticles with high fullerene loading capacity, variable fullerene type, and compatibility towards biological conditions", "journal": "Royal Society of Chemistry (RSC)"}

discovery_of_chemical_markers_for_improving_the_quality_and_safety_control_of_sinomenium_acutum_stem

## Abstract: Sinomenium acutum stem is a popular traditional chinese medicine used to treat bone and joint diseases. Sinomenine is considered the only chemical marker for the quality control of S. acutum stem in mainstream pharmacopeias. However, higenamine in S. acutum stem is a novel stimulant that was banned by the World Anti-Doping Agency in 2017. Therefore, enhancing the quality and safety control of S. acutum stem to avoid potential safety risks is of utmost importance. In this study, a fast, sensitive, precise, and accurate method for the simultaneous determination of 11 alkaloids in S. acutum stem by ultrahigh-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry (UHpLc-QQQ-MS/MS) was established. this method successfully analyzed thirty-five batches of S. acutum stem samples. The average contents of sinomenine, magnoflorine, coclaurine, acutumine, higenamine, sinoacutine, palmatine, magnocurarine, columbamine, 8-oxypalmatine, and jatrorrhizine were 24.9 mg/g, 6.35 mg/g, 435 μg/g, 435 μg/g, 288 μg/g, 44.4 μg/g, 22.5 μg/g, 21.1 μg/g, 15.8 μg/g, 9.30 μg/g, and 8.75 μg/g, respectively. Multivariate analysis, including principal component analysis (PCA), orthogonal partial least square method-discriminant analysis (OPLS-DA), and hierarchical cluster analysis (HCA), were performed to characterize the importance and differences among these alkaloids in S. acutum stem samples. As a result, sinomenine, magnoflorine, coclaurine, acutumine, and higenamine are proposed as chemical markers for quality control. Higenamine and coclaurine are also recommended as chemical markers for safety control. This report provides five alkaloids that can be used as chemical markers for improving the quality and safety control of S. acutum stem. it also alerts athletes to avoid the risks associated with consuming S. acutum stem. Abbreviations EIC Extracted ion chromatogram ESIElectrospray ionization The precision was determined by continuously testing one concentration of the mixed standard solution six times. Sample Q29 was separated into six portions, and each portion was extracted separately to evaluate the repeatability of this method. The test solution of sample Q29 was employed to evaluate the stability over 0, 2, 4, 6, 8, and 12 h in one day at room temperature. The results for all the compounds, which are summarized in Table 2, indicate that the instrument has good precision, the method is repeatable, and the compounds in the sample solution are sufficiently stable for accurate and precise analysis within 12 h at room temperature (Supplementary information). Six portions (approximately 0.25 g each) of sample Q29 were accurately weighed and extracted separately for the recovery test. The results (Table 2) show that the recovery rates of all 11 alkaloids were within the range of 97.7-103.5% with RSDs not more than 5%, indicating that this method is accurate enough to measure the contents of these 11 compounds in S. acutum stem. Multivariate analysis. Principal component analysis (PCA) analysis showed that the cumulative contribution of the first three principal components was 64.3%. The 3D score scatter plot (Fig. 4A) showed that the 11 alkaloids could be divided into three groups, i.e., sinomenine in Group 1, magnoflorine in Group 2, and all the others in Group 3. The orthogonal partial least square method-discriminant analysis (OPLS-DA) model www.nature.com/scientificreports/ resulted in a (1 + 3) component with the variables of R2 (X) of 0.614, R2 (Y) of 0.792, and Q2 of 0.633. As shown in Fig. 5A, the samples were differentiated into two groups by the OPLS-DA model. The low content group is on the left side of the score plot, while the high content group is on the right side. In the loading plot (Fig. 5C), sinomenine, magnoflorine, higenamine, coclaurine, and acutumine were further from the origin. The plot in Fig. 5D displays the variable importance for the projection values (VIPs) of all variables. The VIPs of OPLS-DA demonstrated that sinomenine and magnoflorine had the greatest influence. Hierarchical cluster analysis (HCA) showed that the 11 alkaloids could be divided into three groups, as shown in Fig. 6. Sinomenine and magnoflorine were present at high levels. Higenamine, acutumine, and coclaurine were present at moderate levels. Magnocurarine, sinoacutine, columbamine, jatrorrhizine, palmatine, and 8-oxypalmatine were present at low levels. ## Discussion In this study, thirty-five batches of S. acutum stem samples were acquired from local hospitals or pharmacies in China and were determined by the optimized UHPLC-QQQ-MS/MS method. Hua Zhou authenticated all the samples, and the voucher specimens are stored at the State Key Laboratory of Quality Research in Chinese Medicine (Macau University of Science and Technology). The sample number, sample origin, and collection location for each sample are shown in Table 3, and the samples are highly representative of this herb. The average total content of these 11 alkaloids was 32.6 ± 7.17 mg/g. These alkaloids can be empirically divided into three groups (Fig. 7) based on their abundance. Sinomenine and magnoflorine, which are present at the milligram level and account for 76.6 and 19.5%, respectively, of total content, can be classified as high-abundance compounds. Coclaurine, acutumine, and higenamine, which are present at the microgram level and account for 1.34, 1.34, and 0.885%, respectively, of the total content, can be classified as moderate-abundance compounds. Moreover, the remaining six alkaloids, sinoacutine, palmatine, magnocurarine, columbamine, 8-oxypalmatine, and jatrorrhizine, although at the microgram level, account for less than 0.374% of the total content and therefore can be classified as low-abundance compounds. Magnoflorine is a homolog of sinomenine, and they have similar phenanthrene structures and similar biosynthetic pathways 22 , which could be the reason that they are in the same group. A similar explanation can be applied in the case of higenamine and coclaurine 11 . www.nature.com/scientificreports/ PCA can simplify complex information by replacing the original variable index with a small number of comprehensive indicators utilizing dimension reduction. In the present study, PCA was used to analyze the distribution pattern of the 11 alkaloids in the S. acutum stem. The result of the cumulative contributions meant that the original information of the dataset was basically retained. The 2D loading scatter plot (Fig. 4B), which provided useful information to identify the essential features in the PC1 and PC2 dimensions, showed that sinomenine, magnoflorine, coclaurine, acutumine, and higenamine were located at the edges of the axes, demonstrating the greater correlations with PC1 and PC2 and implying that these five compounds were essential. OPLS-DA is a supervised discrimination method. Figure 5B shows the modeling used to predict the specific components with the most significant influence on the samples. It demonstrated the considerable quality of the model. Samples with more substantial VIP (> 1.0) are generally more related to sample classification. We found that the VIP values of sinomenine and magnoflorine were more than 1.0, and those of higenamine and coclaurine were very close to 1, and these compounds were in the top four. Therefore, as indicated by the plots in Fig. 5C,D, sinomenine, magnoflorine, higenamine, and coclaurine played an essential role in the S. acutum stem samples. The HCA results were also consistent with the results of content determination, PCA, and OPLS-DA above. Therefore, multivariate analysis verified that sinomenine, magnoflorine, higenamine, coclaurine, and acutumine were the main chemical components of the S. acutum stem. According to the Chinese Pharmacopeia, the following three requirements should be used for the selection of potential chemical markers. The content of the marker in the medicinal material should be higher than 0.02%. The corresponding specific or active ingredients selected as markers for content determination should be involved in the function or bioactivity of the Chinese medicines. A multicomponent detection method should be used when a single component cannot reflect the medicinal materials' overall activity 23 . The pharmacological activities of sinomenine 2,24-26 , magnoflorine 14,27 , coclaurine 28 , acutumine 29 , and higenamine 30,31 well represent the main indications or bioactivities of S. acutum stem, which are anti-inflammation, analgesia, anti-hypertension, anti-arrhythmia, anticancer, and immunomodulation 32 . Therefore, based on the multivariate analysis results and the bioactivities of these components, it was reasonable and representative to identify these specific alkaloids, i.e., sinomenine, magnoflorine, higenamine, acutumine, and coclaurine, as chemical markers of S. acutum stem. These alkaloids are recommended for improving the quality control of S. acutum stem in pharmacopeias and for use as relevant standards in the future. The use of sinomenine and magnoflorine as chemical markers was also consistent with the published literature conclusions 2,33 . The other three alkaloids proposed together with sinomenine and magnoflorine better reflect the overall quality of this herb. www.nature.com/scientificreports/ For the safety control of S. acutum stem, higenamine and coclaurine are proposed as chemical markers. Before discussing the safety, we determined that the sinomenine content in each of the samples tested in this study met the requirements of the pharmacopoeias 3,4 . These samples were qualified under the existing standards. In this study, higenamine and coclaurine were found in S. acutum stem at high levels. Therefore, athletes should consider this herbal medicine and its products with caution. The WADA stipulates that all selective and nonselective beta-2 agonists, including all optical isomers, are prohibited. Higenamine is a β-androgenic receptor agonist. It possesses lipolytic activity and can improve cardiac left ventricular function. Therefore, it can promote the growth of skeletal muscle. This is the main reason that higenamine is explicitly banned as a novel stimulant ingredient 8 . The average contents of higenamine and coclaurine in the S. acutum stem were 288 μg/g and 435 μg/g, respectively, in the present study. The recommended oral dosage of S. acutum stem is 6-12 g per day according to the Chinese Pharmacopoeia. A previous study reported that the maximum urinary concentration of higenamine in humans was 0.2-0.4 ng/mL within 10-12 h after oral administration of the herbal product containing 19.8 μg higenamine, and the maximum urinary concentration of coclaurine was 0.3-1.0 ng/mL (corresponding to 4.5 μg of coclaurine) 21 . Therefore, we initially speculated that the maximum urinary concentration ranges of higenamine and coclaurine that could be detected after oral S. acutum stem treatment for 10-12 h were 17.5-69.9 ng/mL and 39.5-264 ng/mL, respectively, if the ingredients were completely extracted. The anti-doping organization required that the concentration of higenamine in urine should not be more than 10 ng/mL 34 . Apparently, when athletes take regular doses of S. acutum stem, the urinary concentration of higenamine exceeds the stimulant detection threshold. Even worse, because of the enzymatic conversion between coclaurine and higenamine, the total concentration of stimulants may also increase in the human body. Therefore, according to the existing www.nature.com/scientificreports/ standards, these qualified herbs still pose a notable safety risk. With the widespread use of S. acutum stem and its products, there is a high risk that unintentional higenamine doping could be detected. This situation also reminds industry and regulatory bodies that higenamine and coclaurine can be used as chemical markers for the safety control for S. acutum stems. It is better to warn athletes to use caution on the packaging of related products. This study also alerts athletes that they should be very cautious when they are seeking assistance from herbal medicines to relieve inflammation and pain caused by sports injuries. ## Materials and methods Reagents and chemicals. Sinomenine, magnoflorine, sinoacutine, columbamine, and coclaurine as chemical reference standards were purchased from Chengdu Chroma Biotechnology Co., Ltd. (Chengdu, P.R. China). Higenamine, acutumine, magnocurarine, jatrorrhizine, palmatine, and 8-oxypalmatine as reference standards were purchased from Shanghai Chenyi Biotechnology Co., Ltd. (Shanghai, P.R. China). All compounds were at a purity of ≥ 98.0%, and the chemical structures are given in Fig. 8. Formic acid from Sigma-Aldrich Corporation (St. Louis, MO, USA), acetonitrile from Anaoua Chemicals Supply (Houston, TX, USA), and methanol from Anaoua Chemicals Supply (Cleveland, OH, USA) were of HPLC grade. Ethyl alcohol from Anaoua Chemicals Supply (Cleveland, OH, USA) and other chemicals and reagents were of analytical grade. instrument and analytical conditions. An ultrasonic cleaner (i-Quip, Shanghai, China) was used to extract the alkaloids from the samples. A Milli-Q ultrapurification system (Millipore, Bedford, MA, USA) was used to produce ultrapure water. A UHPLC system (1290 series, Agilent Technologies, Santa Clara, CA, USA) coupled with a QQQ-MS/MS (6460 series, Agilent Technologies, Santa Clara, CA, USA) was used to quantitatively detect the 11 alkaloids. Chromatographic separation was performed on a Waters ACQUITY UPLC HSS C18 SB column (2.1 mm × 100 mm, 1.7 μm, Waters, Milford, MA, USA) at 30 °C with a mobile phase consisting of 0.1% formic acid (A) and acetonitrile (B) in the following gradient: 0-4 min, 16-16% B; 4.01-7 min, 30-30% B; 7.01-10 min, 70-70% B. The injection volume was 2 μL, and the flow rate was 0.35 mL/min. Mass Hunter software (Agilent Technologies, Santa Clara, CA, USA) was used for optimization and quantification. The 11 alkaloids were detected using multiple reaction monitoring (MRM) and an electrospray ionization (ESI) source in positive ion mode. The transitions of the 11 compounds are shown in Table 2. The other parameters were as follows: drying gas (N 2 ) flow rate, 11.0 L/min; drying gas temperature, 300 °C; nebulizer, 15 psig; and capillary voltage, 4,000 V. ## Standard solution preparation. Appropriate amounts of the reference standards of the 11 alkaloids were dissolved in 70% ethanol to prepare stock solutions. The mixed standard solution was obtained by accurately mixing the 11 stock solutions and diluting them with 70% ethanol. The final concentrations of sinomenine, magnoflorine, coclaurine, acutumine, higenamine, sinoacutine, palmatine, magnocurarine, columbamine, 8-oxypalmatine and jatrorrhizine in the mixed solution were 156, 63.4, 4.90, 7.62, 4.02, 0.656, 0.546, 0.723, 0.437, 0.342 and 0.247 μg/mL, respectively. Two microliters of the mixed standard solution was injected into the UHPLC- The sample powder (0.5 g passed through a 50 mesh sieve) was accurately weighed in a conical flask with a stopper. The flask was supplemented with 20.0 mL of 70% ethanol, well stoppered, and accurately weighed. Next, the flask was ultrasonicated for 20 min (power 150 W, frequency 20 kHz), cooled to room temperature, weighed again, and the weight was restored to its initial value with 70% ethanol. The sample solution was shaken thoroughly and filtered through 0.22-μm microporous filtration membranes. Two hundred microliters of the obtained filtrate was diluted to 1.0 mL with 70% ethanol to afford the test solution.

chemsum

{"title": "Discovery of chemical markers for improving the quality and safety control of Sinomenium acutum stem by the simultaneous determination of multiple alkaloids using UHPLC-QQQ-MS/MS", "journal": "Scientific Reports - Nature"}

a_homoleptic_alkynyl-protected_[ag₉cu₆(^<i>t</i>bucc)₁₂]

## Abstract: We report the first homoleptic alkynyl-protected AgCu superatomic nanocluster [Ag 9 Cu 6 ( t BuC^C) 12 ] + (NC 1, also Ag 9 Cu 6 in short), which has a body-centered-cubic structure with a Ag 1 @Ag 8 @Cu 6 metal core. Such a configuration is reminiscent of the reported AuAg bimetallic nanocluster [Au 1 @Ag 8 @Au 6 ( t BuC^C) 12 ] + (NC 2, also Au 7 Ag 8 in short), which is also synthesized by an anti-galvanic reaction (AGR) approach with a very high yield for the first time in this study. Despite a similar Ag 8 cube for both NCs, structural anatomy reveals that there are some subtle differences between NCs 1 and 2. Such differences, plus the different M 1 kernel and M 6 octahedron, lead to significantly different optical absorbance features for NCs 1 and 2. Density functional theory calculations revealed the LUMO and HOMO energy levels of NCs 1 and 2, where the characteristic absorbance peaks can be correlated with the discrete molecular orbital transitions. Finally, the stability of NCs 1 and 2 at different temperatures, in the presence of an oxidant or Lewis base, was investigated. This study not only enriches the M 15 + series, but also sets an example for correlating the structure-property relationship in alkynyl-protected bimetallic superatomic clusters. ## Introduction Superatomic coinage bimetallic nanoclusters (NCs) with atomic precision are currently being extensively investigated due to their tunable structure, 1,2 enhanced stability, and signifcantly modifed physicochemical properties, compared with homonuclear parent clusters. 9-13 Among these monolayerprotected clusters, the most studied combination is Au-Ag, as they are fully miscible in bulk. 14,15 It is well known that a plethora of stable Au NCs have been synthesized and characterized in the past few decades, 16,17 yet stable Ag NCs comprise a rather recent entry in the coinage metal NC feld. 18,19 In contrast to Au or Ag, they are far fewer examples of Cucontaining coinage alloy NCs, particularly AgCu molecules. As for the capping agent for protection, mixed ligands can yield a more complicated structure and hence obscure the structureproperty relationship establishment; therefore, molecules of single structure type particularly thiolate compounds have been widely employed to prepare AgCu NCs. For instance, in 2016, Zheng's group reported the frst thiolated chiral threeconcentric-shell cluster containing free valence electrons, [Ag 28 Cu 12 (SR) 24 ] 4 (SR ¼ 2,4-dichlorobenzenethiolate). 20 In 2018, Zhu's group showcased the controllable synthesis of the AgCu bimetallic NC [Ag 40.13 Cu 13.87 S 19 ( t BuS) 20 ( t BuSO 3 ) 12 ], which consisted of a Cu 10 Ag 2 S 7 core, a M 42 ( t BuS) 20 ( t BuSO 3 ) 12 shell, and another 12 bare S atoms. 21 Recently, Bao et al. prepared the [Ag 13 Cu 10 (SAdm) 12 ] 3+ (Adm ¼ -SC 10 H 15 ) NC, which has a Ag 13 core and a Cu 10 (SAdm) 12 shell. 22 Recently, alkynyl molecules have been emerging as a new type of ligand for preparing coinage metal NCs, 23,24 mainly because alkynyl molecules can generate more diverse surface binding moieties and some undiscovered molecular clusters with magic numbers, eventually leading to drastically different functionalities. In terms of AuAg NCs, Wang and Zheng groups documented the fabrication of the Au 34 Ag 28 (-PhC^C) 34 NC and its use as a model catalyst to explore the signifcance of surface ligands in promoting catalysis. 34 In another report, Wang et al. discovered that when incorporating alkali metal ions or copper atoms into the alkynyl-protected body-centered cubic (BCC) [Au 7 Ag 8 ( t BuC^C) 12 ] + NC (which is also NC 2 in this study), site preference can be observed. 35 Recently, Yuan et al. found that the alkynyl-protected monomeric (AuAg) 34 can be assembled into 1D polymers with Ag-Au-Ag bonds between neighboring clusters through a solventmediated approach. 36 In the AgCu regime, the alkynyl ligand has also been utilized. For instance, Williams and co-workers reported two halide-ion-templated heterometallic Ag 8 Cu 6 rhombic dodecahedron clusters and investigated the spectroscopic properties and reactivity of these clusters along with those of the parent Ag 14 NCs. 37 Mak's group employed the designed tetranuclear precursors [(R-C^C-C^C)Ag] 4 (R ¼ i Pr, t Bu, and Ch) to construct a series of heteropolynuclear silver(I)copper(I) diynyl complexes that bear a common trigonal-planar CuAg 3 NC core, and such complexes exhibited long-lived emission upon photoexcitation in various media at room temperature and 77 K. 38 In a recent study, Zang's group discovered that the o-carboranealkynyl-protected [Cu 6 Ag 8 (C 4 -B 10 H 11 ) 12 Cl]NO 3 NC can serve as a perfect hypergolic material, as its ignition delay time can be shortened to 15 ms. 39 However, in the above examples of AgCu NCs, all the Ag and Cu atoms are present as the +1 charge state, and no free electrons exist in these clusters. According to the superatom theory, 40 these molecules are regarded as M(I) clusters or complexes rather than superatoms with free electrons. Due to the presence of free electrons, the superatoms have quite different structures and physicochemical properties, and hence they can fnd different applications in catalysis, 41,42 optoelectronic devices, 36 biomedical regimes 31 and so on. To the best of our knowledge, no case of a homoleptic alkynyl-protected superatomic AgCu NC with free electrons has been reported so far. Herein, we report the frst case of a homoleptic alkynylprotected AgCu superatomic NC, namely [Ag 9 Cu 6 ( t BuC^C) 12 ] + (NC 1, also Ag 9 Cu 6 in short), which has a BCC-based structure with a three-layered Ag 1 @Ag 8 @Cu 6 metal core confguration. Such a structure is reminiscent of the reported [Au 7 Ag 8 (t BuC^C) 12 ] + (NC 2, also Au 7 Ag 8 in short) molecule with a Au 1 @Ag 8 @Ag 6 metal architecture, and it is frst-time synthesized by an anti-galvanic reaction (AGR) approach with a ultrahigh yield in this study. The structural differences between NC 1 and NC 2 result in signifcantly different optical absorption properties. Comprehensive DFT calculations disclosed the discrete LUMO and HOMO energy levels of NCs 1 and 2, where the characteristic absorbance peaks of NCs 1 and 2 can be correlated to the specifc molecular orbital transitions. Finally, the stability of NCs 1 and 2 at room temperature and 60 C, in the presence of H 2 O 2 or CH 3 ONa, was investigated and compared. ## Results and discussion NC 1 was frst prepared by following a modifed "Two-in-One" method (see experimental details in the ESI †). 43 Briefly, in the presence of NaSbF 6 , t BuC^CAg(I) is reduced by (PPh 3 ) 2 CuBH 4 in the mixed solvent of dichloromethane and acetonitrile. The reaction was aged for 12 h during which the solution gradually changed from colorless to yellow, and fnally to dark blue. A blue block crystal was obtained by diffusing methanol into the dichloromethane solution containing the crude product. It is worth noting that by introducing one metal in the precursor and another metal in the reducing agent, such a "Two-in-One" method might be universal for synthesizing bimetallic NCs. NC 2 was synthesized by the AGR approach between organometallic t BuC^CAg(I) and Au 22 ( t BuC^C) 18 NC under mild conditions. The synthetic protocols regarding the t BuC^CAg(I) precursor and Au 22 ( t BuC^C) 18 can be found in the ESI. † The chemical compositions of NCs 1 and 2 were verifed by electrospray ionization mass spectrometry (ESI-MS) in positive mode. As shown in Fig. 1 ] + (cal. MW: 3214.8515 Da, deviation: 0.0005 Da), respectively. Also, the isotopic distributions of the two NCs match perfectly with the simulated results (inset in Fig. 1A and B). One may notice that there are some fragments in the ESI-MS spectra of NC 1, and the peak analysis in Fig. S1 ] + , respectively. For NC 2, there is a much less pronounced peak, and the peak assignment analysis in Fig. S2 † shows that it can be assigned to [Au 7 Ag 8 ( t BuC^C) 10 ] + , which is probably generated by losing two ligands (-t BuC^C) from parent NC 2. Moreover, the fngerprint absorbance peaks of NC 1 are located at 544, 579, and 620 nm, and its characteristic absorbance feature along with the simulated pattern will be discussed next. Nevertheless, we monitored the absorbance change during the formation of NC 1. As shown in Fig. S3A, † upon reduction, an absorbance peak at 477 nm gradually appeared with the maximal value reached at 1 h. After that, such an absorbance peak gradually diminished and the characteristic peak at 579 nm from NC 1 gradually emerged. The 477 nm absorbance peak indicates that some intermediate may exist; however, several attempts to isolate it were not successful. As depicted in Fig. S3B, † the sharp color transition from yellow to dark yellow, slight pink and eventually blue can be clearly visualized. To better understand the AGR process from Au 22 (t BuC^C) 18 to NC 2, the reaction process was also monitored using time-resolved UV-visible absorption spectra. As shown in Fig. S4A, † the absorbance features of Au 22 NC disappeared immediately upon the addition of t BuC^CAg(I), while a new absorption band at $537 nm emerged. There are two obvious color changes at the time point of t BuC^CAg(I) addition and in the period from 4 to 8 h (Fig. S4B †). After 2 h, the characteristic peak at 487 nm of NC 2 gradually emerged, meanwhile the absorbance peak at 537 nm gradually diminished. It has been postulated but not ascertained that there might be some critical intermediate during the AGR process, which is still under investigation. The electronic structures of NCs 1 and 2 were subsequently probed by X-ray photoelectron spectroscopy (XPS), and the results are presented in Fig. S5, S6 and Table S1. † As depicted in Fig. S5A, † the XPS survey scan profle confrmed the coexistence of Ag and Cu elements. The Ag/Cu atomic ratio is estimated as 10.33/6.90, in good agreement with the theoretical value (9/6). It can be noted that the binding energy of the Ag 3d 5/ 2 electrons is located at 368.41 eV, higher than that of bulk Ag (367.9 eV) and lower than that of Ag(I) (368.87 eV) (Fig. S5B †). 44 It suggests that the valence state of Ag in NC 1 is between 0 and +1. Meanwhile, the binding energy of Cu 2p 3/2 (933.12 eV) agrees well with that of Cu(I) (932.45-933.48 eV), implying that six Cu atoms are present as Cu(I) (Fig. S5C †). 45,46 For NC 2, from the XPS survey scan profle (Fig. S6A †), the Ag/Au atomic ratio can be estimated as 8.27/7.28, in good agreement with the theoretical value (8/7). As shown in Fig. S6B, † the binding energy of the Au 4f 7/2 electrons is located at 84.50 eV, in between those of bulk Au (84.0 eV) and Au(I) (86.0 eV), 47 suggesting that the M 1 core in NC 2 is Au(0). In addition, the binding energy of the Ag 3d 5/2 electrons is located at 368.83 eV, indicating that the valence state of Ag in NC 2 is +1 (Fig. S6C †). 45 Subsequently, the atomic packing structure of NC 1 was examined using a single-crystal X-ray diffractometer. As illustrated in Fig. S7, † NC 1 crystallizes in the space group R 3, and each unit cell has a SbF 6 counterion, indicating that NC 1 possessed a +1 charge state. More detailed structural parameters are summarized in Table S2. † The structural anatomy of NC 1 is shown in Fig. 2A, which contains nine silver atoms, six copper atoms and twelve tert-butylacetylene ligands, and hence the molecule can be formulated as [Ag 9 Cu 6 ( t BuC^C) 12 ]SbF 6 . The six copper atoms and twelve alkynyl ligands form six t BuC^C-Cu-C^C t Bu motifs on the surface of this quasispherical structure of NC 1 (Fig. 2B). Interestingly, all tertbutylacetylene ligands bind with Cu atoms via s bonding, and with Ag atoms in the p manner (Fig. 2C). Note that such linear motifs were observed for the frst time in the alkynyl-protected AgCu bimetallic NCs, even though similar linear motifs have been previously documented in several alkynyl-protected AuAg NCs, including Au 7 Ag 8 , 35 Au 24 Ag 20 , 48 Au 34 Ag 28 , 34 (AuAg) 34 , 36 Au 57 Ag 53 , 49 and Au 80 Ag 30 . 50 As illustrated in the space-flling structure (Fig. 2D), six Cu sites and eight Ag sites are exposed, which might serve as open active sites for catalysis. Despite the orientation of the rigid ligand on the surface of NC 1 being similar to that of NC 2, the exposure extent of Ag atoms in NC 2 is somehow more than that in NC 1 (Fig. S8A and B †). However, for NC 2, even with the same ligand, it is more expansive, probably owing to the much larger radius of the Au atom (Fig. S8C and D †) than that of the Cu atom. Next, the anatomical structure of NC 1 is compared with that of NC 2 (the detailed structural parameters of NC 2 are summarized in Table S3 †). As shown in Fig. 2E, NC 1 adopts a core-shell-shell confguration of Ag 1 @Ag 8 @Cu 6 , similar to that of NC 2, as both of them can be classifed as the BCC-based M 15 + series with an M 1 kernel@Ag 8 cube@M 6 octahedron architecture. However, the AuAg core in NC 2 is slightly constricted compared to the AgCu core in NC 1, as the average adjacent Ag-Ag bond length is 3.333 and 3.271 in the Ag 8 cube for NC 1 and 2, respectively (Fig. S9 †). In addition, the average bond length from the central Ag to the Ag atoms in the Ag 8 cube is 2.887 , comparable with that of bulk Ag (2.889 ). Note that, compared with the reported Ag-Ag bond lengths in alkynyl-protected Ag NCs listed in Table S4, † there is argentophilic Ag-Ag interaction in the Ag@Ag 8 cube for NC 1. Moreover, as illustrated in Fig. S10A, † the capped Cu atom isn't located exactly above the centre of the Ag 4 plane, which is different from the surface of NC 2 (Fig. S10B †). That means, besides the atom differences in the M 1 kernel and M 6 octahedron, NC 1 and NC 2 have some subtle structural differences, and such subtle differences may affect their physicochemical properties as well. In addition, the structure of the thiolateprotected M 15 NC Au 15 (SR) 13 also has been theoretically proposed, but it is quite different from that of NC 1 and NC 2, as it consists of a tetrahedral Au 4 core, a [Au 7 (SR) 7 ] ring, and two [Au 2 (SR) 3 ] "staple" motifs. 51 Next, we frst compared the optical absorbance properties of NC 1 and NC 2. For NC 1, as shown in Fig. 3A and S11, † there are four prominent peaks at 333 nm (3.72 eV), 544 nm (2.28 eV), 579 nm (2.14 eV), and 620 nm (2.00 eV), a broad absorption peak at 422 nm (2.94 eV), and a weak shoulder at 357 nm (3.47 eV). The energy bandgap derived from the absorbance spectrum is $1.80 eV. For NC 2, as presented in Fig. 3B and S11, † there are also four prominent peaks at 3.13 nm (3.96 eV), 422 nm (2.80 eV), 477 nm (2.60 eV), and 506 nm (2.45 eV), and a weak shoulder at 339 nm (3.65 eV), while the energy bandgap is $2.22 eV. It is worth noting that, despite some similar absorbance patterns, the absorbance features are drastically different for NC 1 and NC 2 in terms of the peak position and optical bandgap. Such huge discrepancies can be probably attributed to the structural differences. As both NCs adopt an M 1 @Ag 8 @M 6 metal confguration, the M 1 kernel (Ag vs. Au) can make a dramatically different contribution to the absorbance, and the outer M 6 octahedron (Ag 6 vs. Au 6 ) not only modulates the geometrical confguration, but also influences the electronic structure (discussed next). Given the standard absorbance curve (Fig. S12A and B †) of the two NCs, according to Lambert-Beer's law, the molecular absorptivity (3) of NC 1 (3 ¼ 0.35 10 4 M 1 cm 1 ) and NC 2 (3 ¼ 0.78 10 4 M 1 cm 1 ) can be determined, as summarized in Table S5. † Therefore, through calculation, the yield of NC 1 was 41.05% (based on Ag, and the yield is 41.74% based on Cu), and the yield of NC 2 was up to 86.72% (based on Au). The details of the calculation process can be found in the ESI † (Fig. S13 and Tables S6, S7 †). It is worth noting that the yield of NC 2 here is much higher than that of the previously reported method. This is mainly due to the fact that the reported method is a "bottom-up" direct reduction approach, in which other polydisperse clusters are also produced, while the AGR method here can yield more homogeneous products, and NC 2 is the main oxidation product (from 4e of Au 22 NC to 2e of Au 7 Ag 8 NC). In addition, we also studied the photo-luminescence properties of the two M 15 NCs. As shown in Fig. S14, † NC 2 strongly emits in the near-IR region (l max ¼ 818 nm), in good agreement with the previous report. 35 However, there is no obvious emission peak for NC 1. To elucidate the relationship between the electronic structure and optical properties of NCs 1 and 2, we carried out time dependent-density functional theory (TD-DFT) calculations. The optimized structure based on the crystal structure is used as a model for TD-DFT calculation. As shown in Fig. 3E and F, there is no change in structure after optimization, except for a slightly distorted orientation of the ligands. The distribution of the electronic cloud map of the highest occupied molecular orbital (HOMO) of NC 1 is quite similar to that of NC 2, whereas the cloud density position in the lowest unoccupied molecular orbital (LUMO) of NC 1 is quite different from that of NC 2. From the cloud density distribution, it can be clearly noted that charge transfer occurs from ligands to the metal core for NC 2, resulting in free electron localization in the metal core. While in stark contrast, charge transfer happens from the metal core to ligands for NC 1, which leads to a non-radiative loss of excited state electron energy. Such different charge transfer behaviors are probably the main cause of the different luminescence properties. From Fig. 3E, the molecular orbitals of NC 1 revealed a jelliumatic shell closing at the HOMO state of 1S with two electrons and the LUMO state of 1P, in which there was conversion from S to P. It is also in good accord with the electron counting results, as the metal core offers ffteen delocalized electrons; while twelve electrons are delocalized at the metalcore bonds, one electron has to be deducted to form a cation, and hence NC 1 can be literally considered as a two-electron jelliumatic molecule. The absorption feature of NCs 1 and 2 was also theoretically simulated. As shown in Fig. 3C and D, four prominent peaks namely a, b, g, and d can be clearly recognized for both NCs, and the detailed transitions corresponding to the signifcant peaks are listed in Tables S8 and S9 † for NCs 1 and 2, respectively. For NC 1, an optical bandgap of ca. 1.80 eV is extrapolated according to the value of the absorption edge, which is close to the absorption peak at 1.72 eV (a) in the simulated spectrum. Note that the band a could not be merely considered as a HOMO to LUMO transition, but might be resulted from three transition modes (HOMO to LUMO, HOMO to LUMO + 1, and HOMO to LUMO + 2) with nearly equal contribution values (96.2%, 96.1%, and 95.6%) (Fig. S15A †). Similar transition modes are observed in NC 2 (Fig. 3D and S16A †), and the excitation energy (DE ¼ 2.04 eV) is also close to the optical bandgap (exp. 2.22 eV). The b peak at 2.22 eV of NC 1 can also be attributed to three transition modes (HOMO to LUMO + 3, HOMO to LUMO + 4, and HOMO to LUMO + 5), whereas the b peak at 2.89 eV in NC 2 can be assigned to two transition modes (HOMO 8 to LUMO and HOMO 8 to LUMO + 1) with the contribution of 42.3% and 12.5%, respectively (Fig. S15B and S16B †). In addition, the g peak of NC 1 comprises ffteen transition modes (e.g., HOMO 6 (67.7%), HOMO 7 (55.9%), HOMO 1 (43.5%) to LUMO + 2 and so on) (Fig. S15C †). In contrast, the g peak of NC 2 can be attributed to six transition modes, including HOMO to LUMO + 4 (69.0%), LUMO + 8 (59.4%), and LUMO + 9 (56.7%) (Fig. S16C †). The d peak of NC 1 is mainly contributed by the HOMO 28 to LUMO + 2 (48.2%) transitions (Fig. S15D †), while the d peak of NC 2 is predominantly contributed by two transition modes (i.e., HOMO 6 to LUMO + 4, 21.4%, and HOMO 19 to LUMO + 2, 24.1%) (see Fig. S16D †). One can conclude that even if NCs 1 and 2bear a similar M 15 + geometrical confguration, the optical absorption properties are quite different, mainly owing to the differences in the M 1 kernel (Ag vs. Au) and M 6 octahedron (Cu vs. Au), and probably the subtle difference in the Ag 8 cube as well. Furthermore, we also calculated the electronic structures of NCs 1 and 2 to unravel the relationship between the electronic structure and the optical absorption properties of M 15 + NCs. According to the Kohn-Sham (KS) molecular orbital energy level diagram (Fig. 4A and B), there is an obvious energy gap between the HOMO and the remaining occupied orbitals for the two NCs, which may be because the two electrons at the HOMO orbital with higher energy tend to relax to the HOMO -1 orbital. It is worth noting that for the frontier unoccupied molecular orbitals (including the LUMO), the Ag(sp) atomic orbital makes the most signifcant contribution in both NCs. That means, for both NCs, Ag 8 cubic atoms are the major contributor to the unoccupied molecular orbitals. It indicates that the unoccupied molecular orbitals of the M 15 + clusters might be localized at some specifc position (i.e., M 8 cube), while the M 1 kernel and M 6 octahedron make less contribution, reminiscent of the case of M 21 (SCH 3 ) 15 . 52 This explains that there are triplet peaks located at high wavelength for both NCs. However, for the remaining occupied molecular orbitals (not including the HOMO) particularly the deep occupied orbitals, the Cu(d) atomic orbital makes the most important contribution in NC 1, while Au(d) and Ag(d) both contributed signifcantly in NC 2. For the HOMO, drastic differences can be observed, Ag(sp) and Au(sp) atomic orbitals make the most signifcant contribution to the HOMO orbital in NCs 1 and 2, respectively. The absorption band a in NC 1 is primarily attributed to intraband Ag(sp) to Ag(sp) transitions, while the interband Au(d) to Ag(sp) transitions lead to the a band in NC 2. This fnding reveals that the central M(0) atom of M 15 NCs plays a signifcant role in the HOMO energy. Moreover, the HOMO energy of NC 1 is higher than that of NC 2, probably due to the fact that, compared with Ag, the central Au atom has higher cohesive force thus causing NC 2 to have lower HOMO energy than NC 1. Despite the similar structural scaffold of the two NCs, the subtle differences in metal composition and structural coordination mode might result in a vast difference of their stability. The stability of NCs 1 and 2 was investigated by monitoring the time-resolved UV-vis absorbance spectra at different temperatures, in the presence of an oxidant (e.g., H 2 O 2 ) or Lewis base (e.g., CH 3 ONa). Fig. 5 shows the intensity of the peaks at 579 nm for NC 1 and 487 nm for NC 2 versus time. At room temperature, the absorbance of NCs 1 and 2 remained almost unchanged for 24 h (Fig. S17A and B †), indicating that the two NCs can be stable under ambient conditions. As shown in Fig. 5A, the relative absorbance intensity of NC 2 preserved 81% of its initial value in 24 h, however, NC 1 retained 94%, indicating much less decomposition. It was because both NCs are situated at the lowest energy state, as confrmed by DFT calculations of the vibration frequency of NCs 1 and 2 (Table S10 †). When incubated at 60 C (Fig. 5B), the relative intensity of NC 1 remained 81% at 24 h, slightly higher than that of NC 2 (78%), indicating excellent thermal stability for both NCs. However, the absorption intensity of NC 1 dropped much faster than that of NC 2 in the frst 5 h (Fig. S17C and D †), indicating that it is more susceptible to heat. However, in the following 5-24 h, the relative intensity of NC 1 remained almost unchanged. Previous studies have documented that the thermal stability of bimetallic NCs not only depends on the extent of free electron centralization, 4,53 but also the metal-ligand interaction needs to be considered, 54 specifcally, the interaction between the surface metal atoms (Ag, Au or Cu) and the alkynyl ligand must be taken into account for these two title NCs. As shown in Fig. S9, † the s and p bonds between the metal and carbon atoms of the surface binding motif in NC 1 (Cu1-C1: 1.856 ; Cu1-C2: 1.884 ; Ag1-C1: 2.395 ; Ag2-C2: 2.355 ) are slightly stronger than those in NC 2 (Au1-C1: 1.983 ; Au1-C2: 1.980 ; Ag1-C1: 2.495 ; Ag2-C2: 2.505 ). Therefore, the more compact structure of NC 1 can prevent decomposition at higher temperature. Furthermore, NC 1 is more stable than NC 2 in the presence of the Lewis base (1 wt% CH 3 ONa in EtOH), but rapidly decomposed upon the addition of the oxidant (30 wt% H 2 O 2 ). As shown in Fig. 5C, NC 1 decomposed slightly faster than NC 2 in the frst 5 h, and both reached the same intensity at 8 h, after that, NC 2 decomposed slightly faster. The higher relative intensity (84% vs. 80%) indicates that NC 1 possessed slightly superior stability in the presence of the Lewis base. During this process, the color of NC 1 showed no change, while the NC 2 solution gradually turned from orange to light orange, as visualized in Fig. S18A and B. † Note that the cohesive force between Ag and Au in NC 2 is higher than that between Cu and Ag in NC 1, making NC 2 more vulnerable to the Lewis base, as CH 3 ONa is a nucleophilic agent, and hence it can attack the surface of NC 2 more favorably. However, NC 2 is more robust than NC 1 upon adding H 2 O 2 aqueous solution (Fig. S18C and D †). As shown in Fig. 5D, NC 1 decomposed completely in 1 h, suggesting that NC 1 is sensitive to H 2 O 2 , probably because the Cu(I) atoms on the surface of NC 1 can be easily oxidized into Cu(II). In contrast, the corresponding Au(I) atoms in NC 2 possessed strong antioxidation capacity. However, it still lost 35% of the initial value, and such a decomposition can be presumably attributed to the fact that the Ag atoms in the Ag 8 cube are easily attacked by the lone pair electrons of the peroxy radical (O 2 2 ). 55 Such a phenomenon occurs even more seriously for NC 1, leading to an accelerated and complete decomposition in 1 h. ## Conclusions In conclusion, a novel homoleptic alkynyl-protected AgCu superatom [Ag 9 Cu 6 ( t BuC^C) 12 ] + was synthesized for the frst time via a one-pot reaction with high yield (>40%). X-ray crystallographic analysis revealed that it possesses a BCC-based Ag 1 @Ag 8 @Cu 6 confguration. Also, the BCC-based [Au 7 Ag 8 (t BuC^C) 12 ] + cluster with a Au 1 @Ag 8 @Au 6 metal core was also prepared by an AGR approach with a ultrahigh yield (>86%). DFT calculations revealed that the different absorption features of the two NCs can be attributed to the differences in the M 1 kernel (Ag vs. Au), M 6 octahedron (Cu vs. Au), and the subtle differences in the Ag 8 cube. The characteristic absorbance peaks of NCs 1 and 2 are successfully correlated with the specifc molecular orbital transitions. NC 1 possessed superior stability to NC 2 at both room temperature and elevated temperature, and NC 1 also showed better tolerance to the Lewis base but is much more sensitive to the oxidant. We envision that this study can stimulate more research efforts on Cucontaining bimetallic superatomic NCs in terms of their synthesis, structural analysis, property exploration and beyond.

chemsum

{"title": "A homoleptic alkynyl-protected [Ag₉Cu₆(^<i>t</i>BuC\ue002C)₁₂]⁺ superatom with free electrons: synthesis, structure analysis, and different properties compared with the Au₇Ag₈ cluster in the M₁₅⁺ series", "journal": "Royal Society of Chemistry (RSC)"}

evaluation_of_3′-phosphate_as_a_transient_protecting_group_for_controlled_enzymatic_synthesis_of_dna

## Abstract: Chemically modified oligonucleotides have advanced as important therapeutic tools as reflected by the recent advent of mRNA vaccines and the FDA-approval of various siRNA and antisense oligonucleotides. These sequences are typically accessed by solid-phase synthesis which despite numerous advantages is restricted to short sequences and displays a limited tolerance to functional groups. Controlled enzymatic synthesis is an emerging alternative synthetic methodology that circumvents the limitations of traditional solid-phase synthesis. So far, most approaches strived to improve controlled enzymatic synthesis of canonical DNA and no potential routes to access xenonucleic acids (XNAs) have been reported. In this context, we have investigated the possibility of using phosphate as a transient protecting group for controlled enzymatic synthesis of DNA and locked nucleic acid (LNA) oligonucleotides. Phosphate is ubiquitously employed in natural systems and we demonstrate that this group displays most characteristics required for controlled enzymatic synthesis. We have devised robust synthetic pathways leading to these challenging compounds and we have discovered a hitherto unknown phosphatase activity of various DNA polymerases. These findings open up directions for the design of protected DNA and XNA nucleoside triphosphates for controlled enzymatic synthesis of chemically modified nucleic acids. X enonucleic acids (XNAs) are synthetic genetic polymers that differ from canonical nucleic acids mainly by the chemical composition of their sugar, phosphate, and nucleobase moieties . The presence of chemical modifications on the scaffold of XNAs endows these biopolymers with enhanced properties compared to natural DNA and RNA. For instance, the presence of modified sugar units massively enhances their resistance against nuclease-mediated degradation which is an important prerequisite for the development of therapeutic oligonucleotides . Similarly, the presence of additional functional groups on nucleobases or the installation of unnatural base pairs improve the binding and catalytic properties of nucleic acids . So far, synthetic access to XNA oligonucleotides is granted by two different approaches: (i) automated solid-phase synthesis using phosphoramidite building blocks and (ii) polymerase-mediated synthesis with modified nucleoside triphosphates (dN*TPs). While the first approach permits to produce larger amounts of XNA oligonucleotides 3,21 it is limited in terms of size (less than 100 nucleotides) and functional group tolerance 6,22 . On the other hand, the chemoenzymatic method grants access to oligonucleotides of virtually any length 23 and permits in vitro selection experiments to identify XNA aptamers and XNAzymes 20, . However, this method also requires the use of specially engineered polymerases that are capable of copying DNA templates into XNA and then back into DNA and all sites will contain the same type of modification. Recently, controlled enzymatic synthesis of DNA, a hybrid method combining elements of both approaches, has emerged and is raising increased attention. In this approach, nucleoside triphosphates are equipped with temporary 3′-protecting groups that can be removed after incorporation into a solid-phase bound primer sequence by a template dependent or independent DNA polymerase . So far, most efforts focused on using the template-independent DNA polymerase Terminal deoxynucleotidyl Transferase (TdT) in conjunction with small, reversible protecting groups such as aminoalkoxyl 39 or 3′-O-azidomethylene 40 placed on the 3′hydroxyl moiety of DNA nucleoside triphosphates (dNTPs). While this strategy culminated in the launch of a prototype of enzymatic DNA synthesizer by the biotechnology company DNA Script 41 , controlled enzymatic synthesis is still mainly restricted to rather short oligonucleotides 42 and to deoxyribose chemistry exclusively. Herein, we have explored the possibility of expanding this method to the synthesis of XNA oligonucleotides. To do so, we have evaluated the use of 3'-phosphate as a simple, biocompatible protecting group for the controlled enzymatic synthesis of DNA and locked nucleic acid (LNA) oligonucleotides. ## Results Rationale and design. The design of a reversible protecting group for controlled DNA and XNA synthesis involves a finely tuned balance between multiple factors. Indeed, polymerases have evolved as finely tuned enzymes capable of specifically recognizing canonical dNTPs or NTPs as substrates and to repel nucleotides with altered sugar moieties including those equipped with functional groups appended on the 2′/3′-OH groups 39,43 . Hence, the protecting group must be a rather small, preferably hydrophilic chemical entity that ensures substrate recognition by the polymerase and does not compromise its incorporation into DNA. In addition, the protecting group must be stable both upon storage of the nucleotide in buffered solution and during the polymerasemediated catalytic step so as to prevent the simultaneous incorporation of multiple nucleotides. Concomitantly, cleavage of the protecting group should proceed in high yields under mild conditions so as not to damage the growing DNA/XNA chain and to permit synthesis of longer oligonucleotides. The installation of a 3′-phosphate group fulfills most of these criteria since it is not a bulky, hydrophilic group that should be stable to hydrolysis under storage and synthesis and can easily be removed by the action of phosphatases. In order to evaluate the possibility of using a 3′-phosphate group to block the addition of nucleotides by polymerases we carried out primer extension (PEX) reactions with a 3′-phosphorylated primer with 10 different DNA polymerases and unmodified DNA dNTPs. Using a 31 nucleotide long template T1 and a 15 nucleotide long, 5′-FAM-labeled primer P1 equipped with a 3′-phosphate moiety 44 (see Supporting Information for sequence composition), all the reactions with the exception of those carried out with Therminator led to a negligible (i.e. <10% conversion) extension of the primer to full length or truncated products (Fig. 1). Importantly, treatment of primer P1 with phosphatases such as the FastAP thermosensitive alkaline phosphatase allowed removal of the 3′-phosphate protecting group and facilitated polymerasemediated DNA synthesis (Supplementary Fig. 1). Similar results were obtained with the TdT polymerase where the 3′-phosphorylated primer P1 prevented the polymerase from adding dT nucleotides and treatment with FastAP thermosensitive alkaline phosphatase restored the tailing reaction capacity of the TdT (Supplementary Fig. 2). Lastly, we performed an Autodock simulation study using the reported X-ray structure of the ternary complex of mouse TdT with ssDNA and an incoming nucleotide (PDB 4I27). In each analysis, we replaced the incoming nucleotide with either 3′-phosphate LNA-TTP or 3′-phosphate-dTTP. This analysis revealed that both modified nucleotides were rather well tolerated within the active site of the TdT polymerase with favorable free energies (−16.94 kcal/mol and −17.40 kcal/mol for the protected dTTP and LNA-TTP, respectively; see Supplementary Figs. 3 and 4) comparable to that of unprotected LNA-TTP 44 . Taken together, these initial experiments suggest that the 3′-phosphate group can efficiently block DNA synthesis, can be removed by the action of phosphatases, and 3′-phosphorlyated dNTPs appear to be rather well tolerated within the active site of certain DNA polymerases at least according to docking experiments. Synthesis of 3′-phosphate-dTTP 5 and 3′-phosphate-LNA-TTP 10. We next synthesized the 3′-phosphorylated versions of dTTP (3′-phos-dTTP 5) and LNA-TTP (3′-phos-LNA-TTP 10) to assess whether these modified nucleotides are compatible with controlled enzymatic DNA and XNA synthesis (Fig. 2 and Supplementary Figs. 46-69). To do so, we envisioned a common synthetic pathway that involved first conversion of the commercially available dT phosphoramidite 1 or the known LNA dT phosphoramidite 6 45,46 to the corresponding H-phosphonates 2 and 7 using ETT as activator . H-phosphonates 2 and 7 were then oxidized to the corresponding P(V) containing nucleotides with iodine under typical oxidation conditions used in solidphase DNA synthesis. The DMTr masking groups of 3 and 8 were then removed under acidic conditions and the deprotected nucleoside analogues were converted to the expected dN*TPs 5 and 10 by application of the 4 step one pot method developed by Ludwig and Eckstein 50 . Biochemical characterization of 3′-phosphorylated nucleotides 5 and 10. With both 3′-phosphorylated nucleotides at hand, we set out to evaluate their substrate acceptance by DNA polymerases under PEX reaction conditions. To do so, we carried out PEX reactions using the P1/T1 primer/template system along with 10 different DNA polymerases and with both 3′-phosphorylated nucleotide analogs (Fig. 3). When PEX reactions were conducted with Taq, the expected n + 3 product (corresponding to the addition of a dA, a dC, and one phosphorylated nucleotide) formed in moderate yields (~50%) in the presence of 3′-phos-dTTP 5 and 3′-phos-LNA-TTP 10, which could be optimized to complete conversion of the primer to n + 3 product (Supplementary Fig. 5). Unexpectedly, all other polymerases extended the primer further and generated 22 nucleotide long oligonucleotides corresponding to n + 7 products. Reactions conducted with Therminator even led to the formation of full length products. Intrigued by these results, we analyzed the products stemming from the PEX reactions conducted with the combination of Taq polymerase and 3′-phos-dTTP 5 as well as that with Therminator and 3′-phos-LNA-TTP 10 by LCMS (Table 1, Supplementary Figs. 23-28, and Supplementary Note 1) using established protocols for products stemming from PEX reactions with natural and modified nucleotides 51,52 . This analysis clearly revealed that 1. no phosphorylated nucleotide was incorporated by polymerases and that 2. dA nucleotides were misincorporated opposite templating dAs instead of the modified triphosphates. Such a preference for dAMP misincorporation was further demonstrated when PEX reactions were performed in the presence of all natural dNTPs except for dTTP (Supplementary Fig. 6). Such a behavior was previously observed for the highly modified XNA nucleotide 7′,5′-bc-DNA since incorporation of the modified nucleotide by DNA polymerases proceeded with much lower efficiency than misincorporation of dAMP opposite templating dT residues 53 . Based on these considerations we next wondered if single incorporations might be observed when the 3′-phosphorylated analogs were used in the absence of competitors such as dATP. Hence, we carried out PEX reactions with template T2 that contains a stretch of dA nucleotides immediately 3′-downstream of the corresponding primer P1 as well as with template T3 which was designed as a universal template for controlled DNA synthesis 34 . The PEX reactions carried out with dN*TPs 5 and 10 individually and template T2 are shown in Fig. 4. Analysis of the reaction products by gel electrophoresis revealed that both nucleotides were seemingly well accepted by a number of DNA polymerases since bands corresponding to n + 1 and n + 2 and sometimes even n + 3 and n + 4 products could be observed with both nucleotides with the polymerases HemoKlenTaq, Bst, Therminator, Vent (exo − ), and Kf (exo − ). Similar results were obtained when the universal template T3 was used instead of T2 (Supplementary Fig. 7). Here as well, we performed an LCMS analysis of the PEX reaction products obtained with the P1/T3 system in order to try to understand the origin of these multiple incorporation events. The results obtained with the n + 1 and n + 2 products with both modified nucleotides are summarized in Table 2 (also see Supplementary Figs. 29-40). This analysis clearly reveals that both modified nucleotides are successfully incorporated when no competitor such as dATP is present. On the other hand, all observed products correspond to the addition of one or two nucleotides onto primer P1 but without the presence of the 3′-phosphate protecting group. These results suggest that both A-family (e.g. Taq) and B-family (e.g. Vent (exo − )) DNA polymerases are capable of removing the 3′phosphate protecting group either at the level of the incoming nucleotide or once installed on the extended primer. We next questioned whether the 3′-phosphate protected nucleotides are accepted as substrates by other polymerase families and whether the phosphate protecting group is also removed by these polymerases. To do so, we performed template-independent PEX reactions using the X-family DNA polymerase TdT along with the 3′-protected nucleotides 5 and 10. In addition, we supplemented the reaction mixtures with three different M 2+ cofactors since the metal preference of TdT is not very strict 33 . After significant optimization of the reaction conditions with 3′-phos-dTTP 5, ~50% conversion of 5′-FAM-labeled primer P2 (Supporting Information for sequence composition) 54 into the corresponding n + 1 product was observed in the presence of Mn 2+ alone or together with Mg 2+ (Fig. 5). On the other hand, 3′-phos-LNA-TTP 10 was not well accepted as a substrate by the TdT polymerase since very modest yields (10-20%) of n + 1 product formed even after long reaction times or when the feed ratio of monomers (i.e., modified triphosphates) to initiator (i.e., primer) was increased (Supplementary Fig. 8) 55 . Collectively these results demonstrate that 3′-phosphate protected nucleotides are not very good substrates for DNA polymerases, particularly for family X polymerases such as the TdT. In the absence of competing nucleotides such as dATP, these nucleotides are readily incorporated into DNA by various family A and B polymerases but at the expense of an incomplete blocking activity of the 3′-phosphate group presumably due to the inherent esterase/phosphatase activity of several DNA polymerases. Certain DNA polymerases were recently shown to display an esterase activity (see Discussion) and hence a phosphatase activity is not totally unexpected. Even though commercially available DNA polymerases are certified by the supplier to display less than 0.0001 unit of alkaline phosphatase activity (New England Biolabs), we have performed an MS analysis on Kf (exo − ) which confirmed the absence of any contaminants including phosphatases (Supplementary Figs. 89 and 90, Supplementary Table 1, Supplementary Note 2, and Supplementary Discussion). Effect of charge: 3′-cyanoethyl-phosphate protecting group. We first hypothesized that the poor acceptance of nucleotides 5 and 10 as polymerase substrates might be ascribed to the presence of the negative charges on the 3′-phosphate moiety. Hence, we rationalized that an additional phosphoester bond on the 3′protecting group might reduce this negative charge and improve the substrate tolerance. Such an additional ester linkage is readily available if the β-cyanoethyl protecting group of the original phosphoramidites is not removed by cleavage with ammonia. Moreover, docking experiments revealed that dTTP and LNA-TTP equipped with 3′-β-cyanophosphate groups fitted well into the active site of the TdT polymerase (Supplementary Figs. 9 and 10). The free energy for the docking of 3′-β-cyanophosphate-dTTP into the active site of the TdT was comparable to that of the corresponding 3-phosphate nucleotide (−16.65 kcal/mol) while a much more favorable free energy was obtained with 3′β-cyanophosphate-LNA-TTP (−18.18 kcal/mol). We thus converted analogues 4 and 9 into the corresponding triphosphates 11 and 12 by the application of the Ludwig Eckstein protocol (Fig. 6 and Supplementary Figs. 70-76). With nucleotides 11 and 12 at hand, we evaluated their substrate capacity for DNA polymerases under PEX reactions with templates T1 and T2 as well as the possibility of using these analogs for TdT-mediated extension reactions. Gel analysis of all the products obtained from PEX reactions revealed that both nucleotides acted as poor substrates for DNA polymerases (Supplementary Figs. 11-14). Indeed, we either observed only very little n + 1 formation or multiple incorporation events were detected at longer reaction times, presumably due to the loss of the β-cyanoethyl protecting group caused by β-elimination in the lower pH of the polymerase buffers. On the other hand, highly contrasting results were obtained when both nucleotides were assayed with the TdT polymerase. Indeed, while the TdT polymerase did not accept the blocked canonical nucleotide 11 (Supplementary Fig. 15), large product distributions were observed when LNA nucleotide 12 was used as substrate (Fig. 7). These results are surprising because i) LNAs are poor substrates for the TdT and usually terminate synthesis after the addition of a single nucleotide 44,56 ; (ii) nucleotide 11 is not recognized as a substrate by the TdT and the primer is not extended by the polymerase; iii) the protecting group is removed either by the polymerase or in the reaction medium. In order to shed some light into these results, we first performed TdT reactions with nucleotide 11 followed by the addition of 3′-unblocked LNA-TTP (Supplementary Fig. 16). This experiment clearly revealed that only a single LNA nucleotide was incorporated by the polymerase, suggesting that nucleotide 11 was not recognized by the enzyme. Next, we analyzed the products stemming from the TdT-mediated tailing reaction in conjunction with LNA nucleotide 12 by LCMS (Table 3 and Supplementary Figs. 43-S45). This analysis reveals that the intermediate bands as well as the n i and n i+1 products correspond to different chemical entities. In particular, bands corresponding to single or multiple addition events consist of the primer with one or multiple dehydrated LNA nucleotides devoid of any protecting groups. Such dehydration events have been observed in MS analysis of modified nucleotides 57 . In addition, bands that run between these bands correspond to similar species albeit with an additional Δm/z of 15 compared to the parent bands. Such a Δm/z is typically observed with misincorporation events (e.g. incorporation of dG instead of dA opposite templating dT or dC instead of dT opposite dA) under standard PEX reaction conditions 58 . However, under our experimental conditions, only modified triphosphate 12 was present as substrate. Moreover, similar double-banding events such as that displayed in Fig. 7 have already been described in the past for TdT primer extension reactions carried out in conjunction with sugar and 5′-phosphate-modified nucleotides 59 . This gel pattern was ascribed to the capacity of TdT to phosphorylate (and phosphonylate) oligonucleotides. While the double-banding pattern appears similar, the LCMS analysis of products does not fit with such a phosphorylation event. Such a Δm/z might potentially be connected to deprotection of the β-cyanoethyl moiety and the concomitant addition of the resulting acrylonitrile on a nucleobase. Hence, in order to shed more light into the nature of these products we first analyzed the stability of the cyanoethyl group on nucleotide 11 in a TdT-mediated reaction -19). This analysis revealed that even after 12 h of incubation, the chemical integrity of nucleotide 11 was not altered and no loss of the cyanoethyl group could be detected under these conditions. On the other hand, when LNA-TTP 44 was incubated with acrylonitrile prior to the extension reaction, only the expected n + 1 products could be observed suggesting that the emergence of additional products might arise via a different, yet unidentified mechanism (Supplementary Fig. 20). Enhancing resistance against hydrolysis: 3′-thiophosphate group. The addition of an additional cyanoethyl moiety reduced the charge present on the 3′-phosphate blocking groups but Table 2 Summary of the results from the LCMS analysis of the PEX reaction products obtained on the P1/T3 system and with modified nucleotides. introduced a steric bulk that precludes efficient incorporation of the resulting nucleotides by most polymerases. Hence, we questioned whether a minimal perturbation of the phosphate moiety such as the substitution of an oxygen moiety by a sulfur atom could improve the substrate capacity of 3'-phosphate-modified LNA nucleotides. We rationalized that the introduction of a sulfur atom could decrease the capacity of polymerases at hydrolyzing the phosphate moiety since reaction at P = S centers is slower than for the native P = O centers 60,61 and concentrating the negative charge on sulfur could increase interactions with polymerases 62 . Docking experiments comforted these assumptions since the sulfur atom is predicted to interact mainly with an arginine of the active site of the TdT and the overall free energy is very favorable (−17.50 kcal/mol; see Supplementary Fig. 21). Synthesis of the 3′-thiophosphate-bearing nucleotide 15 is highlighted in Fig. 8 and makes use of our recently developed method for the synthesis of thiophosphates with the Beaucage reagent 44 . Briefly, H-phosphonate 7 (Fig. 2) is oxidized to the corresponding P(V) nucleotide 13 using the Beaucage reagent. After deprotection of the DMTr group, the 3′-phosphorothioate nucleotide 14 is converted to the corresponding 5′-triphosphate using the Ludwig-Eckstein approach (Supplementary Figs. 77-88). The substrate acceptance of nucleotide 15 for DNA polymerases was investigated in PEX reactions and TdT-mediated tailing reactions (Fig. 9). Clearly, the presence of a 3′-thiophosphate moiety does not improve the substrate acceptance by polymerases since a similar product distribution as with LNA nucleotide 10 (Fig. 3) is observed during PEX reactions with primer P1 and template T1 (Fig. 9A). With the TdT, formation of the expected n + 1 product resulted but in low yields (~20%) and longer reaction times led to the appearance of additional bands, presumably stemming from hydrolytic degradation of the primer (Fig. 9B). These results suggest that the presence of a sulfur atom on the 3′-phosphate moiety does not improve the substrate acceptance by polymerases since misincorporation events might be favored even though the predicted hydrolysis of the protecting group might be reduced. Next, we synthesized nucleotide 16 (Fig. 8) which presents both a sulfur and a βcyanoethyl moiety on the terminal 3′-phosphate group in order to evaluate whether the combination of a sulfur atom and a reduction of the negative charge could improve the incorporation efficiency. However, similar PEX reactions conducted with primer P1 and template T1 (Supplementary Fig. 22) and with the TdT (Supplementary Fig. 23) did only show marginal improvements compared to the incorporation efficiency of the parent compound 15. Interestingly, the presence of the β-cyanoethyl moiety did not lead to multiple incorporation events when the TdT was used as polymerase as was the case for nucleotide 12 that has a P = O center rather than a P = S. Docking experiments reflect these results since a lower free energy (−16.81 kcal/mol) was calculated and unfavorable positioning of the 3′-protecting group within the active site of the polymerase were detected (Supplementary Fig. 24). ## Discussion Controlled enzymatic synthesis of DNA, RNA, and XNAs represents an interesting and versatile alternative to chemical, phosphoramidite-based synthesis since in principle it is devoid of sequence length limitations and should be more tolerant to chemical modifications on nucleotides and oligonucleotides. This approach would be highly beneficial in a number of practical applications including storage of digital information , assembly of synthetic genes and genomes 67,68 , or functional RNA oligonucleotides 69 . However, despite recent progress and increased interest in this methodology, no universal blocking group has been identified yet that allows synthesis of longer stretches of nucleic acids, particularly of XNAs. This difficulty resides in a delicate balance between steric bulk, robustness, and lability of a protecting group which is required to ensure substrate recognition of nucleotides by polymerases, efficient incorporation into oligonucleotides, and high yielding coupling and deprotection steps. In this context, we have explored the possibility of using 3′-phosphate as a temporary protecting group. Indeed, phosphate is ubiquitously used in nature for transient protection/ modifications of proteins but also of nucleotides and oligonucleotides. Phosphate moieties are robust but can easily be removed by phosphatases and do not introduce a massive steric bulk into scaffolds. Nucleotides bearing 3′-phosphate moieties, however, are poor substrates for A-and B-family DNA polymerases since misincorporation of dAMP moieties is favored to incorporation of such modified nucleotides. This poor substrate acceptance by polymerases might be ascribed to the presence of two negative charges-even though partially masked by interaction with mono-or divalent metal cations or by interactions with residues of side chains of the active sites of polymerases. A similar accumulation of negative charge at the 3′-end of nucleotides might also explain the inhibitory effect of magic spot nucleotides or alarmones (i.e. guanosine-3′,5′-bis(diphosphate) ppGpp and guanosine-3′-diphosphate-5′-triphosphate pppGpp) even though these compounds have never been assayed in conjunction with DNA polymerases 70,71 . When reaction mixtures were supplemented with 3′-phosphate containing nucleotides alone, multiple incorporation events were observed which results from abstraction of the protecting group. Since 3′-phosphorylated primers cannot be extended by polymerases, we ascribe these multiple incorporation events to a moonlighting, phosphatase activity of polymerases directly at the level of the incoming, modified nucleotides. This observation is not totally unexpected since various polymerases including HIV-RT, Sequenase 72 , an exonuclease-deficient variant of the archaeal B-family 9°N DNA polymerase 73 , and the large fragment of the A-family DNA polymerase from Bacillus stearothermophilus (BF) 74 possess an efficient 3′-esterase activity once the ester group is installed on the extended primer. Recently, DNA polymerase I fragment (Klenow) was shown to possess a phosphatase activity at the level of nucleotides but this activity consisted in the removal of one or two phosphate groups from 5′-triphosphate entities and only in the strict presence of RNA 75,76 . Lastly, some DNA polymerases (mainly belonging to family X polymerases) such as involved in repair pathways recruit Polymerase Histidinol Phosphatase (PHP) domains to mediate phosphatase activity 77 . While additional work will be necessary to pinpoint the site involved in such activity and to unravel its mechanism, this phosphatase activity of polymerases is unprecedented and further underscores the capacity of polymerases to act as enzymes with promiscuous activities. ## Conclusions Controlled enzymatic synthesis represents an alluring alternative to traditional synthetic methods for the generation of wild type and modified oligonucleotides. While intense research has been dedicated to the development of methods and protecting groups suitable for DNA synthesis, little or no efforts have been devoted to similar strategies but for RNA or XNAs. In this context, we have explored the possibility of using phosphate as a transient 3′blocking group for controlled enzymatic synthesis of DNA and LNA containing oligonucleotides. While this protecting group does not appear suitable for our approach despite meeting most of the required criteria, an unexpected and unprecedented moonlighting activity of various family A and B DNA polymerases was discovered. These results will allow us to refine the design and the chemical nature of other 3′-protecting groups to be explored for the controlled synthesis of XNA oligonucleotides and might have repercussions in understanding the mechanism of alarmones and the effect of phosphorylation of nucleotides in complex systems. ## Methods General protocol of TdT-mediated tailing reactions. Primer P2 (20 pmol) is incubated with the modified nucleoside triphosphate (200 µM) with a suitable metal cofactor (0.25 mM Co 2+ , 1 mM Mn 2+ , or 1 mM Mg 2+ ) and the TdT polymerase (10 U) in 1X reaction buffer (supplied with the polymerase; 10 µL final volume) at 37 °C for given reaction times. The reaction mixtures were then purified by Nucleospin columns and quenched by the addition of an equal volume of loading buffer (formamide (70%), ethylenediaminetetraacetic acid (EDTA, 50 mm), bromophenol (0.1%), xylene cyanol (0.1%)). The reaction products were then resolved by electrophoresis (PAGE 20%) and visualized by phosphorimager analysis. General procedure for primer extension reactions. The template (15 pmol) was annealed to its complementary primer (10 pmol) by heating to 95 °C and slowly (over 30 min) cooling down to room temperature. The annealed oligonucleotides were then supplemented with modified and/or natural dNTPs (all 200 µM final concentrations) and polymerase (2 U) in 1X reaction buffer. The reaction mixtures were then incubated at the recommended temperature for given amounts of time. The reaction mixtures were then purified by Nucleospin columns and quenched by the addition of an equal volume of loading buffer (formamide (70%), ethylenediaminetetraacetic acid (EDTA, 50 mm), bromophenol (0.1%), xylene cyanol (0.1%)). The reaction products were then resolved by electrophoresis (PAGE 20%) and visualized by phosphorimager analysis. Chemical syntheses. Detailed protocols for the synthesis of all nucleoside and nucleotide analogs can be found in the Supporting Information of this article. Docking experiments. AutoDock version 4.2 was used for the docking simulation 78 . The TdT enzyme file was prepared using published coordinates (PDB 4I27). The magnesium atom was retained within the protein structure. A charge of +2 and a solvation value of −30 were manually assigned to the Mg atom. The molecules files were built on Biovia Discovery Studio ® 4.5 and saved as pdb files. The docking area was assigned visually around the presumed active site. A grid of 40 x 40 x 40 with 0.497 spacing was calculated around the docking area using AutoGrid. We selected the Lamarckian genetic algorithm (LGA) for ligand conformational searching, which evaluates a population of possible docking solutions and propagates the most successful individual solution from each generation into the subsequent generation of possible solutions. For each compound, the docking parameters were as follows: trial of 20 dockings, population size of 150, random starting position and conformation, translation step ranges of 1.5 , rotation step ranges of 35°, elitism of 1, mutation rate of 0.02, crossover rate of 0.8, local search rate of 0.06 and 2,500,000 energy evaluations. The docking method was first evaluated by redocking the corresponding ligand of the PDB structure and then docking of the molecules of interest in the TdT active site. The conformation of the obtained results was inspected and compared to the literature and crystal structures. The docking results from each of the compounds were clustered on the basis of the root-mean-square deviation (rmsd) of the Cartesian coordinates of the atoms and were ranked on the basis of free energy of binding. The top-ranked compounds were visually inspected for correct chemical geometry.

chemsum

{"title": "Evaluation of 3\u2032-phosphate as a transient protecting group for controlled enzymatic synthesis of DNA and XNA oligonucleotides", "journal": "Nature Communications Chemistry"}

iron_detection_and_remediation_with_a_functionalized_porous_polymer_applied_to_environmental_water_s

## Abstract: Iron is one of the most abundant elements in the environment and in the human body. As an essential nutrient, iron homeostasis is tightly regulated, and iron dysregulation is implicated in numerous pathologies, including neurodegenerative diseases, atherosclerosis, and diabetes. Endogenous iron pool concentrations are directly linked to iron ion uptake from environmental sources such as drinking water, providing motivation for developing new technologies for assessing iron(II) and iron(III) levels in water. However, conventional methods for measuring aqueous iron pools remain laborious and costly and often require sophisticated equipment and/or additional processing steps to remove the iron ions from the original environmental source. We now report a simplified and accurate chemical platform for capturing and quantifying the iron present in aqueous samples through use of a post-synthetically modified porous aromatic framework (PAF). The ether/thioether-functionalized network polymer, PAF-1-ET, exhibits high selectivity for the uptake of iron(II) and iron(III) over other physiologically and environmentally relevant metal ions. Mössbauer spectroscopy, XANES, and EXAFS measurements provide evidence to support iron(III) coordination to oxygen-based ligands within the material. The polymer is further successfully employed to adsorb and remove iron ions from groundwater, including field sources in West Bengal, India. Combined with an 8-hydroxyquinoline colorimetric indicator, PAF-1-ET enables the simple and direct determination of the iron(II) and iron(III) ion concentrations in these samples, providing a starting point for the design and use of molecularly-functionalized porous materials for potential dual detection and remediation applications. ## Introduction Iron is the fourth most abundant element in the earth's crust and the most abundant transition metal in the human body. 1 It is required for sustaining a range of physiological processes such as electron transfer, oxygen transport, respiration, and gene expression, and iron deficiency leads to anemia. 6 However, excess iron can increase production of reactive oxygen species, resulting in oxidative stress cascades that lead to lipid oxidation and DNA damage. 7,8 Aberrant iron accumulation is implicated in aging and in several diseases, including cardiovascular diseases, neurodegenerative diseases, and cancer. 6, In this context, chronic exposure to elevated iron levels in common drinking water is a potential contributor to abnormal iron accumulation. The World Health Organization (WHO) recommends an upper limit of 0.3 mg/L for iron ions in drinking water. 15 Unfortunately, the reported iron ion concentrations in drinking water sources can vary over several orders of magnitude-for instance from 0.007 to 33.6 mg/L in West Bengal 16 or from undetectable amounts to 950 mg/L based on Groundwater Ambient Monitoring and Assessment data from the San Francisco Bay (see Supporting Information). Because traditional methods for iron(II) and iron(III) detection require expensive instrumentation, such as inductively coupled plasma mass spectrometry or atomic absorption spectroscopy, 17,18 it remains a challenge to rapidly and inexpensively screen drinking water for quantities of iron and other metal ion contaminants, particularly in developing countries and other lower-resource environments. 11,16,17, To meet this challenge, we sought to develop a chemical strategy that would enable simultaneous detection and removal of both iron(II) and iron(III) ions from drinking water and other environmental and biomedical samples, with high selectivity over other metal ion contaminants. In particular, we This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins Please do not adjust margins envisioned a robust, molecularly-tailored solid-state adsorbent that would efficiently capture and remove iron ions from a complex water sample obtained in the field while also permitting a quantitative measure of the iron concentration. We chose to investigate porous aromatic frameworks (PAFs) for this dual remediation and quantitative detection function, owing to their high chemical and thermal stability-particularly in aqueous and biological samples-and because of their ability to be functionalized in a molecular fashion. The polymer PAF-1 is one such material that exhibits a high Brunauer-Emmett-Teller (BET) surface area 38 of up to 5600 m 2 /g and is readily functionalized through post-synthetic modification. Indeed, we recently reported a thioetherfunctionalized variant of this porous polymer, PAF-1-SMe, as a platform for selective copper ion capture from biofluid samples, ultimately as a diagnostic tool for Wilson's disease. 42 Here, we present the synthesis of an iron-selective porous polymer via ether-thioether (ET) functionalization of PAF-1. The polymer PAF-1-ET (Figure 1a) exhibits highly selective iron(II) and iron(III) ion uptake over competing metal ions in laboratory and field water samples. The combination of this polymer with 8-hydroxyquinoline indicator enables rapid and quantitative monitoring of iron levels with a simple colorimetric assay. We highlight the potential utility of this method for remediation and screening of synthetic groundwater as well as field samples of drinking water collected from West Bengal, India. This work provides a starting point for the development of new porous polymers for simplified, accurate, and rapid diagnostic and remediation applications without the need for bulky and expensive instrumentation. ## General methods All reactions were performed under a nitrogen or argon atmosphere and in dry solvents, unless otherwise stated. Tetrakis(4-bromophenyl)methane was purchased from TCI America as a pale yellow powder. All other starting materials and reagents were purchased from Sigma-Aldrich. Nitrogen adsorption isotherms were measured using a Micromeritics ASAP 2020 or 2420 instrument. Samples were transferred to a pre-weighed glass analysis tube that was capped with a Transeal and then evacuated on the ASAP until the outgas rate was less than 3 µbar/min. Ultrahigh-purity grade (99.999%) nitrogen was used for gas adsorption measurements. Nitrogen isotherms were obtained using a 77 K liquid-N2 bath and were used to determine the surface areas and pore volumes using the Micromeritics software, assuming a value of 16.2 2 for the molecular cross-sectional area of N2. Infrared spectra were obtained on a Perkin-Elmer Spectrum 100 Optica FTIR spectrometer furnished with an attenuated total reflectance accessory. Thermal gravimetric analysis data were collected at a ramp rate of 5 °C/min under flowing nitrogen using a TA Instruments TGA Q5000. Scanning electron microscopy (SEM) samples were prepared by dispersing fine polymer powders into methanol and drop casting onto silicon chips. To dissipate charge, the samples were sputter coated with approximately 3 nm of Au (Denton Vacuum). Polymers were imaged at 5 keV and 12 μA by field emission SEM (JEOL FSM6430). Elemental analyses (C, H, N, S) were obtained from the Microanalytical Laboratory at the University of California, Berkeley. Elemental analysis for chlorine was performed at Galbraith Laboratories. UV-Vis spectroscopic measurements were performed in 100 mM HEPES buffer (pH 6.7). Absorption spectra were recorded using a Varian Cary 50 spectrophotometer, and samples for absorption measurements were prepared in 1 × 0.5 cm quartz cuvettes (1.4-mL, Starna). Inductively coupled plasma-mass spectrometry (ICP-MS) was performed on samples that had been diluted into 2% nitric acid (made freshly from concentrated nitric acid [BDH Aristar Ultra] and Milli-Q water) containing 20 µg/L Ga internal standard (Inorganic Ventures, Christiansburg, VA). The samples were analyzed on a ThermoFisher iCAP-Qc ICP-MS in Kinetic Energy Discrimination mode against a calibration curve of known metal concentrations (made from CMS-5, Inorganic Ventures, Christiansburg, VA). Lowtemperature X-band EPR spectra were recorded using a Varian E109 EPR spectrometer equipped with a Model 102 microwave bridge. Sample temperature was maintained at 8 K by using an Air Products LTR liquid helium cryostat. The following spectrometer conditions were used: microwave frequency, 9.22 GHz; field modulation amplitude, 32 G at 100 kHz, and a microwave power of 20 mW. ## Synthesis of PAF-1-ET 2-(methylthio)ethan-1-ol (1.83 mL, 0.021 mol) and 3 equiv. of NaH (1.5 g, 0.063 mol) were mixed with toluene (100 mL) in a 250 mL Schlenk flask under N2. After 5 min, freshly-prepared PAF-1-CH2Cl (260 mg, see the Supporting Information) was added, and the mixture was stirred at 90 °C for 3 days. The resulting solid was collected, washed sequentially with 100 mL each of H2O, ethanol, CHCl3, and THF, and dried in a vacuum oven at 150 °C to produce PAF-1-ET as an off-white powder. Calc. for C32.5H34O2S2 (%): C 74.96, H 6.58, S 12.31, Cl 0.00; observed: C 74.89, H 5.08, S 5.50, Cl 1.97. Based on the sulfur elemental analysis, this preparation resulted in 45% substitution with 2-(methylthio)ethan-1-ol. § Solid-state 13 C NMR NMR sample preparation. Samples of PAF-1-ET (35 mg) and PAF-1-CH2Cl (20 mg) were dried at 100 °C for 3 h before data collection. Iron(III)-loaded PAF-1-ET was prepared by stirring PAF-1-ET (50 mg) at room temperature overnight in a solution of FeCl3 (10 mL, 100 mg/L) dissolved in 100 mM HEPES buffer (pH = 6.7) with 2 equiv. of citric acid. The filtered iron(III)-PAF-1-ET was washed with Milli-Q H2O, ethanol, dichloromethane, and THF (50 mL each) and dried at 100 °C for 3 h before data collection. ## NMR experiments. All experiments were conducted at a 13 C frequency of 75.5 MHz using a Tecmag Discovery spectrometer equipped with a 7.05 T magnet and a Chemagnetics 4 mm HX CP/MAS probe (magic-angle spinning rate of 10 kHz). Crosspolarization from 1 H was used when acquiring spectra for PAF-1-Please do not adjust margins Please do not adjust margins CH2Cl and PAF-1-ET. The Hartmann-Hahn condition 43 for crosspolarization experiments was obtained on solid adamantane, which is also a secondary 13 C chemical shift reference (the methylene signal of adamantane was set to 38.48 ppm relative to TMS). The PAF-1-CH2Cl spectrum was collected using a CP contact time of 10 ms and a pulse delay of 4 s. A two-pulse phase modulation (TPPM) proton decoupling scheme was used, with a TPPM angle of 15 degrees and decoupling field strength of ~60 kHz. The spectrum for PAF-1-ET was obtained using a contact time of 1 ms and a pulse delay of 4 s. Direct polarization (1 s pulse delay) was used to collect the spectrum for iron(III)-loaded PAF-1-ET. Continuous wave proton decoupling was used for both the PAF-1-ET and iron(III)-PAF-1-ET spectra, with a decoupling field strength of ~60 kHz. ## Metal ion adsorption in PAF-1-ET Iron adsorption measurements. Samples of PAF-1-ET (2.0 mg) were added to conical tubes containing 5 mL of (NH4)2Fe(SO4)2•6H2O (dissolved in 100 mM HEPES buffer, pH 6.7) with concentrations ranging from 10 −3 -240 mg/L. Each mixture was capped under air and stored in a shaker at room temperature overnight. Each solution was subsequently filtered through a 0.45-µm membrane filter, and the filtrates were analyzed by ICP-MS to determine the residual iron content. Iron uptake (initial−residual iron concentration) data was fit using a Langmuir model given by: where qe is the adsorption capacity (mg/g), Ce is the equilibrium iron ion concentration (mg/L), qsat is the adsorption saturation capacity (mg/g), and KL is the Langmuir constant (L/mg), which is related to the binding affinity of the adsorption site. Langmuir fits are shown in Figure 2a, and fit parameters are provided in Table S1. Metal ion adsorption selectivity studies. Samples of PAF-1-ET (2 mg) were added to conical tubes containing aqueous solutions of NaCl, KCl, MgCl2, CaCl2, MnCl2, (NH4)2Fe(SO4)2•6H2O, FeCl3, CoCl2, NiCl2, CuCl2, or ZnCl2 at initial concentrations of 0.3, 2, or 20 mg/L in 100 mM HEPES buffer (pH 6.7). Iron(II) samples were prepared and stored under anaerobic conditions until analysis by ICP-MS. In the case of iron(III), one equivalent of citric acid was also added to the samples to prevent Fe(OH)3 precipitation. The slurries were stored in a shaker at room temperature overnight and then filtered through a 0.45-µm membrane. The filtrates were analyzed using ICP-MS, and the amount of metal ion adsorbed was calculated by subtracting the residual iron concentration from the initial iron concentration. The distribution coefficient, Kd, for each metal ion was determined as described in Section 3 of the Supporting Information. Iron adsorption kinetics. An Erlenmeyer flask containing 2 mg of PAF-1-ET was charged with a solution of (NH4)2Fe(SO4)2•6H2O (10 mL, 10.2 ppm) in 100 mM HEPES buffer (pH = 6.7) and 1 equiv. of citric acid. The mixture was stirred at room temperature for 8 h. During this period, aliquots of the mixture were filtered at intervals through a 0.45-µm membrane. The filtrates were analyzed using ICP-MS to determine the iron ion concentration. The amount of iron adsorbed by PAF-1-ET was calculated by subtracting the residual from the initial iron concentration. The adsorption data were fit with the pseudo-second-order kinetic model (see Figure S7): where k is the pseudo-second-order rate constant (g/(mg min)) and qe is the amount of iron adsorbed at equilibrium (mg/g). ## Mössbauer experiments A sample of PAF-1-ET (~50 mg) was added to an aqueous solution of 57 FeCl3 (50 mg/L, see the Supporting Information), and the mixture was stirred overnight at room temperature under N2. The resulting 57 Fe(III)-loaded PAF-1-ET was collected, washed with warm H2O (100 mL) and CHCl3 (100 mL), and then dried in a vacuum oven at 150 °C to yield a white powder. Mössbauer spectra were obtained between 5 and 300 K with a SEE Co. Mössbauer spectrometer equipped with a Co-57 source in Rh matrix. Reported isomer shifts are given relative to α-iron at 295 K. The spectral absorber was prepared in air by packing the sample into a 1.27 cm diameter Nylon washer before transferring to the spectrometer, where the absorber was always maintained under a He atmosphere. See the Supporting Information (Section 6) for full measurement details. ## X-ray absorption measurements X-ray absorption spectra were collected at the Stanford synchrotron radiation light source on beamline 9-3 with ring storage conditions of 3.0 GeV and 500 mA. The iron K-edge absorption spectra of the PAF samples, packed in 0.5 mm thick aluminum sample holders with Kapton film windows, were recorded at room temperature. Reference compounds were analyzed after dilution with boron nitride. The spectral data were collected in transmission mode for Fe, Fe2O3, and FeO and in fluorescence mode for PAF-1-ET with a 100-element Ge monolithic solid-state detector from Canberra. The incident radiation was monochromatized using a Si(220) double crystal monochromator, which was detuned to 50% of flux maximum at the iron K-edge to minimize the higher harmonics and reduce X-ray flux. A harmonic rejection mirror was used to further reduce the contamination from higher harmonics radiation. The incident and transmitted X-ray intensities were monitored with N2filled ion chambers. An iron foil spectrum was concomitantly recorded for energy calibration where the first inflection point was assigned to 7111.2 eV. Even at the low X-ray flux density used, a slight photoreduction of PAF-1-ET was observed even after two scans at a given sample position. As a consequence, the spectral data were collected at multiple spots, and only the first two scans at each position were used for averaging the spectral data over multiple positions. Data reduction was carried out with the SamView software obtained from SixPack software. ‡ Athena software, Demeter version 0.9.25 45 was used for data averaging and removal of the This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins Please do not adjust margins pre-edge and post-edge background absorption. A five-domain cubic spline was used to remove low-frequency background in kspace. The resulting k-space data, as k 3 χ(k), was then Fourier transformed into r-space over a k range of 3.46 to 10.52 −1 and used for the extended X-ray absorption fine structure (EXAFS) refinement. The EXAFS fitting was carried out using the Artemis software 45 with phase and amplitude functions obtained from FEFF, version 6. 46 The average bond distance between the iron and scattering atom (R) and the mean square displacement of the bond distance (σ 2 ) were allowed to vary, while N, the number of atoms in the shell, was systematically varied in integral steps. The value of E0, the energy of the zero value of the photoelectron wave vector k, was also varied but constrained to a common value for every shell in a given fit. The value for S0 2 , the amplitude reduction factor, was extracted from the fit of the Fe foil data and was fixed at 0.95 during all other fits. The best fit parameters for EXAFS fits are given in Table S3. ## Synthesis and characterization Building upon the design of the thioether-functionalized polymer PAF-1-SMe for selective copper ion capture 42 and the related PAF-1-SH for mercury ion adsorption, 40 we initially synthesized a variety of PAF-1 derivative scaffolds. Subsequent testing for metal ion uptake from aqueous solutions showed that the variant with an extended ether-thioether pendant, referred to here as PAF-1-ET, is an effective material for selective capture of both iron(II) and iron(III). This work complements our recent efforts to develop activity-based fluorescent probes for iron(II). The polymer PAF-1-ET was synthesized in three steps, starting with the synthesis of parent PAF-1 and PAF-1-CH2Cl (see the Supporting Information) 40 followed by treatment of the latter with 2-(methylthio)ethan-1-ol to yield PAF-1-ET (Figure 1a). Elemental analysis revealed a decrease in chlorine content from 13.60% in PAF-1-CH2Cl to 1.97±1.5% in PAF-1-ET, and the sulfur content of the latter was found to be 5.50±0.82%-corresponding to grafting of 45±6.7% of the phenyl groups in PAF-1-ET or a loading of 1.7 mmol/g. In the IR spectrum of PAF-1-ET, the absence of a peak at 1270 cm −1 (assigned to the C-H wagging mode of -CH2Cl in PAF-1-CH2Cl) further confirmed functionalization (Figure S2). Solid-state 1 H− 13 C cross-polarization magic angle spinning NMR spectroscopy revealed new 13 C chemical shifts at 73, 48, 39, and 17 ppm for PAF-1-ET, arising from the ether-thioether groups, and the absence of a shift at 43 ppm corresponding to Please do not adjust margins Please do not adjust margins the -CH2Cl groups of PAF-1-CH2Cl (Figure 1b). Nitrogen adsorption isotherms obtained at 77 K revealed that PAF-1-ET retains permanent porosity with a high BET surface area of 1500±420 m 2 /g (Figure 1c; error determined by measuring the N2 adsorption of four different PAF-1-ET samples). The average pore size distribution obtained from the adsorption isotherms was found to be <10 for PAF-1-ET, which is smaller than the average value of 12 for PAF-1 and supports incorporation of the ether-thioether groups (see Figure S3). ## Selectivity and kinetics of iron uptake Equilibrium iron(III) isothermal adsorption data were collected for PAF-1-ET and PAF-1-CH2Cl over aqueous ion concentrations ranging from 1 µg/L to 300 mg/L, and these data were fit using the Langmuir model 29 to assess framework saturation capacities and binding affinities for iron(III) (Figure 2a and Table S1). Notably, PAF-1-ET exhibited much higher iron(III) uptake than PAF-1-CH2Cl over the entire concentration range and a binding affinity twice that of PAF-1-CH2Cl. At saturation, the maximum adsorption capacity (qsat) of PAF-1-ET was found to be 105(4) mg/g, which corresponds to an uptake of 1.8 mmol of iron(III) per gram of material-nearly three times the capacity of PAF-1-CH2Cl (37(2) mg/g). Based on the maximum adsorption capacity of PAF-1-ET, the ET:iron ratio is at least 1.1. We also collected adsorption data using iron(III) chloride, Please do not adjust margins Please do not adjust margins iron(III) sulfate hydrate, or ammonium iron(III) citrate to investigate the effect of the counterion on iron uptake in PAF-1-ET. For all salts, PAF-1-ET showed comparable iron uptake at low and high iron concentrations (see the Supporting Information, Section 4). Importantly, PAF-1-ET also exhibited high selectivity for the adsorption of iron(II) and iron(III) ions over other biologicallyrelevant metal ions at initial concentrations of 0.3, 2, and 20 mg/L (Figure 2b). For example, the distribution coefficient, Kd, for 10 mg/L iron(II) in pH = 6.7 HEPES buffer was found to be 2.6(7) × 10 4 mL/g, over an order of magnitude greater than the Kd values for 10 mg/L of Na + , K + , Mg 2+ , Ca 2+ , Cu 2+ , and Zn 2+ (600, 120, 180, 770, 3300, and 38 mL/g, respectively). Given this exceptional performance, PAF-1-ET should be useful for iron from a variety of water samples. The concentration dependence of iron(III) uptake by PAF-1-ET was also evaluated by examining various pH 6.7 aqueous solutions in HEPES buffer, and it was found that the adsorbed amount increases with increasing ion concentration in solution (Figure 3a). In order to develop a colorimetric assay for detection of the adsorbed iron(III), we evaluated the ability of 8-hydroxyquinoline to bind iron(III) captured within the porous framework. Upon binding free iron(III), 8-hydroxyquinoline undergoes a distinct change from colorless (315 nm absorption, ε = 1.95 × 10 3 M −1 cm −1 ) to blue-green (460 and 560 nm absorption, ε = 750 M −1 cm −1 at 460 nm), which is distinctive of an iron(III) 8-hydroxyquinoline complex. Thus, successful binding of iron(III) within PAF-1-ET by 8hydroxyquinoline should permit a facile and quantitative determination of the quantity adsorbed. To confirm this capability, a 1 mM solution of 8-hydroxyquinoline in dimethyl sulfoxide was added to dried samples of PAF-1-ET that had been exposed to the aqueous iron samples in Figure 3a. Gratifyingly, in the presence of these samples, the 8hydroxyquinoline absorption spectra exhibited two new peaks at 460 and 560 nm (Figure 3b), indicative of iron(III) complex formation. The calculated amount of iron(III) adsorbed by PAF-1-ET-based on the 460 nm absorbance peak for the highest sample concentration-correlated well with amounts determined directly via ICP-MS (Figure 3c). ## Spectroscopic characterization To obtain additional insight into the nature of the interactions between adsorbed iron(III) and the framework functional groups, 57 Fe Mössbauer spectra were collected between 5 and 300 K. Representative spectra at 100 and 5 K are shown in Figure 4a and b (see also Figure S9 and Table S2). At all temperatures, the spectral fits indicated the predominant presence of paramagnetic, high-spin iron(III) adsorbed within PAF-1-ET (red lines), with no evidence of long-range magnetic order. A small constrained component (green lines, 9% by area) was also present in all data, likely due to residual highspin iron(II) from sample preparation. The spectra were found to be very similar between 50 and 300 K, with a predominant bimodal distribution of quadrupole splittings, ΔEQ, between 0.6 and 1.0 mm/s, centered about a unique high-spin iron(III) isomer shift, δ, of 0.385(2) mm/s at 300 K (0.507(1) mm/s at 50 K). These values are consistent with an iron(III) ion residing in a pseudooctahedral coordination with a distribution of nearneighbor oxygen environments, a conclusion that is consistent with the extended X-ray absorption fine structure (EXAFS) data below. Interestingly, upon cooling to 20 K and below, some of the highly dispersed iron(III) ions adsorbed in PAF-1-ET exhibited initial evidence for slow paramagnetic relaxation on the Mössbauer timescale (~10 -8 s), with predominant hyperfine fields of 45 T (17.6(6)% area), 46.6(1) T (50.4(6)% area), and 46.6(1) T (58.8(4)% area), respectively. Please do not adjust margins Please do not adjust margins Iron K-edge X-ray absorption spectroscopy was used to investigate the local coordination environment of the adsorbed iron. The X-ray absorption near edge structure (XANES) spectrum of iron(III) adsorbed in PAF-1-ET is shown in the inset of Figure 4c, along with Fe2O3, FeO, and Fe foil for reference. The rising edge energy of the sample aligns well with that of Fe2O3, supporting the presence of iron(III). Figure 4c shows the k 3 -weighted EXAFS data for iron(III) adsorbed PAF-1-ET in r-space along with the best fit (see Table S3). The horizontal axis represents the apparent distance R′, which is shorter than the actual distance by ~0.5 due to a phase shift. For iron(III) adsorbed PAF-1-ET, 57 the best two-shell fit was achieved with a coordination environment of six oxygen atoms at a distance of 2.00(1) and 12 carbon atoms at a distance of 3.06(4) . We also obtained the 13 C NMR spectrum of iron(III)-loaded PAF-1-ET to compare with that of PAF-1-ET. The coordination of paramagnetic iron(III) resulted in severe peak broadening in addition to an overall shift in the peaks observed for PAF-1-ET (Figure S11). The benzene ring resonances between 148-132 ppm for PAF-1-ET shifted to 147-140 ppm in the spectrum of iron(III)-loaded PAF-1-ET, and all peaks corresponding to the ET functional groups of PAF-1-ET (73, 48, 39, and 17 ppm) shifted upfield in iron(III)-loaded PAF-1-ET (to ~34-5 ppm). The more dramatic shift in the ether-thioether peaks provides additional evidence that this group is indeed bound to iron(III). ## Iron coordination in PAF-1-ET To investigate possible iron coordination environments within PAF-1-ET, we used the program Materials Studio to generate a hypothetical portion of the PAF-1-ET structure, featuring one iron ion within a single diamond net. Based on the EXAFS data, it was assumed that a total of six oxygen atoms-and no sulfur atoms-coordinate to the iron. A monodentate sulfate anion was always included at one of the coordination sites for charge balance, and the remaining sites were coordinated by water and the ET oxygen atoms. The modeling revealed that up to three oxygen atoms from three different functional groups (ET:Fe = 2.9) can coordinate to the same iron ion at the low loading observed for genuine groundwater samples, provided that these groups are located at the 2-, 2′-, and 3-positions of two adjacent biphenyl groups (Figure S12). Importantly, the modeling also showed that the ET group flexibility may enable two groups to bind the same iron ion, regardless of their respective positions on a biphenyl unit (Figure S12a-c). Based on the maximum iron(III) adsorption capacity data, for high iron loading (ET:Fe = 1.1), it is presumed that only a single ET group can bind to the iron. In this case, a coordinated water molecule might participate in a weak CH-π interaction with a benzene ring that stabilizes the iron ions (Figure S13), a hypothesis supported by the benzene ring shift in the 13 C NMR spectrum of iron(III)-loaded PAF-1-ET (Figure S11). To investigate the importance of the ether-thioether orientation and pore environment in PAF-1-ET for iron(III) uptake, we prepared a series of related porous polymers and evaluated their iron adsorption properties. The first of these Please do not adjust margins Please do not adjust margins polymers, PAF-1-TE, was synthesized from PAF-1-CH2Cl using 2-methoxyethane-1-thiol, yielding a material analogous to PAF-1-ET but featuring interchanged positions for the pendant oxygen and sulfur moieties. We also prepared etherfunctionalized porous polymers PAF-1-OMe and -Ethoxy (Figure S14) and a linear polysulfone polymer functionalized with the ether-thioether ligand, PSF-ET (Figure S15). When exposed to a solution of FeCl3 (20 mg/L) dissolved in 100 mM HEPES buffer (pH = 6.7) with one equivalent of citric acid, each of the derivative polymers exhibited significantly lower iron(III) uptake than PAF-1-ET (Figure S16). The much lower uptake of PAF-1-TE, -OMe, and -Ethoxy suggests that both the position of oxygen and the presence of sulfur are crucial for iron adsorption, while the low uptake of PSF-ET emphasizes the importance of a compact pore environment in tandem with the ET functionality. ## Iron capture and detection in synthetic and environmental water samples To verify the detection capability of PAF-1-ET when exposed to iron(III) sources from different regions, synthetic groundwater was prepared according to the Gadgil 61 procedure with iron(III) concentrations of 1.8, 4.7, 6.7, and 37 mg/L. The polymer was also used to treat genuine groundwater samples collected in West Bengal, India, reported to contain 14 mg/L of iron(III) ions (Figure 5a). 16 Notably, PAF-1-ET adsorbed between ~41 and 91% of the iron(III) in the synthetic groundwater samples, from initial concentrations between 37 and 1.8 mg/L, respectively. In the presence of PAF-1-ET, the concentration of iron(III) in the genuine groundwater decreased with time as given by the expression y = Ae −t /t0 + C, where y is the detected amount of iron(III), A is a scale factor, C is a constant, t0 is the decay time, and t is the elapsed time. The best fit shown in the inset to Figure 5b corresponds to A = 9.2(3) mg/L, C = 4.1(1) mg/L, and t0 = 12(1) min. In other words, within 24 min PAF-1-ET captured 72% of the iron(III) ions and was essentially saturated after ~36 min, such that the iron(III) concentration in the genuine groundwater reached a constant value of ~3.92 mg/L. The final concentration in the genuine groundwater sample is higher than that recommended for safe drinking water by the WHO (0.3 mg/L), and the same is true for the synthetic groundwater solution with an initial concentration of 37 mg/L. However, the saturation capacity of PAF-1-ET suggests that the framework is capable of reducing the iron(III) content in both these solutions to levels lower than 0.3 mg/L. It is likely that at higher iron(III) concentrations, precipitation of Fe(OH)3 within the pores blocks some of the accessible coordination sites and reduces the effective capacity. Even still, PAF-1-ET functions exceptionally well in the removal of iron from solutions with relatively low initial concentrations-indeed, the framework was able to reduce the iron(III) ion content to safe drinking levels for solutions with initial iron concentrations of 1.8, 4.7, and 6.7 mg/L. For the effective treatment of water containing higher iron ion concentrations, it may be necessary to use larger quantities of citric acid to prevent Fe(OH)3 precipitation. Analysis of the PAF-1-ET samples using an 8hydroxyquinoline assay revealed an increase in absorbance at 460 and 560 nm with increasing iron(III) concentration, as associated with the original water samples (Figure 5b). The iron(III) concentrations calculated from the absorption at 460 nm were again in good agreement with those determined from direct ICP-MS measurements (Figure 5c). Finally, using the three-sigma method (3σ/k) the iron(III) detection limit for the PAF-1-ET and 8-hydroxyquinoline assay was determined to be 150 µg/L (see Figure S17 and the Supporting Information for details). Importantly, PAF-1-ET retains structural integrity and porosity following the addition of 8hydroxyquinoline and can be cycled at least three times without noticeable loss of adsorption capacity (Figure S18). ## Conclusions We have demonstrated that the ether-thioetherfunctionalized porous aromatic framework PAF-1-ET is capable of selective and efficient iron ion uptake and removal from both synthetic water and environmental groundwater. In this material, captured iron(III) is preferentially bound by oxygen in a pseudooctahedral coordination environment, as confirmed by Mössbauer and X-ray absorption spectroscopy characterization. The introduction of oxygen functionality within the framework is thus responsible for a shift to iron ion selectivity from our previously reported copper-selective thioether-functionalized material, PAF-1-SMe. 42 Finally, the combination of PAF-1-ET with 8-hydroxyquinoline as a colorimetric indicator provides an efficient and accurate tool for directly determining the iron ion concentrations from groundwater samples, with minimal processing and equipment needs. Notes and references ‡ http://www.sams-xrays.com/sixpack. § Less than 100% loading of 2-(methylthio)-ethan-1-ol loading might be attributed to the reactivity of sodium hydride with benzyl chloride and the conversion of some of the -CH2Cl groups to other functionalities such as methyl, or the linkage of these groups to form polymeric 1,2-diphenylethane (see S. Bank, M. C. Prislopski, J. Chem. Soc. Chem. Commun., 1970, 0, 1624; M. I. Watkins, G. A. Olah, J. Am. Chem. Soc., 1981, 103, 6566; and Y. Yuan, Y. Bian. Appl. Organomet. Chem., 2008, 22, 15.)

chemsum

{"title": "Iron detection and remediation with a functionalized porous polymer applied to environmental water samples", "journal": "ChemRxiv"}

press/md:_predictor_of_skin_sensitization_caused_by_chemicals_leaching_from_medical_devices

## Abstract: Safety evaluation for medical devices includes the toxicity assessment of chemicals used in device manufacturing, cleansing and/or sterilization that may leach into a patient. According to international standards on biocompatibility assessments (ISO 10993), chemicals that could be released from medical devices should be evaluated for their potential to induce skin sensitization/allergenicity, and one of the commonly used approaches is the guinea pig maximization test (GPMT). However, there is growing trend in regulatory science to move away from costly animal assays to employing New Approach Methodologies including computational methods. Herein, we developed a new computational tool for rapid and accurate prediction of the GPMT outcome that we named PreSS/MD (Predictor of Skin Sensitization for Medical Devices).To enable model development, we (i) collected, curated, and integrated the largest publicly available dataset for GPMT; (ii) succeeded in developing externally predictive (balanced accuracy of 70-74% as evaluated by both 5-fold external cross-validation and testing of novel compounds) Quantitative Structure-Activity Relationships (QSAR) models for GPMT using machine learning algorithms, including Deep Learning; and (iii) developed a publicly accessible web portal integrating PreSS/MD models that enables the prediction of GPMT outcomes for any molecules using. We expect that PreSS/MD will be used by both researchers and regulatory agencies to support safety assessment for medical devices and help replace, reduce or refine the use of animals in toxicity testing. PreSS/MD is freely available at https://pressmd.mml.unc.edu/. ## Introduction Sensitization is a toxicological endpoint associated with the ability of an offending chemical to cause or elicit an allergic response in some people following repeated exposures to the allergen. 1,2 Traditionally, assessing the sensitization potential for a chemical or material has relied on the use of animal models. The guinea pig maximization test (GPMT) of Magnusson and Kligman 3 and the Buehler test 4 have been predominantly used methods for more than five-decades since their original development. 3,4 Alternative assays, such as the murine Local Lymph Node Assay (LLNA), have been employed for assessing skin sensitization as well. However, more recently, regulatory agencies have been supporting the development of alternative in vitro and in chemico methods that could help reduce, refine or replace testing in animals without compromising the acceptable standards for the identification of sensitizers. 5,6 Medical devices encompass a vast array of products intended to treat patients or diagnose diseases or other health-compromising conditions. 7 For marketing in the United States, the Food and Drug Administration (FDA) has set the definition of a medical device in Section 201(h) of the Food, Drug, and Cosmetic Act. 8 Medical devices require a pre-market biocompatibility assessment described in Guidance for Industry and FDA Staff on Use of International Standard ISO 10993-1, Biological evaluation of medical devices -Part 1: Evaluation and testing within a risk management process. 9 Many medical devices, such as implants and glucose meters, contain chemicals that may leach and cause toxicity. Depending on the type and the duration of the contact with the body, a device may be evaluated for its biocompatibility, including the potential to produce localized sensitization responses. 13 Pre-market submissions for medical devices address sensitization potential with data gathered primarily with the GPMT or Buehler tests as recommended by the International Organization for Standardization (ISO) standard 10993 Part 10. 9 In the last several years, both our and other 17,18 groups have developed computational models for predicting the sensitizing activity of chemicals in LLNA. In an effort to modernize the evaluation of medical devices potential for causing skin sensitization and help reduce in vivo animal testing, we embarked on the development of a unique open-source computational tool and web app that we named PreSS/MD (Predictor of Skin Sensitization caused by Medical Devices). We envisioned a context of use where this tool can be employed to assess the skin sensitization potential of medical devices, to supplement and, potentially, replace the experimental assessments such as animal-based tests currently accepted for regulatory submissions of medical devices. To achieve this goal, we (i) collected, curated, and integrated the largest publicly available dataset for GPMT; (ii) developed and externally validated QSAR models to predict GPMT; and (iii) incorporated GMPT models into the PreSS/MD web portal to help evaluate the skin sensitization potential for medical devices. ## Materials and Methods The workflow employed in the study is depicted in Figure 1. ## European Chemical Agency (ECHA) dataset Experimental animal data on skin sensitization evaluated with the Guinea Pig Maximization Test (GPMT) were retrieved from the ECHA study results database (https://iuclid6.echa.europa.eu/reach-study-results). Unfortunately, there were numerous problems with the collected raw data. For instance, many numerical data were represented as string variables, the units of measurements were not standardized through the datasets, and there were many "free text" data. Therefore, we extensively cleaned and standardized all the data and converted measurements to the same units in each dataset. We also used regex expressions to find essential features for the database that were described in text format; this was key to classifying endpoints into GHS hazard categories. Following this laborious data preparation and standardization, we performed both chemical and biological data curation. After removing inconsistent data and non-modelable compounds (see Data Curation section), 1,023 out of the original 5,727 data points were kept. Among 23 duplicate chemical pairs in the dataset, biological annotations for 20 of them were concordant and for three, were discordant, i.e., duplicative compounds had different annotated classifications (sensitizer vs. non-sensitizer). All the discordant replicates and one of each concordant replicate were removed. The final dataset comprised 995 unique chemical compounds, including 247 sensitizers and 748 non-sensitizers. ## Literature We also collected GPMT skin sensitization experimental data from the scientific literature. After removing mixtures, inorganics, and counter ions, 701 out of the original 745 data points were kept. Only one pair of duplicates showed biological annotation disagreement among 221 chemicals with more than one data point in the dataset. The discordant replicates were removed and only one data point for each concordant replicate was kept. Thus, the final dataset had 374 unique chemical compounds, including 173 sensitizers and 201 non-sensitizers. ## Combined GPMT data from ECHA and the literature We merged the curated data from ECHA and the research literature and examined the content of this combined data. There were 41 pairs of replicates between these two data sets, and the sensitization potential of only six of these pairs was annotated differently. These discordant records were removed, and only one record for each concordant pair of duplicates was kept. The merged data set had 1322 unique compounds including 432 sensitizers and 890 non-sensitizers, i.e., it was imbalanced with the ratio of sensitizers to non-sensitizers of approximately 1:2. ## Case studies sets An additional literature search executed identified nine new compounds with GPMT data that were not part of the training set used for model development. These compounds were standardized and used as an additional validation set. We also collected the 474 compounds available in the Extractables and Leachables Safety Information Exchange (ELSIE) Database 24 After the removal of inorganics, mixtures, and duplicates, 415 compounds remained. We found that 102 compounds were present on our GPMT list and 313 unique compounds were kept for model evaluation. ## Data curation Datasets were thoroughly curated following the workflows developed by us earlier. 25 . First, we performed chemical structure curation and removed mixtures, inorganics, and organometallic compounds, cleaned and neutralized salts, normalized the specific chemotypes, and applied the special treatment to chemicals with multiple replicated records as follows: (i) when replicated records presented the same binary outcome, only one record was kept; (ii) when a majority of replicated records presented the same binary outcome and one had different binary outcome, only one record with the agreeing binary outcome was kept, (iii) when replicated records had different binary outcomes, all of them were removed. All the curated data are available in Supplementary Material. ## QSAR modeling The modelability index (MODI) 26 was calculated to estimate the feasibility of obtaining predictive QSAR models. We developed our models following the best practices of QSAR modeling. 27 The models were developed using open-source chemical descriptors based on ECFP4like circular fingerprints with 2048 bits and an atom radius of 2 (Morgan2) calculated in RDKit 28 . Machine learning approaches included Support Vector Machine (SVM) 29 , Random Forest (RF), 30 and Light Gradient Boosting Machines (lightGBM) algorithms implemented in Scikit-learn. 31 All models were optimized using a Bayesian approach implemented in Scikit-Optimize v.0.7.4. 32 The details of hyperparameters explored in this work are available in the Supporting Information. The Bayesian optimization may be defined as follows (Equation 1): where, xi is the ith sample, and 𝑓(xi) is the observation of the objective function at xi. The observations D1:t = {x1:t, 𝑓(x1:t)} are accumulated. The prior distribution is combined with the likelihood function P(D1:t|𝑓) of overserving D 1:𝑡 given model 𝑓 multiplied by the prior probability of P(𝑓). In doing so, Bayesian optimization finds hyperparameters that maximize the objective function (G-mean score) by building a surrogate function (probabilistic model) based on past evaluation hyperparameters of the objective. 32,33 The geometric (G)-mean was selected as the scorer since it measures the balance between classification performances on both the majority (non-toxic) and minority (toxic) classes. The QSAR models employing deep learning were developed using Keras The predictivity of the models were assessed by the Equations 2-7: Balanced accuracy: Sensitivity: Specificity: Positive Predictive Value (PPV): Negative Predictive Value (NPV): Kappa 𝐾𝑎𝑝𝑝𝑎 = 2×(𝑇𝑃×𝑇𝑁−𝐹𝑁×𝐹𝑃) (𝑇𝑃+𝐹𝑃)×(𝐹𝑃+𝑇𝑁)+(𝑇𝑃+𝐹𝑁)+(𝐹𝑁+𝑇𝑁) (7) where TP are the true positives, FP are the false positives, TN are the true negatives, and FN are the false negatives. ## Mechanistic interpretation of QSAR models Maps of predicted fragment contribution 34,35 were generated from the QSAR models to help identify and visualize the substructure(s) predicted to provide significant contribution to the skin sensitization potential. Here, the contribution of an atom is estimated by a contribution difference obtained when the associated bits in the fingerprint corresponding to the atom are removed. Then, the normalized contributions were used to color-code the atoms in a topographylike map, in which green indicates negative contribution for toxicity (i.e., skin sensitization reduces when the atom is absent), and magenta indicating a positive contribution for toxicity (i.e., skin sensitization increases when the atom is present). 35 ## Model implementation The PreSS/MD web app was implemented on an Ubuntu Server. The app is coded using Flask (http://flask.pocoo.org), uWSGI (https://uwsgi-docs.readthedocs.org), Nginx (http://nginx.org), Python (https://www.python.org), RDKit (http://www.rdkit.org), scikit-learn (http://scikit-learn.org), and JavaScript (http://www.ecma-international.org). PreSS/MD also includes the JSME molecule editor written in JavaScript, 36 supported by the most popular web browsers. Java or Flash plugins are not required to use the app. ## QSAR models for predicting skin sensitization using GPMT data High values of MODI (≥0.7) allowed us to expect that robust and predictive QSAR models could be developed for this dataset. The statistical characteristics of the skin sensitization models built and validated using GPMT data are shown in Table 3. The machine learning models built using RF, SVM, lightGBM, and Deep Learning were able to predict the external set with balanced accuracy of 73%, 74%, 70%, and 72%, respectively. ## PreSS/MD usability PreSS/MD has an intuitive user interface (Figure 2). The user may draw a molecule of interest or directly paste the query chemical structure's SMILES string in the "molecular editor" potential. These predictions are followed by the prediction's confidence, which is estimated by the ratio of predictions made by internal models, 30 the applicability domain (AD), and the maps of predicted fragment contribution. ## Case studies As an example of a practical application, we tested PreSS/MD by employing it to predict the skin sensitization potential of nine medical device ingredients identified internally at the FDA with discordant data between GPMT and human clinical data. We compared this list with the dataset used to build our models and found that all these compounds were new and were not included in the original dataset. Therefore, we performed a blind prediction using the PreSS/MD to predict the skin sensitization potential of these nine compounds. The predicted results are shown in Table 4. PreSS/MD correctly predicted six out of nine compounds (balanced accuracy of 65%, sensitivity of 80%, specificity of 50%, PPV of 66% and NPV of 66%). Although the evaluation of these nine compounds presented low specificity, the NPV indicates the probability of predicted non-sensitizer being truly non-sensitizers is high as 66%. In addition to these nine compounds with GPMT data, we exploited our models to predict a list of 474 chemicals known to leach from MD. After the removal of inorganics, mixtures, and duplicates, 415 compounds remained, and we found that 102 compounds were present in our curated GPMT list. Out of the 313 remaining compounds, our models predicted 98 compounds as sensitizers in the GPMT assay and 215 as non-sensitizers. We analyzed this list's overlap with the expanded skin sensitization dataset of human, LLNA, and three non-animal assays (DPRA, KeratinoSens, and h-CLAT) data described in our previous paper. 14 Out of 313 chemicals, we found that 34 had experimental data in one of the skin sensitization assays. Table 3 shows the concordance of the predicted values using PreSS/MD and the skin sensitization potential available from experimental assays. Although the pool of compounds was small, the results show a high concordance with all assays. This high concordance suggests that integration of PreSS/MD models with non-animal methods, such as DPRA, KeratinoSens, and h-CLAT may be complementary to assess skin sensitization. The use of GPMT to predict human skin sensitization. Previously, we analyzed the correlation of LLNA and Human skin sensitization data to understand how valuable the animal model is for determining risk assessment. 37 As GPMT is still being used to check the sensitization potential of leachable chemicals from medical devices, 9 we decided to conduct a similar analysis we reported before, when comparing LLNA vs. human data. 37 Here we compared the overlap between the 1322 compounds with GPMT data and the 138 compounds with human data we previously reported elsewhere. 14 As seen in Table 4, 109 compounds were both tested in GPMT and had human clinical data. In total, 46 compounds were sensitizers in both tests and 41 compounds were classified as non-sensitizers in both tests, while 22 disagreed in classification. Therefore, our analysis has shown that the accuracy of using GPMT to predict human skin sensitization is estimated to have the balanced accuracy of 80%, sensitivity of 85%, PPV of 77%, specificity of 74%, and NPV of 84%. Out of the 112 compounds shown in Table 4, 14 compounds were labeled to be leaching from medical devices in the ELSIE dataset. Of these there were 9 sensitizers and 5 non-sensitizers with human data. All the non-sensitizers in humans were also non-sensitizers in GPMT and only one sensitizer in humans was labeled as a non-sensitizer in GPMT. Given the small number of compounds with known experimental values from both GPMT and humans, we decided to apply our previously developed QSAR models of human data 16 to the remaining 1210 compounds with GPMT data lacking human data. The use of QSAR-imputed human data allowed us to examine the possible relationships between the two endpoints for a much larger set of compounds. 38 found that GPMT had sensitivity of 70% and specificity of 100%. However, the data analyzed was much smaller, with 57 chemicals and only 3 non-sensitizers. Variability of the GPMT has been documented as dependent on the total number of animals, dosage, and grade patterns of the sensitization response considered in the test. 39 Within the extensive data collected in this work, GPMT data showed high reproducibility. In the ECHA dataset, only three pairs of compounds out of 23 duplicate chemicals had discordant annotations. The data collected from the literature had only one pair of duplicates with discordant annotations among 221 chemicals. Finally, there were 41 pairs of replicates between these two data sets, and the sensitization potential was different for only six of these pairs. Conversely, human tests show high inter-individual variability, especially for compounds tested at a high dose, which can show weak sensitization rates in the tested populations. 40 In our previous analysis, 14 we found the accuracy of LLNA to predict Human skin sensitization was estimated to have a balanced accuracy of 68%, sensitivity of 84%, and specificity of 52%. The low specificity means that LLNA is oversensitive to predict human skin sensitization, i.e., more compounds tend to be skin sensitizers in mice than in humans. Conversely, GMPT showed higher concordance with human data, with specificity as high as 75%. ## An alternative to animal testing for skin sensitization for medical devices The GPMT was first published in 1969 3 and was considered the preferred animal method to assess skin sensitization caused by chemicals for decades. In 1989, the LLNA was first described. 41 Since then, it underwent multiple evaluations and refinements, becoming the preferred animal testing for skin sensitization after the publication of the Organisation for Economic Cooperation and Development (OECD) Testing Guideline No. 429. 42 However, international standards (ISO 10993) 43 still recommend the evaluation of chemicals released from MD for skin sensitization/allergenicity potential using the Guinea Pig Maximization Test (GPMT). 6 Recently, Svobodová et al. 44 evaluated the sensitization potential of chemicals present in MD using a combination of in chemico (DPRA) and in vitro (LuSens) methods in comparison with the LLNA method and suggested a testing strategy for the safety assessment of medical device extracts. The authors reported an overall concordance of 63.9-82.5% between LLNA and DPRA and 80-85.4% between LLNA and LuSens. Unfortunately, no sensitivity and specificity were reported. The results shown in Table 4 of this study reveal that there is a high concordance between GPMT and human data, which is in contrast with our previous findings showing that LLNA tends to be oversensitive as compared to the human response. 14,37 Although GPMT shows a higher concordance with human data than the LLNA, it is important to note that GPMT requires the sacrifice of several animals 45 for each tested chemicals and, therefore, better approaches need to become available soon. Recently, the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) published a Strategic Roadmap, 1 calling for the development of alternative approaches to reduce animal testing of chemical and medical agents. Thus, there is an expressed need to modernize the safety evaluation of MD using alternative methods, shorten the regulatory review time, and ultimately bring safer devices to the market faster. QSAR models developed in this study and implemented in the PreSS/MD web app showed balanced accuracy of 70-74%. Although our analysis of replicates identified only six out of 41 replicated entries to disagree, a previous study has shown that dose, number of animals, and response pattern may influence in the outcome, which is evaluated by a specialist. Therefore, considering the absence of state-of-the-art predictors of GPMT as well as the variability of the assay, we suggest these models can be used to reduce the use of GPMT when used within integrated testing strategies. Moreover, since GPMT has shown higher concordance to human data than the LLNA, we suggest that QSAR models based on GPMT are more appropriate than running GPMT to assess the response to chemicals in humans. ## Discussion and conclusions. Previously, our group has developed the first QSAR models for skin sensitization based on human data. 37 Later, we employed an innovative approach using human, LLNA, and three validated non-animal assays within a Bayesian model to predict the human response. 14 This model showed higher accuracy in predicting the human response than the model built using only human data. 14 These models were implemented in a newer version of the Pred-Skin web app. 16 Since the publication of the OECD Testing Guideline No. 429, 42 LLNA has been regarded as the preferred animal test for evaluating skin sensitization. However, the GPMT is still required for the approval of MD. For this reason, we decided to develop a separate skin sensitization web application focusing on the safety evaluation of these devices. In order to apply in silico methods to predict the toxicity of MD, it is essential to note that a cornerstone in any safety evaluation of FDA-regulated products is an exposure assessment focused on actual conditions of use. Traditional methods to estimate exposure do not apply to all MD. Consequently, the medical device regulatory framework has implemented a chemical characterization and subsequent toxicological risk assessment approach. The chemical characterization involves identifying the device's component or determining chemicals that might leach into a patient during use and corresponding quantities. 46 Toxicologists use this information to conduct a risk assessment to ascertain whether any of the leachable chemicals might pose a health risk to patients at the doses quantitated. Both the chemical characterization and toxicological risk assessment for MD are generally done as recommended by the ISO standard 10993 Parts 18 and 17, respectively. PreSS/MD can predict potential leachable compounds submitted for regulatory pre-market consideration. In summary, in this contribution we described the development of PreSS/MD, a web application to predict the skin sensitization potential of chemicals based on GPMT. This tool is the first publicly available tool based on this assay. Although non-animal assays have been explored to evaluate the potential skin sensitization effects of chemical hazards, 2 animals are still required by regulatory agencies to evaluate MD. Our results here show that GPMT has a good correlation with human data, which is higher than the murine LLNA. However, although the use of guinea pigs is justified as their response to various skin sensitizers is similar to humans, interpretation of these assays' results requires unique expertise. 47 Moreover, the use of guinea pigs raises moral and ethical concerns, defying the principle of the 3Rs -Replacement, Refinement, and Reductionwhose goal is to identify alternative methods that utilize phylogenetically lower species, reduce the number, and refine the use of animals to lessen pain and distress. 1,48 Therefore, there is an imperative need to replace these assays. Our results show that the historical and publicly available GPMT data is sufficient to generate predictive and robust in silico models using machine learning approaches. The PreSS/MD web application fulfills an unmet need to help modernize the evaluation of skin sensitization for MD to reduce the need for animal testing. These models can be employed within integrated testing strategies to provide a weight of evidence of the sensitization potential of chemicals leaching from MD without requiring further animal tests. Moreover, we expect that the models developed in this study are applicable to estimate the toxicity of other industrial chemicals. 49 The PreSS/MD web application is publicly available at https://pressmd.mml.unc.edu/.

chemsum

{"title": "PreSS/MD: Predictor of Skin Sensitization Caused by Chemicals Leaching from Medical Devices", "journal": "ChemRxiv"}

me₃sisime₂(o^<i>n</i>bu):_a_disilane_reagent_for_the_synthesis_of_di

## Abstract: Herein, a readily available disilane Me 3 SiSiMe 2 (O n Bu) has been developed for the synthesis of diverse silacycles via Brook-and retro-Brook-type rearrangement. This protocol enables the incorporation of a silylene into different starting materials, including acrylamides, alkene-tethered 2-(2-iodophenyl)-1Hindoles, and 2-iodobiaryls, via the cleavage of Si-Si, Si-C, and Si-O bonds, leading to the formation of spirobenzosiloles, fused benzosiloles, and p-conjugated dibenzosiloles in moderate to good yields. Preliminary mechanistic studies indicate that this transformation is realized by successive palladiumcatalyzed bis-silylation and Brook-and retro-Brook-type rearrangement of silane-tethered silanols. ## Introduction Silacycles have attracted increasing attention because they have shown unique physical, optoelectronic, and physiological properties in medicinal chemistry and materials science. 1 In this context, considerable efforts have been devoted to the development of synthetic methods for silacycles, which is a prerequisite for fully discovering their application potentials. 2,3 Among them, of particular interest to synthetic chemists is the synthesis of silacycles via the cleavage of C-Si bonds. 3 Typical strategies include direct annulation of silicon-based frameworks 3a-n and C-Si/C-C bond exchange reaction of small ring systems based on the existence of a high ring strain. 3o-r However, these methods generally require transition-metal catalysts to assist the activation of C-Si bonds. Transitionmetal-free catalyzed annulation to assemble silacycles by cleaving C-Si bonds is still elusive so far. The Brook rearrangement enables an intramolecular migration of a silyl group from carbon to oxygen atoms via a hypervalent silicon species, 4 which was initially introduced by Brook 5 and was demonstrated to be a reversible process. 6 Its reverse process, namely retro-Brook rearrangement, can, in turn, be achieved by the transfer of a silyl group from oxygen to carbon atoms (Scheme 1A). 7 Obviously, the Brook and retro- Brook rearrangements allow the cleavage of a C-Si bond and the formation of a C-Si bond under transition-metal-free catalysis. 8 On the other hand, the existing studies are limited to the translocation of a single silyl group from the starting materials. The migration of two different silyl groups in one event has not been reported so far. In this context, we want to explore the synthesis of silacycles via the migration of two different silyl groups in the Brook and retro-Brook rearrangement. The design and synthesis of silane-tethered silanols is undoubtedly the primary task and challenge to achieve this hypothesis. Disilanes have been among the most versatile silylation reagents in organic synthesis. Over the past few decades, a myriad of methods for accessing organosilanes from disilanes have focused on the activation of Si-Si bonds for the development of mono-silylation involving aryl halides or cyanides, alkenes, and C-H bonds and bis-silylation of alkynes, alkenes, carbenes, and palladacycles (Scheme 1B). Particularly, the pioneering work that realizes the bis-silylation of in situ generated palladacycles with hexamethyldisilane via the cleavage of Si-Si bonds has recently been reported by Zhang,Cheng,and us. 12,13 These advances have inspired us to modify hexamethyldisilane, namely the replacement of the methyl group with an oxygen-containing group, for the synthesis of silane-tethered silanols, which were further converted into silacycles via Brook and retro-Brook-type rearrangement. Herein, we disclose a Brook and retro-Brook-type rearrangement strategy for the synthesis of diverse silacycles, including spirobenzosiloles, fused benzosiloles, and p-conjugated dibenzosiloles, by employing a readily available disilane reagent Me 3 SiSiMe 2 (-O n Bu) that could be prepared by a simple treatment of pentamethylchlorodisilane with n-butanol in the presence of NEt 3 at room temperature (Scheme 1C). Notably, the cleavage of Si-Si, Si-C, and Si-O bonds is involved in the transformation. ## Results and discussion We initiated the studies by investigating the reaction of acrylamide 1a with 1-butoxy-1,1,2,2,2-pentamethyldisilane 2a. To our delight, the anticipated spirobenzosilole 3a was indeed afforded in 66% yield by using a simple catalytic system composed of Pd(OAc) 2 and K 2 CO 3 in DMF at 90 C. Encouraged by these initial results, various parameters were screened, and the optimized reaction conditions are as follows: 1a (0.2 mmol), 2a (0.3 mmol), Pd(OAc) 2 (10 mol%), PPh 3 (20 mol%), and K 2 CO 3 (0.6 mmol) in DMA (2 mL) at 90 C under N 2 for 6 h (see the ESI † for details). Next, several disilanes 2b-h were tested. As shown in Scheme 2, when the n-butyl group of disilane 2a was replaced by other functional groups such as n-hexyl, benzyl, cyclohexyl, and 2-oxopropyl, all of them could afford the desired product 3a, albeit in a lower yield. Unexpectedly, disilane 2f was unreactive. Finally, disiloxane 2g was found to produce product 3a in 58% yield. With the optimal reaction conditions and disilane reagent confrmed, the scope of acrylamides 1 was subsequently examined. Gratifyingly, this protocol was applicable to a large variety of acrylamides 1 to afford spiro[benzo [b]silole-3,3 0indolin]-2 0 -ones 3a-u in moderate to good yields (Scheme 3). Note that the replacement of iodine atoms with bromine atoms on the acrylamide showed good reactivity, delivering the product 3a in 66% yield. Moreover, the three substructures of acrylamides 1 were systematically investigated. Regarding different substituents on the nitrogen atom, methyl or ethyl group substituted acrylamides 1b and 1c were competent substrates, while acrylamide 1d with a Ts group could not give the target product 3d under the standard conditions. For the 2iodoaniline fragment, a broad range of functional groups on the benzene ring, including electron-donating groups (Me and OMe), modifable halogen groups (F and Cl), and even strong electron-withdrawing groups (CF 3 , CO 2 Me, and NO 2 ), were well tolerated (3e-n). Meanwhile, the structure of 3g was unambiguously confrmed by X-ray crystallography. Their electronic properties seem to affect the reactivity, since substrates 1l-n with strong electron-withdrawing groups, especially CO 2 Me and NO 2 groups, resulted in a diminished yield. Finally, the compatibility was further demonstrated by testing the key 2phenylalkene moiety. Both the benzene ring containing ortho or para substituents and the naphthalene ring could survive, affording spirobenzosilole 3o-u in moderate to good yields. Nevertheless, a slight modifcation of the reaction conditions was required when substrates bearing F groups were used (3q and 3t). When the reaction was scaled up to 1 mmol, 70% spirocyclic product 3a could also be obtained. To highlight the generality of this domino Heck/ silacyclization, we envision that fused benzosiloles can be synthesized by a domino Heck/ortho C-H functionalization of aryl iodides. Therefore, 2-(2-iodophenyl)-1-(2-methylallyl)-1Hindole was employed to react with disilane 2a under the above reaction conditions. To our delight, indolo[2,1-a]silolo [4,3,2-de] isoquinolines 5a could smoothly be produced in 58% yield by the cleavage of Si-Si and Si-O bonds. Encouraged by these results, the scope of 2-(2-halophenyl)-1-(2-methylallyl)-1Hindoles was then explored (Scheme 4). Bromine atoms instead of iodine atoms on the substrate 4a 0 were subjected to the standard conditions, which could give product 5a, albeit in a lower yield. Satisfactorily, substrates 4b-g being diversely substituted (Me, F, and Cl) on the indole ring were able to undergo this domino Heck/silacyclization with disilane 2a to provide the desired products 5b-g in moderate yields. Differently, using substrate 4h required relatively mild conditions. To emphasize the versatility of the disilane reagent Me 3 -SiSiMe 2 (O n Bu), we next attempted to synthesize p-conjugated dibenzo [b,d]siloles by performing the reaction of 2-iodo-1,1 0biphenyl with Me 3 SiSiMe 2 (O n Bu) 2a. However, no anticipated product was observed under the above standard conditions. Subsequently, PPh 3 was found to suppress the reaction, since dibenzo [b,d]siloles 7a could be afforded in 52% yield by the removal of PPh 3 . Encouraged by these results, the optimal reaction conditions that could furnish 64% of 7a were established by the screening of various parameters (see the ESI † and Scheme 5). Afterward, a series of substituted 2-iodobiphenyls were examined. Delightfully, the electron-donating group (Me and OMe) on the benzene ring could be tolerated for the silacyclization reaction with disilane 2a, thus delivering the desired products 7b-k in moderate yields. Unfortunately, this protocol was not applicable to substrates 6l-o bearing an electronwithdrawing group. The possible reason is that the protonation of palladacycles formed by substrates 6l-o is easier than bis-silylation (see the ESI †). To gain insight into the reaction mechanism, a range of control experiments were performed (Scheme 6). The reaction of acrylamides 1a with disiloxane 2h or 2i could afford product 3a in 27% and 50% yields, respectively, under the optimal reaction conditions (eqn (1) †). Moreover, hexamethyldisiloxane (TMSOTMS) was detected by gas chromatography in the model reaction of 1a with 2a (eqn (2), see the ESI †). These results indicated that the silicon source of product 3a came from the dimethylsilyl group generated by disilane 2a via the cleavage of Si-Si and Si-O bonds. Interestingly, two disilylated products 8a and 8a 0 as well as product 3a were isolated in 43%, 27%, and 16% yields when 1a and 2a were reacted under the standard conditions for 20 minutes (eqn (3) †). Therefore, we speculated that two disilylated products 8a and 8a 0 were the reaction intermediates (the structure of 8a was absolutely confrmed by X-ray crystallography). Finally, a spiropalladacycle 14 that could Scheme 5 Variations of the 2-iodobiphenyls (6). a Reaction conditions: 6 (0.2 mmol), 2a (0.24 mmol), Pd(OAc) 2 (10 mol%), K 2 CO 3 (3 equiv.), and DMF (2 mL) at 90 C under a N 2 atmosphere for 12 h. b 2-Bromobiphenyl was used. c 2-Iodo-4 0 -methyl-1,1 0 -biphenyl was used. d 2-Iodo-3 0 -methyl-1,1 0 -biphenyl was used. be prepared from acrylamides and stoichiometric Pd(PPh 3 ) 4 was employed for the reaction with disilane 2a (eqn (4) †). Unexpectedly, no product 3b was observed in the absence of Pd(OAc) 2 and PPh 3 . Surprisingly, extra addition of Pd(OAc) 2 and PPh 3 could give product 3b in 60% yield. These results suggested that the spiropalladacycle as a reaction intermediate underwent transmetalation with intermediate G, rather than direct oxidative addition with disilane 2a, to furnish disilylated products 8a and 8a 0 , which were then converted into the desired product 3a. To verify our hypothesis and propose the possible formation process of 3a from 8a or 8a 0 , we conducted several control experiments (Scheme 7). The silacyclization of disilylated products 8a and 8a 0 , respectively, was conducted in the absence of Pd(OAc) 2 and PPh 3 , and the product 3a was obtained in 66% and 60% yields as expected (eqn (1) †). Besides, TMSOTMS was also detected (see the ESI †). These results demonstrated that the transformation of 8a and 8a 0 into 3a involved the cleavage of the Me 3 Si-C bond, which did not require the assistance of palladium catalysts. On the basis of these results from eqn (1) † and previous work reported by Smith and Takeda,15 we speculated that 8a and 8a 0 undergo a Brook-and retro-Brook-type rearrangement to afford 3a (path c and path d). To capture carbanion species F and F 0 , two common electrophilic reagents, such as iodomethane and benzyl bromide, were added for the silacyclization of 8a and 8a 0 (eqn ( 2) and (3) †). However, the corresponding products F-1 and F 0 -2 were not observed. Therefore, these results are more favorable to this pathway involving synergetic Brook/retro-Brook-type rearrangement (path d). Based on the results of mechanistic experiments as well as reported work, 12,13,15 a plausible mechanism for the synthesis of silacycles was proposed (Scheme 8). Initially, oxidative addition followed by intramolecular Heck-cyclization of acrylamides 1a to Pd(0) species forms intermediate A, which then undergoes a C-H activation to afford spiropalladacycle B. Next, spiropalladacycle B produces disilylated products 8a/8a 0 and regenerates Pd(0) by sequential transmetalation with intermediate G generated by disilane 2a, reductive elimination and further hydrolysis (path a). 8a and 8a 0 then undergo a synergetic Brook/retro-Brook-type rearrangement to afford 3a and a trimethylsiloxy anion, which could be converted into TMSOTMS (path d). 16 Notably, another possible pathway that synthesizes disilylated products 8a/8a 0 by direct oxidative addition of spiropalladacycle B with disilane 2a is ruled out by the results of eqn ( 4) † (path b). ## Conclusions In conclusion, we have disclosed the frst example of divergent synthesis of silacycles via a Brook-and retro-Brook-type rearrangement strategy by employing a readily accessible disilane reagent Me 3 SiSiMe 2 (O n Bu). In this novel transformation, divergent silacycles, such as spirobenzosiloles, fused benzosiloles, and p-conjugated dibenzosiloles, can be produced in moderate to good yields by an unprecedented complex process composed of a bis-silylation of a palladacycle and a Brook-and retro-Brook-type rearrangement. Notably, mechanistic studies reveal that bis-silylation of the palladacycle is completed by a transmetalation process. Further applications of the disilane reagent Me 3 SiSiMe 2 (O n Bu) and the rearrangement are still in progress in our laboratory.

chemsum

{"title": "Me₃SiSiMe₂(O^<i>n</i>Bu): a disilane reagent for the synthesis of diverse silacycles <i>via</i> Brook- and retro-Brook-type rearrangement", "journal": "Royal Society of Chemistry (RSC)"}

self-assembled_molecular_nanowires_on_prepatterned_ge(001)_surfaces

## Abstract: It is a long-standing goal to fabricate conductive molecular nanowires (NWs) on semiconductor surfaces.Anchoring molecules to pre-patterned surface nanostructures is a practical approach to construct molecular NWs on semiconductor surfaces. Previously, well-ordered inorganic Ge NWs were deduced to spontaneously grow onto Pt/Ge(001) surfaces after annealing at an elevated temperature. In this work, we further demonstrate that organic 7,7,8,8-tetracyanoquinodimethane (TCNQ) molecular NWs can selfassemble onto the atomic NWs on Pt/Ge(001) surfaces. The outer nitrogen atoms in TCNQ molecules hybridize with under-coordinated Ge atoms in Ge NWs with an energy release of $1.14 eV per molecule, and electrons transfer from Ge NWs to the frontier orbitals of anchored TCNQs resulting in a negatively charged state. This largely tailors the electronic configurations of TCNQs and Pt/Ge(001) surfaces, enhancing the electron transport along the dimer row direction. The TCNQ molecular NWs coupled with the Ge NWs represent an exemplary showcase for the fabrication of molecular NWs on semiconductor surfaces. ## Introduction Molecular electronics have been attracting much attention due to the fact that commercially utilized silicon-based electronic devices are approaching their physical limits. Organic molecules can be utilized to design molecular devices. Onedimensional (1D) molecular nanowires (NWs) may serve as conductive channels for molecular devices. Therefore, the fabrication of molecular NWs on semiconductor surfaces becomes important for designing high-performance molecular electronics. To date, various strategies have been proposed to fabricate molecular NWs on semiconductor substrates. 7 Among them, self-assembly may be the simplest yet effective approach. For example, the self-assembly of molecular NWs can occur on a predefned position on Si(001) platforms via a radical chain reaction mechanism. 8 Molecules with terminal C^C, C]C, and C]O groups can easily react with the dangling bonds on silicon surfaces, where the dangling bonds could be created by the removal of H atoms on H-terminated silicon surfaces using an STM tip. These predesigned dangling bonds serve as a starting point for the subsequent radical chain propagation. The reactions involve the breakage of the double or triple bond, thereby resulting in a radical intermediate with a Si-C bond and a C-centred radical. Subsequently, the newly formed radical intermediate is capable of abstracting H atoms from its neighbouring Si atoms, producing a new dangling bond on the nearby Si atom, which continues to interact with another molecule. This series of reactions sets off a chain reaction on hydrogenated silicon surfaces, leading to the growth of molecular NWs on Si(001) and Si(111) surfaces. From the examples stated above, we notice that dangling bonds play critical roles in the self-assembly of molecular NWs on semiconductor surfaces. The presence of dangling bonds is a prerequisite to fabricate covalently adsorbed molecular NWs onto semiconductor surfaces. It was reported that atomic NWs, extending for hundreds of nanometres, can be self-organized on Pt/Ge(001) surfaces. 13 These NWs are constructed of undercoordinated Ge atoms, possibly suggesting the presence of dangling bonds on NWs. In this regard, they are expected to be good platforms for fabricating molecular NWs, because the dangling bonds on Ge NWs on Pt/Ge(001) surfaces can serve as potential points for the assembly of molecular NWs. Moreover, an appropriate space should be provided to accommodate the molecular NWs. On the Pt/Ge(001) platform, Ge NWs are spaced by either 1.6 nm or 2.4 nm. In our experimental observations, we indeed found that 7,7,8,8-tetracyanoquinodimethane (TCNQ) molecular NWs can be self-organized on Ge NWs on Pt/ Ge(001) surfaces. This observation of TCNQ molecular NWs is an exemplary showcase for the fabrication of molecular NWs on semiconducting surfaces. ## Methods Low-temperature scanning tunnelling microscopy (LT-STM) characterization was carried out in a custom-designed Unisoku LT-STM system with the base pressure maintained in a low 10 10 Torr range at 77 K, using the constant current mode with a commercial Pt-Ir tip. The bias voltages were applied to the sample. Ge(001) was cut from one-side-polished n-type wafers (commercially available from AXT Inc.). Samples were ultrasonically cleaned in propanol for 15 min several times and then dried under nitrogen gas. Subsequently, the prepared sample was fxed onto a Mo sample holder. Pristine Ge(001) surfaces with c(4 2) and p(2 2) periodicities were prepared through several cycles of 500 eV Ar + ion sputtering and annealing at around 1100 K. Pt atoms were then resistively evaporated onto dimerized Ge(001) surfaces at about 873 K for 10 min. TCNQ molecules (98%, Aldrich) were deposited on Pt/Ge(001) surfaces at 300 K using a Knudsen cell at 393 K. The calculation details are presented in the ESI. † ## Results and discussion Noble metals (i.e., Au and Pt) are known to trigger the selfassembly of atomic NWs on Ge(001) surfaces. 13, DFT calculations revealed that self-organized NWs on Pt/Ge(001) surfaces are constituted by under-coordinated Ge atoms (Fig. 1b, S1a and b †), suggesting the presence of dangling bonds on the NWs. 30 As aforementioned, dangling bonds on semiconductor surfaces can capture molecules by chemically bonding with them, and then setting off the self-assembly of molecular NWs on the semiconductor surfaces. TCNQ molecules, as archetypal electron acceptors, are widely utilized to prepare charge transfer complexes, showing potential in engineering molecular electronics. The electronic properties of TCNQ are mainly determined by the population of the lowest unoccupied molecular orbital (LUMO) (Fig. 1f). Here, the cyano groups in TCNQ are expected to chemically react with under-coordinated Ge atoms in the NWs on Pt/Ge(001) surfaces, abstracting electrons from the Ge atoms in the NWs. Moreover, Fig. 1e indicates that TCNQ is a planar molecule with a width of 4.0 and a length of 8.3 . The Ge NWs in Fig. 1a are spaced by 1.6 or 2.4 nm, providing adequate room to accommodate TCNQs. The Ge dimers in the NWs are spaced by 5.4 , further suggesting that the outer nitrogen atoms, separated by 4.0 (d N1-N2 ¼ d N3-N4 ¼4.0 ), in a TCNQ molecule possibly react with Ge atoms. As expected, TCNQ molecular NWs were experimentally observed alongside the Ge NWs on Pt/Ge(001) surfaces (see Fig. 1c and d). Furthermore, individual TCNQ molecules can adsorb in another orientation, marked with white dotted rectangles in Fig. 1d, perpendicular to the TCNQ molecular NWs. Fig. 2a shows that the TCNQs anchor onto the Ge NWs with their cyano groups located above the Ge NWs, generating a flat adsorption geometry with respect to the Pt/Ge(001) surface, and Ge NWs could be observed adjacently about 1.6 nm away. DFT calculations indicate that TCNQ molecules prefer to chemically bond to the Ge NWs instead of physisorption. The calculation results in Fig. 2b suggest that the nitrogen atoms on the left side of the TCNQ (i.e., N1 and N2) form covalent bonds with the Ge1 atoms in NWs, and the remaining nitrogen atoms (i.e., N3 and N4) on the right side (see the blue arrow in Fig. 2c) are situated in the trench between two Ge NWs. The formation of Ge-N bonds causes the rupture of the Ge1-Ge2 dimer in Ge NWs. The Ge1 atom chemically interacts with the N1 and N2 atoms, and the Ge2 atom, marked with dotted circles, moves downwards to react with Pt atoms. No covalent bond is formed between the Ge2 atom and TCNQ molecule. Furthermore, the N3 and N4 atoms, indicated with a blue arrow, slightly move downwards due to the presence of dangling bonds of the Ge atoms (red circles in Fig. 2c) near Pt atoms. The conjugated p-system extending across the TCNQ makes it a planar and rigid molecule in the gas-phase state. The adsorbed TCNQ becomes flexible if electrons are transferred to the cyano groups since the peripheral carbon atoms of TCNQ turn into the nonplanar sp 3 hybridization. 35 Moreover, the LUMO of a TCNQ is constructed of antibonding p orbitals gathering around the double bonds in TCNQ; therefore, the electron flling of the LUMO can weaken these double bonds and make TCNQ flexible. 35 The negatively charged TCNQ molecular NWs are no longer rigidly planar, accounting for the bending of anchored TCNQs. The chemical adsorption of TCNQs onto the Ge NWs on Pt/ Ge(001) surfaces is an exothermic reaction with an energy release of 1.14 eV per molecule. Energy variation during this process is calculated using the formula: E ads ¼ E[TCNQ/Ge(001)] E(TCNQ) E[Ge(001)], where E[TCNQ/Ge(001)], E(TCNQ) and E[Ge(001)] are the energies of the anchored TCNQ on Pt/Ge(001) surfaces, the isolated TCNQ molecule, and the Pt/Ge(001) surfaces, respectively. The DFT-simulated occupied state STM in Fig. 2d presents well-ordered TCNQ molecular NWs propagating along the Ge NWs on Pt/Ge(001) surfaces. Two TCNQs are 8.1 apart alongside NWs, double the distance of the neighbour Ge dimers on Ge(001)c(4 2) surfaces, which is consistent with the experimental value of $8.1 (Fig. 2a). As aforementioned, the TCNQ chemisorption leads to the Ge atom rearrangement in Ge NWs. Given that the Ge NWs near the TCNQ NWs (see Fig. 2a) are not fully occupied in the experiment, the slight rotation of anchored TCNQs in experiment may be attributed to the effect from the nearest neighbour Ge NWs. Moreover, a single TCNQ can be adsorbed on either side of Ge NWs; however, TNCQs in Fig. 2a appear on the same side. The nearest distance between two TCNQs is about 3.5 , suggesting a pronounced van der Waals interaction responsible for the low entropy confguration. To further investigate the charge transfer between the TCNQ molecular NWs and Ge NWs, the band structures and charge density differences are illustrated in Fig. 3. Fig. 3a shows the band structure of the dimerized Ge(001) surface (see the atomic structure in Fig. S1e and f †), and this surface is semiconducting with a bandgap around the Fermi level, E F . The red bands in Fig. 3b refer to the Pt/Ge(001) substrates, and the navy blue energy levels correspond to isolated TCNQs (see the atomic structure in Fig. S1c and d †). One can note that the Pt modifed Ge(001) surfaces behave as a metal since the S1 and S2 bands pass through the Fermi level (Fig. 3b), and the energy band dispersions perpendicular to the Ge dimer row direction are weaker than the bands parallel to the Ge dimer row direction, indicating that the NWs are more conducting. Moreover, compared to S1 and S2, the bands S3 and S4 across the Fermi level are more dispersed around the Fermi level after being chemically bonded with TCNQ molecular NWs, as displayed in Fig. 3c. More dispersive bands typically suggest smaller effective masses and higher carrier mobility; therefore, the conductivity along the NW direction is enhanced due to the presence of the TCNQ molecular NWs. Additionally, no band passes through the Fermi level along the X-S and Y-G paths perpendicular to the direction of NWs, possibly suggesting the suppression of conductivity perpendicular to the TCNQ molecular NWs. Moreover, the TCNQ molecular energy level in Fig. 3b is isolated without the chemical interactions between TCNQs and the Pt/Ge(001) surfaces (see the structural model in Fig. S1c and d †). The molecular energy level of TCNQ in close vicinity to the Fermi level is considered as the LUMO. It is mentioned in Fig. 2b that the Ge dimers of NWs on the Pt/Ge(001) surfaces are rearranged after the formation of TCNQ molecular NWs. Therefore, compared to Fig. 3b, the electronic band structures of TCNQ molecular NWs (Fig. 3c) are largely modifed. The pronounced dispersions of the HOMO and LUMO of TCNQs reflect hybridization between TCNQ and Pt/ Ge(001) surfaces (Fig. S2 †). In particular, the LUMO of TCNQ presents a concentrated distribution from 0.1 to 0.5 eV (Fig. 3c) below the Fermi level after chemically reacting with Ge NWs on Pt/Ge(001) surfaces, suggesting substantial electron charge transfer from Ge NWs to TCNQs. This charge transfer is further visualized in Fig. 3d. The Bader charge analysis suggests that each Ge in Ge-N bonds donates about 0.6 electrons to the anchored TCNQ. Notably, the charge transfer occurs between Ge NWs and TCNQs and between the Ge atoms near the Pt arrays and TCNQs indicated by the black arrows in Fig. 3e. The electron transfer of the Ge atoms near the Pt arrays should be partially responsible for the bending of anchored TCNQs on Ge NWs because of the Coulomb interactions. In general, the Bader charge analysis indicates the transfer of a total of 1.1 electrons from the substrate to each anchored TCNQ. A closer inspection of the whole charge transfer pattern in Fig. 3d indicates that the electron accumulated region resembles the LUMO of TCNQs (Fig. 1f). This further confrms the electron transfer to the LUMO of TCNQs, generating negatively charged TCNQ molecular NWs. To sum up, the adsorption of TCNQ molecular NWs causes pronounced alterations in the electronic and geometrical confgurations of both TCNQs and Pt/Ge(001) surfaces. ## Conclusions In this work, the deposition of TCNQs onto Ge NWs on Pt/ Ge(001) surfaces at room temperature results in the selfassembly of TCNQ molecular NWs. The Ge NWs can serve as fences on Pt/Ge(001) surfaces, providing adequate space for the growth of TCNQ molecular NWs. The cyano groups in TCNQs can chemically react with the under-coordinated Ge NWs, generating nearly flat TCNQ molecular NWs along the Ge NWs. Electrons transfer from the Ge NWs to the TCNQs, making the TNCQ molecular NWs negatively charged. Our work not only provides an effective approach for fabricating molecular NWs on prepatterned semiconductor surfaces but also offers important insights into tailoring the surface properties of semiconductor surfaces.

chemsum

{"title": "Self-assembled molecular nanowires on prepatterned Ge(001) surfaces", "journal": "Royal Society of Chemistry (RSC)"}

au(<scp>iii</scp>)-aryl_intermediates_in_oxidant-free_c–n_and_c–o_cross-coupling_catalysis

## Abstract: Au(III)-aryl species have been unequivocally identified as reactive intermediates in oxidant-free C-O and C-N cross coupling catalysis. The crystal structures of cyclometalated neutral and cationic Au(III) species are described and their key role in 2 electron-redox Au(I)/Au(III) catalysis in C-O and C-N cross couplings is shown. Nucleophiles compatible with Au-catalyzed cross couplings include aromatic and aliphatic alcohols and amines, as well as water and amides. ## Introduction In the last few years, the use of gold in homogeneous catalysis has experienced increasing attention and progress. Although typically regarded as superior Lewis acids for the activation of multiple C-C bonds towards nucleophiles, 1 gold complexes have recently found new patterns of reactivity, namely 2-electron redox processes applied to cross-coupling transformations. 2 In spite of the growing number of examples in this feld, the access to key Au(III) intermediates has been limited to harsh sacrifcial oxidants, such as I 3+ derivatives or F + sources (Scheme 1a), 3 as well as using highly electrophilic aryldiazonium salts under photoredox conditions or via light-driven radical chain reactions. 4 On the other hand, the straightforward pathway through the oxidative addition of C sp 2 -X and C sp 3 -X bonds (X ¼ halide) to Au(I) has generally been regarded as highly reluctant, 5 markedly differing from other transitionmetal chemistry. 6 However, recent in-depth organometallic investigations have dismissed this conception with remarkable achievements. The frst evidence for the intramolecular oxidative addition of aryl halides to gold(I) complexes was disclosed by the Bourissou group in 2014,7 showing that phosphinechelation assistance is key to delivering the C sp 2 -X bond in close proximity to the gold center, thus promoting the oxidative addition even at room temperature for X ¼ I. The same group later took advantage of the ability of carborane diphosphines to chelate gold(I) with small P-Au-P bite angles, which render a preorganized architecture closer in energy to the ensuing square-planar geometry of the oxidative addition product. By means of this strategy, intermolecular oxidative addition of aryl iodides and strained C-C bonds under mild conditions was accomplished. 8 Also in 2014, the Toste group substantiated the ability of gold to perform the elementary steps of organometallic cross-coupling chemistry, including oxidative addition, with the frst example of a Au(I)-catalyzed C-C bond formation without the requirement of external oxidants. 9 A tethered Au(I) aryl complex featuring an allyl bromide moiety allowed to support this mechanism via oxidative addition under intramolecular conditions (Scheme 1b). In our group we envisioned that a coordinating environment could favor the oxidative addition of Au(I) salts to aryl halides and the following reactivity towards nucleophiles. This strategy, based on attaching a macrocyclic appendage to aryl halide substrates bearing three available nitrogen coordination sites, had actually proved extremely benefcial in the isolation and exhaustive characterization of square pyramidal aryl-Cu(III) and aryl-Ag(III) complexes in the context of 2e redox M(I)/M(III) cross coupling catalysis. 10 Hence, using this approach we were able to describe the frst oxidant-free gold(I)-catalyzed halide exchange and C sp 2 -O bond forming reactions (Scheme 1c), additionally transferring this novel chemistry to more easily available substrates such as 2-(2-halophenyl)pyridines. 11 In this work we present the expansion of the nucleophile scope from halides and phenols to amines and amides, which stands as the frst example of a gold(I)-catalyzed C-N bond formation, resembling the well-known Cu-based Ullmanntype 12 or Pd-based Buchwald-Hartwig cross-coupling catalysis. 13 Furthermore, the straightforward synthesis and crystallographic characterization of the hitherto new (N,C)-cyclometalated Au(III) complexes 3a and 3b via the oxidative addition of a C sp 2 -I bond is also herein described. Their competency as intermediate species in catalytic C-O and C-N couplings is also demonstrated, therefore confrming the redox Au(I)/Au(III) mechanistic cycle previously postulated. ## Results and discussion In the course of our investigations into the oxidant-free gold(I)catalyzed halogen exchange we found that the applicability of the system could be extended to C-O coupling reactions using sodium p-chlorophenolate and, interestingly, sodium methoxide. The latter required its conjugate acid (MeOH) as solvent to proceed, presumably due to the low solubility of the salt in CH 3 CN. This prompted us to study the related transformations with other alkoxides and protic solvents. In a similar manner, EtONa was reacted with 2-(2-bromophenyl)pyridine 1a-I in ethanol (110 C, [Au(NCMe)IPr]SbF 6 as catalyst) and after 24 h a moderate 56% yield of the desired coupling product 1ac was obtained, which could be increased up to 78% after 48 h (Table 1, entry 6). This can be attributed both to the lower acidity of EtOH compared to MeOH and the larger steric hindrance of the ethoxide. The preference for smaller and more acidic alkoxides was confrmed by carrying out the reaction with MeONa in EtOH, whereby 1ab was the major product. This trend could be further extrapolated to more sterically demanding alkoxides such as 2-propoxide (1ad) and tert-butoxide (1ae) giving yields of 21% and 4%, respectively (Table 1, entries 7 and 8). The use of sodium hydroxide as a nucleophile (in H 2 O solvent) deserves a special mention. Although in terms of reactivity one might anticipate a similar behavior to sodium methoxide in MeOH (Table 1, entry 3) to readily provide 2-(pyridin-2-yl)phenol 1af, this was only formed in a 34% yield (Table 1, entry 9). On the contrary, the diaryl ether homocoupling product 1ag was the major product (26% yield). A simple explanation for this outcome involves the deprotonation in basic media of the early-stage generated phenol, which subsequently acts as the preferred nucleophile in the coupling reaction with 1a-Br starting material. It should be noted that this transformation using NaOH as a base was performed in aqueous media, in the absence of a phase-transfer reagent and without the addition of a co-solvent. In an effort to gain a better understanding of this reactivity, we carried out the same reaction using NaOMe instead of NaOH, and the yield of phenol 1af increased up to 52% (Table 1, entry 10). These results suggest that control over the selectivity and yield of each product might be achieved by the appropriate modifcation of the reaction conditions. Notably, the combination of sodium hydroxide and methanol led to the complete formation of the methoxy insertion product 1ab (Table 1, entry 2). Overall, we have shown that steric and pH effects play a crucial role in the Au(I)-catalyzed ether and phenol formation, with smaller and more basic alkoxides leading to better results. This methodology allows for the synthesis of 2-(pyridin-2-yl) phenol 1af in water and 2-(2-methoxyphenyl)pyridine 1ab and 2-(2-ethoxyphenyl)pyridine 1ac in moderate to excellent yields, and represents the frst example of the Au-catalyzed crosscoupling of aliphatic alcohols to aryl halides. Moreover, gold promotes the coupling of water to form phenols, and the coupling of linear aliphatic alcohols to form ethers, thus presenting a complementary methodology to the Cu-based C-O cross-couplings for these challenging nucleophiles. 14 In light of these promising results, we then sought to expand the Au-catalyzed C-heteroatom reaction scope towards C sp 2 -N bond formation through the combination of aryl halides and N-nucleophiles, typically relying on two different approaches: (a) Cu-catalyzed Ullmann-type and (b) Pd-catalyzed Buchwald-Hartwig coupling reactions. As far as we know, this reactivity has only been successfully transferred to nickel. 15 We initially selected p-nitroaniline, given its major acidity compared to other amines (pK a ¼ 20.9 in DMSO), as the nucleophile to be reacted with 1a-Br in the presence of 10 mol% [Au(NCMe)-IPr](SbF 6 ) at 110 C (Table S1 †). Regardless of the base used, in all cases the starting material was recovered. Thus, afterwards, optimization of the solvent was investigated, whereby we obtained a signifcant and encouraging 39% yield of the desired product in DMSO and with KOt-Bu as a base. The same conditions employing the more reactive 1a-I substrate resulted in almost quantitative yields. The fnal optimization set the preferred experimental conditions at 110 C, 24 h and an excess of the nucleophile and 3 equivalents of base for further substrate scoping. It is worth pointing out the high-performance of KOt-Bu as a base without competing with p-nitroaniline as the nucleophile in the coupling reaction, as foreseen from the C-O bond forming results (Table 1, entry 8). Detailed NMR and X-ray crystallography analysis of the isolated product confrmed the expected structure. Remarkably, a gold-free blank experiment was performed and the reaction did not proceed (0% yield). The sluggishness of the reaction when 1a-Br is used instead of 1a-I is in good agreement with a rate-limiting oxidative addition step. Then the scope of the optimized protocol was examined using different amines and amides. Cyclic aromatic and aliphatic amines and amides (imidazole and 2-hydroxypyridine, Scheme 2, products 1am and 1ai) afforded the best results, most likely owing to their superior acidity (pK a ¼ 18.6 and 17.0 in DMSO, respectively). 16 Good outcomes were obtained for primary aliphatic amines (Scheme 2, products 1ak and 1al), while secondary amines were less prone to arylation, with moderate and low yields for piperidine (1ap) and diethylamine (1aq), respectively. Likewise, benzamide was arylated in a moderate yield (59%, 1aj). Essentially, acidity and steric hindrance seem to be the basis of the observed reactivity trend for the aryl iodide 1a-I. We then turned our attention to the electronic properties of the para-substituents on the aniline ring. In contrast to the excellent results provided by p-nitroaniline, aniline did not exceed 50% yield, while p-methoxyaniline was poorly arylated (17%, 1ao). This observation, together with the data collected for the other N-nucleophiles, indicates sensitivity to less acidic and sterically hindered substrates, which is translated into a decrease in yields. In this system, the Au-based methodology for the arylation of aliphatic amines is superior to Cu-based Ullmann-type couplings. 14 C-N cyclometalated gold(III) complexes have been the subject of deep investigation, ever since the frst report of neutral AuCl 2 (ppy) containing a 2-phenylpyridine-type ligand by Constable and co-workers. 17 This family of complexes has shown potential anticancer activity 18 and photophysical properties, 19 and their use has benefted from their tolerance to both air and water. The methods to prepare them require either transmetallation from toxic organomercury derivatives or formal C-H auration with gold(III) tri or tetrahalide salts. 20 Furthermore, an alternative approach, employing aryldiazonium salts and Au(I) complexes under visible light photoredox conditions, has been developed very recently. 4c Nevertheless, since their synthesis had not been previously realized via the oxidative addition of C sp 2-X bonds, we deemed it worthy to investigate this possibility using the 2-(2-halophenyl)pyridine substrates. Inspired by the work of Bourissou and co-workers, 7 we frst reacted 2-(2-iodophenyl)pyridine 1a-I and AuI in dichloromethane at room temperature. However, the complete recovery of the starting materials was observed, even after changing the solvent to toluene and o-xylene and heating up to 130 C. A cationic [Au(NCCH 3 )IPr]SbF 6 gold(I) source gave identical results, regardless of the reaction conditions. This observation suggests the equilibrium displacement towards more stable reagents (see the ESI, † Section 1.6, for details). To tackle the aforementioned difficulties, we envisaged to block the rotation of the pyridine chelating group by incorporating an extra ring into the substrate, aiming at enhancing the stability of the desired product. Consequently, 10-iodobenzo[h]quinolone 2a-I was prepared following a two-step procedure starting from benzo[h]quinolone (see the ESI †). Gratifyingly, oxidative addition of the C Ar -I bond proceeded readily with AuI at 60 C for 18 h to give the cyclometalated Au(III) complex 3a as a red powder (Scheme 3). Complex 3a withstands air and water indefnitely and is only soluble in CH 2 Cl 2 , CHCl 3 and DMSO. X-ray-quality crystals were obtained by gently stirring 2a-I and AuI in CH 2 Cl 2 at room temperature for 4 days, followed by slow evaporation of the solvent. The solid-state structure of the complex displays a Au-C bond length of 2.055( 7 With neutral 3a in hand, we next investigated the abstraction of iodide using 1 equivalent of AgSbF 6 in order to generate a more soluble and reactive cationic complex (Scheme 4). The reaction was carried out in acetonitrile due to its coordinating properties, but no changes were detected after vigorously stirring for 3 hours at room temperature. At this point we hypothesized that the medium-strength Lewis base character of acetonitrile cannot efficiently stabilize a three-coordinate gold(III) species, and consequently a stronger Lewis base was employed. Upon the addition of pyridine (1.1 equiv.), 3a was immediately consumed and the solution turned bright yellow Scheme 2 Substrate scope of the C-N bond forming reactions. with the formation of an abundant grey precipitate (AgI). Complex 3b was isolated as an orange powder after fltration and solvent removal (91% yield). 3b is also air-and moisturestable and can be stored on the benchtop without noticeable decomposition. The ESI-HRMS mass spectrum of this complex shows one major peak at m/z 580.9782 corresponding to the mass of [(2a)AuI(Py)] + , the cationic fragment of 3b. Lowtemperature 1 H-NMR (248 K) allowed the structural characterization of 3b in solution (see the ESI †). Crystals of this complex, grown from the slow diffusion of diethyl ether in a CH 3 CN solution of the compound, confrmed the removal of the iodide trans to the Au-C bond, as expected from the larger trans effect (Scheme 4). In our previous studies on gold(I)-catalyzed halogen exchange and C-O bond formation we proposed a mechanism operating through the general two-electron-based Au(I)/Au(III) cycle (Scheme 6), 11 albeit the high-valent [Au(2a)(IPr)X] (X ¼ halide or phenolate) species could not be detected. In this regard we reasoned that the cationic Au(III) complex 3b might be helpful in trying to unveil the involvement of aryl-Au(III) species in this oxidant-free transformation. First, 1 equivalent of complex 3b was subjected to the same conditions as used for the p-chlorophenolate insertion with 2-(2-bromophenyl)pyridine 1a-I and the desired 10-(4-chlorophenoxy)benzo[h]quinoline 2aa was obtained in 86% yield (Scheme 5a), validating the feasibility of aryl-Au(III) species in Au(I)-catalyzed cross couplings. Nonetheless, an analogous experiment starting from 10-bromobenzo[h]quinolone 2a-Br and catalytic amounts of 3b (10 mol%) provided almost 2 catalytic turnovers (16% yield of product 2aa, Scheme 5b). Therefore, 10 mol% of the N-heterocyclic carbene donor ligand IPr$ (IPr ¼ 1,3-bis(diisopropylphenyl)imidazol-2-ylidene) employed in the catalytic transformations was also added, and we were pleased to fnd that virtually quantitative yields of 2aa were achieved (Scheme 5b). Equal behavior was found with the p-nitroaniline insertion to 2a-I (Scheme 5c), overall unequivocally substantiating the implication of aryl-Au(III) species as competent catalytic intermediates. 3b rapidly generates the neutral complex [Au(2a)(Nuc)X] (X ¼ Br, I) in the presence of a nucleophile (Scheme 6). Then, the IPr$ carbene exchanges one of the anionic ligands to form the less favored [Au(2a)(IPr)Nuc] intermediate, which rapidly reductively eliminates the crosscoupling product (2ax) and gold(I) as [Au(X)IPr]. 11 Strikingly, 1 H-NMR inspection of the crude product proved that all the Au(I) remained in solution in this resting state form. On the other hand, attempts to isolate the [Au(2a)(IPr)Nuc] species generated in solution when IPr$ is added were fruitless, supporting their short-lived nature in the catalytic cycle. ## Conclusions In conclusion, we have developed the frst examples of gold(I)catalyzed C-N cross-coupling reactions in the absence of sacrifcial oxidants, in parallel to the well-established Cu-and Pd-catalyzed methodologies, and extended the previously described C-O coupling catalysis with phenols to aliphatic alcohols and water. This system allows entry to different arylamine, arylamide, phenol and aryl-ether products under practical synthetic laboratory conditions, with absolute tolerance for both air and water. In either case, the acidity of the nucleophile is at the basis of the reactivity observed. Moreover, we have synthesized novel neutral and cationic C-N cyclometalated Au(III) complexes through mild oxidative addition of a C sp 2 -I bond to gold(I) iodide, and presented conclusive evidence of their competence in the C-O and C-N coupling transformations. To the best of our knowledge, this represents the frst example in which the intermediacy of Au(III) species in an oxidant-free 2-electron coupling processes is demonstrated, clarifying the proposed mechanism operating via oxidative addition and reductive elimination steps. Future work is directed towards investigating other suitable chelating groups for a more versatile system, with special interest in removable directing groups.

chemsum

{"title": "Au(<scp>iii</scp>)-aryl intermediates in oxidant-free C\u2013N and C\u2013O cross-coupling catalysis", "journal": "Royal Society of Chemistry (RSC)"}

speeding_up_biomolecular_interactions_by_molecular_sledding

## Abstract: Numerous biological processes involve association of a protein with its binding partner, an event that is preceded by a diffusion-mediated search bringing the two partners together. Often hindered by crowding in biologically relevant environments, three-dimensional diffusion can be slow and result in long bimolecular association times. Similarly, the initial association step between two binding partners often represents a rate-limiting step in biotechnologically relevant reactions. We demonstrate the practical use of an 11-a.a. DNA-interacting peptide derived from adenovirus to reduce the dimensionality of diffusional search processes and speed up associations between biological macromolecules. We functionalize binding partners with the peptide and demonstrate that the ability of the peptide to onedimensionally diffuse along DNA results in a 20-fold reduction in reaction time. We also show that modifying PCR primers with the peptide sled enables significant acceleration of standard PCR reactions. ## Introduction The crowded intracellular environment presents many challenges for basic molecular processes to occur. Non-specifc interactions between proteins hinder diffusional mobility and increase the time needed for binding partners to fnd each other and associate. 1 Nature displays several examples in which the dimensionality of search processes is reduced to speed up association times. 2 For example, binding partners of certain classes of cell-surface receptors associate with lipid membranes and utilise two-dimensional diffusion to promote association. 3 Many DNA-interacting proteins fnd specifc sequences or lesions in large amounts of nonspecifc DNA by performing onedimensional random walks along the DNA. 4 Every time such a protein associates with DNA, it transiently diffuses along the duplex and thus drastically increases the number of sampled DNA positions per unit of time. It then dissociates from the DNA, undergoes three-dimensional (3D) diffusion through solution to rebind at an entirely different region and again searches a stretch by one-dimensional (1D) diffusion. The combination of 3D and 1D searches gives rise to a drastic increase in the effective bimolecular association rate constant of the protein with its target. 5,6 An example of a naturally occurring system in which 1D diffusion along DNA is used to speed up association between two proteins is found in adenovirus. 7,8 During viral maturation, a large number of proteins within a single viral particle need to be proteolytically processed by the adenovirus protease (AVP) before infection of a cell. 9 Tight packing of protein and DNA within the viral particle makes regular 3D diffusion as a mechanism for the protease to travel from one target to the other impossible. Recent work has shown that the AVP protein 10 recruits the short 11-a.a. pVIc peptide (GVQSLKRRRCF), 11 itself a proteolytic product in early maturation, and uses it to slide along the DNA inside the viral particle and thus effectively reduces the search space for the protease from three dimensions to one. 8 ## Results and discussion In this work we demonstrate that the ability of the pVIc peptide to slide along DNA can be used to speed up a much broader class of biomolecular processes than just those occurring in vivo and that it can be used to dramatically improve the speed of common laboratory reactions (Fig. 1). First, as a proof of principle, we couple each of the two binding partners in a canonical biotin-streptavidin association to the pVIc 'molecular sled' and show that association proceeds more than an order of magnitude faster in the presence of DNA (Fig. 2a). Fluorescence Resonance Energy Transfer (FRET) 12 was used to monitor the time dependence of the bimolecular association. For simplicity, we refer to the functionalised biotin and streptavidin as binding partners B and S, respectively. Binding partner B is formed by reacting a maleimide-functionalised biotin with the cysteine Cys10 of Cy3-labelled pVIc in a Michael-addition reaction (see ESI, Fig. S1 †). The maleimide and biotin units are connected via a high-molecular weight polyethylene glycol (PEG) linker resulting in a total molecular weight for binding partner B of 6.7 kDa. This high molecular weight reduces its diffusional mobility and allows us to more easily gain access to the timescale of association. Binding partner S is prepared by forming a complex between a Cy5-labelled tetrameric streptavidin and an unlabelled biotin-pVIc conjugate (see ESI †). The ability of both B and S to 1D diffuse along DNA was confrmed on a single-molecule level using Total Internal Reflection Fluorescence (TIRF) microscopy (Fig. 2b, see ESI † for experimental conditions and notes). We estimated the binding times s 1D y 0.3 s and the 1D diffusion coefficient D 1D y 3 10 4 nm 2 s 1 . Using these values, we can calculate that S and B are able to explore a DNA segment of length L 1D ¼ ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 2D 1D s 1D p y130 nmy400 bp before dissociating and returning to solution. Binding partners B and S were combined in aqueous solution at fnal concentrations of 150 nM and 37.5 nM, respectively, and ensemble FRET between the Cy3 donor and Cy5 acceptor fluorophores was measured (Fig. S2 †). Fig. 2c shows the time dependencies of bimolecular association in the presence of 2686 bp long double-stranded DNA (dsDNA) at different concentrations. Addition of the DNA up to 1 pM did not have a signifcant effect on the reaction rate, whereas DNA concentrations of higher than 10 pM resulted in a clearly discernable reduction of the reaction time. For a DNA concentration of 300 pM, already after 15 s, 99% of the maximum FRET efficiency was achieved. Fig. 3a shows the reaction times as derived from the FRET traces for different DNA concentrations and lengths. For each length, varying from 2686 to 15 base pairs (Table S1 †), the association times decrease by up to 20-fold at higher concentrations of added DNA. Interestingly, the critical concentration for reaction speed up differs for the different DNA lengths: longer DNA fragments are required at lower concentrations than short DNA molecules to achieve the same catalytic effect. This behaviour can be explained by the fact that the critical number of reaction partners associated with DNA is reached at higher DNA concentrations for short fragments and at low DNA concentrations for longer pieces of DNA. Thus, the main parameter that governs the kinetics of reaction is the total base pair concentration, a unit that describes the total length of DNA per unit volume. This notion is validated by plotting the reaction time against DNA base pair concentration (Fig. 3b), showing the curves cluster together in three distinct regions. These regimes can be understood in terms of the density of binding partners trapped on the catalytic DNA molecules. At low base pair concentrations, the amount of DNA available per binding partner is too low to trap a noticeable fraction of the binding partners and influence the overall reaction rate. In the optimal regime, around 0.1 to 10 mM of base pairs, the binding partners have high probability to be trapped by DNA where they can fnd each other by 1D diffusion. At base pair concentrations higher than 100 mM, the probability for binding partners B and S to bind to the same DNA molecule diminishes, resulting in a deceleration of the association. In an alternative mechanism to explain the increased association rates, DNA-bound binding partners bound to the same DNA molecule could be brought into proximity of each other by bending and looping of the DNA duplex. In such a mechanism, the binding partners would rely on the conformational flexibility of the DNA and use the duplex as a scaffold to bring binding partners together. In order to exclude this pathway, we conducted a series of experiments with DNA of four different lengths (50, 100, 150 and 300 bp), which were chosen such that the corresponding DNA looping probability differed signifcantly from one another. 13,14 Under the low-salt buffer conditions used in this study, DNA molecules of 50 and 100 bp can be regarded as stiff rods whose folding onto itself is excluded (DNA persistence length is estimated to be 250 bp at 2 mM NaCl). 15 In case looping was the main mechanism for reaction speed-up one would expect a considerably lower reaction acceleration in case of 50 and 100 bp long DNA as compared to 300 bp, which is long enough to form loops. However, in all cases we observed the same 20-fold reaction speed-up (Fig. 3a and b), confrming that association is not mediated by DNA bending onto itself. Using a similar reasoning, one could argue that the conformational flexibility of the long PEG linkers attached to both binding partners allows those binding partners that are statically but distally bound to the same DNA to associate without the need for sliding. Fig. 3b shows, however, a 10-fold increase in reaction time at a reaction stoichiometry as low as 1 binding partner per 1000 base pairs, clearly an average molecular separation too high to be bridged by the binding partners statically bound to the same DNA. From the considerations above, one can conclude that sliding along DNA, and not just static binding, is responsible for the increase in association rate. An understanding of the origin of the reaction acceleration effect can be obtained from our recent work in which we formulated a kinetic model for a system with linear sinks (i.e. DNA) that can intermittently trap molecules present in a solution and serve as an assembly line for 1D diffusing molecules. 16 Our model semiquantitatively predicts the experimentally observed speed-up in the presence of DNA molecules of different lengths and concentrations. Moreover, according to our simulations, the relative contribution of the 1D reaction pathway in the optimum speed-up regime can be as high as 90%. In this work, we concluded that although association of the binding partners on DNA without 1D sliding does play a role, the primary contributor to the reaction acceleration is a 1D sliding mechanism. This model also shows that in the case of extremely short DNA molecules (15 bp and 50 bp), the reaction acceleration cannot be explained by 1D sliding alone due to the sizes of the binding partners being comparable to the dimensions of the DNA. Instead, reaction acceleration is introduced by the high diffusional mobility of the short DNA duplexes and their ability to electrostatically capture the cationic peptides. 16 As a next step, we set out to use our method to speed up a standard polymerase chain reaction (PCR) by reducing the time needed for pVIc-coupled primers to anneal to the template DNA. The exponential amplifcation of DNA during PCR can be divided into three distinct steps. 17 The frst step is the melting of the double-stranded DNA template (Fig. 4a), followed by primer annealing and elongation with the polymerase. During the annealing step, primers need to fnd and hybridise to their complementary target sequence on a template. During this annealing step, the DNA will consist of a mixture of denatured and double-stranded regions, providing a large variety of structures for the pVIc-primers to interact with and potentially move along, resulting in a reduction of the time needed for the primer to locate and bind to its target sequence. We covalently coupled (see ESI †) the pVIc peptide to the 5 0 ends of a pair of PCR primers (primer set I, Table S2, Fig. S3 and S4 †) designed to amplify a 807 bp stretch from a linear doublestranded 1970 bp-long template and used real-time PCR (qPCR) experiments with SYBR Green I fluorescence to report on the kinetics of amplicon formation 18 (Fig. 4b and S5 †). The correct length of the PCR product was confrmed by agarose gel electrophoresis (Fig. S6 †). Similar results were obtained for PCR experiments employing a different pair of primers (Table S2 and Fig. S7 †) and a longer 8669 bp-long circular template M13KO7 (Fig. S8 †). The kinetics of amplicon formation were quantifed in an unbiased manner by employing a PCR threshold cycle analysis (see ESI, Fig. S9 †). Remarkably, the PCR reaction containing the pVIc-conjugated primers displayed a signifcant reduction in the number of cycles needed, suggesting the use of a molecular sled as a viable approach to speed up the overall reaction time of PCR. In our experiments we were able to shorten the PCR reaction time by 15-27%. To ensure that the increase in speed is not caused by a nonspecifc electrostatic association between the four positively charged amino acids in the sliding peptide and the negatively charged DNA backbone, we repeated the same PCR experiments using primers conjugated to a scrambled peptide (S-peptide, SFRRCGLRQVK) containing the same residues in a random order, which presumably affects the sliding behaviour of the peptide yet preserving the net charge. Our qPCR data reveals that use of primers conjugated with this scrambled peptide does not result in a decrease of the number of PCR cycles required for amplifcation (Fig. 4b, 'S-peptide'). The performance of the S-peptide-modifed primers is very similar to the unmodifed primers. Furthermore, we observed a signifcant reduction in the number of PCR cycles when using a truncated pVIc variant containing only the last six amino acids of pVIc, four of which are positively charged and are sufficient to support sliding along DNA (Fig. 4b, K-peptide, KRRRCF, Fig. S10 †). Finally, we studied the behaviour of the primer modifcations under different conditions by varying the annealing time t A and primer concentration C primer (Fig. S8 †). In case of the most stringent conditions (short annealing time, low primer concentration) the effect of the sliding peptides was the most pronounced. To exclude a scenario in which the acceleration effect could originate from the enhanced primer-template binding due to cationic nature of the peptides, we compared the melting temperatures T m of the modifed and unmodifed primers that were used in the PCR experiments. When using short complementary oligonucleotides, and thus excluding sliding contributing to affinity, the measured T m values of the peptidefunctionalised primers were identical to those of the nonfunctionalised ones (Fig. S11 †). This observation excludes an enhanced stability of binding to DNA in the PCR reactions because of the peptide. The use of chimeric molecules, where the desired functions of parent moieties are combined within one molecule is a wellestablished approach in biotechnology. In PCR, for example, attempts have been made to increase the affinity of primers and polymerases to DNA by functionalising primers with DNAintercalating molecules 19,20 and expressing the polymerases with an additional cationic peptide motif in the sequence. The enhancement of molecular activity in these cases arises from the increase of the attractive electrostatic and intermolecular forces between the desired molecule and DNA. Another approach that uses the same concept of chimeric molecules is DNA-templated synthesis, where the binding partners are conjugated to single-stranded DNA oligonucleotides and are physically brought into proximity of one another by hybridising them to a DNA template. In our study, however, the mechanism of activity enhancement is different from these approaches: as opposed to increasing the affinity between the binding partners by prolonging the dissociation time, we aimed to speed up association by addition of a different reaction pathway -1D diffusion along DNA. The reduction of search dimensionality makes the binding partners fnd each other faster and, thus, results in the overall reaction acceleration.

chemsum

{"title": "Speeding up biomolecular interactions by molecular sledding", "journal": "Royal Society of Chemistry (RSC)"}

semiconductor-driven_“turn-off”_surface-enhanced_raman_scattering_spectroscopy:_application_in_selec

## Abstract: Semiconductor materials have been successfully used as surface-enhanced Raman scattering (SERS)-active substrates, providing SERS technology with a high flexibility for application in a diverse range of fields. Here, we employ a dye-sensitized semiconductor system combined with semiconductor-enhanced Raman spectroscopy to detect metal ions, using an approach based on the "turn-off" SERS strategy that takes advantage of the intrinsic capacity of the semiconductor to catalyze the degradation of a Raman probe.Alizarin red S (ARS)-sensitized colloidal TiO 2 nanoparticles (NPs) were selected as an example to show how semiconductor-enhanced Raman spectroscopy enables the determination of Cr(VI) in water. Firstly, we explored the SERS mechanism of ARS-TiO 2 complexes and found that the strong electronic coupling between ARS and colloidal TiO 2 NPs gives rise to the formation of a ligand-to-metal chargetransfer (LMCT) transition, providing a new electronic transition pathway for the Raman process.Secondly, colloidal TiO 2 nanoparticles were used as active sites to induce the self-degradation of the Raman probe adsorbed on their surfaces in the presence of Cr(VI). Our data demonstrate the potential of ARS-TiO 2 complexes as a SERS-active sensing platform for Cr(VI) in an aqueous solution. Remarkably, the method proposed in this contribution is relatively simple, without requiring complex pretreatment and complicated instruments, but provides high sensitivity and excellent selectivity in a high-throughput fashion. Finally, the ARS-TiO 2 complexes are successfully applied to the detection of Cr(VI) in environmental samples. Thus, the present work provides a facile method for the detection of Cr(VI) in aqueous solutions and a viable application for semiconductor-enhanced Raman spectroscopy based on the chemical enhancement they contribute. ## Introduction In recent years, an increasing interest in the studies of surfaceenhanced Raman scattering (SERS) on semiconducting materials (that is semiconductor-enhanced Raman spectroscopy) has emerged owing to its potential application in biological and photoelectronic analyses. Several semiconductor materials including TiO 2 , ZnO, graphene, Si, and Ge have been developed as SERS substrates. However, the application of these materials in SERS-based quantitative measurements is still matter of debate, because of their specifc dependence on molecular electronic structure and relatively weak enhancement of SERS signals. Most of the improvement in semiconductor-enhanced Raman spectroscopy is mainly induced by a charge-transfer process, leading to an enhancement of approximately 10 2 to 10 4 , because the surface plasmon resonance of semiconductor NPs typically lies in the infrared region, which does not usually coincide with the optical laser frequency. To date, studies have largely focused on the discovery and interpretation of the SERS phenomena with different semiconductor materials. This represents the most signifcant bottleneck in the application of such a technique for practical analysis and detection. The advantage of semiconductor-enhanced Raman spectroscopy is the performance of the semiconductor, which possesses controllable photoelectric properties, good biocompatibility, and environmental stability. In order to exploit these advantages, metal-semiconductor composites were introduced into SERS-based assays with some ingenious designs. However, the complicated process needed to prepare such composites has strongly limited their application. In addition, semiconductor materials do not respond to the Raman enhancement in these systems. Considering the photocatalytic properties of semiconductors, quantitative analysis by semiconductor-enhanced Raman spectroscopy would be achieved through the detection of signal degradation of a labeled probe on a semiconductor. Here, we report that semiconductor-enhanced Raman spectroscopy can also be developed into a sensing platform for the detection of metal ions, even without the assistance of a noble metal. In particular, we present a novel "turn-off" SERS strategy and demonstrate its use in a SERS-based assay for the determination of Cr(VI) in water. Cr has been extensively used in various industrial processes and has become one of the major environmental hazards. 20 The toxicological and biological properties of Cr are entirely dependent on its electric charge. 21 For instance, Cr(VI) is highly toxic; it generally exists as an oxyanion (CrO 4 2 ) in aqueous systems, and is known to be a strong carcinogen. 22 In contrast, Cr(III) is relatively non-toxic and is regarded as an essential trace element associated with the metabolism of carbohydrates and lipids. 23 Therefore, the reduction of Cr(VI) to Cr(III) is a key process for the detoxifcation of Cr(VI)-contaminated water and wastewater. In drinking water, the Maximum Contaminant Level (MCL) for Cr(VI) has been identifed as 1 mM. However, because no efficient testing method is available for only Cr(VI), the estimated MCL by the World Health Organization (WHO) includes the total amount of Cr. 24 Evidently, this defnition is not conducive to encouraging the intake of Cr(III) from a daily diet and has pushed up the cost of industrial wastewater treatment. So far, several methods, including atomic spectrometric, luminescent, 28,29 electrometric, colorimetric, 34 and X-ray fluorometric techniques, 35 have been developed for the selective determination of Cr(VI). Nevertheless, none of these techniques exhibited the desired sensitivity together with easy manipulation. Herein, charge-transfer complexes, alizarin red S (ARS)sensitized colloidal TiO 2 NPs, with a facile synthetic route are used to demonstrate how semiconductor-enhanced Raman spectroscopy enables the determination of Cr(VI) in water. We explored the SERS mechanism of ARS-TiO 2 complexes and found that the molecular polarizability tensor can be enhanced by a ligand-to-metal charge-transfer (LMCT) transition. Interestingly, the SERS intensities of the ARS-TiO 2 complexes have been found to be sensitive to the Cr(VI) concentration due to co-catalysis, indicating their potential for use in the determination of Cr(VI). Several influencing factors such as response time, laser power, pH of the sensing system, and the loading amount of ARS on the colloidal TiO 2 NPs were taken into account to optimize the determination conditions. Our experimental results revealed that the ARS-TiO 2 complexes exhibit high sensitivity and selectivity toward Cr(VI). The practicality of this proposed method was further validated through the detection of Cr(VI) in real water samples. The method proposed here can be used for the determination of Cr(VI) in aqueous solutions for the accurate assessment of pollution levels. Thus, this work provides a clear proof of concept for extending the applications of semiconductorenhanced Raman spectroscopy. ## Materials Alizarin red S and titanium(IV) butoxide were acquired from Sigma-Aldrich Co. Ltd. and used without further purifcation. All other chemicals, obtained from Wako Co. Ltd, were analytical grade and employed without further purifcation. Ultrapure water (18 MU cm) was used throughout the study. The tap water and pond water were collected from the Gakuen district of Sanda and a pond near Kwansei Gakuin University, respectively. All the water samples were fltered through 0.2 mm membranes prior to use. ## Preparation of colloidal TiO 2 nanoparticles The colloidal TiO 2 NPs were synthesized according to a method described in previous reports. 36,37 Briefly, a solution of titanium(IV) butoxide (5 mL) dissolved in 2-propanol (95 mL) was added dropwise (1 mL min 1 ) to an aqueous HNO 3 solution (500 mL, pH 1.5) maintained at 1 C. The solution was continuously stirred for 10-12 hours until a transparent colloid was formed. ## SERS measurement A stock solution of ARS (0.1 M) was prepared in water. ARS solutions with various concentrations were obtained by serial dilution of the stock solution with sodium acetate buffer solution (0.01 M, pH 3.0). The ARS solutions with different concentrations were mixed with the colloidal TiO 2 NPs at the same volume and shaken thoroughly. For the detection of metal ions, 10 mL of each sample mixed with 10 mL of ARS-TiO 2 was dripped into an aluminum pan (0219-0062, Perkin-Elmer), and the mixture was exposed to a laser beam for 30 s before each SERS measurement. The typical exposure time for each Raman/ SERS measurement in this study was 30 s with two accumulations. The error bars represent standard deviations based on three independent measurements. ## Instrument The image of the sample was measured on a Tecnai G2 transmission electron microscope operating at 200 kV. The UV-vis spectra were recorded on a Shimadzu UV-3600 spectrophotometer. A RS-2100 Raman spectrophotometer (Photon Design, Inc.) equipped with a CCD (Princeton Instruments) was used. Radiation with a wavelength of 514.5 nm from an Ar ion laser (Spectra Physics) was employed for the Raman excitation, with a power of 5 mW at the sample. The Raman band of a silicon wafer at 520.7 cm 1 was used to calibrate the spectrometer. ## Synthesis and characterization Colloidal TiO 2 NPs with an average diameter of 3 nm were prepared by a low-temperature acid hydrolysis route, as described previously (see Fig. 1). 36,37 The absorption spectra of the colloidal TiO 2 NPs before and after modifcation with ARS are shown in Fig. 1b, together with that of ARS for the sake of comparison. In contrast to ARS, the ARS-TiO 2 complexes exhibit a more intense absorption band in the longer wavelength region with a peak maximum centered at 489 nm. This absorption band has been assigned to the LMCT transition, which arises from the strong electronic coupling between ARS and the colloidal TiO 2 NPs. 38 Based on the Benesi-Hildebrand analysis for ARS-TiO 2 complexes (Fig. S1 †), the association constant (K ass ) of the complex was determined to be 3.9 10 3 M 1 , which indicates the relatively strong binding of ARS on the surface of the TiO 2 NPs. An ARS molecule contains many functional groups; the FTIR data of the ARS-TiO 2 complexes unambiguously shows that the mode of grafting is bidentate chelation, which involves two hydroxyl groups (Fig. S2 †). These observations suggest that the ARS-TiO 2 composite material can be used for the development of a semiconductor-supported SERS sensing platform due to the clear charge-transfer transition process. Mechanism for SERS of the ARS-TiO 2 system Fig. 2a compares a Raman spectrum of 0.1 M ARS in aqueous solution and a SERS spectrum of ARS-TiO 2 complexes with 514.5 nm excitation. The vibrational mode assignments listed in Table S1 † are based primarily on earlier IR and Raman studies of related alizarin dyes. The SERS spectrum is characterized by a signifcant enhancement in the 1200-1500 cm 1 region, where the bands are typically assigned to the C]C and C-O-R stretching modes. This observation confrms the presence of strong coupling between the electronic transitions and the C]C/C-O-R bond stretching modes in ARS ligands, which is consistent with the conclusions obtained from the absorption and IR spectra of the ARS-TiO 2 complexes. Such coupling has also been observed on other semiconductor NPs in colloidal suspensions such as CeO 2 , Fe 2 O 3 , ZrO 2 , etc. 43 Furthermore, the concentration-dependent SERS experiments displayed in Fig. 2c clearly demonstrate that a concentration as low as 5 10 7 M ARS can be detected. 44 The intensities of the Raman signals can be ftted with the BET model well and represent a saturation effect (Fig. 2d). In addition, a linear correlation was found between the intensity at 1260 cm 1 and the ARS concentration in the range of 5 10 7 to 2 10 4 M. Of note is that the enhancement arises from the strong coupling interaction between the dye molecules and the colloidal TiO 2 NPs and, more importantly, the formation of charge-transfer complexes opens up a new electronic transition pathway for the Raman process. 45,46 In this case, the groundstate electrons of the ARS-TiO 2 complexes are initially excited from the highest occupied molecular orbital (HOMO) level to the conduction band (CB) of the TiO 2 NPs by the incident light (Fig. 2b). Then, the excited electrons immediately transfer back to the vibrational energy level of the ARS molecule and subsequently release a Raman photon with the ARS molecule at some vibrational state. The molecular polarizability tensor can be enhanced by such a charge-transfer process due to the vibronic coupling of the conduction band states of the semiconductor with the excited states of the probe molecule through a Herzberg-Teller coupling term. 47 Therefore, unlike in resonance Raman spectroscopy, where the molecule itself should reach a resonant state with excitation by the incident light, the enhancement can be considered in this case as a SERS phenomenon, which arises from the chemical enhancement mechanism via the Herzberg-Teller contribution. 2,45,48 Mechanism for responding to Cr(VI) In general, organic ligands are susceptible to decomposition on bulk TiO 2 , owing to their adsorption through physisorption or weak chemisorption. Compared with bulk TiO 2 , colloidal TiO 2 NPs possess abundant under-coordinated Ti defect sites, which provide plenty of coordination sites for ARS to bind via bidentate chelation. This chelation mode is favorable to the recombination between ARS + and an electron, thereby stabilizing the ARS molecule. Consequently, ARS adsorbed on the surface of colloidal TiO 2 NPs cannot be easily oxidized by visible light irradiation and preserves the SERS properties even after exposure to high laser power (Fig. S4 †). However, the SERS intensities are decreased with the addition of Cr(VI), indicating the decomposition of ARS adsorbed on the colloidal TiO 2 NP surfaces (Fig. 3 and S5 †). As shown in Fig. 4 and S6, † the ARS-TiO 2 complexes exhibit a remarkably high selectivity and lower interference in the determination of Cr(VI), particularly in the presence of Cr(III). This specifcity originates from the favorable redox potential of the couple Cr(VI)/Cr(V) (+0.55 V) for the reduction promoted by those electrons trapped in inter-bandgap states, together with the strong interaction between Cr(VI) and Ti(IV) atoms with unflled valence orbitals at the TiO 2 surface. This reduction can result in the formation of Cr(V) and decomposition of ARS-TiO 2 complexes (Fig. S5 †), thus leading to a decrease in the SERS intensities. The redox potential of the Fe(III)/Fe(II) couple (+0.77) is close to that of the Cr(VI)/Cr(V) couple. However, the relatively weak interaction between Fe(III) and the positively charged colloidal TiO 2 NPs only causes minor disturbance. To minimize the interference from Fe(III), 0.2 mM EDTA was added to the sodium acetate buffer as a masking agent. As expected, the interference from Fe(III) was found to be negligible in the presence of EDTA. Based on these results, it was inferred that the SERS intensities of ARS are sensitive to the Optimization of the sensing system Prior to the application of such a SERS sensing platform to the detection of Cr(VI), several influencing factors, such as response . Moreover, an aggregation-sedimentation phenomenon was observed at a pH larger than 5.0, providing us with a simple method to recycle the TiO 2 NPs from the analyte. Finally, the response mechanism was based on a co-catalysis scheme, in which both Cr(VI) and ARS are activated by the available Ti coordination sites on the surface of the colloidal TiO 2 NPs. Thus, the catalytic efficiency with different loading amounts of ARS on the colloidal TiO 2 NPs was also investigated to determine the optimum ARS-sensitized concentration. It was clearly found that increasing the loading amount of ARS results in an increase in the SERS intensity, but the best performance was obtained at 50 mM (Fig. S9 †). ## Application Based on the optimized conditions, the sensitivity and linearity of this sensing system were evaluated with different concentrations of Cr(VI) (Fig. 5). A good inverse proportionality was observed between the SERS intensity and the amount of Cr(VI) in the concentration range of 0.6-10 mM. The lowest concentration at which Cr(VI) could be detected is 0.6 mM. This concentration is lower than the maximum level of Cr(VI) in drinking water allowed by the WHO. In addition, the water samples spiked with different concentrations of Cr(VI) were also measured by employing our sensing system (Fig. 6). The measurements, which accurately reported the concentrations of the added standard Cr(VI) with good recoveries (Table S2 †), confrmed that the sensing system proposed in this work has great potential for the quantitative analysis of Cr(VI) in environmental samples. ## Conclusions In this work, we showed that semiconductor-enhanced Raman spectroscopy can be used as a sensing platform for the detection of metal ions. Firstly, the possibility of utilizing the dye-sensitized TiO 2 system to promote SERS is discussed. It is found that the strong coupling interaction between the dye molecules and the colloidal TiO 2 NPs leads to the formation of charge-transfer complexes and thus opens up a new electronic transition pathway for the charge-transfer process. The molecular polarizability tensor can be enhanced by such a charge-transfer process due to the vibronic coupling of the conduction band states of the semiconductor with the excited states of the probe molecule through a Herzberg-Teller coupling term. Secondly, a novel "turn-off" SERS strategy has been proposed and its use in a SERS-based assay for Cr(VI) has been demonstrated. Colloidal TiO 2 NPs can be employed not only as an effective substrate to elicit the SERS signals of an ARS molecule, but also as a catalytic center to induce the self-degradation of the ARS response to Cr(VI). The "turn off" SERS signal upon laser irradiation allows the development of a facile assay to measure Cr(VI). Of note is that this method does not require complex pretreatment and complicated instruments, but provides a high sensitivity in a high-throughput fashion and excellent selectivity toward Cr(VI) over other common anions. Furthermore, the experiments using solutions spiked with Cr(VI) revealed that our method is effective in monitoring the Cr(VI) in real water samples. Based on this "turn-off" SERS strategy, other metal ions can also be detected by utilizing different semiconductor enhancement systems in which the energy level of the semiconductor is matched with the redox potential of the determined metal ion. Thus, we believe that the data described in this contribution clearly demonstrate that semiconductor-enhanced Raman spectroscopy integrated with the catalysis of semiconductor materials can be used as a reliable detection method for metal ions in practical applications.

chemsum

{"title": "Semiconductor-driven \u201cturn-off\u201d surface-enhanced Raman scattering spectroscopy: application in selective determination of chromium(<scp>vi</scp>) in water", "journal": "Royal Society of Chemistry (RSC)"}

hierarchical_defect_engineering_for_licoo2_through_low-solubility_trace_element_doping

## Abstract: Cation doping is a widely utilized method for modifying LiCoO 2 cathodes in an effort to improve the energy density for Li-ion batteries. However, an in-depth understanding of the underlying mechanisms remains elusive. We quantitatively characterized and thoroughly analyzed the segregation of trace-doped Ti in the LiCoO 2 cathode to reveal the hierarchical structural defects, which promote cycling stability by suppressing the undesired phase transformation at a deeply charged state. ## INTRODUCTION The lithium-ion battery (LIB) is a groundbreaking invention that has enormous economic and social impacts. The operation of LIBs requires the cooperation of multiple battery components, involving thermodynamically uphill and downhill reactions during charging and discharging, respectively. Although there are ongoing research efforts looking into all of the LIB components, the cathode material is currently the most significant limiting factor for further improvement of the energy density, and it is a major research focus in this field. 4,5 The structural and chemical complexity in the composite cathode electrodes is indispensable for the desired functionality, and it therefore requires a delicate control for optimal device performance. While the porous carbon-binder domain (CBD) is responsible for providing continuous diffusion pathways for the transportation of Li ions and electrons, the active materials are ultimately the energy reservoir in which Li ions are stored and released upon electrochemical cycling, and this process is accompanied by redox reactions in the host material. Depending on the targeted applications, different active cathode materials could be chosen due to their respective pros and cons in different performance attributes. Although LCO has been utilized as the cathode material in LIBs for several decades, it is still very competitive and dominates the portable electronics market. Stabilization of LCO's layered lattice at a deeply delithiated state is a frontier challenge that has attracted worldwide research interest. This is because charging the LCO cathode to high voltage is of immediate and significant commercial incentives The Bigger Picture Cation doping has long been regarded as an effective method of modifying LiCoO 2 for withstanding a higher cutoff voltage. In addition, the microstructure and chemical complexity of the cathode active particles could critically affect the overall battery performance. The underlying fundamental mechanisms, however, are often rather complicated and unclear. Herein, we present a simple yet effective multiscale defectengineering approach via lowsolubility trace element doping and demonstrate improved cycling stability. Advanced synchrotron characterization techniques reveal the multiscale segregation of Ti in the LiCoO 2 particle, formulating a hierarchical structural complexity that involves modified particle surface, grain boundaries, and lattice distortion defects. These structural defects play an important role in stabilizing the LiCoO 2 structure at a highly charged state. Moreover, this hierarchical defectengineering strategy is broadly applicable to the other material systems. through increasing the energy density, one of the most critical battery-performance specifications. Upon deep charging, there is a clear tendency for the LCO particles to build up mechanical strain, 19,20 to release lattice oxygen, 21,22 and to undergo structure reconstruction. 11,23 These negative effects are intertwined over different time and length scales and are harmful to stable battery operation at high voltage. To make it most effective, a material modification procedure must mitigate these negative effects in a systematic manner. In our previous work, we demonstrate that trace co-doping of Al, Mg, and Ti (~0.1 wt %) can greatly enhance the cycling performance of LiCoO 2 at 4.6V and reveal that each dopant contributes through different mechanisms for such performance enhancement. 24 Although such a synergistic effect formulated by the three co-existing dopants leads to overall optimal performance, it complicates the system and makes it difficult to single out the fundamental role of each dopant. While both Mg and Al have reasonable solubility in the LCO lattice, the most profound complexity lies with Ti. On the one hand, the majority of Ti segregates at the surface and interface, forming a 3D network. On the other hand, residual Ti can be found inhomogeneously in the host LCO matrix. According to the X-ray diffraction (XRD) patterns and the corresponding refinement results reported in our previous work, Ti has a major role to play in the distortion of the host lattice compared with Al and Mg. Based on these considerations, we choose to focus on Ti in the current study. Besides, because of the chemical complexities induced by different dopants-particularly for Ti that is nonuniformly distributed within particles, including Co-free cathode materials 25 -their impacts on the lattice structure across the entire particle and thus the overall structural stability have yet to be demonstrated. 26,27 Herein, we employ cutting-edge synchrotron characterization techniques, e.g., nanoresolution X-ray microscopy with composition, valence, and lattice defect sensitivities, to elucidate the spontaneous and multiscale dopant segregation in the Ti/ Mg/Al co-doped LCO particles. Our results suggest that, in addition to modifying the LCO particles' bulk and surface and interface properties as a previous study revealed, the inhomogeneous incorporation of Ti into the LCO lattice leads to a significant degree of lattice deformation (e.g., bending, twisting, and d-spacing heterogeneity). All the preexisting hierarchical defects positively contribute to the robustness of the LCO lattice at a deeply charged state as evidenced by the suppression of the LCO's phase transformation (from the O 3 phase to the H 13 and O 1 phases) that occurs at above 4.5 V. We clarify here that, by hierarchical defects, we are referring to the multiscale structural complexities in the material ranging from the secondary-particle level down to the atomic scale. The particle-level microscopic characterization is further supported by bulk-averaged in situ X-ray powder-diffraction data, which cover millions of particles and ensure the statistical representativeness of our conclusions. Our findings highlight a hierarchical selfassemble effect, which is originated from the low solubility of dopant in the host lattice. Such an effect could potentially be used to formulate an unconventional multiscale defect-engineering strategy that is broadly applicable. ## SoC Heterogeneity Effect of the Grain Boundary The morphology and the state of charge (SoC) of a randomly selected bare LCO particle at the charge state are shown in Figure 1. A clear grain boundary with a microcrack at its edge can be observed in Figure 1A, and the grain boundary divides the particle into two domains. Further 3D Co valence distribution through peak-energy mapping using hard X-ray spectro-microscopy suggests that these two domains are in different SoCs. 28,29 Specifically, domain 2 has a higher Co valence state and exhibits a brighter color. The Co K-edge spectroscopic fingerprints of these two domains, together with that of a pristine bare LCO particle, are shown in Figure 1C. From the zoomed-in spectra in Figure 1D, it is clear that the peak energies of the pristine particle, domain 1, and domain 2 are gradually increasing, corresponding to their respective Co valence states. These results suggest that the grain boundary within a bare LCO particle not only is the mechanical weak point but also affects the local chemical states. ## Segregation of Ti in TLCO The above visualized grain boundary in a bare LCO particle appears to be chemically inactive and, consequently, prevents Li diffusion across this barrier. It is, therefore, of practical importance to modify the properties of such buried interfaces. Herein, we tackle this problem by applying trace doping of Ti, which is of a low solubility in the hosting LCO lattice. The morphology of an arbitrarily selected secondary particle of Ti/Mg/Al co-doped LCO (denoted as TLCO since we focus on the heterogeneity of Ti) is shown in Figure 2A. Its compositional heterogeneity is revealed in Figure 2B, which displays the distribution of the dopant (Ti) over a virtual slice through the Article center of the particle. The low solubility of Ti leads to its segregation at the particle surface and the buried interfaces, highlighting the separation of several primary grains as labeled in Figure 2B. Such an effect allows us to further extract and analyze individual primary grain(s). Figure 2C shows the elemental distribution results of an isolated primary TLCO particle. The depth profiles of elemental concentrations of Co and Ti as well as their relative ratios are calculated and plotted in Figure 2D. Our data suggest that the Ti concentration rapidly decreases from the surface to the particle center, while Co is relatively homogeneously distributed. These fine and quantitative analyses clearly confirm that trace doping of low-solubility dopant is an effective interface-engineering approach. ## Formation and Characterization of the Lattice Defects In addition to the modified interfaces, the lattice defects in the TLCO material could also play a significant role in the electrochemical performance. While the Bragg coherent diffraction imaging (BCDI) has been proven a powerful technique for characterizing the topological defects and local elastic properties in cathode crystalline particles, the limited coherence length of a typical third-generation synchrotron source limits BCDI's application to nanosized crystals. 30 To look into the lattice defects within an individual micron-sized primary grain of TLCO, we utilized the scanning hard X-ray nanoprobe technique (Figure 3A). 31 In a hard X-ray nanoprobe setup, the X-rays are focused to a spot of ~30 nm for illuminating the sample, which diffracts the beam in directions where the Bragg condition is satisfied. The diffraction patterns around a selected Bragg peak (101) are recorded. The crystal was rocked over a 2-degree angular range in the vicinity of the (101) Bragg peak, while a 2D raster scan was conducted at each rocking angle. Figure 3B is a typical diffraction pattern, in which we illustrate the orthogonal directions of the Bragg peak shift, relevant to the lattice defects and distortions. For example, the information collected along and perpendicular to the powder-ring direction are corresponding to the Zbending and strain, respectively. In the diffraction contrast images of LCO and TLCO particles, we observe two kinds of features that can be attributed to two different types of lattice defects (see Figure S1). The first type is worm-shaped curves (annotated with red arrows in Figure S1A), which are very similar to the edge-dislocation lines visualized in a Li-rich layered oxide particle using the BCDI method. 32 The line density is significantly higher in the TLCO particle. The second type is annotated with green lines in Figure S1B; these separate regions with different intensities in the diffraction contrast images, indicating different scattering power in different orientations. The second type of feature can therefore be attributed to the crystal twin boundaries. By tracking the diffraction pattern recorded on the pixel-area detector (see Figure 3B for the geometry) and the offsetting of the rocking curve (see Figures 3C and 3D for two representative pixels that show distinct rocking curve), the local lattice distortions can be mapped out. 33,34 To highlight Ti's role in inducing the observed lattice defects, we compare the nanodiffraction data of particles of bare LCO and TLCO in Figure 4. Figures 4A and 4B are the maps of different types of lattice distortions of the bare LCO and TLCO, respectively. The inhomogeneity in Z-bending, Y-twisting, and d-spacing can be reconstructed by conducting this calculation pixel by pixel over the entire scanned TLCO primary grain, as shown in Figure 4B. Visual assessment of these maps suggests that the bare LCO exhibits a higher degree of uniformity. This is confirmed by the histogram plots in Figure 4C, in which the peak broadening is observed in the TLCO data. Such comparison confirms that the Ti doping has induced LCO lattice deformation, which becomes a preexisting condition before the TLCO material is subjected to Article electrochemical cycling. To ensure the statistical representativeness of our particlelevel evaluation, the bulk-averaged laboratory XRD characterization is carried out. Our bulk XRD results of the pristine bare LCO and TLCO samples confirm the observations from nano X-ray diffraction-imaging experiments. Pawley refinements were performed with fundamental parameters (FPs) and pseudo-Voigt peak functions, respectively, for analysis of the lattice strain. It can be seen that TLCO exhibits much larger lattice strain than the bare LCO, indicating a larger amount of lattice defects in TLCO. More discussion on the XRD results can be found in the next section. ## Suppression of O 3 to H 1À3 Phase Transition For a better understanding of how such heterogeneously distributed lattice defects affect the phase transition of LCO over the charge-discharge process, we performed in situ laboratory XRD experiments to study the phase-evolution behavior of the bare LCO and the TLCO (Figure 5). Figure 5A shows the initial charging curve of TLCO under 0.1C condition with a highlighted voltage window of 4.40-4.60 V, in which the O 3 to H 13 phase transition occurs. Figure 5B focuses on the (003) diffraction peak shifts of the bare LCO and the TLCO in the charging voltage range of 4.40-4.60 V. A clear sign of the two-phase reaction can be observed at the end of this charging voltage range for the bare LCO, while the TLCO displays an overall solid-solution reaction. The split of the (003) peak is a typical characteristic of the O 3 to H 13 phase transition. Such structural transformation involves gliding of the TMO 2 slabs, which could cause particle fracturing and, consequently, bring in a series of detrimental effects, leading to the deterioration of the cycle performance. For further quantitative analysis, the evolution of cell-lattice parameters and cell volume were obtained through Pawley XRD refinement (see Tables S1-S3). As shown in Fig- ure 5C, the changes of both the cell-lattice parameters a and c and the cell volume of TLCO are smaller than those of the bare LCO, which strongly demonstrates the significant suppression of the phase transition in TLCO. In connection with the aforementioned analysis of the diffraction images, it can be hinted that the multimodular defects induced by the low solubility of Ti, in addition to homogeneous Al/Mg lattice doping, 24,35 can well suppress the collective O 3 to H 13 phase transition occurring in the bare LCO. Such an effect can partially contribute to the greatly enhanced cycle performances of TLCO at 4.6 V. The underlying mechanism can be understood as shown in Figure 5D, which schematically compares the lattice defect density (top) and the microstrain distribution (bottom) in primary grains of LCO and TLCO crystal. The suppression of the O 3 to H 13 phase transition in TLCO could be attributed to its abundant lattice defects and, subsequently, its highly disordered microstrain distribution. It is well known that strain has a significant impact on the phase-evolution behavior of battery-electrode materials during electrochemical reactions. 30 A recent density functional theory (DFT) calculation study has also demonstrated that the anisotropic microstrain in LCO lattice increases the energy barriers of structural transition during delithiation at high voltage. 36 Another important aspect is the size effect. As the inhomogeneous Ti distribution and the resultant hierarchical lattice defects separate the sample particle into small grains, the size effect may contribute to the energy that can affect the phase transition. The effect of defect energy and size on modification of the phase-transition behavior has been demonstrated in previous literature reports. For example, Wagemaker and co-workers performed very careful studies and revealed that nanosized rutile TiO 2 has a wider Li-solubility range that can accommodate more Li deintercalation and intercalation than a micro-sized one because of the size-induced extra surface and interfacial energy. 37 We also performed DFT calculations to elucidate how size affects the O 3 to H 13 phase transition in LCO. The detailed discussions are expanded in the Supplemental Information. In summary, our experimental results suggest that the intergrain (e.g., grain boundary) and intragrain (e.g., lattice doping) defects synergistically inhibit the O 3 to H 13 phase transition at high voltages. Therefore, hierarchical defect engineering is essential for the design of high-capacity cathodes that can overcome the giant strain due to the large amount of Li deintercalation and intercalation and thus achieve high structural stability. It is worth noting that both bare LCO and TLCO show very similar structural evolution behavior in low voltage range (OCV-4.3 V). In general, the order-disorder phase transition that occurred at around 4.2 V (Li x CoO 2 , x = 0.5) is very sensitive to the element doping and doping content. This indicates that the relatively low doping content of Ti (~0.1 wt %) in TLCO does not affect the bulk structure transition at low voltages. Therefore, the Ti-induced hierarchical doping is effective at promoting the stability at highly delithiated state without affecting the LCO's behavior very much at lower SoC. ## DISCUSSION In this study, we show that trace Ti doping can modify the microstructure and thus the intra-and intergrain defect properties within individual particles as a result of the low solubility of Ti in LCO lattice. Quantitative analysis of the nanoresolution diffraction images on single particles and XRD patterns covering structural information of millions of particles reveal a significant amount of defects that are heterogeneously distributed in TLCO. The construction of such hierarchical defects demonstrates the effectiveness in suppressing the undesired O 3 to H 13 phase transition of LCO at a deeply delithiated state, contributing to the greatly improved cycle performances of TLCO at high voltages. Given that high-energy-density cathode materials require the capability of stable deintercalation and intercalation of a large numbers of alkali ions in their lattices, which will inevitably introduce a high level of strain within the particles, a hierarchical defect-engineering strategy could enable highly disordered dispersion of lattice strain across both primary and secondary particles. Such an approach could be essential for constructing cathode materials with high electrochemical stability over long-termcycling. We would like to remind in the end that the lattice defects not only affect the structural robustness but could also modulate the cation and/or anion redox reactions and rearrange the Li-ion diffusion pathways. The interplay among lattice defects, cation and anion redox, and charge heterogeneity is a frontier research topic that is worth systematic follow-up efforts. ## EXPERIMENTAL PROCEDURES Resource Availability Lead Contact Further information and requests for resources should be directed to and will be fulfilled by the Lead Contact, Y. Liu ([email protected]).

chemsum

{"title": "Hierarchical Defect Engineering for LiCoO2 through Low-Solubility Trace Element Doping", "journal": "Chem Cell"}

accurate_cancer_cell_identification_and_microrna_silencing_induced_therapy_using_tailored_dna_tetrah

## Abstract: Accurate cancer cell identification and efficient therapy are extremely desirable and challenging in clinics.Here, we reported the first example of DNA tetrahedron nanostructures (DTNSs) to real-time monitor and image three intracellular miRNAs based on the fluorescence "OFF" to "ON" mode, as well as to realize cancer therapy induced by miRNA silencing. DTNSs were self-assembled by seven customized singlestranded nucleic acid chains containing three recognition sequences for target miRNAs. In the three vertexes of DTNSs, fluorophores and quenchers were brought into close proximity, inducing fluorescence quenching. In the presence of target miRNAs, fluorophores and quenchers would be separated, resulting in fluorescence recovery. Owing to the unique tetrahedron-like spatial structure, DTNSs displayed improved resistance to enzymatic digestion and high cellular uptake efficiency, and exhibited the ability to simultaneously monitor three intracellular miRNAs. DTNSs not only effectively distinguished tumor cells from normal cells, but also identified cancer cell subtypes, which avoided false-positive signals and significantly improved the accuracy of cancer diagnosis. Moreover, the DTNSs could also act as an anti-cancer drug; antagomir-21 (one recognition sequence) was detached from DTNSs to silence endogenous miRNA-21 inside cells, which would suppress cancer cell migration and invasion, and finally induce cancer cell apoptosis; the result was demonstrated by experiments in vitro and in vivo. It is anticipated that the development of smart nanoplatforms will open a door for cancer diagnosis and treatment in clinical systems. ## Introduction Accurate identifcation of cancer at an early stage plays an important role in cancer diagnosis and treatment. MicroRNAs (miRNAs) are a kind of cancer biomarker; their aberrant expression levels are closely related to the initiation and progression of cancers, and therefore, sensitive detection of tumor related miRNAs holds great promise for cancer diagnostics and prognostics. Furthermore, simultaneous detection of multiple tumor related miRNAs can avoid false-positive signals and enhance the accuracy of cancer diagnosis. With the development of nanomaterials, a variety of nanoprobes have been reported for the detection of multiple miRNAs. For example, the Tang group developed multicolor fluorescent nanoprobes based on gold nanoparticles (GNPs) for evaluating cellular migration and invasion by simultaneously imaging miRNA-221, PTEN mRNA and MMP9 in living cells. 13 The Zhu group assembled multicomponent nucleic acid enzymes onto the surface of mesoporous silica-coated gold nanorods as multifunctional nanodevices for intracellular miRNA-21 and miRNA-145 in situ imaging. 14 However, the preparation processes of these nanoprobes are often complicated and timeconsuming, and their stability, biocompatibility and cell permeation ability are also not satisfactory for in vivo diagnosis, which has driven researchers to fnd other substitutes. DNA tetrahedron nanostructures have attracted enormous interest owing to their unique advantages, such as ease of self-assembly, excellent biocompatibility, high nuclease stability, remarkable transmembrane capability through a caveolin-dependent pathway and availability for multiple modifcations. To signifcantly improve the survival rate of cancer patients, besides accurate cancer identifcation, an efficient treatment strategy is another crucial step. Gene silencing as a kind of gene therapy has now been considered as one of the most promising options to overcome the limitations of traditional cancer therapy. It can induce sequence-specifc inhibition of oncogene expression or translation through the delivery of antagomirs to cancer cells, which makes it possess advantages of high specifcity, improved safety, high efficacy and unrestricted choice of targets. 22,23 For example, leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) is a novel gastric cancer marker, and silencing its expression with antagomirs could efficiently inhibit cancer angiogenesis. 24 miR-10b was overexpressed in metastatic breast tumor patients, and silencing of miR-10b with antagomirs could signifcantly decrease miR-10b levels and suppress breast cancer metastasis. 25 miRNA-21 as a key oncogenic miRNA was widely overexpressed in various tumors and participated in tumor occurrence and development. Inhibition of endogenous miRNA-21 with antagomirs could suppress cancer cell proliferation, migration and invasion, and tumor growth. 26,27 In biomedical science, developing nanomaterials that integrating both ultrasensitive diagnosis and highly efficient therapy functions remains attractive and challenging. 28,29 In this work, we reported the frst example of vertebral-shaped DNA tetrahedron nanostructures (DTNSs) for accurate cancer iden-tifcation and miRNA silencing induced therapy. Based on the fluorescence "OFF" to "ON" mode, three intracellular miRNAs (miRNA-21, miRNA-122 and miRNA-194) were simultaneously monitored and imaged, which not only effectively distinguished tumor cells from normal cells, but also identifed cancer cell subtypes, and thus the accuracy of cancer diagnosis was signifcantly improved. In miRNA-21 overexpressed cancer cells, antagomir-21 (one recognition sequence) was detached from DTNSs to silence endogenous miRNA-21 inside cells, which would suppress cancer cell migration and invasion, and fnally induce cancer cell apoptosis. The prepared DTNSs displayed improved resistance to enzymatic digestion and high cellular uptake efficiency, and exhibited accurate cancer identifcation and efficient cancer therapy ability. ## Preparation and characterization of DNA tetrahedron nanostructures (DTNSs) The DTNSs were prepared with seven customized singlestranded nucleic acid chains (P1-P7) through a simple thermal annealing method (Fig. 1). Four chains (P1-P4), partially complementing each other, would spontaneously and respectively fold into triangles and then assemble into a rigid tetrahedron (named TDN). Three sequences linked with quenchers (BHQ1 for FAM, BHQ2 for TAMRA and Cy5) were distributed in the vertexes of the tetrahedron, respectively, and were complementary to the recognition sequence in P5-P7 chains. Fluorescein FAM labeled P5, TAMRA labeled P6 and Cy5 labeled P7 were introduced into the above tetrahedron based on the principles of Watson-Crick base pairing to form DTNSs. The formation of DTNSs was identifed by agarose gel electrophoresis analysis (inset in Fig. 1). For lanes 1 and 2, only a single band was observed, indicating that fluorescence or quenching group modifed nucleic acid chains maintained good purity. With the step-by-step addition of chains from lane 3 to lane 8, there clearly appeared a gradual reduction of electrophoretic mobility, which could be ascribed to the increasing molecular mass and more complicated spatial construction of assemblies. The AFM image further verifed the successful formation of the DTNSs, as shown in Fig. S1, † and the prepared DTNSs were vertebral-shaped nanoparticles with a diameter of $3 nm. ## Fluorescence quenching and recovery capability of the prepared DTNSs The fluorophores in the vertexes of DTNSs were quenched by the adjacent quenchers, and the fluorescence quenching capability depended on the ratio of TDN to nucleic acid chains (P5/P6/P7) during DTNSs preparation, which was determined by ((F control F DTNSs )/F control ) 100%, where F DTNSs was the fluorescence of DTNSs and F control was the fluorescence of P5/ P6/P7 chains. The fluorescence of FAM/TAMRA/Cy5 was gradually quenched by increasing the ratio of TDN to P5/P6/P7 and reached a constant value at ratios of 1.5, 1.25 and 1.25, respectively, and the quenching efficiency was up to 90% (Fig. S2 and Table S1 †). In the presence of target miRNAs (miRNA-21, miRNA-122 and miRNA-194), P5, P6 and P7 would release from DTNSs, and then hybridize with the targets to form much more stable double strands. In consequence, the corresponding fluorescence signals (F target ) were recovered. The degree of fluorescence recovery (F target /F DTNSs ) was gradually enhanced with the increased ratio of tetrahedron to P5/P6/P7 and reached a platform at ratios of 1.5, 1.5 and 1.5, respectively (Fig. S3 †). Combining the quenching and recovery efficiency, a ratio of 1.5 was selected for TDN to P5/P6/P7. Under the optimized conditions, the fluorescence intensities of FAM, TAMRA, and Cy5 increased by about 6.7-fold, 5.4-fold and 11.0-fold in the presence of the three miRNA targets, respectively. Ability of DTNSs to detect miRNAs in a homogeneous solution Fig. 2A-C show that the fluorescence intensities of FAM, TAMRA and Cy5 all increased linearly with the concentration of miRNA targets from 0.15-37.5 nM. The detection limit was calculated to be 0.13 nM for miRNA-21, 0.64 nM for miRNA-122, and 0.68 nM for miRNA-194 (Fig. 2D-F), which were signifcantly lower than the previous reports (Table S2 †). 15, We next assessed the sequence specifcity and the multiple detection ability for the three miRNA targets. DTNSs were transferred into 21 wells (3 7) and different mixed solutions of various combinations of the three target miRNAs were respectively added. The fluorescence emission spectra of FAM, TAMRA or Cy5 were obtained only in response to miRNA-21, miRNA-122 or miRNA-194 targets and had no notable cross-reactivity with each other (Fig. 2G). And when the three targets co-existed, the fluorescence signals of FAM, TAMRA or Cy5 all continuously increased with the corresponding target addition (Fig. S4 †). In contrast, treatment of DTNSs with miRNAs with a scrambled sequence (shown in Table S3 †) showed no signifcant changes in fluorescence intensity (Fig. S5 †). The above results indicated that the prepared DTNSs allowed high-throughput monitoring of the three miRNA targets with high sensitivity, no notable crossreactivity and good specifcity. To verify the nuclease resistance of the DTNSs, 10% fetal calf serum (FBS, v/v) was used for the preparation of DTNSs solution to closely mimic physiological conditions. As shown in Fig. S6A-C, † no detectable fluorescence changes were observed with time for DTNSs in both FBS and PBS. The fluorescence recovery had no signifcant difference after the addition of target miRNAs for the two groups. Moreover, the bands of DTNSs treated with PBS or FBS solution for 1-4 h in agarose gel electrophoresis still maintained the same position, demonstrating that DTNSs were undecomposed when treated with FBS for 4 h (Fig. S4D †). All the above results confrmed that the DTNSs had good nuclease stability due to their tetrahedron-like spatial structure and were suitable for in vitro and in vivo studies. Cytotoxicity was another vital factor for living cell studies. The cytotoxicity of DTNSs was tested by the MTT assay in normal cells (HEK293). After incubation with DTNSs at a high concentration of 100 nM for 48 h, the HEK293 cells still maintained more than 90% of the viability, revealing the low cytotoxicity of the DTNSs to normal cells (Fig. S7 †). ## Detection and imaging of intracellular miRNAs with DTNSs For this study, the cell permeability of DTNSs was investigated by using non-rigid DNA tetrahedron-based nanoprobes (n-DTNSs) and DNA line nanostructures (DLNSs) as controls. After 4 h incubation, the fluorescence in HepG2 cells incubated with DTNSs was obviously higher than that of HepG2 cells incubated with n-DTNSs or DLNSs (Fig. S8 †), indicating that the rigid DTNSs undoubtedly improved self-delivery capability without the help of transfection agents owing to their rapid internalization through a caveolin-dependent pathway, 34,35 while n-DTNSs and DLNSs had poor permeability and low biostability. The time-dependent response of DTNSs incubated with cells was then studied by monitoring the fluorescence changes with different incubation periods. Huh7 cells with high expression levels of the three target miRNAs were chosen as the model. 36 The fluorescence intensities of green, orange and red all increased before 2 h (Fig. S9 †), and then stayed at the same level for another 2 h due to the complete entry of DTNSs, and thus, 2 h was used for the incubation of cells and DTNSs in the following experiments. We next investigated whether DTNSs could monitor miRNAs in living cells. Three different cell lines, Huh7, HepG2 and HEK293 (human normal cells) were chosen, miRNA-194 was used as a model target. Both confocal images (Fig. 3A) and flow cytometric analysis (Fig. 3B) showed that the fluorescence intensities of Cy5 were obviously different in each cell line, indicating different miRNA-194 expression levels in the three cell lines. In agreement with previous reports, Huh7 had a higher miRNA-194 expression level than HepG2, while HEK293 exhibited a quite low expression level, and quantitative reverse transcription PCR (qRT-PCR) was further used to confrm the relative miRNA-194 expression levels (Fig. S10 †). The expression levels of miRNAs in cancer cells indicated the stage of tumorigenesis, the effect of therapy and prognosis, and dynamic monitoring of miRNA expression levels inside cells was signifcant. To this end, HepG2 cells were treated with a miRNA194 mimic and inhibitor, and non-treated HepG2 cells were used as a control. An miRNA-194 inhibitor with 2 0 -Omethyl modifcation was chosen to transfect HepG2 cells to downregulate the expression level of miRNA-194, 39 while the miRNA-194 mimic was selected to upregulate it. 40 As shown in Fig. 3C, compared with the control group, a higher Cy5 fluorescence signal was observed in the mimic treated group while a lower one was displayed in the inhibitor treated group. Additionally, the flow cytometry assay further confrmed the results of confocal images (Fig. 3D). These results demonstrated that DTNSs had the capability of real-time monitoring miRNAs inside cells. Detection of one kind of miRNA to identify cancers will produce high false positive signals, and thus simultaneous detection of multiple miRNAs inside cells would beneft the diagnostic precision of related cancers. Here we simultaneously detected three miRNAs (miRNA-21, miRNA-122 and miRNA-194) in three cell lines (two cancer cells: Huh7 and HepG2, one normal cell line: HEK293). Confocal images (Fig. 4A) and corresponding grayscale values (Fig. 4B) exhibited three fluorescent signals inside cells with different intensities and spatial distributions. For Huh7 cells, the three target miRNAs were all overexpressed; for HepG2 cells, miRNA-21 was high, and miRNA-122 and miRNA-194 were low; for HEK293, the three target miRNAs were quite low, and the expression levels were in agreement with literature reports. 36,38, qRT-PCR was used to quantitatively measure relative miRNA expression levels in the three cell lines (Fig. 4C), and the results were consistent with confocal images and flow cytometry. The fndings suggested that DTNSs could be used not only to effectively distinguish cancer cells from normal cells, but also to identify cancer cell subtypes. In vitro cancer therapy induced by miRNA-21 silencing with DTNSs Previous reports have demonstrated that silencing intracellular miRNA-21 with antagomir-21 could efficiently suppress cancer cell migration and invasion, and fnally induce cancer apoptosis. 26,44 In this system, DTNSs were used as a smart carrier to deliver antagomir-21 into cells through a caveolindependent pathway. After cell uptake, endogenous miRNA-21 inside cancer cells served as an initiator to cause the release of antagomir-21 from DTNSs, and then suppress cancer cell migration and invasion, and trigger cell apoptosis. The woundhealing assay was employed to assess the inhibition of cancer cell migration and invasion. As shown in Fig. S11, † after Huh7 cells being treated with DTNSs (500 nM) for a period, the cell migration and invasion were obviously inhibited, while for HEK293 normal cells, the cell migration and invasion were almost unaffected, which was ascribed to the endogenous miRNA-21 silencing effect of DTNSs. To investigate the therapeutic effect of DTNSs in vitro, cytotoxicity assessment was accomplished by the MTT assay (Fig. 5A and B); even at an antagomir-21 dose of 2 mM in DTNSs, the HEK293 cell viability still remained over 90%. However, the cell viability of Huh7 cells was down to 58% after treatment with 2 mM DTNSs, which was obviously lower than that of the DTNS-C treated group ($90%). Here, DTNS-C was prepared by replacing antagomir-21 (P5) with normal strand P8, which didn't have a gene silencing function. Therefore, our prepared DTNSs could efficiently induce cancer cell apoptosis and had no side effects to normal cells. And the apoptosis was further confrmed by flow cytometry analysis with the dual fluorescence of Annexin V-FITC/PI (Fig. 5C). To avoid the fluorescence interference of FAM/Cy5 in DTNS-C or DTNSs, DTNS-C or DTNSs without fluorophore modifcation was synthesized for flow cytometry analysis. The Huh7 and HEK293 cells were respectively treated with DTNS-C and DTNSs without fluorophore modifcation (2 mM) for 48 h; the Huh7 cells underwent nearly 50% apoptosis after treatment with DTNSs but no obvious apoptosis was observed in the DTNS-C group. In contrast, no prominent apoptosis was observed in both DTNS-C and DTNSs treated HEK293 cells, and the result was in accordance with the MTT assay. ## In vivo cancer imaging and therapy with DTNSs Before the in vivo study of DTNSs for the liver cancer therapeutic effect, the clinical signifcance of the selected miRNA-21, miRNA-122 and miRNA-194 biomarkers was frstly investigated in clinical patients (Fig. S12 †). It was shown that the expression of miRNA-21 in clinical patients' liver cancer tumors (T) was signifcantly higher than that in non-tumor liver tissues (NT); miRNA-122 showed decreased expression in T compared with that in NT, but there was no distinct difference for the expression of miRNA-194-1/2 in T and NT, which indicated that miRNA-21 and miRNA-122 targets were valuable biomarkers for liver cancer diagnosis while miRNA-194 mainly played a role in the discrimination of hepatocellular carcinoma cell subtypes. The normalized relative expression of miRNA-21, miRNA-122 and miRNA-194 in Huh7 cells compared with that in different tissues of mice was also studied. the tumor region and size in mice were reflected by a luminescence signal, which originated from the D-luciferin catalyzed by luciferase. 45,46 After two weeks, mice injected with DTNS-C showed strong FAM (Green) and TAMRA (Orange) signals in the liver tumor and liver tissues, suggesting the high expression of miRNA-21 and miRNA-122 in the liver tumor and liver tissues. However, a signifcantly weaker FAM (Green) signal and obvious smaller tumor size were observed in the DTNSs injected group. From the result of quantitative analysis of bioluminescence signals, we could see that the tumor growth rate of the DTNSs treatment group was obviously lower than that of the DTNS-C group (Fig. 6B). In addition, the qRT-PCR analysis result of mice liver cancer tumor models also showed that only miRNA-21 was down-regulated in the DTNSs treated group (Fig. 6C), demonstrating that antagomir-21 loaded in DTNSs silenced the expression of miRNA-21 in liver tumors and suppressed the tumor growth. All the above results indicated that the prepared DTNSs could serve as a diagnostic probe and anticancer drug to treat cancers. ## Conclusions In summary, we have successfully developed a smart nanosystem, DNA tetrahedron nanostructures (DTNSs), which can realize simultaneous monitoring of three intracellular miRNAs accompanied by efficient cancer therapy for the frst time. DTNSs exhibited high biostability and cellular uptake efficiency owing to their unique tetrahedron-like spatial structure. Based on the fluorescence "OFF" to "ON" mode, three intracellular miRNA (miRNA-21, miRNA-122 and miRNA-194) expression levels could be quantitatively detected with high sensitivity, specifcity and no notable cross-reactivity, and their dynamic changes could be real-time monitored. This system not only effectively distinguished tumor cells from normal cells, but also identifed cancer cell subtypes, which avoided false-positive signals and signifcantly enhanced the accuracy of cancer diagnosis. Moreover, DTNSs could also act as a kind of cancer drug to treat cancer efficiently through endogenous miRNA-21 silencing, which was demonstrated by experiments in vitro and in vivo. Therefore, we anticipate that our strategy will have potential application in clinical diagnosis and treatment of cancer in future. ## Ethical statement All procedures involving animals were conducted in accordance with the guidelines for Care and Use of Laboratory Animals of Nanjing Medical University and experiments were approved by the Animal Ethics Committee of Nanjing Medical University. ## Conflicts of interest There are no conflicts to declare.

chemsum

{"title": "Accurate cancer cell identification and microRNA silencing induced therapy using tailored DNA tetrahedron nanostructures", "journal": "Royal Society of Chemistry (RSC)"}

dual-graphite_cells_based_on_the_reversible_intercalation_of_bis(trifluoromethanesulfonyl)imide_anio

## Abstract: Recently, dual-ion cells based on the anion intercalation into a graphite positive electrode have been proposed as electrochemical energy storage devices. For this technology, in particular electrolytes which display a high stability vs. oxidation are required due to the very high operation potentials of the cathode, which may exceed 5 V vs. Li/Li + . In this work, we present highly promising results for the use of graphite as both the anode and cathode material in a so-called "dual-graphite" or "dual-carbon" cell. A major goal for this system is to find suitable electrolyte mixtures which exhibit not only a high oxidative stability at the cathode but also form a stable solid electrolyte interphase (SEI) at the graphite anode. As an electrolyte system, the ionic liquid-based electrolyte mixture Pyr 14 TFSI-LiTFSI is used in combination with the SEI-forming additive ethylene sulfite (ES) which allows stable and highly reversible Li + ion and TFSI À anion intercalation/de-intercalation into/from the graphite anode and cathode, respectively. By addition of ES, also the discharge capacity for the anion intercalation can be remarkably increased from 50 mA h g À1 to 97 mA h g À1 . X-ray diffraction studies of the anion intercalation into graphite are conducted in order to understand the influence of the electrolyte additive on the graphite structure and on the cell performance. Broader contextOne of the most challenging issues in the 21 st century is the preservation of a consistent energy supply that meets the world's increasing energy demands. The present energy economy based on fossil fuels is considered to be at serious risk due to several factors, such as the shortages of non-renewable resources or concerns about the environmental impact of energy consumption, and thus gives rise to the development of renewable energy sources. The need for clean and efficient storage of electrical energy will be vast, not only to meet the rising energy demand, but in particular to prevent global warming. Currently, lithium-ion batteries dominate the small format battery market for portable electronic devices and are now being widely regarded as the technology of choice for future automotive and stationary applications. The requirements for stationary batteries are signicantly different from those of power batteries in electric vehicles. For stationary batteries, high safety, low cost and long cycle life are the most important parameters. In this paper, we introduce the dual-graphite/dual-carbon battery technology as a promising option for grid applications, since it displays environmental, safety and cost benets (e.g. free of transition metals, non-ammability of the ionic liquid electrolyte, graphite as a low-cost electrode material, aqueous electrode processing possible for anodes and cathodes) over stateof-the-art lithium-ion batteries. ## Introduction As a redox-amphoteric material, graphite can be reduced as well as oxidized by chemical or electrochemical methods. 1 The resulting positive or negative charge can be compensated by the intercalation of a variety of certain anionic/cationic intercalation guests into the interlayer gaps of the graphite lattice. 1,2 While lithiated graphite is an established anode material in state-of-the-art lithium-ion batteries, 3 graphite intercalation compounds (GICs) intercalated by anions were suggested by Carlin et al. as possible active materials for positive electrodes in so-called "dual intercalating molten electrolyte batteries". 4 In their work, they proposed a dual-graphite battery system which uses different room temperature ionic liquids (RTILs), such as 1-ethyl-3-methylimidazolium-hexafluorophosphate (EMI + PF 6 ). In this set-up, the electrolyte does not only act as a charge carrier but also as a source for intercalation guests, in which EMI + intercalates into the graphite anode and PF 6 intercalates into the graphite cathode during the charge process. 4 However, the reversibility of the intercalation/de-intercalation behavior of this dual-graphite cell was relatively poor. The concept of a dualcarbon cell is also followed in industry, as recently the Japanese start-up "Power Japan Plus" announced plans to commercialize such a kind of dual-carbon battery. 5 Recently, we introduced a novel dual-intercalation battery system using a mixture of N-butyl-N-methylpyrrolidinium bis-(trifluoromethanesulfonyl)imide (Pyr 14 TFSI) and lithium bis-(trifluoromethanesulfonyl)imide (LiTFSI) as an electrolyte. In this "dual-ion cell", where e.g. metallic lithium or Li 4 Ti 5 O 12 (LTO) is used as an anode material, and graphite, which is intercalated by TFSI anions during charge, is used as a cathode material, and shows a highly reversible cycling performance with a Coulombic efficiency exceeding 99%. 6 It was demonstrated that a reversible cathode discharge capacity of more than 50 mA h g 1 at room temperature and above 110 mA h g 1 at 60 C operation temperature can be achieved. 6 X-ray diffraction investigations revealed the mechanism behind the formation of C n + TFSI GICs. 10 The intercalation of TFSI anions proceeds in defnable and consecutive phases. The attainment of these different nphases is dependent on the operation conditions such as the cut-off potential or the temperature. A specifc discharge capacity of 115 mA h g 1 is the maximum we found so far for the TFSI uptake into graphite, which corresponds to a stage-1 GIC with a stoichiometry ranging between C 19 TFSI and C 20 TFSI. 10 In order to differentiate the energy storage mechanism of this battery system from the ion transfer mechanism which is known from lithium-ion batteries, we introduced the term "dual-ion cell". This type of cell mechanism includes all cell reactions where simultaneously anions and cations react with the electrodes. In this dual-ion battery system, which also implies the dual-graphite/dual-carbon system, the electrolyte needs to be considered as an active material as well. This is in contrast to a lithium-ion cell, in which the electrolyte "only" acts as a charge carrier between the anode and the cathode and where the thickness of the electrolyte layer needs to be minimized. According to the work of Dahn and Seel in 2000, 11 there must be a high amount of electrolyte present in the dualgraphite cell to provide enough ions which are needed during charging of the cell. In their work, they reported that an electrolyte with a high molarity is crucial to achieve a sufficiently high energy density of this system. 11 Ionic liquids (ILs), being salts of low temperature melting points, are one of the most promising classes of electrolytes providing new opportunities in electrochemistry. 12,13 ILs show several advantageous properties for electrochemical applications, such as a negligible vapor pressure, a broad liquid range and an ionic conductivity which can be in the same range of organic solvent-based electrolytes. 12,13 It should be noted that the use of ILs as solvents for electrochemical reactions is predominantly driven by their excellent oxidative and reductive stability and thus a large electrochemical stability window, which generally exceeds the ones of conventional electrolyte solvents. 14 In particular, electrolytes displaying a high stability vs. oxidation are required for the dual-ion technology, due to the very high operation potentials of the cathode, which may surpass 5 V vs. Li/Li + . Since the compatibility of the ionic liquid Pyr 14 TFSI with graphite anodes is not sufficient, so far the selection of the anode materials for the dual-ion system has been limited to metallic lithium or LTO. Due to the excellent electrochemical stability of Pyr 14 TFSI against cathodic decomposition, there is no effective solid electrolyte interphase (SEI) formation on the graphite anode. Thus, graphite undergoes exfoliation by intercalation of the relatively large organic cations (Pyr 14 + ) between the graphene layers, leading to irreversible degradation of the negative electrode. 17 This process is illustrated schematically in Fig. 1 for a dual-graphite cell. One solution to enable the use of ILs with graphite anodes is the addition of SEI-forming electrolyte additives such as ethylene sulfte (ES). Sulfur-based electrolyte additives are proposed to support the SEI formation by adsorption of their reduced species onto the catalytically active functional groups of the graphite surface. 18,19,21 Unfortunately, the mechanism of the SEI formation in the presence of ES as an additive has not been well understood so far. Ota et al. proposed that an intermediate, such as SO 2 , is formed during the reductive decomposition of ES, which then reacts with various compounds such as Li 2 SO 3 or Li 2 SO 4 . 22 These compounds may contribute to more effective formation of the inorganic part of the SEI layer. Furthermore, Wrodnigg et al. suggested ES as an effective SEIforming agent in the presence of propylene carbonate (PC) as a solvent, whereby graphite is protected from rapid exfoliation caused by massive co-intercalation of PC, leading to the destruction of the graphite structure. 18 Recently, Leggesse and Jiang reported that ES can undergo a one-and two-electron reduction mechanism in PC-based electrolytes, whereas the formation of Li 2 SO 3 is more favorable in the two-electron reduction process. 23 The major reduction products which are considered to be responsible for the formation of an effective SEI layer are Li 2 SO 3 , (CH 2 OSO 2 Li) 2 , CH 3 CH(OSO 2 Li)-CH 2 OCO 2 Li and ROSO 2 Li. 23 Fig. 1 Schematic illustration of a dual-graphite cell with no effective SEI layer at the graphite anode during the charge process. The negative graphite electrode suffers from exfoliation reactions caused by cointercalation of the relatively large pyrrolidinium (Pyr 14 In this work, we present promising results concerning the use of graphite as both the anode and cathode material in a so-called "dual-graphite cell" using the ionic liquid-based electrolyte mixture Pyr 14 TFSI-LiTFSI in combination with ES as an SEI-forming additive. The energy storage mechanism of this system is based on the simultaneous TFSI anion intercalation into the graphite positive electrode and Li + ion intercalation into the graphite negative electrode during charge. During discharge, both ions are released back into the electrolyte. In this regard, the intercalation of Pyr 14 + cations into the graphite anode needs to be prevented by the formation of a protective SEI layer, which still allows the reversible intercalation/de-intercalation of lithium ions, as depicted in Fig. 2. Recently, Read et al. reported that a dual-graphite cell using an organic solvent-based electrolyte can be cycled reversibly. 24 However, the long-term cycling stability as well as Coulombic efficiency was relatively poor. Herein, we will demonstrate that by the use of the ionic liquid electrolyte mixture, the dualgraphite system displays a stable cycling performance with a high Coulombic efficiency, while both the discharge capacity and efficiency can be tailored by the operation conditions, such as the upper cut-off cell voltage. In addition, X-ray diffraction studies of the anion intercalation into graphite are performed in order to understand the influence of the electrolyte additive on the graphite structure and on the cell performance. ## Experimental The preparation of the graphite positive electrodes was carried out using a composition of 90 wt% of KS6L graphite (Imerys; D90 ¼ 8.5 mm; BET specifc surface area ¼ 19.0 m 2 g 1 ), 5 wt% of conductive carbon black C-nergy Super C65 (Imerys) and 5 wt% of sodium carboxymethyl-cellulose (CMC) as a binder (Walocell CRT 2000 PPA12, Dow Wolff Cellulosics). The details of the electrode preparation have been described previously. 6 The graphite negative electrodes were prepared in the same way using 90% of commercial graphite (further abbreviated as CG; D90 ¼ 40.8 mm; BET specifc surface area ¼ 3.96 m 2 g 1 ) as an active material, 5 wt% of conductive carbon black agent C-nergy™ Super C65 (Imerys), 2.5 wt% of the binder styrenebutadiene rubber (SBR; LIPATON SB 5521, Polymer Latex GmbH) and 2.5 wt% of sodium carboxymethylcellulose (CMC) as a binder (Walocel CRT 2000 PPA 12, Dow Wolff Cellulosics). The electrode paste for the graphite anode was cast on highpurity copper foil (Carl Schlenk AG ® ). The mass loading of the graphite negative electrode was ca. 2.5 mg cm 2 . 20 The ionic liquid N-butyl-N-methylpyrrolidinium bis(tri-fluoromethanesulfonyl)imide (Pyr 14 TFSI, Solvionic, purity: 99.9%) used as an electrolyte solvent was dried under ultra-high vacuum (5 10 8 mbar, 110 C) for 48 hours using a turbo molecular pump TPS-compact (Varian Vacuum Technologies) before use. Under inert conditions, a mixture of dried Pyr 14 TFSI (water content less than 10 ppm, determined by Karl Fischer titration) with 1 M lithium bis(trifluoromethane-sulfonyl)imide (LiTFSI, 3 M, purity: 99.95%) as conductive and electroactive salt (mole fraction of LiTFSI: 0.3) was prepared. To this electrolyte solution, 2 wt% of the SEI-forming additive ethylene sulfte (ES, Sigma Aldrich, purity: 98%) were admixed. The maximum solubility of LiTFSI in the ionic liquid is strongly dependent on the temperature. While at 20 C a LiTFSI mole fraction of ca. 0.32 is possible, this can be strongly enhanced to about 0.68 at 60 C. Phase diagrams of the ionic liquid electrolyte mixtures were reported by Henderson and Passerini. 25 Electrochemical measurements were carried out either in a half-cell set-up (Subsections 3.1 and 3.2) with metallic lithium as counter and reference electrodes or in a full-cell set-up (Subsection 3.4) with the CG-based electrode as an anode, the KS6L-based electrode as a cathode and high-purity metallic lithium foil (Rockwood Lithium ® ) as a reference electrode. For that purpose a custom-made Swagelok ® type T-cell with a threeelectrode confguration was used. A borosilicate glass micro-fber (Whatman ® GF/D) drenched with 120 mL of electrolyte served as the separator. Charge/discharge cycling was performed on a multichannel Maccor 4300 battery test system (MACCOR, INC). After cell assembly, the cells rested for at least 12 hours to ensure homogeneous electrolyte distribution and sufficient electrode wetting. The constant current cycling of CG-based graphite anodes in a half-cell set-up was conducted with a specifc current of 37.2 mA g 1 (0.1C) between cut-off potentials of 0.02 V and 1.5 V vs. Li/Li + (see Subsection 3.1). The charge/discharge cycling of KS6L graphite cathodes in a half-cell set-up was performed with a constant current of 50 mA g 1 between cut-off potentials of 3.0 V and 4.8-5.2 V vs. Li/Li + (see Subsection 3.2). The constant current cycling of dual-graphite cells (Subsection 3.4) was carried out with a constant charge and discharge current of 10 mA g 1 for the frst three cycles in order to provide a homogeneous SEI-formation, while from the 4th cycle the current is increased in certain experiments (e.g. to 50 mA g 1 in Fig. 9 or to 500 mA g 1 in Fig. 11). For the in situ X-ray diffraction (XRD) measurements, a modifed commercial CR2016 coin cell (Hohsen, Japan) with an X-ray transparent window was used. The details of this set-up have been described previously. 10 After assembly of the coin cell using high-purity metallic lithium (Rockwood Lithium ® ) as a counter electrode, the cell was directly placed in an X-ray diffractometer (BRUKER D8 Advance, equipped with a copper target X-ray tube). XRD measurements were conducted in a range between 2q ¼ 20 and 35 with a step size of 0.185 per second resulting in 20 minutes per in situ XRD scan at an accelerating voltage of 40 kV and a current flow of 40 mA. For in situ XRD measurements, cyclic voltammetry was performed using a VSP multichannel potentiostatic-galvanostatic system (Biologic ® Science Instrument, France) with a scan rate of 50 mV s 1 between 3.4 V and 5.2 V at 20 C with a 3 hour constant voltage step at the upper cut-off voltage. ## Results and discussion A major goal for the dual-graphite system is to fnd a suitable electrolyte mixture which exhibits frst a high oxidative stability, in particular with anions that are stable upon intercalation/deintercalation into the graphite cathode, and second is able to form a stable SEI layer at the graphite anode. As an electrolyte system, the ionic liquid-based electrolyte mixture Pyr 14 TFSI-1 M LiTFSI in combination with the SEI-forming additive ethylene sulfte (2 wt%) is studied. At frst, lithium ion intercalation into the graphite anode and TFSI anion intercalation into the graphite cathode are investigated independently in a half-cell set-up (Secitons 3.1 and 3.2). In Seciton 3.4, the electrochemical performance of the dual-graphite system is studied in detail. ## Graphite anode performance in a half-cell set-up As mentioned above, the compatibility of the pure ionic liquid Pyr 14 TFSI-LiTFSI with graphite anodes is relatively poor, which is related to insufficient SEI formation and the co-intercalation of Pyr 14 + cations between the graphene layers, leading to irreversible degradation/exfoliation of graphite. 15,17 Fig. 3 displays the charge/discharge cycling behavior for lithium ion intercalation/de-intercalation into/from graphite as an anode material (CG graphite) with the additive containing ionic liquid-based electrolyte (Pyr 14 TFSI, 1 M LiTFSI + 2 wt% ES). Even though the maximum discharge capacity is only about 100 mA h g 1 , which is signifcantly lower than the theoretical value of fully lithiated graphite (372 mA h g 1 ), a capacity retention of 83% can be obtained after 50 cycles at a C-rate of 0.1 (Fig. 3a). This relatively low reversible discharge capacity was also reported by others 15,26 and is most likely related to the low charge transfer kinetics for lithium intercalation/de-intercalation into/from graphite at the electrode/electrolyte interface, i.e. at the SEI. Furthermore, it can be caused by the high viscosity of the electrolyte and by the relatively low self-diffusion coefficient and transference number of Li + in the ionic liquid. 27 The reversible capacity may be further enhanced by tailoring the SEI layer by the use of certain electrolyte additives. However, these additives must not only build a stable SEI layer but also exhibit a high stability vs. oxidation for the dual-graphite cell. The average Coulombic efficiency is about 99.0% along the cycles 10 to 50 (Fig. 3b). However, the efficiency in the frst cycle with only 49% is quite low. Fig. 3c displays a representative charge/discharge potential profle from the 50 th cycle, showing that lithium ion intercalation reversibly takes place in the potential range from 0.20 V to 0.02 V vs. Li/Li + . Overall, by addition of 2 wt% ES to the ionic liquid electrolyte, a reversible lithiation/de-lithiation of graphite is realized. In Section 3.4, we will show that this electrolyte system displays a highly reversible electrochemical performance in the dual-graphite cell, outperforming any results published so far in the literature for similar systems. ## Graphite cathode performance in a half cell set-up The electrochemical intercalation/de-intercalation behavior of TFSI anions from the pure ionic liquid (Pyr 14 TFSI-LiTFSI) into graphite in a metallic lithium/graphite dual-ion cell was studied in detail in previous publications. 6,7,28 As the use of the additive ES is primarily intended to increase the compatibility of the ionic liquid electrolyte with the graphite anode, no negative influence on the TFSI anion intercalation is desirable in the presence of ES. In order to verify this assumption, electrochemical investigations of the TFSI anion intercalation were performed with the ES-based electrolyte. It is obvious that the discharge capacity of the ES-containing system is remarkably increased to 97 mA h g 1 (50 th cycle) in comparison with the one of the pure ionic liquid, which displays a maximum value of 50 mA h g 1 (Fig. 4a and c). Apart from the frst charge/discharge cycles, the Coulombic efficiency is very similar for both systems and reaches about 99% (Fig. 4b). As we discussed previously, the low Coulombic efficiency in the frst cycles is most likely related to some type of "formation", which is presumably a kinetic hindrance due to the frst intercalation of TFSI between the graphene sheets. 28 This "formation" may take a few cycles until all graphite particles of the porous electrode are wetted and the maximum discharge capacity is achieved. 28 Since the use of the electrolyte additive leads to an increase of the maximum discharge capacity of the graphite cathode, the influence of a varying upper charging end potential, ranging from 4.8 V to 5.2 V vs. Li/Li + on the discharge capacity and Coulombic efficiency was investigated. Table 1 summarizes the discharge capacity and efficiency values obtained at these different potentials. In general, the discharge capacity increases with an increasing upper charging end potential, i.e. from 49 mA h g 1 at 4.8 V vs. Li/Li + to 126 mA h g 1 at 5.1 V vs. Li/Li + , while the Coulombic efficiency slightly decreases, i.e. from 99.5% to 99.0%. If the upper cut-off potential is further enhanced to 5.2 V vs. Li/Li + , the discharge capacity drops to 116 mA h g 1 and the efficiency also deteriorates to 97.8%. Therefore, it can be assumed that potentials above 5.1 V vs. Li/Li + lead to enhanced irreversible reactions, most likely caused by electrolyte degradation and in particular by decomposition of the intercalated anion. Fig. 5 displays the differential capacity profles of the metallic lithium/KS6L graphite dual-ion cell at different charging end potentials ranging from 4.8 V to 5.2 V vs. Li/Li + , each from the 50 th charge/discharge cycle. In Fig. 5a, the dQ/dV profles over the whole potential range are depicted, while Fig. 5b-d are magnifcations for a better visualization of the intercalation/deintercalation peaks. From these profles the correlation between the TFSI intercalation and de-intercalation peaks can be realized, which are marked by Roman numerals. The correlation of these peak positions is also summarized in Table 2. When the upper cut-off potential is set to 5.2 V vs. Li/Li + , six TFSI intercalation peaks are observable, while the main intercalation takes place in the potential range from 4.36 V to 4.65 V vs. Li/Li + (II and III) and at 4.97 V vs. Li/Li + (VI). In relation to that, also six corresponding de-intercalation peaks/potential regions can be identifed (Table 2), ranging from 4.91 V to 4.64 V vs. Li/Li + (III 0 to VI 0 ) and from 4.44 V to 3.90 V vs. Li/Li + (I 0 and II 0 ). If the cut-off potential is decreased, e.g. to 5.0 V or 4.9 V vs. Li/Li + , the TFSI intercalation peaks V and VI diminish and/or disappear, while, in the same way, the deintercalation peaks VI to III weaken or completely disappear. In contrast, the intercalation peaks I to IV and the de-intercalation peaks I 0 and II 0 nearly stay constant, independent of the upper cut-off potential from 4.8 V to 5.2 V vs. Li/Li + . In order to gain a better understanding of the influence of the electrolyte additive on the TFSI anion intercalation/deintercalation behavior, the differential capacity profles from the Li/KS6L graphite dual-ion cell using the pure and the ES-containing IL-electrolyte are compared in Fig. 6. In addition, Table 2 also lists the peak positions for anion intercalation/deintercalation from the pure ionic liquid electrolyte. It is obvious that the electrolyte additive displays a strong influence on the TFSI uptake/release into/from graphite since the potential ranges for intercalation/de-intercalation as well as the intensities of these peaks completely change (Fig. 6). While for the pure ionic liquid fve main intercalation and de-intercalation peaks/ potential regions (I-V and I 0 -V 0 ) can be seen, the IL with ES additive displays six peaks, as described above. In particular, the onset potential for the anion uptake is shifted to a lower value for the ES-containing electrolyte: 4.26 V compared to 4.37 V vs. Li/Li + (peaks I and I). In addition, the main intercalation peak for the additive containing electrolyte is below 5.0 V vs. Li/ Li + (peak VI), while for the pure ionic liquid the main intercalation takes place above 5.0 V vs. Li/Li + (peak V). Anion deintercalation from the additive-based electrolyte starts at a higher potential (4.91 V compared to 4.84 V vs. Li/Li + ) and also takes place at lower potentials (3.80 V vs. 3.98 V vs. Li/Li + , see Table 2). Overall, it can be summarized that the addition of ES as an additive to the Pyr 14 TFSI-LiTFSI ionic liquid results in a strongly enhanced discharge capacity for the anion intercalation into graphite. This enhancement may be related to a reduced coordination of Li + ions with TFSI anions in the presence of ethylene sulfte. Deshpande et al. reported on the mobility and transport properties of ionic liquids, in particular on an enhancement of the lithium ion mobility in ionic liquid-based electrolytes which contain electrolyte additives, such as ethylene carbonate, vinylene carbonate or tetrahydrofuran. 29 They proposed that the inclusion of organic additives decreases the extent of coordination of the lithium ion with the IL-anion. 29 Furthermore, Bayley et al. reported that electrolyte additives will influence the transport properties of cations and anions in ionic liquid electrolytes, i.e. the diffusion coefficient of the anion may be enhanced by addition of certain organic diluents. 30 These effects may also explain the differences, i.e. the increased discharge capacity and reduced onset potential for the anion intercalation process into graphite from the ES containing IL in comparison with the pure ionic liquid electrolyte. However, further investigations are necessary to confrm this assumption. ## In situ X-ray diffraction study of the anion intercalation into graphite X-ray diffraction studies of the TFSI anion intercalation were performed in order to examine whether the increase in discharge capacity also results in a change of the structural composition of the GIC, i.e. in a higher stage formation, for the additive containing electrolyte compared to the pure ionic Table 2 Correlation between the different peaks for TFSI intercalation and de-intercalation into/from KS6L graphite and the corresponding peak potential positions, according to the differential capacity profiles in Fig. 6 Pyr liquid-based system. In a previous publication, we reported on the XRD investigation of the TFSI intercalation into graphite for the pure Pyr 14 TFSI-LiTFSI electrolyte. 10 Here, we showed that the anion intercalation takes place via a stage formation process and calculated the maximum stage number, periodic repeat distance and gallery height for the C n + TFSI GIC. 10 Fig. 7a illustrates the frst cycle of the in situ XRD pattern for TFSI intercalation/de-intercalation from the ES containing electrolyte, which was carried out by cyclic voltammetry (CV). Fig. 7b displays the voltage and specifc current profles in dependence of time from the CV experiment. The scan rate was set to 50 mV s 1 and a constant voltage step of 180 minutes was conducted at the upper cut-off voltage of 5.20 V. For a better visualization, the frst 22 XRD scans in the back of Fig. 7a are not depicted, since the (002) peak of graphite with relatively high intensity remains unaffected until the frst anion intercalation takes place at about 4.55 V (Fig. 7b). To enable a better comparison between the XRD pattern and the cyclic voltammogram, representative colored XRD scans (Fig. 7a) can be correlated with the colored dots (with the same color) in the voltage and current profles (Fig. 7b). In addition, Table 3 summarizes the values for the most dominant stage number n, the periodic repeat distance I c , the TFSI gallery height d i , and the gallery expansion Dd, which are calculated in accordance with our previous publication. 10 For comparison reasons, also the corresponding values for the pure ionic liquid electrolyte are listed in Table 3. The scan 23 for the pristine KS6L graphite electrode shows the (002) peak of graphite as expected at 2q ¼ 26.55 (Fig. 7a). By charging the cell to 4.55 V, the (002) graphite peak decreases in intensity and splits up into two new peaks, one more dominant peak (00n + 1) at 24.7 and the second (00n + 2) at 29.7 (scan 24). 31,32 By determining the ratio of the d (n+2) /d (n+2) peak positions and correlating these with the ratios of stage pure GICs, given in our previous publication, 10 it is possible to calculate the most dominant stage phase of the observed GIC. 31,32 Here, the frst observable intercalation stage is 4 (Table 3). When the voltage is increased to 4.86 V, the stage is raised to the next higher level of 3 (scan 29). Passing stage 2 by scan 33 at 5.10 V, the maximum intercalation stage 1 is reached during the constant voltage step at 5.2 V, exemplarily represented by scan 40 (Fig. 7 and Table 3). As the de-intercalation starts at 5.06 V (compare with peak VI 0 in Fig. 6), the intercalation stage changes to 2 at 4.40 V (scan 57) and to 4 at 4.16 V (scan 61). In contrast, for the pure ionic liquid electrolyte only a maximum stage number of 2 is observed under the same operation conditions (Table 3). These results confrm that by addition of ES to the electrolyte, not only the discharge capacity is increased, but also the composition changes from a stage-2 GIC to a stage-1 GIC. The TFSI gallery height of about 8 and thus the gallery expansion of 4.65 nearly stay unaffected from the type of electrolyte as well as from the stage number (Table 3). ## Dual-graphite cell performance Considering the investigations performed in different half-cell set-ups, it is obvious that the Pyr 14 TFSI-LiTFSI-based electrolyte in combination with the additive ES enables frst the reversible lithiation of graphite and second improves the anion intercalation into the graphite cathode. For this reason, dual-graphite full-cell investigations were conducted. Fig. 8 and 9 illustrate the cycling behavior of the dualgraphite cells, performed with constant charge/discharge currents of 10 mA g 1 and 50 mA g 1 , respectively. In the second case, three formation cycles with a lower current were carried out (10 mA g 1 ) before increasing the current to 50 mA g 1 (Fig. 9). Assuming a theoretical capacity of 100 mA h g 1 , a specifc current of 10 mA g 1 would correspond to a C-rate of 0.1. The upper cut-off voltage for these experiments was varied between 4.8 V and 5.2 V, in order to investigate the influence on the Coulombic efficiency, discharge capacity and cycling stability. The corresponding values of the cycling data from representative cycles, including the Coulombic efficiency and discharge capacity, are given in Table 4. In general, the discharge capacity increases with a rising upper charging end voltage. At a constant current of 10 mA g 1 , the discharge capacity changes from 60 mA h g 1 at 4.8 V to 121 mA h g 1 at 5.2 V (representative value for the 50 th cycle, see Table 4). By the increase of the specifc charge/discharge current to 50 mA g 1 (Fig. 9), the discharge capacity decreases for all cutoff voltages compared to cycling at 10 mA g 1 . At 4.8 V, a discharge capacity of 32 mA h g 1 is measured, which can be enhanced to 113 mA h g 1 at 5.2 V. A detailed study on the influence of the specifc current on the electrochemical performance of the dual-graphite cell was reported in our previous work. 20 Here, we found that the charge process (¼ intercalation of lithium ions and TFSI anions into graphite) is the rate-determining step, while a discharge capacity ranging from 100 mA h g 1 to 30 mA h g 1 is achieved for a specifc current that varies from 10 mA g 1 to 500 mA g 1 . 20 Fig. 8 and 9 depict the frst 50 cycles of the charge/discharge cycling and reveal a stable cycling behavior, independent of the upper cutoff voltage. Fig. 10 displays representative cell voltage (black curve, left yaxis) as well as the anode and cathode potential profles vs. Li/ Li + (red curves, right y-axis) for the CG/KS6L dual-graphite cell during selected cycles (1 st , 2 nd , 49 th and 50 th cycles) of the constant current charge/discharge process. The upper and lower cell voltages are 5.1 V and 3.0 V, respectively, and are given Table 3 The dominant stage index and calculated values for the in situ X-ray diffraction measurement of the TFSI intercalation into graphite from Pyr 14 TFSI, 1M LiTFSI + 2 wt% ES electrolyte for the lithium/KS6L graphite dual-ion cell. Data correspond to Fig. 7. The comparison to the TFSI intercalation from Pyr 14 TFSI, 1 M LiTFSI is given according to ref. by the difference of the cathode and anode potentials. 33 The charge/discharge rate for the frst three cycles was 10 mA g 1 , while the following cycles were performed at 50 mA g 1 (corresponding to Fig. 9; 5.1 V cut-off voltage). From the cell voltage and potential profles of the anode and cathode, it is obvious that the frst cycle differs from the following cycles, which is mainly related to the SEI formation and electrolyte decomposition taking place at the graphite anode as well as to the frst intercalation of TFSI anions into the graphite cathode. In the frst charge process, the anode potential reaches 0.115 V vs. Li/Li + , which means that the graphite anode is not fully intercalated by lithium ions, i.e. stage 2 is observed (LiC 12 ). 34 The transition to stage 1 occurs at a potential of ca. 0.09 V vs. Li/Li + and is thus about 25 mV below the potential plateau of stage 2. 34 In other words this means that the anode is oversized in terms of capacity, which is desired in order to avoid lithium metal plating and therefore safety issues at the anode. 33 According to the behavior of the anode potential, the cathode potential in the frst cycle rises to a maximum of 5.21 V vs. Li/Li + . At an increased rate (50 mA g 1 ), the anode potential drops to 0.04 V vs. Li/Li + and the cathode potential drops to the same extent to 5.14 V vs. Li/Li + , which may be related to an increased cell resistance. The potential profles for the anion intercalation have been discussed in detail in previous publications. 6,28,35 Overall, it can be observed that the intercalation of both lithium ions and TFSI anions into the graphite anode and cathode, respectively, occurs with a high reversibility leading to a high cycling stability. Recently, we reported a capacity retention of more than 98% for this dualgraphite system after 500 charge/discharge cycles at a rate of 50 mA g 1 and at an upper cut-off voltage of 5.0 V. 20 The Coulombic efficiency of the dual-graphite system varies in the frst charge/discharge cycle between 69.5% and 75.0% (Table 4), which is mainly related to SEI formation and electrolyte decomposition at the anode as well as the frst anion intercalation into the graphite cathode (kinetic activation), as described above. The efficiency in the frst cycle seems to be independent of the upper cut-off voltage, as no correlation can be seen. Since the electrolyte in this system can be considered as an active material, an excess of electrolyte is necessary due to the irreversible consumption of lithium for the SEI formation. In the subsequent cycles, the Coulombic efficiency increases and varies, e.g. in the 50 th cycle, between 97.2% and 98.2% at a charge/discharge rate of 10 mA g 1 (Table 4 and Fig. 8), while the highest efficiency is reached at a cut-off voltage of 5.0 V. At an enhanced rate of 50 mA g 1 , the efficiency can be further increased and varies between 98.6% and 99.1% (Table 4 and Fig. 9). Here, the highest efficiency is obtained at 5.0 and 5.1 V. This trend can most likely be explained by a decreased decomposition of intercalated TFSI anions as well as a decreased self-discharge for an increased charge/discharge current, which we observed in current rate investigations for the dual-ion system. Thus a higher rate results in an enhanced Coulombic efficiency. 6,28 The influence of the cycling rate on the coulombic efficiency was studied in more detail by Smith et al. for different lithium-ion full cells based on graphite negative electrodes. 36 They reported that the time of one cycle is the dominant contributor to the irreversible capacity (coulombic inefficiency) for cells that were cycled at low rates (e.g. C/24), which indicated that parasitic reactions consuming charge proceed independent of the cycling rate. 36 Since the electrolyte oxidation and decomposition can be considered as parasitic side reactions for the dual-graphite cell, the departure of the coulombic efficiency from unity (1.0000.) will increase with cycle time and behave inversely with the C-rate. In particular at elevated operation temperatures (e.g. 60 C), the efficiency will decrease remarkably. This is also what we reported in a recent publication, showing the influence of the operation temperature on the electrochemical performance of the dual-ion system. 28 Fig. 11 displays the constant current charge/discharge cycling behavior and efficiency curves for the dual-graphite system cycled at 500 mA g 1 (corresponding to 5C if a theoretical capacity of 100 mA h g 1 is assumed). The system exhibits a stable cycling performance over 500 cycles with a discharge capacity of about 50 mA h g 1 . In addition, the Coulombic efficiency displays an average value of 99.8%. As discussed above, one can assume that the increased charge/discharge rate results in a reduced decomposition of intercalated anions as well as reduced self-discharge reactions. ## Conclusion In this work, we demonstrated promising results for the electrochemical performance of a "dual-graphite cell" using the ionic liquid-based electrolyte mixture Pyr 14 TFSI-LiTFSI in combination with ethylene sulfte (ES) as an SEI-forming additive. The main issue for this dual-graphite cell is to fnd a suitable electrolyte mixture that frst enables reversible lithium ion intercalation/de-intercalation into the graphite negative electrode by formation of a stable solid electrolyte interphase (SEI) as well as second allows the reversible intercalation/deintercalation of the electrolyte salt anions (here: TFSI anions) into the graphite positive electrode with a sufficiently high Coulombic efficiency. This in turn is dependent on a high oxidative stability of the electrolyte mixture, in particular on the stability of the intercalated anion. By the use of the Pyr 14 TFSI-LITFSI ionic liquid electrolyte with 2 wt% of ES, it was possible to cycle the dual-graphite cell with a high reversibility, avoiding graphite exfoliation at the anode which is extensively observed for the pure ionic liquid. Furthermore, we found that the discharge capacity, which is related to the anion intercalation for a metallic lithium/graphite dual-ion cell, is nearly doubled if ES as an additive is used. At an upper cut-off potential of 5.0 V vs. Li/Li + , a discharge capacity of 97 mA h g 1 is reached, while only 50 mA h g 1 is obtained if no electrolyte additive is present. By the use of in situ XRD measurements we studied the anion intercalation into graphite with respect to the stage formation and structural composition. It was possible to determine the maximum stage number as well as to calculate the periodic repeat distance, the TFSI gallery height and the gallery expansion. From these investigations, we found that by the use of ES as an electrolyte additive a maximum stage number of 1 is reached in the frst cycle, while for the pure ionic liquid only a stage 2 is obtained. Therefore, the addition of ethylene sulfte to the electrolyte not only results in an enhanced discharge capacity for the anion uptake, but also in a different structural composition. For the pure ionic liquid, we found a maximum capacity of 115 mA h g 1 for the TFSI uptake into graphite, which corresponds to a stage-1 GIC with a stoichiometry ranging between C 19 TFSI and C 20 TFSI. 10 However, these results were obtained at an increased operation temperature of 60 C. 10 For the Pyr 14 TFSI-LiTFSI electrolyte with ES as an additive we determined a maximum capacity of 126 mA h g 1 , which corresponds to a stage-1 GIC with a stoichiometry of ca. C 18 TFSI. The enhancement in the specifc capacity and the change in the potential profle for intercalation/de-intercalation, e.g. a reduced onset potential for anion intercalation, may be related to a reduced coordination of Li + ions with TFSI anions in the presence of ES, i.e. the diffusion coefficient of the anion may be enhanced by addition of ES. However, these assumptions need to be confrmed in future investigations. The constant current charge/discharge cycling investigations of the dual-graphite system displayed a stable cycling performance over 50 cycles with a specifc discharge capacity varying from 32 mA h g 1 to 121 mA h g 1 depending on the upper charging end voltage and the rate. In a previous publication, we showed that a stable cycling is realized even over 500 cycles for this dual-graphite system. 20 Enhanced charging end potentials and lower charge/discharge rates result in a higher specifc capacity. The Coulombic efficiency can also be tailored by the charging end potential and rate, while here a contrary trend was found. With increasing rate and decreasing upper charging end potential, the Coulombic efficiency can be enhanced and may exceed 99%. Recently, we reported that the charge process (¼ intercalation of lithium ions and TFSI anions into graphite) is the ratedetermining step for the dual-graphite system, while a discharge capacity ranging from 100 mA h g 1 to 30 mA h g 1 can be achieved for a specifc current that ranges from 10 mA g 1 to 500 mA g 1 . 20 Here, we could additionally show that a stable charge/discharge cycling performance over 500 cycles at a specifc current of 500 mA g 1 (corresponds to 5C if a theoretical capacity of 100 mA h g 1 is assumed) is achieved with a specifc

chemsum

{"title": "Dual-graphite cells based on the reversible intercalation of bis(trifluoromethanesulfonyl)imide anions from an ionic liquid electrolyte", "journal": "Royal Society of Chemistry (RSC)"}

a_free_aluminylene_with_diverse_σ-donating_and_doubly_σ/π-accepting_ligand_features_for_transition_m

## Abstract: We report herein the synthesis, characterization, and coordination chemistry of a free N-aluminylene, namely a carbazolylaluminylene 2b. This species is prepared via a reduction reaction of the corresponding carbazolyl aluminium diiodide. The coordination behavior of 2b towards transition metal centers (W, Cr) is shown to afford a series of novel aluminylene complexes 3-6 with diverse coordination modes. We demonstrate that the Al center in 2b can behave as: 1. a σ-donating and doubly π-accepting ligand; 2. a σ-donating, σ-accepting and π-accepting ligand; and 3. a σ-donating and doubly σ-accepting ligand. Additionally, we show ligand exchange at the aluminylene center providing access to the modulation of electronic properties of transition metals without changing the coordinated atoms. Investigations of 2b with IDippCuCl (IDipp = 1,3-bis(2,6-diisopropylphenyl)imidazol-2-ylidene) show an unprecedented aluminylene-alumanyl transformation leading to a rare terminal Cu-alumanyl complex 8. The electronic structures of such complexes and the mechanism of the aluminylene-alumanyl transformation are investigated through density functional theory (DFT) calculations. ## Introduction Ancillary ligands play essential roles in modern synthetic chemistry and materials science. It is well-known that L-type ligands can not only donate electron density to transition metal centers (σ-donating) but also accept d-electrons from the metal centers via π-backdonation (π-accepting). Such ligands in the coordination sphere of transition metals can also exhibit the σaccepting ability to act as a Lewis acid for external ligands. According to the coordination modes of terminal L-type ligands (Figure 1a), they can be classified into four broadly defined categories, namely σ-donating/π-accepting type I, σ-donating and doubly π-accepting type II, σ-donating and doubly σ-accepting type III, and σ-donating, σand π-accepting type IV. Ligands based on Al have attracted considerable attention due to the fundamental significance of the structural and electronic properties as well as their applications in synthetic chemistry. The electropositive nature of aluminium (χ = 1.61) makes such ligands highly electron-releasing, thereby exhibiting unusual bonding and reactivities. In the case of the terminal L-type Al ligands, representative examples include transition metal complexes A and B [3g-j] derived from Schnöckel's (Cp*Al)4 and Roesky's HC[(CMe)(NDipp)]2Al, respectively (Figure 1b). It was independently demonstrated by the Power group [3g] and Crimmin group [3h] that unprecedented low-valent molecular complexes HC[(CMe)(NDipp)]2AlCu[(NMes)(CR)]2CH (R= Me, CF3) feature an unsupported dispersion-enhanced Al−Cu bond. Furthermore, in the late 1990s, the aluminylene complexes C of type III were disclosed by Fischer,Frenking et al. In 2014, Tokitoh and coworkers described the synthesis of terminal Pt-aluminylene complexes D bearing a di-coordinate Al atom via the reaction of a dialumene-benzene adduct with Pt(PCy3)2. The Al ligand in D reveals donor-acceptor interactions with Pt akin to the bonding mode of type II. Additionally, a few of aluminium-transtion metal hydride complexes have been shown to feature aluminylene character. [4k, 11] For transition metal-alumanyl complexes with a terminal X-type Al ligand, in two recent examples, Aldridge, Goicoechea et al. isolated an Au-alumanyl complex t Bu3PAuAl(NON) (NON = 4,5-bis(2,6-diisopropylanilido)-2,7-ditert-butyl-9,9-dimethylxanthene) containing an unprecedented nucleophilic Au center, while Hill, McMullin et al. reported the syntheses of two Cu-alumanyl complexes LCuAl(SiN Dipp ) (L = N,N'-diisopropyl-4,5-dimethyl-2-ylidene and (1-(2,6diisopropylphenyl)-3,3,5,5-tetramethyl-pyrrolidin-2-ylidene, SiN Dipp = (CH2SiMe2NDipp)2) with ambiphilic Cu−Al bonding. Taking advantage of sterically demanding terphenyl ligands, Power, Tuononen et al. very recently disclosed the first and sole example of a room-temperature-stable monomeric aluminylene (alanediyl) : 1b) via a reduction reaction of AlI2Ar iPr8 with 5% w/w Na/NaCl. This breakthrough allowed further explorations into unusual/unprecedented patterns of reactivity of E toward hydrogen and organic azides, in which the latter led to the first stable iminoalane with an Al≡N triple bond. In the present work, we report the synthesis, characterization and coordination chemistry of a free one-coordinate N-aluminylene (Figure 1c). Of note, this aluminylene functions as a σ-donating and doubly σ/πaccepting ligand for transition metals, leading to a series of unprecedented aluminylene and alumanyl complexes with diverse coordination modes via a simple one-step process. ## Results and Discussion Synthesis, Characterization and Bonding Analysis of N-Aluminylene. The installation of Al with bulky π-donor substituents, such as amino, phosphino or carbazolyl, should enhance the stabilization of the inherent electron deficiency of free aluminylenes due to the possible π-donation of a N/P lone pair into an accessible vacant p orbital at Al. We thus chose the carbazolyl-substituted aluminium diiodides 1 as the precursors (Scheme 1). These species were readily accessible from a salt metathesis reaction of the respective potassium carbazolide with AlI3, and their structures were confirmed by single crystal X-ray diffraction analysis (Figure S30). While all attempts of reducing 1a afforded an unidentified mixture, stirring a toluene solution of the more sterically encumbering 1b with excess 5% w/w K/KI (4 equivalents) from −15 to 13 o C for 2 days gave rise to the free aluminylene 2b as a white powder in 67% yield (Scheme 1). Scheme 1. Synthesis of 2b. Single crystals of 2b suitable for X-ray diffraction were obtained from slow evaporation of a concentrated n-hexane solution at room temperature within 12 h. The X-ray diffraction study revealed the N(1) atom adopts a planar environment (sum of angles: 359.3 o ) (Figure 2). The Al(1)−N(1) bond length (1.913(9) ) is slightly shorter than the Pyykkö standard value for an Al−N single bond (1.970 ) whereas much longer than those of typical Al=N double bonds (1.705(2)−1.725(1) ) in terminal aluminum imides, indicative of the presence of a weak N-to-Al π-donation. The Al(1) atom is located nearly symmetrically between the two flanking 3,5-di-tert-butylphenyl rings of the carbazolyl substituent. There is no strong secondary bonding interaction between Al and the two arenes in the solid state (the shortest Al−C distance: 3.015(3) ), which is similar to that observed for Power's :AlAr iPr8 . Infrared spectroscopic studies of 2b show no evidences for Al−H stretching frequencies (Figure S1). Crystalline 2b can be stored at room temperature under an inert atmosphere for over a month. A benzene solution of 2b was heated up to 80 o C for 10 h without noticeable decomposition. However, it is extremely sensitive to moisture and oxygen, leading to the complete scission of the Al−N bond affording the corresponding carbazole and unidentified Al-containing species (Figure S29). The ambiphilic nature of 2b is unambiguously demonstrated by its frontier molecular orbitals (M06-2X/def2-SVP) (Figure 3). The LUMO+6 and LUMO are mainly the in-plane and out-of-plane Al 3p orbitals, respectively (Figures 3a and 3b). The HOMO is composed of the lone pairs at both Al and N atoms as well as some π-bonding orbitals over the carbazolyl substituent, while the HOMO-1 predominantly involves the Al nonbonding lone pair (Figures 3c and 3d). These observations are different from those calculated for :AlAr iPr8 , illustrating that the N-substitution at Al dramatically affects the electronic structure of aluminylenes. Moreover, the natural population analysis (NPA) shows that the Al atom is positively charged (0.79 a.u.) and the N atom carries a negative charge (-0.96 a.u.). The Wiberg bond index (WBI) of the Al-N bond is 0.28 which can be explained by its substantial ionic nature. The second-order perturbation theory of the natural bond orbital (NBO) method reveals that the donor-acceptor interaction from a N lone pair into a vacant p orbital at Al has a small stabilization energy of 16.5 kcal mol -1 due to the electropositive nature of Al (χ = 1.61) (Figure S32). For comparison, the calculated stabilization energies arising from a N-to-Al π-donation in t Bu2AlNMes2 (Mes = mesityl) and (Mes*AlNPh)2 (Mes* = 2,4,6-( t Bu)3C6H2) are 4.4 and 21.3 kcal mol -1 , respectively (Figure S33). Compound 2b shows two absorption maxima in the UV/Vis spectrum in toluene at 346 and 356 nm (Figure S2), which are blue-shifted relative to those of :AlAr iPr8 (351 and 467 nm). These absorptions are attributed to the HOMO-LUMO and HOMO-1-LUMO transitions according to TD-DFT calculations (Figure S35). Isolation of Aluminylene Complexes. We thus speculated that 2b should be an interesting ligand featuring σ-donor and σ/πacceptor properties for transition metals if the Al atom is kinetically accessible. 2b is completely inert upon stirring its benzene solution with an equal molar portion of W(CO)6 at room temperature for 12 h. However, UV lamp (254 nm) exposure is known to facilitate the removal of CO in metal carbonyls, so the solution was irradiated for 24 h which cleanly furnished a new species 3 (Scheme 2). After workup, 3 was isolated as a yellow solid in 85%. The 1 H NMR spectrum of 3 shows two singlets for the t Bu groups of 3,5-di-tert-butylphenyl substitutes at 1.35 and 1.43 ppm, indicating the asymmetric nature with respect to the carbazolyl plane. Two singlet carbonyl resonances at 197.5 and 198.8 ppm are observed via a 13 C NMR spectroscopic study. Slow evaporation of a concentrated hexane solution of 3 at room temperature resulted in X-ray quality yellow crystals after 5 h. The solid-state structure of 3 was determined by X-ray diffraction (Figure 4a). In contrast to 2b, the N(1) atom in 3 is slightly pyramidalized (sum of angles: 351.8 o ), and the Al(1)−N(1) bond (1.841(3) ) is bent out of the carbazolyl plane, which consequently reduces the effective steric bulk of the substituent drastically. It is observed that the Al(1)−W(1) bond length (2.5363(11) ) in 3 is much shorter compared to those of (TMEDA)Al(Et)W(CO)5 (2.670(1) ) (TMEDA = N,N,N',N'tetramethylethylenediamine) and (TMPDA)Al(Cl)W(CO)5 (2.645(2) ) (TMPDA = N,N,N',N'-tetramethylpropanediamine), [9b] indicating the stronger π-backdonation from W to Al in our case. Although the only known examples of terminal base-free aluminylene complexes D (Figure 1b) reveal an almost linear geometry at Al (R = H, 179.2(2) o ; R = t Bu, 174.0(1) o ), the bond angle of N(1)−Al(1)−W(1) (147.31(10) o ) in 3 appears to be bent, likely due to the steric hindrance arising from two 3,5-di-tertbutylphenyl substituents. The aluminylene ligand in 3 acts as σdonor and double π-acceptor (vide infra). Species 3 represents the first example of an early transition metal-aluminylene complex with a di-coordinated Al atom. As the aluminylene ligand in 3 formally contains two vacant p orbitals, 3 should be susceptible to Lewis base coordination. Indeed, 3 rapidly converted to a new product 4 quantitatively in THF (Scheme 2). Alternatively, treatment of 2b with W(CO)6 in THF at room temperature yielded 4 as well in 60% yield. In an analogous fashion, the reaction of 2b with Cr(CO)6 in THF led to a species 5 as a white solid in 62% yield. The NMR spectroscopic features of 4 and 5 are very comparable. The 1 H NMR spectra of Scheme 2. Synthesis of 3-6. both cases display two diagnostic broad singlets (4: 0.96 and 3.30 ppm; 5: 0.96 and 3.32 ppm), integrating to four protons each. This suggests the presence of a coordinated THF molecule. Colorless single crystals of 4 and 5 were obtained via slow evaporation of their concentrated benzene solutions. In the solid state, species 4 and 5 appear to be a W-aluminylene and a Craluminylene complexes, respectively (Figures 4b and S34). The structural parameters of the carbazolyl aluminylene parts in 4 and 5 are similar. The N(1) atoms in both cases are clearly pyramidalized (sum of angles: 311.9 o (4), 316.9 o (5)), and the Al(1)−N(1) bonds (1.898(7) (4), 1.883(3) ( 5)) are bent out of the carbazolyl plane. This allow the coordination of a THF molecule to Al with a Al(1)−O(1) bond length of 1.862(5) (4) or 1.853(3) (5), thereby compensating the electron deficiency of Al. The bond lengths of Al(1)−W(1) (2.601(2) ) and Al(1)−Cr(1) (2.4087(12) ) are shorter than those seen for (TMEDA)Al(Et)W(CO)5 (2.670(1) ) and (TMPDA)Al(Cl)Cr(CO)5 (2.482(1) ), respectively. [9b] These imply the presence of the π-backdonation from W/Cr to Al. 123.63(5) o ). [9b] Of note, the aluminylene ligand in 4 and 5 behaves as σ-donor, σ-acceptor and π-acceptor (vide infra), and such bonding modes are extremely rare for coordination chemistry. 3c, 7 The σ-acceptor property of ligands has been invoked in mechanistic studies using aluminylene ligands [4k, 5n] and related gallylene systems. Importantly, Crimmin et al. disclosed that such property of aluminylenes is crucial to catalytic processes. [4k, 5n-p] In addition, the coordination behavior of free one-coordinate aluminylenes toward transition metals is hitherto unknown. [3g-j, 4a-j, 5a, 5d, 15-16] The formation of 3-5 demonstrates the facile access to metal-aluminylene complexes through this straightforward process. DFT modelling reveals that dissociation of the THF from 4 to produce 3 is only unfavorable by the free energy of 4.5 kcal mol -1 , indicative of the labile nature of the THF. We thus envisioned the possibility for ligand exchange reactions at Al. To this end, 4dimethylaminopyridine (DMAP) was employed (Scheme 2). Addition of 2 equivalents of DMAP to a toluene solution of 4 at room temperature immediately yielded a sole product 6, which was isolated as a yellow powder in 90%. A C6D6 solution of 6 displays a characteristic singlet at 2.13 ppm integrating for twelve protons corresponding to the methyl groups of DMAP in the 1 H NMR spectrum, and there is no evidence for the presence of THF. This suggests that the coordinated THF in 4 is completely replaced by two DMAP molecules. Indeed, in the solid state, 6 bears a tetracoordinate Al(1) center with the tetrahedron geometry (Figure 4c). The bond length of Al(1)−N(1) (1.9549( 17 of 3 (2.5363(11) ) and 4 (2.601(2) ), and slightly longer than that in (TMEDA)Al(Et)W(CO)5 (2.670(1) ). [9b] The formation of 6 undergoes a formal ligand exchange reaction at an aluminylene, reminiscent of scarce examples of ligand exchanges at low-valent main group centers, such as borylene, phosphinidene, carbene, and vinylidene. Moreover, is a rare example of complexes containing a group 13 ligand with the coordination type III (Figure 1a). [3h, 9b, 31] It is interesting to note that the presence of weak semi-bridging carbonyl interactions is observed with the asymmetry parameter (α) taking values of 0.50, 0.55, 0.56 and 0.55 for these complexes 3-6, respectively. Such values are slightly larger than those of HC[(CMe)(NDipp)]2AlFe(CO)3L (L = CO, 0.47; L = Cy3P, 0.49) reported by Crimmin and Kong. [3h] The electronic properties of the aluminylene ligands in 3, 4 and 6 were next established from the carbonyl stretching frequencies (νCO). With respect to the number of ligands at Al in the series N-Al(L)nW(CO)5 (n = 0-2), which can consecutively suppress Wto-Al π-backdonation while enhance Al-to-W σ-donation, there is significant decrease of the frequencies. 3 exhibits distinctly high frequencies (νCO 2060(νCO , 1974(νCO and 1922 cm -1 cm -1 ) indicative of reduced electron releasing ability of the Al ligand in 3 compared to those in 4 (νCO 2046, 1958 1897 cm -1 ) and 6 (νCO 2015, 1916 and 1854 cm -1 ). These modifications at the ligand site (i.e. coordination of THF or DMAP) drastically influence the electronic properties of the transition metal without changing the coordinated Al ligand. Analyses. For a better understanding bonding scenarios of 3, 4 and 6, density functional theory (DFT) calculations, coupled with energy decomposition analyses with natural orbitals for chemical valence (EDA-NOCV) calculations and intrinsic bond orbital (IBO) investigations were carried out. The IBO method is proven to give an exact representation of any Kohn−Sham DFT wave function. Inspections of IBOs of 3 demonstrate that the Al center forms two σ-bonds (Al−N and Al−W σ-bonds) (Figures 5a and 5b). It is observed that two formally vacant 3p orbitals of Al accept electron density from symmetrically accessible filled 5d orbitals of W, forming two apparent π-back-bonding (Figures 5c and 5d). This accounts for the relatively short Al(1)−W(1) bond length (vide supra). In contrast, the Al center of 4 is coordinated with a THF molecule and thus three σ-bonds (Al−N, Al−O and Al−W σ-bonds) at Al are observed (Figures 5e-5g), along with a W-to-Al π-back-bonding (Figure 5h). For 6, the coordination of two DMAP molecules prevents forming π-back-bonding (Figure S36), thereby giving four σ-bonds at Al (Al−W and three Al−N σ-bonds) (Figures 5i-5l). Additionally, EDA-NOCV calculations demonstrate that, in all cases, the orbital interactions ΔEorb are dominant between Al and W with the magnitude of -68.1, -71.1 and -92.3 kcal mol -1 for 3, 4 and 6, respectively (Figures S37-S39). Examinations of the deformation density plots allow visualization of this donoracceptor interaction (Figure S40). In all cases, the Al-to-W σdonation (3: -49.5 kcal mol -1 ; 4: -54.5 kcal mol -1 ; 6: -75.2 kcal mol -1 ) comprises the most significant contribution to ΔEorb, whereas the W-to-Al π-backdonation of 3 and 4 plays a minor role in contributions to ΔEorb (3: -12.8 kcal mol -1 ; 4: -6.0 kcal mol -1 ). Isolation of an Alumanyl Complex. Further reactivity explorations reveal that 2b is highly reducing and can readily react with (THT)AuCl (THT = tetrahydrothiophene) to afford the carbazolyl-substituted aluminium dichloride 7 as well as Au mirror (Figure S31). Repeated crystallization attempts of 7 yielded crystals of poor quality, nonetheless preliminary X-ray studies confirmed its formulation (Figure S31). In a similar vein, upon mixing 2b with IDippCuCl (IDipp = 1,3-bis(2,6diisopropylphenyl)imidazol-2-ylidene) in toluene at ambient temperature, a white solid of the Cu-alumanyl complex 8 was isolated in 71% (Scheme 3). The solid-state structure of 8 exhibits a planar Al(1) center with the sum of angles at 359.9 o (Figure 6). The Al(1)−Cu(1) bond length is 2.3448(13) , which is comparable to that seen for LCuAl(SiN Dipp ) (L = N,N'-diisopropyl-4,5-dimethyl-2-ylidene, 2.3450(6) ) whereas slightly longer with respect to that of HC[(CMe)(NDipp)]2AlCu[(NMes)(CMe)]2CH (2.3011(7) ). [3g] To date, the solid-state structural authentication of terminal Cualumanyl complexes is limited to LCuAl(SiN Dipp ) (L = N,N'diisopropyl-4,5-dimethyl-2-ylidene and (1-(2,6diisopropylphenyl)-3,3,5,5-tetramethyl-pyrrolidin-2-ylidene) and K[Cu[Al(NON)]2]. These species were formed by a salt metathesis reaction of the corresponding potassium aluminyl compound with a ligand-stabilized copper halide. It is important to note that the facile synthesis of 8 showcases a new avenue to terminal alumanyl complexes that are extremely rare and otherwise difficult to prepare. [4c, 4d, 12-13, 36-37] Scheme 3. Synthesis of 8. Mechanistic Investigations. The mechanism of the formation of 8 was probed via DFT calculations (SMD-M06-2X/def2-TZVP//M06-2X/def2-SVP) (Figure 7). The reaction begins with the approach of the aluminylene 2b toward the Cu atom of IDippCuCl. This prompts the slight pyramidalization of N and the formation of an Al−Cu dative bond to generate an intermediate IN1 (free energy of 11.3 kcal mol -1 ) in a barrier-less process (Figure S41). Subsequent oxidative addition of the Cu−Cl bond to Al proceeds via TS1, with the energy barrier of 13.4 kcal mol -1 (2b→TS1), to yield the stable product 8 (-4.8 kcal mol -1 ). Concurrent with this is the increase of the formal oxidation state of Al from +1 to +3. ## Conclusion In summary, a room-temperature-stable N-substituted free aluminylene 2b has been isolated and characterized by spectroscopic, crystallographic and computational techniques. While the planarization of the N atom coupled with two flanking 3,5-di-tert-butylphenyl rings of the carbazolyl substituent in 2b results in the thermodynamic and kinetic stabilization at Al, the facile pyramidalization of the same N atom making the Al kinetically accessible can occur upon treating 2b with a variety of transition metal complexes (i.e. W, Cr). This allows the isolation of a series of unprecedented aluminylene complexes 3-6. Interestingly, this Al ligand showcases σ-donor and σ/π-acceptor properties in diverse manners for transition metals. For 3, the Al is a σ-donating and doubly π-accepting ligand. For 4 and 5, the Al serves as a σ-donating, σ-accepting and π-accepting ligand. Significant to note is that 6 is prepared via an intriguing Alcentered ligand exchange reaction of 4 with DMAP and the Al functions as a σ-donating and doubly σ-accepting ligand. Infrared spectroscopic investigations show that such modifications of ligands at the Al (i.e. coordination of THF or DMAP) significantly affect the electronic properties of transition metals without changing the coordinated atoms. Finally, the first example of aluminylene-alumanyl conversion has been demonstrated, generating a Cu-alumanyl complex 8. Considering DFT calculations, the mechanism leading to 8 involves an initial Al−Cu coordination followed by an oxidative addition of a Cu−Cl bond at Al. We anticipate that these discoveries can pave a way for other unknown metal-alumanyl complexes. The utility of 2b in the production of other intriguing species, the subsequent chemistry of these new complexes and the extension of this Al ambiphilicity to catalysis are the subjects of ongoing work.

chemsum

{"title": "A Free Aluminylene with Diverse \u03c3-Donating and Doubly \u03c3/\u03c0-Accepting Ligand Features for Transition Metals", "journal": "ChemRxiv"}

mechanosynthesis_of_sydnone-containing_coordination_complexes

## Abstract: N-Phenyl-4-(2-pyridinyl) sydnone was shown to act as a fourelectron donor N,O-ligand in unprecedented coordination complexes featuring three different metallic centers (Co, Cu, Zn). Starting from various anilines, the use of ball-mill enabled efficiently the synthesis of N-arylglycines, subsequent nitrosylation and cyclization into sydnone, and further metalation.Sydnones, mesoionic heterocyclic compounds, are versatile building blocks for heterocyclic chemistry, for example in the formation of pyrazoles through copper-catalyzed 1,3-dipolar cycloaddition.1 They also display interesting biological activities, e.g., as antibacterial, antineoplastic and antiinflammatory agents.2 Sydnone synthesis from α-amino acids involves a nitrosylation step, using sodium nitrite in highly acidic media or isoamyl nitrite, 3 and subsequent cyclization using acetic or trifluoroacetic anhydride (TFAA). 4 In 2014, Shih et al. showed that palladium complexes featuring a sydnone-4carbaldehyde N(4)-phenylthiosemicarbazone ligand exhibited higher anticancer activities than 5-fluorouracil (Figure 1). 5 To date, this is the only reported example in which the exocyclic oxygen of the sydnone is shown to coordinate a metal. Indeed, in the literature, sydnones have been modified as phosphine ligands for palladium complexation (Figure 1), 6 directly metalated at C 4 , 7 through deprotonation or carbon-halogen insertion, or modified at N 3 with a 3-pyridine for iron coordination. 8 In this context, we wondered if the presence of a 2-pyridine at C 4 would enable the access to unprecedented coordination structures featuring sydnones as N,O-ligands. Given our expertise in mechanochemistry for organic and organometallic synthesis, 10 we envisioned a solvent-free mechanochemical approach 11 for an efficient access to sydnones and to unprecedented coordination complexes. During our study, we realized that accessing sydnone precursors could also benefit from mechanochemistry in terms of reaction time and yield. N-arylglycines were thus prepared via solvent-free mechanochemistry, using a 15 mL Teflon jar (filled with one 1 cm diameter stainless steel ball) agitated at 25 -30 Hz in a vibratory ball-mill (Scheme 1), through either alkylation of substituted anilines with ethylbromoacetate and subsequent saponification (one-pot two-step, Method A) or reductive amination involving glyoxylic acid and sodium cyanoborohydride (Method B). N-arylglycines featuring in para position electron-donating groups such as methyl (1b) or methoxy (1c) could be isolated in 95% and 80% yield over the two steps, respectively, using method A. When a bromine atom was either in meta or para position, method A was also found efficient, giving corresponding glycines in 85% (1h) and 76% (1d) yield, respectively. Mesitylamine, even though extremely sterically hindered reacted using method A to furnish the expected glycine 1i in 37% yield. Even if modest, this yield is four-fold higher than the 10% yield reported in literature. 12 When using anilines bearing electron-withdrawing groups such as NO 2 , CF 3 or CN, method B was found more ## This work • Novel structures • Reagent-less mechanosynthesis ## • Variety of transition metals Known structures Scheme 1. Mechanosynthesis of N-arylglycines 1a-i efficient, giving corresponding products in 53-74% yield (1e-g). In all the cases, yields obtained using the mechanochemical approaches were similar if not better compared to literature conditions in solution. 13 In addition, reaction time was limited to 2 -3 h, and a simple extraction/precipitation procedure was required to isolate pure compounds. Using N-phenylglycine 1a as model substrate, the synthesis of corresponding sydnone in a mechanochemical one-pot twostep approach was next investigated. Recently, the Taran group developed a one-pot procedure using tert-butyl nitrite and TFAA under solvent-free conditions and magnetic stirring, but sydnones were not isolated and directly engaged in a cycloaddition step. 14 Under mechanochemical activation, tBuONO was found to be efficient and nitrosylation was complete within 5 min, as confirmed by HPLC analysis (Table 1, entry 1). Addition of trifluoroacetic anhydride (2.5 equiv.) to the reaction mixture in the same jar furnished sydnone 2a in 51% yield. Even though this method is sodium nitrite and acid free, tBuONO has to be prepared from tert-butanol using these reagents. We reasoned that sodium nitrite, without the addition of concentrated HCl, could furnish the nitrosylated intermediate. Indeed, the two hydrogen atoms necessary for water release in the nitrosylation mechanism would in this a Total mass of the reagents was calculated to obtain a milling load (ML) of 20 mg.mL -1 . Reactions were performed in a vbm at 25 Hz. case come from the amine and acid functions of 1a. Gratifyingly, milling 1a and NaNO 2 in stoichiometric quantity for 5 min resulted in full conversion of 1a into N-nitroso-Nphenylglycinate. Subsequent addition of TFAA to the crude mixture and milling for 30 min yielded sydnone 2a in 54% yield (Table 1, entry 2). Increasing the milling time of the second step to 1 h enabled an excellent 95% yield (Table 1, entry 3). Notably, changing the stoichiometry of TFAA did not improve the final yield (Table 1, entries 4 and 5). Milling conditions were then applied to N-arylglycines 1a-i (Scheme 2). Reaction time and milling frequency were modulated to obtain in each case fast and full conversion, which enabled the avoidance of chromatography on silica gel. In all cases, total reaction time did not exceed 120 min with yields comparable to those obtained in solution. 13 Sydnones featuring methyl (2b) and methoxy (2c) groups in para position of the phenyl moiety were produced in 91% and 54% yield, respectively. With a bromine atom either in para or meta position, corresponding sydnones 2d and 2h could be obtained in 91% and 81% yield, respectively. Sydnones 2e-g, featuring NO 2 , CN and CF 3 electron-withdrawing groups were isolated in lower yields of 34 -46%. Even though full conversion was reached, a recrystallization step (for 2e-f) or a chromatography on silica gel (only for 2g) that reduced the final yield was required to furnish pure compounds. The same remark applies to sydnone 2i, featuring sterically hindered mesityl group, which was yielded in 53%. To the best of our knowledge, it is the first time that sydnone 2i is synthesized and fully characterized. ## 15, 7f The introduction of a pyridine at C 4 of the sydnone was next performed to obtain a putative bidendate ligand. N-Phenylsydnone 2a was reacted with 2-bromopyridine in the presence of Pd(PPh 3 ) 4 or Pd(OAc) 2 /XPhos as catalytic systems according to literature methods. 16 However, yields obtained were not reproducible and separation of XPhos ligand revealed complicated. Although other palladium-catalysed crosscouplings were successful in the ball-mill, 17 attempts to perform the coupling reaction with 2a under mechanochemical conditions failed. A quick study showed that this coupling could be performed using Pd(OAc) 2 /PPh 3 (1:2) in DMC (dimethylcarbonate), an environmentally friendly solvent, giving corresponding 3-phenyl-4-(2-pyridine)sydnone 3a in 55% yield, with a facilitated purification (Scheme 3). Bidentate compound 3a was then reacted, using a vibratory ball-mill, with various metallic salts (CoCl 2 , CuCl 2 , Cu(OTf) 2 and ZnCl 2 ) under solvent-free conditions. We were pleased to observe that after 1h of milling corresponding complexes were obtained in yields of 66-74%. Conversion was followed in the solid state using IR spectroscopy, as the C=O stretching band in 3a (ν = 1757 cm -1 ) shifts to lower wave numbers upon coordination (ν = 1660 -1726 cm -1 ). The structure of the complexes [Co(µ-Cl)(3a) 2 ] 2 .COCl 4 , [CuCl 2 (3a) 2 ], [Cu(OTf) 2 (3a) 2 ] and [ZnCl 2 (3a)] were confirmed by single crystal X-ray diffraction after crystallisation (Figure 2). 13 Interestingly, cobalt complex possesses a dimeric structure with two sydnone ligands on each metallic center, and bridging chloride atoms. This kind of arrangement was rarely observed in literature with bidentate N,O ligands. 18 For copper containing species, an octahedral geometry with two sydnone ligands and with a trans-arrangement between the two chloride (or triflate) ligands was observed. On the other hand, reaction with zinc(II) chloride provided the complex with only one sydnone ligand and a tetrahedral geometry. In all the X-ray structures, the pyridine and sydnone rings were not found in the same plane and a twist of 22 -35° was observed in the complexes. This non-planarity was also witnessed in the X-ray structure of 3a. 13 Such observation tends to indicate that pyridine and sydnone rings may not be conjugated. Additionally, the exocyclic C-O bond was measured at 1.21 -1.24 , which is consistent with a double bond character. Bidentate sydnone 3a may thus act as a four-electron rather than a three-electron donor ligand. In conclusion, the use of ball mills enabled the efficient reagent-less preparation of a series of N-aryl glycines and of corresponding sydnones. N-Phenyl-4-(2-pyridinyl) sydnone 3a was used for the first time as ligand to coordinate metals, leading to unprecedented compounds via a mechanochemical solvent-free approach. Such results pave the way for the development of novel families of coordination complexes. ## Conflicts of interest There are no conflicts to declare.

chemsum

{"title": "Mechanosynthesis of Sydnone-containing Coordination Complexes", "journal": "ChemRxiv"}

dynamics_of_hydration_water_plays_a_key_role_in_determining_the_binding_thermodynamics_of_protein_co

## Abstract: Interfacial waters are considered to play a crucial role in protein-protein interactions, but in what sense and why are they important? Here, using molecular dynamics simulations and statistical thermodynamic analyses, we demonstrate distinctive dynamic characteristics of the interfacial water and investigate their implications for the binding thermodynamics. We identify the presence of extraordinarily slow (~1,000 times slower than in bulk water) hydrogen-bond rearrangements in interfacial water. We rationalize the slow rearrangements by introducing the "trapping" free energies, characterizing how strongly individual hydration waters are captured by the biomolecular surface, whose magnitude is then traced back to the number of water-protein hydrogen bonds and the strong electrostatic field produced at the binding interface. We also discuss the impact of the slow interfacial waters on the binding thermodynamics. We find that, as expected from their slow dynamics, the conventional approach to the water-mediated interaction, which assumes rapid equilibration of the waters' degrees of freedom, is inadequate. We show instead that an explicit treatment of the extremely slow interfacial waters is critical. Our results shed new light on the role of water in proteinprotein interactions, highlighting the need to consider its dynamics to improve our understanding of biomolecular bindings.Water is an active and indispensable component of cells. Understanding its versatile roles in determining the structure and dynamics of biomolecules and mediating their interactions is of fundamental importance 1-3 . The versatility of water in biological contexts arises from its ability to alter its characteristics depending on its interaction with biomolecules. For example, the DNA sequence-dependent behavior of hydration water serves as a sequence-specific "hydration fingerprint" 4 ; changes in water dynamics during binding of a substrate to an enzyme play a vital role in protein-ligand recognition 5 ; and the non-bulk behavior of water inside the translocon strongly affects the partitioning of hydrophobic segments from the translocon to the membrane 6 . However, although our understanding of the behavior of hydration water around biomolecules has advanced significantly in recent years [7][8][9][10][11][12][13][14] , it remains a challenge to elucidate the extent to which water molecules located between two biomolecules are modified through concurrent interactions with the two binding surfaces and how such altered water molecules in turn affect the binding affinity.In this connection, it has been suggested that water-mediated contacts substantially complement direct protein-protein contacts, providing an additional layer of biomolecular recognition 15,16 . The necessity of an explicit treatment of interfacial water molecules to properly describe such water-mediated interactions has also been noted 17 . Indeed, recent computational studies have reported on the relevance of explicitly handling "key" interfacial waters in protein-protein interaction 18 and protein-ligand binding 19 : for example, including two, rather than all, interfacial water molecules was crucial to correctly obtaining the trends observed in mutation effects on protein-protein binding affinity 18 ; in another study, explicitly taking into account interfacial water molecules ranging in number (N wat ) from 30 to 70 significantly improved the correlation with the experimental binding affinities for four different systems, where the optimum value of N wat depended on the specific system 19 . What, however, distinguishes those key interfacial water molecules from others? Do any distinctive characteristics of the interfacial water emerge upon protein-protein complex formation? In this paper, we investigate the dynamic and thermodynamic features of interfacial water in the barnasebarstar complex 15 . This is a well-studied paradigm for protein-protein interactions and is also an ideal system for analyzing the interfacial water because X-ray measurements indicate the presence of waters filling the gap between the binding surfaces 15,20 . We perform molecular dynamics simulations to explore dynamic characteristics of the interfacial water. We focus on the rearrangements of hydrogen bonds, which are the most important protein-water interaction because the protein-protein binding surfaces comprise mainly polar and charged residues 21 . We then conduct statistical thermodynamic analyses to rationalize the dynamic characteristics of the interfacial water. Finally, we discuss the impact of the interfacial water dynamics on the protein-protein binding affinity. We find that the conventional approach to the water-mediated interaction, which assumes the time-scale separation between the protein and hydration water dynamics, fails owing to the extremely slow dynamics exhibited by the interfacial waters. We show instead that an explicit treatment of those slow waters as an integral part of biomolecules is critical. Thereby, we would like to shed new light on the role of water in protein-protein interactions based on a dynamic view point. ## Methods Molecular dynamics simulations. We conducted explicit-water molecular dynamics simulations for the barnase-barstar complex (Fig. 1) and for the free barnase and barstar proteins. The initial complex structure was modeled based on the X-ray structure (PDB ID: 1 BRS 15 ) as detailed in ref. 22. The starting structures of the free barnase and barstar simulations were taken from their NMR structures (1 BNR 23 and 1 BTA 24 , respectively). The complex structure was solvated by 23,477 waters and neutralized by 4 counter Na + ions in a cubic box of the initial side length 95.4 ; the free barnase (barstar) was solvated by 14,346 (8,397) waters and neutralized by 2 Cl − (6 Na + ) ions in a cubic box of the initial size 81.7 (69.5 ). All the simulations were performed using the AMBER14 suite 25 with the FF99SB force field 26 for proteins and the TIP3P model 27 for water. The temperature and pressure were maintained at T = 300 K and P = 1 bar using the Berendsen's method 28 . We adopted the same simulation procedures as described in ref. 22, and three independent 1 μs production runs were performed for each system starting from different random initial velocities. We also conducted pure-water simulations to obtain dynamical quantities for bulk water. Three independent 100 ns simulations were performed at T = 300 K and P = 1 bar with 2,539 waters. Hydrogen-bond rearrangement dynamics. We analyzed the hydrogen-bond time-correlation function, which quantifies the extent to which hydrogen bonds found at time t = 0 survive to subsequent times t 29 , to investigate hydrogen-bond rearrangements between protein and hydration water. A hydrogen bond is considered formed when the water oxygen is located within 3.5 from heavy atoms in a protein. The hydration water is classified as follows (see Fig. 2a for an illustration). A water molecule forming a single hydrogen bond to a protein is referred to as single HB water. The locations of single HB waters in a simulation snapshot are indicated by cyan spheres in Fig. 2b, and their average number (±standard deviation) computed from the whole simulation trajectories for the complex is 325.7 ± 13.7 (Table 1 summarizes the number of water molecules and the number of water-protein hydrogen bonds). A water molecule making two or more hydrogen bonds to a protein is termed double HB water: the positions of double HB waters in a snapshot are shown by orange spheres in Fig. 2b. There are 133.0 ± 8.0 double HB waters in the system, and the average number of hydrogen bonds to a protein is 2.4 ± 0.1. Finally, a water molecule forming concurrent hydrogen bonds with two proteins is called bridging water. By definition, bridging waters are present only at the interface between two proteins (red spheres in Fig. 2b). We find 19.6 ± 3.0 bridging waters located at the interface, with the average number of water-protein hydrogen bonds being 2.9 ± 0.2. We investigate the hydrogen-bond time-correlation function defined by Trapping free energy. We introduce the trapping free energies of individual hydration waters to quantify how strongly they are bound to the protein surface. The trapping free energy refers to the reversible work (i.e., the potential of mean force) for transferring a water molecule from an infinite separation to a specific position and orientation relative to the protein-protein complex. We consider here the transfer process to a fixed position and orientation relative to the solute for two reasons. First, this allows us to compute the trapping free energies of individual hydration waters solely based on the simulation snapshots such as the one presented in Fig. 2b. Second, we are interested in whether the trapping free energies so computed at time t = 0 serve as a descriptor of the degree of retardation of the subsequent (t > 0) dynamics of individual water molecules. The thermodynamic cycle shown in Fig. 3 is used to obtain this quantity; in this cycle, we consider the transfer of the i-th water molecule to a specific position and orientation around the solute u, which includes the hydration water molecules of interest (e.g., all the waters displayed in Fig. 2b). The solute from which the i-th water molecule is excluded is denoted as u′. Process (1) in Fig. 3 is the independent solvation processes of the i-th water molecule and the solute u′; hence, the associated Gibbs free energy change is given by G G G u (1) w ater solv solv ∆ = + ′ , which consists of the solvation free energies of a single water molecule (G water solv ) and of the solute u′ ( ′ G u solv ). In process (2), the i-th water molecule is transferred to a specific position and orientation around the solute u′ from an infinite separation in vacuum. The reversible work required for this process is given by the interaction energy ′ − E u i between the solute u′ and the i-th . Process (3) is the solvation of the solute u(=u′ + i), and we obtain . From the thermodynamic cycle, one obtains the trapping free energy from -ΔG (1) + ΔG (2) + ΔG (3) ; that is, Table 1. Average number of waters and water-protein hydrogen bonds a . a Average ± standard deviation. We computed the interaction energy ′− E u i from the force field, whereas the solvation free energy G u solv is obtained using the 3D-RISM theory 30 , whose details are presented in Supplementary Methods. An efficient method for computing the contribution G G ′ which is based on the atomic decomposition of the solvation free energy 31,32 is also provided there. The trapping free energies for the hydration waters surrounding free barnase and barstar proteins can be obtained in a similar manner. Recently, several computational methods have been developed for evaluating thermodynamic functions of individual hydration waters . However, these methods typically demand performing additional distinct simulations. For example, the application of the inhomogeneous solvation theory requires conducting simulations in which restrains are added on protein atoms to sample waters' positions and orientations for a given protein conformation. Further complications in analysis will arise when hydration waters exchange with bulk waters during those additional simulations. On the other hand, our computational method for the trapping free energy that employs the integral-equation theory (3D-RISM) is applicable solely based on snapshots taken from unrestrained equilibrium simulations, and it is in this sense more computationally efficient. Standard binding free energy. Conventional approach. The statistical thermodynamic expression for the standard binding free energy is given by refs 22 and 42 Here, ΔX denotes the change in X upon complex formation from two free proteins (labeled 1 and 2), solv comprises the gas-phase energy (E u ) and the solvation free energy (G u solv ) of the solute u (here, u refers to the complex or one of the two free proteins and excludes hydration waters); the bar denotes the average over the simulated configurations; S config is the configurational entropy associated with the solute's internal degrees of freedom; and ΔS ext is the entropy change originating from the reduction in the external (positional and orientational) degrees of freedom upon complex formation. ΔS ext carries the standard-state dependence, which is chosen here to be the one of the standard concentration (1 M). We computed the gas-phase energy E u from the force field adopted in the simulations. (E u for free proteins represents only the intra-protein energy, but E u for the complex includes the inter-protein interaction energy as well). For the solvation free energy G u solv , we employed the 3D-RISM theory 30 (see Supplementary Methods). For the configurational entropy S config , we used an energetic approach 43,44 that expresses S config in terms of the statistical properties of f u . In particular, when the probability distribution W(f u ) of f u is Gaussian, the following holds: where k B is Boltzmann's constant, and f f f u u u δ = − . For the external entropy ΔS ext , we use the estimate TΔS ext = −6.8 ± 0.1 kcal/mol for the barnase-barstar complex, which was computed in ref. 22. by extending the energetic approach to the binding process and is close to the value reported in ref. 45. ## Explicit inclusion of the water molecules of interest. A statistical thermodynamic formulation of the binding free energy which allows one to explicitly include certain solvent molecules was also derived in ref. 42. In essence and using our notation, what is required is to replace In this expression, the solute u now explicitly includes the water molecules of interest (e.g., E u now contains interactions with and among those water molecules), n is the number of water molecules included, and G water solv is the single water molecule's solvation free energy. S config then needs to be evaluated by combining equations ( 4) and (5). ## Results and Discussion Hydrogen-bond rearrangement dynamics. We study the dynamic and thermodynamic features of the hydration water surrounding the barnase-barstar complex by conducting molecular dynamics simulations and statistical thermodynamic analyses. In particular, we aim to uncover the distinctive characteristics of the interfacial water between two proteins that emerge upon complex formation. This is done by contrasting the dynamics of the interfacial water with that of the hydration water surrounding free proteins; for this purpose, we also perform simulations and analyses for the free barnase and barstar proteins. We focus on the rearrangements of hydrogen bonds, which are the most important protein-water interactions because of the largely hydrophilic nature of the protein-protein binding surfaces 21 . Figure 4 shows the hydrogen-bond time-correlation functions, which quantify the extent to which hydrogen bonds found at time t = 0 remain at subsequent times t. For water molecules making a single hydrogen bond to a protein (referred to as single HB water; see Fig. 2 and Table 1), we observe profound slowing down of the relaxation dynamics compared to those of bulk water (see Table 2 for a comparison of the average relaxation times). For water molecules making two or more hydrogen bonds to a protein (double HB water), the hydrogen-bond rearrangement is even slower. For bridging water molecules, that is, interfacial water molecules making concurrent hydrogen bonds with two proteins, the relaxation is extraordinarily slow (~1,000 times slower than the relaxation in bulk water). Furthermore, the relaxation curve is anomalous, exhibiting a logarithmic decay over three orders of magnitude in time. ## Thermodynamic-dynamic relationship diagram. To rationalize the slow relaxations of hydration waters, we conducted statistical thermodynamic analyses. We focused on the long-time region where the time-correlation functions decay from 0.3 to 0.1 (light yellow region in the upper panel of Fig. 5) and extracted the water molecules contributing to the relaxation there by examining the hydrogen-bond survival times (τ i ) of individual molecules. For each of those water molecules, we computed the trapping free energy (G i trap ) using the simulation snapshot at t = 0. The trapping free energy can be considered as the effective potential characterizing how strongly each water molecule is captured by the biomolecular surface: a more negative trapping free energy means that a water molecule is more stable near the protein complex than in the bulk, and hence, is more favorably "trapped" by the protein complex. The lower panel of Fig. 5 shows scatter plots of the relaxation times and trapping free energies of individual water molecules. (The average relaxation times and trapping free energies listed in Table 2 are obtained from these plots. Since the distribution of the individual waters' relaxation times τ i is well represented on the logarithmic axis as shown in the lower panel of Fig. 5, Table 2 also provides the statistics computed with log 10 τ i ). The resulting "thermodynamic-dynamic relationship diagram" clearly illustrates that the trapping free energy (G i trap ) at t = 0 serves as a good descriptor of the degree of retardation (τ i ) of the subsequent dynamics of hydration water. Thermodynamic-dynamic relationship diagram is presented schematically in Fig. 6a. Single HB water exhibits slower dynamics than bulk water because it is more stable near the protein surface, which in turn reflects the fact that the hydrogen bond between water and protein is stronger than the one between waters. The even slower dynamics of double HB water can be understood similarly; further stabilization originates from an additional water-protein hydrogen bond. Why, then, is bridging water, which has the comparable number of hydrogen bonds with proteins as double HB water (Table 1), more strongly trapped than double HB water? We also notice here that the dynamic, as well as thermodynamic, characteristics of single and double HB water molecules are nearly the same, irrespective of whether they are placed near the isolated monomer or protein-protein complex (Supplementary Figs S1 and S2). The emergence of the "red region" for bridging water in the diagram (Fig. 6a) thus arises solely from the formation of the protein-protein interface. Is there any special factor that is effective only at the interface? We notice in this regard the electrostatic complementarity of the barnase-barstar binding surfaces (Fig. 6b), which creates a strong electrostatic field that is exerted on the interfacial water. Indeed, we find that the magnitude of the electrostatic field is stronger and the water's dipole vector is more oriented along the electrostatic field for bridging water than for single and double HB water (Supplementary Fig. S3). Thus, whereas essentially no change in the hydration water dynamics is observed in the non-interfacial region before and after the binding (Supplementary Figs S1 and S2), the strong electrostatic field created at the binding interface produces an extra stabilizing factor for bridging water, causing it to exhibit extremely slow (nanosecond timescale) hydrogen-bond relaxations (Table 2). It would be interesting to investigate how the transition in the hydration water dynamics occurs during the binding process, but for this purpose, one needs to perform spontaneous binding simulations. The trapping free energy introduced in the present work serves as a valuable quantity not only to characterize the formation of the binding interface from the water's perspective, but also to discuss how and whether the hydration water is rearranging to go from the unbound protein to bound complex. While the barnase-barstar complex studied here is known to be a system in which the interfacial waters are particularly immobile 46 , we anticipate the emergence of the extremely slow water relaxations to be a generic feature of hydrophilic protein-protein interfaces because electrostatic complementarity of the binding surfaces has been observed in numerous protein complexes 47,48 . "Conventional" binding thermodynamics. Among the terms that contribute to ∆G bind 0 (see equation ( 3)), the quantity solv plays a special role for two reasons. First, it is generally difficult to compute the configurational (ΔS config ) and external (ΔS ext ) entropies for complex macromolecules such as proteins. Second, ΔS config and ΔS ext are usually negative upon complex formation and thus make unfavorable positive contributions to G bind 0 ## ∆ ; hence, the driving force for binding must originate from Δf u . Indeed, Δf u is the central quantity, termed the effective binding free energy 49 , in computational approaches to biomolecular bindings such as the molecular-mechanics Poisson-Boltzmann surface area (MM-PBSA) method . We computed f u ∆ by averaging Δf u over the simulated protein conformations. (Our approach is referred to as the three-trajectory approach because we conducted separate computations for the complex and for two free proteins. Numerical values for the binding thermodynamics shall be reported with standard errors computed based on the respective independent trajectories of the complex and free proteins and on the rule of error propagation). The energetic contributions (ΔE u ) were calculated directly from the force field, and the solvation contributions ( G u solv ∆ ) were computed using the 3D-RISM theory (see Supplementary Methods). We obtained f 25 7 2 6 u ∆ = + . ± . kcal/mol; this result leads to an unphysical positive value of ∆G bind 0 , which is in obvious disagreement with the experimental observation ( G 18 9 bind 0 ∆ =− . kcal/mol) 53 . Interestingly, positive effective binding free energy has also been reported based on the MM-PBSA calculations for the barnase-barstar complex: ∆ = + f 14 u kcal/mol in ref. 54 and ∆ = + . f 3 6 u kcal/mol in ref. 55. (The difference in these values may originate from the use of the one-trajectory approach in the MM-PBSA calculations, in which both the complex and monomer configurations were taken from simulations of the complex; the use of different force fields; and the use of different approximations for the solvation free energy). Basic assumption behind the conventional approach. At this point, we critically examine the basic assumption behind the expression (3) for the standard binding free energy. To simplify the discussion, we work in the canonical ensemble and ignore the external entropy, which would not alter the essential point here. We start from the configuration integral, the potential part of the partition function, for a solute-solvent system: Here, r u and r v collectively denote the solute and solvent degrees of freedom, respectively; β = 1/(k B T) is the inverse temperature; and E u , E uv , and E v are the solute energy, solute-solvent interaction energy, and solvent-solvent interaction energy, respectively. Z tot is the principal object in free energy simulations: the change in the free energy F tot = −k B T log Z tot , e.g., upon mutation, is computed from simulations in which both the solute and solvent degrees of freedom are explicitly handled. However, equation (6) does not serve as a basis of equation (3): for example, by introducing the probability distribution the entropy that naturally arises from equation ( 6) is , and it is non-trivial to partition the solute and solvent terms from this total entropy. This is in contrast to equation (3) where the solute (S config ) and solvent (contained in G u solv ) entropies are separated. To arrive at equation ( 3) from equation ( 6), one has to resort to a pre-averaging of solvent degrees of freedom. For a given solute configuration r u , this pre-averaging can be performed as in terms of the solute-configuration dependent solvation free energy G r ( ) v v is the configuration integral for the pure solvent. Now, the configuration integral after the pre-averaging of the solvent degrees of freedom is given by , the associated entropy is given by , which is the defining equation for the solute configurational entropy S config . It is therefore clear that equation ( 3) is based on the pre-averaging of solvent degrees of freedom (see refs 22 and 42 for a complete derivation of equation (3) from equation ( 8)). By the "conventional" approach, we mean the one that is based on this pre-averaging, and do not refer to specific methods such as PBSA and 3D-RISM. In practical applications of the conventional approach, one takes only the protein conformations from simulation trajectories, replacing all the explicit water molecules by the equilibrium continuum model (PBSA) or molecular distribution function (3D-RISM). Such a treatment is usually justified because of the timescale separation between the typical water dynamics (picoseconds) and the protein conformational motions (nanoseconds) 16 , i.e., because of the rapid equilibration of surrounding waters. However, the extreme slowness of the bridging-water relaxation may invalidate such a naive treatment of the water at biomolecular interfaces, and we conjectured that this might the origin of the unphysical positive value of u ∆ . Explicit inclusion of bridging water. We therefore investigated the impact of explicit inclusion of the slow bridging waters. For this purpose, we not only take the protein configurations from the simulation trajectories for the complex, but also bridging waters located at the interface: the number (n) of bridging waters depends on the simulation snapshot, and its average value is 19.6 ± 3.0 (Table 1). Now, those bridging waters are considered as a structural part of the complex, and we apply equation ( 5) to compute f u for the complex. We obtain a negative value f 34 2 2 1 u ∆ = − . ± . kcal/mol, which now serves as the driving force for binding. This result indicates the necessity of considering the dynamics of the interfacial water in the binding thermodynamics. To support our explicit inclusion of bridging waters through a comparison with experiment, we computed the binding free energy G bind 0 ∆ . To this end, we need to estimate the configurational (ΔS config ) and external (ΔS ext ) entropy contributions. For the configurational entropy, we used an energetic approach 43,44 that expresses ΔS config in terms of the fluctuations in f u . In particular, when the probability distribution W(f u ) of f u is Gaussian, S config is simply given by the mean-squared fluctuations of f u (see equation ( 4)). Indeed, W(f u ) of the barnase-barstar complex with bridging water is well approximated by Gaussian, as well as that of the free barnase and barstar proteins (Supplementary Fig. S4), from which TΔS config = −4.5 ± 18.5 kcal/mol is obtained. For the external entropy ΔS ext , we use the estimate TΔS ext = −6.8 ± 0.1 kcal/mol, which was obtained using the method developed in ref. 22 and is close to the value reported in ref. 45. Combining all these results, we obtain ∆ =− . ± . G 22 9 18 6 bind 0 kcal/mol, which is in reasonable accord with experiment (−18.9 kcal/mol) 53 . (As can be inferred from the numerical values presented above, the large standard error for G bind 0 originates from that for TΔS config ; indeed, the protein configurational entropy is known as the most difficult thermodynamic parameter to estimate). ## Conclusions Water molecules are ubiquitously found at the interfaces between biomolecules, and it is often stated that the interfacial water must be considered as an integral part of biomolecules. The work presented here sheds new light on this statement based on the dynamic viewpoint. We demonstrate the emergence of "special" waters in the interfacial region that bridge two biomolecules through concurrent hydrogen bonds and exhibit extremely slow hydrogen-bond rearrangements. By analyzing the thermodynamic-dynamic relationship diagram, we find that the extremely slow nature of bridging water is due to not only the number of hydrogen bonds involved, but also the additional stabilization resulting from the strong electrostatic field between the binding surfaces of electrostatic complementarity. The role of such slow interfacial waters in determining the binding affinity cannot be described using the conventional approach to the water-mediated interaction, which assumes rapid equilibration of the waters' degrees of freedom. Indeed, we observe that a meaningful estimate of the binding affinity is achieved only with a unified treatment of both the biomolecules and the interfacial bridging water. Our work thus demonstrates the impact of the hydration dynamics on the protein-protein binding thermodynamics.

chemsum

{"title": "Dynamics of Hydration Water Plays a Key Role in Determining the Binding Thermodynamics of Protein Complexes", "journal": "Scientific Reports - Nature"}

a_proton_shelter_inspired_by_the_sugar_coating_of_acidophilic_archaea

## Abstract: The acidophilic archaeons are a group of single-celled microorganisms that flourish in hot acid springs (usually pH , 3) but maintain their internal pH near neutral. Although there is a lack of direct evidence, the abundance of sugar modifications on the cell surface has been suggested to provide the acidophiles with protection against proton invasion. In this study, a hydroxyl (OH)-rich polymer brush layer was prepared to mimic the OH-rich sugar coating. Using a novel pH-sensitive dithioacetal molecule as a probe, we studied the proton-resisting property and found that a 10-nm-thick polymer layer was able to raise the pH from 1.0 to . 5.0, indicating that the densely packed OH-rich layer is a proton shelter. As strong evidence for the role of sugar coatings as proton barriers, this biomimetic study provides insight into evolutionary biology, and the results also could be expanded for the development of biocompatible anti-acid materials. Several mechanisms have been suggested to act synergistically to maintain the pH homeostasis of acidophilic archaea, including the intracellular positive transmembrane potential that inhibits proton influx and antiporters that pump out excess protons [1][2][3][4] , with the most comprehensive evidence being reported for the extremely low proton permeability of the cell membrane 1,3,5,6 . The plasma membrane of acidophilic archaeons is unique in two aspects (Fig. 1). First, unlike the bilayer structure commonly found in other archaeal, bacterial or eukaryotic cell membranes, it is a monolayer composed of unique ''tetraether lipids'' in which two hydrophilic heads attached to the same hydrophobic tail through ether bonds and is, therefore, physiochemically more stable and less fluid 2,6-10 . Second, in addition to the tetraether core structure, the membrane lipids are also characterized by a substantially high content of glycolipids (as high as . 90% in some species), with one or more sugar units exposed at the outer surface of the cell 8,9 . This structure has been suggested to provide proton resistance because the average number of sugar units attached increases when the environmental pH decreases 11 . However, a deeper understanding of this mechanism had been hindered, mainly due to the lack of proper tools. In the present study, this biological proton shelter was studied within a novel biomimicry context by mimicking the hydroxyl (OH)rich sugar coating with OH-rich polymer brushes. ResultsDesign of the biomimicry regime. Three tools made a detailed study of the above enigma possible: (1) a newly designed acid-probing dithioacetal molecule (Compound 1, Fig. 2a), (2) the surface-initiated polymerization (SIP) to prepare the finely tuned polymer brushes 12 (Fig. 2b) and (3) the quartz crystal microbalance (QCM) that is sensitive to interfacial changes [13][14][15] (Fig. 3a).Compound 1 is a novel pH-sensitive initiator of the SIP that forms self-assembled monolayers 16 (SAMs) on a gold surface that is stable at neutral pH values (down to 5.0), whereas undergoes partial disassembly when exposed to dilute hydrochloric acid (HCl, pH 1.0, Supplementary Fig. S2). The SAM of 1 plays two roles in this study: (1) because of its acid-sensitivity, it was used as a sensing layer that could probe local pH changes, and (2) because of the bromoisobutyryloxy end it contains, it was used as a layer of initiators from which the poly(oligo(ethylene glycol) methacrylate) (hereafter abbreviated as poly(OEGMA)) brushes were grafted via the SIP (Fig. 2b). The monomer OEGMA 526 (M n 5 526 g mol 21 ) was used to prepare the OH-rich brushes to mimic the OH-rich sugar coating on acidophilic archaeons, whereas OEGMA 475 (M n 5 475 g mol 21 ) was used to prepare the OCH 3 -rich brushes as the control. The SAM and polymer brushes were prepared on the gold electrode of a QCM chip for QCM measurements under various pH solutions. QCM is an acoustic-based sensor that detects interfacial mass losses as frequency increases (Df . 0) 15,17 . The polymer-coated chip was treated with HCl (pH 1.0) to mimic the environmental pH of acidophiles (Fig. 2b). If the H 1 from the HCl reached the Au-S bonds, one would observe frequency increases due to the partial disassembly of the SAM and attached polymer chains. If the polymer layer exerted any inhibiting effect on proton permeation, the frequency response would be weakened or silenced. Examination of the effect of the OH-rich polymer as a proton shelter. First, the chips grafted with poly(OEGMA 475 ) were challenged with HCl. As is shown by the red line in Fig. 3a, the QCM frequency remained unresponsive to the pH 5.0 HCl but increased significantly when exposed to the pH 1.0 HCl, indicating that the H 1 from HCl penetrated the poly(OEGMA 475 ) layer and reached the Au-S bonds. As a control, poly(OEGMA 475 ) was also grafted from the acid-treated 1 SAMs and thiol initiator (vmercaptoundecyl bromoisobutyrate) SAMs (Supplementary Figs S5 and S6). The resulting chips showed no acid responses, confirming that the mass loss in Fig. 3a (red line) was driven by the acid-sensitivity of the 1 SAMs. The acid-induced partial loss of surface mass was also evidenced by atomic force microscopy (Supplementary Fig. S7). The poly(OEGMA 526 )-covered chips were also investigated. As shown in Fig. 3a (the black line), no significant Df was recorded when the pH value dropped from 5.0 to 1.0, indicating that the H 1 from HCl did not fully penetrate the poly(OEGMA 526 ) coating and that the local pH near the Au-S bonds was at least . 5.0. This is strong evidence that the replacement of -OCH 3 groups to -OH groups enabled the polymer coating to act as a proton shelter. Similar results were observed when OEGMA 300 (M n 5 300 g mol 21 , -OCH 3 terminated) and OEGMA 360 (M n 5 360 g mol 21 , -OH terminated) were used as another pair of -OCH 3 /-OH-presenting monomers in the SIP (Supplementary Figs S8 and S9). To confirm the inhibition effect of -OH groups on H 1 penetration further, we designed the following series of experiments. First, we copolymerized OEGMA 526 and OEGMA 475 at different feed ratios to tune the relative content between the -OCH 3 and -OH ends (Supplementary Fig. S10). The result demonstrated that the pH response was completely silenced when the proportion of OEGMA 526 in the copolymer was as low as 10%; a significant proton-resistant effect was still observed even when that ratio dropped to 0.1%. Second, when the -OH ends in the 10% OEGMA 526 -containing copolymer were converted to -OCH 3 groups using MeI, the pH-responsiveness was regained, as expected (Supplementary Fig. S11). Cyclic voltammetry (CV) experiments also indicated a higher electronic resistance of poly(OEGMA 526 ) than poly(OEGMA 475 ), consistent with protonblocking property 18 (Supplementary Figs S12-14). All of these results support the notion that the OH-rich polymer brushes could act as a proton shelter. The -OH groups must be presented in a brush form to gain the proton-shelter function, i.e., the -OH groups must be spatially confined. When a poly(OEGMA 475 )-grafted chip was treated with a pH 1.0 HCl solution containing 1 mM OEGMA 526 monomer, a Df . 0 was observed (Supplementary Fig. S15), indicating that the free form of the -OH groups in solution did not suppress the pH response. Therefore, the surface-grafted polymer brushes were necessary for the confinement and enrichment of the -OH groups, a mimic to the conditions in glycolipids. To assess further the effect in three-dimensional compartments, the proton-shelter was lifted up from the SAM surface by block copolymerization in which a ''proton-conductive'' layer containing exclusively poly(OEGMA 475 ) was first prepared as a spacer to set the distance between the ''proton shelter layer'' and the SAM (Fig. 3b). The chips with this two-layer architecture were subjected to acid treatment, and a prominent proton-resisting function was observed, even when the distance increased to 80 nm (24 nm of dry thickness of the poly(OEGMA 475 ) layer corresponded to 80 nm of wet thickness in HCl), proving the proton resistance effect of the -OH groups in three-dimensional space. ## Discussion In dramatic contrast to those in bacteria or eukaryotic cells, the majority of the lipids in the cell membrane of acidophilic archaeons are glycolipids or glycophospholipids that contain at least one sugar residue on the extracellular side (Fig. 1b). When the environmental pH decreased from 3.0 to 1.2, the content of glycolipids in the plasma membrane of Thermoplasma acidophilum increased and the percentage of the lipids that contained 2-4 sugar units rose from 14% to 35%, suggesting an adaptation to low pH values by extending the sugar chains on the cell surface 11 . As a systematic investigation of the effect of -OH groups on proton resistance, our study presents strong evidence for that adaptation mechanism and pinpoints the effective site as -OH groups. The layer remained resistant to proton permeation after 1,000-fold dilution of the -OH group concentration, suggesting that there a redundancy still exists with regard to the amount of -OH groups in our experiments. Thus it is reasonable to suppose that a layer of a few sugar units (1-2 nm in thickness) is capable of exerting moderate effects on acid defense (Fig. 1c). The concentration of -OH groups inside the poly(OEGMA 526 ) brushes was estimated to be 0.5 M (see Supplementary Information for the detailed calculation), which is on the similar order of magnitude with that on the cell surfaces of acidophilic archaeons, thus justifying our simulation 19 . Our study proves the strong proton-resistance of surface confined -OH groups and also paves the way for the investigation of the underlying mechanism. The proton-resistance properties of other groups, such as -NH 2 and epoxy groups, could be studied by changing the monomers, the direction of our ongoing experiments. The results, combined with a theoretical analysis, are expected to reveal the mechanisms. The bioinspired poly(OEGMA 526 ) membrane possesses a strong proton-sheltering effect that has been quantitatively defined as raising the pH from 1.0 to . 5.0. Therefore, our biomimetic study may be expanded for the development of biocompatible anti-acid strategies, such as tooth protection and drug-carriers that withstand gastric acid digestion 20 . Our conclusion also provides information for the design of novel paints that offer protection to vehicles, buildings and infrastructures against acid rain corrosion, an area for which realistic solutions are still lacking. Furthermore, as the first organosulfur compound that has been found to form an acid-sensitive Au-S bond, compound 1 could be used to study the nature of Au-S bonds. The dithioacetal initiator (1) was synthesized using the following procedure: (1) Synthesis of 4-formylphenyl -2-bromo-2-methylpropanoate 21 : Hydroxybenzaldehyde (0.61 g, 5 mmol), triethylamine (0.75 mL, 1.033 mmol), and dry dichloromethane (25 mL) were added to a 50-mL round-bottom flask with a stir bar. The mixture was cooled to 0uC, followed by the dropwise addition of ice-cold bromoisobutyryl bromide (0.67 mL, 5.39 mmol). After stirring at 0uC for 1 h, the reaction was continued for another 12-16 h at room temperature. Water (20 mL) and dichloromethane (10 mL) were added to the mixture for a two-phase extraction. The aqueous phase was further extracted with dichloromethane (2 3 30 mL), and the organic phase was concentrated by rotary evaporation to remove the dichloromethane. The resulting crude extract was dissolved in dichloromethane (40 mL), washed with a saturated sodium bicarbonate solution (3 3 40 mL), and dried over MgSO 4 . The removal of the dichloromethane resulted in a yellowish oil, which was passed through a column (silica gel, neutral, with petroleum ether:ethyl acetate 5 20:1) and then vacuum dried overnight. The final product (4-formylphenyl -2bromo-2-methylpropanoate) was a white solid, obtained in high purity and with a high yield (0.2168 g, 80% yield). 1 (2) Synthesis of dithioacetal initiator 22 : Mercaptoundecanol (0.48 g, 2 mmol): 4formylphenyl -2-bromo-2-methylpropanoate (0.271 g, 1 mmol) and toluene (10 mL) were added to a 50 mL round-bottom flask with a stir bar. The mixture was refluxed to 110uC, followed by the addition of p-toluenesulfonic acid (0.009 mL, 0.05 mmol). After stirring at 110uC for 3 h, the reaction was stopped by the addition of triethylamine (3 mL). The mixture was concentrated by rotary evaporation to remove the toluene. The removal of the toluene resulted in an orange oil, which was passed through a column (silica gel, neutral, with petroleum ether: ethyl acetate5 2:1 with 2% triethylamine as eluent) and then vacuum dried overnight. The final product was a white solid, obtained in high purity and with a high yield (1.4040 g, 93.1% yield). 1 H NMR (400 MHz, CDCl 3 ): d7.493 ppm (d, J 5 6.4, 2H), 7.111 (d, J 5 8.8, 2H), 4.886 (s, 6H), 3.642 (t, J 5 13.2, 4H), 2.542 (m, J 5 43.6, 4H), 2.073 (s, 6H), 1.554 (m, J 5 27.6, 10H), 1.322 (m, J 5 39.6, 26H). 13 SAMs. The QCM chips were incubated in 1 mM ethanol solution of 1 for 18 hours. The reaction was executed at ambient temperature, under nitrogen atmosphere protection and protected from light. SIP. The SIP of OEGMA from the SAMs of 1 as the initiators was performed as reported previously 15 using bipyridine as a ligand and water: ethanol 5 1: 1 as a solvent, with a molar ratio of OEGMA/CuBr 2 /bipyridine/CuBr/ 5 400/1/30/10 (i.e., 10/0.025/0.75/0.25 mM). The reaction was terminated by MilliQ-water (pH 6.2), and the chips were then rinsed thoroughly with MilliQ-water and ethanol to remove any salt particles and dried under a nitrogen flow. The dry thicknesses of the surfacetethered polymer brushes were determined by Ellipsometry. QCM measurements. All of the QCM measurements were conducted with a relative humidity controlled below 25%. The operation temperature was set at 25uC. The chips (AT cut, 5 MHz; HZDW, Hangzhou, China) were placed in a home-built QCM with control software purchased from Resonant Probes GmbH (Goslar, Germany). For the liquid-phase measurements, the QCM was operated in a flow-through mode at a speed of 80 mL min 21 . Milli-Q water (pH 5 6.2) with a resistivity of 18.2 MV cm 21 was used for the solution preparation. AFM. The AFM images of the samples in their dry state were taken in the tapping mode (Digital Instruments, Santa Barbara). The polymer-coated QCM chips were imaged before and after washing with a dilute acid solution. Ellipsometry. The dry film thickness was measured using an M-2000V spectroscopic ellipsometer (J. A. Woollam Co., Inc.) at angles of 65u, 70uand 75uand wavelengths from 400 nm to 800 nm. The ellipsometric data were fitted for thickness using materialspecific models (Cauchy layer model) from a vendor-supplied software, with fixed (A n , B n ) values (1.46, 0.01). Each datum was an average of three measurements.

chemsum

{"title": "A proton shelter inspired by the sugar coating of acidophilic archaea", "journal": "Scientific Reports - Nature"}

spatial_and_temporal_distributions_of_polycyclic_aromatic_hydrocarbons_in_sediments_from_the_canadia

## Abstract: Highlights• The polycyclic aromatic hydrocarbon (PAH) concentrations in Canadian Arctic sediments are low • The PAH input to sediments has remained constant throughout the last century • The PAHs in Canadian Arctic sediments mainly originate from natural sources Abstract 1. The concentrations of 23 polycyclic aromatic hydrocarbons (PAHs; 16 parent and 7 alkylated PAHs) in 113 surface marine sediment samples, 13 on-land sediment samples and 8 subsampled push cores retrieved from the Canadian Arctic Archipelago (CAA) were calculated. PAHs were extracted via accelerated solvent extraction (ASE) and quantified via gas chromatography-mass spectrometry (GC-MS). The sums of the concentrations 16 PAHs in the surface sediments ranged from 7.8 to 247.7 ng g -1 (dry weight [dw]) basis). The PAH inputs to the sediments have remained constant during the last century and agree with the results obtained for the surface sediments. Diagnostic ratios indicated that the PAHs in the CAA mainly originate from natural petrogenic sources, with some pyrogenic sources. Temporal trends did not indicate major source shifts and largely indicated petrogenic inputs.Overall, the sediments retrieved from the CAA have low PAH concentrations that are mainly natural. ## Introduction Within the context of climate change, the Arctic is undergoing major perturbations, and many studies have focused on sea ice conditions and navigability projections in the Arctic Ocean (Lasserre et al., 2010;Askenov et al., 2017). Because the summer sea ice extent is rapidly decreasing, leading to a seasonally ice-free Arctic Ocean, it has been speculated that maritime traffic could increase within the Canadian Arctic Archipelago (CAA); for example, the northwest passage could open to cargo transportation for a longer time period each year by the middle of the century (Lasserre et al., 2010;Smith and Stephenson, 2013). This shipping route connecting Asia and Europe is shorter than the Suez Canal, the Panama Canal or the Cape of Good Hope (Lasserre et al., 2010;Askenov et al., 2017). Hence, maritime companies have shown interest in traveling through the Arctic since this would allow time and fuel savings and consequent cost reductions. However, maritime traffic and oil exploration within the Arctic could also increase the anthropogenic pressure and pollution load in Arctic ecosystems (Jörrundsdóttir, 2014). Shipping-related fuel combustion and anthropogenic activities are local sources of polycyclic aromatic hydrocarbons (PAHs), which constitute a wide class of organic compounds consisting of more than one benzene ring (C6H6) fused in a variety of conformations (AMAP, 2017;Haritash and Kaushik, 2009). Hundreds of these compounds are found in the environment, but since the mid-70s, 16 PAHs have been listed as priority environmental pollutants by the Environmental Protection Agency of the United States (US EPA) and are therefore closely monitored (Keith et al., 2014;Pampanin and Sydnes, 2017). Hence, PAHs are pollutants of great concern, especially since the emissions originating in developed countries have decreased while those originating in developing countries have increased (Zhang and Tao, 2009;Wang et al., 2010). PAHs are introduced to the environment via natural or anthropogenic sources (Lima et al., 2005;Foster et al., 2015;Chen et al., 2018), and anthropogenic activities are major sources of the PAHs occurring in the biosphere (Yanik et al., 2003;Morillo et al., 2008). Seven PAHs have been classified as probably carcinogenic for humans by the International Agency for Research on Cancer (IARC) of the World Health Organization because of the reactivity of their metabolites (IARC, 1987). Inuit communities within the Arctic might be exposed to PAHs via the consumption of traditional foods such as mollusks (Rapinski et al., 2018). However, the Arctic is a region where the seafloor composition is the least studied and understood. Indeed, the vast majority of the channels within the CAA and their adjoining continental shelves and slopes exhibit a substantial knowledge gap regarding sediment composition and associated contaminants (Stein, 2008). Moreover, studies have focused on specific areas of the Arctic (e.g., Beaufort Sea or Baffin Bay; Yunker et al., 1995Yunker et al., , 2002aYunker et al., , 2002b;;Foster et al., 2015) or sites near anthropogenic influences (Boitsov et al., 2009a,b;Zaborska et al., 2011). To our knowledge, no complete baseline information on the PAHs in recent sediments or historic tendencies of the PAH inputs to sediments are available within the CAA. ## PAH sources Pyrogenic PAHs are produced during the incomplete combustion of organic matter, which includes forest and bush fires and fossil fuel and coal combustion (Chen et al., 2018;Yu et al., 2019). These PAHs are mainly emitted into the atmosphere and could occur either in the gaseous phase or bonded to the particulate phase (i.e., mineral dust and salt) (Tobiszewski and Namieśnik, 2012). Owing to their low vapor pressures, the majority of these semivolatile compounds undergoes repeated cycles of volatilization-deposition, travel across long distances and eventually end up in waters, soils and sediments via deposition (AMAP, 2017;Chen et al., 2018;Balmer et al., 2019). The PAHs produced at mid-latitudes could thus reach the Arctic, as shown by modeling studies (Wang et al., 2010;Sofowote et al., 2011). Petrogenic PAHs are hydrocarbons stemming from losses or seepage of oil and petroleum deposits, crude oil spills or rock weathering and are therefore naturally present in sediments and water bodies, and these PAHs are not of great concern because of their very low concentration (Lima et al., 2005;Pampanin and Sydnes, 2017;Chen et al., 2018). They are readily dispersed via water runoff, and since petrogenic PAHs are not directly emitted into the atmosphere, they are slightly influenced by long-range atmospheric transport (Pampanin and Sydnes, 2017). ## Environmental fate In the Arctic, PAHs stemming from distant sources may enter the marine environment via river discharge, but atmospheric long-range transport is believed to be a significant input process (Sofowote et al., 2011;Yu et al., 2019). Regarding the environmental fate of atmospheric PAHs, Lammel et al. (2009) showed via a modeling approach that between 0.5% and 12.8% of the total environmental burden of PAHs might be stored within Arctic ecosystems (i.e., air, soil, vegetation and ocean) depending on the chosen gas/particle partitioning scenario. In addition to the already existing natural PAHs in Arctic soils and sediments, atmospheric deposition of PAHs originating from remote sources, in addition to new local sources such as ship traffic and oil exploration/exploitation, are PAH sources in the CAA (Balmer et al., 2019). PAHs are not easily degraded under natural conditions and are therefore slightly persistent (Pelletier et al., 2008;Haritash and Kaushik, 2009). Photooxidation of PAHs is a chemical pathway of degradation, but biological degradation by bacteria, fungi and algae is accepted as the main process (Roslund et al., 2018;Haritash and Kaushik, 2009;Balmer et al., 2019). Because most vertebrates (e.g., fishes, birds and mammals) readily metabolize PAHs, they do not tend to experience biomagnification through the food chain (Xue et Warshawsky, 2005;Haritash et Kaushik, 2009;AMAP, 2017). However, PAHs could accumulate in benthic species such as clams and mussels (Balmer et al. 2019), and these organisms are an important food source for northern communities (Jörrundsdóttir et al., 2014;Rapinski et al. 2018). If deposited on land, PAHs could leach through soils or could be transported via water runoff and eventually reach aquatic ecosystems (Wang et al., 2007;Klungsøyr et al., 2010). They are poorly soluble in water because of their hydrophobicity and lipophilicity (Chen et al., 2018;Zhao et al., 2016). Consequently, PAHs exhibit a relatively high affinity for suspended and particulate matter and sediments, which is why the latter are considered the main sink of PAHs (Chen et al., 2018). Considering that PAHs are pollutants of interest that could be released by an increase in anthropogenic activities in the Arctic and that could accumulate in sediments and considering that the Arctic sediment PAH composition is not completely known, it is essential to determine the actual baseline. The aim of this study is to (1) characterize the modern spatial distribution patterns of the PAHs within the CAA, (2) determine the temporal trends of the PAH concentration based on 210 Pb-dated box cores collected across the CAA, and (3) establish the origin of PAHs (i.e., petrogenic or pyrogenic) according to two diagnostic ratios, namely, fluoranthene over the sum of fluoranthene and pyrene (Fla/ [Fla+Pyr]) and benz(a)anthracene over the sum of benz(a)anthracene and chrysene (BaA/[BaA+Chr]). ## Study site Since pyrogenic PAHs traveling via long-range atmospheric transport are deposited in soil or water and petrogenic PAHs mainly originate from rock weathering and oil reserves, they both occur in sediments, i.e., sediments are a sink for organic pollutants such as PAHs, and their affinity for fine and organic-rich sediments is well documented (Chiou et al., 1998;Gschwend and Hites, 1981;Stark et al., 2003). Letaïef (2019) reported that the sediments within the CAA ranged from clay (2 µm) to fine silt (4 to 8 µm). The total organic carbon (TOC) content is lower than 2% in most surface sediments retrieved from the CAA (e.g., Letaïef, 2019). More specifically, the Mackenzie Shelf and Delta and the Beaufort Sea/Canada Basin exhibit TOC values ranging from 0.5% to 1.9% (Yunker et al., 2011;Letaïef, 2019). The Queen Maud Gulf and the M'Clintock Channel, central CAA, exhibit relatively low TOC contents, with values ranging from 0.2% to 0.5 % (Letaïef, 2019). Finally, the TOC content in Baffin Bay sediments ranges from 0.2% to 1.5% (Stein, 1991;Madaj, 2016). The CAA counts approximately 36500 islands and numerous waterways, straits, channels and sills formed by glacial action under past climate conditions (Melling et al., 2002;Michel et al., 2006). The recent sedimentary dynamics within the CAA are controlled by the sediment supply stemming from river discharge in the west and central CAA, whereas the east CAA is more influenced by sea ice and coastal erosion (Letaïef, 2019). Indeed, the Mackenzie River alone annually discharges approximately 420 km 3 /yr of sediments onto the continental shelf of the Beaufort Sea and is therefore a major source of continental PAHs (Wagner et al., 2011). Other small rivers exert a cumulative significant impact on the sediment load, such as the Coppermine River, the Ellice and Back Rivers and the Cunningham River, with a total contribution of approximately 110 km 3 /yr (Alkire et al., 2017). Once in the marine ecosystem, sediments are entrained throughout the archipelago by sea ice via suspension freezing and ice anchoring (Reimnitz et al., 1993;Darby et al., 2003Darby et al., , 2011;;Stein, 2008). Coastal erosion by seasonal sea ice, glaciogenic debris flows, meltwater plumes, mass movements along submarine canyons and sea lifting are other dominant sedimentary processes in glacial environments contributing to the dispersal of sediments across great distances within the CAA (Hiscott et al., 1989;Ó Cofaigh et al., 2003;Harris, 2012;Dowdeswell et al., 2015;Lai et al., 2016). Processes involving sea ice are mostly active during the sea ice formation season, and sediments are discharged elsewhere during summer melting (Darby et al., 2011). All of these processes contribute to the dispersion of PAHs originating from distant sources within the CAA before they become trapped in marine sediments. ## Materials and reagents All reagents were of analytical or high-performance liquid chromatography (HPLC) grade. Hexanes and dichloromethane were obtained from Anachemia, methanol was acquired from Millipore and 2-propanol was obtained from Fisher Chemicals. Nitric acid (HNO3) and hydrochloric acid (HCl) were acquired from VWR Analytical. Silica gel (technical grade, 70-230 mesh) and copper powder (<425 µm) were obtained from Sigma-Aldrich. Diatomaceous earth (celite 566) was acquired from UCT Enviro-Clean. Standard reference material NIST-1944 was purchased from the National Institute of Standards and Technology (NIST). PAH Mix manufactured by AccuStandard was adopted for the generation of calibration curves, combined with an alkylated PAH homemade mix (2,6dimethylnaphthalene and 9,10-dimethylanthracene were obtained from Sigma-Aldrich, while 2,3,5-trimethylnaphthalene, 1-methtylnaphthalene and 3,6-dimethylphenanthrene were acquired from Fisher Chemicals). Before analysis, every sample was spiked with deuterated 1-methylnaphthalene and benz(a)anthracene purchased from Sigma. A mixture of deuterated naphthalene (Sigma-Aldrich), anthracene (Cambridge Isotope Laboratories) and perylene (Sigma-Aldrich) was added as an internal standard for quantification purposes. The targeted compounds in this study included 16 parent PAHs and 7 alkylated PAHs: naphthalene, 1-methylnaphthalene, 2-methylnaphthalene, 2,6-dimethylnaphthalene, acenaphthylene, acenaphthene, 2,3,5-trimethylnaphthalene, fluorene, phenanthrene, anthracene, 1-methylphenanthrene, 3,6-dimethylphenanthrene, fluoranthene, pyrene, 9,10dimethylanthracene, benz(a)anthracene, chrysene, benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(a)pyrene, indeno(1,2,3-c,d)pyrene, dibenz(a,h)anthracene and benzo(g,h,i)perylene. Silica gel was activated at 450°C for 2 h and stored in a desiccator. Copper powder, for sulfur removal from sediments, was activated as follows: in a Teflon tube, copper powder was covered with hydrochloric acid (HCl) 6N and shaken for 3 min. It was then rinsed with distilled water until a neutral pH was attained. The copper powder was then washed 3 times with both methanol and dichloromethane and was finally stored in a Teflon tube covered with dichloromethane. ## Sediment samples and chronology A total of 126 sediment samples was analyzed in this study: 113 marine surface sediment samples and 13 terrestrial sediment samples collected from glaciers and rivers (Fig. 1). Additionally, 8 push cores were subsampled to determine temporal trends (Fig. 1). All samples were retrieved from box cores collected between 2016 and 2019 across a large area covering the Canadian Beaufort Sea to Baffin Bay during the ArcticNet summer expeditions onboard the Canadian Coast Guard Ship (CCGS) icebreaker Amundsen. Samples were collected from glaciers and rivers using the ship helicopter as CCGS Amundsen traveled through the CAA. All marine coring sites were targeted using high-resolution seismic profiles, which indicated that Late Holocene sediment accumulation was not influenced by mass wasting events (Montero-Serrano et al., 2016, 2017, 2018, 2019). Surface sediments (uppermost 0.5 to 1 cm) were collected with a spoon and stored in plastic bags (WhirlPack) at 4°C until further analysis. Push cores were collected by pushing a plexiglass tube (10-cm diameter) into sediments. The cores were stored at 4°C until further subsampling. Sediment subsamples were retrieved from the push cores from 0 to 10 cm at 1cm intervals. Age-depth models of the box cores have been previously published in Letaïef (2019), constructed using 210 Pb measurements combined with the constant rate of supply (CRS) model (a constant rate of the 210 Pb supply; Appleby and Oldfield, 1983). The age is reported as common era (CE) and before common era (BCE) hereafter. The sedimentation rates and ages at ~10 cm depth are provided in Table 1 and Fig. S1. Note that no dates are available for core AMD1902-05BC recovered in the Robeson Channel (Fig. 1). However, based on the sedimentation rate (4 cm ka -1 ) determined for a well-dated neighboring core (HLY03-05GC; Jennings et al., 2011), we inferred that the age at a core depth of ~10 cm is approximately 2.5 cal ka BP (550 BCE). All samples were sieved through a 150-µm Nitex® mesh using distilled water. The <150 µm sediment fraction was then stored in a 50-mL Falcon® tube successively rinsed with tap water and soap, distilled water, nitric acid (HNO3) 5% (3 times), distilled water and 2-propanol (3 times). The sediment samples were then frozen at -80°C for at least 12 h and freeze dried. The samples were finally crushed using an agate mortar. Aliquots weighing 5 g of these homogenized sediment samples were used for PAH analysis. ## PAH extraction and analysis PAH extraction was conducted via accelerated solvent extraction (ASE) following the method developed by Choi et al. (2014). Briefly, 22-mL stainless-steel extraction cells were loaded as follows, from bottom to top: cellulose filter, diatomaceous earth, activated silica gel (5 g), activated copper (5 g), freeze-dried sediment sample (5 g) and diatomaceous earth to the top (Fig. S2). A PAH spike (1-methylnaphthalene-d10 and benz(a)anthracene-d12) was directly added onto the sediment sample. The cells were maintained open and protected from dust contamination at room temperature for 2 h to acclimate. Blanks were prepared similar to the samples but with no sediments. To confirm the accuracy of the method, 0.5 g of standard reference material NIST-1944 was processed as a sample. One blank, one standard reference material sample and one duplicate were tested for every 12 samples. The addition of activated silica gel and copper directly to the cell enabled one-step extraction and cleanup. The extraction was performed with a Dionex ASE 200 system (Thermo Co., Sunnyvale, CA, USA). The temperature and pressure were set to 100°C and 1700 psi, respectively. The flush volume and purge time were set to 60% and 100 s, respectively. The extraction was performed with a mixture of hexane and dichloromethane (at a ratio of 4:1 v/v) and two static cycles of 5 min. Extracts were collected in 60-mL clear collection vials (previously rinsed with tap water and soap, distilled water, hexane and dichloromethane (4:1 v/v) mixture and propanol). The extracts were then evaporated to approximately 5 mL with a rotating evaporator and then evaporated to exactly 0.5 mL with a nitrogen stream at room temperature. In regard to standard material NIST-1944, the extracts were evaporated to exactly 1.5 mL. PAH analysis was performed via gas chromatography (GC, Agilent Technologies 6850 series II; Santa Clara, CA, USA) coupled with mass spectrometry (MS, Agilent Technologies 5975B VL MSD) using total ion count (TIC). The injection was performed with an Agilent Technologies 6850 series autosampler. The capillary column used was an Rxi®-5 ms (30 m x 0.25-mm inner diameter (ID) x 0.25 µm ft, 5% diphenyl and 95% polysiloxane from RESTEK). The oven temperature was set as follow: 50°C for 2 min, 15°C/min until 275°C, held for 2 min, 15°C/min until 325°C, held for 15 min, and a postrun of 2 min at 300°C. A sample volume of 1 µL was injected at a temperature of 250°C under splitless injection with helium as the carrier gas at a flow rate of 1 mL/min. ## Quality control and quality assurance (QC/QA) The procedural blanks did not reveal contamination. Hence, the results were not blank corrected. The spike recoveries were 73.7% ± 15.0% for 1-methynaphthalene-d10 and 83.5% ± 23.2% for benz(a)anthracene-d12. Some samples did not meet the generally accepted QC/QA recovery criteria of 70% to 103%. All samples were hence spike corrected. The efficiency of the method was confirmed with standard reference material NIST-1944. The mean recoveries obtained are compared in Table S2 to those obtained by Choi et al. (2014), who developed the ASE method applied in this study. The method detection limit (MDL) for each PAH was calculated as suggested by the US EPA (Oblinger Childress et al., 1999). Briefly, 7 replicates of a spiked solution at the second lowest calibration point were analyzed. Hence, the MDL was determined as 3.143 times (Student's t value for 6 degrees of freedom and the 99% confidence level) the standard deviation of the measured concentration for each compound. The MDL ranged from 8.1 ng/g for benz(a)anthracene to 155.9 ng/g for acenaphthylene (Table S2). ## Data processing Prior to all multivariate analyses, the values below the detection limit (VBDLs) were imputed via multiplicative lognormal replacement with R package zCompositions (Palarea-Albaladejo and Martin-Fernandez, 2015). This method preserves the geometry of the compositional data while accounting for corresponding detection limit thresholds. However, -Fernandez, 2015). Next, a log-centered (clr) transform was applied to the data (Aitchison, 1990). This operation removed the statistical constraints on the compositional variables, such as the constant-unit sum, and enabled the valid application of classical (Euclidean) statistical methods to the compositional data (Aitchison, 1986(Aitchison, , 1990;;Montero-Serrano et al., 2010). We applied fuzzy c-means (FCM) clustering analysis (Kaufman and Rousseeuw, 2009) to identify samples possessing similar PAH compositions within the CAA. We adopted the Aitchison distance as a measure of similarity between the samples and the Ward method (minimum-variance method) for agglomerative calculation purposes. The FCM algorithm requires in-advance specification of the overall number of clusters to be detected. R package NbClust (Charrad et al., 2014) was employed to apply 23 indices and to determine the optimum number of clusters. The FCM clustering results are visualized in silhouette and principal coordinate ordination plots (Kaufman and Rousseeuw, 2009). The silhouette plot allows visualization of the robustness of clusters, where negative values indicate an incorrect and/or questionable assignment (Borcard et al., 2011). Moreover, principal component analysis (PCA) was performed using the PAH data and FCM clustering results with the goal of determining PAH associations with similar relative variation patterns (von Eynatten et al., 2003;Montero-Serrano et al., 2010). FCM clustering analysis was conducted with R software (R Core Team, 2020) using the compositions (van den Boogaart and Tolosana-Delgado, 2008) and cluster packages (Maechler et al., 2019). PCA was conducted with Compositional Data Package (CODAPAK) software (Comas and Thió-Henestrosa, 2011). Finally, the FCM clustering results and PAH concentrations were analyzed to produce distribution maps using Ocean Data View software (Schlitzer, 2015). These maps were generated using a weighted-average gridding algorithm with a quality limit of 1.5. Diagnostic ratios of fluoranthene over the sum of fluoranthene and pyrene (Fla/[Fla+Pyr]) and benz(a)anthracene over the sum of benz(a)anthracene and chrysene (BaA/[BaA+Chr]) were considered to draw boxplots and discriminate PAH sources (i.e., pyrogenic vs petrogenic). ## FCM clustering analysis The FCM clustering analysis results indicate that there are three regional PAH clusters within the CAA (Figs. 2A and S3). Cluster 1 (PAH C#1, red) is mostly representative of the western CAA. Yunker et al. (1996) showed that the Mackenzie River imposed a dominant influence on the sedimentary dynamics in this region, namely, all their samples collected from the Mackenzie River, the Mackenzie Shelf and the Beaufort Sea Shelf edge clustered together, agree with our results. This cluster also seems to be dominated more by both medium molecular weight PAHs (MMW = 4-5 rings) and light molecular weight PAHs (LMW = 2-3 rings). This cluster exhibits a higher influence of high molecular weight PAHs (HMW= 6 rings) than the other clusters; however, HMW PAHs are minor contributors to the clusters (Fig. 2B). Clusters 2 (PAH C#2, green) and 3 (PAH C#3, blue) are slightly less defined. However, cluster 2 tends to be more represented by LMW PAHs and samples from the eastern CAA, while cluster 3 is more represented by LMW to MMW PAHs (and samples retrieved from the central CAA, as shown in Fig. 2B). The ordination diagram (Fig. 3A) and silhouette plot (Fig. 3B) show the robustness of the above clusters, revealing that the samples can be divided into 3 clusters: (1) western CAA, (2) eastern CAA and (3) central CAA. The negative values in Fig. 3B indicate an incorrect and/or questionable assignment. The second group contains the most samples (n=6) that might belong to another group, whereas only 3 samples of the other groups might be incorrectly assigned. Indeed, those samples show a greater mix between the 3 clusters, likely due to their PAHs assemblages and sources (Fig. S4). ## Distribution of the PAHs in recent sediments The sums of the concentrations of the 16 priority PAHs (Σ16PAHs, dry weight [dw]) designated by the US EPA in the surface sediments of the CAA ranged from 7.8 to 247.7 ng g −1 with a mean value of 56.8 ng g −1 (Fig. 4B; Table 2). The highest values of Σ16PAHs are found in the western CAA, with values ranging from 15.7 to 247.7 ng g −1 and a mean value of 107.9 ng g −1 (Fig. 4B). According to the values reported in the literature (Table 2), it seems that the seafloors near the Mackenzie River are naturally rich in PAHs, which has been previously explained by the river discharge of the Mackenzie River (Yunker et al., 2002a). Indeed, the river alone discharges an annual flux of 49 ± 8 tons of both particulate and dissolved PAHs onto the Mackenzie Shelf (Yunker et al., 1991). Additionally, the western CAA is well known for three areas of natural hydrocarbon seeps (oil and/or gas) along the Mackenzie River, Delta and Shelf (Thomas, 1979;Janicki, 2001;Yunker et al., 2002a). The Beaufort Shelf and Mackenzie Shelf are also known for their pockmarks and mud volcanoes releasing fluids and gas into the water column (Blasco et al., 2006;Walsh et al., 2006). Certain on-land seeps, such as the Smoking Hills (Cape Bathurst, Northwest Territories), also release smoke clouds and fumaroles containing PAHs that are then transported by wind (Klungsøyr et al., 2010). The western CAA also has a past history of petroleum exploration. Indeed, from the 1960s to the 1990s, extensive drilling was performed in the Mackenzie/Beaufort Basin, and many sumps for drilling wastes were built, which have leaked since their abandonment. Hence, accidental oil spills have occurred, but the total inputs are much lower than those from other sources (Klungsøyr et al., 2010). This natural hydrocarbon-rich background and petroleum exploration/extraction activity could explain the relatively high concentrations observed in the Mackenzie River area. Overall, the surface sediment concentrations of Σ16PAHs reported in other studies for the Canada Basin (58.9 -75.9 ng g −1 ; Ma et al., 2017) and the Chukchi Sea/Canada Basin (102 ng g −1 ; Yunker et al., 2011 and 8.8 -78.3 ng g −1 ; Ma et al., 2017) are comparable to those reported here for the western CAA but lower than those reported for the Mackenzie Shelf (495 -755 ng g −1 ; Yunker and MacDonald, 1995). In the two other regions of the CAA, the value of Σ16PAHs (dw) remains low: 7.8 to 100.7 ng g −1 with a mean value of 40.8 ng g −1 for the eastern CAA and 9.3 to 79.2 ng g −1 with a mean value of 35.5 ng g −1 for the central CAA (Fig. 4B). These results are similar to other Arctic regions, such as the Kara Sea, the Barents Sea or the Svalbard coast, but higher than the reported values for the Makarov Basin or the Central Arctic Ocean (Table 2). Dong et al. (2015) pointed out a decreasing tendency of the PAH concentration with increasing latitude, which could explain why higher PAH concentrations occur in the CAA than those at other northerly sites (e.g., the Canada and Makarov Basins). Another known hydrocarbon seep is located in the eastern CAA along Baffin Island at Scott Inlet (Levy, 1978), but the sample collected near this area does not exhibit a higher concentration than those exhibited by the other samples collected from the eastern CAA. The values of Σ16PAHs (dw) for the terrestrial samples (i.e., glacier and river samples) are consistent with those for the marine sediments, with values ranging from 22.1 to 108.8 ng g −1 and a mean value of 71.1 ng g −1 (Fig. 4B), which are fairly low values. However, a major exception of 356.1 ng g −1 is found for the sample collected near the Sydkap Glacier, located approximately 60 km west of the Grise Fiord, the northernmost Inuit community in the CAA (Fig. 4A). It is the highest result among all the samples. No anthropogenic activities or historical accidental spills have been recorded in this area. However, Ellesmere Island is known for its numerous coal deposits (Ricketts and Embry, 1984;Kalkreuth, 2004;Harrison et al., 2011). Coal layers outcrop along the Stenkul Fiord, literally meaning the Coal Fiord, which is part of the Eureka Sound Group coals and occurs approximately 60 km north of the Grise Fiord (Kalkreuth et al., 1996). Thus, such outcropping along the watershed and river shores surrounding the Sydkap area could explain the high PAH concentrations observed in this area. In general, the inputs of PAHs during the last century seem to have remained relatively constant. The value of Σ16PAHs (dw) stays within the general concentrations observed in the surface sediments of the CAA, and none of them is higher than the maximum value of 246.6 ng g −1 encountered in the marine sediments in this study. In regard to the surface sediments, the highest sums are found in the western CAA, especially for AMD1603-408, with a mean value of 156.2 ng g −1 . Very low values are obtained for AMD1902-05 (a mean value of 20.0 ng g −1 ), a core collected near Alert (Nunavut, Canada) at the very extreme north of Ellesmere Island. This is consistent with the general trend of the decreasing PAH concentration in the sediments with increasing latitude, since remote locations are far from industrial activities, and the PAH inputs stemming from remote sources are only influenced by long-range atmospheric transport (Dong et al., 2015;Balmer et al., 2019). Additionally, it should be noted that in marine sediments, trends commonly tend to be less defined mainly because of ocean perturbations (e.g., currents or ship traffic; AMAP, 2017). Therefore, variations due to worldwide fluctuations might indicate a time shift, especially because processes involving ocean currents could last years, whereas atmospheric processes are more common on a daily scale (Klungsøyr et al., 2010). The PAH inputs occurring during the last century are therefore relatively stable. In sediments collected from the Barents Sea, Boitsov et al. (2009b) measured a 10-fold increase in the PAH concentration in marine sediments corresponding to the 1910-1940 period, while the inputs prior to 1850 remained constant. ## Historical trends of the PAH inputs into the sediments After approximately 1980, the concentration slightly decreased. This general decreasing tendency has been associated with a reduction in the worldwide PAH emissions since 1995 (Shen et al., 2013). However, we do not observe this situation in our results. Additionally, Foster et al. (2015) studied pre-1900 and post-1900 sediments retrieved from the Baffin Bay area. The majority of their results is within a factor of 10 from those obtained for the post-1900 sediments, indicating a constant PAH concentration over time, which is consistent with our results. preindustrial sediments (e.g., Yunker et al., 2002b;Foster et al., 2015). Typically, Fla/(Fla+Pyr) ratios below 0.4 are representative of petrogenic PAHs, those between 0.4 and 0.5 are representative of fossil fuel combustion and those above 0.5 are representative of biomass combustion (Yunker et al., 2002b). In regard to the ratio benz(a)anthracene over the sum of benz(a)anthracene and chrysene (BaA/[BaA+Chr]), a ratio below 0.2 indicates a petrogenic source, a ratio ranging from 0.2 and 0.35 indicates a mixed source (i.e., either fossil fuel or biomass combustion) and a ratio above 0.35 indicates a pyrogenic source (Yunker et al., 2002b). In regard to the surface samples retrieved from the western CAA, the Fla/Fla+Pyr values ranged from 0.14 to 0.56, and the BaA/(BaA+Chr) values ranged from 0.13 to 0.33, indicating a mainly petrogenic origin (Fig. 6A-B) with a small influence of mixed combustion origins. This is in agreement with the results reported for the Mackenzie River basin (Yunker et al., 2002a(Yunker et al., , 2011)), suggesting that erosion of the organic-rich rocks of the Devonian Canol formation in the lower Mackenzie River valley contributes large amounts of petrogenic hydrocarbons to the shelf. Additionally, the hydrocarbon sources of the Mackenzie Shelf and Canada Basin sediments exhibit a strong signal originating from vascular plants and petrogenic input that is likely to overwhelm a possible combustion signal, leading to a petrogenic signal (Yunker et al., 2011). In the western Arctic Ocean, Ma et al. (2017) also reported a mixed petrogenic and pyrogenic source for the PAHs in the surface sediments of the Chukchi Sea and Canada Basin, in line with our results. In the global Arctic Ocean, it has been reported that the natural background signature of petrogenic PAHs seemed to dominate the signal in sediments (Yunker et al., 2011). Similarly, the surface samples collected from the central CAA appeared to largely exhibit petrogenic signatures, as the Fla/(Fla+Pyr) values ranged from 0.13 to 0.50 (Fig. 6A). However, the BaA/(BaA+Chr) values ranged from 0.20 to 0.6, suggesting a mixed source/pyrogenic source. Regarding the surface samples retrieved from the eastern CAA, the Fla/(Fla+Pyr) values ranged from 0.13 to 0.67, and the BaA/(BaA+Chr) values ranged from 0.20 to 0.50, indicating a well-mixed origin from both petrogenic and pyrogenic sources (Fig. 6A-B). Thus, the central and eastern CAA exhibit a greater pyrogenic influence than the western CAA. It was previously established that the diagnostic ratios for sediments from remote areas might reflect a more pyrogenic influence because the main PAH sources are atmospheric deposition followed by sedimentation (Tsapakis et al., 2003;Tobiszewski and Namieśnik, 2012), which could explain our results. Ma et al. (2017) and Zhao et al. (2016) also reported that the PAHs in samples retrieved from the Arctic Ocean and the Makarov Basin originated from a mixture of petroleum and biomass combustion. However, although diagnostic ratios are useful for discriminating the origins of PAHs, they should be interpreted with caution due to different environmental processing of the isomers during transport processes (e.g., Galarneau, 2008;Yunker et al., 2002aYunker et al., ,b, 2011)). For example, degradation and/or transformation occurring during atmospheric processes and transport through the water column of the less stable fluoranthene and benz(a)anthracene might contribute to bias in old sediments (Yunker et al., 2002a(Yunker et al., , 2002b(Yunker et al., , 2011)). Tobiszewski and Namieśnik (2012) also suggested that the ratio of Fla/(Fla+Pyr) was more conservative than other ratios (e.g., BaA/[BaA+Ch] and anthracene over the sum of anthracene and phenanthrene; Ant/[Ant+Phe]) during atmospheric photoreactions. Pyrogenic PAHs stemming from anthropogenic combustion have been widely detected in atmospheric samples retrieved from remote areas in the Arctic, such as Alert (Yu et al., 2019). Modeling studies have shown that long-range atmospheric transport of PAHs from urban areas to remote Arctic regions occurs (Chen et al., 2018). More specifically, air mass trajectory modeling performed at Alert (Nunavut, Canada) has suggested that the atmospheric PAHs in this region mainly originate from Eurasia, North Europe and North America, while East China is a minor contributor (Wang et al., 2010). Hence, the main anthropogenic PAH source in the Canadian Arctic is the atmospheric deposition of PAHs stemming from worldwide hydrocarbon consumption (Klungsøyr et al., 2010;Yunker et al., 2011). It has also been proposed that local anthropogenic sources are actually negligible compared to deposition from remote sources (e.g., Rose et al., 2004;Wang et al., 2010). In addition, forest fires are also a contributor to atmospheric pyrogenic PAHs, with an annual budget of approximately 9 tons in the Canadian Arctic, and pyrogenic PAHs have become more frequently detected in atmospheric samples since 2005 (Klungsøyr et al., 2010;Yu et al., 2019). Ma et al. (2017) found a pyrogenic influence in deep ocean sediments of the central Arctic Ocean stemming from forest fire events. These events could explain the pyrogenic signal observed in the central and eastern CAA, especially since forest fire events had increased in Canada. For example, the average annual burned area was approximately 1 million hectares in the early 1920s and reached 2 million hectares in the 2010s, with a maximum area of 2.75 million hectares in the 1990s (Wildland Fire Management Working Group, 2013). In Frobisher Bay, near Iqaluit, it appears that anthropogenic activities could locally contribute to pyrogenic PAHs in the bay (Fig. S6). The city, home to more than 7700 people, produces its electricity via imported diesel fuel (Government of Canada, 2017). In 2017, its main greenhouse gas emission sectors were the transportation, industry and electricity sectors (Government of Canada, 2017), all contributing to a pyrogenic signature. Waste burning in Iqaluit is also a common practice (Giroux, 2014), and episodic landfill fire events might contribute to a local PAH input: in 2010, a landfill fire lasted 6 weeks (Harvey, 2018), while another major fire occurred between May and September in 2014 (Weichenthal et al., 2015). Finally, the petrogenic signature recorded near Sydkap Glacier confirms a coal origin (Fla/[Fla+Pyr] = 0.16) rather than an anthropogenic source, as previously reported (Fig. S6). Regarding the core samples, they all seem to exhibit a mainly petrogenic signature combined with mixed sources: the Fla/(Fla+Pyr) values range from 0.13 to 0.88, and the BaA/(BaA+Chr) values range from 0.16 to 0.50 (Figs. 6A-B and S5). These values are consistent with the results obtained for the surface samples, except for the eastern CAA core samples, in which the pyrogenic side is more abundant. Overall, it seems that the PAH sources over time have remained relatively constant since the core sample results are consistent with the surface sample results but feature a greater pyrogenic influence. Finally, the terrestrial samples exhibit Fla/Fla+Pyr values ranging from 0.14 to 0.38, which indicate a petrogenic source, whereas the BaA/(BaA+Chr) values range from 0.12 to 0.50, indicating a relatively wide range of sources, from petrogenic to mixed/pyrogenic sources. Since only 30% (n=4) of the terrestrial samples attained a BaA/(BaA+Chr) value compared to 85% (n=11) of the samples in regard to Fla/(Fla+Pyr), this pyrogenic influence might not be representative of all the terrestrial samples. However, if it is representative of the 4 samples, it might indicate a more direct connection between atmospheric pyrogenic PAHs and soils since the sedimentary processes do not occur and that soils are mainly influenced by atmospheric deposition (Mostert et al., 2010). Finally, the results given by the diagnostic ratios should be considered with care given the unknown effect of environmental processes occurring between the emission and deposition of the PAHs (Tobiszewski and Namieśnik, 2012). Katsoyiannis and Breivik (2014) illustrated that basic conditions, such as distance from the sources and ambient temperature, have a significant influence on the molecular ratios. Additionally, diagnostic ratios established for a certain type of sediment in urban area might not be directly applicable to remote sediments; indeed, old basin sediments in the Artic Ocean are depleted in reactive and LMW PAHs from combustion related sources, and only fluoranthene, pyrene and PAHS with molecular weights greater than 252 could provide usable source ratios (Yunker et al., 2011). Hence, the contradictory results obtained in this study do not mean that they are wrong: a combination of degradation during atmospheric processes and the remote locations might explain why the Fla/(Fla+Pyr) and BaA/(BaA+Chr) values are not exactly the same (Tobiszewski and Namieśnik, 2012). ## Principal component analysis (PCA) PCA based on the Σ16PAH data for all samples (surface, core and terrestrial samples) revealed that PC-1 (24% of the total variance) was positively correlated with 9,10-dimethylanthracene, 3,6-dimethylphenanthrene and pyrene, whereas PC-2 (15% of the total variance) was positively correlated with chrysene, 1-methylphenanthrene and pyrene (Fig. 7A). Finally, PC-3 (14% of the total variance) was positively correlated with 1-methylnaphthalene, 2-methylnaphthalene and acenaphthene (Fig. 7B). Parent PAHs are typically more closely associated with combustion processes, while alkylated PAHs are generally derived from petrogenic PAHs (Yunker and Macdonald, 1995;Lima et al., 2005, Balmer et al., 2019). Hence, each score indicated a mostly petrogenic influence. Additionally, the PC-1 and PC-3 scores were negatively correlated with at least one parent PAH composed of four rings (e.g., pyrene or chrysene). Unsubstituted PAHs containing four to six rings are mainly associated with combustion sources (Laflamme and Hites, 1978). Hence, PCA confirms a mainly petrogenic influence with a small pyrogenic contribution to the PAHs occurring in the surface, core and terrestrial sediments within the CAA. Overall, both diagnostic ratios support the predominant petrogenic nature of the PAHs in the surface sediments of the CAA (Thomas and MacDonald, 2005;Yunker et al., 2011;Foster et al., 2015;Yu et al., 2019). These petrogenic sources are presumably derived from hydrocarbon seeps, weathering of organic-rich rocks, and coastal terrestrially derived material. Strong pyrogenic influences are observed in the central and eastern CAA and are likely due to forest fire events plus long-range atmospheric transport and deposition of PAHs originating from distant sources. Other pyrogenic influence might occur because the remote locations are mainly influenced by deposition of atmospheric pyrogenic PAHs, as previously mentioned. Our results are comparable to those of previous studies pointing to petrogenic sources of the PAHs in the surface sediments of the Mackenzie River/Delta, Beaufort Sea, Nansen Basin, and North Baffin Bay (Yunker et al., 2011, Foster et al., 2015) and mixed sources in the surface sediments of North Baffin Bay, Greenland Sea and north Barents Sea (Yunker et al., 2011;Foster et al., 2015). ## Risk assessment of the PAHs 602 The potential ecological risk of the PAHs in sediments can be determined based on 603 guideline values, such as the effects range-low (ERL, the probability of adverse biological 604 effects is <10%) and effects range-median (ERM, the probability of adverse biological 605 effects is >50%) values, as proposed by Long et al. (1995). The PAH content ranges in the 606 CAA sediments are almost all below the ERL and ERM values (Table 3), indicating that the 607 measured PAHs pose a low ecological risk to the benthic organisms or other organisms living 608 near the sediments. Only fluorene might be an exception and would require greater attention. 609 The sample with a result of 23.7 ng g -1 is located in the Amundsen Gulf. This result excludes 610 the next highest value for fluorene, 15.9 ng g -1 , and all samples are therefore below the above 611 ERL and ERM values. 612 613 Long et al., 1995. 615 616 ## Conclusions This study provides a robust baseline record of the PAHs in surface and core marine sediments and on-land sediments retrieved from the CAA. The results of this research yield the following generalizations and conclusions: 2. The sums of the concentrations of the 16 priority PAHs designated by the US EPA in both the marine and terrestrial sediments of the CAA are fairly low and comparable to other sediment levels reported for Arctic remote regions. Indeed, our results reveal Σ16PAH values ranging from 7.8 to 247.7 ng g −1 for the total CAA, while the terrestrial sediments exhibit values ranging from 23.1 to 108.8 ng g −1 . Overall, regarding Σ16PAHs, the different regions are classified as follows: central CAA < eastern CAA < terrestrial sediments < western CAA. Additionally, the values of Σ16PAHs in the sediments are all below the ERL and ERM guidelines, except for a single sample retrieved from the Amundsen Gulf, which exhibits a fluorene content above the ERL value. 3. The inputs throughout the last century have remained relatively stable and below the maximum sum obtained for the surface sediment samples, with the Σ16PAH values in the core samples ranging from 8.1 to 191.1 ng g −1 with a very low Σ16PAH value in the northernmost core located in the Robertson Channel. 4. The diagnostic ratios of Fla/(Fla+Pyr) and BaA/(BaA+Chr), in addition to the PCA results of the PAH data, suggest that the PAHs mainly have a natural petrogenic origin, but the central and eastern CAA areas also contain PAHs originating from both fossil fuel and biomass burning, likely because of the increase in forest fire events in northern Canada in recent decades and long-range atmospheric deposition of PAHs stemming from urban areas located further south. However, diagnostic ratios should be used with care when applied to sediments from remote locations: PAHs might undergo major environmental processes before their deposition, which could lead to bias and complications in interpreting diagnostic ratio values.

chemsum

{"title": "Spatial and temporal distributions of polycyclic aromatic hydrocarbons in sediments from the Canadian Arctic Archipelago", "journal": "ChemRxiv"}

a_facile,_rapid,_one-pot_regio/stereoselective_synthesis_of_2-iminothiazolidin-4-ones_under_solvent/

## Abstract: A rapid and efficient one pot solvent/scavenger-free protocol for the synthesis of 2-iminothiazolidin-4-ones has been developed. Interestingly, the regio/stereoselective synthesis affords the regioisomeric (Z)-3-alkyl/aryl-2-(2-phenylcyclohex-2-enylimino)thiazolidin-4-one as the sole product in good yield. The selectivities observed have been rationalized based on the relative magnitude of the allylic strains developed during the course of the reaction. This is the first report wherein the impact of allylic strains in directing the regiocyclization has been noted. ## Introduction Thiazolidin-4-one derivatives are well known for their bioactivities such as antidiabetic , anticancer , calcium-channel blocker , platelet activating factor (PAF) antagonist and anti-HIV activity. In addition, 2-iminothiazolidin-4-ones exhibit remarkable hypnotic , antitubercular , cardiovascular and cyclooxygenase (COX) inhibitory activities (Figure 1). A common strategy involved in the prevailing synthetic protocols for 2-iminothiazolidin-4-ones is the cyclization of thioureas with α-halocarboxylic acids or acyl halides or carboxylic esters . These protocols are generally solution phase methods using organic solvents and acid scavengers. In the present scenario, such protocols may not be recommended by the principles of green chemistry. Consequently, the search for simple and efficient environmentally friendly methodologies for the synthesis of 2-iminothiazolidin-4-ones is worth attempting. In this regard, and in continuation of our recent reports on the solvent-free synthesis of amides , thioamides , cyclic imides , thiazolidin-4-ones , spirothiazolidin-4-ones , 1,2,3-triazoles , 1,2,3-triazolylchalcones , and 1,2,3-triazolyldihydropyrimidine-2-thiones , we herein present a one-pot solvent/scavenger-free synthetic protocol for 2-iminothiazolidin-4-ones. This environmentally benign method avoids toxic organic solvents and acid scavengers, the details of which are presented below. ## Results and Discussion At the outset, optimization of the one-pot reaction was attempted by varying the solvents and using triethylamine as the acid scavenger (Table 1). The reaction was also attempted under solvent-free conditions. The latter was more promising in the sense that the reaction was very rapid affording the product 4f in 15-20 min (Table 1, entries 7 and 8) compared to 2-6 h (Table 1, entries 1-6) in solvents. The structure of the product 4f was assigned as (Z)-2-(2-phenylcyclohex-2-enylimino)-3-ptolylthiazolidin-4-one based on the single-crystal XRD data of its analogues (4c and 4j). Though the rate of reaction rate could be accelerated, the yield of 2-iminothiazolidin-4-one 4f was not good (41-66%) under both solution-phase and solvent-free conditions. Hence, as an attempt to optimize the yield, the solvent-free protocol was screened with and without the acid scavenger. Hereto, the yield of the product was poor (Table 2). Thus, the screening indicated that the scavengers had no positive, but rather an impeding effect. To develop an insight in this regard, a plausible mechanism of the reaction in the presence of the acid scavenger was proposed (Scheme 1). From the mechanism, it can be envisaged that the acid scavenger may neutralize the HCl (that is generated during the course of the reaction) or the iminium ions by deprotonation. Also, another possibility is that the use of base as the scavenger may lead to the acid-base reaction resulting in the formation of the carboxylate anion of one the starting materials viz. chloroacetic acid or the thiourea-chloroacetic acid coupled product. This may retard the direct amine-carboxylic acid coupling, thus decreasing the yield of the product. In view of the above perception, the solvent-free protocol was screened with one equivalent or in the absence of acid scavenger and varying equivalents of chloroacetic acid at 100 °C (Table 2). The optimum conditions were found to be with 3 equivalents of chloroacetic acid in the absence of acid scavenger affording a good yield of 2-iminothiazolidin-4-one 4f (Table 2, entry 6). In this context, it is pertinent to mention that while the prevailing solution-phase protocol uses an acid scavenger, such as sodium hydroxide, triethylamine, pyridine or sodium acetate, the solvent-free methodology involved in the present investigation does not require any acid scavenger. The scope of the new synthetic protocol was proved through the synthesis of a library of 2-iminothiazolidin-4-ones (Table 3). However, its limitations were realized when the synthesis of ortho-tolyl/1-napthyl analogues and the para-substituted (NO 2 and COOH) phenyl analogues failed. Apparently, the reason for this can be attributed to the retardation of the nucleophilic attack of the amines on the isothiocyanate due to the steric effect (Figure 2) in the former, and decrease of the nucleo- philicity of the amines by the electron-withdrawing group in the latter, thus not affording the expected thiourea. Having established the new protocol for the synthesis of 2-iminothiazolidin-4-ones, the method was extended to the rapid synthesis of a library of thiazolidinone derivatives (Table 4). Further, it is pertinent to mention here the interesting regio/ stereoselectivity noted in the synthesis. Though the formation of a Yield of isolated product, b regioisomeric mixtures obtained. the four regio/stereoisomeric 2-iminothiazolidin-4-ones 4a, 4b, 5a and 5b is possible, it is novel to note that only one of them, viz. 4b, is formed exclusively (Figure 3). The high regio/stereoselectivity of the reaction can be rationalized based on the relative magnitudes of allylic strains (A 1,2 and A 1,3 ) developed during the course of the regiocyclization (Scheme 2). In this context, it is relevant to recall the literature reports on the factors directing the regioselectivity in the synthesis of 2-iminothiazolidin-4-ones. Only a couple of reports in this regard are available in the literature. While one of these reports suggests that the pK a of amines directs the regioselectivity, another investigation indicates that the chelating effect of the substituent directs the regiochemical outcome. In both the reports, two regioisomeric 2-iminothiazolidin-4-ones are obtained. Thus, the present investigation affording a single regioisomeric product exclusively is the first report wherein the allylic strains are noted to direct the high regioselectivity. Finally, the stereoselective formation of the (Z)-stereoisomer is also explicable based on allylic strain, which is summarized in Figure 4. ## Conclusion In conclusion, a new solvent/scavenger-free synthetic protocol for 2-iminothiazolidin-4-ones has been reported. Unlike the prevailing solution-phase protocols employing organic solvents and acid scavengers, the present study avoids solvents and scavengers. The rate of the reaction is prominently enhanced under solvent-free conditions compared to that in the solution phase. Apparently, the intimacy of the highly polar reactants in the fused state in the absence of solvent may be responsible for the rate enhancement.

chemsum

{"title": "A facile, rapid, one-pot regio/stereoselective synthesis of 2-iminothiazolidin-4-ones under solvent/scavenger-free conditions", "journal": "Beilstein"}

seconds-resolved_pharmacokinetic_measurements_of_the_chemotherapeutic_irinotecan_<i>in_situ</i>_in_t

## Abstract: The ability to measure drugs in the body rapidly and in real time would advance both our understanding of pharmacokinetics and our ability to optimally dose and deliver pharmacological therapies. To this end, we are developing electrochemical aptamer-based (E-AB) sensors, a seconds-resolved platform technology that, as critical for performing measurements in vivo, is reagentless, reversible, and selective enough to work when placed directly in bodily fluids. Here we describe the development of an E-AB sensor against irinotecan, a member of the camptothecin family of cancer chemotherapeutics, and its adaptation to in vivo sensing. To achieve this we first re-engineered (via truncation) a previously reported DNA aptamer against the camptothecins to support high-gain E-AB signaling. We then co-deposited the modified aptamer with an unstructured, redox-reporter-modified DNA sequence whose output was independent of target concentration, rendering the sensor's signal gain a sufficiently strong function of square-wave frequency to support kinetic-differential-measurement drift correction. The resultant, 200 mm-diameter, 3 mm-long sensor achieves 20 s-resolved, multi-hour measurements of plasma irinotecan when emplaced in the jugular veins of live rats, thus providing an unprecedentedly high-precision view into the pharmacokinetics of this class of chemotherapeutics. ## Introduction The goal of personalized medicine is to precisely tailor treatment to the individual. 1,2 To this end, an ability to measure drugs in the living body with seconds resolution would allow clinicians to defne drug dosing based on high-precision, patient-specifc pharmacokinetic measurements rather than on indirect predictors of drug metabolism such as age, body mass, or pharmacogenetics. 3,4 Ultimately, the ability to measure drugs in the body in real-time would enable closed-loop feedback-controlled delivery, 5 vastly improving dosing precision by actively responding to minute-to-minute fluctuations in a patient's metabolism. 4,6 The development of such technology, however, faces signifcant hurdles. 7,8 First, an in vivo sensor must be small enough to be placed in the body without causing undue damage. Second, it cannot require the addition of exogenous reagents or the use of batch processing, such as washing or separations. Third, it must make measurements at a frequency that is rapid relative to the drug's pharmacokinetics. Finally, it must be selective and stable enough to work for prolonged periods in the complex, fluctuating environments found in vivo. To this end we are developing electrochemical aptamer-based (E-AB) sensors, a technology that, by achieving these goals, supports the high frequency, real-time measurement of specifc molecules directly in the living body. 9 E-AB sensors employ an electrode-bound, redox-reporter-modifed aptamer as their recognition element (Fig. 1A). Binding of the target molecule to this aptamer induces a conformational change that produces an easily measured electrochemical output (here we employ square wave voltammetry) without needing reagent additions or wash steps. Because E-AB signaling is generated by a binding-induced conformational change and not, as is the case for most other reagentless biosensor architectures, by the adsorption of target to the sensor surface, 7 E-AB sensors are largely insensitive to non-specifc adsorption and support multi-hour measurements in biological fluids not only in vitro 10 but also in vivo. 9 Finally, because their signaling arises due to target binding alone, and not, as is the case, for example, of the continuous glucose monitor, 11 from the chemical reactivity of the target, E-AB sensors are a platform technology generalizable to a wide variety of analytes, including two of which, the aminoglycosides and doxorubicin, have been measured in vivo. 9 Building on this foundation we describe here the fabrication and characterization of an E-AB sensor adapted to measurements in situ in the body, one directed against the camptothecin family of anticancer drugs, an important class of chemotherapeutic agents used in the treatment of a range of human cancers. 12,13 ## Results and discussion As the recognition element in our sensor we employ a DNA aptamer that binds to the camptothecins. 14,15 Specifcally, a 40base version of this aptamer, termed CA40, which folds into a target-recognizing G-quadruplex flanked by a 12-base-pair stem (Fig. 2A), binds the camptothecin, irinotecan, with a dissociation constant of 475 AE 10 nM when the unmodifed aptamer is free in solution (Fig. 2B and S1 †). To adapt this into an E-AB sensor modifed its 3 0 end with a methylene blue redox reporter and deposited it onto a gold electrode via a six-carbon thiol at its 5 0 end (Fig. 1A). Electrochemically interrogating the resulting sensor in buffer we observe the expected Langmuir isotherm binding with an estimated K D of 126 AE 24 mM (error bars here and, unless otherwise noted, reflect the standard deviation derived using at least three independently fabricated sensors), signal gain (the relative change in signal upon the addition of saturating target) of 84 AE 4% (Fig. 2C, blue curve) and association and dissociation kinetics too rapid to measure (time constants < 5 s at clinically relevant concentrations; Fig. 2D). We presume that the poorer affinity the aptamer exhibits in the context of the sensor arises due to interactions with the electrode surface, as this is known to destabilize the folding (thus hindering binding) surface-attached oligonucleotide. 16 Despite this, when challenged in buffer the sensor supports the detection of irinotecan over the clinically relevant 1 to 15 mM range. 17,18 When challenged in whole blood, however, the (apparent) affinity of the surface-bound aptamer is poorer still (K D ¼ 291 AE 15 mM), presumably because the concentration of the free drug is reduced due to protein binding. 17 Worse, under these conditions the gain falls to 15 AE 1%, pushing its useful dynamic range out of the clinically relevant concentration window (Fig. 2C, red curve). To improve sensor performance in whole blood we reengineered the camptothecin-binding aptamer to better populate its "unfolded" state in the absence of target, thus increasing the sensor's gain. 19,20 To do so we destabilized the aptamer's doublestranded stem (Fig. 3A) via either truncation (CA36, CA32, CA28, CA16) or the introduction of one (CA40_2MM) or two (CA40_2MM) G-T mismatches. As estimated using the nucleic acid folding predictor NUPACK 21 these strategies should decrease the stability of folded aptamer from the 31.8 kJ mol 1 of the C40 parent to as low as 0.3 kJ mol 1 for CA28 (Fig. S2 †). Characterizing sensors fabricated using these variants (Fig. 3A) we obtain dissociation constants ranging from 38 AE 11 mM to 254 AE 48 mM (Table 1 ESI †) and signal gain of up to 755% (Fig. 3B and Table S1 †) when challenged in working buffer. Testing in whole blood (Fig. 3C) once again reduces both gain and apparent affinities (Table 1 ESI †). Even under these more challenging conditions, however, sensors employing the CA32, CA28, and CA16 variants support high-gain E-AB sensing. Having achieved good in vitro performance with a sensor employing the CA32 variant we next set out to adapt this to use in situ in the veins of live rats. Under such conditions E-AB sensors often exhibit signifcant baseline drift. 9,22 We have previously corrected this using "Kinetic Differential Measurements" (KDM) an approach that exploits the generally strong square-wave frequency dependence of E-AB signal gain. 9,22 Specifcally, the signal gain of the E-AB sensors we have previously described is so great that they exhibit a "signal-on" (target binding increases the signaling current) response at some square-wave frequencies no observable gain or even "signal-off" behavior at others. 9,22 Conveniently, the signals obtained under these different regimes drift in concert such that taking their difference via KDM removes the drift seen in vivo. 9 And since the two signaling currents respond in opposition in the presence of their target, taking their difference also improves signal gain. Uniquely in our experience, however, the gain of the camptothecin-detecting E-AB sensors (CA32, CA28, and CA16) is only a weak function of square-wave frequency (Fig. S3 †), necessitating the development of a new approach to performing KDM. To enhance the frequency-dependence of the sensor's gain in support of KDM drift correction we added a second reporter-modifed DNA strand to the sensor that: (1) transfers electrons more rapidly than the aptamer does and (2) does not respond to the presence of the target (Fig. S4A †). Our rational for doing so was that, at frequencies at which this "non-responsive" DNA dominates the signal (i.e., at higher frequencies) the gain of the resultant sensor will be low, and at frequencies at which, instead, the aptamer dominates the signal gain will be higher (Fig. S5B †). To achieve this we co-deposited the CA32 aptamer variant and an unstructured 10-base strand comprised of a random sequence of adenines and thymines (Fig. 4A) that is known to transfer electrons at a rate of 80 s 1 (Fig. S4 †). 23 Per our expectations, sensors employing a 1 : 1 mixture of this sequence and the aptamer achieve sufficiently frequency-dependence gain to enable KDM (Fig. 4B). To our surprise, however, the resultant frequency dependence is so strong that the sensor's gain becomes slightly negative at low frequencies, an observation inconsistent with the expectations described above (if the currents are additive, the gain cannot go below zero). We presume this occurs due to interactions between the two sequences on the surface that alter their electron transfer kinetics. Irrespective of its origins, however, the effect supports accurate KDM drift correction. Specifcally, using the signals obtained at 10 Hz (signal-off) and 120 Hz (signal-on) to perform KDM (Fig. S5 †) we can easily monitor irinotecan in both buffer and whole blood (Fig. 4C) over the entire 0.5 to 15 mM (0.06 to 10 mg mL 1 ) human therapeutic range. 17,18 KDM-corrected indwelling E-AB sensors readily support the real-time, high frequency irinotecan measurements in situ in the bodies of live rats. To demonstrate this we fabricated sensors using 75 mm-diameter gold, platinum and silver wires as the working, counter and reference electrodes, respectively (Fig. 1B). We inserted the resulting sensor in the jugular vein of anesthetized Sprague-Dawley rats via a previously emplaced 22gauge catheter (Fig. 1C). Testing this with a single intravenous injection (20 mg kg 1 ) of irinotecan we fnd that the signal observed at both 10 and 120 Hz respond to the drug, but these are also accompanied by the expected 9 signal drift (Fig. 5A). And the gain observed at 10 Hz becomes, under these conditions, slightly positive. We are nevertheless still able to use KDM to correct the sensor's drift and recover stable baselines, thus enabling continuous, real-time measurements of the drug at therapeutically relevant concentrations (Fig. 5B). To further characterize the performance of the camptothecin sensor we used it to monitor sequential intravenous injections of irinotecan (at 10 and 20 mg kg; Fig. 6A). The resultant maximum concentrations (C MAX ¼ 39.8 AE 3.2 mM and 20.9 AE 2.0 mM, respectively; here and below the confdence intervals reflect standard errors calculated from the fts) and distribution rates (a ¼ 0.58 AE 0.07 min and 0.48 min (fxed value, see ESI Table 2 †)) are comparable to those seen in previous studies that employed ex-vivo drug-level measurements (Fig. S6A and Table S2 †). In contrast, the elimination rates (b ¼ 9.2 AE 1.4 min and 8.4 AE 2.2 min) we observe are more rapid than those Fig. 3 We reengineered the parent aptamer to produce higher-gain E-AB signaling. (A) We did so by destabilizing the aptamer's stem-loop (thus increasing the population of unfolded molecules poised to respond to target) via either introduction of one (CA40_1MM) or two (CA40_2MM) mismatches or via truncation (CA36, CA32, CA28, CA16) of the stem. (B) When challenged in a simple buffered solution all of the re-engineered variants exhibited higher gain than that of the parent aptamer (see ESI Table 1 †), with the most destabilized (CA40_2MM, CA32, CA28, CA16) producing the greatest signal gain. (C) When tested in whole blood their gain and affinity are reduced, but the best performing nevertheless still support high-gain E-AB sensing. reported previously and, thus, the resulting "areas under the curve" for the drug are reduced (Fig. S7 and Table S2 †). We believe this discrepancy (Fig. S7 †) occurs because the prior work used chromatographic and mass spectrometric methods to Fig. 4 To correct the drift of seen during in vivo deployment we modified the E-AB sensor so that it better supports "Kinetic Differential Measurements" (KDM). (A) KDM requires that the gain of an E-AB sensor be a strong function of square wave frequency. 22 To induce this we codeposited the aptamer with a redox-reporter-modified linear DNA sequence that does not respond to target. (B) The signal gain (relative signal change between no target and saturating target -i.e., 100 mM) of the original E-AB sensor (100 : 0 black curve) is a relatively minor function of square-wave frequency. Upon co-deposition with increasing amounts of the linear-strand (to a maximum ratio of 50 : 50, red curve) we observe increasingly strong frequency dependence, albeit with a corresponding reduction in the maximum gain. (C) A sensor fabricated using a 50 : 50 mixture of the two strands and employing KDM drift correction (here the difference in the relative signals seen at 10 and 120 Hz) responds to target over the clinically-relevant range (0.5 mM to 15 mM; 0.06 and 10 mg mL 1 ) 17,18 in both buffer and in undiluted whole blood. Fig. 5 The KDM-corrected E-AB sensor supports real time, secondsresolved measurements of plasma irinotecan levels. 9,22 (A) In the absence of KDM signals collected at high (120 Hz) and low (10 Hz) frequencies both drift significantly, but because they drift in concert (B) taking their difference (KDM) produces a stable baseline. (C) As expected, control injections of either a saline "blank" or a second chemotherapeutic (5-fluorouracil, which is often co-administered with irinotecan) 31,32 do not produce any measurable sensor response. This journal is © The Royal Society of Chemistry 2019 Chem. Sci., 2019, 10, 8164-8170 | 8167 measure total drug levels (which requires removal of blood samples from the animal's body and the extraction of the total drug into buffer). 24-28 E-AB sensors, in contrast, measure the free drug, which is the fraction of the drug that is pharmacologically active. 29 And, in general, the elimination and clearance of free drug are more rapid than those of total drug as drugs that interact strongly with plasma proteins tend to clear more slowly than those that do not. 30 E-AB-derived measurements of irinotecan pharmacokinetics represent a signifcant advance over prior pharmacokinetic studies of the camptothecins. For example, the 20 s temporal resolution of our measurements (defned by the time required to take the two square wave scans necessary to perform KDM) is at least an order of magnitude better than that of the most highly time resolved prior study. Moreover, all prior studies reported plasma level measurements averaged over multiple animals, thus eliminating their ability to explore subject-to-subject pharmacokinetic variability. The present E-AB-derived measurement parameters, in contrast, provide 300 time points per hour in each animal, and thus determine the pharmacokinetics of individuals with exceptional precision. Because the excretion phase of irinotecan exhibits signifcant inter-patient variability (due to drug-drug interactions, variations in health status, and pharmacogenetics), 17,18,33,34 this latter Fig. 7 (A) To determine irinotecan's pharmacokinetics with more precision we performed an intravenous injection at a much higher dose (60 mg kg 1 ). (B) The higher peak concentrations reached in this experiment lead to longer measurement runs, in turn improving the precision of our estimates of the relevant pharmacokinetic parameters. point is likely of clinical signifcance. To illustrate our ability to measure such variability we performed sequential 10 and 20 mg kg 1 irinotecan injections in three rats (Fig. 6). The resulting measurements reveal only small ($10 to 20%) variation in either C MAX or the rate of the distribution phase (Fig. S6B and C, and Table S2 †). In contrast, however, the rate of drug excretion and its clearance values vary many fold from individual to individual. As all of the animals we employed in these experiments were healthy male rats these pharmacokinetic differences arose solely due to metabolic variability between the animals. The elimination rate and clearance of irinotecan are the pharmacokinetic parameters used to determine its optimized, personalized dosing during chemotherapy. 17,18,33,34 To measure these parameters with greater precision we administered a large dose (60 mg kg 1 ) of the drug over a longer period. The higher plasma concentrations this produces lead, in turn, to a longer measurement period (after delivery ceases) before the sensor's limit of detection is reached (Fig. 7). The $150 plasma drug measurements we thus achieve in a single pharmacokinetic profle produces estimates of the drug's elimination half-life (10.4 AE 0.4 min) and clearance (18.6 AE 1.4 mL min 1 ) that are far more precise than those produced in prior, ex vivo studies, which typically achieve less than a dozen measurements per profle, much less the two measurements used in typical "peaks-and-troughs" clinical measurements. ## Conclusions Here we describe an indwelling E-AB sensor supporting the seconds-resolved measurement of the anticancer drug irinotecan in situ in the living body over the course of hours. Design of the sensor required the reengineering of a parent aptamer to support high-gain E-AB signaling and the development of a novel method to ensure sufficient frequency-dependent signal gain to support KDM-based drift correction. Using the resulting sensors we measured plasma irinotecan levels with micromolar concentration resolution and seconds temporal resolution, with the latter representing an orders of magnitude improvement over that of prior studies. The resulting measurements defne the pharmacokinetics of irinotecan of individual animals, providing an unprecedented high precision view of the drug's inter-subject pharmacokinetic variability. E-AB sensors are a platform technology that supports the high frequency, real-time measurement of specifc molecules (irrespective of their chemical reactivity) in situ in the living body. When coupled with the platform's convenience and precision this versatility provides signifcant opportunities to improve drug dosing. As noted above, for example, irinotecan suffers from signifcant inter-patient metabolic variability, 35,36 leading to toxicity and increasing side effects. 17,34 But because current methods for measuring plasma drug levels are slow and cumbersome, 37 the FDA has invoked pharmacogenetic estimates of metabolism as the primary means of reducing the risk associated with this variation. 35 In this light, the ease with which E-AB sensors provide high precision, patient-specifc measurements of drug elimination (as opposed to indirect estimates), suggests the platform could provide a valuable adjunct to chemotherapeutic treatment. In addition to improving the precision and accuracy of personalized dose determination, E-AB-derived measurements may also support a new paradigm for personalized drug delivery. Specifcally, we have recently used the real-time concentration information provided by E-AB sensors to inform closed-loop feedback controlled drug delivery. 5 In this the rate of drug administered is optimized multiple times a minute, enabling the maintenance of plasma drug concentrations at a pre-defned value with precision of better than 20% despite $3-fold hour-to-hour changes in drug pharmacokinetics. This approach to drug delivery provides an unprecedented means of overcoming pharmacokinetic variability, improving the overall efficacy and safety of treatment. Given this, drug-detecting E-AB sensors could prove a powerful new tool in the clinician's arsenal. ## Conflicts of interest One author (K. W. P.) has a fnancial interest in and serves on the scientifc advisory boards of two companies attempting to commercialize E-AB sensors. A. I., N. A. C. and K. W. P. have fled a provisional patent based on the work presented in this paper.

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{"title": "Seconds-resolved pharmacokinetic measurements of the chemotherapeutic irinotecan <i>in situ</i> in the living body", "journal": "Royal Society of Chemistry (RSC)"}