The structural properties of full-length annexin A11.

Annexin A11 (ANXA11) is a calcium-dependent phospholipid-binding protein belonging to the annexin protein family and implicated in the neurodegenerative amyotrophic lateral sclerosis. Structurally, ANXA11 contains a conserved calcium-binding C-terminal domain common to all annexins and a putative intrinsically unfolded N-terminus specific for ANXA11. Little is known about the structure and functions of this region of the protein. By analogy with annexin A1, it was suggested that residues 38 to 59 within the ANXA11 N-terminus could form a helical region that would be involved in interactions. Interestingly, this region contains residues that, when mutated, may lead to clinical manifestations. In the present study, we have studied the structural features of the full-length protein with special attention to the N-terminal region using a combination of biophysical techniques which include nuclear magnetic resonance and small angle X-ray scattering. We show that the N-terminus is intrinsically disordered and that the overall features of the protein are not markedly affected by the presence of calcium. We also analyzed the 38-59 helix hypothesis using synthetic peptides spanning both the wild-type sequence and clinically relevant mutations. We show that the peptides have a remarkable character typical of a native helix and that mutations do not alter the behaviour suggesting that they are required for interactions rather than being structurally important. Our work paves the way to a more thorough understanding of the ANXA11 functions.

Strong structuring arising from weak cooperative O-H···π and C-H···O hydrogen bonding in benzene-methanol solution.

Weak hydrogen bonds, such as O-H···π and C-H···O, are thought to direct biochemical assembly, molecular recognition, and chemical selectivity but are seldom observed in solution. We have used neutron diffraction combined with H/D isotopic substitution to obtain a detailed spatial and orientational picture of the structure of benzene-methanol mixtures. Our analysis reveals that methanol fully solvates and surrounds each benzene molecule. The expected O-H···π interaction is highly localised and directional, with the methanol hydroxyl bond aligned normal to the aromatic plane and the hydrogen at a distance of 2.30 Å from the ring centroid. Simultaneously, the tendency of methanol to form chain and cyclic motifs in the bulk liquid is manifest in a highly templated solvation structure in the plane of the ring. The methanol molecules surround the benzene so that the O-H bonds are coplanar with the aromatic ring while the oxygens interact with C-H groups through simultaneous bifurcated hydrogen bonds. This demonstrates that weak hydrogen bonding can modulate existing stronger interactions to give rise to highly ordered cooperative structural motifs that persist in the liquid phase.

Sleep-related benefits to transitive inference are modulated by encoding strength and joint rank.

Transitive inference is a measure of relational learning that has been shown to improve across sleep. Here, we examine this phenomenon further by studying the impact of encoding strength and joint rank. In experiment 1, participants learned adjacent premise pairs and were then tested on inferential problems derived from those pairs. In line with prior work, we found improved transitive inference performance after retention across a night of sleep compared with wake alone. Experiment 2 extended these findings using a within-subject design and found superior transitive inference performance on a hierarchy, consolidated across 27 h including sleep compared with just 3 h of wake. In both experiments, consolidation-related improvement was enhanced when presleep learning (i.e., encoding strength) was stronger. We also explored the interaction of these effects with the joint rank effect, in which items were scored according to their rank in the hierarchy, with more dominant item pairs having the lowest scores. Interestingly, the consolidation-related benefit was greatest for more dominant inference pairs (i.e., those with low joint rank scores). Overall, our findings provide further support for the improvement of transitive inference across a consolidation period that includes sleep. We additionally show that encoding strength and joint rank strongly modulate this effect.

SERS Sensing of Dopamine with Fe(III)-Sensitized Nanogaps in Recleanable AuNP Monolayer Films.

Sensing of neurotransmitters (NTs) down to nm concentrations is demonstrated by utilizing self-assembled monolayers of plasmonic 60 nm Au nanoparticles in close-packed arrays immobilized onto glass substrates. Multiplicative surface-enhanced Raman spectroscopy enhancements are achieved by integrating Fe(III) sensitizers into the precisely-defined <1 nm nanogaps, to target dopamine (DA) sensing. The transparent glass substrates allow for efficient access from both sides of the monolayer aggregate films by fluid and light, allowing repeated sensing in different analytes. Repeated reusability after analyte sensing is shown through oxygen plasma cleaning protocols, which restore pristine conditions for the nanogaps. Examining binding competition in multiplexed sensing of two catecholamine NTs, DA and epinephrine, reveals their bidentate binding and their interactions. These systems are promising for widespread microfluidic integration enabling a wide range of continuous biofluid monitoring for applications in precision health.

In-Situ Spectro-Electrochemistry of Conductive Polymers Using Plasmonics to Reveal Doping Mechanisms.

Conducting polymers are a key component for developing wearable organic electronics, but tracking their redox processes at the nanoscale to understand their doping mechanism remains challenging. Here we present an in-situ spectro-electrochemical technique to observe redox dynamics of conductive polymers in an extremely localized volume (<100 nm3). Plasmonic nanoparticles encapsulated by thin shells of different conductive polymers provide actively tuned scattering color through switching their refractive index. Surface-enhanced Raman scattering in combination with cyclic voltammetry enables detailed studies of the redox/doping process. Our data intriguingly show that the doping mechanism varies with polymer conductivity: a disproportionation mechanism dominates in more conductive polymers, while sequential electron transfer prevails in less conductive polymers.

Collective Mid-Infrared Vibrations in Surface-Enhanced Raman Scattering.

Surface-enhanced Raman scattering (SERS) is typically assumed to occur at individual molecules neglecting intermolecular vibrational coupling. Here, we show instead how collective vibrations from infrared (IR) coupled dipoles are seen in SERS from molecular monolayers. Mixing IR-active molecules with IR-inactive spacer molecules controls the intermolecular separation. Intermolecular coupling leads to vibrational frequency upshifts up to 8 cm-1, tuning with the mixing fraction and IR dipole strength, in excellent agreement with microscopic models and density functional theory. These cooperative frequency shifts can be used as a ruler to measure intermolecular distance and disorder with angstrom resolution. We demonstrate this for photochemical reactions of 4-nitrothiophenol, which depletes the number of neighboring IR-active molecules and breaks the collective vibration, enabling direct tracking of the reaction. Collective molecular vibrations reshape SERS spectra and need to be considered in the analysis of vibrational spectra throughout analytical chemistry and sensing.

Optical suppression of energy barriers in single molecule-metal binding.

Transient bonds between molecules and metal surfaces underpin catalysis, bio/molecular sensing, molecular electronics, and electrochemistry. Techniques aiming to characterize these bonds often yield conflicting conclusions, while single-molecule probes are scarce. A promising prospect confines light inside metal nanogaps to elicit in operando vibrational signatures through surface-enhanced Raman scattering. Here, we show through analysis of more than a million spectra that light irradiation of only a few microwatts on molecules at gold facets is sufficient to overcome the metallic bonds between individual gold atoms and pull them out to form coordination complexes. Depending on the molecule, these light-extracted adatoms persist for minutes under ambient conditions. Tracking their power-dependent formation and decay suggests that tightly trapped light transiently reduces energy barriers at the metal surface. This opens intriguing prospects for photocatalysis and controllable low-energy quantum devices such as single-atom optical switches.

Carboxylate Catalyzed Isomerization of ß,γ-Unsaturated N-Acetylcysteamine Thioesters.

We demonstrate herein the capacity of simple carboxylate salts - tetrametylammonium and tetramethylguanidinium pivalate - to act as catalysts in the isomerization of ß,γ-unsaturated thioesters to α,ß-unsaturated thioesters. The carboxylate catalysts gave reaction rates comparable to those obtained with DBU, but with fewer side reactions. The reaction exhibits a normal secondary kinetic isotope effect (k1H /k1D =1.065±0.026) with a ß,γ-deuterated substrate. Computational analysis of the mechanism provides a similar value (k1H /k1D =1.05) with a mechanism where γ-reprotonation of the enolate intermediate is rate determining.

Combined Free-Energy Calculation and Machine Learning Methods for Understanding Ligand Unbinding Kinetics.

The determination of drug residence times, which define the time an inhibitor is in complex with its target, is a fundamental part of the drug discovery process. Synthesis and experimental measurements of kinetic rate constants are, however, expensive and time consuming. In this work, we aimed to obtain drug residence times computationally. Furthermore, we propose a novel algorithm to identify molecular design objectives based on ligand unbinding kinetics. We designed an enhanced sampling technique to accurately predict the free-energy profiles of the ligand unbinding process, focusing on the free-energy barrier for unbinding. Our method first identifies unbinding paths determining a corresponding set of internal coordinates (ICs) that form contacts between the protein and the ligand; it then iteratively updates these interactions during a series of biased molecular dynamics (MD) simulations to reveal the ICs that are important for the whole of the unbinding process. Subsequently, we performed finite-temperature string simulations to obtain the free-energy barrier for unbinding using the set of ICs as a complex reaction coordinate. Importantly, we also aimed to enable the further design of drugs focusing on improved residence times. To this end, we developed a supervised machine learning (ML) approach with inputs from unbiased "downhill" trajectories initiated near the transition state (TS) ensemble of the string unbinding path. We demonstrate that our ML method can identify key ligand-protein interactions driving the system through the TS. Some of the most important drugs for cancer treatment are kinase inhibitors. One of these kinase targets is cyclin-dependent kinase 2 (CDK2), an appealing target for anticancer drug development. Here, we tested our method using two different CDK2 inhibitors for the potential further development of these compounds. We compared the free-energy barriers obtained from our calculations with those observed in available experimental data. We highlighted important interactions at the distal ends of the ligands that can be targeted for improved residence times. Our method provides a new tool to determine unbinding rates and to identify key structural features of the inhibitors that can be used as starting points for novel design strategies in drug discovery.

Resolving sub-angstrom ambient motion through reconstruction from vibrational spectra.

Metal/organic-molecule interactions underpin many key chemistries but occur on sub-nm scales where nanoscale visualisation techniques tend to average over heterogeneous distributions. Single molecule imaging techniques at the atomic scale have found it challenging to track chemical behaviour under ambient conditions. Surface-enhanced Raman spectroscopy can optically monitor the vibrations of single molecules but understanding is limited by the complexity of spectra and mismatch between theory and experiment. We demonstrate that spectra from an optically generated metallic adatom near a molecule of interest can be inverted into dynamic sub-Å metal-molecule interactions using a comprehensive model, revealing anomalous diffusion of a single atom. Transient metal-organic coordination bonds chemically perturb molecular functional groups > 10 bonds away. With continuous improvements in computational methods for modelling large and complex molecular systems, this technique will become increasingly applicable to accurately tracking more complex chemistries.

Nanoparticle surfactants for kinetically arrested photoactive assemblies to track light-induced electron transfer.

Nature controls the assembly of complex architectures through self-limiting processes; however, few artificial strategies to mimic these processes have been reported to date. Here we demonstrate a system comprising two types of nanocrystal (NC), where the self-limiting assembly of one NC component controls the aggregation of the other. Our strategy uses semiconducting InP/ZnS core-shell NCs (3 nm) as effective assembly modulators and functional nanoparticle surfactants in cucurbit[n]uril-triggered aggregation of AuNCs (5-60 nm), allowing the rapid formation (within seconds) of colloidally stable hybrid aggregates. The resultant assemblies efficiently harvest light within the semiconductor substructures, inducing out-of-equilibrium electron transfer processes, which can now be simultaneously monitored through the incorporated surface-enhanced Raman spectroscopy-active plasmonic compartments. Spatial confinement of electron mediators (for example, methyl viologen (MV2+)) within the hybrids enables the direct observation of photogenerated radical species as well as molecular recognition in real time, providing experimental evidence for the formation of elusive σ-(MV+)2 dimeric species. This approach paves the way for widespread use of analogous hybrids for the long-term real-time tracking of interfacial charge transfer processes, such as the light-driven generation of radicals and catalysis with operando spectroscopies under irreversible conditions.

The Influence of Secondary Interactions on the [N-I-N]+ Halogen Bond.

[Bis(pyridine)iodine(I)]+ complexes offer controlled access to halonium ions under mild conditions. The reactivity of such stabilized halonium ions is primarily determined by their three-center, four-electron [N-I-N]+ halogen bond. We studied the importance of chelation, strain, steric hindrance and electrostatic interaction for the structure and reactivity of halogen bonded halonium ions by acquiring their 15 N NMR coordination shifts and measuring their iodenium release rates, and interpreted the data with the support of DFT computations. A bidentate ligand stabilizes the [N-I-N]+ halogen bond, decreasing the halenium transfer rate. Strain weakens the bond and accordingly increases the release rate. Remote modifications in the backbone do not influence the stability as long as the effect is entirely steric. Incorporating an electron-rich moiety close by the [N-I-N]+ motif increases the iodenium release rate. The analysis of the iodine(I) transfer mechanism highlights the impact of secondary interactions, and may provide a handle on the induction of stereoselectivity in electrophilic halogenations.

Joint neutron/X-ray crystal structure of a mechanistically relevant complex of perdeuterated urate oxidase and simulations provide insight into the hydration step of catalysis.

Cofactor-independent urate oxidase (UOX) is an ∼137 kDa tetrameric enzyme essential for uric acid (UA) catabolism in many organisms. UA is first oxidized by O2 to de-hydro-isourate (DHU) via a peroxo intermediate. DHU then undergoes hydration to 5-hy-droxy-isourate (5HIU). At different stages of the reaction both catalytic O2 and water occupy the 'peroxo hole' above the organic substrate. Here, high-resolution neutron/X-ray crystallographic analysis at room temperature has been integrated with molecular dynamics simulations to investigate the hydration step of the reaction. The joint neutron/X-ray structure of perdeuterated Aspergillus flavus UOX in complex with its 8-azaxanthine (8AZA) inhibitor shows that the catalytic water molecule (W1) is present in the peroxo hole as neutral H2O, oriented at 45° with respect to the ligand. It is stabilized by Thr57 and Asn254 on different UOX protomers as well as by an O-H⋯π interaction with 8AZA. The active site Lys10-Thr57 dyad features a charged Lys10-NH3 + side chain engaged in a strong hydrogen bond with Thr57OG1, while the Thr57OG1-HG1 bond is rotationally dynamic and oriented toward the π system of the ligand, on average. Our analysis offers support for a mechanism in which W1 performs a nucleophilic attack on DHUC5 with Thr57HG1 central to a Lys10-assisted proton-relay system. Room-temperature crystallography and simulations also reveal conformational heterogeneity for Asn254 that modulates W1 stability in the peroxo hole. This is proposed to be an active mechanism to facilitate W1/O2 exchange during catalysis.

Optical probes of molecules as nano-mechanical switches.

Molecular electronics promises a new generation of ultralow-energy information technologies, based around functional molecular junctions. Here, we report optical probing that exploits a gold nanoparticle in a plasmonic nanocavity geometry used as one terminal of a well-defined molecular junction, deposited as a self-assembled molecular monolayer on flat gold. A conductive transparent cantilever electrically contacts individual nanoparticles while maintaining optical access to the molecular junction. Optical readout of molecular structure in the junction reveals ultralow-energy switching of ∼50 zJ, from a nano-electromechanical torsion spring at the single molecule level. Real-time Raman measurements show these electronic device characteristics are directly affected by this molecular torsion, which can be explained using a simple circuit model based on junction capacitances, confirmed by density functional theory calculations. This nanomechanical degree of freedom is normally invisible and ignored in electrical transport measurements but is vital to the design and exploitation of molecules as quantum-coherent electronic nanodevices.

O-I-O halogen bond of halonium ions.

The reactivity of halonium ions is conveniently modulated by three-center, four-electron halogen bonds. Such stabilized halonium complexes are valuable reagents for oxidations and halofunctionalization reactions. We report the first example of the stabilization of a halenium ion in a three-center, four-electron halogen bond with two oxygen ligands. The influence of electron density and solvent on the stability of the complexes is assessed. O-I-O halogen bond complexes are applicable as synthetic reagents and as supramolecular synthons.

Host-Guest Chemistry Meets Electrocatalysis: Cucurbit[6]uril on a Au Surface as a Hybrid System in CO2 Reduction.

The rational control of forming and stabilizing reaction intermediates to guide specific reaction pathways remains to be a major challenge in electrocatalysis. In this work, we report a surface active-site engineering approach for modulating electrocatalytic CO2 reduction using the macrocycle cucurbit[6]uril (CB[6]). A pristine gold surface functionalized with CB[6] nanocavities was studied as a hybrid organic-inorganic model system that utilizes host-guest chemistry to influence the heterogeneous electrocatalytic reaction. The combination of surface-enhanced infrared absorption (SEIRA) spectroscopy and electrocatalytic experiments in conjunction with theoretical calculations supports capture and reduction of CO2 inside the hydrophobic cavity of CB[6] on the gold surface in aqueous KHCO3 at negative potentials. SEIRA spectroscopic experiments show that the decoration of gold with the supramolecular host CB[6] leads to an increased local CO2 concentration close to the metal interface. Electrocatalytic CO2 reduction on a CB[6]-coated gold electrode indicates differences in the specific interactions between CO2 reduction intermediates within and outside the CB[6] molecular cavity, illustrated by a decrease in current density from CO generation, but almost invariant H2 production compared to unfunctionalized gold. The presented methodology and mechanistic insight can guide future design of molecularly engineered catalytic environments through interfacial host-guest chemistry.

Organocatalytic Access to a cis-Cyclopentyl-γ-amino Acid: An Intriguing Model of Selectivity and Formation of a Stable 10/12-Helix from the Corresponding γ/α-Peptide.

In this study, we have developed a highly enantioselective organocatalytic route to the (1S,2R)-2-(aminomethyl)cyclopentane-1-carboxylic acid monomer precursor, which has a cis-configuration between the C- and N-termini around the cyclopentane core. Kinetic measurements show that the product distribution changes over time due to epimerization of the C1 center. Computations suggest the cis-selectivity is a result of selective C-C bond formation, while subsequent steps appear to influence the selectivity at higher temperature. The resulting γ-amino acid residue was incorporated into a novel γ/α-peptide, which forms a well-ordered 10/12-helix with alternate H-bond directionality in spite of the smallest value of the ζ-angle yet observed for a helix of this type. This highly defined structure is also a result of the narrow range of potential ζ-angles in our monomer. In contrast, the larger range of potential ζ-values observed for the corresponding trans-system can be fulfilled by several competing helical structures.

Dynamic Refolding of Ion-Pair Catalysts in Response to Different Anions.

Four distinct folding patterns are identified in two foldamer-type urea-thiourea catalysts bearing a basic dimethylamino unit by a combination of X-ray crystallography, solution NMR studies, and computational studies (DFT). These patterns are characterized by different intramolecular hydrogen bonding schemes that arise largely from different thiourea conformers. The free base forms of the catalysts are characterized by folds where the intramolecular hydrogen bonds between the urea and the thiourea units remain intact. In contrast, the catalytically relevant salt forms of the catalyst, where the catalyst forms an ion pair with the substrate or substrate analogues, appear in two entirely different folding patterns. With larger anions that mimic the dialkyl malonate substrates, the catalysts maintain their native fold both in the solid state and in solution, but with smaller halide anions (fluoride, chloride, and bromide), the catalysts fold around the halide anion (anion receptor fold), and the intramolecular hydrogen bonds are disrupted. Titration of catalyst hexafluoroacetylacetonate salt with tetra-n-butylammonium chloride results in dynamic refolding of the catalyst from the native fold to the anion receptor fold.

Halogen Bonding Helicates Encompassing Iodonium Cations.

The first halonium-ion-based helices were designed and synthesized using oligo-aryl/pyridylene-ethynylene backbones that fold around reactive iodonium ions. Halogen bonding interactions stabilize the iodonium ions within the helices. Remarkably, the distance between two iodonium ions within a helix is shorter than the sum of their van der Waals radii. The helical conformations were characterized by X-ray crystallography in the solid state, by NMR spectroscopy in solution and corroborated by DFT calculations. The helical complexes possess potential synthetic utility, as demonstrated by their ability to induce iodocyclization of 4-penten-1-ol.

Mechanistic Insight into Asymmetric Hetero-Michael Addition of α,ß-Unsaturated Carboxylic Acids Catalyzed by Multifunctional Thioureas.

Carboxylic acids and their corresponding carboxylate anions are generally utilized as Brønsted acids/bases and oxygen nucleophiles in organic synthesis. However, a few asymmetric reactions have used carboxylic acids as electrophiles. Although chiral thioureas bearing both arylboronic acid and tertiary amine were found to promote the aza-Michael addition of BnONH2 to α,ß-unsaturated carboxylic acids with moderate to good enantioselectivities, the reaction mechanism remains to be clarified. Detailed investigation of the reaction using spectroscopic analysis and kinetic studies identified tetrahedral borate complexes, comprising two carboxylate anions, as reaction intermediates. We realized a dramatic improvement in product enantioselectivity with the addition of 1 equiv of benzoic acid. In this aza-Michael reaction, the boronic acid not only activates the carboxylate ligand as a Lewis acid, together with the thiourea NH-protons, but also functions as a Brønsted base through a benzoyloxy anion to activate the nucleophile. Moreover, molecular sieves were found to play an important role in generating the ternary borate complexes, which were crucial for obtaining high enantioselectivity as demonstrated by DFT calculations. We also designed a new thiourea catalyst for the intramolecular oxa-Michael addition to suppress another catalytic pathway via a binary borate complex using steric hindrance between the catalyst and substrate. Finally, to demonstrate the synthetic versatility of both hetero-Michael additions, we used them to accomplish the asymmetric synthesis of key intermediates in pharmaceutically important molecules, including sitagliptin and α-tocopherol.