A modular phase transfer and ligand exchange protocol for quantum dots

In this paper, we describe a quantum dot (qdot) phase transfer protocol using ligand exchange and the amino acid histidine. The phase transfer from nonpolar solvents to aqueous buffers is homogeneous, and no appreciable precipitation occurs. The molecule histidine was chosen in order to first displace the organic encapsulation and second to provide a weakly chemisorbing intermediate at the qdot ionic interface. This allows the histidine to act as an intermediate shell upon which further direct ligand exchange can occur. Since this intermediate encapsulation is easily displaced by an assortment of different molecules while in aqueous buffers, we refer to this approach as modular. Characterization via FTIR and NMR revealed the extent of ligand exchange, and provides insights into the interfacial binding mechanism. The colloidal stability and photostability of the qdots was probed via UV-vis and steady state fluorescence, which revealed promising quantum yield stability of greater than 1 year. The qdots have hydrodynamic diameters of <12 nm and surface charges dependent upon ligand type and coverage. The modularity of this approach is shown by tailoring the qdot surface charge via sequential ligand exchange using mixed monolayers of carboxylic acid and poly(ethylene glycol)-terminated thiols.     

Characterisation by immobilised metal ion affinity chromatographic procedures of the binding behaviour of several synthetic peptides designed to have high affinity for Cu(II) ions.

In this investigation, several peptides containing an increasing number of histidine residues have been designed and synthesised. The peptides involved repeat units of either the pentameric EAEHA or the tetrameric HLLH sequence motifs. Adsorption isotherms for these synthetic peptides and hexahistidine (hexa-His) as a control substance were measured under batch equilibrium binding conditions with an immobilised Cu(II)-iminodiacetic acid (IDA) sorbent. The experimental data were analysed in terms of Langmuirean binding behaviour. In common with previous studies with synthetic peptides, these investigations have demonstrate that the sequential organisation of the histidine side chains in these peptides can affect the selectivity of the coordination interactions with borderline metal ions in immobilised metal ion affinity chromatographic systems. The results also confirm that peptides selected on the basis of their potential to form amphipathic secondary structures with their histidine residues presented on one face of the molecule can exhibit equivalent or higher affinity constants towards copper ions than hexa-His, although they contain fewer histidine residues. These findings are thus relevant to the selection of peptides produced inter alia by combinatorial synthetic procedures to have enhanced binding properties for Cu(II) or Ni(II) ions, or intended for use as peptide tags in the fusion handle approach for the affinity chromatographic purification of recombinant proteins.     

Bayesian analysis of heterogeneity in the distribution of binding properties of immobilized surface sites

Once a homogeneous ensemble of a protein ligand is taken from solution and immobilized to a surface, for many reasons the resulting ensemble of surface binding sites to soluble analytes may be heterogeneous. For example, this can be due to the intrinsic surface roughness causing variations in the local microenvironment, nonuniform density distribution of polymeric linkers, or nonuniform chemical attachment producing different protein orientations and conformations. We previously described a computational method for determining the distribution of affinity and rate constants of surface sites from analysis of experimental surface binding data. It fully exploits the high signal/noise ratio and reproducibility provided by optical biosensor technology, such as surface plasmon resonance. Since the computational analysis is ill conditioned, the previous approach used a regularization strategy assuming a priori all binding parameters to be equally likely, resulting in the broadest possible parameter distribution consistent with the experimental data. We now extended this method in a Bayesian approach to incorporate the opposite assumption, i.e., that the surface sites a priori are expected to be uniform (as one would expect in free solution). This results in a distribution of binding parameters as close to monodispersity as possible given the experimental data. Using several model protein systems immobilized on a carboxymethyl dextran surface and probed with surface plasmon resonance, we show microheterogeneity of the surface sites in addition to broad populations of significantly altered affinity. The distributions obtained are highly reproducible. Immobilization conditions and the total surface density of immobilized sites can have a substantial impact on the functional distribution of the binding sites.     

Binding maps for the study and prediction of bimetallic catalyst surface reactions: The case of methanol oxidation

Focusing on the competing pathways of methanol oxidation on platinum and platinum/gold bimetallic catalysts, we explore a novel density functional theory (DFT)‐based approach to the study of reactions on catalyst surfaces. Traditionally, DFT has been used to compute binding energies of products and intermediates as proxies for catalytic activity, and to compute full reaction pathways and their activation energy barriers. Merging the computational simplicity and intuitive clarity of binding energy calculations with the site sensitivity of transition state calculations, we construct maps of the binding energies of relevant atoms and molecules at all sites on a surface. We show that knowledge of the arrangement of strong and weak binding sites on a surface is powerful in rationalizing the ease with which a reaction step proceeds on a given local motif of surface atoms. We highlight the prospects and challenges of this approach toward catalyst screening and prediction.     

Robust identification of binding hot spots using continuum electrostatics: application to hen egg-white lysozyme

Binding hot spots, protein regions with high binding affinity, can be identified by using X-ray crystallography or NMR spectroscopy to screen libraries of small organic molecules that tend to cluster at such hot spots. FTMap, a direct computational analogue of the experimental screening approaches, uses 16 different probe molecules for global sampling of the surface of a target protein on a dense grid and evaluates the energy of interaction using an empirical energy function that includes a continuum electrostatic term. Energy evaluation is based on the fast Fourier transform correlation approach, which allows for the sampling of billions of probe positions. The grid sampling is followed by off-grid minimization that uses a more detailed energy expression with a continuum electrostatics term. FTMap identifies the hot spots as consensus clusters formed by overlapping clusters of several probes. The hot spots are ranked on the basis of the number of probe clusters, which predicts their binding propensity. We applied FTMap to nine structures of hen egg-white lysozyme (HEWL), whose hot spots have been extensively studied by both experimental and computational methods. FTMap found the primary hot spot in site C of all nine structures, in spite of conformational differences. In addition, secondary hot spots in sites B and D that are known to be important for the binding of polysaccharide substrates were found. The predicted probe-protein interactions agree well with those seen in the complexes of HEWL with various ligands and also agree with an NMR-based study of HEWL in aqueous solutions of eight organic solvents. We argue that FTMap provides more complete information on the HEWL binding site than previous computational methods and yields fewer false-positive binding locations than the X-ray structures of HEWL from crystals soaked in organic solvents.     

Binding affinity of a small molecule to an amorphous polymer in a solvent. Part 2: preferential binding to local sites on a surface

Crystallization, a separation and purification process, is commonly used to produce a wide range of materials in various industries, and it usually begins with heterogeneous nucleation on a foreign surface in industrial practice and most other circumstances. Recent studies show that amorphous polymeric substrates are useful in controlling crystallization and selectively producing pharmaceutical polymorphs. In our previous publication, we investigated the possible correlation of the binding affinity of one molecule to key binding sites (local binding), and the possibility of using this binding affinity to guide the selection of polymers promoting heterogeneous nucleation. The studied systems were aspirin binding to four nonporous cross-linked polymers in ethanol-water 38 v% mixture. Cross-linked with divinylbenzene (DVB), these polymers were poly(4-acryloylmorpholine) (PAM), poly(2-carboxyethyl acrylate) (PCEA), poly(4-hydroxylbutyl acrylate) (PHBA), and polystyrene (PS). We discovered that the trend of the magnitudes of the average free energies of binding to the best sites is very similar to that of heterogeneous nucleation activities. This Article aims to investigate whether or not local binding to key sites is the important variable to describe heterogeneous nucleation as opposed to the overall/average binding affinity of molecules to a surface, and to investigate the possibility of using the overall binding affinity to guide the selection of polymers. We used the polymer surfaces generated from our previous study to calculate the overall binding affinity of aspirin molecules to the surface as measured by the preferential interaction coefficients of aspirin (1 m) to these polymers. We discovered that the trend of the average preferential interaction coefficients does not correlate as well to that of heterogeneous nucleation activities as the free energies of binding to the best sites. We also computed the average numbers of aspirin molecules associated with the areas of the surfaces' best binding sites and found that they correlate better to heterogeneous nucleation activities than the average preferential interaction coefficients. These results further support that local binding is indicative of heterogeneous nucleation. Moreover, we found a weak trend of the distance order parameters of the aspirin molecules to be similar that of heterogeneous nucleation activities. Our results from the two-part study suggest the importance of local binding to heterogeneous nucleation as well as the possibility of using the binding affinity to the local area (the free energy of binding to the best site and the number of nucleating molecules associated with the area of the best binding site) and the distance order parameters to guide the selection of polymers.     

A luminescent receptor with affinity for N-terminal histidine in peptides in aqueous solution

Crown ethers of suitable size are the perfect artificial host compounds for ammonium ion binding, but the rather low affinity in aqueous solution prevents their use at physiological conditions. We report here the synthesis and properties of a luminescent benzo crown ether with a pendant copper imidodiacetic acid complex, which coordinates with high affinity to histidine. The emission intensity of the benzo crown ether increases significantly in the presence of ammonium ions in methanol. At physiological conditions in buffered water at pH 7.5 these interactions are too weak to be detected. If an ammonium ion and an imidazole moiety are present in the analyte, such as in His-Lys-OMe or His-OMe, high binding affinity in aqueous solution is restored. The binding event is signaled by an increase in emission intensity, which can even be observed with the naked eye. This allows the selective detection of small peptides containing N-terminal histidine or histidine among all other amino acids at physiological conditions.     

A Novel pH/Light‐Triggered Surface for DNA Adsorption and Release

A simple strategy for the immobilization of Cy3‐labeled single strand DNA (Cy3‐ssDNA) on a Si(001) surface and its release under control of both light and pH stimuli is presented. In order to prepare a dual pH/light‐triggered surface, positively chargeable azobenzene molecules are self‐assembled on the Si(001) surface. The surface wettability of this substrate can be changed under influence of both light and pH conditions. The substrates can be positively charged under mildly acidic conditions. The pH‐sensitive behavior of the film allows binding of Cy3‐ssDNA on the functionalized Si(001) surface through e?ective electrostatic interactions with the negatively charged polynucleotide backbone. Moreover, irradiation of the film with UVA light induces trans–cis isomerization of the azobenzene units on the surface. As a result, the binding a?nity for DNA decreases due to the changing surface hydrophilicity. In order to understand and control the reversible photoswitchable mechanism of this surface, water contact angles are measured after UVA and visible light irradiation. The release of DNA from a dual pH/light‐sensitive sample is performed using fluorescence microscopy. The results show that irradiation of the film with UVA light induces trans–cis isomerization of the photoresponsive azobenzene units; this leads to significant changes in the surface hydrophilicity and reduces the binding affinity for DNA.     

Applications of on-line weak affinity interactions in free solution capillary electrophoresis

The impressive selectivity offered by capillary electrophoresis can in some cases be further increased when ligands or additives that engage in weak affinity interactions with one or more of the separated analytes are added to the electrophoresis buffer. This on-line affinity capillary electrophoresis approach is feasible when the migration of complexed molecules is different from the migration of free molecules and when separation conditions are nondenaturing. In this review, we focus on applying weak interactions as tools to enhance the separation of closely related molecules, e.g., drug enantiomers and on using capillary electrophoresis to characterize such interactions quantitatively. We describe the equations for binding isotherms, illustrate how selectivity can be manipulated by varying the additive concentrations, and show how the methods may be used to estimate binding constants. On-line affinity capillary electrophoresis methods are especially valuable for enantiomeric separations and for functional characterization of the contents of biological samples that are only available in minute quantities.     

Robust Microporous Metal–Organic Frameworks for Highly Efficient and Simultaneous Removal of Propyne and Propadiene from Propylene

Simultaneous removal of trace amounts of propyne and propadiene from propylene is an important but challenging industrial process. We report herein a class of microporous metal–organic frameworks ( NKMOF‐1‐M ) with exceptional water stability and remarkably high uptakes for both propyne and propadiene at low pressures. NKMOF‐1‐M separated a ternary propyne/propadiene/propylene (0.5 : 0.5 : 99.0) mixture with the highest reported selectivity for the production of polymer‐grade propylene (99.996 %) at ambient temperature, as attributed to its strong binding affinity for propyne and propadiene over propylene. Moreover, we were able to visualize propyne and propadiene molecules in the single‐crystal structure of NKMOF‐1‐M through a convenient approach under ambient conditions, which helped to precisely understand the binding sites and affinity for propyne and propadiene. These results provide important guidance on using ultramicroporous MOFs as physisorbent materials.     

Drug screening of pharmaceutical discovery compounds by micro-size exclusion chromatography/mass spectrometry.

Micro-size exclusion chromatography coupled with capillary liquid chromatography (capLC) and mass spectrometry (MS) provides a rapid and simple approach to the preliminary screening of active ligands toward a specific target macromolecule. In this study, the effectiveness of this technique is demonstrated by a number of small molecule ligands with known binding affinities towards the protein target. All ligands were incubated together with a target protein under native conditions. Separation was then achieved by microcentrifugation where the high molecular weight (MW) compounds were selectively passed through the size-exclusion material. The retained low MW compounds were then recovered and analyzed by capLC/MS. The absence of the ligand indicated strong affinity towards the target, while ligand detection indicated inactivity. This assay demonstrated the drugs that were acting as strong inhibitors of Co-PDF from those showing to be comparatively inactive. The relative binding rank order of the drugs towards Co-PDF was also determined. The results were validated by a corresponding set of control experiments in which the target molecules were excluded from the process. In principle, high-throughput micro-size exclusion chromatography, coupled with capLC/MS, offers a powerful technique as a preliminary screen in determining both the strong binding affinity and the relative affinity rank ordering of ligands towards a specific target macromolecule, and is complementary with other analytical drug screening techniques.     

Reproducing crystal binding modes of ligand functional groups using Site-Identification by Ligand Competitive Saturation (SILCS) simulations

The applicability of a computational method, Site Identification by Ligand Competitive Saturation (SILCS), to identify regions on a protein surface with which different types of functional groups on low-molecular weight inhibitors interact is demonstrated. The method involves molecular dynamics (MD) simulations of a protein in an aqueous solution of chemically diverse small molecules from which probability distributions of fragments types, termed FragMaps, are obtained. In the present application, SILCS simulations are performed with an aqueous solution of 1 M benzene and propane to map the affinity pattern of the protein for aromatic and aliphatic functional groups. In addition, water hydrogen and oxygen atoms serve as probes for hydrogen-bond donor and acceptor affinity, respectively. The method is tested using a set of 7 proteins for which crystal structures of complexes with several high affinity inhibitors are known. Good agreement is obtained between FragMaps and the positions of chemically similar functional groups in inhibitors as observed in the X-ray crystallographic structures. Quantitative capabilities of the SILCS approach are demonstrated by converting FragMaps to free energies, termed Grid Free Energies (GFE), and showing correlation between the GFE values and experimental binding affinities. For proteins for which ligand decoy sets are available, GFE values are shown to typically score the crystal conformation and conformations similar to it more favorable than decoys. Additionally, SILCS is tested for its ability to capture the subtle differences in ligand affinity across homologous proteins, information which may be of utility toward specificity-guided drug design. Taken together, our results show that SILCS can recapitulate the known location of functional groups of bound inhibitors for a number of proteins, suggesting that the method may be of utility for rational drug design.     

Improving the study of proton transfers between amino acid side chains in solution: choosing appropriate DFT functionals and avoiding hidden pitfalls

We have studied the influence of implicit solvent models, inclusion of explicit water molecules, inclusion of vibrational effects, and density functionals on the quality of the predicted pK _a of small amino acid side chain models. We found that the inclusion of vibrational effects and explicit water molecules is crucial to improve the correlation between the computed and the experimental values. In these micro-solvated systems, the best agreement between DFT-computed electronic energies and benchmark values is afforded by BHHLYP and B97-2. However, approaching experimental results requires the addition of more than three explicit water molecules, which generates new problems related to the presence of multiple minima in the potential energy surface. It thus appears that a satisfactory ab initio prediction of amino acid side chain pK _a will require methods that sample the configurational space in the presence of large solvation shells, while at the same time computing vibrational contributions to the enthalpy and entropy of the system under study in all points of that surface. Pending development of efficient algorithms for those computations, we strongly suggest that whenever counterintuitive protonation states are found in a computational study (e.g., the presence of a neutral aspartate/neutral histidine dyad instead of a deprotonated aspartate/protonated histidine pair), the reaction profile should be computed under each of the different protonation micro-states by constraining the relevant N–H or O–H bonds, in order to avoid artifacts inherent to the complex nature of the factors contributing to the pK _a.     

Conformational adaptation and selective adatom capturing of tetrapyridyl-porphyrin molecules on a copper (111) surface

We present a combined low-temperature scanning tunneling microscopy and near-edge X-ray adsorption fine structure study on the interaction of tetrapyridyl-porphyrin (TPyP) molecules with a Cu(111) surface. A novel approach using data from complementary experimental techniques and charge density calculations allows us to determine the adsorption geometry of TPyP on Cu(111). The molecules are centered on "bridge" sites of the substrate lattice and exhibit a strong deformation involving a saddle-shaped macrocycle distortion as well as considerable rotation and tilting of the meso-substituents. We propose a bonding mechanism based on the pyridyl-surface interaction, which mediates the molecular deformation upon adsorption. Accordingly, a functionalization by pyridyl groups opens up pathways to control the anchoring of large organic molecules on metal surfaces and tune their conformational state. Furthermore, we demonstrate that the affinity of the terminal groups for metal centers permits the selective capture of individual iron atoms at low temperature.     

Protein surface-assisted enhancement in the binding affinity of an inhibitor for recombinant human carbonic anhydrase-II

We elaborate on a novel strategy for enhancing the binding affinity of an active-site directed inhibitor by attaching a tether group, designed to interact with the surface-exposed histidine residue(s) of enzymes. In this approach, we have utilized the recombinant form of human carbonic anhydrase-II (hCA-II) as the enzyme source and benzenesulfonamide and its derivatives as inhibitors. The steady-state kinetic and the ligand binding data revealed that the attachment of iminodiacetate (IDA)-Cu(2+) to benzenesulfonamide (via a triethylene glycol spacer) enhanced its binding affinity for hCA-II by about 40-fold. No energetic contribution of either IDA or triethylene glycol spacer was found (at least in the ground state of the enzyme-inhibitor complex) when Cu(2+) was stripped off from the tether group-conjugated sulfonamide derivative. Arguments are presented that the overall strategy of enhancing the binding affinities of known inhibitors by attaching the IDA-Cu(2+) groups to interact with the surface-exposed histidine residues will find a general application in designing the isozyme-specific inhibitors as potential drugs.     

Steric and allosteric effects of fatty acids on the binding of warfarin to human serum albumin revealed by molecular dynamics and free energy calculations

Human serum albumin (HSA) binds with drugs and fatty acids (FAs). This study was initiated to elucidate the relationship between the warfarin binding affinity of HSA and the positions of bound FA molecules. Molecular dynamics simulations of 11 HSA-warfarin-myristate complexes were performed. HSA-warfarin binding free energy was then calculated for each of the complexes by the molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) method. The results indicated that the magnitude of the binding free energy was smaller in HSA-warfarin complexes that had 4 or more myristate molecules than in complexes with no myristate molecules. The unfavorable effect on the HSA-warfarin binding affinity was caused sterically by the binding of a myristate molecule to the FA binding site closest to the warfarin binding site. On the other hand, the magnitude of HSA-warfarin binding free energy was largest when 3 myristate molecules were bound to the high-affinity sites. The strongest HSA-warfarin binding was attributable to favorable entropic contribution related to larger atomic fluctuations of the amino acid residues at the warfarin binding site. In the binding of 2 myristate molecules to the sites with the highest and second-highest affinities, allosteric modulation that enhanced electrostatic interactions between warfarin and some of the amino acid residues around the warfarin binding site was observed. This study clarified the structural and energetic properties of steric/allosteric effects of FAs on the HSA-warfarin binding affinity and illustrated the approach to analyze protein-ligand interactions in situations such that multiple ligands bind to the other sites of the protein.     

Evaluation of designed ligands by a multiple screening method: Application to glycogen phosphorylase inhibitors constructed with a variety of approaches

Glycogen phosphorylase (GP) is an important enzyme that regulates blood glucose level and a key therapeutic target for the treatment of type II diabetes. In this study, a number of potential GP inhibitors are designed with a variety of computational approaches. They include the applications of MCSS, LUDI and CoMFA to identify additional fragments that can be attached to existing lead molecules; the use of 2D and 3D similarity-based QSAR models (HQSAR and SMGNN) and of the LUDI program to identify novel molecules that may bind to the glucose binding site. The designed ligands are evaluated by a multiple screening method, which is a combination of commercial and in-house ligand-receptor binding affinity prediction programs used in a previous study (So and Karplus, J. Comp.-Aid. Mol. Des., 13 (1999), 243–258). Each method is used at an appropriate point in the screening, as determined by both the accuracy of the calculations and the computational cost. A comparison of the strengths and weaknesses of the ligand design approaches is made.     

Atomistic De-novo Inhibitor Generation-Guided Drug Repurposing for SARS-CoV-2 Spike Protein with Free-Energy Validation by Well-Tempered Metadynamics

Computational drug design is increasingly becoming important with new and unforeseen diseases like COVID-19. In this study, we present a new computational de novo drug design and repurposing method and applied it to find plausible drug candidates for the receptor binding domain (RBD) of SARS-CoV-2 (COVID-19). Our study comprises three steps: atom-by-atom generation of new molecules around a receptor, structural similarity mapping to existing approved and investigational drugs, and validation of their binding strengths to the viral spike proteins based on rigorous all-atom, explicit-water well-tempered metadynamics free energy calculations. By choosing the receptor binding domain of the viral spike protein, we showed that some of our new molecules and some of the repurposable drugs have stronger binding to RBD than hACE2. To validate our approach, we also calculated the free energy of hACE2 and RBD, and found it to be in an excellent agreement with experiments. These pool of drugs will allow strategic repurposing against COVID-19 for a particular prevailing conditions.     

人血白蛋白结合位点II的分子结合模式研究

人血白蛋白是血浆中含量最高的蛋白,是一种重要的载体蛋白,能与多种内源性和外源性物质结合。人血白蛋白主要有两个药物结合位点,位点I和位点II,其中位点II的柔性较大,对药物分子的亲和性较高。本文采用分子模拟方法,基于12种结合于位点II的小分子-人血白蛋白晶体结构,分析了相互作用能,发现12种分子的结合以疏水作用为主,静电作用为辅。进一步采用丙氨酸扫描和结合能评价,分析结合部位的关键氨基酸残基,探究结合模式的规律性,发现从位点入口到空腔内部存在静电、疏水和杂合的三层相互作用分布,共同形成了稳定的分子结合。最后采用分子对接和分子动力学模拟,预测了L-色氨酸的结合模式。研究结果有助于深入了解人血白蛋白药物位点II的小分子结合模式,为基于位点II的药物和分离配基的优化设计提供指导。