Unique Conformation Research Articles

Fractionation, chemical characterization and immunostimulatory activity of β-glucan and galactoglucan from Russula vinosa Lindblad

Water-extracted polysaccharides from Russula vinosa Lindblad (WRP) were separated into three fractions (WRP-1, WRP-2 and WRP-3) by gradient ethanol precipitation and gel chromatography. Structural characterization indicated that WRP-1 was a branched β-(1→3)-glucan and exhibited rigid helical conformation in aqueous solution with Mw of 2,180 kDa and radius of gyration (Rg) of 123.4 nm. The galactoglucan of WRP-2 and WRP-3 were mainly composed of →6)-Galp-(1→ and →4)-Glcp-(1→ terminated by glucose and mannose, presenting much lower Mw (392 and 93.6 kDa) and Rg (57.6 and 42.6 nm), and more incompact flexible conformation than WRP-1. All fractions showed potential immunostimulatory activity by promoting macrophage proliferation, phagocytosis, as well as the release of nitric oxide and cytokines (TNF-α and IL-1β). WRP-1 with unique structure and conformation showed the best immunostimulatory effects among them. This study suggests that WRP could be explored as natural immunostimulator used in the food and pharmaceutical industry.

Shear-Induced Amyloid Aggregation in the Brain: V. Are Alzheimer's and Other Amyloid Diseases Initiated in the Lower Brain and Brainstem by Cerebrospinal Fluid Flow Stresses?

Amyloid-β (Aβ) and tau oligomers have been identified as neurotoxic agents responsible for causing Alzheimer’s disease (AD). Clinical trials using Aβ and tau as targets have failed, giving rise to calls for new research approaches to combat AD. This paper provides such an approach. Most basic AD research has involved quiescent Aβ and tau solutions. However, studies involving laminar and extensional flow of proteins have demonstrated that mechanical agitation of proteins induces or accelerates protein aggregation. Recent MRI brain studies have revealed high energy, chaotic motion of cerebrospinal fluid (CSF) in lower brain and brainstem regions. These and studies showing CSF flow within the brain have shown that there are two energetic hot spots. These are within the third and fourth brain ventricles and in the neighborhood of the circle of Willis blood vessel region. These two regions are also the same locations as those of the earliest Aβ and tau AD pathology. In this paper, it is proposed that cardiac systolic pulse waves that emanate from the major brain arteries in the lower brain and brainstem regions and whose pulse waves drive CSF flows within the brain are responsible for initiating AD and possibly other amyloid diseases. It is further proposed that the triggering of these diseases comes about because of the strengthening of systolic pulses due to major artery hardening that generates intense CSF extensional flow stress. Such stress provides the activation energy needed to induce conformational changes of both Aβ and tau within the lower brain and brainstem region, producing unique neurotoxic oligomer molecule conformations that induce AD.

A Chemical Probe for Dark Kinase STK17B Derives Its Potency and High Selectivity through a Unique P-Loop Conformation.

STK17B is a member of the death-associated protein kinase family and has been genetically linked to the development of diverse diseases. However, the role of STK17B in normal and disease pathology is poorly defined. Here, we present the discovery of thieno[3,2-d] pyrimidine SGC-STK17B-1 (11s), a high-quality chemical probe for this understudied "dark" kinase. 11s is an ATP-competitive inhibitor that showed remarkable selectivity over other kinases including the closely related STK17A. X-ray crystallography of 11s and related thieno[3,2-d]pyrimidines bound to STK17B revealed a unique P-loop conformation characterized by a salt bridge between R41 and the carboxylic acid of the inhibitor. Molecular dynamic simulations of STK17B revealed the flexibility of the P-loop and a wide range of R41 conformations available to the apo-protein. The isomeric thieno[2,3-d]pyrimidine SGC-STK17B-1N (19g) was identified as a negative control compound. The >100-fold lower activity of 19g on STK17B was attributed to the reduced basicity of its pyrimidine N1.

Application of Novel Calix[4]arene Metal-free Sensitizers in Dye-sensitized Photoelectrochemical Cells for Water Splitting

A series of novel calix[4]arene metal-free dyes, featuring macrocyclic structure and unique conical confor-mation, has been introduced into photoanode-based dye-sensitized electrochemical cell system as photosensitizers. The electrochemical properties of the corresponding sensitized photoanodes were systematically studied in the absence/ presence of water oxidation catalyst(WOC). Furthermore, the visible-light-driven overall water-splitting reactions were conducted by fully assembled devices, obtaining a performance trend of Calix-3 Calix-2 Calix-1. The corresponding device of Calix-3 exhibited the best photoactivity, giving an initial photocurrent density of ca. 300 μA/cm2, an IPEC peak value of ca. 9.0% at 365 nm and a wide photo-respond band up to ca. 620 nm. The best performance of Calix-3 can be attributed to its most effective light-harvesting ability, best ICT transition property, highest oxidation potential and thus best ability of activating WOC. This work offers an inspiration for the application of new-type effective metal-free sensitizers in photocatalytic water-splitting device.

Nanoparticles as vectors for antibiotic resistance: The association of silica nanoparticles with environmentally relevant extracellular antibiotic resistance genes

A relevant but yet unconsidered subset of particles that may alter the fate of extracellular antibiotic resistance genes (eARGs) are nano-scale particles (NPs), which are ubiquitous in natural environments and have unique properties. In this study, sorption isotherms were developed describing the association of linear DNA fragments isolated from widespread eARGs (blaI and nptII) with either micon-sized kaolinite or silica nanoparticles (SNPs), to determine if sorption capacity was enhanced at the nanoscale. For each isotherm, eARG fragments were added at five starting concentrations (5–40 μg/mL) to mixed batch systems with 0.25 g of particles and nuclease-free water. Sorption was quantified by the removal of DNA from solution, as detected by a Qubit fluorimeter. Isotherms were developed for eARGs of various fragment lengths (508, 680 and 861 bp), guanine-cytosine (GC) contents (34%, 47% and 54%) and both double and single stranded eARGs, to assess the impact of DNA properties on particle association. Sorption isotherms were also developed in systems with added humic acid and/or CaCl2, to assess the impact of these environmental parameters on sorption. FTIR analysis was performed to analyze the conformation of sorbed eARGs. Desorption of eARGs was studied by quantifying the removal of eDNA from washed and vortexed post-sorption particles. Statistically significant irreversible sorption of eARGs to environmentally relevant NPs (humic acid functionalized silica nanoparticles) was demonstrated for the first time. Nano-emergent properties did not increase sorption capacity of eARGs, but led to a unique compressed conformation of sorbed eARGs. The addition of humic acid, increased CaCl2 concentration and small DNA fragment size favored sorption. NPs showed a slight preference for the sorption of single-stranded DNA over double-stranded DNA. These findings suggest that NP association with eARGs may be a significant and unique environmental phenomenon that could influence the spread of antibiotic resistance.

Esterase-Responsive Polypeptide Vesicles as Fast-Response and Sustained-Release Nanocompartments for Fibroblast-Exempt Drug Delivery.

Enzyme-responsive polypeptide vesicles have attracted considerable attention for precision theranostics because of their biocompatibility, biodegradability, and unique secondary conformation transition triggered by the catalytic actions of enzymes. These promising potentials of polypeptide vesicles could be limited in a drug delivery system by the very slow enzyme diffusion rate into vesicles that could reduce the efficacy of the drug. On the other hand, stimuli-responsive polymeric vesicles that respond to stimuli can undergo microstructure destruction for the burst release of drugs, which would penetrate through the membrane of dead cells and the tumor extracellular matrix, inducing acute toxicity to neighboring cells. Here, we designed amphiphilic PEG-polypeptide copolymers containing esterase-labile carbamate-caged primary amines. It was found that the diblock can self-assemble into vesicular structures. Esterase-triggered self-immolative decaging reactions could quickly release the primary amine moiety of monomers that can undergo an amidation reaction for transition of the bilayer of vesicles from hydrophobic to partially hydrophilic. This esterase-responsive process retains the nanostructure of vesicles but permeabilizes the vesicle membrane, which can afford the sustained release of encapsulating drugs. These esterase-responsive polypeptide vesicles mediate selective cytotoxicity in cancer cells with high esterase expression over normal fibroblasts with low esterase, enabling the potent anticancer chemotherapy with minimized side effects.

Controlled steric selectivity in molecular doping towards closest-packed supramolecular conductors

Recent developments in molecular doping technologies allow extremely high carrier densities in polymeric semiconductors, exhibiting great diversity because of the unique size, conformation, and steric effect of molecular dopants. However, it is controversial how steric effects can limit the doping efficiency and to what extent dopants can be accommodated in polymers. Here, we employ two distinct conjugated polymers with different alkyl side-chain densities, where polymers are doped via anion-change, allowing greater variation in the incorporation of molecular dopants having different electrostatic potentials and shapes. We characterize the doping efficiency with regard to steric effects, considering the unique void space in the conjugated polymers. Our study reveals that doping efficiency of polymers with sparse alkyl side-chains is significantly greater than that with dense side-chains. A closest-packed supramolecule is realized with a particular combination of a sparse polymer and a large dopant, giving rise to high conductivity, air stability, and remarkably high work function. This work provides a critical insight into overcoming steric effects in molecular doping.

Current Advances in the Development of Diagnostic Tests based on Aptamers in Parasitology: A Systematic Review.

Aptamers are single-stranded DNA or RNA sequences of 20–80 nucleotides that interact with different targets such as: proteins, ions, viruses, or toxins, through non-covalent interactions and their unique three-dimensional conformation. They are obtained in vitro by the systematic evolution of ligands by exponential enrichment (SELEX). Because of their ability of target recognition with high specificity and affinity, aptamers are usually compared to antibodies. However, they present many advantages that make them promising molecules for the development of new methods for the diagnosis and treatment of human diseases. In medical parasitology, aptamers also represent an attractive alternative for the implementation of new parasite detection methods, easy to apply in endemic regions. The aim of this study was to describe the current advances in the development of diagnostic tests based on aptamers in parasitology. For this, articles were selected following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, with specific inclusion and exclusion criteria. The 26 resulting articles deal with the use of aptamers for the detection of six important protozoa that affect human health. This systematic review clearly demonstrates the specificity, sensitivity and selectivity of aptamers and aptasensors, that certainly will soon become standard methods in medical parasitology.

Efficient approach for exploring the multiple-channel bimolecular interactions of conformationally flexible reagents. Epoxide ring opening reaction

Algorithm for generation and assessment of probability of possible reaction pathways for multiple-channel bimolecular interactions is presented. The proposed algorithm comprises a combination of few steps. They include conformational search for reaction intermediate using the molecular mechanics (MMX) approach, based on the obtained conformation construction of structures of transition states and pre-reaction complexes, and calculation activation energies to further determine the probable reaction pathways. The proposed algorithm could be adopted for investigation of chemical and biochemical reactions of different types. Here, we have considered the reaction of bicyclo[2.2.1]hept-5-en-endo-2-ylmethylamine (1) with glycidyl ether (2) in a neutral environment that proceeds through SN2-like mechanism forming bipolar ion (3) which is a good starting point for identification of the reaction channels. Conformational properties of intermediate (3) have been investigated using stochastic conformational search. From the 95 localized conformations within 10 kcal/mol of global minimum that have been obtained, 63 unique transition state conformations were generated and optimized by using the PM7 and M062X/6-31G(d) methods for accurate estimation of overall rate constant of reaction. The most energetically favorable pathways have been investigated at the M062X/6-31G(d) level of theory taking into account the influence of solvent.

A new medium-sized abelisaurid (Theropoda, Dinosauria) from the late cretaceous (Maastrichtian) Allen Formation of Northern Patagonia, Argentina

Abelisaurid theropods are well known in Cretaceous beds along South America, particularly Patagonia. However, the record of latest Cretaceous abelisauroids is still far from satisfactory. Until recently, few taxa were described from Maastrichtian beds: Carnotaurus sastrei and Quilmesaurus curriei, but also perhaps Abelisaurus comahuensis. In this contribution, we describe a new genus and species (Niebla antiqua gen. et sp. nov.), of medium-sized abelisaurid coming from Late Cretaceous (Maastrichtian) beds from Río Negro province, northern Patagonia, Argentina. The paleohistological analysis shows that this individual has reached the somatical maturity and that it represents an adult of a mid-sized abelisaurid. The specimen is represented by a nearly complete braincase, fragmentary jaw and teeth, relatively complete scapulocoracoid, dorsal ribs and incomplete vertebrae. The new taxon is relatively small, much smaller than other coeval abelisaurids such as Carnotaurus and Abelisaurus. The braincase shows autapomorphic features such a dorsoventrally tall basal tuber and postemporal foramen enclosed by parietal and exoccipitals. The scapulocoracoid is notably similar to that of Carnotaurus in having a posterodorsally oriented glenoid, a dorsoventrally expanded and wide coraco-scapular plate, and a very narrow and straight scapular blade. These features are very different from those of other abelisaurids, which may indicate a unique conformation of the pectoral girdle among these South American theropods.

DSS1 allosterically regulates the conformation of the tower domain of BRCA2 that has dsDNA binding specificity for homologous recombination

DSS1 is an evolutionary conserved, small intrinsically disordered protein that regulates various cellular functions. Although several studies have elucidated the role of DSS1 in stabilizing BRCA2 and its importance in homologous recombination repair (HRR), yet the structural mechanism behind the stability and HRR remains elusive. In this study, using molecular dynamics simulation we show that DSS1 stabilizes linearly arranged DNA/DSS1 binding domains of BRCA2 with many native contacts. These contacts are absent in the complexes with two missense DSS1 mutants associated with germline breast cancer and somatic mouth carcinoma. Most importantly, our protein energy-based network models show DSS1 allosterically regulates the conformation of the distant tower domain of BRCA2 that has dsDNA binding specificity for HRR. We further postulate that the unique conformation of the tower domain with kinked-helices might be responsible for DNA strand invasion and initiation of HRR. Induced conformation of the tower domain by the kinked-helices is absent in the unbound BRCA2, as well as in the two mutant DSS1-BRCA2 complexes. This suggests that DSS1 allosterically regulates the tower domain conformations of BRCA2 that affects dsDNA binding, essential for HRR. Our results add a new dimension to the function of DSS1 and its role in regulating HRR.

Cryo-EM structures reveal two distinct conformational states in a picornavirus cell entry intermediate.

The virions of enteroviruses such as poliovirus undergo a global conformational change after binding to the cellular receptor, characterized by a 4% expansion, and by the opening of holes at the two and quasi-three-fold symmetry axes of the capsid. The resultant particle is called a 135S particle or A-particle and is thought to be on the pathway to a productive infection. Previously published studies have concluded that the membrane-interactive peptides, namely VP4 and the N-terminus of VP1, are irreversibly externalized in the 135S particle. However, using established protocols to produce the 135S particle, and single particle cryo-electron microscopy methods, we have identified at least two unique states that we call the early and late 135S particle. Surprisingly, only in the "late" 135S particles have detectable levels of the VP1 N-terminus been trapped outside the capsid. Moreover, we observe a distinct density inside the capsid that can be accounted for by VP4 that remains associated with the genome. Taken together our results conclusively demonstrate that the 135S particle is not a unique conformation, but rather a family of conformations that could exist simultaneously.

The origin of the high stability of 3'-terminal uridine tetrads: contributions of hydrogen bonding, stacking interactions, and steric factors evaluated using modified oligonucleotide analogs.

RNA G-quadruplexes fold almost exclusively into parallel-stranded structures and thus display much less structural diversity than their DNA counterparts. However, also among RNA G-quadruplexes peculiar structural elements can be found which are capable of reshaping the physico-chemical properties of the folded structure. A striking example is provided by a uridine tetrad (U-tetrad) placed on the 3′-terminus of the tetramolecular G-quadruplex. In this context, the U-tetrad adopts a unique conformation involving chain reversal and is responsible for a tremendous stabilization of the G-quadruplex (ΔTm up to 30°C). In this report, we attempt to rationalize the origin of this stabilizing effect by concurrent structural, thermal stability, and molecular dynamics studies of a series of G-quadruplexes with subtle chemical modifications at their 3′-termini. Our results provide detailed insights into the energetics of the “reversed” U-tetrad motif and the requirements for its formation. They point to the importance of the 2′OH to phosphate hydrogen bond and preferential stacking interactions for the formation propensity and stability of the motif.

An Ultrahigh-Affinity Complement C4b-Specific Nanobody Inhibits In Vivo Assembly of the Classical Pathway Proconvertase.

The classical and lectin pathways of the complement system are important for the elimination of pathogens and apoptotic cells and stimulation of the adaptive immune system. Upon activation of these pathways, complement component C4 is proteolytically cleaved, and the major product C4b is deposited on the activator, enabling assembly of a C3 convertase and downstream alternative pathway amplification. Although excessive activation of the lectin and classical pathways contributes to multiple autoimmune and inflammatory diseases and overexpression of a C4 isoform has recently been linked to schizophrenia, a C4 inhibitor and structural characterization of the convertase formed by C4b is lacking. In this study, we present the nanobody hC4Nb8 that binds with picomolar affinity to human C4b and potently inhibits in vitro complement C3 deposition through the classical and lectin pathways in human serum and in mouse serum. The crystal structure of the C4b:hC4Nb8 complex and a three-dimensional reconstruction of the C4bC2 proconvertase obtained by electron microscopy together rationalize how hC4Nb8 prevents proconvertase assembly through recognition of a neoepitope exposed in C4b and reveals a unique C2 conformation compared with the alternative pathway proconvertase. On human induced pluripotent stem cell-derived neurons, the nanobody prevents C3 deposition through the classical pathway. Furthermore, hC4Nb8 inhibits the classical pathway-mediated immune complex delivery to follicular dendritic cells in vivo. The hC4Nb8 represents a novel ultrahigh-affinity inhibitor of the classical and lectin pathways of the complement cascade under both in vitro and in vivo conditions.

Poly-Amido-Saccharides (PASs): Functional Synthetic Carbohydrate Polymers Inspired by Nature.

Carbohydrates are ubiquitous in nature, playing vital roles in all organisms ranging from metabolism to intercellular signaling. Polysaccharides, repeating units of small molecule carbohydrates, are hydrophilic, densely functionalized, stereoregular, and rigid macromolecules, and these characteristics are simultaneously advantageous in biomedical applications while presenting major hurdles for synthetic methodology and development of structure property relationships. While naturally obtained polysaccharides are widely utilized in the biochemical and medical literature, their poor physicochemical definition and the potential for contaminated samples hinders the clinical translation of this work. To address the need for new methods to synthesize carbohydrate polymers, we reported a novel class of biomaterials (Poly-Amido-Saccharides; PAS) in 2012. PASs share many properties with natural polysaccharides, such as hydrophilicity, dense hydroxyl functionality, stereoregularity, and a rigid backbone. PASs are connected by an α-1,2-amide linkage, instead of an ether linkage, that confers resistance to enzymatic and hydrolytic degradation and leads to a unique helical conformation. Importantly, our synthetic methodology affords control over molecular weight distribution resulting in pure, well-defined polymers. This Account provides an overview of the development of PAS, from the factors that initially motivated our research to current efforts to translate functional PAS to biomedical applications. We detail the synthesis of glucose- and galactose-based PAS and their biophysical properties including conformation analysis, lectin interactions, cell internalization, and water solubility. Additionally, we describe postpolymerization modification strategies to afford PASs that act as protein stabilizers. We also highlight our recent efforts toward a mechanistic understanding of monomer synthesis via [2 + 2] cycloaddition reactions in order to develop novel monomers with different stereochemistry and amine or alkyl functionality, thereby accessing functional carbohydrate polymers. Throughout our work, we apply computational and theoretical analysis to explain how properties at the monomer level (e.g., stereochemistry, functionality) significantly impact polymer properties, helical conformation, and bioactivities. Collectively, the results from the theoretical, synthetic, and applied aspects of this research advance us toward our goal of utilizing PASs in key biomedical applications as alternatives to natural polysaccharides. The importance of carbohydrates in nature and the versatility of their functions continue to inspire our investigation of new monomers, polymers, and copolymers, leveraging the advantageous properties of PAS to develop potential therapies.

Evidence for Many Unique Solution Structures for Chymotrypsin Inhibitor 2: A Thermodynamic Perspective Derived from vT-ESI-IMS-MS Measurements.

Chymotrypsin inhibitor 2 (CI-2) is a classic model for two-state cooperative protein folding and is one of the most extensively studied systems. Alan Fersht, a pioneer in the field of structural biology, has studied the wild-type (wt) and over 100 mutant forms of CI-2 with traditional analytical and biochemical techniques. Here, we examine wt CI-2 and three mutant forms (A16G, K11A, L32A) to demonstrate the utility of variable-temperature (vT) electrospray ionization (ESI) paired with ion mobility spectrometry (IMS) and mass spectrometry (MS) to map the free energy folding landscape. As the solution temperature is increased, the abundance of each of the six ESI charge states for wt CI-2 and each mutant is found to vary independently. These results require that at least six unique types of CI-2 solution conformers are present. Ion mobility analysis reveals that within each charge state there are additional conformers having distinct solution temperature profiles. A model of the data at ∼30 different temperatures for all four systems suggests the presence of 41 unique CI-2 solution conformations. A thermodynamic analysis of this system yields values of ΔCp as well as ΔG, ΔH, and ΔS for each state at every temperature studied. Detailed energy landscapes derived from these data provide a rare glimpse into Anfinsen's thermodynamic hypothesis and the process of thermal denaturation, normally thought of as a cooperative two-state transition involving the native state and unstructured denatured species. Specifically, as the temperature is varied, the entropies and enthalpies of different conformers undergo dramatic changes in magnitude and relative order to maintain the delicate balance associated with equilibrium.

Stereoselective access to [5.5.0] and [4.4.1] bicyclic compounds through Pd-catalysed divergent higher-order cycloadditions.

Medium-sized rings, including those embedded in bridged and fused bicyclic scaffolds, are common core structures of myriad bioactive molecules. Among various synthetic strategies towards their synthesis, intermolecular higher-order cycloaddition provides great potential to build complex medium-sized rings from simple building blocks. Unfortunately, such transformations are often plagued with competitive reaction pathways and low levels of site- and stereoselectivity. Herein, we report catalyst-controlled divergent access to three classes of medium-sized bicyclic compounds in high efficiency and stereoselectivity, by palladium-catalysed cycloadditions of tropones with γ-methylidene-δ-valerolactones. Mechanistic studies and density functional theory calculations disclosed that the divergent reactions stem from the different reaction profiles of the diastereomeric intermediates. While one undergoes either O- or C-allylation to provide [5.5.0] or [4.4.1] bicyclic compounds, the unique conformation of the other diastereomer allows an unconventional alkene isomerization to deliver bridgehead alkene-containing bicyclo[4.4.1] compounds. The conversion of these products to diverse complex polycyclic scaffolds has also been demonstrated.

Novel Mechanism of Cholesterol Transport by ABCA5 in Macrophages and Its Role in Dyslipidemia

Cholesterol homeostasis results from a delicate interplay between influx and efflux of free cholesterol primarily mediated by ABCA1. Here we report downregulation of ABCA1 in hyper-cholesterol conditions in macrophages, which might be responsible for compromised reverse cholesterol transport and hyperlipidemia. Surprisingly, this is countered by the upregulation of a lesser known family member ABCA5 to maintain cholesterol efflux. The relative contribution of ABCA1 and ABCA5 toward cholesterol efflux was evaluated and revealed ABCA5 as the primary efflux mediator under high cholesterol load. These observations were correlated to cholesterol load in circulation in vivo, and we observed an inverse expression profile in mice models of atherosclerosis (ApoE−/−) and hyperlipidemia (PPARα−/−) in response to high cholesterol diet. Observations were further validated in human plasma samples. Simulation studies revealed a unique conformation of ABCA5 proposing a favored route for cholesterol loading onto high-density lipoproteins for reverse cholesterol transport. Thus, our study implicates a functional complementation between these two transporters, formulating an efficient strategy to maintain efflux in cholesterol excess conditions in macrophages.

Ab Initio Quantitative Prediction of Tacticity in Radical Polymerization of Poly(methyl methacrylate) by a Molecular Simulation Technique with the Conformation Indexing for Multiple Transition States

Free-radical polymerization is a critical route for generating vinyl polymers owing to its versatility and productivity, while the realization of stereospecific propagation has been considered very difficult and demanding. Therefore, a concrete investigation on the mechanism of tacticity obtained in radical polymerization is urgent. Experimentally, tacticity of poly(methyl methacrylate) produced by radical polymerization in bulk was reported to be syndiotactic and temperature-dependent. In this study, we intended to rationalize those behaviors on the microscopic scale via density functional theory calculations and a hybrid Monte Carlo/molecular dynamics simulation [Red Moon methodology]. We devised a unique conformation indexing based on the multidimensional conformational analysis and obtained as many as 576 transition state conformations for the propagation reaction. Boltzmann factors of the 576 conformations implied the overall higher stability of pro-racemo conformations than pro-meso ones and, in addition, the more syndiotactic tendency at low temperature. By the Red Moon simulations utilizing the energy barriers of those conformations, we successfully simulated the propagation reaction process and achieved quantitative agreement of the product tacticity. These results exhibit the validity of our ab initio molecular simulation procedure with the conformation indexing to realize quantitative analysis of the selectivity of reactions.

Metabolite Structure Assignment Using In Silico NMR Techniques.

A major challenge for metabolomic analysis is to obtain an unambiguous identification of the metabolites detected in a sample. Among metabolomics techniques, NMR spectroscopy is a sophisticated, powerful, and generally applicable spectroscopic tool that can be used to ascertain the correct structure of newly isolated biogenic molecules. However, accurate structure prediction using computational NMR techniques depends on how much of the relevant conformational space of a particular compound is considered. It is intrinsically challenging to calculate NMR chemical shifts using high-level DFT when the conformational space of a metabolite is extensive. In this work, we developed NMR chemical shift calculation protocols using a machine learning model in conjunction with standard DFT methods. The pipeline encompasses the following steps: (1) conformation generation using a force field (FF)-based method, (2) filtering the FF generated conformations using the ASE-ANI machine learning model, (3) clustering of the optimized conformations based on structural similarity to identify chemically unique conformations, (4) DFT structural optimization of the unique conformations, and (5) DFT NMR chemical shift calculation. This protocol can calculate the NMR chemical shifts of a set of molecules using any available combination of DFT theory, solvent model, and NMR-active nuclei, using both user-selected reference compounds and/or linear regression methods. Our protocol reduces the overall computational time by 2 orders of magnitude over methods that optimize the conformations using fully ab initio methods, while still producing good agreement with experimental observations. The complete protocol is designed in such a manner that makes the computation of chemical shifts tractable for a large number of conformationally flexible metabolites.