Structural Assignment Research Articles

Structural Elucidation and Complete NMR Spectral Assignments of Monascus Monacolin Analogs.

One new monacolin analog, monacolin V (1), together with two new monacolin-like natural products, 6-hydroxyl monacolin P (2) and 3-keto monacolin S (3), were isolated from the ethyl acetate portion of red yeast rice ethanol extract. Their structures were identified by HRESIMS and NMR experiments, and the complete assignments of 1H and 13C NMR data for three compounds were obtained by the aid of HSQC, HMBC, 1H-1H COSY, and NOESY data. This is the first time that the NMR data of compounds 2 and 3 have been fully assigned.

Atomically resolved imaging of the conformations and adsorption geometries of individual β-cyclodextrins with non-contact AFM

Glycans, consisting of covalently linked sugar units, are a major class of biopolymers essential to all known living organisms. To better understand their biological functions and further applications in fields from biomedicine to materials science, detailed knowledge of their structure is essential. However, due to the extraordinary complexity and conformational flexibility of glycans, state-of-the-art glycan analysis methods often fail to provide structural information with atomic precision. Here, we combine electrospray deposition in ultra-high vacuum with non-contact atomic force microscopy and theoretical calculations to unravel the structure of β-cyclodextrin, a cyclic glucose oligomer, with atomic-scale detail. Our results, established on the single-molecule level, reveal the different adsorption geometries and conformations of β-cyclodextrin. The position of individual hydroxy groups and the location of the stabilizing intramolecular H-bonds are deduced from atomically resolved images, enabling the unambiguous assignment of the molecular structure and demonstrating the potential of the method for glycan analysis.

Protein Quakes in Redox Metalloenzymes: Clues to Molecular Enzyme Conductivity Triggered by Binding of Small Substrate Molecules.

Multicentre redox metalloproteins undergo conformational changes on electrochemical surfaces, or on enzyme substrate binding. The two-centre copper enzymes, laccase (Type I and TypeII/III Cu) and nitrite reductase (CuNIR) (Type I and Type II Cu) are examples. With some exceptions, these enzymes show no non-turnover voltammetry on Au(111)-surfaces modified by thiol based self-assembled molecular monolayers, but dioxygen or nitrite substrate triggers strong electrocatalytic signals. Scanning tunnelling microscopy also shows high conductivity only when dioxygen or nitrite is present. Atomic force microscopy shows constant CuNIR height but pronounced structural expansion in the electrocatalytic range on nitrite binding. We have recently offered a rationale, based on ab initio quantum chemical studies of water/nitrite substitution in a 740-atom CuNIR fragment. Presently we provide much more detailed structural assignment mapped to single-residue resolution. NO2 --binding induces both a 2 Å Cu-Cu distance increase, and pronounced frontier orbital delocalization strongly facilitating ET between the Cu regions. The conformational changes transmit from the catalytic Type II centre to the electron inlet Type I centre, via the His129-Cys130 ligands, and via Type I-Cys130 or Type I-His129 ending at Type II Asp92. The ET patterns are reflected in different atomic Mulliken charges in the water and nitrite CuNIR fragment.

The scent gland composition of the Mangshan pit viper, Protobothrops mangshanensis.

The Mangshan pit viper, Protobothrops mangshanensis, is a rare, highly endangered snake native to the mountainous regions of Hunan Province in China. Snakes possess abdominal scent glands, which have been chemically studied in several species. These glands can contain various lipids and peptides, but very often also complex mixtures of carboxylic acids. We report here the occurrence of novel methyl-branched unsaturated acids found in the secretions of six captive individuals living in a zoo. The structures of these compounds, 4.6-dimethylalk-5-enoates in a homologous series from C11-C16, were characterized by GC-MS and GC-IR analysis and various microderivatization reactions including hydrogenation and esterification leading to methyl and pyridylmethyl esters. In addition, dimethyloxazoline formation helped to localize the double bond. The synthesis of methyl 4,6-dimethyldodec-5-enoate allowed the correct assignment of structures and showed the (E)-configuration of the double bond for the major naturally occurring diastereomers. These acids occur in small amounts compared to the major glandular components, cholesterol, and 1-O-hexadecylglycerol, as well as other common long-chain alcohols and amides. Although a general defensive function has been proposed for snake abdominal scent glands, the specific chemistry and moderate amounts of acids reported here may suggest a function in chemical signaling for the Mangshan pit viper. In addition, proline-containing diketopiperazines were identified for the first time in snake scent glands, although an artificial formation from amino acids likely present in the secretion cannot be excluded.

Mining expressed sequence tag (EST) microsatellite markers to assess the genetic differentiation of five Hynobius species endemic to Taiwan

Taiwan harbors five endemic species of salamanders (Hynobius spp.) that inhabit distinct alpine regions, contributing to population fragmentation across isolated “sky islands”. With an evolutionary history spanning multiple glacial-interglacial cycles, these species represent an exceptional paradigm for exploring biogeography and speciation. However, a lack of suitable genetic markers applicable across species has limited research efforts. Thus, developing cross-amplifying markers is imperative. Expressed sequence-tag simple-sequence repeats (EST-SSRs) that amplify across divergent lineages are ideal for species identification in instances where phenotypic differentiation is challenging. Here, we report a suite of cross-amplifying EST-SSRs from the transcriptomes of the five Hynobius species that exhibit an interspecies transferability rate of 67.67%. To identify individual markers exhibiting cross-species polymorphism and to assess interspecies genetic diversity, we assayed 140 individuals from the five species across 84 sampling sites. A set of EST-SSRs with a high interspecies polymorphic information content (PIC = 0.63) effectively classified these individuals into five distinct clusters, as supported by discriminant analysis of principal components (DAPC), STRUCTURE assignment tests, and Neighbor-joining trees. Moreover, pair-wise FST values > 0.15 indicate notable between-cluster genetic divergence. Our set of 20 polymorphic EST-SSRs is suitable for assessing population structure within and among Hynobius species, as well as for long-term monitoring of their genetic composition.

Polar Stacking of Dipole Parallel-Aligned Monolayers of Unsymmetrical 1,4-Diphenyl-1,3-butadienes Creates Nonlinear Optical Materials: Insights from Experiments Guide Structure Assignments

Polar Stacking of Dipole Parallel-Aligned Monolayers of Unsymmetrical 1,4-Diphenyl-1,3-butadienes Creates Nonlinear Optical Materials: Insights from Experiments Guide Structure Assignments

Suppressing sidechain modes and improving structural resolution for 2D IR spectroscopy via vibrational lifetimes.

Vibrational spectroscopy of protein structure often utilizes 13C18O-labeling of backbone carbonyls to further increase structural resolution. However, sidechains such as arginine, aspartate, and glutamate absorb within the same spectral region, complicating the analysis of isotope-labeled peaks. In this study, we report that the waiting time between pump and probe pulses in two-dimensional infrared spectroscopy can be used to suppress sidechain modes in favor of backbone amide I' modes based on differences in vibrational lifetimes. Furthermore, differences in the lifetimes of 13C18O-amide I' modes can aid in the assignment of secondary structure for labeled residues. Using model disordered and β-sheet peptides, it was determined that while β-sheets exhibit a longer lifetime than disordered structures, amide I' modes in both secondary structures exhibit longer lifetimes than sidechain modes. Overall, this work demonstrates that collecting 2D IR data at delayed waiting times, based on differences in vibrational lifetime between modes, can be used to effectively suppress interfering sidechain modes and further identify secondary structures.

Structural elucidation and complete NMR spectral assignments of Monascus monacolin analogues.

Three new monacolin analogues, 3,6-dihydroxy-monacolin P (1), 6-methoxy monacolin S (2), and 6-methoxy dehydromonacolin S (3), were isolated from a fraction that strongly inhibited 3-hydroxy-3-methylglutaryl-CoA reductase from the ethyl acetate portion of red yeast rice ethanol extract. Their structures were determined through a combination of 1D and 2D NMR experiments, mass spectrometry analysis, and known literature reports.

A chemoenzymatic method for simultaneous profiling N- and O-glycans on glycoproteins using one-pot format

Glycosylation is generally characterized and controlled as a critical quality attribute for therapeutic glycoproteins because glycans can impact protein drug-product efficacy, half-life, stability, and safety. Analytical procedures to characterize N-glycans are relatively well established, but the characterization of O-glycans is challenging due to the complex workflows and lack of enzymatic tools. Here, we present a simplified chemoenzymatic method to simultaneously profile N- and O-glycans from the same sample using a one-pot format by mass spectrometry (MS). N-glycans were first released by PNGase F, followed by O-glycopeptide generation by proteinase K, selective N-glycan reduction, and O-glycan release by β-elimination during permethylation of both N- and O-glycans. Glycan structural assignments and determination of N- to O-glycan ratio was obtained from the one-pot mass spectra. The streamlined, one-pot method is a reliable approach that will facilitate advanced characterizations for quality assessments of therapeutic glycoproteins.

From mundane to surprising nonadditivity: drivers and impact on ML models.

Nonadditivity (NA) in Structure-Activity and Structure-Property Relationship (SAR) data is a rare but very information rich phenomenon. It can indicate conformational flexibility, structural rearrangements, and errors in assay results and structural assignment. While purely ligand-based conformational causes of NA are rather well understood and mundane, other factors are less so and cause surprising NA that has a huge influence on SAR analysis and ML model performance. We here report a systematic analysis across a wide range of properties (20 on-target biological activities and 4 physicochemical ADME-related properties) to understand the frequency of various different phenomena that may lead to NA. A set of novel descriptors were developed to characterize double transformation cycles and identify trends in NA. Double transformation cycles were classified into "surprising" and "mundane" categories, with the majority being classed as mundane. We also examined commonalities among surprising cycles, finding LogP differences to have the most significant impact on NA. A distinct behavior of NA for on-target sets compared to ADME sets was observed. Finally, we show that machine learning models struggle with highly nonadditive data, indicating that a better understanding of NA is an important future research direction.

Cross-Validation of Lipid Structure Assignment Using Orthogonal Ion Activation Modalities on the Same Mass Spectrometer.

The onset and progression of cancer is associated with changes in the composition of the lipidome. Therefore, better understanding of the molecular mechanisms of these disease states requires detailed structural characterization of the individual lipids within the complex cellular milieu. Recently, changes in the unsaturation profile of membrane lipids have been observed in cancer cells and tissues, but assigning the position(s) of carbon-carbon double bonds in fatty acyl chains carried by membrane phospholipids, including the resolution of lipid regioisomers, has proven analytically challenging. Conventional tandem mass spectrometry approaches based on collision-induced dissociation of ionized glycerophospholipids do not yield spectra that are indicative of the location(s) of carbon-carbon double bonds. Ozone-induced dissociation (OzID) and ultraviolet photodissociation (UVPD) have emerged as alternative ion activation modalities wherein diagnostic product ions can enable de novo assignment of position(s) of unsaturation based on predictable fragmentation behaviors. Here, for the first time, OzID and UVPD (193 nm) mass spectra are acquired on the same mass spectrometer to evaluate the relative performance of the two modalities for lipid identification and to interrogate the respective fragmentation pathways under comparable conditions. Based on investigations of lipid standards, fragmentation rules for each technique are expanded to increase confidence in structural assignments and exclude potential false positives. Parallel application of both methods to unsaturated phosphatidylcholines extracted from isogenic colorectal cancer cell lines provides high confidence in the assignment of multiple double bond isomers in these samples and cross-validates relative changes in isomer abundance.

How hot are weakly-bound cluster ions in a blackbody field? Insights from master equation modeling

Measuring accurate energetics in mass spectrometry thermochemical and calorimetry experiments depends on detailed knowledge of the energetics of the ion populations. For example, in cases where blackbody infrared radiative dissociation (BIRD) kinetics are not in the rapid energy exchange (REX) limit, threshold dissociation energies obtained directly from experiment will be too low. When ions are not in the REX limit, the ion internal energy distributions can be modeled using a master equation (ME). The ME allows evaluation of ion internal energies over time with a set of rate equations that describe the transfer of energy from one energy state to another. Here, ME modeling that accounts for the radiative absorption and emission, and dissociation rate constants is performed to determine the energetics of two model systems, M2+(H2O)n with n = 24, 55, 96, 178 and H+(AlaGly)n with n = 4, 8, 16, 32, activated by a blackbody field at temperatures between 120 and 200 K. The hydrated cluster and oligopeptide sizes are chosen such that respective ions have comparable number of internal degrees-of-freedom. The effects of blackbody temperature and inherent properties, such as frequencies and infrared (IR) intensities, molecule size, and dissociation parameters (threshold dissociation energy, E0, and high-pressure pre-exponential factor, A∞) on the resulting ion effective temperatures, steady-state energy distributions, and BIRD kinetics are explored. ME results show that at low blackbody temperatures (<∼140 K), the steady-state internal energy distributions of the ion populations resemble those of Boltzmann distributions at the blackbody temperature. At higher blackbody temperatures (>∼140 K), rapid dissociation causes the steady-state internal energy distributions to equilibrate to lower energies where absorption and emission are competitive with dissociation. This results in ion effective temperatures that deviate from and are “colder” than the blackbody temperatures. The temperature where this transition occurs depends on the competition among absorption, emission, and dissociation, and is controlled by the dissociation parameters, vibrational frequencies, and IR intensities, as illustrated for M2+(H2O)n and H+(AlaGly)n. This work shows that, under certain conditions, the ion effective temperatures can deviate significantly from those of the blackbody field temperatures. ME modeling can be used to determine the energy content of ion complexes in mass spectrometry experiments to improve the accuracy of thermochemical and calorimetry measurements of weakly-bound clusters and for more confident assignments of conformations and structures in action spectroscopy.

Structure Determination of 1,3-Dioxolane-Containing Lipids from the Marine Sponge Leucetta sp. Using Chiral 1H NMR Analysis of Model Systems.

Chemical investigation of n-hexane extract from the marine sponge Leucetta sp. led to the isolation of five new lipids, 1-5, each characterized by a substituted dioxolane core. The structures of 1-5 were established based on the interpretation of NMR and HRESIMS data. To assign the absolute configuration at C-1', model systems consisting of diastereomers at C-2, C-4, and C-1' of the dioxolane core were prepared from a chiral glycerol dimethylacetal. 1H NMR inspection of model compounds revealed that a pair of C-1' epimers, 11a/c and 11b/d, was indistinguishable, restricting structural assignment by direct comparison of NMR data. In addition, the lack of chromophores in the dioxolane core resulted in unreliable ECD results, with Cotton effects appearing below 190 nm. As an alternative, a chiral NMR method using Eu(hfc)3 revealed notable lanthanide-induced shifts, allowing the spectroscopic discrimination of 11a/c and ent-11a/c. Therefore, the absolute configuration of all five new lipids was determined to be 2S, 4S, 1'S by direct comparison with the Eu(hfc)3-induced 1H NMR data.

Spectator Exciton Effects in Nanocrystals III: Unveiling the Stimulated Emission Cross Section in Quantum Confined CsPbBr3 Nanocrystals.

Quantifying stimulated emission in semiconductor nanocrystals (NCs) remains challenging due to masking of its effects on pump-probe spectra by excited state absorption and ground state bleaching signals. The absence of this defining photophysical parameter in turn impedes assignment of band edge electronic structure in many of these important fluorophores. Here we employ a generally applicable 3-pulse ultrafast spectroscopic method coined the "Spectator Exciton" (SX) approach to measure stimulated-emission efficiency in quantum confined inorganic perovskite CsPbBr3 NCs, the band edge electronic structure of which is the subject of lively ongoing debate. Our results show that in 5-6 nm CsPbBr3 NCs, a single exciton bleaches more than half of the intense band edge absorption band, while the cross section for stimulated emission from the same state is nearly 6 times weaker. Discussion of these findings in light of several recent electronic structure models for this material proves them unable to simultaneously explain both measures, proving the importance of this new input to resolving this debate. Along with femtosecond time-resolved photoluminescence measurements on the same sample, SX results also verify that biexciton interaction energy is intensely attractive with a magnitude of ∼80 meV. In light of this observation, our previous suggestion that biexciton interaction is repulsive is reassigned to hot phonon induced slowdown of carrier relaxation leading to direct Auger recombination from an excited state. The mechanism behind the extreme slowing of carrier cooling after several stages of exciton recombination remains to be determined.

Structural Characterization of Pyruvic Acid Dimers Formed inside Helium Nanodroplets by Infrared Spectroscopy and Ab Initio Study.

The structural arrangements of α-keto acid complexes hold significant interest across various fields of chemistry such as enzyme modeling, drug design, or polymer blending. Herein, we report mass-selective infrared (IR) spectra of pyruvic acid monomers and dimers in the range 1720-1820 cm-1 recorded in helium nanodroplets at 0.37 K. The monomer features IR bands at 1807.1 and 1734.5 cm-1, which are assigned to the carboxylic and ketonic C═O stretching vibrations, respectively. Furthermore, the pyruvic acid dimers generated inside the helium nanodroplets are characterized by carboxylic and ketonic C═O stretch vibrations appearing at 1799.2 and 1737.0 cm-1, respectively. This frequency shift of ±7 cm-1 for both C═O stretching bands from the monomer to the dimer demonstrates that the structural motif of the monomer is maintained upon dimer aggregation in helium nanodroplets. The structural assignments were supported by a comparison of the MP2/aug-cc-pVDZ-predicted harmonic vibrational spectra at the C═O stretching region with the experiments. The global minimum monomer structure with an intramolecular hydrogen bond and its dimer stabilized by both inter- and intramolecular hydrogen bonding interactions reproduce the experimental spectra from the monomer and dimer. This assigned dimer structure lies ca.11 kJ/mol above the corresponding global minimum and is favored in helium nanodroplets due to the long-range realignment of molecules via dipole-dipole interaction, followed by short-range stabilization upon intermolecular hydrogen bond formation. The barrier for reconfiguration of the precooled monomer conformer leading to the formation of the most stable dimer structure is around 58 kJ/mol, which is infeasible at 0.37 K.

Structural diversity in nudibranch chemistry: elucidation of norditerpenes with a dendrillane scaffold from the Australian nudibranch Goniobranchus coi

In addition to three known compounds (1–3), two new rearranged spongian diterpenes (4 and 5) with perhydroazulene and dioxabicyclooctane ring systems were isolated from the mantle and viscera of the Australian nudibranch Goniobranchus coi. The relative configuration of the major spiroepoxide 4 was explored by comparison with those of m-chloroperbenzoic acid oxidation products derived from dendrillolide A (1). Aldehydes 6 and 7 were identified as the ring-opened artefacts of the spiroepoxides 5 and 4 respectively, and yielded ketone 3 on storage. The relative configurations of lactols 8 and 9, isolated as an inseparable mixture of diastereomers, were deduced by molecular modelling and computational studies. Acetylation of the lactol mixture provided dendrillolide A (1), further confirming the structural assignments of 8 and 9. Dissection of animal tissue established that the norditerpenoid metabolites were present in both mantle and viscera tissues.

Experimentally Determined Diagnostic Ions for Identification of Peptide Glycotopes.

The analysis of the structures of glycans present on glycoproteins is an essential component for determining glycoprotein function; however, detailed glycan structural assignment on glycopeptides from proteomics mass spectrometric data remains challenging. Glycoproteomic analysis by mass spectrometry currently can provide significant, yet incomplete, information about the glycans present, including the glycan monosaccharide composition and in some circumstances the site(s) of glycosylation. Advancements in mass spectrometric resolution, using high-mass accuracy instrumentation and tailored MS/MS fragmentation parameters, coupled with a dedicated definition of diagnostic fragmentation ions have enabled the determination of some glycan structural features, or glycotopes, expressed on glycopeptides. Here we present a collation of diagnostic glycan fragments produced by traditional positive-ion-mode reversed-phase LC-ESI MS/MS proteomic workflows and describe the specific fragmentation energy settings required to identify specific glycotopes presented on N- or O-linked glycopeptides in a typical proteomics MS/MS experiment.

Dielectric barrier discharge ionization procures characteristic adducts of polyfluoroalkyl substances (PFAS) amenable by gas chromatography-mass spectrometry

Fluorotelomer alcohols (FTOHs) and acrylates (FTACs) have been reported as precursors of perfluoroalkyl acids and are known to contaminate biotic and abiotic compartments. Here, a detailed study was carried out for assessing the capability of dielectric barrier discharge ionization (DBDI) to be used as soft ionization technique, as alternative to the more energetic electron ionization (EI), for the quantitative determination of four FTOHs and two FTACs in environmental, biological or food samples by gas chromatography-mass spectrometry. While gas chromatography-EI-mass spectrometry (GC-EI-MS) shows significant fragmentation of the compounds, the ionization obtained by gas chromatography-DBDI-high resolution mass spectrometry (GC-DBDI-HRMS) affords dominant molecular adducts on the spectra. In the positive ion mode, the ion [M + H3O]+ and, of lower intensity, [M + H]+ are observed in the spectra of FTOHs, while the ion [M + H]+ is the most abundant in the spectra of FTACs and accompanied by a lower signal attributed to the ion [M + NO]+. In the negative ion mode, the FTOHs give rise to carbon dioxide and oxygen adducts observed with ions [M − H + CO2]- and [M + O2]-. Structure assignments were confirmed with stable isotope-labelled FTOH analogs and 13CO2. Gas chromatographic separation of the six compounds reveals room for development of methods applicable to soils, plants, water, food and biological materials.

Accurate Prediction of NMR Chemical Shifts: Integrating DFT Calculations with Three-Dimensional Graph Neural Networks.

Computer prediction of NMR chemical shifts plays an increasingly important role in molecular structure assignment and elucidation for organic molecule studies. Density functional theory (DFT) and gauge-including atomic orbital (GIAO) have established a framework to predict NMR chemical shifts but often at a significant computational expense with a limited prediction accuracy. Recent advancements in deep learning methods, especially graph neural networks (GNNs), have shown promise in improving the accuracy of predicting experimental chemical shifts, either by using 2D molecular topological features or 3D conformational representation. This study presents a new 3D GNN model to predict 1H and 13C chemical shifts, CSTShift, that combines atomic features with DFT-calculated shielding tensor descriptors, capturing both isotropic and anisotropic shielding effects. Utilizing the NMRShiftDB2 data set and conducting DFT optimization and GIAO calculations at the B3LYP/6-31G(d) level, we prepared the NMRShiftDB2-DFT data set of high-quality 3D structures and shielding tensors with corresponding experimentally measured 1H and 13C chemical shifts. The developed CSTShift models achieve the state-of-the-art prediction performance on both the NMRShiftDB2-DFT test data set and external CHESHIRE data set. Further case studies on identifying correct structures from two groups of constitutional isomers show its capability for structure assignment and elucidation. The source code and data are accessible at https://yzhang.hpc.nyu.edu/IMA.

Structure and mechanism of the K+/H+ exchanger KefC

Intracellular potassium (K+) homeostasis is fundamental to cell viability. In addition to channels, K+ levels are maintained by various ion transporters. One major family is the proton-driven K+ efflux transporters, which in gram-negative bacteria is important for detoxification and in plants is critical for efficient photosynthesis and growth. Despite their importance, the structure and molecular basis for K+-selectivity is poorly understood. Here, we report ~3.1 Å resolution cryo-EM structures of the Escherichia coli glutathione (GSH)-gated K+ efflux transporter KefC in complex with AMP, AMP/GSH and an ion-binding variant. KefC forms a homodimer similar to the inward-facing conformation of Na+/H+ antiporter NapA. By structural assignment of a coordinated K+ ion, MD simulations, and SSM-based electrophysiology, we demonstrate how ion-binding in KefC is adapted for binding a dehydrated K+ ion. KefC harbors C-terminal regulator of K+ conductance (RCK) domains, as present in some bacterial K+-ion channels. The domain-swapped helices in the RCK domains bind AMP and GSH and they inhibit transport by directly interacting with the ion-transporter module. Taken together, we propose that KefC is activated by detachment of the RCK domains and that ion selectivity exploits the biophysical properties likewise adapted by K+-ion-channels.