Unambiguous Assignment Research Articles

Sn-Position-Resolved Quantification of Aminophospholipids by Isotopic N,N-Dimethyl Leucine Labeling and High-Resolution Ion Mobility Mass Spectrometry.

Aminophospholipids (APLs), composed of phosphatidylethanolamines (PEs) and phosphatidylserines (PSs), are vital components of mammalian cell membranes and lipoproteins, participating in both homeostasis and cellular signaling. Their structural changes, including the permutation of fatty acid connectivity (sn-positions), due to dysfunctional metabolic processes have been linked to many diseases. However, the accurate quantification of APLs with unambiguous fatty acyl assignment through routine label-free LC-MS/MS lipidomic analysis remains a major challenge. In this study, we explore the functionalization of the free primary amine groups of APLs using amine-reactive isotopic N,N-dimethyl leucine (iDiLeu) and employ high-resolution ion mobility MS (IM-MS) to develop a novel method for sensitive discernment and accurate quantification of APL sn-isomers. With high-resolution demultiplexing (HRdm) providing IM resolving power >200, labeled sn-isomeric pairs of APLs (ΔCCS ≈ 1%) demonstrate excellent, near baseline separation. In addition to greatly enhanced sensitivity, 5-plex iDiLeu labeling enables the construction of an internal 4-point calibration curve and therefore absolute quantification of APL sn-isomers in a single run. This strategy enabled precise annotation and quantification of 239 APLs including 60 pairs of sn-isomers in the mouse cortex. Additionally, we were able to find ratio changes in multiple APL sn-isomer pairs between wild type and APP/PS1 Alzheimer's disease (AD) model mice at different ages, indicating their strong correlation to AD progression. This strategy could provide universal utility in unraveling the alteration of APL sn-isomers, which have long been considered as the "dark matter" of traditional lipidomic analyses, leading to more precise elucidation of molecular mechanisms of various diseases.

Cryo-EM structures of apo-APC/C and APC/CCDH1:EMI1 complexes provide insights into APC/C regulation

APC/C is a multi-subunit complex that functions as a master regulator of cell division. It controls progression through the cell cycle by timely marking mitotic cyclins and other cell cycle regulatory proteins for degradation. The APC/C itself is regulated by the sequential action of its coactivator subunits CDC20 and CDH1, post-translational modifications, and its inhibitory binding partners EMI1 and the mitotic checkpoint complex. In this study, we took advantage of developments in cryo-electron microscopy to determine the structures of human APC/CCDH1:EMI1 and apo-APC/C at 2.9 Å and 3.2 Å resolution, respectively, providing insights into the regulation of APC/C activity. The high-resolution maps allow the unambiguous assignment of an α-helix to the N-terminus of CDH1 (CDH1α1) in the APC/CCDH1:EMI1 ternary complex. We also identify a zinc-binding module in APC2 that confers structural stability to the complex, and we confirm the presence of zinc ions experimentally. Finally, due to the higher resolution and well defined density of these maps, we are able to build, aided by AlphaFold predictions, several intrinsically disordered regions in different APC/C subunits that likely play a role in proper APC/C assembly and regulation of its activity.

Structure and dynamics of the active site of hen egg-white lysozyme from atomic resolution neutron crystallography.

Hen egg-white lysozyme (HEWL) is a widely used model protein in crystallographic studies and its enzymatic mechanism has been extensively investigated for decades. Despite this, the interaction between the reaction intermediate and the catalytic Asp52, as well as the orientation of Asn44 and Asn46 side chains, remain ambiguous. Here, we report the crystal structures of perdeuterated HEWL and D2O buffer-exchanged HEWL from 0.91 and 1.1Å resolution neutron diffraction data, respectively. These structures were obtained at room temperature and acidic pH, representing the active state of the enzyme. The unambiguous assignment of hydrogen positions based on the neutron scattering length density maps elucidates the roles of Asn44, Asn46, Asn59, and nearby water molecules in the stabilization of Asp52. Additionally, the identification of hydrogen positions reveals unique details of lysozyme's folding, hydrogen (H)/deuterium (D)exchange, and side chain disorder.

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.

Synthesis and Application of D- and 13C-Labelled tert-Butyl Hoechst Dye.

Herein, the successful syntheses of D3- and 13C-N-methyl and D9-tert-butyl Hoechst dyes are presented. This includes the preparation of the labelled D3- and 13C-N-methyl piperazines and D9-tert-butylated hydroxytoluene precursors. The tert-butyl Hoechst dye is known to bind a specific RNA aptamer. Spectroscopic NMR studies of the labelled Hoechst dye-aptamer complexes allowed for the unambiguous assignment of chemical shifts, as well as the dynamics of the bound dye.

Design, synthesis, structural, in silico, and in vitro exploration of structurally modified hydrazones of piperidin-4-one with acetyl & ester moieties

The accessibility and bioavailability of medicinally important molecules like piperidones can be improvised by specific structural modifications and such modifications can be effected by acetylation & esterification of core moiety, as it changes physicochemical properties. Synthesis of hydrazinecarboxylate derivatives of 1-(2-chloroacetyl)-3-methyl-2,6-diphenylpiperidin-4-ones (3a-i) which are structural hybrids of hydrazone of piperidone with acetyl and carboxylate templates is being reported in this paper. The synthesized compounds were structurally characterized by IR, 1H & 13C NMR, mass (3a-d, 3f) spectral techniques, and single crystal X-ray diffraction study (3b and 3d). For compound 3b, 2D NMR (HSQC & HMBC) was recorded for unambiguous assignments of chemical shifts. In addition to the SC-XRD for compounds 3b and 3d, the DFT study revealed the distorted boat conformation of the piperidone ring having E – configuration about the hydrazone moiety in both molecules. Hirshfeld surface analysis on 3b and 3d, exposed the H…H interaction in both the molecules supports their crystal stability to the maximum extent (≥ 53 %) while other interactions viz., O…H, Cl…H, C…H also contribute to the crystal stabilization. In silico bio-study for (3a-i) was done by AutoDock4 with human estrogen receptor (3ERT) protein and found the docking scores ranging from − 7.18 to − 8.64 kcal/mol. The compound 3d identified to possess pronounced biological potential towards the tested protein through the molecular docking studies, was subjected to in vitro (MTT) assay against MCF-7 cell line to explore its anticancer potential.

Heterometallic Molecular and Ionic Isomers.

Numerous descriptions of structural isomerism in metal complexes do not list any molecular vs ionic isomers. At the same time, one of the most striking examples of structural isomerism in organic chemistry is molecular urea, which has the same atomic composition as the chemically distinct ionic ammonium cyanate. This iconic organic couple now meets its inorganic heterometallic counterpart. We introduce a new class of structural isomers, molecular vs ionic, that can be consummated in complex and coordinatively unsaturated polynuclear/heterometallic compounds. We report inorganic molecular and ionic isomers of the composition [NaCrFe (acac)3(hfac)3] (acac = acetylacetonate; hfac = hexafluoroacetylacetonate). Heterometallic molecular [CrIII(acac)3-Na-FeII(hfac)3] (1m) and ionic {[CrIII(acac)3-Na-CrIII(acac)3]+[FeII(hfac)3-Na-FeII(hfac)3]-} (1i) isomers have been isolated in pure form and characterized. While both ions are heterobimetallic trinuclear entities, the neutral counterpart is a heterotrimetallic trinuclear molecule. The two isomers exhibit distinctly different characteristics in terms of solubility, volatility, mass spectrometry ionization, and thermal behavior. Unambiguous assignment of the positions and oxidation/spin states of the Periodic Table neighbors, Fe and Cr, in both isomers have been made by a combination of characterization techniques that include synchrotron X-ray resonant diffraction, synchrotron X-ray fluorescence spectroscopy, Mössbauer spectroscopy, and DART mass spectrometry. The transformation between the two isomers that does take place in solutions of noncoordinating solvents has also been tested.

1H NMR Chemical Shift Changes as a Result of Introduction of Carbonyl-Containing Protecting Groups Observed in Bornol and Isoborneol

Complete assignments of the 1H NMR chemical shifts for monoterpenes, borneol, and isoborneol, and their derivatives in which the secondary hydroxy group is protected with an acetyl group or a benzoyl group, have been made in CDCl3 and C6D6. Upon the protection of the hydroxy group with the carbonyl functional groups, or acetyl or benzoyl groups, many protons constituting the bicyclic ring exhibited downfield and upfield shifts in the chemical shift values, aiding in the unambiguous assignments of protons and carbons.

How Vibrational Notations Can Spoil Infrared Spectroscopy: A Case Study on Isolated Methanol.

Unraveling methanol's infrared spectrum has challenged spectroscopists for a century, with numerous loose ends still to be explored. We engage in this exploration based on experiments of isolating single methanol molecules in solid argon and neon matrices. We report infrared spectra of methanol in its natural isotopic composition and with partial and full deuteration. These experiments are accompanied by calculating wavenumbers involving anharmonicity and mode-coupling based on the vibrational configuration interaction approach. This allows for an unambiguous assignment of all fundamentals and resonances in the mid-infrared spectrum. An increasing degree of deuteration lifts resonances and aids in assigning bands uniquely. It also becomes evident that different notations typically used in chemistry or physics to describe molecular vibration from spectroscopy fail to describe the spectra appropriately. We highlight the shortcomings and suggest a more elaborate analysis using Sankey diagrams to unambiguously identify spectral features. Consequently, we demystify debated resonances occurring from various stretches and deformations of the methyl group.

Automated Mixture Analysis via Structural Evaluation.

The determination of chemical mixture components is vital to a multitude of scientific fields. Oftentimes spectroscopic methods are employed to decipher the composition of these mixtures. However, the sheer density of spectral features present in spectroscopic databases can make unambiguous assignment to individual species challenging. Yet, components of a mixture are commonly chemically related due to environmental processes or shared precursor molecules. Therefore, analysis of the chemical relevance of a molecule is important when determining which species are present in a mixture. In this paper, we combine machine-learning molecular embedding methods with a graph-based ranking system to determine the likelihood of a molecule being present in a mixture based on the other known species and/or chemical priors. By incorporating this metric in a rotational spectroscopy mixture analysis algorithm, we demonstrate that the mixture components can be identified with extremely high accuracy (≥97%) in an efficient manner.

Controlled single-electron transfer enables time-resolved excited-state spectroscopy of individual molecules.

An increasing number of scanning-probe-based spectroscopic techniques provides access to diverse electronic properties of single molecules. Typically, these experiments can only study a subset of all electronic transitions, which obscures the unambiguous assignment of measured quantities to specific quantum transitions. Here we develop a single-molecule spectroscopy that enables the access to many quantum transitions of different types, including radiative, non-radiative and redox, that is, charge-related, transitions. Our method relies on controlled alternating single-charge attachment and detachment. For read-out, the spin states are mapped to charge states, which we can detect by atomic force microscopy. We can determine the relative energies of ground and excited states of an individual molecule and can prepare the molecule in defined excited states. After a proof-of-principle demonstration of the technique on pentacene, we apply it to PTCDA, the scanning-probe luminescence of which has been interpreted controversially. The method may be used to guide, understand and engineer tip-induced chemical reactions as well as phosphorescence and fluorescence of individual molecules.

Exotic Spin Excitations in a Polar Magnet VOSe_{2}O_{5}.

Magnetic resonance dynamics has been studied for a polar magnet VOSe_{2}O_{5}, which hosts several nontrivial magnetic phases including Néel-type skyrmion lattice (SkL). In both cycloidal and SkL spin states, two excitation modes active to oscillating magnetic field B_{ν}⊥c and one mode active to B_{ν}∥c are identified. The subsequent micromagnetic simulations well reproduce the observed selection rules and relative resonance frequencies, which allows the unambiguous assignment of the spin oscillation manner for each mode. Interestingly, the IC-2 phase with a potential double-q character was found to host similar excitation modes as the SkL state. We also discovered the existence of the novel B' phase with four modes active to B_{ν}⊥c. The present results provide a fundamental basis for the comprehensive understanding of resonant spin dynamics in polar magnets, and highlight VOSe_{2}O_{5} as a unique material platform to host a rich variety of nontrivial spin excitations.

Expanding the scope of resonance Raman spectroscopy in hydrogenase research: New observable states and reporter vibrations

Oxygen-tolerant [NiFe] hydrogenases are valuable blueprints for the activation and evolution of molecular hydrogen under application-relevant conditions. Vibrational spectroscopic techniques play a key role in the investigation of these metalloenzymes. For instance, resonance Raman spectroscopy has been introduced as a site-selective approach for probing metal-ligand coordinates of the [NiFe] active site and FeS clusters. Despite its success, this approach is still challenged by a limited number of detectable active-site states – due to missing resonance enhancement or intrinsic light sensitivity – and difficulties in their assignment. Utilizing two oxygen-tolerant [NiFe] hydrogenases as model systems, we illustrate how these challenges can be met by extending excitation and detection wavelength regimes in resonance Raman spectroscopic studies. Specifically, we observe that this technique does not only probe low-frequency metal-ligand vibrations but also high-frequency intra-ligand modes of the diatomic CO/CN− ligands at the active site of [NiFe] hydrogenases. These reporter vibrations are routinely probed by infrared absorption spectroscopy, so that direct comparison of spectra from both techniques allows an unambiguous assignment of states detected by resonance Raman spectroscopy. Moreover, we find that a previously undetected state featuring a bridging hydroxo ligand between Ni and Fe can be probed using higher excitation wavelengths, as photoconversion occurring at lower wavelengths is avoided. In summary, this study expands the applicability of resonance Raman spectroscopy to hydrogenases and other complex metalloenzymes by introducing new strategies for probing and assigning redox-structural states of the active site.

Synthesis and full characterization of tert-butyl (4-ethynylphenyl)carbamate

Structure and conformation of tert-butyl(4-ethynylphenyl)carbamate, the compound combining carbamate and alkyne parts within the molecule, were studied using single X-ray diffraction analysis, NMR, FTIR and UV–vis spectroscopies. For unambiguous assignment of the signals 1H and 13C-APT-NMR, HMQC and HMBC-NMR experiments were realized. The compound crystallized in orthorhombic crystal system, displaying NHO hydrogen bonds and CH-π interactions. The X-ray structure has been recreated by means of theoretical calculations in order to assess the proposed arrangement and discuss the source of the intra and intermolecular relationships. The use of Grimme, Wiberg and Hirshfeld surface tools were applied to have a good confirmation of the presence and importance of hydrogen bonds.

Density functional theory and spectroscopic analysis of stigmasterol from Garcinia wightii: Deciphering its inhibitory potential against drug-resistant tuberculosis via insilico molecular docking and molecular dynamics simulations

This study investigates the molecular structure and electronic properties of stigmasterol, isolated from Garcinia wightii, using density functional theory (DFT) alongside experimental techniques including FT-IR, FT-Raman, UV–Vis, 1H and 13C NMR spectroscopy. Theoretical predictions are compared with experimental results, facilitated by vibrational energy distribution analysis for unambiguous vibrational wavenumber assignments. Natural bond orbitalanalyzes reveal donor-acceptor bond energies and electron densities, while Fukui functions identify reactive sites susceptible to electrophilic and nucleophilic attacks. Electronic properties are further explored through various analyzes, unveiling reactive surface sites. Non-covalent interactions are examined using reduced density gradient analysis. Assessment of absorption, distribution, metabolism, and excretion attributes aids in drug discovery efforts. Molecular docking studies against 20 target proteins for antitubercular screening show a stable glide score of -9.52 kcal/mol for the protein 4FDO (Mycobacterium tuberculosis DprE1 in complex with CT319). Molecular dynamics simulations over 100 ns indicate the stability of the ligand-protein complex, suggesting its potential as an antituberculosis agent. Stigmasterol's structural, vibrational, electronic, and topological properties are thoroughly investigated, revealing its ring skeleton with a flexible isooctyl side chain, adhering to C1 point group symmetry. Steric and stereo electronic interactions play crucial roles in conformation and binding to target proteins. Theoretical and experimental analyzes align, confirming the compound's bioactive properties. This comprehensive investigation underscores stigmasterol's potential as a tuberculosis treatment option, providing valuable insights into its structural characteristics and therapeutic possibilities.

Enhanced Declustering Enables Native Top-Down Analysis of Membrane Protein Complexes using Ion-Mobility Time-Aligned Fragmentation.

Native mass spectrometry (MS) is proving to be a disruptive technique for studying the interactions of proteins, necessary for understanding the functional roles of these biomolecules. Recent research is expanding the application of native MS towards membrane proteins directly from isolated membrane preparations or from purified detergent micelles. The former results in complex spectra comprising several heterogeneous protein complexes; the latter enables therapeutic protein targets to be screened against multiplexed preparations of compound libraries. In both cases, the resulting spectra are increasingly complex to assign/interpret, and the key to these new directions of native MS research is the ability to perform native top-down analysis, which allows unambiguous peak assignment. To achieve this, detergent removal is necessary prior to MS analyzers, which allow selection of specific m/z values, representing the parent ion for downstream activation. Here, we describe a novel, enhanced declustering (ED) device installed into the first pumping region of a cyclic IMS-enabled mass spectrometry platform. The device enables declustering of ions prior to the quadrupole by imparting collisional activation through an oscillating electric field applied between two parallel plates. The positioning of the device enables liberation of membrane protein ions from detergent micelles. Quadrupole selection can now be utilized to isolate protein-ligand complexes, and downstream collision cells enable the dissociation and identification of binding partners. We demonstrate that ion mobility (IM) significantly aids in the assignment of top-down spectra, aligning fragments to their corresponding parent ions by means of IM drift time. Using this approach, we were able to confidently assign and identify a novel hit compound against PfMATE, obtained from multiplexed ligand libraries.

The effects of thickness, polarization, and strain on vibrational modes of 2D Fe3GeTe2

In this study, we investigated the effects of thickness, light polarization, and strain on the Raman spectra of two-dimensional (2D) Fe3GeTe2 (FGT) crystals synthesized via chemical vapor transport. The crystals are thoroughly characterized using a combination of microscopic, diffraction, and spectroscopic techniques. Particularly, a systematic angle-resolved polarized Raman spectroscopy study reveals a clear polarization dependence of the Raman intensity in both parallel and crossed polarization directions, with the Ag1 mode completely disappearing in the crossed polarization direction. The angle-dependent intensity of both the Ag1 and E2g2 modes in parallel polarization and the intensity of the E2g2mode in the crossed polarization remain constant at all angles. These findings align with predictions from Raman tensor analysis, providing compelling evidence for the unambiguous assignment of the Ag1 and E2g2 modes to specific peaks observed in the Raman spectrum of FGT, resolving existing confusion in the literature regarding their assignment. Furthermore, we examined the effect of strain on the Raman spectrum of 2D FGT in-situ using a bending device. Our study, conducted on a monolayer to few-layer 2D FGT deposited onto polyethylene terephthalate and subjected to outward (inward), i.e., tensile (compressive) bending, demonstrates appreciable downshifting (upshifting) of the Raman peak position of both Ag1 and E2g2 modes. These findings are particularly significant given that strain engineering represents an effective approach to modulate the magnetic properties of FGT and other 2D magnetic materials.

A Gated TIMS FTICR MS Instrument to Decipher Isomeric Content of Complex Organic Mixtures.

Modern research faces increasingly complex materials with a constant need for new analytical strategies that can provide deeper levels of chemical insight. Ultrahigh resolution mass spectrometry (MS), particularly Fourier transform ion cyclotron resonance (FTICR) MS, has provided a robust analytical foundation. However, MS alone offers limited structural information. Here, we present the first implementation and results from an FTICR MS with fully integrated dual accumulation analysis with gated trapped ion mobility spectrometry (gTIMS) capability. The drastically extended charge capacity and parallel accumulation facilitate the analysis of complex mixtures. We achieved a high dynamic range of 4 orders of magnitude within a single FTICR acquisition event. Simultaneously, the valuable linear relationship between the TIMS elution voltage and reduced mobility was retained over a wide mobility range. Benchmarking the instrument performance with Suwannee River fulvic acid (SRFA) by variable ramp gTIMS analysis allowed separation and unambiguous assignment of different charge state distributions. Application to bio-oils has proven the capability to distinguish the isomeric diversity in these ultracomplex samples, while maintaining the expected FTICR MS resolving power and mass accuracy. Valuable information about the molecular distribution, isomeric diversity, and main molecular differences could directly be extracted within the analysis time of a classical "dilute and shoot" direct infusion experiment. The development of this fully integrated and flexible gTIMS with FTICR MS analysis possesses the potential to significantly change the current landscape of high-resolution mass spectrometric analysis of complex mixtures through the added insight of isomeric complexity afforded by TIMS. The exploration of the added IMS dimension promises transformative effects across diverse fields including energy transition, environmental studies, and biological research.

Total Synthesis and Stereochemical Assignment of Enteropeptin A.

The total synthesis and structural elucidation of the antimicrobial sactipeptide enteropeptin A is reported. Enteropeptin A contains a thioaminoketal group with an unassigned stereochemical configuration that is embedded in a highly unusual thiomorpholine ring. In this synthesis, a linear peptide containing a dehydroamino acid and a pendant cysteine residue is subjected to Markovnikov hydrothiolation by a dithiophosphoric acid catalyst. This cyclization reaction forms the central thiomorpholine ring found in the enteropeptins. Both diastereomers at the unassigned thioaminoketal stereocenter of enteropeptin A were prepared, and their comparison to an authentic standard allowed for the unambiguous stereochemical assignment of the natural product to be of the D configuration. This inaugural total synthesis of enteropeptin A represents the first total synthesis of a sactipeptide reported to date. Moreover, the strategy disclosed herein serves as a general platform for the synthesis of stereochemically defined thiomorpholine-containing peptides, which may enable the discovery of new cyclic peptide antibiotics.

Exploring uncommon Fe-oxides in non-metallic inclusions in ultra-high-strength steel

Investigating non-metallic inclusions within ultra-high-strength-steel via conventional methods is a known: however, the challenge is to obtain chemical information of such inclusions at the sub-micrometer level. In this context, probing Fe-based oxide in inclusions is a vital aspect for guiding steel’ performance. The vibrational properties of sub micrometer size Fe-based oxides were investigated by Raman mapping along with chemometric analysis with the aim of probing their chemical composition. Highly contrasted Raman spectra were recorded from several inclusions embedded at different spatial locations. The observed spectral features were identified as specific markers of hematite (α-Fe2O3) and magnetite (Fe3O4). Principal Component Analysis was used to confirm the presence of these markers and potentially revealing additional patterns. Their unambiguous assignment has been inferred by comparing our experimental findings with the literature data recorded either in single crystals of iron oxides or oxyhydroxides. Micro-Raman spectroscopy is proven to be a reliable, cost-effective, and non-invasive tool for the unambiguous identification of subsurface regions of steel.