Spin Coupling in a Lewis Trisaccharide as Evidence of Inter-Residue C-H···O Hydrogen Bonding in Aqueous Solution.

Prior studies of the solution conformation of the Lewis (Le) trisaccharide, αFuc-(1→3)[βGal-(1→4)]-βGlcNAc, suggest that nonclassical inter-residue C-H···O hydrogen bonding in aqueous solution contributes to the stabilization of its 3D structure and affects its biological properties. Experimental evidence for this hydrogen bond in aqueous solution has been reported in the form of a NMR spin-coupling constant between C5'Fuc and H1″Gal measured by 2D NMR methods in unlabeled samples. A methyl glycoside of Le (MeβLe) was prepared containing selective C-labeling at C5'Fuc, and the H1″Gal signal was examined in high-field H NMR spectra for evidence of splitting or line-broadening caused by the C at C5'Fuc.

Context Effects on Human Milk Oligosaccharide Linkage Conformation and Dynamics Revealed by Analysis.

An emerging NMR method, analysis, has been applied to investigate context effects on the conformational properties of several human milk oligosaccharides (HMOs). The model of the β-(1→4) linkage in the disaccharide, methyl β-lactoside (MeL), was compared to those obtained for the same linkage in the HMO trisaccharides, methyl 2'-fucosyllactoside (Me2'FL) and methyl 3-fucosyllactoside (Me3FL), and in the tetrasaccharide, methyl 2',3-difucosyllactoside (Me2',3DFL). analysis revealed significant context effects on the mean values and circular standard deviations (CSDs) of the psi (ψ) torsion angles in these linkages.

analysis of the -glycosidic linkages of oligosaccharides using nonconventional NMR -couplings: and MD models of .

analysis (Meredith , 2022, , 3135-3141) is a new NMR-based method to treat ensembles of redundant NMR spin-coupling constants (-couplings) to obtain experiment-based probability distributions of molecular torsion angles in solution. Work reported to date on modeling the conformations of -glycosidic linkages of oligosaccharides using three conventional -coupling constraints ( , , ) has shown that the method gives mean torsion angles and circular standard deviations (CSDs) for in very good agreement with those obtained by MD simulation. On the other hand, CSDs for determined by analysis have consistently been much larger than those determined by MD, calling into question either the reliability of analysis or MD to accurately predict this behavior.

Analysis: Unbiased Multi-State Conformational Modeling of Exocyclic Hydroxymethyl Group Conformation in Methyl Aldohexopyranosides.

analysis (. , , 3135-3141) has been applied to model exocyclic hydroxymethyl group conformation in methyl β-D-glucopyranoside (βGlcOMe), methyl β-D-galactopyranoside (βGalOMe), and methyl β-D-mannopyranoside (βManOMe) in an unbiased manner. Using up to eight NMR -couplings sensitive to rotation about the C5-C6 bond (torsion angle ω), two-state models of ω were obtained that are qualitatively consistent with the relative populations of the , and rotamers reported previously.

Methyl α-D-galactopyranosyl-(1→3)-β-D-galactopyranoside and methyl β-D-galactopyranosyl-(1→3)-β-D-galactopyranoside: Glycosidic linkage conformation determined from MA'AT analysis.

MA'AT analysis has been applied to two biologically-important O-glycosidic linkages in two disaccharides, α-D-Galp-(1→3)-β-D-GalpOMe (3) and β-D-Galp-(1→3)-β-D-GalpOMe (4). Using density functional theory (DFT) to obtain parameterized equations relating a group of trans-O-glycosidic NMR spin-couplings to either phi (ϕ') or psi (ψ'), and experimental J, J, and J spin-couplings measured in aqueous solution in C-labeled isotopomers, probability distributions of ϕ' and ψ' in each linkage were determined and compared to those determined by aqueous 1-μs molecular dynamics (MD) simulation. Good agreement was found between the MA'AT and single-state MD conformational models of these linkages for the most part, with modest (approximately <15°) differences in the mean values of ϕ' and ψ', although the envelope of allowed angles (encoded in circular standard deviations or CSDs) is consistently larger for ϕ' determined from MA'AT analysis than from MD for both linkages.

Analysis: Probability Distributions of Molecular Torsion Angles in Solution from NMR Spectroscopy.

ConspectusMonosaccharides adopt multiple conformations in solution, and this structural complexity increases significantly when they are assembled into oligosaccharides and polysaccharides. Characterization of the conformational properties of saccharides in solution by NMR spectroscopy has been hampered by several complicating factors, including difficulty interpreting spectra because of significant signal overlap, population averaging of NMR parameters, and unique properties of the spectra that make accurate measurements of NMR parameters prone to error (e.g.

One-bond C-C spin-coupling constants in saccharides: a comparison of experimental and calculated values by density functional theory using solid-state C NMR and X-ray crystallography.

Methyl aldohexopyranosides were C-labeled at contiguous carbons, crystallized, and studied by single-crystal X-ray crystallography and solid-state C nuclear magnetic resonance (NMR) spectroscopy to examine the degree to which density functional theory (DFT) can calculate one-bond C-C spin-coupling constants () in saccharides with sufficient accuracy to permit their use in ' analysis, a newly-reported hybrid DFT/NMR method that provides probability distributions of molecular torsion angles in solution (Zhang , , 2017, , 3042-3058; Meredith , , 2022, , 3135-3141). Experimental values in crystalline samples of the doubly C-labeled compounds were measured by solid-state C NMR and compared to those calculated from five different DFT models: (1) values calculated from single structures identical to those observed in crystalline samples by X-ray crystallography (all atom refinement); (2) values calculated from the single structures in (1) but after Hirshfeld atom refinement (HAR); (3) values calculated from the single structures in (1) after DFT-optimization of hydrogen atoms only; and (4 and 5) values calculated in rotamers of torsion angle (C1-C2-O2-O2H) or (C4-C5-C6-O6) from which either specific or generalized parameterized equations were obtained and used to calculate values in the specific or rotamers observed in crystalline samples. Good qualitative agreement was observed between calculated values and those measured by solid-state C NMR regardless of the DFT model, but in no cases were calculated values quantitative, differing (over-estimated) on average by 4-5% from experimental values.

Unveiling the Mechanism of Photodamage to Sphingolipid Molecules via Laser Flash Photolysis and EPR.

Sphingolipids are involved in the maintenance of the skin barrier function and regulate cellular processes of keratinocytes. The work reported here is designed to uncover details of the mechanism of damage to such lipids by UV radiation. Our approach employs laser flash photolysis and electron paramagnetic resonance (EPR) spectrometry to explore the mechanism of the decay reactions, and to determine the associated kinetic parameters.

One-Bond C-H and C-C Spin-Coupling Constants as Constraints in Analysis of Saccharide Conformation.

analysis uses ensembles of redundant experimental NMR spin-coupling constants, parametrized -coupling equations obtained from density functional theory (DFT) calculations, and circular statistics to produce probability distributions of molecular torsion angles in solution and information on librational motions about these angles (Meredith et al., . , , 3135-3141).

Reactivity of Zn in high-temperature water radiolysis.

Reactivity of transients involving Zn in high-temperature water radiolysis has been studied in the temperature range of 25-300 °C. The reduced monovalent zinc species were generated from an electron transfer process between the hydrated electron and Zn ions using pulse radiolysis. The Zn species can subsequently be oxidized by the radiolytically-produced oxidizing species: ˙OH, HO and ˙H.

Nonconventional NMR Spin-Coupling Constants in Oligosaccharide Conformational Modeling: Structural Dependencies Determined from Density Functional Theory Calculations.

Nonconventional NMR spin-coupling constants were investigated to determine their potential as conformational constraints in modeling of the -glycosidic linkages of oligosaccharides. Four ( , , , and ) and eight ( , , , , , , , and ) spin-couplings in methyl β-d-galactopyranosyl-(1→4)-β-d-glucopyranoside (methyl β-lactoside) were calculated using density functional theory (DFT) to determine their dependencies on -glycosidic linkage C-O torsion angles, ϕ and ψ, respectively. Long-range was also examined as a potential conformational constraint of either ϕ or ψ.

-Acetyl Side-Chain Conformation in Saccharides: Solution Models Obtained from Analysis.

analysis has been applied to model the conformational properties of -acetyl side-chains in biologically important GlcNAc and ManNAc monosaccharides and in a βGlcNAc-(1→4)-βGlcNAc disaccharide. Density functional theory calculations were conducted to obtain parameterized equations that relate the magnitudes and signs of 10 spin-coupling constants to conformations of the C2-N2 bonds of GlcNAc and ManNAc. Six of these equations were used with experimental -couplings, measured in HO/HO and DMSO- solvents in selectively C-labeled compounds, to model the C1-C2-N2-C1' torsion angle (θ) in GlcNAc and ManNAc residues.

Analysis of Aldofuranosyl Rings: Unbiased Modeling of Conformational Equilibria and Dynamics in Solution.

analysis has been applied to methyl β-d-ribofuranoside () and methyl 2-deoxy-β-d--pentofuranoside () to demonstrate the ability of this new experimental method to determine multi-state conformational equilibria in solution. Density functional theory (DFT) was used to obtain parameterized equations for >20 NMR spin-coupling constants sensitive to furanose ring conformation in and , and these equations were used in conjunction with experimental spin-couplings to produce unbiased models of ring pseudorotation. These models describe two-state north-south conformational exchange consistent with results obtained from traditional treatments of more limited sets of NMR spin-couplings (e.

Two-bond C-C spin-coupling constants in saccharides: dependencies on exocyclic hydroxyl group conformation.

Seven doubly C-labeled isotopomers of methyl β-D-glucopyranoside, methyl β-D-xylopyranoside, methyl β-D-galactopyranoside, methyl β-D-galactopyranosyl-(1→4)-β-D-glucopyranoside and methyl β-D-galactopyranosyl-(1→4)-β-D-xylopyranoside were prepared, crystallized, and studied by single-crystal X-ray crystallography and solid-state C NMR spectroscopy to determine experimentally the dependence of values in aldopyranosyl rings on the C1-C2-O2-H torsion angle, , involving the C2 carbon of the C1-C2-C3 coupling pathway. Using X-ray crystal structures to determine in crystalline samples and by selecting compounds that exhibit a relatively wide range of values in the crystalline state, values measured in crystalline samples were plotted against and the resulting plot compared to that obtained from density functional theory (DFT) calculations. For values ranging from ∼90° to ∼240°, very good agreement was observed between the experimental and theoretical plots, providing strong validation of DFT-calculated spin-coupling dependencies on exocyclic C-O bond conformation involving the central carbon of geminal C-C-C coupling pathways.

Isopropyl 3-deoxy-α-D-ribo-hexopyranoside (isopropyl 3-deoxy-α-D-glucopyranoside): evaluating trends in structural parameters.

Isopropyl 3-deoxy-α-D-ribo-hexopyranoside (isopropyl 3-deoxy-α-D-glucopyranoside), CHO, (I), crystallizes from a methanol-ethyl acetate solvent mixture at room temperature in a C chair conformation that is slightly distorted towards the S twist-boat form. A comparison of the structural parameters in (I), methyl α-D-glucopyranoside, (II), α-D-glucopyranosyl-(1→4)-D-glucitol (maltitol), (III), and 3-deoxy-α-D-ribo-hexopyranose (3-deoxy-α-D-glucopyranose), (IV), shows that most endocyclic and exocyclic bond lengths, valence bond angles and torsion angles in the aldohexopyranosyl rings are more affected by anomeric configuration, aglycone structure and/or the conformation of exocyclic substituents, such as hydroxymethyl groups, than by monodeoxygenation at C3. The structural effects observed in the crystal structures of (I)-(IV) were confirmed though density functional theory (DFT) calculations in computed structures (I)-(IV).

Pulse Radiolysis Investigation of Radicals Derived from Water-Soluble Cyanine Dyes: Implications for Super-resolution Microscopy.

Light-induced blinking, an inherent feature of many forms of super-resolution microscopy, has been linked to transient reduction of the fluorescent cyanine dye used as an imaging agent. There is, however, only scant literature information related to one-electron reduced cyanine dyes, especially in an aqueous environment. Here, we examine a small series of cyanine dyes, possessing disparate π-conjugation lengths, under selective reducing or oxidizing conditions.

OH radical reactions with the hydrophilic component of sphingolipids.

In this work, using the example of model compounds, we studied the reactions resulting from the interaction of OH radicals with the hydrophilic part of sphingolipids. We compared the stopped-flow EPR spectroscopy and pulse radiolysis with optical detection methods to characterize radical intermediates formed in the reaction of OH radicals with glycerol, serinol and N-boc-serinol. Quantum chemical calculations were also performed to help interpret the observed experimental data.

Dissociative electron attachment to amide bond containing molecules: N-ethylformamide and N-ethylacetamide.

To advance our quest to understand the role of low energy electrons in biomolecular systems, we performed investigations on dissociative electron attachment (DEA) to gas-phase N-ethylformamide (NEF) and N-ethylacetamide (NEA) molecules. Both molecules contain the amide bond, which is the linkage between two consecutive amino acid residues in proteins. Thus, their electron-induced dissociation can imitate the resonant behavior of the DEA process in more complex biostructures.

Reconciling MA'AT and molecular dynamics models of linkage conformation in oligosaccharides.

MA'AT conformational models of the phi torsion angles of O-glycosidic linkages differ from those obtained from MD simulation. To determine the source of the discrepancy, MA'AT analyses were performed using DFT-derived equations obtained with and without psi constraints. The resulting phi models were essentially the same, indicating a force-field problem.

Glycosidic linkage, N-acetyl side-chain, and other structural properties of methyl 2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→4)-β-D-mannopyranoside monohydrate and related compounds.

The crystal structure of methyl 2-acetamido-2-deoxy-β-D-glycopyranosyl-(1→4)-β-D-mannopyranoside monohydrate, CHNO·HO, was determined and its structural properties compared to those in a set of mono- and disaccharides bearing N-acetyl side-chains in βGlcNAc aldohexopyranosyl rings. Valence bond angles and torsion angles in these side chains are relatively uniform, but C-N (amide) and C-O (carbonyl) bond lengths depend on the state of hydrogen bonding to the carbonyl O atom and N-H hydrogen. Relative to N-acetyl side chains devoid of hydrogen bonding, those in which the carbonyl O atom serves as a hydrogen-bond acceptor display elongated C-O and shortened C-N bonds.

Radiation damage and dose limits in serial synchrotron crystallography at cryo- and room temperatures.

Radiation damage limits the accuracy of macromolecular structures in X-ray crystallography. Cryogenic (cryo-) cooling reduces the global radiation damage rate and, therefore, became the method of choice over the past decades. The recent advent of serial crystallography, which spreads the absorbed energy over many crystals, thereby reducing damage, has rendered room temperature (RT) data collection more practical and also extendable to microcrystals, both enabling and requiring the study of specific and global radiation damage at RT.

C-C spin-coupling constants in crystalline C-labeled saccharides: conformational effects interrogated by solid-state C NMR spectroscopy.

Solid-state C NMR spectroscopy has been used in conjunction with selectively C-labeled mono- and disaccharides to measure C-C spin-couplings (J) in crystalline samples. This experimental approach allows direct correlation of J values with specific molecular conformations since, in crystalline samples, molecular conformation is essentially static and can be determined by X-ray crystallography. J values measured in the solid-state in known molecular conformations can then be compared to corresponding J values calculated in the same conformations using density functional theory (DFT).

Radiation-damage investigation of a DNA 16-mer.

In macromolecular crystallography, a great deal of effort has been invested in understanding radiation-damage progression. While the sensitivity of protein crystals has been well characterized, crystals of DNA and of DNA-protein complexes have not thus far been studied as thoroughly. Here, a systematic investigation of radiation damage to a crystal of a DNA 16-mer diffracting to 1.

Dipole-Supported Electronic Resonances Mediate Electron-Induced Amide Bond Cleavage.

Dissociative electron attachment (DEA) plays a key role in radiation damage of biomolecules under high-energy radiation conditions. The initial step in DEA is often rationalized in terms of resonant electron capture into one of the metastable valence states of a molecule followed by its fragmentation. Our combined theoretical and experimental investigations indicate that the manifold of states responsible for electron capture in the DEA process can be dominated by core-excited (shake-up) dipole-supported resonances.