Structural Analysis of Glycans by NMR Chemical Shift Prediction

Structural determination of N- and O-linked glycans as well as polysaccharides is hampered by the limited spectral dispersion. The computerized approach CASPER, an acronym for computer assisted spectrum evaluation of regular polysaccharides, uses liquid state NMR data to elucidate carbohydrate structure based on agreement with predicted ¹H and ¹³C chemical shifts. We here demonstrate developments based on multiple through-bond <i>J</i>-based correlations that significantly enhance the credence to the sequence connectivities proposed in the analysis exemplified by an oligosaccharide and a bacterial polysaccharide. The approach is also suitable for predicting ¹H and ¹³C NMR chemical shifts of synthesized oligosaccharides and glycoconjugates, thereby corroborating a proposed structure

Automatic Structure Determination of Regular Polysaccharides Based Solely on NMR Spectroscopy

The structural analysis of polysaccharides requires that the sugar components and their absolute configurations are determined. We here show that this can be performed based on NMR spectroscopy by utilizing butanolysis with (+)- and (−)-2-butanol that gives the corresponding 2-butyl glycosides with characteristic ¹H and ¹³C NMR chemical shifts. The subsequent computer-assisted structural determination by CASPER can then be based solely on NMR data in a fully automatic way as shown and implemented herein. The method is additionally advantageous in that reference data only have to be prepared once and from a user’s point of view only the unknown sample has to be derivatized for use in CASPER

Conformational Dynamics and Exchange Kinetics of <i>N</i>‑Formyl and <i>N</i>‑Acetyl Groups Substituting 3‑Amino-3,6-dideoxy-α‑d‑galactopyranose, a Sugar Found in Bacterial O‑Antigen Polysaccharides

Three dimensional shape and conformation of carbohydrates are important factors in molecular recognition events and the <i>N</i>-acetyl group of a monosaccharide residue can function as a conformational gatekeeper whereby it influences the overall shape of the oligosaccharide. NMR spectroscopy and quantum mechanics (QM) calculations are used herein to investigate both the conformational preferences and the dynamic behavior of <i>N</i>-acetyl and <i>N</i>-formyl substituents of 3-amino-3,6-dideoxy-α-d-galactopyranose, a sugar and substitution pattern found in bacterial O-antigen polysaccharides. QM calculations suggest that the amide oxygen can be involved in hydrogen bonding with the axial OH4 group primarily but also with the equatorial OH2 group. However, an NMR <i>J</i> coupling analysis indicates that the θ₁ torsion angle, adjacent to the sugar ring, prefers an <i>ap</i> conformation where conformations <180° also are accessible, but does not allow for intramolecular hydrogen bonding. In the formyl-substituted compound ⁴<i>J</i>_HH coupling constants to the <i>exo</i>-cyclic group were detected and analyzed. A van’t Hoff analysis revealed that the <i>trans</i> conformation at the amide bond is favored by Δ<i>G</i>° ≈ – 0.8 kcal·mol^–1 in the formyl-containing compound and with Δ<i>G</i>° ≈ – 2.5 kcal·mol^–1 when the <i>N</i>-acetyl group is the substituent. In both cases the enthalpic term dominates to the free energy, irrespective of water or DMSO as solvent, with only a small contribution from the entropic term. The <i>cis</i>–<i>trans</i> isomerization of the θ₂ torsion angle, centered at the amide bond, was also investigated by employing ¹H NMR line shape analysis and ¹³C NMR saturation transfer experiments. The extracted transition rate constants were utilized to calculate transition energy barriers that were found to be about 20 kcal·mol^–1 in both DMSO-<i>d</i>₆ and D₂O. Enthalpy had a higher contribution to the energy barriers in DMSO-<i>d</i>₆ compared to in D₂O, where entropy compensated for the loss of enthalpy

Conformational Preferences of the O‑Antigen Polysaccharides of Escherichia coli O5ac and O5ab Using NMR Spectroscopy and Molecular Modeling

Escherichia coli serogroup O5 comprises two different subgroups (O5ab and O5ac), which are indiscernible from the point of view of standard immunological serotyping. The structural similarities between the O-antigen polysaccharides (PSs) of these two strains are remarkable, with the only difference being the glycosidic linkage connecting the biological tetrasaccharide repeating units. In the present study, a combination of NMR spectroscopy and molecular modeling methods were used to elucidate the conformational preferences of these two PSs. The NMR study was based on the analysis of intra- and inter-residue proton–proton distances using NOE build-up curves. Molecular models of the repeating units and their extension to polysaccharides were obtained, taking into account the conformational flexibility as assessed by the force field applied and a genetic algorithm. The agreements between experimentally measured and calculated distances could only be obtained by considering an averaging of several low energy conformations observed in the molecular models

Direct Evidence for Hydrogen Bonding in Glycans: A Combined NMR and Molecular Dynamics Study

We introduce the abundant hydroxyl groups of glycans as NMR handles and structural probes to expand the repertoire of tools for structure–function studies on glycans in solution. To this end, we present the facile detection and assignment of hydroxyl groups in a wide range of sample concentrations (0.5–1700 mM) and temperatures, ranging from −5 to 25 °C. We then exploit this information to directly detect hydrogen bonds, well-known for their importance in molecular structural determination through NMR. Via HSQC-TOCSY, we were able to determine the directionality of these hydrogen bonds in sucrose. Furthermore, by means of molecular dynamics simulations in conjunction with NMR, we establish that one out of the three detected hydrogen bonds arises from intermolecular interactions. This finding may shed light on glycan–glycan interactions and glycan recognition by proteins

Conformational Preferences of the O‑Antigen Polysaccharides of Escherichia coli O5ac and O5ab Using NMR Spectroscopy and Molecular Modeling

Escherichia coli serogroup O5 comprises two different subgroups (O5ab and O5ac), which are indiscernible from the point of view of standard immunological serotyping. The structural similarities between the O-antigen polysaccharides (PSs) of these two strains are remarkable, with the only difference being the glycosidic linkage connecting the biological tetrasaccharide repeating units. In the present study, a combination of NMR spectroscopy and molecular modeling methods were used to elucidate the conformational preferences of these two PSs. The NMR study was based on the analysis of intra- and inter-residue proton–proton distances using NOE build-up curves. Molecular models of the repeating units and their extension to polysaccharides were obtained, taking into account the conformational flexibility as assessed by the force field applied and a genetic algorithm. The agreements between experimentally measured and calculated distances could only be obtained by considering an averaging of several low energy conformations observed in the molecular models

Synthesis of β‑(1→2)-Linked 6‑Deoxy‑l‑altropyranose Oligosaccharides via Gold(I)-Catalyzed Glycosylation of an <i>ortho</i>-Hexynylbenzoate Donor

The β-(1→2)-linked 6-deoxy-l-altropyranose di- to pentasaccharides <b>2</b>–<b>5</b>, relevant to the O-antigen of the infectious <i>Yersinia enterocolitica</i> O:3, were synthesized for the first time. The challenging 1,2-<i>cis</i>-altropyranosyl linkage was assembled effectively via glycosylation with 2-<i>O</i>-benzyl-3,4-di-<i>O</i>-benzoyl-6-deoxy-l-altropyranosyl <i>ortho</i>-hexynylbenzoate (<b>7</b>) under the catalysis of PPh₃­AuNTf₂. NMR and molecular modeling studies showed that the pentasaccharide (<b>5</b>) adopted a left-handed helical conformation

Conformational Preferences of the O‑Antigen Polysaccharides of Escherichia coli O5ac and O5ab Using NMR Spectroscopy and Molecular Modeling

Escherichia coli serogroup O5 comprises two different subgroups (O5ab and O5ac), which are indiscernible from the point of view of standard immunological serotyping. The structural similarities between the O-antigen polysaccharides (PSs) of these two strains are remarkable, with the only difference being the glycosidic linkage connecting the biological tetrasaccharide repeating units. In the present study, a combination of NMR spectroscopy and molecular modeling methods were used to elucidate the conformational preferences of these two PSs. The NMR study was based on the analysis of intra- and inter-residue proton–proton distances using NOE build-up curves. Molecular models of the repeating units and their extension to polysaccharides were obtained, taking into account the conformational flexibility as assessed by the force field applied and a genetic algorithm. The agreements between experimentally measured and calculated distances could only be obtained by considering an averaging of several low energy conformations observed in the molecular models

Bifurcated Hydrogen Bonding and Asymmetric Fluctuations in a Carbohydrate Crystal Studied via X‑ray Crystallography and Computational Analysis

The structure of the <i>O</i>-methyl glycoside of the naturally occurring 6-<i>O</i>-[(<i>R</i>)-1-carboxyethyl]-α-d-galactopyranose, C₁₀H₁₈O₈, has been determined by X-ray crystallography at 100 K, supplementing the previously determined structure obtained at 293 K (<i>Acta Crystallogr.</i> <b>1996</b>, <i>C52</i>, 2285–2287). Molecular dynamics simulations of this glycoside were performed in the crystal environment with different numbers of units cells included in the primary simulation system at both 100 and 293 K. The calculated unit cell parameters and the intramolecular geometries (bonds, angles, and dihedrals) agree well with experimental results. Atomic fluctuations, including B-factors and anisotropies, are in good agreement with respect to the relative values on an atom-by-atom basis. In addition, the fluctuations increase with increasing simulation system size, with the simulated values converging to values lower than those observed experimentally indicating that the simulation model is not accounting for all possible contributions to the experimentally observed B-factors, which may be related to either the simulation time scale or size. In the simulations, the hydroxyl group of O7 is found to form bifurcated hydrogen bonds with O6 and O8 of an adjacent molecule, with the interactions dominated by the HO7–O6 interaction. Quantum mechanical calculations support this observation

Kinetics and Mechanism of the Palladium-Catalyzed Oxidative Arylating Carbocyclization of Allenynes

Pd-catalyzed C–C bond-forming reactions under oxidative conditions constitute a class of important and widely used synthetic protocols. This Article describes a mechanistic investigation of the arylating carbocyclization of allenynes using boronic acids and focuses on the correlation between reaction conditions and product selectivity. Isotope effects confirm that either allenic or propargylic C–H activation occurs directly after substrate binding. With an excess of H₂O, a triene product is selectively formed via allenic C–H activation. The latter C–H activation was found to be turnover-limiting and the reaction zeroth order in reactants as well as the oxidant. A dominant feature is continuous catalyst activation, which was shown to occur even in the absence of substrate. Smaller amounts of H₂O lead to mixtures of triene and vinylallene products, where the latter is formed via propargylic C–H activation. The formation of triene occurs only in the presence of ArB­(OH)₂. Vinylallene, on the other hand, was shown to be formed by consumption of (ArBO)₃ as a first-order reactant. Conditions with sub-stoichiometric BF₃·OEt₂ gave selectively the vinylallene product, and the reaction is first order in PhB­(OH)₂. Both C–H activation and transmetalation influence the reaction rate. However, with electron-deficient ArB­(OH)₂, C–H activation is turnover-limiting. It was difficult to establish the order of transmetalation vs C–H activation with certainty, but the results suggest that BF₃·OEt₂ promotes an early transmetalation. The catalytically active species were found to be dependent on the reaction conditions, and H₂O is a crucial parameter in the control of selectivity