Glycan OH Exchange Rate Determination in Aqueous Solution: Seeking Evidence for Transient Hydrogen Bonds

Hydrogen bonds (Hbonds) are important stabilizing forces in biomolecules. However, for glycans in aqueous solution, direct NMR detection of Hbonds is elusive because of their transient nature. Here, we present Isotope-based Natural-abundance TOtal correlation eXchange SpectroscopY (INTOXSY), a new ¹H–¹³C heteronuclear single quantum coherence–total correlation spectroscopy based method, to extract OH groups’ exchange rate constants (<i>k</i>_ex) for molecules in natural ¹³C abundance and show that OH Hbonds can be inferred from “slower” H/D <i>k</i>_ex. We evaluate <i>k</i>_ex measured with INTOXSY in light of those extracted with line-shape analysis. Subsequently, we use a set of common glycans to establish a <i>k</i>_ex reference basis set and to infer the existence of transient Hbonds involving OH donor groups. Then, we report <i>k</i>_ex values for a series of mono- and disaccharides, as well as for oligosaccharides sialyl Lewis X and β-cyclodextrin, and compare the results with those from the reference set to extract Hbond information. Finally, we utilize NMR experimental data in conjunction with molecular dynamics simulations to establish donor and acceptor Hbond pairs. Our exchange rate measurements indicate that OH/OD exchange rates, <i>k</i>_HD, values <10 s^–1 are consistent with transient Hbond OH groups and potential acceptor groups can be uncovered through MD simulations

Uncovering Nonconventional and Conventional Hydrogen Bonds in Oligosaccharides through NMR Experiments and Molecular Modeling: Application to Sialyl Lewis‑X

We describe the direct NMR detection of a C–H···O nonconventional hydrogen bond (Hbond) and provide experimental and theoretical evidence for conventional Hbonds in the pentasaccharide sialyl Lewis-X (sLe^X-5) between 5 and 37 °C in water. Extensive NMR structural studies together with molecular dynamics simulations offer strong evidence for significant local dynamics in the Le^X core and for previously undetected conventional Hbonds in rapid equilibrium that modulate structure. These NMR studies also showed temperature-dependent ¹H and ¹³C line broadening. The resulting model emerging from this study is more complex than a simple rigid core description of Le^X-like molecules and improves our understanding of stabilizing interactions in glycans

Glycan Stability and Flexibility: Thermodynamic and Kinetic Characterization of Nonconventional Hydrogen Bonding in Lewis Antigens

We provide evidence for CH-based nonconventional hydrogen bonds (H-bonds) for 10 Lewis antigens and two of their rhamnose analogues. We also characterize the thermodynamics and kinetics of the H-bonds in these molecules and present a plausible explanation for the presence of nonconventional H-bonds in Lewis antigens. Using an alternative method to simultaneously fit a series of temperature-dependent fast exchange nuclear magnetic resonance (NMR) spectra, we determined that the H-bonded conformation is favored by ∼1 kcal/mol over the non-H-bonded conformation. Additionally, a comparison of temperature-dependent 13C linewidths in various Lewis antigens and the two rhamnose analogues reveals H-bonds between the carbonyl oxygen of the N-acetyl group of N-acetylglucosamine and the OH2 group of galactose/fucose. The data presented herein provide insight into the contribution of nonconventional H-bonding to molecular structure and could therefore be used for the rational design of therapeutics