Experimental evidence of chemical exchange over the β(1→3) glycosidic linkage and hydrogen bonding involving hydroxy protons in hyaluronan oligosaccharides by NMR spectroscopy

The hydroxy protons of unsaturated di-, tetra-, hexa- and octa-saccharides of hyaluronan (Delta HA(2), Delta HA(4), Delta HA(6) and Delta HA(8)) in 85% H2O/15% acetone-d(6) have been studied by NMR spectroscopy. The chemical shifts (delta), chemical shift differences (Delta delta), temperature coefficients (d delta/dT) and nuclear or rotating-frame Overhauser effects (NOEs or ROEs) of hydroxy protons were measured to gain insight into hydration, hydrogen bonds and flexibility of the HA structure. The NMR data give the first experimental evidence that weak hydrogen bonds exist between O(4)H of N-acetyl-D-glucosamine (GlcNAc) and O(5) of D-glucuronic acid (GlcA) across the beta(1 -> 3) glycosidic linkage and between O(3)H of GlcA and O(5) of GlcNAc across the beta(1 -> 4)-linkage. A chemical exchange was observed between O(4)H of GlcNAc and O(2)H of GlcA over the beta(1 -> 3)-linkage. The interaction could be mediated through water bridge(s) and thus contribute to the water-retaining ability of hyaluronan. In this study it was also demonstrated how the chemical shifts of exchangeable hydroxy or amide proton signals can be used to describe small structural and conformational perturbations within large oligosaccharides.