1H NMR of glycosaminoglycans and hyaluronic acid oligosaccharides in aqueous solution: The amide proton environment

Abstract

The exchangeable amide protons of hyaluronic acid (HA) oligosaccharides and a higher-molecular-weight segment dissolved in H 2O at pH 2.5 or 5.5 were examined by 1H NMR spectroscopy at 250 MHz. The HA segment preparation showed a single amide resonance, near the chemical shift for the amide proton of the monosaccharide 2-acetamido-2-deoxy-β- d-glucopyranose 3 3 Abbreviations used: HA, hyaluronic acid; GlcNAc, 2-acetamido-2-deoxy-β- d-glucopyranose; GlcUA, β- d-glucopyranuronic acid; IdUA, α- l-idopyranuronic acid; Me β-GlcNAc, methyl 2-acetamido-2-deoxy-β- d-glucose; TMS, trimethylsilane. (β-GlcNAc). Smaller HA oligosaccharides showed two or three separate amide proton resonances, corresponding in relative peak area to interior or end GlcNAc residues. The interior GlcNAc amide resonance occurred at the same chemical shift as the single resonance of the HA segment. For the end GlcNAc residues, linkage to d-glucuronopyranose (GlcUA) through C1 resulted in an upfield shift relative to the β-anomer of GlcNAc, whereas linkage through C3 resulted in a downfield shift relative to the corresponding anomer of GlcNAc. These chemical-shift perturbations appeared to be approximately offsetting in the case of linkage at both positions. The amide proton vicinal coupling constant (ca. 9 Hz) was found to be essentially independent of chain length, residue position, or solution pH. These data favor a nearly perpendicular orientation for the acetamido group with respect to the sugar ring, little affected by linkage of GlcNAc to GlcUA. No evidence for the existence of a stable hydrogen bond linking the amide proton with the carboxyl(ate) oxygen of the adjacent uronic acid residue was found. The amide proton resonances for chondroitin, chondroitin 4-sulfate, and dermatan sulfate were compared to that of HA. The chemical shifts of these resonances deviated no more than 0.1 ppm from that of HA. A small dependence on the identity of the adjacent uronic acid residue was noted, based on the observation of two resonances for dermatan sulfate.