Solution conformational analysis of hyaluronan with different chain lengths by NMR spectroscopy

The solution conformation of hyaluronan (HA), a biopolymer in the glycosaminoglycan (GAG) family, is still not well understood despite its simple primary structure. The main origin of the structural flexibility of HA is its two alternating β(1 → 4) and β(1 → 3) glycosidic linkages and several studies have reported apparently contrasting evidence on the structural and dynamic behavior of these two linkages. Meanwhile, only short HA oligosaccharides have been investigated thoroughly by NMR spectroscopy and few studies have focused on comparisons between short and longer HA chains. We report herein a detailed conformational investigation of HA octasaccharide and 20-mer by NMR spectroscopy based on NOEs and ³J-couplings, focusing on the glycosidic linkages and the N-acetyl group. This is the first NMR study that provides experimental support for the existence of minor conformations of the β(1 → 4) glycosidic linkage and validation of the predominant conformation of the β(1 → 3) glycosidic linkage previously established by MD simulations. This approach provides methodologies for conformational analysis among different chain lengths in other glycans.