¹³C-NMR studies of hyaluronan: Conformational sensitivity to varied environments

Hyaluronan (HA) samples ranging in size from small oligosaccharides to high molecular weight polymer have been studied by ¹³C-NMR spectroscopy. In neutral aqueous solutions, the chemical shifts of carbons directly involved in the β-1,3 glucuronidic linkage are found to be sensitive to (1) residue linkage position in short chains. (2) oligomer degree of polymerization. (3) solvent ionic strength, and (4) monovalent vs divalent counterions. The carbons of the β-1,4-glucosaminidic linkage show less sensitivity to the above conditions. Thus conformational versatility for HA in aqueous solution is correlated with a chemical shift change primarily in carbons of the β-1,3 linkage. We have also compared the ¹³C spectrum of HA in neutral aqueous salt solutions to spectra observed in dimethyl sulfoxide J (DMSO) solution (ordered 2- or 4-fold HA form) or the solid state (Na⁺ counterion, tetragonal 4-fold helical HA form). The solid state spectrum is similar to that found in DMSO but differs substantially from the aqueous solution spectrum. The differences are attributed to 111 rotation of the acetamido group, with concomitant change in hydrogen bonding and average conformation at the β-1,4 linkage, and (2) loes of hydrogen bonds in aqueous solution and consequnt change in average conformation at the β-1,3 linkage.