1H- and 13C-NMR studies of solutions of hyaluronic acid esters and salts in methyl sulfoxide: comparison of hydrogen-bond patterns and conformational behaviour.

The 1H- and 13C-NMR spectra of the ethyl and benzyl esters and the tetrabutylammonium and tetraethylammonium salts of hyaluronic acid [[symbol: see text]2)-beta-D-GcpA+-1----3)-beta-D-GlcpNAc-(1[symbol: see text]n] in Me2SO-d6 have been assigned using 1D and 2D techniques. The chemical shifts of the resonance of GlcNAc C-3 suggest that the relative orientations of the monosaccharides at the (1----3) linkage in the esters and salts are different. Small differences in the chemical shifts of the resonance GlcA C-4 suggest only a slight conformational variation around the (1----4) linkage. The 13C-NMR data also suggest similarities in conformation between the esters in Me2SO-d6 and the salts in water. The chemical shifts of the 1H resonances for NH and OH groups and their temperature dependence for the esters and salts in Me2SO reveal markedly stronger inter-residue hydrogen bonds between the carboxyl and NH groups and between HO-4 of GlcA and O-5 of GlcNAc for the salts. The 3J2,NH values indicate a slightly different orientation for the acetamido group. For solutions in Me2SO, the higher segmental flexibility of the esters is supported by the line widths, whereas the reduced viscosity for the tetrabutylammonium salt showed a sigmoidal concentration dependence and suggests association of chains which could contribute to the segmental rigidity. The linear concentration dependence for the benzyl ester suggests a higher overall flexibility without chain association.