A DETAILED H-1 AND C-13 NMR-STUDY OF A REPEATING DISACCHARIDE OF HYALURONAN - THE EFFECTS OF TEMPERATURE AND COUNTERION TYPE

For the first time, a detailed NMR study of the conformation of methyl 2-acetamido-2-deoxy-3-O-(beta-D-glucopyranosyluronic acid)-beta-D-glucopyranoside (disaccharide 1) in aqueous solution is reported. This disaccharide is a repeating unit of hyaluronan, a polysaccharide with widespread biological and pharmaceutical applications. Relatively small changes in temperature, over typical experimental conditions (0-37-degrees-C), completely change the appearance of its one-dimensional H-1 NMR spectrum at 500 MHz. To determine the underlying cause for this temperature sensitivity, we analyzed H-1 and C-13 chemical shifts, temperature coefficients (DELTAdelta/DELTAT), H-1-H-1 coupling constants, and interglycosidic H-1-C-13 coupling constants for 1 as a function of temperature. For comparison, we measured the temperature dependence of H-1 chemical shifts and coupling constants for related monosaccharides: glucuronate (GlcUA or U) and N-acetylglucosamine (GlcNAc or N), and glucose (Glc). The temperature sensitivity of the H-1 spectrum of 1 is caused by relatively larger values of DELTAdelta/DELTAT for some ring protons, rather than a conformational change. The effect is mediated by strong coupling. To detect the presence of long-lived intramolecular hydrogen bonds in the disaccharide, we measured chemical shifts, DELTAdelta/DELTAT, and coupling constants for hydroxyl protons of 1, GlcUA, and GlcNAc in 1:1 H2O-acetone-d6 at low temperature. We compared H-1 NMR parameters for 1, GlcUA, and GlcNAc in water with published values measured in Me2SO-d6 and concluded that interactions with water predominated. We found no evidence for long-lived intramolecular hydrogen bonds occurring in 1 in aqueous solution.