Source:Carbohydrate Research, 242:29 - 51, 1993
For the first time, a detailed NMR study of the conformation of methyl 2-acetamido-2-deoxy-3-O-(β-d-glucopyranosyluronic acid)-β-d-glucopyranoside (dissacharide 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°C), completely change the appearance of its one-dimensional 1H NMR spectrum at 500 MHz. To determine the underlying cause for this temperature sensitivity, we analyzed 1H and 13C chemical shifts, temperature coefficients (Δδ/ΔT), 1H-1H coupling constants, and interglycosidic 1H-13C coupling constants for 1 as a function of temperature. For comparison, we measured the temperature dependence of 1H 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 1H spectrum of 1 is caused by relatively larger values of Δδ/ΔT 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, Δδ/ΔT, and coupling constants for hydroxyl protons of 1, GlcUA, and GlcNAc in 1:1H2O-acetone-d6 at low temperature. We compared 1H 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.