A detailed 1H and 13C 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 1H NMR spectrum at 500 MHz. To determine the underlying cause for this temperature sensitivity, we analyzed 1H and 13C chemical shifts, temperature coefficients (delta gamma/delta T), 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 delta delta/delta T 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, delta delta/delta T, and coupling constants for hydroxyl protons of 1, GlcUA, and GlcNAc in 1:1 H2O-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.
Department of Chemistry, University of Wisconsin-Madison 53706.
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Sicinska
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Adams
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Lerner
D000117:Acetylglucosamine / Q000737:chemistry; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002247:Carbon Isotopes; D002414:Cations, Monovalent; D004187:Disaccharides / Q000737:chemistry*; D005965:Glucuronates / Q000737:chemistry; D020723:Glucuronic Acid; D006801:Humans; D006820:Hyaluronic Acid / Q000737:chemistry*; D006859:Hydrogen; D007202:Indicators and Reagents; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008969:Molecular Sequence Data; D013816:Thermodynamics
D002247:Carbon Isotopes; D002414:Cations, Monovalent; D004187:Disaccharides; D005965:Glucuronates; D007202:Indicators and Reagents; C081213:methyl 2-acetamido-2-deoxy-3-O-glucopyranosyluronoylglucopyranoside; D006859:Hydrogen; D020723:Glucuronic Acid; D006820:Hyaluronic Acid; D000117:Acetylglucosamine