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Structural Characterization of Chemically Modified GAG Oligosaccharides through 1H NMR Measurements of the Labile Nitrogen-bound Protons


Glycosaminoglycans (GAGs) mediate a variety of biological processes through their interactions with proteins. To advance our understanding of these interactions, it is necessary to explore the structural characteristics and physico-chemical properties of GAGs. The major focus of the research presented in this dissertation is the structural analysis of GAG oligosaccharides with emphasis on the chemical modification and isolation of oligosaccharides with unique structures. Reactions for the solvolytic de-N-sulfation, chemoselective N-acetylation, and reduction of reducing end residues with sodium borohydride were employed to obtain oligosaccharides containing amide and free amine moieties with unique structural elements. The impact of oligosaccharide primary structure on elements of secondary structure in aqueous solution was explored using 1H NMR.

The role of rare, but biologically important 3-O-sulfated glucosamine residues in heparan sulfate related oligosaccharides was examined. Possible salt bridge formation was probed through the measurement of amino proton temperature coefficients, amino proton solvent exchange rates, and comparison of the carboxylate and amino group pKa values of de-N-sulfated Arixtra (dNSA). Reduced amino proton temperature coefficients, accelerated exchange rates measured by exchange spectroscopy (EXSY), and altered pKa values of amino and carboxylate groups provided evidence that the dNSA 3-O-sulfated glucosamine amino group is likely involved in salt bridge with the neighboring 3-O-sulfo group and possibly with carboxylate group of the adjacent iduronic acid residue. This hypothesis was supported by the results of molecular dynamics simulations.

The effect of oligosaccharide structure on the solvent exchange behavior of GAG oligosaccharide amide group protons was evaluated through comparison of temperature coefficients, activation energy barriers, and pH titrations of oligosaccharides containing amide groups. A library of 20 oligosaccharides differing in size, glycosidic linkage, saccharide type, and sulfation position and extent, allowed the examination of the effect of charge repulsion by nearby negatively charged sulfate and carboxylate groups on the hydroxyl ions responsible for catalyzing amide proton exchange. This charge repulsion effect calls into question the validity of using exchange rates and activation energies for detection of hydrogen bonds without support from temperature coefficients measured for labile protons.

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