Glycoproteins are formed by the covalent attachment of monosaccharides or their oligomers to proteins. The intermediates through which glycoproteins are biosynthesized are nucleotide sugars. The research described within this thesis focuses on the use of chemical methods by which the structures of glycans (the carbohydrate portion of a glycoconjugate) can be experimentally manipulated. Most of the glycan-altering compounds discussed herein prevent the normal biosynthesis of glycoproteins either because of their metabolism into non-natural nucleotide sugars or by preventing the normal utilization of nucleotide sugars. The perturbation of glycan structures is one of the main ways in which the biological functions of glycans can be assessed. Three different classes of glycoproteins were investigated, namely, glycoproteins containing asparagine- or serine/threonine-linked glycans, and those containing only serine/threonine-linked N-acetylglucosamine residues. Two thiosugar-containing disaccharide analogues were discovered, in a cell-based screening assay, to significantly impair the biosynthesis of asparagine-linked glycans within treated cells. These compounds, generally called 5-thiomannosides, were extensively characterized in the context of their effects on proprotein convertases, a class of proteases that are responsible for the production of a wide variety of bioactive peptides. The 5-thiomannosides investigated inhibited the production of endorphin in pituitary cells and were used to demonstrate the importance of correct glycosylation on the regulated peptide-hormone-targeting inside cells. Furthermore, this class of compounds was found to inhibit another key proprotein convertase involved in cholesterol biosynthesis. Several different techniques were used to investigate the structures of these glycans isolated from treated cells, the most significant of which was capillary electrophoresis (CE). This technique was used to deduce the structures of glycans obtained from cells by comparison with known standards or on the basis of their sensitivity in various enzyme assays. Furthermore, CE methods for the analysis of nucleotide sugars or nucleotides, and carbohydrate-like natural products were developed. These methods were used to develop an assay for an important drug target in the bacterium Mycobacterium tuberculosis and to investigate the biosynthesis of unusual nucleotide sugars within cultured cells. Of particular interest was the biosynthesis of nucleotide sugars containing thiosugar moieties, that is, sugars that contain an endocyclic sulfur atom.
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Thesis advisor: Pinto, B. Mario
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