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The structure of sialic acid on living cells can be modulated by metabolism of unnatural biosynthetic precursors. Here we investigate the conversion of a panel of azide-functionalized mannosamine and glucosamine derivatives into cell-surface sialosides. A key tool in this study is the Staudinger ligation, a highly selective reaction between modified triarylphosphines and azides that produces an amide-linked product. A preliminary study of the mechanism of this reaction, and refined conditions for its in vivo execution, are reported. The reaction provided a means to label the glycoconjugate-bound azidosugars with biochemical probes. Finally, we demonstrate that the cell-surface Staudinger ligation is compatible with hydrazone formation from metabolically introduced ketones. These two strategies provide a means to selectively modify cell-surface glycans with exogenous probes.

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The polypeptide N-acetyl-alpha-galactosaminyltransferases (ppGalNAcTs, also abbreviated ppGaNTases) initiate mucin-type O-linked glycosylation and therefore play pivotal roles in cell-cell communication and protection of tissues. In order to develop new tools for studying mucin-type O-linked glycosylation, we screened a 1338 member uridine-based library to identify small molecule inhibitors of ppGalNAcTs. Using a high-throughput enzyme-linked lectin assay (ELLA), two inhibitors of murine ppGalNAcT-1 (K(I) approximately 8 microM) were identified that also inhibit several other members of the family. The compounds did not inhibit other mammalian glycosyltransferases or nucleotide sugar utilizing enzymes, suggesting selectivity for the ppGalNAcTs. Treatment of cells with the compounds abrogated mucin-type O-linked glycosylation but not N-linked glycosylation and also induced apoptosis. These uridine analogs represent the first generation of chemical tools to study the functions of mucin-type O-linked glycosylation.