Abstract: In this work, I focus on the use of azidosugars as substrates for metabolic oligosaccharide engineering. Azide-labeled glycans can be selectively tagged with phosphine probes in a reaction termed the Staudinger ligation. Chapter 2 focuses on studies of azidosugar metabolism in vivo . Mice were administered peracetylated N-α-azidoacetylmannosamine (Ac₄ManNAz) and its metabolic products were probed ex vivo using the Staudinger ligation. The results demonstrate that Ac ₄ManNAz is a competent metabolic precursor for the delivery of azido sialic acid (SiaNAz) to glycans in mice. SiaNAz analogs were also found to be competent metabolic precursors of glycoconjugate-bound SiaNAz, albeit to different extents depending on the tissue type. Chapter 3 describes the use of the azidosugar peracetylated N-α-azidoacetylgalactosamine (Ac₄GalNAz) as a chemical reporter of mucin-type O-linked glycosylation. Mice were administered Ac₄GalNAz, and the metabolic fate of the azidosugar was analyzed in various tissues and cell populations using the Staudinger ligation. Interestingly, GalNAz is incorporated at high levels into glycans in B-cells but not T-cells, a phenomenon that might be caused by differences in cell cycling and de novo protein biosynthesis. The results presented in Chapter 4 demonstrate that azide-labeled glycans can be chemically tagged using the Staudinger ligation in vivo. The ability of the Staudinger ligation to probe glycosylation in living animals has significant implications for imaging healthy and disease-associated glycans, and for identifying disease-associated glycans and their scaffolds. Next, progress toward non-invasive imaging of cancer-associated glycans using metabolic oligosaccharide engineering is reported (Chapter 5). This strategy for tumor imaging relies on either differential levels of steady-state glycan expression or changes in metabolic activity in order to distinguish cancer cells from normal healthy tissue. Preliminary non-invasive imaging experiments were conducted in tumor-bearing mice using Ac₄ManNAz and phosphine-based reporters. Finally, progress toward enhancing selective incorporation of azidosugars into tumor cell glycans is described (Chapter 6). Briefly, 'caged' azidosugars were designed for liberation and metabolism in cells that express tumor-specific enzymes. Synthesis and initial biological test results are reported. (Abstract shortened by UMI.)