| Aberrant glycosylation is a hallmark of numerous disease states. Techniques that enable visualization of glycosylation changes could benefit the development of new diagnostics. Proteins can be readily visualized in living systems using genetically encoded reporters, such as the green fluorescent protein (GFP), or antibodies linked to imaging probes. By contrast, glycans are not amenable to genetically encoded tags, and only a handful of antibodies are known to bind glycans with high affinity. This thesis describes a strategy to equip glycans with reporter tags for visualization and isolation from living systems. As outlined in Chapter 1, the approach relies on the installation of metabolic precursor sugars endowed with unique chemical functionality (e.g., an azide) into target glycans using the cell's own biosynthetic machinery. Azides are non-native functional groups that are inert to biological species, but can be covalently modified via "bioorthogonal" chemical reactions with exogenously delivered probes. Only two such reactions are suitable for use in living systems: the Staudinger ligation of azides with triarylphosphines and the [3 + 2] cycloaddition of azides with cyclooctyne reagents. This two-step labeling process can be used to track glycans in their native environment.;Chapter 2 describes the metabolic labeling of murine tissues with an azido analog of sialic acid (SiaNAz). This was achieved by administration of its precursor, peracetylated N-azidoacetylmannosamine (Ac4ManNAz), to laboratory mice. Chapter 3 describes a similar series of studies using peracetylated N-azidoacetylgalactosamine (Ac4GalNAz) as a metabolic label of mucin-type O-linked glycoproteins. In Chapter 4, the metabolic labeling studies of the previous two chapters were extended to in vivo targeting of azide-labeled cells with phosphine probes via Staudinger ligation. Chapter 5 introduces an alternative to the Staudinger ligation, the strain-promoted [3 + 2] cycloaddition with cyclooctyne analogs, for tagging azidosugars with probes in vivo. Finally, Chapter 6 presents preliminary work toward the use of metabolic labeling and Staudinger ligation for imaging glycosylation in living systems. |
