Fluorescent imaging of biomolecules in a cellular context is a widely used approach to interrogate biological systems. In particular, immunofluorescence- and fluorescent fusion protein-based approaches are extensively utilized to image specific proteins. These provide information about the absolute and relative localization of proteins of interest and augment biochemical and genetic techniques. Unfortunately, these approaches are largely limited to protein imaging. Other important classes of biomolecules such as glycans and nucleic acids require a novel imaging approach.;The Bertozzi laboratory has developed a method, termed metabolic labeling, to label glycans in live cells. In this method, a monosaccharide bearing a bioorthogonal chemical reporter moiety is incorporated into cellular glycans by the endogenous biosynthetic machinery. This chemical reporter, either an azide or alkyne, is then specifically ligated with an exogenous reagent which may bear an affinity tag or fluorophore for detection. Three different azide- or alkyne-compatible ligation chemistries have been developed with varying properties, and each is useful in particular 1 contexts. This dissertation describes advances in the use of metabolic labeling to image biomolecules with all three ligation chemistries.;First, I discuss a phosphine reagent which is nonfluorescent until covalent ligation to a metabolically incorporated azido biomolecule. Importantly, this reagent displays no background fluorescence in the absence of azides, but is brightly fluorescent upon azide ligation. Next, I focus on the development of a cell permeant reagent for azide imaging in live cells. I developed an assay to immobilize high concentrations of azides inside living cells in order to screen azide-reactive fluorescent reagents for cell permeability. My collaborators and I identified a cyclooctyne-fluorophore conjugate which is the first reagent capable of labeling intracellular azides. Last, I describe progress toward the development of an azido fluorophore which is intended to be nonfluorescent until ligation to metabolically incorporated alkynyl biomolecules.
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