Modulating cell surface immunoreactivity and cellular targeting of diagnostic probes using bio -orthogonal chemistry

Traditional biochemical approaches to understanding biomolecular function involve reconstitution of the biochemical system in a cell-free environment. Unfortunately, the biological functions of many biomolecules (i.e. glycoproteins) are not readily apparent when studied in a cell-free system, necessitating their study in an intact cellular or tissue environment. If functional groups that are chemically orthogonal to the cell's endogenous biochemistry can be incorporated into a cell's molecules, then it should be possible to directly detect the modified biomolecule in a cellular environment using this "bio-orthogonal" chemistry. This dissertation describes the results of three projects that sought to discover the potential and limitations of introducing orthogonal chemistry in cellular glycoproteins.;In Chapter 1, we explored a strategy for the selective delivery of magnetic resonance contrast reagents to tumor cells that is dependent on differential sialic acid expression. By exploiting the intrinsic promiscuity of the sialic acid biosynthetic pathway and the highly selective reaction of ketones with aminooxy groups, we were able to distinguish cells solely on the basis of subtle alterations in sialic acid metabolism.;Since sialic acid is a component of many tumor-associated oligosaccharide antigens, we explored whether unnatural cell surface sialosides produced by metabolism can act as neo-antigens and modulate the immunogenicity of cells. We found that structural alteration of sialic acids can replace a tolerated self-antigen with an antigenic determinant. Thus, incorporation of unnatural sialosides into cell surface glycoconjugates through biosynthetic means can alter the immunoreactivity of cells, providing new possibilities for tumor immunotherapy.;In the final chapter, we developed a phosphine-derivatized dye which becomes fluorescent after undergoing a Staudinger ligation with an azide. The dye is based on a coumarin scaffold that is nonfluorescent due to phosphine-induced quenching. Reaction with an azide converts the phosphine to a phosphine oxide, rendering the dye fluorescent. Since phosphines and azides are chemically orthogonal to the cellular environment, we anticipate this probe and others based on the concept of ligation-activated fluorescence will facilitate new biochemical experiments.