| Chapter 1. My research has focused on developing chemical tools to aid the investigation of glycobiology, one example of which is the approach termed metabolic oligosaccharide engineering. This chapter summarizes efforts by our laboratory and others towards the development of this approach.;Chapter 2. This chapter describes a series of experiments designed to investigate the scope of metabolic oligosaccharide engineering using azide-functionalized N-acetylmannosamine (ManNAc), N-acetylglucosamine (GlcNAc) and sialic acid analogs. The metabolism of azido analogs was probed using a reaction developed in our laboratory called the Staudinger ligation. To understand the metabolic products resulting from the treatment of cells with azido sugars, the biosynthetic fate of these compounds was probed with competition experiments and by employing mutant cell lines.;Chapter 3. In this chapter, I applied metabolic oligosaccharide engineering to study the effects of GlcNAc 2-epimerase expression on sialic acid production in cells. A key tool developed for this study was a cell-permeable, small molecule inhibitor of G1cNAc 2-epimerase designed based on mechanistic principles. My results indicated that, unlike UDP-GlcNAc 2-epimerase which promotes the biosynthesis of sialic acid, GlcNAc 2-epimerase can serve a catabolic role, diverting metabolic flux away from the sialic acid pathway.;Chapter 4. In this chapter, I used metabolic oligosaccharide engineering to introduce a bioorthogonal functional group, the azide, into cellular and recombinant glycoproteins for subsequent chemical elaboration via Staudinger ligation. I targeted sialic acid as a host for azides using ManNAz as a biosynthetic precursor. SiaNAz was found to comprise between 4% and 41% of total sialosides, depending on the system. Metabolic labeling of recombinant interferon-beta and GlyCAM-Ig was achieved, demonstrating the utility of the method for functionalizing N-linked and O-linked glycoproteins of therapeutic interest, respectively.;Chapter 5. In this chapter, I showed that a ManNAc-deficient cell line is a suitable host for investigating cell surface binding to natural and modified sialic acids. Using these cells, I discovered that SiaNAz is recognized by the lectins MAA, SNA and E-selectin. By contrast, SiaNAz is not able to bind the monoclonal antibody HECA-452 nor the lectins LFA and Siglec-1. The accrued knowledge of lectin specificity for unnatural sialic acids will aid in predictions of the physiological consequences of unnatural sialic acid biosynthesis in vivo. (Abstract shortened by UMI.). |
