Mechanisms and applications of unnatural carbohydrate biosynthesis.

摘要

Cell-surface carbohydrates are vital in cellular processes including signaling, adhesion and infection by pathogens. However, methods for studying them have lagged behind those for the other major classes of biopolymers, proteins and nucleic acids. The method of cell-surface oligosaccharide metabolic engineering, wherein an unnatural monosaccharide precursor is utilized by cells to generate unnatural cell-surface epitopes, has been demonstrated to be a useful tool in glycobiology. Introduction of unnatural residues into cell-surface glycans has been used to modify the binding properties of the epitopes and to introduce chemically reactive groups onto cell surfaces for further modification.; This dissertation focuses on elucidation of mechanisms by which unnatural N-acylmannosamine compounds are converted to cell-surface sialosides, and applications of the process to studies of tumor cell behavior. In addition, I explored the possibility of unnatural fucoside biosynthesis.; The first chapter of this dissertation provides an overview of the field of cell-surface oligosaccharide engineering and introduces the techniques of metabolic engineering and metabolic inhibition. Chapter 2 describes investigations into the intrinsic tolerance of the human sialic acid biosynthetic pathway toward substrate modifications. Although certain N-acyl modifications were tolerated, the extent of incorporation was found to correlate strongly to side-chain length. Further experiments, outlined in Chapter 3, sought to define a mechanism for exclusion of untolerated unnatural mannosamine analogs using in vitro enzyme assays and whole-cell studies. A "bottleneck" enzyme in unnatural sialic acid biosynthesis was identified and bypassed. In Chapter 4, I attempted to adapt the technique of unnatural metabolic engineering to the fucose biosynthetic pathway. Azide-containing fucose analogs were synthesized and tested in cellular assays as potential vehicles for the expression of cell-surface azides. Finally, Chapter 5 details the application of metabolic inhibition with unnatural mannosamines to the study of polysialic acid and its role in cancer. In an effort to directly address the role of polysialic acid in the tumorigenicity and metastatic potential of tumor cells, I studied the effect of metabolic inhibitors on the proliferation, invasion and morphology of human and mouse breast cancer model cell lines.