SYNTHESIS AND BIOLOGICAL EVALUATION OF AZIDE- TAGGED BIOMIMETIC PROBES FOR GLOBAL LABELING OF THE SACCHAROMYCES CEREVISIAE GLYCEROLIPIDOME

Lipid metabolism is an extremely complex, interconnected process that, when dysregulated, is strongly linked to cellular disease states including cancer. Study of these metabolic processes is complicated by the characteristic intricacy of these systems; nonetheless, it is achievable through metabolic labeling. Visualization of biosynthesis is an advantage offered by metabolic labeling through the incorporation of fluorescent tags. In this technique, synthetic probes bearing a clickable tag such as azide are fed into live cells where they are directly incorporated using the native enzymatic and cellular processes to metabolically label lipid products. These labeled lipid products are capable of being studied by direct visualization after an alkyne-azide click reaction. The metabolic intermediates of glycerol, monoacylglycerol, glycerol-3-phosphate, and phosphatidic acid were chosen as targets for the development of synthetic probes bearing clickable azide tags due to their central role in lipid metabolism which will be discussed more in the text. Additionally, the azide tag was placed on the glycerol backbone of these metabolic probes to ensure that it is not cleaved during metabolism but can continually be incorporated into a broad range of lipid products for global labeling. Metabolically labeled lipids were evaluated by fluorescence microscopy after a Cu-free click reaction to visualize probe incorporation at the cellular membrane. Fluorescence TLC analysis for visualization of individually labeled lipid products have been conducted with lipid extracts that had been treated with Cu-catalyzed click reaction for incorporation of the fluorescent tag. Efforts to identify and quantify azide-tagged lipid products by HPLC-MS/MS have been initiated. This thesis describes progress towards the design and synthesis of azide-tagged biomimetic molecular probes and their application toward developing fluorescence labeling via click chemistry as a means for direct imaging and analysis of lipid products.