Design and Synthesis of Analogs of <i>myo</i>-Inositol, Serine, and Cysteine to Enable Chemical Biology Studies

Phosphorylated myo-inositol compounds including inositol phosphates (InsPs) as well as the phosphatidylinositol polyphosphate lipids (PIP_ns) are critical biomolecules that regulate many of the most important biological processes and pathways. They are aberrant in many disease states due to their regulatory function. The same is true of the phospholipid phosphatidylserine (PS) which can serve as a marker to begin apoptosis. However, the full scope of activities of these structures is not clear, particularly since techniques that enable global detection and analysis of the production of these compounds spatially and temporally are lacking. With all of these obstacles in mind, devising a system that enables selective enrichment and detection of inositol and serine lipid products would allow the tracking of the various products formed during myo-inositol and serine metabolism and biological activity. Towards this end, the goal of this project is to design and synthesize azide-tagged myo-inositol precursors that enable the selective labeling and quantitation of inositol products via copper-mediated azide-alkyne (CuAAC) and strain–promoted (SPAAC) click chemistry, enrichment, mass spectrometry (MS) identification and fluorescence imaging. Successfully synthesized 2-propylazido inositol ether was the lead compound for these studies. The compound was incubated with and metabolically incorporated both into S. cerevisiae and C. albicans. Evidence of incorporation was proven by fluorescence microscopy after SPAAC conjugation to a dibenzocyclooctyne dye. Lipid extraction followed by CuAAC with an alkyne dye was used for TLC validation and showed labeling of tagged inositol containing lipids in C. albicans. Also explored were serine precursors that can be used to irreversibly bind to the PS synthase active site in order to investigate the active site substrates and mechanism. Serine and cysteine synthesized with a bromopropyl chain tethered to the side chain thiol served as an electrophilic trap in the PS synthase active site. The modified cysteine compound has shown competition for active site infiltration by decreasing the production of tritiated PS by >50% at 5 mM.