Our group synthesizes novel small molecule chemical probes designed to covalently bind proteins with activities of interest. These probes are also designed with moieties for reporter tag (fluorophore or biotin) appendage that allow for visual or LC-MS analysis. One such approach is being applied to trap polycyclic aromatic hydrocarbon (PAH) metabolizing enzymes. A variety of PAHs have been shown to be carcinogens. Metabolism of PAH may play a key role in this toxicity, and gaining a better understanding of this process will be important for developing prevention strategies. Phase II metabolism of PAH is mediated by enzymes including GST and UGT. The goal of this project was to develop chemical probes for these enzyme activities. 1,4-naphthoquinones are excellent Michael acceptors and have been shown to be potent irreversible inhibitors of most human and rodent GST. We have modified 1,4-naphthoquninoes to synthesize 1 (figure A) by incorporating a terminal alkyne. To facilitate the irreversible binding of UGT, the sodium salt of uridine diphosphate (UDP) was passed over a cation exchange resin, into a solution of tri-n-butyl amine and further reacted with an anhydride to form alkynalated-UDP (2). Both 1 and 2 can be used to label GST and UGT respectively. Validation of the selectivity of new probes was performed on purified enzymes and mouse liver and lung extracts using fluorescent gel and proteomic analysis. The synthesis of probes, their application in studying phase II metabolism via click-chemistry and proteomics studies will be discussed.

Design and synthesis of novel probes for irreversible binding to glutathione S-transferase (GST) and uridine 5’-diphospho-glucoronosyl transferase (UGT) enzymes

251st National Meeting (2016)

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