Properties of a dehydroalanine analog of glutathione: A reactive electrophilic busulfan metabolite

Gamma-Glutamyldehydroalanylglycine (EdAG) is a dethiolated, electrophilic metabolite of glutathione (GSH) derived from the Phase II conjugation of GSH with busulfan catalyzed by glutathione S-transferase (GST). It was demonstrated in this dissertation that EdAG could be successfully synthesized and shown to be a metabolite of busulfan from in vitro metabolism by enzymes in human liver cytosol. The electrophilic EdAG was reactive toward the cellular thiols cysteine and glutathione (GSH), and toward cysteine residues in reduced bovine insulin B-chain and bovine serum albumin in vitro , as well. EdAG was demonstrated to be a reversible noncompetitive inhibitor of GSTs, specifically GSTA1-1 at micromolar concentrations up to 1 mM. EdAG at high concentration (10 mM) was found to be an irreversible inhibitor of human GSTA1-1, although an EdAG-modified peptide was not observed. Inhibition of GST by a busulfan metabolite has the potential of altering busulfan pharmacokinetics or GSH cellular function. Since conversion of GSH to EdAG represents a loss of thiol-related redox properties and the gain of a captodative radical scavenging dehydroalanine group, the reactivity of EdAG with hydroxyl radical was evaluated. EdAG was shown to scavenge hydroxyl radical generated in the Fenton reaction in a concentration-dependent manner. The results indicate a stabilized carbon-based captodative radical intermediate in the reaction of EdAG with hydroxyl radical. In support of a captodative mechanism was the identification of a dimerized gamma-glutamylserylglycine as a product in the reaction. Conversion of GSH from a redox active nucleophile to an electrophilic dehydroalanine is a significant change in chemical reactivity that may have biological implications for GSH and free radical biochemistry as well as busulfan therapeutics