Background: The study of novel sites of metabolism is important in understanding new mechanisms of biotransformation of a particular moiety by metabolic enzymes. This information is valuable in designing metabolically-stable compounds with drug-like properties. It may also provide insights into the existence of active and reactive metabolites.
Methods: We utilized small scale incubations to generate adequate amounts of the metabolite of interest. After purification, LC-MS/MS and Proton Nuclear Magnetic Resonance (1H-NMR) were utilized to unequivocally assign the novel site of glutathione conjugation on the purine ring system.
Results: A proposed novel site of glutathione conjugation was investigated on a diaminopurine-containing molecule. It was demonstrated that the formation of the glutathione conjugate at the C-6 position of the purine ring system was due to the bioactivation of the compound to a di-imine intermediate by CYP3A4, followed by the nucleophilic addition of glutathione.
Conclusion: S-glutathionylation at C-6 position of a purine was proven unequivocally. This previously unreported mechanism constitutes a novel biotransformation for purines.