Abacavir is a nucleoside reverse transcriptase inhibitor marketed since 1999 for the treatment of infection with the human immunodeficiency virus type 1 (HIV). Despite its clinical efficacy, abacavir administration has been associated with serious and sometimes fatal toxic events. Abacavir has been reported to undergo bioactivation in vitro, yielding reactive species that bind covalently to human serum albumin, but the haptenation mechanism and its significance to the toxic events induced by this anti-HIV drug have yet to be elucidated. Abacavir is extensively metabolized in the liver, resulting in inactive glucuronide and carboxylate metabolites. The metabolism of abacavir to the carboxylate involves a two-step oxidation via an unconjugated aldehyde, which under dehydrogenase activity isomerizes to a conjugated aldehyde. Concurrently with metabolic oxidation, the two putative aldehyde metabolites may be trapped by nucleophilic side groups in proteins yielding covalent adducts, which can be at the onset of the toxic events associated with abacavir. To gain insight into the role of aldehyde metabolites in abacavir-induced toxicity and with the ultimate goal of preparing reliable and fully characterized prospective biomarkers of exposure to the drug, we synthesized the two putative abacavir aldehyde metabolites and investigated their reaction with the α-amino group of valine. The resulting adducts were subsequently stabilized by reduction with sodium cyanoborohydride and derivatized with phenyl isothiocyanate, leading in both instances to the formation of the same phenylthiohydantoin, which was fully characterized by NMR and MS. These results suggest that the unconjugated aldehyde, initially formed in vivo, rapidly isomerizes to the thermodynamically more stable conjugated aldehyde, which is the electrophilic intermediate mainly involved in reaction with bionucleophiles. Moreover, we demonstrated that the reaction of the conjugated aldehyde with nitrogen bionucleophiles occurs exclusively via Schiff base formation, whereas soft sulfur nucleophiles react by Michael-type 1,4-addition to the α,β-unsaturated system. The synthetic phenylthiohydantoin adduct was subsequently used as standard for LC-ESI-MS monitoring of N-terminal valine adduct formation, upon modification of human hemoglobin in vitro with the conjugated abacavir aldehyde, followed by reduction and Edman degradation. The same postmodification strategy was applied to investigate the products formed by incubation of abacavir with rat liver cytosol, followed by trapping with ethyl valinate. In both instances, the major adduct detected corresponded to the synthetic phenylthiohydantoin standard. These results suggest that abacavir metabolism to the carboxylate(s) via aldehyde intermediate(s) could be a factor in the toxic events elicited by abacavir administration. Furthermore, the availability of a reliable and fully characterized synthetic standard of the abacavir adduct with the N-terminal valine of hemoglobin and its easy detection in the model hemoglobin ...
