Trapping Labile Adducts Formed Between an ortho-Quinone Methide and DNA

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McCrane, Michael Patrick
Rokita, Steven E
Exogenously generated electrophiles are capable of alkylating DNA. If not repaired, the resulting DNA adducts can lead to mutations and either cancer or cell death. Electrophilic ortho-quinone methides (o-QM) are reactive intermediates that alkylate DNA and are generated during xenobiotic metabolism of a variety of compounds including environmental toxins and therapeutic agents. Identifying the full alkylation profile of o-QM towards DNA would allow for the genotoxicity of o-QM precursors to be better understood. From model studies based on nucleosides, o-QMs react most readily, but reversibly with the strong nucleophiles 2'-deoxycytidine (dC) N3, 2'-deoxyguanosine (dG) N7, and 2'-deoxyadenosine (dA) N1 and less efficiently, but irreversibly with the weak nucleophiles dG N1, dG N2, and dA N6. The reverse reactions complicate analysis of their products in DNA, which requires enzymatic digestion and chromatographic separation. Selective oxidation by bis[(trifluoroacetoxy)iodo]benzene (BTI) can transform the reversible o-QM-DNA adducts into irreversible derivatives capable of surviving such analysis. To facilitate this analysis, a series of oxidized o-QM-dN adducts were synthesized as analytical standards. Initial oxidative trapping studies with an unsubstituted o-QM and dC demonstrated the necessity of an alkyl substituent para to the phenolic oxygen to block over-oxidation. A novel o-QM included a methyl group para to the phenolic oxygen that successfully blocked the over-oxidation allowing for generation of a stable MeQM-dC N3 oxidized product. Further oxidative trapping studies with MeQM and dG resulted in the formation of three stable MeQM-dG oxidized products (guanine N7, dG N1, and dG N2). Initial studies with duplex DNA optimized the enzymatic digestion and confirmed that the assay conditions were compatible with oxidative trapping. The low yielding MeQM alkylation of duplex DNA needs to be scaled up prior to the oxidative trapping studies. Alternative studies quantified the release of MeQM from DNA with the use of 2-mercaptoethanol as a nucleophilic trap. These studies revealed single stranded DNA as a superior carrier of MeQM than duplex DNA and, therefore, a better target DNA for the oxidative trapping studies due to increased yield of MeQM adducts. With the increased MeQM-DNA yield, the intrinsic selectivity and reactivity of MeQM towards DNA can be determined.