Oxidative quenching of quinone methide adducts reveals transient products of reversible alkylation in duplex DNA.

ortho-Quinone methides (ortho-QM) and para-quinone methides are generated by xenobiotic metabolism of numerous compounds including environmental toxins and therapeutic agents. These intermediates are highly electrophilic and have the potential to alkylate DNA. Assessing their genotoxicity can be difficult when all or some of their resulting adducts form reversibly. Stable adducts are most easily detected but are not necessarily the most prevalent products formed initially as DNA repair commences. Selective oxidation of ortho-QM-DNA adducts by bis[(trifluoroacetoxy)iodo]benzene (BTI) rapidly quenches their reversibility to prevent QM regeneration and allows for observation of the kinetic products. The resulting derivatives persist through standard enzymatic digestion, chromatography, and mass spectral analysis. The structural standards required for this approach have been synthesized and confirmed by two-dimensional NMR spectroscopy. The adducts of dA N(6), dG N1, dG N(2), and guanine N7 are converted to the expected para-quinol derivatives within 5 min after addition of BTI under aqueous conditions (pH 7). Concurrently, the adduct of dA N1 forms a spiro derivative comparable to that characterized previously after oxidation of the corresponding dC N3 adduct. By application of this oxidative quenching strategy, the dC N3 and dA N1 adducts have been identified as the dominant products formed by both single- and double-stranded DNA under initial conditions. As expected, however, these labile adducts dissipate within 24 h if not quenched with BTI. Still, the products favored by kinetics are responsible for inducing the first response to ortho-QM exposure in cells, and hence, they are also key to establishing the relationship between biological activity and molecular structure.

Trapping a labile adduct formed between an ortho-quinone methide and 2'-deoxycytidine.

Selective oxidation by bis[(trifluoroacetoxy)iodo]benzene (BTI) provides an effective trap for quenching adducts formed reversibly between dC and an ortho-quinone methide (QM) under physiological conditions. A model adduct generated by 4-methyl-o-QM and 2'-deoxycytidine is rapidly converted by intramolecular cyclization and loss of aromaticity to a characteristic product for quantifying QM alkylation. However, BTI induces a surprising rearrangement driven by overoxidation of a derivative lacking an alkyl substituent at the 4-position of the QM.