Oxidative Quenching of Quinone Methide Adducts Reveals Transient Products of Reversible Alkylation in Duplex DNA

<i>ortho</i>-Quinone methides (<i>ortho</i>-QM) and <i>para</i>-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 <i>ortho</i>-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 N⁶, dG N1, dG N², and guanine N7 are converted to the expected <i>para</i>-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 <i>ortho</i>-QM exposure in cells, and hence, they are also key to establishing the relationship between biological activity and molecular structure