2,7-Diaminomitosene, a Monofunctional Mitomycin C Derivative, Alkylates DNA in the Major Groove. Structure and Base-Sequence Specificity of the DNA Adduct and Mechanism of the Alkylation

Abstract

Reductive activation of the bifunctional alkylator and DNA cross-linking agent mitomycin C (MC) yields 2,7-diaminomitosene (2,7-DAM) as the major product of its activation both in cell-free systems and in vivo. 2,7-DAM lacks one of the alkylating functions of MC, the aziridine. We show that 2,7-DAM itself alkylates DNA monofunctionally upon reductive activation, to form a heat-labile adduct. A guanine-N7−2,7-DAM adduct was isolated from the drug−DNA complex upon heating. Nuclease digestion yielded this adduct both in the nucleoside (dG-2,7-DAM) and dinucleoside phosphate [d(GpG)-2,7-DAM] forms. The structures of all three forms were determined by LC−ESIMS and differential UV spectroscopy. The adduct is sequence-specific to guanines in (G) n tracts of DNA. A guanine-N7−2,7-DAM adduct in DNA was indirectly observed previously by Prakash et al. (Prakash, A. S.; Beall, H.; Ross, D.; Gibson, N. W. Biochemistry 1993, 32, 5518−5525). The results indicate that selective removal of the aziridine function of MC results in a switch from minor to major groove alkylation of DNA, and a switch of sequence specificity from guanines in CpG to guanines in (G) n tracts. They also show that the mitosene C-10 carbamate intrinsically reacts as an SN2 alkylating agent, specific to guanine-N7.

Interdependent Sequence Selectivity and Diastereoselectivity in the Alkylation of DNA by Decarbamoylmitomycin C

“…The relative low abundance of 6 in culture cells treated with DMC compared to MC correlates with DMC highert oxicity.T his is in agreement with the hypothesis stating that formation of adduct 6,anoncytotoxic DNA adduct, is part of the reductive detoxification pathway of mitomycins. [22,40]…”

A New Mechanism of Action for the Anticancer Drug Mitomycin C: Mechanism-Based Inhibition of Thioredoxin Reductase

“…Differential UV spectroscopy and CD spectroscopy are two ideally suited tools for the structural characterization of biological adducts formed by MMC, as both the MMC and the mitosenes resulting from activation of the drug show distinct bands in spectral regions where biological molecules do not interfere significantly. − The UV spectrum of MMC shows a characteristic absorption band at 364 nm that disappears when MMC is converted to mitosene derivatives, while the 7-aminomitosene chromophore presents a distinctive band at 300–320 nm. Subtracting the spectrum of unmodified TrxR from the UV spectrum of MMC-inactivated, TrxR should therefore reveal if the inactivated enzyme contains MMC or mitosene chromophores in its structure.…”

Structure of Adduct X, the Last Unknown of the Six Major DNA Adducts of Mitomycin C Formed in EMT6 Mouse Mammary Tumor Cells

“…We reported earlier that 2,7-DAM is capable of alkylating DNA when it is reduced to the hydroquinone stage, as evidenced by isolating guanine N7 adducts of 2,7-DAM (adducts Y and 8 ) formed in reactions of 2,7-DAM with DNA in the presence of various reducing agents (). The mechanism of formation of adduct X and adduct Y from MC and DNA involves, therefore, conversion of reduced MC to 2,7-DAM, which then undergoes a second round of reductive activation to alkylate DNA, at both guanine N 2 and guanine N-7 ( , ) (Scheme ). N-7 alkylation occurs with high selectively for G runs, while N 2 alkylation is shown here to favor the GpC sequence.…”