Sequence-specific DNA interstrand cross-linking by an aziridinomitosene in the absence of exogenous reductant

The aziridinomitosene derivative (1S,2S)-6-desmethyl(methylaziridino) mitosene (4) was shown to alkylate plasmid DNA at pH 7.4 in the absence of a reducing agent [Vedejs, E., Naidu, B. N., Klapars, A., Warner, D. L., Li, V.-s., Na, Y., and Kohn, H. (2003) J. Am. Chem. Soc. 125, 15796-15806], an activity not found in the parent mitomycins. We sought to evaluate aziridinomitosene 4 for the presence of DNA interstrand cross-linking activity using nonreductive reaction conditions. Radiolabeled DNA treated with 4 was analyzed by denaturing polyacrylamide gel electrophoresis (DPAGE), a technique that readily separates the less mobile cross-linked ds DNA from the more mobile ss DNA products. Nonreduced 4 produced an interstrand cross-link (ICL) in duplex DNA containing 5′-d(CG) sites, and the yield of ICL was comparable to that obtained from reduced MC under similar conditions. A ds DNA having the central tetranucleotide 5′-d(ACGT) provided the greatest ICL yield from both nonreduced 4 and reduced MC. Substitution of 5′-d(CG) with the inverted sequence 5′-d(GC) completely abolished interstrand cross-linking by 4, revealing 5′-d(CG) as its specific site of ICL formation. Replacement of dG at 5′-d(CG) with 2′-deoxyinosine (dI), which lacks the exocyclic C ₂ amino group present in dG, also prevented DNA ICL formation by 4, revealing an essential role for the dG C₂ amino group in the interstrand cross-linking reaction between 4 and duplex DNA. This report directly demonstrates the presence of bifunctional alkylating activity in a nonreduced aziridinomitosene and clearly shows that unreduced 4 alkylates residues in the minor groove of ds DNA, cross-linking with the same 5′-d(CG) sequence specificity displayed by reduced MC.