DNA Interstrand Cross-Linking Reactions of Pyrrole-Derived, Bifunctional Electrophiles: Evidence for a Common Target Site in DNA

The site of DNA interstrand cross-linking identified by a family of pyrrole-derived bifunctional electrophiles was studied in vitro in synthetic DNA duplexes. This family includes reductively activated mitomycin C (l), oxidatively activated pyrrolizidine alkaloids (e.g. 2), the simple pyrroles 2,3and 3,4-bis-(acetoxymethyl)1-methylpyrrole (3 and 4), and the antitumor substance 2,3-dihydro-5-(3’,4’-dichlorophenyl)-6,7-bis(hydroxymethyl)-1H-pyrrolizine bis(isopropylcarbamate) (IPP, 5). It is demonstrated herein that these agents preferentially cross-link a common target site in duplex DNA, the exocyclic amino groups of deoxyguanosine residues at the sequence 5’-d(CG). Specificially, 4 and 5 are shown to form interstrand cross-links in synthetic DNA, 4 and 5 (like 1 and 3) are shown to preferentially cross-link deoxyguanosine residues at the duplex nucleotide sequence 5’-d(CG), and 3,4, and 5 are shown to require both N2 exocyclic amino groups of the potentially bridged deoxyguanosine residues for efficient interstrand crosslinking. Taken together, these results strongly implicate N2 of deoxyguanosine residues at the sequence 5’-d(CG) as the common site of the covalent interstrand cross-linking reaction of these agents. This connectivity was unequivocally proven for the cross-link caused by 5 through isolation and characterization of the relevant lesion from an enzymatic digest of interstrand cross-linked DNA and comparison to an authentic sample prepared by rational chemical synthesis. New syntheses of 3 and 4 are reported.