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 (1), oxidatively activated pyrrolizidine alkaloids (e.g. 2), the simple pyrroles 2,3- and 3,4-bis-(acetoxymethyl)-l-methylpyrrole (3 and 4), and the antitumor substance 2,3-dihydro-5-(3′,4′-dichlorophenyl)-6,7-bis(hydroxymethyl)-lH-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 cross-linking. 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.