THE CHEMICAL EVOLUTION OF DNA-DNA INTERSTRAND CROSS-LINKERS THAT RECOGNIZE DEFINED MIXED AT AND GC SEQUENCES

The monoalkylation and cross-linking reactivities of a group of four s tructurally related DNA-DNA interstrand cross-linkers have been determ ined on restriction enzyme fragments and select oligomers. These highl y potent cytotoxic DNA-DNA cross-linkers consist of two cyclopropa[c]p yrrolo[3,4-3]indol-4(5H)-one indoles [(+)-CPI-I] joined by a urea (Biz elesin) or a bisamido furan, bisamido pyrrole, or bisamido N-methylpyr role linker. Using a thermal cleavage assay in combination with radio- labeled restriction enzyme fragments, we have shown that these compoun ds cross-link duplex DNA six or seven base pairs apart on opposite str ands, but they differ among themselves for both alkylation reactivity and DNA sequence selectivity. Bizelesin and the [(+)-CPI-I](2) bisamid o furan and [(+)-CPI-I](2) bisamido N-methyl pyrrole compounds prefer purely AT-rich sequences (e.g., 5'-(T) under bar(A/T)(4) or (5)A-3', where (T) under bar represents the cross-strand adenine alkylation and A represents an adenine alkylation), while the [(+)-CPI-I](2) bisami do pyrrole requires a centrally positioned GC base pair for high cross -linking reactivity (i.e., 5'-(T) under bar(A/T)(2)G(A/T)(2)A-3'). By comparison of the cross-linking reactivity of the four compounds in 2 1-mer duplex oligomers containing strategically placed GC or IC base p airs, the sequence and linker requirements for high reactivity of the six- and seven-base-pair cross-linkers in 5'-(T) under bar(N)(4) or (5 )A-3' sequences were determined. In the duplex, to attain highest rea ctivity, a centrally placed GC base pair and the exocyclic 2-amino gro up were required, while for the linker in the bisamido pyrrole compoun d, an unsubstituted amine in the pyrrole ring was necessary. On the ba sis of the known requirements for monoalkylation of duplex DNA by (+)- CPI-derived compounds and the structural consequences of monoalkylatio n, together with the information gleaned from this study, we are able to provide a rationale for the structural requirements for the specifi c sequence cross-linked with high reactivity by the pyrrole compound. We propose that, because monoalkylation of the duplex produced a bent DNA duplex that is unsuitable for cross-linking, the duplex has to fir st undergo a ligand-induced rearrangement involving two hydrogen-bondi ng donor-acceptor pairs, which reinstates the requirements necessary f or the second alkylation reaction.