SELECTIVE ABSTRACTION OF H-2 FROM C-1' OF THE C-RESIDUE IN AGC.ICT BY THE RADICAL CENTER AT C-2 OF ACTIVATED NEOCARZINOSTATIN CHROMOPHORE - STRUCTURE OF THE DRUG DNA COMPLEX RESPONSIBLE FOR BISTRANDED LESION FORMATION

Glutathione-activated neocarzinostatin chromophore (NCS-Chrom) generates bistranded lesions at AGC.GCT sequences in DNA, consisting of an abasic site at the C residue and a strand break at the T residue on the complementary strand, due to hydrogen atom abstraction from C-1' and C-5', respectively. Earlier work showed that H-2 from C-5' of T was selectively abstracted by the radical center at C-6 of activated NCS-Chrom, supporting a proposed model of the active-drug/DNA complex. However, since under the conditions used breaks at the T exceeded their inclusion in bistranded lesions, it was not clear what fraction of the hydrogen transfer represented bistranded lesions. Since virtually all abasic sites at the C are part of a bistranded lesions, hydrogen transfer from C-1' of C into the drug should reflect only the bistranded reaction. Accordingly, a self-complementary oligodeoxynucleotide 5'-GCAGCICTGC-3' was synthesized in which the C contained H-2 at the C-1' position. In order to eliminate an H-2 isotope effect on the transfer and to increase the extent of the bistranded reaction, an I residue was substituted for the G opposite the C residue. Sequencing gel electrophoretic analysis revealed that under one-hit kinetics, 37% of the damage reaction was associated with abasic site (alkali-labile break) formation at the C residue and 48% with direct strand breaks at the T residue. Thus, 74% of the damage involved a bistranded lesion. H-1 NMR spectroscopic analysis of the reacted chromophore showed that H-2 had been selectively transferred into the C-2 position to the extent of approximately 22%. Among other possible sources of hydrogen transfer, accounting for the low H-2 incorporation from DNA, are the hydrogen of the solvent (methanol/water), and possibly other components, present in the reaction mixture (intermolecular) and the carbon alpha to the sulfur of the adducted glutathione (intramolecular). The finding that solvent deuterium selectively quenches the radical at C-2 of NCS-Chrom in the presence of DNA helps to explain the excess of single-stranded over double-stranded lesions. The results in this report provide experimental support for an earlier proposed model [Galat, A., & Goldberg, I. H. (1990) Nucleic Acids Res. 18, 2093-2099] for the activated NCS-Chrom/DNA complex that generates bistranded lesions at AGC.GCT.