NEOCARZINOSTATIN-INDUCED HYDROGEN-ATOM ABSTRACTION FROM C-4' AND C-5' OF THE T-RESIDUE AT A D(GT) STEP IN OLIGONUCLEOTIDES - SHUTTLING BETWEEN DEOXYRIBOSE ATTACK SITES BASED ON ISOTOPE SELECTION EFFECTS

The thiol-activated neocarzinostatin chromophore cleaves duplex oligonucleotides containing the sequence -TGTTTGA-, producing 3'-phosphoglycolate and 3'-phosphate fragments at T, indicating the involvement of 4'- as well as 5'-chemistry at this residue. Substitution of deuterium for hydrogen at the C-4' position of the affected T leads to a kinetic isotope effect (k(H)/k(D)) of 4.0 on the formation of the glycolate-ended product, whereas deuterium at C-5' of the same T reveals k(H)/k(D) of 1.6 in the formation of the phosphate-ended product. The proportion of the products representing 4'- and 5'-chemistry can be shifted on the basis of isotope selection effects. A second product resulting for 4'-chemistry, the abasic site associated with 4'-hydroxylation, has been identified as an alkali-labile site, and as a pyridazine derivative formed after cleavage by hydrazine. A comparable isotope effect on its production (k(H)/k(D) = 3.7) relative to that of 3'-phosphoglycolate production is consistent with a common intermediate, a putative 4'-peroxy radical, in their formation. The formation of both products of 4'-chemistry is oxygen-dependent, and the internal partitioning between them (3'-phosphate or 3'-phosphoglycolate) is influenced by thiols. Moreover, the nitroaromatic radiation sensitizer misonidazole can substitute for dioxygen, yielding 3'-phosphoglycolate and alkali-labile 3'-phosphate ends, indicative of 4'-chemistry. In addition to the internal partitioning of 4'-chemistry, thiols also affect the overall extent of cleavage (4' and 5') and the relative partitioning between both sites of attack (4' or 5').