Determination of the major tautomeric form of the covalently modified adenine in the (+)-CC-1065-DNA adduct by sup 1 H and sup 15 N NMR studies

({plus})-CC-1065 is an extremely potent antitumor antibiotic produced by Streptomyces zelensis. The potent cytotoxic effects of the drug are thought to be due to the formation of a covalent adduct with DNA through N3 of adenine. Although the covalent linkage sites between ({plus})-CC-1065 and DNA have been determined, the tautomeric form of the covalently modified adenine in the ({plus})-CC-1065-DNA duplex adduct was not defined. The (6-{sup 15}N)deoxyadenosine-labeled 12-mer duplex adduct was then studied by {sup 1}H and {sup 15}N NMR. One-dimensional NOE difference and two-dimensional NOESY {sup 1}H NMR experiments on the nonisotopically labeled 12-mer duplex adduct demonstrate that the 6-amino protons of the covalently modified adenine exhibit two signals at 9.19 and 9.08 ppm. Proton NMR experiments on the (6-{sup 15}N)deoxyadenosine-labeled 12-mer duplex adduct show that the two resonance signals for adenine H6 observed on the nonisotopically labeled duplex adduct were split into doublets by the {sup 15}N nucleus with coupling constants of 91.3 Hz for non-hydrogen-bonded and 86.8 Hz for hydrogen-bonded amino protons. The authors conclude that the covalently modified adenine N6 of the ({plus})-CC-1065-12-mer duplex adduct is predominantly in the doubly protonated form, in which calculations predict that the C6-N6 bond is shortened and the positive charge is delocalized over the entire adenine molecule.