Determination of the Major Tautomeric Form of the Covalently Modified Adenine in the (+)-CC-1065-DNA Adduct by ^lH and ¹⁵N NMR Studies

(+)-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 (+)-CC-1065 and DNA have been determined, the tautomeric form of the covalently modified adenine in the (+)-CC-I065-DNA duplex adduct was not defined. A [6-¹⁵N]deoxyadenosine-labeled 12 base pair non-self-complementary oligomer, d(GGCGGAGTT*AGG)·d(CCTAACTCCGCC) (asterisk indicates ¹⁵N-labeled base), containing the (+)-CC-1065 most preferred binding sequence 5′AGTTA, was synthesized and modified with (+)-CC-1065. This [6-¹⁵N]deoxyadenosine-labeled 12-mer duplex adduct was then studied by ¹H and ¹⁵N NMR. Onedimensional NOE difference and two-dimensional NOESY ¹H 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 [6-¹⁵N]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 ¹⁵N nucleus with coupling constants of 91.3 Hz for non-hydrogen-bonded and 86.8 Hz for hydrogen-bonded amino protons. Parallel ¹⁵N NMR experiments on the [6-¹⁵N]deoxyadenosine-labeled (+)-CC-1065-12-mer duplex adduct show a triplet-like signal around -−76.9 ppm and coupling constants of 91.5 and 85.6 Hz. The large downfield shift (24.05 ppm) of the ¹⁵N signal of the (+)-CC-l 065-12-mer duplex adduct is in accord with the formation of an extra positive charge on the exocyclic nitrogen at the N6 position and a partial double bond character between N6 and C6 atoms of the covalently modified adenine upon drug modification. We conclude that the covalently modified adenine N6 of the (+)-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.