A MITOMYCIN-N-6-DEOXYADENOSINE ADDUCT ISOLATED FROM DNA

A minor N-6-deoxyadenosine adduct of mitomycin C (MC) was isolated fro m synthetic oligonucleotides and calf thymus DNA, representing the fir st adduct of MC and a DNA base other than guanine. The structure of th e adduct (8) was elucidated using submilligram quantities of total ava ilable material. UV difference spectroscopy, circular dichroism, and e lectrospray mass spectroscopy as well as chemical transformations were utilized in deriving the structure of 8. A series of synthetic oligon ucleotides was designed to probe the specificities of the alkylation o f adenine by MC. The nature and frequency of the oligonucleotide-MC ad ducts formed under conditions of reductive activation of MC were deter mined by their enzymatic digestion to the nucleoside level followed by quantitative analysis of the products by HPLC. The analyses indicated the following: (i) (A)(n) sequence is favored over (AT)(n) for adduct formation; (ii) the alkylation favors the duplex structure; (iii) at adenine sites only monofunctional alkylation occurs; (iv) the adenine- to-alkylation frequency in the model oligonucleotides was 0.3-0.6 rela tive to guanine alkylation at the 5'-ApG sequence but only 0.02-0.1 re lative to guanine alkylation at 5'-CpG. The 5'-phosphodiester linkage of the MC-adenine adduct is resistant to snake venom diesterase. The o verall ratio of adenine to guanine alkylation in calf thymus DNA was 0 .03, indicating that 8 is a minor MC-DNA adduct relative to MC-DNA add ucts at guanine residues in the present experimental residues in the p resent experimental system. However, the HPLC elution time of 8 coinci des with that of a major, unknown MC adduct detected previously in mou se mammary tumor cells treated with radiolabeled MC [Bizanek, R., Chow dary, D., Arai, H., Kasai, M., Hughes, C. S., Sartorelli, A. C., Rockw ell, S., and Tomasz, M. (1993) Cancer Res. 53, 5127-5134]. Thus, 8 may be identical or closely related to this major adduct formed in vivo. This possibility can now be tested by further comparison.