A Mitomycin−N⁶-Deoxyadenosine Adduct Isolated from DNA

Abstract

A minor N6-deoxyadenosine adduct of mitomycin C (MC) was isolated from synthetic oligonucleotides and calf thymus DNA, representing the first adduct of MC and a DNA base other than guanine. The structure of the adduct (8) was elucidated using submilligram quantities of total available material. UV difference spectroscopy, circular dichroism, and electrospray mass spectroscopy as well as chemical transformations were utilized in deriving the structure of 8. A series of synthetic oligonucleotides was designed to probe the specificities of the alkylation of adenine by MC. The nature and frequency of the oligonucleotide-MC adducts formed under conditions of reductive activation of MC were determined 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 relative to guanine alkylation at the 5'-ApG sequence but only 0.02-0.1 relative to guanine alkylation at 5'-CpG. The 5'-phosphodiester linkage of the MC-adenine adduct is resistant to snake venom diesterase. The overall 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 adducts at guanine residues in the present experimental residues in the present experimental system. However, the HPLC elution time of 8 coincides with that of a major, unknown MC adduct detected previously in mouse mammary tumor cells treated with radiolabeled MC [Bizanek, R., Chowdary, D., Arai, H., Kasai, M., Hughes, C. S., Sartorelli, A. C., Rockwell, 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.

Mitomycinoid Alkaloids: Mechanism of Action, Biosynthesis, Total Syntheses, and Synthetic Approaches

“…[121] At the carbamate carbon (C10) 2,7-DAM ( 36 ) monoalkylates DNA in the major groove at guanine N7 of (G) n tracts. [122,123] The selectivity correlates with the sequence specificity of the negative molecular electrostatic potential of the major groove, suggesting that the alkylation selectivity of 2,7-DAM is determined by sequence-specific variation of the reactivity of the DNA.…”

Synthesis of Oligonucleotides Containing Trans Mitomycin C DNA Adducts at N6 of Adenine and N2 of Guanine

“…In the case of the dG monoadduct (10), the mitosene is positioned either in the minor groove (cluster 1 and 3, Figure S14) or more centrally between the two strands (cluster 2 and 4, Figure S15). The only disrupted standard base pairing is between the adducted guanine and the opposite cytosine, with the cytosine being displaced and stabilized via H-bonding with the phosphate's oxygen.…”

“…Although MC is inert in its native form, it is reduced enzymatically in vivo to reactive mitosenes. [1][2][3][4] These species undergo nucleophilic attack by the guanine [5][6][7] or adenine [8][9][10] bases in DNA to form monoadducts as well as interstrand crosslinks (ICLs) (Figure 1: Adducts 1 a, 1 b, 2 a, 2 b are generated at N 2 of dG; ICLs 3 a and 3 b are formed between opposing dG; adducts 4 a and 4 b are found at N 6 of dA; adducts 5 and 6 result from the alkylation of DNA at dG by 2,7-diaminomitose (2,7-DAM), the major metabolite of MC). ICLs are considered the most cytotoxic lesions because, if not repaired, they prevent cell replication and may lead to mitotic catastrophe.…”

Isolation and Rationale for the Formation of Isomeric Decarbamoylmitomycin C-N6-deoxyadenosine Adducts in DNA

“…Both spectra are similar to the spectrum of a previously isolated and characterized mitomycin C-N 6 -deoxyadenosine adduct. 23 This prompted us to investigate if these new adducts were stereoisomeric N 6 -deoxyadenosine adducts of DMC.…”