Solution structure of a guanine-N7-linked complex of the mitomycin C metabolite 2,7-diaminomitosene and DNA. Basis of sequence selectivity

2,7-Diaminomitosene (2,7-DAM), the major metabolite of the antitumor antibi otic mitomycin C, forms DNA adducts in tumor cells. 2,7-DAM was reacted wit h the deoxyoligonucleotide d(GTGGTATACCAC) under reductive alkylation condi tions. The resulting DNA adduct was characterized as d(G-T-G-[M]G-T-A-T-A-C -C-A-C) (5), where [M]G stands for a covalently modified guanine, linked at its N7-position to C10 of the mitosene. The adducted oligonucleotide compl ements with itself, retaining 2-fold symmetry in the 2:1 drug-duplex comple x, and provides well-resolved NMR spectra, amenable for structure determina tion. Adduction at the N7-position of G4 ([M]G. 4) is characterized by a do wnfield shift of the G4(H8) proton and separate resonances for G4(NH2) Prot ons. We assigned the exchangeable and nonexchangeable proton resonances of the mitosene and the deoxyoligonucleotide in adduct duplex 5 and identified intermolecular proton-proton NOEs necessary for structural characterizatio n. Molecular dynamics computations guided by 126 intramolecular and 48 inte rmolecular distance restraints were performed to define the solution struct ure of the 2,7-DAM-DNA complex 5. A total of 12 structures were computed wh ich exhibited pairwise rmsd values in the 0.54-1.42 A range. The 2,7-DAM mo lecule is anchored in the major groove of DNA by its C10 covalently linked to G4(N7) and is oriented 3' to the adducted guanine. The presence of 2,7-D AM in the major groove does not alter the overall B-DNA helical structure. Alignment in the major groove is a novel feature of the complexation of 2,7 -DAM with DNA; other known major groove alkylators such as aflatoxin, posse ssing aromatic structural elements, form intercalated complexes. Thermal st ability properties of the 2,7-DAM-DNA complex 5 were characteristic of noni ntercalating guanine-N7 alkylating agents. Marked sequence selectivity of t he alkylation by 2,7-DAM was observed, using a series of oligonucleotides i ncorporating variations of the 5'-TGGN sequence as substrates. The selectiv ity correlated with the sequence specificity of the negative molecular elec trostatic potential of the major groove, suggesting that the alkylation sel ectivity of 2,7-DAM is determined by sequence-specific variation of the rea ctivity of the DNA. The unusual, major groove-aligned structure of the addu ct 5 may account for the low cytotoxicity of 2,7-DAM.