ISOLATION AND STRUCTURE OF AN INTRASTRAND CROSS-LINK ADDUCT OF MITOMYCIN-C AND DNA

A new covalent mitomycin C-DNA adduct (4) was isolated from DNA expose d to reductively activated mitomycin C (MC) in vitro. The MC-treated D NA was hydrolyzed enzymatically under certain conditions, and the new adduct was isolated from the hydrolysate by HPLC. Its structure was de termined by ultraviolet and circular dichroism spectroscopy and chemic al and enzymatic transformations conducted on microscale. In the struc ture, a single 2"beta,7"-diaminomitosene residue is linked bifunctiona lly to two guanines in the dinucleoside phosphate d(GpG). The guanines are linked at their N2 atoms to the C1" and C10" positions of the mit osene, respectively. A key to the structure was a finding that removal of the mitosene from the adduct by hot piperidine yielded d(GpG); ano ther was that the adduct was slowly converted to the known interstrand cross-link adduct 3 by snake venom diesterase and alkaline phosphatas e. Adduct 4 represents an intrastrand cross-link in DNA formed by MC. Of the two possible strand-polarity isomers of 4, 4a in which the mito sene 1"-position is linked to the 3'-guanine of d(GpG) is designated a s the proper structure, on the basis of the mechanism of the cross-lin king reaction. The same adduct 4 was isolated from poly(dG).poly(dC), synthetic oligonucleotides containing the GpG sequence, and Micrococcu s luteus and calf thymus DNAs. The relative yields of interstrand and intrastrand cross-links (3 and 4) were determined under first-order ki netic conditions; an average 3.6-fold preference for the formation of 3 over that of 4 was observed. An explanation for this preference is p roposed. Energy-minimized structural models were generated for the two strand-polarity isomers of the intrastrand cross-link (4a and 4b), in corporated in a duplex decanucleotide. Both models indicate bending of DNA near the cross-link site. The identification and structural prope rties of the new, intrastrand cross-link lesion of DNA by MC are signi ficant with respect to cancer drug development as well as structural c hemistry of DNA-damaging agents.