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.