Mitomycin C (MC), a potent antitumor drug, and decarbamoylmitomycin C (DMC), a derivative lacking the carbamoyl group, form highly cytotoxic DNA interstrand crosslinks. The major interstrand crosslink formed by DMC is the C1'' epimer of the major crosslink formed by MC. The molecular basis for the stereochemical configuration exhibited by DMC was investigated using biomimetic synthesis. The formation of DNA-DNA crosslinks by DMC is diastereospecific and diastereodivergent: Only the 1''S-diastereomer of the initially formed monoadduct can form crosslinks at GpC sequences, and only the 1''R-diastereomer of the monoadduct can form crosslinks at CpG sequences. We also show that CpG and GpC sequences react with divergent diastereoselectivity in the first alkylation step: 1"S stereochemistry is favored at GpC sequences and 1''R stereochemistry is favored at CpG sequences. Therefore, the first alkylation step results, at each sequence, in the selective formation of the diastereomer able to generate an interstrand DNA-DNA crosslink after the "second arm" alkylation. Examination of the known DNA adduct pattern obtained after treatment of cancer cell cultures with DMC indicates that the GpC sequence is the major target for the formation of DNA-DNA crosslinks in vivo by this drug.

Mitomycin C (MC), an antitumor drug, and decarbamoylmitomycin C (DMC), a derivative of MC, alkylate DNA and form deoxyguanosine monoadducts and interstrand crosslinks (ICLs). Interestingly, in mammalian culture cells, MC forms primarily deoxyguanosine adducts with a 1"-R stereochemistry at the guanine-mitosene bond (1"-α) whereas DMC forms mainly adducts with a 1"-S stereochemistry (1"-β). The molecular basis for the stereochemical configuration exhibited by DMC has been investigated using biomimetic synthesis. Here, we present the results of our studies on the monoalkylation of DNA by DMC. We show that the formation of 1"-β-deoxyguanosine adducts requires bifunctional reductive activation of DMC, and that monofunctional activation only produces 1"-α-adducts. The stereochemistry of the deoxyguanosine adducts formed is also dependent on the regioselectivity of DNA alkylation and on the overall DNA CG content. Additionally, we found that temperature plays a determinant role in the regioselectivity of duplex DNA alkylation by mitomycins: At 0 °C, both deoxyadenosine (dA) and deoxyguanosine (dG) alkylation occur whereas at 37 °C, mitomycins alkylate dG preferentially. The new reaction protocols developed in our laboratory to investigate DMC-DNA alkylation raise the possibility that oligonucleotides containing DMC 1"-β-deoxyguanosine adducts at a specific site may be synthesized by a biomimetic approach.

Mitomycin C (MC) is an anticancer agent that alkylates DNA to form monoadducts and interstrand cross-links. Decarbamoylmitomycin C (DMC) is an analogue of MC lacking the carbamate on C10. The major DNA adducts isolated from treatment of culture cells with MC and DMC are N-deoxyguanosine (dG) adducts and adopt an opposite stereochemical configuration at the dG-mitosene bond. To elucidate the molecular mechanisms of DMC-DNA alkylation, we have reacted short oligonucleotides, calf thymus, and M. luteus DNA with DMC using biomimetic conditions. These experiments revealed that DMC is able to form two stereoisomeric deoxyadenosine (dA) adducts with DNA under bifuntional reduction conditions and at low temperature. The dA-DMC adducts formed were detected and quantified by HPLC analysis after enzymatic digestion of the alkylated DNA substrates. Results revealed the following rules for DMC dA alkylation: (i) DMC dA adducts are formed at a 48- to 4-fold lower frequency than dG adducts, (ii) the 5'-phosphodiester linkage of the dA adducts is resistant to snake venom diesterase, (iii) end-chain dA residues are more reactive than internal ones in duplex DNA, and (iv) nucleophilic addition by dA occurs on both faces of DMC and the ratio of stereoisomeric dA adducts formed is dependent on the end bases located at the 3' or 5' position. A key finding was to discover that temperature plays a determinant role in the regioselectivity of duplex DNA alkylation by DMC: at 0 °C, both dA and dG alkylation occur, whereas at 37 °C, DMC preferentially alkylates dG residues.

Mitomycin C(MC) an antitumord rug and decarba-moylmitomycinC (DMC), ad erivative of MC lacking the carbamoyl moiety,a re DNA alkylating agents whichc an form DNA interstrand crosslinks (ICLs)b etween deoxyguanosine residues located on opposing DNA strands. MC formsp rimarily deoxyguanosine adducts with a1 "-R stereochemistry at the guanine-mitosene bond (1"-a, trans)w hereas DMC forms mainly adducts with a1 "-S stereochemistry (1"-b, cis). The crosslinking reaction is diastereospecific: trans-crosslinks are formed exclusively at CpG sequences, while cis-crosslinks are formed only at GpC sequences. Until now,o ligonucleotides containing 1"-b-deoxyguanosinea dducts or ICL at a specific site could not be synthesized, thus limiting the investigation of the role played by the stereochemical configuration at C1'' in the toxicity of thesec ompounds.H ere, a novel biomimetic synthesist oa ccess these substrates is presented. Structural proof of the adducted oligonucleotides and ICL were provided by enzymatic digestion to nucleosides, high resolution mass spectrala nalysis, CD spectroscopy and UV melting temperature studies. Finally,avirtual model of the 25-mer 1"-b ICL synthesized was createdt oe xplore the conformational space and structuralf eatures of the crosslinked duplex.

Mitomycins are important anti‐cancer drugs. Treatment of cancer cells with mitomycins generates stereoisomeric (trans and cis) DNA interstrand crosslinks (ICLs), which prevent these highly proliferating cells from replicating. trans‐Crosslinks are formed exclusively at CpG sequences, while cis‐crosslinks are formed only at GpC sequences. This work describes a synthetic methodology to generate oligonucleotides containing the cis‐ICL (green ICL in the picture). Structural proof of the crosslinked oligonucleotide was obtained, and a virtual model of the duplex was created. More information can be found in the Full Paper by E. Champeil et al. on page 12570.