Chemical modification of DNA with muta-carcinogens. III. Reductive alkylation of DNA with mitomycin C.

Mitomycin C (MMC) binds to DNA after its reductive activation by catalytic hydrogenation with Pd on charcoal. Three modified nucleotides, named MG-1, MG-2, and MA, were isolated from the modified DNA after enzymatic hydrolysis to 5'-nucleotides. The structures of these modified nucleotides were deduced from their 1H-NMR and UV spectra, and from studies of the chemically transformed derivatives (hydrolysis, methylation, diazotization, and thioketonization). These three modified nucleotides were concluded to be 1,2-trans-2,7-diamino-1-(N2-deoxyguanylyl)mitosene (MG-1), 2,7-diamino-1-(O6-deoxyguanylyl)mitosene (MG-2) and 2,7-diamino-1-(N6-deoxyadenylyl)mitosene (MA). The same modified nucleotides were identified in DNA extracted from the livers of rats treated with MMC.


Introduction
Mitomycin C (MMC) is a potent antibiotic and is also used clinically as an antitumor agent. Recently (1), a revised structure of MMC, the antipode of the structure reported previously, was proposed (I). It is well estab- lished that MMC requires reductive activation to become an alkylating agent of DNA (2). The action of MMC as a bioreductive alkylating agent of DNA is believed to be, at least in part, responsible for its effectiveness as an antitumor agent. Bifunctional alkylation of DNA leading to crosslinking of the two strands was considered to be the direct cause of the cytotoxicity of the drug (3), although the monofunctional attachment, which occurs 10 to 20 times more frequently than the crosslinking, has also been implicated as biologically significant damage to DNA (2). Elucidation of molecular aspects of this alkylation reaction of DNA by MMC *Faculty of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan should provide basic information for the development of more effective antitumor agents. Recently, Moore suggested that positions 1 and 10 of MMC are binding sites of MMC with DNA (4), though no unambiguous evidence for the chemical structure of the modified DNA has yet been obtained. Very recently, we reported (5) the alkylation of 5'-guanylic acid by reductively activated MMC and showed the structure of MMC-bound 5'-guanylic acid to be 1,2-cis-2,7-diamino-1-(5'-guanylyl)mitosene (Fig. 1). The other nucleic acid derivatives modified with MMC are uridyl derivatives ( Fig. 1) reported by Tomasz and Lipman (6), but the products were obtained by reaction of MMC and uridylic acid derivatives under acidic conditions and without reductive activation of MMC.
In this article, we review the isolation and structural identification of monofunctional alkylation products of DNA obtained with reductive activated MMC (7,8). DNA was modified chemically by catalytic reduction, as well as in vivo in male Wistar rats (8).°C Structure of the Nucleotides Modified with MMC MMC binds to DNA only after reductive activation under neutral conditions. We used catalytic hydrogenation with 5% Pd on charcoal and H2 gas for reductive activation of MMC. About one molecule of MMC was bound per 200-300 nucleotides in DNA, as estimated from the UV spectrum of the modified DNA. Analysis of an enzymatic hydrolysate of the modified DNA showed the presence of three modified nucleotides, MG-1, MG-2, and MA. More than 80% of the binding of MMC to DNA could be accounted for by the formation of MG-1, MG-2, and MA.
The UV spectra of MG-1, MG-2, and MA have absorption maxima at ca. 310 nm, suggesting that these modified nucleotides contain a mitosene moiety, because the mitosene chromophore has an absorption maximum at 310 nm (9).
When these modified nucleotides were treated with alkaline phosphatase, less polar products (probably the corresponding nucleosides) were obtained quantitatively. This finding and the observation that these nucleotides were all resistant to phosphodiesterase suggest that these compounds have a monophosphate group at the 5'-position of 2'-deoxyribose.
1H-NMR spectra were obtained in DMSO-d6-CF3COOD. The use of completely anhydrous solvent and exchange of hydrogen of NH and OH made it possible to assign all the signals. Assignment of proton signals was performed by homonuclear decoupling experiments and by comparing the spectra obtained with those of model compounds such as mitosenes and nucleotides. The results are shown in Figure 2.
The 1H-NMR spectra and the results of hydrolysis of MG-1, MG-2, and MA suggest that the binding sites of nucleic acid base moieties are at heteroatoms of the purine rings. These modified nucleotides were hydrolyzed with 1 N HCI after methylation with trimethylsulfoxonium iodide in DMSO. Both MG-1 and MG-2 gave guanine (4-10%), 3-methylguanine (1%), 1-methylguanine (2-3%), and 7-methylguanine (4-5%). MG-1 also gave xanthine (4%). MA gave adenine (trace), 1-methyladenine (14%), 3-methyladenine (11%), and 7-methyladenine (0.5%). In all cases, some other products were observed, but not 06-methylguanine from MG-2 and not N6-methylor N6,N6-dimethyladenine from MA. These results suggest that the methylated positions (the 1-, 3-, and 7-positions) and the glycosylated position (the 9-position) of the purine rings are not the binding sites. Therefore, the binding site of MA is at the N6 atom of adenine, and those of MG-1 and MG-2 are at the O6 or MG-1 5  N2 atom of guanine. For unambiguous determination of the binding sites of the guanine moieties of MG-1 and MG-2, the guanine moieties were chemically transformed. Acid hydrolysis of MG-1 and MG-2 after treatment with P2S5 gave 6-thioguanine from MG-1 (30%), but this was not obtained from MG-2. This suggests that the 06 atom of the guanine moiety is blocked in MG-2 but not in MG-1. In addition, MG-1 and MG-2 were hydrolyzed with HCI after treatment with NaNO2. The free amino group of guanine should be diazotized easily. Guanine (10%) together with xanthine (20%) was obtained from MG-1, but only xanthine (30%), not guanine, from MG-2. From these results, the binding sites of MG-1 and MG-2 were determined to be at the N2 and 06 atom, respectively. Therefore, the structures of MG-1, MG-2, and MA are as shown in Figure 2 (7,8). The structure of MG-2 was confirmed by Tomasz et al (9). They isolated MG-2 from the hydrolysate of the dinucleotide GpC modified with reductively activated MMC, and determined the configuration at positions 1 and 2 to be trans.

Modification of DNA in Vivo
DNA extracted from the liver of a rat treated with MMC was modified with MMC. About one molecule of MMC was bound per 1-2 x 104 nucleotides, as estimated from the UV spectrum of the modified DNA (ratio of absorbance at 260 nm to that at 310 nm). Enzymatic hydrolysis gave MG-1, MG-2, and MA in a ratio of 1:5:2 ( Fig. 3) (8). The yields of these three modified nucleotides accounts for most of the MMC bound to DNA; the amounts of other nucleotides modified with MMC, if any, were very small. MMC bound preferentially to guanine in DNA, especially at the 06 atom.

Reaction Mechanism
The mechanism of formation of these modified nucleotides was not established unequivocally, but in view of previous results, we propose that the mechanism of the reaction is as shown in Figure 4. MMC is reduced to the corresponding hydroquinone (MMC-H2). This reduction enhances the electron-donating ability and aids elimination of the 9a-methoxy group. In the demethoxy product (demethoxy-MMC-H2), cleavage ofthe aziridine ring is facilitated: position 1 is now benzylic and is activated by many electron-donating groups.   I aziridine ring has a planar structure which can intercalate into DNA and can thus approach position 2 or 6 of the purine bases. Covalent binding then occurs between position 1 of mitosene and heteroatom at position 2 or 6 of the purine bases. The products might be oxidized by excess MMC or MMC derivatives (or by other cellular components in vivo and in vitro) or during the work-up procedures to give MG-1, MG-2, and MA.

Conclusion
The structures of the nucleotides modified with reductively activated MMC were determined to be as shown in Figure 2. The reaction established chemically occurs both in vitro and in vivo. The present findings represent a first step in understanding the molecular basis of the action of MMC as a bioreductive alkylating agent. The reductive activation and the mode of modification of nucleotides in DNA must, at least in part, play a role in the carcinostatic or carcinogenic mechanisms of MMC. These findings may contribute to the development of more effective antitumor-active MMC derivatives.