ORGN 728 |
| The guanine (G)-N2 DNA monoadduct of mitomycin C (MC), a cytotoxic anticancer drug, inhibits translesion bypass by DNA polymerases as shown previously. The non-cytotoxic MC metabolite 2,7-diaminomitosene (2,7-DAM) forms a G-N7 DNA monoadduct in vitro and in vivo. We synthesized 24, 27 and 36mer DNA templates, adducted at a single guanine either with MC or 2,7-DAM using a combination of bioorganic chemistry and molecular biology approaches in order to perform in vitro translesion synthesis using T7 exo-, Klenow exo- and eta DNA polymerases. Synthetic oligonucleotides (9 and 12 mers) were incubated as DNA duplexes with MC or 2,7-DAM under reductive conditions using Na2S2O4 or catalytic hydrogenation (H2(g)+ PtO2) in aqueous buffer (pH 5.0) to obtain (G) N2 or (G) N7 monoalkylated products which were purified by reversed phase high pressure liquid chromatography (HPLC). The short adducted oligonucleotides were ligated to 5'-phosphorylated oligonucleotides by T4 DNA ligase in the presence of a complementary oligomer in order to obtain 24, 27 and 36 mer adducted templates. The constructed alkylated templates were purified from the other oligonucleotides in the ligation mix using a 16% polyacrylamide-8M urea gel and were analyzed for the presence of the MC or 2,7-DAM adduct by digestion with snake venom phosphodiesterase and alkaline phosphatase followed by nucleosides analysis of the digests by HPLC. The HPLC together with UV-VIS spectroscopy and negative electrospray mass spectroscopy ((-) ESIMS) analysis confirmed the structural identity and the purity of the (G) N2 or (G) N7 monoalkylated templates. |
|
Combinatorial, Parallel and Process Chemistry, Heterocycles, Aromatics, New Reactions and Methodology
8:00 PM-10:00 PM, Wednesday, August 22, 2007 BCEC -- Exhibit Hall - B2, Poster
Division of Organic Chemistry |