Synthesis of Oligonucleotides Containing the Ethylene Dibromide-Derived DNA Adducts S-[2-(N7-Guanyl)ethyl]glutathione, S-[2-(N2-Guanyl)ethyl]glutathione, and S-[2-(O6-Guanyl)ethyl]glutathione at a Single Site

The carcinogen ethylene dibromide (EDB) is activated by enzymatic conjugation with GSH to form S-(2-bromoethyl)GSH, which reacts with DNA via an episulfonium ion. The major DNA adduct derived from EDB was previously characterized as S-[2-(N7-guanyl)ethyl]GSH, and S-[2-(N2-guanyl)ethyl]GSH and S-[2-(O6-guanyl)ethyl]GSH are minor adducts [Cmarik, J. L., Humphreys, W. G., Bruner, K. L., Lloyd, R. S., Tibbetts, C., and Guengerich, F. P. (1992) J. Biol. Chem. 267, 6672-6679]. S-[2-(N7-Guanyl)ethyl]GSH has been incorporated at the G* site in d(5'-TGCTG*CAAG-3'), a site previously found to show GC to AT transitions following treatment of M13 phage with S-(2-chloroethyl)GSH, and the desired product was separated by HPLC. This was ligated to d(5'-GGTACCGAG-3') to yield d(5'-TGCTG*CAAGGGTACCGAG-3'). S-[2-(N2-Guanyl)ethyl]GSH was incorporated into the G* site of the oligonucleotide in d(5'-TGCTG*CAAGGGTACCGAG-3') by reacting S-(2-aminoethyl)GSH with an oligomer containing 2-fluoro-O6-[(trimethylsilyl)ethoxy]deoxyinosine at the target site. The 5'-(dimethoxytrityl)-N2-(phenoxyacetyl)-N-[(fluorenylmethyl)formyl ] derivative of S-[2-(O6-deoxyguanosyl)-ethyl]GSH dimethyl ester was synthesized by Mitsunobu alkylation of 5'-(dimethoxytrityl)-N2-(phenoxyacetyl)deoxyguanosine with N-[(fluorenylmethyl)formyl]-S-(2-hydroxyethyl)GSH dimethyl ester, modified to form the phosphoramidite derivative, and incorporated at the G* site of d(5'-TGCTG*CAAGGGTACCGAG-3'). The protective groups were removed with 0.10 N NaOH to give the modified oligonucleotide containing S-[2-(O6-guanyl)ethyl]GSH. Although the overall yields were low, the synthesis of a single set of target site oligonucleotides containing these overall yields were low, the synthesis of a single set of target site oligonucleotides containing these three known guanyl adducts allows for in vitro site-specific misincorporation studies.