Synthesis and restriction enzyme analysis of oligodeoxyribonucleotides containing the anti-cancer drug 2', 2'-difluoro-2'-deoxycytidine

Abstract

The anti-cancer drug 2', 2'-difluoro-2'-deoxycytidine (dFdC) is internally incorporated into DNA in vitro. To determine the effects of this incorporation on DNA structure and function, the β-cyanoethyl phosphor-amidite of dFdC was synthesized and oligodeoxyribo-nucleotides containing dFdC were made using automated solid phase DNA synthesis techniques. Extension of the coupling time was required to achieve high coupling efficiency, suggesting a significant reduction in the rate of phosphotriester formation. Insertion of dFdC 5' into the recognition sequence of restriction enzymes Hpall and Kpnl reduced the rate of cutting by 4% and 14% over 60 minutes. This reduction is similar to the effects seen with arabinofuranosylcyti-dine (ara-C) but small compared to the reductions caused by base analogues and phosphothioates. Insertion of dFdC into the BamHI recognition sequence, but not 5' to the cut site, did not alter the rate of cutting/recognition. The presence of a single dFdC reduced the Tm's of oligomers by 2-4°C, depending on sequence and location. These results demonstrate that, once incorporated into DNA, dFdC does not greatly alter recognition between DNA and restriction enzymes; however, it does significantly alter duplex stability. © 1992 Oxford University Press.