Kinetic analysis of the deamination reactions of cyclobutane dimers of thymidylyl-3',5'-2'-deoxycytidine and 2'-deoxycytidylyl-3',5'-thymidine

Abstract

The cyclobutane dimer photoproducts of dTpdC and dCpdT have been produced by acetophenone photosensitization and separated by reverse-phase HPLC. Each dinucleoside monophosphate was shown to produce one cis,syn isomer and two trans,syn isomers. Three of these photoproducts, namely, the cis,syn isomers of dTpdC and dCpdT and one trans,syn isomer (the syn-anti glycosidic isomer) of dTpdC were selected to study the deamination kinetics. Analysis of the pH dependence indicates that the deamination proceeds by the hydrolysis of the imido amide group of the 5,6-saturated cytosine base with the formation of a carbinolamine intermediate. Determination of the kinetic parameters showed that, for these three cyclobutane dimers, the rate-determining step at physiological pH is a cyclobutane dimers, the rate-determining step at physiological pH is a nucleophilic attack of hydroxide ion on the protonated 5,6-saturated cytosine base. The kinetic analysis showed that the cis,syn isomers deaminate approximately 3 times faster than the trans,syn isomer, which is due to a large difference in pKa of the 5,6-saturated cytosine moiety. An electrostatic interaction between the iminium group of cytosine and the carbonyl group of thymine is proposed to account for the increase in pKa for the cis,syn isomers relative to the trans,syn isomer. A similar interaction is proposed to explain the relative difference in reactivity between the cis,syn isomers and the trans,syn isomer with regard to the breakdown of the carbinolamine intermediate.