One-Electron Oxidation of Gemcitabine and Analogs: Mechanism of Formation of C3′ and C2′ Sugar Radicals

Gemcitabine is a modified cytidine analog having two fluorine atoms at the 2'-position of the ribose ring. It has been proposed that gemcitabine inhibits RNR activity by producing a C3 center dot intermediate via direct H3'-atom abstraction followed by loss of HF to yield a C2' with 3'-keto moiety. Direct detection of C3 center dot and C2 center dot during RNR inactivation by gemcitabine still remains elusive. To test the influence of 2'- substitution on radical site formation, electron spin resonance (ESR) studies are carried out on one-electron oxidized gemcitabine and other 2'-modified analogs, i.e., 2'-deoxy-2'-fluoro-2'-C-methylcytidine (MeFdC) and 2'-fluoro-2'-deoxycytidine (2'-FdC). ESR line components from two anisotropic beta-2'-F-atom hyperfine couplings identify the C3 center dot formation in one-electron oxidized gemcitabine, but no further reaction to C2 center dot is found. One-electron oxidized 2 -FdC is unreactive toward C3 center dot or C2 center dot formation. In one-electron oxidized MeFdC, ESR studies show C2 center dot production presumably from a very unstable C3 center dot precursor. The experimentally observed hyperfine couplings for C2 center dot and C3 center dot match well with the theoretically predicted ones. C3 center dot to C2 center dot conversion in one-electron oxidized gemcitabine and MeFdC has theoretically been modeled by first considering the C3 center dot and H3O+ formation via H3'-proton deprotonation and the subsequent C2' formation via HF loss induced by this proximate H3O+. Theoretical calculations show that in gemcitabine, C3 center dot to C2 center dot conversion in the presence of a proximate H3O+ has a barrier in agreement with the experimentally observed lack of C3 center dot to C2 center dot conversion. In contrast, in MeFdC, the loss of HF from C3' in the presence of a proximate H3O+ is barrierless resulting in C2 center dot formation which agrees with the experimentally observed rapid C2 center dot formation.