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′• intermediate via direct H3′-atom abstraction followed by loss of HF to yield a C2′• with 3′-keto moiety. Direct detection of C3′• and C2′• 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 β-2′-F-atom hyperfine couplings identify the C3′• formation in one-electron oxidized gemcitabine, but no further reaction to C2′• is found. One-electron oxidized 2′-FdC is unreactive toward C3′• or C2′• formation. In one-electron oxidized MeFdC, ESR studies show C2′• production presumably from a very unstable C3′• precursor. The experimentally observed hyperfine couplings for C2′• and C3′• match well with the theoretically predicted ones. C3′• to C2′• conversion in one-electron oxidized gemcitabine and MeFdC has theoretically been modeled by first considering the C3′• 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′• to C2′• conversion in the presence of a proximate H3O+ has a barrier in agreement with the experimentally observed lack of C3′• to C2′• conversion. In contrast, in MeFdC, the loss of HF from C3′• in the presence of a proximate H3O+ is barrierless resulting in C2′• formation which agrees with the experimentally observed rapid C2′• formation.