RESUMO
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 ß-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.
Assuntos
Carboidratos/química , Desoxicitidina/análogos & derivados , Desoxicitidina/química , Espectroscopia de Ressonância de Spin Eletrônica , Elétrons , Radicais Livres/química , Oxirredução , GencitabinaRESUMO
In this work, iminyl σ-radical formation in several one-electron-oxidized cytosine analogs, including 1-MeC, cidofovir, 2'-deoxycytidine (dCyd), and 2'-deoxycytidine 5'-monophosphate (5'-dCMP), were investigated in homogeneous, aqueous (D2O or H2O) glassy solutions at low temperatures by employing electron spin resonance (ESR) spectroscopy. Upon employing density functional theory (DFT) (DFT/B3LYP/6-31G* method), the calculated hyperfine coupling constant (HFCC) values of iminyl σ-radical agree quite well with the experimentally observed ones, thus confirming its assignment. ESR and DFT studies show that the cytosine iminyl σ-radical is a tautomer of the deprotonated cytosine π-cation radical [cytosine π-aminyl radical, C(N4-H)(â¢)]. Employing 1-MeC samples at various pHs ranging from ca. 8 to 11, ESR studies show that the tautomeric equilibrium between C(N4-H)(â¢) and the iminyl σ-radical at low temperature is too slow to be established without added base. ESR and DFT studies agree that, in the iminyl σ-radical, the unpaired spin is localized on the exocyclic nitrogen (N4) in an in-plane pure p-orbital. This gives rise to an anisotropic nitrogen hyperfine coupling (Azz = 40 G) from N4 and a near isotropic ß-nitrogen coupling of 9.7 G from the cytosine ring nitrogen at N3. Iminyl σ-radical should exist in its N3-protonated form, as the N3-protonated iminyl σ-radical is stabilized in solution by over 30 kcal/mol (ΔG = -32 kcal/mol) over its conjugate base, the N3-deprotonated form. This is the first observation of an isotropic ß-hyperfine ring nitrogen coupling in an N-centered DNA radical. Our theoretical calculations predict that the cytosine iminyl σ-radical can be formed in double-stranded DNA by a radiation-induced ionization-deprotonation process that is only 10 kcal/mol above the lowest energy path.