The reactions of cytidine and 2′-deoxycytidine with SO4˙– revisited. Pulse radiolysis and product studies

Abstract

The reactions of SO4.- with 2'-deoxycytidine 1a and cytidine 1b lead to very different intermediates (base radicals with 1a, sugar radicals with 1b). The present study provides spectral and kinetic data for the various intermediates by pulse radiolysis as well as information on final product yields (free cytosine). Taking these and literature data into account allows us to substantiate but also modify in essential aspects the current mechanistic concept (H. Catterall, M. J. Davies and B. C. Gilbert, J. Chem. Soc., Perkin Trans. 2, 1992, 1379). SO4.- radicals have been generated radiolytically in the reaction of peroxodisulfate with the hydrated electron (and the H. atom). In the reaction of SO4.- with 1a (k = 1.6 x 10(9) dm3 mol-1 s-1), a transient (lambda max = 400 nm, shifted to 450 nm at pH 3) is observed. This absorption is due to two intermediates. The major component (lambda max approximately 385 nm) does not react with O2 and has been attributed to an N-centered radical 4a formed upon sulfate release and deprotonation at nitrogen. The minor component, rapidly wiped out by O2, must be due to C-centered OH-adduct radical(s) 6a and/or 7a suggested to be formed by a water-induced nucleophilic replacement. These radicals decay by second-order kinetics. Free cytosine is only formed in low yields (G = 0.14 x 10(-7) mol J-1 upon electron-beam irradiation). In contrast, 1b gives rise to an intermediate absorbing at lambda max = 530 nm (shifted to 600 nm in acid solution) which rapidly decays (k = 6 x 10(4) s-1). In the presence of O2, the decay is much faster (k approximately 1.3 x 10(9) dm3 mol-1 s-1) indicating that this species must be a C-centered radical. This has been attributed to the C(5)-yl radical 8 formed upon the reaction of the C(2')-OH group with the cytidine SO4(.-)-adduct radical 2b. This reaction competes very effectively with the corresponding reaction of water and the release of sulfate and a proton generating the N-centered radical. Upon the decay of 8, sugar radical 11 is formed with the release of cytosine. The latter is formed with a G value of 2.8 x 10(-7) mol J-1 (85% of primary SO4.-) at high dose rates (electron beam irradiation). At low dose rates (gamma-radiolysis) its yield is increased to 7 x 10(-7) mol J-1 due to a chain reaction involving peroxodisulfate and reducing free radicals. Phosphate buffer prevents the formation of the sugar radical at the SO4(.-)-adduct stage by enhancing the rate of sulfate release by deprotonation of 2b and also by speeding up the decay of the C(5)-yl radical into another (base) radical. Cytosine release in cytidine is mechanistically related to strand breakage in poly(C). Literature data on the effect of dioxygen on strand breakage yields in poly(C) induced by SO4.- (suppressed) and upon photoionisation (unaltered) lead us to conclude that in poly(C) and also in the present system free radical cations are not involved to a major extent. This conclusion modifies an essential aspect of the current mechanistic concept.

One-electron oxidation reactions of purine and pyrimidine bases in cellular DNA

“…In agreement with these thermodynamic characteristics, the radical cations of all four free natural nucleosides (dAdo, dGuo, dCyd and dThd) can be generated by one-electron oxidation with sulfate radical anions, SO 4 • − (Steenken 1989, von Sonntag 1991). The latter radicals are strong one-electron oxidants with the standard reduction potential of E ∘ = 2.43 V vs. NHE (Huie et al 1991), and rapidly oxidize free nucleosides with rate constants close to the diff usion-controlled limit (O’Neill and Davies 1987, Candeias and Steenken 1989, 1993, Deeble et al 1990, Aravindakumar et al 2003). The inorganic carbonate and dibromine radical anions, CO 3 •− and Br 2 • − , with the reduction potentials E ∘ (CO 3 •− /CO 3 2− ) = 1.59 V and E ∘ (Br 2 • − /2Br − ) = 1.62 V vs. NHE, are weaker oxidants than SO 4 • − radicals, and they only oxidize guanine out of all four DNA bases (Candeias and Steenken 1989, Shafirovich et al 2001).…”

“…Cytosine and 5-methylcytosine 2′-Deoxycytidine (dCyd) is the most resistant DNA nucleoside to one-electron oxidation that reacts rapidly only with very strong oxidants such as the SO 4 • − and • OH radicals (von Sonntag and Schuchmann 1986, O’Neill and Davies 1987, von Sonntag 1987a, b, 1991, Hildenbrandt et al 1989). The radiolytically generated SO 4 • − radicals produce a mixture of radical intermediates detected by ESR and pulse radiolysis (Hildenbrandt et al 1989, Catteral et al 1992, Aravindakumar et al 2003). The major intermediate, which shows an absorption band at 385 nm by transient absorption spectroscopy does not react with O 2 , has been identified as the N -centered radical dC( N 4 -H) • derived by deprotonation from the exocyclic amino group of cytosine radical cations, C •+ (Decarroz et al 1987, Aravindakumar et al 2003) (Figure 4).…”

“…The radiolytically generated SO 4 • − radicals produce a mixture of radical intermediates detected by ESR and pulse radiolysis (Hildenbrandt et al 1989, Catteral et al 1992, Aravindakumar et al 2003). The major intermediate, which shows an absorption band at 385 nm by transient absorption spectroscopy does not react with O 2 , has been identified as the N -centered radical dC( N 4 -H) • derived by deprotonation from the exocyclic amino group of cytosine radical cations, C •+ (Decarroz et al 1987, Aravindakumar et al 2003) (Figure 4). …”

“…These radicals are also formed via diffusion-controlled addition of • OH to the C5-C6 double bond of 2′-deoxycytidine Aravindakumar et al 2003). …”

“…Therefore, several one-electron oxidants that are described in the following section have been considered to partly mimic the direct effect of ionizing radiation in model mechanistic studies involving aqueous solutions. In this respect, the contribution of Clemens von Sonntag to the delineation of one- is of electron oxidation reactions of pyrimidine bases using peroxodisulfate as the source of SO 4 •− major important importance (von Sonntag 1987b, 1991, Schuchmann et al 1987, Rashid et al 1991, Aravindakumar et al 2003). …”