Repair Reactions of Pyrimidine-Derived Radicals by Aliphatic Thiols Aleksandra Wo ´ jcik, † Sergej Naumov, ‡ Bronislaw Marciniak, § and Ortwin Brede* ,† Interdisciplinary Group of Time-ResolVed Spectroscopy, UniVersity of Leipzig, Permoserstrasse 15, 04303 Leipzig, Germany, Institute of Surface Modification, 04303 Leipzig, Germany, and Faculty of Chemistry, Adam Mickiewicz UniVersity, Grunwaldzka 6, 60-780 Poznan ˜ , Poland ReceiVed: March 14, 2006; In Final Form: April 25, 2006 Pyrimidinyl radicals of various structures (Pyr • ) were generated in aqueous and alcohol-containing solutions by means of pulse radiolysis to determine the rate constants of their repair reactions by different thiols (RSH ) cysteamine, 2-mercaptoethanol, cysteine, and penicillamine): Pyr • + RSH f PyrH + RS • . C5-OH and C6-OH adduct radicals of the pyrimidines react with thiols with k 9 ) (1.2-10.0) × 10 6 dm 3 mol -1 s -1 . Similar repair rate constants were found for uracil- and thymine-derived N1-centered radicals, k 31 ) (1.5- 6.1) × 10 6 dm 3 mol -1 s -1 . However, pyrimidine radical anions protonated at their C6 position and C6- uracilyl radicals, with carbonyl groups at their C5 position, react with thiols faster, with k 24 ) (0.5-7.6) × 10 7 dm 3 mol -1 s -1 and k 14 ) (1.4-4.8) × 10 7 dm 3 mol -1 s -1 , respectively. Quantum chemical calculations, at the B3LYP/6-31G(d,p) and self-consistent reaction field polarizable continuum model level point to the combined effects of the energy gap between interacting molecular orbitals, charge distribution within different pyrimidine-derived radicals, and the coefficients of the atomic orbitals as the possible reasons for the differences in the rate constants of repair. 1. Introduction Thiols (RSH) act as antioxidants. Therefore they are capable of protecting cells, in a certain sense, against the damages induced by ionizing radiation. 1,2 In model systems, one can speak about radiation protection based on preventing the attack of the reactive species, for example, • OH radicals, on the molecule of interest according to the competition between reactions 1 and 2 3 In living cells, however, this kind of competition is unlikely to occur on a large scale since it requires extremely high concentrations of thiols, greatly exceeding their physiological levels. However, thiols can act through “chemical repair” of damage already induced in biomolecules, for example This kind of “repair” brings no restoration of the original structure of DNA. One can speak about “protection” or “repair” only in the sense of converting more reactive C-centered radicals into less reactive, although not completely harmless, S-centered radicals. 4-7 Accordingly, protection of DNA by thiols can occur also by electron transfer 8 Although investigated in the past, 9,10 the mechanism of DNA protection by thiols still attracts attention. 11-13 The rate constants for repair reactions are of particular interest as far as under- standing of this mechanism is concerned. It appears that the net charge on the thiols strongly influences the repair rate constants observed for DNA (investigated both in aqueous solution and in prokaryotic cells) and for its model system, polyuracil. This effect is explained by counterion condensation and the co-ion depletion phenomenon. 14-16 The fundamental mechanisms of direct and indirect DNA damage seem to be known. 1 Furthermore, the probable structures of the radicals involved are known (i.e., radical cation centers at guanine, radical anion centers at thymine or cytosine, 17 and • OH radical and H • atom adducts to unsaturated bonds of nucleobases 1 ). Despite these advances in understanding, it is still difficult to state precisely which radical structure is actually being repaired. There are four different nucleobases in the DNA molecule. Their primary radicals can undergo further reactions, including protonation or reaction with the sugar moiety. 18 Moreover, in double-stranded DNA, considerable attack of reactive species at the sugar moiety itself can occur due to the hindered accessibility of the nucleobases. Considering how thoroughly investigated the thiol-repair effect has been in systems mimicking biological ones as well as in cells themselves (see previous paragraph), surprisingly little is known about the rate constants for the thiol-repair reactions with pyrimidine-derived radicals having well-defined structures (Pyr • ) The source of the difficulty is that direct observation of the repair reaction is complicated by many competing reactions of thiols. By monitoring the absorption of the disulfide radical anion, Adams et al. 19 determined, in an indirect manner, the upper limit of the repair rate constant k repair < 1 × 10 7 dm 3 mol -1 s -1 for • OH radical adducts of uracil and deoxythymidine being repaired by cysteamine. Nucifora et al. 20 used pulse radiolysis with electron para- magnetic resonance (EPR) detection to investigate the repair reaction of • OH radical adducts of thymine and uracil by * Author to whom correspondence should be addressed. E-mail: brede@ mpgag.uni-leipzig.de. † University of Leipzig. ‡ Institute of Surface Modification. § Adam Mickiewicz University. DNA + • OH f DNA • OH (1) RSH + • OH f RS • + H 2 O (2) DNA • OH + RSH f DNA(H)OH + RS • (3) DNA •+ + RSH f DNA + RS • + H + (4) Pyr • + RSH f PyrH + RS • (5) 12738 J. Phys. Chem. B 2006, 110, 12738-12748 10.1021/jp061574e CCC: $33.50 © 2006 American Chemical Society Published on Web 06/02/2006