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The interaction of thiols and thiyl radicals with DNA model systems

Final Activity Report Summary - DNA-PROTDAM (The interaction of thiols and thiyl radicals with DNA model systems)

The main aim of this work was to study the photosensitised oxidation reactions of aliphatic thiols (cysteamine, 2-mercaptoethanol, cysteine, penicillamine) using various sensitisers, as possible ways of generating thiyl radicals. Measurements were carried out in aqueous and acetonitrile solutions using the technique of laser flash photolysis with optical detection. Bimolecular rate constants and quantum yields of the intermediate products were determined for all the investigated reactions. Possible reaction mechanisms were proposed.

In neutral and alkaline aqueous solutions, thiols react with triplet 4-carboxybenzophenone (CB) with rate constants close to the diffusion-controlled limit, kq = (1.3-3.4) × 109 M-1 s-1. For cysteine and penicillamine, however, triplet-quenching rate constants in neutral pH are lower by one order of magnitude.

Deconvoluted spectra confirm the presence of electron-transfer intermediates, namely CB ketyl radicals, CB ketyl radical anions and disulfide radical anions formed in a reaction of thiyl radicals with thiolates. The quantum yields for the formation of the CB ketyl radical anion were found to be approximately equal to 0.10 whereas the quantum yields of the disulfide radical anion were equal to 0.05. The lower yields were due to the reversibility of the reaction between thiyl radicals and thiolates. Triplet CB quenching by thiols occurs via electron-transfer and the formation of a radical-ion pair. The radical-ion pair decays by a back electron-transfer process (circa 90 %), by separation of the radical ions and by proton transfer within the radical-ion pair with further separation of the neutral radicals.

Triplets of benzophenone, 4,4'-dimetoxybenzophenone, 4-trifluoromethylbenzophenone and xanthone react with mercaptoethanol with rate constants well below the diffusion-controlled limit, kq = (1.2-17.4) × 106 M-1 s-1. Xanthone is the most reactive photosensitiser, whereas the least reactive is 4,4'-dimetoxybenzophenone. All the determined quenching rate constants show a good correlation with the redox potentials of the photosensitisers in their excited states. This is indirect evidence that the primary photochemical step involves triplet quenching via electron transfer.

The quantum yields of ketyl radicals formation were equal to 0.13 for BP and DMBP, 0.12 for TFMBP and 0.32 for xanthone. The determined values indicate a high contribution for the back electron-transfer process in the decay of the radical-ion pair.

Thiols react with triplet 9,10-anthraquinone-2,6-disulphonate (AQ26DS) in the mixture water / acetornitrile forming two longer-lived species identified as the AQ26DS radical and the AQ26DS radical anion. For mercaptoethanol, cysteine and penicillamine the AQ26DS triplet-quenching rate constants are in the range of kq = (1.8-3.2) × 109 M-1 s-1, but for cysteamine the measured kq value is as high as 9.9 × 109 M-1 s-1. These high quenching rate constants and presence of the electron-transfer intermediates indicate that the triplet of AQ26DS reacts with thiols via an electron-transfer process.

Although photochemical generation of thiyl radicals still requires further studies, our investigations confirm that the photochemical pathway may be a relatively efficient way for generating thiyl radicals. The mechanism of photosensitised oxidation of thiols strongly depends on the reaction environment, which is reflected by the rate constants of the quenching reaction. In aqueous solutions electron-transfer intermediates were observed directly, whereas in acetonitrile a charge-transfer mechanism for the triplet quenching was proven by the correlation between kq and the redox potentials of the photosensitisers in their excited states.

Understanding the reactions of thiols with ketone triplets is a preliminary step in the study of the interactions of photochemically generated thiyl radicals with unsaturated bonds in purines, which is relevant to an understanding of free-radical damage occurring in DNA.