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5-Thiocyanato-2'-deoxyuridine as a possible radiosensitizer: electron-induced formation of uracil-C5-thiyl radical and its dimerization.

ABSTRACT: In this work, we have synthesized 5-thiocyanato-2'-deoxyuridine (SCNdU) along with the C6-deuterated nucleobase 5-thiocyanatouracil (6-D-SCNU) and studied their reactions with radiation-produced electrons. ESR spectra in ?-irradiated nitrogen-saturated frozen homogeneous solutions (7.5 M LiCl in H2O or D2O) of these compounds show that electron-induced S-CN bond cleavage occurs to form a thiyl radical (dU-5-S? or 6-D-U-5-S?) and CN(-)via the initial ?-anion radical (SCNdU?(-)) intermediate in which the excess electron is on the uracil base. HPLC and LC-MS/MS studies of ?-irradiated N2-saturated aqueous solutions of SCNdU in the presence of sodium formate as a OH-radical scavenger at ambient temperature show the formation of the dU-5S-5S-dU dimer in preference to dU by about 10 to 1 ratio. This shows that both possible routes of electron-induced bond cleavage (dUC5-SCN and S-CN) in SCNdU?(-) and dU-5-S? formation are preferred for the production of the ?-type uracilyl radical (dU?) by 10 fold. DFT/M06-2x/6-31++G(d,p) calculations employing the polarizable continuum model (PCM) for aqueous solutions show that dU-5-S? and CN(-) formation was thermodynamically favored by over 15 kcal mol(-1) (?G) compared to dU? and SCN(-) production. The activation barriers for C5-S and S-CN bond cleavage in SCNdU?(-) amount to 8.7 and 4.0 kcal mol(-1), respectively, favoring dU-5-S? and CN(-) formation. These results support the experimental observation of S-CN bond cleavage by electron addition to SCNdU that results in the formation of dU-5-S? and the subsequent dU-5S-5S-dU dimer. This establishes SCNdU as a potential radiosensitizer that could cause intra- and inter-strand crosslinking as well as DNA-protein crosslinking via S-S dimer formation.

SUBMITTER: Zdrowowicz M

PROVIDER: S-EPMC4481187 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature

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5-Thiocyanato-2'-deoxyuridine as a possible radiosensitizer: electron-induced formation of uracil-C5-thiyl radical and its dimerization.

Zdrowowicz Magdalena M Chomicz Lidia L Żyndul Michał M Wityk Paweł P Rak Janusz J Wiegand Tyler J TJ Hanson Cameron G CG Adhikary Amitava A Sevilla Michael D MD

Physical chemistry chemical physics : PCCP 20150701 26

In this work, we have synthesized 5-thiocyanato-2'-deoxyuridine (SCNdU) along with the C6-deuterated nucleobase 5-thiocyanatouracil (6-D-SCNU) and studied their reactions with radiation-produced electrons. ESR spectra in γ-irradiated nitrogen-saturated frozen homogeneous solutions (7.5 M LiCl in H2O or D2O) of these compounds show that electron-induced S-CN bond cleavage occurs to form a thiyl radical (dU-5-S˙ or 6-D-U-5-S˙) and CN(-)via the initial π-anion radical (SCNdU˙(-)) intermediate in wh ...[more]

PMID: 26059609

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Project description:Previous studies have demonstrated that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) undergoes NAD(H) linkage to an active site thiol when it comes into contact with .NO-related oxidants. We found that a free-radical generator 2,2'-azobis-(2-amidinopropane) hydrochloride (AAPH), which does not release either .NO or .NO-related species, was indeed able to induce the NAD(H) linkage to GAPDH. We performed spin-trapping studies with purified apo-GAPDH to identify a putative thiol intermediate produced by AAPH as well as by .NO-related oxidants. As .NO sources we used .NO gas and two .NO-donors, S-nitroso-N-acetyl-D,L-penicillamine and 3-morpholinosydno-nimine hydrochloride (SIN-1). Because SIN-1 produces .NO and a superoxide radical simultaneously, we also tested the effects of peroxynitrite. All the .NO-related oxidants were able to induce the linkage of NAD(H) to GAPDH and the formation of a protein free-radical identified as a thiyl radical (inhibited by N-ethylmaleimide). .NO gas and the .NO-donors required molecular oxygen to induce the formation of the GAPDH thiyl radical, suggesting the possible involvement of higher nitrogen oxides. Thiyl radical formation was decreased by the reconstitution of GAPDH with NAD+. Apo-GAPDH was a strong scavenger of AAPH radicals, but its scavenging ability was decreased when its cysteine residues were alkylated or when it was reconstituted with NAD+. In addition, after treatment with AAPH, a thiyl radical of GAPDH was trapped at high enzyme concentrations. We suggest that the NAD(H) linkage to GAPDH is mediated by a thiyl radical intermediate not specific to .NO or .NO-related oxidants. The cysteine residue located at the active site of GAPDH (Cys-149) is oxidized by free radicals to a thiyl radical, which reacts with the neighbouring coenzyme to form Cys-NAD(H) linkages. Studies with the NAD+ molecule radio-labelled in the nicotinamide or adenine portion revealed that both portions of the NAD+ molecule are linked to GAPDH.

Dataset Information

5-Thiocyanato-2'-deoxyuridine as a possible radiosensitizer: electron-induced formation of uracil-C5-thiyl radical and its dimerization.

Publications

5-Thiocyanato-2'-deoxyuridine as a possible radiosensitizer: electron-induced formation of uracil-C5-thiyl radical and its dimerization.

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