Article pubs.acs.org/JPCB
5‑Selenocyanatouracil: A Potential Hypoxic Radiosensitizer. Electron Attachment Induced Formation of Selenium Centered Radical Marta Sosnowska, Samanta Makurat, Magdalena Zdrowowicz, and Janusz Rak* Department of Chemistry, University of Gdańsk, Wita Stwosza 63, 80-308 Gdańsk, Poland S Supporting Information *
ABSTRACT: The propensity of 5-selenocyanatouracil (SeCNU) to decomposition induced by attachment of electron was scrutinized with the G3B3 composite quantum-chemical method and radiolytic studies. Favorable thermodynamic (Gibbs free reaction energy of −13.65 kcal/mol) and kinetic (Gibbs free activation energy of 1.22 kcal/mol) characteristics revealed by the G3B3 free energy profile suggest SeCNU to be sensitive to electron attachment. The title compound was synthesized in the reaction between uracil and selenocyanogen chloride in acetic acid. Then, an aqueous and deoxygenated solution of the HPLC purified compound containing tert-butanol as a hydroxyl radical scavenger was irradiated with X-rays. SeCNU radio-degradation results in two major products: the U−Se−Se−U dimer and the adduct of the ●OtBu radical to the U−Se● radical, U−Se−OtBu. The effects of radiolysis as well as the results of G3B3 calculations point to U−Se● as the primary product of dissociative electron attachment to SeCNU. The MTT test shows that SeCNU is nontoxic in vitro in concentrations equal to or lower than 10−6 M. Ionizing radiation will probably induce cytotoxic intra- and interstrand DNA cross-links as well as protein−DNA cross-links in the genomic DNA labeled with SeCNU.
1. INTRODUCTION Radiotherapy is one of the most common modalities for treating human cancer.1 However, the oxygen level in solid tumors is very low, and as a result, tumor cells become resistant to ionizing radiation.2 Therefore, effective radiotherapy should be combined with the use of chemical agentsradiosensitizerscapable of sensitizing tumor to the effects of highenergy radiation and, as a consequence, effectively reducing the therapeutic dose of ionizing radiation. It is worth emphasizing that ionizing radiation is not neutral toward normal cells adjacent to the tumor and is even able to initiate carcinogenesis.3 Surprisingly, the range of radiosensitizers currently employed in the clinical practice is quite narrow.4 Efficient radiosensitizers should selectively sensitize tumor rather than healthy cells. Taking into account this criterion, Hall distinguished only two classes of radiosensitizing agents which could find practical use in clinical radiotherapy.5 The first group consists of hypoxic cell sensitizers. Here, selectivity results from the fact that hypoxia occurs only in cancer cells. The second type of radiosensitizers are pyrimidine analogues that could be incorporated into DNA due to their structural © 2017 American Chemical Society
similarity to native nucleosides. The selectivity of this group of sensitizers is based on the ability of the cancer cells to undergo fast and uncontrolled cell division. As a consequence, cancer cells incorporate more of the drug than the surrounding normal tissues. Thus, nucleoside derivatives seem to be promising, nontoxic candidates for effective radiosensitizers of DNA damage.3 The structural modifications of nucleosides should rely on the introduction of suitable substituents to a nucleobase that would increase their sensitivity to degradation induced by solvated electrons, which are one of the most abundant products of water radiolysis under hypoxia.4 One could doubt, however, that solvated electrons (e−aq) are able to attach to nucleobases, since they are strongly bound by water. Yet, it is wellestablished that e−aq, formed in pulse radiolysis experiments, adds to nucleobases with a near diffusion controlled rate.6 The adiabatic electron affinity (AEA) of solvated nucleobases Received: April 18, 2017 Revised: June 1, 2017 Published: June 2, 2017 6139
DOI: 10.1021/acs.jpcb.7b03633 J. Phys. Chem. B 2017, 121, 6139−6147
Article
The Journal of Physical Chemistry B amounts to 1.7−2.2 eV.7 On the other hand, the AEA of e−aq was estimated to be only 1.6 eV.8 Thus, the transfer of e−aq to a native nucleobase is thermodynamically favorable, which explains the diffusion controlled rate of the formation of nucleobase electron adducts. It is, however, worth emphasizing that, althought DNA in an aqueous solution attaches solvated electrons, it does not undergo further secondary reactions, leading to lethal strand breaks.9−11 Therefore, the incorporation of modified electrophilic nucleosides unstable after electron attachment into the genome should lead to the increase of radiosensitivity of the labeled cells. One of the best known radiosensitizers belonging to nucleoside analogues is 5-bromo-2′-deoxyuridine (BrdU).12−14 In the past, a number of investigations have demonstrated that BrdU is easily incorporated into the genetic material and caused the enhancement of DNA damage induced by ionizing radiation.15,16 Despite these promising in vitro studies, this derivative is not used in clinical practice.17 Hence, there is still a necessity of searching for new, more effective nucleosides of this type. The sensitivity of modified nucleobases to electron attachment is governed by two factors: nucleobases’ electron affinity and their ability to undergo electron-induced dissociation leading to reactive radicals that in secondary steps may produce strand breaks or other types of DNA damage. Our quantum chemical studies of these two factors, carried out for several derivatives of uracil, led to the development of a methodology that enables radiosensitizing potential of any substituted nucleobase, a 5-substituted uracil in particular, to be evaluated.18 This theoretical model allowed us to propose 5thiocyanatouracil (SCNU) as a promising candidate for a hypoxic radiosensitizer. For this reason, we synthesized SCNU and confirmed the computational predictions using negative ion photoelectron spectroscopy (PES). The anionic mass spectra demonstrated a rapid decomposition of SCNU caused by electron attachment.13 The mechanism of electron-induced degradation of the described above derivative has been further investigated on 5-thiocyanato-2′-deoxyuridine (SCNdU) using low-temperature electron spin resonance spectroscopy (ESR), steady-state radiolysis at ambient temperature, and molecular modeling at the density functional theory (DFT) level.19 Very recently, the employment of our theoretical model against an extended set of 5-substituted uracils allowed us to identify several potential radiosensitizers worth further experimental studies.20 Before expensive and time-consuming DNA labeling (PCR,21−23 enzymatic,24 in vitro12), studies on the response of the labeled cell to ionizing radiation, and tests on animal models, one should synthesize a chosen nucleoside and prove that it does not undergo hydrolysis, is radiosensitive in aqueous solution, and is relatively nontoxic. Therefore, in the following, we describe the chemical synthesis of 5-selenocyanatouracil (SeCNU) in the reaction of in situ generated selenocyanogen chloride with uracil.25 Furthermore, we prove the sensitivity of SeCNU to electron attachment in aqueous solution with the use of steady-state radiolysis at ambient temperature followed by the liquid chromatography−mass spectrometry (LC-MS) analysis of radiolytes. We also demonstrate, using the Gaussian-G3 method variant using B3LYP structures and frequencies (G3B3) quantum-chemical method of chemical accuracy, that the electron-induced cleavage of the Se−C bond is the main channel of SeCNU dissociation. Finally, the cellular MTT test carried out on the
MCF-7 breast cancer line proves no cytotoxicity of the studied derivative at concentrations usually used for cell labeling.
2. EXPERIMENTAL AND THEORETICAL METHODS 2.1. Synthesis of 5-Selenocyanatouarcil. Dried KSeCN (1.5 g, 10.4 mmol) was added to a cooled solution of gaseous chlorine in glacial acetic acid (0.54 g of Cl2 in 150 mL of acetic acid), and stirring was maintained for 1.5 h at room temperature. To the resulting solution of selenocyanogen chloride (SeCNCl), uracil (129 mg, 1.15 mmol) was added and the reaction mixture was stirred at room temperature for 20 h. The resulting suspension was filtered, and the solvent was evaporated in vacuo at
