Low-Energy Electron Induced Reactions in Metronidazole at Different Solvation Conditions.

Metronidazole belongs to the class of nitroimidazole molecules and has been considered as a potential radiosensitizer for radiation therapy. During the irradiation of biological tissue, secondary electrons are released that may interact with molecules of the surrounding environment. Here, we present a study of electron attachment to metronidazole that aims to investigate possible reactions in the molecule upon anion formation. Another purpose is to elucidate the effect of microhydration on electron-induced reactions in metronidazole. We use two crossed electron/molecular beam devices with the mass-spectrometric analysis of formed anions. The experiments are supported by quantum chemical calculations on thermodynamic properties such as electron affinities and thresholds of anion formation. For the single molecule, as well as the microhydrated condition, we observe the parent radical anion as the most abundant product anion upon electron attachment. A variety of fragment anions are observed for the isolated molecule, with NO2- as the most abundant fragment species. NO2- and all other fragment anions except weakly abundant OH- are quenched upon microhydration. The relative abundances suggest the parent radical anion of metronidazole as a biologically relevant species after the physicochemical stage of radiation damage. We also conclude from the present results that metronidazole is highly susceptible to low-energy electrons.

Guanosine Dianions Hydrated by One to Four Water Molecules.

Intermolecular interactions such as those present in molecule···water complexes may profoundly influence the physicochemical properties of molecules. Here, we carried out an experimental-computational study on doubly deprotonated guanosine monophosphate···water clusters, [dGMP - 2H]2-·nH2O (n = 1-4), using a combination of negative anion photoelectron spectroscopy (NIPES) with molecular dynamics (MD) and quantum chemical (QM) calculations. Successive addition of water molecules to [dGMP - 2H]2- increases the experimental adiabatic detachment (ADE) and vertical detachment energy (VDE) by 0.5-0.1 eV, depending on the cluster size. In order to choose the representative conformations, we combined MD simulations with a clustering procedure to identify low energy geometries for which ADEs and VDEs were computed at the CAM-B3LYP/6-31++G(d,p) level. Our results demonstrate that the assumed approach leads to sound geometries and energetics of the studied microsolvates since the calculated ADEs and VDEs are in pretty good agreement with the experimental characteristics. The evolution of hydrogen bonding with cluster size indicates the possibility of the occurrence of proton transfer for clusters comprising a larger number of water molecules.

Inactive-to-Active Transition of Human Thymidine Kinase 1 Revealed by Molecular Dynamics Simulations.

Despite its importance in the nucleoside (and nucleoside prodrug) metabolism, the structure of the active conformation of human thymidine kinase 1 (hTK1) remains elusive. We perform microsecond molecular dynamics simulations of the inactive enzyme form bound to a bisubstrate inhibitor that was shown experimentally to activate another TK1-like kinase, Thermotoga maritima TK (TmTK). Our results are in excellent agreement with the experimental findings for the TmTK closed-to-open state transition. We show that the inhibitor induces an increase of the enzyme radius of gyration due to the expansion on one of the dimer interfaces; the structural changes observed, including the active site pocket volume increase and the decrease in the monomer-monomer buried surface area and of the number of hydrogen bonds (as compared to the inactive enzyme control simulation), indicate that the catalytically competent (open) conformation of hTK1 can be assumed in the presence of an activating ligand.

Photoelectron Spectroscopy and Theoretical Investigations of Gaseous Doubly Deprotonated 2'-Deoxynucleoside 5'-Monophosphate Dianions.

A better understanding of the mechanism of oxidative DNA damage requires obtaining a molecular level description of nucleotides in various charge states. Herein, we report a systematic photoelectron spectroscopy and theoretical investigation of the electronic and geometric structures of four doubly deprotonated 2'-deoxynucleoside 5'-monophosphate dianions, the smallest quintessential DNA building block. These dianions are intrinsically stable with their adiabatic/vertical detachment energies (ADE/VDE) ranging from 0.85/1.07 (A) and 1.05/1.30 (G) to 1.20/1.50 (C) and 1.80/2.10 eV (T). The repulsive Coulomb barrier against electron detachment is 2.0 eV for purines and 2.5 eV for pyrimidines. Dianions are deprotonated at the phosphate group and the amino group of a nucleobase. The π-type HOMO orbital resides on the nucleobase moiety for each dianion. This spatial distribution of HOMO suggests that the most loosely bound electron is detached along the direction perpendicular to the nucleobase. When combined with the previous results, this work makes complete the depiction of basic building blocks of DNA at the molecular level.

5-(N-Trifluoromethylcarboxy)aminouracil as a Potential DNA Radiosensitizer and Its Radiochemical Conversion into N-Uracil-5-yloxamic Acid.

Hypoxia-a hallmark of solid tumors-dramatically impairs radiotherapy, one of the most common anticancer modalities. The adverse effect of the low-oxygen state can be eliminated by the concomitant use of a hypoxic cell radiosensitizer. In the present paper, we show that 5-(N-trifluoromethylcarboxy) aminouracil (CF3CONHU) can be considered as an effective radiosensitizer of DNA damage, working under hypoxia. The title compound was synthesized in the reaction of 5-aminouracil and trifluoroacetic anhydride in trifluoroacetic 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. Radiodegradation in a 26.67 ± 0.31% yield resulted in only one major product-N-uracil-5-yloxamic acid. The mechanism that is possibly responsible for the formation of the observed radioproduct has been elucidated with the use of DFT calculations. The cytotoxic test against the PC3 prostate cancer cell line and HDFa human dermal fibroblasts confirmed the low cytotoxicity of CF3CONHU. Finally, a clonogenic assay and flow cytometric analysis of histone H2A.X phosphorylation proved the radiosensitization in vitro.

Uracil-5-yl O-Sulfamate: An Illusive Radiosensitizer. Pitfalls in Modeling the Radiosensitizing Derivatives of Nucleobases.

Efficient radiotherapy requires the concomitant use of ionizing radiation (IR) and a radiosensitizer. In the present work uracil-5-yl O-sulfamate (SU) is tested against its radiosensitizing potential. The compound possesses appropriate dissociative electron attachment (DEA) characteristics calculated at the M06-2X/6-31++G(d,p) level. Crossed electron-molecular beam experiments in the gas phase demonstrate that SU undergoes efficient DEA processes, and the single C-O or S-O bond dissociations account for the majority of fragments induced by electron attachment. Most DEAs proceed already for electrons with kinetic energies of ∼0 eV, which is supported by the exothermic thresholds calculated at the M06-2X/aug-cc-pVTZ level. However, in water solution under reductive conditions and physiological pH, SU does not undergo radiolysis, which demonstrates the crucial influence of aqueous environment on the radiosensitizing properties of modified nucleosides.

Why Does the Type of Halogen Atom Matter for the Radiosensitizing Properties of 5-Halogen Substituted 4-Thio-2'-Deoxyuridines?

Radiosensitizing properties of substituted uridines are of great importance for radiotherapy. Very recently, we confirmed 5-iodo-4-thio-2'-deoxyuridine (ISdU) as an efficient agent, increasing the extent of tumor cell killing with ionizing radiation. To our surprise, a similar derivative of 4-thio-2'-deoxyuridine, 5-bromo-4-thio-2'-deoxyuridine (BrSdU), does not show radiosensitizing properties at all. In order to explain this remarkable difference, we carried out a radiolytic (stationary and pulse) and quantum chemical studies, which allowed the pathways to all radioproducts to be rationalized. In contrast to ISdU solutions, where radiolysis leads to 4-thio-2'-deoxyuridine and its dimer, no dissociative electron attachment (DEA) products were observed for BrSdU. This observation seems to explain the lack of radiosensitizing properties of BrSdU since the efficient formation of the uridine-5-yl radical, induced by electron attachment to the modified nucleoside, is suggested to be an indispensable attribute of radiosensitizing uridines. A larger activation barrier for DEA in BrSdU, as compared to ISdU, is probably responsible for the closure of DEA channel in the former system. Indeed, besides DEA, the XSdU anions may undergo competitive protonation, which makes the release of X- kinetically forbidden.

5-Iodo-4-thio-2'-Deoxyuridine as a Sensitizer of X-ray Induced Cancer Cell Killing.

Nucleosides, especially pyrimidines modified in the C5-position, can act as radiosensitizers via a mechanism that involves their enzymatic triphosphorylation, incorporation into DNA, and a subsequent dissociative electron attachment (DEA) process. In this paper, we report 5-iodo-4-thio-2'-deoxyuridine (ISdU) as a compound that can effectively lead to ionizing radiation (IR)-induced cellular death, which is proven by a clonogenic assay. The test revealed that the survival of cells, pre-treated with 10 or 100 µM solution of ISdU and exposed to 0.5 Gy of IR, was reduced from 78.4% (for non-treated culture) to 67.7% and to 59.8%, respectively. For a somewhat higher dose of 1 Gy, the surviving fraction was reduced from 68.2% to 54.9% and to 40.8% for incubation with 10 or 100 µM ISdU, respectively. The cytometric analysis of histone H2A.X phosphorylation showed that the radiosensitizing effect of ISdU was associated, at least in part, with the formation of double-strand breaks. Moreover, the cytotoxic test against the MCF-7 breast cancer cell line and human dermal fibroblasts (HDFa line) confirmed low cytotoxic activity of ISdU. Based on the results of steady state radiolysis of ISdU with a dose of 140 Gy and quantum chemical calculations explaining the origin of the MS detected radioproducts, the molecular mechanism of sensitization by ISdU was proposed. In conclusion, we found ISdU to be a potential radiosensitizer that could improve anticancer radiotherapy.

Electron-Induced Dissociation of the Potential Radiosensitizer 5-Selenocyanato-2'-deoxyuridine.

5-Selenocyanato-2'-deoxyuridine (SeCNdU) is a recently proposed radiosensitizer based on 2'-deoxyuridine (dU) with the electron-affinic selenocyanato (-SeCN) side group attached at the C5 position of uracil. Since electron interaction processes may be an important source of DNA damage by ionizing radiation, we have studied low-energy dissociative electron attachment to SeCNdU in the gas phase. Negative ion formation has been obtained by means of mass spectrometry, where a rich fragmentation pattern is observed even at ∼0 eV. The reaction pathways exhibiting the highest ion yields are C4N2O2H2Se•- and CN-, both involving a cleavage of the Se-CN bond. The heaviest fragment anion observed is C9N2O5H10Se•-, where besides the charged species, the hydrogen and cyano radicals are also formed. Further decomposition channels also yield the highly reactive hydroxyl radical, which possesses a high DNA damage potential. All observed channels have experimentally determined onsets at 0 eV, which are supported by calculations performed at the M06-2X/aug-cc-pVTZ level. The calculations comprise the thermochemical thresholds at standard and experimental (428.15 K, 3 × 10-11 atm) conditions together with the adiabatic electron affinities. The present study shows that low-energy electrons very effectively decompose SeCNdU upon attachment of thermal electrons, producing a large variety of charged fragments and radicals.

5-Selenocyanato and 5-trifluoromethanesulfonyl derivatives of 2'-deoxyuridine: synthesis, radiation and computational chemistry as well as cytotoxicity.

5-Selenocyanato-2'-deoxyuridine (SeCNdU) and 5-trifluoromethanesulfonyl-2'-deoxyuridine (OTfdU) have been synthesized and their structures have been confirmed with NMR and MS methods. Both compounds undergo dissociative electron attachment (DEA) when irradiated with X-rays in an aqueous solution containing a hydroxyl radical scavenger. The DEA yield of SeCNdU significantly exceeds that of 5-bromo-2'-deoxyuridine (BrdU), remaining in good agreement with the computationally revealed profile of electron-induced degradation. The radiolysis products indicate, in line with theoretical predictions, Se-CN bond dissociation as the main reaction channel. On the other hand, the DEA yield for OTfdU is slightly lower than the degradation yield measured for BrdU, despite the fact that the calculated driving force for the electron-induced OTfdU dissociation substantially overpasses the thermodynamic stimulus for BrdU degradation. Moreover, the calculated DEA profile suggests that the electron attachment induced formation of 5-hydroxy-2'-deoxyuridine (OHdU) from OTfdU, while 2'-deoxyuridine (dU) is mainly observed experimentally. We explained this discrepancy in terms of the increased acidity of OTfdU resulting in efficient deprotonation of the N3 atom, which brings about the domination of the OTfdU(N3-H)- anion in the equilibrium mixture. As a consequence, electron addition chiefly leads to the radical dianion, OTfdU(N3-H)˙2-, which easily protonates at the C5 site. As a result, the C5-O rather than O-S bond undergoes dissociation, leading to dU, observed experimentally. A negligible cytotoxicity of the studied compounds toward the MCF-7 cell line at the concentrations used for cell labelling calls for further studies aiming at the clinical use of the proposed derivatives.

Dominant Pathways of Adenosyl Radical-Induced DNA Damage Revealed by QM/MM Metadynamics.

Brominated nucleobases sensitize double stranded DNA to hydrated electrons, one of the dominant genotoxic species produced in hypoxic cancer cells during radiotherapy. Such radiosensitizers can therefore be administered locally to enhance treatment efficiency within the solid tumor while protecting the neighboring tissue. When a solvated electron attaches to 8-bromoadenosine, a potential sensitizer, the dissociation of bromide leads to a reactive C8 adenosyl radical known to generate a range of DNA lesions. In the current work, we propose a multiscale computational approach to elucidate the mechanism by which this unstable radical causes further damage in genomic DNA. We employed a combination of classical molecular dynamics conformational sampling and QM/MM metadynamics to study the thermodynamics and kinetics of plausible reaction pathways in a realistic model, bridging between different time scales of the key processes and accounting for the spatial constraints in DNA. The obtained data allowed us to build a kinetic model that correctly predicts the products predominantly observed in experimental settings-cyclopurine and ß-elimination (single strand break) lesions-with their ratio and yield dependent on the effective lifetime of the radical species. To date, our study provides the most complete description of purine radical reactivity in double stranded DNA, explaining the radiosensitizing action of electrophilic purines in molecular detail as well as providing a conceptual framework for the computational modeling of competing reaction pathways in biomolecules.

The Sequence Dependence of Photoinduced Single Strand Break in 5-Bromo-2'-deoxyuridine Labeled DNA Supports That Electron Transfer Is Responsible for the Damage.

The UVB irradiation of DNA labeled with 5-bromo-2'-deoxyuridine (BrdU) leads to single-strand breaks (SSBs) as a major photochemical damage. Some time ago, we demonstrated that SSB is a secondary damage forming due to thermal dissociation of 2'-deoxyribonolactone generated photochemically in DNA labeled with BrdU. For the first time, we study here the variation of the yield of UVB generated SSBs with the alteration of 3'-neighbor nucleobase of electron donor (2'-deoxyguanine (dG)) and acceptor (excited BrdU) in double-stranded DNA. We showed that the experimental damage yields can be explained by the calculated ionization potentials of dG and electron affinities of excited BrdU via a kinetic scheme based on the Marcus model of electron transfer (ET). Hence, our studies on the sequence dependence of photochemical damage in DNA labeled with BrdU constitute a further argument that photochemically generated SSBs occur as a result of long-range ET.

5-Selenocyanatouracil: A Potential Hypoxic Radiosensitizer. Electron Attachment Induced Formation of Selenium Centered Radical.

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.

Electrophilic 5-Substituted Uracils as Potential Radiosensitizers: A Density Functional Theory Study.

Although 5-bromo-2'-deoxyuridine (5BrdU) possesses significant radiosensitizing power in vitro, clinical studies do not confirm any advantages of radiotherapy employing 5BrdU. This situation calls for a continuous search for efficient radiosensitizers. Using the proposed mechanism of radiosensitization by 5BrdU, we propose a series of 5-substituted uracils, XYU, that should undergo efficient dissociative electron attachment. The DFT-calculated thermodynamic and kinetic data concerning the XYU degradations induced by electron addition suggests that some of the scrutinized derivatives have much better characteristics than 5BrdU itself. Synthesis of these promising candidates for radiosensitizers, followed by studies of their radiosensitizing properties in DNA context, and ultimately in cancer cells, are further steps to confirm their potential applicability in anticancer treatment.

Mechanisms of Damage to DNA Labeled with Electrophilic Nucleobases Induced by Ionizing or UV Radiation.

Hypoxia--a hallmark of solid tumors--makes hypoxic cells radioresistant. On the other hand, DNA, the main target of anticancer therapy, is not sensitive to the near UV photons and hydrated electrons, one of the major products of water radiolysis under hypoxic conditions. A possible way to overcome these obstacles to the efficient radio- and photodynamic therapy of cancer is to sensitize the cellular DNA to electrons and/or ultraviolet radiation. While incorporated into genomic DNA, modified nucleosides, 5-bromo-2'-deoxyuridine in particular, sensitize cells to both near-ultraviolet photons and γ rays. It is believed that, in both sensitization modes, the reactive nucleobase radical is formed as a primary product which swiftly stabilizes, leading to serious DNA damage, like strand breaks or cross-links. However, despite the apparent similarity, such radio- and photosensitization of DNA seems to be ruled by fundamentally different mechanisms. In this review, we demonstrate that the most important factors deciding on radiodamage to the labeled DNA are (i) the electron affinity (EA) of modified nucleoside (mNZ), (ii) the local surroundings of the label that significantly influences the EA of mNZ, and (iii) the strength of the chemical bond holding together the substituent and a nucleobase. On the other hand, we show that the UV damage to sensitized DNA is governed by long-range photoinduced electron transfer, the efficiency of which is controlled by local DNA sequences. A critical review of the literature mechanisms concerning both types of damage to the labeled biopolymer is presented. Ultimately, the perspectives of studies on DNA sensitization in the context of cancer therapy are discussed.