Intramolecular Proton Transfer in the Radical Anion of Cytidine Monophosphate Sheds Light on the Sensitivities of Dry vs Wet DNA to Electron Attachment-Induced Damage.

Single-strand breaks (SSBs) induced via electron attachment were previously observed in dry DNA under ultrahigh vacuum (UHV), while hydrated electrons were found not able to induce this DNA damage in an aqueous solution. To explain these findings, crossed electron-molecular beam (CEMB) and anion photoelectron spectroscopy (aPES) experiments coupled to density functional theory (DFT) modeling were used to demonstrate the fundamental importance of proton transfer (PT) in radical anions formed via electron attachment. Three molecular systems were investigated: 5'-monophosphate of 2'-deoxycytidine (dCMPH), where PT in the electron adduct is feasible, and two ethylated derivatives, 5'-diethylphosphate and 3',5'-tetraethyldiphosphate of 2'-deoxycytidine, where PT is blocked due to substitution of labile protons with the ethyl residues. CEMB and aPES experiments confirmed the cleavage of the C3'/C5'-O bond as the main dissociation channel related to electron attachment in the ethylated derivatives. In the case of dCMPH, however, electron attachment (in the aPES experiments) yielded its parent (intact) radical anion, dCMPH-, suggesting that its dissociation was inhibited. The aPES-measured vertical detachment energy of the dCMPH- was found to be 3.27 eV, which agreed with its B3LYP/6-31++G(d,p)-calculated value and implied that electron-induced proton transfer (EIPT) had occurred during electron attachment to the dCMPH model nucleotide. In other words, EIPT, subduing dissociation, appeared to be somewhat protective against SSB. While EIPT is facilitated in solution compared to the dry environment, the above findings are consistent with the stability of DNA against hydrated electron-induced SSB in solution versus free electron-induced SSB formation in dry DNA.

Why 6-Iodouridine Cannot Be Used as a Radiosensitizer of DNA Damage? Computational and Experimental Studies.

Previous density functional theory (DFT) studies on 6-brominated pyrimidine nucleosides suggest that 6-iodo-2'-deoxyuridine (6IdU) should act as a better radiosensitizer than its 5-iodosubstituted 2'-deoxyuridine analogue. In this work, we show that 6IdU is unstable in an aqueous solution. Indeed, a complete disappearance of the 6IdU signal was observed during its isolation by reversed-phase high-performance liquid chromatography (RP-HPLC). As indicated by the thermodynamic characteristics for the SN1-type hydrolysis of 6IdU obtained at the CAM-B3LYP/DGDZVP++ level and the polarizable continuum model (PCM) of water, 6-iodouracil (6IU) was already released quantitatively at ambient temperatures. The simulation of the hydrolysis kinetics demonstrated that a thermodynamic equilibrium was reached within seconds for the title compound. To assess the reliability of the calculations carried out, we synthesized 6-iodouridine (6IUrd), which was, unlike 6IdU, sufficiently stable in an aqueous solution at room temperature. The activation barrier for the N-glycosidic bond dissociation in 6IUrd was estimated experimentally using an Arrhenius plot. The stabilities in water calculated for 6IdU, 6IUrd, and 5-iodo-2'-deoxyuridine (5IdU) could be explained by the electronic and steric effects of the 2'-hydroxy group present in the ribose moiety. Our studies highlight the issue of the hydrolytic stability of potentially radiosensitizing nucleotides which, besides having favorable dissociative electron attachment (DEA) characteristics, must be stable in water to have any practical application.

Electron Attachment to 5-Fluorouracil: The Role of Hydrogen Fluoride in Dissociation Chemistry.

We investigate dissociative electron attachment to 5-fluorouracil (5-FU) employing a crossed electron-molecular beam experiment and quantum chemical calculations. Upon the formation of the 5-FU- anion, 12 different fragmentation products are observed, the most probable dissociation channel being H loss. The parent anion, 5-FU-, is not stable on the experimental timescale (~140 µs), most probably due to the low electron affinity of FU; simple HF loss and F- formation are seen only with a rather weak abundance. The initial dynamics upon electron attachment seems to be governed by hydrogen atom pre-dissociation followed by either its full dissociation or roaming in the vicinity of the molecule, recombining eventually into the HF molecule. When the HF molecule is formed, the released energy might be used for various ring cleavage reactions. Our results show that higher yields of the fluorine anion are most probably prevented through both faster dissociation of an H atom and recombination of F- with a proton to form HF. Resonance calculations indicate that F- is formed upon shape as well as core-excited resonances.

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.

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.

Electron-Induced Decomposition of Uracil-5-yl O-(N,N-dimethylsulfamate): Role of Methylation in Molecular Stability.

The incorporation of modified uracil derivatives into DNA leads to the formation of radical species that induce DNA damage. Molecules of this class have been suggested as radiosensitizers and are still under investigation. In this study, we present the results of dissociative electron attachment to uracil-5-yl O-(N,N-dimethylsulfamate) in the gas phase. We observed the formation of 10 fragment anions in the studied range of electron energies from 0-12 eV. Most of the anions were predominantly formed at the electron energy of about 0 eV. The fragmentation paths were analogous to those observed in uracil-5-yl O-sulfamate, i.e., the methylation did not affect certain bond cleavages (O-C, S-O and S-N), although relative intensities differed. The experimental results are supported by quantum chemical calculations performed at the M06-2X/aug-cc-pVTZ level of theory. Furthermore, a resonance stabilization method was used to theoretically predict the resonance positions of the fragment anions O- and CH3-.

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.

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.

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.