Simulation of UV absorption spectra and relaxation dynamics of uracil and uracil-water clusters.

Despite many studies, the mechanisms of nonradiative relaxation of uracil in the gas phase and in aqueous solution are still not fully resolved. Here we combine theoretical UV absorption spectroscopy with nonadiabatic dynamics simulations to identify the photophysical mechanisms that can give rise to experimentally observed decay time constants. We first compute and theoretically assign the electronic spectra of uracil using the second-order algebraic-diagrammatic-construction (ADC(2)) method. The obtained electronic states, their energy differences and state-specific solvation effects are the prerequisites for understanding the photodynamics. We then use nonadiabatic trajectory-surface-hopping dynamics simulations to investigate the photoinduced dynamics of uracil and uracil-water clusters. In contrast to previous studies, we found that a single mechanism - the ethylenic twist around the C[double bond, length as m-dash]C bond - is responsible for the ultrafast component of the nonradiative decay, both in the gas phase and in solution. Very good agreement with the experimentally determined ultrashort decay time constants is obtained.

Radiation-Induced Oxidation Reactions of 2-Selenouracil in Aqueous Solutions: Comparison with Sulfur Analog of Uracil.

One-electron oxidation of 2-selenouracil (2-SeU) by hydroxyl (●OH) and azide (●N3) radicals leads to various primary reactive intermediates. Their optical absorption spectra and kinetic characteristics were studied by pulse radiolysis with UV-vis spectrophotometric and conductivity detection and by the density functional theory (DFT) method. The transient absorption spectra recorded in the reactions of ●OH with 2-SeU are dominated by an absorption band with an λmax = 440 nm, the intensity of which depends on the concentration of 2-SeU and pH. Based on the combination of conductometric and DFT studies, the transient absorption band observed both at low and high concentrations of 2-SeU was assigned to the dimeric 2c-3e Se-Se-bonded radical in neutral form (2●). The dimeric radical (2●) is formed in the reaction of a selenyl-type radical (6●) with 2-SeU, and both radicals are in equilibrium with Keq = 1.3 × 104 M-1 at pH 4 (below the pKa of 2-SeU). Similar equilibrium with Keq = 4.4 × 103 M-1 was determined for pH 10 (above the pKa of 2-SeU), which admittedly involves the same radical (6●) but with a dimeric 2c-3e Se-Se bonded radical in anionic form (2●-). In turn, at the lowest concentration of 2-SeU (0.05 mM) and pH 10, the transient absorption spectrum is dominated by an absorption band with an λmax = 390 nm, which was assigned to the ●OH adduct to the double bond at C5 carbon atom (3●) based on DFT calculations. Similar spectral and kinetic features were also observed during the ●N3-induced oxidation of 2-SeU. In principle, our results mostly revealed similarities in one-electron oxidation pathways of 2-SeU and 2-thiouracil (2-TU). The major difference concerns the stability of dimeric radicals with a 2c-3e chalcogen-chalcogen bond in favor of 2-SeU.

Experimental and theoretical studies on thymine photodimerization mediated by oxidatively generated DNA lesions and epigenetic intermediates.

Interaction of nucleic acids with light is a scientific question of paramount relevance not only in the understanding of life functioning and evolution, but also in the insurgence of diseases such as malignant skin cancer and in the development of biomarkers and novel light-assisted therapeutic tools. This work shows that the UVA portion of sunlight, not absorbed by canonical DNA nucleobases, can be absorbed by 5-formyluracil (ForU) and 5-formylcytosine (ForC), two ubiquitous oxidatively generated lesions and epigenetic intermediates present in living beings in natural conditions. We measure the strong propensity of these molecules to populate triplet excited states able to transfer the excitation energy to thymine-thymine dyads, inducing the formation of cyclobutane pyrimidine dimers (CPDs). By using steady-state and transient absorption spectroscopy, NMR, HPLC, and theoretical calculations, we quantify the differences in the triplet-triplet energy transfer mediated by ForU and ForC, revealing that the former is much more efficient in delivering the excitation energy and producing the CPD photoproduct. Although significantly slower than ForU, ForC is also able to harm DNA nucleobases and therefore this process has to be taken into account as a viable photosensitization mechanism. The present findings evidence a rich photochemistry crucial to understand DNA damage photobehavior.

Selenium substitution effects on excited-state properties and photophysics of uracil: a MS-CASPT2 study.

The photophysics of selenium-substituted nucleobases has attracted recent experimental attention because they could serve as potential photosensitizers in photodynamic therapy. Herein, we present a comprehensive MS-CASPT2 study on the spectroscopic and excited-state properties, and photophysics of 2-selenouracil (2SeU), 4-selenouracil (4SeU), and 2,4-selenouracil (24SeU). Relevant minima, conical intersections, crossing points, and excited-state relaxation paths in the lowest five electronic states (i.e., S0, S1, S2, T2, and T1) are explored. On the basis of these results, their photophysical mechanisms are proposed. Upon photoirradiation to the bright S2 state, 2SeU quickly relaxes to its S2 minimum and then moves in an essentially barrierless way to a nearby S2/S1 conical intersection near which the S1 state is populated. Next, the S1 system arrives at an S1/T2/T1 intersection where a large S1/T1 spin-orbit coupling of 430.8 cm-1 makes the T1 state populated. In this state, a barrier of 6.8 kcal mol-1 will trap 2SeU for a while. In parallel, for 4SeU or 24SeU, the system first relaxes to the S2 minimum and then overcomes a small barrier to approach an S2/S1 conical intersection. Once hopping to the S1 state, there exists an extended region with very close S1, T2, and T1 energies. Similarly, a large S1/T1 spin-orbit coupling of 426.8 cm-1 drives the S1→ T1 intersystem crossing process thereby making the T1 state populated. Similarly, an energy barrier heavily suppresses electronic transition to the S0 state. The present work manifests that different selenium substitutions on uracil can lead to a certain extent of different vertical and adiabatic excitation energies, excited-state properties, and relaxation pathways. These insights could help understand the photophysics of selenium-substituted nucleobases.

Time-resolved dynamics in iodide-uracil-water clusters upon excitation of the nucleobase.

The dynamics of iodide-uracil-water (I-·U·H2O) clusters following π-π* excitation of the nucleobase are probed using time-resolved photoelectron spectroscopy. Photoexcitation of this cluster at 4.77 eV results in electron transfer from the iodide moiety to the uracil, creating a valence-bound anion within the cross correlation of the pump and probe laser pulses. This species can decay by a number of channels, including autodetachment and dissociation to I- or larger anion fragments. Comparison of the energetics of the photoexcited cluster and its decay dynamics with those of the bare iodide-uracil (I-·U) complex provides a sensitive probe of the effects of microhydration on these species.

Influence of Cooking (Microwaving and Broiling) on Cylindrospermopsin Concentration in Muscle of Nile Tilapia (Oreochromis niloticus) and Characterization of Decomposition Products.

Cylindrospermopsin (CYN) has become increasingly important as a freshwater algal toxin, showing cytotoxic effects. This toxin is able to bioaccumulate in freshwater food webs, representing a serious human health problem. Normally, fish is cooked before consumption, and CYN concentration can be altered. For the first time, the effects of microwaving and broiling for 1 and 2 min on CYN concentration and its decomposition products in fish muscle (Oreochromis niloticus) contaminated in the laboratory were investigated, using UPLC-MS/MS and Orbitrap. The results show that cooking the fish reduced unconjugated CYN levels by 11, 10 and 15% after microwaving for 1 and 2 min, and broiling for 2 min, respectively, compared to control fish. Different CYN decomposition products with m/z 416.1234 (7-epi-CYN) and m/z 336.16663 (diasteroisomers C-3A, C-3C, C-3D, C-3E, C-3F) are generated in fish samples submitted to cooking. Based on the relative abundance of the decomposition products, the possible degradation pathways taking place by microwaving may be through the formation of 7-epi-CYN and m/z 336.16663 compounds, whereas in the case of broiling the last route is the only one observed in this study. The influence of cooking and the toxicity characterization of the degradation products generated in CYN-contaminated fish are of importance for more realistic risk evaluation related to their consumption.

Photoinduced C-I bond homolysis of 5-iodouracil: A singlet predissociation pathway.

5-Iodouracil (5-IU) can be integrated into DNA and acts as a UV sensitive chromophore suitable for probing DNA structure and DNA-protein interactions based on the photochemical reactions of 5-IU. Here, we perform joint studies of time-resolved Fourier transform infrared (TR-FTIR) spectroscopy and ab initio calculations to examine the state-specific photochemical reaction mechanisms of the 5-IU. The fact that uracil (U) is observed in TR-FTIR spectra after 266 nm irradiation of 5-IU in acetonitrile and ascribed to the product of hydrogen abstraction by the uracil-5-yl radical (U·) provides experimental evidence for the C-I bond homolysis of 5-IU. The excited state potential energy curves are calculated with the complete active space second-order perturbation//complete active space self-consistent field method, from which a singlet predissociation mechanism is elucidated. It is shown that the initially populated 1(ππ*) state crosses with the repulsive 1(πσ*) or 1(nIσ*) state, through which 5-IU undergoes dissociation to the fragments of (U·) radical and iodine atom. In addition, the possibility of intersystem crossing (ISC) is evaluated based on the calculated vertical excitation energies. Although a probable ISC from 1(ππ*) state to 3(nOπ*) and then to the lowest triplet 3(ππ*) could occur in principal, there is little possibility for the excited state populations bifurcating to triplet manifold, given that the singlet state predissociation follows repulsive potential and should occur within dozens to hundreds of femtoseconds. Such low population of triplet states means that the contribution of triplet state to photoreactions of 5-IU should be quite minor. These results demonstrate clearly a physical picture of C-I bond homolysis of 5-IU and provide mechanistic illuminations to the interesting applications of 5-IU as photoprobes and in radiotherapy of cancer.

Excitation of nucleobases from a computational perspective II: dynamics.

This chapter is devoted to unravel the relaxation processes taking place after photoexcitation of isolated DNA/RNA nucleobases in gas phase from a time-dependent perspective. To this aim, several methods are at hand, ranging from full quantum dynamics to various flavours of semiclassical or ab initio molecular dynamics, each with its advantages and its limitations. As this contribution shows, the most common approach employed up to date to learn about the deactivation of nucleobases in gas phase is a combination of the Tully surface hopping algorithm with on-the-fly CASSCF calculations. Different dynamics methods or, even more dramatically, different electronic structure methods can provide different dynamics. A comprehensive review of the different mechanisms suggested for each nucleobase is provided and compared to available experimental time scales. The results are discussed in a general context involving the effects of the different applied electronic structure and dynamics methods. Mechanistic similarities and differences between the two groups of nucleobases - the purine derivatives (adenine and guanine) and the pyrimidine derivatives (thymine, uracil, and cytosine) - are elucidated. Finally, a perspective on the future of dynamics simulations in the context of nucleobase relaxation is given.

Photoionization spectroscopy of nucleobases and analogues in the gas phase using synchrotron radiation as excitation light source.

We review here the photoionization and photoelectron spectroscopy of the gas phase nucleic acid bases adenine, thymine, uracil, cytosine, and guanine, as well as the three base analogues 2-hydroxyisoquinoline, 2-pyridone, and δ-valerolactam in the vacuum ultraviolet (VUV) spectral regime. The chapter focuses on experimental work performed with VUV synchrotron radiation and related ab initio quantum chemical calculations of higher excited states beyond the ionization energy. After a general part, where experimental and theoretical techniques are described in detail, key results are presented by order of growing complexity in the spectra of the molecules. Here we concentrate on (1) the accurate determination of ionization energies of isolated gas phase NABs and investigation of the vibrational structure of involved ionic states, including their mutual vibronic couplings, (2) the treatment of tautomerism after photoionization, in competition with other intramolecular processes, (3) the study of fragmentation of these molecular systems at low and high internal energies, and (4) the study of the evolution of the covalent character of hydrogen bonding upon substitution, i.e., examination of electronic effects (acceptor, donor, etc.).

Photochemistry of nucleic acid bases and their thio- and aza-analogues in solution.

The steady-state and time-resolved photochemistry of the natural nucleic acid bases and their sulfur- and nitrogen-substituted analogues in solution is reviewed. Emphasis is given to the experimental studies performed over the last 3-5 years that showcase topical areas of scientific inquiry and those that require further scrutiny. Significant progress has been made toward mapping the radiative and nonradiative decay pathways of nucleic acid bases. There is a consensus that ultrafast internal conversion to the ground state is the primary relaxation pathway in the nucleic acid bases, whereas the mechanism of this relaxation and the level of participation of the (1)πσ*, (1) nπ*, and (3)ππ* states are still matters of debate. Although impressive research has been performed in recent years, the microscopic mechanism(s) by which the nucleic acid bases dissipate excess vibrational energy to their environment, and the role of the N-glycosidic group in this and in other nonradiative decay pathways, are still poorly understood. The simple replacement of a single atom in a nucleobase with a sulfur or nitrogen atom severely restricts access to the conical intersections responsible for the intrinsic internal conversion pathways to the ground state in the nucleic acid bases. It also enhances access to ultrafast and efficient inter-system crossing pathways that populate the triplet manifold in yields close to unity. Determining the coupled nuclear and electronic pathways responsible for the significantly different photochemistry in these nucleic acid base analogues serves as a convenient platform to examine the current state of knowledge regarding the photodynamic properties of the DNA and RNA bases from both experimental and computational perspectives. Further investigations should also aid in forecasting the prospective use of sulfur- and nitrogen-substituted base analogues in photochemotherapeutic applications.

UV-excitation from an experimental perspective: frequency resolved.

Electronic spectroscopy of DNA bases in the gas phase provides detailed information about the electronic excitation, which places the molecule in the Franck-Condon region in the excited state and thus prepares the starting conditions for excited-state dynamics. Double resonance or hole-burning spectroscopy in the gas phase can provide such information with isomer specificity, probing the starting potential energy landscape as a function of tautomeric form, isomeric structure, or hydrogen bonded or stacked cluster structure. Action spectroscopy, such REMPI, can be affected by excited-state lifetimes.

Excited states in DNA strands investigated by ultrafast laser spectroscopy.

Ultrafast laser experiments on carefully selected DNA model compounds probe the effects of base stacking, base pairing, and structural disorder on excited electronic states formed by UV absorption in single and double DNA strands. Direct π-orbital overlap between two stacked bases in a dinucleotide or in a longer single strand creates new excited states that decay orders of magnitude more slowly than the generally subpicosecond excited states of monomeric bases. Half or more of all excited states in single strands decay in this manner. Ultrafast mid-IR transient absorption experiments reveal that the long-lived excited states in a number of model compounds are charge transfer states formed by interbase electron transfer, which subsequently decay by charge recombination. The lifetimes of the charge transfer states are surprisingly independent of how the stacked bases are oriented, but disruption of π-stacking, either by elevating temperature or by adding a denaturing co-solvent, completely eliminates this decay channel. Time-resolved emission measurements support the conclusion that these states are populated very rapidly from initial excitons. These experiments also reveal the existence of populations of emissive excited states that decay on the nanosecond time scale. The quantum yield of these states is very small for UVB/UVC excitation, but increases at UVA wavelengths. In double strands, hydrogen bonding between bases perturbs, but does not quench, the long-lived excited states. Kinetic isotope effects on the excited-state dynamics suggest that intrastrand electron transfer may couple to interstrand proton transfer. By revealing how structure and non-covalent interactions affect excited-state dynamics, on-going experimental and theoretical studies of excited states in DNA strands can advance understanding of fundamental photophysics in other nanoscale systems.

Excited states behavior of nucleobases in solution: insights from computational studies.

We review the most significant results obtained in the study of isolated nucleobases in solution by quantum mechanical methods, trying to highlight also the most relevant open issues. We concisely discuss some methodological issues relevant to the study of molecular electronic excited molecular states in condensed phases, focussing on the methods most commonly applied to the study of nucleobases, i.e. continuum models as the Polarizable Continuum Model and explicit solvation models. We analyse how the solvent changes the relative energy of the lowest energy excited states in the Franck-Condon region, their minima and the Conical Intersections among the different states, interpreting the experimental optical spectra, both steady state and time-resolved. Several methods are available for accurately including solvent effects in the Franck-Condon region, and for most of the nucleobases the solvent shift on the different excited states can be considered assessed. The study of the excited state decay, both radiative and non-radiative, in solution still poses instead significant theoretical challenges.

Computational modeling of photoexcitation in DNA single and double strands.

The photoexcitation of DNA strands triggers extremely complex photoinduced processes, which cannot be understood solely on the basis of the behavior of the nucleobase building blocks. Decisive factors in DNA oligomers and polymers include collective electronic effects, excitonic coupling, hydrogen-bonding interactions, local steric hindrance, charge transfer, and environmental and solvent effects. This chapter surveys recent theoretical and computational efforts to model real-world excited-state DNA strands using a variety of established and emerging theoretical methods. One central issue is the role of localized vs delocalized excitations and the extent to which they determine the nature and the temporal evolution of the initial photoexcitation in DNA strands.

Modified nucleobases.

Various molecules which are similar to the natural nucleobases exist in nature or have been synthetically developed. In this chapter we review work on the photophysical properties of several modified nucleobases, focusing particularly on how these properties differ from those of the natural nucleobases. We discuss studies that give physical insight into how the molecular structure can be related to photophysical properties with many of these studies being theoretical. One useful photophysical property is the ability to fluoresce with high quantum yields. Natural bases practically do not fluoresce, so being able to design molecules that fluoresce is a goal of practical importance. Many of the modified nucleobases discussed in this review are fluorescent analogues, analogues that have very different fluorescent properties from the natural bases. The studies reviewed here may provide ways to design other analogues with a set of desired properties.

Photosynthesis and photo-stability of nucleic acids in prebiotic extraterrestrial environments.

Laboratory experiments have shown that the UV photo-irradiation of low-temperature ices of astrophysical interest leads to the formation of organic molecules, including molecules important for biology such as amino acids, quinones, and amphiphiles. When pyrimidine is introduced into these ices, the products of irradiation include the nucleobases uracil, cytosine, and thymine, the informational sub-units of DNA and RNA, as well as some of their isomers. The formation of these compounds, which has been studied both experimentally and theoretically, requires a succession of additions of OH, NH2, and CH3groups to pyrimidine. Results show that H2O ice plays key roles in the formation of the nucleobases, as an oxidant, as a matrix in which reactions can take place, and as a catalyst that assists proton abstraction from intermediate compounds. As H2O is also the most abundant icy component in most cold astrophysical environments, it probably plays the same roles in space in the formation of biologically relevant compounds. Results also show that although the formation of uracil and cytosine from pyrimidine in ices is fairly straightforward, the formation of thymine is not. This is mostly due to the fact that methylation is a limiting step for its formation, particularly in H2O-rich ices, where methylation must compete with oxidation. The relative inefficiency of the abiotic formation of thymine to that of uracil and cytosine, together with the fact that thymine has not been detected in meteorites, are not inconsistent with the RNA world hypothesis. Indeed, a lack of abiotically produced thymine delivered to the early Earth may have forced the choice for an RNA world, in which only uracil and cytosine are needed, but not thymine.

Excitation of nucleobases from a computational perspective I: reaction paths.

The main intrinsic photochemical events in nucleobases can be described on theoretical grounds within the realm of non-adiabatic computational photochemistry. From a static standpoint, the photochemical reaction path approach (PRPA), through the computation of the respective minimum energy path (MEP), can be regarded as the most suitable strategy in order to explore the electronically excited isolated nucleobases. Unfortunately, the PRPA does not appear widely in the studies reported in the last decade. The main ultrafast decay observed experimentally for the gas-phase excited nucleobases is related to the computed barrierless MEPs from the bright excited state connecting the initial Franck-Condon region and a conical intersection involving the ground state. At the highest level of theory currently available (CASPT2//CASPT2), the lowest excited (1)(ππ*) hypersurface for cytosine has a shallow minimum along the MEP deactivation pathway. In any case, the internal conversion processes in all the natural nucleobases are attained by means of interstate crossings, a self-protection mechanism that prevents the occurrence of photoinduced damage of nucleobases by ultraviolet radiation. Many alternative and secondary paths have been proposed in the literature, which ultimately provide a rich and constructive interplay between experimentally and theoretically oriented research.

Kinetics and mechanisms of cylindrospermopsin destruction by sulfate radical-based advanced oxidation processes.

Cylindrospermopsin (CYN) is a potent cyanobacterial toxin frequently found in water bodies worldwide raising concerns over the safety of drinking and recreational waters. A number of technologies have been investigated to remove and/or degrade cyanotoxins with advanced oxidation processes (AOPs) being among the most promising and effective for water detoxification. In this study, the degradation of CYN by sulfate radical-based UV-254 nm-AOPs was evaluated. The UV/S2O8(2-) (UV/peroxydisulfate) was more efficient than UV/HSO5(-) (UV/peroxysulfate) and UV/H2O2 (UV/hydrogen peroxide) processes when natural water samples were used as reaction matrices. The observed UV fluence based pseudo-first-order rate constants followed the expected order of radical quantum yields. The presence of 200 µM natural organic matter (NOM) as carbon slightly inhibited the destruction of CYN; 1.24 mg L(-1)NO3(-) (nitrate) had no significant influence on the removal efficiency and 50 µg L(-1) Fe(2+) [iron (2+)] or Cu(2+) [copper (2+)] improved the performance of UV/S2O8(2-). The addition of tert-butyl alcohol (t-BuOH; hydroxyl radical scavenger) in the reaction yielded byproducts that indicated specific sites in CYN preferentially attacked by sulfate radicals (SRs). The predominant CYN degradation byproduct was P448 consistent with fragmentation of the C5C6 bond of the uracil ring. The subsequent formation of P420 and P392 through a stepwise loss of carbonyl group(s) further supported the fragmentation pathway at C5C6. The byproduct P432 was identified exclusively as mono-hydroxylation of CYN at tricyclic guanidine ring, whereas P414 was detected as dehydrogenation at the tricyclic ring. The elimination of sulfate group and the opening of tricyclic ring were also observed. The possible degradation pathways of CYN by SR-AOP were presented.

Electron-induced hydrogen loss in uracil in a water cluster environment.

Low-energy electron-impact hydrogen loss due to dissociative electron attachment (DEA) to the uracil and thymine molecules in a water cluster environment is investigated theoretically. Only the A(')-resonance contribution, describing the near-threshold behavior of DEA, is incorporated. Calculations are based on the nonlocal complex potential theory and the multiple scattering theory, and are performed for a model target with basic properties of uracil and thymine, surrounded by five water molecules. The DEA cross section is strongly enhanced when the attaching molecule is embedded in a water cluster. This growth is due to two effects: the increase of the resonance lifetime and the negative shift in the resonance position due to interaction of the intermediate negative ion with the surrounding water molecules. A similar effect was earlier found in DEA to chlorofluorocarbons.

Degradation mechanism of cyanobacterial toxin cylindrospermopsin by hydroxyl radicals in homogeneous UV/H2O2 process.

The degradation of cylindrospermopsin (CYN), a widely distributed and highly toxic cyanobacterial toxin (cyanotoxin), remains poorly elucidated. In this study, the mechanism of CYN destruction by UV-254 nm/H2O2 advanced oxidation process (AOP) was investigated by mass spectrometry. Various byproducts identified indicated three common reaction pathways: hydroxyl addition (+16 Da), alcoholic oxidation or dehydrogenation (-2 Da), and elimination of sulfate (-80 Da). The initiation of the degradation was observed at the hydroxymethyl uracil and tricyclic guanidine groups; uracil moiety cleavage/fragmentation and further ring-opening of the alkaloid were also noted at an extended reaction time or higher UV fluence. The degradation rates of CYN decreased and less byproducts (species) were detected using natural water matrices; however, CYN was effectively eliminated under extended UV irradiation. This study demonstrates the efficiency of CYN degradation and provides a better understanding of the mechanism of CYN degradation by hydroxyl radical, a reactive oxygen species that can be generated by most AOPs and is present in natural water environment.