Influence of microhydration on the structures and proton-induced charge transfer in RNA intermediates
Solvation effects are of major interest in the context of radiation damage, due to their potential applications in cancer therapy. Reliable modeling of the solvent is, however, quite challenging, and numerous studies have been devoted to isolated biomolecules and stepwise-hydrated molecules in which the amount of solvent is controlled one molecule at a time. The influence of stepwise hydration on radiation damage is investigated here using the example of proton-induced charge transfer in two biomolecular targets. Uracil has been widely investigated both experimentally and theoretically in this context, and 2-aminooxazole was recently shown to be a potentially important intermediate in prebiotic chemistry. Focusing here on doubly hydrated biomolecules, stable structures and infrared spectra were obtained by combining the results of molecular dynamics simulations with those of quantum chemistry calculations performed at the density-functional theory level with the double hybrid M06-2X functional. The charge-transfer cross-sections upon proton impact were obtained from ab initio molecular calculations and after applying a semi-classical approach to investigate the collision. Our results suggest a significant relationship between the detailed hydration structure and the efficacy of proton-induced charge transfer, highlighting the competing roles of inter- and intramolecular hydrogen bonding.
KeywordsWater clusters Microhydration Charge transfer Proton collisions Molecular calculations RNA chemistry IR spectra Structure
Radiation damage in biological media has been widely investigated due to its applicability to cancer therapy treatments. Research efforts have increasingly been directed into understanding the different mechanisms at play at the molecular level—not only the interactions with the primary radiation but also those with secondary particles . Among such secondary processes, low-energy electrons have been shown to cause single- or double-strand breaks through dissociative electron attachment [2, 3], and secondary ions may ionize the biomolecules, possibly causing them to fragment. These indirect processes can contribute significantly to the decay of the medium [4, 5, 6].
In the context of ion-induced damage, gas-phase experiments provide a wealth of information about single collision events, providing access to the fragmentation pattern associated with the biomolecular target for a given impinging ion [7, 8]. The fragmentation cross-sections may be deduced from mass spectra and interpreted theoretically by determining the molecular orbitals involved in the process. Theory and modeling are invaluable for unraveling the dynamical and chemical mechanisms of the ionized target [9, 10]. Although ionization occurs very rapidly compared to the subsequent molecular rearrangement, it drives the entire mechanism either directly or through delayed charge transfer from the incident ion to the biomolecular target. This charge-transfer process is analyzed here, using a molecular representation and focusing on proton impact collisions, which have been widely investigated for related systems in previous studies [11, 12, 13]. In terms of gas-phase experiments, this approach has been successfully applied to isolated biomolecules—mainly DNA and RNA building blocks, pyrimidine nucleobases [14, 15], sugars [16, 17], and 5-halouracil targets . Other interesting molecules of prebiotic relevance such as 2-aminooxazole  have also been studied in this manner. Damage due to ion bombardment is a natural process in astrophysical environments, and charge transfer caused by the impact of protons can be an important source of decay in such environments, particularly in ionized clouds, where protons are particularly abundant. However, it is difficult to scale up the properties of isolated molecules to actual, incredibly complex biological media. In order to account for the presence of the water solvent, it is useful to disentangle the specific features of the solute from the solute–solvent interactions by hydrating the compound of interest one water molecule at a time. Mass spectrometry experiments have thus tried to bridge the gap between the gas and condensed phases by analyzing how hydration modifies the properties of the solute [20, 21]. In the context of radiation damage, where the contribution of the solvent may be very important, this bottom-up approach of controlled microhydration is of particular relevance. In the impact of low-energy electrons on uracil and thymine, dissociative electron attachment cross-sections have indeed been shown to be significantly enhanced if the molecule is embedded in a water cluster . We have also demonstrated that, in the case of uracil, this effect even occurs when just one water molecule is present . In contrast, the microhydration of 2-aminooxazole does not lead to such an enhancement , which we interpreted as resulting from the very different hydration patterns exhibited by the two molecules. These results suggest intriguing effects of the detailed hydrogen-bonding structure on the charge-transfer efficiency upon proton impact, which are all the more important because multiple isomers could be populated at a finite temperature—as in a real liquid solvent. In view of those results, we undertook in the present paper a systematic exploration of the influence of the conformer on the charge transfer in uracil and 2-aminooxazole following proton collisions. Doubly hydrated compounds were selected as they provide a wide variety of possible hydration patterns with competing solute–solvent and intrasolute hydrogen-bonding networks.
We followed a similar protocol to that used in our earlier works [23, 24] by first exploring the conformational diversity of the dihydrated biomolecules, using a well-established hierarchical protocol that relies on a force field to generate many candidate geometries which are subsequently refined at a more accurate level of quantum chemistry using an explicit description of electronic structure. The various theoretical approaches, including those used to compute the charge-transfer cross-sections, are detailed in the next section of the paper. The results for uracil and 2-aminooxazole are then presented and discussed in the third section, before some concluding remarks are made in the final section.
Structures and IR spectra
The complex energy landscapes of the doubly hydrated uracil and 2-aminooxazole molecules were first explored using the Amber force field (ff99 parameters)  in replica-exchange molecular dynamics (REMD) simulations, periodically saving and locally minimizing the configurations that occurred during the simulations. The REMD strategy is particularly well suited to moderately large systems, as it allows large energy barriers to be surmounted in a much shorter time than when a conventional molecular dynamics approach is used. Although reasonable, the Amber force field does not possess chemical accuracy, so the resulting structures were subsequently locally reoptimized using a more realistic method with an explicit description of electronic structure. Here, density-functional theory (DFT) with the global hybrid meta-GGA functional M06-2X  was employed as our method of choice, and the triple-zeta basis set aug-cc-pVTZ was applied; diffuse functions were included mainly to obtain better descriptions of the ionic systems.
For all stable structures, we considered the relative binding energies relative to the global minimum. Adiabatic and vertical ionization energies as well as adiabatic and vertical electron affinities were calculated at the same level of density-functional theory. Electronic structure calculations also provided information about the vibrational spectra of the microhydrated compounds for possible future experimental characterization. Infrared (IR) spectra were determined in the double harmonic approximation, and the frequencies in the mid-IR range were scaled by 0.95 to account for anharmonicities and nuclear delocalization corrections at the most basic level.
The collisional system formed by the impinging proton and the biomolecular target was treated in the one-dimensional reaction coordinate framework conventionally used for complex systems [27, 28]. The collision dynamics were simulated with a semi-classical approach using the EIKONXS code  to an accuracy of 10−4 for the symmetry of the S matrix. As the collision process is very fast, the dynamics were developed using the sudden approximation hypothesis, which considers vibrational and rotational motions to be frozen during the collision. This approximation is valid for keV impact energies, for which the nuclear vibrational time and (a fortiori) the rotational periods are about two orders of magnitude longer than the collision time . Building upon a recent analysis of time-dependent quantum wavepacket and semiclassical approaches to charge-transfer processes , the present semiclassical approach may be extended to lower collision energies. We developed the calculations in the 10 eV to 10 keV collision energy range. For all dihydrated compounds, the biomolecules exhibit an almost planar ring, and it has been widely shown that charge transfer is strongly enhanced perpendicular to the ring [14, 15]. To achieve a better comparison of the various isomers, the proton-induced charge transfer was only evaluated in the perpendicular z orientation, averaging the results for the two directions (+z and −z) when calculating the final value of the cross-section.
In practice, a step size Δ = 0.0012 a.u. was found to lead to a stable differentiation procedure .
Results and discussion
The effects of hydration on the uracil nucleobase have been widely investigated using various experimental and theoretical approaches [35, 36, 37, 38, 39, 40]. In the context of radiation damage, interest has recently focused on reactions in which electrons or protons are generated along the radiation track. These particles drive biomolecule excitation, resulting in the ionization of the biomolecule and possibly its subsequent decay via fragmentation. The dissociative electron attachment cross-sections appear to be strongly modified by solvation . Our earlier analysis  of charge transfer following the collision of protons with a series of uracil@(H2O)n = 1–5 clusters showed that the charge-transfer efficiency increases sharply when just one water molecule is added to the bare uracil. This enhancement can be associated with the polarization of the molecular orbital that is mainly involved in the charge-transfer mechanism in the presence of water. Considering the peripheral hydration patterns observed for uracil@(H2O)n clusters, such polarization effects remain largely unchanged as the microhydration level is increased, despite a decrease in the cross-section as the number of water molecules reaches n = 5 . Therefore, a more detailed analysis of this microhydration effect is required to understand this process in depth. The case with n = 2 studied here provides a great diversity of hydration patterns, allowing us to gain further insight into the specific role of structure in the charge-transfer process.
Relative energies, vertical and adiabatic electron affinities (EA), and vertical and adiabatic ionization energies (IE) of uracil@(H2O)2 compounds, as obtained from quantum chemical calculations performed at the DFT/M06-2X/aug-cc-pVTZ level. The relative values obtained with the Amber ff99 force field are also reproted
Energy (kcal mol−1) using Amber ff99
Energy (kcal mol−1) using DFT/M06-2X
Vertical EA (eV)
Adiabatic EA (eV)
Vertical IE (eV)
Adiabatic IE (eV)
The structures of the different families A, B, C, or AC are separated by larger energies, which confirms that the hydrogen-bonding topology is the factor that determines the stabilities of the various conformers. The most favorable hydration pattern (A and A′) is clearly associated with hydrogen bonds that include N–H as the proton donor and oxygen as the proton acceptor. It is worth noting an interesting evolution between structures A, AC, and C from structural and energetic aspects, the stepwise displacement of a water molecule from region A to region C destabilizing the cluster by about 1.0 kcal mol−1. Both A and C are, however, relatively similar regions, offering an N–H group as a proton donor and an oxygen as a proton acceptor, with the proximity of a free C=O bond acting as a destabilizing factor. A similar remark could be made for the B family, which is clearly energetically disfavored and shows a free C=O bond together with a free N–H bond, which is closely related to the locked-chain arrangement of water molecules.
For the different uracil@(H2O)2 clusters, the electron affinities and ionization energies are not very sensitive to the region occupied by the water molecules. However, noticeable variations occur between the vertical and adiabatic values for the ionization energies, and especially for the electron affinities. While the adiabatic EAs are positive and rather low, vertical values are markedly negative. This indicates that a major rearrangement occurs upon the addition of an electron to the cluster, leading to significant stabilization of the anion. Likewise, noticeable rearrangement occurs upon the removal of an electron, as evidenced by the difference of about 0.6 eV between the vertical and adiabatic values of the ionization energy. The planar uracil@(H2O)2 structures thus appear to very efficiently stabilize an extra electron and (albeit to a lesser extent) the cationic cluster.
In general, the two conformers of the same topological family A, B, C, or AC exhibit nearly identical spectra, and it is sufficient to discuss the differences among families. The spectrum for family A appears to be very similar to the bare uracil spectrum, with the N–H stretching mode at almost the same position (around 3100 cm−1) and the OH modes in the 3300–3400 cm−1 range. The hydrogen bonds created by the presence of the water chain in this region do not significantly influence the IR spectrum, which looks nearly identical to the spectrum of the bare biomolecule across the entire spectral domain. In contrast, for the locked-chain structures in the B and C regions, clear shifts are observed towards lower wavelengths for the N–H stretching mode and towards higher wavelengths for the O–H modes (due to the increased sensitivity upon pivoting one out-of-plane O–H mode to the other side of the uracil plane). The spectrum is quite different for the AC–AC′ species, in which both N–H bonds are connected to water molecules, and N–H stretching modes near 3000–3100 cm−1 vanish during the process. Structures from the AC family also show the strongest contrast with the bare molecule in the bending mode region, with the absence of intrawater bonding clearly demonstrated by the presence of the intense peak near 1600 cm−1, which is exclusive to these conformers.
Although they are all planar, the different conformers exhibit significant variations in charge transfer depending on the hydration region. The results obtained for the various structures can be grouped into two main sets depending on the magnitude of the charge-transfer cross-section. All conformers of the types A, AC, and C, irrespective of the relative orientations of the two water molecules away from the uracil plane, show charge-transfer cross-sections of the same order of magnitude as bare uracil. The charge-transfer process is somewhat more efficient for the A and A′ compounds, in particular at lower collision energies, but the mean cross-section values are roughly similar for A′, C–C′, and bare uracil across the entire collision energy domain, with the cross-section increasing slightly with increasing energy. For the AC conformers, the cross-section values are between those of A and C. Interestingly, the successive jumping of water molecules from A to C through AC, which energetically destabilizes the cluster, also decreases the charge-transfer cross-section in a stepwise fashion. From a chemical point of view, the A and C regions are very similar, and both can induce efficient hydrogen bonds with an N–H group as proton donor and an oxygen as proton acceptor. The main difference comes from the presence of a free C=O bond in the C geometry, which happens to be a destabilizing factor and is found to slightly reduce the charge-transfer efficiency. However, the effect is quite minor. Conversely, isomers from the B family display approximately one magnitude higher cross-sections than that of bare uracil, which remain almost constant with increasing collision energy. In these higher-energy conformers, the presence of equidistant C=O and N–H bonds thus seems to favor charge transfer despite destabilizing the system. The process is clearly very sensitive to the structure of the target, and may be employed as a tool for structural analysis.
Interest has recently focused on 2-aminooxazole in the context of prebiotic chemistry. This molecule has been suggested as a possible key intermediate in an efficient reaction sequence leading to the formation of RNA under prebiotic conditions . The chemical mechanism—which initially involves simple molecules such as cyanamide and glycolaldehyde—could be related to the chemistry of HCN, which is pivotal to our understanding of the processes that led to the development of life . The microwave spectrum of 2-aminooxazole has been investigated theoretically , and photochemical processes have been proposed to explain the formation of the isolated molecule as well as hydrated clusters in astrophysical environments [44, 45, 46]. In this respect, we recently studied the evolution of 2-aminooxazole in microhydrated environments  and, notably, its response to proton impact, focusing on the effects of stepwise hydration rather than the details of the microhydrated structures. Microhydration effects are particularly relevant for prebiotic compounds which have been hypothesized to form in extraterrestrial environments—a theory supported by the discovery of aminoacids on meteorites . However, it is unclear how they could survive in space and, in particular, how they could resist from proton damage. This motivated us to investigate radiation damage on aminooxazole in bare  and microhydrated  forms. Our results indicate that 2-aminooxazole@(H2O)n clusters exhibit a segregated structure with a water cluster on the amino side group, which leads to a weaker effect on the charge-transfer efficiency than observed for uracil@(H2O)n . Smaller 2-aminooxazole@(H2O)n structures were found to be very similar to the corresponding microhydrated uracil compounds, with a locked-chain arrangement on the side of the biomolecule. However, these results are in significant conflict with those obtained in another recent theoretical study by Szabla and coworkers , who, despite predicting a clear segregation between the biomolecule and the cluster, found that this cluster preferred to be on the oxygen side instead of the nitrogen side. This provides additional justification for investigating the detailed microhydrated structures of dihydrated 2-aminooxazole and discriminating the various hydration patterns.
Relative energies, vertical and adiabatic electron affinities (EA), and vertical and adiabatic ionization energies (IE) of 2-aminooxazole@(H2O)2, as obtained from quantum chemical calculations performed at the DFT/M06-2X/aug-cc-pVTZ level. The relative values obtained with the Amber ff99 force field are also reported
Energy (kcal mol−1) using Amber ff99
Energy (kcal mol−1) using DFT/M06-2X
Vertical EA (eV)
Adiabatic EA (eV)
Vertical IE (eV)
Adiabatic IE (eV)
Clearly the conformers A and A′ are much lower in energy than all the other local minima, a result which supports the data we obtained in a previous investigation in which we explored a broader range of cluster sizes but fewer competing structures . Placing the water molecules on the oxygen side is significantly energetically disfavored (by more than 5 kcal mol−1). As previously observed for uracil@(H2O)2, the cluster is stepwise destabilized when each water molecule jumps from A to C. More generally, the formation of simultaneous hydrogen bonds with the nitrogen of the ring and the amino group appears to be energetically preferred, although we note that the near-planar configurations (A and A′) are much more favorable than the out-of-plane arrangement A2, with the single-bonded conformer A1 being (unsurprisingly) even less stable. Finally, moving the water molecule connected to the amino group in A–A′ to the opposite carbon in the cycle in B–B′ destabilizes the cluster by more than 5.5 kcal mol−1, which again emphasizes the importance of the nitrogen component of the biomolecule.
The ionization energies given in Table 2 show similar behavior to that presented in Table 1 for uracil@(H2O)2 clusters. Despite being about 1 eV lower than for dihydrated uracil, the ionization energies of 2-aminooxazole@(H2O)2 also show rather low sensitivity to the conformer, as well as a comparable difference of about 0.7 eV between the vertical and adiabatic IEs. These results suggest that there is rearrangement upon the removal of an electron, as also noted for uracil@(H2O)2 clusters. The electron affinity depends also only weakly on the conformer of 2-aminooxazole@(H2O)2, and the adiabatic and vertical values are about the same. The addition of one electron therefore does not induce major rearrangement, which indicates the presence of a diffuse electron cloud at the periphery of the cluster. This behavior contrasts markedly with our findings for uracil@(H2O)2 clusters, which were found to accommodate the extra electron very efficiently. This difference is likely related to the presence of the amino group on the five-membered ring of oxazole. Indeed, since the presence of this chemical group was found to lead to segregated structures with a water cluster on the side of the amino group , it may also stabilize a peripheral extra electron, which would explain the contrasting trends.
A comparative analysis of the 2-aminooxazole@(H2O)2 conformers was also carried out in relation to proton-induced charge transfer, focusing only (as also done for dihydrated uracil) on collisions perpendicular to the biomolecular cycle and averaging the cross-sections in the two directions +z and −z. It is more important to account for both orientations here than for uracil, because the amino side group leads now to a nonplanar molecule.
As the temperature increases, populations of the higher-energy conformers should become more prominent. As previously noted for uracil clusters, stepwise jumping of a water molecule from region A to region C should therefore occur for 2-aminooxazole. Also similar to uracil, this thermal excitation leads to a decrease in the charge-transfer cross-section. However, in contrast to uracil, the two-water chain on the oxygen side promotes charge transfer. The presence of the hydrogen-bond chain between the ring oxygen and the amine group thus appears to be a favorable destabilizing factor. This is consistent with the mechanism of electron-driven proton transfer along H2O chains proposed by Szabla and coworkers .
Microhydrated biomolecules bridge the gap between isolated compounds in the gas phase and their counterparts in a biological medium. The insight attained by studying stepwise hydration with a controlled number of water molecules is particularly useful for disentangling the roles of solute–solvent and solvent–solvent interactions, although extrapolation to the bulk limit is never straightforward. The present work was performed to theoretically investigate two small biomolecules of relevance to RNA chemistry, namely uracil and 2-aminooxazole (recently suggested to be a prebiotic precursor), both of which were placed in contact with just two water molecules so that a near-exhaustive exploration of their energy landscapes could be achieved. The stable structures generated using density-functional theory with the M06-2X functional and the triple-zeta basis set aug-cc-pVTZ have the water chain localized at the side of each molecule, and contrast with earlier findings present in the literature, at least in the case of 2-aminooxazole. There is generally a marked correlation between the hydration pattern, the electronic properties (the electron affinity and ionization potential in particular), and the IR spectrum, although it is unclear whether the vibrational response could be used to experimentally characterize the most stable conformers. In many cases, the localization of the water molecules in the biomolecular plane should also lead to some partial disorder, as the dangling water O–H bonds that are not connected to the biomolecule are relatively free to move between the sides of the plane.
The charge-transfer cross-sections following the impact of energetic protons were also calculated for collisions perpendicular to the biomolecular cycle and along the two possible directions. We generally found that the cross-section significantly depended on the hydrogen-bonding pattern, but that there were interesting differences between the two biomolecular examples chosen. In uracil, the addition of a couple of water molecules markedly increases the charge-transfer efficiency for all conformers. Conversely, 2-aminooxazole is more chemically heterogeneous, and charge transfer can be enhanced or disfavored depending on the conformer, with the net effect of the addition of water molecules being much weaker than for uracil.
The contrasting behaviors of the two systems considered here highlight the complexity of the charge-transfer processes involved in radiation damage and their interplay with the chemical structure of the complex involved. They also emphasize the need to carefully evaluate the diversity of conformers in the energy landscape, given that the most stable structure is essentially impossible to predict based on intuition only, and hydrogen-bonding patterns involving both the solute and solvent have similar orders of magnitude. The present work could be extended along several possible lines, starting with investigations of larger sizes and bulk icy surfaces , which are particularly relevant in the context of interstellar grains .
We acknowledge computational support from the CCIN2P3 in Villeurbanne and the CCRT/CINES/IDRIS under the allocation x2016087662 made by GENCI. The authors also acknowledge also support from the COST actions TD1308 Life-Origins, CM1204 XLIC, and CM1401 Astrochemical History.
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