State-Dependent Fragmentation of Protonated Uracil and Uridine

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A: Spectroscopy, Molecular Structure, and Quantum Chemistry

State-Dependent Fragmentation of Protonated Uracil and Uridine Martin Pitzer, Christian Ozga, Catmarna Küstner-Wetekam, Philipp Reiß, André Knie, Arno Ehresmann, Till Jahnke, Alexandre Giuliani, and Laurent Nahon J. Phys. Chem. A, Just Accepted Manuscript • DOI: 10.1021/acs.jpca.9b01822 • Publication Date (Web): 03 Apr 2019 Downloaded from http://pubs.acs.org on April 6, 2019

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The Journal of Physical Chemistry

State-dependent Fragmentation of Protonated Uracil and Uridine Martin Pitzer,∗,† Christian Ozga,‡ Catmarna Küstner-Wetekam,‡ Philipp Reiß,‡ André Knie,‡ Arno Ehresmann,‡ Till Jahnke,¶ Alexandre Giuliani,§ and Laurent Nahon∗,§ †Department of Chemical and Biological Physics, Weizmann Institute of Science, Rehovot, Israel ‡Institute of Physics and Center for Interdisciplinary Nanostructure Science and Technology (CINSaT), University of Kassel, Germany ¶Institute for Nuclear Physics, Goethe-University, Frankfurt, Germany §Synchrotron Soleil, Gif-sur-Yvette, France E-mail: [email protected]; [email protected] Phone: +972 (0)8 934 2927. Fax: +972 (0)8 934 4123

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Abstract Action spectroscopy using photon excitation in the VUV range (photon energy 4.5−9 eV) was performed on protonated uracil (UraH+ ) and uridine (UrdH+ ). The precursor ions with m/z 113 and m/z 245 respectively were produced by an Electrospray Ionization source (ESI) and accumulated inside a quadrupole ion trap mass spectrometer. After irradiation with tunable synchrotron radiation, product ion mass spectra were obtained. Fragment yields as a function of excitation energy show several maxima that can be attributed to the photo-excitation into different electronic states. For uracil, vertically excited states were calculated using the equation-of-motion coupled cluster approach (EOM-CCSD) and compared to the observed maxima. This allows to establish correlations between electronic states and resulting fragment masses and can thus help to disentangle the complex de-excitation and fragmentation pathways of nucleic acid building blocks. Photofragmentation of the nucleoside uridine shows a significantly lower variety of fragments, indicating stabilization of the nucleobase by the attached sugar.

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Introduction Many experimental techniques have been used to investigate the interaction of electromagnetic radiation with building blocks of DNA (desoxyribonucleic acid) and RNA (ribonucleic acid). Understanding the response of these small molecular species to the absorption of an energetic photon (from near to vacuum ultra-violet) may provide insight into radiation damage of living tissue and may also have astrobiological implications regarding the stability of these compounds in the interstellar medium. For better comparison with theoretical models, investigating isolated - i.e. solvent-free - molecules in the gas phase is often the method of choice. Early works on nucleobases and related compounds include absorption measurements of neutral gas phase molecules with lamp-based spectrophotometers in a vapor cell 1 or resonanceenhanced multiphoton ionization (REMPI) from a supersonic jet; 2 the observed absorption maxima have been interpreted with the help of quantum chemical calculations. 3 Photoelectron spectroscopy has revealed details of the electronic structure, 4 by means of time-resolved 5 or highly energy-resolved 6–8 measurements. Mass spectrometry has been used in various approaches to determine ionization energies of biologically relevant molecules and appearance energies of their fragments. 9 For recent reviews on the interaction of neutral nucleic acid building blocks with UV and X-ray radiation, see Ref. 10,11 As an intermediate between neutral gas-phase molecules and species in solution, protonated and microhydrated molecules are often considered to better represent in vivo environmental conditions than their neutral counterparts. 12 Cation photodissociation processes provide an additional benefit for mass spectrometric (MS) investigation: Even at low excitation energy, charged fragments are produced that can easily be detected and quantified by MS, contrary to the dissociation products of neutral molecules. Many works have been based on quadrupole traps to select a charged precursor which is then photoexcited by a UV-laser (see e.g. the reviews 13,14 ). Protonated nucleobases have extensively been studied with such set-ups, and in particular the role of microsolvation was investigated, 15,16 as well as the effect 3



of different tautomers 17–20 and the excited state relaxation dynamics. 21 However, by using near and middle UV photons available from these sources, only the first and the onset of the second singlet excited electronic state can be probed. As a consequence, only the break-ups with relatively low dissociation energy are visible. In this paper we present a complete study over the entire VUV range, showing the photodynamics of uracil and its nucleoside uridine up to 9 eV photon energy.

Experiment The experiments were performed with the SRMS2 setup 22 on the DESIRS beamline 23 at Soleil synchrotron facility (France), using photons of variable energy for activation - an approach frequently designated as action spectroscopy. The protonated species were created upon electrospray ionization. For uracil, 0.3 mM aqueous solution, and for uridine, 1 mM aqueous solution were used. The precursor ions were accumulated for 50 ms in the trap and then irradiated with linearly polarized photons for 5 s. Even with this irradiation time, the most abundant fragments reached less than 1 % of the precursor yield, ruling out absorption of a second photon by the fragments. In order to maximize the photon flux (∼ 1012 photons/s), we used wide monochromator slits corresponding to a bandwidth of typically 9 meV. Several photon energy scans were performed in 100 meV steps; at each photon energy, mass spectra were recorded for four minutes, yielding 45 fragment spectra per photon energy that were averaged. All spectra were normalized for the photon flux that was measured with a photo-diode prior to the experiments. To avoid ionization and fragmentation by higher orders of the synchrotron radiation, a suprasil window was used in the lower energy range (4.5 − 7.1 eV), and a krypton gas filter in the higher energy range (7 − 9.5 eV). This change in settings leads to differences in photon flux for the two scan regions. The relative scaling of the measured fragment yields between these two photon energy regions was found by requiring consistent values at the overlapping

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photon energies (7.0 − 7.1 eV). Below 5 eV, the photon flux drops significantly (see SI, Fig. S1) so that only the strongest fragmentation channels yield meaningful results; above 9.5 eV, ionization of neutral molecules from the residual gas or from contaminants in the source leads to a background which prevents a quantitative analysis. We therefore limited our study to the 4.5 − 9.5 eV range. The main source of uncertainty in the present measurements are fluctuations between individual mass spectra recorded at the same photon energy, i.e. statistical variations in particle numbers due to the low abundance (“shot noise”). We therefore use the standard deviation √ of the mean (σ/ N ) of the fragment yield for the error bars. It can be seen that the uncertainty is higher for low photon energy where the fragment yield is low, and that it is comparable for different masses at a given photon energy. Another source of error can originate from the calibration of the photon flux: Since the transmission of the suprasil window drops drastically above 7eV (see Supporting Information (SI), figure S1), slightly different conditions for the reference and the actual measurement can introduce additional errors in the normalization particularly in this region. For an overview, the resulting data can be displayed in a two-dimensional fragment map (see Results and Discussion), with the fragment mass-to-charge ratio m/z on the x-axis and the excitation energy on the y-axis. This representation allows to identify interesting fragment masses, while plotting the yield of selected fragments as a function of photon energy enables a more quantitative spectroscopic analysis.

Computational Details Calculation of excited state energies was performed with GAMESS(US). 24 So far, only few theoretical works are available that deal with electronically excited states of nucleobases in the measured energy range. Epifanovsky et al. 25 studied in detail the effect of the basis set and the choice of the excitation algorithm for neutral uracil. Before calculating the excitation

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energies of protonated uracil (which has the same number of electrons and - as is generally assumed for protonated nucleobases 16,21,26 - the same planar Cs symmetry) we benchmarked our numerical settings against those results. For geometry optimization of the ground state, density functional theory (DFT) with the B3LYP functional 27–29 and the 6-311++G(2d,2p) basis set 30 was used. Vertical excitation energies were calculated with EOM-CCSD (equation-of-motion coupled cluster with single and double excitation) 31–33 and the 6-311+G(d,p) basis set. These parameters were found to yield reliable results for the first few states (both for singlet and triplet states). States with larger Rydberg state contribution (n ≥ 3) are very sensitive to the basis set and the level of theory 25 are therefore omitted here. Excited state optimization or vibrational analysis was beyond the scope of this work due to the expected complex shape of the potential energy landscape. 17

Figure 1: Structures of uracil and uridine. Top row: The two energetically lowest tautomers of protonated uracil. The enol–enol/2H–4H form (u178) is considered the ground state, while the enol–keto/3H–4H (u138) contributes 1 to 20 % of the population at room temperature, depending on the model. Bottom row: Fischer projection of neutral uridine. For a discussion of protonation sites and conformations of uridine, see Ref. 34

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Protonated uracil exists in several tautomers. 16,19,26 In our calculation, we consider the two structures that are depicted in Figure 1. They have been consistently identified as the two energetically lowest tautomers in the cited works. Estimates of the enthalpy difference and the relative population vary strongly depending on the theoretical model used. The higher-lying tautomer u138 (enol–keto form) is estimated to contribute between 1 and 20 % to the population at room temperature. 16

Results and discussion Uracil Figure 2 gives an overview of the fragmentation pattern of protonated uracil (C4H5N2O2+) as a function of excitation energy. The fragment mass-to-charge ratio m/z is plotted on the x-axis, the excitation energy on the y-axis. The left panel shows a projection of the spectrum, i.e. the total fragment yield as a function of excitation energy (see SI, Fig. S2 for a comparison to extinction measurements). A mass spectrum at fixed energy (6.7 eV) is given in the SI as well (Fig. S3). On the lower end of the energy spectrum, several peaks

Figure 2: Two-dimensional fragment map after photoexcitation of protonated uracil (precursor m/z 113, not shown because of high abundance). The fragment mass is plotted on the x-axis, the excitation energy on the y-axis. The color indicates the relative yield, normalized for the photon flux. Several masses with appearance energies between 5 and 7 eV can be identified. The left panel displays the integrated signal over all fragments with 30 < m/z < 111.

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around 5 eV and a broader region from 6 to 7.5 eV can be discerned. Figure 3 displays the yield of four fragments with low appearance energy. The repeated measurements at 7 eV mark the overlap of two energy scans with different settings (see Experimental section). The fragment masses shown here are usually considered as NH3 loss (m/z 96), H2O loss (m/z 95), HNCO loss (m/z 70); 16,19,26,35 the fragment m/z 43 is attributed to either HNCO+ 16 or C2H3O+. 35 Due to an instrumental low-mass cut-off at m/z 30, we could not observe the fragment m/z 18 that was reported previously. 16 The vertical bars at the top of the figure indicate the calculated vertical excitation energies

Figure 3: Fragment yields of protonated uracil as a function of excitation energy. Only fragments that exhibit a significant yield at low energy (5 eV) are displayed. The error bars represent the standard deviation of the mean of the individual spectra (see Experimental Section); the overlapping values at 7 eV are due to a change in beamline settings. The vertical bars at the top indicate the vertical excitation energies calculated with EOM-CCSD for the two tautomers. The observed fragments are ascribed as follows: m/z 96: NH3 loss; m/z 95: H2O loss; m/z 70: HNCO loss; m/z 43: HNCO+ or C2H3O+. for the two considered tautomers. The calculated excitation energies and oscillator strengths with respect to the ground state are summarized in Table 1. For completeness, triplet state energies are shown in the table as well; since the electronic ground state has singlet character, transitions into these states are forbidden and omitted in the figures. The strong peak 8


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Table 1: Vertical excitation energies ∆E (singlet and triplet) and oscillator strengths f (singlet) for excitation from the ground electronic state. Calculations were performed for the two energetically lowest tautomers (see Fig. 1), using EOM-CCSD with the 6-311+G(d,p) basis set. state 1 3 A0 2 3 A0 1 1 A0 1 3 A00 1 1 A00 2 1 A0 2 1 A00

tautomer u178 ∆E (eV) f 4.332 forbidden 5.060 forbidden 5.564 0.096 6.018 forbidden 6.169 8 · 10−4 6.498 0.069 6.996 0.005

tautomer u138 ∆E (eV) f 3.695 forbidden 5.420 forbidden 4.607 0.108 5.994 forbidden 6.459 8 · 10−6 6.913 0.094 7.411 0.005

around 5 eV that has been observed previously 16,19 is usually attributed to the first excited singlet state S1 with the term symbol 1 1 A0 . By cooling the protonated ions to cryogenic temperatures (20 − 50 K), Berdakin et al. 19 were able to disentangle the contributions of the two tautomers and the vibrational structure of this state. Using the empirical formula Eexp ≈ Evert − 0.5 eV, they attribute the region between 4.8 and 5.4 eV to the first excited singlet state of the u178-tautomer, and a region between 3.9 and 4.5 eV (not visible in our data) to the u138-tautomer. This rule accounts for the fact that no excited state optimization has been performed when calculating the vertical excitation energy, and seems to be in line with our observations. Note that it is, however, contradictory to the observation that fragment yields can peak at higher energies than the vertical excitation energy due to activation barriers for the dissociation and a kinetic shift of the reaction. 11 Such processes, from our experiment/model comparison, seem negligible in the present case of uracil. It should be noted that the lowest excited state of neutral uracil is a so-called dark state, due to its 1 A00 symmetry and the concomitant small oscillator strength for transition from the symmetric ground state. For both considered tautomers of protonated uracil, this dark state is higher in energy than the bright 1 1 A0 state, so that the first maximum can be discussed without referring to complications such as intensity-borrowing by a lower-lying state. 25,36

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Figure 4: Fragment yields of protonated uracil with appearance energies above 5.5 eV, with error bars analogous to 3. The calculated vertical excitation energies are again drawn as gray bars. The fragment m/z 70 is sketched for better comparison with the low-energy fragments. Concerning the identification of the observed masses, see Table 2. In addition to this first peak, three partly overlapping peaks can be distinguished at 6.0, 6.6 and 7.2 eV. Considering the small oscillator strength for the transition to the 1 1 A00 state, the most likely assignment for the first two peaks is 6.0 eV ⇔ 2 1 A0 (u178) and 6.6 eV ⇔ 2 1 A0 (u138), shifted again by ≈ 0.5 and ≈ 0.3 eV respectively compared to the calculated vertical excitation energy. The third peak could be related to the 2 1 A00 of either tautomer, but additional higher-lying states or extended vibrational bands could play a role as well. Although all four masses follow the trend, differences are visible: While m/z 70, m/z 95 and m/z 96 show the strongest yield for the first observed excitation, m/z 43 is much more pronounced for higher energy. The large uncertainty at low photon energies, however, prevents a more quantitative analysis.

Figure 4 shows additional fragments that have previously not been observed after photoexcitation of protonated uracil. They do not occur below 5.5 eV, indicating that they

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cannot originate from the first electronically excited state. From DFT calculations, 26 the fragment cation m/z 42 was attributed to H2CCNH2+ and m/z 67 as C3N2H3+; in that work, the two isomers HOCNH+ and OCNH2+ are proposed for m/z 44 and various isomers of C3NOH2 for m/z 68. This chemical formulae are consistent with an early study using isotope-labelled species 35 that additionally identified m/z 53 as C3OH+. It should be noted that Sadr-Arani et al. 26 assumed partly different tautomers. The previously discussed fragment m/z 70 is inserted to allow a better comparison with Figure 3. In addition to the higher appearance energy, these fragments show slightly different features between 6 and 8 eV compared to the fragments in Figure 3: Most strikingly, the peak at 7.2 eV is as prominent or even higher than the one at 6.6 eV whereas for the fragments with low appearance energy, the latter is dominant. Following the previous assignment, this means that the “high-energy” fragments predominantly decay from 2 1 A00 state. Since the calculations suggest a much higher transition probability into the 2 1 A0 state, we cannot exclude that high vibrational excitations play also a role in producing these fragments. In addition, it is remarkable that the yields of the “high-energy” fragments show a similar trend as function of the excitation energy. Finally, we compare the grouping into “high-energy” and “low-energy” fragments to primary Table 2: Identification of fragments and fragmentation pathways in collisioninduced dissociation (CID) 26,35 grouped according to appearance at low photon energy (top) or high photon energy (bottom) in the current study. m/z 96 95 70 43 68 67 53 44 42

chem. formula (CID) C4H2NO2+ C4H3N2O+ C3H4NO+ C2H3O+ C3NOH2+ C3N2H3+ C3OH+ H2NCO+ C2NH4+

suggested C4H5N2O2+ C4H5N2O2+ C4H5N2O2+ C4H5N2O2+ C4H5N2O2+ C4H5N2O2+ C4H5N2O2+ C4H5N2O2+ C4H5N2O2+

decay mechanism (CID) - NH3 - H 2O - HNCO - HNCO - HCN - NH3 - CO - H 2O - CO - HNCO - NH3 - C3H3NO - HNCO - CO

and secondary fragments as classified in a thorough CID study using various combinations 11



of oxygen and carbon isotopes, as well as thio- and methyl-derivatives of uracil: 35 There, the cations with masses m/z 96, 95, 70 and possibly 44 were attributed to a primary loss of neutral groups as discussed above. The cations m/z 68, 67, 53, 52, 43, 42 and 28 were identified as products from a secondary decay of the former fragments. Except for the cations with m/z 43, 44 and the unobserved fragments m/z 52, 28, the primary fragments appear at lower photon energy whereas the secondary fragments only occur at higher excitation energies. This is remarkable since collision-induced dissociation is expected to primarily excite rovibrational states. Table 2 provides a summary of the assignment of observed masses to the chemical formulae and fragmentation pathways found in CID.

Uridine Fragmentation of the nucleoside of uracil, uridine, was measured in the same excitation energy range (Fig. 5). Due to instrumental limitations, masses below m/z 65 cannot be recorded for the uridine precursor m/z 245. Very few fragments are detected and dominant break-ups of protonated uracil such as m/z 70 or m/z 95 barely occur here (for a fragment mass spectrum at 6.7 eV, see SI, figure S3). This can be clearly seen by comparing Figure 5 to the much richer fragment map of uracil in Figure 2. This difference indicates stabilization of the nucleobase by the N-glycosidic bond. Below 5 eV, the photon flux was too low to extract any meaningful fragment data. The main fragments for photon energies below 9 eV are m/z 227 and m/z 113 that can be attributed to a water loss and the splitting into uracil and sugar moiety respectively. Similar fragment patterns have been observed in collision-induced dissociation of protonated uridine 37 and in photon-induced near-threshold ionization of neutral uridine. 38,39 The N-glycosidic bond cleavage has also been found to be the only decay channel in an infrared multiple photon dissociation (IRMPD) study of protonated uridine at low dissociation energies. 34 The fragment m/z 115 is tentatively assigned to a glycosidic bond cleavage followed by a water loss of the protonated sugar; m/z 86 could 12


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Figure 5: Two-dimensional fragment map after photoexcitation of protonated uridine (m/z 245). Below 9 eV, fragments from ring opening reactions are not present (cf. Figure 2). The ion with m/z 86 could not be assigned. The left panel displays the integrated signal over all fragments with 65 < m/z < 240. not be assigned, but fragments in that mass range were also present in a photoionization study of neutral uridine. 38 Figure 6 shows in more detail the yield of these two fragments as a function of the photon energy. The splitting into nucleobase and sugar roughly follows the trend that was observed for the uracil fragments with peaks at 5, 6.5 and 7.2 eV. This indicates that the corresponding electronic states of the nucleobase “chromophore” are excited, but decay differently than in the bare nucleobase. An additional peak occurs at 8 eV where the uracil fragmentation shows a minimum. A meaningful calculation of excited electronic states of this molecule with 29 atoms and various conformers, was beyond our resources. However, it has previously been suspected that nucleosides internally convert into the electronic ground state via conical intersections, thus driving the excess energy into fragmentation of the weakest bond, the sugar-nucleobase link. 40 Tautomeric changes due to the attached sugar might also play a role in stabilizing the nucleobase. 34

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Figure 6: Fragment yield for the two most abundant fragments of protonated uridine (m/z 245) as a function of excitation energy, with error bars again representing the standard deviation of the mean of the individual spectra. For low energies, water loss m/z 227 is the strongest decay channel. The yield of the protonated nucleobase UraH+ with m/z 113 follows the trend observed in the fragment spectra of UraH+ .

Conclusions The fragment mass spectra of protonated uracil and protonated uridine have been recorded as a function of the excitation energy in the range of 4.5 − 9.5 eV. This approach combines two appealing features: First, single photon excitation precisely defines the energy deposited in the system. Second, the additional charge present in the protonated form leads to charged fragments at lower excitation energy than for the neutral, making low-lying states accessible to mass spectrometry. The experimental data show that various fragment masses exhibit an energy-dependent yield with distinct maxima. Calculations allowed correlating some of these peaks to certain electronically excited states. In particular, fragments could be grouped into those that can already decay from the first singlet excited state (m/z ∈ {43, 70, 95, 96}) and those originating from higher excitations (m/z ∈ {42, 44, 53, 67, 68}). This grouping corresponds very well to a distinction into primary and secondary fragments in collisioninduced dissociation. 26,35

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It should be kept in mind that the fragmentation process occurs on a complex potential landscape. Many groups have investigated in detail the relaxation dynamics of neutral uracil, e.g., 36,41–46 and related species (for a recent review, see 47 ). These works illustrate that fast internal conversion to the electronic ground state can also prevent fragmentation, and how these processes depend on isomerization and environment of the molecule. The present results only show the start and end point of this complex trajectory on the potential energy surfaces. Nevertheless, they can serve as benchmark for numerical investigation of the break-up mechanisms, since the excitation energy (= the photon energy) and the reaction products (except for possible neutral fragmentation) are determined precisely. Energy dependent fragment mass spectra of uridine, the nucleoside of uracil, in the same photon energy range show a very different fragmentation pattern. The absence of low-mass fragments indicates that the sugar group provides deexcitation mechanisms that compete with ring opening reactions and thus stabilize the nucleobase itself, a finding which appears very relevant for the field of radiobiology.

Supporting Information Additional data available: Photon flux curves as a function of energy; comparison of total fragment yield with extinction measurements; mass spectra of protonated uracil and protonated uridine at 6.7 eV excitation energy.

Acknowledgments We thank the whole SOLEIL staff for running smoothly the facility and for granting beamtime under proposal no. 20160870. For the computational work, we acknowledge support from the ChemFarm High Performance computing at Weizmann Institute, in particular from Dr. Mark Vilensky. This work was supported by the Agence Nationale de la Recherche (France), under project number ANR-08-BLAN-0065 and by the Deutsche Forschungsge15



meinschaft (DFG, German Research Foundation) - Projektnummer 328961117 - SFB 1319 "Extreme Light for Sensing and Driving Molecular Chirality" (ELCH).

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State-Dependent Fragmentation of Protonated Uracil and Uridine

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