Structures of Protonated Thymine and Uracil and Their Monohydrated

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Structures of Protonated Thymine and Uracil and Their Monohydrated Gas-Phase Ions from Ultraviolet Action Spectroscopy and Theory Sara Øvad Pedersen,† Camilla Skinnerup Byskov,† Frantisek Turecek,*,‡ and Steen Brøndsted Nielsen*,† †

Department of Physics and Astronomy, Aarhus University, DK-8000 Aarhus C, Denmark Department of Chemistry, Bagley Hall, Box 351700, University of Washington, Seattle, Washington 98195-1700, United States

‡

S Supporting Information *

ABSTRACT: The strong UV chromophores thymine (Thy) and uracil (Ura) have identical heteroaromatic rings that only differ by one methyl substituent. While their photophysics has been elucidated in detail, the effect on the excited states of base protonation and single water molecules is less explored. Here we report gas-phase absorption spectra of ThyH+ and UraH+ and monohydrated ions and demonstrate that the substituent is not only responsible for spectral shifts but also influences the tautomer distribution, being different for bare and monohydrated ions. Spectra interpretation is aided by calculations of geometrical structures and transition energies. The lowest free-energy tautomer (denoted 178, enol−enol form) accounts for 230−280 nm (ThyH+) and 225−270 nm (UraH+) bands. ThyH+ hardly absorbs above 300 nm, whereas a discernible band is measured for UraH+ (275−320 nm), ascribed to the second lowest free-energy tautomer (138, enol−keto form) comprising a few percent of the UraH+ population at room temperature. Band widths are similar to those measured of cold ions in support of very short excited-state lifetimes. Attachment of a single water increases the abundance of 138 relative to 178, 138 now clearly present for ThyH+. 138 resembles more the tautomer present in aqueous solution than 178 does, and 138 may indeed be a relevant transition structure. The band of ThyH+(178) is unchanged, that of UraH+(178) is nearly unchanged, and that of UraH+(138) blue-shifts by about 10 nm. In stark contrast to protonated adenine, more than one solvating water molecule is required to reestablish the absorption of ThyH+ and UraH+ in aqueous solution.

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INTRODUCTION DNA and RNA nucleobases are some of the most important building blocks responsible for life, and they are also the primary UV chromophores of nucleic acids. Their particular photophysical properties are associated with quick conversions of electronic energy into vibrational energy that is dissipated as harmless heat to the surroundings.1−3 This behavior accounts for the high photostability of nucleotides in spite of their high absorption cross sections. The situation is more complicated in the case of strands, and there is much controversy regarding the character of the excited states, and how the complicated return to the ground state actually occurs.1−3 The bases themselves exist in many tautomeric forms that have been spectroscopically characterized in the gas phase in great detail as has work on isolated Watson−Crick base-pair dimers recently reviewed by Kleinermanns et al.4 In contrast, there is a sparse amount of absorption spectroscopy data on protonated bases5−10 or protonated nucleotides.8,11−14 The neutral tautomers have high but different proton affinities, and their relative significance can therefore change after protonation. Also the immediate environment can favor one protonated form over the other.5 Extensive theoretical © 2014 American Chemical Society

calculations of low-energy structures of protonated bases have been reported, but often many tautomers are rather close in energy,5,15−23 and the relative order is somewhat dependent on the theoretical method. While ground-state calculations can be done to a high accuracy, predictions of excited states are challenging, and experimental data to benchmark theory are highly useful. Importantly, earlier work by Marian et al.,5 Cheong et al.,7 and Pedersen et al.8 on protonated adenine (AdeH+) showed that two different tautomers may display quite different electronic absorption spectra. Gas-phase absorption spectra were obtained as the yield of fragment ions after photon absorption versus excitation wavelength. This indirect and approximate method is denoted action spectroscopy. It was found that AdeH+ ions produced by electrospray ionization absorb to the blue of those produced after ionization of cold neutral adenine dimers. These findings suggest that, along with vibrational spectroscopy, electronic absorption spectroscopy Received: April 29, 2014 Revised: May 29, 2014 Published: May 29, 2014 4256

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doubles the visible light in a barium borate (BBO) crystal to produce ultraviolet light pulses (widths of a few nanoseconds). The laser was operated at 20 Hz, which implies that only every second ion bunch was irradiated in order to identify the photoinduced signal (difference between “laser on” and “laser off” signals). The length of the ion bunches in time was about 10 μs. Three different experiments were performed. (1) Photoinduced dissociation mass spectroscopy: A hemispherical electrostatic analyzer was scanned to identify the fragment ion masses, and the ions were counted by a channeltron detector. (2) Power-dependence experiments: The photoinduced yield of each fragment ion was monitored as a function of the power of the laser light. (3) Action spectroscopy: The photoinduced yield of each fragment ion was monitored as a function of excitation wavelength from 210 to 340 nm. The data-taking program was written in LabView, and it was also used to synchronize the laser irradiation with the emptying of the ion trap. Absorption spectra of protonated thymine and uracil in aqueous solutions were obtained using an Evolution 300 Thermo spectrometer. The pH was adjusted to 1 by addition of concentrated HCl(aq).

could be a useful method to shed light on gas-phase ion structures. Presumably, within the relevant experimental time scale the energetic UV photons induce dissociation more easily than do lower-energy IR photons, lowering the number of required photon absorptions. Even less is known about the perturbation by a single water molecule of the electronic transition energies of protonated bases.8 An interesting question is how many local water molecules are needed to reach the situation in aqueous solution. Spectroscopy of microhydrated ions is nontrivial due to low ion-beam currents, and a starting point is monohydrated ions. Also, the effect of the first water molecule is expectedly the largest and is the focus of this work. In this paper, we have investigated spectroscopy of protonated thymine (ThyH+) and uracil (UraH+) (see Figure 1). Important information is gained from action-spectroscopy

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Figure 1. Dominant tautomers of ThyH+ and UraH+.

CALCULATIONS All density functional theory (DFT) and ab initio calculations were carried out using the Gaussian 09 suite of programs.28 Geometries were optimized with the B3LYP29 and M06-2X30 hybrid functionals and the 6-311+G(2d,p) basis set. Local energy minima were characterized by harmonic frequency calculations. Single-point energies were obtained with the B3LYP and M06-2X functionals and the larger 6-311+ +G(3df,2p) basis set. Relative enthalpies and free energies included zero-point vibrational corrections and 298 K enthalpies and entropies that were calculated with the rigidrotor harmonic-oscillator approximation with corrections for hindered rotor modes.31,32 The relative energies for uracil ion tautomers from the DFT calculations were benchmarked on coupled-cluster single-point calculations with single, double, and disconnected triple excitations, CCSD(T),33,34 and the aug-cc-pVTZ basis set.35 These showed close agreement with the energies based on the M06-2X/6-311++G(3df,2p) calculations which are discussed in the main text. Vertical excitation energies were calculated with time-dependent DFT36 using the B3LYP and M06-2X functionals and the 6-311+ +G(3df,2p) basis set. The TD-DFT excitation energies for uracil, thymine, and low-energy uracil water cluster ions were benchmarked on equation-of-motion coupled-cluster single and double (EOM-CCSD)37,38 excited-state calculations using the 6-311++G(2d,p) basis set. For a systematic comparison of TDDFT and EOM-CCSD calculations of excited-state energies, see ref 39. EOM-CCSD/6-311++G(2d,p) calculations on thymine water cluster ions with 19 atoms exceeded our computational resources and could not be performed.

experiments in combination with theoretical calculations of low-energy tautomer structures and excited-state energies. The two nucleobase ions in this study differ only by the methyl substituent present in thymine. We report on the effect of a single water molecule on the absorption and compare to the situation in bulk aqueous solution where ions are fully hydrated. This work builds upon our recent work on protonated adenine and its monohydrated ions.8 In the former work the spectral interpretation was simple as electrospray ionization produces one dominant tautomer,5 and this one binds water more strongly than other tautomers.5,15,16 As we will show, the situation is much more complicated for protonated thymine and uracil, and the methyl substituent is certainly not without an effect. It should also be mentioned that our results on the bare ions are in full accordance with very recent findings by Jouvet and co-workers9,10 on cryogenically cooled ThyH+ and UraH+ and earlier IR multiphoton dissociation spectroscopy ̂ experiments by Salpin, Maitre, and co-workers.24,25 Photoinduced dissociation (PID) mass spectra are first presented as these form the foundation for the following action-spectroscopy experiments, followed by the results from the theoretical calculations and a comparison to the experimental work.

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EXPERIMENTAL SECTION Experiments were performed on a home-built mass spectrometer that has been described in detail elsewhere.26,27 Thymine and uracil were purchased from Sigma-Aldrich. They were dissolved in a water:methanol (1:1) solution with 10% acetic acid added and electrosprayed. All ions were accumulated in an octopole-ion trap that was emptied every 25 ms (40 Hz repetition rate). To produce monohydrated ions, the octopole was filled with water vapor at a pressure of at least 10−2 mbar. Ion bunches were accelerated to 50 keV kinetic energies and the ions of interest selected by a bending magnet according to their mass-to-charge ratios. These were then photoexcited by light from a pulsed tunable laser system. The laser is a Nd:YAG that produces 1064 nm infrared light, frequency triples it to 355 nm (UV light), splits it into visible light and an idler beam in an optical parametric oscillator (OPO), and finally frequency

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RESULTS AND DISCUSSION Photoinduced Dissociation Mass Spectra. PID mass spectra of the protonated molecules revealed that the two dominant dissociation channels for both ions are loss of 17 Da (NH3) and loss of 43 Da (HNCO) (see Figure 2). These are low-energy dissociation channels of protonated nucleobases.18,23,24 In the case of ThyH+ a smaller peak was seen at m/z 56 corresponding to loss of 59 Da (CHNO2) while for 4257

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Figure 2. PID mass spectra of (a) ThyH+ (m/z 127) and (b) UraH+ (m/z 113). The excitation wavelength was 260 nm.

UraH+ two peaks were seen at m/z 18 and 43 corresponding to NH4+ and HNCO+. It should be mentioned that the spectra cannot be corrected for ion-beam variations, and that the relative importance of the fragment ions can therefore not be read from the spectra. This is particularly an issue as it takes more than an hour to record a full mass scan. The yields of the fragment ions increased linearly with the intensity of the laser light (Supporting Information), and fragmentation was here ascribed to the absorption of one photon. The flight time of the ions to the electrostatic analyzer after photoexcitation was up to 10 μs, which is the maximum time scale on which we can monitor dissociation. The time constant for dissociation after one-photon absorption is likely similar to this time or shorter. Three important fragment ions were identified for the ThyH+(H2O) and UraH+(H2O) water complexes corresponding to loss of H2O, H2O and NH3, and H2O and HNCO. Again these fragment ions were produced after the absorption of one photon according to power-dependence measurements. Action Spectra. First we present the results for protonated thymine and its water complex. Figure 3 shows the photofragment ion yields versus excitation wavelength for ThyH+. At 260 nm the percentages of the three fragment ions with m/z 56, 84, and 110 are 12%, 48%, and 40%; these were established within a few minutes to avoid complications from ion-beam fluctuations (vide supra). The three spectra are quite similar except that the importance of the lowest mass ion increases with excitation energy. In all three cases, there is clear absorption from 210 nm to about 280 nm. However, very weak absorption is observed above 300 nm as is evident from the m/z 110 ion bunch profiles obtained with “laser on” and “laser off” and their difference (Supporting Information). The “laser off” signal is due to the decay of metastable parent ions or to dissociation after collisions between parent ions and residual gas in the beamline. The fact that the difference signal is positive over nearly the whole length of the ion bunch profile unequivocally reveals that light is absorbed. The individual action spectra (Si(λ)) were combined according to the relative importance of each fragment ion (i)

Figure 3. Action spectra for the formation of (a) m/z 56, (b) m/z 84, and (c) m/z 110 fragment ions from protonated thymine (m/z 127).

at 260 nm (Fi(260 nm)) to give the total action spectrum (Figure 4), total spectrum (λ) =

∑ Si(λ)Fi(260nm)/Si(260nm) i

Figure 4. Action spectra of ThyH+ and ThyH+(H2O) obtained after summing the yields from all dissociation channels. The absorption spectrum of thymine in an acidic aqueous solution is also included for comparison. The bars are TD-DFT-calculated vertical excitation energies; (□) ThyH+ and (■) ThyH+(H2O). 4258

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the more computationally demanding EOM-CCSD method. For example, the dipole allowed S0 → S2 transition in neutral thymine had the resonant wavelength at 250, 233, and 244 nm from B3LYP, M06-2X, and EOM-CCSD calculations, respectively. A similar trend in the calculated data was obtained for the ionic systems (Tables 1−3), indicating that the much less expensive TD-DFT methods can be used for reliable calculations of electronic excitation energies in larger nucleobase systems. Computational results for bare protonated thymine ions are summarized in Table 1 including the geometrical structures of the two most stable tautomers (th178 and th138). The nomenclature indicating the protonation on the ring positions N-1 and N-3 and oxygen atoms O-7 and O-8 was introduced in ref 20 and is kept here for the sake of consistency. The thymine ion should be predominantly the th178 tautomer in accordance with previous work;21 th138 contributes 4% or 0.3% (dependent on the theoretical method) to the ion-beam population assuming equilibrium conditions at 298 K. We note that we assume thermal equilibrium to have been established in the octopole ion trap in our experiment, and we neglect some collisional heating that may occur during extraction from the trap and during acceleration to 50 keV energies. The experimental spectrum shows practically a single band with maximum at ca. 250−265 nm, while theory provides values of 237, 248, and 235 nm (corrected values from EOM-CCSD, B3LYP, and M06-2X, respectively) for the th178 absorption. Very weak absorption was identified above 300 nm in agreement with th138 absorbing to the red of that of th178: Theory predicts absorption values of 288 nm, 297 nm, and 280 nm for th138 from the three models. The position of the absorption maximum relative to the wavelength of the resonant vertical transition was judged on the basis of a detailed dynamic analysis by Lischka and coworkers.40 These authors reported a consistent red shift of ca. 0.15 eV in the absorption maxima, which we applied to correct the excitation energies calculated for the nucleobase ions and water clusters. According to the EOM-CCSD calculations, the band with maximum below 210 nm is ascribed to excitations to the second excited state of a predicted absorption maximum at 207 nm. While the calculated oscillator strength (f) was high (0.14), the TD-DFT calculations gave a value of only 0.001. Calculations of protonated thymine with one water molecule attached predict several isomers/tautomers at equilibrium, with th178w7 dominating (Table 2). The calculated absorptions of th178w7, w1, and w8 are very similar, 232−251 nm, so the water molecule position has only a small effect. The calculated transition wavelength is close to the dominant band in the experimental absorption spectrum at 255 nm. The longwavelength isomer th138w8 (calculated at 276−294 nm) shows as a weak band at ca. 300−310 nm in the experimental spectrum. It is noteworthy that the importance of the th138 tautomer increases after water attachment (now accounting for 6% or 2% of the population, from either B3LYP or M06-2X), which is again in accordance with experiment where absorption is clearly evident for the water complex. At a gas-phase equilibrium, protonated uracil is calculated to have a major tautomer (u178) and a minor one (u138) (Table 3) in agreement with earlier work.19 These are the same as those found for ThyH+ but with a smaller bias toward 178. Thus, at room temperature, u138 comprises 1−23% of the ion population (the range is based on different theoretical models).

This spectrum is taken to represent the absorption spectrum of bare ThyH+. We will return to band assignments after presenting the theoretical results. The action spectra for ThyH+(H2O) are shown in the Supporting Information for the three dominant fragment ions. All spectra reveal absorption between 210 and 280 nm, and for water loss also in the region 280 to 335 nm. This is also evident from the ThyH+ fragment ion bunch profiles (Supporting Information), as there are more ion counts when the laser is on than when it is off in the 300−310 nm region. The importance of the lowest mass ion increases with excitation energy. At 260 nm the percentages of the three fragment ions are 13.5% (m/z 84), 33% (m/z 110), and 53.5% (m/z 127). Based on these percentages and the three individual action spectra, we calculated the total action spectrum (Figure 4). This spectrum is nearly identical to that of the bare ion in the region from 210 to 280 nm. However, at higher wavelengths the monohydrated ions show clear absorption. Next we consider the results for protonated uracil and its water complexes. The action spectra for the bare ions are shown in Supporting Information. Absorption between 210 and 265 nm is seen in all four spectra, and absorption at higher wavelengths for the two high-mass fragment ions. Again the low-mass fragment ion is formed most easily at the lowest wavelengths. At 250 nm, the percentages of the fragment ions are 6% (m/z 18), 13% (m/z 43), 53% (m/z 70), and 28% (m/z 96). The spectra were combined according to these percentages to give the total action spectrum (Figure 5).

Figure 5. Action spectra of UraH+ and UraH+(H2O) obtained after summing the yields from all dissociation channels. The absorption spectrum of uracil in an acidic aqueous solution is also included for comparison. The bars are TD-DFT-calculated vertical excitation energies; (□) UraH+ and (■) UraH+(H2O).

Finally, we present the action spectra for UraH+(H2O) (Supporting Information). They are quite different with absorption spanning over a broad range from 210 to 330 nm. The percentages at 285 nm are 43.5% (m/z 70), 26.5% (m/z 96), and 30% (m/z 113). Again combining the spectra provides the total action spectrum (Figure 5). Theoretical Calculations. Excited state calculations were carried out at three levels of theory. The excitation energies obtained by TD-DFT methods closely bracketed those from 4259

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Table 1. Theoretical Results for Thy and ThyH+ Tautomers th178 and th138 Including Relative Energies (in kJ/mol), Vertical Excitation Energies (λmax in nm), and Oscillator Strengths (f)

a

Calculated transitions. bEstimated absorption maxima corrected according to Lischka et al.40 are in brackets.

similar to the first transition. This could explain the band we measure with maximum below 210 nm. Comparing results for UraH+ with those for ThyH+, theory confirms the red-shifting effect of the methyl substituent on the electronic transition energies seen experimentally. As was the case for ThyH+, theory gives a vertical excitation energy of u178 that is to the blue of the experimental band maximum. Calculations for UraH+(H2O) indicate two tautomers, u178w7 and u138w8 (Table 4). Several more isomers were found, but these two have by far the lowest energies, in agreement with other theoretical work.25 The computational results are summarized in Table 4. The most stable tautomer (ur178) binds water rather promiscuously through O-7-H, O-8H, and N-1-H with the largest binding free energy of 45 kJ mol−1 for u178w7. The u138 ion tautomer prefers binding to water through O-8-H with a free energy of 45 kJ mol−1. The increased relative abundance of the u138w8 tautomer (20−40% from the most reliable theoretical calculations) indicates that

Tautomer u178 absorbs at 229−234 nm ( f = 0.1−0.23), and u138 absorbs at 267−283 nm (f = 0.1−0.27) (all adjusted maxima according to Lischka40). Recent calculations that were carried out with the RI-ADC(2) method reported vertical transitions at 248 nm (5.00 eV) and 322 nm (3.85 eV) for the two lowest-energy uracil ion tautomers.10 The experimental spectrum shows two bands, a stronger one with maximum at ca. 240−250 nm and a weaker one at ca. 290−305 nm. The agreement between theory and experiment is acceptable for wavelength band positions. While the theoretical equilibrium is much in favor of u178, the experimental spectrum shows a visible portion of u138. The signal for the latter could be enhanced by its larger f (from EOM-CCSD the ratio is almost three), a nonequilibrium population formed by electrospray, or combination thereof. As for ThyH+, the EOM-CCSD model predicts a band for UraH+ with maximum at 205 nm and an oscillator strength 4260

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Table 2. Theoretical Results for ThyH+(H2O)

a

Calculated transitions (in nm). bEstimated absorption maxima (in nm) corrected according to Lischka et al.40 are in brackets.

we assign the absorption bands to be due to the 178 and 138 tautomers for both thymine and uracil; the former dominates the population and more so for ThyH+ than for UraH+, but 138 increases in importance after water binding for both ions. The 138 tautomers absorb further to the red than the 178 ones. We note that our findings are in full agreement with very recent spectroscopy work by Jouvet and co-workers9,10 on cold (few tens of kelvins) protonated bases. They found the same two tautomers as we did, and concluded that 138 was more important for uracil than for thymine. Interestingly, while the cold-ion spectrum displayed well-resolved vibronic transitions, the absorption bands were still broad (ca. 227−256 nm and ca. 260−317 nm for UraH+ and ca. 226−268 nm for ThyH+); they were very similar to those we obtain for room-temperature ions, maybe except for a small tail to higher wavelengths in our experiment (hot bands). It therefore seems possible that the widths of the broad bands are determined by rapid deexcitation of the excited state (cf., uncertainty principle) as discussed in detail in ref 9. Based on infrared multiphoton dissociation (IRMPD) spectroscopy of room-temperature ions, Salpin et al.24 identified both tautomers and concluded that 178 was

u138 binds water more preferably than u178. Note that u138w8 is slightly preferred against u178w7 by enthalpy at all levels of theory, but shows a lower entropy which increases its relative free energy. This is consistent with the optimized structure of u138w8 which has the water molecule-coordinating hydroxyl (O-8-H) in the ring plane. This alignment slightly increases the frequencies of the pertinent H−O−C and O−H−O twisting and bending modes, lowering the ion entropy. The calculated absorptions are at 226−238 nm for u178w7 and 262−278 nm for u138w8. The experimental spectrum shows two bands with maxima at ca. 250 nm and 280−295 nm. These can be assigned to the two most stable isomers, and the increase of the second band compared to the bare ion spectrum is accounted for by the predicted increase in u138 tautomers after water attachment. Again the signal from u138w8 could also be enhanced by its greater f. Importantly, both experiment and theory show that the absorption by the u138 water complex is to the blue of that by the bare ion. Taken together, there is a good agreement between theory and experimental gas-phase spectra, providing for the systematically higher predicted excitation energies. Based on theory, 4261

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Table 3. Theoretical Results for Ura and UraH+ Tautomers u178 and u138

a

Calculated transitions (in nm). bEstimated absorption maxima (in nm) corrected according to Lischka et al.40 are in brackets.

dominant. Finally, Bakker et al.25 also found, from IRMPD experiments, isomers of UraH+(H2O) originating from the two lowest-energy tautomers of UraH+, in accordance with our results. Gas-Phase versus Solution-Phase Spectra. Included in Figures 4 and 5 are the recorded absorption spectra of acidic solutions of thymine and uracil where the bases are protonated. One broad band is seen for both hydrated ions. The maximum of UraH+(aq) is at 258 nm while that of ThyH+(aq) is at 264 nm. Hence, for both isolated bare and monohydrated ions, as well as fully hydrated ions in solution, UraH+ absorbs to the blue of ThyH+. However, the solution-phase spectra are very different from the gas-phase spectra, with the broad band positioned between those of gas-phase 178 and 138 tautomers.

Clearly, more than one water molecule is needed to obtain the solution-phase characteristics, but how many are needed is unknown. In aqueous solution, it seems that the preferred structure is the double ketone form with N-3 protonated.41−44 This structure is indeed more similar to 138 than 178 in qualitative accordance with the increase in importance of 138 upon attachment of a single water molecule. We speculate that 138 is a transition structure toward the fully hydrated one. The behavior of UraH+ and ThyH+ contrasts with that of protonated adenine (AdeH+) where the chromophore microenvironment plays a minor role as evidenced by the almost identical absorption spectra of bare AdeH+, AdeH+(H2O), and AdeH+(aq).8 4262

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Table 4. Theoretical Results for UraH+(H2O)

a

Calculated transitions (in nm). bEstimated absorption maxima (in nm) corrected according to Lischka et al.40 are in brackets.

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CONCLUSIONS In conclusion, we have spectroscopically characterized protonated thymine and uracil and established the effect of a single water molecule on the absorption spectra and tautomer distribution. The spectra were compared to those obtained for the nucleobases in acidic aqueous solution revealing significant differences. More than one water molecule is clearly needed for these protonated bases to obtain solution-phase characteristics in stark contrast to protonated adenine that earlier was found to show identical absorption when bare, monohydrated, or fully hydrated. Our interpretation of the results and band assignments are based on theoretical calculations of both minimum-energy structures and excited

state energies. While theory systematically predicts somewhat higher excitation energies, all trends are well reproduced allowing for band assignments. Hence electronic absorption spectroscopy in unison with theory provides important information on tautomer structures, and as such serves as a useful alternative to gas-phase vibrational spectroscopy. As more advanced theories are developed for better predicting transition energies, this approach would be even stronger. Future work addressing the spectral changes as more water molecules are added in a controlled manner would be highly illuminating in helping establish the transition to the solutionphase tautomer and the involved tautomers on the way. 4263

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(12) Liu, B.; Hvelplund, P.; Brøndsted Nielsen, S.; Tomita, S. Photodissociation of Singly Charged Oligonucleotide Cations: Arrhenius Parameters and Identification of Nonstatistical Processes. Phys. Rev. A 2006, 74, 052704−1−6. (13) Worm, E. S.; Andersen, I. H.; Andersen, J. U.; Holm, A. I. S.; Hvelplund, P.; Kadhane, U.; Brøndsted Nielsen, S.; Poully, J. C.; Støchkel, K. Photodissociation of Dinucleotide Ions in a Storage Ring. Phys. Rev. A 2007, 75, 042709−1−7. (14) Aravind, G.; Antoine, R.; Klaerke, B.; Lemoine, J.; Racaud, A.; Rahbek, D. B. Rajput, J.; Dugourd, P.; Andersen, L. H. SubMicrosecond Photodissociation Pathways of Gas Phase Adenosine 5′-Monophosphate Nucleotide Ions. Phys. Chem. Chem. Phys. 2010, 12, 3486−3490. (15) Chen, X.; Syrstad, E. A.; Nguyen, M. T.; Gerbaux, P.; Turecek, F. Distonic Isomers and Tautomers of the Adenine Cation Radical in the Gas Phase and Aqueous Solution. J. Phys. Chem. A 2004, 108, 9283−9293. (16) Turecek, F.; Chen, X. Protonated Adenine: Tautomers, Solvated Clusters, and Dissociation Mechanisms. J. Am. Soc. Mass Spectrom. 2005, 16, 1713−1726. (17) Yao, C.; Cuadrado-Peinado, M. L.; Polásek, M.; Turecek, F. Gas-Phase Tautomers of Protonated 1-Methylcytosine. Preparation, Energetics, and Dissociation Mechanisms. J. Mass Spectrom. 2005, 40, 1417−1428. (18) Yao, C.; Turecek, F.; Polce, M.J.; Wesdemiotis, C. Proton and Hydrogen Atom Adducts to Cytosine. An Experimental and Computational Study. Int. J. Mass Spectrom. 2007, 265, 106−123. (19) Colominas, C.; Luque, F. J.; Orozco, M. Tautomerism and Protonation of Guanine and Cytosine. Implications in the Formation of Hydrogen-Bonded Complexes. J. Am. Chem. Soc. 1996, 118, 6811− 6821. (20) Wolken, J. K.; Turecek, F. Proton Affinity of Uracil. A Computational Study of Protonation Sites. J. Am. Soc. Mass Spectrom. 2000, 11, 1065−1071. (21) Jiao, D.; Wang, H.; Zhang, Y.; Tang, Y. A DFT Study of Thymine and Its Tautomers. Can. J. Chem. 2009, 87, 406−415. (22) Hass, E. C.; Mezey, P. G. Non-Empirical SCF-MO Studies on the Protonation of Bio-Polymer Constituents. 3. Protonation of Cytosine, Thymine, and Their Tautomeric Forms. Theor. Chim. Acta 1981, 60, 283−297. (23) Qian, M.; Yang, S.; Wu, H.; Majumdar, P.; Leigh, N.; Glaser, R. Ammonia Elimination from Protonated Nucleobases and Related Synthetic Substrates. J. Am. Soc. Mass Spectrom. 2007, 18, 2040−2057. (24) Salpin, J.-Y.; Guillaumont, S.; Tortajada, J.; MacAleese, L.; Lemaire, J.; Maître, P. Infrared Spectra of Protonated Uracil, Thymine, and Cytosine. ChemPhysChem 2007, 8, 2235−2244. (25) Bakker, J. M.; Sinha, R. K.; Besson, T.; Brugnara, M.; Tosi, P.; Salpin, J.-Y.; Maître, P. Tautomerism of Uracil Probed via Infrared Spectroscopy of Singly Hydrated Protonated Uracil. J. Phys. Chem. A 2008, 112, 12393−12400. (26) Støchkel, K.; Milne, B. F.; Brøndsted Nielsen, S. Absorption Spectrum of the Firefly Luciferin Anion Isolated in Vacuo. J. Phys. Chem. A 2011, 115, 2155−2159. (27) Wyer, J. A.; Brøndsted Nielsen, S. Absorption by Isolated Ferric Heme Nitrosyl Cations In Vacuo. Angew. Chem., Int. Ed. 2012, 124, 10402−10406. (28) Frisch, M. J. et al. Gaussian 09, Revision A.02; Gaussian, Inc.: Wallingford, CT, 2009. (29) Becke, A. D. A New Mixing of Hartree-Fock and Local DensityFunctional Theories. J. Chem. Phys. 1993, 98, 1372−1377. (30) Zhao, Y.; Truhlar, D. G. The M06 Suite of Density Functionals for Main Group Thermochemistry, Thermochemical Kinetics, Noncovalent Interactions, Excited States, and Transition Elements: Two New Functionals and Systematic Testing of Four M06-Class Functionals and 12 Other Functionals. Theor. Chem. Acc. 2008, 120, 215−241. (31) McClurg, R. B.; Flagan, R. C.; Goddard, W. A. The Hindered Rotor Density-of-States Interpolation Function. J. Chem. Phys. 1997, 106, 6675−6680.

ASSOCIATED CONTENT

S Supporting Information *

Additional data available: Yields of fragment ions versus laser power and ion bunch profiles for ThyH+ and ThyH+(H2O) with laser on and laser off. Action spectra associated with the formation of each fragment ion. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS S.B.N. gratefully acknowledges support from Lundbeckfonden. F.T. thanks the Chemistry Division of the National Science Foundation (Grant CHE-1055132) and the Klaus and Mary Ann Saegebarth Endowment for financial support.

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ABBREVIATIONS Ade, adenine; CC, coupled cluster; DFT, density functional theory; EOM, equation-of-motion; IR, infrared; IRMPD, infrared multiphoton dissociation; MP, Møller−Plesset; PID, photoinduced dissociation; Thy, thymine; Ura, uracil; UV, ultraviolet

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REFERENCES

(1) Crespo-Hernandez, C. E.; Cohen, B.; Hare, P. M.; Kohler, B. Ultrafast Excited-State Dynamics in Nucleic Acids. Chem. Rev. 2004, 104, 1977−2019. (2) Markovitsi, D. Interaction of UV Radiation with DNA Helices. Pure Appl. Chem. 2009, 81, 1635−1644. (3) Nielsen, L. M.; Hoffmann, S. V.; Brøndsted Nielsen, S. Electronic Coupling Between Photo-Excited Stacked Bases in DNA and RNA Strands with Emphasis on the Bright States Initially Populated. Photochem. Photobiol. Sci. 2013, 12, 1273−1285. (4) Kleinermanns, K.; Nachtigallová, D.; de Vries, M. S. Excited State Dynamics of DNA Bases. Int. Rev. Phys. Chem. 2013, 32, 308−342. (5) Marian, C.; Nolting, D.; Weinkauf, R. The Electronic Spectrum of Protonated Adenine: Theory and Experiment. Phys. Chem. Chem. Phys. 2005, 7, 3306−3316. (6) Nolting, D.; Weinkauf, R.; Hertel, I. V.; Schultz, T. Excited-State Relaxation of Protonated Adenine. ChemPhysChem 2007, 8, 751−755. (7) Cheong, N. R.; Nam, S. H.; Park, H. S.; Ryu, S.; Song, J. K.; Park, S. M.; Pérot, M.; Lucas, B.; Barat, M.; Fayeton, J. A.; Jouvet, C. Photofragmentation in Selected Tautomers of Protonated Adenine. Phys. Chem. Chem. Phys. 2011, 13, 291−295. (8) Pedersen, S. Ø.; Støchkel, K.; Byskov, C. S.; Baggesen, L. M.; Brøndsted Nielsen, S. Gas-Phase Spectroscopy of Protonated Adenine, Adenosine 5′-Monophosphate and Monohydrated Ions. Phys. Chem. Chem. Phys. 2013, 15, 19748−19752. (9) Berdakin, M.; Féraud, G.; Dedonder-Lardeux, C.; Jouvet, C.; Pino, G. A. Excited States of Protonated DNA/RNA bases. Phys. Chem. Chem. Phys. 2014, 16, 10643−10650, DOI: 10.1039/ C4CP00742E. (10) Feraud, G.; Dedonder, C.; Jouvet, C.; Inokuchi, Y.; Haino, T.; Sekiya, R.; Ebata, T. Development of Ultraviolet Hole-Burning Spectroscopy for Cold Gas-Phase Ions. J. Phys. Chem. Lett. 2014, 5, 1236−1240. (11) Brøndsted Nielsen, S.; Andersen, J. U.; Forster, J. S.; Hvelplund, P.; Liu, B.; Pedersen, U. V.; Tomita, S. Photodestruction of Adenosine 5′-Monophosphate (AMP) Nucleotide Ions in Vacuo: Statistical versus Nonstatistical Processes. Phys. Rev. Lett. 2003, 91, 048302−1−4. 4264

dx.doi.org/10.1021/jp504153p | J. Phys. Chem. A 2014, 118, 4256−4265

The Journal of Physical Chemistry A

Article

(32) Ayala, P. Y.; Schlegel, H. B. Identification and Treatment of Internal Rotation in Normal Mode Vibrational Analysis. J. Chem. Phys. 1998, 108, 2314−2325. (33) Cizek, J.; Paldus, J.; Sroubkova, L. Cluster Expansion Analysis for Delocalized Systems. Int. J. Quantum Chem. 1969, 3, 149−167. (34) Purvis, G. D.; Bartlett, R. J. A Full Coupled-Cluster Singles and Doubles Model - The Inclusion of Disconnected Triples. J. Chem. Phys. 1982, 76, 1910−1918. (35) Dunning, T. H. Gaussian-Basis Sets for Use in Correlated Molecular Calculations. 1. The Atoms Boron through Neon and Hydrogen. J. Chem. Phys. 1989, 90, 1007−1023. (36) Furche, F.; Ahlrichs, R. Adiabatic Time-Dependent Density Functional Methods for Excited State Properties. J. Chem. Phys. 2002, 117, 7433−7447. (37) Koch, H.; Jørgensen, P. Coupled Cluster Response Functions. J. Chem. Phys. 1990, 93, 3333−3344. (38) Comeau, D. C.; Bartlett, R. J. The equation-of-motion coupledcluster method. Applications to open- and closed-shell reference states. Chem. Phys. Lett. 1993, 207, 414−423. (39) Sonk, J. A.; Schlegel, H. B. TD-CI simulation of the electronic optical response of molecules in intense fields II: comparison of DFT functionals and EOM-CCSD. J. Phys. Chem. A 2011, 115, 11832− 11840. (40) Barbatti, M.; Aquino, A. J. A.; Lischka, H. The UV Absorption of Nucleobases: Semi-Classical Ab Initio Spectra Simulations. Phys. Chem. Chem. Phys. 2010, 12, 4959−4967. (41) Jardetzky, O.; Pappas, P.; Wade, N. G. Proton Magnetic Resonance Studies of Purine and Pyrimidine Derivatives.9. Protonation of Pyrimidines in Acid Solution. J. Am. Chem. Soc. 1963, 85, 1657−1658. (42) Christensen, J. J.; Rytting, J. H.; Izatt, R. M. Thermodynamics of Proton Dissociation in Dilute Aqueous Solution.8. pK, ΔHo, and ΔSo Values for Proton Ionization from Several Pyrimidines and Their Nucleosides at 25°. J. Phys. Chem. 1967, 71, 2700−2705. (43) Izatt, R. M.; Christensen, J. J.; Rytting, J. H. Sites and Thermodynamic Quantities Associated with Proton and Metal Ion Interaction with Ribonucleic Acid, Deoxyribonucleic Acid, and Their Constituent Bases, Nucleosides, and Nucleotides. Chem. Rev. 1971, 71, 439−481. (44) Ganguly, S.; Kundu, K. K. Protonation/Deprotonation Eneregetics of Uracil, Thymine, and Cytosine in Water from EMF/ Spectrophotometric Measurements. Can. J. Chem. 1994, 72, 1120− 1126.

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