Comprehensive Core-Level Study of the Effects of Isomerism

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Comprehensive Core-Level Study of the Effects of Isomerism, Halogenation, and Methylation on the Tautomeric Equilibrium of Cytosine Vitaliy Feyer,*,† Oksana Plekan,†,# Antti Kivim€aki,‡ Kevin C. Prince,†,‡ Tatyana E. Moskovskaya,§ Irina L. Zaytseva,§ Dmitriy Yu. Soshnikov,§ and Alexander B. Trofimov§,^ †

Sincrotrone Trieste, in Area Science Park, Basovizza (Trieste) I-34149, Italy Laboratorio TASC, CNR-IOM, Basovizza (Trieste) I-34149, Italy § Laboratory of Quantum Chemistry, Irkutsk State University, 664003 Irkutsk, Russia ^ Favorsky Institute of Chemistry, SB RAS, 664033 Irkutsk, Russia ‡

ABSTRACT: Core-level X-ray photoemission and near-edge X-ray absorption fine structure spectra of 5-methylcytosine, 5-fluorocytosine, and isocytosine are presented and discussed with the aid of highlevel ab initio calculations. The effects of the methylation, halogenation, and isomerization on the relative stabilities of cytosine tautomers are clearly identified spectroscopically. The hydroxyoxo tautomeric forms of these molecules have been identified, and their quantitative populations at the experimental temperature are calculated and compared with the experimental results and with previous calculations. The calculated values of Gibbs free energy and Boltzmann population ratios are in good agreement with the experimental results characterizing tautomer equilibrium.

1. INTRODUCTION Tautomers of DNA bases can cause genetic mutations by pairing incorrectly with wrong complementary bases, and these mutations can be the precursors to some molecular-based diseases. Therefore, the tautomerism of cytosine and its biologically active modifications have been the subject of a great number of theoretical and experimental investigations due to their biochemical significance. Furthermore, there is intrinsic interest in understanding the fundamentals of tautomerism and how substituents influence tautomeric equilibria. Gas-phase experimental data serve as benchmarks for quantum chemical calculations, whereas computational results help interpret and guide experiment.1,2 Microwave and low-temperature matrix isolation infrared spectroscopy are the most commonly used experimental techniques in studies dedicated to molecular tautomerism.29 They are high-resolution methods, and the spectra give detailed structural information about the isolated molecules. However, the intensity of the observed signal is not directly proportional to the tautomer population, and for the determination of the latter, demanding calculations are required.2 The calculated energies of the tautomers are strongly dependent on the level of theoretical treatment and basis set,1016 so that some quantitative discrepancies r 2011 American Chemical Society

can occur in the predicted ratios of the different tautomeric forms. The advantage of X-ray photoemission spectroscopy (XPS) for thermally evaporated compounds is that the corelevel photoemission intensities well above threshold are directly proportional to the population of the corresponding chemical state while the sample is in thermal equilibrium.1719 On the other hand, XPS can be used as a technique complementary to well-established spectroscopies such as microwave and infrared in the study of biomolecular tautomerism.20 Recently, using XPS, we have shown that in the gas phase at experimental temperatures, a few tautomers of guanine and cytosine are significantly populated, whereas thymine, adenine, and uracil exist in a single form.18,19,21 The calculated chemical shifts and measured core level spectra showed the fingerprint features associated with the three tautomeric forms of cytosine, such as aminohydroxy (I), aminooxo (II), and iminooxo (III), and their populations were determined.18 The electronic structure of rotamers (hydroxy or imino Received: February 22, 2011 Revised: May 13, 2011 Published: May 17, 2011 7722

dx.doi.org/10.1021/jp2017506 | J. Phys. Chem. A 2011, 115, 7722–7733

The Journal of Physical Chemistry A

Figure 1. Schematic structures of the most stable tautomeric forms of (a) isocytosine, (b) 5-methylcytosine, and (c) 5-fluorocytosine.

groups) is only slightly different, so that their ionization energies do not differ significantly, and the corresponding features in the measured spectra could not be resolved. The schematic structures of the lowest-energy tautomeric forms of isocytosine, 5-methylcytosine, and 5-fluorocytosine are shown in Figure 1. These systems have been previously investigated by matrix isolation infrared and microwave techniques.47 The oxohydroxy equilibrium of isocytosine and halo-cytosine in the gas phase was shown to shift toward the hydroxy tautomeric form compared with the cytosine result. The three tautomers, aminohydroxy, aminooxo, and iminooxo of 5-methylcytosine in the matrix were present simultaneously. In the present work, the effects of isomerism, halogenation, and methylation on the oxohydroxy equilibrium of cytosine tautomers have been investigated using core-level spectroscopy and accurate ab initio calculations.

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2. EXPERIMENTAL AND THEORETICAL DETAILS The measurements were carried out at the gas-phase photoemission beamline, Elettra, Trieste,22 using the apparatus described in detail elsewhere.21 The photoemission spectra were measured with total resolutions (photon þ analyzer) of 0.25, 0.35, and 0.55 eV at 382 (C 1s), 495 (N 1s), and 628 eV (O 1s) photon energies, respectively. The binding energy (BE) scale of the photoemission spectra was referred to the following core levels and gases: 297.6 eV (C 1s, CO2);23 409.9 eV (N 1s, N2);24 and 541.3 eV (O 1s, CO2).24,25 The NEXAFS spectra were recorded by collecting the ion yield signal using a channel electron multiplier placed close to the ionization region, and they were normalized to the photon flux measured by a photodiode. The photon energy scales of the spectra were calibrated by taking simultaneous spectra of the samples and a calibrant gas introduced into the experimental chamber. The energies of the peaks of the calibrating gases were 290.77 (C 1s f π, CO2),26 400.87 (N 1s f π, N2),27 and 535.4 eV (O 1s f π, CO2).28 The NEXAFS spectra were recorded with energy resolutions of 0.07, 0.06, and 0.10 eV at the C, N, and O K-edges, respectively. Isocytosine was purchased with g99% purity from Sigma Aldrich, while 5-methylcytosine and 5-fluorocytosine were obtained from Alfa Aesar with 97 and 98þ% purities, respectively. All samples were used without any further purification. They were sublimated in vacuum from a noncommercial furnace wound with a spiral, noninductive Thermocoax heating element, equipped with a chromel/alumel thermocouple. The materials used were aluminum or stainless steel for the crucible, stainless steel for the heated parts of the furnace and heat shield. The evaporation temperatures for 5-methylcytosine, isocytosine, and 5-fluorocytosine were 445, 425, and 420 K, respectively. Valence band (VB) spectra collected at 100 eV photon energy were used to check the quality of the samples during the experiment. The VB spectra remained constant over the course of the experiment and did not show evidence of decomposition products, such as water or carbon dioxide, which are easily identified as sharp peaks in valence spectra.29 The local vapor pressure of the samples in the interaction region can be estimated by comparing the intensity (peak areas) of the N 1s core-level photoionization spectra associated with the measured molecules and the signal from nitrogen gas (N2) admitted to the chamber, whose pressure is known. We assume the same cross section, and we calibrated the size of the field of view of the analyzer. The local pressures for 5-methylcytosine, isocytosine, and 5-fluorocytosine are 8 106, 4 106, and 2 106 mbar, respectively. There may be a systematic error associated with this measurement as the nitrogen gas is distributed uniformly whereas the effusive source is inhomogeneous. We estimate this error to be about a factor of 2. A similar theoretical approach to calculation of the XPS spectra as that in our previous study of nucleic acid bases18,19,21 was employed, and we summarize here only the main details. The energies Ω and the relative intensities (pole strengths) P of the vertical ionization transitions were computed using the algebraicdiagrammatic construction (ADC) approximation scheme for the one-particle Green’s function3032 and cc-pVDZ basis sets.3335 In contrast to the previous work, here, the thirdorder (ADC(3)) level of approximation was used rather than the more accurate, though computationally rather expensive, ADC(4) method. The ADC(3) treatment of core-level ionization overestimates the total ionization energies quite significantly, 7723

dx.doi.org/10.1021/jp2017506 |J. Phys. Chem. A 2011, 115, 7722–7733

The Journal of Physical Chemistry A Table 1. Relative Electronic Energies (kcal/mol) of Isocytosine, 5-Methylcytosine, and 5-Fluorocytosine Tautomers Computed Using MP2 and CCSD Methods and cc-pVTZ Basis Setsa, Relative Gibbs Free Energies (kcal/mol), and BPRs for a Given Tb

a

Molecular geometry optimized at the MP2/cc-pVTZ level of theory. Gibbs free energies and BPRs obtained by combining the CCSD/ cc-pVTZ//MP2/cc-pVTZ electronic energies with the thermodynamic quantities computed at the B3LYP/cc-pVTZ level of theory. T1 = 298.15 K, and T2 = 425, 445, and 420 K for isocytosine, 5-methylcytosine, and 5-fluorocytosine, respectively. c Isocytosine: MP4(SDQ)/6-31þG* results from ref 12; 5-methylcytosine: B3LYP/6-31þþG** results from ref 13; 5-fluorocytosine: MP2/DZPþZPVE(HF/3-21G) results from ref 10. d Tautomer IV of 5-methylcytosine was not included in the present ADC(3) calculations. The tautomer ratio [Ia]/[Ib]/[II]/[III] was 0.49:0.22:0.14:0.15. b

the discrepancies being on the order of 7.5 (O 1s), 6.5 (N 1s), and 4.5 eV (C 1s). Within a specific X 1s spectrum (X = O, N, C), however, the shifts are largely uniform, so that the relative ADC(3) energies afford a rather satisfactory description. The ADC(3) calculations of the ionization spectra were performed using the original code36 linked to the GAMESS ab initio program package.37 The planar molecule approximation was adopted in the ADC(3) calculations. This approximation introduces only minor errors to the calculated spectra but makes it possible to use the molecular symmetry of the Cs point group to simplify both the computations and the interpretation of the results. The planar molecular geometries were obtained from the constrained geometry optimization carried out using the second-order MøllerPlesset perturbation theory (MP2) using cc-pVTZ basis set.3335 The theoretical core-level ionization spectra were constructed as Boltzmann population ratio (BPR) weighted sums over all tautomers. The spectral envelopes were generated by convoluting the discrete transition lines with Lorentzian functions. The values of the Lorentzian widths were selected to best match the experimental intensity, and in an approximate manner, they account for experimental resolution, unresolved vibrational structure, and natural life times of the respective corehole states.

3. RESULTS AND DISCUSSION 3.1. Thermochemistry of the Most Stable Tautomers. Separate quantum chemical calculations were carried out in

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order to obtain the structures and relative stability of tautomers under study (Figure 1). The molecular geometries were fully optimized at the MP2/cc-pVTZ level of theory. The total ground-state electronic energies for the optimal structures were then finalized using the coupled clusters singles and doubles approach (CCSD). The resulting energies were combined with thermochemical corrections computed using the density functional theory (DFT) approach with the Becke three-parameter LeeYangParr (B3LYP) potential38,39 and the cc-pVTZ basis sets to yield the Gibbs free energies and BPRs for the required temperatures. The calculations were performed with the GAUSSIAN package of programs.40 The calculated quantities are summarized in Table 1, where the previous theoretical results for the relative electronic energies of isocytosine, 5-methylcytosine, and 5-fluorocytosine tautomers are also shown for comparison.10,12,13 The electron correlation plays a very important role in the description of the molecular tautomers. The present MP2 and CCSD energies differ from each other quite appreciably, with maximal difference reaching 2.4 kcal/mol. From this, it can be seen that the electron correlation plays an important role here. The latter fact is probably responsible for the disagreements seen between the present results and previous theoretical predictions obtained using schemes with a lower level of electron correlation treatment. The qualitative picture concerning the tautomer ordering is however quite consistently described by the present and previous calculations. The predicted relative tautomer stabilities do not change much when the true Gibbs free energies are considered. Here, we note that the present results are not only theoretically very rigorous but also more complete with respect to the manifold of the considered tautomer and rotamer forms. 3.2. Core-Level Photoemission Spectra. 3.2.1. O 1s Photoelectron Spectra. The oxygen 1s photoemission spectra of 5-methylcytosine, 5-fluorocytosine, and isocytosine are shown in Figure 2 and manifest several features. These molecules contain only one oxygen atom each; therefore, the observation of two or three features directly indicates the presence of more than one tautomeric species. On the basis of the present ADC(3) calculation, the three features labeled A, B, and C in 5-methylcytosine and 5-fluorocytosine spectra can be assigned to the ionization of amino hydroxy (I), aminooxo (II), and iminooxo (III) tautomers, respectively. The measured O 1s spectrum of isocytosine shows only two maxima A and C, each with a full width at half-maximum (fwhm) of 1.0 eV. This suggests that only two tautomeric forms of the isocytosine molecule (aminohydroxy and one oxo form) are significantly populated under the present experimental conditions. Our ADC(3) and thermochemical calculations support such a conclusion and allow assignment of the observed signals A and C to tautomers I and II of isocytosine, respectively. Interestingly, according to the calculations, only rotamer Ia is populated with the ratio [Ia]/[Ib] of 91:1 under the experimental conditions (Table 2). This is apparently a result of specific interaction of the hydroxyl hydrogen atom with the lone pair of the N3 atom, stabilizing the Ia form. Such a stabilization mechanism is absent in form Ib, which leads to its higher relative energy. It has to be noted that the observed prevalence of the single rotamer is characteristic of isocytosine only and is not observed in normal cytosine species where both orientations of the OH group lead to stabilized forms due to the interactions with N1 or N3 atoms and thus, to the more uniformly populated 7724



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Figure 2. Experimental (a) and theoretical (b) O 1s photoionization spectra of isocytosine, 5-methylcytosine, and 5-fluorocytosine. Experiment: points, data; line, fitted curves. The theoretical calculations were convoluted with a Lorentzian curve of half width of 0.9 eV and shifted by 7.5, 7.66, and 7.3 eV to lower binding energy for isocytosine, 5-methylcytosine, and 5-fluorocytosine, respectively.

Table 2. Relative Energies Ω (eV), Pole Strengths P, and Relative Photoelectron Intensities I Including BPRs for Vertical O 1s Ionization Transitions of Isocytosine, 5-Methylcytosine and 5-Fluorocytosine Tautomers Calculated at the ADC(3)/cc-pVDZ Level in Comparison with the Experimental Data (Ωexp and Iexp)

a For easier comparison with experiment, the ADC(3) ionization energies have been corrected by 7.5, 7.66, and 7.3 eV for isocytosine, 5-methylcytosine, and 5-fluorocytosine, respectively (which is the overall shift of the theoretical spectrum with respect to experiment). b Only transitions with I g 0.01 are shown. c The sums of intensities have been normalized to 1.

rotamers. The present observation of the two tautomer species is also in agreement with published infrared studies.4 Stepanian and co-workers investigated the isocytosine molecule in an argon matrix, and they found two tautomeric forms, aminohydroxy and aminooxo.4 Later, using different approaches, this experimental evidence was also confirmed theoretically.12 To extract the relative populations (Iexp), we fitted the experimental spectra with Gaussian functions. The present calculations do not include the vibrational wave functions, and vibronic states are not resolved experimentally due to the large number of states and the lifetimes of the core levels. Within the

BornOppenheimer approximation, electronic and nuclear motion are decoupled, and thus, it is valid to integrate over all vibronic states for a particular electronic transition to obtain an experimental value to be compared with the calculated pole strength. The ionization energies obtained by fitting are then vertical, rather than adiabatic, energies. The Iexp are listed in Table 2, assuming an equal cross section of O 1s, along with the theoretical intensities. They agree very well with the experimental results, and the calculated and measured values for the observed bands differ by at most 4%. The calculated relative ionization energies also agree very satisfactorily with the experimental 7725



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Figure 3. Experimental (a) and theoretical (b) N 1s photoionization spectra of isocytosine, 5-methylcytosine, and 5-fluorocytosine. The theoretical calculations were convoluted with a Lorentzian curve of half width 0.7 eV and shifted by 6.3, 6.9, and 6.2 eV to lower binding energy for isocytosine, 5-methylcytosine, and 5-fluorocytosine, respectively.

data in isocytosine but show some larger deviations from experiment in 5-methylcytosine and 5-fluorocytosine. The theoretical and experimental results for 5-methylcytosine show that at the present experimental temperature, the amino hydroxy form Ia is the most stable, and its rotamer Ib is 0.67 kcal/mol higher in energy. The features due to the aminooxo (II) and iminooxo (III) tautomers in the O 1s spectrum are present with nearly the same intensities (0.15 and 0.16, respectively). The results of the present calculations (0.14 and 0.15, respectively) are in excellent agreement with these data and correspond to the ΔG445 values of 1.11 and 1.04 kcal/mol, respectively (Table 1). These data are in agreement with population ratios observed for infrared spectra of 5-methylcytosine measured in an argon matrix.6 The relative stabilities of 5-methylcytosine in the gas phase were calculated by Les and Adamowicz.11 The calculation was performed at the second-order level of the many-body perturbation theory (MBPT2), and it showed that the most stable tautomeric form is aminohydroxy. The two oxo forms, amino (II) and imino (III and IV), were estimated to be less stable than the main form (I) by about 3.49 and 3.51 kcal/mol, respectively. Sambrano et al., using the B3LYP/6-31þþG** approach, predicted the iminooxo form (III and IV) to be more stable compared with aminooxo tautomers (II).13 A later comprehensive theoretical study of tautomers of methylated derivatives of DNA bases was performed by Ford et al. using DFT and MP2 methods with the 6-31G(d,p) basis set.16 Both theoretical methods predicted that the hydroxy tautomeric form (I) has the lowest energy; however, the DFT calculation showed that the aminooxo form (II) is only 0.07 kcal/mol higher in energy. The MP2 data are in better agreement with the present XPS results. For aminooxo, ΔG was calculated to be 1.16 kcal/mol and agrees with the value reported here of 1.11 kcal/mol, but it overestimates the stability of the iminooxo form, calculated to be 0.5 kcal/mol versus the present value of 1.03 kcal/mol (see Table 1).

On the basis of our recent studies of cytosine tautomers in the gas phase using XPS, we conclude that the methylation of the C5 carbon atom clearly changes the iminooxo versus aminooxo tautomeric equilibrium.18 In the case of cytosine, the populations of the iminooxo and aminooxo forms are 11 and 26%, respectively, while for 5-methylcytosine both oxo-type tautomers are present in nearly the same concentration (see Table 2). The change in the hydroxyoxo tautomeric equilibrium of cytosine derivatives due to halogenation is much stronger than that induced by methylation. The O 1s photoelectron spectrum of 5-fluorocytosine is shown in Figure 2, and the feature A related to aminohydroxy tautomers is dominant in the spectrum. Using the same fitting procedure (see above), the populations of aminohydroxy, aminooxo, and iminooxo are equal to 0.92, 0.05, and 0.03, respectively, with the sums of intensities normalized to 1. These numbers are in excellent agreement with the calculated Boltzmann population ratios reported in Table 1. These numbers correspond to average values of ΔG420 of 0.84 (aminohydroxy Ib), 2.04 (aminooxo), and 3.05 kcal/mol (iminooxo), referred to the most stable aminohydroxy form Ia. The earlier calculations using the MBPT approach predicted correctly that the aminohydroxy form of 5-fluorocytosine is the most stable in the gas phase.10 However, the stability of this tautomer with respect to the aminooxo form, second lowest in energy, was overestimated. The relative energy difference was calculated to be ΔG ≈ 1.1 kcal/mol with respect to the value of 2.04 kcal/mol obtained in the current work. The numbers obtained in the ab initio calculations with the larger basis set 6-31G** and 3-21G optimized geometries agree well with the present data. It has been shown that aminooxo and iminooxo forms are less stable than the aminohydroxy by 3.1 and 3.87 kcal/mol, respectively.5 These energies are close to those that we have observed (see Table 1). 7726



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Table 3. Ionization Energies Ω (eV), Pole Strengths P, and Relative Photoelectron Intensities I Including BPRs for Vertical N 1s Ionization Transitions of Isocytosine, 5-Methylcytosine, and 5-Fluorocytosine Tautomers Calculated at the ADC(3)/cc-pVDZ Level in Comparison with the Experimental Data

a For easier comparison with experiment, the ADC(3) ionization energies have been corrected by 6.3, 6.9, and 6.2 eV for isocytosine, 5-methylcytosine, and 5-fluorocytosine, respectively (which is the overall shift of the theoretical spectrum with respect to experiment).

Published infrared spectra of halogenated cytosine molecules isolated in an argon matrix also showed the dominance of the aminohydroxy tautomer.5 An increase in the relative stabilities of this form on going from substitution of the hydrogen atom at C5 by iodine to fluorine was observed and correlated with the changes of the electronegativities of the halogen atom. The infrared studies of isocytosine performed by Vranken and co-authors showed that the relative free energy of the two tautomeric forms is ΔG400 = 1.74 kcal/mol in favor of the aminohydroxy with respect to the aminooxo.7 This value is in good agreement with our thermochemical calculation and XPS

data (see Tables 1 and 2). The theoretical values of the relative energy were more scattered, and they depended on the theoretical approach used.12 3.2.2. N 1s Photoelectron Spectra. Figure 3 shows the measured and theoretical N 1s spectra of 5-methylcytosine, 5-fluorocytosine, and isocytosine. The calculated relative energies, pole strengths, and relative intensities for vertical N 1s ionization transitions of molecular tautomers are also shown in Table 3, where they are compared with the binding energies of the features observed in the experimental spectra. The cytosine derivatives contain three nitrogen atoms, and the N 1s ionization 7727



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Figure 4. Experimental (a) and theoretical (b) C 1s photoionization spectra of isocytosine, 5-methylcytosine, and 5-fluorocytosine. The theoretical calculations were convoluted with a Lorentzian curve of half width 0.5 eV and shifted by 4.5, 4.8, and 4.55 eV to lower binding energy for isocytosine, 5-methylcytosine, and 5-fluorocytosine, respectively.

spectra manifest two intense maxima A and B and a shoulder C at a higher BE. Using our theoretical calculations, the intense feature A is assigned to the contribution from imino nitrogen atoms in the tautomeric forms, while peak B and shoulder C are related to the amino nitrogen atoms in different tautomeric forms (see Table 3). The ratios of the integrated intensities of the A and B bands in the spectra of 5-methylcytosine, 5-fluorocytosine, and isocytosine are equal to 1.56:1.44, 1.83:1.17, and 1.69:1.31, respectively. Because each molecule contains three nitrogen atoms, the nonstoichiometric ratios of the peak intensities in the spectra are direct evidence of existence of oxo- and hydroxy-type tautomeric forms. The low-energy peak A contains the signal of imino nitrogen atoms. The signal from amino-type nitrogen atoms of amino hydroxy tautomers mainly contribute to the feature B, while the amino-nitrogen atoms present in the oxo forms are responsible for the shoulder C at higher BE (see Figure 3). The observed intensity ratio of A/B indicates a reduction of oxo tautomeric population in halogenated cytosine compared with methylated molecules. These results are consistent with those observed in the O 1s photoionization spectra. The ionization energies of N 1s are summarized in Table 3 and compared with those of cytosine taken under similar experimental conditions. The results show that, due to the higher electronegativity of the halogen atom compared with hydrogen, the N 1s ionization energies are shifted toward higher energies. In contrast, a shift to a lower BE was observed in the spectrum of 5-methylcytosine. This suggests that the presence of the halogen atom affects the aromatic ring by withdrawing charge density, while the methyl group donates charge to the ring, as expected. In the case of 5-methylcytosine and 5-fluorocytosine, N atoms are in meta positions with respect to the substituted C5 carbon atom. Therefore, the energy shifts in the N 1s core-level spectra are mainly due to isotropic inductive effects, charge donation by CH3, and withdrawal by F substitution. The resonance effects are

very weak as they will occur in the ortho and para positions of the pyrimidine ring. The present results are consistent with the recent studies of halogenated pyridine molecules in the gas phase, where similar energy shifts toward higher BE were observed due to the halogenation.41 Isocytosine is an isomer of cytosine with the amino and carbonyl groups interchanged (see Figure 1). In the cytosine molecule, the nitrogen atoms in the pyrimidine ring are in meta positions with respect to carboxyl/carbonyl groups and in ortho and para positions to the amino group. However, for the isocytosine molecule, this situation is reversed. The energy separation between the main features A and B in the N 1s spectrum of cytosine is equal to 1.6 eV, while in the isocytosine spectrum, the energy separation between A and B is only 0.95 eV. These differences in the chemical shifts are well reproduced by the present theoretical calculations (Table 3). Here, we have observed a small difference, 0.1 eV, between the binding energies of the imino nitrogen atoms (peaks A) in the cytosine and isocytosine spectra. The NH2 and OH groups in hydroxy tautomers of cytosine and isocytosine induce increased electron density on the pyrimidine ring at the ortho and para positions through the resonance effect. In oxo-type tautomers, the CdO group decreases the electron density on the ring through the resonance effect on the same positions in the pyrimidine ring. However, for both cytosine and isocytosine, the nitrogen atoms in the pyrimidine ring remain in ortho and para positions with respect to the NH2 and OH/CdO groups. Therefore, the chemical shifts for the ring nitrogen atoms in cytosine and isocytosine due to the resonance effects are expected to be small. Another strong factor that influences the ionization energies is an inductive effect. This effect occurs due to the difference in the electronegativity of the OH, CdO, and NH2 groups attached to the different atoms in the ring. However, the inductive effect will be strongly pronounced for carbon atoms directly involved in the bonding and less so for atoms farther away in the ring, such as nitrogen. 7728



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Table 4. Ionization Energies Ω (eV), Pole Strengths P, and Relative Photoelectron Intensities I Including BPRs for Vertical C 1s Ionization Transitions of Isocytosine, 5-Methylcytosine, and 5-Fluorocytosine Tautomers Calculated at the ADC(3)/cc-pVDZ Level in Comparison with the Experimental Data

For easier comparison with experiment, the ADC(3) ionization energies have been corrected by 4.5, 4.8, and 4.55 eV for isocytosine, 5-methylcytosine, and 5-fluorocytosine, respectively (which is the overall shift of the theoretical spectrum with respect to experiment).

a

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The Journal of Physical Chemistry A In contrast to nitrogen atoms in the ring, the N 1s ionization energies of amino (NH2) and O 1s ionization energies of OH/ CdO groups attached to the pyrimidine ring in cytosine and isocytosine show significant differences. The feature B in the N 1s spectrum of isocytosine is shifted by 0.55 eV to a lower BE with respect to the corresponding feature B in the cytosine spectrum (see Table 2). For the O 1s spectra, this trend has been reversed, and the features associated with hydroxy and oxo groups were at 0.350.4 eV higher energy with respect to the peaks in the cytosine spectrum (Table 2). 3.2.3. C 1s Photoelectron Spectra. The C 1s spectra are reported in Figure 4, and the calculated and measured ionization energies are summarized in Table 4. The molecules have four inequivalent carbon atoms in the pyrimidine ring, while 5-methylcytosine has an additional methyl group linked to the C5 carbon atom. The spectra of 5-methylcytosine and isocytosine show multiple features with different intensity ratios, while the 5-fluorocytosine spectrum manifests four strong peaks. This is further direct experimental evidence of the existence of several tautomers for 5-methylcytosine and isocytosine and the dominance of one tautomeric form for 5-fluorocytosine, in agreement with the O 1s and N 1s core-level results. The observed features in the C 1s core-level spectra are assigned using the present high-level theoretical calculation and by comparison with reported experimental and theoretical spectra of pyrimidine and cytosine.18,41 The lowest band A in 5-methylcytosine and isocytosine contains a signal from the C5 atoms of all tautomers. The peak B is associated with the ionization of the C5 carbon atom in the 5-fluorocytosine spectrum and is clearly shifted to a higher BE. The corresponding peak in the cytosine spectrum is at 290.6 eV.18 By comparison of this value with the ionization energies observed here, we conclude that the methylation in the C5 position and isomerization by the exchange of the hydroxy/oxy and amino groups led to charge donation to the C5 carbon atom. As a result, the peaks in the 5-methylcytosine and isocytosine spectra shifted to lower BE by about 0.2 and 0.4 eV, respectively. The halogen substitution strongly affects the ionization energy of the C5 carbon atom as a chemical shift of 2.12 eV to higher binding energy is observed. These chemical shifts are related to the different electronegativity of the substituted groups. The influence of halogen substitution on the ionization energy of the atom directly involved in the bonding is in agreement with the studies by Bolognesi et al.41 and Carroll et al.42,43 These authors provided detailed descriptions of the effect of halogenation on the screening of carbon core holes in the aromatic pyrimidine and benzene rings. Ionization of C6 atoms in the cytosine spectrum gives rise to two peaks at 291.70 and 292.40 eV associated with hydroxy and oxo tautomeric forms, respectively.18 The difference in binding energy in the C 1s spectra of cytosine tautomers is related to the fact that the C6 atom in the hydroxy form is adjacent to the imino-type nitrogen N1, which in the oxo tautomer forms becomes an amino-type N atom with a lower degree of aromaticity. We use the same assignment for the features B and C in the 5-methylcytosine spectrum. Due to the presence of the CH3 moiety, a general shift by 0.4 eV is observed compared with the values for cytosine. The peak B shows higher intensity associated with the contribution of C 1s ionization of the CH3 group;44 however, it is not well reproduced by the present theoretical calculation (see Figure 4). In the spectrum of isocytosine, the peak B shows a single structure assigned to ionization of the C6

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atom. This is expected because in the hydroxy/oxo tautomerization of isocytosine, as for the case of cytosine, the N3 atom is involved but not the N1 atom. The single peak is also supported by the absence of the iminooxo form of isocytosine (III) in the gas phase. This is in agreement with the O 1s data, present thermochemical calculation, and published infrared spectra discussed above.4 Peaks D and C in 5-methylcytosine and 5-fluorocytosine spectra include the signals from the C4 atoms. The features E and D in the C 1s spectra are associated with the C2 carbon atom. In addition, F is also related to the C2 carbon atom in the iminooxo form of 5-methylcytosine, and this peak is a fingerprint of this tautomer in the C 1s spectrum (see Figure 4 and Table 4). The presence of a halogen atom affects the ionization energies of all carbon atoms in the pyrimidine ring. Compared with the corresponding experimental ionization energies of carbon atoms in cytosine, for 5-fluorocytosine, we observed core-level shifts to higher BE by 0.18, 2.12, 0.12, and 0.05 eV for C6, C5, C4 and C2, respectively. The shift is largest for the C5 carbon atom that is directly bonded to fluorine and weaker for other carbons in the pyrimidine ring. In the N 1s spectrum of 5-fluorocytosine, the peak due to the ionization of imino nitrogen atoms is shifted by 0.35 to a higher BE compared with the corresponding feature in the cytosine spectrum. The chemical shifts for the C6 and C4 carbons are significantly smaller, although these atoms are located in the ring closer to the halogen compared with the nitrogen atoms. This observation can be explained by resonance effects. The halogen atom can resonantly induce a positive charge in the aromatic system at the ortho and para positions with respect to the halogen.4143,45 In the case of 5-fluorocytosine, the resonance effect reduces the inductive effect of F on the C4 and C6 carbon atoms that occupy the ortho positions in the pyrimidine ring with respect to the halogenated C5 atom. The resonance effect produces better screening of the core hole of C4 and C6 atoms, and it results in a decrease of the chemical shift. Similar results have been reported by Clark et al. for chloro- and dichlorobenzenes and by Bolognesi et al. for halogenated pyrimidines.41,45 Our results are also in good agreement with the comprehensive studies of the chemical shifts of carbon 1s ionization energies for the methyl-substituted and fluoro-substituted benzenes by Carroll et al. and Myrseth et al.42,43,46 Using high-resolution XPS, they showed that the methyl group donates charge to the benzene ring and increases the reactivity of the ring in the positions ortho and para relative to the methyl group. Fluorine is an overall strongly electron-withdrawing atom but also a weak π electron-donating atom. In the isocytosine spectrum, the features due to the C2 and C4 carbon atoms overlap. The calculation of relative energies shows that the ionization energy of C4 is higher by 0.1 eV. The stronger peak C shows a prominent shoulder D at higher BE. We associate the band D with the aminooxo tautomeric forms of isocytosine. The difference in binding energies is related to the fact that the C2 atoms in the hydroxy forms (I, see Figure 1) are bonded to the imino-type nitrogen N3, while in aminooxo tautomers (II, Figure 1), this nitrogen becomes an amino-type with a lower degree of aromaticity. The intensity ratio D/C was obtained by fitting the spectrum with two Gaussian functions and is equal to 0.10. This corresponds to populations of aminohydroxy and aminooxo equal to 80 and 20%, respectively. These values are in reasonable agreement with those observed in the O 1s data but indicate an overestimation of the population of the aminooxo 7730



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Figure 5. O K-edge (a), N K-edge (b), and C K-edge (c) photoabsorption spectra of 5-methylcytosine (1), 5-fluorocytosine (2), and isocytosine (3).

tautomeric form. The various two-hole/one-particle photoelectron satellites can contribute to the feature D and cause this overestimation. 3.3. Photoabsorption Spectra. Figure 5 presents the O, N, and C 1s NEXAFS spectra of all three molecules. The resonances can be assigned by comparison with recently reported experimental and theoretical spectra of pyrimidine and cytosine.4749 The O 1s NEXAFS spectra (Figure 5a) of the three samples are dominated by two sharp features A and B and a broad highenergy feature C. The maximum A in all spectra is assigned to transitions of O(oxo) 1s to the antibonding π-type unoccupied valence orbital V1.49 These resonances are the fingerprint of oxo tautomeric forms, and indeed, the feature A is stronger in the spectrum of 5-methylcytosine and weaker in the case of 5-fluorocytosine. This evidence is consistent with the XPS results where the population of oxo tautomers of 5-methylcytosine is 31.5%, while it is only about 8% for the 5-fluorocytosine molecule. In the spectrum of isocytosine, the maximum A is shifted by ∼0.2 eV toward lower energy compared with the corresponding peak in the spectrum of 5-methylcytosine. Peak B is more complex, with the main contribution coming from transitions of hydroxy O 1s to the V1 unoccupied valence orbital and strong Rydberg-like excitations to 3s. This peak also contains a very weak contribution from the O(oxo) 1s f 3s transition. This assignment is supported by measured O 1s absorption spectra of uracil and thymine, which showed only oxo tautomers in the XPS data, and no intense feature in the higher-energy range of the absorption spectra was observed.21,49

We associate the broad feature C in the spectra with a combination of 3p, 4s, and 4p Rydberg transitions, and the more intense lines are related to the hydroxy tautomer.49 The N 1s NEXAFS spectra (see Figure 5b) present three strong features. The spectrum of 5-methylcytosine resembles the cytosine spectrum49 because for both molecules, similar populations of the tautomers occur, and the methyl group represents a small perturbation. The spectrum of 5-fluorocytosine shows some differences due to the dominance of the hydroxy tautomer under the present experimental conditions. The strong feature B at ∼399.2 eV in all spectra is attributed to excitations N1 1s f V1 and N3 1s f V1 of hydroxy tautomers. This feature is the strongest peak in the N K-edge spectra of biomolecules containing a pyrimidine ring. In the N K-edge spectrum of pyrimidine, this band was observed to be 0.4 eV lower in energy.49 The transitions N7 1s f V1 and N3 1s f V1 for iminooxo and aminooxo tautomers, respectively, are responsible for the shoulder of the main feature in the spectrum of 5-methylcytosine. The peak B and its shoulder are the fingerprint of the existence of hydroxy and oxo tautomers of pyrimidine-containing molecules. The shoulder A in the 5-fluorocytosine spectrum is very weak compared with the 5-methylcytosine spectrum. This is consistent with XPS data, where it has been shown that the oxo tautomeric form is less populated for 5-fluorocytosine than for 5-methylcytosine. The peak C was not observed in the N K-edge spectrum of pyrimidine.48 Pyrimidine contains two imino nitrogen atoms in its structure. This suggests that N 1s excitation of amino nitrogen atoms to the valence V1 orbital contributes to this band in the 7731



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Table 5. Fractional Tautomer Populations at the Given Temperatures compound

hydroxy

oxo

other

cytosine (450 K)

0.63

0.26

0.11

5-methylcytosine (445 K) 5-fluorocytosine (420 K)

0.68 0.92

0.13 0.06

0.19 0.02

isocytosine (425 K)

0.92

0.08

0

spectra (N1 and N7 in the oxo and N7 in the hydroxy forms of 5-methylcytosine and 5-fluorocytosine and N3 and N7 in the oxo and N7 in hydroxy forms of isocytosine). Part of the spectral intensity of the feature C comes from the excitations of the 1s amino nitrogen atom of both oxo and hydroxy tautomeric forms to the 3s Rydberg state. These assignments are supported by published theoretical calculations of cytosine absorption spectra using the extended second-order ADC approximation scheme for the polarization propagator.49 The features D and E were also observed in the spectrum of pyrimidine measured by Bolognesi et al.,48 and they are very complex. These bands are associated with the 3p, 4p, and 4s Rydberg states and higher members of the series.49 Transitions of 1s electrons of amino and imino nitrogen atoms of all tautomers to the lowest unoccupied V2 and V3 molecular orbitals also contribute to the intensities of features D and E.49 In summary, the N 1s NEXAFS spectra confirm that several tautomers are populated. The C 1s absorption spectra are shown in Figure 5 and manifest rich fine structures. The spectrum of 5-methylcytosine contains four clear maxima and a few shoulders. The shoulder A is absent in the spectrum of 5-fluorocytosine, and features C and D overlap in the spectrum of isocytosine. The lowest photoelectron band A in the 5-methylcytosine and isocytosine spectra can be attributed to the C5 1s f V1 transition associated with both oxo and hydroxy tautomers. In the 5-fluorocytosine spectrum, this feature is chemically shifted to higher photon energy and overlaps with the more intensive peaks (see Figure 5b). The shift is due to the effect of the higher electronegativity of the halogen that substitutes the hydrogen atom bound to C5. The proposed assignments are supported by theoretical and experimental studies of cytosine, pyrimidine, and halogenated pyrimidines in the gas phase.4749 The next three strong features B, C, and D in the carbon NEXAFS spectra are related mainly to the excitation of 1s electrons of C6, C4, and C2, respectively to the valence V1 molecular orbital. In the case of isocytosine, the peaks due to the C4 1s f V1 and C2 1s f V1 transitions overlap, giving a broad peak (see Figure 5c). This is consistent with the XPS data, where the C 1s chemical core-level shifts of C4 and C2 were not resolved experimentally. The carbon atoms are not directly involved in the hydrogen migration (see Figure 1); therefore, in general, each tautomer contributes to all C 1s excitation bands, and there are no bands due to individual tautomers. As was shown in the case of cytosine, this is because the valence configurations of carbon do not change from one tautomer to another, and the C 1s excitation spectra of different tautomers are similar. However, some assignments to individual tautomers seem to be possible based on the published cytosine data,49 where differences appear mainly as shoulders on strong peaks. For example, the band B is mainly influenced by C4 1s f V1 excitations of hydroxy tautomers,

while the shoulder on its high-energy side is due to the same transition in the oxo tautomers. In the case of feature C, the lowenergy flank is associated with the C4 1s f V1 transition of oxo tautomers, while the same transition of the hydroxy form is slightly shifted to higher energy. A large number of less-intense 3s, 3p, and 4p Rydberg and V2 and V3 valence transitions contribute to the spectral intensity at higher energy.49

4. DISCUSSION: TAUTOMERIC EQUILIBRIA The above experimental tautomer populations can be compared with those of the parent compound, cytosine18 (see Table 5). The effect of the electronegativity of the ligand groups on oxohydroxy equilibria appears straightforward; substitution of hydrogen by the mildly electron-donating methyl group hardly changes the total hydroxy population from 63 to 68%. However, substitution by fluorine increases the hydroxy population to 92%. Fluorine is strongly electronegative, but it is also a π donor, and both properties appear to stabilize the aromatic ring, that is, there are both inductive and resonance effects. The effect of pure isomerism (cytosine compared to isocytosine) is also to change the equilibrium from 63% hydroxy to 92%, comparable to the effect of fluorination. The effect here is more complex but can only be due to anisotropic resonance effects because the functional groups are the same but rearranged, and there is no net change in the sum of inductive effects. 5. CONCLUSIONS The relative energies of the tautomers of cytosine derivatives have been calculated using MP2 and CCSD methods and ccpVTZ basis sets to yield quantitative predictions of the Boltzmann population ratios. The XPS and NEXAFS spectra of 5-methylcytosine, 5-fluorocytosine, and isocytosine have been measured at the C 1s, N 1s, and O 1s edges. The vertical corelevel ionization energies and corresponding intensities have been calculated using the ADC(3) approximation scheme for the oneparticle Green’s function and the cc-pVDZ basis set. The results of the ADC(3) calculations have been combined with the calculated BPRs for different tautomers, and a consistent theoretical model of XPS spectra of the cytosine derivatives was obtained. The measured and calculated core-level ionization spectra are in good quantitative agreement with each other. The agreement of the calculated and experimental intensities confirms the calculated Boltzmann population ratios. By comparison with cytosine spectra, we conclude that the methylation of C5 carbon atoms hardly changes the fraction of hydroxy tautomers but changes significantly the iminooxo versus aminooxo tautomeric equilibrium. In 5-methylcytosine, these tautomers are present with nearly the same population, while in the case of cytosine, the concentration of the aminooxo form was higher by a factor of 2. The substitution of hydrogen by fluorine in the C5 position leads to a strong dominance of the hydroxy tautomer. For isocytosine, the tautomeric aminooxo and aminohydroxy forms coexist, with the aminooxo form less stable by 2.07 kcal/mol at 425 K. ’ AUTHOR INFORMATION Corresponding Author

*Tel.: þ39 0403758287. Fax: þ390403758565. E-mail: vitaliy. [email protected]. 7732


The Journal of Physical Chemistry A Present Addresses #

Department of Physics and Astronomy, Aarhus University, Ny Munkegade 120, 8000 Aarhus C, Denmark.

’ ACKNOWLEDGMENT We gratefully acknowledge the assistance of our colleagues at Elettra for providing good-quality synchrotron light. The theoretical part of this study was supported by a grant of the Russian Foundation for Basic Research (RFBR). The authors are indebted to Prof. J. Schirmer for interest in this work and valuable discussions. ’ REFERENCES (1) de Vries, M. S.; Hobza, P. Annu. Rev. Phys. Chem. 2007, 58, 585. (2) Stanovnik, B.; Tisler, M.; Katritzky, A. R.; Denisko, O. V. Adv. Heterocycl. Chem. 2006, 91, 1. (3) Brown, R. D.; Godfrey, P. D.; McNaughton, D.; Piertol, A. P. J. Am. Chem. Soc. 1989, 111, 2208. (4) Stepanian, S. G.; Radchenko, E. D.; Sheina, G. G.; Blagoi, Yu. P. J. Mol. Struct. 1990, 216, 77. (5) Jaworski, A.; Szczesniak, M.; Szczesniak, K.; Kubulat, K.; Person, W. B. J. Mol. Struct. 1990, 223, 63. (6) Lapinski, L.; Nowak, M. J.; Fulara, J.; Les, A.; Adamowicz, L. J. Phys. Chem. 1990, 94, 6555. (7) Vranken, H.; Smets, J.; Maes, G.; Lapinski, L.; Nowak, M. J. Spectrochim. Acta, Part A 1994, 50, 875. (8) Caminati, W. Angew. Chem., Int. Ed. 2009, 48, 9030. (9) Alonso, J. L.; Pe~na, I.; Lopez, J. C.; Vaquero, V. Angew. Chem., Int. Ed. 2009, 48, 6141. (10) Adamowicz, A. Chem. Phys. Lett. 1988, 153, 147. (11) Les, A.; Adamowicz, L. J. Mol. Struct. 1990, 221, 209. (12) Gorb, L.; Podolyan, Y.; Leszczynski, J. J. Mol. Struct.: THEOCHEM 1999, 487, 47. (13) Sambrano, J. R.; de Souza, A. R.; Queralt, J. J.; Oliva, M.; Andres J. Chem. Phys. 2001, 264, 333. (14) Trygubenko, S. A.; Bogdan, T. V.; Rueda, M.; Orozco, M.; Luque, F. J.; Sponer, J.; Slavicek, P.; Hobza, P. Phys. Chem. Chem. Phys. 2002, 4, 4192. (15) Fogarasi, G. J. Phys. Chem. A 2002, 106, 1381. (16) Forde, G. K.; Forde, A. E.; Hill, G.; Ford, A.; Nazario, A.; Leszczynski, J. J. Phys. Chem. B 2006, 110, 15564. (17) Brown, R. S.; Tse, A.; Vederas, J. C. J. Am. Chem. Soc. 1980, 102, 1174. (18) Feyer, V.; Plekan, O.; Richter, R.; Coreno, M.; Vall-llosera, G.; Prince, K. C.; Trofimov, A. B.; Zaytseva, I. L.; Moskovskaya, T. E.; Gromov, E. V.; Schirmer, J. J. Phys. Chem. A. 2009, 113, 5736. (19) Plekan, O.; Feyer, V.; Richter, R.; Coreno, M.; Vall-llosera, G.; Prince, K. C.; Trofimov, A. B.; Zaytseva, I. L.; Moskovskaya, T. E.; Gromov, E. V.; Schirmer, J. J. Phys. Chem. A 2009, 113, 9376. (20) Melandri, S.; Evangelisti, L.; Maris, A.; Caminati, W.; Giuliano, B. M.; Feyer, V.; Prince, K. C.; Coreno, M. J. Am. Chem. Soc. 2010, 132, 10269. (21) Plekan, O.; Feyer, V.; Richter, R.; Coreno, M.; de Simone, M.; Prince, K. C.; Trofimov, A. B.; Gromov, E. V.; Zaytseva, I. L.; Schirmer, J. Chem. Phys. 2008, 347, 360. (22) Prince, K. C.; Blyth, R. R.; Delaunay, R.; Zitnik, M.; Krempasky, J.; Slezak, J.; Camilloni, R.; Avaldi, L.; Coreno, M.; Stefani, G.; Furlani, C.; de Simone, M.; Stranges, S. J. Synchrotron Radiat. 1998, 5, 565. (23) Myrseth, V.; Bozek, J. D.; Kukk, E.; Sæthre, L. J.; Thomas, T. D. J. Electron Spectrosc. Relat. Phenom. 2002, 122, 57. (24) Thomas, T. D.; Shaw, R. W., Jr. J. Electron Spectrosc. Relat. Phenom. 1974, 5, 1081. (25) Hatamoto, T.; Matsumoto, M.; Liu, X.-J.; Ueda, K.; Hoshino, M.; Nakagawa, K.; Tanaka, T.; Tanaka, H.; Ehara, M.; Tamaki, R.; Nakatsuji, H. J. Electron Spectrosc. Relat. Phenom. 2007, 155, 54.

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Comprehensive Core-Level Study of the Effects of Isomerism

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