Near-IR-Induced, UV-Induced, and Spontaneous

Article pubs.acs.org/JPCB

Near-IR-Induced, UV-Induced, and Spontaneous Isomerizations in 5‑Methylcytosine and 5‑Fluorocytosine Leszek Lapinski,† Igor Reva,‡ Hanna Rostkowska,† Rui Fausto,‡ and Maciej J. Nowak*,† †

Institute of Physics, Polish Academy of Sciences, Al. Lotnikow 32/46, 02-668 Warsaw, Poland Department of Chemistry, University of Coimbra, 3004-535 Coimbra, Portugal

‡

S Supporting Information *

ABSTRACT: Monomeric 5-methylcytosine (5mCyt) and 5fluorocytosine (5FCyt) were studied using the matrix-isolation method. In 5mCyt and 5FCyt, the most stable form, dominating in low-temperature matrixes, is the amino-hydroxy (AH) tautomer. For both compounds, irradiation of the matrixes with near-IR laser light or with broadband near-IR or mid-IR light induces interconversions between the two rotamers of tautomer AH. In addition, for matrixes kept in darkness, a spontaneous tunneling conversion of the higherenergy hydroxy conformer (with the OH group directed toward the N3 atom) into the lower-energy form (OH directed toward N1) was occurring, with half-life time of 70 min for 5mCyt and 127 min for 5FCyt. These tunneling processes are much faster than that found for unsubstituted cytosine, where the half-life time is more than 30 h. UV irradiation of 5mCyt (at 316 nm) led to phototautomeric conversion of the amino-oxo form into the amino-hydroxy tautomer. Another phototransformation induced by irradiation of 5mCyt at 316 nm was the cleavage of the C−N bond in the amino-oxo form, resulting in generation of the open-ring conjugated isocyanate product. Irradiation of 5mCyt at shorter waves (λ ≤ 310 nm) induced the syn−anti photoisomerization within the imino-oxo forms of the compound. For matrix-isolated 5FCyt, the amount of the amino-oxo form was very small (with respect to the amino-hydroxy tautomer), while the imino-oxo isomers were not detected at all.

1. INTRODUCTION Ultrafast deactivation of electronically excited states, reported during the past decade for nucleic acid bases,1−4 had a profound influence on the common opinion about the photostability of this class of compounds. The reports suggested that there are very effective deactivation pathways, allowing extremely fast (∼1 ps) depopulation of low-energy excited states of nucleic acid bases. Many authors concluded that such a very rapid dissipation of the excited state energy should prohibit any rearrangement of the atoms in the structure of nucleic acid bases. These conclusions were in contrast with UV-induced hydrogen-atom-transfer and ring-opening processes experimentally found in isolated monomers of nucleic acid bases4−8 as well as for these compounds in solution9−11 and in the solid state.12 Very recently, lifetimes have been determined for the excited 1 ππ state vibronic levels of the amino-oxo form of cytosine in the gas phase.13,14 It has been shown that the lifetime of the 0− 0 level is as long as 44 ps and that up to 437 cm−1 of energy excess the lifetime does not decrease below 25 ps. Such lifetimes (20−40 times longer than those previously observed for higher-energy excitations)1−4 should allow rearrangements of atoms and generation of photoproducts with altered structure. Hence, the observation of non-ultrafast lifetimes14 © 2014 American Chemical Society

makes the occurrence of photoisomerizations in nucleic acid bases, in particular in cytosines, comprehensible. In the present work, we studied light-induced structural changes in 5-methylcytosine and 5-fluorocytosine. Both compounds are relevant from the biological point of view. 5Methylcytosine is a product of DNA methylation,15,16 which is the most important epigenetic alteration in eukaryotes. Methylation of cytosine residues in genomic DNA plays a key role in the regulation of gene expression by blocking transcription and causing gene silencing.17−23 5-Fluorocytosine (“flucytosine”) is an antifungal agent.24 It is also an antitumor prodrug, which converts to the highly toxic metabolite, 5fluorouracil.25,26 Treatment with 5-fluorocytosine, combined with a tumor-specific gene therapy, results in more selective elimination of tumor cells without significant toxicity to the patient.27,28 Because of the biological importance of 5methylcytosine and 5-fluorocytosine, numerous investigations were devoted to the physicochemical and spectroscopic properties of these compounds.13,29−33 Received: November 20, 2013 Revised: February 19, 2014 Published: February 24, 2014 2831

dx.doi.org/10.1021/jp411423c | J. Phys. Chem. B 2014, 118, 2831−2841

The Journal of Physical Chemistry B

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Table 1. Relative Electronic Energies (kJ mol−1) of Isomeric Forms of Cytosine, 5-Methylcytosine, and 5-Fluorocytosined

a Calculated in ref 34 using the cc-pVQZ basis set. bCalculated in the current work using the 6-31++G(d,p) basis set. cCalculated in ref 29 using the cc-pVTZ basis set. Zero-point vibrational energy calculated at the DFT(B3LYP)//DFT(B3LYP) level using the 6-31++G(d,p) basis set. dThe values corrected for zero-point vibrational energy are given in parentheses. (method A)//(method B) means that electronic energy was calculated using (method A) at geometry optimized using (method B).

Cryogenics DE-202A closed-cycle helium refrigerator. The mid-IR spectra were recorded with 0.5 cm−1 resolution using a Thermo Nicolet Nexus 6700 FTIR spectrometer equipped with a KBr beam splitter and a DTGS detector. Near-IR spectra were recorded using the same spectrometer but equipped with a CaF2 beam splitter and an InGaAs detector. Monomers of 5fluorocytosine or 5-methylcytosine isolated in Ar matrixes were irradiated using narrowband tunable near-IR light of the idler beam of the Quanta-Ray MOPO-SL pulsed (10 ns) optical parametric oscillator (full width at half-maximum 0.2 cm−1, repetition rate 10 Hz, pulse energy 10 mJ) pumped with a pulsed Nd:YAG laser. For UV irradiations, the frequencydoubled signal beam of the same Quanta-Ray MOPO-SL optical oscillator was used. In order to protect matrixes from higher-frequency infrared light, some spectra were recorded using a standard Edmund Optics long-pass filter (with a cutoff at 2200 cm−1, see Figure S2 in the Supporting Information) placed between the spectrometer source and the matrix sample.

The studies carried out in the present work concern photoisomerizations occurring in isolated monomers of 5methylcytosine and 5-fluorocytosine upon excitation with nearinfrared (near-IR) or ultraviolet (UV) narrowband laser light. The experimental methods, applied for this purpose, represent the state of the art in matrix-isolation photochemistry. Apart from the light-induced transformations, hydrogen-atom tunneling was also observed, for both compounds, and the rates of this spontaneous process were analyzed.

2. EXPERIMENTAL SECTION The sample of 5-fluorocytosine (5FCyt), purity 98%, used in the present study was a commercial product supplied by SigmaAldrich. 5-Methylcytosine (5mCyt) was purchased from AlfaAesar, but the compound, as a free base, was found to readily convert to thymine when exposed to the air (see Figure S1 in the Supporting Information). Therefore, for the experiments reported in the current paper, we used a sample of 5methylcytosine hydrochloride, supplied by Sigma-Aldrich. Free base 5-methylcytosine was prepared immediately before the matrix experiment, by vacuum sublimation in an evacuated, sealed glass tube. By this procedure, free base of 5methylcytosine was condensing from the gas phase onto the glass walls above the heated hydrochloride crystals, whereas the evolving gaseous HCl was trapped in a remote part of the glass tube, kept in liquid nitrogen. In order to obtain the low-temperature matrixes, crystals of the studied compounds were heated [to ca. 450 K (5mCyt) and ca. 425 K (5FCyt)] in a miniature glass oven placed in the vacuum chamber of a helium-cooled cryostat. The vapors coming out of the oven were codeposited with a large excess of argon onto a CsI window, cooled to 14 K by an APD

3. COMPUTATIONAL SECTION Relative energies of the five isomeric forms of cytosine, 5methylcytosine, and 5-fluorocytosine (see the structures presented in Table 1) were calculated using the QCISD35 and CCSD36 methods. The barriers for rotation of the OH group in the amino-hydroxy tautomers of cytosine, 5methylcytosine, and 5-fluorocytosine in the electronic ground state were calculated using the minimum-energy-path approach. At each of the points, intermediate between the minima corresponding to the rotameric structures AH1 and AH2, all geometry parameters (except for the torsional angle N1−C−O−H, which served as a driving coordinate) were optimized using the MP2 method.37 2832



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The geometries of the isomers of 5-methylcytosine and 5fluorocytosine were fully optimized using the density functional theory DFT(B3LYP) method38−40 with the Becke threeparameter exchange functional38 and the Lee, Yang, Parr correlation functional.39 At the optimized geometries, the harmonic vibrational frequencies and IR intensities were calculated at the same level. The 6-31++G(d,p) basis set was used in all calculations. All quantum-mechanical computations were carried out with the Gaussian 03 program.41

4. RESULTS AND DISCUSSION 4.1. Relative Energies and Relative Populations of Isomeric Forms of 5mCyt and 5FCyt. The relative energies of the isomers of 5mCyt and 5FCyt were calculated at the QCISD/6-31++G(d,p) and CCSD/6-31++G(d,p) levels of theory. The results of these calculations are compared with relative energies obtained for cytosine (Cyt) isomers in Table 1. All the calculations presented in Table 1 indicate that the amino-hydroxy AH1 form should be the most stable isomer of 5mCyt as well as of 5FCyt. For both compounds, the AH1 form should be most abundant in the gas phase and in lowtemperature inert gas matrixes. According to the calculations, the other amino-hydroxy rotameric AH2 forms of 5mCyt and 5FCyt should be only slightly (by ca. 3 kJ mol−1) higher in energy than the corresponding AH1 rotamers. Hence, the AH2 forms of 5mCyt and 5FCyt should be populated in the gas phase and trapped in low-temperature matrixes. As far as the canonical amino-oxo (AO) form of 5mCyt is concerned, the calculations predict its relative energy, with respect to AH1, to be significantly higher than that for unsubstituted cytosine (see Table 1). For 5FCyt, the relative energy of AO isomer is even higher. Consequently, in comparison to the gas-phase equilibrium in cytosine, the amino-oxo AO form should be less abundant in 5mCyt and only very slightly populated in 5FCyt. The high-frequency regions of the experimental mid-infrared spectra of Cyt, 5mCyt, and 5FCyt isolated in argon matrixes are presented in Figure 1 (lower panel). The infrared bands due to the OH and N1H stretching vibrations, present in this spectral region, have similar absolute intensities (close to 100 km mol−1, see Table S1 in the Supporting Information). Moreover, these bands do not overlap with the bands due to any other vibrations. Hence, the relative intensities of the experimental bands due to these vibrations can serve as an approximate estimate of relative populations of the amino-hydroxy, aminooxo, and imino-oxo forms of the studied compounds trapped in low-temperature matrixes. In particular, the bands due to νOH vibrations (at ca. 3600 cm−1) should represent the population of the amino-hydroxy AH form in the matrix, whereas the bands due to νN1H vibrations (at ca. 3460 and at 3490 cm−1) should represent the populations of the amino-oxo AO and imino-oxo IO isomers, respectively. Comparison of relative intensities of the band ascribed to νN1H vibration in AO forms of Cyt, 5mCyt, and 5FCyt (see Figure 1) shows that the population of this form in 5mCyt is lower than that in Cyt, while the population of the AO form in 5FCyt is very small. These observations are then in a qualitative agreement with the theoretical relative energies presented in Table 1. Comparison of the theoretically predicted relative energies of IO1 and AO forms in 5mCyt and Cyt suggests that the relative population of IO1 should be higher in 5mCyt than in unsubstituted Cyt. Accordingly, the intensity of the νNH

Figure 1. Fragments of the experimental near-IR and mid-IR spectra of cytosine (Cyt), 5-methylcytosine (5mCyt), and 5-fluorocytosine (5FCyt) isolated in Ar matrixes. In the presented spectral ranges, there appear absorption bands due to the fundamental transitions (lower panel) and first overtones (upper panel) of the OH and NH stretching vibrations. The bands marked (AH + AO) are due to antisymmetric νaNH2 and symmetric νsNH2 stretching vibrations of the NH2 group. The theoretical νaNH2 and νsNH2 frequencies predicted for AH1, AH2, and AO forms are very similar (see Table S1, Supporting Information), and the corresponding experimental bands overlap.

band at ca. 3490 cm−1, with respect to bands characteristic of other forms, shows that the IO tautomer is more populated in 5mCyt than in Cyt. On the other hand, in the spectrum of 5FCyt, the analogous band is missing. This indicates a very small population of IO in 5FCyt, which is in agreement with the high relative energy (more than 12 kJ mol−1) calculated for IO forms of this compound. The assignment of the IR absorption bands observed in the spectra of 5-fluorocytosine and 5-methylcytosine isolated in Ar matrixes is shown in Figures S3 and S4 (in the Supporting Information). The experimental IR bands were ascribed to isomeric forms of 5mCyt and 5FCyt on the basis of their intensity changes induced by UV or near-IR irradiation of the matrixes (as described below). 4.2. Photoisomerizations Induced by Excitation with UV Light. Monomers of 5-methylcytosine isolated in argon matrixes were excited at a series of chosen UV wavelengths using tunable, narrowband UV laser light. The frequencydoubled signal beam of the optical parametric oscillator was used for this purpose. The UV irradiation started at λ = 322 nm. It was followed by consecutive irradiations at gradually 2833



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Figure 2. Identification of the amino-oxo form of 5-methylcytosine isolated in an argon matrix. (a−c) Fragments of the experimental infrared spectra of 5mCyt: (blue) recorded before any irradiation; (red) after narrowband UV irradiation at λ = 316 nm; (black) difference spectrum: trace (red) minus trace (blue). (d) (green) theoretical spectrum of the AO form calculated at the DFT(B3LYP)/6-31++G(d,p) level. The theoretical frequencies were scaled by a single factor of 0.98, and the theoretical intensities were multiplied by −1. The tilde indicates the theoretical νCO band (truncated); the predicted absolute intensity of this band is 893 kJ mol−1.

shorter UV wavelengths. After each irradiation, the matrix was monitored by taking its IR spectrum. UV irradiations with λ ≥ 318 nm did not induce any transformations of matrix-isolated 5mCyt. The first changes in the IR spectrum of the compound were observed only after the irradiation at 317−316 nm. Upon irradiation at these wavelengths, the population of AO tautomer of 5mCyt diminished (see Figure 2). The applied wavelengths (317−316 nm) are only slightly shorter than the 0−0 origin (at 319.7 nm) of the absorption spectrum measured by REMPI spectroscopy for AO isomer of 5mCyt in the gas phase.13,31,32 The decrease of AO population occurring upon irradiation at 316 nm was accompanied by a slight increase of the population of the amino-hydroxy tautomer (Figure 2). Another product growing at the cost of the AO form manifested itself by a band with maxima at 2273 and 2261 cm−1 (Figure 3). Also, for 1,5dimethylcytosine, where the amino-oxo form dominates in the matrix before any irradiation, a new structured band at 2247 cm−1 appeared in the infrared spectrum recorded after exposure to UV (λ > 314 nm) light. Photoproducts having characteristic bands at ca. 2260−2270 cm−1 in their IR spectra were previously observed in photochemical investigations on 1-methyl-2(1H)pyrimidinone and cytosine. These products were assigned to open-ring structures with a conjugated isocyanate moiety.5,6,42 It is very probable that a Norrish type I ring-opening process occurs also for 5mCyt and that conjugated isocyanate is generated, upon UV irradiation, from the AO form of this compound. Very

Figure 3. Fragment of the infrared spectrum of 5-methylcytosine monomers isolated in an Ar matrix recorded (black) after deposition of the matrix and (red) after irradiation of the matrix with monochromatic λ = 316 nm light. The schemes present the two most stable structures of the open-ring conjugated isocyanate, the plausible carrier of the new band.

recently, ring-opening by Norrish type I cleavage of the CN bond (α-bond with respect to the CO group) was observed for a series of oligonucleotides and mononucleosides, both in solid films and in solution.12 Isocyanates were identified as products of these photoreactions. It looks then that UVinduced ring-opening constitutes an important channel in the 2834



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photochemistry of nucleic acid bases that has been recognized only recently. Upon subsequent UV (λ ≤ 310 nm) irradiation of 5mCyt isolated in an argon matrix, the imino-oxo IO1 form of the compound converted into IO2 isomer. Spectral manifestations (Figure 4) of this syn−anti isomerization allowed identification

Figure 4. Effect of UV-induced syn−anti isomerization in the iminooxo form of 5-methylcytosine isolated in an argon matrix. (bottom) Difference spectrum obtained as the spectrum recorded after irradiation at λ = 308 nm minus the spectrum recorded before this irradiation but after the previous irradiations at λ = 316−310 nm. (top) Theoretical spectra of the IO1 and IO2 isomers calculated at the DFT(B3LYP)/6-31++G(d,p) level. All theoretical frequencies were scaled by a single factor of 0.98, and the theoretical intensities of the bands due to form IO1 were multiplied by −1.

Figure 5. Fragments of the infrared spectra of 5-methylcytosine isolated in an argon matrix: (black) recorded after a series of irradiations at λ = 316−310 nm; (red) recorded after the subsequent irradiation at λ = 308 nm.

of both imino-oxo isomers of 5mCyt. The initial population of form IO2 was very small, but it increased several times upon irradiation at λ ≤ 310 nm (Figure 5). The identification of IO2 as the product growing upon UV (λ ≤ 310 nm) irradiation at the cost of IO1 is based on the good agreement between the experimental and theoretical IR spectra of those species (Figure 4). The photoreaction transforming the IO1 form of 5mCyt into IO2 is analogous to the syn−anti photoisomerizations transforming the IO1 form of cytosine5 or 1-methylcytosine6 into the IO2 product. This analogy additionally supports the assignment of the set of bands, growing upon UV (λ ≤ 310 nm) irradiation of 5mCyt, to the IO2 form of the compound. For 5FCyt isolated in low-temperature argon matrixes, the amino-hydroxy tautomer dominates very strongly. Nearly all of the bands observed in the IR spectrum of 5FCyt are due to AH1 and AH2 rotamers of the amino-hydroxy tautomer. These bands either grow or decrease in the AH1 ↔ AH2 photorotamerization process induced by near-IR excitations; see sections 4.3 and 4.4. Apart from the IR bands ascribed to the amino-hydroxy AH1 and AH2 forms, only few lowintensity IR bands were observed in the spectrum of 5FCyt. Such bands, assigned to AO tautomer, are not affected by nearIR irradiations of the matrix. The small population of the AO form of 5FCyt was found to decrease upon exposure of the matrix-isolated compound to UV (λ ≤ 313 nm) light (Figure 6). At the cost of the decreasing population of AO, some tiny increase of the population of AH tautomer was observed. The set of IR features decreasing upon UV (λ ≤ 313 nm) irradiation

includes the characteristic AO bands due to the stretching vibrations of the NH group at 3465 cm−1 and of the CO group at 1734, 1730, and 1715 cm−1. This observation further supports the assignment of the spectrum in question to the AO tautomer of 5FCyt. 4.3. Conformational AH1−AH2 Conversions Induced by Monochromatic Near-IR Light. Prior to an attempt to induce a transformation between conformers AH1 and AH2 by excitation of matrix-isolated cytosines with near-IR light, the 2νOH and 2νNH overtone regions of the spectra were recorded for 5mCyt and 5FCyt isolated in argon matrixes. In the 7100−6700 cm−1 range of those spectra, presented in Figure 1 (upper panel), absorption features due to overtones of νOH, νNH2, and νNH vibrations were found. The bands of the fundamental νOH, νNH2, and νNH transitions were found in the 3620−3400 cm−1 spectral region (Figure 1, lower panel). It seems very likely that the comparatively strong absorptions at 7035 cm−1 (5FCyt), 7029 cm−1 (5mCyt), and 7033, 7016 cm−1 (Cyt) are due to the overtones of the νOH vibrations. Note that the νOH overtone bands do not appear at the highest frequencies of all the bands in the 7100−6700 cm−1 region, despite that fundamental νOH bands have the highest frequencies of all the absorptions found in the 3620−3400 cm−1 range. This must be related with higher anharmonicity of the OH vibrations, with respect to the anharmonicities of the NH stretching modes. In the spectra of 5mCyt and 5FCyt, the bands due to the νOH fundamental transitions in forms AH1 and AH2 overlap 2835



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Figure 7. Fragment of the experimental mid-IR spectra of 5fluorocytosine (5FCyt) isolated in an argon matrix: (a) recorded after formation of the matrix before any irradiation; (b) recorded after 1 h of near-IR irradiation at 7030 cm−1; (c) experimental difference spectrum: trace b minus trace a; (d) theoretical spectra predicted for AH1 and AH2 forms of 5FCyt. The theoretical spectra were calculated at the DFT(B3LYP)/6-31++G(d,p) level. The theoretical wavenumbers were scaled by a single factor of 0.98. The intensities calculated for AH1 were multiplied by −1.

Figure 6. Fragments of the infrared spectra of 5-fluorocytosine isolated in an argon matrix: (a) recorded before any irradiation; (b) recorded after narrowband UV irradiation at λ = 310 nm. Note that spectrum a is presented here twice: in survey ordinate scale (lower trace) and in expanded ordinate scale (upper trace). The ordinate scale of part b is the same as upper trace a.

energy barrier for AH1 ↔ AH2 rotamerization (about 40 kJ mol−1, see Figure 8). Irradiations at 7030 cm−1 (or at any other frequency within the shape of the 2νOH absorption band) did not lead to total conversion of AH1 into AH2 (see Figure 7a,b). Because of the overlap of the 2νOH bands in the spectra of AH1 and AH2, both forms were simultaneously excited and rotamerizations in both AH1 → AH2 and AH2 → AH1 directions were induced.

tightly. The same concerns the bands due to the νOH overtone transitions in AH1 and AH2. Hence, simultaneous near-IR excitation of both AH1 and AH2 forms is inevitable, even when narrowband light of a tunable laser is used for this purpose. Monomers of 5FCyt isolated in an argon matrix were exposed to the monochromatic near-IR idler beam of an optical parametric oscillator (OPO) tuned to 7030 cm−1. This frequency corresponds to the center of the 2νOH overtone absorption (see Figure 1). As it is evident from the comparison of the spectra recorded before and after such irradiation (Figure 7), excitation of 5FCyt molecules at 7030 cm−1 led to substantial changes in the relative populations of the isomeric forms of the compound. Specifically, the bands due to the initially dominating AH1 form decreased considerably, while those ascribable to AH2 increased several times. The experimental spectra of the bands diminishing and growing upon irradiation (Figure 7c) are compared with the spectra calculated for AH1 and AH2 (see Figure 7d). Good agreement between the experimental and theoretical spectra confirms the assignment of the reactant to rotamer AH1 and the identification of AH2 as the product of the 7030 cm−1 induced phototransformation. The reason why AH1 and AH2 rotamers of matrix-isolated 5FCyt convert into one another upon irradiation at 7030 cm−1 is that the energy (84 kJ mol−1) introduced to the molecule with this excitation exceeds more than twice the height of the

Figure 8. Comparison of the relaxed potential-energy profiles for the interconversion between conformers AH1 and AH2 of cytosine (red), 5-fluorocytosine (green), and 5-methylcytosine (blue). The N1−C−O−H torsional angles were used as driving coordinates. The MP2/6-31++G(d,p) calculations were carried out for a series of points with constrained values of the torsional N1−C−O−H angle and all other degrees of freedom fully optimized. 2836



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transformations are limited to the OH rotamerizations in the amino-hydroxy structures. 4.4. Conformational AH1−AH2 Conversions Induced by Broadband Near-IR/Mid-IR Light. Periodical monitoring of the population ratio of AH1 and AH2 rotamers of matrixisolated 5FCyt, exposed to the unfiltered beam of the FTIR spectrometer, revealed that irradiation with near-IR/mid-IR light (emitted by the Ever-Glo, ceramic-bar IR source) induces changes in the relative abundances of AH1 and AH2 forms. The fragments of the mid-IR spectra presented in Figure 10 show that conversions in both directions, AH1 → AH2 and AH2 → AH1, occur upon excitation with broadband near-IR/ mid-IR light. The direction of the net population shift was found to depend on the initial ratio of AH1 and AH2 forms. When the changes were monitored for a freshly deposited matrix (Figure 10A−C), then, upon irradiation with broadband near-IR/mid-IR light, the population of AH2 was increasing at the cost of decreasing abundance of AH1. However, when the changes were monitored for a matrix previously irradiated at 7030 cm−1, the population of AH1 was increasing while that of AH2 was diminishing (Figure 10D−F). In the two sequences presented in Figure 10, the common feature is the final ratio of AH1 and AH2 rotamers, obtained upon prolonged exposure to broadband near-IR/mid-IR light (compare parts B and E of Figure 10; see also Figure 10C and F). This photostationary ratio obviously corresponds to a photoequilibrium between the photoprocess transforming AH1 into AH2 and that transforming AH2 into AH1. Similar processes leading to photostationary states were observed for 5mCyt (see Figure S5 in the Supporting Information). 4.5. Spontaneous AH2 → AH1 Conversion by Hydrogen Atom Tunneling in the Dark. Irradiation of isolated 5FCyt monomers with monochromatic near-IR light at 7030 cm−1 leads to a significant increase of AH2 population in the matrix. After 60 min of such irradiation, the amount of AH2 reached 72% of the combined AH1 + AH2 population. Analogously, for matrix-isolated 5mCyt, upon 60 min of nearIR irradiation at 7034 cm−1, the population of AH2 grew to 64% of the combined population of AH1 + AH2. After the irradiation with monochromatic near-IR light, the matrixisolated compounds (5FCyt or 5mCyt), with the majority of the amino-hydroxy molecules in the AH2 rotameric form, were kept at 14 K and periodically monitored by taking the mid-IR spectra. By recording of these spectra, an infrared cutoff filter, transmitting only in the 2200−400 cm−1 range (see Figure S2 in the Supporting Information), was put between the spectrometer source and the low-temperature matrix to prevent transformations induced by the spectrometer beam. In the time between collections of the consecutive spectra, the spectrometer beams were completely blocked. Under such conditions, a transformation of the higher-energy AH2 form into the lower-energy AH1 rotamer was observed for 5FCyt as well as for 5mCyt (see Figures 11 and 12). This transformation proceeded monotonously and led, within several hours, to a significant depletion of AH2. Simultaneously, the population of the most stable AH1 isomer systematically grew at the cost of AH2 and, within 5 h, exceeded 80% (in 5FCyt) and 90% (in 5mCyt) of the total population of the amino-hydroxy tautomer. The rate of the spontaneous AH2 → AH1 conversion in 5FCyt and in 5mCyt was much higher than it was in the AH2 → AH1 conversion previously observed for unsubstituted cytosine.43 Whereas for Cyt the population of AH2 was

Therefore, the total process was leading to a photostationary state. The ratio of AH1 and AH2 populations at the photostationary state is a combined result of different coefficients of absorption at 7030 cm−1 and of the yields of the AH1 → AH2 and AH2 → AH1 conversions. In an analogous experiment, monomers of 5mCyt isolated in an argon matrix were irradiated at 7034 cm−1. Also, in this case, substantial changes in the relative populations of AH1 and AH2 forms were induced by excitation with near-IR light (see the spectra recorded before and after irradiation presented in Figure 9a,b). In spite of the large scale of the near-IR-induced

Figure 9. Fragment of the experimental mid-IR spectra of 5methylcytosine (5mCyt) isolated in an argon matrix: (a) recorded before any irradiation; (b) recorded after 1 h of near-IR irradiation at 7034 cm−1; (c) experimental difference spectrum: trace b minus trace a; (d) theoretical spectra predicted for AH1 and AH2 forms of 5mCyt. The theoretical spectra were calculated at the DFT(B3LYP)/6-31+ +G(d,p) level. The theoretical wavenumbers were scaled by a single factor of 0.98. The intensities calculated for AH1 were multiplied by −1. Note that the absorptions observed in the 1760−1680 cm−1 region do not change upon near-IR irradiation. These bands are due to the νCO vibrations of AO and IO forms of 5mCyt.

population changes, the process did not lead to total consumption of one of the forms and its transformation to the other rotamer. The reason for that is the tight overlap of the 2νOH bands of AH1 and AH2, which implies simultaneous excitation of both forms and a photostationary state as the final stage of the near-IR-induced process. The form with the population growing in the observed photoconversion was identified as the amino-hydroxy rotamer AH2. Consequently, AH1 structure was assigned to the form with population diminishing in this process. The assignment of these structures was based on the comparison of the spectra decreasing and increasing upon near-IR irradiation with the spectra predicted for AH1 and AH2 (see Figure 9c,d). Note that, in the 1760−1680 cm−1 region of the spectra presented in Figure 9, bands due to the νCO vibrations of AO and IO forms of 5mCyt were observed. No changes in intensities and shapes of these bands were induced by exposure of the matrix to near-IR light. This result certifies that the near-IR-induced 2837



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Figure 10. Fragments of the infrared spectra of 5-fluorocytosine isolated in an Ar matrix recorded: (A) after deposition of the matrix; (B) after 60 min of exposure to the spectrometer near-IR/mid-IR beam; (C) evolution of AH1 and AH2 abundances with time of broadband near-IR/mid-IR irradiation (the initial point corresponds to part A, and the final point corresponds to part B); (D) spectrum recorded after 60 min of near-IR narrowband irradiation at 7030 cm−1; (E) after subsequent 80 min of exposure to the spectrometer near-IR/mid-IR beam; (F) evolution of AH1 and AH2 abundances with time of broadband near-IR/mid-IR irradiation (the initial point corresponds to part D, and the final point corresponds to part E).

Figure 12. Evolution of abundances of AH1 and AH2 amino-hydroxy rotamers of 5-methylcytosine with time of keeping the matrix in the dark at 14 K and monitoring only through a filter transmitting in the 2200−400 cm−1 spectral range.

Figure 11. Evolution of abundances of AH1 and AH2 amino-hydroxy rotamers of 5-fluorocytosine with time of keeping the matrix in the dark at 14 K and monitoring only through a filter transmitting in the 2200−400 cm−1 spectral range.

(e.g., of a methyl group) can affect tunneling dynamics. Hydrogen-atom tunneling observed in acetic acid44 was considerably faster than the analogous process in formic acid.46 Also in cytosines, a massive substituent (methyl group or fluorine atom) introduced at position 5, causes increase of the density of low-energy vibrational states. This looks as a plausible reason that can explain the substantial difference between the tunneling rate in cytosine and the corresponding rates in 5FCyt and 5mCyt.

decreasing with a half-life time longer than 1800 min, the analogous half-life time estimated for 5FCyt and 5mCyt was only 127 and 70 min, respectively. The slight differences in the height of the barrier for the rotation of the OH group, calculated for Cyt, 5mCyt, and 5FCyt (Figure 8), cannot explain the impressive differences in the rates of the spontaneous AH2 → AH1 tunneling in these compounds. Though the barrier calculated for cytosine is somewhat (by 2.5 kJ mol−1) higher than those predicted for 5FCyt and 5mCyt, such a difference cannot be the sole reason for drastic differences in the rates of the AH2 → AH1 tunneling processes. Previous studies on tunneling processes involving internal rotation of OH groups in simple carboxylic acids isolated in cryogenic inert matrixes44−48 have reached the conclusion that, besides the height of the barrier, the density of low-energy vibrational states and coupling with intramolecular rotations

5. CONCLUSIONS Photoinduced processes studied in the current work concern transformations of all isomeric forms of 5-methylcytosine and 5-fluorocytosine (see Scheme 1). Thanks to the observed photoconversions, five isomers were identified for monomeric 5-methylcytosine. Two rotameric forms of the dominating 2838



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Scheme 1. Summary of the UV-Induced and Near-IR-Induced Transformations Observed for Monomers of 5-Methylcytosine (X = CH3) and 5-Fluorocytosine (X = F).

cytosine. The drastic difference in the tunneling rates (more than 1 order of magnitude) was experimentally observed in spite of the very similar heights of the barriers for OH group torsion predicted theoretically for 5-methylcytosine, 5-fluorocytosine, and cytosine. Light-induced changes in populations of different isomeric forms of 5-methylcytosine and 5-fluorocytosine allowed assignment of the bands observed in the infrared spectra of these compounds isolated in low-temperature argon matrixes. This assignment is graphically presented in Figures S3 and S4 in the Supporting Information.

amino-hydroxy tautomer were easily detected, because the rotamers convert to one another upon narrowband or broadband near-IR excitations. Identification of the canonical amino-oxo form of the compound was also easy: the population of this tautomer was depleted upon UV (λ ≤ 316 nm) irradiation of 5-methylcytosine. The main photoprocess consuming the amino-oxo form was the oxo → hydroxy phototautomerism of the type observed for 4(3H)-pyrimidinone and related compounds.49,50 The minor channel of the photochemical transformations induced by irradiation at λ ≤ 316 nm was the opening of the heterocyclic ring of the aminooxo form. In this photoprocess, conjugated isocyanate product was generated. Exposure of matrix-isolated 5-methylcytosine to shorter-wave UV (λ ≤ 310 nm) light promotes also syn−anti transformations, leading to the large scale population shift between the two imino-oxo isomers of the compound. Due to this effect, the two minor imino-oxo forms of 5-methylcytosine were reliably identified. Similar syn−anti photoisomerizations were previously observed for the imino-oxo forms of cytosine and 1-methylcytosine.5,6 The experiments reported in the current paper provide clear experimental evidence that UV-induced structural reorganizations in 5-methylcytosine are possible. This contradicts the widespread belief1,4 that any photoinduced rearrangements of atoms within nucleic acid bases would be impossible because of the very short lifetime of excited states. Analogous photoprocesses were also observed in the case of 5-fluorocytosine (see Scheme 1). Irradiation of matrix-isolated molecules of the compound with near-IR light allowed separation of two sets of the bands belonging to the spectra of the two rotamers of the amino-hydroxy tautomer. The very weak bands unaffected by near-IR light but decreasing upon excitation with the UV light at λ ≤ 313 nm were ascribed to the amino-oxo form. No spectral signatures of the imino-oxo form of 5-fluorocytosine could be observed for the compound isolated in argon matrixes. The experiments carried out within the present work demonstrated that the relative populations of the aminohydroxy rotamers can change not only upon excitation with light but also spontaneously, by tunneling of the higher-energy rotamer to the lower-energy one. The tunneling processes observed for 5-methylcytosine and 5-fluorocytosine occurred much faster than the analogous process in unsubstituted

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ASSOCIATED CONTENT

S Supporting Information *

Figure S1 illustrates the transformation of 5-methylcytosine to thymine on air; Figure S2 shows the transmission range of the Edmund Optics long-pass filter used in this study; Figures S3 and S4 contain IR spectra of 5-fluorocytosine and 5methylcytosine isolated in an Ar matrix and assignment of the bands to isomeric forms; Figure S5 illustrates the evolution of abundances of AH1 and AH2 forms of 5mCyt with time of broadband near-IR/mid-IR irradiation; Table S1 shows calculated frequencies and infrared intensities of the OH and NH stretching vibrations of isomeric forms of cytosine, 5methylcytosine, and 5-fluorocytosine. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: (+48) 22 116 3219. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work has been supported by the National Science Center (Poland) under the grant 2011/01/B/ST4/00718 and by the European Community’s Seventh Framework Programme under the Grant Agreement No. 228334. This work was also supported by the Portuguese “Fundaçaõ para a Ciência e a Tecnologia” (FCT), Research Projects PTDC/QUI-QUI/ 111879/2009 and PTDC/QUI-QUI/118078/2010 (FCOMP01-0124-FEDER-021082), cofunded by QREN-COMPETE2839



Article

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UE, and bilateral project N° 1691 for cooperation between Poland and Portugal.

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Near-IR-Induced, UV-Induced, and Spontaneous

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