Near-IR-Induced, UV-Induced, and Spontaneous ...reva/FULL/108.pdf · of nucleic acid bases. These conclusions were in contrast with UV-induced hydrogen-atom-transfer and ring-opening

Near-IR-Induced, UV-Induced, and Spontaneous Isomerizations in5‑Methylcytosine and 5‑FluorocytosineLeszek 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 5-fluorocytosine (5FCyt) were studied using the matrix-isolationmethod. In 5mCyt and 5FCyt, the most stable form,dominating in low-temperature matrixes, is the amino-hydroxy(AH) tautomer. For both compounds, irradiation of thematrixes with near-IR laser light or with broadband near-IR ormid-IR light induces interconversions between the tworotamers of tautomer AH. In addition, for matrixes kept indarkness, a spontaneous tunneling conversion of the higher-energy hydroxy conformer (with the OH group directedtoward the N3 atom) into the lower-energy form (OHdirected toward N1) was occurring, with half-life time of 70min for 5mCyt and 127 min for 5FCyt. These tunnelingprocesses are much faster than that found for unsubstituted cytosine, where the half-life time is more than 30 h. UV irradiation of5mCyt (at 316 nm) led to phototautomeric conversion of the amino-oxo form into the amino-hydroxy tautomer. Anotherphototransformation 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 amountof the amino-oxo form was very small (with respect to the amino-hydroxy tautomer), while the imino-oxo isomers were notdetected at all.

1. INTRODUCTION

Ultrafast deactivation of electronically excited states, reportedduring the past decade for nucleic acid bases,1−4 had aprofound influence on the common opinion about thephotostability of this class of compounds. The reportssuggested that there are very effective deactivation pathways,allowing extremely fast (∼1 ps) depopulation of low-energyexcited states of nucleic acid bases. Many authors concludedthat such a very rapid dissipation of the excited state energyshould prohibit any rearrangement of the atoms in the structureof nucleic acid bases. These conclusions were in contrast withUV-induced hydrogen-atom-transfer and ring-opening pro-cesses experimentally found in isolated monomers of nucleicacid 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 excited1ππ state vibronic levels of the amino-oxo form of cytosine inthe 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 energyexcess the lifetime does not decrease below 25 ps. Suchlifetimes (20−40 times longer than those previously observedfor higher-energy excitations)1−4 should allow rearrangementsof atoms and generation of photoproducts with alteredstructure. Hence, the observation of non-ultrafast lifetimes14

makes the occurrence of photoisomerizations in nucleic acidbases, in particular in cytosines, comprehensible.In the present work, we studied light-induced structural

changes in 5-methylcytosine and 5-fluorocytosine. Bothcompounds are relevant from the biological point of view. 5-Methylcytosine is a product of DNA methylation,15,16 which isthe most important epigenetic alteration in eukaryotes.Methylation of cytosine residues in genomic DNA plays akey role in the regulation of gene expression by blockingtranscription and causing gene silencing.17−23 5-Fluorocytosine(“flucytosine”) is an antifungal agent.24 It is also an antitumorprodrug, which converts to the highly toxic metabolite, 5-fluorouracil.25,26 Treatment with 5-fluorocytosine, combinedwith a tumor-specific gene therapy, results in more selectiveelimination of tumor cells without significant toxicity to thepatient.27,28 Because of the biological importance of 5-methylcytosine and 5-fluorocytosine, numerous investigationswere devoted to the physicochemical and spectroscopicproperties of these compounds.13,29−33

Received: November 20, 2013Revised: February 19, 2014Published: February 24, 2014

Article

pubs.acs.org/JPCB

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

pubs.acs.org/JPCB

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

2. EXPERIMENTAL SECTIONThe sample of 5-fluorocytosine (5FCyt), purity 98%, used inthe present study was a commercial product supplied by Sigma-Aldrich. 5-Methylcytosine (5mCyt) was purchased from Alfa-Aesar, but the compound, as a free base, was found to readilyconvert to thymine when exposed to the air (see Figure S1 inthe Supporting Information). Therefore, for the experimentsreported in the current paper, we used a sample of 5-methylcytosine hydrochloride, supplied by Sigma-Aldrich. Freebase 5-methylcytosine was prepared immediately before thematrix experiment, by vacuum sublimation in an evacuated,sealed glass tube. By this procedure, free base of 5-methylcytosine was condensing from the gas phase onto theglass walls above the heated hydrochloride crystals, whereas theevolving gaseous HCl was trapped in a remote part of the glasstube, kept in liquid nitrogen.In order to obtain the low-temperature matrixes, crystals of

the studied compounds were heated [to ca. 450 K (5mCyt) andca. 425 K (5FCyt)] in a miniature glass oven placed in thevacuum chamber of a helium-cooled cryostat. The vaporscoming out of the oven were codeposited with a large excess ofargon onto a CsI window, cooled to 14 K by an APD

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

3. COMPUTATIONAL SECTIONRelative energies of the five isomeric forms of cytosine, 5-methylcytosine, and 5-fluorocytosine (see the structurespresented in Table 1) were calculated using the QCISD35

and CCSD36 methods. The barriers for rotation of the OHgroup in the amino-hydroxy tautomers of cytosine, 5-methylcytosine, and 5-fluorocytosine in the electronic groundstate were calculated using the minimum-energy-path approach.At each of the points, intermediate between the minimacorresponding to the rotameric structures AH1 and AH2, allgeometry parameters (except for the torsional angleN1−C−O−H, which served as a driving coordinate) wereoptimized using the MP2 method.37

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

aCalculated 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 thecc-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 valuescorrected 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).

The Journal of Physical Chemistry B Article

dx.doi.org/10.1021/jp411423c | J. Phys. Chem. B 2014, 118, 2831−28412832

The geometries of the isomers of 5-methylcytosine and 5-fluorocytosine were fully optimized using the density functionaltheory DFT(B3LYP) method38−40 with the Becke three-parameter exchange functional38 and the Lee, Yang, Parrcorrelation functional.39 At the optimized geometries, theharmonic vibrational frequencies and IR intensities werecalculated 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 theGaussian 03 program.41

4. RESULTS AND DISCUSSION4.1. Relative Energies and Relative Populations of

Isomeric Forms of 5mCyt and 5FCyt. The relative energiesof the isomers of 5mCyt and 5FCyt were calculated at theQCISD/6-31++G(d,p) and CCSD/6-31++G(d,p) levels oftheory. The results of these calculations are compared withrelative energies obtained for cytosine (Cyt) isomers in Table 1.All the calculations presented in Table 1 indicate that theamino-hydroxy AH1 form should be the most stable isomer of5mCyt as well as of 5FCyt. For both compounds, the AH1form should be most abundant in the gas phase and in low-temperature inert gas matrixes. According to the calculations,the other amino-hydroxy rotameric AH2 forms of 5mCyt and5FCyt should be only slightly (by ca. 3 kJ mol−1) higher inenergy than the corresponding AH1 rotamers. Hence, the AH2forms of 5mCyt and 5FCyt should be populated in the gasphase 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, withrespect to AH1, to be significantly higher than that forunsubstituted cytosine (see Table 1). For 5FCyt, the relativeenergy of AO isomer is even higher. Consequently, incomparison to the gas-phase equilibrium in cytosine, theamino-oxo AO form should be less abundant in 5mCyt andonly very slightly populated in 5FCyt.The high-frequency regions of the experimental mid-infrared

spectra of Cyt, 5mCyt, and 5FCyt isolated in argon matrixes arepresented in Figure 1 (lower panel). The infrared bands due tothe OH and N1H stretching vibrations, present in this spectralregion, have similar absolute intensities (close to 100 km mol−1,see Table S1 in the Supporting Information). Moreover, thesebands do not overlap with the bands due to any othervibrations. Hence, the relative intensities of the experimentalbands due to these vibrations can serve as an approximateestimate of relative populations of the amino-hydroxy, amino-oxo, and imino-oxo forms of the studied compounds trapped inlow-temperature matrixes. In particular, the bands due to νOHvibrations (at ca. 3600 cm−1) should represent the populationof the amino-hydroxy AH form in the matrix, whereas thebands due to νN1H vibrations (at ca. 3460 and at 3490 cm−1)should represent the populations of the amino-oxo AO andimino-oxo IO isomers, respectively. Comparison of relativeintensities of the band ascribed to νN1H vibration in AO formsof Cyt, 5mCyt, and 5FCyt (see Figure 1) shows that thepopulation 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 thetheoretical 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 relativepopulation of IO1 should be higher in 5mCyt than inunsubstituted Cyt. Accordingly, the intensity of the νNH

band at ca. 3490 cm−1, with respect to bands characteristic ofother forms, shows that the IO tautomer is more populated in5mCyt than in Cyt. On the other hand, in the spectrum of5FCyt, the analogous band is missing. This indicates a verysmall population of IO in 5FCyt, which is in agreement withthe high relative energy (more than 12 kJ mol−1) calculated forIO forms of this compound.The assignment of the IR absorption bands observed in the

spectra of 5-fluorocytosine and 5-methylcytosine isolated in Armatrixes is shown in Figures S3 and S4 (in the SupportingInformation). The experimental IR bands were ascribed toisomeric forms of 5mCyt and 5FCyt on the basis of theirintensity changes induced by UV or near-IR irradiation of thematrixes (as described below).

4.2. Photoisomerizations Induced by Excitation withUV Light. Monomers of 5-methylcytosine isolated in argonmatrixes were excited at a series of chosen UV wavelengthsusing tunable, narrowband UV laser light. The frequency-doubled signal beam of the optical parametric oscillator wasused for this purpose. The UV irradiation started at λ = 322nm. It was followed by consecutive irradiations at gradually

Figure 1. Fragments of the experimental near-IR and mid-IR spectraof cytosine (Cyt), 5-methylcytosine (5mCyt), and 5-fluorocytosine(5FCyt) isolated in Ar matrixes. In the presented spectral ranges, thereappear absorption bands due to the fundamental transitions (lowerpanel) and first overtones (upper panel) of the OH and NH stretchingvibrations. The bands marked (AH + AO) are due to antisymmetricνaNH2 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, SupportingInformation), and the corresponding experimental bands overlap.



shorter UV wavelengths. After each irradiation, the matrix wasmonitored by taking its IR spectrum. UV irradiations with λ ≥318 nm did not induce any transformations of matrix-isolated5mCyt. The first changes in the IR spectrum of the compoundwere observed only after the irradiation at 317−316 nm. Uponirradiation at these wavelengths, the population of AOtautomer of 5mCyt diminished (see Figure 2). The appliedwavelengths (317−316 nm) are only slightly shorter than the0−0 origin (at 319.7 nm) of the absorption spectrum measuredby REMPI spectroscopy for AO isomer of 5mCyt in the gasphase.13,31,32

The decrease of AO population occurring upon irradiation at316 nm was accompanied by a slight increase of the populationof the amino-hydroxy tautomer (Figure 2). Another productgrowing at the cost of the AO form manifested itself by a bandwith maxima at 2273 and 2261 cm−1 (Figure 3). Also, for 1,5-dimethylcytosine, where the amino-oxo form dominates in thematrix before any irradiation, a new structured band at 2247cm−1 appeared in the infrared spectrum recorded after exposureto UV (λ > 314 nm) light.Photoproducts having characteristic bands at ca. 2260−2270

cm−1 in their IR spectra were previously observed inphotochemical investigations on 1-methyl-2(1H)pyrimidinoneand cytosine. These products were assigned to open-ringstructures with a conjugated isocyanate moiety.5,6,42 It is veryprobable that a Norrish type I ring-opening process occurs alsofor 5mCyt and that conjugated isocyanate is generated, uponUV irradiation, from the AO form of this compound. Very

recently, ring-opening by Norrish type I cleavage of the CNbond (α-bond with respect to the CO group) was observedfor a series of oligonucleotides and mononucleosides, both insolid films and in solution.12 Isocyanates were identified asproducts of these photoreactions. It looks then that UV-induced ring-opening constitutes an important channel in the

Figure 2. Identification of the amino-oxo form of 5-methylcytosine isolated in an argon matrix. (a−c) Fragments of the experimental infrared spectraof 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 theoreticalfrequencies were scaled by a single factor of 0.98, and the theoretical intensities were multiplied by −1. The tilde indicates the theoretical νCOband (truncated); the predicted absolute intensity of this band is 893 kJ mol−1.

Figure 3. Fragment of the infrared spectrum of 5-methylcytosinemonomers isolated in an Ar matrix recorded (black) after depositionof the matrix and (red) after irradiation of the matrix withmonochromatic λ = 316 nm light. The schemes present the twomost stable structures of the open-ring conjugated isocyanate, theplausible carrier of the new band.



photochemistry of nucleic acid bases that has been recognizedonly recently.Upon subsequent UV (λ ≤ 310 nm) irradiation of 5mCyt

isolated in an argon matrix, the imino-oxo IO1 form of thecompound converted into IO2 isomer. Spectral manifestations(Figure 4) of this syn−anti isomerization allowed identification

of both imino-oxo isomers of 5mCyt. The initial population ofform IO2 was very small, but it increased several times uponirradiation at λ ≤ 310 nm (Figure 5). The identification of IO2as the product growing upon UV (λ ≤ 310 nm) irradiation atthe cost of IO1 is based on the good agreement between theexperimental and theoretical IR spectra of those species (Figure4). The photoreaction transforming the IO1 form of 5mCytinto IO2 is analogous to the syn−anti photoisomerizationstransforming the IO1 form of cytosine5 or 1-methylcytosine6

into the IO2 product. This analogy additionally supports theassignment of the set of bands, growing upon UV (λ ≤ 310nm) 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 ofthe bands observed in the IR spectrum of 5FCyt are due toAH1 and AH2 rotamers of the amino-hydroxy tautomer. Thesebands either grow or decrease in the AH1 ↔ AH2photorotamerization process induced by near-IR excitations;see sections 4.3 and 4.4. Apart from the IR bands ascribed tothe amino-hydroxy AH1 and AH2 forms, only few low-intensity IR bands were observed in the spectrum of 5FCyt.Such bands, assigned to AO tautomer, are not affected by near-IR irradiations of the matrix. The small population of the AOform of 5FCyt was found to decrease upon exposure of thematrix-isolated compound to UV (λ ≤ 313 nm) light (Figure6). At the cost of the decreasing population of AO, some tinyincrease of the population of AH tautomer was observed. Theset of IR features decreasing upon UV (λ ≤ 313 nm) irradiation

includes the characteristic AO bands due to the stretchingvibrations of the NH group at 3465 cm−1 and of the COgroup at 1734, 1730, and 1715 cm−1. This observation furthersupports the assignment of the spectrum in question to the AOtautomer of 5FCyt.

4.3. Conformational AH1−AH2 Conversions Inducedby Monochromatic Near-IR Light. Prior to an attempt toinduce a transformation between conformers AH1 and AH2 byexcitation of matrix-isolated cytosines with near-IR light, the2νOH and 2νNH overtone regions of the spectra wererecorded for 5mCyt and 5FCyt isolated in argon matrixes. Inthe 7100−6700 cm−1 range of those spectra, presented inFigure 1 (upper panel), absorption features due to overtones ofνOH, νNH2, and νNH vibrations were found. The bands of thefundamental νOH, νNH2, and νNH transitions were found inthe 3620−3400 cm−1 spectral region (Figure 1, lower panel). Itseems very likely that the comparatively strong absorptions at7035 cm−1 (5FCyt), 7029 cm−1 (5mCyt), and 7033, 7016 cm−1

(Cyt) are due to the overtones of the νOH vibrations. Notethat the νOH overtone bands do not appear at the highestfrequencies of all the bands in the 7100−6700 cm−1 region,despite that fundamental νOH bands have the highestfrequencies of all the absorptions found in the 3620−3400cm−1 range. This must be related with higher anharmonicity ofthe OH vibrations, with respect to the anharmonicities of theNH stretching modes.In the spectra of 5mCyt and 5FCyt, the bands due to the

νOH fundamental transitions in forms AH1 and AH2 overlap

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

Figure 5. Fragments of the infrared spectra of 5-methylcytosineisolated in an argon matrix: (black) recorded after a series ofirradiations at λ = 316−310 nm; (red) recorded after the subsequentirradiation at λ = 308 nm.



tightly. The same concerns the bands due to the νOH overtonetransitions in AH1 and AH2. Hence, simultaneous near-IRexcitation of both AH1 and AH2 forms is inevitable, even whennarrowband 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 opticalparametric oscillator (OPO) tuned to 7030 cm−1. Thisfrequency corresponds to the center of the 2νOH overtoneabsorption (see Figure 1). As it is evident from the comparisonof the spectra recorded before and after such irradiation (Figure7), excitation of 5FCyt molecules at 7030 cm−1 led tosubstantial changes in the relative populations of the isomericforms of the compound. Specifically, the bands due to theinitially dominating AH1 form decreased considerably, whilethose ascribable to AH2 increased several times. Theexperimental spectra of the bands diminishing and growingupon irradiation (Figure 7c) are compared with the spectracalculated for AH1 and AH2 (see Figure 7d). Good agreementbetween the experimental and theoretical spectra confirms theassignment of the reactant to rotamer AH1 and theidentification of AH2 as the product of the 7030 cm−1 inducedphototransformation.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 moleculewith this excitation exceeds more than twice the height of the

energy barrier for AH1 ↔ AH2 rotamerization (about 40 kJmol−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 totalconversion of AH1 into AH2 (see Figure 7a,b). Because of theoverlap of the 2νOH bands in the spectra of AH1 and AH2,both forms were simultaneously excited and rotamerizations inboth AH1 → AH2 and AH2 → AH1 directions were induced.

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

Figure 7. Fragment of the experimental mid-IR spectra of 5-fluorocytosine (5FCyt) isolated in an argon matrix: (a) recordedafter formation of the matrix before any irradiation; (b) recorded after1 h of near-IR irradiation at 7030 cm−1; (c) experimental differencespectrum: trace b minus trace a; (d) theoretical spectra predicted forAH1 and AH2 forms of 5FCyt. The theoretical spectra were calculatedat the DFT(B3LYP)/6-31++G(d,p) level. The theoretical wave-numbers were scaled by a single factor of 0.98. The intensitiescalculated for AH1 were multiplied by −1.

Figure 8. Comparison of the relaxed potential-energy profiles for theinterconversion between conformers AH1 and AH2 of cytosine (red),5-fluorocytosine (green), and 5-methylcytosine (blue). TheN1−C−O−H torsional angles were used as driving coordinates. TheMP2/6-31++G(d,p) calculations were carried out for a series of pointswith constrained values of the torsional N1−C−O−H angle and allother degrees of freedom fully optimized.



Therefore, the total process was leading to a photostationarystate. The ratio of AH1 and AH2 populations at thephotostationary state is a combined result of differentcoefficients of absorption at 7030 cm−1 and of the yields ofthe 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 AH2forms were induced by excitation with near-IR light (see thespectra recorded before and after irradiation presented inFigure 9a,b). In spite of the large scale of the near-IR-induced

population changes, the process did not lead to totalconsumption of one of the forms and its transformation tothe other rotamer. The reason for that is the tight overlap of the2νOH bands of AH1 and AH2, which implies simultaneousexcitation of both forms and a photostationary state as the finalstage of the near-IR-induced process.The form with the population growing in the observed

photoconversion was identified as the amino-hydroxy rotamerAH2. Consequently, AH1 structure was assigned to the formwith population diminishing in this process. The assignment ofthese structures was based on the comparison of the spectradecreasing and increasing upon near-IR irradiation with thespectra predicted for AH1 and AH2 (see Figure 9c,d). Notethat, in the 1760−1680 cm−1 region of the spectra presented inFigure 9, bands due to the νCO vibrations of AO and IOforms of 5mCyt were observed. No changes in intensities andshapes of these bands were induced by exposure of the matrixto near-IR light. This result certifies that the near-IR-induced

transformations are limited to the OH rotamerizations in theamino-hydroxy structures.

4.4. Conformational AH1−AH2 Conversions Inducedby Broadband Near-IR/Mid-IR Light. Periodical monitoringof the population ratio of AH1 and AH2 rotamers of matrix-isolated 5FCyt, exposed to the unfiltered beam of the FTIRspectrometer, revealed that irradiation with near-IR/mid-IRlight (emitted by the Ever-Glo, ceramic-bar IR source) induceschanges in the relative abundances of AH1 and AH2 forms.The fragments of the mid-IR spectra presented in Figure 10show that conversions in both directions, AH1 → AH2 andAH2 → AH1, occur upon excitation with broadband near-IR/mid-IR light. The direction of the net population shift wasfound to depend on the initial ratio of AH1 and AH2 forms.When the changes were monitored for a freshly depositedmatrix (Figure 10A−C), then, upon irradiation with broadbandnear-IR/mid-IR light, the population of AH2 was increasing atthe cost of decreasing abundance of AH1. However, when thechanges were monitored for a matrix previously irradiated at7030 cm−1, the population of AH1 was increasing while that ofAH2 was diminishing (Figure 10D−F). In the two sequencespresented in Figure 10, the common feature is the final ratio ofAH1 and AH2 rotamers, obtained upon prolonged exposure tobroadband near-IR/mid-IR light (compare parts B and E ofFigure 10; see also Figure 10C and F). This photostationaryratio obviously corresponds to a photoequilibrium between thephotoprocess transforming AH1 into AH2 and that trans-forming AH2 into AH1. Similar processes leading tophotostationary states were observed for 5mCyt (see FigureS5 in the Supporting Information).

4.5. Spontaneous AH2 → AH1 Conversion by Hydro-gen Atom Tunneling in the Dark. Irradiation of isolated5FCyt monomers with monochromatic near-IR light at 7030cm−1 leads to a significant increase of AH2 population in thematrix. After 60 min of such irradiation, the amount of AH2reached 72% of the combined AH1 + AH2 population.Analogously, for matrix-isolated 5mCyt, upon 60 min of near-IR irradiation at 7034 cm−1, the population of AH2 grew to64% of the combined population of AH1 + AH2. After theirradiation with monochromatic near-IR light, the matrix-isolated compounds (5FCyt or 5mCyt), with the majority ofthe amino-hydroxy molecules in the AH2 rotameric form, werekept at 14 K and periodically monitored by taking the mid-IRspectra. By recording of these spectra, an infrared cutoff filter,transmitting only in the 2200−400 cm−1 range (see Figure S2in the Supporting Information), was put between thespectrometer source and the low-temperature matrix to preventtransformations induced by the spectrometer beam. In the timebetween collections of the consecutive spectra, the spectrom-eter beams were completely blocked.Under such conditions, a transformation of the higher-energy

AH2 form into the lower-energy AH1 rotamer was observedfor 5FCyt as well as for 5mCyt (see Figures 11 and 12). Thistransformation proceeded monotonously and led, withinseveral hours, to a significant depletion of AH2. Simulta-neously, the population of the most stable AH1 isomersystematically grew at the cost of AH2 and, within 5 h,exceeded 80% (in 5FCyt) and 90% (in 5mCyt) of the totalpopulation 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 unsubstitutedcytosine.43 Whereas for Cyt the population of AH2 was

Figure 9. Fragment of the experimental mid-IR spectra of 5-methylcytosine (5mCyt) isolated in an argon matrix: (a) recordedbefore any irradiation; (b) recorded after 1 h of near-IR irradiation at7034 cm−1; (c) experimental difference spectrum: trace b minus tracea; (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 singlefactor 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 tothe νCO vibrations of AO and IO forms of 5mCyt.



decreasing with a half-life time longer than 1800 min, theanalogous half-life time estimated for 5FCyt and 5mCyt wasonly 127 and 70 min, respectively. The slight differences in theheight of the barrier for the rotation of the OH group,calculated for Cyt, 5mCyt, and 5FCyt (Figure 8), cannotexplain the impressive differences in the rates of thespontaneous AH2 → AH1 tunneling in these compounds.Though the barrier calculated for cytosine is somewhat (by 2.5kJ mol−1) higher than those predicted for 5FCyt and 5mCyt,such a difference cannot be the sole reason for drasticdifferences 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 incryogenic inert matrixes44−48 have reached the conclusion that,besides the height of the barrier, the density of low-energyvibrational states and coupling with intramolecular rotations

(e.g., of a methyl group) can affect tunneling dynamics.Hydrogen-atom tunneling observed in acetic acid44 wasconsiderably faster than the analogous process in formicacid.46 Also in cytosines, a massive substituent (methyl groupor fluorine atom) introduced at position 5, causes increase ofthe density of low-energy vibrational states. This looks as aplausible reason that can explain the substantial differencebetween the tunneling rate in cytosine and the correspondingrates in 5FCyt and 5mCyt.

5. CONCLUSIONSPhotoinduced processes studied in the current work concerntransformations of all isomeric forms of 5-methylcytosine and5-fluorocytosine (see Scheme 1). Thanks to the observedphotoconversions, five isomers were identified for monomeric5-methylcytosine. Two rotameric forms of the dominating

Figure 10. Fragments of the infrared spectra of 5-fluorocytosine isolated in an Ar matrix recorded: (A) after deposition of the matrix; (B) after 60min of exposure to the spectrometer near-IR/mid-IR beam; (C) evolution of AH1 and AH2 abundances with time of broadband near-IR/mid-IRirradiation (the initial point corresponds to part A, and the final point corresponds to part B); (D) spectrum recorded after 60 min of near-IRnarrowband irradiation at 7030 cm−1; (E) after subsequent 80 min of exposure to the spectrometer near-IR/mid-IR beam; (F) evolution of AH1 andAH2 abundances with time of broadband near-IR/mid-IR irradiation (the initial point corresponds to part D, and the final point corresponds to partE).

Figure 11. Evolution of abundances of AH1 and AH2 amino-hydroxyrotamers of 5-fluorocytosine with time of keeping the matrix in thedark at 14 K and monitoring only through a filter transmitting in the2200−400 cm−1 spectral range.

Figure 12. Evolution of abundances of AH1 and AH2 amino-hydroxyrotamers of 5-methylcytosine with time of keeping the matrix in thedark at 14 K and monitoring only through a filter transmitting in the2200−400 cm−1 spectral range.



amino-hydroxy tautomer were easily detected, because therotamers convert to one another upon narrowband orbroadband near-IR excitations. Identification of the canonicalamino-oxo form of the compound was also easy: the populationof this tautomer was depleted upon UV (λ ≤ 316 nm)irradiation of 5-methylcytosine. The main photoprocessconsuming the amino-oxo form was the oxo → hydroxyphototautomerism of the type observed for 4(3H)-pyrimidi-none and related compounds.49,50 The minor channel of thephotochemical transformations induced by irradiation at λ ≤316 nm was the opening of the heterocyclic ring of the amino-oxo form. In this photoprocess, conjugated isocyanate productwas generated. Exposure of matrix-isolated 5-methylcytosine toshorter-wave UV (λ ≤ 310 nm) light promotes also syn−antitransformations, leading to the large scale population shiftbetween the two imino-oxo isomers of the compound. Due tothis effect, the two minor imino-oxo forms of 5-methylcytosinewere reliably identified. Similar syn−anti photoisomerizationswere previously observed for the imino-oxo forms of cytosineand 1-methylcytosine.5,6

The experiments reported in the current paper provide clearexperimental evidence that UV-induced structural reorganiza-tions in 5-methylcytosine are possible. This contradicts thewidespread belief1,4 that any photoinduced rearrangements ofatoms within nucleic acid bases would be impossible because ofthe very short lifetime of excited states.Analogous photoprocesses were also observed in the case of

5-fluorocytosine (see Scheme 1). Irradiation of matrix-isolatedmolecules of the compound with near-IR light allowedseparation of two sets of the bands belonging to the spectraof the two rotamers of the amino-hydroxy tautomer. The veryweak bands unaffected by near-IR light but decreasing uponexcitation with the UV light at λ ≤ 313 nm were ascribed to theamino-oxo form. No spectral signatures of the imino-oxo formof 5-fluorocytosine could be observed for the compoundisolated in argon matrixes.The experiments carried out within the present work

demonstrated that the relative populations of the amino-hydroxy rotamers can change not only upon excitation withlight but also spontaneously, by tunneling of the higher-energyrotamer to the lower-energy one. The tunneling processesobserved for 5-methylcytosine and 5-fluorocytosine occurredmuch faster than the analogous process in unsubstituted

cytosine. The drastic difference in the tunneling rates (morethan 1 order of magnitude) was experimentally observed inspite of the very similar heights of the barriers for OH grouptorsion predicted theoretically for 5-methylcytosine, 5-fluo-rocytosine, and cytosine.Light-induced changes in populations of different isomeric

forms of 5-methylcytosine and 5-fluorocytosine allowedassignment of the bands observed in the infrared spectra ofthese compounds isolated in low-temperature argon matrixes.This assignment is graphically presented in Figures S3 and S4in the Supporting Information.

■ ASSOCIATED CONTENT*S Supporting InformationFigure S1 illustrates the transformation of 5-methylcytosine tothymine on air; Figure S2 shows the transmission range of theEdmund Optics long-pass filter used in this study; Figures S3and S4 contain IR spectra of 5-fluorocytosine and 5-methylcytosine isolated in an Ar matrix and assignment ofthe bands to isomeric forms; Figure S5 illustrates the evolutionof abundances of AH1 and AH2 forms of 5mCyt with time ofbroadband near-IR/mid-IR irradiation; Table S1 showscalculated frequencies and infrared intensities of the OH andNH stretching vibrations of isomeric forms of cytosine, 5-methylcytosine, and 5-fluorocytosine. This material is availablefree of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*E-mail: [email protected]. Phone: (+48) 22 116 3219.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis work has been supported by the National Science Center(Poland) under the grant 2011/01/B/ST4/00718 and by theEuropean Community’s Seventh Framework Programme underthe Grant Agreement No. 228334. This work was alsosupported by the Portuguese “Fundacao para a Ciencia e aTecnologia” (FCT), Research Projects PTDC/QUI-QUI/111879/2009 and PTDC/QUI-QUI/118078/2010 (FCOMP-01-0124-FEDER-021082), cofunded by QREN-COMPETE-

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).



http://pubs.acs.org

mailto:[email protected]

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

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Near-IR-Induced, UV-Induced, and Spontaneous ...reva/FULL/108.pdf · of nucleic acid bases. These conclusions were in contrast with UV-induced hydrogen-atom-transfer and ring-opening