Quantum Chemical Investigation of the Electronic Spectra of the Keto, Enol, and Keto-Imine Tautomers of Cytosine

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1 8410 J. Phys. Chem. A 2005, 109, Quntum Chemicl Investigtion of the Electronic Spectr of the Keto, Enol, nd Keto-Imine Tutomers of Cytosine Ktrin Tomić, Jo1rg Ttchen, nd Christel M. Mrin* Institute of Theoreticl nd Computtionl Chemistry, Heinrich-Heine-UniVersity, UniVersitätsstrsse 1, D Düsseldorf, Germny ReceiVed: Mrch 23, 2005; In Finl Form: July 20, 2005 Downloded by UNIVERSITAT WIEN BIBLIOTHEKS on October 22, Publiction Dte (Web): August 30, 2005 doi: jp051510o 1. Introduction The low-lying excited singlet sttes of the keto, enol, nd keto-imine tutomers of cytosine hve been investigted employing combined density functionlmultireference configurtion interction (DFTMRCI) method. Unconstrined geometry optimiztions hve yielded out-of-plin distorted structures of the π f π* nd n f π* excited sttes of ll cytosine forms. For the keto tutomer, the DFTMRCI dibtic excittion energy of the π f π* stte (4.06 ev including zero-point vibrtionl energy corrections) supports the resonnt two-photon ioniztion (R2PI) spectrum (Nir et l. Phys. Chem. Chem. Phys. 2002, 5, 4780). On its S 1 potentil energy surfce, conicl intersection between the 1 ππ* stte nd the electronic ground stte hs been identified. The brrier height of the rection long constrined minimum energy pth mounts to merely 0.2 ev bove the origin nd explins the brek-off of the R2PI spectrum. The 1 ππ* minimum of the enol tutomer is found t considerbly higher excittion energies (4.50 ev). Becuse of significnt geometry shifts with respect to the ground stte, long vibrtionl progressions re expected, in ccord with experimentl observtions. For the keto-imine tutomer, crossing of the 1 ππ* potentil energy surfce with the ground-stte surfce hs been found, too. Its n f π* minimum (3.27 ev) is locted well below the conicl intersection between the π f π* nd S 0 sttes, but it will be difficult to observe becuse of its smll trnsition moment. The identified conicl intersections of the π f π* excited sttes of the keto cytosine tutomers re mde responsible for the ultrfst decy to the electronic ground sttes nd thus my explin their subpicoseconds lifetimes. The nucleobses represent one of the min building blocks of life. Becuse of their gret biologicl importnce, they hve been the subject of numerous theoreticl nd experimentl investigtions tht were reviewed recently by Crespo-Hernández et l. 1 Detiled knowledge of their electronic structure nd spectrl properties is indispensble for complete understnding of the biologicl functionlity of polynucleosides, in prticulr with respect to photostbility. Solution spectr of cytosine re brod nd structureless. 2,3 It is therefore very difficult to extrct informtion bout the electronic nd nucler structure of electroniclly excited cytosine from these spectr. Time-resolved trnsient bsorption 4,5 or fluorescence up-conversion 6 spectr in queous solution yield decy rtes tht re best fitted by biexponentil functions with femtosecond component nd picosecond component. The origin of the biexponentil decy is not cler. It might be tutomer effect (s ssumed for denine), might involve two electroniclly excited sttes of the sme tutomer, or my even be cused by rpid chnge of electronic trnsition moments with nucler geometry relxtion in the electroniclly excited stte. Fundmentl insight is gined from the exmintion of the excited sttes of the free molecule in the gs phse. Here the pyrimidine nucleobse cytosine ppers in three different tutomeric forms: the keto, enol, nd keto-imine forms, which re represented in Figure 1. Differences in the electronic * Corresponding uthor. E-mil: Christel.Mrin@uni-duesseldorf.de. URL: Figure 1. Tutomeric forms of cytosine: keto (left), enol (middle), keto-imine (right). The numbering of the centers grees with the conventions for pyrimidine. structures of the tutomers produce differences in their spectr. High-resolution resonnt two-photon ioniztion (R2PI) spectr of cytosine were recorded close to the bnd origins of the lowestlying trnsitions. 7 The experimentlly observed gs-phse vibronic spectrum of cytosine 8 is divided in two spectrl regions which re seprted by bout 4000 cm -1. The spectrum round cm -1 hs been ssigned to the keto tutomer nd the spectrum round cm -1 to the enol tutomer. Femtosecond trnsient ioniztion signls of electroniclly excited cytosine prepred t 267 nm ( cm -1 ) in moleculr bem show biexponentil decy of the excited-stte popultion. 9 The tutomer(s) responsible for this behvior hve not been identified so fr. In moleculr bem microwve spectroscopy study 10 the popultion of the keto-imine tutomer is estimted to be only one-qurter of the popultion of the other tutomers. Its electronic excittion spectrum hs not been observed in experiment. Previous theoreticl work by Sobolewski nd Domcke on the gunine-cytosine (GC) bse pir 11 opened the question whether the geometry of photoexcited cytosine is plnr or nonplnr. In this pper, two fst relxtion pthwys of the electroniclly excited bse pir to the electronic ground stte re described jp051510o CCC: $ Americn Chemicl Society Published on Web

2 Quntum Chemicl Investigtion J. Phys. Chem. A, Vol. 109, No. 37, Downloded by UNIVERSITAT WIEN BIBLIOTHEKS on October 22, Publiction Dte (Web): August 30, 2005 doi: jp051510o The mechnism dubbed SPT involves n electron trnsfer from gunine to cytosine followed by single proton trnsfer. The second pthwy nmed ETH proceeds on potentil energy hypersurfce corresponding to loclly excited electronic structure of cytosine. Here, n intersection between the S 1 nd S 0 potentil energy hypersurfces of the bse pir tkes plce t moderte energies for structure in which the C 5 dc 6 bond (see Figure 1) of the cytosine ring is twisted. The rpid decy of the excited-stte popultion vi these pthwys is believed to be responsible for the ultrshort lifetime of the Wtson- Crick bse pir observed in experiment. 12 The detection of the ETH pthwy 11 led us to the question whether similr conicl intersection cn be found in the bre cytosine. Merchán nd Serrno-Andrés 13 observed different conicl intersection between the ππ* stte nd the ground stte of the bre ketocytosine. In the ltter cse, the decisive coordinte ppers to be the CO bond stretch. Guided by these findings, we study here the behvior of the photoexcited cytosine tutomers. The min im of our study is to compute the verticl nd dibtic electronic excittion spectr of the bove-mentioned tutomers nd to compre the results with the experimentl ones. To this end, we hve clculted geometries nd reltive energies of the ground nd low-lying excited sttes s well s norml modes in the ground sttes. 2. Methods nd Technicl Detils All clcultions hve been performed employing the vlence triple-ζ bsis set with polriztion functions (d,p) from the TURBOMOLE librry. 14 This bsis set is denoted by TZVP in the following. The procedure tht we hve used to obtin geometries nd reltive energies hs been recently pplied to the investigtion of denine by Mrin. 15 Full geometry optimiztions hve been crried out utilizing the TURBOMOLE progrm pckge, version ,17 Optimiztions of the ground nd excited sttes hve been performed t the level of density functionl theory (DFT) nd time dependent density functionl theory (TDDFT), respectively. To check the results with respect to the influence of different density functionls, clcultions of the verticl electronic spectr hve been performed employing the equilibrium nucler rrngements obtined for both the BH-LYP nd the B3-LYP hybrid functionls. 18,19 For the clcultion of the dibtic spectr, the BH-LYP optimized geometries were employed. Constrined minimum energy pths (CMEP) were obtined from prtil optimiztions in internl coordintes, employing the EF.X progrm. 20 We hve used the SNF progrm 21 to clculte norml modes in the ground sttes of the tutomers. The vibrtionl frequencies hve been computed t the B3-LYP geometry nd hve been scled by common fctor of For the determintion of the singlet electronic sttes of cytosine, we pplied the combined density functionl theory multireference configurtion interction (DFTMRCI) method by Grimme nd Wletzke. 23 This semiempiricl method ws shown to yield excellent electronic spectr of orgnic molecules t resonble computtionl cost. Using n effective, prmetrized Hmiltonin, the DFTMRCI includes both dynmic (DFT) nd sttic (MRCI) electron correltion. The problem of double counting of electron correltion is voided mostly by introducing n exponentil dmping fctor into the Hmiltonin. The configurtion stte functions (CSFs) in the MRCI expnsion re built up from Kohn-Shm (KS) orbitls. Optimized prmeter sets for the effective DFTMRCI Hmiltonin re vilble in combintion with the BH-LYP functionl. The initil Figure 2. Ground-stte geometry of the keto tutomer optimized with the BH-LYP functionl. DFTMRCI energy: E H. Dipole moment t the DFTMRCI level: 6.82 D. set of CSFs cn be generted utomticlly in complete ctive spce type procedure nd is then itertively improved. The MRCI expnsion is kept moderte by extensive configurtion selection. The DFTMRCI pproch gives reltive energies of high qulity, with root-men-squre devition from experimentl reference dt by bout 0.15 ev. 23 Beside n excellent description of the energy, the DFTMRCI provides relible prediction of properties. More detils bout the method cn be found in the originl publiction by Grimme nd Wletzke. 23 Moleculr KS orbitls, optimized for the dominnt closed shell determinnt of the electronic ground stte, were employed s one-prticle bsis for the MRCI runs. In the ltter step, ll vlence electrons were correlted. For plnr nucler rrngements, eigenvlues nd eigenvectors of four singlet sttes were determined in ech irreducible representtion (A, A ). At lower symmetry points, the eight lowest singlet sttes were determined. The DFTMRCI dibtic excittion energies re improved by tking into ccount zero-point vibrtionl energy (ZPVE) corrections. These re obtined by performing norml-mode nlysis in both ground nd excited sttes. The zero-point energies re scled with common fctor for the BH-LYP functionl of The scling fctor is estimted on the bsis of DFT study of vibrtionl spectr, 24 in which observed nd clculted fundmentl vibrtionl frequencies t the BH-LYP level re compred for set of molecules. 3. Results nd Discussion Crtesin coordintes of ground nd excited-stte minim of ll tutomers cn be found in the Supporting Informtion. This dt collection lso includes the coordintes of nucler structures t or close to conicl intersections between the ground stte nd the first excited singlet π f π* sttes of the keto nd the keto-imine tutomers Keto Tutomer of Cytosine. We begin the investigtion of the keto tutomer with the ground-stte geometry optimiztion. The DFTMRCI energy, geometry prmeters optimized t the BH-LYP level of theory, nd the dipole moment of the ground stte of keto cytosine re presented in Figure 2. As generl trend, the B3-LYP functionl gives somewht lrger bond distnces (1 pm). In the nucler rrngement optimized with the BH-LYP functionl ll nuclei lie in common plin. The B3-LYP minimum structure shows slight out-of-plne distortions which include mino group pyrmidliztion, but the energy difference between the C 1 -symmetric minimum nd the C s -symmetric sddle-point mounts to merely 1 cm -1 t this level of theory. From the clculted vibrtionl frequency of the pyrmidliztion mode (166 cm -1 ), we conclude tht the

3 8412 J. Phys. Chem. A, Vol. 109, No. 37, 2005 Tomić etl Downloded by UNIVERSITAT WIEN BIBLIOTHEKS on October 22, Publiction Dte (Web): August 30, 2005 doi: jp051510o TABLE 1: Hrmonic Vibrtionl Frequencies [cm -1 ] for the NH Stretching Modes in the Ground Stte of the Keto Cytosine DFT(B3-LYP, scled) experiment 25 experiment 26 NH 2(s) N 1H NH 2() Key: (s) symmetric vibrtions; () ntisymmetric vibrtions. TABLE 2: Verticl Singlet Excittion Energies E [ev] (DFTMRCI, TZVP Bsis, BH-LYP Geometry) nd Dipole Trnsition Oscilltor Strengths f(r) of Keto Cytosine DFTMRCI, present work stte E f(r) dominnt excittion(s) S A π H f π L S A n O f π L S A n N f π L S A π H-1 f π L S A n O f π L +1, n N f π L +1 S A (π H f Ryd) S A π H f π L +3 When diffuse functions re dded to the bsis, the energy of the Rydberg excittions is expected to decrese considerbly. first vibrtionl level (V )0) should be locted well bove the brrier to plnrity. Therefore, we hve computed the verticl excittion spectrum in C s symmetry. According to our investigtions the keto form is the second most stble tutomer in the gs phse. The dipole moment of this form of cytosine is the lrgest one mong the three tutomers. It will, therefore, be preferentilly stbilized in polr solvents. The scled frequencies of the NH stretching modes in the ground stte re given in Tble 1. They compre fvorbly with the vlues mesured in IR-UV double resonnce experiments 25 nd IR spectr recorded in liquid helium nnodroplets The Verticl Absorption Spectrum. To describe the electronic structure of the excited sttes, it is necessry to chrcterize the frontier moleculr orbitls. The highest occupied moleculr orbitl (HOMO) is π-type orbitl with lrge mplitude t the oxygen site nd in the following we denote it by π H. The second highest occupied moleculr orbitl (HOMO- 1) is lso of π-type nd we designte s π H-1. The third highest occupied moleculr orbitl (HOMO-2) is n n-type orbitl with lrge mplitude for the oxygen lone-pir orbitl, denominted by n O in the following. The fourth highest occupied moleculr orbitl is lso of n-type, but the mplitude is concentrted on the nitrogen (N 3 ) lone-pir orbitl nd we designte it s n N. Concerning the virtul orbitls, the lowest-lying unoccupied moleculr orbitl (LUMO) is π-type orbitl, designted by π L. It is deloclized over the whole molecule. Using the sme nottion for the higher vlence moleculr orbitls, we denote them by π L+1, π L+2, etc. The verticl electronic spectrum t the minimum of the ground-stte potentil energy surfce of the keto tutomer ws computed using C s point group symmetry. The reltive DFT MRCI excittion energies, oscilltor strengths nd dominnt excittions re shown in Tble 2. The lowest-lying excited stte corresponds to the HOMO-LUMO trnsition. The trnsition is ccompnied by chrge trnsfer from the oxygen into the ring nd hs medium oscilltor strength. The second excited singlet stte is n n f π* stte nd rises from the excittion of the n O to the π L orbitl. This trnsition is wek, which is expected for n f π* singlet excittions of heteroromtic compounds. The third singlet stte (S 3 ) results from n N f π L excittion nd is found to hve smll oscilltor strength, too. S 4 rises from π H-1 f π L excittion. It is ccompnied by chrge trnsfer from the NH 2 group into the ring nd ppers s very strong trnsition. When we compre the verticl spectr obtined t the B3- LYP nd BH-LYP geometries, we conclude tht there is no difference in the ordering of the low-lying excited sttes with respect to different functionls used in the optimiztions. Nevertheless, we notice tht the B3-LYP geometry exhibits lower excittion energies. The energy of the lowest π f π* stte chnges from 4.83 ev t the BH-LYP geometry to 4.68 ev t the B3-LYP geometry. At the BH-LYP geometry the lowest n f π* stte hs the energy of 5.02 ev, while t the B3-LYP geometry its energy is 4.90 ev. These findings re consequence of the elongted bond lengths of the B3-LYP equilibrium geometry with respect to the BH-LYP optimized nucler rrngement. They re therefore expected s generl trend. Here we compre our results with previous theoreticl tretments of the verticl spectrum of cytosine. For the comprison, we use only few low-lying excited sttes. In the higher-energy region vlence sttes mix with the Rydberg sttes nd to describe such sttes it would be necessry to use extended bsis sets. We find good greement with the results of Petke et l. 27 obtined by extended-bsis RPA clcultions. They find tht the lowest-lying 1 ππ* stte ppers t 4.61 ev. This vlue shows better greement with the DFTMRCI energy t the B3- LYP geometry (4.68 ev) thn with the one t the BH-LYP geometry (4.83 ev). They lso predict tht the two next sttes of n f π* chrcter pper t 4.95 nd 5.43 ev, respectively. This is in ccordnce with our findings for the two n f π* sttes computed t the BH-LYP geometry (Tble 2). Fülscher nd Roos 28 clculted CASPT2 excittion energies of keto cytosine. They found the lowest π f π* excited stte to occur t 4.39 ev. This vlue is much lower thn ours. The first excited singlet n f π* stte is computed by CASPT2 to pper t 5.00 ev. This vlue grees with the energy of 5.02 ev tht we hve computed for the 1 nπ* stte. Nevertheless, the first stte rises from n excittion of nitrogen lone-pir electron (n N ) in their CASPT2 study, while we find it to originte from n excittion of n oxygen lone-pir electron (n O ). Our finding is supported by mny other theoreticl investigtions. 13,29 In nother CASPT2 study by Merchán nd Serrno-Andrés, 13 the lowest-lying n f π* stte ppers to be the n O π* stte with n excittion energy of 4.88 ev, which is in ccord with our result. The energies of the π f π* stte (4.64 ev) nd the n f π* stte (4.77 ev) obtined by Shukl nd Leszczynski 30 using the TDDFT (B3- LYP) method gree well with our DFTMRCI vlues t the equilibrium geometry optimized with the B3-LYP functionl. As lredy noted, these vlues re somewht lower thn the corresponding ones t the BH-LYP geometry. The dipole moment of the keto tutomer clculted t the LDA-DFT level 31 mounts to 6.71 D. Kwitkowski nd Leszczyński 32 obtined similr results with the HF method (7.12 D) nd the DFT (B3-LYP) method (6.29 D). In photodetchment-photoelectron spectroscopy, 33 the dipole moment of the keto tutomer ws predicted to be pproximtely 7 D, in good greement with our DFTMRCI vlue of 6.82 D. There is lso number of experimentl spectr reported for cytosine. In solution of trimethyl phosphte Clrk nd Tinoco 2 observed n bsorption bnd t pproximtely 277 nm (4.5 ev). Absorption mesurements in queous solution by Voelter et l. 3 revel n bsorption bnd with mximum t 266 nm (4.66 ev). These bnds cn be ssigned to π f π* trnsition in

4 Quntum Chemicl Investigtion J. Phys. Chem. A, Vol. 109, No. 37, Downloded by UNIVERSITAT WIEN BIBLIOTHEKS on October 22, Publiction Dte (Web): August 30, 2005 doi: jp051510o Figure 3. Spectrum of the keto cytosine (DFTMRCI energies) t selected nucler geometries. The nucler rrngement ws optimized t the BH-LYP level using C s symmetry constrints nd without ny constrints. Minimum geometries re lbeled by subscript min, sddlepoints by sp. The lbel CI denotes point close to the conicl intersection. The conicl intersection occurs between the excited singlet π f π* stte nd the electronic ground stte. Solid lines: 1 ππ* stte. Dshed lines: 1 nπ* stte. keto cytosine. They show greement with the DFTMRCI excittion energy of the lowest 1 ππ* stte. A mgnetic circulr dichroism (MCD) spectrum of cytosine, mesured by Voelter et l., yields n bsorption bnd t bout 270 nm (4.6 ev). Clrk et l. 34 mesured the vpor spectr of cytosine nd found shoulder in the long wvelength region t bout 290 nm (4.28 ev). This much lower vlue is somewht uncertin becuse the cytosine decomposes during these mesurements nd gives mny other unidentified bnds. 34 During the course of the present quntum chemicl investigtion, EELS (electron energy loss spectroscopy) mesurements were crried out on cytosine under gs-phse conditions. 35 In these experiments the verticl S 1 r S 0 excittion cme out t 4.65 ( 0.1 ev. The comprison with erlier solution spectr implies tht the solvent shift is smll for this trnsition The Adibtic Spectrum nd Conicl Intersections. The experimentl R2PI spectr of cytosine were recorded close to the bnd origin nd cn therefore not be compred directly with verticl bsorption spectr. For tht purpose, we need dibtic spectr. The order nd the energies of the singlet excited sttes of keto cytosine depend strongly on the nucler geometry (Figure 3). At first, we hve performed the energy minimiztion of the two lowest excited singlet sttes with C s symmetry constrints by mens of TDDFT (BH-LYP). The dibtic DFTMRCI excittion energy for the n O f π L stte t the resulting sttionry point mounts to 4.40 ev compred to verticl bsorption energy of 5.02 ev. In this region of the coordinte spce the 1 nπ* stte corresponds to the lowest-lying excited singlet (S 1 ). When the singlet excited π H f π L stte is optimized under C s symmetry constrints, its energy decreses from 4.83 ev t the ground-stte geometry to 4.19 ev. The order of electronic sttes is similr to tht in the verticl bsorption region. However, the plnr structures of the 1 ππ* nd the 1 nπ* sttes correspond only to sddle points on the potentil energy surfce of these sttes. Geometry optimiztion of the n O f π L stte without symmetry constrints results in strongly distorted excited-stte structure (Figure 3, left side). The geometry chnge involves pyrmidliztion of the mino group nd significnt out-of-plne deformtions of the ring. The energy of this stte is decresed from 4.40 ev in the C s symmetric structure to 4.24 ev t the minimum. Figure 4. DFTMRCI energies of the keto tutomer clculted long the TDDFT rection pth connecting the singlet π f π* minimum nd the conicl intersection between the 1 ππ* stte nd the electronic ground stte. The dihedrl ngle is the N 1C 6C 5H ngle. Below vlue of 110 constrined optimiztion of the excited stte leds to conicl intersection t the TDDFT level. The TDDFT CMEP chnges direction t the conicl intersection. Tringles: electronic ground stte. Dimonds: π f π* stte. Unconstrined geometry optimiztion of the first excited 1 ππ* stte results in n out-of-plne structure (Figure 3, right side). The nonplnrity is not only due to the pyrmidliztion of the mino group, but lso the ring of cytosine is out-of-plne deformed nd possesses butterfly conformtion. The dibtic excittion energy of the π f π* stte mounts to 4.18 ev. C s nd C 1 energies of the singlet π f π* stte re very similr while the geometries differ strongly. The excited-stte potentil energy surfce in tht region is very flt. For strict comprison with the experiment, ZPVE corrections hve to be included. With these corrections, the 1 ππ* dibtic excittion energy becomes 4.06 ev. The bnd origin of the experimentl R2PI spectrum of the keto tutomer ppers t round cm -1 (3.97 ev). Tking into ccount the usul error-brs of the DFT MRCI method, the spectrl region of the keto form is predicted in good ccord with experimentl observtions. Motivted by the results of recent quntum chemicl study on the gunine-cytosine (GC) bse pir 11 where strongly deformed nucler structure of the cytosine ring ws obtined in the loclly excited stte, we studied the potentil energy surfce of the singlet π H f π L stte for lrge out-of-plne distortions. The deformtion involved twisting of the C 5 dc 6 double bond. This kind of out-of-plne distortion destbilizes the energy of the ground stte which ppers to be very sensitive to the twisting of the C 5 dc 6 bond. Strting t highly distorted geometry, the TDDFT grdient leds the system towrd conicl intersection with the electronic ground stte (Figure 3, right side). Beside the twisting of the C 5 dc 6 bond, the deformtion involves n elongtion of this bond from 1.34 Å in the ground stte to 1.44 Å in the excited π f π* stte. These results resemble the behvior of the excited singlet stte in the GC bse pir. 11 To obtin n estimte of the brrier height between the minimum nd conicl intersection res on the potentil energy surfce of the S 1 stte we performed constrined energy optimiztion t the TDDFT level. The proper internl coordinte for this purpose ws found to be the dihedrl ngle N 1 -C 6 - C 5 -H. We kept this ngle fixed while relxing ll other internl degrees of freedom. Note tht for dihedrl ngles of 110 nd below, the geometry optimiztion breks off when the electronic ground-stte hits the intersection sem. These points might, therefore, not be locted on the true TDDFT minimum energy pth. After prtil optimiztions, single-point DFTMRCI clcultions were performed. The CMEP is presented in Figure 4. For dihedrl ngles between 170 nd 120, the first excited stte corresponds to the π H f π L trnsition. For smller vlues of the ngle the order of the 1 ππ* stte nd the ground stte is interchnged. The lowest point of the conicl intersection sem

5 8414 J. Phys. Chem. A, Vol. 109, No. 37, 2005 Tomić etl TABLE 3: Hrmonic Vibrtionl Frequencies [cm -1 ] for the NH Stretching Modes in the Ground Stte of the Enol Cytosine DFT(B3-LYP, scled) experiment 25 experiment 26 NH 2(s) NH 2() OH Key: (s) symmetric vibrtions; () ntisymmetric vibrtions. Downloded by UNIVERSITAT WIEN BIBLIOTHEKS on October 22, Publiction Dte (Web): August 30, 2005 doi: jp051510o Figure 5. Ground-stte geometry of the enol tutomer optimized with the BH-LYP functionl. DFTMRCI energy: E H. Dipole moment t the DFTMRCI level: 3.55 D. is reched t bout 70 (3.98 ev). The energy difference between the minimum in the FC region nd the highest point on the CMEP mounts to merely 0.2 ev. The presence of conicl intersections between the 1 (π f π*) stte nd the electronic ground stte tht is seprted from the 1 (π f π*) minimum in the FC region by smll brrier, explins the experimentl finding tht only few shrp vibronic peks close to the bnd origin could be observed in the R2PI spectrum of keto cytosine. 1,36 As soon s the conicl intersection cn be ccesseds either directly or vi tunneling processsthe excited stte popultion is rpidly trnsferred to the electronic ground-stte surfce nd the spectrum breks off. Merchán nd Serrno-Andrés in their CASSCFCASPT2 study 13 found nother (gsππ*) conicl intersection of the keto tutomer. According to these clcultions, the crossing occurs long the CdO stretching mode t bond distnce of Å nd very little energy is needed to overcome brrier (0.1 ev) nd the excited-stte surfce rpidly decys to the ground-stte surfce. The conicl intersection obtined in our study is not identicl with the one described by Merchán nd Serrno- Andrés. Its nucler structure exhibits CdO bond length of 1.19 Å, similr to the ground-stte equilibrium distnce. The conicl intersection of the potentil energy surfces long the C 5 dc 6 twisting coordinte rther represents n lterntive pthwy for the ultrfst decy of the excited stte of the keto cytosine to its electronic ground stte Enol Tutomer of Cytosine. Our investigtion of the electronic ground stte of the enol tutomer hs shown tht it is the most stble form in the gs phse. Among ll the tutomers of cytosine, the enol form hs the smllest dipole moment. Upon DFT (B3-LYP) optimiztion without symmetry constrints, the geometry exhibits deformtions which include slight pyrmidliztion of the mino group. However, the energy chnge with respect to the C s -symmetric structure obtined t the sme level of theory is negligible (0.001 ev). Employing the BH-LYP functionl, plnr nucler rrngement hs been found for the ground stte minimum. For further clcultions of verticl nd dibtic bsorption spectr we hve used the BH-LYP equilibrium structure of the ground stte geometry which is presented in Figure 5. Utilizing the SNF progrm we hve clculted the hrmonic vibrtionl frequencies for the ground stte (Tble 3). The scled frequencies show good greement with the experimentlly observed ones, displyed longside in Tble The Verticl Absorption Spectrum. We begin the description of the excited-stte electronic structure with the chrcteriztion of the moleculr orbitls. The highest occupied moleculr orbitl (HOMO) is π-type orbitl, designted by π H. It belongs to the irreducible representtion nd is TABLE 4: Verticl Singlet Excittion Energies E [ev] (DFTMRCI, TZVP Bsis, BH-LYP Geometry) nd Dipole Trnsition Oscilltor Strengths f(r) of Enol Cytosine DFTMRCI, present work stte E f(r) dominnt excittion(s) S A π H f π L S A n N f π L S A n N f π L +1 S A π H f π L +1, π H-1 f π L S A (π H f Ryd) S A π H-1 f π L, π H f π L +1 S A n O f π L When diffuse functions re dded to the bsis, the energy of the Rydberg excittion is expected to decrese considerbly. deloclized over the whole molecule. The second highest occupied moleculr orbitl (HOMO-1) is n n-type orbitl. It exhibits lrge mplitudes for the N 1,N 3, nd N 8 lone-pir orbitls nd we designte it s n N. The third highest occupied moleculr orbitl (HOMO-2) is of π-type, denoted by π H-2. The fourth highest occupied moleculr orbitl (n O ) is nonbonding (ntype) orbitl with lrge mplitudes for the oxygen, N 1, nd N 3 lone-pir orbitls. We begin the chrcteriztion of the virtul orbitls with the lowest-lying unoccupied moleculr orbitl (LUMO). This is π-type orbitl which we denote by π L. The next higher vlence orbitl is π L+1. Both orbitls belong to the irreducible representtion. The DFTMRCI excittion energies, oscilltor strengths, nd dominnt excittions hve been computed t the BH-LYP geometry nd re presented in Tble 4. The first singlet excited stte is π f π* stte, nd it corresponds to the HOMO- LUMO trnsition. It ppers to be very strong. The second excited singlet stte 1 1 A rises from n excittion out of the highest occupied nonbonding orbitl (n N ) to the π L. This trnsition hs smll oscilltor strength. The third excited singlet stte S 3 results from n excittion out of the n N orbitl to π L+1. The oscilltor strength for this trnsition is found to be very smll. The fourth excited singlet stte S 4 is gin π f π* stte. The MRCI expnsion of this stte is mixed nd contins two dominnt excittions: π H fπ L+1 nd π H-1 fπ L. It ppers s very strong trnsition. When we compre the verticl spectr t different geometries we notice tht the spectrum t the B3-LYP geometry is slightly shifted to lower energies. The first excited π f π* stte t the BH-LYP geometry hs n energy of 5.14 ev while t the B3- LYP geometry its energy is 5.02 ev. The lowest n f π* stte exhibits the sme behvior. Its energy chnges from 5.27 ev t the BH-LYP geometry to 5.20 ev t the B3-LYP geometry. However, the ordering of the excited sttes remins the sme. The stbility of the enol tutomer nd its dipole moment in the ground-stte gree very well with the results of previous theoreticl studies. 37,38 The RI-MP2 clcultions for the gs phse by Hobz et l. 38 predict tht the enol is the most stble form of cytosine. They obtin dipole moment in the ground stte of 3.25 D. Yng nd Rodgers 37 clculted the dipole

6 Quntum Chemicl Investigtion J. Phys. Chem. A, Vol. 109, No. 37, Downloded by UNIVERSITAT WIEN BIBLIOTHEKS on October 22, Publiction Dte (Web): August 30, 2005 doi: jp051510o Figure 6. Spectrum of the enol cytosine (DFTMRCI energies) t selected nucler geometries. The sketched distorted geometry on the right side corresponds to the minimum of the first excited π f π* stte nd on the left side to the minimum of the first n f π* excited stte. Solid lines: 1 ππ* stte. Dshed lines: 1 nπ* stte. See Figure 3 for further explntions. Figure 7. Ground-stte geometry of the keto-imine tutomer optimized with the BH-LYP functionl. DFTMRCI energy: E H. Dipole moment t the DFTMRCI level: 4.92 D. moment of the enol form to be 3.72 D t the MP2(full)6-31G* level. The LDA-DFT method 31 predicted vlue of 3.51 D. Kwitkowski nd Leszczyński 32 using the HF nd DFT (B3- LYP) pproches clculted the dipole moment to be 3.39 nd 3.16 D, respectively. The experimentl finding from the photodetchment-photoelectron spectroscopy 33 mounts to 3.4 D. Our DFTMRCI vlue of 3.55 D is in ccordnce with these results. The verticl bsorption spectrum for enol cytosine ws predicted by the semiempiricl INDOs method. 39 The π f π* trnsition energies re underestimted nd differ from our DFT MRCI vlues. According to the INDOs pproch, the lowestlying π f π* stte ppers t cm -1 (4.59 ev) while we find n excittion energy of 5.14 ev t the BH-LYP geometry. The uthors lso suggested tht the lowest-lying n f π* stte is the third excited stte with n energy of cm -1 (5.80 ev). In the DFTMRCI, the n f π* stte ppers s the second trnsition with n energy of 5.27 ev The Adibtic Spectrum nd Excited-Stte Geometry. The dibtic excittion energy represents the energy difference between the ground stte equilibrium nd the optimized excited-stte structure. When ZPVE corrections re tken into ccount, this energy is roughly comprble to the 0-0 trnsition in the experimentl R2PI spectrum. By mens of TDDFT, we hve first optimized excited sttes under C s - symmetry constrints (Figure 6). The DFTMRCI excittion energy of the n f π* stte decreses from 5.27 ev t the ground-stte minimum to 4.66 ev t the optimized plnr structure. Here, the n f π* stte becomes the lowest-lying excited stte. The miniml excittion energy of the plnr π f π* stte is 4.83 ev. The optimiztion stbilizes this stte by bout 0.3 ev with respect to its energy t the ground-stte geometry (5.14 ev). Unconstrined geometry optimiztion of the lowest n f π* stte leds to n out-of-plne distorted minimum structure (Figure 6, left side). The deformtion refers to the pyrmidliztion of the NH 2 group while the ring geometry remins plnr. Becuse of the smll geometry chnge, the dibtic excittion energy of the n f π* stte (4.61 ev) is only slightly decresed with respect to the excittion energy of the C s symmetric structure (4.66 ev). The optimized C s symmetric structure of the 1 ππ* stte represents only sddle point on the potentil energy surfce. Unconstrined geometry optimiztion of this stte leds to n out-of-plne distorted structure (Figure 6, right side). Comprison of the C s - nd C 1 -symmetric structures shows tht the geometry chnge upon electronic excittion involves not only pyrmidliztion of the mino group, but lso deformtion of the ring itself. The dibtic excittion energy of the distorted 1 ππ* stte of enol cytosine mounts to 4.60 ev, corresponding to stbiliztion by more thn 0.5 ev with respect to its vlue t the ground-stte minimum. Tking into ccount the scled ZPVE of the ground nd excited stte, the dibtic excittion energy of the 1 ππ* stte of the enol tutomer becomes 4.50 ev. The origin of the experimentl R2PI spectrum ws observed round cm -1 (4.46 ev). Compring these results, we conclude tht the DFTMRCI pproch predicts the spectrl region of enol cytosine in good greement with experiment. The geometric relxtion on the 1 ππ* potentil energy surfce involves twisting of the C 5 dc 6 double bond nd lso its elongtion from 1.36 Å in the ground stte to 1.44 Å t the singlet ππ* minimum. The energy of the ground stte t this nonplnr geometry is significntly incresed nd shows strong dependence on the out-of-plne deformtions. However, no conicl intersection ws encountered during the optimiztion. We therefore expect tht low-lying vibrtionl levels in the 1 ππ* stte of the enol form of cytosine decy by norml internl conversion, possibly through coupling to nerby 1 nπ* levels. This process is slow compred to decy vi conicl intersection nd should llow the observtion of mny shrp spectrl lines, in ccord with experimentl findings Keto)Imine Tutomer of Cytosine. The third structurl isomer of cytosine tht we investigte is known s the ketoimine tutomer. Energeticlly, this is the lest stble form in the gs phse mong the three tutomers. The dipole moment of the keto-imine lies between the vlues for the keto nd the enol tutomers. The ground-stte optimiztion ws performed with nd without C s symmetry constrints. In the ltter cse, the structure converged to the plnr form nd the energy remined the sme s for the C s -symmetric structure. The ketoimine does not possess the NH 2 group nd thus exhibits no pyrmidliztion effect. The equilibrium ground-stte geometry, the dipole moment, nd the DFTMRCI energy of the ketoimine re shown in Figure 7. Employing the SNF progrm, we hve clculted the hrmonic vibrtionl frequencies in the ground stte of this tutomer. The scled frequencies re presented in Tble 5. As experimentl vlues re not vilble, these dt cn be used s led to identify this tutomer in forthcoming IR-UV double-resonnce experiments The Verticl Absorption Spectrum. For the description of the electronic structure of the excited sttes, it is necessry t first to ssign the orbitl nture. The highest

7 8416 J. Phys. Chem. A, Vol. 109, No. 37, 2005 Tomić etl Downloded by UNIVERSITAT WIEN BIBLIOTHEKS on October 22, Publiction Dte (Web): August 30, 2005 doi: jp051510o TABLE 5: Hrmonic Vibrtionl Frequencies [cm -1 ] for the NH Stretching Modes in the Ground Stte of the Keto-Imine Cytosine DFT(B3-LYP, scled) N 8H 3370 N 3H 3458 N 1H 3500 TABLE 6: Verticl Singlet Excittion Energies E [ev] (DFTMRCI, TZVP Bsis, BH-LYP Geometry) nd Dipole Trnsition Oscilltor Strengths f(r) of Keto-Imine Cytosine DFTMRCI, present work stte E f(r) dominnt excittion(s) S A n N f π L S A π H f π L S A π H-1 f π L S A n O f π L, n O f π L +1 S A (π H f Ryd) S A π H f π L +1 S A n N f π L + 1, n O f π L When diffuse functions re dded to the bsis, the energy of the Rydberg excittions is expected to decrese considerbly. occupied moleculr orbitl (HOMO) is π-type orbitl, designted by π H. The second highest occupied moleculr orbitl (HOMO-1) belongs lso to the π-type orbitls nd we denote it by π H-1. The third highest occupied moleculr orbitl is nonbonding orbitl with lrge mplitude for the N 3 lone-pir orbitl. We designte it by n N. The fourth highest occupied moleculr orbitl (HOMO-3) is n n-type orbitl with lrge mplitude for the crbonyl oxygen, N 1, nd N 3 lone-pir orbitls nd is lbeled by n O. We begin the ssignment of the virtul orbitls with the lowest unoccupied moleculr orbitl (LUMO). This is π-type orbitl designted by π L. In the energy gp between the LUMO nd the next vlence moleculr orbitl we find one diffuse orbitl. Nevertheless, the higher vlence orbitls will be denominted π L+1, π L+2, etc. Verticl excittion energies of the keto-imine, oscilltor strengths, nd dominnt excittions re given in Tble 6. The lowest-lying singlet stte does not correspond to HOMO- LUMO trnsition. It results from n excittion out of the highest occupied nonbonding moleculr orbitl (n N )totheπ L orbitl. As is expected for n f π* trnsitions, its oscilltor strength is smll. The second excited singlet stte S 2 rises from the HOMO-LUMO trnsition. This trnsition is found to be very strong. The third excited singlet stte is lso π f π* stte. The trnsition is ccompnied by chrge trnsfer from the oxygen into the ring. Its oscilltor strength is smll. The fourth excited-stte S 4 origintes from two dominnt excittions: one corresponds to the promotion of n electron out of the n O orbitl to the π L, nd the other to the promotion of n electron from the n O to the π L+1 orbitl. The oscilltor strength for this trnsition is found to be very smll. Compring the verticl spectr t the BH-LYP nd the B3- LYP geometries, we confirm our expecttions tht the B3-LYP geometry exhibits longer bond distnces nd yields lower excittion energies. The verticl excittion energy of the lowest n f π* stte chnges from 5.19 ev t the BH-LYP geometry to 5.10 ev t the B3-LYP geometry. The energy of the first excited π f π* stte is lowered from 5.26 ev t the BH-LYP to 5.12 ev t the B3-LYP geometry. Here we mke comprison of our results with the previous theoreticl tretments of the keto-imine tutomer. The reltive stbility of isolted cytosine tutomers in the gs phse, including the imino form, hs been exmined by Hobz et l. 38 They performed ground-stte optimiztions t the RIMP2 TZVPP level of theory nd found tht the keto-imine form is the lest stble in the gs phse with dipole moment of 4.63 D. The DFTMRCI vlue mounts to 4.92 D. In greement with our results, Hobz et l. 38 find tht the keto-imine form is plnr in the ground stte wheres the keto nd enol forms exhibit pyrmidliztion of the mino groups. Employing the HF nd DFT (B3-LYP) methods, 32 its dipole moment is found to be 5.19 nd 4.57 D, respectively. The reltive stbilities of the cytosine tutomers determined t the MP2(full)6-31 G* level of theory by Yng nd Rodgers 37 gree well with our results, wheres the dipole moment of the keto-imine tutomer (5.46 D) is somewht lrger thn the one we obtin with the DFT MRCI method. The clculted vlue in the LDA-DFT pproch 31 (4.83 D) is in good greement with our finding. Investigtions by Shukl nd Leszczynski 30 employing the TDDFT (B3-LYP) method, confirm the order of stbility in the gs phse for cytosine tutomers. Their computed dipole moment of the keto-imine form (5.12 D) is in very good greement with the DFTMRCI result. Shukl nd Leszczynski 30 lso clculted verticl ππ* nd nπ* excittion energies. In the TDDFT study the lowest-lying π f π* stte ppers t 4.77 ev. This vlue is lower thn the one we find in the DFTMRCI t the BH-LYP geometry (5.26 ev). The second excited 1 ππ* stte is computed to pper t 5.97 ev. This vlue grees very well with the one we obtin for the second π f π* trnsition (5.96 ev). The lowest n f π* stte in the TDDFT method hs the energy of 5.13 ev. This vlue grees with our excittion energy for the first excited 1 nπ* stte (5.19 ev). Becuse the bsorption spectrum of the ketoimine form could not be mesured yet, comprison of the verticl excittion energies with experiment is not possible The Adibtic Spectrum nd Excited-Stte Geometry. We hve optimized the geometries of the singlet excited n f π* nd π f π* sttes. In the beginning we restrict the symmetry to C s. The miniml excittion energy (confined to plnr structure) for the n f π* trnsition is 4.22 ev, compred to verticl bsorption energy of 5.19 ev. The optimized structure of the 1 ππ* stte hs n energy of 4.50 ev which is considerbly decresed with respect to its vlue t the groundstte minimum (5.26 ev). As for the other tutomers, we wnted to investigte possible out-of-plne structures of the excited sttes. Therefore, we performed unconstrined geometry optimiztions. At smll devitions from plnrity the first excited stte is the n f π* stte. Optimizing the energy of this stte, we obtin structure which contins plnr ring with pronounced out-of-plne devition of the NH group (Figure 8, left side). The C 4 N 8 H ngle is 108 with the NH group lmost perpendiculr to the ring itself. The dibtic excittion energy of the 1 nπ* stte mounts to merely 3.32 ev. Including the scled ZPVE corrections, it is decresed to 3.27 ev. This is the lowest-lying excited stte of the keto-imine tutomer. Its energy is much lower thn the one for the plnr structure, confirming tht the C s symmetric structure represents only sddle point on the potentil energy surfce of this stte. The lrge difference between the verticl nd dibtic excittion energies implies tht the Frnck-Condon fctor for the 0-0 trnsition will be very smll. The unconstrined geometry optimiztion of the singlet π f π* stte stbilizes the energy of this stte, but destbilizes strongly the energy of the ground stte. To find the minimum of this stte we hve employed norml-mode nlysis. Smll distortions were mde long the norml mode with the negtive

8 Quntum Chemicl Investigtion J. Phys. Chem. A, Vol. 109, No. 37, Downloded by UNIVERSITAT WIEN BIBLIOTHEKS on October 22, Publiction Dte (Web): August 30, 2005 doi: jp051510o Figure 8. Spectrum of the keto-imine cytosine (DFTMRCI energies) t selected nucler geometries. The sketched geometry on the left side corresponds to the minimum of the first excited n f π* stte. The structure of the conicl intersection which occurs between the singlet π f π* excited stte nd the electronic ground stte is shown on the right side. Solid lines: 1 ππ* stte. Dshed lines: 1 nπ* stte. See Figure 3 for further explntions. frequency nd the DFTMRCI energies were clculted t these points. The minimum structure of the π f π* stte is lmost plnr, with slight out-of-plne devitions. Its energy mounts to 4.46 ev nd the potentil energy surfce in the region between the plnr sttionry point nd the minimum is very flt. The ground stte ppers to be very sensitive to the out-of-plne distortions. The excited-stte grdient leds the system towrd conicl intersection with the electronic ground stte. Energeticlly, the crossing occurs t bout 4 ev. The distortion involves twisting of the C 5 dc 6 bond ccompnied by n elongtion from 1.35 Å t the ground-stte equilibrium to 1.45 Å in the excited π f π* stte. The out-of-plne deformtion nd the observed crossing in the 1 ππ* stte resemble the behvior of the first excited π f π* stte of the keto tutomer of cytosine. The dibtic spectrum of the keto-imine including the distorted excited-stte structures is presented in Figure 8. After detection of crossing between the 1 ππ* stte nd the ground stte, question bout the rection energy pth nd brrier height rose. For tht purpose constrined energy optimiztions on the S 1 potentil energy surfce were performed. An pproprite rection coordinte ws found to be the dihedrl ngle N 1 -C 6 -C 5 -H. After hving ccomplished the prtil TDDFT optimiztions, single-point DFTMRCI clcultions were crried out. Note tht the geometry optimiztion breks off when the electronic ground stte hits the sem of conicl intersections. The points obtined for dihedrl ngles below 130, might therefore, not be locted on the true TDDFT minimum energy pth. The clculted rection pth is presented in Figure 9. For dihedrl ngles smller thn 130, the system runs into conicl intersection. The lowest point on the intersection sem is reched t pproximtely 70 (4.04 ev). When the chosen dihedrl ngle is between 130 nd 160, the first excited stte corresponds to π H f π L excittion. For the geometries close to the plnr one, the order of the 1 ππ* nd 1 nπ* sttes is interchnged. The n f π* minimum is locted well below the conicl intersection between the S 1 nd S 0 sttes. At more distorted geometries, the higher-lying singlet π f π* stte cn decy to the ground stte vi n lmost brrierless rection pth. In high-resolution R2PI mesurements this cytosine tutomer hs not been observed so fr, lthough its popultion is estimted to be bout one-qurter of the popultion of the keto nd enol tutomers. 10 On the bsis of our theoreticl results, we ttribute the lck of its observtion to the following resons. The lowestlying excited singlet stte of the keto-imine is the n f π* stte. The DFTMRCI clcultions hve shown tht its dibtic Figure 9. DFTMRCI energies of the keto-imine tutomer clculted long the TDDFT rection pth connecting the singlet n f π* minimum nd the conicl intersection between the 1 ππ* stte nd the electronic ground stte. The dihedrl ngle is the N 1C 6C 5H ngle. Below vlue of 130 constrined optimiztion of the excited stte leds to conicl intersection t the TDDFT level. The TDDFT CMEP chnges directions t the conicl intersections. Tringles: electronic ground stte. Dimonds: π f π* stte. Strs: n f π* stte. excittion energy lies in the low-frequency spectrl region corresponding to lser wvelength of 373 nm. This spectrl region hs not been scnned by Nir et l. 7 In ddition to the smll electronic dipole trnsition moment, the FC-fctors re smll due to the lrge geometry shift. The first excited 1 (π f π*) stte undergoes conicl intersections with the 1 (n f π*) stte nd the electronic ground stte which cuse its very short lifetime nd possibly disble it to be observed in the experimentl spectrum. It remins to be seen whether the experiment will confirm these findings. 4. Summry nd Conclusions In the present work the keto, enol, nd keto-imine tutomers of cytosine hve been investigted by combined density functionl nd multireference configurtion interction methods. It is found tht the enol form is the most stble tutomeric form in the gs phse. The order of stbility for the tutomers ccording to our findings is s follows: keto-imine < keto < enol. Nevertheless, due to its lrger dipole moment, the keto tutomer my be preferentilly stbilized in queous environments nd in other polr solvents. Our min im ws to study the excited sttes of the bovementioned cytosine tutomers nd to explin their experimentlly observed high-resolution resonnt two-photon ioniztion (R2PI) vibronic spectr. For the comprison with these spectr the computed dibtic excittion energies were used. As response to electronic excittion, significnt chnges in geometry tke plce. Upon relxtion, loss of plnrity occurs for the lowest-lying singlet π f π* nd n f π* sttes of ll three tutomers investigted. For the keto tutomer, the form in which cytosine ppers in DNA, distorted butterfly conformtion of the 1 ππ* stte is found. Its dibtic excittion energy computed t the DFT MRCI level (4.06 ev including ZPVE corrections) supports the ssignment of the R2PI bnds round cm -1 ( 4 ev) to the keto tutomer. We hve explored n energy rection pth for twisting the C 5 dc 6 double bond in the cytosine ring. A conicl intersection between the lowest excited π f π* stte nd the ground stte hs been locted on this pth. After excittion of the S 1 stte, the conicl intersection becomes ccessible vi rection with smll energy brrier bove the origin, thus enbling rpid internl conversion to the electronic ground stte. For the 1 ππ* stte of the enol tutomer we hve obtined minimum t distorted nucler rrngement which involves twisting nd n elongtion of the C 5 dc 6 bond. It is locted t considerbly higher excittion energies (4.50 ev including ZPVE corrections) thn the corresponding trnsition in the keto