1. The elusive question of tautomerism in cytosine: Quantum chemical and matrix isolation spectroscopic investigations Gábor Bazsó, Géza Fogarasi, Péter G. Szalay and György Tarczay Institute of Chemistry, Eötvös Loránd University, Budapest, H-1518, Pf. 32. Introduction The biological importance of possible tautomerization of nucleotide bases has been recognized from the beginnings of molecular genetics [1]. Still, there is much uncertainty about the relative abundance of their tautomers. Specifically about cytosine, there is general consensus that it exists in the “canonical” amino-oxo form (1) in condensed phases. In the gas phase, however, all spectroscopic and theoretical studies indicate that two or three tautomers coexist, with dominance of the amino- hydroxy form (2). High level quantum chemical calculations predict extremely low free energy differences of  G o ≤ 1 kcal/mol for 1 and, more surprisingly, also for the imino-oxo form 3a, relative to 2b [2]. This would imply an abundance of up to 20% for both. Spectroscopic studies do „see” these isomers but in much smaller mole ratios. Accepting that commercial, crystalline cytosine consists of molecules of tautomer 1 (connected by hydrogen bonds), and the barrier to unassisted tautomerization is very high (all calculations put it to around 40 kcal/mol), a fundamental question is how tautomerization can take place during evaporation in the gas phase experiments. Recently, we have calculated the electronic spectrum, including vibrational structure, of tautomer 1 and analyzed the available solution and solid-phase spectra on the basis of this single isomer [3]. It was indicated that the remaining discrepancies may be due to the difference between gas-phase (calculated) and condensed phases (measured). No gas-phase spectrum exists on cytosine but matrix spectra may be of help. Thus, for the present study we have measured the UV spectrum in an Ar matrix, and recorded also the IR spectrum under similar conditions. The quantum chemistry computations have been extended to tautomers 2 and 3. On this basis, we will try to interpret the spectra as composite spectra of various tautomers/isomers. Scheme 1. Three tautomers of cytosine (with two rotamers). _____________________________________________________________________ [1] J.D. Watson and F.H.C. Crick, Nature 171, 964-967 (1953). [2] G. Fogarasi, J. Phys. Chem. A 106, 1381-1390 (2002). [3] A. Tajti, G. Fogarasi and P. G. Szalay, ChemPhysChem, 10, 1603-1606 (2009). [4] M. Szczesniak, K. Szczepaniak, J.S. Kwiatkowski, K. Kubulat and W.B. Person, J. Am. Chem. Soc. 110, 8319-8330 (1988). [5] M.J. Nowak, L. Lapinski, J. Fulara, Spectrochim. Acta A, 45A, 229 (1989). [6] G.G. Sheina, E. D. Radchenko, A. M. Plohotnichenko and Yu. P. Blagoi, Biofizika, XXVII, 983-986 (1982), in Russian. [7] G. Fogarasi, Chem. Phys., 349, 204-209 (2008). [8] Z. Yang and M. T. Rodgers, Phys. Chem. Chem. Phys. 6, 2749-2757 (2004). [9] O. Kostko, K. Bravaya, A. Krylov, M. Ahmed, Phys. Chem. Chem. Phys. 12, 2860 – 2872 (2010). [10] CFOUR, a quantum chemical program package written by J. F. Stanton, J. Gauss, M. E. Harding, P. G. Szalay. [11] H. Köppel, W. Domcke, L. S. Cederbaum, Adv. Chem. Phys. 1984, 57, 59-246. [12] PQS version 2.3, Parallel Quantum Solutions, 2013 Green Acres Road, Fayetteville, Arkansas 72703. Figure 2. The UV-spectra of cytosine tautomers Acknowledgement. Financial support has been provided by Hungarian science grants NKTH-OTKA-A07, no. K 68427 and OTKA K72423. The authors thank Prof. J.F. Stanton for providing access to and assistance with the SIM code. Figure 1. The IR-spectrum of cytosine tautomers Figure 3. Three H-bonded dimers derived from 1.  : E(TS) = 6.3 kcal/mol  : E(TS) = 4.90 kcal/mol  : E(TS) = 7.7 kcal/mol Given that crystalline cytosine contains only the keto form the basic question is: how do the tautomers form under the experimental circumstances? As mentioned above, all quantum chemical calculations predict a high barrier of about 40 kcal/mol for unassisted transformation of cytosine monomer. Traces of water may play a crucial role as water reduces the barrier by about a half [7]. In addition, the interesting possibility of bimolecular tautomerization was brought up recently by Rodgers [8,9]. We have made our own calculations to test this idea. Three H-bonded forms of 1:1 are shown in Fig. 3. From dimer  a new dimer 2b:3a can be derived,  leads to 2b:2b and  to 3a:3a [5]. Each tautomerization has its own transition state barrier as indicated under the picture. Results The Ar-matrix spectra are shown, together with the theoretical simulations, in Figs 1 and 2. For estimating the tautomer ratios, previous Methods Experimental details Cytosine sample from Sigma-Aldrich (solid sample, 99% purity), evaporation at 155 o C, matrix: Argon (99.9997%) and Krypton (99.998%). UV Spectrometer: Varian Cary 3E UV-VIS (1 nm resolution), IR spectrometer: Bruker FTS 55 (1 cm -1 resolution). For details of the matrix isolation setup see poster P-75. Computational details Calculation of the UV spectra was based on equation of motion coupled cluster theory (EOM-CC) including the effect of triple substitutions at EOM-CC3 level, using the program package CFOUR [10]. The basis set was aug-cc-pVDZ. The vibrational structure was obtained by the Linear Vibronic Coupling (LVC) method [11]. The IR spectra and the dimers were calculated at B3LYP/6-31++G(d,p) level using the PQS package [12]. Concerning the matrix isolation UV spectrum, we have found a measurement that went largely unobserved in the literature [6]. This early result is in basic agreement with our better quality spectrum. In Fig. 2, the left graph shows the simulation by the keto form only, while the right side simulation is based on a mixture. The exact details are obviously uncertain but one statement seems justified: The UV spectrum does not contradict the IR spectrum in that it may quite well indicate the presence of 2-3 tautomers. Considering the IR-spectrum in Fig. 1, in agreement with previous studies [4,5], it is obvious that there is a mixture of tautomers in the matrix: the keto and hydroxy forms are comparable in quantity, while 3a can just be identified. theoretical results were used. Transforming the data from ref. [2] to the present experimental temperature of 428 K, the ratios for 1 : 2a : 2b : 3a are 0.55 :0.44 : 1.00 : 0.60. These ratios conform to other literature results except for the imino form 3a which is generally estimated to be a few percent only. As explained further below, the latter ratio was thus reduced to 0.10 in the simulated spectra. From our point of view the most remarkable result is the following. The lowest TS leads to the hydroxy form 2b. The transition states which lead, partly or fully, to the imino form 3a lie significantly higher. Thus, this model may explain why the imino form appears in the gas state with much smaller abundance than expected. Isolated Biomolecules and Biomolecular Interactions Fritz-Haber-Institut der Max Planck-Gesellschaft and Tagungsstätte Harnack-Haus, 12-17 June 2010, Berlin, Germany.