Tautomeric Equilibrium of Uracil and Thymine in Model Protein-Nucleic Acid Contacts. Spectroscopic and Quantum Chemical Approach Svitlana P. Samijlenko, Yevgen P. Yurenko, Andriy V. Stepanyugin, and Dmytro M. Hovorun* Department of molecular and quantum biophysics, Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, Vul. Zabolotnoho150, KyiV, 03143, Ukraine ReceiVed: September 21, 2009; ReVised Manuscript ReceiVed: NoVember 14, 2009 This work deals with tautomeric transformations of uracil (Ura) and thymine (Thy) in their model complexes with the deprotonated carboxylic group. Essential changes in the UV spectra of the bases upon their interaction with NaAc, vanishing signals of both imino protons in 1 H NMR spectra, and a perceptible decrease in intensity of both IR bands, related to the stretching vibrations ν(CdO) of the carbonyl groups, imply involvement of enolic tautomers. Results of quantum chemical calculations of the double complexes of the Ura(Thy) tautomers with CH 3 COO - at the MP2/6-311++G(2df,pd)//B3LYP/ 6-311++G(d,p) level of theory proved to be incompatible with the spectral features: despite the fact that the complexes of the enolic tautomers are much closer in energy to the diketo ones as compared to isolated tautomers, the energy gap between them is such that in tautomeric equilibrium dominate diketo forms. Calculations of triple complexes of the type CH 3 COO - :Ura(Thy) tautomer:Na + , taking into account the effect of the Na + coordination with tautomers, show that three triple complexes formed by enolic tautomers appeared more stable than those formed by diketo ones. This makes the UV and 1 H NMR data understandable, but the high residual intensity of the ν(CdO) bands in the IR spectra remains unclear. At that ion, Na + itself was not able to disturb the tautomeric equilibrium in the coordination complexes of the type Ura(Thy) tautomer:Na + . To evaluate the DMSO effect, the CPCM solvation model was applied to triple complexes of the Ura tautomers. It appeared that in the solution there is coexistence between the diketo and enolic tautomers in a ratio of 53%:47%. This makes possible reconciliation of our experimental data. The biological significance of high-energy tautomers of nucleotide bases is discussed. I. Introduction Uracil (Ura) and thymine (Thy), differing only by a methyl group in Thy instead of a hydrogen atom in Ura at the ring position 5, are pyrimidine nucleotide bases occurring as canoni- cal ones against their complementary base adenine (Ade) in double helices of RNA and DNA, respectively. However, there are a few exceptions. In the case of phage TPBS, deoxyuridine (dU), a deoxynucleoside of Ura, is a normal constituent of DNA instead of thymidine (T). 1 Deoxyuridine was also identified among four canonical DNA nucleosides, isolated from the herring sperm hydrolysate. 2 Generally speaking, the presence of Ura in DNA is harmful for a cell. Deamination of cytosine (Cyt) to Ura, as well as 5-methylcytosine (m 5 Cyt) to Thy, is the most often occurring form of the DNA endogenous damage with mutagenic consequences, if the G:T (G: nucleotide of guanine, Gua) resulting mispairing remains unrepaired. 3,4 Such point mutations are frequent in human cancer cells (ref 6 in work 4). The Ura incorporation (under some conditions) in DNA against Ade during replication is not directly mutagenic but cytotoxic. 5 A propos, in itself, Ura is believed to be a “fascinating” molecule due to not only its biological significance but also its numerous therapeutic effects and applications in industrial chemistry and agriculture. 6 Tautomerism is one of the most intriguing features of the nucleic acid bases. Dozens of publications were devoted to this subject. Up to now, tautomerism of the bases was perceived only in the context of DNA spontaneous mutations (see works 7-9 and references therein). Tautomerization of nucleotide bases in vacuum and a water environment was a subject of numerous publications (e.g., see works 10-15). The problem of modeling xanthine tautomers by corresponding methyl derivatives has been considered in the work. 16 The N9 T N7 tautomerism of purine bases (purine, adenine, hypoxanthine, and mercaptopurine) was investigated in methanol and N1N- dimethylformamide solutions by low-temperature 1 H and 13 C NMR spectroscopy. 17 The authors came across the problem of the tautomerism of the nucleotide bases in the course of experimental investigations of elementary processes of protein-nucleic acid recognition Via amino acid carboxylic groups in model systems. 18-22 Highly specific interactions of proteins with nucleic acids, named “recognition” due to their extreme selectivity, is a distinctive attribute of living organisms. Protein-nucleic acid recognition is the very essence of such significant biological processes as storage, replication, transcription, and translation of genetic information, 23 as well as repair of the DNA damage, both exo- and endogenic. According to von Hippel, 24 a search for a physicochemical basis of protein-nucleic acid recognition is a key line of investigations in molecular biophysics and molecular biology. Interactions between the biopolymers proved to depend on the size and relief of their contact surface and, to a large extent, on the affinity between them, determined mainly by hydrogen bonds. 25 At that, certain structural motives of the * To whom the correspondence should be addressed. E-mail: dhovorun@imbg.org.ua. J. Phys. Chem. B 2010, 114, 1454–1461 1454 10.1021/jp909099a  2010 American Chemical Society Published on Web 01/04/2010