Hydrophobicities of the Nucleic Acid Bases: Distribution Coefficients from Water to Cyclohexane Phoebe Shih 1 , Lee G. Pedersen 2,3 , Paul R. Gibbs 1 and Richard Wolfenden 1 * 1 Department of Biochemistry and Biophysics and 2 Department of Chemistry, University of North Carolina, Chapel Hill NC, 27599-7260, USA 3 Laboratory of Quantitative and Computational Biology, National Institute of Environmental Health Sciences, Research Triangle Park, NC, 27709, USA To establish an experimental scale of hydrophobicities for the nucleic acid bases, comparable with a scale developed earlier for amino acid side-chains, these bases and their parent compounds (purine and pyrimi- din-2-one) were converted to n-butylated and tetrahydrofurylated deriva- tives that are appreciably soluble in cyclohexane, a truly non-polar solvent that dissolves negligible water at saturation. Distribution measurements between neutral aqueous solution and cyclohexane, at varying solute concentrations, showed no evidence of self-association of the solute in either solvent, and the possibility of speci®c entrainment of water by solutes entering cyclohexane was ruled out by the results of experiments with tritiated water. In both the n-butyl and tetrahydrofuryl series, the bases span a range of 5.3 kcal mol 1 in their free energies of transfer from water to cyclohexane, and are arranged in the following rank, in order of decreasing hydrophobicity: purine > thymine > ade- nine > uracil > pyrimidin-2-one > hypoxanthine 5 cytosine 5 guanine. In both series of pyrimidin-2-ones, hydrophobicity decreases with introduc- tion of an amino substituent, but addition of an exocyclic keto group results in a modest enhancement of hydrophobicity; and free energies of transfer are relatively insensitive to the position of N-alkyl substitution. In both series of purines, hydrophobicity decreases with the introduction of exocyclic amino and keto groups, the keto group having the greater effect; and free energies of transfer vary substantially depending on the position of N-alkyl substitution. Several additional compounds were examined to test recent predictions based on SM5.4/A, a quantum mech- anical self-consistent-®eld solvation model; and that model was found to yield values in reasonable agreement with the experimental results. # 1998 Academic Press Keywords: nucleic acid bases; hydrophobicity; cyclohexane; partition coef®cients; free energy of transfer *Corresponding author Introduction The major nucleic acid bases participate in many kinds of interactions that involve structural com- plementarity, including base-pairing during tran- scription and translation of genetic information; enzyme-RNA recognition (particularly in class I aminoacyl-tRNA synthetases; Cusack, 1995); and binding of substrates, coenzymes and effectors by many enzymes. Non-covalent binding interactions of the nucleic acid bases, like those of other biologi- cal molecules, involve the stripping away of sol- vent water from regions of contact between the binding partners. Accordingly, the observed strength of their interactions with other molecules can be considered to include the cost of removing the interacting molecules (at least those parts that make contact with each other) from the solvent to which they were previously exposed (Wolfenden, 1983). In attempts to detect the presence of speci®c attractive or repulsive interactions between the Abbreviations used: THF, tetrahydrofuryl; Bu, butyl; Me, methyl; HPLC, high pressure liquid chromatography; A, adenine; C, cytosine; G, guanine; H, hypoxanthine; P, purine; T, thymine; U, uracil; Z, pyrimidin-2-one; chx, cyclohexane; chf, chloroform; ÁG tr , free energy of transfer; K dist , distribution coef®cient; r, correlation coef®cient; FTIR, Fourier transform infrared spectroscopy. Article No. mb981880 J. Mol. Biol. (1998) 280, 421±430 0022 ± 2836/98/280421±10 $30.00/0 # 1998 Academic Press