Determination of pK_a values for deprotonable nucleobases in short model oligonucleotides

The deprotonation of ionizable nucleobases centrally placed in short model oligonucleotides was examined under different physical conditions, using UV absorption spectroscopy. The oligonucleotide sequences were designed so that only the central base would be ionized over the pH range examined. pK_a values of 9.90 ± 0.01 and 9.34 ± 0.04 were determined for the guanine group in the oligomer d-ACAGCAC and 2′-deoxyguanosine, respectively, both at 25 °C and 0.1 M NaCl. Lengthening the oligonucleotide up to the tridecamer stage further increases the pK_a of the central guanine moiety. Electrolyte concentration, temperature, and mixed water-ethanol solvents affect the acidity of the central base. Changes in the sequence surrounding the central guanine can also have a significant effect, especially in the case of strongly stacking sequences. The pK_a values were also determined for the hepta(2′-O-methyl)ribonucleotide and the heptamer PNA of identical sequence, as well as for oligodeoxyribonucleotides with different deprotonable bases, viz. thymine, uracil, or hypoxanthine, in the central position. The results are interpreted in terms of the electric-field effect exerted on the departing proton by the negative electric charges located on the internucleotide phosphate groups, and calculations show this effect to approximately explain the magnitude of the pK_a difference observed between the deoxyriboheptanucleotide and its electroneutral PNA analogue.