The acid/base character of nucleobases affects phenomena such as self-association, interaction with metal ions, molecular recognition by proteins, and nucleic acid base-pairing. Therefore, the investigation of proton-transfer equilibria of natural and synthetic nucleos(t)ides is of great importance to obtain a deeper understanding of these phenomena. For this purpose, a set of ATP prototypes was investigated using 15N NMR spectroscopy, and the corresponding adenine bases were investigated by theoretical calculations. 15N NMR measurements provided not only acidity constants but also information on the protonation site(s) on the adenine ring and regarding the ratio of the singly protonated species in equilibrium. Substituents of different nature and position on the adenine ring did not change the preferred protonation site, which remained N1. However, for 2-thioether-ATP derivatives a mixed population of N1 and N7 singly protonated species was observed. Reduction of basicity of 0.4-1 pKa units relative to ATP was also observed for all evaluated ATP derivatives, except for 2-Cl-ATP, for which Ka was ca. 10,000-fold lower. To explain the substitution-dependent variations in the experimental pKa values of the ATP analogues, gas-phase proton affinities (PA), ΔΔGhyd, and pKa values of the corresponding adenine bases were calculated using quantum mechanical methods. The computed PA and ΔΔGhyd values successfully explained the experimental pKa values. A computational procedure for the prediction of accurate pKa values was developed using density functional theory and polarizable continuum model calculations. In this procedure, we developed a set of parameters for the polarizable continuum model that was fitted to reproduce experimental pKa values of nitrogen heterocycles. This method is proposed for the prediction of pKa values and protonation site(s) of purine analogues that have not been synthesized or analyzed.