Macroscopic pKa Calculations for Fluorescein and Its Derivatives

This study describes the calculation of the microscopic dissociation and tautomerization constants of fluorescein and its derivatives, 2',7'-dichlorofluorescein (DCF) and 2',7'-difluorofluorescein (DFF), in an aqueous environment. In vacuo free energies were obtained using complete basis set (CBS) and DFT-based methods, while free energies of solvation were calculated with the CPCM implicit solvation protocol using the UAHF, UAKS, and Pauling radii sets. Our results indicate that the different vacuum protocols give free energy changes upon dissociation within 1 kcal/mol of each other for a given molecule. Therefore, we suggest that the computationally less intensive PBE1PBE/6-311+G(2d,2p)//PBE1PBE/6-31+G(d) model chemistry may reasonably be used in pKa calculations of larger molecules. The calculations also provided a rigorous test of the implicit solvation models. Relative calculations of dissociation constants gave results in good agreement with experiment; absolute values deviated from experimental data by 1-3 pKa units. Consistently better results were obtained with the Pauling radii set. The influence of geometry relaxation on going from vacuum to solvent is negligible for pKa2 and larger for pKa1 but still smaller than the variation due to the radii set. Calculation of tautomerization constants gave more variable results, with none of the solvation methods able to reproduce experimental values consistently, although certain individual constants were correctly calculated.