Determination of dissociation constants of folic acid, methotrexate, and other photolabile pteridines by pressure-assisted capillary electrophoresis

Pressure-assisted CE (PACE) was applied to determine the previously inaccessible complete set of pK values for folic acid and eight related multiprotic compounds. PACE allowed the determination of all acidity macroconstants at low (<= 0.1 mM) concentration without interferences of selfassociation or photodegradation throughout the pH range. The accuracy of the constants was verified by NMR-pH, UV-pH, and potentiometric titrations and the data could be converted into physiological ionic strength. It was shown that even three overlapping pK values can be determined by CE with good precision (< 0.06) and accuracy if an appropriately low sample throughput is used. Experimental aspects of PACE for the quantitation of acid-base properties are analyzed. The site-specific basicity data obtained for folic acid and methotrexate (MTX) reveal that apparently slight constitutional differences between folic acid and MTX carry highly different proton-binding propensities at analogous moieties, especially at the pteridine N-1 locus, providing straightforward explanation for the distinctive binding to dihydrofolate reductase at the molecular level.