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

Abstract

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 (<or=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.

Toward resolving the catalytic mechanism of dihydrofolate reductase using neutron and ultrahigh-resolution X-ray crystallography

“…This indicates the presence of a deuterium atom bound to the N3 atom of folate; thus, the N3 atom of folate is protonated and Asp27 remains ionized in the folate-NADP + complex. Given the experimentally determined pK a values of N3 in folate and DHF of ∼8 and 10.8, respectively (21,34,35), we predict that N3 will be protonated in a DHF ternary complex. The distance between the N3 deuterium atom and the Oδ2 of Asp27 is 1.7 Å, and the angle between N3, its bound D atom, and Oδ2 is 172.5°, indicating a strong hydrogen bond between folate and the catalytic aspartate.…”

Interaction of folic acid and some matrix metalloproteinase (MMP) inhibitor folate-γ-hydroxamate derivatives with Zn(II) and human serum albumin

“…The three pK of the folate--carboxylic acids show overlapping proton dissociation steps, but the order of -COOH, -COOH and Pte moiety is the most probable based on the findings related to the model compounds above and literature data [46,47]. The deprotonation steps are much better separated in the case of the folate-γ-hydroxamic acids and the dissociation sequence of the -COOH, the Pte and the hydroxamic acid moieties is the most likely.…”

“…Namely, the carboxylic and hydroxamic acid groups exhibit higher pK in the presence of DMSO, since the pK of anionic bases are increased as the dielectric permittivity of the solvent is decreased [48]. The pK of the pteroyl moiety, however, show merely a minor drift in the various media due to the strong hydrogen binding interaction between the enolate and the solvent molecules [47,49].…”

Statistical data processing of mobility curves of univalent weak bases

“…They were developed to circumvent the above-mentioned drawbacks of the linearized forms. Most of them are based on the use of the Boltzman sigmoidal decay function [5,11,[16][17][18] and, especially on its special form called exponential form of electrophoretic Henderson-Hasselbach equation [7,[19][20][21]. For weak bases it is:…”