Proton Magnetic Resonance Studies of Folate, Dihydrofolate, and Methotrexate

From the variation of ultraviolet absorbance with pH, the following pK values at 25° were determined: methotrexate, 5.71; folate, 8.38; and dihydrofolate, 9.54. The dependence of the proton magnetic resonance spectrum of these three folates on pH and concentration was measured. At infinite dilution, the folate spectra had little dependence on pH, but had a large dependence on pH at non-zero concentrations. The dependencies of chemical shift on pH and concentration were successfully fitted to dimerization and protonation equations with the following association constants which were determined at 23° in 2H2O at ionic strength of 0.1 to 0.5 m; folate, basic form 340 ± 40 mm, neutral form 19.4 ± 1.4 mm; dihydrofolate, neutral form, 38.5 ± 1.5 mm; methotrexate, neutral form 61.5 ± 1.5 mm, acidic form 11.0 ± 1.5 mm. A molecular structure for the neutral dimer of folate is proposed; the structure has two folates in a fully stretched out configuration with vertical ring stacking in a coplanar fashion, one molecule above the other in a head-to-tail arrangement. Similar structures for the other dimers are proposed. The dimers appear to be in rapid equilibrium with the monomers.