Spectrophotometric titrations of pyridoxal Schiff's bases in methanol using glass electrode for control of acidity

Abstract

Schiff's bases prepared by mixing pyridoxal, pyridoxal 5′-phosphate (PLP), or salicylaldehyde (SA) with amino compounds, having a different structure at carbon-1, were spectrophotometrically titrated in methanol by using glass electrode and operational pH ∗ scale for control of the acidity. The titrations were carried out with methanolic HCl from alkaline solutions, obtained by adding sodium methoxide. The solutions were titrated so rapidly that the acetal forms of the Schiff's bases did not appear in considerable amounts. When the Schiff's bases were prepared from 2-amino acids, the p K ∗ values of phenolic hydroxyls were higher than those of the Schiff's bases made from 3- or 4-amino acids or from alkylamines. Carboxylate of the 2-amino acid also increased the relative amount of the ketoenamine tautomer at pH ∗ 6–10, as compared to the other amino compounds studied. The effect of carboxylate on the tautomerism was interpreted to be due to internal hydrogen bonding between carboxylate anion, azomethine nitrogen and phenolic hydroxyl. In addition, phosphate of pyridoxal 5′-phosphate was deduced to force carboxylate, by ionic repulsion, to a favorable conformation for the internal hydrogen bonding. The protonation of carboxylate of 2-amino acid imines (p K ∗ about 5) brought about a great change toward enolimine tautomer due to a rupture of the internal hydrogen bonding. Below p K ∗ of about 3, pyridine nitrogen protonized and concomitantly the tautomeric equilibrium reshifted on ketoenamine. With salicylaldehyde imines an immonium ion appeared at about the same pH ∗. The present results are applicable to the predicting of spectral changes in hydrophobic active sites of pyridoxal 5′-phosphate enzymes.