Acid-base chemistry of vitamin B6 compounds in methanol

The enzymatic reactions involving pyridoxal 5'-phosphate (PLP) can be simulated in solutions; thus, this system forms a favorable model for understanding the requirements of the enzymatic catalysis. We have studied in methanol protonic equilibria of the imines formed between PLP or salicylaldehyde (SA) and various amino acids, using UV and NMR spectroscopy. A glass electrode and an operational pH* scale were used to control acidity. The first protonation of the phosphate of PLP imines can be detected by UV spectroscopy with pK* at 10.8, proved by [31P]-NMR. The second protonation of phosphate (pK* at 4.8) is accompanied by increased hydrolysis of the imines. The imines of aspartate deviate from the imines of nondicarboxylic amino acids indicating that the beta-carboxyl of aspartate is internally hydrogen-bonded. PLP-2-aminobutanol Schiff base does not show with [1H]-NMR at pH* 7 separate peaks for ketoenamine-enolimine tautomers even at -90 degrees C, SA-phenylalanine shows an unidentified absorption at 350-380 nm. This was tentatively assigned a trans structure.