Abstract
The vitamins, pyridoxine, pyridoxal, pyridoxamine, pyridoxal‐5′‐phosphate and pyridoxamine‐5′‐phosphate, have been studied in aqueous solution over a pH range of 2–12 by ^13^C nuclear magnetic resonance spectroscopy. Resonance assignments are made primarily by the spin–spin coupling constants of carbons with protons and with phosphorus. The proton–carbon coupling constants show a marked conformational dependence in the hemiacetal form of pyridoxal. Furthermore, the H‐6C‐5 coupling constant in the vitamins is much smaller than the corresponding constant in pyridine. This may be due either to an effect of the C‐5 substituent in vitamins or to a different electronic configuration of the zwitterionic hydroxypyridine ring. The addition of manganese to a solution of pyridoxal phosphate causes line broadenings consistent with the interaction of the metal ion with this vitamin at the formyl and phenolic oxygens. The chemical shifts of the aromatic carbons of pyridoxine have been calculated, as a function of pH, by summing shielding parameters which were estimated empirically from pyridine derivatives. The calculated shifts agree well with the experimental data for C‐3, C‐5 and C‐6, less well for C‐2, and poorly for C‐4. The deviation from additivity for C‐4 indicates a preferred orientation for the 4‐hydroxymethyl substituent caused by internal hydrogen bonding between the substituents at C‐3 and C‐4. Evidence is presented for the existence of the free aldehyde form of pyridoxal at alkaline pH. Aldimine complexes of pyridoxal and pyridoxal phosphate with amines and amino acids have also been studied. Characteristic chemical shift changes caused by both pyridinium and aldimine nitrogen deprotonations are seen. Additionally, the chemical shifts of carbons of the pyridine ring are dependent upon the structure of the imine, especially when the aldimine nitrogen is protonated. We conclude that this dependency is due to steric effects in an aldimine complex which is constrained by internal hydrogen bonding. We also discuss the merits of carbons 3 and 4 as possible sites of cofactor labeling for enzymatic studies.