NMR Studies of Solvent-Assisted Proton Transfer in a Biologically Relevant Schiff Base: Toward a Distinction of Geometric and Equilibrium H-Bond Isotope Effects

The tautomeric equilibrium in a Schiff base, N-(3,5-dibromosalicylidene)-methylamine 1, a model
for the hydrogen bonded structure of the cofactor pyridoxal-5‘-phosphate PLP which is located in the active
site of the enzyme, was measured by means of 1H and 15N NMR and deuterium isotope effects on 15N
chemical shifts at variable temperature and in different organic solvents. The position of the equilibrium
was estimated using the one-bond 1J(OHN) and vicinal 3J(HαCNH) scalar coupling constants. Additionally,
DFT calculations of a series of Schiff bases, N-(R1-salicylidene)-alkyl(R2)amines, were performed to obtain
the hydrogen bond geometries. The latter made it possible to investigate a broad range of equilibrium
positions. The increase of the polarity of the aprotic solvent shifts the proton in the intramolecular OHN
hydrogen bond closer to the nitrogen. The addition of methanol and of hexafluoro-2-propanol to 1 in aprotic
solvents models the PLP−water interaction in the enzymatic active site. The alcohols, which vary in acidity
and change the polarity around the hydrogen bond, also stabilize the equilibrium, so that the proton is
shifted to the nitrogen.