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

Abstract

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(H(alpha)CNH) 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.

A review with comprehensive data on experimental indirect scalar NMR spin–spin coupling constants across hydrogen bonds

“…[67] The last value of 1h J NH is consistent with an earlier paper of Parello et al on sugar osazones (Scheme 23), where 1h J NH = 5 Hz was measured. [71] If some of the 1 J NH couplings of Table 22 (for CD 2 Cl 2 and Freon, the value for 190 K has been averaged) are plotted against the temperature, a sigmoid curve is obtained (red circles, Fig.…”

“…The cofactor of pyridoxal-5 -phosphate (PLP) (31) was modeled with a Schiff base (32) (Scheme 22) to study first the intramolecular HB (discussed here for convenience and not in Section 2). [67] The 1 J NH coupling constant of 32 15 N-labeled (two tautomeric forms: enolimine OH 32a and ketoamine NH 32b) was studied in toluene, CD 2 Cl 2 , CDCl 3 and Freon at different temperatures (Table 22) in pure form or in the presence of acids.…”

“…In several places of this review the problem of tautomerism in relation with nh J XY couplings has appeared. [67,71,83,87,88] Usually, to study tautomerism by NMR the tautomerizable proton is replaced by a methyl group and the properties of the resulting isomers compared with those of the tautomer. Obviously, this is not possible here since the proton is essential for the HB to exist.…”

“…[108] This hypothesis was not challenged until very recently; [67] for instance, Kurkovskaya [109] reported a J NH = 1.55 Hz ( 15 N-labeled, CDCl 3 at 0 • C), which by applying Dudek's equation was assigned to 1.7% of tautomer 102a.…”

Syntheses and Structural Studies of Schiff Bases Involving Hydrogen Bonds

“…Within the framework of a general project investigating mechanisms of enzymatic reactions involving hydroxypyridines [11], we have already studied the new Schiff bases 5a-5f and 6a-6d shown in Scheme 2, obtained from 3-hydroxy-4-pyridinecarboxaldehyde (1), and 4-R-substituted anilines 2a-2f or N-aminoazoles 3a-3d, including their preparation and structural studies in solution ( 1 H-, 13 C-and 15 N-NMR spectroscopy) and in solid state ( 13 C-and 15 N-CPMAS NMR) [6,12]. As an extension of that work, we now present our results concerning derivatives 7a-7e, obtained from 1 and the corresponding N-aminobenzazoles 4a-4e.…”