Ionisation, self association, and proton exchange studies of nicotinamide in aqueous solution using nuclear magnetic resonance spectroscopy

Abstract

From an analysis of the single resonance ¹³C n.m.r. spectrum of nicotinamide at 25·2 MHz at high (pH > 6·0) and low (pH < 1·0) pH values the C–H spin coupling constants have been obtained. The C–H coupling constants for the charged nicotinamide (pH < 1·0) agree well with those which could be measured in NAD⁺. The unusually large C–H coupling constants (ca. 12 Hz) observed between C(2) and H(6) across the nitrogen atom in the uncharged nicotinamide have been observed in the pteridine ring of folic acid and the adenine ring of NAD⁺: in both cases aromatic carbon atoms without directly bonded protons could be assigned on the basis of such coupling constants observed in their single resonance ¹³C spectra. The ¹³C chemical shift differences observed on protonation of the nicotinamide ring were measured and compared with values calculated on the basis of the differences in the calculated total electron densities at the ring carbons: excellent agreement was found for C(3)–C(5) but C(2) and C(6) which are near the site of protonation showed no agreement probably due to intramolecular electric field effect contributions to their shielding. Carbon-13 relaxation time measurements on nicotinamide and pyridine suggest that nicotinamide is associated at high pH (ca. pH 6·0) and concentration (ca. 2M solutions). At low pH values (< 1·0) less association is observed. ¹H and ¹³C chemical shift measurements on nicotinamide aqueous solutions at different concentrations also support this conclusion. Furthermore the largest shift changes on dilution were centred on the CONH₂ group of nicotinamide. The ¹H chemical shift changes on dilution were in the opposite direction to those expected from association involving parallel stacking of the nicotinamide rings. Thus there is strong evidence that the association results from interamide interactions. Interactions involving the CONH₂ group are not thought to be of importance in the intramolecular stacking found for the nicotinamide and adenine rings of NAD⁺ at neutral pH values. From measuring the concentrations and pH dependence of the line widths of the CONH₂ protons in H₂O solution it was possible to estimate the separate exchange rates between the cis- and trans-NH protons with water protons. Similar measurements could be made on NAD⁺ where it was found that at pH values < 5·5 the exchange rates are slower than found for nicotinamide under the same conditions.