Spectroscopic characterization of a functionalized NAD + analogue and its polyethyleneimine-bound derivative

Abstract

The 1H and 13C NMR spectra of deuterium oxide solutions of two NAD + derivatives, i.e. nicotinamide-6-(2-hydroxy-3-carboxypropylamino)purine dinucleotide and 4(NAD +-N 6)-3-hydroxybutyryl-polyethylememine, have been examined over a range of pH, as well as the fluorescence spectra of their reduced forms. The results have been compared with those obtained with NAD +. Small shifts have been observed in both the proton and carbon magnetic resonance spectra in passing from low to high pH. This behavior, as well as the fluorescence studies, indicate that, analogously to NAD +, the two NAD + derivatives exist in solution as a pH dependent equilibrium of folded and unfolded forms, with the folded form prevailing at basic pH. As consequence of the folding, an intramolecular interaction takes place between the adenine and nicotinamide rings. The variation of proton and carbon resonances with pH changes has been utilized to evaluate the strength of this intramolecular interaction.