NMR and MS study of the formation of β-d-glucopyranosylamine uronic acid in aqueous solution

Abstract

The products of the reaction of d-glucuronic acid with various combinations of ammonia and volatile ammonium salts in water were studied by NMR and MS spectroscopy. For long reaction times (~24 h), the expected products β-d-glucopyranosylamine uronic acid and ammonium N-(β-d-glucopyranosyluronic acid)carbamate were obtained in good-to-high yield, whereas seven intermediate species were identified in samples taken at earlier reaction times. 1H–1H homonuclear and 1H–13C heteronuclear correlation experiments enabled a complete assignment of the 1H and 13C NMR spectra of the starting and final compounds, and a partial assignment of the peaks of intermediate species. Based on these results, a 1H NMR protocol for the quantification of the different compounds taking part in the reaction was developed, which was used to monitor the evolution of the composition of an early reaction sample redissolved in D2O. It was thus established that two of the observed intermediate species are actually the α anomer of the main products, whereas the others are precursors to the formation of α/β-d-glucopyranosylamine uronic acid and ammonium N-(α/β-d-glucopyranosyluronic acid)carbamate. The correct assignments for the 1H and 13C spectra of d-glucuronic acid in D2O are also reported.