Acid dissociation constants of uridine-5'-diphosphate compounds determined by 31phosphorus nuclear magnetic resonance spectroscopy and internal pH referencing.

The acid dissociation constant (pK(a)) of small, biological molecules is an important physical property used for investigating enzyme mechanisms and inhibitor design. For phosphorus-containing molecules, the (31)P nuclear magnetic resonance (NMR) chemical shift is sensitive to the local chemical environment, particularly to changes in the electronic state of the molecule. Taking advantage of this property, we present a (31)P NMR approach that uses inorganic phosphate buffer as an internal pH reference to determine the pK(a) values of the imide and second diphosphate of uridine-5'-diphosphate compounds, including the first reported values for UDP-GlcNAc and UDP-S-GlcNAc. New methods for using inorganic phosphate buffer as an internal pH reference, involving mathematical correction factors and careful control of the chemical shift reference sample, are illustrated. A comparison of the newly determined imide and diphosphate pK(a) values of UDP, UDP-GlcNAc, and UDP-S-GlcNAc with other nucleotide phosphate and thio-analogs reveals the significance of the monosaccharide and sulfur position on the pK(a) values.