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

The acid dissociation constant (pKa) of small, biological molecules is an important physical property used for investigating enzyme mechanisms and inhibitor design. For phosphorus-containing molecules, the 31P 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 31P NMR approach that uses inorganic phosphate buffer as an internal pH reference to determine the pKa 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 pKa 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 pKa values.

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► The first reported phosphate and imide pKa values of UDP-GlcNAc and UDP-S-GlcNAc.
► New role for the monosaccharide in the imide pKa of uridine-5′-phosphate compounds.
► UDP-S-GlcNAc and UDP-GlcNAc have the same phosphate pKa, unlike thioyl analogs.
► The 31P chemical shift of inorganic phosphate is a viable internal pH reference.
► Stability of the external 31P chemical shift reference is essential.