Boronic acids for affinity chromatography: spectral methods for determinations of ionization and diol-binding constants.

Arylboronic acids attached to solid matrices have proved useful for the diol-specific chromatography of biomolecules and affinity purification of enzymes by exchangeable-ligand chromatography. The latter use has been limited by the intrinsic ionization constant (pKa approximately 9) of the most common commercial products. The synthesis of several arylboronic acids with ionization constants near neutrality are described, and the application of a new general, spectral-difference method for determining acid ionization constants and formation constants with fructose is developed. In particular 4-(N-methyl) carboxamido-benzeneboronic acid was found to have a pKa of 7.86 and a formation constant with D-fructose of 8600. It was stable toward acid or base hydrolysis. We suggest that 4-carboxybenzeneboronic acid might be useful for preparing matrices for enzyme affinity chromatography.