Boronate Affinity Chromatography

The use of boronate affinity chromatography for separation of nucleic acid components and carbohydrates was first reported by Weith and colleagues in 1970 ( 1 ). Since then, the specificity of boronate has been exploited for the separation of a wide variety of cis-diol-containing compounds, including catechols, nucleosides, nucleotides, nucleic acids, carbohydrates, glycoproteins, and enzymes ( 2 ) (see Note 1 ). The basic interaction for boronate chromatography is esterification between boronate ligands and cis-diols. The major structural requirement for boronate/cis-diol esterification is that the two hydroxyl groups of a diol should be on adjacent carbon atoms and in an approximately coplanar configuration, that is, a 1,2-cis-diol. Although interaction of boronate with 1,3-cis-diols and trident interactions with cis-inositol or triethanolamine can also occur, 1,2-cis-diols give the strongest boronate ester bonds ( 3 ). In aqueous solution, under basic conditions, boronate, which normally has a trigonal coplanar geometry, is hydroxylated, yielding a tetrahedral boronate anion, which can then form esters with cis-diols (Fig. 1 ). The resulting cyclic diester can be hydrolyzed under acidic conditions, reversing the reaction. The boronate diester bond strength has not been well studied and only a few dissociation constants for phenylboronic acid diesters have been reported. Those reported include adonitol, 2.2 � 10 -3 M; dulcitol, 1.1 � 10 -3 M (4); mannitol, 3.3 � 10 -3 M ( 5 ); and NADH, 5.9 � 10 -3 M ( 6 ). The dissociation constant of 4-(N-methyl)-carboxamido-benzeneboronic acid and D-fructose diester is 1.2 � 10 -4 M ( 7 ). These dissociation constants are relatively high compared to the constants of 10 -4 –10 -8 M observed in most affinity ligand/protein interactions.   Fig. 1.  The proposed mechanism of esterification between a phenylboronic acid and a cis-diol in aqueous solution.