NEW LIGANDS FOR BORONATE AFFINITY-CHROMATOGRAPHY - SYNTHESIS AND PROPERTIES

In order for a boronate ligand to be useful in affinity chromatography for the purification of biomolecules, it must be able to form a stable complex in an environment (pH) in which the affinity molecule is stable. A major limitation of the widely used ligand, 3-aminophenylboronate, is its high ionization constant (pK(a) 8.75). To make this complex under more favorable pH conditions, different methods have been explored here in order to introduce an electron-withdrawing (nitro) group in the phenyl ring. Reagents and procedures for the preparation of ortho-, meta- and para-nitro derivatives of succinamidophenylboronic acid using nitronium trifluoromethanesulfonate are described. Preferential substitution of the nitro functionality into the ortho position of the boronic acid is exploited by selective use of acetic anhydride for the reaction medium. This method yields mostly an ortho-nitro derivative (pK(a) 7.4) under selected reaction conditions. The ionization and solute-ligand interaction of several phenylboronates are studied in solution by using B-11 NMR and spectrophotometric methods. The results indicate the presence of specific chemical shifts for the neutral (delta 30), the boronate anions (delta 3), and the cis-diol-complexed boronate species (delta 7.5). In the presence of a cis-diol derivative, the complex formation is favored over anionization of neutral species. Moreover, the complex is formed approximately one pH unit below the ionization constant of the ligand and is stable, i.e. fails to break down in boronate anion, even when the solution pH is raised appreciably. Two boronate affinity column matrices were examined for their binding capacity and apparent dissociation constant. The results clearly indicate that the formation and also the breakdown of the complex are greatly enhanced because of the presence of the electron-withdrawing group in the boronate ligand. The results further demonstrate that small structural differences in affinity molecules have significant differences on their binding capacities. A comparison of binding between alkyl-cis-diols and aryl-cis-diols to different boronate matrices indicates that the aryl affinity molecules not only form a complex but do so very effectively. The significance of this work lies in the demonstration that the best environment for the ligand-solute interaction can be established by carrying out studies in solution, without prior immobilization of the ligand. The results derived from in-solution studies and those from the affinity columns are in very good agreement. The new nitrophenylboronate matrix offers enhanced binding of most affinity molecules over those examined with the phenylboronate matrix. In addition, the new matrix offers chromatographic separations of alkali-unstable biomolecules.