Boronic acid complexes with amino phenolic N,O-ligands and their use for non-covalent protein fluorescence labeling

Phenylboronic acid (PBA) forms neutral tetrahedral N,O-coordinated 6-membered cyclic complexes with stability constants reaching the values as large as 1.3 × 10⁴ M^-1 at pH 7.4 in water with amino phenolic compounds including 2-(2'-hydroxyphenyl)-1H-benzimidazole (HPBI) often used for protein probing and labeling. The crystal structures of isolated complexes demonstrate unusually high for boronate adducts degree of the tetrahedral character of the boron atom with short B-N bonds in agreement with their high solution stability. The complexation of PBA with HPBI, causes a strong enhancement of the fluorescence of the "enol" form of the ligand, increases the affinity of the dye to a protein (bovine serum albumin) and makes more pronounced the shift in emission maximum induced by the protein binding. Similar, but larger effects are observed with an amino HPBI derivative and with a stronger boronic acid benzoxaborole. Thus, the binding constant to the protein about 2 × 10⁴ M^-1 for free HPBI increases to 1.2 × 10⁶ M^-1 for the complex of 5-amino-HPBI with benzoxaborole making it suitable for an efficient non-covalent protein labeling or bioconjugation.