A Tetrahedral Boronic Acid Diester Formed by an Unnatural Amino Acid in the Ligand Pocket of an Engineered Lipocalin

Abstract

Boronic acids have long been known to form cyclic diesters with cis‐diol compounds, including many carbohydrates. This phenomenon was previously exploited to create an artificial lectin by incorporating p‐borono‐l‐phenylalanine (Bpa) into the ligand pocket of an engineered lipocalin, resulting in a so‐called Borocalin. Here we describe the X‐ray analysis of its covalent complex with 4‐nitrocatechol as a high‐affinity model ligand. As expected, the crystal structure reveals the formation of a cyclic diester between the biosynthetic boronate side chain and the two ortho‐hydroxy substituents of the benzene ring. Interestingly, the boron also has a hydroxide ion associated, despite an only moderately basic pH 8.5 in the crystallization buffer. The complex is stabilized by a polar contact to the side chain of Asn134 within the ligand pocket, thus validating the functional design of the Borocalin as an artificial sugar‐binding protein. Our structural analysis demonstrates how a boronate can form a thermodynamically stable diester with a vicinal diol in a tetrahedral configuration in aqueous solution near physiological pH. Moreover, our data provide a basis for the further engineering of the Borocalin with the goal of specific recognition of biologically relevant glycans.