Slow-Onset, Potent Inhibition of Mandelate Racemase by 2-Formylphenylboronic Acid. An Unexpected Adduct Clasps the Catalytic Machinery

o-Carbonyl arylboronic acids such as 2-formylphenylboronic acid (2-FPBA) are employed in biocompatible conjugation reactions with the resulting iminoboronate adduct stabilized by an intramolecular N–B interaction. However, few studies have utilized these reagents active site-directed enzyme inhibitors. We show that 2-FPBA is a potent reversible, slow-onset inhibitor of mandelate racemase (MR), has served valuable paradigm for understanding enzyme-catalyzed abstraction α-proton from carbon substrate high pKa. Kinetic analysis progress curves slow onset inhibition wild-type MR using two-step kinetic mechanism gave Ki and Ki* values 5.1 ± 1.8 0.26 0.08 μM, respectively. Hence, binds affinity exceeds ∼3000-fold. K164R was inhibited 2-FPBA, while K166R not inhibited, indicating Lys 166 essential inhibition. Unexpectedly, mass spectrometric NaCNBH3-treated enzyme–inhibitor complex did yield evidence adduct. 11B nuclear magnetic resonance spectroscopy MR·2-FPBA indicated boron atom sp3-hybridized (δ 6.0), consistent dative bond formation. Surprisingly, X-ray crystallography revealed formation Nζ–B between cyclization to form benzoxaborole, rather than expected iminoboronate. Thus, when o-carbonyl modify proteins, structure product depends on protein architecture at site modification.