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

Abstract

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

Capturing the free energy of transition state stabilization: insights from the inhibition of mandelate racemase

“…Since the ε-NH 2 group of Lys 166 did not form an N–B interaction with the boron atom of PBA ( vide supra ), we anticipated that Lys 166 might form a Schiff base with the 2-formyl group that would subsequently be stabilized by direct coordination of the lone pair of electrons on the imine nitrogen to the boron atom to form an iminoboronate [107,110]. In accord with these expectations, we discovered that 2-formyl-PBA is a slow-onset inhibitor of MR, exhibiting K i and K i * values of 5.1 µM and 0.26 µM, respectively [105], making it one of the most potent inhibitors of MR identified to-date that does not have any additional substituents on the phenyl ring ( K m / K i * ≈ 3000, figure 3). Furthermore, substitution of Lys 166 by Arg obviated inhibition, confirming that the ε-NH 2 group of Lys was essential for inhibition.…”

“…As shown in panel ( c ), the boronic acid hydroxyl groups of PBA form multiple H-bonds with the side chains of active site residues, in addition to the His 297 N ε 2 –B dative bond. Similarly, the potent inhibition exhibited by 2-formyl-PBA arises from formation of multiple H-bonds between the benzoxaborole adduct and the side chains of active site residues, as well as the Lys 166 N ζ –B dative bond (panel ( d ), PDB ID: 7MQX [105]). In all panels, the ligand and active site residues are shown in stick representation, and the Mg 2+ is represented as a sphere.…”

Synthesis of 5′-Thymidine-Conjugated Formylphenylboronic Acids as Potential Lysine Targeting Iminoboronate Reversible Covalent Enzyme Probes

“…5′-O-tert-Butyldimethylsilyl-3′-O,3-dibenzoylthymidine (21). 38 To a solution of 5′-O-tert-butyldimethylsilylthymidine (20, 5.33 g, 15.0 mmol), 4-dimethylaminopyridine (370 mg, 3.0 mmol), and triethylamine (7.7 mL, 55.2 mmol) in anhydrous dichloromethane (80 mL) at 0 °C was added benzoyl chloride (5.33 mL, 45.8 mmol) over 10 min.…”

“…Recently, Bearne and colleagues identified the formation of a benzoxaborole adduct (Scheme 1B) when 2-formylphenylboronic acid complexed with an active site lysine residue in mandelate racemase, demonstrating that alternative reaction pathways are possible within enzymes. 21 In concert with the chemistry of formylphenylboronic acids, boron-containing drugs have emerged as relatively new clinical agents, sampling new chemical space, often with the boron atom invoking a critical interaction with the drug target. 22 This underpins the relevance of boron-containing molecules to intervene in a biological setting.…”

“…There have been relatively few examples of iminoboronate formation within enzymes, and the literature is limited to targeting either a noncatalytic or a catalytic lysine residue in protein kinases. Recently, Bearne and colleagues identified the formation of a benzoxaborole adduct (Scheme B) when 2-formylphenylboronic acid complexed with an active site lysine residue in mandelate racemase, demonstrating that alternative reaction pathways are possible within enzymes . In concert with the chemistry of formylphenylboronic acids, boron-containing drugs have emerged as relatively new clinical agents, sampling new chemical space, often with the boron atom invoking a critical interaction with the drug target .…”