Boron-11 nuclear magnetic resonance studies of the structure of the transition-state analog phenylboronic acid bound to chymotrypsin

Adebodun, Foluso; Jordan, Frank

doi:10.1021/ja00209a060

Cited by 20 publications

(12 citation statements)

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“…To obtain information about the hybridization state of the boron of PBA when bound at the active site of MR in the solution state, we utilized 11 B NMR spectroscopy. This approach has been employed to study the interaction of boronic acid-bearing inhibitors with β-lactamase, γ-glutamyl transpeptidase, and a variety of proteases. ,− In the absence of MR, the boron of PBA exhibited a signal with a chemical shift of 28.2 ppm in the assay buffer (Figure A), which is in accord with previous studies , and arises from the neutral trigonal boronic acid species [Ph−B(OH) 2 ]. Upon addition of MR, a new peak was observed at 0.97 ppm (Figure B – D), which was also the sole peak present when the enzyme and PBA were at equal concentrations (Figure E), and suggested that there was slow exchange between the MR-bound PBA and free PBA .…”

Section: Resultssupporting

confidence: 82%

Potent Inhibition of Mandelate Racemase by Boronic Acids: Boron as a Mimic of a Carbon Acid Center

et al. 2020

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Boronic acids have been successfully employed as inhibitors of hydrolytic enzymes. Typically, an enzymatic nucleophile catalyzing hydrolysis adds to the electrophilic boron atom forming a tetrahedral species that mimics the intermediate(s)/transition state(s) for the hydrolysis reaction. We show that para-substituted phenylboronic acids (PBAs) are potent competitive inhibitors of mandelate racemase (MR), an enzyme that catalyzes a 1,1-proton transfer rather than a hydrolysis reaction. The K i value for PBA was 1.8 ± 0.1 μM, and p-Cl-PBA exhibited the most potent inhibition (K i = 81 ± 4 nM), exceeding the binding affinity of the substrate by ∼4 orders of magnitude. Isothermal titration calorimetric studies with the wild-type, K166M, and H297N MR variants indicated that, of the two Brønsted acid−base catalysts Lys 166 and His 297, the former made the greater contribution to inhibitor binding. The X-ray crystal structure of the MR•PBA complex revealed the presence of multiple H-bonds between the boronic acid hydroxyl groups and the side chains of active site residues, as well as formation of a His 297 N ε2 −B dative bond. The dramatic upfield change in chemical shift of 27.2 ppm in the solution-phase 11 B nuclear magnetic resonance spectrum accompanying binding of PBA by MR was consistent with an sp 3 -hybridized boron, which was also supported by density-functional theory calculations. These unprecedented findings suggest that, beyond substituting boron at carbon centers participating in hydrolysis reactions, substitution of boron at the acidic carbon center of a substrate furnishes a new approach for generating inhibitors of enzymes catalyzing the deprotonation of carbon acid substrates.

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Section: Resultssupporting

confidence: 82%

Potent Inhibition of Mandelate Racemase by Boronic Acids: Boron as a Mimic of a Carbon Acid Center

et al. 2020

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“…Reactions of chymotrypsin with boric acid, phenylboronic acid, and 2-phenylethaneboronic acid gave complexes displaying 1 H NMR signals at 15.9, 16.3, and 17.2 ppm, well upfield from the signals in free chymotrypsin. , Deeply penetrating 11 B-, 15 N-, and 1 H NMR analysis of peptide boronate complexes of chymotrypsin and subtilisin as functions of pH and peptide structure revealed information about hydrogen bonding and ligation of boron. − In general, the complexes were found to be tetrahedral about boron, all complexes involved bonding of serine to boron, the most downfield histidinium proton was N δ1 –H bonded to aspartate-β-COO – , and the basicity of the active site histidine was elevated. Ligation of histidine to boron could be observed with a less specific boronic acid. ,,, The most downfield proton appeared at 17.2–17.4 ppm, depending on the enzyme and inhibitor, upfield from the signal for chymotrypsin at low pH.…”

Section: Discussionmentioning

confidence: 99%

Serine Protease Catalysis: A Computational Study of Tetrahedral Intermediates and Inhibitory Adducts

2016

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Peptide boronic acids and peptidyl trifluoromethyl ketones (TFKs) inhibit serine proteases by forming monoanionic, tetrahedral adducts to serine in the active sites. Investigators regard these adducts as analogs of monoanionic, tetrahedral intermediates. Density functional theory (DFT) calculations and fractional charge analysis show that tetrahedral adducts of model peptidyl TFKs are structurally and electrostatically very similar to corresponding tetrahedral intermediates. In contrast, the DFT calculations show the structures and electrostatic properties of analogous peptide boronate adducts to be significantly different. The peptide boronates display highly electrostatically positive boron, with correspondingly negative ligands in the tetrahedra. In addition, the computed boron-oxygen and boron-carbon bond lengths in peptide boronates (which are identical or very similar to the corresponding bonds in a peptide boronate adduct of α-lytic protease determined by X-ray crystallography at subangstrom resolution) are significantly longer than the corresponding bond lengths in model tetrahedral intermediates. Since protease-peptidyl TFKs incorporate low-barrier hydrogen bonds (LBHBs) between an active site histidine and aspartate, while the protease-peptide boronates do not, these data complement the spectroscopic and chemical evidence for the participation of LBHBs in catalysis by serine proteases. Moreover, while the potency of these classes of inhibitors can be correlated to the structures of the peptide moieties, the present results indicate that the strength of their bonds to serine contribute significantly to their inhibitory properties.

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“…There was some interest in using boron containing substances as analogues for naturally occurring molecules, applying 11 B NMR to study their binding properties to biological macromolecules. [2][3][4] However, the majority of publications in this field and devoted to investigations related to molecules used in an experimental cancer treatment known as boron neutron capture therapy (BNCT), and these will be the main subject of the present review.…”

Section: Introductionmentioning

confidence: 99%

Biomedical applications of10B and11B NMR

Bendel

2005

NMR Biomed.

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This review focuses mainly on the detection and investigation of molecules used for boron neutron capture therapy (BNCT) by 10 B and 11 B NMR. In this binary radiation treatment, boron-containing molecules (also called 'BNCT agents') enriched in the 10 B isotope, are targeted to the tumor, and irradiated with thermal or epithermal neutrons. Capture of these neutrons by 10 B nuclei generates cell-damaging radiation, confined to single cell dimensions. NMR research efforts have primarily been applied in two directions: first, to investigate the metabolism and pharmaco-kinetics of BNCT agents invivo, and second, to use localized NMR spectroscopy and/or MRI for non-invasive mapping of the administered molecules in treated animals or patients. While the first goal can be pursued using 11 B NMR for natural-abundance samples (80% 11 B / 20% 10 B), molecules used in the actual treatment are >95% enriched in 10 B, and must therefore be detected by 10 B NMR. Both 10 B (spin 3) and 11 B (spin 3/2) are quadrupolar nuclei, and their typical relaxation times, in common BNCT agents in biological environments, are rather short. This poses some technical challenges, particularly for MRI, which will be reviewed, along with possible solutions. The first attempts at 11 B NMR and MRI detection of BNCT agents in biological tissue were conducted over a decade ago. Since then, results from 11 B MRI in laboratory animals and in humans have been reported, and 11 B NMR spectroscopy provided interesting and unique information about the metabolism of some BNCT agents in cultured cells. 10 B NMR was applied either 'indirectly' (in double-resonance experiments involving coupled protons), but also by direct 10 B MRI in mice. However, no results involving the NMR detection of 10 B-enriched compounds in treated patients have been reported yet.

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Boron-11 nuclear magnetic resonance studies of the structure of the transition-state analog phenylboronic acid bound to chymotrypsin

Cited by 20 publications

References 0 publications

Potent Inhibition of Mandelate Racemase by Boronic Acids: Boron as a Mimic of a Carbon Acid Center

Potent Inhibition of Mandelate Racemase by Boronic Acids: Boron as a Mimic of a Carbon Acid Center

Serine Protease Catalysis: A Computational Study of Tetrahedral Intermediates and Inhibitory Adducts

Biomedical applications of10B and11B NMR

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