Structural basis of metallo-β-lactamase, serine-β-lactamase and penicillin-binding protein inhibition by cyclic boronates

β-Lactamases enable resistance to almost all β-lactam antibiotics. Pioneering work revealed that acyclic boronic acids can act as ‘transition state analogue' inhibitors of nucleophilic serine enzymes, including serine-β-lactamases. Here we report biochemical and biophysical analyses revealing that cyclic boronates potently inhibit both nucleophilic serine and zinc-dependent β-lactamases by a mechanism involving mimicking of the common tetrahedral intermediate. Cyclic boronates also potently inhibit the non-essential penicillin-binding protein PBP 5 by the same mechanism of action. The results open the way for development of dual action inhibitors effective against both serine- and metallo-β-lactamases, and which could also have antimicrobial activity through inhibition of PBPs.


Supplementary Synthesis
Synthetic Routes

General method A: Trimethylsilyl (TMS) group deprotection and amide coupling
The desired TMS protected amine (1.2 eq) was dissolved in THF (20 mL); to this solution was added anhydrous MeOH (2 mL). The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was concentrated in vacuo to afford a pale brown oil which was used in the next step without further purification.
The desired carboxylic acid (1.0 eq) and HATU (1.1 eq) were dissolved in CH 2 Cl 2 (DCM) (30 mL). The reaction mixture was cooled to 0 °C, then Et 3 N (1.5 eq) was added dropwise. The reaction mixture was stirred at 0 °C for 30 mins and then at room temperature for 1 h. The solution was then cooled to −20 °C and the desired amine (1.2-2.0 eq) added dropwise. The reaction mixture was allowed to warm to room temperature overnight. After this time the reaction was quenched with water (50 mL). The aqueous layer was extracted with EtOAc (3 × 50 mL). The combined organics were dried (MgSO 4 ) and concentrated in vacuo to give the desired amide, which was purified using flash column chromatography eluting with Hexane/EtOAc to afford the coupled products. and brine (20 mL), dried (MgSO 4 ) and concentrated in vacuo to afford the crude product.
The crude product was purified by preparative HPLC to afford the desired product as an oil.

General method C: Global deprotection to give cyclic boronate inhibitors.
Boron tribromide (1M in DCM, 7.0 eq) was added drop-wise to a solution of the boronic acid derivative (1.0 eq) in DCM (10 mL) at −78 °C. The reaction mixture was stirred at −78 °C for 1 h before warming to room temperature and quenched with water (6 mL).
The DCM layer was evaporated in situ. Water (75 mL) was then added and the mixture was extracted with diethyl ether (3 × 50 mL). The aqueous layer was concentrated to ~5 mL and the pH adjusted to ~pH1 with 2M HCl (aq). The product was purified by biotage chromatography on a C 18 reverse phase cartridge eluting with 2:98 Isopropanol-water to afford the desired product as a colourless solid.
The reaction mixture was then concentrated in vacuo. The residue was partitioned between EtOAc (10 mL) and water (10 mL