Following the Reactions of Mechanism-Based Inhibitors with β-Lactamase by Raman Crystallography

Citations Over TimeTop 19% of 2003 papers

Abstract

The reactions between three clinically relevant inhibitors, tazobactam, sulbactam, and clavulanic acid, and SHV beta-lactamase (EC 3.5.2.6) have been followed in single crystals using a Raman microscope. The data are far superior to those obtained for the enzyme in aqueous solution and allow us to identify species on the reaction pathway and to measure the rates of the accumulation and decay of these species. A key intermediate on the reaction pathway is an acyl enzyme formed between Ser70 and the lactam ring's C=O group. By using the E166A deacylation deficient variant of the enzyme, we were able to focus on the process of acyl enzyme formation. The Raman data show that all three inhibitors form an enamine-type acyl enzyme reaching maximal populations at 10, 22, and 29 min for sulbactam, clavulanic acid, and tazobactam, respectively. The enamine intermediate exhibits a characteristic and relatively intense band near 1595 cm(-1) due to a stretching motion of the O=C-C=C-NH moiety that shifts to lower frequency upon NH ND exchange. This feature was used to follow the kinetics of enamine buildup and decay in the crystal. Quantum mechanical calculations support the assignment of the 1595 cm(-1) band, as well as several other bands, to a trans-enamine species. The Raman data also demonstrate that the lactam ring opens prior to enamine formation since the lactam ring carbonyl (C=O) peak disappears prior to the appearance of the enamine 1595 cm(-1) band. Tazobactam appears to form approximately twice as much enamine intermediate as sulbactam and clavulanic acid, which correlates with its superior performance in the clinic, a finding that may bear on future drug design.

Related Papers

• → Synthesis and Shuttling Behavior of [2]Rotaxanes with a Pyrrole Moiety(2016)26 cited
• → The Reaction of Enamine with Amide·Phosphorylchloride Complex and the Reduction of Enamine(1973)1 cited
• → Synthesis of Pyrrolo[2,3‐c]2,7‐naphthyridine Derivatives by Cascade Heterocyclization Reaction of 2‐Amino‐4‐cyanomethyl‐6‐dialkylamino‐3,5‐pyridinedicarbonitriles.(2004)
• Design, synthesis and evaluation of novel hydroxyethylamine derivatives with nitrogen heterocyclic moiety at N-terminal as BACE1 inhibitors(2010)
• → Enamine launches Frankfurt site(2023)