| Abstract
| - A new β-lactamase inhibitor, a methylidene penem having a 5,6-dihydro-8H-imidazo[2,1-c][1,4]oxazine heterocyclic substituent at the C6 position with a Z configuration, irreversibly inhibits bothclass A and class C serine β-lactamases with IC50 values of 0.4 and 9.0 nM for TEM-1 and SHV-1 (classA), respectively, and 4.8 nM in AmpC (class C) β-lactamases. The compound also inhibits irreversiblythe class C extended-spectrum GC1 β-lactamase (IC50 = 6.2 nM). High-resolution crystallographic structuresof a reaction intermediate of (5R)-(6Z)-6-(5,6-dihydro-8H-imidazo[2,1-c][1,4]oxazin-2-ylmethylene)-7-oxo-4-thia-1-azabicyclo[3.2.0]hept-2-ene-3-carboxylic acid 1 with the SHV-1 β-lactamase and with theGC1 β-lactamase have been determined by X-ray diffraction to resolutions of 1.10 and 1.38 Å, respectively.The two complexes were refined to crystallographic R-factors (Rfree) of 0.141 (0.186) and 0.138 (0.202),respectively. Cryoquenching of the reaction of 1 with each β-lactamase crystal produced a common,covalently bound intermediate. After acylation of the serine, a nucleophilic attack by the departing thiolateon the C6‘ atom yielded a novel seven-membered 1,4-thiazepine ring having R stereochemistry at thenew C7 moiety. The orientation of this ring in each complex differs by a 180° rotation about the bond tothe acylated serine. The acyl ester bond is stabilized to hydrolysis through resonance stabilization withthe dihydrothiazepine ring and by low occupancy or disorder of hydrolytic water molecules. In the classA complex, the buried water molecule on the α-face of the ester bond appears to be loosely bound orabsent. In the class C complex, a water molecule on the β-face is disordered and poorly activated forhydrolysis. Here, the acyl intermediate is unable to assist its own hydrolysis, as is thought to occur withmany class C substrates.
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