On-line Abstract Book

Mechanism of Inactivation of Beta-lactamases by Novel 6-Methylidene Penems

Marshall M. Siegel¹, Keiko Tabei¹, Xidong Feng¹, Aranapakam M. Venkatesan¹, Takao Abe², Ushirogochi Hideke², Tarek S. Mansour¹

Correspondence address: Marshall M. Siegel, Wyeth Research, Chemical Technologies, 401 N. Middletown Rd, Pearl River, New York, 10965 United States.

Keywords: Antibiotics; MS/MS, Structure Determination; Protein Structure; Rearrangement.

Novel aspect: ESI and MALDI mass spectrometry were used to characterize the inhibition reactions of 6-methylidene penems with beta-lactamases (~30 kDa proteins) to form a covalent complex. Trypsin digests of the reaction product was analyzed by ESI-MS-MS.

 

The reactions of substituted 6-methylidene penems with beta-lactamases (TEM-1, SHV-1, Amp-C) were characterized by electrospray ionization mass spectrometry (ESI-MS). The kinetics of the reactions were monitored demonstrating that only one penem molecule reacts to form an acyl-enzyme complex. For a substituted 6-methylidene penem, the ESI-MS/MS spectrum of the hydrolysis product produced in the reaction was identical to the spectrum generated from a synthesized substituted dihydro[1,4]thiazepine, confirming the rearrangement of the 5-membered thiazole ring to a 7-membered dihydro[1,4]thiazepine structure. Gas phase ESI-MS/MS fragmentation data were rationalized due to tautomerization between imine and enamine substructures. ESI-MS/MS analysis of the T-6 trypsin-digested fragments of TEM-1 and SHV-1 demonstrated that the penems were only attached to Ser-70 of these class A beta-lactamases and that the penem five-membered thiazole structures were rearranged to seven-membered dihydro[1,4]thiazepines.