Mechanism of inactivation of beta-lactamases by novel 6-methylidene penems elucidated using electrospray ionization mass spectrometry.
The reactions of 6-methylidene penems 4-7 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 penem 5, the ESI-MS/MS spectrum of the hydrolysis product produced in the reaction was identical to the spectrum generated from a synthesized dihydro[1,4]thiazepine 10, confirming the rearrangement of the penem ring system to a seven-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 ring structures were rearranged to seven-membered dihydro[1,4]thiazepines.
Tabei
Keiko
K
Wyeth Research, Chemical and Screening Sciences, Chemical Technologies and Medicinal Chemistry, Pearl River, NY 10965, USA.
Feng
Xidong
X
Venkatesan
Aranapakam M
AM
Abe
Takao
T
Hideki
Ushirogochi
U
Mansour
Tarek S
TS
Siegel
Marshall M
MM
D001426:Bacterial Proteins / Q000737:chemistry; D001665:Binding Sites; D006868:Hydrolysis; D021241:Spectrometry, Mass, Electrospray Ionization; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000737:chemistry*; D047090:beta-Lactams / Q000138:chemical synthesis / Q000737:chemistry*
D001426:Bacterial Proteins; D065093:beta-Lactamase Inhibitors; D047090:beta-Lactams; C054882:beta-lactamase PIT-2; C048795:AmpC beta-lactamases; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1; C063912:carbapenemase