Design of specific compounds for beta-lactamase which afford stable acyl enzyme

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The resistance of microorganism to beta-lactam antibiotics is caused by the secretion of beta-lactamase, which catalyzes rapid hydrolysis of beta-lactam ring. Consequently, there is considerable interest in beta-lactamase inhibitors.Compounds which afford stable acyl enzyme intermediate are expected to be good candidates as inhibitors for such enzymes. In our previous work, specific and efficient production of acyl enzymes from trypsin and trypsin-like enzymes was achieved by means of "inverse substrates". It seems promising approach to design compounds that can carry out efficient acylation at the beta-lactamase catalytic residue by reference to the observation of "inverse substrates".In this respect, the concept of "inverse substrates" has further-been extended by designing optically active "inverse substrates" and the spatial requirement of the enzyme active site for catalytic efficiency has been analyzed. Differentiation of tryptic enzymes based on the spatial requirement has also been carried out.For the second stage of this research project, design of beta-lactamase inhibitors has been carried out. Compounds having a structural analogy with diketene have been synthesized and their potencies as inhibitors have been studied. Among six compounds so far tested, alpha-phenyl-beta-benzylidene-3-propanolide was shown to be irreversible inhibitor of the enzyme. Inhibitors of "mechanism-based" type has also been designed. N-Nitroso-beta-phenyl-beta-lactam has been found to be a specific irreversible inhibitor which is comparable to one of the most potent inhibitors, clavulanic acid.