(I) BIOSYNTHESIS OF KETOMYCIN. (II) BIOMIMETIC MODEL FOR BETA-LACTAMASE CATALYSIS: HOST-GUEST INTERACTIONS IN CYCLODEXTRIN-PENICILLIN INCLUSION COMPLEX

HSIAO-WUN MAK, Purdue University

Abstract

I. The antibiotic ketomycin is formed from shikimic acid via chorismic acid and prephenic acid. Phenylalanine and 2',5'-dihydrophenylalanine derived from shikimic acid are not intermediates in the biosynthesis. Degradation of ketomycin derived from 1,6-('14)C shikimic acid showed that prephenic acid is converted into ketomycin with stereospecific discrimination between the two enantiotopic edges of the ring, the pro-S-R edge giving rise to the C-2', C-3' side of the cyclohexane ring of ketomycin. II. The resistance of pathogenic bacteria to the action of (beta)-lactam antibiotics is mainly ascribed to their ability to produce (beta)-lactamase to cleave the (beta)-lactam ring. It is essential to understand the molecular nature of (beta)-lactamase-penicillin recognition for designing and formulating more effective (beta)-lactam antibiotics. A biomimetic study of (beta)-lactamase is therefore initiated. To meet the requirements of hydrophobic and "serine" protease characteristics of (beta)-lactamase, (alpha)-cyclodextrin is chosen as a biomimetic model for (beta)-lactamase. The structural specificity and the chemical dynamics of (alpha)-cyclodextrin-phenoxymethyl penicillin inclusion complex in solid state and in solution have been determined by IR and NMR (470 ('1)HNMR, 50 MHz solution ('13)CNMR and 25 MHz CP/MAS solid-state ('13)CNMR) spectroscopy. The spectral results strongly indicate that the phenyl portion of the phenoxymethyl penicillin forms a stable inclusion complex with the hydrophobic cavity of (alpha)-cyclodextrin in solution as well as in the solid state. A similar hydrophobic interaction of the acyl side chain of penicillins with (beta)-lactamase has been demonstrated to play an important role in the initial enzyme-substrate recognition. Kinetic studies followed by ('1)HNMR and HPLC analyses under alkaline condition have shown that the (alpha)-cyclodextrin mimics the catalytic function of serine of (beta)-lactamase in the stereospecific hydrolysis of the (beta)-lactam ring of phenoxymethyl penicillin. The hydrolysis is initiated by forming a six-membered ring transition complex between the (beta)-lactam carbonyl group and the vicinal diols of cyclodextrin to yield a penicilloyl-(alpha)-cyclodextrin intermediate, reminiscent of an acyl-(beta)-lactamase intermediate. The proposed (alpha)-cyclodextrin-catalyzed penicillin hydrolysis mechanism is supported by the kinetic studies of selectively modified (alpha)-cyclodextrins and the identification of the acyl intermediate. The structure of the key penicilloyl-(alpha)-cyclodextrin intermediate has been elucidated by FAB-MS, and ('1)HNMR analysis. The C-2 hydroxyl oxygen of (alpha)-cyclodextrin forms an ester linkage with the (beta)-lactam carbonyl carbon. This finding further substantiates that (alpha)-cyclodextrin can be a good biomimetic model for elaborating the molecular mechanism of (beta)-lactamase catalysis.

Degree

Ph.D.

Subject Area

Biochemistry

COinS