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Antibody-catalyzed formation of a 14-membered ring lactone

University of British Columbia

A number of macrolide antibiotics have a 14-membered ring lactone skeleton. Besides the stereochemical challenges associated with the syntheses of these macrocyclic compounds, the difficulty in controlling the ring forming step has provided the basis for many synthetic organic methodology studies. Attempts to use enzymes in aqueous and organic solvents to catalyze medium to large ring lactonizations has met with limited success, particularly with secondary alcohols. We report monoclonal antibody F123, raised against transition state analogue 50, which catalyzed the intramolecular transesterification of hydroxyester 57 to give the 14-membered ring lactone 19. The reaction of antibody F123 with substrate 57 displayed enzyme-like [Chemical model] Michaelis-Menten kinetics. The kinetic parameters of this antibody reaction were determined by two methods, namely a multiwell method and the spectrophotometric cuvette method. Analysis of the reaction of F123 with 57 via the multiwell method yielded a Km of 250 ± 10 uM, Vmax of 0.62 ± 0.01 ixmol/min mg and a kcat of 1.1 min"1. The spectrophotometric cuvette method yielded a rCr, of 330 ± 50 uM, V m a x of 1.4 ± 0.1 umol/min mg and a kcat of 2.2 min'1. The results obtained from these two methods were found to be in good agreement once the delay times in determining initial rates, characteristic of the multiwell method, were accounted for. In both methods, the observed rates were [chemical model] corrected for the background hydrolysis in buffer. Substrate specificity and competitive inhibition by the hapten derivative 60 (K = 2.9 ± 0.4 u.M) demonstrated that the catalytic activity was associated with binding in the antibody-combining site. Finally, the lactone product was isolated from pooled antibody-catalyzed reactions by ether extraction and identified using gas chromatography-mass spectroscopy by comparison with an authentic lactone sample.

Thesis/Dissertation

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2009-04-20

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http://hdl.handle.net/2429/7424

Chemistry

University of British Columbia

UBCV

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