| Summary: | 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.
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