A Fluorescence‐Based Assay for Baeyer–Villiger Monooxygenases, Hydroxylases and Lactonases

Alkylation of umbelliferone and nitrophenol with chloroacetone, 3‐chlorobutanone, 2‐chlorocyclopentanone and 2‐chlorocyclohexanone gave the corresponding 2‐coumaryloxy and 2‐nitrophenoxy ketones. The 2‐coumaryloxy ketones were used as fluorogenic substrates to detect Baeyer–Villiger monooxygenases activities of microbial cultures in high‐throughput using microtiter plates. The 2‐coumaryloxy ketones were oxidized by microorganisms producing Baeyer–Villiger monooxygenases (BVMO), releasing umbelliferone as a fluorescent signal. The substrates were also biotransformed by a microbial monooxygenase (Trichosporon cutaneum). Chemical Baeyer–Villiger oxidation of 2‐coumaryloxy ketones using meta‐chloroperbenzoic acid proceeded regioselectively to the corresponding acyloxyalkyl derivatives of umbelliferone and nitrophenol. These chiral lactones underwent a fluorogenic and chromogenic reaction upon hydrolysis by esterases, in particular pig liver esterase. Enantioselectivity of the ester hydrolysis reaction was determined by chiral‐phase analysis of the unreacted lactones.