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

Abstract

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 highthroughput 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.

Fluorescence Assays for Biotransformations

“…The substrates can also be prepared in a two -step sequence involving alkylation of umbelliferone with 2 -chloroketones followed by Baeyer -Villiger oxidation. The sequence is particularly interesting for obtaining the fl uorogenic lactones 28 and 29 , which only react with esterases [40] .…”

“…It has recently been shown that 2 -aryloxyketones 47 -50 act as fl uorogenic substrates for these enzymes (Scheme 1.15 ) [40] . The natural selectivity of oxidative cleavage leads to insertion of the oxygen atom on the side of the aryl ether to form an unstable lactone or acetal ester, which is rapidly hydrolyzed to form umbelliferone.…”

Sn-based catalysts for Baeyer-Villiger oxidations by using hydrogen peroxide as oxidant

“…Generally, percarboxylic acids (i.e., meta-chloroperbenzoic acid (m-CPBA), peracetic acid, etc.) are the most common and suitable oxidants used in BV oxidation, but they are becoming problematic for industrial scale use due to their generating amounts of carboxylic acid waste as side product and resulting in low ester selectivity [7][8][9] . Moreover, percarboxylic acids are expensive, and unstable in solution and typically require chlorinated solvents for improved performance [10].…”

Esterase screening using whole cells of Brazilian soil microorganisms

“…21, No. 8, 2010 Several methodologies have been applied to screen hydrolases 11 however the fluorogenic substrates coupled to umbelliferone are stable at high temperature and over a broad range of pH-values 12 and have been reported as screening probes for epoxide hydrolases 13 and BaeyerVilliger monooxygenases, 14 using microorganism whole cells. This methodology has been applied to the screening of a great number of activities in strain collections.…”

“…This methodology has been applied to the screening of a great number of activities in strain collections. 13,14 The general principle of the method involves a sequence of steps in which a fluorescent probe is released after enzymatic transformation (Scheme 1).…”