A Fluorescence-Based Assay for Baeyer – Villiger Monooxygenases, Hydroxylases and Lactonases Renaud Sicard, a Lu S. Chen, b Anita J. Marsaioli, b, * Jean-Louis Reymond a, * a Department of Chemistry & Biochemistry, University of Berne, Freistrasse 3, 3012 Berne, Switzerland Fax: ( þ 41)-31-631-80-57, e-mail: reymond@ioc.unibe.ch b Chemistry Institute, State University of Campinas, CP 6154, 13083-970 Campinas – SP, Brazil Received: January 21, 2005; Accepted: March 30, 2005 Supporting Information for this article is available on the WWW under http://asc.wiley-vch.de/home/. Abstract: Alkylation of umbelliferone and nitrophe- nol with chloroacetone, 3-chlorobutanone, 2-chloro- cyclopentanone and 2-chlorocyclohexanone gave the corresponding 2-coumaryloxy and 2-nitrophenoxy ke- tones. The 2-coumaryloxy ketones were used as fluo- rogenic substrates to detect Baeyer–Villiger mono- oxygenases activities of microbial cultures in high- throughput using microtiter plates. The 2-coumary- loxy ketones were oxidized by microorganisms pro- ducing 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 regiose- lectively to the corresponding acyloxyalkyl deriva- tives 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 hy- drolysis reaction was determined by chiral-phase analysis of the unreacted lactones. Keywords: biotransformations; enzyme catalysis; fluo- rescent probes; lactones; oxidation Introduction Most enzymes of industrial interest are currently isolat- ed from microbial sources or from genetic libraries. [1] In the search for novel enzymes, it is necessary to apply functional tests for high-throughput screening that are capable of detecting enzyme activities with high selec- tivity and sensitivity. [2] Recently we developed enzyme substrates that release either umbelliferone as a blue- fluorescent product or nitrophenol as a yellow colored product by b-elimination from an intermediate carbonyl product formed after the enzyme-catalyzed step. This assay methodology detects various enzyme types with high selectivity, including alcohol dehydrogenases, [3] al- dolase catalytic antibodies, [4] acylases and phosphatas- es, [5] proteases, [6] lipases and esterases, [3,5,7] epoxide hy- drolases, [5,8] and transaldolases. [9] These assays may also be applied in parallel using arrays of structurally di- verse substrates to produce enzyme-specific activity fin- gerprints. [10,11] The same fluorescence release principle has also been used by other laboratories in fluorescence assays for transketolases [12] and Baeyer–Villiger mono- oxygenases. [13] In the latter case Baeyer–Villiger mono- oxygenase activity was detected with umbelliferone 4- oxopentyl ether and an auxiliary enzyme, an alcohol de- hydrogenase. In all of these assays a sequence of secondary reaction steps converts the primary product of the enzyme-cata- lyzed reaction to a fluorescent product. While this fea- ture provides highly selective assays for reactions that often cannot be rendered fluorogenic otherwise, it also complicates the reaction set-up and the kinetic analysis. Recently we reported that acyloxymethyl ethers of um- belliferone such as 3, which we originally used to discov- er catalytic antibodies by screening cell culture superna- tants, [14] are useful fluorogenic probes for lipases and es- terases (Scheme 1). [15] In this case the primary hydroxy- methyl ether product is very unstable and immediately releases umbelliferone 4 without any kinetic delay or auxiliary reagent. Herein we report the preparation and evaluation of cyclic and acyclic 2-coumaryloxy-ketones 1 and 5 – 7 as fluorogenic substrates for detecting Baeyer–Villiger monooxygenase activities using the same principle (Scheme 1). The Baeyer–Villiger oxidation, the oxida- tive transformation of a ketone to the corresponding es- ter or lactone, is a useful transformation in organic syn- thesis. An asymmetric version of this reaction is possible applying biocatalytic methods with BVMO allowing the FULL PAPERS Adv. Synth. Catal. 2005, 347, 1041 – 1050 DOI: 10.1002/adsc.200505040  2005 WILEY-VCH Verlag GmbH&Co. KGaA, Weinheim 1041