DOI: 10.1002/cctc.200900190 A High-Throughput Screening Method for Chiral Alcohols and its Application to Determine Enantioselectivity of Lipases and Esterases Ismael Bustos-Jaimes, [a] Werner Hummel,* [a] Thorsten Eggert, [a] Eliane Bogo, [a] Michael Puls, [a] Andrea Weckbecker, [a] and Karl-Erich Jaeger* [a] Chiral alcohols are valuable intermediates in the synthesis of pharmaceutical, agricultural, and fine chemicals, [1] which can be produced either by hydrocarbon oxidation, ketone reduc- tion, or ester hydrolysis. Nevertheless, these reactions usually produce non-enantiopure compounds. For this reason, several methods for the enantioselective synthesis of alcohols have been developed, which range from the synthesis of catalysts by combinatorial chemistry to the in vitro directed evolution of enzymes. [2] In any case, high-throughput methods need to be applied to measure the enantiomeric excess (ee) or enantiopur- ity of the produced alcohols within a large number of samples. Several methods for high-throughput screening for enantiose- lectivity of catalysts have been reported, including electrospray ionization coupled to mass spectrometry, HPLC coupled to cir- cular dichroism, FTIR spectroscopy, [3] and enzymatic methods. [4] Some of these sophisticated methods require, however, isotop- ically labeled pseudo-enantiomers for the assay and occasion- ally expensive equipment. Herein, we report a new colorimetric method for the evaluation of the ee values of alcohols based on enantioselective alcohol dehydrogenases (ADHs) coupled to a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (diaphorase) and its successful application in directed evolution for the screening of mutant libraries of lipases for enantioselective ester hydrolysis. The assay is based on the enantioselective oxidation of alco- hols by two different ADHs assayed separately in parallel assays: the (R)-specific ADH from Lactobacillus kefir (LKADH) and the (S)-specific ADH from Rhodococcus erythropolis (READH), of which enantioselectivities and catalytic properties have been reported. [5] The oxidation of either (R)-1 or (S)-1 pro- duces NAD(P)H, which is again oxidized to NAD(P) by diaphor- ase from Clostridium kluyveri with the concomitant reduction of 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium (INT) 3 to its corresponding red formazan derivative 4 (Scheme 1). The formation of this dye can be easily followed at 492 nm. The reaction is carried out within five minutes, during which the slope of color development over time is linear. The regeneration of the oxidized form of the coenzyme also ensures high reaction rates of ADHs. As in any other coupled assay, the amount of diaphorase, the coupling enzyme, was kept in excess relative to the ADH enzymes in order to follow first-order kinetics. [6] Previous studies using one or two enzymes to measure ee values have been reported and their accuracy was well demon- strated. Berkowitz et al. coupled (S)- and (R)-ADHs with the Co III -salen-catalyzed hydrolytic kinetic resolution of epoxides, whereas the group of Turner coupled R- or S-selective alcohol oxidases with the enzymatic reduction of ketones to determine the enantioselectivity of ketoreductases. [6] The mathematical framework for the analysis of these data has been developed based on the Michaelis–Menten equation. [7] Nevertheless, en- zymes may display different kinetic behaviors in the presence of diverse compounds. [8] In our study, neither LKADH nor READH display hyperbolic initial-rate curves. Instead, both enzymes displayed sigmoidally shaped kinetic curves (Figure 1). Scheme 1. Enantioselective reactions catalyzed by READH and LKADH and its coupling to the diaphorase redox system. Oxidation of either alcohol (S)-1 or (R)-1 correspondingly produces a molecule of coenzyme, NADH or NADPH, which in turn are oxidized by diaphorase through the reduction of 3 to produce the corresponding formazan red dye 4. [*] Dr. I. Bustos-Jaimes, [+] Prof. Dr. W. Hummel, Dr. T. Eggert, [#] Dr. E. Bogo, Dr. M. Puls, [#] Dr. A. Weckbecker, [#] Prof. Dr. K.-E. Jaeger Institut für Molekulare Enzymtechnologie Heinrich-Heine Universität Düsseldorf Forschungszentrum Jülich, Stetternicher Forst 52426 Jülich (Germany) Fax: (+ 49) 2461-612490 E-mail: w.hummel@fz-juelich.de karl-erich.jaeger@fz-juelich.de [ + ] Present address: Departamento de Bioquímica, Fac. de Medicina Universidad Nacional Autónoma de MØxico P.O. Box 70-159, C.U., MØxico DF 04510 (Mexico) [ # ] Present address: Evocatal GmbH, Merowingerplatz 1a 40225 Düsseldorf (Germany) ChemCatChem 2009, 1, 445 – 448  2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 445