Fluorogenic kinetic assay for high-throughput discovery of stereoselective ketoreductases relevant to pharmaceutical synthesis |
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Authors: | Yen-Chi Thai Anna Szekrenyi Yuyin Qi Gary W Black Simon J Charnock Wolf-Dieter Fessner |
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Institution: | 1. Institut für Organische Chemie und Biochemie, Technische Universität Darmstadt, Alarich-Weiss-Str. 4, 64287 Darmstadt, Germany;2. Prozomix Ltd, Station Court, Haltwhistle, Northumberland NE49 9HN, UK;3. Department of Applied Sciences, Northumbria University, Faculty of Health and Life Sciences, Newcastle-upon-Tyne NE1 8ST, UK |
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Abstract: | Enantiomerically pure 1-(6-methoxynaphth-2-yl) and 1-(6-(dimethylamino)naphth-2-yl) carbinols are fluorogenic substrates for aldo/keto reductase (KRED) enzymes, which allow the highly sensitive and reliable determination of activity and kinetic constants of known and unknown enzymes, as well as an immediate enantioselectivity typing. Because of its simplicity in microtiter plate format, the assay qualifies for the discovery of novel KREDs of yet unknown specificity among this vast enzyme superfamily. The suitability of this approach for enzyme typing is illustrated by an exemplary screening of a large collection of short-chain dehydrogenase/reductase (SDR) enzymes arrayed from a metagenomic approach. We believe that this assay format should match well the pharmaceutical industry’s demand for acetophenone-type substrates and the continuing interest in new enzymes with broad substrate promiscuity for the synthesis of chiral, non-racemic carbinols. |
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Keywords: | Alcohol dehydrogenase Asymmetric synthesis Biocatalysis Chiral alcohols Enantioselectivity |
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