Lipases and esterases are versatile biocatalysts and find increasing application in organic syntheses.[1] The main interest stems for their broad substrate specificity and exceptionally high stability in organic solvents. Thus, not only hydrolysis, but also synthesis reactions are possible. Indeed, numerous reactions have been performed in organic media and many of them have been commercialized. These include the synthesis of optically pure building blocks, flavors and fragrances, lipid modification, and also simple esters, such as myristyl myristate for cosmetic applications.Active (and stereoselective) biocatalysts can be identified by means of hydrolytic assays, for which a considerable number of useful high-throughput methods have been developed.[2] However, it often turns out that enzymes identified by these methods are not suitable for the desired synthetic reactions. An alternative can be small-scale esterification reactions in organic solvents, for which conversion (and optical purity) is then determined by GC or HPLC analysis. Broad investigations of a large number of enzymes and the identification of best reaction conditions (different alcohols or acyl donors, solvents, carriers for immobilization, temperature) easily can lead to several hundred individual experiments and are therefore very time-consuming.Herein we describe an assay format, which allows highthroughput determination of synthetic activity of hydrolases. This method is based on lipase-or esterase-catalyzed transesterification reactions between a vinyl ester and an alcohol. Acetaldehyde released stoechiometrically by keto-enol tautomerization of the vinyl alcohol released during ester synthesis, [3] reacts in situ with hydrazine 1 to produce a fluorogenic derivative 2, which can be quantified by fluorescence measurements (Scheme 1).

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