Substrate Arrays for Fluorescence-Based Enzyme Fingerprinting and High-Throughput Screening

摘要

Indirect release of fluorogenic phenols such as umbelliferone was used as a chemical principleto prepare fluorogenic substrates for a variety of enzymes in which the enzyme-reactive groupis separated from the fluorescent reporter group, allowing structural and functional diversifica-tion. Fluorescent probes were obtained for enzymes useful for asymmetric synthesis includingaldolase catalytic antibodies, transaldolases, alcohol dehydrogenases, lipases, epoxide hydrolases,proteases, and Bayer-Villiger monoxygenases. Arrays of structurally related substrates were pre-pared and used to record the activity of enzymes on multiple substrates simultaneously in parallelformats such as microtiter plates, microarrays, and substrate cocktails, leading to enzyme-specificactivity patterns. Arrays of nonlabeled substrates were assayed using indirect product sensors suchas the adrenaline-periodate back-titration assay for 1,2-diols or the copper—calcein assay for aminoacids. Many of the fluorogenic substrates and sensors described here are either commerciallyavailable or accessible in one or two simple synthetic steps and can be used for high-throughputscreening of enzyme activities.