Substrate arrays for fluorescence-based enzyme fingerprinting and high-throughput screening.

Indirect release of fluorogenic phenols such as umbelliferone was used as a chemical principle to prepare fluorogenic substrates for a variety of enzymes in which the enzyme-reactive group is separated from the fluorescent reporter group, allowing structural and functional diversification. Fluorescent probes were obtained for enzymes useful for asymmetric synthesis including aldolase catalytic antibodies, transaldolases, alcohol dehydrogenases, lipases, epoxide hydrolases, proteases, and Bayer-Villiger monoxygenases. Arrays of structurally related substrates were prepared and used to record the activity of enzymes on multiple substrates simultaneously in parallel formats such as microtiter plates, microarrays, and substrate cocktails, leading to enzyme-specific activity patterns. Arrays of nonlabeled substrates were assayed using indirect product sensors such as the adrenaline-periodate back-titration assay for 1,2-diols or the copper-calcein assay for amino acids. Many of the fluorogenic substrates and sensors described here are either commercially available or accessible in one or two simple synthetic steps and can be used for high-throughput screening of enzyme activities.