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

Abstract

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.

Flow-Based Systems for Rapid and High-Precision Enzyme Kinetics Studies

“…Searching for new enzymes or identifying a particular enzyme through its fingerprint, in which many parallel enzyme assays under various conditions and types of substrate are combined to gain a reactivity profile of a particular enzyme, has become an important trend in developing enzyme systems that are suitable for certain applications [5, 89]. The high-throughput feature of flow-based analysis should be very welcome for this area of study.…”

Monoamine oxidase and transaminase screening: biotransformation of 2-methyl-6-alkylpiperidines by Neopestalotiopsis sp. CBMAI 2030

“…Most HTS methodologies detect signals from a fluorogenic or chromogenic probe in 96-well microplates, which reveal that the enzymatic reaction occurred (Reymond 2008). Assays detecting fluorescent signals are more sensitive and provide a linear response to the reaction progress without interference of colored products (Reymond 2006).…”

Structure–activity relationship studies using peptide arrays: the example of HIV-1 Rev–integrase interaction

“…1 Peptide arrays are used for specic detection of individual proteins in complex mixtures, 2 for identifying binding sites between proteins, [3][4][5] for determining the contribution of individual amino acid residues to the binding 6,7 and for high throughput measurements of enzymatic activites. 8,9 Here we demonstrate the use of peptide arrays for performing detailed SAR studies of lead peptide inhibitors of the interaction between HIV integrase and HIV Rev.…”