A Förster Resonance Energy Transfer Assay for Investigating the Reactivity of Thioesters

27 March 2023, Version 1
This content is a preprint and has not undergone peer review at the time of posting.


The sulfur–carbonyl bond in a thioester is an “energy-rich” functionality but is hydrolytically stable, while susceptible to attack by thiolate and amine nucleophiles, enabling thioester chemistry to take place in aqueous medium. In this work, we developed a fluorogenic assay format for the direct and continuous investigation of the rate of reaction between thioesters and nucleophiles under various conditions. We designed three different substrates, mimicking the chemical environment in acyl-CoA, S-acylcysteine, and an activated thioester commonly used in chemical protein synthesis by native chemical ligation. The data resulting from monitoring of the reaction of these substrates with a series of nucleophiles at varying pH values generally recapitulated previously reported reactivity of thioesters. We investigated key aspects of native chemical ligation reaction conditions. Our data revealed a profound effect of the tris-(2-carboxyethyl)phosphine (TCEP) commonly used in systems where thiol–thioester exchange occurs, including a potentially harmful hydrolysis side reaction. These data provide insight for potential optimization of native chemical ligation chemistry.


acyl transfer
native chemical ligation
acyl coenzyme A

Supplementary materials

Supporting Information
Supplementary methods, chemical synthesis, compounds characterisation data, rate equations, FRET assay data, UPLC assay data, and copies of UPLC chromatograms, 1H and 13C NMR spectra.


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