Design of chromogenic probes for efficient screening and evaluation of feruloyl esterase-like activities

• Hydroxycinnamate-based chromogenic substrates were synthesized in good yields.
• Detection of esterase activity was achieved in both solid and liquid media.
• Indolyl- and 4NTC-based synthetic probes are suitable for high-throughput screening.
• 4NTC ferulate is a new tool for the characterization of feruloyl esterases.

Feruloyl esterases (FAEs) constitute an important sub-group of hydrolytic enzymes involved in the deconstruction of plant cell wall polysaccharides. However, in the current era of genomics and metagenomics, finding and characterizing FAEs is not straightforward, mainly due to a lack of suitable compounds for high-throughput assays. To remedy this, indolyl and 4-nitrocatechol hydroxycinnamates (i.e. trans-ferulate and p-coumarate derivatives) were synthesized in good overall yields – between 46 and 56% after 4 steps – and their usefulness as substrates for FAEs was ascertained. The hydrolysis of the ester bond of these chromogenic compounds leads to a colour change, which can be readily monitored. Overall, these compounds considerably improve upon the current situation and enable the measurement of FAE activities in both qualitative solid medium-based and quantitative liquid assays.

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