Regioselective chemo-enzymatic syntheses of ferulate conjugates as chromogenic substrates for feruloyl esterases

TL;DR

This study presents a chemoenzymatic method to synthesize cost-effective, regioselective ferulate conjugates that serve as chromogenic substrates for feruloyl esterases, improving synthesis efficiency and substrate utility.

Contribution

Developed a streamlined chemoenzymatic synthesis route for ferulate conjugates, enhancing yields and creating novel substrates for enzyme characterization.

Findings

Synthesized ferulate conjugates with reduced steps and increased yields.

Created a new feruloylated-butanetriol substrate for enzyme analysis.

Demonstrated the substrate's effectiveness in characterizing feruloyl esterases.

Abstract

Generally, carbohydrate-active enzymes are studied using chromogenic substrates that provide quick and easy color-based detection of enzyme-mediated hydrolysis. In the case of feruloyl esterases, commercially available chromogenic ferulate derivatives are both costly and limited in terms of their experimental application. In this study, we describe solutions for these two issues, using a chemoenzymatic approach to synthesize different ferulate compounds. The overall synthetic routes towards commercially available 5-bromo-4-chloro-3-indolyl and 4-nitrophenyl O-5-feruloyl-$\alpha$-l-arabinofuranosides 1a and 1b were significantly shortened (7-8 steps reduced to 4-6) and transesterification yields enhanced (from 46 to 73% for 1a and 47 to 86 % for 1b). This was achieved using enzymatic (immobilized Lipolase 100T from Thermomyces lanuginosus) transesterification of unprotected vinyl ferulate to the primary hydroxyl group of $\alpha$-l-arabinofuranosides. Moreover, a novel feruloylated-butanetriol 4-nitrocatechol-1-yl analog 12, containing a cleavable hydroxylated linker was also synthesized in 29% overall yield in 3 steps (convergent synthesis). The latter route combined regioselective functionalization of 4-nitrocatechol and enzymatic transferuloylation. The use of 12 as a substrate to characterize type A feruloyl esterase from Aspergillus niger reveals the advantages of this substrate for the characterizations of feruloyl esterases.