Study of phenoxy radical couplings using the enzymatic secretome of Botrytis cinerea

Abstract

Phenoxy radical coupling reactions are widely used in nature for the synthesis of complex molecules such as lignin. Their use in the laboratory has great potential for the production of high value compounds from the polyphenol family. While the enzymes responsible for the generation of the radicals are well known, the behavior of the latter is still enigmatic and difficult to control in a reaction flask. Previous work in our laboratory using the enzymatic secretome of B. cinerea containing laccases has shown that incubation of stilbenes leads to dimers, while incubation of phenylpropanoids leads to dimers as well as larger coupling products. Building on these previous studies, this paper investigates the role of different structural features in phenoxy radical couplings. We first demonstrate that the presence of an exocyclic conjugated double bond plays a role in the generation of efficient reactions. In addition, we show that the formation of phenylpropanoid trimers and tetramers can proceed via a decarboxylation reaction that regenerates this reactive moiety. Lastly, this study investigates the reactivity of other phenolic compounds: stilbene dimers, a dihydro-stilbene, a 4-O-methyl-stilbene and a simple phenol with the enzymatic secretome of B. cinerea. The observed efficient dimerization reactions consistently correlate with the presence of a para-phenol conjugated to an exocyclic double bond. The absence of this structural feature leads to variable results, with some compounds showing low conversion or no reaction at all. This research has allowed the development of a controlled method for the synthesis of specific dimers and tetramers of phenylpropanoid derivatives and novel stilbene derivatives, as well as an understanding of features that can promote efficient radical coupling reactions.

Keywords: Botrytis cinerea; coupling; enzymatic secretome; laccase; phenol; phenoxy radical; phenylpropanoids; stilbene.

FIGURE 1

Phenoxy radical coupling reactions of stilbenes and phenylpropanoids. (A) Delocalization of the radical formed on the phenolic function. (B, C) Products obtained by phenoxy radical coupling using stilbenes (e.g., resveratrol) (Righi et al., 2020; Huber et al., 2022a) (B) or phenylpropanoids (e.g., caffeic acid) (Huber et al., 2022b) (C) as starting materials. Dimeric structures are shown with one monomer in black and another in blue, with the new bonds and atoms in red.

FIGURE 2

Directed radical coupling reaction of ferulic acid (1). UHPLC-ELSD chromatograms are shown for each step. (A) ferulic acid 1 is used as starting material. (B) Esterification leads to ethyl ferulate (2). (C) Ethyl ferulate (2) is efficiently converted to 3 using the enzymatic secretome of Botrytis cinerea (D) Ester cleavage—decarboxylation process of 3 results in the formation of poacic acid (4). Purification is necessary at this step as side products are also formed. (E) Poacic acid (4) is dimerized into diastereoisomers 5 and 6 using the enzymatic secretome of B. cinerea. (F) 5 and 6 are easily separated to give the pure diastereoisomers.

FIGURE 3

Biotransformation reactions performed with different phenolic compounds (A–H) using the enzymatic secretome of Botrytis cinerea. The results of each reaction were evaluated by UHPLC-PDA-ELSD-MS analysis after 48 h. The detailed chromatograms for each reaction are shown in Supplementary Figure S4.

FIGURE 4

Dimeric products obtained with the incubation of dihydro-resveratrol (10) with the enzymatic secretome of Botrytis cinerea.

FIGURE 5

Left: Structure of compound 15 showing COSY (bond in bold), HMBC (black arrows) and ROESY correlations (red arrows). Right: HMBC spectrum of 15.

FIGURE 6

Left: Structure of compound 16 showing COSY (bond in bold), HMBC (black arrows) and ROESY correlations (red arrows). Right: HMBC spectrum of 16.