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

Olga Gherbovet, Fernando Ferreira, Apolline Clément, Mélanie Ragon, Julien Durand, Sophie Bozonnet, Michael O'Donohue, Régis Fauré

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## To cite this version:

Olga Gherbovet, Fernando Ferreira, Apolline Clément, Mélanie Ragon, Julien Durand, et al. *Regioselective chemo-enzymatic syntheses of ferulate conjugates as chromogenic substrates for feruloyl esterases*. Beilstein Journal of Organic Chemistry, 2021, 17, pp.325-333. 10.3762/bxiv.2020.107.v1 hal-02988548v2

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# Regioselective chemoenzymatic syntheses of ferulate conjugates as chromogenic substrates for feruloyl esterases

Olga Gherbovet, Fernando Ferreira, Apolline Clément, Mélanie Ragon, Julien Durand, Sophie Bozonnet, Michael J. O'Donohue and Régis Fauré*

Full Research Paper

Open Access

Address:
Toulouse Biotechnology Institute, Bio & Chemical Engineering (TBI),
Université de Toulouse, CNRS 5504, INRAE 792, INSA de Toulouse,
135 Avenue de Rangueil, 31077 Toulouse, France

Email:
Régis Fauré* - regis.faure@insa-toulouse.fr

Keywords:
esterase; feruloylated conjugates; hydrolysis; lipase;
transesterification

Beilstein J. Org. Chem. **2021**, *17*, 325–333.
https://doi.org/10.3762/bjoc.17.30

Received: 15 September 2020

Accepted: 15 January 2021

Published: 01 February 2021

Associate Editor: K. N. Allen

© 2021 Gherbovet et al.; licensee Beilstein-Institut.
License and terms: see end of document.

## Abstract

Generally, carbohydrate-active enzymes are studied using chromogenic substrates that provide quick and easy color-based detection of enzyme-mediated hydrolysis. For 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 5-O-feruloyl-α-L-arabinofuranosides were significantly shortened (from 7 or 8 to 4–6 steps), and the transesterification yields were enhanced (from 46 to 73% and from 47 to 86%, respectively). This was achieved using enzymatic (immobilized Lipozyme® TL IM from *Thermomyces lanuginosus*) transesterification of unprotected vinyl ferulate to the primary hydroxy group of α-L-arabinofuranosides. Moreover, a novel feruloylated 4-nitrocatechol-1-yl-substituted butanetriol analog, containing a cleavable hydroxylated linker, was also synthesized in 32% overall yield in 3 steps (convergent synthesis). The latter route combined the regioselective functionalization of 4-nitrocatechol and enzymatic transferuloylation. The use of this strategy to characterize type A feruloyl esterase from *Aspergillus niger* reveals the advantages of this substrate for the characterizations of feruloyl esterases.

## Introduction

The development of “white biotechnology” is underpinned by advances in enzyme discovery and engineering, areas that are being driven by metagenomics and in vitro-directed enzyme evolution. These techniques procure massive discovery or the creation of new enzyme-encoding sequences, filling up data-

bases with a wealth of information. However, while resolving an early step in the discovery pipeline, these techniques progressively create a new bottleneck regarding enzyme characterization. Therefore, there is a pressing need to extend the enzymologist's toolbox, providing informationally rich high-

<footnote>* Corresponding author</footnote>
throughput screens that can not only attribute an activity to putative enzymes but also procure some qualitative details on enzyme properties. In this respect, the availability of easy-to-use chromogenic substrates that can provide both qualitative and quantitative assays and are compatible with automatized protocols is a crucial issue.

Feruloyl esterases (Faes; EC 3.1.1.73 and the family CE1 of the CAZy classification [1]) are of interest, both because of their role in the deconstruction of complex plant-based materials and also as synthetic tools for the preparation of bioactive compounds with potential antioxidant properties [2-5]. Operating via a two-step serine protease mechanism involving a conserved Ser-His-Asp/Glu catalytic triad [6,7], Faes catalyze the hydrolysis of ester bonds linking hydroxycinnamoyl groups to the glycosyl moieties of plant-based polysaccharides, such as arabinoxylans and arabinans. In this respect, Faes are important components of plant-cell-wall-degrading enzymatic arsenals since the hydrolysis of *trans*-ferulate–polysaccharide linkages contributes to the breakdown of intermolecular bonds that structure the lignocellulosic matrix. Moreover, Faes are useful tools to obtain commercially relevant ferulic acid, which represents up to 3 wt % of plant cell wall dry weight [8].

So far, the detection and characterization of Faes has mainly relied on either the use of HPLC or on UV-visible spectrophotometry, using natural or synthetic compounds [9,10]. The latter, which are used in high-throughput screening (HTS) assays, fall into three categories. The simplest are feruloyl esters of chromogenic moieties [11-14], such as *p*-nitrophenol or short-chain alkyl groups (e.g., methyl ferulate). More elaborate and biologically relevant substrates contain a feruloylated *l*-arabinofuranosyl moiety [12,15-17]. These structurally more complex compounds are obtained using multistep syntheses, considerably limiting the availability. Moreover, they might be specific to certain subcategories of feruloyl esterases [18-20], and their use involves a challenging tandem reaction [21]. Finally, the synthesis of other, more generic esters that can be used to assay esterases, including Faes, and lipases have been reported [22,23].

In this work, we revisit the preparation of simple feruloylated substrates, such as the 5-bromo-4-chloro-3-indolyl and 4-nitrophenyl 5-O-feruloylated α-L-arabinofuranosides **1a** and **1b**. Although these substrates are commercially available, their synthesis involves 7 or 8 steps [15-17]. This contributes to their rather high retail costs (e.g., as of July 29th, 2020, 2500 and 778 € per 100 mg for **1a** and **1b**, respectively), which are approximately 19- and 14-fold higher than non-feruloylated precursors. Therefore, our aim was to simplify the synthesis in order to reduce the cost. Furthermore, we describe the short

synthesis of the new feruloylated chromogenic substrate **12**, a molecule that obviates the need for a glycosyl moiety while containing a cleavable hydroxylated linker that mimics the natural geometry and physicochemical properties of glycosidic linkages.

## Results and Discussion

### Chemoenzymatic synthesis of 5-O-feruloylated α-L-arabinofuranosides

The synthesis of the chromogenic 5-O-feruloylated α-L-arabinofuranosides **1a** and **1b** is usually achieved using a multistep pathway that involves trapping the furanose conformation, anomeric activation, glycosidation, regioselective deprotection of the primary hydroxy group, feruloylation, and final deprotection to yield the target molecule [12,15-17]. Additionally, the temporary protection of the functional groups is sometimes used during the synthesis in order to facilitate certain steps. Using an alternative approach, we employed a one-step regioselective transesterification of the unprotected vinyl ferulate **2** (synthesized in 56% in-house yield and in up to 77% previously reported yield [24,25] in one step on a gram scale) using Lipozyme® TL IM (a commercially available immobilized lipase from *T. lanuginosus* that efficiently catalyzes the transesterification of cinnamates) [26-28] and readily available and reasonably cheap 5-bromo-4-chloro-3-indolyl or 4-nitrophenyl α-L-arabinofuranosides. This afforded the corresponding feruloylated derivatives **1a** and **1b** (Figure 1). The yields (73 and 86% for the indolyl and 4-nitrophenyl derivatives, respectively) characterizing the regioselective enzymatic feruloylation of the primary hydroxy group compare favorably with the previously reported overall yield (46 and 47%, respectively, in three steps) [15-17,29], which relate to the selective enzymatic O-5-deacetylation and esterification of the primary hydroxy group and a final deprotection of the 2,3-O-acetyl groups of the glycoside and the O-acetyl group of the ferulate moiety. The fact that lipase-catalyzed transesterification obviates the need for protection and deprotection is a considerable advantage because the final deprotection in the chemical pathway is complicated by the presence of another ester linkage within the molecules [12-15]. In principle, the method described herein is generic, and thus applicable to other chromogenic α-L-arabinofuranosidic compounds, such as 4-nitrocatechol (4NTC), 2-chloro-4-nitrophenyl, and umbelliferyl derivatives.

To demonstrate the suitability of **1a** for qualitative in situ screening of microbial colonies growing on solid agar medium, the compound (300 µg/mL) was incorporated into top agar, along with an α-L-arabinofuranosidase from *Thermobacillus xylanilyticus* (*TxAbf*). This provided the means to reveal colored microbial colonies expressing the type A Fae from
<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /><figcaption>**Figure 1:** Alternative syntheses (A) and full structures (B) of the 5-bromo-4-chloro-3-indolyl or 4-nitrophenyl 5-O-feruloyl-α-L-arabinofuranosidic chromogenic substrates **1a** and **1b** and (C) detection of type A Fae activity on solid agar medium using **1a**. The overall yield of the transesterifications of **1a** and **1b** are reported for both pathways. <sup>a</sup>3-step pathway. <sup>b</sup>1-step pathway.</figcaption></figure>

Aspergillus niger (AnFaeA, Figure 1C). Coloration is the result of successive reactions: (i) release of the free 5-bromo-4-chloroindoxyl-3-ol by an enzyme cascade, wherein TxAbf-catalyzed cleavage of the glycosidic bond was made possible by the prior release by AnFaeA of the ferulate moiety [21] and (ii) spontaneous oxidation and subsequent dimerization forming the insoluble blue 5-bromo-4-chloro-substituted indigo dye [15]. In an alternative demonstration, **1a** was also used for colony level detection of FAE using yeast cells (Yarrowia lipolytica [30]) that actually coexpressed AnFaeA and TxAbf (data not shown).

## Investigating the enzymatic transferuloylation reaction of non-glycosidic motifs

While lipase-catalyzed transesterifications of cinnamate or phenolic derivatives onto glycosidic structures have been exten-

sively described [25,27,28,31-33], data related to the regioselectivity of transesterifications of hydroxylated alkyl and/or aryl moieties are sparser [26,34-36]. The extent to which Lipozyme® TL IM catalyzes feruloyl transfer reactions involving substituted benzylic alcohols was thus investigated to establish its usefulness for the preparation of both various polyhydroxylated molecules of interest (e.g., antioxidants) [4,26] and of novel chromogenic feruloylated substrates with various physicochemical features for screening applications. Accordingly, we observed that transesterifications only occurred when using primary benzylic alcohols; no phenol acylation was detected with hydroxynitrobenzylic alcohol (Table 1, no side product of **4** and **9** with transfer on an aromatic secondary alcohol; i.e., phenol), 2-chloro-4-nitrophenol, or 4NTC (data not shown). Additionally, the exact position of the benzyl alcohol affected the transfer, with *ortho*-substitutions ($R^1$) displaying a hindered electron-withdrawing group. This led to a low target product
<figure><img src="image_7.png" /><figcaption>**Table 1:** Enzymatic transferuloylation of substituted nitrobenzylic alcohols.</figcaption></figure>

<table><thead><tr><th>R<sup>1</sup></th><th>R<sup>2</sup></th><th>R<sup>3</sup></th><th>product</th><th>yield (%)<sup>a</sup></th><th>ratio (%)<sup>b</sup></th></tr></thead><tbody><tr><td>H</td><td>H</td><td>NO<sub>2</sub></td><td><b>3</b></td><td>79</td><td>10:28:62</td></tr><tr><td>OH</td><td>H</td><td>NO<sub>2</sub></td><td><b>4</b></td><td>76</td><td>19:32:49</td></tr><tr><td>Cl</td><td>H</td><td>NO<sub>2</sub></td><td><b>5</b></td><td>44</td><td>31:38:31</td></tr><tr><td>F</td><td>H</td><td>NO<sub>2</sub></td><td><b>6</b></td><td>46</td><td>33:28:39</td></tr><tr><td>H</td><td>NO<sub>2</sub></td><td>H</td><td><b>7</b></td><td>97</td><td>10:24:66</td></tr><tr><td>NO<sub>2</sub></td><td>H</td><td>H</td><td><b>8</b></td><td>—<sup>c</sup></td><td>28:48:24</td></tr><tr><td>NO<sub>2</sub></td><td>H</td><td>OH</td><td><b>9</b></td><td>—<sup>c</sup></td><td>27:64:9</td></tr></tbody></table>

yield (i.e., **5**, **6**, **8**, and **9**), a lower reactivity, and/or a higher hydrolysis of the vinyl ferulate into ferulic acid.

## Synthesis of L-arabinofuranoside-free 4-nitro-catechol-1-yl-linker-ferulate chromogenic substrate 12 and evaluation of 12 as a chromogenic substrate for Fae assays

To synthesize chromogenic (±)-4-O-(2-hydroxy-4-nitrophenyl)-1-O-trans-feruloyl-1,2,4-butanetriol (4NTC-linker-Fe, **12**), which contains 4NTC bound via a cleavable linker to a ferulate motif, a multistep route was devised (Scheme 1). First, a shorter, more practical pathway towards racemic (±)-4-O-(2-hydroxy-4-nitrophenyl)-1,2,4-butanetriol (**11**) was developed. Compared to the previously reported 4-step synthesis [37], two

drawbacks were circumvented, notably avoiding (i) the preparation of the volatile (S)-1-iodo-3,4-O-isopropylidene-3,4-butanediol intermediate and (ii) the use of a protected version of the chromogenic linker **11**, either for the extra hydroxy group of the catecholyl moiety or the secondary hydroxy group of the linker [23,37]. It is noteworthy that a racemic mixture of this linker has previously been used to prepare chromogenic substrates for esterases [23,38].

The alkylation of 4NTC with homoallylic bromide under basic conditions gave a mixture of mono- and dialkylated 4NTC derivatives, with **10** (38%) predominating because of the preferential formation of the phenolate at the *para*-position (relative to the nitro group) [39]. Osmium tetroxide-mediated dihydroxylation in the presence of *N*-methylmorpholine *N*-oxide (NMMO)

<figure><img src="image_8.png" /><figcaption>**Scheme 1:** Chemoenzymatic synthesis of (±)-4-O-(2-hydroxy-4-nitrophenyl)-1-O-trans-feruloyl-1,2,4-butanetriol (4NTC-linker-Fe, **12**).</figcaption></figure>

<footnote><sup>a</sup>Yield of isolated ferulates after the purification step. <sup>b</sup>Determined by ¹H NMR spectroscopy, ratio of the different feruloyl derivatives in the crude reaction mixture: remaining vinyl ferulate, ferulic acid (hydrolysis product), and the ferulates **3–9**. <sup>c</sup>The expected ferulates were confirmed by HRMS analysis, but the low purity of the samples after purification prevented a fine-structural characterization by NMR.</footnote>
afforded **11** in 90% yield. Finally, the regioselective transferferu-
loylation of the primary hydroxy group of the triol derivative **11**
with Lipozyme® TL IM was performed, and the expected
chromogenic substrate **12** was isolated in a high yield of 94%.
Accordingly, the synthesis of the chromogenic ferulate **12** was
achieved in 32% overall yield in 3 steps from commercial reac-
tants (convergent synthesis using a slight excess of the synthe-
sized vinyl ferulate **2**) and without the requirement to perform a
final deprotection.

As expected, the investigation of the stability of the chromo-
genic substrate **12** using UV-visible spectrophotometry
revealed that, unlike 4-nitrocatechol-1-yl ferulate (4NTC-Fe),
which undergoes spontaneous hydrolysis even at a neutral pH
value and 40 °C [13], the presence of the alkyl linker procures a
higher stability over a wide pH value range (up to pH 9.0), irre-
spective of the temperature. This is because in compound **12**,
the ferulate moiety is not directly linked to the good leaving
group 4NTC (pKₐ = 6.61 [40]). Instead, it is bonded to the
linker with a pKₐ value that can be compared either to that of
glycerol (pKₐ = 13.61) or L-arabinose (pKₐ = 11.31) [41],
meaning that it is a poor leaving group. Moreover, our observa-
tions regarding the linker stability are consistent with the known
stability of the ester linkages under basic conditions.

The usefulness of 4NTC–linker–Fe (12) for the characteriza-
tion of Faes was investigated (Figure 2), measuring 4NTC
release by AnFaeA [42] at 40 °C. The enzyme-catalyzed reac-
tion leads to the cleavage of the ester bond linking the ferulate
to the linker–4NTC moiety, and thus to the accumulation of

linker-4NTC. Therefore, working in a discontinuous mode,
4NTC is quantified by submitting samples removed from the
reaction to the oxidative action of sodium periodate at 0 °C and
registering the absorbance at 530 nm (under alkaline conditions)
[23,37,38,43]. Importantly, it is vital to include a stoichiometric
amount of ethylene glycol to avoid further oxidation of free
4NTC by sodium periodate (Figure 2B).

The specific activity (SA) of AnFaeA on 4NTC–linker–Fe (12) was determined to be 3 IU/mg of the protein (with IU corresponding to the international unit of the Fae hydrolytic activity), a value comparable to that measured on destarched wheat bran (3 IU/mg) [44] containing 5-O-feruloylated α-L-arabinofuranosyl moieties, but lower than that (40 IU/mg, unpublished data) measured using the more labile 4NTC–Fe. Therefore, although 4NTC–Fe is a practical synthetic probe for both the high-throughput screening and the preliminary characterization of the Fae activity [13], 4NTC–linker–Fe (12) is almost certainly a better analogue of ferulate linkages found in plant-based structures.

Conclusion

The use of immobilized Lipozyme® TL IM provides the means
to perform the regioselective transesterification of the vinyl
ferulate **2** to the primary hydroxy group of benzylic alcohols
and polyhydroxylated compounds. Three compounds suitable
for the detection and/or characterization of Fae activity were
synthesized in a straightforward protocol that holds the poten-
tial to greatly reduce the cost of the substrates **1a** and **1b**. More-
over, the enzyme-driven convergent synthesis of **12** affords a

<figure><img src="image_9.png" /><figcaption>**Figure 2:** (A) Spectrometric monitoring (at 530 nm) of 4NTC released after the action of Fae on **12** in the presence of sodium periodate and (B) control reactions of the discontinuous assay of the Fae-mediated hydrolysis of **12**. The optical density (OD) was measured during the colorimetric assays. + and – indicate whether the reaction conditions (e.g., pH > 10 or the addition of ethylene) were satisfied in the liquid reaction medium.</figcaption></figure>
novel substrate that is highly suitable for the characterization of
feruloyl esterases.

Experimental

Materials and general methods

4-Nitrophenyl α-L-arabinofuranosides and 5-bromo-4-chloro-3-
indolyl α-L-arabinofuranosides were purchased from
Carbosynth and Lipozyme® TL IM (immobilized lipase from
*T. lanuginosus*, 250 IUN/g, IUN = interesterification unit) was
supplied by Novozymes. The reaction evolution was monitored
by analytical thin-layer chromatography using silica gel 60 F254
precoated plates (Merck). Spots were visualized using UV light
of 254 nm wavelength, followed by soaking in a 0.1% (w/v)
orcinol solution containing a sulfuric acid/ethanol/water
3:72.5:22.5, v/v/v mixture, followed by charring. Purification
by column chromatography was performed using a Reveleris®
flash chromatography automated system (Büchi) equipped with
prepacked irregular silica gel 40–63 µm cartridges (FlashPure
EcoFlex, Büchi). NMR spectra were recorded on a Bruker
Avance II 500 spectrometer at 298 K. Chemical shifts (δ) are
given in ppm, with the signals of the residual solvents as the
internal references [45]. The coupling constants (J) are reported
in Hertz (Hz), with singlet (s), doublet (d), triplet (t), doublet of
doublets (dd), doublet of doublets of doublets (ddd), broad (br),
and quadruplet of triplets (qt) as abbreviations. Analyses and as-
signments were made using 1D (¹H, ¹³C, and J-modulated
spin–echo (J<sub>mod</sub>)) and 2D NMR experiments (COSY and
HSQC). High-resolution mass spectrometry (HRMS) analyses
were performed at PCN-ICMG (Grenoble, France). Optical
rotations were measured using a JASCO P-2000 polarimeter at
20 °C.

General procedure for enzymatic transesterifications

The enzymatic transesterification steps were performed accord-
ing to a published protocol [32]. Briefly, Lipozyme® TL IM
(1 g, reusable) was added to a solution of alcohol (0.30 mmol,
1 equiv) and the vinyl ferulate 2 (100 mg, 0.45 mmol,
1.5 equiv) in acetonitrile (6 mL). The reaction mixture was
stirred overnight at 37 °C, then filtered, the filter cake was
washed with acetone, and the filtrate was evaporated to dryness.
The residue was recovered in ethyl acetate, and washed three
times with saturated aqueous sodium hydrogen carbonate. The
combined organic phases were dried over anhydrous sodium
sulfate, filtered, and concentrated under reduced pressure. Flash
chromatography (gradient of ethyl acetate in petroleum ether
from 0 to 50%) afforded the pure ferulates **1a, 1b, 3–9**, and **12**.

5-Bromo-4-chloro-3-indolyl 5-*O*-trans-feruloyl-α-L-arabinofuranoside (1a, 107 mg, 0.19 mmol, 73%). Green-yellow foam. The NMR data (CD3OD) were consistent with those previously reported [15].

4-Nitrophenyl 5-*O*-trans-feruloyl-α-L-arabinofuranoside (1b). The application of the general procedure for enzymatic transesterifications was used with commercial 4-nitrophenyl α-L-arabinofuranoside (50 mg, 0.18, mmol, 1 equiv) and the vinyl ferulate 2 (61 mg, 0.28 mmol, 1.5 equiv) to give 1b (71 mg, 0.16 mmol, 86%) as a yellowish powder. The NMR data (CD3OD) were consistent with those previously reported [16,17].

3-Nitrobenzyl *trans*-ferulate (3, 78 mg, 0.24 mmol, 79%), white powder. ¹H NMR (500 MHz, (CD₃)₂CO, δ) 8.32 (t, *J* = 1.7, 1H, CH of Bn), 8.21 (ddd, *J* = 8.3, 2.2, 1.1, 1H, CH of Bn), 7.90 (ddd, *J* = 7.6, 1.7, 1.0, 1H, CH of Bn), 7.71 (t, *J* = 7.6, 1H, CH of Bn), 7.68 (d, *J* = 15.8, 1H, CH=CHCO₂), 7.37 (d, *J* = 2.0, 1H, CH of Fe), 7.17 (dd, *J* = 8.3, 2.0, 1H, CH of Fe), 6.87 (d, *J* = 8.3, 1H, CH of Fe), 6.50 (d, *J* = 15.8, 1H, CH=CHCO₂), 5.38 (s, 2H, CH₂ of Bn), 3.92 (s, 3H, OMe); ¹³C NMR (125 MHz, (CD₃)₂CO, δ) 167.2 (C=O), 150.3 (Cq), 149.4 (Cq), 148.8 (Cq) 146.7 (CH=CHCO₂), 140.2 (Cq), 135.1 (CH of Bn), 130.8 (CH of Bn), 127.3 (2Cq), 124.2 (CH of Fe), 123.6 (CH of Bn), 123.4 (CH of Bn), 116.1 (CH of Bn), 115.1 (CH=CHCO₂), 111.4 (CH of Fe), 65.1 (CH₂ of Bn), 56.4 (OMe); HRESIMS (*m/z*): [M − H]⁻ calcd for C₁₇H₁₄NO₇, 328.0821; found, 328.0833.

2-Hydroxy-5-nitrobenzyl *trans*-ferulate (4, 79 mg, 0.23 mmol, 76%), white powder. ¹H NMR (500 MHz, (CD₃)₂CO, δ) 8.29 (d, *J* = 2.8, 1H, CH of Bn), 8.14 (dd, *J* = 8.9, 2.8, 1H, CH of Bn), 7.68 (d, *J* = 15.8, 1H, CH=CHCO₂), 7.38 (d, *J* = 1.8, 1H, CH of Fe), 7.17 (dd, *J* = 8.0, 1.8, 1H, CH of Fe), 7.12 (d, *J* = 8.9, 1H, CH of Bn), 6.88 (d, *J* = 8.0, 1H, CH of Fe), 6.52 (d, *J* = 15.8, 1H, CH=CHCO₂), 5.33 (s, 2H, CH₂ of Bn), 3.92 (s, 3H, OMe); ¹³C NMR (125 MHz, (CD₃)₂CO, δ) 167.5 (C=O), 162.0 (Cq) 150.3 (Cq), 148.8 (Cq), 148.7 (Cq), 146.6 (CH=CHCO₂), 141.6 (Cq), 127.4 (Cq), 126.3 (2CH of Bn), 124.3 (CH of Fe), 116.5 (CH of Bn), 116.1 (CH of Fe), 115.2 (CH=CHCO₂), 111.3 (CH of Fe), 61.1 (CH₂ of Bn), 56.4 (OMe); HRESIMS (*m/z*): [M − H]⁻ calcd for C₁₇H₁₄NO₇, 344.0770; found, 344.0778.

2-Chloro-5-nitrobenzyl *trans*-ferulate (5, 48 mg, 0.13 mmol, 44%), white powder. ¹H NMR (500 MHz, (CD₃)₂CO, δ) 8.42 (br d, *J* = 3.0, 1H, CH of Bn), 8.26 (dd, *J* = 9.0, 3.0, 1H, CH of Bn), 7.81 (d, *J* = 9.0, 1H, CH of Bn), 7.71 (d, *J* = 16.0, 1H, CH=CHCO₂), 7.40 (d, *J* = 1.8, 1H, CH of Fe), 7.19 (dd, *J* = 8.0, 1.8, 1H, CH of Fe), 6.88 (d, *J* = 8.0, 1H, CH of Fe), 6.56 (d, *J* = 16.0, 1H, CH=CHCO₂), 5.43 (s, 2H, CH₂ of Bn), 3.93 (s, 3H, OMe); ¹³C NMR (125 MHz, (CD₃)₂CO, δ) 167.1 (C=O), 164.6 (Cq), 150.4 (Cq), 148.8 (Cq), 147.1 (CH=CHCO₂), 140.6 (Cq), 137.4 (Cq), 131.7 (CH of Bn), 127.3 (Cq), 125.2 (2CH of Bn), 124.4 (CH of Fe), 116.1 (CH of Fe), 114.7 (CH=CHCO₂),
111.4 (CH of Fe), 63.0 (CH₂ of Bn), 56.4 (OMe); HRESIMS (*m/z*): [M − H]⁻ calcd for C₁₇H₁₃ClNO₆, 362.0431; found, 362.0431.

2-Fluoro-5-nitrobenzyl *trans*-ferulate (6, 48 mg, 0.14 mmol, 46%), white powder. ¹H NMR (500 MHz, (CD₃)₂CO, δ) 8.43 (dd, *J* = 3.2, 2.9, 1H, CH of Bn), 8.33 (ddd, *J* = 9.0, 4.3, 3.0 Hz, 1H, CH of Bn), 7.67 (d, *J* = 16.0, 1H, CH=CHCO₂), 7.49 (t, *J* = 9.0, 1H, CH of Bn), 7.36 (d, *J* = 1.8, 1H, CH of Fe), 7.16 (dd, *J* = 8.0, 1.8, 1H, CH of Fe), 6.87 (d, *J* = 8.0, 1H, CH of Fe), 6.49 (d, *J* = 16.0, 1H, CH=CHCO₂), 5.39 (s, 2H, CH₂ of Bn), 3.91 (s, 3H, OMe); ¹³C NMR (125 MHz, (CD₃)₂CO, δ) 167.1 (C=O), 165.1 (d, *J* = 257.4, Cq), 150.4 (Cq), 148.8 (Cq), 146.9 (CH=CHCO₂), 127.2 (Cq), 127.1 (d, *J* = 6.1, CH of Bn), 126.9 (d, *J* = 10.5, CH of Bn), 126.7 (d, *J* = 17.1, Cq), 124.3 (CH of Fe), 117.7 (d, *J* = 24.3, CH of Bn), 116.1 (CH of Fe), 114.7 (CH=CHCO₂), 111.4 (CH of Fe), 59.6 (d, *J* = 3.9, CH₂ of Bn), 56.3 (OMe); HRESIMS (*m/z*): [M − H]⁻ calcd for C₁₇H₁₃FNO₆, 346.0727; found, 346.0731.

4-Nitrobenzyl *trans*-ferulate (7, 96 mg, 0.29 mmol, 97%), white powder. ¹H NMR (500 MHz, (CD₃)₂O, δ) 8.27 (m, 2H, CH of Bn), 7.72 (m, 2H, CH of Bn), 7.69 (d, *J* = 16.0, 1H, CH=CHCO₂), 7.35 (d, *J* = 2.0, 1H, CH of Fe), 7.17 (dd, *J* = 8.2, 2.0, 1H, CH of Fe), 6.88 (d, *J* = 8.2, 1H, CH of Fe), 6.51 (d, *J* = 16.0, 1H, CH=CHCO₂), 5.38 (s, 2H, CH₂ of Bn), 3.92 (s, 3H, OMe); ¹³C NMR (125 MHz, (CD₃)₂CO, δ) 167.1 (C=O), 150.3 (Cq), 148.8 (Cq), 148.6 (Cq) 146.7 (CH=CHCO₂), 145.4 (Cq), 129.4 (CH of Bn), 127.3 (Cq), 124.4 (CH of Fe), 124.2 (CH of Bn), 116.0 (CH of Fe), 115.0 (CH=CHCO₂), 111.4 (CH of Fe), 65.1 (CH₂ of Bn), 56.4 (OMe); HRESIMS (*m/z*): [M − H]⁻ calcd for C₁₇H₁₄NO₆, 328.0821; found, 328.0825.

## Synthesis of the feruloylated 4-nitrocatechol-1-yl-substituted butanetriol 4NTC-linker-Fe (12)

2-*O*--(But-3-enyloxy)-5-nitrophenol (10). To a solution of 4-nitrocatechol (4NTC, 1.00 g, 6.44 mmol, 1 equiv) in dry DMF (8 mL) were added potassium carbonate (1.06 g, 7.67 mmol, 1.2 equiv) and homoallylic bromide (670 µL, 6.51 mmol, 1 equiv) at 40 °C. After overnight stirring at 40 °C, the reaction mixture was concentrated under reduced pressure. The residue was redissolved in ethyl acetate, washed with a saturated aqueous sodium hydrogen carbonate solution, and brine. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Flash chromatography (gradient of ethyl acetate in petroleum ether from 0 to 50%) afforded 10 (516 mg, 2.47 mmol, 38%) as a solid. ¹H NMR (500 MHz, CDCl₃, δ) 7.82 (dd, *J* = 9.0, 3.0, 1H, CH of 4NTC), 7.80 (d, *J* = 3.0, 1H, CH of 4NTC), 6.90 (d, *J* = 9.0, 1H, CH of 4NTC), 5.91–5.83 (m, 1H, =CH), 5.23–5.16 (m, 2H, CH₂=), 4.21 (t, *J* = 6.6, 2H, CH₂O), 2.63 (br

qt, *J* = 6.6, 1.6, 2H, CH₂); ¹³C NMR (500 MHz, CDCl₃, δ) 151.3 (Cq), 145.8 (Cq), 142.1(Cq), 133.2 (=CH), 118.1 (CH₂=), 116.8 (CH of 4NTC), 110.4 (CH of 4NTC), 110.1 (CH of 4NTC), 68.5 (CH₂O), 33.2 (CH₂); HRESIMS (*m/z*): [M − H]⁻ calcd for C₁₀H₁₀NO₄, 208.0610; found, 208.0615.

(±)-4-*O*--(2-Hydroxy-4-nitrophenyl)-1,2,4-butanetriol (11). A solution of 10 (157 mg, 0.75 mmol, 1 equiv) in acetone/water 2.5–1, v/v (3.5 mL) was treated at 25 °C under stirring with NMMO (106 mg, 0.90 mmol, 1.2 equiv) and osmium tetroxide (2.5 wt % solution in *tert*-butanol, 38 µL) and stirred at room temperature for 18 h. 10% (w/v) aqueous sodium sulfite (0.5 mL) was added, and stirring was continued for 30 min. The product was extracted with ethyl acetate (three times with 10 mL) and washed with brine. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, which afforded the expected compound 11 (165 mg, 0.68 mmol, 90%) as a solid. [α]D²⁰ 0 (c 2.0, CH₃OH). The NMR data (CD₃OD) of the racemate 11 were consistent with those previously reported for the pure (S)-enantiomer [37] ([α]D²⁰ -52 (c 2.0, CH₃OH)).

(±)-4-*O*--(2-Hydroxy-4-nitrophenyl)-1-*O*-trans-feruloyl-1,2,4-butanetriol (12). The application of the general procedure for the enzymatic transesterifications was performed with 11 (70 mg, 0.29, mmol, 1 equiv) and the vinyl ferulate 2 (94 mg, 0.43 mmol, 1.5 equiv) to give 12 (113 mg, 0.27 mmol, 94%) as a white powder. ¹H NMR (500 MHz, (CD₃)₂CO, δ) 7.79 (dd, *J* = 9.0, 2.8, 1H, CH of 4NTC), 7.67 (d, *J* = 2.8, 1H, CH of 4NTC), 7.63 (d, *J* = 16.0, 1H, CH=CHCO₂), 7.33 (d, *J* = 2.0, 1H, CH of Fe), 7.20 (d, *J* = 9.0, 1H, CH of 4NTC), 7.13 (dd, *J* = 8.2, 2.0, 1H, CH of Fe), 6.87 (1H, d, *J* = 8.2, CH of Fe), 6.40 (d, *J* = 16.0, 1H, CH=CHCO₂), 4.47–4.42 (m, 1H), 4.40–4.36 (m, 1H), 4.23–4.16 (m, 3H), 3.95 (s, 3H, OMe), 2.18–2.11 (m, 1H), 2.01–1.94 (m, 1H); ¹³C NMR (125 MHz, (CD₃)₂CO, δ) 167.4 (C=O), 153.6 (Cq), 150.2 (Cq), 148.8 (Cq), 147.7 (Cq), 146.0 (CH=CHCO₂), 142.4 (Cq), 127.4 (Cq), 124.0 (CH of Fe), 117.1 (CH of 4NTC), 116.1 (CH of Fe), 115.7 (CH=CHCO₂), 112.4 (CH of 4NTC), 111.3 (CH of Fe), 110.8 (CH of 4NTC), 69.0 (CH₂), 67.1 (CH₂), 66.9 (CH), 56.3 (OMe), 33.7 (CH₂); HRESIMS (*m/z*): [M − H]⁻ calcd for C₂₀H₂₀NO₉, 418.1138; found, 418.1139.

## Screening of Fae(+) microorganisms in solid medium using X-α-L-Araf-Fe (1a)

The *Y. lipolytica* AnFaeA(+) strain was used to inoculate solid YNB medium (1.7 g/L YNB without casamino acid, 5 g/L ammonium chloride, 20 mL/L oleic acid, 10 g/L D-glucose, 2 g/L casamino acid, and 15 g/L bacto agar in 100 mM citrate–phosphate buffer at pH 5). The petri dishes were incubated for 48 h at 30 °C and then overlayed with a prepara-
tion of 1% (w/v) molten top agar containing the chromogenic
substrate **1a** (300 µg/mL and 0.5% DMSO) and *TxAbf*
(2 IU/mL). Once the top agar was solid, incubation at 37 °C for
1 h allowed the color to develop. *Y. lipolytica* strains that
contained no *AnFaeA* gene were also checked to remain color-
less after the addition of the chromogenic substrate and the
auxiliary enzyme.

Liquid-medium-based colorimetric assays using
4NTC–linker–Fe (12)

In a typical experiment, discontinuous enzyme assays were per-
formed in triplicates under buffered conditions (100 mM sodi-
um phosphate at pH 6.0) in the presence of 1.8 mM **12** and
3.6% DMSO, final concentrations. For the assay, this solution
was preincubated at 40 °C before *AnFaeA* addition. Aliquots
(25 µL) were stopped by cooling (at 0 °C) every 6 min over a
24 min period and mixed with 45 µL of a cooled 10 mM NaIO₄
solution (pH 2.0). After being kept for 5 min at 0 °C through-
hout, 45 µL of ethylene glycol were added, followed by 135 µL
of 2 M Na₂CO₃ after 5 min. The OD values at 530 nm were re-
corded on an Infinite M200 PRO (TECAN) microplate reader.
One international unit (IU) of Fae specific activity (SA,
expressed in µmol/min/mg or IU/mg) corresponds to the
amount of released 4NTC (in µmol) per minute per milligram
of protein. Negative controls containing all of the reactants
except the enzyme were always included in order to monitor
and correct for spontaneous hydrolysis of the substrate. Control
reactions containing **12**, **11** and 4NTC without enzyme, and
4NTC without both enzyme and ethylene glycol were also pre-
pared.

Acknowledgements

The NMR work carried out in this work at TBI (Toulouse,
France) was performed with the equipment of Meta-Toul
(Metabolomics & Fluxomics Facitilities, Toulouse, France,
http://www.metatoul.fr). MetaToul is part of the national infra-
structure MetaboHUB (The French National Infrastructure for
Metabolomics and Fluxomics, http://www.metabohub.fr) and is
supported by grants from the Région Midi-Pyrénées, the Euro-
pean Regional Development Fund, SICOVAL, IBiSa-France,
CNRS and INRAE. We thank the ICEO facility dedicated to en-
zyme screening and discovery and part of the Integrated
Screening Platform of Toulouse (PICT, IBiSA) for providing
access to their equipment. The authors wish to acknowledge the
support from the ICMG Chemistry Nanobio Platform
(Grenoble, France) for HRMS analyses. Moreover, the authors
acknowledge that this work is based on a manuscript that is
included in the PhD thesis by J. D. titled “Approches multiples
d’ingénierie pour l’utilisation d’enzymes hydrolytiques comme
outils de synthèse”. INSA de Toulouse, France, 2017 (https://
tel.archives-ouvertes.fr/tel-02011218).

Funding

This work was supported by the European Union's Seventh
Programme for Research, Technological Development and
Demonstration under Grant Agreement No 613868, the
OPTIBIOCAT project (to O. G. and J. D.) by the French
National Research Agency, grant ANR-05-PNRB-002, the proj-
ect SPPECABBE (to M. R.), and the Région Midi-Pyrénées
grant DAER-Recherche 07009817 (to F. F.).

ORCID® iDs

Mélanie Ragon - https://orcid.org/0000-0002-1676-4764

Julien Durand - https://orcid.org/0000-0002-5631-6210

Sophie Bozonnet - https://orcid.org/0000-0001-5091-2209

Michael J. O'Donohue - https://orcid.org/0000-0003-4246-3938

Régis Fauré - https://orcid.org/0000-0002-5107-9009

Preprint

A non-peer-reviewed version of this article has been previously published as a preprint: https://doi.org/10.3762/bxiv.2020.107.v1

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