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

Article abstract of DOI:10.1016/j.molcatb.2016.01.012

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 straightf

Full text of DOI:10.1016/j.molcatb.2016.01.012

Journal of Molecular Catalysis B: Enzymatic 126 (2016) 24–31
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Design of chromogenic probes for efficient screening and evaluation
of feruloyl esterase-like activities
Olga Gherboveta,b,c,1, Régis Fauréa,b,c,∗,1, Fernando Ferreiraa,b,c, Julien Duranda,b,c
,
Mélanie Ragona,b,c, Guillaume Hostyna,b,c, Eric Recordd,e, Sophie Bozonneta,b,c
Michael J. O’Donohuea,b,c,∗
,
a Université de Toulouse, INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France
b INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France
c CNRS, UMR5504, F-31400 Toulouse, France
d INRA, UMR1163 Biotechnologie des Champignons Filamenteux, Aix-Marseille Université, Polytech Marseille, 163 Avenue de Luminy, CP 925, F-13288
Marseille, France
e Aix-Marseille Université, UMR1163 Biotechnologie des Champignons Filamenteux, Faculté des Sciences de Luminy-Polytech, CP 925, F-13288 Marseille
Cedex 9, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Feruloyl esterases (FAEs) constitute an important sub-group of hydrolytic enzymes involved in the decon-
struction 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.
Received 16 November 2015
Received in revised form 15 January 2016
Accepted 25 January 2016
Available online 28 January 2016
Keywords:
Feruloyl esterase
High-throughput screening
Chromogenic substrate
Indolyl hydroxycinnamates
Nitrocatechol ferulate
© 2016 Elsevier B.V. All rights reserved.
1. Introduction
useful for high-throughput screening [3], because these are gener-
ally relatively easy to use and can often serve in liquid assays.
Enzyme discovery is being increasingly empowered by high-
throughput technologies, such as functional metagenomics and
directed evolution techniques, which in turn are creating a new bot-
tleneck linked to screening and further characterization of newly
identified biocatalysts [1,2]. Therefore, there is a pressing need to
develop compounds that can provide the basis for informationally-
rich high-throughput screens that will not only pinpoint target
activities but also provide more detailed qualitative data on enzyme
properties. So far, chromogenic substrates have proven to be very
The development of enzymes for biorefinery purposes is cur-
rently a major target for enzyme discovery, with techniques such as
metagenomics are providing a vast reservoir of candidate enzymes.
In this area, a lot of focus has been on the discovery and engi-
neering of glycoside hydrolases (GHs) [4,5], notably cellulases.
However, there is increasing recognition that other so-called aux-
iliary enzymes are also required for biomass hydrolysis, with
these including carbohydrate esterases and particularly feruloyl
esterases (FAEs) [6–8].
Hydroxycinnamoyl esters (e.g. trans-ferulic acid and p-coumaric
acid) can constitute up to 3% (w/w, dry weight) of plant cell
walls where they play vital roles in maintaining cell-wall integrity
[9,10]. Trans-ferulic acid, which is the most widespread and abun-
dant hydroxycinnamic acid in plants, is usually ester-linked to
polysaccharides, such as arabinoxylans (attached to O-5 or O-
2 of ␣-l-arabinofuranosyl side chain), xyloglucans (attached to
O-4 to ␤-d-xylopyranosyl residues), arabinans (attached to O-
2 of ␣-l-arabinofuranosyl units) and arabinogalactan (attached
to O-6 of ␤-d-galactopyranosyl residues) [7,9,11]. When linked
Abbreviations:
BI-Co, 5-bromoindol-3-yl coumarate; CinnAE, cinnamoyl
esterase; CSA, camphor sulphonic acid; DIPC, N,Nꢀ-diisopropylcarbodiimide; DMAP,
4-(dimethylamino)pyridine; FAE, feruloyl esterase; GH, glycoside hydrolase;
HTS, high-throughput screening; 4NTC, 4-nitrocatechol ferulate; 4NTC-Fe, 4-
nitrocatechol-1-yl ferulate; X-Fe, 5-bromo-4-chloroindol-3-yl ferulate.
∗
Corresponding author.
E-mail addresses: regis.faure@insa-toulouse.fr (R. Fauré),
michael.odonohue@insa-toulouse.fr (M.J. O’Donohue).
1
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.molcatb.2016.01.012
1381-1177/© 2016 Elsevier B.V. All rights reserved.

O. Gherbovet et al. / Journal of Molecular Catalysis B: Enzymatic 126 (2016) 24–31
25
Fig. 1. Previously reported synthetic substrates (A), with (1a,d–f,h and 2a) or without sugar moiety (3d,e,g,h and 4h), and substrates synthesized in this work (B). All of
these substrates can be used to screen for and measure FAE activities. The sites of enzymatic cleavage are marked with dashed lines.
to polysaccharides, feruloyl moieties can be engaged in di-
ferulic dehydrodimers that constitute either intermolecular ester
crosslinks between polysaccharides or ester-ether bridges between
polysaccharides and lignin, both contribute to the overall plant
cell wall architecture. To release hydroxycinnamic acids from plant
cell material and thus, at least in the case of ferulic acid, disrupt
the cross-linking of cell wall components many lignocellulolytic
microorganisms (exemplified by some filamentous fungi) produce
carbohydrate esterases (FAEs and cinnamoyl esterases, CinnAEs,
E.C. 3.1.1.73) [11,12] that cleave the ester bonds linking feruloyl
moieties (i.e. monomeric and/or dimeric motifs) to sugars. FAEs
and CinnAEs belong to the larger category of carboxylic ester hydro-
lases, and, within the CAZy classification (www.cazy.org) [13], are
mostly found in family CE1. Thus far, the only mechanism attributed
to FAEs and CinnAEs is a two-step reaction involving the formation
of a covalent acyl-enzyme intermediate. The reaction is catalysed
by a Ser-His-Asp catalytic triad, with Ser acting as a nucleophile
and His as general base for the acylation step. Presently, carbohy-
drate esterases are classified into four subgroups based on substrate
specificity and sequence similarity [14–16].
5-O-(trans-feruloyl)-␣-l-arabinofuranoside moiety connected to a
chromogenic (e.g. 5-bromo-4-chloro-3-indolyl 1a, 4-nitrophenyl
1d, 2-chloro-4-nitrophenyl 1e) [18–20] or a fluorescent (umbellif-
eryl 1f) [21] group. In addition to the multi-step synthetic pathways
required to procure these compounds, their use entails a tandem
reaction involving ester bond cleavage and almost concomitant
release of the chromogenic group. This second reaction requires
the use of a sufficient quantity of ␣-l-arabinofuranosidase, which
implies that the overall reaction must be properly optimized to
avoid a situation in which the second (auxiliary enzyme-catalyzed)
reaction becomes rate-limiting [18,22]. Consequently, although
these substrates are useful they are not particularly convenient
for high-throughput screening (HTS) assays, where simplicity and
rapidity are paramount.
Interestingly, structure examination of a mutant of feruloyl
esterase A from Aspergillus niger (AnFAEA) complexed with a feru-
loylated arabinoxylo-trisaccharide revealed that this enzyme does
not interact with the glycosidic part of the substrate, suggesting
that artificial substrates might not need to harbour sugar moieties
to be properly recognized by FAEs [23]. This knowledge confirms
that the preparation of ␣-l-arabinofuranoside-free substrates (e.g.
3e, Fig. 1A) is a well-founded strategy and is fortunate because
such compounds, combining shorter synthetic pathways and direct
detection, are better adapted to HTS assays [21,24–26]. Finally, it
is noteworthy that despite the fact that previously described com-
pounds have been shown to be potentially suitable for the detection
of FAEs in solid medium [18,27], so far none of these have been
successfully employed in a HTS strategy.
So far the detection and characterization of FAE activities has
mostly relied on the use of UV spectrophotometry (i.e. exploiting
the difference in molar absorption coefficient between ester-
ified and free ferulic acid) or HPLC, using various natural or
synthetic compounds, such as biomass-derived hydroxycinnamic-
esterified oligosaccharides, methyl ferulate (3h, Fig. 1A) and its
analogues [17]. Recently, several substrates for FAEs have been
reported in the literature (Fig. 1A), most of them containing a

26
O. Gherbovet et al. / Journal of Molecular Catalysis B: Enzymatic 126 (2016) 24–31
3a (70 mg, 166 ␮mol, 85%) as green foam. 1H NMR (500 MHz;
CDCl3): ı 8.13 (1H, br s, NH), 7.88 (1H, d, J 15.9 Hz, CH CHCO2), 7.38
(1H, dd, J 0.4, 8.7 Hz, CH of X), 7.33 (1H, dd, J 0.4, 2.8 Hz, CH of X), 7.17
(1H, ddd, J 0.5, 2.0, 8.2 Hz, CH of Fe), 7.12 (1H, br d, J 2.0 Hz, CH of
Fe), 7.10 (1H, d, J 8.7 Hz, CH of X), 6.97 (1H, d, J 8.2 Hz, CH of Fe), 6.55
(1H, d, J 15.9 Hz, CH CHCO2), 3.97 (3H, s, OMe); 13C NMR (125 MHz;
CDCl3): ı 165.9 (C O), 148.4 (Cq), 147.0 (CH CHCO2), 146.8 (Cq),
133.4 (Cq), 129.6 (Cq), 127.3 (CH of X), 126.8 (Cq), 124.3 (Cq), 123.6
(CH of Fe), 119.3 (Cq), 116.1 (CH of X), 114.8 (CH of Fe), 114.3, 114,3
(Cq and CH CHCO2), 111.4 (CH of X), 109.5 (CH of Fe), 56.0 (OMe);
ES–HRMS calc. for C18H13BrClNO4Na ([M + Na]+) m/z 443.96142;
found: 443.9615. Elemental analysis calc. for C18H13BrClNO4: C,
51.15; H, 3.10; N, 3.31; found: C, 50.19; H, 3.33; N, 3.22%.
The ambition to design simple-to-use compounds harbour-
ing a hydroxycinnamic acid moiety that can be deployed in HTS
strategies to detect FAEs is central to the present study. Herein,
we report the synthesis and the evaluation of several types
of compounds including indolyl- (i.e. 5-bromo-4-chloroindol-3-
yl or 5-bromoindol-3-yl) and 4-nitrocathechol-based derivatives
(4NTC) coupled to either ferulic or p-coumaric acids, 3a, 3c and 4b
(Fig. 1B). We demonstrate that these versatile chromogenic sub-
strates can provide the basis for HTS assays for FAEs, working in
either solid (qualitative assay) or liquid (quantitative assay) media.
2. Material and methods
2.1. Chemical synthesis of chromogenic hydroxycinnamates
2.1.3. 4-Nitrocatechol-1-yl 4-O-acetyl-ferulate (7c)
2.1.1. N-Acetyl-5-bromo-4-chloroindol-3-yl 4-O-acetyl-ferulate
(7a)
Coupling method C: Thionyl chloride (0.92 mL, 12.63 mmol, 3
equiv.) and a drop of DMF were added to a solution of the 4-O-
acetyl-ferulic acid 5a [17] (1.0 g, 4.21 mmol, 1 equiv.) in toluene
(18 mL). The resulting mixture was refluxed (95 ◦C) for 1 h, and
then evaporated to dryness, providing the expected acyl chloride
derivative as brown foam in quantitative yield. 4-Nitrocathechol
sulfate dipotassium salt (1.31 g, 4.21 mmol, 1 equiv.) diluted in DMF
(6 mL) and triethylamine (0.48 mL, 3.58 mmol, 0.85 equiv.) and a
catalytic amount of DMAP (0.13 mg, 1.05 mmol, 0.25 equiv.) were
added to a solution of the previous acyl chloride in dichloromethane
(12 mL). After stirring for 3 h at room temperature, the reac-
tion mixture was poured into a solution of camphor sulphonic
acid (CSA; 1.15 g, 5.04 mmol, 1.2 equiv.) in acetone (315 mL). Sol-
vents were removed under reduced pressure and the residue was
recovered in a saturated aqueous sodium hydrogencarbonate solu-
tion, then extracted with ethyl acetate. The combined organic
extracts were washed sequentially in water then brine, dried on
magnesium sulfate, filtered and evaporated. Flash chromatogra-
phy (dichloromethane–acetone, 1:0 to 9:1 v/v) afforded pure 7c
(779 mg, 2.08 mmol, 50%) as white powder: 1H NMR (500 MHz,
(CD3)2CO): ı 8.12 (1H, s, CH of 4NTC), 8.11 (1H, dd, J 2.8, 8.8, CH
of 4NTC), 7.89 (1H, d, J 16.0 Hz, CH CHCO2), 7.59 (1H, d, J 2.0 Hz,
CH of Fe.), 7.38 (1H, ddd, J 0.5, 2.0, 8.1, CH of Fe), 7.21 (1H, dt, J
1.5, 8.8 Hz, CH of 4NTC), 7.17 (1H, d, J 8.1 Hz, CH of Fe), 6.84 (1H,
d, J 16.0 Hz, CH CHCO2), 3.94 (s, 3H, OMe), 2.27 (s, 3H, OAc); 13C
NMR (125 MHz, (CD3)2CO): ı 168.8 (C O), 164.9 (C O), 156.5 (Cq),
152.8 (Cq), 147.2 (CH CHCO2), 143.2 (Cq), 141.2 (Cq), 139.2 (Cq),
133.9 (Cq), 124.3 (CH of Fe), 123.8 (CH of 4NTC), 122.6 (CH of Fe),
120.6 (CH of 4NTC), 117.7 (CH CHCO2), 117.6 (CH of 4NTC), 112.7
(CH of Fe), 56.5 (OMe), 20.5 (OAc); ES–HRMS calc. for C18H15NO8Na
([M + Na]+) m/z 396.0695; found: 396.0696.
Coupling method A: N,Nꢀ-Diisopropylcarbodiimide (DIPC;
95 ␮L, 0.60 mmol,
4 equiv.) and a catalytic amount of 4-
(dimethylamino)pyridine (DMAP) were added to a solution of
4-O-acetyl-ferulic acid 5a [17] (71 mg, 0.30 mmol, 2 equiv.) and
N-acetyl-5-bromo-4-chloroindoxyl-3-ol [28] (43 mg, 0.15 mmol,
1 equiv.) in dichloromethane (2 mL). After stirring for 1 h at room
temperature, the reaction was diluted with ethyl acetate, filtered
through Celite and washed with brine. The organic layers were
dried and evaporated to dryness.
Coupling method B: Thionyl chloride (1.27 mL, 17.3 mmol, 3
equiv.) and a drop of dimethylformamide (DMF) were added to a
solution of 4-O-acetyl-ferulic acid [17] (1.37 g, 5.8 mmol, 1 equiv.)
in toluene (15 mL). The resulting mixture was refluxed (95 ◦C)
for 1 h, and then evaporated to dryness, providing the expected
acyl chloride derivative [24] as yellow foam in quantitative yield.
N-Acetyl-5-bromo-4-chloroindoxyl-3-ol [28] (0.56 g, 1.9 mmol, 1
equiv.) diluted in pyridine (5 mL) and a catalytic amount of DMAP
were added to a solution of the previous acyl chloride (1.48 g,
5.8 mmol, 3 equiv.) in pyridine (5 mL). After stirring for 1 h at room
temperature, the reaction was diluted with ethyl acetate, washed
with saturated aqueous sodium hydrogencarbonate (three times)
and brine, dried and concentrated.
Flash chromatography (using gradient dichloromethane-
petroleum ether, 95:5 to 100:0 v/v) followed by recrystallization
from ethyl acetate afforded pure 7a (0.58 g, 1.1 mmol, 60%) as
white needles. mp 201–203 ◦C (from EtOAc); 1H NMR (500 MHz;
CDCl3): ı 8.34 (1H, d, J 8.9 Hz, CH of X), 7.92 (1H, d, J 16.0 Hz,
CH CHCO2), 7.72 (1H, s, CH of X), 7.60 (1H, d, J 8.9 Hz, CH of X),
7.22 (1H, dd, J 1.8, 8.1 Hz, CH of Fe), 7.18 (1H, d, J 1.8 Hz, CH of Fe),
7.11 (1H, d, J 8.1 Hz, CH of Fe), 6.61 (1H, d, J 16.0 Hz, CH CHCO2),
3.91 (3H, s, OMe), 2.64 (3H, s, NAc), 2.34 (3H, s, OAc); 13C NMR
(125 MHz; CDCl3): ı 168.7 (C O,), 168.4 (C O), 164.3 (C O) 151.6
(Cq), 147.0 (CH CHCO2), 142.1 (Cq), 133.3 (Cq), 133.1 (Cq), 132.8
(Cq), 130.6 (CH of X), 124.6 (Cq), 123.5 (CH of Fe), 122.6 (Cq), 121.7
(CH of Fe), 118.7 (Cq), 116.5 (CH CHCO2), 116.4 (CH of X), 116.1
(CH of X), 111.5 (CH of Fe), 56.0 (OMe), 23.8 (NAc), 20.6 (OAc);
ES–HRMS calc. for C22H17BrClNO6Na ([M + Na]+) m/z 527.98255;
measured 527.9825. Elemental analysis calc. for C22H17BrClNO6:
C, 52.15; H, 3.38; N, 2.76; found: C, 51.80; H, 3.34; N, 2.72%.
2.1.4. 4-Nitrocatechol-1-yl ferulate (3c)
Deacetylation method F: Amano lipase PS (3.46 g) was added to
a solution of 13 (778 mg, 2.08 mmol) in dichloromethane (38 mL)
and 2-propanol (19 mL). The reaction mixture was stirred for 3 days
at 37 ◦C. The reaction was terminated by enzyme removal using fil-
tration. The filter cake was washed with dichloromethane and the
filtrate was concentrated by evaporation of the solvents at reduced
pressure to afford the product 3c as yellow powder (635 mg,
1.91 mmol, 92%). 1H NMR (500 MHz; (CD3)2CO): ı 8.11–8.08 (2H,
m, CH of 4NTC), 7.81 (1H, d, J 15.9 Hz, CH CHCO2), 7.45 (1H, d, J
2.0 Hz, CH of Fe), 7.26 (1H, ddd, J 0.5, 2.0, 8.2 Hz, CH of Fe.), 7.20
(1H, dd, J 0.5, 9.9 Hz, CH of 4NTC), 6.92 (1H, d, J 8.2 Hz, CH of Fe),
6.64 (1H, d, J 15.9 Hz, CH CHCO2), 3.96 (3H, s, OMe); 13C NMR
(125 MHz; (CD3)2CO): ı 165.3 (C O), 156.6 (Cq), 150.7 (Cq), 148.8
(Cq), 148.4 (CH CHCO2), 141.2 (Cq), 139.3 (Cq), 127.2 (Cq), 124.6
(CH of Fe), 123.7 (CH of 4NTC), 120.6 (CH of 4NTC), 117.6 (CH of
4NTC), 116.2 (CH of Fe), 114.0 (CH CHCO2), 111.6 (CH of Fe), 56.4
2.1.2. 5-Bromo-4-chloroindol-3-yl ferulate (3a)
Deacetylation method E: Hydrazine acetate (45 mg, 489 ␮mol,
2.5 equiv.) was added to a cooled solution of N-acetyl-5-bromo-
4-chloroindol-3-yl 4-O-acetyl-ferulate 7a (100 mg, 197 ␮mol, 1
equiv.) in DMF (2 mL). After stirring for 2 h at 0 ◦C, the reaction
was quenched by addition of ethyl acetate, washed with brine,
dried, and concentrated. Flash chromatography (using gradient
petroleum ether–ethyl acetate, 9:1–7:3 v/v) procured compound

O. Gherbovet et al. / Journal of Molecular Catalysis B: Enzymatic 126 (2016) 24–31
27
(OMe); ES–HRMS calc. for C16H13NO7Na ([M + Na]+) m/z 354.0590;
found: 354.0589.
for 30 min. The optical densities at 650 nm were recorded on a
VersaMax microplate reader (Molecular Devices). Negative con-
trols containing all of the reactants except the enzyme were always
included in order to correct for spontaneous hydrolysis of the sub-
strates.
2.1.5. N-Acetyl-5-bromoindol-3-yl 4-O-acetyl-coumarate (8b)
Coupling method A: DIPC (0.64 mL, 4.0 mmol, 4 equiv.) and
a catalytic amount of DMAP were added to a solution of 4-O-
acetyl-coumaric acid 6b [29] (412 mg, 2.0 mmol, 2 equiv.) and
N-acetyl-5-bromo-indoxyl-3-ol [30] (254 mg, 1.0 mmol, 1 equiv.)
in dichloromethane (15 mL). After stirring for 2 h at room tempera-
ture, the reaction was filtrated through Celite, washed with water,
dried and concentrated under reduced pressure. Flash chromatog-
raphy (dichloromethane-petroleum ether, 9:1 v/v) afforded pure
8b (416 mg, 0.94 mmol, 94%), which crystallized in ethyl acetate as
white needles. mp 156–158 ◦C (from EtOAc); 1H NMR (500 MHz;
CDCl3): ı 8.38 (1H, d, J 8.9 Hz, CH of BI), 7.90 (1H, d, J 16.0 Hz,
CH CHCO2), 7.84 (1H, s, CH of BI), 7.76 (1H, dd, J 0.5, 2.0 Hz, CH
of BI), 7.67–7.64 (2H, m, CH of Co), 7.50 (1H, ddd, J 0.5, 2.0, 8.9 Hz,
CH of BI), 7.21–7.19 (2H, m, CH of Co), 6.64 (1H, d, J 16.0 Hz,
CH CHCO2), 2.63 (3H, s, NAc), 2.34 (3H, s, OAc); 13C NMR (125 MHz;
CDCl3): ı 169.0 (C O), 168.6 (C O), 163.6 (C O), 152.7 (Cq), 146.4
(CH CHCO2), 133.7 (Cq), 131.6 (Cq), 131.5 (Cq), 129.6 (2C, CH of
Co), 129.1 (CH of BI), 125.4 (Cq), 122.4 (2C, CH of Co), 120.4 (CH
of BI), 118.3 (CH of BI), 117.1 (Cq), 116.2 (CH CHCO2), 114.4 (CH
of BI), 23.8 (NAc), 21.1 (OAc); ES–HRMS calc. for C21H16BrNO5Na
([M + Na]+) m/z 464.01095; found: 464.0113. Elemental analysis
calcd for C21H16BrNO5 C, 57.03; H, 3.65; N, 3.17; found: C, 56.73;
H, 3.56; N, 3.10%.
2.4. Liquid medium-based colorimetric assays using 4NTC-Fe
First an aliquot of the stock solution of 3c (50 mM 4NTC-Fe) in
DMSO was diluted in 100 mM sodium phosphate buffer pH 6.0 to
achieve a final concentration of 2 mM 4NTC-Fe in 4% (v/v) DMSO.
For the assay, 400 ␮L of this solution was incubated at 40 ◦C for
4 min and then combined with 50 ␮L of enzyme solution. After
enzyme addition, 4NTC release was monitored in a discontinuous
assay, removing 50 ␮L aliquots every 3 min over a 21 min-period.
Upon removal, the aliquots were immediately mixed in 96-well
microplates with 200 ␮L of an aqueous solution of ice-cold 2 M
Na2CO3 and absorbance at 530 nm was recorded using a microplate
reader (Tecan Infinite 200 PRO). Afterwards, activity was calculated
using a standard absorbance curve of 4NTC (from 0.01 to 1 mM)
in an aqueous solution of 2 M Na2CO3 containing an appropriate
amount of sodium phosphate buffer (i.e. 20 mM final concentra-
tion). 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.
2.5. Screening of FAE(+) microorganisms in solid medium
A mixture of the strains Yarrowia lipolytica zeta (Ura−) and Y.
lipolytica AnFaeA(Ura3+) were grown for 27 h at 28 ◦C with shaking
at 650 rpm in 96-well microtiter plates containing YPD medium
(composed of 10 g/L yeast extract, 20 g/L bactopeptone and 20 g/L
glucose). With the aid of a colony picker QPixII, (Genetix), the 96
cultures were then used to inoculate OmniTray plates containing
solid YPD (i.e. containing 1% w/v agar) overlaid with a nitrocellulose
membrane. OmniTrays were incubated at 28 ◦C for 36 h and then
the membranes were transferred onto fresh OmniTrays containing
150 ␮g/mL (0.2% final of DMSO, v/v) of either 4NTC-Fe 3c, X-Fe 3a
or BI-Co 4b in 50 mM sodium phosphate buffer pH 6.8, containing
1% (w/v) agarose. For 4NTC-Fe, ammonium iron citrate (0.5 mM)
was also added to the medium. OmniTrays were incubated at 37 ◦C
in order to follow the coloration of colonies producing FAE activity.
2.1.6. 5-Bromoindol-3-yl coumarate (4b)
Deacetylation method D: N-acetyl-5-bromoindol-3-yl 4-O-
acetyl-coumarate 8b (277 mg, 0.63 mmol, 1 equiv.) was treated
in methanol (15 mL) with sodium methoxide (1 M in methanol,
0.63 mL, 0.63 mmol, 1 equiv.) for 2 h at 0 ◦C. The solution was neu-
tralized with Amberlite IR-120 (H+), filtered, and then concentrated
under reduced pressure. Flash chromatography (using gradient
dichloromethane then dichloromethane–methanol, 99.5:0.5 to
99:1 v/v) gave compound 4b (146 mg, 0.41 mmol, 65%) as a slightly
dark foam. 1H NMR (500 MHz; (CD3)2CO): ı 7.87 (1H, d, J 16.0 Hz,
CH CHCO2), 7.71 (1H, td, J 0.6, 2.0 Hz, CH of BI), 7.69–7.66 (2H,
m, CH of Co), 7.57 (1H, dd, J 0.4, 2.7 Hz, CH of BI), 7.40 (1H, dd,
J 0.6, 8.7 Hz, CH of BI), 7.27 (1H, ddd, J 0.4, 2.0, 8.7 Hz, CH of BI),
6.96–6.93 (2H, m, CH of Co), 6.66 (1H, d, J 16.0 Hz, CH CHCO2);
13C NMR (125 MHz; (CD3)2CO): ı 165.4 (C O), 161.0 (Cq), 147.2
(CH CHCO2), 133.1 (Cq), 131.4 (2C, CH of Co), 130.5 (Cq), 127.0
(Cq), 125.7 (CH of BI), 122.9 (Cq), 120.7 (CH of BI), 116.9 (2C, CH
of Co), 116.7 (CH of BI), 114.6 (CH of BI), 114.5 (CH CHCO2), 112.9
(Cq); ES–HRMS calc. for C17H12BrNO3Na ([M + Na]+) m/z 379.98982;
found: 379.9902.
3. Results and discussion
3.1. Synthesis of substrates
The synthetic routes yielding chromogenic compounds 3a, 3c
and 4b are summarized in Scheme 1 and Table 1.
2.2. Enzymes
The 4-bromo-5-chloro-3-indolyl ferulate (X–Fe, 3a) was synthe-
sized starting from the 4-O-acetyl-ferulic acid 5a [17] (obtained
from almost-quantitative acetylation of the ferulic acid). Two dif-
ferent pathways were used for the esterification of the carboxylic
acid function. In the first strategy, coupling involved direct esterifi-
cation of carboxylic acid 5a using DIPC activation in the presence of
a catalytic amount of DMAP in dichloromethane following an adap-
tation of the previously reported Steglich reaction protocol [25].
The crystallized ferulate 7a was thus obtained in 65% yield. The
second strategy involved an initial treatment of 5a with thionyl
chloride catalyzed by DMF in refluxing toluene, to quantitatively
yield the 4-acetoxyferuloyl chloride 5c [17], followed by coupling
to N-acetyl-5-bromo-4-chloroindoxyl-3-ol [28] in pyridine, leading
to the protected ferulate 7a in 60% yield over two steps. Catalytic
deacetylation was first attempted using sodium methoxide in a
methanol/dichloromethane mixture, but this afforded the target
compound 3a in poor yield (26%) due to transesterification. When
Feruloyl esterases from A. niger, AnFAEA and AnFAEB, were pro-
duced and purified as previously described [31,32].
2.3. Liquid medium-based, colorimetric assays using indolyl
compounds
The semi-quantitative measurement of FAE activity was per-
formed in a continuous assay carried out in buffered conditions in
the presence of either X-Fe 3a or BI-Co 4b (100 ␮g/mL). Solutions
of the 3a or 4b (100 ␮g/mL) were prepared by diluting aliquots of
the initial stock solution (20 mg/mL in DMSO) with buffer (i.e. with
DMSO at a final concentration of 0.5%, v/v). Prior to the addition
of enzyme solution, the reaction mixture was pre-incubated at the
optimal temperature for 5 min. Reaction mixtures (200 ␮L) were
incubated in 96-well microtiter plates under stirring at 500 rpm

28
O. Gherbovet et al. / Journal of Molecular Catalysis B: Enzymatic 126 (2016) 24–31
Scheme 1. Synthesis of ferulate (3a and 3c) and p-coumarate (4b) derivatives. Reagents and conditions: (i) 5a or 6b, N-acetyl-5-bromo-4-chloroindoxyl-3-ol or N-acetyl-
5-bromoindoxyl-3-ol, DIPC, DMAP, CH2Cl2, 2 h, 7a: 65%, 8b: 94%; or 5c, N-acetyl-5-bromo-4-chloroindoxyl-3-ol, pyridine, DMAP, 1 h, 7a: 60%; or 5c, 4-nitrocatechol
sulfate dipotassium salt, Et3N, DMAP, CH2Cl2/DMF, 3 h then CSA, acetone, 30 min, 7c: 50% (2 steps); (ii) 7a, hydrazine acetate, DMF, 2 h, 0 ◦C, 3a: 85%; or 7c, lipase PS,
CH2Cl2/isopropanol, 72 h, 37 ◦C, 3c: 92%; or 8b, MeONa, MeOH, 2 h, 0 ◦C, 4b: 65%.
Table 1
Synthesis of ferulate (3a and 3c) and p-coumarate (4b) derivatives: summary of tested pathways and yields for the coupling and deacetylation steps.
Starting compound
(i) Coupling
Methoda
A
(ii) Deacetylation
Methoda
Protected compound
Yield (%)
65
Deprotected compound
Yield (%)
5a
7a
D
E
3a
26
85
5c
5a
5c
5c
6b
B
A
B
C
A
60
b
Fd
7c
−
3c
92
c
−
50
94
E
D
<10
65
8b
4b
a
Couplig method A: N-acetyl-indoxyl-3-ol derivative or 4NTC sulfate dipotassium salt, DIPC, DMAP, CH2Cl2; Coupling method B: N-acetyl-indoxyl-3-ol derivative or 4NTC
sulfate dipotassium salt, pyridine, DMAP; Coupling method C: 4NTC sulfate dipotassium salt, Et3N, DMAP, CH2Cl2/DMF then CSA, acetone; Deacetylation method D: MeONa,
MeOH; Deacetylation method E: hydrazine acetate, DMF; and Deacetylation method F: lipase PS, CH2Cl2/isopropanol.
b
No trace of the expected compound 7c was detected, but formation of the corresponding N-(ferulic acid) acylcarbamide in 76% yield was obtained (see Supplementary
data (Appendix A)).
c
No trace of the desired ferulate 7c was detected, but di-feruloylated 4NTC was obtained in 49% yield. This was successfully deacetylated using the enzymatic method (E),
procuring 4NTC–1,2-yl bis-ferulate in 87% yield (see Supplementary data (Appendix A)).
d
Other attempts to obtain 3c using method (D) or with anhydrous K2CO3 in a mixture of CH2Cl2/MeOH, led to its decomposition.
milder conditions were applied (i.e. hydrazine acetate in cold DMF),
the desired chromogenic substrate 3a was obtained in 85% yield.
The same strategy was applied to access 4-bromo-indol-3-yl p-
coumarate (BI-Co, 4b) from p-coumaric acid. The latter was first
acetylated in 92% yield using acetic anhydride in pyridine [29]. Effi-
cient esterification of 6b with N-acetyl-5-bromo-indoxyl-3-ol [33]
using DIPC/DMAP activation in dichloromethane gave p-coumarate
8b in 94% yield. Catalytic deacetylation using sodium methoxide in
methanol afforded the targeted chromogenic coumarate 4b in 65%
yield. The synthesis of indolyl derivatives 3a and 4b was achieved
in 4 steps starting from commercial sources and provided overall
yields of 55 and 56%, respectively.
The synthesis of a single regioisomer of 4-nitrocatechol-1-
yl ferulate (4NTC-Fe, 3c) was achieved through the selective

O. Gherbovet et al. / Journal of Molecular Catalysis B: Enzymatic 126 (2016) 24–31
29
Fig. 2. Detection in solid medium of FAE activity using 3a (A), 3c (B) and 4b (C). Y. lipolytica cells producing AnFAEA were characterized by the formation of turquoise (A)
or dark blue (C) haloes, or the development of a brown colour (B) within and around the colony. Cells not producing AnFAEA failed to display haloes and thus remained
uncoloured. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 3. Detection of FAE activity in liquid medium: hydrolysis of 3a, 4b and 3c in 96-well microtiter plates. (A) Buffered, 30-min reactions in the presence or absence of FAE
(+ and − respectively); (B) and (C) discontinuous time-dependent monitoring of both autohydrolysis ( ) and AnFAEA-mediated hydrolysis ( ) of 3c.
esterification of one hydroxyl position of 4NTC using the commer-
cially available dipotassium salt of 4-nitrocatechol sulfate. Since
direct esterifications failed (see Supplementary data (Appendix A)),
coupling of the feruloyl chloride derivative 5c to 4NTC sulfate salt
in DMF in the presence of catalytic amounts of trimethylamine
and DMAP was performed and one-pot deprotection, using a solu-
tion of CSA in acetone [34], led to 7c in 50% overall yield. Selective
deacetylation was achieved using Amano lipase PS from Burkholde-
ria cepacia [24] (originally classified as Pseudomonas cepacia) in a
high 92% yield. Finally, 3c was obtained in 46% overall yield (in 4
steps) from commercial ferulic acid and 4NTC sulfate derivative.
435 or Amano lipase PS, lipases Lip2 and Lip8 from Y. lipolytica, or
lipases Lip1 and Lip4 from Candida rugosa—data not shown), but
were cleaved by FAEs (see below).
The suitability of 3a, 4b and 3c for the detection in solid medium
of microorganisms expressing FAE was evaluated using wild-type
Y. lipolytica Zeta cells [35] as negative control and recombinant Y.
lipolytica Zeta producing AnFAEA as the positive sample (unpub-
lished data). To complete the evaluation, the microbial samples
were also cultured on non-selective (i.e. YPD, which supports the
growth of both yeast strains) and selective media (i.e. YNB, which
only supports the growth of the URA3+, AnFAEA-producing strain),
in the absence of chromogenic substrates (Fig. A.5). When grown
in the presence of Y. lipolytica Zeta cells producing AnFAEA 3a were
characterized by the formation of turquoise haloes, while growth
in the presence of 4b and 3c led to the formation of dark blue and
brown coloration respectively (Fig. 2). Coloration in the case of 3a
and 4b was the result of enzymatic ester bond cleavage and sub-
sequent release of free indoxyl-3-ol groups that upon oxidation
dimerized forming indigo-like insoluble dyes [33]. However, in the
case of 3c coloration was due to enzymatic ester bond cleavage
followed by the chelation of free 4NTC with ferric ions [36,37].
Although all three compounds appeared to be suitable for quali-
tative HTS on solid medium, the compromise between sensitivity
of 3c and diffusion of 4NTC-iron(III) complex in the operating con-
ditions limits its use to short assays (<30 min), whereas thanks to
precipitation of their related dehydroindigo 3a and 4b are more
useful for longer assays, for example to detect weakly-expressed
FAE activity (Fig. A.6). It is also noteworthy that the usefulness of
3a, 4b and 3c was established in different assay conditions (i.e.
with or without top agar, or when using a membrane) and using
3.2. Evaluation and applications for hydroxycinnamoyl esterases
The stability of the three esters was evaluated over the pH range
4.0–10.0, using UV spectrophotometry to monitor the state of the
compounds (Figs. A.1–A.3). Indolyl derivatives 3a and 4b showed
very high stability over a wide pH range and up to high tempera-
ture, the first signs of degradation becoming apparent at pH 9.0 and
above 70 ◦C. These observations are consistent with the known sta-
bility of ester linkages under basic conditions. On the other hand,
the ferulate 3c was only moderately stable with hydrolysis being
observed even at neutral pH and 40 ◦C (Fig. A.4). Nevertheless,
4NTC-Fe 3c was stable enough to be used for the characterization
of FAEs operating in the pH range 5.0–6.0 and up to 40 ◦C, since
the appearance of free 4NTC due to spontaneous hydrolysis can be
easily corrected for by the use of proper controls without enzyme.
Furthermore, it is noteworthy that although FAEs and fungal lipase
share a very similar overall fold, catalytic triad and mechanism
[11], the ester linkage of 3a, 4b and 3c was stable towards vari-
ous lipases (with >1 IU/mL of commercial immobilized Novozyme

30
O. Gherbovet et al. / Journal of Molecular Catalysis B: Enzymatic 126 (2016) 24–31
different FAE(+)-microorganisms (i.e. A. niger or Y. lipolytica) (Fig. 2
and Figs. A.7,A.8).
Appendix A. Supplementary data
The indolyl-based compounds 3a and 4b and the 4NTC-ester
derivative 3c also proved to be suitable for use in liquid assays
aimed at semi-quantitative and quantitative analyses (Fig. 3).
Although the quantification of dehydroindigo in solution is quite
tricky, due to their relative insolubility in aqueous solutions and the
lack of suitable standards (i.e. pure dehydroindigo), 3a and 4b are
nevertheless well-adapted for primary HTS in 96-well microtiter
plates and spectrophotometric detection. The reproducibility of
this type of assay was evaluated using 3a and 4b, and 20 mIU/mL of
AnFAEs A and B in standard conditions (at 50 ◦C in 100 mM citrate-
phosphate buffer pH 6.0). After 30 min, the mean absorbance values
at 650 nm were 0.31 0.03 (33 repeats) and 0.12 0.01 (24 repeats)
respectively, thus the coefficient of variance (CV) of the assays
was 10.8 and 11.3% respectively. Moreover, since the optimum
concentration of 3a and 4b in this assay is 100 ␮g/mL, only 2 mg
per microtiter plate is required, assuming a detection threshold of
2 mIU/mL.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.molcatb.2016.01.
012.
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