Chromogenic substrates for feruloyl esterases

Article abstract of DOI:10.1016/j.carres.2007.06.004

Chromogenic mono- and diferuloyl-butanetriol analogs were prepared by chemical syntheses and their efficiency was evaluated as substrates for feruloyl esterases from Aspergillus niger.

Full text of DOI:10.1016/j.carres.2007.06.004

Carbohydrate Research 342 (2007) 2316–2321
Note
Chromogenic substrates for feruloyl esterases
a
b
b
b
`
Laurence Marmuse, Michele Asther, David Navarro, Laurence Lesage-Meessen,
b
a
a,
*
´
Marcel Asther, Sebastien Fort and Hugues Driguez
aCentre de Recherches, sur les Macromole´cules Ve´ge´tales, (CERMAV-CNRS ), BP 53, F-38041 Grenoble, France
bUMR-1163 INRA/Universite´ de Provence/Universite´ de la Me´diterrane´e de Biotechnologie des Champignons Filamenteux,
ESIL, 163 avenue de Luminy, CP 965, F-13288 Marseille, France
Received 13 April 2007; received in revised form 31 May 2007; accepted 1 June 2007
Available online 9 June 2007
Abstract—Chromogenic mono- and diferuloyl-butanetriol analogs were prepared by chemical syntheses and their efficiency was
evaluated as substrates for feruloyl esterases from Aspergillus niger.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Keywords: Feruloyl esterase; Chromogenic substrate
The enzymatic degradation of plant cell wall into fer-
mentable sugars involves not only glycanases but also
accessory enzymes like acetylxylan (AcXEs) and feruloyl
esterases (FAE).The first set of enzymes is able to O-
deacetylate xylans while the second one hydrolyzes phe-
nolic groups that play a major role in the cross-linkage
between hemicelluloses, pectin and lignin via ester
bonds.1,2 A growing interest in the search for new FAEs
with enhanced enzymatic and physicochemical proper-
ties has recently emerged with the need to improve bio-
ethanol production. Most of FAEs of eukaryotic and
prokaryotic origins belong to family 1 of the carbo-
hydrate esterase classification,3 and were recently orga-
nized in four classes A–D, which take into account
substrate specificities against synthetic methyl esters of
hydroxycinnamic acids, growth substrate requirements
of the microorganisms, and protein sequence identity.
In practice, enzymatic assay of FAEs has been carried
out using commercially available methyl and ethyl esters
of hydroxycinnamic acids or using naturally feruloyl-
ated oligosaccharides isolated by controlled enzymatic
digestion of arabinoxylan.4 In the same context, Hatfield
et al.5 synthesized methyl 5-O-trans-feruloyl-a-L-arabino-
furanoside (FA Ara). In these examples ultraviolet
spectrometric measurement was used to detect FAE
activity since ferulic acid has a maximum absorption
at 340 nm (pH 11) distinct from its esters at 375 nm.
Recently, 4-nitrophenyl 2- and 5-O-trans-feruloyl-a-L-
arabinofuranosides were found to be new chromogenic
substrates for various feruloyl esterases.6 The assay
was based on tandem reaction involving the splitting
of ester bond with almost concomitant release of
4-nitrophenol through addition of a-L-arabinofuran-
osidase in large quantity. However, all of these com-
pounds suffer from major and notable drawbacks like
the poor sensibility or stability under extreme pH and
temperature. Furthermore, it is clear that new substrates
for high-throughput screening of enzyme activities have
to be designed and synthesized. It was shown that esters
of 4-(nitrophenoxy)-1,2-butanediol provide excellent
chromogenic substrates for the detection of lipase and
esterase activities in microtiter plate setup.7 The reaction
product obtained after enzymatic treatment was sub-
jected to periodate oxidation and b-elimination trigger-
ing p-nitrophenolate liberation (Chart 1).
*
Corresponding author. Tel.: +33 476 037 664; fax: +33 476 547
203; e-mail: Hugues.Driguez@cermav.cnrs.fr
Following an equivalent strategy, we decided to inves-
tigate the synthesis of mono- and diferuloyl chromogenic
esters of (2-chloro-4-nitrophenyl)-1,2,4-butanetriol to
´
Affiliated with Universite Joseph Fourier, and member of the Institut
´
de Chimie Moleculaire de Grenoble FR-CNRS 2607.
0008-6215/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2007.06.004

L. Marmuse et al. / Carbohydrate Research 342 (2007) 2316–2321
2317
O
led the expected compound 8 in 74% yield over the
two steps. Selective protection of the primary hydroxyl
group of 3 was achieved by tritylation. Compound 9
was obtained in 81% yield and was then esterified in
92% yield by treatment with feruloyl chloride to afford
10. Removal of the acetyl group under basic conditions
and then acidic hydrolysis of the trityl group gave target
molecule 12.
O
esterase
RCO
O
HO
OH
OH
NO2
B-
NO2
NaIO4
O
H
-O
BSA
pH>7
NO2
O
NO2
O
O
NO2
Chart 1. Release of nitrophenolate after action of the esterase or lipase
on esters of 4-(nitrophenoxy)-1,2-butanediol with the presence of
NaIO4 and bovine serum albumin (BSA).
These three compounds 5, 8 and 12 were tested as po-
tential substrates for the feruloyl esterases FAEA and
FAEB isolated from A. niger. On the opposite of what
is described for lipases and esterases, a one-step proce-
dure could not be performed, probably due to inhibition
of feruloyl esterase activities at neutral or basic pH. Our
assay required to be first carried out at pH 6 for optimal
feruloylesterase activity before quantifying at pH 9 the
chloro-nitrophenolate ion released upon periodate oxi-
dation followed by b-elimination catalyzed by BSA.
Although this two-step procedure precludes its use in a
high-throughput manner, we analyzed the hydrolysis
of compounds 5, 8, 12 by pure FAEA and FAEB en-
zymes from A. niger and demonstrated a linear relation-
ship between the release of chloronitrophenol and the
incubation time. Moreover, the rate of the released chlo-
ronitrophenol was proportional to enzyme concentra-
tion (Fig. 1).
test them with feruloyl esterases from Aspergillus niger
FAEA and FAEB.
As shown in Scheme 1, tosylate displacement of com-
pound 18 by 2-chloro-4-nitrophenol led to 2 in 86%
yield. Removal of the isopropylidene group by acidic
treatment and esterification of the key diol 3 with an ex-
cess of feruloyl chloride afforded the protected disubsti-
tuted compound 4 in 64% yield. O-Deacetylation gave
target molecule 5 in 81% yield. Esterification of the pri-
mary hydroxyl of 3 under milder condition was achieved
in 58% yield. However, direct O-deacetylation of 6
occurred with ester migration. To solve this problem, the
secondary hydroxyl of 6 was temporarily blocked with
a tetrahydropyranyl (THP) moiety, and then this inter-
mediate was treated in basic conditions. THP removal
Only FAEA showed detectable activity on all sub-
strates with catalytic efficiencies in the order
Cl
ii
i
OH
Cl
O
O
2
a
c
HO
O
b
O
d
O
NO2
OTs
O
O
NO2
3
1
MeO
MeO
HO
AcO
O
O
O
Cl
Cl
O
O
5
O
iii
iv
O
O
O
NO2
NO2
MeO
HO
MeO
4
AcO
O
O
O
Cl
OTHP
7
Cl
Cl
O
OH
8
OH
6
vi
MeO
3
vii
v
O
O
O
O
NO2
O
NO2
NO2
MeO
HO
MeO
AcO
AcO
MeO
ix
MeO
MeO
AcO
HO
HO
O
O
O
OH
9
Cl
Cl
Cl
Cl
O
O
O
O
TrO
TrO
TrO
HO
x
viii
xi
NO2
O
NO2
O
NO2
NO2
O
10
11
12
Scheme 1. Reagents and conditions: (i) 2-chloro-4-nitrophenol, 18-Crown-6, K2CO3, acetone, 86%; (ii) AcOH aq, 94%; (iii) C5H5N, DMAP,
4-acetoxyferuloyl chloride, 64%; (iv) K2CO3, CH2Cl2–MeOH, 81%; (v) C5H5N, CH3CN, 4-acetoxyferuloyl chloride, 58%; (vi) Dihydropyran,
camphor-10-sulfonic acid, 98%; (vii) K2CO3, CH2Cl2–MeOH then, HCl, MeOH, 74%; (viii) TrCl, C5H5N, DMAP, 81%; (ix) C5H5N,
4-acetoxyferuloyl chloride, 92%; (x) K2CO3, CH2Cl2–MeOH then, HBF4 35%, CH3CN, 84%.

2318
L. Marmuse et al. / Carbohydrate Research 342 (2007) 2316–2321
Silica Gel 60 was used. NMR spectra were recorded on
0.25
0.2
0.15
0.1
0.05
0
Bruker AC 300 or Bruker Avance 400 at 298 K. Proton
chemical shifts are reported in ppm relative to external
SiMe4 (0 ppm). Low-resolution mass spectra were re-
corded in the positive FAB mode on a R1010C quadri-
polar mass spectrometer (model 2000, Nermag, Reuil-
Malmaison, France). MALDI-TOF measurements were
performed on a Bruker Daltonics Autoflex apparatus.
ESI experiments were performed on a Waters Micromass
ZQ spectrometer. The spectrophotometric measure-
ments were recorded on UVIKON XS spectrometer
equipped with a thermostated cuvette holder.
0
20
40
60
80
100
Time (s)
Figure 1. Time course of the liberation of 4-nitrophenol from
compound 8 by A. niger FAEA. Three different enzyme concentrations
were assayed: 5.6 nM (m), 14 nM (j) and 28 nM (d).
1.2. ( )-1,2-O-Isopropylidene-4-O-(2-chloro-4-nitro-
phenyl)-1,2,4-butanetriol (2)
( )-1,2-O-Isopropylidene-4-O-(4-tolylsulfonyl)-1,2,4-but-
anetriol 110 (730 mg, 2.42 mmol) was dissolved in dry
acetone (10 mL), 2-chloro-4-nitrophenol (405 mg,
2.90 mmol), 18-C-6 (44 mg, 0.17 mmol) and K2CO3
(670 mg, 4.84 mmol) were added to the soln. The reac-
tion was heated under reflux for 24 h. Acetone was then
removed under diminished pressure, the residue was dis-
solved in diethyl ether and washed with 0.1 M NaOH
and brine, dried over Na2SO4. The crude product was
purified by column chromatography (1:0!19:1 tolu-
ene–diethyl ether) to give title compound 2 as a syrup
(633 mg, 86%); 1H NMR (300 MHz, CDCl3): d 8.25
(1H, d, J 2.9 Hz, H–Ar), 8.11 (1H, dd, J 2.9 Hz, J 9.3
Hz, H–Ar), 6.97 (1H, d, J 9.3 Hz, H–Ar), 4.24 (4H, m,
H-a/H-a0, H-c, Hd/H-d0), 3.68 (1H, t, J 8.3 Hz, H-a/
H-a0), 2.12 (2H, m, H-b), 1.40 (3H, s, CH3), 1.34 (3H,
s, CH3); 13C NMR (75 MHz, CDCl3): d 126.0, 123.9,
123.3, 111.7, 108.9 (C–CH3, C–Ar), 72.9, 69.4, 66.6,
33.2 (C-a, C-b, C-c, C-d), 26.9, 25.5 (C–CH3); DCIMS:
m/z 302 [M+H]+, 319 [M+NH4]+.
Table 1. Kinetic parameters for the hydrolysis of the different
synthesized substrates
Catalytic efficiencies kcat/Km (Mꢁ1 sꢁ1
)
FAEA
FAEB
MFA
FA
8
0.52 · 105
2 · 106
0.25 · 105
2.1 · 105
ND
ND
ND
0.5 · 105
0.36 · 105
0.0041 · 105
12
5
MFA, methyl ferulate; FA, 5-O-trans-feruloyl-a-L-Araf; ND: activity
not detected.
8 > 12 ꢀ 5 (Table 1). The preferential hydrolysis of ester
of primary hydroxyl group found in this work has also
recently been observed by Biely and co-workers. on
4-nitrophenyl 2-, 3- and 5-O-feruloyl-a-L-arabino-
furanosides.9
On the other hand, FAEB showed no activity on 5
and a very weak activity on 8 and 12 when great
amounts of enzyme were added, while ferulic acid
methyl ester is hydrolyzed in a similar way by both
enzymes.
1.3. ( )-4-O-(2-Chloro-4-nitrophenyl)-1,2,4-butanetriol
(3)
In conclusion, we have described the synthesis of three
new chromogenic substrates for feruloyl esterases based
on a (2-chloro-4-nitrophenyl)-1,2,4-butanetriol scaffold.
All the compounds act as moderate but specific sub-
strate for FAEA with the same affinity as methyl feru-
late while they remain unhydrolyzed by FAEB.
Compound 2 (4.27 g, 14.1 mmol) was dissolved in acetic
acid (30 mL) and water (2 mL) was added to the soln.
The reaction mixture was stirred at room temperature
overnight. Then the acid was removed under diminished
pressure with several coevaporations with toluene. The
residue was dissolved in EtOAc and precipitated with
hexane to give the desired product 3 as a white solid
(3.45 g, 94%); 1H NMR (400 MHz, CD3OD): d 8.20
(1H, J 2.7 Hz, H–Ar) 8.13 (1H, dd, J 2.7 Hz, J 9.2 Hz,
H–Ar), 7.19 (1H, d, J 9.2 Hz, H–Ar), 4.28 (2H, m, H-
a), 3.85 (1H, m, H-c), 3.49 (2H, m, H-d), 2.05 (1H, m,
H-b0), 1.82 (1H, m, H-b); 13C NMR (100 MHz,
CD3OD): d 126,4, 125.1, 113.5 (C–Ar), 69.7 (C-c),
67.7, 67.3 (C-a, C-d), 33.7 (C-b); ESIMS: m/z 284.2
[M+Na]+; HRESIMS: calcd for C10H12NO5ClNa+,
284.0302; found m/z 284.0291.
1. Experimental
1.1. General method
Reactions were monitored by TLC using Silica Gel 60
F254 precoated plates (E. Merck, Darmstadt) and detec-
tion by charring with sulfuric acid soln 3:45:45 H2SO4–
MeOH–H2O. For flash chromatography, E. Merck

L. Marmuse et al. / Carbohydrate Research 342 (2007) 2316–2321
2319
1.4. ( )-4-O-(2-Chloro-4-nitrophenyl)-di-1,2-O-(4-acet-
oxyferuloyl)-1,2,4-butanetriol (4)
oxyferuloyl chloride (390 mg, 1.53 mmol) was added per
portion to the reaction. Once the addition is complete
the bath was removed. After 2 h acetoxyferuloyl chlo-
ride (80 mg, 0.30 mmol) and pyridine (370 lL,
4.60 mmol) were added once again to the mixture. Then
1 h later acetoxyferuloyl chloride (80 mg, 0.30 mmol)
was added. The reaction mixture was stirred for 1 h,
then diluted with CH2Cl2, washed with water and dried
over Na2SO4. The crude product was purified by column
chromatography (1:4!3:7, EtOAc–petroleum ether)
and crystallized with EtOAc to give the desired com-
pound 6 (430 mg, 58%); mp 145–146 ꢁC; 1H NMR
(300 MHz, CDCl3): d 8.25 (1H, d, J 2.4 Hz, H–Ar)
8.11 (1H, dd, J 2.9 Hz, J 12.2 Hz, H–Ar), 7.64 (1H, d,
HC@CH), 7.09–6.97 (4H, m, H–Ar), 6.37 (1H, d, J
16.2 Hz, HC@CH), 4.35–4.19 (4H, m, H-a, H-c, H-d),
3.82 (3H, s, OMe), 2.28 (3H, s, CH3C@O), 2.00–2.12
(2H, m, H-b); 13C NMR (75 MHz, CDCl3): d 168.9,
167.1 (C@O, CH3C@O) 159.45–111.6 (C–Ar, C@C),
68.8, 67.5, 66.7 (C-a, C-c, C-d), 56.1 (OMe), 32.7 (C-
b), 20.8 (CH3C@O); HRESIMS: calcd for
C22H22NO9ClNa+, 502.0881; found m/z 502.0871.
Compound 3 (500 mg, 1.91 mmol) was dissolved in dry
pyridine (25 mL) and DMAP (23 mg, 0.19 mmol) was
added to the reaction. Then freshly prepared 4-acetoxy-
feruloyl chloride (1.9 g, 7.48 mmol) was added portion-
wise over 12 h. The reaction mixture was stirred at
room temperature for 19 h. Then the reaction was con-
centrated and coevaporated with toluene (3 times). The
residue was dissolved in CH2Cl2, and washed with HCl
(1 M), a satd aq NaHCO3, water and dried over
Na2SO4. The crude product was purified by flash col-
umn chromatography (9:1 toluene–diethyl ether) to give
1
title compound 4 as a white foam (855 mg, 64%); H
NMR (400 MHz, CDCl3): d 8.29 (1H, d, J 2.7 Hz, H–
Ar) 8.14 (1H, dd, J 2.7 Hz, J 9.0 Hz, H–Ar), 7.66 (2H,
d, J 16 Hz, HC@CH), 6.98–7.26 (7H, m, H–Ar), 6.40
(2H, d, J 16 Hz, HC@CH), 5.506 (1H, m, H-c), 4.55
0
0
(1H, dd, Jcd 3.6 Hz, Jdd , H-d ), 4.48 (1H, dd, Jcd
0
6.1 Hz, Jdd 12.1 Hz, H-d), 4.28 (2H, m, H-a), 3.85
(6H, s, 2 · OMe), 2.33–2.39 (8H, m, H-b, 2 ·
CH3C@O); 13C NMR (100 MHz, CDCl3): d 169.0–
110.4 (C–Ar, C@O) 69.6 (C-c), 66.3 (C-a), 65.52 (C-d),
56.33 (2 · OMe), 31.06 (C-b), 21.05 (2 · CH3C@O);
ESIMS: m/z 720.24 [M+Na]+; HRESIMS: calcd for
C34H32NO13ClNa+, 720.1460; found m/z 720.1470.
1.7. ( )-4-O-(2-Chloro-4-nitrophenyl)-1-O-feruloyl-1,2,4-
butanetriol (8)
1.7.1. ( )-4-O-(2-Chloro-4-nitrophenyl)-1-O-(4-acetoxy-
feruloyl)-2-O-tetrahydropyranyl-1,2,4-butanetriol
(7).
1.5. ( )-4-O-(2-Chloro-4-nitrophenyl)-di-1,2-O-(ferulo-
yl)-1,2,4-butanetriol (5)
Compound 6 (80 mg, 0.17 mmol) was suspended in
dry CH2Cl2 (1 mL), dihydropyran (75 lL, 0.85 mmol)
and a catalytic amount of camphorsulfonic acid were
added to the mixture. The reaction mixture was stirred
at room temperature for 2 h, then diluted with CH2Cl2,
washed with satd aq NaHCO3 and water and dried over
Na2SO4. The crude product was purified by column
chromatography (1:4!3:7 EtOAc–petroleum ether) to
give title compound 7 as a yellow solid (92 mg, 98%);
ESIMS: m/z 586.28 [M+Na]+. The compound was used
without any further characterization.
To a soln of 4 (300 mg, 0.43 mmol) in 1:1 CH2Cl2–
MeOH, K2CO3 (75 mg, 0.54 mmol) was added. The
reaction mixture was stirred at room temperature for
1 h, then diluted with CH2Cl2, washed with NH4Cl in
water and dried over Na2SO4. The crude product was
purified by flash column chromatography (9:1!4:1 tolu-
ene–acetone) to give title compound 5 as a yellowish
1
foam (215 mg, 81%); H NMR (400 MHz, CDCl3): d
8.24 (1H, d, J 2.5 Hz, H–Ar), 8.10 (1H, dd, J 2.5 Hz, J
9.1 Hz, H–Ar), 7.62 (2 · 1H, d, J 15.8 Hz, HC@CH),
7.24–6.88 (6H, m, H–Ar), 6.29 (2 · 1H, d, J 15.6 Hz,
HC@CH), 5.54 (1H, m, H-c), 4.46 (2H, m, H-d), 4.26
(2H, m, H-a), 3.88 (6H, s, 2 · OMe), 2.34 (2H, m, H-
b); 13C NMR (100 MHz, CDCl3): d 167.0, 166.8
(C@O), 159.4–109.5 (C–Ar, C@C), 69.1 (C-c), 66.1,
65.1 (C-a, C-d), 56.1 (2 · OMe), 30.8 (C-b); ESIMS:
m/z 336.1 [M+Na]+; HRESIMS: calcd for
C30H28NO11ClNa+ 636.1248; found m/z 636.1245.
1.7.2.
( )-4-O-(2-Chloro-4-nitrophenyl)-1-O-feruloyl-
1,2,4-butanetriol (8). To a soln of 7 (82 mg, 0.14 mmol)
in 1:1 CH2Cl2–MeOH (4 mL), K2CO3 (40 mg,
0.29 mmol) was added. The reaction mixture was stirred
at room temperature for 5 h, then diluted with CH2Cl2,
washed with water and dried over Na2SO4. Without any
further purification and characterization, the resulting
crude product was used in the next step. The compound
was dissolved in MeOH (0.5 mL) and 1 M HCl (50 lL)
was added to the soln, the reaction mixture was stirred
at room temperature for 2.5 h, then neutralized with
Et3N and evaporated. The crude product was purified
by flash column chromatography (3:7!1:1 EtOAc–
petroleum ether) to give title compound 8 as a yellowish
foam (47 mg, 74%); 1H NMR (300 MHz, CD3OD):
d 8.14 (1H, d J 3 Hz, H–Ar) 8.07 (1H, dd, J 3 Hz,
1.6. ( )-4-O-(2-Chloro-4-nitrophenyl)-1-O-(4-acetoxy-
feruloyl)-1,2,4-butanetriol (6)
Compound 3 (400 mg, 1.53 mmol) was suspended in
acetonitrile (7 mL) with pyridine (370 lL, 4.60 mmol),
the reaction was ice cooled and freshly prepared 4-acet-

2320
L. Marmuse et al. / Carbohydrate Research 342 (2007) 2316–2321
J 9 Hz, H–Ar), 7.53 (1H, d, J 12 Hz, HC@CH); 7.15
(1H, d, J 9 Hz, H–Ar), 7.05 (1H, d, J 1.5 Hz, H–Ar),
6.94 (1H, dd, J 1.5 Hz, J 8 Hz, H–Ar), 6.70 (1H, d,
J 8 Hz, H–Ar), 6.26 (1H, d, J 16 Hz, HC@CH), 4.29–
4.09 (5H, m, H-a, H-c, H-d), 3.79 (3H, s, OMe), 2.08
(1H, m, H-b0), 1.93 (1H, m, H-b); 13C NMR
(C-b), 20.8 (CH3C@O); FABMS m/z 745 [M+Na]+;
HRESIMS: calcd for C41H36NO9ClNa+, 744.1976;
found m/z 744.1969.
1.10. ( )-4-O-(2-Chloro-4-nitrophenyl)-2-O-feruloyl-1-O-
(triphenylmethyl)-1,2,4-butanetriol (11)
(75 MHz, CD3OD):
d 169.2 (C@O) 161.0–111.5
(C–Ar, C@C), 69.4, 67.6, 67.3 (C-a, C-c, C-d),
56.53 (OMe), 34.1 (C-b); ESIMS: m/z 460.10 [M+Na]+;
HRESIMS: calcd for C20H20NO8ClNa+, 460.0775;
found m/z 460.0774.
To a soln of 10 (250 mg, 0.34 mmol) in 1:1 CH2Cl2–
MeOH (4 mL), K2CO3 (96 mg, 0.68 mmol) was added.
The reaction mixture was stirred at room temperature
for 2 h, then diluted with CH2Cl2, washed with a
NH4Cl, water and dried over Na2SO4. The crude prod-
uct was purified by flash column chromatography
(9:1!7:3 petroleum ether–EtOAc) to give title com-
1.8. ( )-4-O-(2-Chloro-4-nitrophenyl)-1-O-(triphenyl-
methyl)-1,2,4-butanetriol (9)
1
pound 11 as a white foam (225 mg, 97%); H NMR
Diol 3 (300 mg, 1.15 mmol) was dissolved in pyridine
(5 mL), DMAP (28 mg, 0.23 mmol) and triphenyl
methyl chloride (480 mg, 1.72 mmol) were added to
the soln. Additional triphenylmethyl chloride (400 mg,
1.44 mmol) was added in portion over 12 h. Then the
reaction was evaporated and coevaporated with toluene.
The residue was dissolved in CH2Cl2, washed with water
and dried over Na2SO4. The crude product was purified
by flash column chromatography (3:7 EtOAc–toluene)
to give title compound 9 as a white solid (470 mg,
81%): 1H NMR (300 MHz, CDCl3): d 8.3 (1H, d, J
2.9 Hz, H–Ar), 8.12 (1H, dd, J 2.5 Hz, J 8.8 Hz, H–
Ar), 7.44–7.21 (18H, m, H–Ar), 6.94 (1H, d, J 9.3 Hz,
H–Ar), 4.27–4.06 (4H, m, H-a, H-c), 3.25 (2H, m, H-
d), 1.99 (2H, m, H-b); 13C NMR (75 MHz, CDCl3):
d 144.0–112.1 (C–Tri, C–Ar) 68.3, 67.8, 67.0 (C-a, C-
c, C-d), 33.0 (C-b); ESIMS m/z 526.06 [M+Na]+;
HRESIMS: calcd for C29H26NO5ClNa+, 526.1397;
found m/z 526.1391.
(300 MHz, CDCl3): d 8.24 (1H, d, J 2.8 Hz, H–Ar),
7.64 (1H, dd, J 2.9 Hz, J 8.8 Hz, H–Ar), 7.47–6.87
(20H, m, H–Ar), 6.32 (1H, d, J 16 Hz, HC@CH), 5.40
(1H, m, H-c), 4.13 (2H, m, H-a), 3.93 (3H, s, OMe),
3.39 (1H, dd, Jc;d 4.4 Hz, Jd;d 10.2 Hz, H-d0), 3.28 (1H,
0
0
0
dd, Jc,d 4.9 Hz, Jd;d 10.2 Hz, H-d), 2.32 (2H, m, H-b);
13C NMR (75 MHz, CDCl3): d 166.8 (C@O) 159.55–
109.6 (C–Ar, C@C), 86.9 (C–Tr), 65.0, 66.7, 70.3
(C-a, C-c, C-d), 56.2 (OMe), 31.0 (C-b); FABMS m/z
680 [M+H]+; HRESIMS: calcd for C39H34NO8ClNa+,
702.1870; found m/z 702.1866.
1.11. ( )-4-O-(2-Chloro-4-nitrophenyl)-2-O-feruloyl-
1,2,4-butanetriol (12)
Compound 11 (190 mg, 0.28 mmol) was dissolved in
acetonitrile (4 mL), then 35% HBF4 in water (60 lL)
was added. The reaction mixture was stirred at room
temperature for 1 h and neutralized with Et3N. The
crude product was purified by column chromatography
(3:2!1:1 petroleum ether–EtOAc) to give title com-
pound 12 as a yellowish solid (113 mg, 84%); 1H
NMR (300 MHz, CD3OD): d 8.17 (1H, d, J 2.4 Hz,
H–Ar), 8.04 (1H, dd, J 2.4 Hz, J 8.8 Hz, H–Ar), 7.52
(1H, d, HC@CH), 7.05–7.05 (3H, m, H–Ar), 6.77 (1H,
d, J 8.8 Hz, H–Ar), 6.20 (1H, d, J 15.6 Hz, HC@CH),
5.16 (1H, m, H-c), 4.17 (2H, m, H-a), 3.81 (3H, s,
OMe), 3.71 (2H, m, H-d), 2.21 (2H, m, H-b); 13C
NMR (75 MHz, CD3OD): d 168.7 (C@O) 73.4 (C-a),
160.6–111.6 (C–Ar), 67.6, 64.5 (C-c, C-d), 56.4 (OMe),
31.4 (C-b); ESIMS m/z 460.03 [M+Na]+; HRESIMS:
calcd for C20H20NO8ClNa+, 460.07751; found m/z
460.0781.
1.9. ( )-4-O-(2-Chloro-4-nitrophenyl)-2-O-(4-acetoxy-
feruloyl)-1-O-(triphenylmethyl)-1,2,4-butanetriol (10)
Compound 9 (380 mg, 0.75 mmol) was dissolved in pyr-
idine (10 mL). DMAP (18 mg, 0.15 mmol) and 4-acet-
oxyferuloyl chloride (380 mg, 1.50 mmol) were added
to the soln. Then the reaction mixture was stirred over-
night. The soln was evaporated and coevaporated with
toluene. The crude product was purified by column
chromatography (1:0!17:3 EtOAc–petroleum ether)
to give title compound 10 as a white foam (500 mg,
1
92%); H NMR (400 MHz, CDCl3): d 8.26 (1H, d, J
2.7 Hz, H–Ar), 8.10 (1H, dd, J 2.7 Hz, J 9.1 Hz, H–
Ar), 7.69 (1H, d, HC@CH), 7.48–7.07 (18H, m, H–
Ar), 6.90 (1H, d, J 9.1 Hz, H–Ar), 6.45 (1H, d, J
16 Hz, HC@CH), 5.43 (1H, m, H-c), 4.15 (2H, m, H-
1.12. Kinetic experiments
0
0
,
a), 3.88 (3H, s, OMe), 3.43 (1H, dd, Jc;d 4.0 Hz, Jd;d
Pure recombinant feruloyl esterases FAEA and FAEB
from A. niger were obtained according to the procedures
already described.11,12
Methyl ferulate (MFA) was purchased from Apin
Chemicals and the feruloylated arabinose FA, 5-O-
H-d0), 3.31 (1H, dd, Jc,d 4.9 Hz, Jd;d 10.2 Hz, H-d),
2.35 (5H, m, CH3C@O, H-b); 13C NMR (100 MHz,
CDCl3): d 168.9–111.4 (C–Ar, C@O), 86.8 (C–Tri),
66.5 (C-c), 64.9 (C-a), 64.8 (C-d), 56.1 (OMe), 30.9
0

L. Marmuse et al. / Carbohydrate Research 342 (2007) 2316–2321
2321
trans-feruloyl-a-L-Araf, was purified as already de-
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´
Ms. Patricia Chaud and Stephanie Boullanger are
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0401C0042 from ADEME and PNRB2005-11 from
ANR.
`
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Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.carres.
2007.06.004.