A new affinity ligand for the isolation of a single 'feruloyl esterase' (FAE-III) from Aspergillus niger

Article abstract of DOI:10.1016/S0968-0896(00)00037-7

8-Aminooctyl 5'-S-coniferyl-5'-deoxy-thio-α-L-arabinofuranoside has been synthesised and shown to be a selective affinity ligand for the feruloyl esterase III of Aspergillus niger. The hydrolyses of methyl 5-O-coumaroyl, feruloyl, or sinapoyl α-L-arabinofuranosides by this enzyme proceed at comparable rates. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(00)00037-7

Bioorganic & Medicinal Chemistry 8 (2000) 917±924
A New Anity Ligand for the Isolation of a Single `Feruloyl
Esterase' (FAE-III) from Aspergillus niger
Yongxin Zhao a and Michael L. Sinnott a,b,
*
aDepartment of Chemistry (M/C111), University of Illinois at Chicago, 845 West Taylor Street, Chicago, Illinois 60607-7061, USA
bDepartment of Paper Science, UMIST, PO Box 88, Sackville Street, Manchester M60 1QD, UK
Received 22 July 1999; accepted 29 November 1999
AbstractÐ8-Aminooctyl 50-S-coniferyl-50-deoxy-thio-a-l-arabinofuranoside has been synthesised and shown to be a selective anity
ligand for the feruloyl esterase III of Aspergillus niger. The hydrolyses of methyl 5-O-coumaroyl, feruloyl, or sinapoyl a-l-arabino-
furanosides by this enzyme proceed at comparable rates. # 2000 Published by Elsevier Science Ltd. All rights reserved.
Introduction
fungus, the best-understood organism which degrades
all components of lignocellulose. In the event, we failed
to ®nd growth conditions under which this particular
organism produced workable quantities of the enzyme.
However, we produced readily-synthesised substrates
and and an anity ligand (II), which we validated with
respect to the better-characterised ferulic esterase of
Aspergillus niger. The ligand was designed to have a
non-®ssile linkage connecting the ferulic and arabino-
furanosyl moieties, and also a spacer arm between the
arabinofuranosyl moiety and the site of attachment to
the support. The thioether, rather than ether linkage,
less than optimal from the viewpoint of the ligand
geometry, was necessitated by synthetic problems.
Attempts to connect the two halves of the recognition
function as an ether linkage via Williamson-type SN2
reactions with either O5 of a protected arabinofuranosyl
unit or Og of a protected coniferyl alcohol as nucleo-
philes failed, we suspect because of competing electron-
transfer reactions when the electrophile was derived
from coniferyl alcohol, and competing elimination
reactions when it was derived from aminooctyl arabi-
noside. Nonetheless, the ligand II when attached to
cyanogen-bromide-activated Sepharose, selectively
bound FAE-III, the A. niger enzyme with speci®city for
ferulic acid bound to O-5 of the a-l-arabinofuranosyl
moiety, and not other ferulic acid esterases in the same
culture ®ltrate.
The molecular architecture of the cell walls of plants,
and the enzymology of the degradation of the molecular
structures found there, is a subject of great fundamental
and practical importance. Although species such as
pectic substances and xyloglucans play important roles,
discussion of plant biomass can usefully be structured
round the concept of three major components of lig-
nocelluloseÐcellulose lignin, and the various alkali-
soluble polysaccharides known for historical reasons as
hemicellulose. There is increasing evidence that covalent
linkages between these three components are important.
Ester linkages between acids corresponding to the lignin
alcohols and polysaccharides, and the enzymes which
hydrolyse them therefore demand investigation. The
structures of the lignin alcohols and the corresponding
acids (I) are given below.
It is found that, in general, in the plant cell walls of
monocots such as cereals and grasses the ferulic acid
moiety occurs esteri®ed to O-5 of the a-l-arabinofur-
anosyl moiety of the arabinoxylan, whereas in dicots it
occurs esteri®ed to the side chains of pectic substances,
at O-2 of the arabinofuranosyl moiety and at O-6 of the
galactopyranosyl moiety.1,2
As part of a programme into the lignocellulolytic
enzymes of Phanerochaete chrysosporium,3 we wished to
isolate and characterise any ferulic esterase from this
As with most lignocellulolytic enzymes, the choice of
substrate is problematical, with a three-way tension
between ease of monitoring the enzymic reaction, ease
of synthesis of the assay substrate, and ®delity to the
structure of the natural substrate, lignocellulose. Recent
*Corresponding author. Tel.: +44-161-200-3897; fax: +44-161-200-
3858; e-mail: michael.sinnott@umist.ac.uk
0968-0896/00/$ - see front matter # 2000 Published by Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00037-7

918
Y. Zhao, M. L. Sinnott / Bioorg. Med. Chem. 8 (2000) 917±924
studies have employed methyl4,5 and ethyl esters,6 as
well as cell wall extracts released from `cellulase-treated'
plant cell walls.7 9 Methyl and ethyl ferulate provide
the proper phenolic residue but lack a realistic alcohol
moiety. The procedures for the puri®cation of the
coumaryl and feruloyl arabinoxylan oligomers FAXX
and PAXX (III) are low-yielding and tedious.8 We
elected to use the 5-O-feruloyl, coumaroyl, and sina-
poyl esters of methyl a-l-arabinofuranoside (IV), which
are chromogenic, resemble the natural substrate on
both sides of the site of cleavage, and which have been
previously described in terms of the coumaroyl (IVa)10
and feruloyl (IVb)11 compounds. We also synthesised
the methyl 5-O-furylacryloyl arabinofuranoside in the
hope that a highly sensitive ¯uorogenic assay substrate
of the type that had been used for zinc proteinases12
would be produced.
or the nucleophile in SN2-type reactions failed. The
synthetic pathway is set out in Scheme 1.
When attached to CNBr-activated Sepharose, the ligand
proved successful in adsorbing the ferulic acid esterase.
The Faulds and Williamson protocol for isolation of
FAE-III4 from A. niger culture ®ltrates involved
ammonium sulfate fractionation, two hydrophobic
chromatography steps, and an anion exchange chroma-
tography. After one hydrophobic chromatography step,
the anity ligand proved capable of leading to pure
protein. A puri®cation table is given as Table 1.
A typical anity chromatogram is given in Figure 1.
Whatever the loading, some esterase activity was not
adsorbed, and it is now clear that this is probably due to
ferulic acid esterases with other leaving group speci®-
cities. The enzyme was eluted with a mixture of ferulic
acid and arabinose. The absorbance of ferulic acid pre-
cluded the conventional use of A280 as a measure of
protein concentration: fractions had to be assayed indi-
vidually by the Bradford assay.
Results and Discussion
The synthesis of the anity ligand (II) proved relatively
straightforward; the sulfur atom was introduced for
synthetic reasons since, attempts to construct an ether
linkage with the ferulic moiety as either the electrophile
On SDS-PAGE, the feruloyl esterase preparation was
electrophoretically homogenous and migrated as a

Y. Zhao, M. L. Sinnott / Bioorg. Med. Chem. 8 (2000) 917±924
919
Scheme 1.
Table 1. Puri®cation of feruloyl esterase FAE-III from A. niger
Step
Vol. (mL)
Speci®c activity (U/mg protein)
Total activity (U)
Puri®cation (n-fold)
Yield (%)
Crude supernatant
85% (NH4)2SO4
Hydrophobic chromatography
Anity chromatography
930
45
30a
20a
1.89
3.12
23.4
53.7
317
237
212a
81a
1
100
75
67a
25a
1.65
12.4
28.4
aValue is derived from total of the feruloyl esterase protein and activity at each separation step.
Figure 1. Anity chromatogram of feruloyl esterase, using 50 mM potassium phosphate bu€er, pH 6.0 (bu€er A). Bu€er B also contains 25 mM
arabinose, 50 mM methyl a-l-arabinofuranoside, and 50 mM ferulic acid. FA-Ara is substrate IVb.

920
Y. Zhao, M. L. Sinnott / Bioorg. Med. Chem. 8 (2000) 917±924
Table 2. Speci®city of the hydrolyses of the hydroxycinnamyl sub-
strates by the feruloyl esterase from A. niger at 30 ꢀC, in 0.1 M potas-
sium phosphate bu€er, pH 6.0, containing 1.0 mM EDTA and 3 mM
sodium azide
1
Substrates
Speci®c activity
(U/(mg protein))
kcat (s
)
Km (mM)
IVb (feruloyl)
53.7
58.3
19.7
67.2a
156a
0.07a
33
35
12
ND
ND
ND
20
7.0
1230
2080a
1440a
ND
IVc (sinapinoyl)
IVa (coumaroyl)
Methyl ferulate
Methyl sinapinate
Methyl coumarate
aTaken from ref 4.
Figure 2. Variation of activity of FAE-II against substrate IVb
([S]ꢁKm, at least at optimum pH).
single band corresponding to Mr=35.8 kD. Gel per-
meation chromatography of the native enzyme indi-
cated a single active peak correlating to Mr=36±34.4 K.
FAE±III was reported as a monomer of Mr 36 kD (SDS
PAGE) and 28 kD (gel ®ltration).4 The gene-sequence
however indicates a protein Mr of 29 kD;13 the protein
is known to be N-glycosylated.
isolated, in accord with its identi®cation of FAE-III.
This particular esterase has a requirement for a phenolic
hydroxy group on the acyl portion,14 whereas the Mr
2Â76 kD esterase (CinnAE)15 does not. The furyl-
acryloyl substrate may prove useful as a substrate for
this enzyme.
The puri®ed feruloyl esterase exhibited Michaelis±Men-
ten kinetics towards the three substrates IV. Michaelis±
Menten parameters are given in Table 2, kcat values
being calculated on the assumption that the enzyme as
®rst isolated were 100% active protein of Mr=36 kD.
The kinetic parameters for substrate IVb at 30 ꢀC can
be compared with those for 5-O-feruloyl arabinose,
kcat=63 s 1, Km=75 mM at 37 ꢀC, determined for
conventionally isolated enzyme.1 These data indicate
the enzyme as isolated from the anity column is
largely active; the <2-fold lower kcat of substrate IVb
with our preparation must re¯ect the lower assay tem-
perature, the lower Km a combination of the lower assay
temperature and the freezing of the arabinose into the
a-furanose form by the methyl glycoside.
Experimental
Substrates
5-O-Coumaroyl, feruloyl, sinapinoyl esters of methyl ꢀ-
L-arabinofuranoside (IV). The coumaroyl and feruloyl
esters IVa and IVb were prepared according to Helm et
al.10 and Hat®eld et al.,11 respectively, by selective 5-O-
acylation of methyl arabinofuranoside by 4-O-acetyl
feruloyl chloride, followed by selective removal of the
aromatic 4-O-acetyl group with piperidine. The syn-
thesis of substrate IVc proceeded exactly analogously,
with the selective acylation of methyl arabinofuranoside
proceeding in 49% yield, and the aromatic 4-O-deace-
tylation with piperidine in 79% yield.
The Km values for substrates IV are, unsurprisingly,
some 102 lower than those of the methyl esters, but, at
least from the speci®c activities, kcat values are also
reduced; this decrease in kcat with increasingly speci®c
leaving groups was observed previously.1 Although
detailed mechanistic work requires an active site titra-
tion, at this stage the higher kcat values that can be
inferred for the methyl esters indicate that the hydro-
lysis of any acyl-enzyme intermediate does not limit the
hydrolysis of substrates IVb and IVc.
Characterisation data for (IVc): mp 140±142.5 ꢀC
d
‰aŠ25= 39.8ꢀ (c 0.56, CH3OH); H NMR (CD3OD) d
7.60 (d, 1H, J=15.9 Hz), 6.87 (s, 2H), 6.39 (d, 1H,
J=15.9 Hz), 4.75 (d, 1H, J=1.3 Hz), 4.37 (dd, 1H,
J=3.3 Hz, 11.8 Hz), 4.23 (q, 1H), 4.05 (m, 1H), 3.95 (m,
1H); 3.85 (m, 1H); 3.83 (s, 6H), 3.34 (s, 3H); 13C NMR
(CD3OD) d 169.0, 149.6, 139.7, 126.7, 115.7, 110.8, 107,
83.5, 82.8, 79.4, 65.2, 56.9 (2C), 55.6; GC±MS 370
(M+), 338, 310, 266, 224, 207, 175, 176, 147, 121, 105,
91, 73, 61, 45; FT±IR (KBr) 3630±3100 (s), 2939, 2842
(w), 1700 (s), 1632, 1604, 1516, 1460, 1426, 1339, 1286,
1
The pH-activity pro®le for the enzyme against IVb is
displayed in Figure 2: activity in this case means kcat, at
least at optimum pH, since the substrate concentration
was 50ÂKm.
l
Detailed analysis of the present data is unwarranted,
but from simple inspection it is clear the variation is not
that of a classical `bell-shaped' pro®le, since it is not
symmetrical about the pH of maximal activity.
1180, 1114, 1013, 951, 825, 626 cm . Anal. calcd for
C17H22O9: C, 55.13; H, 5.99. Found: C, 54.83; H, 5.96.
Methyl 5-O-(E-20 furylacryloyl)-ꢀ-L-arabinofuranoside.
This was prepared by selective 5-O-acylation of methyl
arabinofuranoside with the acyl chloride in a similar
way to substrates IV. Yield 79% mp 69±71 ꢀC;
In the event methyl 5-O-(20-furyl)acryloyl a-l-arabino-
furanoside proved inert to the ferulic acid esterase we

Y. Zhao, M. L. Sinnott / Bioorg. Med. Chem. 8 (2000) 917±924
921
d
‰aŠ25= 71.1ꢀ (c 1.0, CH3OH); Rf 0.51 (EtOAc); 1H
7.82 (dd, 2H, J=3.1 Hz, 5.4 Hz), 7.69 (dd, 2H, J=
3.0 Hz, 5.4 Hz), 4.98 (s, 1H), 4.13 (d, 1H, J=2.1 Hz),
3.99 (s, 2H), 3.81 (m, 2H), 3.71±3.60 (m, 4H), 3.43 (m,
1H), 3.11 (m, 1H), 1.64±1.29 (m, 12H).
NMR ((CD3)2CO) d 7.72 (d, 1H, J=1.7 Hz), 7.50 (d,
1H, J=15.6 Hz), 6.88 (d, 1H, J=3.4 Hz), 6.60 (dd, 1H,
J=1.7 Hz, 3.5 Hz), 6.28 (d, 1H, J=15.8 Hz), 4.76 (d,
1H, J=1.6 Hz), 4.54 (m, 1H), 4.39 (dd, 1H, J=3.7 Hz,
11.8 Hz), 4.24 (dd, 1H, J=5.9 Hz, 11.7 Hz), 4.09 (m,
1H), 3.99 (m, 1H), 3.90 (m, 1H), 3.30 (s, 3H); 13C NMR
d 146.4, 132.2, 116.3, 116.0, 113.4, 110.5, 83.4, 82.4,
79.4, 65.0, 55.0; MS 284 (M+), 253, 224, 213, 206, 181,
138, 121, 110, 94, 87, 73, 65, 57; IR (KBr), 3500±3200 (s),
2969, 2916, 1716 (s), 1643, 1559, 1481, 1302, 1211, 1170,
1095, 1003, 936, 860, 758, 595 cm 1. Anal. calcd for
C13H16O7: C, 54.93; H, 5.67. Found: C, 54.89; H, 5.66.
80-Phthalimidooctyl 5-O-(p-toluenesulfonyl)-ꢀ-L-arabino-
furanoside (VII). To a stirred solution of 80-phthalimi-
dooctyl a-l-arabinofuranoside (1.02 g, 2.50 mmol) in
dry pyridine (10 mL) with 4 A molecular sieve under N2
at 0 ꢀC, was added p-toluenesulfonyl chloride (0.53 g,
2.75 mmol). The mixture was stirred at 0 ꢀC for 4 h, at
4±7 ꢀC for 24 h and at room temperature 5 h. The
reaction mixture was ®ltered by suction and the solvent
was removed in vacuo. The crude product was puri®ed
by silica gel chromatography (CH3OH:CHCl3 1:35).
Yield 1.08 g (77.7%) of the title product. 1H NMR
(CDCl3) d 7.82±7.66 (m, 6H), 7.32 (d, 2H, J=8.2 Hz),
4.91 (s, 1H), 4.21±4.04 (m, 4H), 3.87 (s, 1H), 3.67±3.59
(m, 3H), 3.34 (m, 1H), 3.15 (s, 1H), 2.41 (s, 3H), 1.67±
1.47 (m, 4H), 1.27 (m, 8H); 13C NMR d 168.5, 145.1,
133.9, 132.2, 132.0, 129.9, 128.0, 123.1, 107.6, 83.1, 80.0,
69.1, 67.8, 37.9, 29.2, 28.9, 28.4, 26.6, 25.8, 21.6; IR
3570±3350 (s), 1771 (m), 1711 (s), 1610 (w), 1438 (m),
Synthesis of anity ligand II
N-80-Hydroxyoctyl phthalimide (V). Potassium phthal-
imide (4.80 g, 25.4 mmol) was added to a solution of 8-
bromo-1-octanol (4.90 g, 22.2 mmol) in dry DMF
(25 mL). The reaction was heated to 90 ꢀC for 30 min,
then kept at room temperature overnight. After the
addition of chloroform (50 mL), the mixture was poured
into water (100 mL). The aqueous phase was separated
and extracted with two 15 mL portions of chloroform.
The combined chloroform extract was washed with 0.2
M NaOH (25 m1), to remove unreacted phthalimide,
and water (25 mL). The chloroform was dried and eva-
porated and the residue was recrystallised from ethyl
acetate/hexane to a€ord 5.6 g (91%) of the title product;
mp 66±68 ꢀC; 1H NMR (CDCl3) d 7.83 (dd, 2H,
J=3.1 Hz, 5.5 Hz), 7.69 (dd, 2H, J=3.1 Hz, 5.5 Hz),
3.70±3.58 (q, 4H), 1.68±1.29 (m, 12H); IR 3510 (s),
1398 (s), 1357 (s), 1176 (s), 1095 (s), 971 (s), 722 (s), 666
1
(m) cm
.
E-4-O-(tert-Butyldimethylsilyl)-3-methoxycinnamaldehyde.
To a stirred solution of E-4-hydroxy-3-methoxycinna-
maldehyde (9.0 g, 50.5 mmol) in dry pyridine (80 mL)
under N2, was added tert-butyldimethylsilyl chloride
(9.0 g, 59.8 mmol). The reaction was allowed to stir
overnight at room temperature. After removal of the
solvent in vacuo, the crude product was re-dissolved in
ethyl acetate:hexane (1:1), ®ltered, evaporated and
crystallized from ethyl acetate:pentane. ꢀYield 10.5 g
2946, 2923, 2853, 1773, 1707 (s), 1469, 1406, 1376, 1342,
1
1190, 1063, 940, 721 cm
.
80-Phthalimidooctyl 2, 3, 5-tri-O-benzoyl-ꢀ-L-arabinofur-
anoside (VI). To a mixture of N-80-hydroxyoctyl phthal-
imide (6.07 g, 22.1 mmol), Hg(CN)2 (4.0 g) and Na2CO3
(2.0 g) in dry THF (60 mL), stirred under nitrogen in the
presence of 4 A molecular sieve, was added 14.0 g
(26.6 mmol) of 2, 3, 5-tri-O-benzoyl-a-l-arabinofur-
anosyl bromide.16 The reaction was allowed to stir 24 h
at ambient temperature. The reaction was ®ltered by
suction and the solvent was removed in vacuo. The
crude product was subjected to ¯ash chromatography
on silica gel using EtOAc:CHCl3 (1:20) as eluant. Con-
centration of the appropriate fractions and recrystalli-
zation from dry benzeneꢀa€orded 9.62 g (60.6%) of the
(72%) of the title product, mp 131±134 C: H NMR
1
(CDC13) d 9.69 (d, 1H, J=7.5 Hz), 7.70 (d, 1H, J=15.8
Hz), 7.00 (d, 2H, J=6.5 Hz), 6.85 (d, 1H, J=8.7 Hz),
6.31 (d,1H, J=15.8 Hz), 3.85 (s, 3H), 1.01 (s, 9H), 0.18
(s, 6H).
E-4-O-(tert-Butyldimethylsilyl)-3-methoxycinnamyl alco-
hol. To a stirred solution of E-4-O-(tert-butyldimethyl-
silyl)-3-methoxycinnamaldehyde (6.13 g, 21.0 mmol)
and CeCl3 (6.0 g) in 80 mL dry THF under N2, was
added 1.3 g (34.4 mmol) of NaBH4. The mixture was
stirred overnight, ®ltered with ®lter agent, evaporated
and puri®ed with silica gel chromatography (ethyl acet-
1
1
title product. Mp 90±92 C; H NMR (CDCl3) d 8.09±
ate:hexane 1:1). Yield 5.0 g (81%). H NMR (CDCl3)
7.98 (m, 6H), 7.82 (dd, 2H, J=3.0 Hz, 5.6 Hz), 7.67 (dd,
2H, J=3.1 Hz, 5.5 Hz), 7.59±7.25 (m, 9H), 5.64 (d, 1H,
J=5.1 Hz, H-3), 5.50 (d, 1H, J=0.8 Hz, H-2), 5.27 (d,
1H, J=0.5 Hz, H-1), 4.83±4.54 (m, 3H), 3.85±3.58 (m,
4H), 3.48 (m, 1H), 1.70±1.21 (m, 12H).
d 6.90±6.78 (m, 3H), 6.53 (d, 1H, J=15.8 Hz), 6.24
(ddd, 1H), 4.28 (dd, 1H, J=0.85 Hz, 5.9 Hz), 3.81 (s,
3H), 0.99 (s, 9H), 0.15 (s, 6H); 13C NMR 150.9, 145.0,
131.3, 130.5, 126.4, 120.8, 119.5, 109.7, 63.8 (OCH2),
55.3 (OCH3), 25.6, 18.4.
80-Phthalimidooctyl ꢀ-L-arabinofuranoside. Removal of
benzoyl groups was achieved under standard Zemplen
conditions. The crude product was puri®ed by silica
gel chromatography (CH3OH:CHCl3 1:13 as eluent)
a€ording 2.90 g (81%) of the title compound. The pro-
duct was co-evaporated several times with dry toluene,
dry ether and recrystallized from dry ethanol/pentane to
E-4-O-(tert-Butyldimethylsilyl)-3-methoxycinnamyl thiol-
acetate. To an eciently stirred solution of triphenyl-
phosphine (7.0 g, 26.6 mmol) in dry THF (60 mL) at
0 ꢀC was added diisopropyl azodicarboxylate (5.3 g,
26.2 mmol) under N2. The mixture was stirred at 0 ꢀC
for 30 min. A white precipitate resulted. To the mixture
was added dropwise over 10 min a mixture of thioacetic
acid (Aldrich, 2.0 mL, 25.3 mmol) and E-4-O-(tert-
form white solid. Mp 79±81 ꢀC; H NMR (CDCl3) d
1

922
Y. Zhao, M. L. Sinnott / Bioorg. Med. Chem. 8 (2000) 917±924
butyldimethylsilyl)-3-methoxycinnamyl alcohol (3.9 g,
13.3 mmol) in dry THF (25 mL) over 10min. The reac-
tion was kept at 0 ꢀC for 1 h and room temperature for
1 h. After removal of the solvent in vacuo, the red crude
product was re-dissolved in hexane, ®ltered, evaporated
and puri®ed by silica gel chromatography (CH2Cl2:
hexane 1:2). Yield 4.40 g (95%) of the title product as
solution of 80-phthalimidooctyl 5-deoxy-5-thio-[E-400-O-
(tert butyldimethylsilyl)-300-methoxycinnamyl)-a-l-ara-
binofuranoside (0.30 g, 0.43 mmol) in methanol (15 mL)
was added ammonium ¯uoride (30 mg, 0.81 mmol). The
mixture was stirred in an oil bath at 60 ꢀC for 3 h. To
the mixture was added silica gel (ꢂ0.5 g). After removal
of the solvent in vacuo, the dry adsorbed silica gel was
added to the top of a silica column. The column was
eluted by chloroform:methanol (20:1). Yield 0.24 g
1
red oil. H NMR (CDCl3) d 6.85 (d, 1H, J=1.7 Hz),
6.78 (m, 2H), 6.49 (d, 1H, J=15.7 Hz), 6.02 (q, 1H,
J=7.7 Hz, 8.0 Hz, 15.7 Hz), 3.80 (s, 3H), 3.68 (m, 2H),
2.35 (s, 3H), 0.98 (s, 9H), 0.14 (s, 6H); 13C NMR d
195.2, 150.9, 144.9, 132.9, 130.4, 127.6, 122.2, 120.8,
119.4, 109.6, 55.3, 31.8, 31.5, 30.4, 25.6, 18.4, 14.0, 4.7.
1
(95%). H NMR (CDCl3) d 7.83 (dd, 2H, J=3.0 Hz,
5.7 Hz), 7.70 (dd, 2H, J=3.1 Hz, 5.7 Hz), 6.89 (s, 1H),
6.83 (t, 2H), 6.38 (d, 1H, J=15.7 Hz), 6.00 (p, 1H), 4.97
(s, 1H), 4.22 (m, 1H), 4.03 (d, 1H, J=l.2 Hz), 3.89 (s,
3H), 3.86 (s, 1H), 3.72±3.62 (m, 3H), 3.39 (m, 3H), 2.82
(t, 2H), 1.70±1.51 (m, 4H), 1.32±1.25 (m, 8H); 13C NMR
d 168.5, 144.8, 133.8, 132.9, 127.2, 123.2, 122.6, 120.2,
114.3, 108.0, 107.7, 85.8, 80.3, 80.0, 67.5, 55.8, 38.1,
35.7, 32.5, 29.0, 28.9, 26.6, 26.0, 24.8.
E-4-O-(tert-Butyldimethylsilyl)-3-methoxycinnamyl thiol
(VIII). To a stirred slurry of LiAlH4 (1.0 g, 26.3 mmol)
and dry cesium chloride (1.5 g, 6.08 mmol) in dry ether
(25 mL) was added dropwise E-4-O-(tert-butyldimethyl-
silyl)-3-methoxycinnamyl thiolacetate (4.4 g, 12.5 mmol)
in ether (25 mL). After addition, the reaction was kept
overnight at ambient temperature. The excess lithium
aluminum hydride was destroyed by the careful addi-
tion of water (10 mL). 0.1 M HOAc was added carefully
until the pH of the solution became 7.0. The ether layer
was separated, dried over MgSO4, ®ltered and evapo-
rated. The crude product was puri®ed by silica gel
chromatography (THF:hexane 1:25). Yield 3.6 g (93%)
80-Aminoooctyl 5-deoxy-5-thio-(E-400-hydroxy-300-meth-
oxycinnamyl)-ꢀ-L-arabinofuranoside (II). To a stirred
solution of 80-phthalimidooctyl 5-deoxy-5-thio-(400-
hydroxy-300-methoxycinnamyl)-a-l-arabinofuranoside
(50 mg, 0.090 mmol) in methanol (2 mL) was added
methylamine (2 mL) under N2. The mixture was stirred
for 15 min under nitrogen. The mixture was evaporated
and redissolved in 0.1 M disodium hydrogen phosphate
(pH 8.5) and evaporated under oil pump vacuum, and
puri®ed with silica gel chromatography (the silica gel
was washed thoroughly with Et3N ®rst) using THF:
methanol:triethylamine (10:10:1) asdeluent. Yield 37 mg
(87%) of the title product ‰aŠ25= 36.0ꢀ (c 1.1,
CH3OH); 1H NMR (CD3OD) d 6.98 (d, 1H, J=1.8 Hz),
6.82 (dd, 1H, J=1.8, 8.1 Hz), 6.71 (d, 1H, J=8.1 Hz),
6.39 (d, 1H, J=15.7 Hz), 6.03 (p, 1H), 4.82 (d, 1H,
J=1.7 Hz), 4.01±3.91 (m, 2H), 3.85 (s, 3H), 3.81±3.64
(m, 3H), 3.44±3.35 (m, 3H), 2.83±2.69 (m, 4H), 1.89 (s,
1H), 1.69±1.30 (m, 12H); 13C NMR d 149.1, 133.7,
130.4, 128.3, 124.1, 120.9, 116.2, 110.3, 109.3, 84.2, 83.8,
81.8, 68.8, 56.3, 41.4, 35.9, 33.9, 30.4, 30.3, 27.6, 27.1.
Anal. calcd for C23H37O6NS: C, 60.63; H, 8.19; N, 3.07;
S, 7.04. Found: C, 60.37; H, 8.02; N, 2.98; S, 6.87.
1
of the title compound as clear oil; H NMR (CDCl3) d
6.88 (d, 1H, J=1.5 Hz), 6.80 (m, 2H), 6.41 (d, 1H,
J=15.5 Hz), 6.17 (ddd, 1H), 3.82 (s, 3H), 3.33 (t, 2H),
1.50 (t, 1H, J=7.6 Hz, 15.2 Hz), 0.99 (s, 9H), 0.15 (s,
6H); 13C NMR 153.4, 151.0, 130.7, 130.5, 126.7, 120.9,
119.5, 109.6, 55.4, 27.3, 25.6, 18.4, 4.7; IR 3027, 2997,
2895, 1600, 1512 (s), 1415, 1307, 1281, 1159, 1037, 904,
1
839, 782 cm
.
80-Phthalimidooctyl 5-deoxy-5-(E-400-O-(tert-butyldimeth-
ylsilyl)-300-methoxycinnamyl)-thio-ꢀ-L-arabinofuranoside
(IX). Sodium (49 mg) was dissolved in dry ethanol
(35 mL) under a blanket of helium. To this mixture was
added E-4-O-(tert-butyldimethylsilyl)-3-methoxycinna-
myl thiol (VIII) (0.67 g, 2.16 mmol) also under helium.
The mixture was kept to stir for 2 h. To this mixture
was added 80-phthalimidooctyl 5-O-(p-toluenesulfonyl)-
a-l-arabinofuranoside (VII) (1.21 g, 2.15 mmol). The
reaction was re¯uxed under He for 18 h, evaporated and
puri®ed with silica gel chromatography (chloroform:
Enzyme and protein assays during puri®cation
Spectrophotometric activity assays. The assay solution
consisted of 100±200 mL (10.0 mM or 5.0 mM) of com-
pound IVb, 10±50 mL of enzyme solution (colourless)
and bu€er to a ®nal volume of 1.0 mL. The bu€ers were
used pH 5.0 (0.1 M sodium acetate), or pH 6.0 or 7.0
(0.1 M potassium phosphate) or universal bu€er
(pH=2±12 see pH optimum). Enzyme activity values
were obtained by continuously monitoring the absor-
bance decrease with Perkin±Elmer Lambda 6 or 3 spec-
trometer over 20±300 min, and the absorbances were
read at 350±390 nm. Blank controls consisted of the
above mixtures without enzyme additions or with boiled
enzyme preparations.
1
methanol 1:40). Yield 0.41 g (27%). H NMR (CDCl3)
d 7.82 (dd, 2H, J=3.2 Hz, 5.5 Hz), 7.68 (dd, 2H,
J=3.1 Hz, 5.5 Hz), 6.88 (d, 1H, J=1.5 Hz), 6.79 (m,
2H), 6.38 (d, 1H, J=15.7 Hz), 6.00 (ddd, 1H, J=7.7 Hz,
8.0 Hz, 15.7 Hz), 4.97 (s, 1H), 4.21 (m, 1H), 4.04 (s, 1H),
3.87 (s, 1H), 3.80 (s, 3H), 3.77±3.61 (m, 5H), 3.41±3.33
(m, 3H), 2.85±2.79 (2H), 1.69±1.50 (m, 4H), 1.29 (m,
8H), 0.97 (s, 9H), 0.13 (s, 6H); 13C NMR d 168.4, 150.9,
144.9, 133.8, 132.9, 132.0, 130.4, 127.1, 123.1, 123.0,
120.8, 119.5, 109.6, 107.7, 85.5, 80.3, 80.2, 67.5, 55.4,
37.9, 35.6, 32.9, 29.3, 28.4, 26.6, 25.9, 25.6, 18.4, 4.71;
IR 3500±3300 (s), 2930, 1714 (s), 1684 (w), 1508 (m),
1
1397 (m), 1281 (m), 1035 (m), 720 (m) cm
.
Chromatographic assay. The dark colour and con-
sequent high absorbances of some enzyme preparations
made the spectrophotometric assay impracticable.
HPLC separation of the esterase substrate IVb and
80-Phthalimidooctyl 5-deoxy-5-thio-(E-400-hydroxy-300-
methoxycinnamyl)-ꢀ-L-arabinofuranoside. To a stirred

Y. Zhao, M. L. Sinnott / Bioorg. Med. Chem. 8 (2000) 917±924
923
its hydrolysis product was carried out on a ternary
HPLC pump system (Spectra-Physics, SP 8800) using a
Econosphere Si 5 U column (Alltech, 4.6Â150 mm)
with a matching guard column (All-guard Cartridge
System, Econosphere Silica 5 U, Alltech). The mobile
phase was CH2Cl2:CH3CN:AcOH (80:50:1) and the
¯ow rate was 0.5 mL/min. The substrates and the
hydrolytic products were detected with a UV detector
(Spectra 100 UV Detector, Spectra-Physics) set at
320 nm. After substrate and enzyme mixtures as for the
UV assay were incubated for 3±1800 min, the reactions
were stopped by adding 1 equivalent volume of methanol.
phenyl Sepharose (Pharmacia), and the medium pres-
sure protein puri®cation system used was Waters 650.
An ammonium sulfate gradient (0±2.0 M) in 50 mM
sodium phosphate (pH 7.0) with 1.0 mM EDTA and
0.02% NaN3 was applied at 3.0 mL/min. Fractions of
3.0 mL were collected.
3. Anity chromatography. The pooled and con-
centrated feruloyl esterase fractions from the hydro-
phobic interaction chromatography step were passed
down the anity column equilibrated with 50 mM of
KH2PO4, pH 6.0 containing 2 mM arabinose (¯ow rate
0.5 mL/min). After some protein eluted, the anity col-
umn was washed with 50 mM of KH2PO4, pH 6.0, con-
taining 25 mM l-arabinose, 50 mM ferulic acid, 50 mM
methyl arabinofuranoside, 1 mM EDTA at 0.75 mL/min.
The desorbed protein fractions were checked using
Bradford reagent because ferulic acid has strong
absorption around 280 nm and it screens detection of
the absorbance around this wavelength. After desorption,
the protein was concentrated and dialyzed against
20 mM KH2PO4, pH 6.0 containing 1.0 mM EDTA and
0.02% NaN3.
One unit of activity is de®ned as the amount of enzyme
releasing 1 mmol of ferulic acid per min at 30 ꢀC. All
assays were performed in duplicate, with blanks to cor-
rect for background reactions in enzyme and substrate
samples.
Proteins. These were estimated using the Bradford
method or Lowry method.17
Growth of Aspergillus niger and isolation of enzyme
The growth of the organism and initial stages of the
puri®cation followed Faulds and Williamson.4
4. Concentration. Enzyme solutions were normally con-
centrated using a Minitan (Millipore) ultra®ltration unit
or Amicon unit with a 10 KDa cut-o€ membrane. The
®ltrate was assayed for residual protein to check for the
correct membrane fraction.
Growth of A. niger. Strain CBS 120.49 (American Type
Culture Collection) was grown from spores. After a 4-
day growth in shake-¯ask culture, solids were removed
by centrifugation at 8000 g for 15 min. The protein
solution was ®ltered with a glass micro®bre ®lter
(Whatman) and stored at 4 ꢀC for further puri®cation.
Enzyme kinetics
The substrates IVa±c were preincubated in a cuvette in
the cell-block of a Perkin±Elmer Lambda 6 for 5 min.
After addition of enzyme, the UV absorbance was con-
tinuously monitored between 350 and 390 nm, the
wavelength being chosen for a convenient initial absor-
bance and total rates. Extinction coecient di€erences
for the three substrates as a function of wavelength and
pH are given in Table 3.
Preparation of the anity column. (1) Activation of
Sepharose. Sepharose 4B (100 mL) was ®ltered from the
liquid in which it was supplied, rinsed extensively with
ice±water, and then placed into gently stirred 100 mL of
2 M sodium carbonate solution at 0 ꢀC. Cyanogen bro-
mide (10 g) in acetonitrile (20 mL) was added to this
stirred solution. After 15 min reaction at room tem-
perature, the activated material was ®ltered o€ and
rinsed extensively with ice±water using suction.
Michaelis±Menten parameters were estimated by direct
®tting of initial rates to a rectangular hyperbola using
KaleidaGraph (Synergy, Inc, Philadelphia, PA).
(II) Coupling. To a completely dissolved solution of
ligand II (30 mg), was added the activated Sepharose.
The slurry was stirred gently overnight and the next day
it was poured in a column (1.6Â20 cm), rinsed exten-
sively with a solution of sodium bicarbonate (0.1 M)
and then solution of glycine (0.2 M).
Bu€ers for kinetic studies. The pH optimum for the
hydrolysis of substrate IVb (1.0 mM) was determined
in universal bu€er: citric acid (0.0286 M), boric acid
(0.0286 M), and `bis-tris propane' (Sigma) (0.0286 M).
The mixture was titrated with 0.2 M KOH or 0.2 M
HCl to give the required pH. This universal bu€er is a
Britton±Robinson type,18 except that `bis-tris propane'
replaces diethyl barbituric acid.
1. Ammonium sulfate precipitation. The proteins from A.
niger culture supernatant were precipitated by adding
(NH4)2SO4 powder to 85% (608 g/L) saturation at 0±
4 ꢀC centrifuged at 4000 g for 20 min. The supernatant
was decanted, the precipitate was resuspended in 1/100
of the original volume of bu€er (25 mM KH2PO4,
1.0 mM EDTA and 0.02% NaN3 pH 7.0), ®ltered with
glass ®ber ®lter papers and stored at 4 ꢀC.
Other kinetic parameters were determined in 0.1 M
potassium phosphate bu€er, pH 6, 1.0 mM in EDTA.
Molecular weight determination
SDS-PAGE was performed using a Pharmacia Phast-
System, using a 8±25% gradient PhastGel (Pharmacia):
protein bands were determined by Coomassie staining.
Mr values were estimated from a plot of log Mr versus
2. Hydrophobic interaction chromatography. The 85%
pellet was fractionated by hydrophobic interaction
chromatography: the column (2.6Â25 cm) contained

924
Y. Zhao, M. L. Sinnott / Bioorg. Med. Chem. 8 (2000) 917±924
Table 3. Extinction coecient di€erences (ÁE/10, i.e., M mm 1, or 10
M
cm 1) at 30 ꢀC between substrates (IV) and the product acid,
1
1
1
measured in 0.1 M bu€er (sodium acetate pH 5.0 and potassium phosphate pH 6.0 and 7.0)a
Substrate
IVa
5.0
IVa
6.0
IVa
7.0
IVb
IVb
6.0
112
72
348
637
879
989
957
830
634
431
268
160
95
IVb
7.0
120
67
IVc
5.0
152
54
IVc
6.0
81
169
IVc
l/(pH)
310
315
320
325
330
335
340
345
350
355
360
365
370
5.0
19.3
183
7.0
1.5
247
533
822
1053
1167
1167
1074
898
684
493
335
221
337
1177
480
716
856
851
726
551
390
249
130
65
688
976
1141
1117
943
714
500
322
189
97
474
31
16.5
322
1153
673
969
1136
1114
947
724
520
352
218
133
865
65
421
672
868
945
904
784
606
408
251
140
79
346
636
880
992
970
848
651
456
302
195
134
336
999
303
558
776
896
922
869
742
566
406
271
167
339
926
461
759
1001
1128
1139
1055
884
668
472
313
195
245
12.3
5.8
322
871
50
322
1146
lmax/(nm)
336
946
336
992
338
1147
ÁEmax
aA positive value corresponds to a decrease in absorbance on hydrolysis.
4. Faulds, C. B.; Williamson, G. Microbiology (UK) 1994,
140, 779.
5. Kroon, P. A.; Faulds, C. B.; Brezillon, C.; Williamson, G.
Eur. J. Biochem. 1997, 248, 245.
mobility using commercially available protein molecular
weight standards (MW-SDS-70L, Sigma).
Gel permeation chromatography determination of mole-
cular weight. Pooled feruloyl esterase active fractions
from the anity chromatography were fractionated on
a Waters 650 rapid protein puri®cation system, using a
gel ®ltration column (0.8Â30 cm, Protein PAK 300SW,
Waters) previously equilibrated with 50 mM KH2PO4,
1.0 mM EDTA and 0.02% NaN3 pH 6.0, and calibrated
with MW-GF-70 and MW-GF-200 protein molecular
weight standard kits (Sigma). Fractions of 0.2 mL were
collected.
6. McDermid, K. P.; Mackenzie, C. R.; Forsberg, C. W. Appl.
Environ. Microbiol. 1990, 56, 127.
7. Mackenzie, C. R.; Bilous, D. Appl. Environ. Microbiol.
1988, 54, 1170.
8. Borneman, W. S.; Hartley, R. D.; Himmelsbach, D. S.;
Ljungdahl, L. G. Anal. Biochem. 1990, 190, 129.
9. Borneman, W. S.; Ljungdahl, L. G.; Hartley, R. D. Appl.
Environ. Microbiol. 1992, 58, 3762.
10. Helm, R. F.; Ralph, J.; Hat®eld, R. D. Carbohydr. Res.
1992, 229, 183.
11. Hat®eld, R. D.; Helm, R. F.; Ralph, J. Anal. Biochem.
1991, 194, 25.
Acknowledgements
12. Mock, W. L. In Comprehensive Biological Catalysis; Sin-
nott, M. L., Ed.; Academic: London, 1998; Vol I, pp 425±453.
13. De Vries, R. P.; Michelsen, B.; Poulsen, C. H.; van den
Heuvel, R. H. H.; van den Hombergh, J. P. T. W.; Visser, J.
Appl. Environ. Microbiol. 1997, 63, 4638.
Financial support from the United States Department
of Agriculture and the University of Illinois Foundation
is gratefully acknowledged.
14. Williamson, G.; Fauls, C. B.; Kroon, P. A. Biochem. Soc.
Trans. 1998, 26, 205.
15. Kroon, P. A.; Faulds, C. B.; Williamson, G. A. Biotechnol.
Appl. Biochem. 1996, 23, 252.
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3. Broda, P.; Birch, P.; Brooks, P.; Copa-Patino, J. L.; Sinnott,
M. L.; Tempelaars, C.; Wang, Q.; Wyatt, A.; Sims, P. FEMS
Microbiol. Rev. 1994, 13, 189.
17. Bollag, D. M.; Edelstein, S. J. Protein Methods. Wiley Liss:
New York, 1991; pp 56±59.
18. Dawson, R. M. C. D., Elliott, D. C., Elliott, W. H.; Jones,
K. M. Data for Biochemical Research, 2nd ed.; Oxford Uni-
versity Press: London, 1989; p 485.