Chemo-enzymatic synthesis of 4-methylumbelliferyl β-(1→4)-D-xylooligosides: New substrates for β-D-xylanase assays

Article abstract of DOI:10.1039/b409583a

Transglycosylation catalyzed by a β-D-xylosidase from Aspergillus sp. was used to synthesize a set of 4-methylumbelliferyl (MU) β-1→4-D-xylooligosides having the common structure [β-D-Xyl-(1→4)]_2-5-β-D-Xyl-MU. MU xylobioside synthesized chemica

Full text of DOI:10.1039/b409583a

View Article Online / Journal Homepage / Table of Contents for this issue
A R T I C L E
Chemo-enzymatic synthesis of 4-methylumbelliferyl
b-(1→4)-D-xylooligosides: new substrates for b-D-xylanase assays
Elena V. Eneyskaya,a Dina R. Ivanen,a Konstantin A. Shabalin,a Anna A. Kulminskaya,*a
Leon V. Backinowsky,b Harry Brumer IIIc and Kirill N. Neustroev†a
a Petersburg Nuclear Physics Institute, Russian Academy of Science, Molecular and Radiation
Biology Division, Gatchina, 188300, Russia. E-mail: kulm@omrb.pnpi.spb.ru;
Fax: +7 81371 32 303; Tel: +7 81371 32 014
b N.D.Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky prosp.
47, Moscow, 119991, Russia
c Department of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University
Centre, S-106 91, Stockholm, Sweden
Received 24th June 2004, Accepted 28th October 2004
First published as an Advance Article on the web 26th November 2004
Transglycosylation catalyzed by a b-D-xylosidase from Aspergillus sp. was used to synthesize a set of
4-methylumbelliferyl (MU) b-1→4-D-xylooligosides having the common structure [b-D-Xyl-(1→4)]2–5
-
b-D-Xyl-MU. MU xylobioside synthesized chemically by the condensation of protected MU b-D-xylopyranoside
with ethyl 2,3,4-tri-O-acetyl-1-thio-b-D-xylopyranoside was used as a substrate for transglycosylation with the
b-D-xylosidase from Aspergillus sp. to produce higher MU xylooligosides. The structures of oligosaccharides
obtained were established by 1H and 13C NMR spectroscopy and electrospray tandem mass spectrometry. MU
b-D-xylooligosides synthesized were tested as fluorogenic substrates for the GH-10 family b-D-xylanase from
Aspergillus orizae and the GH-11 family b-D-xylanase I from Trichoderma reesei. Both xylanases released the
aglycone from MU xylobioside and the corresponding trioside. With substrates having d.p. 4 and 5, the enzymes
manifested endolytic activities, splitting off MU, MUX, and MUX2 primarily.
were studied with the aid of PNP b-acetyllactosamine-repeating
Introduction
oligosides.11 Fluorogenic substrates for glycosidases have certain
advantages over chromogenic analogues: they are both more
sensitive and can be used for samples in highly colored media.12–14
For example, MU xylobioside enables detection of b-xylanase
activity on agar plates.15 Since synthetic organic methods are
difficult for the production of MU xylosides with a d.p. more
than 2,15 an enzymatic approach can serve as an alternative and
effective way to manufacture such compounds. In our previous
related work, we described the enzymatic synthesis of a set of
PNP b-D-xylooligosides with d.p. up to 7 produced with the
aid of a b-D-xylosidase from Aspergilus sp.16 The chromophore-
bearing substrates were produced in good yields and absolute
stereoselectivity; only b-1→4-D-xylosidic linkages were formed.
Here, we used the same b-D-xylosidase for the enzymatic syn-
thesis of new b-D-xylanase substrates, namely, 4-methylumbel-
liferyl b-D-xylooligosides (MUXn) with d.p. 3–6, and tested two
b-D-xylanases with the fluorescent xylosides produced.
b-D-Xylanase (1,4-b-xylanase, 1,4-b-D-xylan xylanohydrolase,
E.C.3.2.1.8) is a glycoside hydrolase, which hydrolyses b-1→4-
xylosidic linkages in the xylan component of plant cell walls.
These enzymes have been isolated mainly from microorganisms
and are of a great interest because of numerous biotechnological
applications.1,2 Xylanases are currently used widely in pulp
and paper manufacture, alcohol, brewery, and food industries,
and the need for these enzymes continuously grows.3,4 Exten-
sive use of b-D-xylanases stimulates both the search for new
sources of the enzymes, and detailed studies of their enzymatic
properties. Assay methods for b-D-xylanases based on tracking
the liberation of reducing ends during xylan hydrolysis have
some restrictions. Xylans obtained from natural sources are
polydisperse and contain arabinose and glucuronic acid side-
chains.5 Furthermore, these methods have insufficient sensitivity
for detailed mechanistic analysis and use less desirable stopped
assay conditions.6,7 The use of polymeric substrates, such as
xylans and glucans, precludes studies of the transglycosylating
activities of b-D-xylanases due to difficulties in distinguishing
transglycosylation and hydrolysis products.8 For these reasons,
detailed kinetic and thermodynamic analyses and investigations
into the action patterns of xylanases require more conve-
nient substrates which enable high sensitivity assays. For such
purposes, a range of xylooligosides bearing chromogenic or
fluorescent groups at the reducing end of a chain can be
employed; analogous substrates are widely used for detailed
enzymatic investigations of various glucanases. For example, the
modes of action of a-amylases from different origins have been
studied with p-nitrophenyl (PNP) maltooligosides with degree
of polymerization (d.p.) 2–7.9 A set of 4-methylumbelliferyl
(MU) b-1,3(4)-glucosides were successfully applied for subsite
mapping of the active site of b-1,3(4)-glucanases based on the Hi-
romi approach.10 Kinetic properties of an endo-b-galactosidase
Results and discussion
Synthesis of MU b-D-xylooligosides and the structure of the
transxylosylation products
To produce MU b-D-xylooligosides (MUXn), we took advantage
of the transglycosylating ability of Aspergillus sp. b-D-xylosidase,
which had previously been used for the synthesis of PNP b-D-
xylooligosides of various lengths.16 MU xyloside (MUX) is a
substrate for the b-D-xylosidase, which splits the glycoside with
a catalytic efficiency comparable with that for the well-known
PNPX (the values for kcat/Km are 220 and 480 s mM−1 for MUX
and PNPX, respectively). However, it appeared impossible to
use MUX in the synthesis of MU b-D-xylooligosides due to low
solubility of the substrate in aqueous solutions (<5 mM), which
significantly limits the use of this compound in transglycosyla-
tion reactions. Transglycosylation reactions catalyzed by glycosi-
dases result from involvement of a glycosyl acceptor molecule
† In memoriam of Dr Kirill N. Neustroev.
1 4 6
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 4 6 – 1 5 1
T h i s j o u r n a l i s
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
©

View Article Online
other than water, and are thus favored by high concentrations of
the alternative acceptor. Previously, the optimum concentration
of PNPX in the synthesis of PNPXn by the Aspergillus b-D-
xylosidase was found to be 110 mM.16 The addition of up
to 30% (v/v) DMF or DMSO to increase the solubility of
MUX led to inactivation of the enzyme. Therefore, to produce a
set of extended MU xylooligosaccharides, we chose the more
water-soluble 4-methylumbelliferyl b-D-xylopyranosyl-(1→4)-
b-D-xylopyranoside (MUX2) as a transglycosylation acceptor
(Scheme 1).
MU xylobioside (MUX2) was synthesized by the reaction
of the known ethyl 2,3,4-tri-O-acetyl-1-thio-b-D-xylopyranoside
1 (glycosyl donor) as described17 with a suitably protected
acceptor and subsequent deacylation (Scheme 2). The NMR
data strongly support the b-configuration of the (1→4)-xylosidic
bond formed. Selective, dibutyltin oxide-mediated chloroacety-
lation of commercially available MUX (2→3), subsequent
benzoylation (→4), and dechloroacetylation with thiourea in
methanol resulted in the target xyloside acceptor (5) with the
free OH group at C-4. Its coupling with the donor, ethyl
1-thioxyloside 1, (methyl fluorosulfonate as a promoter18),
afforded the peracylated MU xylobioside 6 in a good yield.
This was deacylated into the target 4-methylumbelliferyl b-D-
xylobioside (MUX2, 7). Compared with the previously reported
protocol of the synthesis of MUX2 from a mixture of xylose,
xylobiose, and xylotriose,15 our procedure avoids the need of
isolation of a rather moisture-sensitive per-O-acetylxylobiosyl
bromide by crystallization.
Scheme 2
with high stereospecificity is rather unique; oligosaccharide
chains produced by enzymatic transglycosylation often contain
two or three monosaccharide residues with different linkage
configurations.21,22 Only one successful synthesis of a highly
stereoregular product, viz., PNP b-D-galactopyranosyl-(1→4)-
b-D-galactopyranoside, by the b-galactosidase from Bacillus
circulans has been reported.23 Other attempts to prepare MU
oligosides using various exo- or endo-glycosidases have rarely
been effective. This can be explained by poor acceptor prop-
erties of MU glycosides in transglycosylation reactions due
to their low solubility and poor binding affinity. Therefore,
chemical methods are traditionally used for the synthesis of MU
oligosaccharides with d.p. more than 2.17,24 To our knowledge,
only one enzyme, namely, the laminarinase from Oerskovia
sp., has been reported, which was able to produce a range
of chromo/fluorogenic oligosides (in this case, MU b-(1→3)-
D-glucooligosides).25 In the present work, we expanded this
repertoire by employing an exo-glycoside hydrolase, viz., b-
D-xylosidase from Aspergillus sp., to synthesize MU 1→4-b-
xylooligosides in good yields.
The transglycosylation reaction conditions which gave rise to
the maximum yields of MUX3–6 were selected using analytical
HPLC. Under optimum reaction conditions (pH 5.5–7.0; 37 ◦C;
acceptor concentration, 140–160 mM), MU xylooligosides of
d.p. 3–6 were obtained in 21, 12, 6, and 3%, respectively, at
50–60% conversion of MUX2. The structures of individual
transglycosylation products isolated by HPLC were established
by MS and NMR.
1
Assignment of the signals in the H and 13C NMR spectra
followed the same methodology as described in ref. 16 with
reference to the spectra of PNPXn. Thus, a comparative analysis
of 1H and 13C NMR spectroscopic data for MUX3 and MUX4
16,19
and those for PNPX2–4
showed that the transglycosyla-
tion catalyzed by Aspergilus b-xylosidase occurs with absolute
stereospecificity giving only b-1→4-linkages. Furthermore, the
stoichiometry of xylose residues in all isolated transglycosylation
products, MUX3–6, was unambiguously confirmed by CID
MS/MS analysis (Fig. 120). Trace amounts of longer MU
xylooligosides produced by the enzyme were also detected by
TLC (not shown). The ability to produce higher oligosaccharides
Application of MU xylooligosides as mechanistic probes. All
known xylanases are classified into two glycoside hydrolase
families (GH10 and GH11) on the basis of sequence homologies
and action mechanisms according to the Henrissat classifica-
tion (http://afmb.cnrs-mrs.fr/CAZY/).26 We chose the enzyme
isolated from Aspergillus orizae (XynA)27 as a representative
Scheme 1
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 4 6 – 1 5 1
1 4 7

View Article Online
Fig. 1 CID MS/MS spectra of monosodiated adducts of MU xylooligosaccharides. A. MUX2; B. MUX3; C. MUX4; D. MUX5; E. MUX6.
Calculated m/z values of the monosodiated MU xylooligosaccharide adducts (bold italics) were used as reference values to recalibrate all spectra.
Table 1 The yields of transfer products produced by the b-D-xylosidase
of GH10 and a b-xylanase from Trichoderma reesei (XynT)
belonging to GH1128 to test their modes of action towards
synthesized MU 1→4-b-D-xylooligosides with d.p. 2–6. Virtu-
ally complete hydrolysis of MUX2 by both XynA and XynT
led to the liberation of aglycon from xylobiose, which was
identified by HPLC using an authentic sample of xylobiose
as a standard. XynA and XynT were observed to split off the
aglycon moiety from MUX3 at up to 40% conversion of the
substrate giving free MU and xylotriose (Table 1). This indicated
that the above substrates can be used for direct b-xylanase
assays by detecting the liberated fluorophore by conventional
methods. The concentrations of both substrates used in these
experiments were in the range of 0.4 to 1 mM, and the results
obtained match those reported for the action of XynA and
XynT on PNPX2 and PNPX3.16 The Km values in the xylanase-
catalyzed hydrolysis of MUX2 and MUX3, calculated from the
Lineweaver–Burk equations, are given in Table 2. The kinetic
in the reactions of substrate transglycosylation.
Product
Yield (%)
MUX3
MUX4
MUX5
MUX6
21.0
12.0
6.0
3.0
data for both MUX2, 3 and PNPX2, 3 are in the same range of
values as for artificial substrates traditionally used for exo- and
endo-glycoside hydrolase assays.9–14 The b-xylanase-catalyzed
hydrolysis of MU xylosides occurs in the same pH optimum
range as the hydrolysis of xylans.
MUX4 and MUX5 also serve as substrates for both b-D-
xylanases, which produce lower MU b-xylooligosides along with
1 4 8
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 4 6 – 1 5 1

View Article Online
Table 2 The Michaelis constants (Km) of the hydrolysis of MU and
PNP b-D-xylooligosides by XynA and XynT
of the glycoside hydrolase family 11, was kindly donated by
Dr A. Miasnikov, Danisco Global Innovation, Finland, and
separated from contaminating b-xylosidase activity as reported
previously.16 The specific activity of the purified protein was
about 300 5 U mg−1. The Aspergillus orizae b-xylanase (XynA)
was isolated from culture liquid according to the procedures
Km/mM
Substrate
XynA (GH10)
XynT (GH11)
described by Kitamoto et al.28 resulting in 250
5 U mg−1
MUX2
MUX3
PNPX2
PNPX3
0.1
0.15
0.083
0.3
0.054
0.25
0.162
activity. Both xylanase preparations contained less than 0.01%
of extrinsic b-D-xylosidase activity.
0.189
Analytical methods
Table 3 Yieldsa of products from hydrolysis of MU b-D-xylooligosides
by XynA
Protein concentrations were measured following the Lowry
procedure with BSA as a standard.29
Products (mol per mol of MU products)b
All 1H and 13C NMR spectra were recorded with an AMX-500
Bruker spectrometer (1H at 500.13 MHz, 13C at 125.13 MHz) at
ambient temperature for solutions in different solvents. Me4Si
was used as an external standard and calibration was performed
using the signal of the residual protons of the solvents. Chemical
shifts (d in ppm) are given relative to those for CDCl3 (dH 7.25),
30% solution of CD3CN in D2O (dH 2.21), 30% solution of
(CD3)2CO in D2O (dH 2.19); J-values are given in Hz.
Substrate
MU
MUX
MUX2
MUX3
MUX4
MUX2
MUX3
MUX4
MUX5
0.99
0.98
0.79
0.59
traces
traces
0.13
traces
0.07
0.12
0.29
0.1
a The yields are an average of at least 3 determinations. b Experimental
error is 5% of values given.
The oligosaccharides obtained upon enzymatic hydrolysis and
enzymatic synthesis were analyzed qualitatively by TLC on
Kieselgel 60 plates (Merck) with a butan-1-ol–acetic acid–water
(3 : 1 : 1, v/v) solvent system.
Table 4 Yieldsa of products from hydrolysis of MU b-D-xylooligosides
by XynT
Mass spectrometric analysis was performed with a Q-TofTM
2 mass spectrometer fitted with a nanoflow ion source (Waters
Corporation, Micromass MS Technologies, Manchester, U.K.).
Calibration of the TOF analyser (single-reflectron mode, reso-
lution >10000 FWHM) was obtained over the m/z range 50–
1000 using a solution of NaI (1.5 g L−1) in 1 : 1 propan-2-ol–
water. A scan time of 2.5 s with an interscan delay of 0.1 s was
used in all MS modes. Solutions of MU xylooligosides (typical
concentration 10–20 lM in 1 : 1 MeOH–water containing
0.5 mM NaCl) were infused into the ion source at 200 nL min−1
(syringe pump). The cone voltage was set at 45 V and the
electrospray capillary voltage was varied (2.2 kV to 3.0 kV)
to obtain less than 500 counts s−1 for the base peak ion signal.
These conditions favored the production of [M + Na]+ adducts;
the formation of the [M + 2Na]2+ adduct was only significant
for Xyl6-MU. For accurate mass determinations in the TOF MS
mode, centroid spectra were generated from continuum spectra
using the (M + Na)+ adduct of raffinose (a-Galp-(1→6)-a-Glcp-
(1↔2)-b-Fruf ) as an internal standard (calc’d. mass 527.1588 u).
The raffinose (raffinose pentahydrate, R-0250, Sigma-Aldrich,
minimum purity 99%) concentration in each sample was
adjusted empirically to give a peak intensity similar to that
of the MU xylooligoside analyte. For MS/MS experiments,
the collision energy was varied (30–55 V), depending on the
resilience of the (M + Na)+ ion, to achieve an optimal balance
between parent and fragment ion intensities. Data were collected
until an acceptable signal-to-noise ratio was achieved after
the combination of individual spectra (typical 15–30 spectra).
Centroid MS/MS spectra were produced from continuum
spectra using the calculated m/z of the monosodiated adduct
of the appropriate MU xylooligosaccharide as a reference mass.
Products (mol/mol of MU products)b
Substrate
MU
MUX
MUX2
MUX3
MUX4
MUX2
MUX3
MUX4
MUX5
0.91
0.88
0.61
0.35
0.09
traces
traces
traces
0.1
0.09
0.25
0.15
0.21
a The yields are an average of at least 3 determinations. b Experimental
error is 5% of values given.
trace quantities of free MU upon hydrolysis of these compounds.
Reproducible values for the yields of MU xylooligosides and free
MU for all the specimens tested are given in Table 3 and Table 4.
The xylanase subsite structure can be characterized with the aid
of MU xylooligosides since one can easily separate the hydrolysis
products by HPLC.
Thus, the synthesised MU b-D-xylooligosides (d.p. 2–6) are
promising tools for studies of subsite architecture of the active
centre of b-xylanases on the basis of procedures developed
for other glycosidases with artificial substrates.9,10,28 Since b-
D-xylanase splits off the aglycon moiety from MUX2 and
MUX3, these compounds are ideal substrates for detailed kinetic
studies of the enzyme due to a higher sensitivity of detection
when compared with the methods of detecting reducing sugars
released during the b-D-xylan hydrolysis or the use of alternative
substrates.6,7
Experimental
Substrates and enzymes
Enzyme assay
b-D-Xylan from birchwood and MUX (2) were purchased from
Sigma Chemical Co., St. Louis, USA; D-xylopyranosyl-(1→4)-
b-D-xylopyranose was prepared by enzymatic digestion of xylan
by b-D-xylanase from Trichoderma reesei followed by chromato-
graphic purification on a Biogel P2 column (25 × 200 mm)
equilibrated in water. p-Nitrophenyl b-D-xylobioside (PNPX2)
and p-nitrophenyl b-D-xylotrioside (PNPX3) were synthesized
enzymatically by the b-D-xylosidase from Aspergillus sp. as
described earlier.16
The b-xylosidase activity towards PNPX was determined at
◦
37 C in 50 mM sodium acetate buffer, pH 4.5.16 One unit
of b-D-xylosidase was defined as the amount of the enzyme
releasing 1 lmol of p-nitrophenol per min in the above con-
ditions. b-Xylanase activity was evaluated by the Somogyi–
Nelson method30 using xylan as a substrate. One unit of the
b-xylanase activity was defined as the amount of the enzyme
capable of releasing 1 lmol of reducing sugar from xylan with
concentration of 5 mg mL−1 at 37 ◦C in 20 mM sodium acetate
buffer, pH 4.0, per min.
The b-D-xylosidase from Aspergillus sp. was purified accord-
ing to ref. 16. b-Xylanase I from Trichoderma reesei (XynT)
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 4 6 – 1 5 1
1 4 9

View Article Online
Modes of action of XynT and XynA
d, J1,2 4.03, H-1), 5.55 (1 H, dd, H-2), 5.23 (1 H, ddd, J4,5a 3.53,
J4,5b 5.54, H-4), 4.40 (1 H, dd, J5a,5b 12.8, H-5a), 4.11 (1 H, d,
JCH2-a, CH2-b 14.83, ClCH2CO-a), 4.06 (1 H, d, ClCH2CO-b), 3.86
(1 H, dd, H-5b), 2.38 (3 H, 4-Me-MU).
The action patterns of both xylanases were investigated in
20 mM sodium acetate buffer, pH 4.0, at 37 ◦C, using substrate
(MUX2–5) concentrations in the range from 0.8 to 1.2 mM,
the course of the reactions being followed by TLC. To initiate
the reaction, about 0.01 U of the xylanase preparation was
added to the reaction mixture. The reaction was terminated
by freezing and lyophilization. Analysis of MU-containing
hydrolysis products was performed on a Waters Hypersil ODS
column (200 × 4.8 mm) using a linear gradient (0–90%) of
MeCN in water with spectrophotometric detection at 254 nm.
4-Methylumbelliferyl 2,3-di-O-benzoyl-b-D-xylopyranoside 5
A mixture of peracylated glycoside 4 (10 mmol; 4.66 g) and
thiourea (50 mmol; 3.8 g) in MeOH (100 ml) was boiled under
reflux for 6 h. The mixture was concentrated, the residue was
dissolved in CH2Cl2 and subjected to a column chromatography
(9 : 1 → 2 : 1 PhMe–acetone) to give dibenzoate 5 (4.47 g; 87%);
dH (CDCl3): 8.04 (2 H, d, J2,3 8.06, m-OBz), 7.99 (2 H, d, J2,3
7.91, m-OBz), 7.57 (2 H, dd, J3,4 7.48, o-OBz), 7.54 (2 H, dd, J3,4
7.48, o-OBz), 7.48 (1 H, d, J5,6 8.78, H-5 MU), 7.43 (1 H, dd,
p-OBz), 7.40 (1 H, dd, p-OBz), 7.00 (1 H, d, J6,8 2.45, H-8 MU),
6.94 (1 H, dd, H-6-MU), 6.16 (1 H, s, H-3 MU), 5.60 (1 H, dd,
J1,2 5.25, J2,3 7.12, H-2), 5.49 (1 H, d, H-1), 5.40 (1 H, dd, J3,4
6.9, H-3), 4.33 (1 H, dd, J4,5a 4.13, J5a,5b 12.23, H-5a), 4.09 (1 H,
ddd, J4,5b 6.4, H-4), 3.73 (1 H, dd, H-5b), 3.21 (1H, s, OH-4),
2.37 (3 H, s, 4-Me MU).
Determination of kinetic parameters of the hydrolysis of
MU-b-D-xylooligosides by XynT and XynA
The activities of the XynT and XynA in the hydrolysis of
MUX2 and MUX3 were evaluated spectrofluorometrically after
incubating the reaction mixture in 50 mM sodium acetate buffer,
pH 4.0, by measuring the released MU in accordance with ref.
25. The Michaelis constants were determined for each substrate
from the Michaelis-Menten equation by non-linear regression
analysis.31 The rates were determined for at least eight different
substrate concentrations ranging from about 0.1 × the Km value
determined to 4–6 × Km.
4-Methylumbelliferyl 2,3,4-tri-O-acetyl-b-D-xylopyranosyl-
(1→4)-2,3-di-O-benzoyl-b-D-xylopyranoside 6
A solution of ethyl 1-thioxyloside 1 (2 mmol; 640 mg) and the
dibenzoate 5 (1 mmol; 514 mg) in dry CH2Cl2 (25 ml) was
4-Methylumbelliferyl b-D-xylopyranosyl-(1→4)-b-D-
˚
xylopyranoside (MUX2)
stirred with freshly activated MS 4 A (1.5 g) for 1 h, methyl
fluorosulfonate (10 mmol; 1.14 g) was added, and stirring was
continued for 20 h at room temperature. Triethylamine (5 ml)
was added, the mixture was filtered through a layer of Celite
into a mixture of ice–H2O and CH2Cl2. The organic layer was
washed with 10% aq HCl, water, aq NaHCO3, water, dried, and
concentrated. Column chromatography (20 : 1 → 9 : 1 PhMe–
acetone) of the residue gave the protected bioside 6 (613 mg;
79%); dH (CDCl3): 8.06–7.97 (4 H, m, o-OBz), 7.58–7.51 (4 H,
m, m-OBz), 7.47 (1 H, d, J5,6 8.78, H-5-MU), 7.45–7.38 (2 H,
m p-OBz), 6.97 (1 H, d, J6,8 2.45, H-8 MU), 6.91 (1 H, dd, H-6
MU), 6.15 (1 H, m, J3,4-Me 1.44, H-3 MU), 5.66 (1 H, dd, J2,3 6.96,
MU xylobioside was synthesized by the condensation of suit-
ably protected MUX derivative 5, having 4-OH unsubstituted,
with ethyl 2,3,4-tri-O-acetyl-1-thio-b-D-xylopyranoside (1) us-
ing methyl fluorosulfonate as a thiophilic activator (Scheme 2).
Ethyl 2,3,4-tri-O-acetyl-1-thio-b-D-xylopyranoside 1
Compound 1 was prepared by boron trifluoride-catalyzed con-
densation of b-D-xylose peracetate with thiourea as described.17
4-Methylumbelliferyl 4-O-chloroacetyl-b-D-xylopyranoside 3
J3,4 6.76, H-3), 5.50 (1 H, dd, J1,2 5.15, H-2), 5.45 (1 H, d, H-1),
Compound 2 (10 mmol; 3.24 g) and dibutyltin oxide (15 mmol;
3.72 g) were suspended in PriOH (200 ml) and heated under
reflux for 3 h, and the solvent was evaporated. The residue
was dissolved in dry CH2Cl2 (100 ml), cooled to 0 ◦C, and a
solution of ClCH2COCl (11 mmol; 876 ll) in CH2Cl2 (10 ml)
was added dropwise. The mixture was stirred for 1 h at 0 ◦C and
concentrated. Chromatography (toluene : acetone, 9 : 1→1 : 1) of
the residue on a silica gel afforded 3 (2.55 g, 68%); dH(500 MHz,
CD3CN): 7.63 (1 H, d, J5,6 8.8, H-5 MU), 6.99 (1 H, dd, J6,8 2.28,
H-6 MU), 6.97 (1 H, d, H-8 MU), 6.14 (1 H, m, J3,4-Me 1.1, H-3
MU), 5.06 (1 H, d, J1,2 7.24, H-1), 4.85 (1H, ddd, J3,4 9.14, J4,5a
5.35, J4,5b 9.55, H-4), 4.23 (2H, s, ClH2CO), 4.03 (1 H, dd, J5a,5b
11.6, H-5a), 3.91 (0.7 H, s, OH), 3.77 (0.7 H, s, OH), 3.67 (1 H,
dd, J2,3 8.96, H-3), 3.55 (1 H, dd, H-2), 3.52 (1 H, dd, H-5b),
2.38 (3 H, d, 4-Me MU).
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
5.05 (1 H, dd, J2 ,3 7.98, J3 ,4 7.74, H-3 ), 4.86 (1 H, dd, H1 ,2
6.19, H-2ꢀ), 4.68 (1 H, ddd, J4 ,5a 4.75, J4 ,5b 7.87, H-4ꢀ), 4.66 (1
ꢀ
ꢀ
ꢀ
ꢀ
H, d, H-1ꢀ), 4.22 (1 H, dd, J4,5a 4.17, J5a,5b 12.38, H-5a), 4.07 (1
H, ddd, J4,5b 6.65, H-4), 3.79 (1H, dd, J5a ,5b 12.05, H-5aꢀ), 3.71
(1 H, dd, H-5b), 3.20 (1 H, dd, H-5bꢀ), 2.36 (3 H, d, 4-Me MU),
2.04 (3 H, s, OAc), 1.98 (6H, s, OAc).
ꢀ
ꢀ
4-Methylumbelliferyl b-D-xylopyranosyl-(1→4)-b-D-
xylopyranoside (MUX2, 7)
A solution of 6 (500 mg) in dry MeOH (10 ml) was treated with
methanolic NaOMe (100 ll) according to ref. 32 and purified by
reverse-phase chromatography on an INERTSIL PREP-ODS
column (20 × 250 mm) using a linear gradient (0–90%) of MeCN
1
in water to afford the bioside 7. H NMR (D2O) dH 5.12 (1 H,
d, J1,2 7.52, H-1), 4.51 (1 H, d, J1,2 7.84, H-1ꢀ), 4.19 (1 H, dd,
4-Methylumbelliferyl 2,3-di-O-benzoyl-4-O-chloroacetyl-b-D-
xylopyranoside 4
J5a,5b 11.72, J4,5a 5.27, H-5a), 4.01 (1 H, dd, J5a,5b 11.60, J4,5a 5.40,
H-5aꢀ), 3.88 (1H, ddd, J4,6b 10.1, J3,4 8.8, H-4), 3.74 (1 H, t, J2,3
9.4, H-3), 3.66 (1H, ddd, J4,6b 10.3, J3,4 9.2, H-4ꢀ), 3.64 (1H, dd,
H-2), 3.62 (1 H, dd, H-5b), 3.48 (1 H, t, J2,3 9.2, H-3ꢀ), 3.35 (1
H,dd, H-5bꢀ), 3.32 (1 H, dd, H-2ꢀ); 13C NMR (D2O) dC 103.98
(C-1ꢀ), 102.15 (C-1), 78.25 (C-4ꢀ), 77.68 (C-3ꢀ), 75.55 (C-3), 74.82
(C-2ꢀ), 74.57 (C-2), 71.23 (C-4), 67.30 (C-5ꢀ), 65.12 (C-5); ESI+
MS [M + Na]+ m/z 463.1216 calcd. for C20H24NaO11, observed:
463.1209.
Compound 3 (10 mmol; 3.82 g) was dissolved in dry CH2Cl2
(100 ml) containing pyridine (0.1 mol; 8 ml) and cooled to
0 ◦C. Benzoyl chloride (0.03 mol; 3.51 ml) was added dropwise
and stirring was continued overnight at room temperature. The
solution was washed with cold dil HCl, aq NaHCO3, H2O, dried,
and concentrated. Chromatography (toluene : acetone, 20 : 1→4
: 1) of the residue on a column with silica gel gave the peracylated
xyloside 4 (5.84 g, 93%); dH (CDCl3): 8.06 (2 H, dd, J 8.06, m-
OBz), 8.05 (2 H, dd, J 7.9, m-OBz), 7.50 (1 H, d, J5,6 8.78, H-5
MU), 7.46 (2 H, d, J3,4 7.6, o-OBz), 7.43 (2 H, d, J3,4 7.8, o-OBz),
7.24 (1 H, dd, p-OBz), 7.16 (1 H, dd, p-OBz), 7.01 (1 H, d, J6,8
2.45, H-8 MU), 6.95 (1 H, dd, H-6 MU), 6.17 (1 H, m, J3,4-Me
1.44, H-3 MU), 5.64 (1 H, dd, J2,3 5.83, J3,4 5.83, H-3), 5.60 (1 H,
Synthesis of MU b-D-xylooligosides with d.p. 3–6
A solution of MUX2 (80 mM) and appropriate amount of the
b-D-xylosidase corresponding to 8 U were, incubated in 6 mL
of 30 mM sodium phosphate buffer, pH 6.5, for 180 min at
37 ◦C. Following termination of the reaction by freezing and
1 5 0
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 4 6 – 1 5 1

View Article Online
freeze-drying, individual components of a mixture of transgly-
cosylation products were initially separated on an INERTSIL
PREP-ODS column (20.0 × 250 mm) and then purified on a
TSK-NH2-60 column (4.6 × 250 mm, Pharmacia) using isocratic
elution with 80% MeCN. Finally, the appropriate fractions were
freeze-dried and the products obtained were used in further
investigations.
References
1 H. J. Gilbert and G. P. Hazlewood, J. Gen. Microbiol., 1993, 139,
187–194.
2 A. P. Garg, J. C. Roberts and A. J. McCarthy, Enzyme Microb.
Technol., 1998, 22, 594–598.
3 A. Gessesse and G. Mamo, Enzyme Microb. Technol., 1999, 25, 68–
72.
4 Q. K. Beg, M. Kapoor, L. Mahajan and G. S. Hoondal, Appl.
Microbiol. Biotechnol., 2001, 56, 326–338.
5 L. Weinstein and P. Albersheim, Plant Physiol., 1979, 63, 425–432.
6 H. Taguchi, T. Hamasaki, T. Akamatsu and H. Okada, Biosci.
Biotech. Biochem., 1996, 60, 983–985.
7 Y. Zhao, C. J. Chany II, P. F. G. Sims and M. L. Sinnott, J. Biotechnol.,
1997, 57, 181–190.
8 B. O. Petersen, M. Krah, J. Ø. Duus and K. K. Thomsen,
Eur. J. Biochem., 2000, 267, 361–369.
9 L. Kandra, G. Gye´ma´nt, J. Remenyik, G. Hova´nszki and A. Lipta´k,
FEBS Lett., 2002, 518, 79–82.
10 C. Malet and A. Planas, Biochemistry, 1997, 36, 13838–13848.
11 T. Murata, T. Hattori, S. Amarume, A. Koichi and T. Usui,
Eur. J. Biochem., 2003, 270, 3709–3719.
12 Y. Honda, S. Tanimori, M. Kirihata, S. Kaneko, K. Tokuyasu, M.
Hashimoto, T. Watanabe and T. Fukamizo, FEBS Lett., 2000, 476,
194–197.
4-Methylumbelliferyl b-D-xylopyranosyl-(1→4)-b-D-xylopy-
ranosyl-(1→4)-b-D-xylopyranoside (MUX3). dH 5.21 (1 H, d,
J1,2 7.52, H-1), 4.52 (1 H, d, J1,2 7.63, H-1ꢀ), 4.48 (1 H, d, J1,2
7.84, H-1ꢀꢀ), 4.19 (1 H, dd, Ja5,b6 11.82, J4,5a 5.16, H-5a), 4.14 (1
H, dd, J5a,6b 11.76, J4,5a 5.32, H-5ꢀa), 3.98 (1 H, dd, J5a,6b 11.60,
J4,5a 5.48, H-5aꢀꢀ), 3.88 (1 H, ddd, J3,4 8.65, J4,5b 10.0, H-4), 3.80
(1 H, ddd, J3,4 8.86, J4,5b 10.20, H-4ꢀ), 3.73 (1 H, t, J2,3 9.24, H-3),
3.65 (1 H, dd, H-2), 3.64 (1 H, dd, H-5b), 3.63 (1 H, ddd, J3,4 9.2,
J4,5b 10.39, H-4ꢀꢀ), 3.58 (1 H, t, J2,3 9.13, H-3ꢀ), 3.44 (1 H, t, J2,3
9.28, H-3ꢀꢀ), 3.41 (1 H, dd, H-5bꢀ), 3.34 (1 H, dd, H-2ꢀ), 3.32 (1
H, dd, H-5bꢀꢀ), 3.27 (1 H, dd, H-2ꢀꢀ); dC 165.93 (C-2 MU), 161.60
(C-7 MU), 158.01 (C-4 MU), 155.99 (C-8a MU), 128.76 (C-4a
MU), 117.21 (C-5 MU), 116.10 (C-6 MU), 113.41 (C-3 MU),
105.63 (C-8 MU), 103.94 (C-1ꢀꢀ), 103.79 (C-1ꢀ), 102.17 (C-1),
78.47 (C-4ꢀ), 78.21 (C-4), 77.68 (C-3ꢀꢀ), 75.74 (C-3ꢀ), 75.56 (C-3),
74.82 (C-2ꢀꢀ), 74.71 (C-2ꢀ), 74.59 (C-2), 71.24 (C-4ꢀꢀ;), 67.28 (C-
5ꢀꢀ), 65.11 (C-5), 65.05 (C-5ꢀ), 19.93 (4-Me-MU); ESI+ MS [M +
Na]+ m/z 595.1639 calcd. for C25H32NaO15, observed 595.1627.
13 V. M. Chernoglazov, A. N. Jafarova and A. A. Klyosov, Anal.
Biochem., 1989, 179, 186–189.
14 S. Armand, S. Drouillard, M. Schu¨lein, B. Henrissat and H. Driguez,
J. Biol. Chem., 1997, 272, 2709–2713.
15 S. Kaneko, M. Kitaoka, A. Kuno and K. Hayashi, Biosci. Biotech.
Biochem., 2000, 64, 741–745.
16 E. V. Eneyskaya, H. Brumer III, L. V. Backinowsky, D. R. Ivanen,
A. A. Kulminskaya, K. A. Shabalin and K. N. Neustroev, Carbohydr.
Res., 2003, 338, 313–325.
17 F. M. Ibatullin, K. A. Shabalin, J. V. Ja¨nis and A. G. Shavva,
Tetrahedron Lett., 2003, 44, 7961–7964.
18 R. W. Alder, Chem. Ind. (London), 1973, 983–986.
19 K. Takeo, Y. Ohguchi, R. Hasegawa and S. Kitamura, Carbohydr.
Res., 1995, 277, 231–244.
20 B. Domon and C. E. Costello, Glycoconjugate J., 1988, 5, 397–409.
21 K. G. I. Nilsson, Carbohydr. Res., 1987, 167, 95–103.
22 E. V. Eneyskaya, A. M. Golubev, A. M. Kachurin, A. N. Savel’ev
and K. N. Neustroev, Carbohydr. Res., 1998, 305, 83–91.
23 X. Zeng, R. Yoshimo, T. Murata, K. Ajisaka and T. Usui, Carbohydr.
Res., 2000, 325, 120–131.
4-Methylumbelliferyl b-D-xylopyranosyl-(1→4)-bis[O-b-D-
xylopyranosyl-(1→4)]-b-D-xylopyranoside (MUX4). dC 161.02
(C-7), 157.41 (C-4 MU), 155.40 (C-8a MU), 128.16 (C-5 MU),
116.62 (C-4a MU), 115.40 (C-6 MU), 112.72 (C-3 MU), 104.96
(C-8 MU), 19.24 (4-Me-MU), 103.19 (C-1ꢀꢀꢀ), 103.05 (C-1ꢀꢀ),
102.99 (C-1ꢀ), 101.42 (C-1), 77.72 (C-4ꢀ), 77.66 (C-4ꢀꢀ), 77. 44
(C-4), 76.94 (C-3ꢀꢀꢀ), 74.98 (C-3ꢀ and C-3ꢀꢀ) 74.79 (C-3), 74.08
(C-2ꢀꢀꢀ), 73.97 (C-2ꢀ and C-2ꢀꢀ), 73.85 (C-2), 70.49 (C-4ꢀꢀꢀ), 66.53
(C-5ꢀꢀꢀ), 64.38 (C-5), 64.28 (C-5ꢀ and C-5ꢀꢀ); ESI+ MS [M + Na]+
m/z 727.2062 calcd. for C30H40NaO19, observed 727.2075.
4-Methylumbelliferyl b-D-xylopyranosyl-(1→4)-tris[O-b-D-
xylopyranosyl-(1→4)]-b-D-xylopyranoside (MUX5). ESI+ MS
[M + Na]+ m/z 859.2484 calcd. for C35H48NaO23, observed
859.2401.
24 H. De Boeck, K. L. Matta, M. Claeyssens, N. Sharon and F. G.
Loontiens, Eur. J. Biochem., 1983, 131, 453–460.
25 R. Borriss, M. Krah, H. Brumer III, M. A. Kerzhner, L. A. Elyakova,
D. R. Ivanen, E. V. Eneyskaya, S. M. Shishlyannikov, K. A. Shabalin
and K. N. Neustroev, Carbohydr. Res., 2003, 338, 1455–1467.
26 B. Henrissat, Biochem. J., 1991, 280, 309–316.
27 P. Ntarima, W. Nerinckx, K. Klarskov, B. Devreese, M. K. Bhat, J.
Van Beeumen and M. Claeyssens, Biochem. J., 2000, 347, 865–873.
28 N. Kitamoto, S. Yoshino, M. Ito, T. Kimura, K. Ohmiya and N.
Tsukagoshi, Appl. Microbiol. Biotechnol., 1998, 50, 558–563.
29 O. H. Lowry, N. J. Rosenbrough, A. L. Farr and R. J. Randall, J. Biol.
Chem., 1951, 193, 265–275.
30 M. Somogyi, J. Biol. Chem., 1952, 195, 19–23.
31 R. T. Leatherbarrow, Enzifitter., Elsevier Biosoft, Cambridge, 1987.
32 A. Thompson and M. Y. Wolfrom, Methods Carbohydr. Chem., 1963,
2, 215.
4-Methylumbelliferyl b-D-xylopyranosyl-(1→4)-tetra[O-b-D-
xylopyranosyl-(1→4)]-b-D-xylopyranoside (MUX6). ESI+ MS
[M + Na]+ m/z 991.2907 calcd. for C40H56NaO27, observed
991.2803.
Acknowledgements
The present work was supported by the grant of the Program
for Basic Research in Molecular and Cell Biology from the
Presidium of Russian Academy of Sciences (RAS); by the
grant no. 03-04-48756 from the Russian Foundation for Basic
Research; by the Agreement Rostech-PNPI 585-400-1/2003.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 4 6 – 1 5 1
1 5 1