Chemoenzymic synthesis of (1→3,1→4)-β-D-glucooligosaccharides for subsite mapping of (1→3,1→4)-β-D-glucan endohydrolases

Article abstract of DOI:10.1039/a804711a

A series of unsubstituted (1→3,1→4)-β-D-glucooligosaccharides, designed for subsite mapping in which the number of glucosyl-binding subsites and the subsite-binding/transition state activation affinities at individual subsites of plant and bacterial (1→3,1→4)-β-D-glucan 4-glucanohydrolases (EC 3.2.1.73) can be determined, has been synthesised through chemical and enzymic procedures. A recombinant (1→3,1→4)-β-D-glucan 4-glucanohydrolase from Bacillus licheniformis has been used in organic media to catalyse the condensation of 3-O-β-D-glucopyranosyl-β-D-glucopyranosyl fluoride (Glcβ3GlcβF, compound 1) with cellobiose (Glcβ4Glc, 2), cellotriose (Glcβ4Glcβ4Glc, 3), cellotetraose (Glcβ4Glcβ4Glcβ4Glc, 4) and cellopentaose (Glcβ4Glcβ4Glcβ4Glcβ4Glc, 5), to produce the (1→3,1→4)-β-D-glucooligosaccharides, Glcβ3Glcβ4Glcβ4Glc 6, Glcβ3Glcβ4Glcβ4Glcβ4Glc 7, Glcβ3Glcβ4Glcβ4Glcβ4Glcβ4Glc 8, Glcβ3Glcβ4Glcβ4Glcβ4Glcβ4Glcβ4Glc 9. Synthesised oligosaccharides 6-9 were isolated in yields of 15-45%, compared with compound 1. In a second series of syntheses, a cellodextrin phosphorylase (EC 2.4.1.49) from Clostridium thermocellum was used to sequentially transfer glucosyl residues from α-D-glucopyranosyl phosphate 10 to the 4-position of the non-reducing terminus of the trisaccharide Glcβ3Glcβ4Glc 11, to generate the (1→3,1→4)-β-D-glucooligosaccharides, Glcβ4Glcβ3Glcβ4Glc 12, Glcβ4Glcβ4Glcβ3Glcβ4Glc 13, Glcβ4Glcβ4Glcβ4Glcβ3Glcβ4Glc 14 in 14, 10 and 5% yield, respectively, from compound 11.

Full text of DOI:10.1039/a804711a

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Chemoenzymic synthesis of (1→3,1→4)-â-D-glucooligosaccharides
for subsite mapping of (1→3,1→4)-â-D-glucan endohydrolases
Maria Hrmova,*a Geoffrey B. Fincher,a Josep-Luis Viladot,b Antoni Planasb and
Hugues Driguez†c
a Department of Plant Science, University of Adelaide, Waite Campus, Glen Osmond, SA 5064,
Australia
b Laboratori de Bioquímica, Institut Quimic de Sarrià, Universitat Ramon Llul,
08017-Barcelona, Spain
c Centre de Recherche sur les Macromolécules Végétales, CNRS, BP 53,
38041 Grenoble cedex 9, France
Received (in Cambridge) 22nd June 1998, Accepted 1st September 1998
A series of unsubstituted (1→3,1→4)-β--glucooligosaccharides, designed for subsite mapping in which the
number of glucosyl-binding subsites and the subsite-binding/transition state activation affinities at individual
subsites of plant and bacterial (1→3,1→4)-β--glucan 4-glucanohydrolases (EC 3.2.1.73) can be determined, has
been synthesised through chemical and enzymic procedures. A recombinant (1→3,1→4)-β--glucan 4-glucano-
hydrolase from Bacillus licheniformis has been used in organic media to catalyse the condensation of 3-O-β--
glucopyranosyl-β--glucopyranosyl fluoride (Glcβ3GlcβF, compound 1) with cellobiose (Glcβ4Glc, 2), cellotriose
(Glcβ4Glcβ4Glc, 3), cellotetraose (Glcβ4Glcβ4Glcβ4Glc, 4) and cellopentaose (Glcβ4Glcβ4Glcβ4Glcβ4Glc, 5),
to produce the (1→3,1→4)-β--glucooligosaccharides, Glcβ3Glcβ4Glcβ4Glc 6, Glcβ3Glcβ4Glcβ4Glcβ4Glc 7,
Glcβ3Glcβ4Glcβ4Glcβ4Glcβ4Glc 8, Glcβ3Glcβ4Glcβ4Glcβ4Glcβ4Glcβ4Glc 9. Synthesised oligosaccharides 6–9
were isolated in yields of 15–45%, compared with compound 1. In a second series of syntheses, a cellodextrin
phosphorylase (EC 2.4.1.49) from Clostridium thermocellum was used to sequentially transfer glucosyl residues from
α--glucopyranosyl phosphate 10 to the 4-position of the non-reducing terminus of the trisaccharide Glcβ3Glcβ4Glc
11, to generate the (1→3,1→4)-β--glucooligosaccharides, Glcβ4Glcβ3Glcβ4Glc 12, Glcβ4Glcβ4Glcβ3Glcβ4Glc 13,
Glcβ4Glcβ4Glcβ4Glcβ3Glcβ4Glc 14 in 14, 10 and 5% yield, respectively, from compound 11.
the disposition of catalytic amino acids in the substrate-binding
region of the enzyme. Considerable effort is also being directed
Introduction
Polymeric (1→3,1→4)-β--glucans are major constituents of
cell walls in monocotyledonous plants of the Order Poales,
which include commercially valuable pasture grasses and cereal
species. The (1→3,1→4)-β--glucans consist of unbranched
towards understanding the molecular mechanisms of catalysis
of related (1→3,1→4)-β--glucanases from Bacillus spp.6–9 The
process of subsite mapping is based on observations that poly-
saccharide hydrolases bind individual glucosyl residues in
chains of 1000 or more β--glucosyl residues, polymerised
their substrates via a series of tandemly arranged subsites.10,11
through both (1→3)- and (1→4)-glycosidic linkages.1–3 In (1→3,
Kinetic analyses of the hydrolysis of well defined oligosacchar-
1→4)-β--glucans from walls of the starchy endosperm of bar-
ides and the determination of initial products of the reactions
ley (Hordeum vulgare), single (1→3)-β--glucosyl residues are
allow a precise definition of the number of individual glucosyl
usually separated by blocks of two or three adjacent (1→4)-β-
binding subsites involved in the enzyme–substrate association
to be made, they allow the position of the catalytic site to be
defined in relation to the glycosyl-binding subsites, and they
enable binding energies at each subsite to be calculated.
Subsite mapping of the (1→3,1→4)-β--glucanases requires
a series of oligomeric substrates that contain both (1→3)- and
(1→4)-β-glucosyl residues (see Scheme 3). Although the protein
-glucosyl residues.3 Longer blocks of up to 14 contiguous
(1→4)-β--glucosyl residues also occur, but these account for
less than 10% of the polysaccharide overall.3
During the mobilisation of endosperm reserves in germin-
ated barley grain, (1→3,1→4)-β--glucan endohydrolases (EC
3.2.1.73) play a central role in the depolymerisation of cell wall
(1→3,1→4)-β--glucans. These enzymes hydrolyse (1→4)-β-
glucosyl linkages on 3-O-substituted glucopyranosyl resi-
dues.1,4,5 The major products of (1→3,1→4)-β--glucanase
action on this polysaccharide are therefore the trisaccharide
3-O-β--cellobiosyl -glucose (Glcβ4Glcβ3Glc) and the tetra-
saccharide 3-O-β-cellotriosyl -glucose (Glcβ4Glcβ4Glc-
β3Glc).
As part of a broader programme aimed at defining in detail
the molecular basis of barley (1→3,1→4)-β--glucanase sub-
strate specificity, the mechanism of binding of substrate to the
enzyme, and the molecular mechanism of catalysis, we wish to
use subsite mapping procedures to determine subsite affinities
for individual β-glucosyl residues of the substrate and to define
folds of the Bacillus (classified in glycosyl hydrolase family 1612)
and the barley (family 1712) (1→3,1→4)-β-glucanases differ,
both enzymes have deep clefts 30–40 Å in length that extend
over their surfaces. These clefts are long enough to accom-
modate 6–9 individual β-glucosyl residues.13–15 This suggests
that there may be up to nine β-glucosyl-binding subsites on the
enzymes and it has been necessary to design oligomeric sub-
strates to map regions on both the non-reducing (donor) and
reducing (acceptor) terminal side of the linkage that is hydro-
lysed (Scheme 3). Some of the oligosaccharides shown in
Scheme 3 could be purified from enzymic hydrolysates of
the barley (1→3,1→4)-β--glucan, but many are not easily
obtained. Furthermore, to ensure that binding of the substrates
would most accurately mimic (1→3,1→4)-β--glucan binding
to the enzyme, it was considered essential to avoid substrates
† Affiliated with the University Joseph Fourier in Grenoble.
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OH
O
HO
OH
O
HO
OH
OH
O
HO
O
HO
HO
O
HO
HO
O
HO
HO
O
F
O
+
HO
OH
OH
HO
HO
n
2
3
4
5
n = 0
n = 1
n = 2
n = 3
1
OH
O
OH
OH
O
HO
HO
OH
O
HO
O
HO
O
HO
O
HO
HO
HO
O
O
O
HO
HO
HO
OH
OH
n
6
n = 0
n = 1
n = 2
n = 3
7
8
9
Scheme 1 Preparative condensation reaction catalysed by the (1→3,1→4)-β--glucanase from B. licheniformis. Condensation of the glucosyl donor
Glcβ3GlcβF 1 and (1→4)-β--linked glucooligosaccharides of DP 2-5 (compounds 2–5), which serve as acceptor molecules: (1→3,1→4)-β--
glucanase 0.1% w/v, 50 mmol lϪ1 sodium maleate buffer, pH 7.0, containing 1 mmol lϪ1 CaCl2, 40 ЊC, 20 min, 3–7 molar excess of compounds 2–5.
OH
O
series of unsubstituted (1→3,1→4)-β--glucooligosaccharides
which could subsequently be used for subsite mapping of
(1→3,1→4)-β--glucan endohydrolases (EC 3.2.1.73). To avoid
possible complications in kinetic analyses imposed by multiple
hydrolytic events during a single enzyme–substrate encounter
(multiple attack), each substrate was synthesised with only one
scissile linkage; these enzymes have an absolute requirement
for adjacent (1→3)- and (1→4)-linked β--glucosyl residues
(Scheme 3). Similar oligosaccharides have been synthesised in
the past, but usually carry methyl, 4-nitrophenyl or 4-methyl-
umbelliferyl groups at their reducing termini.8,16–21 Although
the substituted oligosaccharides are bound and hydrolysed by
(1→3,1→4)-β--glucan endohydrolases, and have been used for
subsite mapping of bacterial (1→3,1→4)-β--glucanases,8,17,21
the methyl, 4-nitrophenyl and 4-methylumbelliferyl substitu-
ents are likely to influence substrate-binding affinities during
subsite-mapping experiments, particularly the affinities of
glucosyl-binding subsites near the reducing end of the sub-
strate, where substrate-stacking interactions might occur.22,23
Here, chemical and enzymic procedures have been used to
synthesise the seven unsubstituted (1→3,1→4)-β--glucooligo-
saccharides shown in Scheme 3; the eighth oligosaccharide
needed for subsite mapping (Glcβ3Glcβ4Glc) was isolated from
hydrolysates of barley (1→3,1→4)-β--glucan. In all cases,
reaction products were purified by high-performance liquid
chromatography (HPLC) and analysed. Not only did this
facilitate the isolation of highly purified oligosaccharides, it
also enabled unchanged acceptor and donor molecules to be
recovered for additional syntheses. Structures of these com-
pounds were routinely confirmed by 13C NMR spectroscopy,
optical rotation and mass spectrometry. The NMR measure-
ments were performed in mixtures of D2O or mixtures of
[2H6]DMSO and D2O, and recorded at 350 MHz at 303 K,
except for compounds 13 and 14 (Scheme 2), where significantly
decreased or attenuated signals for C-3 glycosidic linkage were
observed. However, by increasing the temperature to 333 K for
compound 13 and 353 K for compound 14, and by using dry
[2H6]DMSO as a solvent, the relaxation of the C-3 signal
improved and became fully detectable (results not shown).24
The first-order chemical shifts of the synthesised compounds
were assigned from chromatograms by analysing the relative
intensities of individual signals. For example, immediately after
dissolving 10 mg of compound 11 in 350 µl D2O and after 30
min of spectrum accumulation, we could detect 24 first-order
chemical shifts. Compound 11 was predominantly in its β-
anomeric form, as found by comparison of relative intensities
of the C-1 signals (δC 92.60 and 96.55) of its respective α- and β-
OH
O
OH
O
HO
OH
HO
HO
HO
HO
HO
HO
O
+
O
O
HO
HO
HO
OH
2–
OPO3
10
11
OH
O
OH
O
HO
HO
OH
HO
O
HO
HO
O
O
HO
O
O
H
O
HO
HO
OH
OH
n
12 n = 1
13 n = 2
14 n = 3
Scheme 2 Preparative transglycosylation reaction catalyzed by cello-
dextrin phosphorylase from Cl. thermocellum. G-1P (compound 10)
served as the glucosyl donor and Glcβ3Glcβ4Glc (compound 11) was
the acceptor: 0.4 U cellodextrin phosphorylase in 100 mmol lϪ1 MOPS
buffer, pH 7.0, containing 4 mmol lϪ1 EDTA, 2 mmol lϪ1 DTT, 25 ЊC, 22
h, 10-molar excess of compound 10.
which carried any chemical substituents or were chemically dis-
tinct in any other way from the native oligosaccharides.
Here we describe the synthesis, purification and characteris-
ation of eight (1→3,1→4)-β--glucooligosaccharides which will
allow the future characterisation of (1→3,1→4)-β--glucanases
by subsite-mapping procedures. The oligosaccharides required
for mapping the reducing terminal region of the enzyme–
substrate complex (acceptor subsites) (Scheme 3) were syn-
thesised through transglycosylation reactions between lamin-
arabiosyl fluoride 1 and various acceptors in reactions catalysed
in acetonitrile–water by a highly stable (1→3,1→4)-β--glucan-
ase from B. licheniformis (see Scheme 1). Oligosaccharides
required to map the non-reducing terminal region (donor
subsites) (Scheme 3) were synthesised from 4-O-β--lamin-
arabiosyl--glucose (Glcβ3Glcβ4Glc) using a cellodextrin
phosphorylase (EC 2.4.1.49) from Clostidium thermocellum and
α-glucopyranosyl phosphate (G-1P) (see Scheme 2). This reac-
tion results in the transfer of glucose units from the glucose
1-phosphate to the 4-position on the non-reducing end glucosyl
residue of the primer oligosaccharide, Glcβ3Glcβ4Glc.
Results and discussion
The primary objective of the present study was to synthesise a
3572
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"Reducing-end-side"
Acceptor side
"Non-reducing-end-side"
Donor side
Scissile bond
Scissile bond
Glcβ3Glcβ4Glcβ4Glc
Glcβ4Glcβ3Glcβ4Glc
Glcβ4Glcβ4Glcβ3Glcβ4Glc
12
13
14
6
7
8
9
Glcβ3Glcβ4Glcβ4Glcβ4Glc
Glcβ3Glcβ4Glcβ4Glcβ4Glcβ4Glc
Glcβ3Glcβ4Glcβ4Glcβ4Glcβ4Glcβ4Glc
Glcβ4Glcβ4Glcβ4Glcβ3Glcβ4Glc
Scheme 3 The two series of (1→3,1→4)-β--glucooligosaccharides required for subsite mapping of donor subsites (Ϫ5 to Ϫ2) and acceptor
subsites (ϩ2 to ϩ5) of (1→3,1→4)-β--glucan endohydrolases. The glycosidic linkage hydrolysed by these enzymes is indicated by arrows.
reducing terminals. The C-3 singlet of the unit II was typically
observed around δC 84.86 and the C-4 α- and β-anomeric
signals were located at δC 79.56 and 79.42, respectively. The
group of signals between δC 66.8 and 76.77 was assigned to
atoms C-2 to C-5, and the group of signals between δC 60.74
and 61.49 belonged to C-6, which were not involved in glyco-
sidic linkages. Similarly, the singlets of C-1 of units II and III,
at δC 103.08 and 103.56, respectively, were fully resolved. There-
fore, the fact that the compound 11 exhibited a single resonance
for C-3 in unit II, which corresponded to that of β-lamin-
arabiose (δC 86.0), indicated that this unit was 3-O-substituted.
Thus, the compound was identified as Glcβ3Glcβ4Glc.
Analogously, the 13C NMR chemical shifts for compounds
6–9 and 12–14 were assigned and the data are listed in sec-
tions describing semi-preparative syntheses of the relevant
compounds.
Different strategies were required for the synthesis of
(1→3,1→4)-β--glucooligosaccharides that will be used to cal-
culate binding energies of glucosyl residues bound on the
reducing-end side or on the non-reducing-end side of the link-
age that is hydrolysed (Scheme 3). For the ‘reducing end’ oligo-
saccharides, β--laminarabiosyl fluoride (Glcβ3GlcβF 1), fresh-
ly prepared at 0 ЊC and under strictly anhydrous conditions,
was used as the laminarabiosyl donor in condensation reactions
with the acceptor molecules cellobiose, cellotriose, cellotetraose
and cellopentaose. The reactions were effected with a highly
stable (1→3,1→4)-β--glucanase from B. licheniformis, which
retains activity in the presence of acetonitrile.20,25 Although this
enzyme normally acts as a hydrolase in aqueous media, its
hydrolytic action can be reversed in solutions containing aceto-
nitrile, in favour of the synthesis of higher oligosaccharides.26–28
Furthermore, the enzyme provides the specificity that ensures
that the laminarabiosyl residues are added only to the C-4
positions of acceptor cellodextrins.
During the preparation of acceptor cellodextrins, which were
obtained by acid-catalysed acetolysis of cellulose followed by
de-O-acetylation of the per-O-acetylated oligomeric products, a
marked degradation of cellotriose was observed in the de-O-
acetylation reaction, under conditions where yields of the other
cellodextrins were relatively high. The reason for this is not
clear, but good yields of cellotriose were eventually obtained by
reducing the time of the de-O-acetylation, by ensuring that
reaction mixtures remained free of water, and by keeping the
temperature at 25 ЊC. Another critical factor in setting up
the condensation reactions was the need to use both donor
and acceptor molecules at close to saturating concentrations.
Cellodextrins were used at 3 (cellopentaose)- to 7 (cellobiose)-
times the concentration of Glcβ3GlcβF, on a molar basis, to
prevent autocondensation of donor molecules. Under these
conditions, molar yields of Glcβ3Glcβ4Glcβ4Glc 6, Glcβ3-
Glcβ4Glcβ4Glcβ4Glc 7, Glcβ3Glcβ4Glcβ4Glcβ4Glcβ4Glc 8
and Glcβ3Glcβ4Glcβ4Glcβ4Glcβ4Glcβ4Glc 9 ranged from
15–45% from Glcβ3GlcβF 1. To obtain these yields two succes-
sive additions of the (1→3,1→4)-β--glucanase to the reaction
mixture were necessary; the second addition increased yields by
5–10%.
(Scheme 3), a different strategy was employed. The starting
compound was Glcβ3Glcβ4Glc 11, which was purified from
hydrolysates generated by hydrolysis of barley (1→3,1→4)-β--
glucan with a commercial (1→4)-β--glucanase (EC 3.2.1.4;
‘endo-cellulase’) preparation. Maximum yields of Glcβ3Glc-
β4Glc were obtained after 3 h at 37 ЊC in 10 m sodium acetate
buffer, pH 5.0. As the incubation time was increased, yields of
Glcβ3Glcβ4Glc decreased markedly and after 22 h only cel-
lobiose (Glcβ4Glc) could be detected. The structure of
Glcβ3Glcβ4Glc was confirmed by its 13C NMR spectrum,
which was identical with a previously published spectrum29
and by the products formed following hydrolysis with purified
barley β--glucosidase and β--glucan exohydrolase, both
of which release glucose from the non-reducing terminus of
oligomeric substrates.30–32 In the enzymic assays cellobiose
and glucose were observed initially, as expected for the
hydrolysis of Glcβ3Glcβ4Glc from the non-reducing end, and
both enzymes produced glucose as the final hydrolysis
product.
Following the purification of Glcβ3Glcβ4Glc 11, 4-linked
β-glucosyl residues were sequentially added to its non-reducing
terminus by a cellodextrin phosphorylase (EC 2.4.1.49) from Cl.
thermocellum. This enzyme specifically transfers glucosyl resi-
dues from G-1P 10 to the C-4 atom of β--glucoside accep-
tors.16 To avoid cellodextrin synthesis from dephosphorylated
G-1P, glucose oxidase and catalase were added to destroy free
glucose.16,19 Time-course studies showed that maximum yields
of the three target molecules were achieved after 22 h of incu-
bation and these yields remained unchanged for an additional
24 h. Under these conditions, the three (1→3,1→4)-β--
glucooligosaccharides Glcβ4Glcβ3Glcβ4Glc 12, Glcβ4Glcβ4-
Glcβ3Glcβ4Glc 13 and Glcβ4Glcβ4Glcβ4Glcβ3Glcβ4Glc 14
(Scheme 3) were obtained in 14%, 10% and 5% yield, respect-
ively, from the initial acceptor molecule Glcβ3Glcβ4Glc 11.
Oligosaccharides with a degree of polymerisation higher than 6
were never observed.
In the course of these studies, attempts were made to
synthesise Glcβ4Glcβ4Glcβ3Glcβ4Glc 13 via an alternative
reaction in which the condensation of Glcβ4GlcF and Glcβ-
3Glcβ4Glc was catalysed by a recombinant Humicola insolens
(1→4)-β--glucan 4-glucanohydrolase (EC 3.2.1.4).33 Although
this synthetic strategy was successful, yields of the Glcβ4Glc-
β4Glcβ3Glcβ4Glc 13 were low and the approach was therefore
abandoned (data not shown).
In conclusion, we have successfully used two distinct strat-
egies to synthesise a series of specific (1→3,1→4)-β--gluco-
oligosaccharides. The 13C NMR spectroscopic data, together
with mass spectrometric data and, in some instances, with
enzymic analyses, were used to identify the structures of the
compounds. The oligosaccharides will be used in the immediate
future to explore the kinetics of hydrolysis and substrate bind-
ing by barley and Bacillus (1→3,1→4)-β--glucan endohydro-
lases, and to define binding/transition-state activation affinities
of individual glucosyl-binding subsites in the substrate-binding
regions of the enzymes. Furthermore, the newly synthesised
oligosaccharides could prove useful in X-ray crystallographic
studies on the disposition of amino acid residues that are
For the synthesis of the ‘non-reducing end’ oligosaccharides
J. Chem. Soc., Perkin Trans. 1, 1998, 3571–3576
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involved in substrate binding and catalysis. The three-
dimensional structures of both the Bacillus and the barley
(1→3,1→4)-β--glucanases have been solved.6,13 Although they
have quite different Cα folds, their overall molecular shapes are
very similar.6,13,15 However, it is not clear from the crystallo-
graphic data why kcat-values for the Bacillus enzyme exceed
those for the barley enzyme by more than 2 orders of magni-
tude (M. Hrmova, B. A. Stone and G. B. Fincher, unpublished
data). The oligosaccharides synthesised in the present work
might allow detailed comparisons of substrate binding between
the two enzymes and could, in turn, offer structural and chem-
ical explanations for the dramatic differences in their catalytic
efficiencies.
3-O-â-D-Glucopyranosyl-â-D-glucopyranosyl fluoride or
Glcâ3GlcâF, 1
Compound 1 was prepared as described previously20 from
1,2,4,6-tetra-O-acetyl-3-O-(2,3,4,6-tetra-O-acetyl-β--gluco-
pyranosyl)-α-glucopyranose, which was obtained by controlled
acid-catalysed acetolysis of Antigum CS6.19 The molecular
mass and purity of the O-acetylated form of Glcβ3GlcβF,
together with the extent of de-O-acetylation of per-O-
acetylated Glcβ3GlcβF, were routinely checked by LR-FAB-
MS, 13C NMR and TLC.
Cellobiose 2, cellotriose 3, cellotetraose 4 and cellopentaose 5
While cellobiose 2 is commercially available, the cellooligo-
saccharides 3–5 were prepared by a de-O-acetylation of their
acetylated forms, which were obtained by controlled acid-
catalysed acetolysis of cellulose.17,36 The acetylated derivatives
α-cellotriose undecaacetate, α-cellotetraose tetradecaacetate
and α-cellopentaose heptadecaacetate were isolated by flash
column chromatography using 1:1 → 1:1.5 (v/v) light
petroleum–EtOAc gradients, and were de-O-acetylated at
25 ЊC, with constant vigorous stirring. Freshly prepared
NaOMe (1 mol lϪ1) was added to solutions of per-O-acetylated
sugars (1.9–3.2 mmol) in anhydrous MeOH (150 ml), and the
mixture was stirred for 1.5 h (per-O-acetylated cellotriose) or
for 41 h (per-O-acetylated cellotetraose and cellopentaose). The
free cellooligosaccharides precipitated and were recovered by
filtration. Precipitates were filtered off, dissolved in water and
neutralised with Amberlite IRN 77(Hϩ). The resin was removed
by filtration and filtrates were concentrated and lyophilised. MS
and 13C NMR spectra and TLC analyses were in agreement
with the proposed structures of compounds 3–5.
Experimental
Materials
Silica gel 60, TLC plates and other chemicals of p.a. grade were
obtained from Merck (Darmstadt, Germany). Cellulose
powder (20 µm diameter) and cellobiose were from Aldrich
(Milwaukee, WI, USA), Actigum CS6 containing scleroglucan
was from System Bio-Industries (Paris, France), dithiothreitol
(DTT), orcinol, α--glucopyranosyl phosphate (Glc-1P), glu-
cose oxidase, catalase, Amberlite IRN 77(Hϩ) and MB-3 resin
were from Sigma (St. Louis, MO, USA), Durapore HVLP 0.45
µm filters were from Millipore (Bedford, MA, USA) and D2O
and [2H6]DMSO were purchased from Peypin (Paris, France).
The per-O-acetylated saccharides used as standards, α--gluco-
pyranose pentaacetate and α-cellobiose octaacetate, were from
Fluka (Buchs, Switzerland), barley (1→3,1→4)-β--glucan was
from Megazyme International (Bray, Wicklow, Ireland) and the
oligosaccharide standards, (1→4)-β-linked glucooligosacchar-
ides (2–5) and laminarabiose were from Seikagaku Kogyo
(Tokyo, Japan).
Preliminary chemoenzymic syntheses of mixed-linkage
(1→3,1→4)-â-D-glucooligosaccharides 6–9
A recombinant B. licheniformis (1→3,1→4)-β--glucan 4-
glucanohydrolase (EC 3.2.1.73), which is referred to more
simply as (1→3,1→4)-β--glucanase and had an activity of 510
U cmϪ3, and a cellodextrin phosphorylase (EC 2.4.1.49) from
Cl. thermocellum, with an activity of 1.2 U cmϪ3, were partially
purified as reported previously.16,34 A crude Aspergillus niger
β--glucanase enzyme preparation (Finizyme) with an activity
of 500 U cmϪ3 was kindly provided by Dr Martin Schülein
(Novo Nordisk, Copenhagen, Denmark).
To a freshly prepared solution of compound 1 (10 mg, 0.028
mmol) in 200 µl of 50 mmol lϪ1 sodium maleate buffer, pH 7.0,
containing 1 mmol lϪ1 CaCl2 and a 7–3 molar excess of (1→4)-
β-linked oligoglucosides of DP 2–5 (compounds 2–5) was
added 26 µl of a 0.5 mg mlϪ1 (1→3,1→4)-β--glucanase
solution (corresponding to 0.1% w/v of compound 2) from B.
licheniformis in the buffer described above, together with 300 µl
of MeCN. The mixture was stirred vigorously for 10 min at
40 ЊC and, when a precipitate formed after 2–5 min, another 26
µl of (1→3,1→4)-β--glucanase solution was added. After a
total of 20 min incubation time, the enzyme was inactivated in a
boiling water-bath for 3 min, the reaction mixture desalted with
Amberlite IRN 77(Hϩ), and the resin was removed by filtration.
The supernatant was concentrated by evaporation and filtered
through a 0.45 µm Millipore filter. The presence of (1→3,1→4)-
β--glucooligosaccharides 6–9 was checked by TLC and
HPLC. The (1→3,1→4)-β--glucooligosaccharides 6–9 were
further purified by HPLC, as described above, and eluted with
MeCN–water (65:35 v/v for compounds 6, 7 or 60:40 v/v for
compounds 8, 9).
General methods
13C NMR spectra were recorded in D2O and [2H6]DMSO with a
Bruker 300 AC (350 MHz) spectrometer at 303, 333 or 353 K.
Low-resolution mass measurements were performed in a
Nermag R-1010C spectrometer in a fast-atom bombardment
negative mode (LR-FAB-MS), using glycerol matrices. High-
resolution mass measurements (HR-MS) were obtained in a
VG ZAB spectrometer in time-of-flight (TOF) geometry, using
MeCN–water (1:1, v/v) as a solvent. Optical rotations were
measured at 20 ЊC in DMSO with a Perkin-Elmer 241 polari-
meter, and [α]D-values are given in units of 10Ϫ1 deg cm2 gϪ1.
Flash and open-column chromatographies were performed on a
silica gel 60 (230–400 mesh) matrix at flow rates of 10–20 ml
minϪ1. Light petroleum was the 60–80 ЊC fraction. Time
courses of enzymic reactions and the purification of mixed-
linkage oligosaccharides (compounds 6–9 and 12–14) were per-
formed on an HPLC system equipped with a refractometric
detector (Waters, Milford, USA). Samples were eluted iso-
cratically with MeCN–water (60–65:40–35, v/v) from a semi-
preparative 250 × 21 mm Bondapak 8 µm NH2 column. TLC
was performed on Silica gel 60 plates with EtOAc–acetic acid–
water (30:20:15, v/v) or EtOAC–MeOH–water (8:4:3, v/v),
and mono- and oligo-saccharides were detected with the
orcinol reagent35 or by charring with 50% (v/v) sulfuric acid.
Semi-preparative syntheses of Glcâ3Glcâ4Glcâ4Glc 6;
Glcâ3Glcâ4Glcâ4Glcâ4Glc 7; Glcâ3Glcâ4Glcâ4Glcâ4Glcâ4Glc
8; and Glcâ3Glcâ4Glcâ4Glcâ4Glcâ4Glcâ4Glc 9
The syntheses of (1→3,1→4)-β--glucooligosaccharides 6–9
were effected as described above, except that reaction mixtures
were scaled up 10-fold. Yields of purified compounds 6–9 were
85, 78, 77 and 49 mg, respectively, corresponding to molar
yields of 45, 33, 27 and 15% from compound 1 used in the
condensation reactions.
Compound 6. (85 mg, 0.128 mmol, 45% from 1). Analytical
data: [α]D ϩ37.65 (c 2.47, DMSO); HR-MS, Calc. for C24H42O21
[M ϩ Na]ϩ: 689.2116; Found: m/z, 689.2123; δC ([2H6]DMSO;
3574
J. Chem. Soc., Perkin Trans. 1, 1998, 3571–3576

View Article Online
D2O; 303 K) 61.2 (C-6β, -6II), 61.3 (C-6α), 61.8 (C-6IV), 61.9
(C-6III), 69.4 (C-4III), 71.0–77.4 (C-2–C-5), 80.1 (C-4II), 80.3
(C-4β), 80.5 (C-4α), 86.1 (C-3III), 93.1 (C-1α), 97.2 (C-1β), 103.5
(C-1II), 103.7 (C-1III) and 104.3 (C-1IV).
aliquots were desalted with mixed-bed resin MB-3 and filtered
through 0.45 µm Millipore filters, and the simultaneous
presence of (1→3,1→4)-β--glucooligosaccharides transgluco-
sylation products 12–14 was determined by HPLC using an
isocratic elution with MeCN–water (65:35, v/v).
Compound 7. (78 mg, 0.094 mmol, 33% from 1). Analytical
data: [α]D ϩ16.00 (c 2.75, DMSO); HR-MS, Calc. for C30H52O26
[M ϩ Na]ϩ: 851.2645; Found: m/z, 851.2601; δC ([2H6]DMSO;
D2O; 303 K) 61.0 (C-6β), 61.1 (C-6II), 61.2 (C-6α), 61.8 (C-6V),
62.0 (C-6III,IV), 68.8 (C-4V), 69.3–80.1 (C-2–C-5), 80.3 (C-4β),
80.5 (C-4α), 86.3 (C-3IV), 93.0 (C-1α), 97.1 (C-1β), 103.4 (C-1II),
103.6 (C-1III,IV) and 104.2 (C-1V).
Semi-preparative syntheses of Glcâ4Glcâ3Glcâ4Glc 12; Glcâ-
4Glcâ4Glcâ3Glcâ4Glc 13; and Glcâ4Glcâ4Glcâ4Glcâ3Glc-
â4Glc 14
The syntheses of the (1→3,1→4)-β--glucooligosaccharides
12–14 were performed as described above, except that reaction
mixtures were scaled up 8-fold and the reaction time was 22 h.
Compound 8. (77 mg, 0.078 mmol, 27% from 1). Analytical
data: [α]D ϩ14.39 (c 2.78, DMSO); HR-MS, Calc. for C36H62O31
[M ϩ Na]ϩ: 1013.3173; Found: m/z, 1013.3142; δC ([2H6]-
DMSO; D2O; 303 K) 61.1 (C-6β, -6II–IV), 61.8 (C-6α), 62.0
(C-6V), 69.3–80.6 (C-2–C-5), 86.3 (C-3V), 93.0 (C-1α), 97.2
(C-1β), 103.5 (C-1II), 103.7 (C-1III–V) and 104.2 (C-1VI).
Compound 12. (76 mg, 0.114 mmol, 14% from 11). Analytical
data: [α]D ϩ13.19 (c 2.73, DMSO); HR-MS, Calc. for C24H42O21
[M ϩ Na]ϩ: 689.2116; Found: m/z, 689.2068; δC ([2H6]DMSO;
D2O; 303 K) 61.4 (C-6I,III), 62.1 (C-6II,IV), 69.5 (C-4II), 71.0-77.5
(C-2–C-5), 79.9 (C-4β, -4III), 80.0 (C-4α), 85.6 (C-3II), 93.3
(C-1α), 97.2 (C-1β), 103.8 (C-1II,IV) and 104.1 (C-1III).
Compound 9. (49 mg, 0.042 mmol, 15% from 1). Analytical
data: [α]D ϩ7.33 (c 3.00, DMSO); HR-MS, Calc. for C42H72O36
[M ϩ Na]ϩ: 1175.3701; Found: m/z, 1175.3697; δC ([2H6]-
DMSO; D2O; 303 K) 60.1–60.9 (C-6II–IV,VI,VII), 61.4 (C-6V,
-6β), 61.6 (C-6α), 69.0–80.8 (C-2–C-5), 87.0 (C-3VI), 92.8
(C-1α), 97.0 (C-1β), 103.1 (C-1II), 103.3 (C-1III–VI) and 104.2
(C-1VII).
Compound 13. (45 mg, 0.078 mmol, 10% from 11). Analytical
data: [α]D ϩ8.06 (c 2.73, DMSO); HR-MS, Calc. for C30H52O26
[M ϩ Na]ϩ: 851.2645; Found: m/z, 851.2651; δC ([2H6]DMSO;
333 K) 60.3–61.0 (C-6I–VI), 70.0 (C-4II), 73.0–80.4 (C-2–C-5),
87.7 (C-3II), 91.9 (C-1α), 96.6 (C-1β) and 102.8–103.7 (C-1II–VI).
Compound 14. (37 mg, 0.037 mmol, 5% from 11). Analytical
data: [α]D ϩ4.40 (c 2.73, DMSO); HR-MS, Calc. for C36H62O31
[M ϩ Na]ϩ: 1013.3173; Found: m/z, 1013.3147; δC ([2H6]-
DMSO; 353 K) 60.2–60.8 (C-6I–VI), 69.9 (C-4II), 72.1–79.7
(C-2–C-5), 86.9 (C-3II), 91.7 (C-1α), 96.5 (C-1β) and 102.4–
103.3 (C-1II–VI).
Compound Glcâ3Glcâ4Glc 11
Barley (1→3,1→4)-β--glucan (60 mg) was dissolved in 12 ml
of 50 mmol lϪ1 sodium acetate buffer, pH 5.0. Finizyme enzyme
preparation (600 µl in the same buffer) was added and the
mixture was incubated at 37 ЊC. After 1, 2, 3, 5 and 22 h, 2 ml
aliquots were removed, placed in a boiling water-bath for 5 min,
precipitated in 10 ml of ice-cold acetone and filtered through
Celite. After ca. 18 h the filtrates were concentrated by evapor-
ation, desalted with Amberlite 77(Hϩ), and filtered through
0.45 µm Millipore filters. The formation of compound 11 in the
reaction mixtures was detected by HPLC, which showed that
after 3 h of incubation, optimal yields of trisaccharide 11 were
obtained.
For preparative-scale syntheses of compound 11, the reac-
tion mixture was scaled up 34-fold. Barley (1→3,1→4)-β--
glucan (2 g) was hydrolysed for 3 h. The compound was isolated
by flash chromatography (gradient 3:1 → 1:1.5 light
petroleum–EtOAc) after the mixture had been fully O-
acetylated as described.20 Eluted per-O-acetylated compound
11 (2.4 g) was de-O-acetylated by the Zemplen transesterifi-
cation procedure, as described above. The de-O-acetylation
afforded 1.1 g (54% yield) of compound 11.
Acknowledgements
We thank Mr C. Gey and Dr C. Bosso from Centre de
Recherche sur les Macromolécules Végétales for expert assist-
ance with various aspects of the project. The work was
supported by grants from the Australian Research Council
(to G. B. F.), Centre National de la Recherche Scientifique (to
H. D.) and Comisión Interministerial de Cienncia y Tecnologia
(grant number BIO97-0511-C02-02 to A. P.). J. L. V. acknow-
ledges a postdoctoral fellowship from DGU, Generalitat de
Catalunya.
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