A Chemoenzymatic route to conjugatable β(1→3)-Glucan Oligosaccharides

Article abstract of DOI:10.1071/CH08517

3^II-O-Allyl-α laminaribiosyl fluoride was prepared as a key synthon for the enzymatic synthesis of β(1→3)-glucan oligosaccharides, catalyzed by a mutated β(1→3)-glucanase (E231G) from barley (Hordeum vulgare L.). A strategy was developed for en

Full text of DOI:10.1071/CH08517

RESEARCH FRONT
Full Paper
CSIRO PUBLISHING
www.publish.csiro.au/journals/ajc
Aust. J. Chem. 2009, 62, 575–584
A Chemoenzymatic Route to Conjugatable β(1→3)-Glucan
Oligosaccharides∗
Emilie Montel,A Maria Hrmova,B Geoffrey B. Fincher,B Hugues Driguez,A
and Sylvain CottazA,C
ACentre de Recherches sur les Macromolécules Végétales (CERMAV-CNRS)#, BP 53,
38041 Grenoble Cedex 9, France.
BSchool of Agriculture, Food and Wine, Australian Centre for Plant Functional Genomics,
University of Adelaide, Waite Campus, Glen Osmond, SA 5064, Australia.
CCorresponding author. Email: cottaz@cermav.cnrs.fr
3II-O-Allyl-α-laminaribiosyl fluoride was prepared as a key synthon for the enzymatic synthesis of β(1→3)-glucan
oligosaccharides, catalyzed by a mutated β(1→3)-glucanase (E231G) from barley (Hordeum vulgare L.). A strategy was
developed for enzymatic elongation of the β(1→3)-glucan chain from the reducing end, using a single glucoside acceptor.
When β-glucoside phenyl disulfide was used as the acceptor, this methodology generated laminari-oligosaccharides
conjugatable at both their reducing and non-reducing ends.
Manuscript received: 26 November 2008.
Final version: 6 March 2009.
Introduction
oligosaccharides, through mutated glycoside hydrolases lacking
hydrolytic activity, but with an ability to catalyze the transfer of
an α-glycosyl fluoride to a suitable glycosidic acceptor.[9–11]
Mutated β(1→3)-glucanase (E231G) from barley (Hordeum
vulgare L.) has proved to be a powerful catalyst for the
synthesis of artificial β(1→3)-glucans by polymerization of
α-laminaribiosyl fluoride, and by its condensation with various
mono- or disaccharide acceptors.[12,13]
In the present study, we report the investigation of a
glycosynthase-based methodology for the synthesis of β(1→3)-
glucan oligosaccharides that are conjugatable on one or both
extremities (reducing and non-reducing ends). This opens the
way to biological studies such as isolation and characteriza-
tion of receptors through the use of biotinylated or fluorescent
oligosaccharide probes.
β(1→3)-Glucans are widely distributed in fungi, where they rep-
resent up to half of the mass of cell walls, and also in other
organisms such as bacteria, yeast, algae, and higher plants.
These polysaccharides consist of a backbone of β(1→3)-linked
d-glucopyranosyl residues, most of which are associated with
(1→6) branches, and with an average molecular mass of up to
2.106 g mol−1. It is well known that poly- and oligo-β(1→3)-
glucans exhibit immunomodulating, antibacterial, and antiviral
activities in animals as well as in humans, thus being classified as
biological response modifiers.[1–4] They act also as elicitors that
could induce natural defence mechanisms and germination in
plants.[5,6] Numerous publications describing these fascinating
biological activities can be found, but their precise mechanisms
of action remain unsolved, owing in part to the fact that the glu-
cans are usually purified from natural sources, and consequently
they are not well defined and characterized. The preparation of
highly purified β(1→3)-glucans from natural sources usually
involves tedious purification procedures, and does not allow the
isolation of β(1→3)-glucan oligosaccharides derivatives, which
would constitute valuable probes for the studies of receptors
and enzymes associated with β(1→3)-glucans. Another source
of β(1→3)-glucans would be their de novo synthesis, employ-
ing chemical or enzymatic strategies. An efficient multistep
chemical synthesis for unbranched β(1→3)-glucan oligosac-
charides has been established, but with limitations concerning
the presence of activatible protecting groups such as the allyl
group.[7,8]
Results and Discussion
A convenient approach to the enzymatic synthesis of linear
β(1→3)-glucan oligosaccharides is proposed in Scheme 1: this
strategy suggests an iterative glycosynthase-based synthesis
requiring a key donor, 3II-O-allyl-α-laminaribiosyl fluoride 1,
and a monosaccharide acceptor such as 4-methoxyphenyl β-
d-glucopyranoside 19. A cycle of glycosylation followed by
deprotection of the allyl group leads to a novel acceptor, a lami-
naritrioside derivative, which can be used in turn as an acceptor
molecule to give the pentamer. Repeated rounds of this reac-
tion lead to an odd-numbered series of linear β(1→3)-glucans.
Starting from a laminaribioside acceptor instead of a glucoside
acceptor would lead to the even-numbered series. This strat-
egy of elongation by the non-reducing end has successfully
Glycosynthase technology offers an alternative for the
production of well-defined, pure β(1→3)-glucans or related
∗Dedicated to Professor Robert V. Stick on the occasion of his retirement.
#Affiliated with Université Joseph Fourier, and member of the Institut de Chimie Moléculaire de Grenoble.
© CSIRO 2009
10.1071/CH08517
0004-9425/09/060575

RESEARCH FRONT
576
E. Montel et al.
OH
O
OH
O
HO
O
HO
O
OH
HO
F
1
(a)
OH
O
OH
HO
O
O
HO
O
O
O
H
OCH3
OH
OCH3
OH
n ꢁ 2
n
(b)
19 n ꢀ 1
Scheme 1. (a) Mutated barley β(1→3)-glucanase (E231G), phosphate buffer. (b) PdCl2.
H
Ph
O
O
O
HO
H
BzO
OAc
O
OR
O
OAc
OCH
2
Ph
BzO
O
3
O
6
O
O
AcO
R O
O
O
AcO
R
1
2
BzO
O
O
O
O
OR
1
(e)
(d)
AcO
R
OR
OAc
BzO
2
OBz
2
OCH
3
R
H
R
2
7
R
H
R
H
1
1
2
8
OCH
3
2
3
4
5
(a)
X
Bz
H
Bz
Bz
9
H, OAc
(b)
(c)
TCA Bz
Scheme 2. (a) BzCl, pyridine. (b) Methylamine/EtOH, THF. (c) Cl3CCN, DBU, CH2Cl2. (d) 4-Å sieves, TMSOTf, CH2Cl2. (e) (i) MeONa, MeOH;
(ii) AcOH; (iii) Ac2O, pyridine.
been employed for the glycosynthase-assisted synthesis of
cellodextrins,[14] and in the present study, the same concept
was preliminarily investigated for the preparation of linear
β(1→3)-glucan oligosaccharides.
activation by hydrogen bromide in acetic acid followed by
nucleophilic displacement with silver acetate in a mixture of
acetic anhydride/acetic acid led to the β-heptaacetate 13,[21]
and hydrogen fluoride/pyridine treatment[22] following Zemplén
deacetylationyieldedto3II-O-allyl-α-laminaribiosefluoride1in
50% yield over the four steps.
Having the key donor 1 in hand, we focussed on the prepa-
ration of the acceptor 4-methoxyphenyl-β-laminaribioside 17
(Scheme 4), which could be prepared in four steps starting
from scleroglucan acetolysis (scleroglucan is available from
Cargill) to give laminaribiose peracetate 15 in 14% yield (this
molar yield was calculated assuming that scleroglucan consti-
tutes a repetitive laminaritriosyl unit branched with a single
β(1→6)-glucosyl unit).[23] Anomeric activation of 15 with
hydrogen bromide in acetic acid, followed by glycosylation
with 4-methoxyphenol under phase-transfer conditions in aque-
ous sodium hydroxide/chloroform, catalyzed by benzyltriethyl-
ammonium chloride[24] following Zemplén deacetylation, gave
laminaribioside 17 in 65% over the three steps.
In a first approach to the synthesis of 3II-O-allyl-
hepta-O-acetyl-β-laminaribiose 13, precursor of 3II-O-allyl-α-
laminaribiosyl fluoride 1, we followed the same methodology
as for the preparation of 3II-azido-α-laminaribiosyl fluoride,[13]
starting from 3-O-allyl glucose 2,[15,16] and methyl gluco-
side 6 (Scheme 2).[17] Tribenzoylated trichloracetimidate 5 was
obtained by regioselective deprotection of tetrabenzoate 3 with
methylamine,[18] followed by activation with trichloroaceto-
nitrile in the presence of DBU, in 80% yield from 2. Trimethyl-
silyl trifluoromethanesulfonate-promoted glycosylation of
donor 5 with acceptor 6 gave rise to laminaribioside derivative 7
in 83% yield. A sequence of deprotection–acetylation cycles led
to methyl laminaribioside 8, but all attempts of acetolysis failed
to produce 3II-O-allyl-hepta-O-acetyl-laminaribiose 9.[19,20]
We thus investigated the glycosylation of dicyclohexyli-
dene glucose 10, which proved to be an excellent acceptor
with donor 5 to give the laminaribiose derivative 11 in 96%
yield (Scheme 3). Cyclohexylidene groups and benzoyl groups
were successively removed by the action of trifluoroacetic acid
and sodium methoxide, respectively, followed by acetic anhy-
dride/pyridine acetylation to give 3II-O-allyl-hepta-O-acetyl-
laminaribiose 9 in 45% yield over the three steps. Anomeric
Glucoside 19, obtained in two steps from penta-O-
acetyl-β-d-glucopyranose by a sequence of glycosylation–
deacetylation,[25,26] was coupled with fluoride 1 catalyzed by
the glycosynthase (E231G) β(1→3)-glucanase to give 3III-O-
allyl-β-laminaritrioside 20 in a very good yield (79%, Table 1).
The regio- and stereochemistry of the newly formed glyco-
sidic linkage was determined from the acetylated derivative

RESEARCH FRONT
Conjugatable β(1→3)-Glucan Oligosaccharides
577
O
O
O
OBz
OR3
O
OR4
O
O
OH
5
O
O
(b)
O
R3O
R4O
R1
O
O
O
BzO
(a)
O
O
O
R3O
OR4
O
R2
OBz
O
R1
R2
R3
R4
11
10
12
9
H, OH
H
Bz
Ac
Ac
Ac
H
(c)
H, OAc Ac
(d)
(e)
13
14
1
OAc
H
H
F
F
Ac
Ac
H
(f)
H
Scheme 3. (a) 4-Å sieves, TMSOTf, CH2Cl2. (b) TFA/H2O, CH2Cl2. (c) (i) MeONa, MeOH; (ii) Ac2O, pyridine. (d) (i) HBr, AcOH, CH2Cl2; (ii) AcOAg,
Ac2O/AcOH. (e) HF, pyridine. (f) MeONa, MeOH.
could be condensed with glucoside 19 to afford 3IV-O-allyl-β-
laminaritetraoside 26 in 70% yield, validating this approach as
a convenient route to produce linear β(1→3)-glucan oligosac-
charides, protected at the non-reducing end by a conjugatable
group. That is, the allyl group can be subjected to thiol radical
addition with cysteamine under UV radiation to yield an amino
derivative, which can covalently be linked to various probes or
biomolecules under mild conditions.[31]
OR3
OR3
O
(a)
O
R3O
R3O
R3O
R1
Scleroglucan
O
R3O
OR3
R2
R1
R2
R3
Ac
Ac
H
15
16
17
H, OAc
(b)
(c)
OMP H
OMP H
In view of developing β(1→3)-glucan oligosaccharides con-
jugatable at the reducing end (or both at the reducing and
non-reducing ends), it was necessary to introduce a conjugat-
able β-glucoside derivative as an acceptor at the last enzymatic
coupling step instead of glucoside derivative 19. β-Glucosyl
phenyl disulfide 27, obtained from the condensation of S-
phenyl benzethiosulfonate and 2,3,4,6-tetra-O-acetyl-1-thio-β-
d-glucopyranose,[32] was a good candidate. This glucoside
displays an aromatic aglycon, which is required for an effective
binding in acceptor subsites of the glycosynthase, and is readily
activated through mild reductive conditions. Glycosynthase-
catalyzed coupling of laminaritriosyl donor 25 with glucosyl
disulfide 27 was performed in the presence of benzoquinone
to prevent spontaneous reduction of the disulfide bridge, to
give the 3IV-O-allyl-β-laminaritetraosyl disulfide 28 in 56%
yield (Table 1). Reductive coupling of tetrasaccharide 28 with
N-(iodoacetamidoethyl)-1-naphtylamine-5-sulfonic acid (1,5-
IAEDANS) gave the fluorescent 3IV-O-allyl-β-laminaritetraosyl
derivative 29 in high yield, demonstrating that the activated
oligosaccharide is effective in conjugation with an electrophilic
probe.
OMP: 4-methoxyphenyl
Scheme 4. (a) H2SO4, Ac2O. (b) (i) HBr, AcOH, CH2Cl2; (ii) 4-
methoxyphenol, benzyltriethylammonium chloride, aq. NaOH, CHCl3.
(c) MeONa, MeOH.
21, where H-3III at 3.51 ppm and C-3II at 78.5 ppm indi-
cated unambiguously the formation of a β(1→3)-linkage. When
laminaribioside 17 was evaluated as an acceptor with an equi-
molar amount of donor 1, no reaction was detected (Table 1).
This behaviour had already been observed when 4-nitrophenyl
β-laminaribioside was used as an acceptor.[13] 4-Methoxyphenyl
β-laminaritrioside 22, obtained by palladium chloride-promoted
de-O-allylation followed by Zemplén deacetylation of acetylated
derivative 21,[27] was assayed in turn as an acceptor but with
the same negative result (Table 1). These observations could
be attributed to a possible competition between the donor and
acceptor disaccharides in the donor sugar-binding subsites of the
glycosynthase, or to a non-productive positioning of the accep-
tor oligosaccharide in the acceptor subsites due to the presence
of an aromatic aglycon, which may cause preferential stacking
interactions with aromatic amino acids in subsites +2 and +3.
With these results in hand, the original synthetic strategy had
to be re-evaluated to elongate the β(1→3)-glucan chain from
the reducing end by using a single glucoside acceptor 19, and a
glycosyl fluoride donor of increasing length (Scheme 5). Lami-
naritrioside 21 was deprotected at the anomeric position by con-
ventional oxidative treatment with ceriumammonium nitrate,[28]
and activated by diethylaminosulfur trifluoride,[29] followed by
anomerization with hydrogen fluoride/pyridine,[30] following
Zemplén deacetylation that gave 3III-O-allyl-α-laminaritriosyl
fluoride 25 in 57% yield over four steps (Table 1). This donor 25
Conclusions
In the present work, we have established a glycosynthase-
based methodology for the synthesis of β(1→3)-oligoglucoside
derivatives, which are conjugatable on both their reducing and
non-reducing ends. The protecting/activatible groups (allyl and
phenyl disulfide) were chosen with respect to their ability to
retain, or even to improve, the binding of the modified oligosac-
charides to the glycosynthase catalyst, and also because of their
abilities to become activated under orthogonal and mild condi-
tions. These molecules will be useful as probes for studies of
biological systems that are involved with β(1→3)-glucans, such
as receptors and enzymes.

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E. Montel et al.

RESEARCH FRONT
Conjugatable β(1→3)-Glucan Oligosaccharides
579
OH
O
HO
HO
O
OH
OCH3
19
(a)
OH
O
OH
O
OH
O
OH
O
HO
O
HO
HO
O
HO
O
O
O
HO
OH
OH
HO
F
OCH3
n
n ꢁ 1
(b)
1 n ꢀ 1
20 n ꢀ 1
26 n ꢀ 2
25 n ꢀ 2
Scheme 5. (a) Mutated barley β(1→3)-glucanase (E231G), phosphate buffer. (b) (i) Ac2O, pyridine; (ii) cerium ammonium nitrate, toluene/CH3CN/H2O;
(iii) DAST, CH2Cl2; (iv) HF, pyridine; (v) MeONa, MeOH.
anomer could be crystallized from EtOH. m/z (FAB) 659
Experimental
[M + Na]+.
General Experimental Procedures
β anomer: Mp 192◦C (EtOH). [α]D +22 (c 1.0, CHCl3).
NMR spectra were recorded on a Bruker AC 300 or a Bruker
Avance 400. Carbon and proton chemical shifts (δ) are reported
in ppm downfield from TMS, using solvent as the inter-
nal reference. Coupling constants (J) are in Hertz (Hz) and
peaks are described as singlet (s), doublet (d), doublet of dou-
blets (dd), triplet (t), quadruplet (q), multiplet (m), broad (b).
High-resolution mass spectra (HRESI-MS) were recorded on a
Micromass ZABSpec-TOF at the Centre Régional de Mesures
Physiques de l’Ouest (CRMPO – Rennes). Low-resolution mass
spectra were recorded on a Waters Micromass ZQ spectrometer
(electrospray ionization, ESI-MS), or on a NERMAG R10–10C
2000, ionization mode by fast-atom bombardment (FAB), chem-
ical ionization (CI), or desorption chemical ionization (DCI).
TLC was performed on silica gel 60F254 (Merck) with detec-
tion by UV light or charring with dil. H2SO4. Purification by
flash chromatography was performed on columns of silica gel
(Geduran SI 60, 40–60 µm, Merck). Light petroleum refers
to the fraction with boiling range of 40–65◦C. After workup,
organic phases were dried over anhydrous Na2SO4. 1,2:5,6-Di-
O-cyclohexylidene-α-d-glucofuranose (CAS 23397–76–4) was
from Sigma–Aldrich. Scleroglucan (Actigum CS6) was from
Cargill France SAS (Saint-Germain en Laye). Optical rotations
were performed at 20◦C using a Perkin–Elmer 341 polarimeter.
Melting points were measured on a Büchi 535 apparatus and
are uncorrected. Microanalyses were performed by the Labora-
toire Central d’Analyses du Centre National de la Recherche
Scientifique (CNRS) (Vernaison). Enzymatic reactions were
performed in a plastic vessel, and gently shaken on a rota-
tory shaker at 37◦C. The construction and purification of the
mutated barley β(1→3)-endoglucanase isoenzyme GII E231A
are described in ref. [12].
(Calc. for C37H32O10: C 69.80, H 5.07. Found C 69.66, H
5.09%.) δH (CDCl3) 8.10–7.35 (m, 20H, Harom), 6.18 (d, J1,2 8,
1H, H1), 5.71–5.55(m, 3H, H-2, H-4, =CH–), 5.11–4.94(m, 2H,
CH2=), 4.63 (dd, 1H, H-6a), 4.45 (dd, 1H, H-6b), 4.27 (m, 1H,
H-5), 4.42–4.10 (m, 3H, H-3, –CH2–). δC (CDCl3) 166.2–164.8
(C=O), 134.0 (=CH–), 133.7–128.3 (Carom), 117.8 (CH2=),
92.6 (C-1), 79.2 (C-3), 73.4 (–CH2–), 73.2 (C-5), 72.0 (C-2),
70.4 (C-4), 63.0 (C-6).
α anomer: δH (CDCl3) 8.10–7.35 (m, 20H, Harom), 6.76 (d,
J1,2 3.7, 1H, H-1), 5.71–5.55 (m, 2H, H-4, =CH–), 5.52 (dd,
1H, H-2), 5.11–4.94 (m, 2H, CH2=), 4.60 (dd, 1H, H-6a), 4.50
(m, 1H, H-5), 4.41 (dd, 1H, H-6b), 4.38 (t, 1H, H-3), 4.17 (m,
2H, –CH2–). δC (CDCl3) 166.2–164.4 (C=O), 134.2 (=CH–),
133.7–128.3 (Carom), 117.4 (CH2=), 90.4 (C-1), 76.9 (C-3),
73.9–70.5 (C-2, C-4, C-5, –CH2–), 62.7 (C-6).
3-O-Allyl-2,4,6-tri-O-benzoyl-D-glucopyranose 4
Methylamine (9.5 mL, 33% in EtOH) was added to a stirred
solution of tetrabenzoate 3 (9 g, 14 mmol) in THF (75 mL), and
the reaction mixture was stirred for 3 h at 20◦C. The mixture
was concentrated under reduced pressure, and purification by
flash chromatography (50:1 CHCl3/acetone) of the residue gave
hydroxy compound 4 as a mixture of α and β anomers (6.5 g,
86%).The α anomer was crystallized from EtOH. m/z (DCI) 550
[M + NH4]+.
α anomer: Mp 167◦C (EtOH). [α]D +89 (c 1.0, CHCl3).
(Calc. for C30H28O9: C 67.66, H 5.30. Found C 67.49, H
5.41%.) δH (CDCl3) 8.28–7.38 (m, 15H, Harom), 5.67–5.58 (m,
2H, =CH–, H-1, J1,2 3.6), 5.53 (t, 1H, H-4), 5.15 (dd, 1H, H-2),
5.09–4.91 (m, 2H, CH2=), 4.60 (dd, 1H, H-6a), 4.45 (m, 1H,
H-5), 4.35 (dd, 1H, H-6b), 4.30 (t, 1H, H-3), 4.19–4.08 (m,
2H, –CH2–). δC (CDCl3) 166.4, 165.7, 165.1 (C=O), 134.4
(=CH–), 133.4–128.5 (Carom), 117.3 (CH2=), 90.5 (C-1), 76.4
(C-3), 74.0 (–CH2–), 73.8 (C-2), 70.9 (C-4), 67.8 (C-5), 63.0
(C-6).
3-O-Allyl-1,2,4,6-tetra-O-benzoyl-D-glucopyranose 3
Benzoyl chloride (30 mL, 240 mmol) was added dropwise to a
stirred solution of 3-O-allyl-d-glucose (8.9 g, 40 mmol)[15,16] in
dry pyridine (150 mL) at 0◦C, and the reaction was allowed to
reach 20◦C overnight. The solution was diluted with CH2Cl2,
washed successively with aq. KHSO4, brine, and water. The
organic layers were concentrated under reduced pressure afford-
ing 3 as a mixture of α and β anomers (25.4 g, 100%). The β
3-O-Allyl-2,4,6-tri-O-benzoyl-D-glucopyranosyl
Trichloroacetimidate 5
Trichloroacetonitrile (3.7 mL, 36.6 mmol) and 1,8-diazabicyclo
[5.4.0]undec-7-ene (DBU) (0.56 mL, 3.6 mmol) were added to

RESEARCH FRONT
580
E. Montel et al.
a stirred solution of tribenzoate 4 (6.5 g, 12.2 mmol) in dry
CH2Cl2 (45 mL) at 0◦C, and the reaction was allowed to reach
20◦C overnight under argon. The mixture was concentrated
under reduced pressure, and purification by flash chromato-
graphy (6:1 light petroleum/EtOAc) of the residue gave imidate
5 as a mixture of α and β anomers (7.7 g, 93%). m/z (CI) 695
[M + NH4]+.
8 (674 mg, 97%). Mp 181◦C (EtOH). [α]D +35 (c 1.0, CHCl3).
m/z (DCI) 666 [M + NH4]+. (Calc. for C28H40O17: C 51.85,
H 6.22. Found C 51.97, H 6.25%.) δH (CDCl3) 5.72 (m, 1H,
=CH–), 5.19–5.07 (m, 2H, CH2=), 5.01 (t, 1H, H-4I), 4.96 (t,
1H, H-4II), 4.89–4.83 (m, 3H, H-2II, H-2I, H-1I, J1,2 4.0), 4.55 (d,
1H, H-1II, J1,2 8.1), 4.25 (dd, 1H, H-6aI), 4.17 (dd, 1H, H-6aII),
4.12 (dd, 1H, H-6bII), 4.09 (t, 1H, H-3II), 4.02 (dd, 1H, H-6bI),
4.00 (d, 2H, –CH2–), 3.90 (m, 1H, H-5II), 3.53 (m, 1H, H-5I),
3.50 (t, 1H, H-3I), 3.39 (s, 3H, –O–CH3), 2.17–2.01 (m, 9H,
COCH3). δC (CDCl3) 170.7–168.8 (6 × C=O), 134.1 (=CH–),
116.8 (CH2=), 101.0 (C-1II), 96.7 (C-1I), 79.9 (C-3I), 75.9
(C-3II), 72.8 (C-2I), 72.2 (–CH2–), 72.1 (C-2II), 71.9 (C-5I),
69.2 (C-4I), 68.0 (C-4II), 67.3 (C-5II), 62.1 (C-6I, C-6II), 55.3
(–O–CH3).
Methyl O-(3-O-Allyl-2,4,6-tri-O-benzoyl-
β-D-glucopyranosyl)-(1→3)-2-O-benzoyl-
4,6-O-benzylidene-α-D-glucopyranoside 7
To a stirred solution of imidate 5 (2.5 g, 3.7 mmol) and
2-O-benzoyl-4,6-O-benzylidene-α-d-glucopyranoside[17] 6(1 g,
2.59 mmol) in dry CH2Cl2 (40 mL), powdered 4-Å molecular
sieves were added, followed by trimethylsilyl trifluoromethane-
sulfonate (TMSOTf) (0.13 mL, 0.7 mmol). The mixture was
stirred vigorously for 45 min at −15◦C under argon and then
Et3NH (0.2 mL) was added to decompose excess TMSOTf. The
mixture was filtered over a bed of diatomaceous earth, the fil-
trate concentrated under reduced pressure, and purification by
flash chromatography (4:1 light petroleum/EtOAc) gave disac-
charide 7 (1.94 g, 83%). Mp 199◦C (EtOH). [α]D +28 (c 1.0,
CHCl3). m/z (ESI) 923 [M + Na]+. (Calc. for C51H48O15: C
67.99, H 5.37. Found C 67.84, H 5.41%.) δH (CDCl3) 8.00–7.15
(m, 20H, Harom), 5.61 (s, 1H, H-7I), 5.45 (t, 1H, H-4II), 5.44 (m,
1H, =CH–), 5.33 (dd, 1H, H-2II), 5.01 (2d, 2H, H-1I, J1,2 3.9,
H-1II, J1,2 7.6), 4.98 (dd, 1H, H-2I), 4.86 (m, 2H, CH2=), 4.53
(dd, 1H, H-6aII), 4.42 (t, 1H, H-3I), 4.35 (dd, 1H, H-6bII), 4.28
(dd, 1H, H-6aI), 3.91 (d, 2H, –CH2–), 3.84 (t, 1H, H-4I), 3.87–
3.75 (m, 3H, H-5I, H-5II, H-6bII), 3.77 (t, 1H, H-3II), 3.40 (s,
3H, –O–CH3). δC (CDCl3) 166.3, 165.2, 165.0, 164.7 (C=O),
137.2 (=CH–), 134.1–126.0 (Carom), 117.4 (CH2=), 101.2 (C-
7I, C-1II), 97.3 (C-1I), 79.5, 79.3 (C-4I, C-3II), 75.7 (C-3I), 74.0
(C-2I), 73.2 (C-2II), 72.8 (–CH2–), 71.9 (C-5II), 71.1 (C-4II),
68.8 (C-6I), 63.6 (C-6II), 62.5 (C-5I), 55.3 (–O–CH3).
(3-O-Allyl-2,4,6-tri-O-benzoyl-β-D-glucopyranosyl)-(1→3)-
1,2:5,6-di-O-cyclohexylidene-α-D-glucofuranose 11
To a stirred solution of imidate 6 (1.88 g, 2.78 mmol) and
1,2:5,6-di-O-cyclohexylidene-α-d-glucofuranose 10 (0.945 g,
2.78 mmol) in dry CH2Cl2 (20 mL), powdered 4-Å molecular
sieves were added, followed by TMSOTf (0.3 mL, 1.6 mmol).
The mixture was stirred vigorously at −5◦C under argon
for 45 min and then Et3NH (0.5 mL) was added to decom-
pose excess TMSOTf. The mixture was filtered over a bed
of diatomaceous earth, the filtrate concentrated under reduced
pressure, and purification by flash chromatography (5:1 light
petroleum/EtOAc) of the residue gave disaccharide 11 (2.29 g,
96%). Mp 187◦C (EtOH). [α]D −3 (c 1.0, CHCl3). m/z (ESI)
877 [M + Na]+. (Calc. for C48H54O14: C 67.43, H 6.37. Found
C 67.36, H 6.38%.) δH (CDCl3) 8.09–7.36 (m, 15H, Harom),
5.58 (m, 1H, =CH–), 5.52 (t, 1H, H-4II), 5.45 (d, 1H, H-1I,
J1,2 3.7), 5.32 (dd, 1H, H-2II), 5.06–4.90 (m, 2H, CH2=), 4.83
(d, 1H, H-1II, J1,2 7.7), 4.59 (dd, 1H, H-6aII), 4.43 (dd, 1H, H-
6bII), 4.35 (q, 1H, H-5I), 4.29 (d, 2H, H-2I, H-3I), 4.18 (dd,
1H, H-4I), 4.07–3.89 (m, 6H, H-6aI, H-6bI, H-3II, H-5II, –CH2–
O), 1.60–1.20 (20H, –CH2–). δC (CDCl3) 166.2, 165.0, 164.6
(C=O), 134.1 (=CH–), 133.4–128.2 (Carom), 117.5 (CH2=),
112.5, 109.1 (Cq), 104.6 (C-1I), 100.0 (C-1II), 82.4–70.9 (C-
2I, C-2II, C-3I, C-3II, C-4I, C-4II, C-5I, C-5II, –CH2–O), 66.1
(C-6I), 63.3 (C-6II), 36.3–23.5 (–CH2–).
Methyl O-(2,4,6-Tri-O-acetyl-3-O-allyl-
β-D-glucopyranosyl)-(1→3)-2,4,6-tri-O-acetyl-
α-D-glucopyranoside 8
MeONa (10 mL, 1 M in MeOH) was added dropwise to an
ice-cooled solution of disaccharide 7 (1.02 g, 1.13 mmol) in
MeOH (100 mL). The mixture was stirred for 4 h at 20◦C,
and then neutralized by an addition of strong acidic cation-
exchange resin, filtered over a plug of cotton wool, and the
filtrate was concentrated under reduced pressure. The result-
ing solid containing methyl (3-O-allyl-β-d-glucopyranosyl)-
(1→3)-4,6-O-benzylidene-α-d-glucopyranoside was dissolved
in diluted AcOH (20 mL, 20% in H2O), and the mixture
was stirred for 0.5 h at 90◦C. The solution was concen-
trated under reduced pressure, and the traces of acetic acid
were removed by co-evaporation with toluene (3 × 5 mL).
The resulting solid residue containing methyl (3-O-allyl-β-d-
glucopyranosyl)-(1→3)-α-d-glucopyranoside was dissolved in
dry pyridine (6 mL), and Ac2O (6 mL) together with a catalytic
amount of 4-dimethylaminopyridine (DMAP). The solution was
stirred for 12 h at 20◦C, protected from light, and cooled in an ice
bath. MeOH (5 mL) was added to decompose excess Ac2O, and
the solution was concentrated under reduced pressure. The solid
residue was dissolved in CH2Cl2, and washed with saturated
aq. NaHCO3, and with H2O. The organic layers were concen-
trated under reduced pressure and flash chromatography of the
residue (2:1 cyclohexane/EtOAc) gave acetylated disaccharide
(3-O-Allyl-2,4,6-tri-O-benzoyl-β-D-glucopyranosyl)-
(1→3)-D-glucopyranose 12
Diluted trifluoroacetic acid (40 mL, 50% in H2O) was added
to an ice-cooled solution of 11 (3.95 g, 4.63 mmol) in CH2Cl2
(4 mL). The mixture was stirred for 12 h at 40◦C, and the reac-
tion mixture was partly concentrated under reduced pressure
to remove CH2Cl2 and to induce the formation of a precipi-
tate of crude 12. H2O (40 mL) was added and the precipitate
was washed several times with H2O. The resulting white solid
was dissolved in acetone (20 mL), and the solution was con-
centrated under reduced pressure in the presence of silica gel
(50 g). The resulting solid was applied on a silica gel column,
and flash chromatography (2:1 CHCl3/MeOH) gave disaccha-
ride 12 (1.79 g, 56%). m/z (ESI) 717 [M + Na]+. HRESI-MS:
calc. for C36H38O14Na [M + Na]+ m/z 717.21593. Found m/z
717.2160. δH (CD3OD) 8.15–7.42 (m, 15H, Harom), 5.56 (m, 1H,
=CH–), 5.45 (t, 1H, H-4II), 4.06 (d, 2H, –CH2–). δC (CD3OD)
168.5–167.6 (C=O), 136.3 (=CH–), 135.6–130.3 (Carom), 118.2
(CH2=), 103.6, 103.5 (C-1IIα, C-1IIβ), 99.2 (C-1Iβ), 94.8 (C-1Iα),
85.3, 82.2 (C-3I, C-3II), 78.4–70.7 (C-2I, C-2II, C-4I, C-4II, C-5I,
C-5II, –CH2–), 65.2, 63.4 (C-6I, C-6II).

RESEARCH FRONT
Conjugatable β(1→3)-Glucan Oligosaccharides
581
(2,4,6-Tri-O-acetyl-3-O-allyl-β-D-glucopyranosyl)-
(1→3)-1,2,4,6-tetra-O-acetyl-D-glucopyranose 9
twice with H2O, and concentrated under reduced pressure. Flash
chromatography of the residue (2:1 cyclohexane/EtOAc) gave
fluoride 14 (0.7 g, 90%). Mp 158◦C (EtOAc/Et2O). [α]D +12 (c
1.0, CHCl3). m/z (ESI) 659 [M + Na]+. (Calc. for C27H37FO16:
C 50.94, H 5.86, F 2.98. Found C 50.58, H 5.84, F 2.99%.)
δH (CDCl3) 5.74 (m, 1H, =CH–), 5.66 (dd, 1H, H-1I, J1,2 2.8,
JH,F53.5), 5.21–5.10 (m, 2H, CH2=), 5.07 (t, 1H, H-4I), 5.04 (t,
1H, H-4II), 4.92 (ddd, 1H, H-2I), 4.89 (dd, 1H, H-2II), 4.56 (d,
1H, H-1II, J1,2 8.1), 4.29 (dd, 1H, H-6aII), 4.23–4.09 (m, 3H, H-
6aI, H-6bI, H-5I), 4.12 (t, 1H, H-3I), 4.05 (dd, 1H, H-6bII), 4.02
(m, 2H, CH2), 3.59 (m, 1H, H-5II), 3.54 (t, 1H, H-3II), 2.20–
2.03 (CH3). δC (CDCl3) 170.6–168.8 (C=O), 134.1 (=CH–),
117.0 (CH2=), 104.0 (d, C-1I, JC,F226.0), 101.1 (C-1II), 79.8
(C-3II), 75.4 (C-3I), 72.4 (CH2), 72.1 (C-2I, C-2II), 72.0 (C-5II),
70.1 (C-5I), 69.2 (C-4II), 66.9 (C-4I), 62.0 (C-6II), 61.4 (C-6I),
20.8–20.4 (CH3).
MeONa (9 mL, 1 M in MeOH) was added dropwise to an
ice-cooled solution of disaccharide 12 (1.3 g, 1.87 mmol) in
MeOH (90 mL). The mixture was stirred for 4 h at 20◦C,
and then neutralized by the addition of strong acidic cation-
exchange resin, filtered over a plug of cotton wool, and the
filtrate was concentrated under reduced pressure. The resulting
solid residue containing methyl (3-O-allyl-β-d-glucopyranosyl)-
(1→3)-d-glucopyranose was dissolved in dry pyridine (2 mL)
and Ac2O (2 mL) together with a catalytic amount of DMAP.
The solution was stirred for 12 h at 20◦C, protected from light,
andcooledinanicebath. MeOH(5 mL)wasaddedtodecompose
excess acetic anhydride, and the solution was concentrated under
reduced pressure. The solid residue was dissolved in CH2Cl2,
and washed with saturated aq. NaHCO3, and then H2O. The
organic layers were concentrated under reduced pressure and
flashchromatographyoftheresidue(2:3lightpetroleum/EtOAc)
gave peracetylated 3II-O-allyl-laminaribiose 9 as a mixture of α
and β anomers (1.02 g, 80%). m/z (FAB) 699 [M + Na]+.
(3-O-Allyl-β-D-glucopyranosyl)-(1→3)-α-D-glucopyranosyl
Fluoride 1
MeONa (0.48 mL, 1 M in MeOH) was added dropwise to an ice-
cooled solution of disaccharide 14 (0.31 g, 0.49 mmol) in MeOH
(48 mL). The mixture was stirred for 2.5 h at 20◦C, and neu-
tralized by the addition of strong acidic cation-exchange resin,
filtered over a plug of cotton wool, and the filtrate was concen-
trated under reduced pressure. The resulting solid residue was
dissolved in H2O, immediately frozen and lyophilized to give
fluoride 1 as a white powder (0.19 g, 100%). m/z (FAB) 407
[M + Na]+.
(2,4,6-Tri-O-acetyl-3-O-allyl-β-D-glucopyranosyl)-(1→3)-
1,2,4,6-tetra-O-acetyl-β-D-glucopyranose 13
HBr (12 mL, 33% inAcOH) was added to an ice-cooled solution
of peracetylated 3II-O-allyl-laminaribiose 9 (1.74 g, 2.6 mmol)
in dry CH2Cl2 (24 mL). The reaction was stirred for 50 min at
0◦C, then diluted with CH2Cl2 (50 mL), and washed succes-
sively with a mixture of ice and H2O, saturated aq. NaHCO3,
and H2O. The organic layers were concentrated under reduced
pressure to give crude peracetylated 3II-O-allyl-laminaribiosyl
bromide, which was directly used without further purification
by dissolution in a mixture of Ac2O (12 mL), AcOH (12 mL)
and AcOAg (1.6 g, 10.4 mmol), and the reaction was stirred for
12 h at 20◦C protected from light. The mixture was diluted with
CH2Cl2 (100 mL), filtered over a bed of diatomaceous earth,
and the filtrate was washed with saturated aq. NaHCO3, and
H2O. The organic layers were concentrated under reduced pres-
sure to give crude peracetylated 3II-O-allyl-β-laminaribiose 13,
which was purified by crystallization from EtOH (0.95 g, 55%).
Mp 164◦C (EtOH). [α]D −23 (c 1.0, CHCl3). m/z (ESI) 699
[M + Na]+. (Calc. for C29H40O18: C 51.48, H 5.96. Found C
51.08, H 5.91%.) δH (CDCl3) 5.73 (m, 1H, =CH–), 5.61 (d,
1H, H-1I, J1,2 8.3), 5.20–5.08 (m, 2H, CH2=), 5.10 (dd, 1H,
H-2I), 5.01 (t, 1H, H-4II), 4.99 (t, 1H, H-4I), 4.88 (dd, 1H, H-
2II), 4.50 (d, 1H, H-1II, J1,2 8.1), 4.28 (dd, 1H, H-6aII), 4.20 (dd,
1H, H-6aI), 4.13 (dd, 1H, H-6bI), 4.04 (dd, 1H, H-6bII), 4.02 (d,
2H, –CH2–), 3.92 (t, 1H, H-3I), 3.80 (m, 1H, H-5I), 3.57 (m,
1H, H-5II), 3.53 (t, 1H, H-3II), 2.12–2.01 (m, 12H, CH3). δC
(CDCl3) 170.7–168.7 (C=O), 134.1 (=CH–), 117.0 (CH2=),
101.2 (C-1II), 91.7 (C-1I), 79.8 (C-3II), 78.5 (C-3I), 72.8 (C-5I),
72.4 (–CH2–), 72.0 (C-2I, C-5II, C-2II), 69.2 (C-4II), 67.7 (C-4I),
62.0 (C-6II), 61.7 (C-6I), 20.8–20.4 (CH3).
(2,3,4,6-Tetra-O-acetyl-β-D-glucopyranosyl)-(1→3)-
1,2,4,6-tetra-O-acetyl-D-glucopyranose 15
A solution of 95% H2SO4 (3.5 mL) in Ac2O (10 mL) was added
dropwise to an ice-cooled mixture of scleroglucan (20 g) inAc2O
(130 mL).The reaction was vigorously stirred for 5 days at 55◦C,
then another portion of 95% H2SO4 (3.5 mL) in Ac2O (10 mL)
was added at 0◦C. The reaction was stirred again for 5 days
at 55◦C, and the mixture was then poured into a mixture of
ice and H2O (1.5 L), and the precipitate was stirred for 12 h.
The white precipitate was dissolved in CHCl3, and washed suc-
cessively with brine, saturated aq. NaHCO3 and H2O, and the
organic layer was concentrated under reduced pressure. Flash
chromatography (1:1 light petroleum/EtOAc) of the residue gave
peracetylated laminirabiose 15 as a mixture of α and β anomers
(4.6 g). The α anomer could be crystallized from EtOH. m/z
(DCI) 696 [M + NH4]+.
α anomer: Mp 80◦C (EtOH) [lit.[33] 78–80◦C]. δH (CDCl3)
6.23 (d, 1H, H-1I, J1,2 3.7), 5.14 (t, 1H, H-3II), 5.08 (t, 1H,
H-4II), 5.05 (dd, 1H, H-4I), 5.04 (dd, 1H, H-2I), 4.89 (dd, 1H,
H-2II), 4.64 (d, 1H, H-1II, J1,2 8.1), 4.38 (dd, 1H, H-6IaI), 4.18
(dd, 1H, H-6Ia), 4.12–4.03 (m, 3H, H-6bI , H-6IbI, H-5I), 4.05 (t,
1H, H-3I), 3.74 (m, 1H, H-5II), 2.18–1.96 (m, 24H, CH3). δC
(CDCl3) 170.7–168.7 (C=O), 100.8 (C-1II), 89.2 (C-1I), 76.1
(C-3I), 72.8 (C-3II), 71.6 (C-5II), 71.3, 71.2 (C-2I, C2II), 69.9
(C-5I), 68.0 (C-4II), 67.4 (C-4I), 61.6 (C-6I, C-6II), 20.9–20.3
(COCH3).
(2,4,6-Tri-O-acetyl-3-O-allyl-β-D-glucopyranosyl)-(1→3)-
2,4,6-tri-O-acetyl-α-D-glucopyranosyl Fluoride 14
Warning: hydrogen fluoride/pyridine is extremely corrosive to
human tissue and must be handled with caution.
4-Methoxyphenyl O-(2,3,4,6-Tetra-O-acetyl-β-D-
glucopyranosyl)-(1→3)-2,4,6-tri-O-acetyl-
β-D-glucopyranoside 16
In a plastic vessel, compound 13 was dissolved in a mixture
of HF and pyridine (7:3, 5 mL), and the reaction was stirred
for 45 min at 0◦C. The solution was diluted with CH2Cl2, and
then carefully added to a mixture of ice/CH2Cl2/NH4OH (33%
in H2O) (1:1:1 by vol., 150 mL). The organic layer was washed
HBr (2 mL, 33% in AcOH) was added to an ice-cooled solu-
tion of peracetylated laminaribiose 15 (0.25 g, 0.37 mmol) in
dry CH2Cl2 (4 mL). The reaction was stirred for 30 min at 0◦C,

RESEARCH FRONT
582
E. Montel et al.
followed by 1 h at 20◦C, and then diluted with CH2Cl2 (25 mL),
and washed successively with a mixture of ice and H2O, sat-
urated aq. NaHCO3, and then H2O. The organic layers were
concentrated under reduced pressure to give crude hepta-O-
acetyl-α-laminaribiosyl bromide, which was purified by flash
chromatography (1:1 cyclohexane/EtOAc) to give pure bromide
(0.22 g, 84%) that was immediately used in the next step. To a
solution of the bromide (0.22 g, 0.31 mmol) in CHCl3 (1.3 mL),
4-methoxyphenol (0.08 g, 0.64 mmol) and benzyltriethylammo-
nium chloride (0.058 g, 0.25 mmol) in aq. NaOH (0.46 mL,
1.25 M) were added, and the reaction was vigorously stirred at
60◦C for 1.5 h. The reaction was diluted with CHCl3 (3 mL)
and washed with aq. NaOH (2 × 5 mL, 1.25 M), then H2O. The
organic layer was concentrated under reduced pressure and flash
chromatography (1:1 cyclohexane/EtOAc) of the residue gave
laminaribioside 16 (0.15 g, 65%). Mp 85◦C (EtOH). [α]D −27 (c
1.0, CHCl3). m/z (DCI) 760 [M + NH4]+; HRESI-MS: calc. for
C33H42O19Na [M + Na]+ m/z 765.2218. Found m/z 765.2209.
δH (CDCl3) 6.92–6.78 (m, 4H, Harom), 5.23 (dd, 1H, H-2I), 5.15
(t, 1H, H-4II), 5.06 (t, 1H, H-4I), 5.01 (t, 1H, H-2II), 4.91 (t,
1H, H-3II), 4.81 (d, 1H, H-1I, J1,2 7.8), 4.62 (d, 1H, H-1II, J1,2
7.8), 4.36 (m, 1H, H-6aII), 4.88 (m, 2H, H-6aI, H-6bI), 4.05 (dd,
1H, H-6bII), 3.95 (t, 1H, H-3I), 3.78 (m, 1H, H-5I), 3.76 (s, 3H,
–O–CH3), 3.69 (m, 1H, H-5II), 2.15–1.98 (m, 21H, COCH3).
δC (CDCl3) 170.6–169.2 (C=O), 155.7, 151.1 (Cq arom), 118.5,
114.5 (CHarom), 100.9 (C-1II), 100.3 (C-1I), 78.7 (C-3I), 72.9
(C-2I), 72.4 (C-3II), 71.9 (C2II), 71.7 (C-5I), 71.1 (C-5II), 68.3
(C-4I), 68.1 (C-4II), 62.2 (C-6I), 61.7 (C-6II), 55.6 (–O–CH3),
20.7–20.3 (COCH3).
(C=O), 155.8, 150.9 (Cq arom), 118.7, 114.6 (CHarom), 100.3 (C-
1), 72.8, 72.0, 71.3, 68.4 (C-2, C-3, C-4, C-5), 62.0 (C-6), 55.7
(–O–CH3), 20.7–20.6 (COCH3).
4-Methoxyphenyl O-β-D-Glucopyranoside 19
The same procedure was applied as for 1, starting from 18
(0.49 g, 1.1 mmol), to give 19 (0.31 g, 100%). [α]D −52 (c
0.5, H2O) [lit.[26] [α]D20 −35.7 (c 1.1, H2O)]. 2m/z (ESI) 595
[2M + Na]+. δH (D2O) 7.00–6.82 (m, 4H, Harom), 4.86 (d, 1H,
H-1, J1,2 6.9), 3.81–3.58 (m, 2H, H-6a, H-6b), 3.67 (s, 3H, –O–
CH3), 3.46–3.35 (m, 4H, H-2, H-3, H-4, H-5). δC (D2O) 152.4,
148.5 (Cq arom), 115.9, 112.7 (CHarom), 98.9 (C-1), 73.8, 73.3,
70.7, 67.1 (C-2, C-3, C-4, C-5), 58.3 (C-6), 53.5 (–O–CH3).
4-Methoxyphenyl O-(3-O-Allyl-β-D-glucopyranosyl)-
(1→3)-β-D-glucopyranosyl-(1→3)-
β-D-glucopyranoside 20
The isoenzyme GII E231G glycosynthase (1 mg mL−1 in 0.2 M
sodium phosphate buffer pH 7, 0.8 mL) was added to a solu-
tion of fluoride 1 (60 mg, 0.16 mmol) and 4-methoxyphenyl
glucoside 19 (45 mg, 0.16 mmol) in sodium phosphate buffer
(0.2 M, pH 7, 3.2 mL). The solution was rotated for 17 h at
37◦C, then purified by loading onto a C-18 cartridge and eluted
with H2O to 9:1 H2O/MeOH. The appropriate fractions were
pooled, concentrated under reduced pressure and freeze-dried to
give 3III-O-allyl-β-laminaritrioside 20 (82 mg, 79%). [α]D −28
(c 1.0, H2O). m/z (ESI) 673 [M + Na]+; HRESI-MS: calc. for
C28H42O17Na [M + Na]+ m/z 673.2320. Found m/z 673.2322.
4-Methoxyphenyl O-β-D-Glucopyranosyl-(1→3)-
β-D-glucopyranoside 17
4-Methoxyphenyl O-(2,4,6-Tri-O-acetyl-3-O-allyl-
β-D-glucopyranosyl)-(1→3)-(2,4,6-tri-O-acetyl-
β-D-glucopyranosyl)-(1→3)-2,4,6-tri-O-acetyl-
β-D-glucopyranoside 21
The same procedure was applied as for 1, starting from 16
(0.12 g, 0.16 mmol), to give 17 (72 mg, 100%). [α]D −37 (c
1.0, H2O). m/z (ESI) 471 [M + Na]+; HRESI-MS: calc. for
C19H28O12Na [M + Na]+ m/z 471.14785. Found m/z 471.1481.
δH (D2O) 7.06–6.88 (m, 4H, Harom), 4.95 (d, 1H, H-1I, J1,2 7.9),
4.70 (d, 1H, H-1II, J1,2 7.9), 3.87–3.82 (m, 2H, H-6aI , H-6IaI),
3.80–3.62 (m, 3H, H-3I, H-6bI , H-6bII), 3.73 (s, 3H, –O–CH3),
3.65 (dd, 1H, H-2I), 3.53–3.50 (m, 2H, H-4I, H-5I), 3.46 (dd,
1H, H-3II), 3.41–3.39 (m, 1H, H-5II), 3.34 (dd, 1H, H-4II), 3.30
(dd, 1H, H-2II). δC (D2O) 156.1, 152.1 (Cq arom), 119.5, 116.3
(CHarom), 104.1 (C-1II), 102.3 (C-1I), 85.5 (C-3I), 77.3, 77.0
(C-4I, C-5I), 76.8 (C-3II), 74.7 (C-2I), 74.0 (C-2II), 70.9 (C-4II),
69.2 (C-5II), 62.0, 61.8 (C-6I, C-6II), 57.1 (–O–CH3).
3III-O-Allyl-β-laminaritrioside 20 (57 mg, 88 µmol) was dis-
solved in dry pyridine (1 mL), and Ac2O (1 mL) with catalytic
amounts of DMAP. The solution was stirred for 5 h at 20◦C, pro-
tected from light, and cooled in an ice bath. MeOH (2 mL) was
added to decompose excess Ac2O, and the solution was concen-
trated under reduced pressure.The solid residue was dissolved in
CH2Cl2, and washed with saturated aq. NaHCO3 and with H2O.
The organic layers were concentrated under reduced pressure and
flashchromatographyoftheresidue(2:3lightpetroleum/EtOAc)
yielded peracetylated 3III-O-allyl-laminaritrioside 21 (85 mg,
95%). [α]D −43 (c 1.0, CHCl3). m/z (FAB) 1051 [M + Na]+;
HRESI-MS: calc. for C46H60O26Na [M + Na]+ m/z 1051.3271.
Found m/z 1051.3275. δH (D2O) 6.91–6.79 (m, 4H, Harom), 5.73
(m, 1H, =CH–), 5.20 (dd, 1H, H-2I), 5.20–5.10 (m, 2H, CH2=),
5.03 (t, 1H, H-4III), 4.98 (t, 1H, H-4I), 4.90 (t, 2H, H-4II, H-2II),
4.87 (t, 1H, H-2III), 4.81 (d, 1H, H-1I, J1,2 7.9), 4.49 (d, 1H,
H-1II, J1,2 8.1), 4.42 (d, 1H, H-1III, J1,2 8.1), 4.33–4.26 (m, 2H,
H-6aIII, H-6aII), 4.19 (m, 2H, H-6aI, H-6bI), 4.08–4.00 (m, 4H,
H-6bII, H-6bIII, CH2), 3.94 (t, 1H, H-3I), 3.82 (t, 1H, H-3II),
3.78 (m, 1H, H-5I), 3.76 (s, 3H, –O–CH3), 3.68 (m, 1H, H-5II),
3.56 (m, 1H, H-5III), 3.51 (t, 1H, H-3III), 2.17–2.00 (m, 27H,
CH3). δC (D2O) 170.6–168.7 (C=O), 155.7, 151.0 (Cq arom),
134.1 (=CH–), 118.4, 114.5 (CHarom), 117.0 (CH2=), 101.2
(C-1III), 100.6 (C-1II), 100.1 (C-1I), 79.7 (C-3III), 78.5 (C-3II),
78.0 (C-3I), 73.0 (C-2I), 72.4 (CH2), 71.9 (C-2III), 71.8 (C-5III,
C-5II), 71.7 (C-5I), 69.2 (C-4III), 68.4 (C-2II, C-4II), 68.3 (C-
4I), 62.2 (C-6I), 62.0 (C-6II, C-6III), 55.6 (–O–CH3), 21.0–20.5
(COCH3).
4-Methoxyphenyl O-2,3,4,6-Tetra-O-acetyl-
β-D-glucopyranoside 18
To a stirred solution of penta-O-acetyl-β-d-glucopyranose
(1 g, 2.56 mmol) in dry CH2Cl2 (28 mL), 4-methoxyphenol
(0.64 g, 5.16 mmol), and boron etherate trifluoride (BF3-Et2O)
(0.65 mL, 5.12 mmol) were added, and the reaction was stirred
for 2.5 h at 0◦C. CH2Cl2 (50 mL) was added, and the solution was
washed twice with aq. NaHCO3 and with H2O.The organic layer
was concentrated under reduced pressure, and flash chromato-
graphy (2:1 cyclohexane/EtOAc) of the residue gave glucoside
18 (1.09 g, 93%). Mp 100◦C (EtOH) [lit.[25] 106–107◦C]. [α]D
−16 (c 1.0, CHCl3) [lit.[25] [α]D20 −8.4 (c 1.0, CHCl3)]. m/z
(DCI) 472 [M + NH4]+. δH (CDCl3) 6.96–6.79 (m, 4H, Harom),
5.30–5.10 (m, 3H, H-2, H-3, H-4), 4.95 (d, 1H, H-1, J1,2 7.2),
4.27–4.11 (m, 2H, H-6a, H-6b), 3.78 (m, 1H, H-5), 3.77 (s, 3H,
–O–CH3), 2.07–2.02 (m, 12H, CH3). δC (CDCl3) 170.5–169.3

RESEARCH FRONT
Conjugatable β(1→3)-Glucan Oligosaccharides
583
4-Methoxyphenyl O-β-D-Glucopyranosyl-(1→3)-β-D-
glucopyranosyl-(1→3)-β-D-glucopyranoside 22
concentrated under reduced pressure. Flash chromatography of
the residue (1:1 toluene/EtOAc) gave α-fluoride 24 (78 mg,
67%). [α]D −13 (c 1.0, CHCl3). m/z (DCI) 942 [M + NH4]+;
HRESI-MS: calc. for C39H53O24FNa [M + Na]+ m/z 947.2809.
Found m/z 947.2806. δH (CDCl3) 5.72 (m, 1H, =CH–), 5.64 (dd,
1H, H-1I, J1,2 2.7, JH,F 53.4), 5.19–5.10 (m, 2H, CH2=), 5.05 (t,
1H, H-4I), 5.02 (t, 1H, H-4III), 4.91 (ddd, 1H, H-2I), 4.89 (t, 1H,
H-4II), 4.88 (t, 1H, H-2II), 4.86 (t, 1H, H-2III), 4.50 (d, 1H, H-1II,
J1,2 8.1), 4.40 (d, 1H, H-1III, J1,2 8.0), 4.30 (m, 2H, H-6aIII, H-
6aII), 4.17 (m, 2H, H-6aI, H-6bI), 4.14–4.08 (m, 1H, H-5I), 4.11
(t, 1H, H-3I), 4.05–4.00 (m, 2H, H-6bII, H-6bIII), 4.00 (d, 2H,
CH2), 3.80 (t, 1H, H-3II), 3.69 (m, 1H, H-5II), 3.57 (m, 1H, H-
5III), 3.51 (t, 1H, H-3III), 2.21–2.01 (m, 27H, CH3). δC (CDCl3)
170.6–168.4 (C=O), 134.1 (=CH–), 117.0 (CH2=), 104.0 (d,
C-1I, JC,F 226.1), 101.2 (C-1III), 100.1 (C-1II), 79.7 (C-3III),
78.6 (C-3II), 75.1 (C-3I), 73.0 (C-2II), 72.4 (CH2), 72.1 (C-2I),
71.9 (C-2III), 71.8 (C-5III), 71.7 (C-5II), 70.1 (C-5I), 69.2 (C-
4III), 68.3 (C-4II), 66.7 (C-4I), 62.0 (C-6III, C-6II), 61.4 (C-6I),
20.8–20.4 (CH3).
PdCl2 (3 mg, 17 µmol) was added to a solution of trisaccha-
ride 21 (18 mg, 18 µmol) in MeOH (1 mL), and the reaction
mixture was stirred for 5 h at 20◦C. The suspension was per-
colated through a short column of silica gel and eluted with
1:4 cyclohexane/EtOAc and with 1:1 EtOAc/MeOH. The filtrate
was concentrated under reduced pressure, dissolved in MeOH
and treated with MeONa (0.2 mL, 1 M in MeOH). The reaction
mixture was stirred for 1 h at 0◦C, and neutralized by the addi-
tion of strong acidic cation-exchange resin, filtered over a plug
of cotton wool, and the filtrate was concentrated under reduced
pressure. The resulting solid was dissolved in H2O and puri-
fied by flash chromatography over C-18 reverse phase silica
(H2O to 1:1 H2O/MeOH). Appropriate fractions were pooled,
concentrated under reduced pressure, and freeze-dried to give 4-
methoxyphenyl laminaritrioside 22 (7 mg, 66%). m/z (ESI) 633
[M + Na]+; HRESI-MS: calc. for C25H38O17Na [M + Na]+ m/z
633.2007. Found m/z 633.1990. δH (D2O) 7.06–6.89 (m, 4H,
Harom), 4.96 (d, 1H, H-1I, J1,2 8.0), 4.75 (d, 1H, H-1II, J1,2 8.0),
4.68 (d, 1H, H-1III, J1,2 8.0), 3.88–3.61 (m, 9H, H-2I, H-3I, H-
3II, H-6aI, H-6bI, H-6aII, H-6bII, H-6aIII, H-6bIII), 3.74 (s, 3H,
CH3), 3.55–3.38 (m, 7H, H-2II, H-4I, H-4II, H-4III, H-5I, H-5II,
H-5III), 3.35–3.26 (m, 2H, H-2III, H-3III). δC (D2O) 155.2, 151.2
(Cq arom), 118.6, 115.4 (CHarom), 103.2 (C-1II), 102.9 (C-1III),
101.4 (C-1I), 84.6, 84.3 (C-3I, C-3II), 76.4 (C-3III), 76.1, 76.0,
75.9 (C-4I, C-4II, C-4III), 73.8, 73.6, 73.2 (C-2I, C-2II, C-2III),
69.9, 68.5, 68.3 (C-5I, C-5II, C-5III), 61.1–60.9 (C-6I, C-6II,
C-6III), 56.2 (CH3).
(3-O-Allyl-β-D-glucopyranosyl)-(1→3)-β-D-glucopyranosyl-
(1→3)-α-D-glucopyranosyl Fluoride 25
The same procedure was applied as for 1, starting from 24
(33 mg, 36 µmol), to give 25 (20 mg, 100%). m/z (ESI) 569
[M + Na]+; HRESI-MS: calc. for C21H35O15FNa [M + Na]+
m/z 569.1858. Found m/z 569.1855.
4-Methoxyphenyl O-(3-O-Allyl-β-D-glucopyranosyl)-
(1→3)-β-D-glucopyranosyl-(1→3)-β-D-glucopyranosyl-
(1→3)-β-D-glucopyranoside 26
(2,4,6-Tri-O-acetyl-3-O-allyl-β-D-glucopyranosyl)-(1→3)-
(2,4,6-tri-O-acetyl-β-D-glucopyranosyl)-(1→3)-
2,4,6-tri-O-acetyl-β-D-glucopyranose 23
The same procedure was applied as for 20, starting from
25 (7.7 mg, 14 µmol) and 19 (5.2 mg, 18 µmol) to give 26
(8 mg, 70%). m/z (ESI) 835 [M + Na]+; HRESI-MS: calc. for
C34H52O22Na [M + Na]+ m/z 835.2848. Found m/z 835.2846.
δH (D2O) 7.11–6.95 (m, 4H, Harom), 5.59 (m, 1H, =CH–), 5.35–
5.20 (m, 2H, CH2=), 5.01 (d, 1H, H-1I, J1,2 8.0), 4.80 (d, 1H,
Cerium ammonium nitrate (653 mg, 1.2 mmol) was added to a
solution of laminaritrioside 21 (123 mg, 0.12 mmol) in a mixture
of toluene/CH3CN/H2O (14:19.5:14, 9.5 mL), and the solu-
tion was stirred for 2 h at 20◦C. The solution was diluted with
CH2Cl2, washed successively with brine, aq. NaHCO3, and
water. The organic layers were concentrated under reduced pres-
sure, and the residue was purified by flash chromatography (1:2
cyclohexane/EtOAc) to give 3III-O-allyl laminaritriose deriva-
tive 23 (94 mg, 85%). m/z (FAB) 945 [M + Na]+; HRESI-MS:
calc. for C39H54O25Na [M + Na]+ m/z 945.2852. Found m/z
945.2862.
H-1II, J1,2 8.1), 4.78 (d, 1H, H-1III, J1,2 8.5), 4.74 (d, 1H, H-1IV
,
J1,2 7.5), 4.34 (m, 2H, CH2), 3.89–3.71 (m, 12H, H-2I, H-3I,
H-3II, H-3III, H-6aI , H-6bI , H-6aII, H-6bII, H-6IaII, H-6bIII, H-6aIV, H-
6bIV), 3.79 (s, 3H, –O–CH3), 3.59–3.46 (m, 12H, H-2II, H-2III,
H-2IV, H-3IV, H-4I, H-4II, H-4III, H-4IV, H-5I, H-5II, H-5III,
H-5IV). δC (D2O) 156.2, 152.2 (Cq arom), 135.6 (=CH–), 119.8
(CH2=), 119.6, 116.4 (CHarom), 104.1 (C-1IV), 103.9 (C-1II, C-
1III), 102.4 (C-1I), 85.5–85.4 (C-3I, C-3II, C-3III), 84.6 (C-3IV),
77.2–77.0 (C-2II, C-2III, C-2IV), 74.1 (C-2I), 70.6–69.3 (C-5I,
C-5II, C-5III, C-5IV), 62.0–61.2 (C-6I, C-6II, C-6III, C-6IV), 57.2
(–O–CH3).
(2,4,6-Tri-O-acetyl-3-O-allyl-β-D-glucopyranosyl)-(1→3)-
(2,4,6-tri-O-acetyl-β-D-glucopyranosyl)-(1→3)-2,4,6-
tri-O-acetyl-α-D-glucopyranosyl Fluoride 24
β-D-Glucopyranosyl Phenyl Disulfide 27
Diethylaminosulfur trifluoride (84 µL, 631 µmol) was added
dropwise to the stirred solution of hemiacetal 23 (117 mg,
127 µmol) in CH2Cl2 (4.1 mL) at −45◦C, and the reaction mix-
ture was allowed to reach −20◦C for 2.5 h.A mixture of ice/H2O
was then added, followed by CH2Cl2, and the organic layer was
washed three times with H2O, and concentrated under reduced
pressure.
The residue containing a mixture of α/β laminaritriosyl flu-
oride was transferred to plastic vessel and dissolved in HF and
pyridine (7:3, 4.5 mL) at −52◦C and the reaction mixture was
stirred for 2.5 h, the temperature slowly rising to −12◦C. The
solution was diluted with CH2Cl2, and carefully added to a
mixture of ice/CH2Cl2/NH4OH (33% in H2O) (1:1:1 by vol.,
100 mL). The organic layer was washed with H2O (twice), and
MeONa (1 M in MeOH, 1.4 mL) was added dropwise to
an ice-cooled solution of 2,3,4,6-tetra-O-acetyl-1-thio-β-d-
glucopyranose[32] (505 mg, 1.24 mmol) in MeOH (60 mL), and
the reaction mixture was stirred for 12 h at 20◦C. The solution
was cooled to 0◦C, before the addition of S-phenyl benzethio-
sulfonate (466 mg, 1.86 mmol), and stirred for 12 h at 20◦C.
Silica gel was added, then after concentration under reduced
pressure, the resulting solid was subjected to flash chromato-
graphy (CH3CN to 9:1 CH3CN/H2O) to give the disulfide 27
(280 mg, 74%). m/z (FAB)327[M + Na]+;HRESI-MS:calc. for
C12H16O5NaS2 [M + Na]+ m/z 327.0340. Found m/z 327.0337.
δH (CD3OD) 7.71–7.25 (m, 5H, Harom), 4.47 (d, 1H, H-1, J1,2
9.3), 3.86–3.30 (m, 6H, H-2, H-3, H-4, H-5, H-6a, H-6b). δC

RESEARCH FRONT
584
E. Montel et al.
(CD3OD) 140.0–128.9 (Carom), 93.0 (C-1), 83.2, 80.4, 74.1, 72.2
(C-2, C-3, C-4, C-5), 63.8 (C-6).
[5] O. Klarzynski, B. Plesse, J. M. Joubert, J. C. Yvin,
M. Kopp, B. Kloareg, B. Fritig, Plant Physiol. 2000, 124, 1027.
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V. Veticka, Glycobiology 2005, 15, 393. doi:10.1093/GLYCOB/
CWI020
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Glycosci. 2008, 1, 19. doi:10.2174/1875398100801010019
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5429. doi:10.1021/JA9936520
(3-O-Allyl-β-D-glucopyranosyl)-(1→3)-
β-D-glucopyranosyl-(1→3)-β-D-glucopyranosyl-
(1→3)-β-D-glucopyranosyl Phenyl Disulfide 28
The same procedure was applied as for 20, starting from 25
(26.9 mg, 49 µmol) and 27 (15 mg, 49 µmol) in presence of ben-
zoquinone(5.3 mg, 49 µmol), togive28(23 mg, 56%). m/z (ESI)
853 [M + Na]+.
[2-[2-((3-O-Allyl-β-D-glucopyranosyl)-(1→3)-β-D-
glucopyranosyl-(1→3)-β-D-glucopyranosyl-(1→3)-
β-D-glucopyranosyl)-thioacetyl]-aminoethyl]-
5-aminonaphtalene-1-sulfonic Acid Sodium Salt 29
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V. Bulone, H. Driguez, G. B. Fincher, J. Biol. Chem. 2002, 277, 30102.
doi:10.1074/JBC.M203971200
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Chem. Eur. J. 2003, 9, 2603. doi:10.1002/CHEM.200304733
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193. doi:10.1016/S0008-6215(00)85898-X
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A solution containing disulfide 28 (23 mg, 28 µmol) and dithio-
threitol (43 mg, 0.28 mmol) in DMF (0.5 mL) was stirred for
10 min at 20◦C, followed by the addition of Et3NH (40 µL,
0.29 mmol) and by N-(iodoacetamidoethyl)-1-naphthylamine-5-
sulfonic acid (30 mg, 72 µmol), and the reaction mixture was
stirredfor12hat20◦C. Silicagelwasadded, followedbyconcen-
tration under reduced pressure, and the resulting solid was sub-
jected to flash chromatography (CH3CN to 8:2 CH3CN/H2O)
to give the EDANS conjugate 29 (28 mg, 96%). m/z (ESI)
1027 [M − H]−; HRESI-MS: calc. for C41H59N2O24Na2S2
[M + Na]+ m/z 1073.2695. Found m/z 1073.2699. δH (D2O)
8.37–6.78 (m, 6H, CHarom), 5.93 (m, 1H, =CH–), 5.23 (m, 2H,
CH2=), 4.70 (d, 1H, H-1II, J1,2 8.0), 4.68 (d, 1H, H-1III, J1,2
8.0), 4.43 (d, 1H, H-1IV, J1,2 8.0), 4.32 (d, 1H, H-1I, J1,2 9.8),
4.28 (d, 2H, –CH2–O), 3.90–3.75 (m, 4H, H-6aI, H-6aII, H-
6aIII, H-6aIV), 3.75–3.14 (m, 24H, H-2I, H-2II, H-2III, H-2IV
,
H-3I, H-3II, H-3III, H-3IV, H-4II, H-4III, H-4IV, H-5II, H-5III,
H-5IV, H-6bI, H-6bII, H-6bIII, H-6bIV, CH2–O, S–CH2, NH–
CH2–CH2–NH), 3.14–2.99 (m, 2H, H-4I, H-5I). δC (D2O) 174.4
(C=O), 145.2, 139.6 (Cq–NH, Cq–SO3Na), 135.5 (=CH–),
130.4, 125.7 (Cq arom), 129.8, 127.6, 126.4, 124.9, 116.3, 107.5
(CHarom), 119.8 (CH2=), 104.0 (C-1IV), 103.8 (C-1II, C-1III),
87.0 (C-3I), 85.6 (C-1I), 85.5, 85.3 (C-3II, C-3III), 84.6 (C-3IV),
80.6 (C-4I), 77.1, 76.9, 76.8 (C-4II, C-4III, C-4IV), 75.2 (CH2–
O), 74.6–74.5 (C-2II, C-2III, C-2IV), 73.0 (C-2I), 70.6, 69.4, 69.3,
69.1 (C-5I, C-5II, C-5III, C-5IV), 61.9 (C-6I, C-6II, C-6III, C-6IV),
44.3, 39.9, 34.4 (S–CH2–, NH–CH2–CH2–NH).
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Acknowledgement
We thank Cargill France SAS Co. (formerly known as Degussa Texturant
Systems France SAS) for a kind gift of scleroglucan.
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