β(1,3) Branched heptadeca- and linear hexadeca-saccharides possessing an aminoalkyl group as a strong ligand to dectin-1

Article abstract of DOI:10.1039/c0cc03153d

In this report, we describe the convergent synthesis of β(1,3) oligosaccharides containing an aminoalkyl group. The branched heptadecasaccharide and linear hexadecasaccharide acted as ligands of dectin-1 whose binding affinity was only 10-fold weaker than

Full text of DOI:10.1039/c0cc03153d

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b(1,3) Branched heptadeca- and linear hexadeca-saccharides possessing
an aminoalkyl group as a strong ligand to dectin-1w
Hiroshi Tanaka,*a Tetsuya Kawai,a Yoshiyuki Adachi,b Naohito Ohnob and
Takashi Takahashi*a
Received 10th August 2010, Accepted 14th September 2010
DOI: 10.1039/c0cc03153d
In this report, we describe the convergent synthesis of b(1,3)
oligosaccharides containing an aminoalkyl group. The branched
heptadecasaccharide and linear hexadecasaccharide acted as
ligands of dectin-1 whose binding affinity was only 10-fold
weaker than that of natural SPG and exhibited dectin-1 agonist
activity.
However, the binding of these synthetic glucans is still
considerably weak in comparison with that of the poly-
saccharides. In addition, modulation of the biological action
of dectin-1 by the synthetic glycans is still an unsolved subject.
Herein we report on the synthesis of b(1,3) branched
heptadeca- and linear hexadeca-saccharides 1 and 2, which
show a strong binding affinity to dectin-1 and their dectin-1
agonist activity.
Polysaccharides are important natural materials that some-
times have potent and unique biological activities. Elucidation
of the mechanism of their biological action and the identification
of target proteins and their minimum binding structure are
important issues, in terms of our understanding of poly-
saccharide-induced biological events. Hydrolysates derived
from such materials can be used as effective chemical probes
for the elucidation of the role of their partial structures.
However, it is difficult to completely purify structurally
defined oligosaccharides from hydrolysates. In addition, the
possibility that isolated polysaccharides could be hetero-
geneous and/or be contaminated with antigenic compounds
cannot be excluded. Therefore, confirmation of biological
activity using chemically synthesized and structurally defined
oligosaccharides is indispensable.
Scheme 1 shows the synthesis of the b(1,3) branched and
linear oligosaccharides 1 and 2. The chemo- and regio-selective
glycosylation of diol 3 protected with a 4,6 benzylidene acetal
at the 3 position with the glycosyl imidate 4 provided
disaccharide 5 in 69% yield along with the C2 glycosylated
product in 6% yield.5 Protection of the C4 and C6 hydroxyl
groups with a benzylidene acetal and unprotection of the C2
hydroxyl group minimized steric hindrance of the C3 hydroxyl
group.6 In addition, the sulfide substituent at the anomeric
position accelerated the selective glycosylation at the C2
hydroxyl group because the glycosylations of the corresponding
methyl and allyl 4,6-O-benzylidene-b-glucopyranosides
resulted in the production of mixtures of O2 and O3 glycosylated
products.7 After acylation of the unprotected C2 hydroxyl
groups of 5 with a benzoyl group, disaccharide 6 was
converted into the glycosyl imidate 7 and diol 8 by anomeric
modification8 and selective deprotection of the Lev group,
Certain types of b-glucans containing b(1,3) and b(1,6)
glucosidic linkages exhibit biological activity, in the sense that
they stimulate innate immunity. Dectin-1, originally identified
as a dendritic cell receptor in the mouse,1 is a b-glucan
receptor that mediates phagocytosis and the production of
inflammatory mediators in the development of immunity to
fungal pathogens.2 Therefore, oligosaccharides that bind
strongly to dectin-1 would be expected to act as effective
modulators of innate immunity. A previous study, using
hydrolysates from b-glucans, revealed that oligosaccharides
containing more than 10 glucose units are required for binding
to dectin-1.3 Williams and co-workers recently reported on the
importance of a b(1,6) branching glucosyl unit on b(1,3)
oligosaccharides in binding to dectin-1 by using a fully
synthetic branched nonasaccharide and linear decasaccharide.4
respectively. The glycosylation of
8 with 7 provided
tetrasaccharide 9 in 90% yield in a stereo- and regioselective
manner, followed by acetylation of the remaining hydroxyl
groups to provide thioglycoside 10. The steric hindrance of the
disaccharide at the anomeric position prevented the donor 7
from glycosidation at the O2 position. The thioglycoside 10
was converted into the acceptor 11 bearing an azido alkyl
group by glycosylation with a primary alcohol possessing a
tosylate group, displacement of the tosylate with an azido
group, followed by removal of the Lev group.9 The glycosylation
of the acceptor 11 with thioglycoside 10 provided the octa-
saccharide 12 in 97% yield as a single isomer. The octa-
saccharide 12 was converted to diol 13 by acetylation of the
remaining unprotected hydroxyl groups, followed by removal
of the Lev group. Glycosylation of the acceptor 13 with
thioglycoside 10 provided dodecasaccharide 14 in 88% yield
as a single isomer. The dodecasaccharide 14 was converted to
diol 15 using the same procedure as was used for 12 to 13.
Glycosylation of dodecasaccharide 15 with the linear tetra-
saccharide 10 and the branched pentasaccharide 16 provided
the linear hexadecasaccharide 18 and the branched hepta-
decasaccharide 17 in 82% and 82% yields, respectively.
Regioselective glycosylation at the C3 position was confirmed
a Department of Applied Chemistry, Graduate School of Science and
Engineering, Tokyo Institute of Technology, 2-12-1-H-101
Ookayama, Meguro, Tokyo, 152-8552, Japan.
E-mail: thiroshi@apc.titech.ac.jp; Fax: +81 3-5734-2884;
Tel: +81 3-5734-2471
b Laboratory for Immunopharmacology of Microbial Products,
School of Pharmacy, Tokyo University of Pharmacy and Life
Sciences, 1432-1 Horinouchi Hachioji, Tokyo, 192-0392, Japan.
E-mail: ohnonao@ps.toyaku.ac.jp; Fax: +81 42-676-5570
w Electronic supplementary information (ESI) available: Procedures
for the synthesis of the oligosaccharides 19–23 and their 1H and 13C
NMR and MS spectra. See DOI: 10.1039/c0cc03153d
c
This journal is The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 8249–8251 8249

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Scheme 1 Reagents and conditions; (a) cat. TMSOTf, CH2Cl2, MS4A, À40 1C, 69%; (b) BzCl, cat. DMAP, Py., 120 1C, 4 h, 96%;
(c) benzenesulfinyl morpholine, Tf2O, CH2Cl2, À50 1C then NEt3, 86%; CCl3CN, Cs2CO3, CH2Cl2, 96%, a : b = 84 : 16; (d) NH2NH2ÁH2O,
AcOH, THF, 99%; (e) cat. TMSOTf, CH2Cl2, MS4A, À50 1C, 90%; (f) Ac2O, cat. DMAP, Py., 96%; (g) HO(CH2)6OTs, NIS, cat. TfOH, MS4A,
À35 1C, 31%; (h) NaN3, DMF; (i) NH2NH2ÁH2O, AcOH, THF, 73%; (j) 10, NIS, cat. TfOH, MS4A, À35 1C, 97% for 12, 88% for 14, 82% for 18;
(k) Ac2O, cat. DMAP, Py.; (l) NH2NH2ÁH2O, AcOH, THF, 93% for 13, 93% for 15; (m) 16, NIS, cat. TfOH, MS4A, À35 1C, 82%; (n) Li, NH3(l),
À30 1C, then MeOH, rt, 13.5 h, 51% for 1, 80% for 2.
by 2D 1H NMR analysis of coupling products 12, 14, 17, and
18, indicating that the remaining hydroxyl groups were located
at the C2 position. The heptadeca- and hexadeca-saccharides
17 and 18 were completely deprotected by a Birch reduction to
provide the fully deprotected oligosaccharides 1 and 2
containing an amino alkyl group, in 51% and 80% overall
yield, respectively.
Competitive effects of the synthetic glucans 1, 2 and 19–23
on binding to a soluble form of dectin-1 attached to a
solid-supported Schizophyllan were examined (Fig. 1 and 2).10
The branched heptadecasaccharide 1 and the linear hexadeca-
Fig. 1 Structures of branched and linear b(1,3) oligosaccharides 19–23.
saccharide 2 inhibited the binding of the soluble dectin-1 to the
solid-supported SPG. The binding activity of 1 and 2 was only
10-fold weaker than that of natural SPG, whose molecular
weight is 450 kD. No significant differences between the
affinity of the branched and linear oligosaccharides 1 and 2
were observed. The remaining oligosaccharides 19–22, which
contained less tridecasaccharide units showed dramatically
reduced inhibitory activities. In addition, a-methyl glucoside
c
8250 Chem. Commun., 2010, 46, 8249–8251
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heptadecasaccharide 1 and the linear hexadecasaccharide 2.
On the other hand, the low-affinity ligands 19–22 failed to
stimulate NF-kB activation. However, the difference in the
activities of the synthetic oligosaccharides and SPG in
promoting NF-kB activation cannot be completely explained
by the differences in their binding affinity to dectin-1.
In conclusion, we describe an effective convergent synthesis
of b(1,3) oligosaccharides using tetra- and pentasaccharide
building blocks. The synthetic b(1,3) hepta- and hexadeca-
saccharides 1 and 2 bound to dectin-1 and promoted the
activation of NF-kB. To the best of our knowledge, this is
the first report to demonstrate that purely synthetic oligo-
saccharides have the ability to stimulate innate immunity
mediated by dectin-1. In addition, the b(1,3) hexadecasaccharide
represents a potentially important structurally-defined chemical
probe for studies related to immunity mediated by dectine-1.
This work was supported by Grant-in-Aid for Scientific
Research (C) (Grant-in-aid No. 20550149) from the Ministry
of Education, Culture, Sports, Science, and Technology. We
thank EIWEISS Chemical Corporation for generous gift of
EDCI and Central Glass Co., Ltd. for their gift of trifluoro-
methanesulfonic acid anhydride.
Fig. 2 Competitive effects of the oligosaccharides 1, 2, and 19–23 on
binding of dectin-1 to SPG. Error bar shows standard deviation.
23 composed of sixteen b(1,3) glucose units did not affect the
binding of the soluble dectin-1 to the solid-supported SPG.
These results indicate that the affinity of the oligosaccharide
probes to the soluble dectin-1 was clearly dependent on the
length of the b(1,3) glucose sequence of 1 and 2. However, the
role of the primary amino group on their enhanced binding
affinity to dectin-1 in comparison with that of 23 is not clear.
In another series of experiments, a luciferase-assisted
NF-kB assay of oligosaccharides 1,
2 and 19–22 was
Notes and references
conducted (Fig. 3). 293T cells, transfected with a plasmid
DNA mixture encoding dectin-1 and signaling molecules
including CARD9 and Bcl10, were stimulated with oligo-
saccharides 1, 2 and 19–22. Luciferase activity, expressed
as the ratio of the NF-kB-assisted firefly luciferase activity
to thymidine kinase (TK) promoter-assisted renillaluciferase
activity, was measured using a Dual-Luciferase Reporter
Assay System. NF-kB activation was observed during
the >stimulation of the 293T cells with the branched
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10 Details of the synthesis of the oligosaccharides 19–23 were in ESIw.
Fig. 3 A luciferase-assisted NF-kB assay of the oligosaccharides 1, 2,
and 19–22. Error bar shows standard deviation.
c
This journal is The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 8249–8251 8251