Synthesis of β(1,3) oligoglucans exhibiting a Dectin-1 binding affinity and their biological evaluation

Article abstract of DOI:10.1016/j.bmc.2012.04.017

In this report, we describe the synthesis and biological evaluation of β(1,3) oligosaccharides that contain an aminoalkyl group and their biological evaluation. A 2,3 diol glycoside with a 4,6 benzylidene protecting group was used as an effective glycosyl

Full text of DOI:10.1016/j.bmc.2012.04.017

Bioorganic & Medicinal Chemistry 20 (2012) 3898–3914
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of b(1,3) oligoglucans exhibiting a Dectin-1 binding affinity
and their biological evaluation
Hiroshi Tanaka a, , Tetsuya Kawai a, Yoshiyuki Adachi b, Shinya Hanashima c, Yoshiki Yamaguchi c,
⇑
Naohito Ohno b, Takashi Takahashi a,
⇑
a Department of Applied Chemistry, Graduate School of Science and Engineering, Tokyo Institute of Technology, 2-12-1-S1-35 Ookayama, Meguro, Tokyo 152-8552, Japan
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
c Chemical Biology Department, RIKEN Advanced Research Institute, 2-1 Hirosawa Wako, Saitama 351-0198, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 February 2012
Revised 6 April 2012
Accepted 7 April 2012
Available online 16 April 2012
In this report, we describe the synthesis and biological evaluation of b(1,3) oligosaccharides that contain
an aminoalkyl group and their biological evaluation. A 2,3 diol glycoside with a 4,6 benzylidene protect-
ing group was used as an effective glycosyl acceptor for the synthesis of some b(1,3) linked glycosides.
The use of a combination of a linear tetrasaccharide and a branched pentasaccharide as glycosyl donors
led to the preparation of b(1,3) linear octa- to hexadecasaccharides and branched nona- to heptadecasac-
charides in good total yields. Measurements of the competitive effects of the oligosaccharides on the
binding of a soluble form of Dectin-1 to a solid-supported Schizophyllan (SPG) revealed that the branched
heptadecasaccharide and the linear hexadecasaccharides also have binding activity for Dectin-1. In addi-
tion, the two oligosaccharides, both of which contain a b(1,3) hexadecasaccharide backbone, exhibited
Keywords:
Innate immunity
Polysaccharides
STD-NMR
Chemoselecitve glycosylation
b-Glucan
agonist activity in a luciferase-assisted NF-jB assay. STD-NMR analyses of complexes of Dectin-1 and
the linear hexadecasaccharides clearly indicate Dectin-1 specifically recognizes the sugar part of the oli-
gosaccharides and not the aminoalkyl chain.
Ó 2012 Published by Elsevier Ltd.
1. Introduction
fined oligosaccharides from hydrolysates and the possibility that
isolated glucan derivatives could be heterogeneous and/or be
Certain types of b-glucans containing b(1,3) and b(1,6) linkages
exhibit biological activity, in the sense that they stimulate innate
immunity. Dectin-1, originally identified as a dendritic cell recep-
tor in the mouse,1 is a b-glucan receptor that mediates phagocyto-
sis and the production of inflammatory mediators that are involved
in the development of immunity to fungal pathogens.2 The elucida-
tion of the minimum structure required for binding to Dectin-1
and for exhibiting Dectin-1 agonist or antagonist activity is an
important issue, in terms of our understanding of b-glucan-in-
duced biological events and would strongly assist in the develop-
ment of artificial modulators for innate immunity. A previous
study using naturally occurring b-glucans and hydrolysates de-
rived from them revealed that oligosaccharides containing more
than 10 glucose units are required for binding to Dectin-1.3 In addi-
tion, particulate b-glucans would need to activate Dectin-1 signal-
ling.4 However, for structure–activity relationship studies, the use
of chemically synthesized b-glucans as biochemical probes would
be highly desirable, because it is difficult to purify structurally de-
contaminated with antigenic compounds cannot be excluded.
Williams and co-workers recently elucidated the importance of a
b(1,6) branching glucosyl unit for binding to Dectin-1 based on
the experiments using a purely synthetic nonasaccharide contain-
ing b(1,3) and b(1,6) branching sugar units.5
b(1,3) oligosaccharides are, in practice, challenging targets ow-
ing to the considerable steric hindrance that results in their fre-
quent adoption of non-chair conformers.6 The synthesis of b(1,3)
glucan related oligosaccharides have been investigated by several
research groups.7 The 4,6-benzylidene and 2-benzoyl protected
glucosides have been reported to be effective blocking groups for
the elongation of b(1,3) glucosides.8 The 4,6-benzylidene acetal
effectively blocks the C4 hydroxyl group with minimum steric hin-
drance to the C3 hydroxyl group. The C2 acyl protecting group as-
sists in b-selective glycosidation reactions, but slightly reduces the
reactivity of the C3 hydroxyl group towards glycosylation. Takeo
et al. successfully prepared a b(1,3) octasaccharide using a disac-
charide donor in a blockwise synthesis.8a We also investigated
the combinatorial synthesis of branched b(1,3) glucan oligosaccha-
rides.9 However most of the established methodologies are direc-
ted at the synthesis of oligosaccharides composed of less than 10
glucose units. In addition, coupling of the complex branched
⇑
Corresponding authors. Tel.: +81 3 5734 2471; fax: +81 3 5734 2884.
E-mail addresses: thiroshi@apc.titech.ac.jp (H. Tanaka), ttak@apc.titech.ac.jp
(T. Takahashi).
0968-0896/$ - see front matter Ó 2012 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmc.2012.04.017

H. Tanaka et al. / Bioorg. Med. Chem. 20 (2012) 3898–3914
3899
oligosaccharide building blocks at the C3 position resulted in the
unexpected production of b-isomers.10 Therefore, an effective
method for the synthesis of complex or large b(1,3) glucans contin-
ues to be needed. Herein, we report on the biological evaluation of
the b(1,3) heptadeca to octasaccharides that were synthesized
based on a convergent strategy. We also report on an analysis of
saturated transfer difference (STD)-NMR spectra of complexes of
a synthetic b(1,3) hexadecasaccharide and Dectin-1.11
pure coupling products could be easily separated from the donors
and accepters by gel permeation chromatography.11
Scheme 2 shows the synthesis of the tetrasaccharide building
blocks 7 and 8. The thioglycoside 10a was converted to the diester
11 in quantitative yield. Treatment of the thioglycoside 11 with
benzenesulfinyl morpholine and Tf2O under basic conditions,14 fol-
lowed by quenching the reaction mixture with water provided the
hemiacetal 12 in 96% yield. Hydrolysis of the thioglcyoside 11 un-
der oxidative conditions with NBS in acetone containing water re-
sulted in an incomplete reaction, in which the removal of the
benzyliden acetal group occurred. The hemiacetal 12 was con-
verted to the glycosyl trichloroimidate 13 in 96% yield. The imidate
13 was sufficiently stable that it could be purified by column chro-
matography on silica gel. Chemo- and regio-selective glycosylation
of thioglycoside 10a with the glycosyl imidate 13 was examined.
Treatment of the glycosyl imidate 13 and a 1.1 equiv of acceptor
2. Results and discusstion
We originally planned to synthesize the linear b(1,3) hexa-
deca-, dodeca- and octasaccharides 2, 4, and 6 and branched hep-
tadeca-, trideca- and nonasaccharides 1, 3, and 5 bearing an
alkylamino group at the reducing end (Scheme 1). The presence
of an amino group enhances the solubility of the compounds
and serves as a connecting group for a wide variety of functional-
ized molecules. The synthesis of b(1,3) glucan oligosaccharides 2,
4, and 6 involved the regio- and stereoselecitve glycosylation of
the 4,6-benzyliden-protected 2,3 diol acceptors 7 at the 3 position
with the linear tetrasaccharide donor 8. Although 4,6-benzyliden-
protected 2,3 diol S-glycosides are known to undergo regiosele-
citve glycosylation at the 3 position, the corresponding methyl
and allyl O-glycoside provided a significant amount of the unde-
sired O2 glyosylated product.12 In our experience, the steric hin-
drance of the sugar moiety at the anomeric position can prevent
glycosylation at the 2 position.13 The resulting coupling product
can be converted into the 2,3 diol acceptor by protecting the
remaining hydroxyl group followed by the removal of the levuli-
nyl (Lev) protecting group for further glycosylation. Repetition of
the coupling of the diol with the linear tetrasaccharide 8 and
deprotection of the Lev group provided the linear oligosaccharides
2 and 4. The use of the branched pentasaccharide 9 as a glycosyl
donor enabled the preparation of the branched oligosaccharides 1,
3 and 5. Because of the large difference in molecular weights, the
a
Ph
O
Ph
O
O
O
O
O
SR
X
HO
LevO
OH
OLev
10a : R = Et
10b : R = Ph
11 : X = β-SEt
12 : X = OH
b
c
13 : X = OC(=NH)CCl3
14 : X = α-Br
d
e or f
Ph
O
O
HO
O
O
O
O
SEt
Ph
O
O
O
Ph
Ph
O
O
O
SEt
O
O
OR
LevO
LevO
OLev
OLev
15 : R = H
17 : R = Bz
g
16
i
h
Ph
O
O
O
Ph
O
O
O
X
OBz
O
Ph
O
O
O
Ph
O
HO
O
SEt
O
O
LevO
OBz
OLev
OH
HO
O
HO
O
20
18 X = OH
19 X = OC(=NH)CCl3
j
HO
HO
HO
O
OH
HO
HO
O
m
O
NH2
O
HO
HO
5
O
OH
O
n
O
OH
HO
O
HO
HO
O
O
HO
HO
HO
k
OH
O
OH
O
OH
OH
Ph
O
O
O
Ph
O
O
OBz
O
O
SEt
Ph
O
O
O
O
Ph
O
5 : m = 1, n = 2
6 : m = 0, n = 2
O
OBz
1 : m = 1, n = 5
2 : m = 0, n = 5
3 : m = 1, n = 3
4 : m = 0, n = 3
O
O
OR
LevO
OLev
21 : R = H
8 : R = Ac
l
Ph
O
O
O
O
O
Ph
O
O
SEt
m
O
Ph
O
O
OBz
Ph
O
O
O
OAc
O
O
OBz
LevO
Ph
O
O
OLev
O
Ph
O
O
O
OBz
O
Ph
O
O
O
X
5
O
O
Ph
O
O
8
O
OAc
O
OBz
Ph
O
O
RO
O
OR
O
Ph
O
O
N3
Ph
O
O
O
O
O
5
Ph
HO
O
O
OBz
O
O
OAc
O
22 : R = Lev, X = OTs
23 : R = Lev, X = N3
7 : R = H, X = N3
OBz
n
o
OH
7
BnO
Scheme 2. Reagents and conditions: (a) levulinic acid, EDC-HCl, DMAP, CH2Cl2,
quant.; (b) benzenesulfinyl morpholine, Tf2O, CH2Cl2, ꢀ50 °C then NEt3, 96%,
O
BnO
O
BnO
Ph
O
O
OBz
BnO
O
O
a
:b = 61:39; (c) CCl3CN, Cs2CO3, CH2Cl2, 96%, a:b = 50:50; (d) IBr, MS-4A, CH2Cl2; (e)
O
SEt
Ph
O
O
O
O
cat. TMSOTf, CH2Cl2, MS4A, ꢀ40 °C, 69% for 15, 6% for 16; (f) AgOTf, MS-4A, CH2Cl2,
Ph
O
O
LevO
O
OBz
OBz
O
28%; (g) BzCl, cat. DMAP, Py., 120 °C, 4 h, 96%; (h) benzenesulfinyl morpholine, Tf2O,
OBz
OLev
CH2Cl2, ꢀ50 °C then NEt3, 86%,
CCl3CN, Cs2CO3, CH2Cl2, 96%,
a:b = 66.34; (i) NH2NH2ꢁH2O, AcOH, THF, 99%; (j)
a
:b = 84:16; (k) cat. TMSOTf, CH2Cl2, MS4A, ꢀ50 °C,
9
90%; (l) Ac2O, cat. DMAP, Py., 96%; (m) HO(CH2)6OTs, NIS, cat. TfOH, MS4A, ꢀ35 °C,
Scheme 1. Strategy for the synthesis of b-glucan oligosaccahrides 1–6.
31%; (n) NaN3, DMF; (o) NH2NH2ꢁH2O, AcOH, THF, 73%.

3900
H. Tanaka et al. / Bioorg. Med. Chem. 20 (2012) 3898–3914
RO
BnO
significant reduction in the coupling yield. The use of phenylthio-
b
AZMBO
O
O
SEt
BnO
glycoside 10b as a diol acceptor instead of ethylthioglycoside 10a
reduced the yield of the coupling product due to the reduced solu-
bility of the acceptor 10b to the reaction solvent. In addition, gly-
cosyl bromide 14 did not worked well in the chemoselective
glycosylation. Treatment of the remaining alcohol 15 with benzoyl
chloride in the presence of a catalytic amount of DMAP in pyridine
at 120 °C for 4 h provided the fully protected thioglycoside 17 in
84% yield. Removal of the Lev groups by treatment with NH2NH2
provided the disaccharide acceptor 20 in 99% yield. Conversion of
the thioglycoside 17 to the glycosyl imidate 19 via the hemiacatal
18 was achieved by hydrolysis of thioglycoside (96%) and acylation
of the resulting hydroxyl group with trichloroacetonitrile (94%).
Glycosylation of the disaccharide acceptor 20 with the disaccha-
ride donor 19 with a catalytic amount of TMSOTf provided the tet-
rasaccharide 21 in 90% yield. The remaining hydroxyl group was
acylated by treatment with acetic anhydride. Glycosylation of the
1,6-hexanediol monotosylate with the tetrasaccharide donor 8
provided tosylate 22 in 31% yield. Treatment of tosylate 22 with so-
dium azide, followed by hydrolysis of the Lev esters afforded the
diol acceptor 7 in 73% yield based on 22.
Scheme 3 shows the procedure used for the synthesis of the
branched pentasaccharide 9. Acylation of the primary alcohol with
2-(azidomethyl)benzoyl chloride provided 2-(azidomethyl)benzo-
ate (AZMB) ester 25 in 92% yield. Oxidative hydrolysis of the thio-
glycoside 25 to the hemiacetal 26 (76%), followed by reaction with
trichloroacetonitrile provided the glycosyl imidate 27 in 93% yield.
The chemo- and regio-selecitve glycosylation of diol 10a with the
glycosyl imidate 27 provided the disaccharide 28 in 94% yield.
The remaining hydroxyl group at the C2 position was protected
by reaction with benzoyl chloride to afford the protected disaccha-
ride 29. Removal of the TBS group at the 3 position of 29, provided
the disaccharide acceptor 30 in 84% yield based on 28.
Glycosylation of the C3 hydroxyl group of 30 with the glycosyl
imdiate 19 provided the linear tetrasaccharide 31 in 59% yield.
Treatment of the tetrasaccharide 31 with tetra-n-butylphosphine
at room temperature resulted in the removal of the AZMB ester
to provide the primary alcohol 32 in 93% yield. Glycosylation of
the resulting primary alcohol was achieved by treatment with
the super-armed glycosyl imitate15 33 in the presence of a catalytic
amount of TMSOTf at 0 °C for 3 h provided the pentasaccharide 9 in
91% yield.
TBSO
TBSO
BzO
BzO
X
26 : X = OH
27 : X = O(C=NH)CCl3
24 : R = H
25 : R =
N3
c
a
O
d
Ph
O
AZMBO
Ph
O
O
O
O
O
O
SEt
OR1
O
SEt
BnO
R2O
HO
OH
BzO
28 : R1 = H, R2 = TBS
29 : R1 = Bz, R2 = TBS
30 : R1 = Bz, R2 = H
10a
e
f
Ph
O
O
O
O
Ph
O
O
CCl3
O
O
OBz
g
LevO
NH
OLev
19
Ph
O
O
RO
BnO
O
O
SEt
OBz
O
Ph
O
O
O
O
Ph
O
O
OBz
O
O
OBz
LevO
OLev
31 : R = AZMB
32 : R = H
h
BnO
BnO
O
O
CCl3
NH
i
BnO
OBz
33
BnO
O
O
BnO
BnO
Ph
O
O
BzO
O
O
SEt
OBz
BnO
Ph
O
O
O
O
O
Ph
O
O
OBz
O
O
OBz
LevO
OLev
9
Scheme 3. Reagents and conditions: (a) 2-(azidomethyl)benzoyl chloride, DMAP,
CH2Cl2, rt, 92%; (b) NBS, acetone, H2O, rt, 10 min, then NEt3, rt, 1 h, 76%; (c) CCl3CN,
Cs2CO3, CH2Cl2, rt, 1 h. 93%; (d) 10a, cat. TMSOTf, CH2Cl2, MS4A, ꢀ15 °C, 94%; (e)
BzCl, cat. DMAP, Py., 120 °C, 7 h; (f) HFꢁPy, Py, 100 °C, 12 h, 84% (g) cat. TMSOTf,
CH2Cl2, MS4A, rt, 2 h, 59%; (h) nBu3P, THF, H2O, rt, 30 min, 93%. (i) cat. TMSOTf,
CH2Cl2, MS4A, 0 °C, 3 h, 91%.
10a in the presence of a catalytic amount of TMSOTf provided the
C3 glycosylated product 15 in 69% yield along with the C2
glycosylated product 16 in 6% yield.12 This method permitted us
to use 39.7 g of donor for the glycosylation of diol 10a without a
Ph
O
O
Ph
O
O
O
O
OBz
O
Ph
O
O
Ph
O
O
O
O
a
Ph
O
O
O
O
O
N
5
3
O
Ph
O
O
O
N
5
3
O
Ph
O
O
Ph
O
O
O
OBz
O
O
OAc
O
O
O
OAc
O
Ph
O
O
OBz
OBz
Ph
O
O
HO
OH
OR1
Ph
O
O
O
O
O
Ph
O
O
OBz
O
O
OAc
O
7
OBz
34 : R1 = H, R2 = Lev
35 : R1 = Ac, R2 = H
R2O
OR2
b
Ph
O
O
O
Ph
O
O
O
O
Ph
O
O
O
N
5
3
O
a
Ph
O
O
O
OBz
O
O
OAc
O
Ph
O
O
O
O
OBz
Ph
O
O
OAc
Ph
O
O
O
O
Ph
O
O
O
OBz
O
OBz
O
O
OAc
O
Ph
O
O
Ph
O
O
O
OR1
Ph
O
O
O
O
O
36 : R1 = H, R2 = Lev
37 : R1 = Ac, R2 = H
Ph
O
O
OBz
O
O
OAc
b
O
Ph
O
OBz
R2O
O
O
OBz
Ph
O
O
O
OR2
O
Ph
O
O
N
5
3
O
Ph
O
O
O
O
O
a
Ph
O
O
O
OAc
O
O
Ph
O
O
O
O
OBz
Ph
O
O
O
O
OAc
Ph
O
O
OBz
O
O
OAc
O
Ph
O
O
OBz
Ph
O
O
O
O
O
OAc
Ph
O
O
O
Ph
O
O
O
OBz
O
O
OAc
Ph
O
O
Ph
O
OBz
O
O
O
OH
Ph
O
O
O
O
O
Ph
O
O
O
OBz
O
38
OAc
O
OBz
LevO
OLev
Scheme 4. Reagents and conditions: (a) 8, NIS, cat. TfOH, MS4A, ꢀ35 °C, 97% for 34, 88% for 36, 82% for 38; (b) (i) Ac2O, cat. DMAP, Py.; (ii) NH2NH2ꢁH2O, AcOH, THF, 93% for
35, 92% for 37.

H. Tanaka et al. / Bioorg. Med. Chem. 20 (2012) 3898–3914
3901
Ph
O
O
O
BnO
Ph
O
O
O
O
O
O
Ph
O
O
O
N
5
3
BnO
O
O
Ph
O
O
O
OBz
a
O
BnO
O
OAc
BzO
BnO
Ph
O
O
OBz
Ph
O
O
O
O
O
O
OH
Ph
O
O
O
O
O
Ph
O
O
OLev
O
O
N
5
O
3
Ph
OBz
O
O
Ph
O
O
O
OBz
O
OBz
OAc
Ph
O
O
O
O
OAc
n
O
LevO
OBz
HO
39
OH
7 : n = 1
Ph
O
O
O
O
35 : n = 3
37 : n = 5
Ph
O
O
O
O
O
a
Ph
O
O
O
N
5
3
Ph
O
O
O
OBz
O
O
OAc
Ph
O
O
O
O
OBz
Ph
O
O
O
O
BnO
BnO
BnO
OAc
Ph
O
O
O
O
O
O
Ph
O
O
O
OBz
O
O
OAc
O
Ph
O
O
BzO
OBz
O
O
OH
a
Ph
O
OR2
O
O
BnO
O
Ph
O
O
OBz
O
40
O
OAc
R2O
OBz
Ph
O
O
O
Ph
O
O
O
O
O
Ph
O
O
N
5
3
Ph
O
O
O
OBz
O
O
O
OAc
O
Ph
O
OBz
Ph
O
O
O
O
OAc
Ph
O
O
O
O
Ph
O
O
O
OBz
O
O
O
OAc
O
Ph
O
BnO
BnO
BnO
O
O
O
OBz
Ph
O
O
O
O
OAc
Ph
O
O
O
O
O
O
Ph
O
O
OBz
O
BzO
BnO
O
OAc
Ph
O
O
OBz
O
O
O
OH
Ph
O
O
O
O
Ph
O
OBz
O
41
O
OAc
O
O
OBz
LevO
OLev
Scheme 5. Reagents and conditions: (a) 9, NIS, cat. TfOH, MS4A, ꢀ35 °C, 89% for 39, 79% for 40, 82% for 41.
Ph
O
O
O
O
n
Ph
O
O
O
O
N3
Ph
O
O
O
O
5
Ph
O
O
O
O
O
BzO
R3O
O
OAc
Ph
O
O
R2O
BzO
O
O
OH
Ph
O
O
O
BzO
Ph
LevO
O
O
O
OR1
O
O
BzO
OLev
41 : n = 1, R1 = Ac, R2-R3 = CHPh
40 : n = 3, R1 = Ac, R2-R3 = CHPh
39 : n = 5, R1 = Ac, R2-R3 = CHPh
34 : n = 1, R1 = Bz, R2 = Bn, R3
36 : n = 3, R1 = Bz, R2 = Bn, R3
38 : n = 5, R1 = Bz, R2 = Bn, R3
=
=
=
BnO
BnO
O
BnO
BzO
HO
HO
HO
O
O
HO
HO
HO
O
HO
O
O
NH2
O
5
HO
OH
O
O
OH
HO
HO
m
HO
n
OH
O
O
HO
HO
O
OH
O
O
HO
O
OH
HO
O
O
O
HO
HO
HO
OH
O
OH
OH
HO
OH
4 : m = 0, n = 3
5 : m = 1, n = 2
6 : m = 0, n = 2
1 : m = 1, n = 5
2 : m = 0, n = 5
3 : m = 1, n = 3
Scheme 6. Strategy for the synthesis of b-glucan oligosaccahrides 1–6.
Table 1
Deprotection of the protected oligosaccharides 34, 36 and 38–41
Substrate
Products
Yield
39
38
40
36
41
34
1
2
3
4
5
6
51
80
81
90
Quant.
43
The synthesis of the linear b(1,3) hexadeca- dodeca- and octa-
saccharides 38, 36 and 34 from the tetrasaccharide donor 8 and
acceptor 7 is shown in Scheme 4. The glycosylation of the acceptor
7 with thioglycoside 8 provided the octasaccharide 34 in 97% yield
as a single isomer. The octasaccharide 34 was converted to diol 35
by acetylation of the remaining unprotected hydroxyl group, fol-
lowed by removal of the Lev groups. Glycosylation of the acceptor
35 with thioglycoside 8 provided the dodecasaccharide 36 in 88%
yield as a single isomer. The dodecasaccharide 33 was converted
Figure 1. Competitive effects of the oligosaccharides 1–6 on binding of Dectin-1 to
SPG.
to diol 37 using the same procedure as was used for 34 to 35. Gly-
cosylation of dodecasaccharide 37 with the linear tetrasaccahride 8

3902
H. Tanaka et al. / Bioorg. Med. Chem. 20 (2012) 3898–3914
6
4
2
the branched pentasaccharide 9 provided branched oligosaccha-
rides 39, 40 and 41 in 89%, 79% and 82% yields, respectively. The
regioselective glycosylation at the C3 position was confirmed by
2D NMR analysis of coupling products 34, 36, 38 and 39–41 indi-
cating that the remaining hydroxyl groups were located at the C2
position. Deprotection of the protected oligosaccharides 34, 36,
38 and 39–41 was achieved by exposure of the protected oligosac-
charides 34, 36, 38 and 39–41 to Birch reduction conditions, fol-
lowed by reaction with LiOMe at room temperature for 12 h to
provide the corresponding oligosaccharides 1–6 in good to moder-
ate yields (Scheme 6 and Table 1).
SPG
1
2
3
4
5
6
Competitive effects of the synthetic glucans 1–6 on binding to a
soluble form of Dectin-1 attached to a solid-supported Schizophy-
llan (SPG) were examined (Fig. 1). The branched heptadecasaccha-
ride 1 and the linear hexadecasaccharide 2 inhibited the binding of
the soluble form of Dectin-1 to the solid-supported SPG. The bind-
ing activity of 1 and 2 was only 10-fold weaker than that of natural
SPG. No significant differences in affinity between the branched
and linear oligosaccharides 1 and 2 were observed. The inhibitory
activities of the remaining oligosaccharides 3–6, which contained
less than tridecasaccharide units were dramatically reduced. These
results indicate that the affinity of the oligosaccharide probes to
the soluble Dectin-1 is clearly dependent on the length of the
b(1,3) glucose sequences of 1 and 2.
0
0.001
0.01
0.1
1
10
100
1000
SPG or oligosaccharides (μg/ml)
Figure 2. Activation of luciferase-assisted NF-
oligosaccharides 1–6.
jB in Dectin-1 transfectant by the
In another series of experiments, a luciferase-assisted NF-jB as-
say was conducted for oligosacchrides 1–6 ( Fig. 2). 293T cells,
transfected with a plasmid DNA mixture, encoding Dectin-1 and
signaling molecules including CARD9 and Bcl10, were stimulated
with oligosaccharides 1–6. Luciferase activity, expressed as the ra-
tio of NF-
(TK) promoter-assisted renillaluciferase activity, was measured
using a Dual-Luciferase Reporter Assay System. NF- B activation
jB-assisted firefly luciferase activity to thymdine kinase
j
was observed during the stimulation of the 293T cells with the
branched heptadecasacccharide 1 and the linear hexadecasacccha-
ride 2. On the other hand, the low-affinity ligands 3–6 failed to
stimulate NF-jB activation. However, the difference in the activi-
Figure 3. Activation of luciferase-assisted NF-jB in TLR4 transfectant by combi-
nation with the oligosaccharides 2, 4, 6 and LPS.
ties 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 addition, the linear oligosac-
provided the linear hexadecasaccharide 38 in 82% yield (Scheme
5). In addition, Glycosylation of the diol acceptors 7, 35 and 37 with
charides 2,4,6 failed to enhance LPS-mediated NF-
through TLR4 (Fig. 3). These results strongly support the view that
jB activation
Figure 4. Saturation transfer difference (STD)-NMR spectra of 2 with Dectin-1. (A) 1H NMR of 2 with Dectin-1, which was off-resonance spectrum irradiated at 40 ppm. (B)
STD-NMR spectrum subtracts the on-resonance spectrum irradiated at 7.5 ppm from the off-resonance spectrum. Experiments were performed at 5 °C in PBS (pH 7.4)
including 10% D2O, and the molar ratio of Dectin-1 (10 l
M) to 2 was 1:25. Spectra were obtained with 500 MHz spectrometer. ⁄: impurity.

H. Tanaka et al. / Bioorg. Med. Chem. 20 (2012) 3898–3914
3903
Figure 5. Binding epitope of 2 for Dectin-1. H-1 of inner glucose residue is calculated as 100%, and relative signal intensities were determined based on the STD amplification
factors (STD-AF). ⁄1: Averaged value of all 4-positon signals including the sugar units at reducing and non-reducing ends. ⁄2: The average value of all 5-positon signals
including sugar units at reducing and non-reducing ends, and 3-positon of the non-reducing end sugar unit.⁄3: Averaged value of all 6-positon signals including sugar units at
reducing and non-reducing ends, and one proton at /-position of alkyl-chain.
the stimulation of innate immunity by a b-glucan is likely initiated
by the recognition of Dectin-1.
of a b(1,3) linked glycosides. The use of a combination of a linear
tetrasaccharide and a branched pentasaccharide as glycosyl donors
permitted b(1,3) linear octa- to hexadecasaccharides and branched
nona- to heptadecasaccharides to be prepared in good total yields.
Measurements of the competitive effects of oligosaccharides on the
binding of a soluble form of Dectin-1 to a solid-supported Schizo-
phyllan (SPG) revealed that the branched heptadecasaccharide 1
and the linear hexadecasaccharides 2 also have binding activity
for Dectin-1. The binding affinity of the trideca- and dodecasaccha-
rides were dramatically reduced compared to 1 and 2. In addition,
oligosaccharides 1 and 2, containing a b(1,3) hexadecasaccharide
backbone, exhibited agonist activity in a luciferase-assisted NF-
The binding between the extracellular C-type lectin-like do-
main (CTLD) of Dectin-1 and the linear hexadecasaccharide 2
was analyzed in detail by NMR spectroscopy. CTLD of Dectin-1
was prepared by means of an E. coli expression system as an insol-
uble fraction, and the refolded protein was purified prior to use
(see Experimental procedure). Titration with the linear hexadeca-
saccharide 2 into the protein solution was initially performed from
0 to 25 M equiv. The results showed that the 1H NMR signals orig-
inating from Dectin-1 were slightly perturbed and dependent on
the molar ratio (data not shown). Based on the findings from the
titration experiments as well as ELISA assays, we further analyzed
the binding event using a saturation transfer difference (STD)-NMR
jB assay. An STD-NMR analysis of complexes of Dectin-1 and the
linear hexadecasacccharides was also examined and the results
clearly show that Dectin-1 specifically recognizes the sugar part
of the oligosaccharides and not the aminoalkyl chain. Further stud-
ies of the nature of the interactions between b(1,3)-glucans and
Dectin-1 are ongoing.
technique (Fig. 4).16 The CTLD of Dectin-1 (10
lM) with the linear
hexadecasaccharide 2 (25 equiv) in PBS with 10% D2O was used in
the STD-NMR experiment. The saturation pulse was irradiated to
the protein signal at 7.5 ppm, which consists of aromatic and
amide protons. STD spectrum (Fig. 4B) was obtained by subtraction
from a reference spectrum irradiated at 40 ppm. The spectrum
clearly indicated the binding epitope of Dectin-1 (Fig. 5). The pro-
tons contained by the inner residues of the glucose-chain showed
higher signal intensities, whereas the 6-aminohexyl-chain at the
reducing termini did not receive a significant saturation transfer
effect from Dectin-1. Further, the H-1 and H-3 signals of the inner
glucose residues provided especially higher STD-amplification fac-
tors (100% and 87%) than the H-2, H-4 and H-6 signals (72–75%).
On the other hand, glucose residues at both reducing and non-
reducing termini appeared to include a small contribution to the
binding, based on their STD results originating from H-1 and H-2
(11–15%). The above spectral evidence suggests that Dectin-1 spe-
4. Experimental section
4.1. Chemistry
4.1.1. Ethylthio 4,6-O-benzylidene-2,3-di-O-levulinyl-b-D-
glucopyranoside (11)
To a stirred solution of ethylthio 4,6-O-benzylidene-b-D-gluco-
pyranoside (10a) (9.50 g, 30.4 mmol) in CH2Cl2 (60.8 mL) was
added EDCI-HCl (12.8 g, 66.9 mmol,), levulinic acid (6.85 mL,
66.9 mmol) and a catalytic amount of DMAP (371 mg, 3.04 mmol)
at 0 °C. After being stirred at room temperature for 3 h, the reaction
mixture was poured into 1 M HCl. The aqueous layer was extracted
with two portions of ethyl acetate. The combined extract was
washed with saturated aq NaHCO3 and brine, and dried over
MgSO4, filtered and concentrated in vacuo. The residue was puri-
fied by chromatography on silica gel with 80:20 toluene:ethyl ace-
cifically recognizes H-1 and H-3 at the a-face of the inner glucose
residues. Daniellou’s group recently reported comparable results
using a laminarine polysaccharide as a probe. They also reported
that significant STD effects were not observed when laminarin-
hexaose was used as a probe.17 An earlier mutagenesis study indi-
cated that Trp221 and His223 of Dectin-1 are key amino acids for
b(1,3)-glucan binding.18 The indole moiety of Trp221 may interact
tate to give diester 11 (15.4 g, 30.3 mmol, quant.). ½a D24
ꢀ74.7
ꢂ
(c = 1.20, CHCl3); 1H NMR (400 MHz, CDCl3) d 7.42–7.45 (m, 2H),
7.33–7.36 (m, 3H), 5.50 (s, 1H), 5.36 (dd, 1H, J = 9.2, 9.7 Hz), 5.06
(dd, 1H, J = 9.2, 9.7 Hz), 4.56 (d, 1H, H-1, J = 9.7 Hz), 4.36 (dd, 1H,
J = 4.8, 10.6 Hz), 3.77 (dd, 1H, J = 9.7, 10.6 Hz), 3.68 (dd, 1H,
J = 9.2, 9.7 Hz), 3.55 (ddd, 1H, J = 4.8, 9.2, 9.7 Hz), 2.52–2.87 (m,
10H), 2.17 (s, 3H), 2.13 (s, 3H), 1.26 (t, 3H, J = 7.2 Hz); 13C NMR
(100 MHz, CDCl3) d 206.3, 206.2, 171.8, 171.5, 136.8, 129.0,
128.2, 126.2, 101.4, 84.2, 78.4, 72.6, 70.8, 70.5, 68.5, 37.8 ꢃ 2,
29.7 ꢃ 2, 27.9, 24.3, 23.5, 14.9; IR (solid): 2971, 2873, 1746,
around the
a-face of the inner type stacking interactions, because
this type of glucose residues by CH-
p
binding has frequently been
identified in lectin–glycan interactions.
3. Conclusion
In conclusion, we describe an effective convergent synthesis of
b(1,3) oligosaccharides using linear tetra- and the branched penta-
saccharides. The 2,3 diol glycoside with a 4,6 benzylidene protect-
ing group acted as an effective glycosyl acceptor for the synthesis
1718, 1369, 1208, 1152, 1080, 1026, 918, 753, 701, 544 cmꢀ1
;
HRMS (ESI-TOF) calcd for C25H36NO9S [M+NH4]+ m/z = 526.2111,
found: 526.2112.

3904
H. Tanaka et al. / Bioorg. Med. Chem. 20 (2012) 3898–3914
4.1.2. 4,6-O-Benzylidene-2,3-di-O-levulinyl-
D
-glucopyranose
(m, 10H), 5.55 (s, 1H), 5.44 (s, 1H), 5.33 (dd, 1H, J = 9.7, 9.7 Hz),
(12)
5.10 (dd, 1H, J = 7.7, 9.7 Hz), 4.85 (d, 1H, J = 7.7 Hz), 4.47 (d, 1H,
J = 9.7 Hz), 4.36 (dd, 1H, J = 4.8, 10.6 Hz), 4.28 (d, 1H, J = 4.8 Hz),
4.20 (dd, 1H, J = 4.8, 10.6 Hz), 3.73–3.79 (m, 3H), 3.68 (dd, 1H,
J = 9.7, 9.7 Hz), 3.59–3.64 (m, 2H), 3.41–3.49 (m, 2H), 2.44–3.01
(m, 10H), 2.22 (s, 3H), 2.13 (s, 3H), 1.32 (t, 3H, J = 7.2 Hz); 13C
NMR (100 MHz, CDCl3) d 208.9, 206.3, 171.7 ꢃ 2, 137.4, 136.9,
129.1 ꢃ 2, 128.3, 128.2, 126.3, 126.1, 103.1, 101.5, 101.0, 87.0,
84.7, 79.2, 78.3, 72.8, 72.4, 71.9, 70.9, 68.7, 66.3, 37.8, 30.1, 29.8,
27.9, 27.7, 24.3, 15.1; IR (solid): 3488, 2868, 2457, 2272, 1719,
1387, 1147, 1106, 917, 693, 524 cmꢀ1; HRMS (ESI-TOF) calcd for
To a stirred solution of thioglycoside 11 (102 mg, 0.201 mmol)
in CH2Cl2 (2.01 mL) was added benzenesulfinyl morpholine
(46.7 mg, 0.221 mmol) and Tf2O (40.6
l
L, 0.241 mmol) at ꢀ40 °C.
After being stirred at the same temperature for 5 min, the reaction
mixture was poured into a mixture of saturated aq NaHCO3 and
10% aq Na2S2O3. The aqueous layer was extracted with two por-
tions of ethyl acetate. The combined extract was washed with a
mixture of saturated aq NaHCO3 and 10% aq Na2S2O3, and brine,
and dried over MgSO4, filtered and concentrated in vacuo. The res-
idue was used for the next reaction without further purification. To
a stirred solution of the residue in CH2Cl2 (2.00 mL) was added tri-
ethylamine (0.500 mL) at room temperature. After being stirred at
the same temperature for 1 h, the reaction mixture was concen-
trated in vacuo. The residue was purified by chromatography on
silica gel with 40:60 hexane:ethyl acetate to give hemiacetal 12
C
38H50NO14S [M+NH4]+ m/z = 776.2952, found: 776.2944. b(1,2)
isomer 16: ½a 2D6
ꢂ
ꢀ65.8 (c = 1.14, CHCl3); 1H NMR (400 MHz, CDCl3)
d 7.34–7.52 (m, 10H), 5.53 (s, 1H), 5.49 (s, 1H), 5.35 (dd, 1H, J = 9.2,
9.2 Hz), 5.04–5.11 (m, 2H), 4.52 (d, 1H, J = 9.7 Hz), 4.31–4.38 (m,
2H), 3.93 (ddd, 1H, J = 3.9, 8.7, 8.7 Hz), 3.81–3.87 (m, 2H),
3.71–3.76 (m, 2H), 3.65 (dd, 1H, J = 8.7, 9.7 Hz), 3.48–3.54 (m,
2H), 3.44 (ddd, 1H, J = 4.8, 9.7, 9.7 Hz), 2.46–2.84 (m, 10H), 2.19
(s, 3H), 2.13 (s, 3H), 1.28 (t, 3H, J = 7.2 Hz); 13C NMR (100 MHz,
CDCl3) d 208.0, 206.3, 171.8, 171.5, 137.2, 136.9, 129.2, 129.1,
128.3, 128.2, 126.4, 126.2, 101.8, 101.7, 101.5, 84.1, 80.4, 80.3,
78.2, 75.2, 72.7, 71.9, 70.3, 68.7, 68.5, 66.3, 37.8 ꢃ 2, 29.9, 29.8,
27.9, 24.4, 14.7; IR (solid): 3425, 2856, 2351, 1742, 1706, 1368,
(89.9 mg, 0.194 mmol, 2 steps 96%,
a:b = 61:39). The a:b ratio
was determined by 1H NMR (400 MHz, CDCl3) d 7.34–7.46 (m,
5H), 5.63 (dd, 0.61H, J = 9.2, 9.7 Hz), 5.50 (s, 1H), 5.40 (br-d,
0.61H, J = 2.4 Hz), 5.37 (dd, 0.39H, J = 9.7, 9.7 Hz), 4.89–4.93 (m,
1H), 4.82 (br-d, 0.39H, J = 8.2 Hz), 4.37 (dd, 0.39H, J = 4.8,
10.6 Hz), 4.30 (dd, 0.61H, J = 4.8, 10.1 Hz), 4.18 (ddd, 0.61H,
J = 4.8, 9.7, 10.1 Hz), 3.95 (br-s, 0.39H), 3.52–3.83 (m, 3H), 2.50–
2.84 (m, 8H), 2.14–2.19 (m, 6H); IR (solid): 3457, 2920, 2871,
1758, 1391, 974, 919, 769, 648, 509 cmꢀ1; HRMS (ESI-TOF) calcd
for C23H32NO10 [M+NH4]+ m/z = 482.2026, found: 482.2027.
1307, 1154, 1097, 971, 749, 698, 547 cmꢀ1
.
4.1.5. Ethylthio 2-O-benzoyl-4,6-O-benzylidene-3-O-(4,6-O-
benzylidene-2,3-di-O-D-glucopyranosyl)-b-levulinyl-b-D-
glucopyranoside (17)
4.1.3. O-(4,6-O-Benzylidene-2,3-di-O-levulinyl-
syl)trichloroacetimidate (13)
D
-glucopyrano-
To a stirred solution of 15 (1.02 g, 1.34 mmol) in pyridine
(6.70 mL) was added benzoyl chloride (0.471 mL, 4.02 mmol) and
a catalytic amount of DMAP (16.4 mg, 0.134 mmol) at room tem-
perature. After being stirred at 120 °C for 4 h, the reaction mixture
was poured into 1 M HCl. The aqueous layer was extracted with
two portions of ethyl acetate. The combined extract was washed
with 1 M HCl, saturated aq NaHCO3 and brine, and dried over
MgSO4, filtered and concentrated in vacuo. The residue was puri-
fied by chromatography on silica gel with 97:3 toluene:acetone
To a stirred solution of hemiacetal 12 (31.7 g, 68.3 mmol) in
CH2Cl2 (68.3 mL) was added trichloroacetonitrile (20.5 mL,
205 mmol) and a catalytic amount of Cs2CO3 (222 mg, 0.683 mmol)
at room temperature. After being stirred at the same temperature
for 1 h, the reaction mixture was filtered through a pad of CeliteÒ
and concentrated in vacuo. The residue was purified by chromatog-
raphy on silica gel with 60:40 hexane:ethyl acetate to give imidate
13 (39.7 g, 65.2 mmol, 96%,
a:b = 50:50). The
a
:b ratio was
to give benzoate 17 (1.11 g, 1.28 mmol, 96%).
½
a 2D4
ꢂ
ꢀ42.7
determined by 1H NMR analysis. 1H NMR (400 MHz, CDCl3) d
8.73 (s, 0.50H), 8.67 (s, 0.50H), 7.35–7.47 (m, 5H), 6.50 (d, 0.50H,
J = 3.9 Hz), 5.96 (d, 0.50H, J = 7.7 Hz), 5.69 (dd, 0.50H, J = 9.2,
9.7 Hz), 5.54 (s, 0.50H), 5.52 (s, 0.50H), 5.42 (dd, 0.50H, J = 9.2,
9.7 Hz), 5.32 (dd, 0.50H, J = 7.7, 9.2 Hz), 5.19 (dd, 0.50H, J = 3.9,
9.7 Hz), 4.43 (dd, 0.50H, J = 4.8, 10.1 Hz), 4.35 (dd, 0.50H, J = 4.8,
10.1 Hz), 4.12 (ddd, 0.50H, J = 4.8, 9.7, 10.1 Hz), 3.70–3.85 (m,
2.5H), 2.49–2.84 (m, 8H), 2.14–2.16 (m, 6H); IR (solid): 3316,
2920, 1751, 1718, 1677, 1364, 1146, 921, 797, 648 cmꢀ1; HRMS
(ESI-TOF) calcd for C25H32N2O10 [M+NH4]+ m/z = 625.1123, found:
625.1124.
(c = 1.05, CHCl3); 1H NMR (400 MHz, CDCl3)
d 8.04 (d, 2H,
J = 7.7 Hz), 7.32–7.65 (m, 13H), 5.57 (s, 1H), 5.38 (s, 1H), 5.31 (dd,
1H, J = 9.2, 9.7 Hz), 5.08 (dd, 1H, J = 8.7, 9.2 Hz), 5.01 (dd, 1H,
J = 7.7, 8.7 Hz), 4.70 (d, 1H, J = 7.7 Hz), 4.60 (d, 1H, J = 9.7 Hz),
4.38 (dd, 1H, J = 4.8, 10.6 Hz), 4.24 (dd, 1H, J = 4.8, 10.1 Hz), 4.18
(dd, 1H, J = 8.7, 9.2 Hz), 3.81 (dd, 1H, J = 10.1, 10.6 Hz), 3.75 (dd,
1H, J = 8.7, 9.7 Hz), 3.68 (dd, 1H, J = 10.1, 10.1 Hz), 3.63 (dd, 1H,
J = 9.2, 9.7 Hz), 3.56 (ddd, 1H, J = 4,8, 9.7, 10.1 Hz), 3.37 (ddd, 1H,
J = 4.8, 9.7, 10.1 Hz), 2.33–2.76 (m, 10H), 2.08 (s, 6H), 1.20 (t, 3H,
J = 7.2 Hz); 13C NMR (100 MHz, CDCl3) d 206.4 ꢃ 2, 171.8, 171.5,
165.1, 137.3, 136.9, 133.6, 129.9, 129.6, 129.2, 129.1, 128.8,
128.3, 128.2, 126.2, 126.1, 101.4, 101.2, 101.1, 84.4, 79.6, 78.9,
78.4, 72.2, 71.9, 71.8, 71.2, 68.6, 66.3, 37.9, 37.8, 29.7 ꢃ 2, 28.0,
27.5, 24.1, 14.8; IR (solid): 2972, 1753, 1717, 1401, 1364, 1149,
4.1.4. Ethylthio 4,6-O-benzylidene-3-O-(4,6-O-benzylidene-2,3-
di-O-levulinyl-b-D-glucopyranosyl)-b-D-glucopyranoside (15)
A mixture of thioglycoside 10 (6.99 g, 22.4 mmol), imidate 13
(12.4 g, 20.4 mmol) (azeotroped twice with dry toluene) and pul-
verized activated MS-4A (20.0 g) in dry CH2Cl2 (407 mL) was stir-
red at room temperature for 30 min under argon to remove a
trace amount of water. Then the reaction mixture was cooled to
ꢀ40 °C. A catalytic amount of trimethylsilyl trifluoromethanesulfo-
nate (0.369 mL, 2.04 mmol) was added to the reaction mixture.
After being stirred at the same temperature for 30 min, the reac-
tion mixture was neutralized with triethylamine, filtered through
a pad of CeliteÒ and concentrated in vacuo. The residue was puri-
fied by chromatography on silica gel with 48:52 hexane:ethyl ace-
tate to give the b(1,3) isomer 15 (10.6 g, 14.0 mmol, 69%) and the
1095, 1015, 917, 751, 698, 540 cmꢀ1
.
4.1.6. 2-O-Benzoyl-4,6-O-benzylidene-3-O-(4,6-O-benzylidene-
2,3-di-O-levulinyl-b- -glucopyranosyl)- -glucopyranose (18)
D
D
To a stirred solution of thioglycoside 17 (103 mg, 0.119 mmol)
in CH2Cl2 (1.19 mL) was added benzenesulfinyl morpholine
(27.8 mg, 0.131 mmol) and Tf2O (24.0
ll, 0.143 mmol) at ꢀ50 °C.
After being stirred at the same temperature for 5 min, the reaction
mixture was poured into a mixture of saturated aq NaHCO3 and
10% aq Na2S2O3. The aqueous layer was extracted with two por-
tions of ethyl acetate. The combined extract was washed with a
mixture of saturated aq NaHCO3 and 10% aq Na2S2O3, and brine,
and dried over MgSO4, filtered and concentrated in vacuo. The res-
idue was used for the next reaction without further purification. To
b(1,2) isomer 16 (0.994 g, 1.31 mmol, 6%). b(1,3) isomer 15: ½a D24
ꢂ
ꢀ54.0 (c = 1.03, CHCl3); 1H NMR (400 MHz, CDCl3) d 7.31–7.51

H. Tanaka et al. / Bioorg. Med. Chem. 20 (2012) 3898–3914
3905
a stirred solution of the residue in CH2Cl2 (2.00 mL) was added tri-
ethylamine (0.500 mL) at room temperature. After being stirred at
the same temperature for 1 h, the reaction mixture was concen-
trated in vacuo. The residue was purified by chromatography on
silica gel with 91:9 toluene:acetone to give hemiacatal 18
4.1.9. Ethylthio 2-O-benzoyl-4,6-O-benzylidene-3-O-(4,6-O-ben
zylidene-3-O-(2-O-benzoyl-4,6-O-benzylidene-3-O-(4,6-O-benzy
lidene-2,3-di-O–O-levulinyl-b-
D-glucopyranosyl)-b-D-glucopyra
nosyl)-b- -glucopyranosyl)-b-
D
D-glucopyranoside (21)
A mixture of diol 20 (46.8 mg, 0.0702 mmol), imidate 19
(56.1 mg, 0.0600 mmol) (azeotroped twice with dry toluene) and
pulverized activated MS-4A (120 mg) in dry CH2Cl2 (2.40 mL)
was stirred at room temperature for 30 min under argon to remove
a trace amount of water. Then the reaction mixture was cooled to
ꢀ50 °C. A catalytic amount of trimethylsilyl trifluoromethanesulfo-
(84.2 mg, 0.103 mmol, 2 steps 86%,
a:b = 66:34). The a:b ratio
was determined by 1H NMR analysis. 1H NMR (400 MHz, CDCl3) d
8.04–8.07 (m, 2H), 7.31–7.64 (m, 13H), 5.55 (s, 0.66H), 5.53 (s,
0.34H), 5.46 (br-s, 0.66H), 5.37 (s, 0.66H), 5.35 (s, 0.34H), 5.06–
5.17 (m, 2H), 4.98–5.03 (m, 1H), 4.85 (d, 0.66H, J = 7.7 Hz), 4.78
(dd, 0.34H, J = 8.2, 8.7 Hz), 4.75 (d, 0.34H, J = 7.7 Hz), 4.44 (dd,
0.66H, J = 9.2, 9.7 Hz), 4.34 (dd, 0.34H, J = 4.8, 10.1 Hz), 4.11–4.30
(m, 3H), 3.89 (d, 0.66H, J = 2.4 Hz), 3.59–3.81 (m, 4H), 3.38–3.54
(m, 1.34H), 2.01–2.71 (m, 14H); IR (solid): 3493, 2921, 2862,
nate (1.09 lL, 6.00 lmol) was added to the reaction mixture. After
being stirred at the same temperature for 30 min, the reaction mix-
ture was neutralized with triethylamine, filtered through a pad of
CeliteÒ and concentrated in vacuo. The residue was purified by
chromatography on silica gel with 94:6 toluene:acetone to give
2056, 1895, 1709, 1601, 1413, 1265, 975, 772, 672, 534 cmꢀ1
;
HRMS (ESI-TOF) calcd for C43H50NO16 [M+NH4]+ m/z = 836.3130,
tetrasaccharide 21 (79.2 mg, 0.0540 mmol, 90%).
½
a 2D4
ꢂ
ꢀ32.8
found: 836.3127.
(c = 1.11, CHCl3); 1H NMR (400 MHz, CDCl3) d 8.00–8.02 (m, 4H),
7.20–7.64 (m, 26H), 5.56 (s, 1H), 5.44 (s, 1H), 5.42 (s, 1H), 5.23–
5.29 (m, 2H,), 5.11 (dd, 1H, J = 4.3, 4.8 Hz), 5.06 (dd, 1H, J = 7.7,
9.2 Hz), 5.01 (d, 1H, J = 4.8 Hz), 4.88 (d, 1H, J = 7.7 Hz), 4.85 (s,
1H), 4.66 (d, 1H, J = 9.7 Hz), 4.39–4.41 (m, 2H), 4.23 (dd, 1H,
J = 4.8, 10.1 Hz), 4.18 (dd, 1H, J = 8.7, 9.2 Hz), 4.03–4.08 (m, 2H),
3.93 (dd, 1H, J = 4.3, 8.2 Hz), 3.39–3.86 (m, 13H), 3.25 (ddd, 1H,
J = 4.8, 9.2, 10.1 Hz), 3.07 (d, 1H, J = 3.4 Hz), 2.49–2.80 (m, 10H),
2.14 (s, 3H, Lev), 2.12 (s, 3H), 1.23 (t, 3H, J = 7.2 Hz); 13C NMR
(100 MHz, CDCl3) d 207.0, 206.4, 171.9, 171.3, 165.7, 165.2,
137.5, 137.3, 137.0, 136.7, 133.6, 133.4, 129.9, 129.8, 129.6,
129.4, 129.1, 128.9, 128.7, 128.5, 128.3 ꢃ 3, 128.0, 126.4, 126.2,
126.1, 126.0, 103.1, 101.9, 101.6, 101.5, 100.5 ꢃ 2, 99.2, 84.4,
79.1, 78.9, 78.4, 77.7, 77.2, 74.2, 73.9, 72.0, 70.8, 68.8, 68.6 ꢃ 2,
68.5, 66.9, 66.3, 65.5, 37.9 ꢃ 2, 29.8 ꢃ 3, 29.7, 28.1, 27.8, 24.3,
14.9; IR (solid): 3523, 2923, 2853, 2362, 1720, 1380, 1069, 917,
4.1.7. O-(2-O-Benzoyl-4,6-O-benzylidene-3-O-(4,6-O-benzylide
ne-2,3-di-O-levulinyl-b-
D-glucopyranosyl)-D-glucopyranosyl)
trichloroacetimidate (19)
To a stirred solution of hemiacatal 18 (4.34 mg, 5.30 mmol) in
CH2Cl2 (26.5 mL) was added trichloroacetonitrile (2.66 mL, 26.5
mmol) and a catalytic amount of Cs2CO3 (173 mg, 0.530 mmol) at
room temperature. After being stirred at the same temperature
for 1 h, the reaction mixture was filtered through a pad of CeliteÒ
and concentrated in vacuo. The residue was purified by chromatog-
raphy on silica gel with 94:6 toluene:acetone to give imidate 19
(4.86 g, 5.06 mmol, 96%,
a:b = 84:16). The a:b ratio was deter-
mined by 1H NMR analysis. 1H NMR (400 MHz, CDCl3) d 8.65 (s,
0.16H), 8.57 (s, 0.84H), 7.99–8.03 (m, 2H), 7.33–7.64 (m, 13H),
6.61 (d, 0.84H, J = 4.3 Hz), 6.05 (d, 0.16H, J = 7.2 Hz), 5.62 (s,
0.84H), 5.60 (s, 0.16H), 5.56 (dd, 0.16H, J = 7.2, 7.7 Hz), 5.37–5.40
(m, 1.84H), 5.13–5.18 (m, 1H), 5.02–5.08 (m, 1H), 4.84 (d, 0.84H,
J = 7.7 Hz), 4.77 (d, 0.16H, J = 7.7 Hz), 4.44–4.48 (m, 1H), 4.37 (dd,
0.84H, J = 4.8, 10.6 Hz), 4.33 (dd, 0.84H, J = 4.8, 10.6 Hz), 4.22–
4.26 (m, 0.32H), 4.11 (ddd, 0.84H, J = 4.8, 9.7, 10.1 Hz), 3.96 (dd,
0.16H, J = 9.2, 9.2 Hz), 3.63–3.88 (m, 4H), 3.51 (ddd, 0.84H, J = 4.8,
9.7, 9.7 Hz), 3.42 (ddd, 0.16H, J = 4.8, 9.7, 10.6 Hz), 2.06–2.75 (m,
734, 672, 507 cmꢀ1
[M+NH4]+ m/z = 1484.5159, found: 1484.5149.
; HRMS (ESI-TOF) calcd for C78H86NO26S
4.1.10. Ethylthio 2-O-benzoyl-4,6-O-benzylidene-3-O-(2-O-acet
yl-4,6-O-benzylidene-3-O-(2-O-benzoyl-4,6-O-benzylidene-3-O-
(4,6-O-benzylidene-2,3-di-O-levulinyl-b-
D-glucopyranosyl)-b-
D-
glucopyranosyl)-b- -glucopyranosyl)-b-
D
D-glucopyranoside (8)
14H); IR (solid): 2886, 1720, 1368, 1142, 959, 793, 664, 530 cmꢀ1
.
To a stirred solution of 21 (3.53 g, 2.40 mmol) in pyridine
(12.0 mL) was added acetic anhydride (2.25 mL, 24.0 mmol) and
a catalytic amount of DMAP (29.3 mg, 0.240 mmol) at room tem-
perature. After being stirred at the same temperature for 30 min,
the reaction mixture was poured into 1 M HCl. The aqueous layer
was extracted with two portions of ethyl acetate. The combined
extract was washed with 1 M HCl, saturated aq NaHCO3 and brine,
and dried over MgSO4, filtered and concentrated in vacuo. The res-
idue was purified by chromatography on silica gel with 95:5 tolu-
4.1.8. Ethylthio 2-O-benzoyl-4,6-O-benzylidene-3-O-(4,6-O-
benzylidene-b-D-glucopyranosyl)-b-D-glucopyranoside (20)
To a stirred solution of the levulinyl ester 17 (4.03 g, 4.67 mmol)
in THF (50.0 mL) was added acetic acid (5.00 mL) and hydrazine
monohydrate (2.00 mL) at 0 °C. After being stirred at the same
temperature for 20 min, the reaction mixture was poured into
1 M HCl. The aqueous layer was extracted with two portions of
ethyl acetate. The combined extract was washed with saturated
aq NaHCO3 and brine, and dried over MgSO4, filtered and concen-
trated in vacuo. The residue was purified by chromatography on
silica gel with 89:11 toluene:acetone to give diol 20 (3.09 g,
ene:acetone to give benzoate 8 (3.48 g, 2.30 mmol, 96%). ½a D24
ꢂ
ꢀ33.5 (c = 1.13, CHCl3); 1H NMR (400 MHz, CDCl3) d 8.04 (d, 2H,
J = 7.2 Hz), 7.98 (d, 2H, J = 7.2 Hz), 7.29–7.68 (m, 25H), 7.21 (t,
1H, J = 7.2 Hz), 5.53 (s, 1H), 5.38 (s, 1H), 5.17–5.21 (m, 2H), 5.11
(dd, 1H, J = 8.7, 9.2 Hz), 4.99 (dd, 1H, J = 7.7, 8.7 Hz), 4.93 (dd, 1H,
J = 9.2, 9.7 Hz), 4.90 (s, 1H), 4.88 (d, 1H, J = 6.8 Hz), 4.83 (dd, 1H,
J = 5.3, 5.8 Hz), 4.70 (d, 1H, J = 7.7 Hz), 4.64 (d, 1H, J = 5.3 Hz),
4.50 (d, 1H, J = 9.7 Hz), 4.34 (dd, 1H, J = 4.8, 10.6 Hz), 4.22 (dd,
1H, J = 4.8, 10.6 Hz), 4.09–4.18 (m, 3H), 4.05 (dd, 1H, J2,3 = 8.7,
9.2 Hz), 3.90 (dd, 1H, J = 9.2, 9.2 Hz), 3.84 (dd, 1H, J = 9.2, 9.2 Hz),
3.54–3.77 (m, 6H), 3.32–3.50 (m, 5H), 2.34–2.72 (m, 10H), 2.09
(s, 3H), 2.05 (s, 3H), 1.73 (s, 3H, Ac), 1.20 (t, 3H, J = 7.2 Hz); 13C
NMR (100 MHz, CDCl3) d 206.5, 206.4, 171.8, 171.4, 169.2, 164.9,
164.8, 137.4, 137.3, 137.2, 136.9, 133.8, 133.4, 129.9, 129.8,
129.7, 129.3, 129.1, 129.0, 128.9, 128.6, 128.3, 128.2 ꢃ 2,
126.3 ꢃ 2, 126.2, 126.1, 101.6, 101.4, 101.1, 100.9, 100.7, 99.0,
98.4, 84.4, 78.6, 78.4 ꢃ 2, 78.1, 76.7, 76.2, 73.9, 72.8, 72.5,
71.8 ꢃ 2, 71.2, 68.8, 68.6 ꢃ 2, 66.2, 66.1, 65.7, 37.9, 37.8, 29.7 ꢃ 2,
4.63 mmol, 99%). ½a D24
ꢂ
½
a 2D4
ꢂ
ꢀ49.6 (c = 1.05, CHCl3); 1H NMR
(400 MHz, CDCl3) d 8.05 (d, 2H, J = 7.7 Hz), 7.31–7.61 (m, 13H),
5.61 (s, 1H), 5.41 (s, 1H), 5.35 (dd, 1H, J = 9.2, 9.7 Hz), 4.72 (d, 1H,
J = 9.7 Hz), 4.46 (d, 1H, J = 7.7 Hz), 4.42 (dd, 1H, J = 4.8, 10.1 Hz),
4.20 (dd, 1H, J = 8.7, 9.2 Hz), 3.77–3.86 (m, 3H), 3.57–3.63 (m,
2H), 3.42–3.47 (m, 3H), 3.28 (ddd, 1H, J = 4.8, 9.7, 9.7 Hz), 2.92
(d, 1H, J = 2.4 Hz), 2.66–2.80 (m, 2H), 2.54 (d, 1H, J = 1.0 Hz), 1.25
(t, 3H, J = 7.2 Hz); 13C NMR (100 MHz, CDCl3) d 165.9, 137.0,
136.7, 133.6, 130.0, 129.7, 129.5, 129.3, 128.6, 128.4, 128.3,
126.4, 126.2, 103.1, 101.9, 101.7, 84.4, 80.3, 79.4, 79.1, 73.5, 72.8,
72.0, 71.0, 68.6, 68.4, 66.7, 24.2, 14.9; IR (solid): 3488, 2980,
2868, 1721, 1452, 1388, 1287, 1183, 1071, 971, 922, 751,
710, 572 cmꢀ1; HRMS (ESI-TOF) calcd for C35H42NO11S [M+NH4]+
m/z = 684.2479, found: 684.2487.

3906
H. Tanaka et al. / Bioorg. Med. Chem. 20 (2012) 3898–3914
28.0, 27.6, 24.3, 20.5, 14.8; IR (solid): 2983, 2884, 1753 1719, 1374,
1267, 1095, 1006, 746, 696, 515 cmꢀ1; HRMS (ESI-TOF) calcd for
C
J = 8.2 Hz), 7.25–7.62 (m, 26H), 5.51 (s, 1H), 5.42 (s, 1H), 5.27 (s,
1H), 5.23 (dd, 1H, J = 6.3, 6.3 Hz), 5.06 (dd, 1H, J = 8.2, 8.7 Hz),
5.02 (d, 1H, J = 6.3 Hz), 4.96 (s, 1H), 4.90 (dd, 1H, J = 5.3, 5.8 Hz),
4.69 (d, 1H, J = 5.3 Hz), 4.53 (d, 1H, J = 8.2 Hz), 4.50 (d, 1H,
J = 8.2 Hz), 4.32 (dd, 1H, Ja = 3.4, 9.7 Hz), 4.05–4.17 (m, 4H), 3.35–
3.92 (m, 17H), 3.31 (ddd, 1H, J = 4.8, 9.2, 9.7 Hz), 3.03–3.06 (m,
3H), 2.60 (br-s, 1H), 1.74 (s, 3H), 1.14–1.50 (m, 8H); 13C NMR
(100 MHz, CDCl3) d 208.3, 169.5, 165.5, 164.8, 137.3, 137.2,
137.1, 136.9, 133.7, 133.4, 130.0, 129.8, 129.7, 129.6, 129.3 ꢃ 2,
128.8, 128.7, 128.3 ꢃ 2, 126.4 ꢃ 2, 126.3, 126.1, 102.5, 101.9,
101.6, 101.4 ꢃ 2, 101.3, 99.1, 98.4, 80.3, 78.8, 78.7, 78.6, 78.5,
78.2, 76.1, 74.4, 73.7, 73.5, 72.9, 72.3, 69.9, 68.8, 68.7 ꢃ 2, 68.5,
66.7, 65.8, 65.6, 51.3, 29.3, 28.6, 26.3, 25.4, 20.5; IR (solid): 3625,
2974, 2884, 2831, 2089, 1760, 1719, 1380, 1275, 1220, 1095,
80H88NO27S [M+NH4]+ m/z = 1526.5264, found: 1526.5272.
4.1.11. 6-(((4-Methylphenyl)sulfonyl)oxy)hexyl 2-O-benzoyl-4,
6-O-benzylidene-3-O-(2-O-acetyl-4,6-O-benzylidene-3-O-(2-O-
benzoyl-4,6-O-benzylidene-3-O-(4,6-O-benzylidene-2,3-di-O-le
vulinyl-b-
glucopyranosyl)-b-
A mixture of 1,6-hexanediol mono-p-toluenesulfonate (21.7 mg,
79.5 mol), thioglycoside 8 (100 mg, 66.2 mol) (azeotroped twice
D-glucopyranosyl)-b-D-glucopyranosyl)-b-D-
D
-glucopyranoside (22)
l
l
with dry toluene) and pulverized activated MS-4A (265 mg) in dry
CH2Cl2 (2.65 mL) was stirred at room temperature for 30 min under
argon to remove a trace amount of water. Then the reaction mixture
was cooled to ꢀ35 °C. N-Iodosuccinimide (17.9 mg, 79.5
l
mol) and
catalytic amount oftrifluoromethanesulfonic acid (2.93 L,
mol) were added to the reaction mixture. After being stirred
765, 697, 515 cmꢀ1
.
a
l
33.1
l
4.1.13. 6-O-(2-(azidomethyl)benzoyl)-2-O-benzoyl-4-O-benzyl-
3-O-tert-butyldimethylsilyl-b- -glucopyranoside (25)
To a stirred solution of ethylthio 2-O-benzoyl-4-O-benzyl-3-O-
tert-butyldimethylsilyl-b- -glucopyranoside (24) (778 mg,
at the same temperature for 30 min, the reaction mixture was neu-
tralized with triethylamine and filtered through a pad of CeliteÒ.
The filtrate was poured into a mixture of saturated aq NaHCO3
and 10% aq Na2S2O3 with cooling. The aqueous layer was extracted
with two portions of ethyl acetate. The combined extract was
washed with a mixture of saturated aq NaHCO3 and 10% aq
Na2S2O3, and brine, and dried over MgSO4, filtered, and evaporated
in vacuo in vacuo. The residue was purified by chromatography on
silica gel with 92:8 toluene:acetone to give tosyalte 22 (35.2 mg,
D
D
1.46 mmol) in CH2Cl2 (7.26 mL) was added 2-(azidomethyl)ben-
zoyl chloride (947 mg, 4.84 mmol) and DMAP (1.48 g, 12.1 mmol)
at room temperature. After being stirred at the same temperature
for 30 min, the reaction mixture was poured into saturated aq
NaHCO3. The aqueous layer was extracted with two portions of
ethyl acetate. The combined extract was washed with saturated
aq NaHCO3 and brine, and dried over MgSO4, filtered and concen-
trated in vacuo. The residue was chromatographed on silica gel
with 90:10 hexane:ethyl acetate to give 2-(azidomethyl)benzoate
20.5
l
mol, 31%). ½a 2D4
ꢂ
ꢀ31.9 (c = 0.290, CHCl3); 1H NMR (400 MHz,
CDCl3) d 8.01 (d, 2H, J = 8.2 Hz), 7.97 (d, 2H, J = 7.7 Hz), 7.76 (d,
2H, J = 7.7 Hz), 7.24–7.64 (m, 28H), 5.51 (s, 1H), 5.38 (s, 1H), 5.09–
5.18 (m, 3H), 4.94–5.01 (m, 3H), 4.87 (d, 1H, J = 6.3 Hz), 4.84 (dd,
1H, J = 6.3, 6.3 Hz), 4.70 (d, 1H, J = 7.7 Hz), 4.62 (d, 1H, J = 6.3 Hz),
4.47 (d, 1H, J = 7.7 Hz), 4.32 (dd, 1H, J = 4.8, 10.6 Hz), 4.22 (dd, 1H,
J = 4.8, 10.6 Hz), 4.17 (dd, 1H, J = 4.8, 10.6 Hz), 4.01–4.14 (m, 3H),
3.57–3.87 (m, 11H), 3.32–3.47 (m, 6H), 2.38–2.76 (m, 11H), 2.09
(s, 3H), 2.05 (s, 3H), 1.70 (s, 3H), 1.07–1.56 (m, 8H); 13C NMR
(100 MHz, CDCl3) d 206.5, 206.4, 171.9, 171.5, 169.1, 164.8, 144.7,
137.4, 137.3, 133.7, 133.5, 133.4, 129.9, 129.8, 129.7, 129.6, 129.3,
129.2, 129.1, 128.9, 128.7, 128.3, 128.2 ꢃ 2, 127.9, 126.3, 126.2,
126.1, 101.7, 101.5, 101.4, 101.3, 100.8, 100.7, 99.4, 98.6, 78.7 ꢃ 2,
78.4 ꢃ 2, 77.7, 76.3, 76.1, 74.3, 73.9, 72.6, 71.8 ꢃ 2, 70.5, 69.8,
68.8, 68.7 ꢃ 2, 68.6, 66.7, 66.3, 66.1, 65.9, 38.0, 37.8, 29.8, 29.7,
29.2, 28.7, 28.0, 27.6, 25.3, 25.0, 21.7, 20.5; IR (solid): 2873, 2350,
ester 25 (927 mg, 1.34 mmol, 92%). ½a D20
ꢂ
= +24.6 (c = 0.855, CHCl3);
1H NMR (400 MHz, CDCl3) d 8.01–8.06 (m, 3H), 7.30–7.61 (m, 11H),
5.25 (dd, 1H, J = 9.2, 9.7 Hz), 4.89 (d, 1H, J = 11.1 Hz), 4.80 (s, 2H),
4.63 (dd, 1H, J = 2.4, 12.1 Hz), 4.58 (d, 1H, J = 11.1 Hz), 4.56 (d,
1H, J = 9.7 Hz), 4.35 (dd, 1H, J = 4.8, 12.1 Hz), 4.01 (dd, 1H, J = 8.7,
9.2 Hz), 3.74 (ddd, 1H, J = 2.4, 4.8, 9.2 Hz), 3.63 (dd, 1H, J = 8.7,
9.2 Hz), 2.60–2.78 (m, 2H), 1.18 (t, 3H, J = 7.2 Hz), 0.80 (s, 9H),
0.04 (s, 3H), -0.13 (s, 3H); 13C NMR (100 MHz, CDCl3) d 166.2,
165.6, 137.6, 137.5, 133.2, 133.0, 121.4, 130.3, 130.0, 129.8,
128.5, 128.4, 128.2, 128.0, 127.8, 83.7, 78.8, 77.0, 75.4, 72.9, 63.9,
53.1, 25.9, 25.7, 23.9, 17.9, 14.9, -3.9, -4.1; IR (solid): 2959, 2928,
2856, 2104, 1724, 1452, 1262, 1070, 838, 711, 519 cmꢀ1; HRMS
(ESI-TOF) calcd for
found: 709.3082.
C
36H49N4O7SiS [M+NH4]+ m/z = 709.3091,
2111, 1720, 1450, 1366, 1266, 1084, 916, 752, 634, 494 cmꢀ1
.
4.1.12. 6-Azidohexyl 2-O-benzoyl-4,6-O-benzylidene-3-O-(2-O-
acetyl-4,6-O-benzylidene-3-O-(2-O-benzoyl-4,6-O-benzylidene-
4.1.14. 6-O-(2-(Azidomethyl)benzoyl)-2-O-benzoyl-4-O-benzyl-
3-O-tert-butyldimethylsilyl- -glucopyranose (26)
To a stirred solution of ethylthio 6-O-(2-(azidomethyl)benzoyl)-
2-O-benzoyl-4-O-benzyl-3-O-tert-butyldimethylsilyl-b- -glucopy-
D
3-O-(4,6-O-benzylidene-b-
yl)-b- -glucopyranosyl)-b-
To a stirred solution of 22 (52.2 mg, 30.4
(1.52 mL) was added sodium azide (3.95 mg, 60.7
D
-glucopyranosyl)-b-
D-glucopyranos
D
D
-glucopyranoside (7)
D
l
mol) in DMF
mol) at room
ranoside (25) (682 mg, 0.986 mmol) in acetone (4.93 L) and water
(49.3 mL) was added N-bromosuccinimide (211 mg, 1.18 mmol) at
0 °C. After being stirred at the same temperature for 10 min, the
reaction mixture was added triethylamine (1.00 mL). After being
stirred at room temperature for 1 h, the reaction mixture was
poured into a mixture of saturated aq NaHCO3 and 10% aq Na2S2O3.
The aqueous layer was extracted with two portions of ethyl ace-
tate. The combined extract was washed with a mixture of satu-
rated aq NaHCO3 and 10% aq Na2S2O3, and brine, and dried over
MgSO4, filtered and concentrated in vacuo. The residue was chro-
matographed on silica gel with 85:15 hexane:ethyl acetate to give
l
temperature. After being stirred at the same temperature for 1 h,
the reaction mixture was poured into water. The aqueous layer
was extracted with two portions of ethyl acetate. The combined
extract was washed with water and brine, and dried over MgSO4,
filtered and concentrated in vacuo. The residue was used for the
next reaction without further purification. To a stirred solution of
the residue in THF (1.00 mL) was added acetic acid (0.300 mL)
and hydrazine monohydrate (0.100 mL) at 0 °C. After being stirred
at the same temperature for 20 min, the reaction mixture was
poured into 1 M HCl. The aqueous layer was extracted with two
portions of ethyl acetate. The combined extract was washed with
saturated aq NaHCO3 and brine, and dried over MgSO4, filtered
and concentrated in vacuo. The residue was purified by chromatog-
raphy on silica gel with 88:12 toluene:acetone to give diol 7
hemiacetal 26 (488 mg, 0.753 mmol, 76%, a:b = 77:23). The a:b ra-
tio was determined by 1H NMR analysis. 1H NMR (400 MHz, CDCl3)
d 8.00–8.10 (m, 3H), 7.22–7.66 (m, 11H), 5.41 (br-s, 0.77H), 5.14
(dd, 0.77H, J = 3.4, 9.2 Hz), 5.10 (dd, 0.23H, J = 8.7, 8.7 Hz), 4.88–
4.93 (m, 1H), 4.73–4.83 (m, 2.23H), 4.57–4.65 (m, 2H), 4.37–4.42
(m, 1.77H), 4.30 (ddd, 0.77H, J = 1.9, 9.7, 4.3 Hz), 4.06 (dd, 0.23H,
J = 8.7, 8.7 Hz), 3.77 (ddd, 0.23H, J = 2.4, 4.3, 9.7 Hz), 3.58–3.67
(31.0 mg, 22.2
l
mol, 2 steps 73%). ½a D24
ꢀ34.6 (c = 1.10, CHCl3);
ꢂ
1H NMR (400 MHz, CDCl3) d 8.03 (d, 2H, J = 8.2 Hz), 8.00 (d, 2H,

H. Tanaka et al. / Bioorg. Med. Chem. 20 (2012) 3898–3914
3907
(m, 1H), 3.34 (d, 0.23H, J = 9.7 Hz), 2.97 (br-s, 0.77H), 0.79 (s, 9H), -
0.05–0.08 (m, 6H); IR (solid): 3427, 2928, 2856, 2411, 2101, 1724,
1453, 1264, 1097, 838, 713, 520 cmꢀ1; HRMS (ESI-TOF) calcd for
was poured into 1 M HCl. The aqueous layer was extracted with
two portions of ethyl acetate. The combined extract was washed
with 1 M HCl, saturated aq NaHCO3 and brine, and dried over
MgSO4, filtered and concentrated in vacuo. The residue was used
for the next reaction without further purification. To a stirred solu-
tion of the residue in pyridine (16.2 mL) was added HF in pyridine
(1.62 mL) at room temperature. After being stirred at 100 °C for
12 h, the reaction mixture was poured into 1 M HCl. The aqueous
layer was extracted with two portions of ethyl acetate. The com-
bined extract was washed with 1 M HCl, saturated aq NaHCO3
and brine, and dried over MgSO4, filtered and concentrated in va-
cuo. The residue was purified by chromatography on silica gel with
78:22 hexane:ethyl acetate to give 30 (2.53 g, 2.72 mmol, 2 steps
C
34H45N4O8Si [M+NH4]+ m/z = 665.3007, found: 665.3002.
4.1.15. O-(6-O-(2-(Azidomethyl)benzoyl)-2-O-benzoyl-4-O-ben
zyl-3-O-tert-butyldimethylsilyl-D-glucopyranosyl)trichloroace
timidate (27)
To a stirred solution of 6-O-(2-(azidomethyl)benzoyl)-2-O-ben-
zoyl-4-O-benzyl-3-O-tert-butyldimethylsilyl- -glucopyranose (26)
D
(960 mg, 1.48 mmol) in CH2Cl2 (2.96 mL) was added trichloroace-
tonitrile (0.742 mL, 7.40 mmol) and a catalytic amount of Cs2CO3
(48.0 mg, 0.148 mmol) at room temperature. After being stirred
at the same temperature for 1 h, the reaction mixture was filtered
through a pad of Celite and concentrated in vacuo. The residue was
chromatographed on silica gel with 90:10 hexane:ethyl acetate to
84%). ½a 1D8
ꢂ
ꢀ8.44 (c = 1.07, CHCl3); 1H NMR (400 MHz, CDCl3) d
7.96 (d, 1H, J = 7.7 Hz), 7.83 (d, 2H, J = 8.2 Hz), 7.73 (d, 2H,
J = 8.2 Hz), 7.21–7.59 (m, 19H,), 5.55 (s, 1H), 5.30 (dd, 1H, J = 8.7,
9.7 Hz,), 5.01 (dd, 1H, J = 7.2, 8.2 Hz), 4.86 (d, 1H, J = 7.2 Hz),
4.65–4.78 (m, 3H), 4.50–4.60 (m, 3H), 4.32–4.37 (m, 2H), 4.27
(dd, 1H, J = 8.7, 9.2 Hz), 3.71–3.83 (m, 3H), 3.62 (dd, 1H, J = 8.7,
9.2 Hz), 3.49–3.55 (m, 2H), 2.61–2.68 (m, 2H), 2.57 (d, 1H,
J = 4.8 Hz), 1.16 (t, 3H, J = 7.2 Hz); 13C NMR (100 MHz, CDCl3)
d166.4, 164.8, 137.6, 137.2, 137.1, 133.2, 132.9, 131.6, 130.1,
130.0, 129.8, 129.5, 129.2 ꢃ 2, 128.7, 128.6 ꢃ 2, 128.5, 128.3 ꢃ 3,
128.1, 126.1, 101.6, 99.9, 84.3, 79.4, 78.7, 77.8, 76.5, 75.3, 74.9,
72.8, 72.2, 71.1, 68.7, 63.7, 53.1, 24.1, 14.8; IR (solid): 3499,
2967, 2875, 2105, 1732, 1602, 1452, 1273, 1096, 913, 712,
542 cmꢀ1; HRMS (ESI-TOF) calcd for C50H53N4O13S [M+NH4]+ m/
z = 949.3330, found: 949.3322.
give imidate 27 (1.09 g, 1.38 mmol, 93%, a:b = 59:41). The a:b ratio
was determined by 1H NMR analysis. 1H NMR (400 MHz, CDCl3) d
8.59 (s, 0.41H), 8.51 (s, 0.59H), 8.00–8.05 (m, 3H), 7.22–7.61 (m,
11H), 6.55 (d, 0.59H, J = 3.4 Hz), 5.97 (d, 0.41H, J = 7.2 Hz), 5.51
(dd, 0.41H, J = 7.2, 8.2 Hz), 5.40 (dd, 0.59H, J = 3.4, 9.7 Hz), 4.93
(d, 0.59H, J = 11.1 Hz), 4.89 (d, 0.41H, J = 11.6 Hz), 4.74–4.83 (m,
2H), 4.56–4.65 (m, 2H), 4.38–4.49 (m, 1.59H), 4.25 (ddd, 0.59H,
J = 1.9, 3.4, 10.1 Hz), 4.12 (dd, 0.41H, J = 8.2, 8.7 Hz), 3.96 (ddd,
0.41H, J = 2.4, 4.8, 9.2 Hz), 3.72–3.79 (m, 1H), 0.80–0.83 (m, 9H), -
0.08–0.11 (m, 6H); IR (solid): 2959, 2855, 2105, 1725, 1672,
1452, 1255, 1070, 712, 519 cmꢀ1
.
4.1.16. Ethylthio 3-O-(6-O-(2-(azidomethyl)benzoyl)-2-O-benzo
yl-4-O-benzyl-3-O-tert-butyldimethylsilyl-b- -glucopyranosyl)-
4,6-O-benzylidene-b- -glucopyranoside (28)
D
4.1.18. Ethylthio 3-O-(6-O-(2-(azidomethyl)benzoyl)-2-O-benzo
yl-3-O-(2-O-benzoyl-4,6-O-benzylidene-3-O-(4,6-O-benzylide
D
A mixture of 10a (5.64 g, 18.1 mmol), 27 (11.9 g, 15.1 mmol)
(azeotroped twice with dry toluene) and pulverized activated MS-
4A (15.1 g) in dry CH2Cl2 (151 mL) was stirred at room temperature
for 30 min under argon to remove a trace amount of water. Then the
reaction mixture was cooled to ꢀ15 °C. A catalytic amount of trim-
ethylsilyltrifluoromethanesulfonate (0.544 mL, 3.01 mmol) was
added to the reaction mixture. After being stirred at the same tem-
perature for 1 h, the reaction mixture was neutralized with trieth-
ylamine, filtered through a pad of CeliteÒ and concentrated in
vacuo. The residue was purified by chromatography on silica gel
with 90:10 hexane:ethyl acetate to give 28 (13.3 g, 14.2 mmol,
ne-2,3-di-O-levnulinyl-b-
syl)-4-O-benzyl-b- -glucopyranosyl)-2-O-benzoyl-4,6-O-benzy
lidene-b- -glucopyranoside (31)
A mixture of 30 (521 mg, 55.9 mol), 19 (592 mg, 61.5 lmol)
D-glucopyranosyl)-b-D-glucopyrano
D
D
l
(azeotroped twice with dry toluene) and pulverized activated
MS-4A (280 mg) in dry CH2Cl2 (2.80 mL) was stirred at room tem-
perature for 30 min under argon to remove a trace amount of
water. Then the reaction mixture was cooled to ꢀ50 °C. A catalytic
amount of trimethylsilyl trifluoromethanesulfonate (10.1 lL,
55.9 lmol) was added to the reaction mixture. After being stirred
at room temperature for 2 h, the reaction mixture was neutralized
with triethylamine, filtered through a pad of CeliteÒ and concen-
trated in vacuo. The residue was purified by chromatography on
silica gel with 85:15 toluene:ethyl acetate and further purified by
gel permeation chromatography (GPC) to give 31 (573 mg,
94%). ½a 1D9
ꢂ
= -3.94 (c = 1.05, CHCl3); 1H NMR (400 MHz, CDCl3) d
8.08 (d, 2H, J = 7.2 Hz), 7.97 (d, 1H, J = 7.7 Hz), 7.15–7.60 (m, 16H),
5.48 (s, 1H), 5.23 (dd, 1H, J = 7.7, 8.7 Hz), 4.93 (d, 1H, J = 7.7 Hz),
4.83 (d, 1H, J = 11.1 Hz), 4.74 (d, 1H, J = 14.5 Hz), 4.67 (d, 1H,
J = 14.5 Hz), 4.50 (d, 1H, J = 11.1 Hz), 4.46 (dd, 1H, J = 1.9, 11.6 Hz),
4.25–4.31 (m, 3H), 4.00 (dd, 1H, J = 8.7, 9.2 Hz), 3.79 (dd, 1H,
J = 8.7, 8.7 Hz), 3.54–3.72 (m, 4H), 3.31–3.42 (m, 2H), 2.48–2.64
(m, 2H), 2.42 (d, 1H, J = 1.9 Hz), 1.18 (t, 3H, J = 7.2 Hz), 0.79 (s,
9H), 0.03 (s, 3H), -0.11 (s, 3H); 13C NMR (100 MHz, CDCl3) d 166.3,
165.9, 137.5, 137.3, 137.1, 133.3, 132.8, 131.6, 130.2, 129.9, 129.7,
129.0, 128.6, 128.5 ꢃ 2, 128.2 ꢃ 2, 127.9, 127.8, 126.0, 101.4,
101.3, 86.2, 81.8, 79.2, 78.7, 75.4, 75.3, 72.9, 72.8, 71.0, 68.6, 63.7,
53.1, 25.7, 24.0, 17.9, 15.2, -4.0, -4.2; IR (solid): 3478, 2928, 2855,
0.330 mmol, 59%).
½
a 1D8
ꢂ
ꢀ17.0 (c = 1.19, CHCl3); 1H NMR
(400 MHz, CDCl3) d 7.99 (d, 1H, J = 7.7 Hz), 7.92 (d, 2H, J = 7.7 Hz),
7.81 (d, 2H, J = 7.7 Hz), 7.14–7.72 (m, 34H), 5.52 (s, 1H), 5.49 (s,
1H), 5.37 (s, 1H), 5.25 (dd, 1H, J = 8.2, 8.7 Hz), 4.93–5.06 (m, 4H),
4.69–4.81 (m, 4H), 4.63 (d, 1H, J = 10.6 Hz), 4.55 (d, 1H,
J = 7.7 Hz), 4.37–4.44 (m, 3H), 4.16–4.32 (m, 4H), 4.02 (dd, 1H,
J2,3 = 5.8, 7.2 Hz), 3.93–3.98 (m, 2H), 3.77 (dd, 1H, J = 7.2,
10.1 Hz), 3.62–3.70 (m, 5H), 3.58 (dd, 1H, J = 9.2, 9.7 Hz), 3.36–
3.44 (m, 2H), 3.25–3.32 (m, 2H), 2.14–2.71 (m, 10H), 2.08 (s, 3H),
2.03 (s, 3H), 1.14 (t, 3H, J = 7.2 Hz); 13C NMR (100 MHz, CDCl3) d
206.4, 206.3, 171.8, 171.4, 166.5, 165.0, 164.8, 164.5, 138.0,
137.3, 137.2, 137.1, 136.9, 133.8, 133.3, 133.2, 132.8, 131.7,
130.0, 129.9, 129.8, 129.6 ꢃ 2, 129.3, 129.2, 129.1, 129.0, 128.9,
128.4, 128.3, 128.2, 127.8, 126.5, 126.2, 126.0, 102.0, 101.3,
101.2, 101.0, 99.5, 98.0, 84.3, 79.9, 79.2, 78.8, 78.4 ꢃ 2, 76.0, 75.4,
74.3, 73.5, 73.3, 72.9, 72.0, 71.8, 71.6, 70.9, 68.7, 68.6, 66.6,
66.2, 64.6, 53.1, 37.9, 37.7, 29.8, 29.7, 27.9, 27.4, 24.4, 14.8; IR
(solid): 2881, 2101, 1716, 1367, 1261, 1096, 994, 879, 702,
2102, 1724, 1600, 1452, 1264, 1071, 985, 837, 700, 518 cmꢀ1
;
HRMS (ESI-TOF) calcd for C49H63N4O12S [M+NH4]+ m/z = 959.3932,
found: 959.3979.
4.1.17. Ethylthio 3-O-(6-O-(2-(azidomethyl)benzoyl)-2-O-benzo
yl-4-O-benzyl-b-
D-glucopyranosyl)-2-O-benzoyl-4,6-O-benzyli
dene-b- -glucopyranoside (30)
D
To a stirred solution of 28 (3.05 g, 3.23 mmol) in pyridine
(16.2 mL) was added benzoyl chloride (1.14 mL, 9.69 mmol) and
a catalytic amount of DMAP (39.5 mg, 0.323 mmol) at room tem-
perature. After being stirred at 120 °C for 7 h, the reaction mixture
;
517 cmꢀ1 HRMS (ESI-TOF) calcd for C93H97N4O28S [M+NH4]+
m/z = 1749.6010, found: 1749.6022.

3908
H. Tanaka et al. / Bioorg. Med. Chem. 20 (2012) 3898–3914
4.1.19. Ethylthio 2-O-benzoyl-3-O-(2-O-benzoyl-3-O-(2-O-benz
oyl-4,6-O-benzylidene-3-O-(4,6-O-benzylidene-2,3-di-O-levnuli
128.6 ꢃ 2, 128.4, 128.3 ꢃ 2, 128.2 ꢃ 3, 128.0 ꢃ 2, 127.9,
127.8 ꢃ 2, 127.7, 127.6 ꢃ 2, 126.6, 126.1, 126.0, 125.4, 101.6,
101.3, 101.0, 100.9, 100.8, 99.4, 98.2, 84.0, 82.7, 79.3 ꢃ 2, 79.0,
78.9, 78.4, 78.3, 78.1, 75.7, 75.1, 75.0, 74.8, 74.3, 74.0, 73.8, 73.6,
73.5, 73.4, 72.3, 71.7, 71.6, 71.0, 68.6, 68.5 ꢃ 2, 68.4, 66.4, 66.1,
37.9, 37.7, 29.7, 29.6, 27.9, 27.4, 24.1, 14.8; IR (solid): 2967,
nyl-b-
ucopyranosyl)-4,6-O-benzylidene-b-
To a stirred solution of 31 (251 mg, 0.145 mmol) in THF
(1.45
L, 0.725 mmol) and water (13.1 mL) was added nBu3P
D
-glucopyranosyl)-b-
D-glucopyranosyl)-4-O-benzyl-b-
D-gl
D
-glucopyranoside (32)
l
(0.109 mL, 0.435 mmol) at 0 °C. After being stirred at room temper-
ature for 30 min, the reaction mixture was poured into saturated
aq NaHCO3. The aqueous layer was extracted with two portions
of ethyl acetate. The combined extract was washed with saturated
aq NaHCO3 and brine, dried over MgSO4, filtered and concentrated
in vacuo. The residue was purified by chromatography on silica gel
with 91:9 toluene:acetone to give 32 (212 mg, 0.134 mmol, 93%).
2318, 1955, 1730, 1602, 1451, 1265, 1096, 772, 702, 527 cmꢀ1
;
HRMS (ESI-TOF) calcd for
2126.7776, found: 2126.7778.
C
119H124NO33
S
[M+NH4]+ m/z =
4.1.21. 6-Azidohexyl 2-O-benzoyl-4,6-O-benzylidene-3-O-(2-O-
acetyl-4,6-O-benzylidene-3-O-(2-O-benzoyl-4,6-O-benzylidene-
3-O-(4,6-O-benzylidene-3-O-(2-O-benzoyl-4,6-O-benzylidene-3-
O-(2-O-acetyl-4,6-O-benzylidene-3-O-(2-O-benzoyl-4,6-O-benzy
½
a 1D7
ꢂ
ꢀ13.1 (c = 1.04, CHCl3); 1H NMR (400 MHz, CDCl3) d 7.85 (d,
2H, J = 7.2 Hz), 7.73 (d, 2H, J = 7.2 Hz), 7.22–7.69 (m, 31H,), 5.47
(s, 2H), 5.37 (s, 1H), 5.21 (dd, 1H, J = 7.7, 8.7 Hz), 5.01 (dd, 1H,
J2,3 = 9.2, 9.7 Hz), 4.88–4.96 (m, 3H), 4.86 (d, 1H, J = 7.7 Hz), 4.67–
4.71 (m, 2H), 4.52 (d, 1H, J = 7.7 Hz), 4.46 (d, 1H, J = 10.1 Hz),
4.32 (dd, 1H, J = 4.8, 10.6 Hz), 4.24 (dd, 1H, J = 4.8, 10.6 Hz), 4.18
(dd, 1H, J = 4.8, 10.6 Hz), 4.12 (d, 1H, J = 10.6 Hz), 4.09 (dd, 1H,
J = 9.2, 9.2 Hz), 4.01 (dd, 1H, J = 6.8, 7.7 Hz), 3.88 (dd, 1H, J = 8.7,
9.2 Hz), 3.54–3.70 (m, 7H), 3.33–3.51 (m, 5H), 3.26 (ddd, 1H,
J = 4.8, 9.7, 9.7 Hz), 2.17–2.67 (m, 10H), 2.07 (s, 3H), 2.03 (s, 3H),
1.93 (br-s, 1H), 1.14 (t, 3H, J = 7.2 Hz); 13C NMR (100 MHz, CDCl3)
d 206.4, 206.3, 171.8, 171.7, 171.4, 165.0, 164.6, 164.2, 143.7,
138.2, 137.1, 136.8, 133.6, 133.2, 133.1, 132.1, 131.8, 129.9,
129.8, 129.7, 129.5, 129.4, 129.2 ꢃ 2, 129.0, 128.8, 128.4, 128.3,
128.2 ꢃ 3, 128.0, 127.8, 126.3, 126.1, 126.0, 123.8, 123.2, 102.0,
101.3, 101.0, 100.9, 99.7, 98.8, 84.4, 79.5, 79.0, 78.8, 78.4, 78.3,
75.6, 74.5, 74.2, 73.6, 73.4, 72.4, 71.6 ꢃ 2, 71.0, 68.6, 68.5, 66.5,
66.1, 63.7, 62.3, 45.6, 37.8, 37.6, 29.7, 29.6, 28.1, 27.9, 27.3, 27.2,
24.3, 24.1 ꢃ 2, 24.0 ꢃ 2, 18.9, 14.7, 13.7, 13.6; IR (solid): 3531,
lidene-3-O-(4,6-O-benzylidene-2,3-di-O-levnulinyl-b-
ranosyl)-b- -glucopyranosyl)-b- -glucopyranosyl)-b-
anosyl)-b- -glucopyranosyl)-b- -glucopyranosyl)-b-
nosyl)-b- -glucopyranoside (34)
D
-glucopy
-glucopyr
D-glucopyra
D
D
D
D
D
D
A mixture of 7 (631 mg, 0.453 mmol), 8 (752 mg, 0.498 mmol,
1.10 equiv) (azeotroped twice with dry toluene) and pulverized
activated MS-4A (906 mg) in dry CH2Cl2 (18.1 mL) was stirred at
room temperature for 30 min under argon to remove a trace
amount of water. Then the reaction mixture was cooled to ꢀ35 °C.
N-iodosuccinimide (135 mg, 0.598 mmol) and a catalytic amount
of trifluoromethanesulfonic acid (20.0 lL, 0.227 lmol) were added
to the reaction mixture. After being stirred at the same temperature
for 30 min, the reaction mixture was neutralized with triethylamine
and filtered through a pad of CeliteÒ. The filtrate was poured into a
mixture of saturated aq NaHCO3 and 10% aq Na2S2O3 with cooling.
The aqueous layer was extracted with two portions of ethyl acetate.
The combined extract was washed with a mixture of saturated aq
NaHCO3 and 10% aq Na2S2O3, and brine, and dried over MgSO4, fil-
tered, and evaporated in vacuo. The residue was purified by chro-
matography on silica gel with 92:8 toluene:acetone and further
purified by gel permeation chromatography (GPC) to give 34
2875, 1737, 1602, 1452, 1376, 1264, 984, 750, 703, 484 cmꢀ1
;
HRMS (ESI-TOF) calcd for C85H89O27S [M+H]+ m/z = 1573.5312,
found: 1573.5287.
(1.25 g, 0.441 mmol, 97%). ½a D24
ꢂ
ꢀ28.3 (c = 0.840, CHCl3); 1H NMR
4.1.20. Ethylthio 2-O-benzoyl-3-O-(2-O-benzoyl-6-O-(2-O-benzo
(400 MHz, CDCl3) d 7.98–8.03 (m, 8H), 7.18–7.63 (m, 52H), 5.50
(s, 1H), 5.47 (s, 1H), 5.39 (s, 1H), 4.85–5.21 (m, 16H), 4.71–4.74
(m, 2H), 4.67 (d, 1H, J = 6.3 Hz), 4.52 (d, 1H, J = 7.7 Hz), 4.32–4.37
(m, 2H), 3.22–4.24 (m, 41H), 3.05 (t, 2H, J = 7.2 Hz), 2.35–2.74 (m,
9H), 2.09 (s, 3H), 2.01 (s, 3H), 1.80 (s, 3H), 1.77 (s, 3H), 1.10–1.50
(m, 8H); 13C NMR (100 MHz, CDCl3) d 206.5 ꢃ 2, 171.9, 171.4,
169.6, 169.0, 165.5, 165.2, 164.8 ꢃ 3, 137.4 ꢃ 3, 137.3 ꢃ 2, 137.1,
137.0, 136.9, 133.9, 133.8, 133.6, 133.5, 130.0, 129.9, 129.8 ꢃ 2,
129.7 ꢃ 2, 129.6, 129.5, 129.4, 129.3, 129.2 ꢃ 2, 129.1, 129.0,
128.9, 128.7 ꢃ 2, 128.3 ꢃ 2, 128.2 ꢃ 2, 126.4, 126.3 ꢃ 2, 126.2,
126.1, 126.0, 102.9, 101.7, 101.6 ꢃ 2, 101.5, 101.4, 101.3, 101.1,
101.0, 100.8, 100.6, 99.8, 99.6, 98.9, 98.7, 98.6, 78.8, 78.7,
78.6 ꢃ 2, 78.5, 78.4, 78.3, 78.0, 77.8, 77.7, 77.6, 76.6, 76.5, 76.4,
76.2, 74.5, 74.4, 74.3 ꢃ 2, 73.9, 73.8, 72.6, 72.4, 71.8 ꢃ 2, 69.9,
68.8 ꢃ 2, 68.7 ꢃ 2, 68.6 ꢃ 2, 68.5 ꢃ 2, 66.8, 66.7, 66.2, 66.1,
65.9 ꢃ 2, 65.8, 65.7 ꢃ 2, 51.3, 38.0, 37.8, 29.8, 29.7, 29.3, 28.6,
28.0, 27.6, 26.3, 25.4, 20.8, 20.5; IR (solid): 2989, 2883, 2822,
2267, 1725, 1386, 1262, 1091, 675, 517 cmꢀ1; HRMS (ESI-TOF) calcd
for C152H161N4O51 [M+NH4]+ m/z = 2858.0128, found: 2858.0181.
yl-3,4,6-tri-O-benzyl-b-
6-O-benzylidene-3-O-(4,6-O-benzylidene-2,3-di-O-levnulinyl-b-
-glucopyranosyl)-b- -glucopyranosyl)-4-O-benzyl-b- -glucopy
ranosyl)-4,6-O-benzylidene-b- -glucopyranoside (9)
mixture of 32 (212 mg, 0.134 mmol), 33 (188 mg,
D-glucopyranosyl)-3-O-(2-O-benzoyl-4,
D
D
D
D
A
0.269 mmol) (azeotroped twice with dry toluene) and pulverized
activated MS-4A (134 mg) in dry CH2Cl2 (1.34 mL) was stirred at
room temperature for 30 min under argon to remove a trace
amount of water. Then the reaction mixture was cooled to
ꢀ10 °C. A catalytic amount of trimethylsilyl trifluoromethanesulfo-
nate (2.43 lL, 13.4 lmol) was added to the reaction mixture. After
being stirred at 0 °C for 3 h, the reaction mixture was neutralized
with triethylamine, filtered through a pad of CeliteÒ and concen-
trated in vacuo. The residue was purified by chromatography on
silica gel with 94:6 toluene:acetone to give 9 (259 mg, 0.123 mmol,
91%). ½a 1D7
ꢂ
ꢀ10.4 (c = 1.19, CHCl3); 1H NMR (400 MHz, CDCl3) d 7.98
(d, 2H, J = 7.2 Hz), 7.84 (d, 2H, J = 7.7 Hz), 7.69 (d, 2H, J = 7.2 Hz),
7.10–7.67 (m, 49H,), 5.47 (s, 1H), 5.41 (s, 1H), 5.36 (s, 1H), 5.26
(dd, 1H, J = 7.7, 8.7 Hz), 5.15 (dd, 1H, J = 7.7, 8.7 Hz), 5.00 (dd, 1H,
J = 9.2, 9.2 Hz), 4.93 (dd, 1H, J = 7.7, 9.2 Hz), 4.88 (dd, 1H, J = 6.3,
6.3 Hz), 4.73–4.80 (m, 4H), 4.54–4.66 (m, 6H), 4.51 (d, 1H,
J = 7.7 Hz), 4.46 (d, 1H, J = 12.1 Hz), 4.13–4.26 (m, 4H), 4.05 (d,
1H, J = 12.1 Hz), 3.85–3.96 (m, 4H), 3.78 (dd, 1H, J = 8.7, 9.2 Hz),
3.69 (dd, 1H, J = 10.1 Hz, J = 10.1 Hz), 3.45–3.65 (m, 9H), 3.40 (dd,
1H, J = 7.7, 9.2 Hz), 3.15–3.32 (m, 5H), 2.22–2.68 (m, 10H), 2.07
(s, 3H), 2.02 (s, 3H), 1.15 (t, 3H, J = 7.2 Hz); 13C NMR (100 MHz,
CDCl3) d 206.4 ꢃ 2, 171.8, 171.4, 165.4, 165.0, 164.7, 164.2, 138.3,
138.2, 137.9, 137.4, 137.1, 136.8, 133.5, 133.1, 133.0, 130.2,
129.9 ꢃ 2, 129.8, 129.6 ꢃ 2, 129.2, 129.1, 129.0, 128.8, 128.7,
4.1.22. 6-Azidohexyl 2-O-benzoyl-4,6-O-benzylidene-3-O-(2-O-
acetyl-4,6-O-benzylidene-3-O-(2-O-benzoyl-4,6-O-benzylidene-
3-O-(2-O-acetyl-4,6-O-benzylidene-3-O-(2-O-benzoyl-4,6-O-ben
zylidene-3-O-(2-O-acetyl-4,6-O-benzylidene-3-O-(2-O-benzoyl-
4,6-O-benzylidene-3-O-(4,6-O-
ne-b- -glucopyranosyl)-b- -glucopyranosyl)-b-
yl)-b- -glucopyranosyl)-b- -glucopyranosyl)-b-
syl)-b- -glucopyranoside (35)
To a stirred solution of 34 (57.9 mg, 20.4
(0.408 mL) was added acetic anhydride (19.1
D
-glucopyranosyl)-b-benzylide
D
D
D-glucopyranos
D
D
D-glucopyrano
D
lmol) in pyridine
l
L, 0.204 mmol)

H. Tanaka et al. / Bioorg. Med. Chem. 20 (2012) 3898–3914
3909
and a catalytic amount of DMAP (1.25 mg, 10.2
l
mol) at room
gel permeation chromatography (GPC) to give 36 (170 mg,
temperature. After being stirred at the same temperature for
1 h, the reaction mixture was poured into 1 M HCl. The aqueous
layer was extracted with two portions of ethyl acetate. The com-
bined extract was washed with 1 M HCl, saturated aq NaHCO3
and brine, and dried over MgSO4, filtered and concentrated in va-
cuo. The residue was used for the next reaction without further
41.1
l
mol, 88%). ½a 1D6
ꢂ
ꢀ24.6 (c = 0.950, CHCl3); 1H NMR (400 MHz,
CDCl3) d 7.97–8.03 (m, 12H), 7.16–7.62 (m, 78H), 5.51 (s, 1H),
5.47 (s, 1H), 5.38 (s, 1H), 5.10–5.21 (m, 6H), 4.91–5.04 (m, 11H),
4.78–4.88 (m, 9H), 4.68–4.72 (m, 3H), 4.63 (br-d, 2H, J = 6.3 Hz),
4.48 (d, 1H, J = 7.7 Hz), 4.31–4.37 (m, 2H), 3.22–4.24 (m, 61H),
3.05 (t, 2H, J = 7.2 Hz), 2.35–2.75 (m, 9H), 2.09 (s, 3H), 2.01 (s,
3H), 1.85 (s, 3H), 1.78 (s, 3H), 1.76 (s, 3H), 1.72 (s, 3H), 1.10–1.43
(m, 8H); 13C NMR (100 MHz, CDCl3) d 206.5, 206.4, 171.8, 171.4,
169.6, 169.1, 169.0 ꢃ 2, 165.5, 165.1, 164.9, 164.8 ꢃ 2, 164.7,
137.4, 137.3, 137.2, 137.0, 136.9, 134.0, 133.9, 133.7, 133.6, 133.4,
129.9, 129.8 ꢃ 3, 129.7 ꢃ 2, 129.6, 129.5 ꢃ 2, 129.4, 129.3 ꢃ 2,
129.2 ꢃ 2, 129.1, 129.0, 128.9, 128.8 ꢃ 2, 128.7, 128.6, 128.3,
128.2 ꢃ 3, 128.1, 126.3 ꢃ 2, 126.2, 126.1, 126.0, 102.9, 101.7,
101.6, 101.5, 101.4 ꢃ 2, 101.3, 101.2, 101.1, 100.9, 100.8, 100.6,
99.9, 99.2, 98.8, 98.5, 98.4, 98.2, 97.9, 78.7, 78.6 ꢃ 2, 78.5, 78.4,
78.2, 78.1, 78.0, 76.3, 76.1, 75.9 ꢃ 2, 75.7, 75.5, 74.5, 74.4 ꢃ 2,
74.2, 74.1, 73.8 ꢃ 2, 72.5, 72.4, 72.2, 71.8, 71.7, 69.9, 68.8, 68.7,
68.6, 68.5, 66.8, 66.7, 66.2, 66.0 ꢃ 2, 65.8, 65.7, 51.2, 37.9, 37.7,
29.7 ꢃ 2, 29.3, 28.6, 28.0, 27.6, 26.3, 25.4, 20.7 ꢃ 3, 20.5; IR (solid):
3492, 2971, 2877, 2417, 2097, 1732, 1602, 1452, 1373, 1262, 1222,
purification. To
a stirred solution of the residue in THF
(1.00 mL) was added acetic acid (0.300 mL) and hydrazine mono-
hydrate (0.100 mL) at 0 °C. After being stirred at the same tem-
perature for 20 min, the reaction mixture was poured into 1 M
HCl. The aqueous layer was extracted with two portions of ethyl
acetate. The combined extract was washed with saturated aq
NaHCO3 and brine, and dried over MgSO4, filtered and concen-
trated in vacuo. The residue was purified by chromatography on
silica gel with 88:12 toluene:acetone to give 35 (50.8 mg,
18.9
l
mol, 2 steps 93%). ½a D23
ꢂ
ꢀ23.8 (c = 1.04, CHCl3); 1H NMR
(400 MHz, CDCl3) d 7.96–8.06 (m, 8H), 7.17–7.63 (m, 52H), 5.52
(s, 1H), 5.42 (s, 1H), 5.32 (s, 1H), 5.26 (dd, 1H, J = 6.8 Hz,
6.8 Hz), 5.12 (s, 2H), 5.06 (d, 1H, J = 6.8 Hz), 4.94–5.00 (m, 3H),
4.76–4.88 (m, 8H), 4.70 (d, 1H, J = 5.3 Hz), 4.63 (br-d, 2H,
J = 5.3 Hz), 4.48–4.52 (m, 2H), 4.33 (dd, 1H, J = 4.3 Hz, 10.1 Hz),
3.25–4.19 (m, 42H), 3.06 (br-s, 1H), 3.05 (t, 2H, J = 7.2 Hz), 2.71
(br-s, 1H), 1.79 (s, 3H), 1.77 (s, 3H), 1.71 (s, 3H), 1.14–1.48 (m,
8H); 13C NMR (100 MHz, CDCl3) d 169.2, 169.0, 168.9, 165.5,
164.8, 164.6 ꢃ 2, 137.8, 137.2, 137.0, 136.8, 133.9, 133.8, 133.5,
133.3, 129.8, 129.7 ꢃ 2, 129.6, 129.5 ꢃ 2, 129.4, 129.3, 129.2 ꢃ 2,
129.1, 129.0, 128.9, 128.8, 128.7, 128.5, 128.4 ꢃ 2, 128.2 ꢃ 2,
128.1 ꢃ 2, 128.0 ꢃ 2, 127.8, 126.3, 126.2, 126.1, 126.0, 125.3,
102.5, 101.7, 101.6, 101.5, 101.4 ꢃ 2, 101.3 ꢃ 2, 101.0, 100.9,
100.8, 99.1, 98.2 ꢃ 2, 98.0, 97.8, 80.2, 78.7, 78.5, 78.2 ꢃ 2, 78.0,
77.9, 75.9, 75.7 ꢃ 2, 75.4, 75.2, 74.2, 74.1, 73.4, 73.3, 72.7, 72.2,
72.0 ꢃ 2, 71.9 ꢃ 2, 69.7, 68.6, 68.5, 68.4 ꢃ 2, 68.3, 66.5, 65.9,
65.7, 65.5, 65.3, 51.1, 29.1, 28.5, 26.1, 25.3, 21.4, 20.5 ꢃ 2, 20.3;
IR (solid): 3046, 2883, 2320, 1732, 1373, 1266, 1097, 997, 700,
517 cmꢀ1; HRMS (ESI-TOF) calcd for C144H151N4O48 [M+NH4]+
m/z = 2703.9498, found: 2703.9475.
1096, 996, 765, 697, 508 cmꢀ1
; HRMS (ESI-TOF) calcd for
C222H229N4O75 [M+NH4]+ m/z = 4150.4228, found: 4150.4053.
4.1.24. 6-Azidohexyl 2-O-benzoyl-4,6-O-benzylidene-3-O-(2-O-
acetyl-4,6-O-benzylidene-3-O-(2-O-benzoyl-4,6-O-benzylidene-
3-O-(2-O-acetyl-4,6-O-benzylidene-3-O-(2-O-benzoyl-4,6-O-
benzylidene-3-O-(2-O-acetyl-4,6-O-benzylidene-3-O-(2-O-
benzoyl-4,6-O-benzylidene-3-O-(2-O-acetyl-4,6-O-benzylidene-
3-O-(2-O-benzoyl-4,6-O-benzylidene-3-O-(2-O-acetyl-4,6-O-
benzylidene-3-O-(2-O-benzoyl-4,6-O-benzylidene-3-O-(4,6-O-
benzylidene-b-D-glucopyranosyl)-b-D-glucopyranosyl)-b-D-
glucopyranosyl)-b-
glucopyranosyl)-b-
glucopyranosyl)-b-
D
D
D
-glucopyranosyl)-b-
-glucopyranosyl)-b-
-glucopyranosyl)-b-
D
D
D
-glucopyranosyl)-b-
-glucopyranosyl)-b-
-glucopyranosyl)-b-
D-
D-
D
-
glucopyranoside (37)
To a stirred solution of 36 (72.6 mg, 17.6
(1.76 mL) was added acetic anhydride (16.4 L, 0.176 mmol) and
a catalytic amount of DMAP (1.07 mg, 8.80 mol) at room temper-
l
mol) in pyridine
l
4.1.23. 6-Azidohexyl 2-O-benzoyl-4,6-O-benzylidene-3-O-(2-O-
acetyl-4,6-O-benzylidene-3-O-(2-O-benzoyl-4,6-O-benzylidene-
3-O-(2-O-acetyl-4,6-O-benzylidene-3-O-(2-O-benzoyl-4,6-O-ben
zylidene-3-O-(2-O-acetyl-4,6-O-benzylidene-3-O-(2-O-benzoyl-
4,6-O-benzylidene-3-O-(4,6-O-benzylidene-3-O-(2-O-benzoyl-4,
6-O-benzylidene-3-O-(2-O-acetyl-4,6-O-benzylidene-3-O-(2-O-
benzoyl-4,6-O-benzylidene-3-O-(4,6-O-benzylidene-2,3-di-O-le
l
ature. After being stirred at the same temperature for 1 h, the reac-
tion mixture was poured into 1 M HCl. The aqueous layer was
extracted with two portions of ethyl acetate. The combined extract
was washed with 1 M HCl, saturated aq NaHCO3 and brine, and
dried over MgSO4, filtered and concentrated in vacuo. The residue
was used for the next reaction without further purification. To a
stirred solution of the residue in THF (1.00 mL) was added acetic
acid (0.300 mL) and hydrazine monohydrate (0.100 mL) at 0 °C.
After being stirred at the same temperature for 20 min, the reac-
tion mixture was poured into 1 M HCl. The aqueous layer was ex-
tracted with two portions of ethyl acetate. The combined extract
was washed with saturated aq NaHCO3 and brine, and dried over
MgSO4, filtered and concentrated in vacuo. The residue was puri-
fied by chromatography on silica gel with 88:12 toluene:acetone
vnulinyl-b-
anosyl)-b- -glucopyranosyl)-b-
nosyl)-b- -glucopyranosyl)-b- -glucopyranosyl)-b-
osyl)-b- -glucopyranosyl)-b- -glucopyranosyl)-b- -glucopyrano
side (36)
A mixture of 35 (125 mg, 46.6 mol), 8 (84.3 mg, 55.9 lmol)
D
-glucopyranosyl)-b-
D
-glucopyranosyl)-b-
-glucopyranosyl)-b-
-glucopyran
D-glucopyr
D
D
D-glucopyra
D
D
D
D
D
D
l
(azeotroped twice with dry toluene) and pulverized activated MS-
4A (279 mg) in dry CH2Cl2 (2.79 mL) was stirred at room tempera-
ture for 30 min under argon to remove a trace amount of water.
Then the reaction mixture was cooled to ꢀ35 °C. N-iodosuccinimide
to give 37 (65.2 mg, 16.4
l
mol, 2 steps 93%). ½a D16
ꢀ24.2 (c = 1.43,
ꢂ
CHCl3); 1H NMR (400 MHz, CDCl3) d 7.96–8.06 (m, 12H), 7.17–
7.62 (m, 78H), 5.52 (s, 1H), 5.42 (s, 1H), 5.32 (s, 1H), 5.26 (dd,
1H, J = 6.8 Hz, 7.2 Hz), 5.16 (s, 2H), 5.13 (s, 1H), 5.12 (s, 1H), 5.06
(d, 1H, J = 6.3 Hz), 4.94–4.98 (m, 3H), 4.77–4.88 (m, 16H), 4.71 (d,
1H, J = 5.3 Hz), 4.63–4.64 (m, 4H), 4.47–4.52 (m, 2H), 4.33 (dd,
1H, J = 4.3 Hz, 10.1 Hz), 3.18–4.19 (m, 62H), 3.03–3.08 (m, 3H),
2.65 (br-s, 1H), 1.79 (s, 3H), 1.77 (s, 3H), 1.76 (br-s, 6H), 1.72 (s,
3H), 1.14–1.46 (m, 8H); 13C NMR (100 MHz, CDCl3) d 169.4,
169.1 ꢃ 3, 165.6, 164.9 ꢃ 2, 164.8 ꢃ 2, 137.4 ꢃ 3, 137.3 ꢃ 2,
137.1, 136.9, 134.0 ꢃ 2, 133.6, 129.9, 129.8 ꢃ 2, 129.7, 129.6,
129.5, 129.3 ꢃ 4, 129.2 ꢃ 2, 129.1 ꢃ 5, 129.0 ꢃ 3, 128.9,
128.8 ꢃ 2, 128.6 ꢃ 2, 128.4, 128.3 ꢃ 3, 128.2 ꢃ 4, 128.1 ꢃ 2,
(15.1 mg, 67.0
lmol) and a catalytic amount of trifluoromethane-
sulfonic acid (2.10
lL, 23.3 lmol) were added to the reaction mix-
ture. After being stirred at the same temperature for 30 min, the
reaction mixture was neutralized with triethylamine and filtered
through a pad of CeliteÒ. The filtrate was poured into a mixture of
saturated aq NaHCO3 and 10% aq Na2S2O3 with cooling. The aque-
ous layer was extracted with two portions of ethyl acetate. The
combined extract was washed with a mixture of saturated aq NaH-
CO3 and 10% aq Na2S2O3, and brine, and dried over MgSO4, filtered,
and evaporated in vacuo. The residue was purified by chromatogra-
phy on silica gel with 91:9 toluene:acetone and further purified by

3910
H. Tanaka et al. / Bioorg. Med. Chem. 20 (2012) 3898–3914
126.4 ꢃ 2, 126.3, 126.2, 102.7, 101.9, 101.7, 101.6 ꢃ 3, 101.5, 101.4,
101.2, 101.1, 101.0 ꢃ 2, 98.5, 98.4, 98.3 ꢃ 3, 97.9, 78.9, 78.7, 78.4,
78.3 ꢃ 3, 78.2 ꢃ 3, 78.1, 76.6, 75.9, 75.8, 75.7, 74.4, 74.3 ꢃ 2,
74.2 ꢃ 3, 73.6, 72.8, 72.1 ꢃ 3, 69.9, 68.8, 68.7, 68.6 ꢃ 4, 68.4, 66.7,
66.1, 66.0 ꢃ 2, 65.8, 65.6, 51.3, 29.8, 29.3, 28.6, 26.3, 25.4, 20.7,
20.6 ꢃ 2, 20.5; IR (solid): 2971, 2877, 2825, 1733, 1374, 1262,
4.1.26. 6-Azidohexyl 2-O-benzoyl-4,6-O-benzylidene-3-O-(2-O-
acetyl-4,6-O-benzylidene-3-O-(2-O-benzoyl-4,6-O-benzylidene-
3-O-(3-O-(2-O-benzoyl-3-O-(2-O-benzoyl-6-O-(2-O-benzoyl-3,4,
6-tri-O-
nzylidene-3-O-(4,6-O-benzylidene-2,3-di-O-levnulinyl-b-
opyranosyl)-b- -glucopyranosyl)-4-O-benzyl-b- -glucopyran
osyl)-4,6-O-benzylidene-b- -glucopyranosyl)-4,6-O-benzylide
ne-b- -glucopyranosyl)-b- -glucopyranosyl)-b- -glucopyrano
syl)-b- -glucopyranoside (39)
A mixture of 7 (87.0 mg, 62.4 lmol), 9 (158 mg, 74.9 lmol)
D
-glucopyranosyl)-3-b-benzyl-O-(2-O-benzoyl-4,6-O-be
D
-gluc
D
D
1222, 1091, 695, 505 cmꢀ1
;
HRMS (ESI-TOF) calcd for
D
C
214H219N4O72 [M+NH4]+ m/z = 3996.3598, found: 3996.3635.
D
D
D
D
4.1.25. 6-Azidohexyl 2-O-benzoyl-4,6-O-benzylidene-3-O-(2-O-
acetyl-4,6-O-benzylidene-3-O-(2-O-benzoyl-4,6-O-benzylidene-
3-O-(2-O-acetyl-4,6-O-benzylidene-3-O-(2-O-benzoyl-4,6-O-
benzylidene-3-O-(2-O-acetyl-4,6-O-benzylidene-3-O-(2-O-ben
zoyl-4,6-O-benzylidene-3-O-(2-O-acetyl-4,6-O-benzylidene-3-
O-(2-O-benzoyl-4,6-O-benzylidene-3-O-(2-O-acetyl-4,6-O-
benzylidene-3-O-(2-O-benzoyl-4,6-O-benzylidene-3-O-(4,6-O-
benzylidene-3-O-(2-O-benzoyl-4,6-O-benzylidene-3-O-(2-O-
acetyl-4,6-O-benzylidene-3-O-(2-O-benzoyl-4,6-O-benzylidene-
(azeotroped twice with dry toluene) and pulverized activated MS-
4A (374 mg) in dry CH2Cl2 (3.74 mL) was stirred at room tempera-
ture for 30 min under argon to remove a trace amount of water.
Then the reaction mixture was cooled to ꢀ35 °C. N-iodosuccinimide
(20.2 mg, 89.9
lmol) and a catalytic amount of trifluoromethane-
sulfonic acid (1.66
lL, 18.7 lmol) were added to the reaction mix-
ture. After being stirred at the same temperature for 30 min, the
reaction mixture was neutralized with triethylamine and filtered
through a pad of CeliteÒ. The filtrate was poured into a mixture of
saturated aq NaHCO3 and 10% aq Na2S2O3 with cooling. The aqueous
layer was extracted with two portions of ethyl acetate. The com-
bined extract was washed with a mixture of saturated aq NaHCO3
and 10% aq Na2S2O3, and brine, and dried over MgSO4, filtered,
and evaporated in vacuo. The residue was purified by chromatogra-
phy on silica gel with 91:9 toluene:acetone and further purified by
gel permeation chromatography (GPC) to give 39 (191 mg,
3-O-(4,6-O-benzylidene-2,3-di-O-levnulinyl-b-
yl)-b- -glucopyranosyl)-b- -glucopyranosyl)-b-
syl)-b- -glucopyranosyl)-b- -glucopyranosyl)-b-
yl)-b- -glucopyranosyl)-b- -glucopyranosyl)-b-
osyl)-b- -glucopyranosyl)-b- -glucopyranosyl)-b-
anosyl)-b- -glucopyranosyl)-b- -glucopyranosyl)-b-
ranoside (38)
A mixture of 37 (20.4 mg, 5.12
D
-glucopyranos
-glucopyrano
-glucopyranos
-glucopyran
-glucopyr
-glucopy
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
l
mol), 8 (9.30 mg, 6.15 lmol)
(azeotroped twice with dry toluene) and pulverized activated
MS-4A (51.2 mg) in dry CH2Cl2 (0.512 mL) was stirred at room
temperature for 30 min under argon to remove a trace amount of
water. Then the reaction mixture was cooled to ꢀ35 °C. N-iodosuc-
55.5
l
mol, 89%). ½a 2D1
ꢂ
ꢀ5.37 (c = 0.980, CHCl3); 1H NMR (400 MHz,
CDCl3) d 7.98–8.01 (m, 4H), 7.91 (d, 2H, J = 7.2 Hz), 7.81 (d, 2H,
J = 7.2 Hz), 7.76 (d, 2H, J = 7.7 Hz), 7.63 (d, 2H, J = 7.2 Hz), 7.03–
7.61 (m, 73H), 5.48 (s, 1H), 5.41 (s, 1H), 5.37 (s, 1H), 5.30 (s, 1H),
5.28 (s, 1H), 5.25 (dd, 1H, J = 8.7 Hz, 8.7 Hz), 5.09–5.17 (m, 2H),
4.88–5.05 (m, 8H), 4.76–4.84 (m, 5H), 4.64–4.69 (m, 3H), 4.54–
4.61 (m, 3H), 4.50 (br-d, 2H, J = 7.7 Hz), 4.40–4.46 (m, 2H), 4.29–
4.34 (m, 2H), 4.05–4.23 (m, 7H), 3.98 (dd, 1H, J = 4.8 Hz, 10.1 Hz),
3.20–3.94 (m, 36H), 3.14 (ddd, 1H, J = 4.8 Hz, 9.7 Hz, 10.1 Hz),
3.01–3.06 (m, 3H), 2.25–2.72 (m, 10H), 2.07 (s, 3H), 2.01 (s, 3H),
1.72 (s, 3H), 1.10–1.48 (m, 8H); 13C NMR (100 MHz, CDCl3) d
206.3 ꢃ 2, 171.7, 171.3, 169.3, 165.4, 165.2, 165.1, 164.8, 164.7,
163.9, 138.7, 138.4, 138.3, 138.0, 137.6, 137.2 ꢃ 2, 136.9, 136.8,
133.7, 133.4, 133.3, 133.2 ꢃ 2, 133.0 ꢃ 2, 129.8 ꢃ 2, 129.7, 129.6,
129.5 ꢃ 2, 129.2 ꢃ 3, 129.1 ꢃ 2, 129.0, 128.9, 128.8 ꢃ 2, 128.7,
128.6, 128.5 ꢃ 2, 128.4, 128.3, 128.2 ꢃ 2, 128.1 ꢃ 2, 128.0, 127.9,
127.8, 127.6, 127.5 ꢃ 2, 127.4, 126.4, 126.3, 126.2 ꢃ 2, 126.1,
126.0 ꢃ 2, 102.6, 101.6 ꢃ 2, 101.5, 101.4, 101.3, 101.2, 101.0,
100.9, 100.8, 100.6, 99.9, 99.2, 98.7, 98.5, 97.7, 82.8, 79.5, 78.8,
78.6, 78.5, 78.4, 78.3, 78.0, 77.8, 76.3, 76.0, 75.9, 75.7, 75.2, 74.8,
74.3 ꢃ 2, 74.1 ꢃ 2, 74.0, 73.9, 73.8, 73.4, 73.3, 72.1, 71.6, 69.8,
69.2, 68.7 ꢃ 3, 68.6 ꢃ 2, 68.5 ꢃ 2, 68.4 ꢃ 3, 68.3, 66.6 ꢃ 2, 66.3,
66.1, 65.9, 65.6, 65.2, 51.2, 37.8, 37.6, 29.6 ꢃ 2, 29.2, 28.5, 27.9,
27.3, 26.2, 25.3, 20.4; IR (solid): 3513, 2869, 2092, 1734, 1451,
cinimide (1.70 mg, 7.37
lmol) and a catalytic amount of trifluoro-
methanesulfonic acid (0.317
l
L, 3.58 mol) were added to the
l
reaction mixture. After being stirred at the same temperature for
30 min, the reaction mixture was neutralized with triethylamine
and filtered through a pad of CeliteÒ. The filtrate was poured into
a mixture of saturated aq NaHCO3 and 10% aq Na2S2O3 with cool-
ing. The aqueous layer was extracted with two portions of ethyl
acetate. The combined extract was washed with a mixture of satu-
rated aq NaHCO3 and 10% aq Na2S2O3, and brine, and dried over
MgSO4, filtered, and evaporated in vacuo. The residue was purified
by chromatography on silica gel with 91:9 toluene:acetone and
further purified by gel permeation chromatography (GPC) to give
38 (22.8 mg, 4.20
l
mol, 82%). ½a 2D4
ꢂ
ꢀ17.6 (c = 2.02, CHCl3); 1H
NMR (400 MHz, CDCl3) d 7.97–8.03 (m, 16H), 7.14–7.62 (m,
104H), 5.51 (s, 1H), 5.47 (s, 1H), 5.38 (s, 1H), 4.63–5.21 (m, 43H),
4.48 (d, 1H, J = 7.7 Hz), 4.31–4.37 (m, 2H), 3.17–4.24 (m, 81H),
3.05 (t, 2H, J = 7.2 Hz), 2.35–2.70 (m, 9H, Lev, OH), 2.08 (s, 3H),
2.00 (s, 3H), 1.86 (s, 3H), 1.78 (s, 3H), 1.76 (s, 9H), 1.71 (s, 3H),
1.08–1.50 (m, 8H); 13C NMR (100 MHz, CDCl3) d 206.5 ꢃ 2, 171.8,
171.4, 169.6, 169.1 ꢃ 3, 169.0, 165.6 165.2, 164.9 ꢃ 2, 164.8 ꢃ 3,
137.4, 137.3, 137.2, 137.1, 137.0, 136.9, 134.1 ꢃ 2, 134.0 ꢃ 3,
133.9 ꢃ 3, 133.7, 133.6, 133.4, 129.9, 129.8 ꢃ 2, 129.7 ꢃ 2, 129.6,
129.5 ꢃ 2, 129.4, 129.3 ꢃ 2, 129.2, 129.1, 129.0, 128.9, 128.8 ꢃ 2,
128.7, 128.6, 128.3 ꢃ 2, 128.2 ꢃ 3, 128.1, 128.0, 126.4, 126.3 ꢃ 3,
126.2, 126.1, 126.0, 125.4, 102.9, 102.8, 101.7, 101.6, 101.5 ꢃ 2,
101.4, 101.3 ꢃ 2, 101.2 ꢃ 2, 101.1 ꢃ 3, 101.0, 100.9, 100.8, 100.6,
99.9, 99.2, 98.8, 98.4 ꢃ 2, 98.2, 98.0 ꢃ 2, 97.8 ꢃ 2, 78.7 ꢃ 3, 78.6,
78.5 ꢃ 2, 78.4, 78.2 ꢃ 2, 78.1, 78.0 ꢃ 3, 77.9, 77.8, 77.7, 76.6 ꢃ 2,
76.4, 76.3 ꢃ 2, 76.0, 75.9, 75.8, 75.7 ꢃ 2, 75.6 ꢃ 3, 75.4, 75.3,
74.5 ꢃ 2, 74.4 ꢃ 2, 74.3 ꢃ 2, 74.2 ꢃ 2, 74.1, 73.9, 73.8, 72.5, 72.3,
72.2 ꢃ 2, 72.1 ꢃ 2, 71.8 ꢃ 2, 71.7, 69.9, 68.9 ꢃ 3, 68.8, 68.7, 68.6,
68.5, 68.4 ꢃ 2, 68.2, 66.8, 66.7, 66.2, 66.1, 66.0 ꢃ 2, 65.9, 65.8,
65.7, 65.6, 51.2, 38.8, 37.9, 37.7, 32.0, 29.8, 29.7, 29.6 ꢃ 3,
29.5 ꢃ 2, 29.4, 29.3, 29.0, 28.6, 28.0, 27.6, 26.3, 25.4, 23.0, 22.7,
21.5, 20.7 ꢃ 2, 20.6, 20.5; IR (solid): 3536, 2926, 2093, 1720,
1374, 1264, 1069, 752, 696, 654, 501 cmꢀ1
.
4.1.27. 6-Azidohexyl 2-O-benzoyl-4,6-O-benzylidene-3-O-(2-O-
acetyl-4,6-O-benzylidene-3-O-(2-O-benzoyl-4,6-O-benzylidene-
3-O-(2-O-acetyl-4,6-O-benzylidene-3-O-(2-O-benzoyl-4,6-O-ben
zylidene-3-O-(2-O-acetyl-4,6-O-benzylidene-3-O-(2-O-benzoy l-
4,6-O-benzylidene-3-O-(3-O-(2-O-benzoyl-3-O-(2-O-benzoyl-6-
O-(2-O-benzoyl-3,4,6-tri-O-benzyl-b-
O-benzoyl-4,6-O-benzylidene-3-O-(4,6-O-benzylidene-2,3-di-O-
levnulinyl-b- -glucopyranosyl)-4-b- -glucopyranosyl)-O-benzyl-
b- -glucopyranosyl)-4,6-O-benzylidene-b- -glucopyrano syl)-4,
6-O-benzylidene-b- -glucopyranosyl)-b- -glucopyranos yl)-b-
-glucopyranos yl)-b-
-glucopyrano side (40)
mol)
(azeotroped twice with dry toluene) and pulverized activated
D-glucopyranosyl)-3-O-(2-
D
D
D
D
D
D
D-
glucopyranosyl)-b-
D
-glucopyranosyl)-b-
-glucopyranosyl)-b-
D
D
D-
glucopyranosyl)-b-
D
A mixture of 35 (73.0 mg, 27.2 lmol), 9 (86.0 mg, 40.8 l
1369, 1256, 1093, 909, 647, 496 cmꢀ1
.

H. Tanaka et al. / Bioorg. Med. Chem. 20 (2012) 3898–3914
3911
MS-4A (163 mg) in dry CH2Cl2 (1.63 mL) was stirred at room tem-
perature for 30 min under argon to remove a trace amount of
water. Then the reaction mixture was cooled to ꢀ35 °C. N-iodo-
methanesulfonic acid (0.467
l
L, 5.28
l
mol) were added to the
reaction mixture. After being stirred at the same temperature for
30 min, the reaction mixture was neutralized with triethylamine
and filtered through a pad of CeliteÒ. The filtrate was poured into
a mixture of saturated aq NaHCO3 and 10% aq Na2S2O3 with cool-
ing. The aqueous layer was extracted with two portions of ethyl
acetate. The combined extract was washed with a mixture of sat-
urated aq NaHCO3 and 10% aq Na2S2O3, and brine, and dried over
MgSO4, filtered, and evaporated in vacuo. The residue was purified
by chromatography on silica gel with 85:15 toluene:ethyl acetate
and further purified by gel permeation chromatography (GPC) to
succinimide (11.0 mg, 48.9
lmol) and a catalytic amount of tri-
fluoromethanesulfonic acid (1.20
lL, 13.6 lmol) were added to
the reaction mixture. After being stirred at the same temperature
for 30 min, the reaction mixture was neutralized with triethyl-
amine and filtered through a pad of CeliteÒ. The filtrate was
poured into a mixture of saturated aq NaHCO3 and 10% aq
Na2S2O3 with cooling. The aqueous layer was extracted with
two portions of ethyl acetate. The combined extract was washed
with a mixture of saturated aq NaHCO3 and 10% aq Na2S2O3,
and brine, and dried over MgSO4, filtered, and evaporated in va-
cuo. The residue was purified by chromatography on silica gel
with 91:9 toluene:acetone and further purified by gel permeation
give 41 (87.0 mg, 14.4
l
mol, 82%). ½a 2D1
ꢀ10.6 (c = 2.16, CHCl3);
ꢂ
1H NMR (400 MHz, CDCl3) d 7.96–8.01 (m, 14H), 7.90 (d, 2H,
J = 7.7 Hz), 7.81 (d, 2H, J = 7.7 Hz), 7.74 (d, 2H, J = 7.7 Hz), 6.99–
7.63 (m, 125H), 5.52 (s, 1H), 5.41 (s, 1H), 5.36 (s, 1H), 5.33 (s,
1H), 5.30 (s, 1H), 5.25 (dd, 1H, J = 8.7 Hz, 8.7 Hz), 5.09–5.17 (m,
6H), 4.76–5.03 (m, 29H), 4.54–4.70 (m, 10H), 4.48 (br-d, 2H,
J = 7.7 Hz), 4.40–4.45 (m, 2H), 4.31–4.35 (m, 2H), 3.12–4.22 (m,
85H), 3.00–3.07 (m, 3H), 2.17–2.67 (m, 10H), 2.07 (s, 3H), 2.00
(s, 3H), 1.76 (s, 12H), 1.72 (s, 3H), 1.10–1.48 (m, 8H); 13C NMR
(100 MHz, CDCl3) d 206.4 ꢃ 2, 171.8, 171.4, 169.3, 169.1 ꢃ 2,
165.7 ꢃ 3, 165.3, 165.1, 165.0, 164.9 ꢃ 2, 164.8, 164.0, 138.7,
138.5, 138.4, 138.1, 137.7, 137.5 ꢃ 2, 137.4, 137.3 ꢃ 2, 137.1,
136.9, 134.1, 134.0 ꢃ 3, 133.7, 133.6, 133.4 ꢃ 2, 133.3 ꢃ 3, 133.2,
133.1 ꢃ 2, 130.3, 130.2 ꢃ 2, 130.1, 129.8 ꢃ 2, 129.7, 129.6,
129.5 ꢃ 2, 129.4, 129.3, 129.2, 129.1, 128.9, 128.8, 128.7,
128.6 ꢃ 2, 128.5, 128.4, 128.3, 128.2 ꢃ 2, 128.1, 128.0, 127.9,
127.7, 127.6 ꢃ 2, 127.4, 126.6 ꢃ 3, 126.4 ꢃ 2, 126.3, 126.2, 126.1,
102.7 ꢃ 2, 102.5, 101.8 ꢃ 3, 101.7 ꢃ 2, 101.6 ꢃ 2, 101.5 ꢃ 3,
101.4 ꢃ 3, 101.3 ꢃ 2, 101.2 ꢃ 2, 101.1 ꢃ 3, 101.0, 100.9,
100.7 ꢃ 2, 100.1, 100.0, 99.2, 98.7, 98.6, 98.5, 98.4 ꢃ 3, 98.3 ꢃ 2,
98.2, 98.1, 98.0 ꢃ 3, 97.9 ꢃ 2, 82.9, 79.6, 79.5, 79.0, 78.9, 78.8,
78.7, 78.5, 78.4, 78.3, 78.2, 78.1, 77.9, 77.8 ꢃ 2, 77.7 ꢃ 3,
76.5 ꢃ 2, 76.4, 76.0 ꢃ 3, 75.9, 75.8, 75.7, 75.6 ꢃ 2, 75.5 ꢃ 2,
75.3 ꢃ 2, 75.2 ꢃ 2, 74.9, 74.4 ꢃ 2, 74.2 ꢃ 3, 73.9 ꢃ 2, 73.8, 73.5,
73.4, 72.4, 72.1, 71.7, 70.6, 69.9, 68.9, 68.7, 68.6 ꢃ 2, 68.4, 66.7,
66.6, 66.4, 66.1 ꢃ 2, 66.0, 65.8, 65.7, 65.6, 51.3, 37.9, 37.7, 29.7,
29.6, 29.3, 28.6, 28.0, 27.4, 26.3, 25.4, 20.7, 20.6, 20.5; IR (solid):
3729, 2863, 2344, 2138, 1968, 1734, 1496, 1368, 1218, 1023,
chromatography (GPC) to give 40 (102 mg, 21.5 lmol, 79%).
½
a 2D1
ꢂ
= ꢀ9.17 (c = 0.960, CHCl3); 1H NMR (400 MHz, CDCl3)
d
7.96–8.00 (m, 8H), 7.90 (d, 2H, J = 7.2 Hz), 7.81 (d, 2H,
J = 7.2 Hz), 7.75 (d, 2H, J = 7.2 Hz), 6.99–7.63 (m, 101H), 5.51 (s,
1H), 5.40 (s, 1H), 5.36 (s, 1H), 5.33 (s, 1H), 5.30 (s, 1H), 5.24
(dd, 1H, J = 8.2 Hz, 8.7 Hz), 5.11–5.16 (m, 4H), 4.76–5.02 (m,
21H), 4.54–4.70 (m, 8H), 4.49 (d, 1H, J = 6.8 Hz), 4.48 (d, 1H,
J = 7.7 Hz), 4.40–4.45 (m, 2H), 4.31–4.34 (m, 2H), 3.22–4.22 (m,
64H), 3.14 (ddd, 1H, J = 4.8 Hz, 8.7 Hz, 9.2 Hz), 3.00–3.07 (m,
3H), 2.22–2.67 (m, 10H), 2.07 (s, 3H), 2.04 (s, 3H), 1.76 (s, 3H),
1.75 (s, 3H), 1.71 (s, 3H), 1.10–1.48 (m, 8H); 13C NMR (100 MHz,
CDCl3) d 208.1, 206.4, 171.8, 171.4, 169.3, 169.1, 169.0, 165.3,
165.1, 164.9, 164.8 ꢃ 2, 138.7, 138.4, 138.3, 138.0, 137.7, 137.4,
137.3 ꢃ 3, 137.0, 136.9, 133.4 ꢃ 2, 133.3 ꢃ 2, 133.2, 133.1,
130.0 ꢃ 2, 129.8 ꢃ 2, 129.7 ꢃ 2, 129.6 ꢃ 2, 129.5 ꢃ 2, 129.4,
129.3 ꢃ 2, 129.2 ꢃ 3, 129.1 ꢃ 2, 129.0 ꢃ 2, 128.9 ꢃ 2, 128.8,
128.7, 128.6 ꢃ 2, 128.5 ꢃ 2, 128.3, 128.2 ꢃ 2, 128.1 ꢃ 2, 128.0,
127.9 ꢃ 2, 127.8, 127.7, 127.6, 127.5, 127.4, 126.5, 126.4, 126.3,
126.2, 126.1 ꢃ 3, 102.7, 101.8, 101.7, 101.6 ꢃ 2, 101.5 ꢃ 2, 101.4,
101.3, 101.2 ꢃ 2, 101.1 ꢃ 2, 101.0, 100.8, 100.6, 100.0, 99.2,
98.6 ꢃ 2, 98.5 ꢃ 2, 98.2, 98.0, 97.8, 82.9, 78.8, 78.7, 78.5 ꢃ 2,
78.4, 78.3, 78.2 ꢃ 2, 78.0, 77.9, 76.5, 76.1, 76.0, 75.9 ꢃ 2, 75.7,
75.2, 74.9, 74.4, 74.3, 74.2 ꢃ 2, 74.1, 73.9 ꢃ 2, 73.8, 73.5, 73.4,
72.4, 72.1, 71.6, 69.9, 68.8, 68.7 ꢃ 3, 68.6 ꢃ 3, 68.5, 68.4 ꢃ 2,
66.7, 66.6, 66.4, 66.1 ꢃ 2, 66.0 ꢃ 2, 65.8, 65.6 ꢃ 2, 65.2, 53.5,
51.2, 37.9, 37.7, 29.7, 29.6, 29.3, 28.6, 28.0, 27.4, 26.3, 25.4,
20.6 ꢃ 2, 20.5; IR (solid): 3631, 2964, 2093, 1734, 1412, 1258,
876, 770, 654, 508 cmꢀ1
.
4.1.29. 6-Aminohexyl 3-O-(3-O-(3-O-(3-O-(3-O-(6-O-(b-
D-gluco
1089, 773, 696, 500 cmꢀ1
.
pyranosyl)-3-O-(3-O-(b- -glucopyranosyl)-b- -glucopyranosyl)-
D
D
b-
b-
D
D
-glucopyranosyl)-b-
-glucopyranosyl)-b-
D
-glucopyranosyl)-b-
-glucopyranosyl)-b-
D
-glucopyranosyl)-
-glucopyranoside
4.1.28. 6-Azidohexyl 2-O-benzoyl-4,6-O-benzylidene-3-O-(2-O-
acetyl-4,6-O-benzylidene-3-O-(2-O-benzoyl-4,6-O-benzylidene-
3-O-(2-O-acetyl-4,6-O-benzylidene-3-O-(2-O-benzoyl-4,6-O-ben
zylidene-3-O-(2-O-acetyl-4,6-O-benzylidene-3-O-(2-O-benzoyl-
4,6-O-benzylidene-3-O-(2-O-acetyl-4,6-O-benzylidene-3-O-(2-
O-benzoyl-4,6-O-benzylidene-3-O-(2-O-acetyl-4,6-O-benzylide
ne-3-O-(2-O-benzoyl-4,6-O-benzylidene-3-O-(3-O-(2-O-benzo
D
D
(5)
To a stirred solution of the protected nonasaccharide 39
(33.9 mg, 9.85 mol) in NH3/THF/EtOH (8.50 mL/1.00 mL/
l
0.500 mL) was added a large amount of lithium (100 mg) at
ꢀ78 °C. After being stirred under reflux for 1.5 h, the reaction mix-
ture was added methanol (1.00 mL). After being stirred at room
temperature for 12 h, the reaction mixture was concentrated in va-
cuo. The residue was purified by size exclusion column chromatog-
raphy on Sephadex LH-20 eluted with water and further purified by
revese-phase column chromatography (Bond Elut-C18) with 90:10
yl-3-O-(2-O-benzoyl-6-O-(2-O-benzoyl-3,4,6-tri-O-benzyl-b-
glucopyranosyl)-3-O-(2-O-benzoyl-4,6-O-benzylidene-3-O-(4,6-
O-benzylidene-2,3-di-O-levnulinyl-b- -glucopyranosyl)-b-
glucopyranosyl)-4-O-benzyl-b- -glucopyranosyl)-4,6-O-benzyli
dene-b- -glucopyranosyl)-4,6-O-benzylidene-b- -glucopyranos
D-
D
D-
D
D
D
water:methanol to give nonasaccharide 5 (15.5 mg, 9.83 lmol,
yl)-b-
yl)-b-
D
-glucopyranosyl)-b-
D
-glucopyranosyl)-b-
-glucopyranosyl)-b-
D
-glucopyranos
-glucopyrano
quant.). ½a 2D4
ꢂ
ꢀ24.2 (c = 0.335, H2O); 1H NMR (400 MHz, D2O) d
D-glucopyranosyl)-b-
D
D
4.70–4.80 (m, 7H), 4.49 (d, 1H, J = 7.7 Hz), 4.44 (d, 1H, J = 7.7 Hz),
4.18 (br-d, 1H, J = 10.6 Hz), 3.27–3.91 (m, 55H), 2.80 (t, 2H,
J = 7.2 Hz), 1.51–1.63 (m, 4H), 1.35–1.36 (m, 4H); 13C NMR
(100 MHz, D2O) d 103.6 ꢃ 2, 103.4 ꢃ 2, 102.7, 85.7, 85.5, 85.2,
85.1, 84.8, 76.8, 76.7, 76.5, 76.4, 75.4, 74.3, 74.1, 74.0 ꢃ 2, 73.9,
73.8, 73.7, 71.2, 70.4, 69.6, 69.0 ꢃ 2, 68.9, 61.5, 40.5, 29.3,
28.5, 26.2, 25.4; IR (solid): 3481, 2884, 1846, 1669, 1438, 1314,
syl)-b-
syl)-b-
D
-glucopyranosyl)-b-
D
-glucopyranosyl)-b-
-glucopyranoside (41)
mol,), 9 (55.7 mg, 26.4 lmol)
D-glucopyrano
D-glucopyranosyl)-b-
D
A mixture of 37 (70.0 mg, 17.6
l
(azeotroped twice with dry toluene) and pulverized activated
MS-4A (176 mg) in dry CH2Cl2 (1.76 mL) was stirred at room tem-
perature for 30 min under argon to remove a trace amount of
water. Then the reaction mixture was cooled to ꢀ35 °C. N-iodosuc-
1153, 1032, 855, 664, 526 cmꢀ1
60H106NO46 [M+H]+ m/z = 1576.5986, found: 1576.5981.
; HRMS (ESI-TOF) calcd for
cinimide (7.13 mg, 31.7
lmol) and a catalytic amount of trifluoro-
C

3912
H. Tanaka et al. / Bioorg. Med. Chem. 20 (2012) 3898–3914
4.1.30. 6-Aminohexyl 3-O-(3-O-(3-O-(3-O-(3-O-(3-O-(3-O-(3-O-
(3-O-(6-O-(b- -glucopyranosyl)-3-O-(3-O-(b- -glucopyrano
syl)-b- -glucopyranosyl)-b- -glucopyranosyl)-b- -glucopyran
by revese-phase column chromatography (Bond Elut-C18) with
D-
D
D
90:10 water:methanol to give octasaccharide
6
(7.00 mg,
D
D
D
4.58
l
mol, 43%). ½a 2D4
ꢂ
ꢀ33.7 (c = 0.255, H2O); 1H NMR (400 MHz,
osyl)-b-
osyl)-b-
D
-glucopyranosyl)-b-
D
-glucopyranosyl)-b-
-glucopyranosyl)-b-
D
-glucopyran
-glucop
D2O) d 4.70–4.76 (m, 7H), 4.44 (d, 1H, J = 7.7 Hz), 3.88–3.91 (m,
9H), 3.70–3.85 (m, 16H), 3.33–3.56 (m, 27H), 2.99 (t, 2H,
J = 7.2 Hz), 1.59–1.68 (m, 4H), 1.37–1.40 (m, 4H); 13C NMR
(100 MHz, D2O) d 103.7, 103.6, 103.5, 103.4, 102.8, 85.5 ꢃ 2,
85.4, 85.3, 85.2 ꢃ 2, 85.0, 76.8, 76.6, 76.5, 76.4, 74.3, 74.2, 74.1,
74.0, 73.7, 71.2, 70.4, 69.0, 68.9, 61.5, 40.5, 29.3, 28.7, 26.2, 25.4;
IR (solid): 3307, 2897, 2341, 1591, 1438, 1307, 1154, 1042, 669,
D-glucopyranosyl)-b-
D
D
yranosyl)-b-
To a stirred solution of the protected tridecasaccharide 40
(16.1 mg, 3.40 mol) in NH3/THF/EtOH (8.50 mL/1.00 mL/
D-glucopyranosyl)-b-D-glucopyranoside (3)
l
0.500 mL) was added a large amount of lithium (100 mg) at
ꢀ78 °C. After being stirred under reflux for 1.5 h, the reaction mix-
ture was added methanol (1.00 mL). After being stirred at room
temperature for 12 h, the reaction mixture was concentrated in va-
cuo. The residue was purified by size exclusion column chromatog-
raphy on Sephadex LH-20 eluted with water and further purified
by revese-phase column chromatography (Bond Elut-C18) with
525 cmꢀ1
;
HRMS (ESI-TOF) calcd for C54H96NO41 [M+H]+
m/z = 1414.5458, found: 1414.5460.
4.1.33. 6-Aminohexyl 3-O-(3-O-(3-O-(3-O-(3-O-(3-O-(3-O-(3-O-
(3-O-(3-O-(3-O-(b-
D-
D
-glucopyranosyl)-b-
D
-glucopyranosyl)-b-
D
-
80:20 water:methanol to give tridecasaccharide
3
(6.10 mg,
glucopyranosyl)-b-
glucopyranosyl)-b-
glucopyranosyl)-b-
D
D
D
-glucopyranosyl)-b-
-glucopyranosyl)-b-
-glucopyranosyl)-b-
D
D
D
-glucopyranosyl)-b-
-glucopyranosyl)-b-
-glucopyranosyl)-b-
D
D
D
-
-
-
2.74
l
mol, 81%). ½a 2D4
ꢂ
ꢀ22.9 (c = 0.135, H2O); 1H NMR (400 MHz,
D2O) d 4.70–4.76 (m, 11H), 4.49 (d, 1H, J = 7.7 Hz), 4.45 (d, 1H,
J = 8.7 Hz), 4.18 (br-d, 1H, J = 11.1 Hz), 3.27–3.91 (m, 79H), 2.79
(t, 2H, J = 7.2 Hz), 1.52–1.63 (m, 4H), 1.35–1.37 (m, 4H); 13C NMR
(100 MHz, D2O) d 103.6, 103.4 ꢃ 2, 102.5, 85.1, 76.5, 76.4, 74.1,
74.0, 70.4, 69.0, 68.9, 61.5, 40.6, 29.3, 29.0, 26.2, 25.4; IR (solid):
3413, 2885, 1821, 1648, 1591, 1368, 1154, 1077, 1042, 652,
glucopyranoside (4)
To stirred solution of the protected decasaccharide 36
(30.8 mg, 7.45 mol) in NH3/THF/EtOH (8.50 mL/1.00 mL/
a
l
0.500 mL) was added a large amount of lithium (100 mg) at
ꢀ78 °C. After being stirred under reflux for 1.5 h, the reaction mix-
ture was added methanol (1.00 mL). After being stirred at room
temperature for 12 h, the reaction mixture was concentrated in va-
cuo. The residue was purified by size exclusion column chromatog-
raphy on Sephadex LH-20 eluted with water and further purified
by revese-phase column chromatography (Bond Elut-C18) with
;
519 cmꢀ1 HRMS (ESI-TOF) calcd for C84H146NO66 [M+H]+
m/z = 2224.8099, found: 2224.8054.
4.1.31. 6-Aminohexyl 3-O-(3-O-(3-O-(3-O-(3-O-(3-O-(3-O-(3-O-
(3-O-(3-O-(3-O-(3-O-(3-O-(6-O-(b-
D
-
D-glucopyranosyl)-3-O-(3-O-
(b- -glucopyranosyl)-b- -glucopyranosyl)-b-
D
D
D
-glucopyranosyl)-
90:10 water:methanol to give dodecasaccharide 4 (13.8 mg,
b-
b-
b-
b-
b-
D
-glucopyranosyl)-b-
-glucopyranosyl)-b-
-glucopyranosyl)-b-
-glucopyranosyl)-b-
D
D
D
D
-glucopyranosyl)-b-
-glucopyranosyl)-b-
-glucopyranosyl)-b-
-glucopyranosyl)-b-
D
D
D
D
-glucopyranosyl)-
-glucopyranosyl)-
-glucopyranosyl)-
-glucopyranosyl)-
6.69
l
mol, 90%). ½a 2D4
ꢂ
ꢀ13.9 (c = 0.315, H2O); 1H NMR (400 MHz,
D
D
D
D
D2O) d 4.70–4.76 (m, 11H), 4.44 (d, 1H, J = 8.2 Hz), 3.88–3.91 (m,
13H), 3.63–3.81 (m, 24H), 3.32–3.56 (m, 37H), 2.77 (t, 2H,
J = 7.2 Hz), 1.49–1.63 (m, 4H), 1.28–1.36 (m, 4H); 13C NMR
(100 MHz, D2O) d 103.6, 103.4, 102.8, 85.5, 85.2, 85.0, 76.8, 76.5,
76.4, 74.3, 74.1, 73.7, 71.2, 70.4, 69.0, 68.9, 61.5, 40.4, 30.9, 30.7,
30.5, 30.3, 30.1, 29.9, 29.7, 29.3, 28.1, 26.1, 25.4; IR (solid): 3341,
2912, 2352, 2014, 1836, 1592, 1439, 1309, 1154, 1077, 1044,
-glucopyranoside (1)
To a stirred solution of the protected heptadecasacchairde 41
(40.7 mg, 6.75 mol) in NH3/THF/EtOH (8.50 mL/1.00 mL/0.500
l
mL) was added a large amount of lithium (100 mg) at ꢀ78 °C. After
being stirred under reflux for 1.5 h, the reaction mixture was added
methanol (1.00 mL). After being stirred at room temperature for
12 h, the reaction mixture was concentrated in vacuo. The residue
was purified by size exclusion column chromatography on Sepha-
dex LH-20 eluted with water and further purified by revese-phase
column chromatography (Bond Elut-C18) with 70:30 water:meth-
911, 671, 526 cmꢀ1
; HRMS (ESI-TOF) calcd for C78H136NO61
[M+H]+ m/z = 2062.7571, found: 2062.7598.
4.1.34. 6-Aminohexyl 3-O-(3-O-(3-O-(3-O-(3-O-(3-O-(3-O-(3-O-
(3-O-(3-O-(3-O-(3-O-(3-O-(3-O-(3-O-(b-D-glucopyranosyl)-b-D-
glucopyranosyl)-b-
glucopyranosyl)-b-
glucopyranosyl)-b-
glucopyranosyl)-b-
glucopyranosyl)-b-
D
D
D
D
D
-glucopyranosyl)-b-
D
-glucopyranosyl)-b-
-glucopyranosyl)-b-
-glucopyranosyl)-b-
-glucopyranosyl)-b-
D
D
D
D
-
-
-
-
anol to give heptadecasacchairde (1) (9.80 mg, 3.41
lmol, 51%).
-glucopyranosyl)-b-
-glucopyranosyl)-b-
-glucopyranosyl)-b-
-glucopyranosyl)-b-
D
D
D
D
½
a 2D4
ꢂ
ꢀ31.9 (c = 0.125, H2O); 1H NMR (400 MHz, D2O) d 4.70–4.76
(m, 15H), 4.49 (d, 1H, J = 7.7 Hz), 4.44 (d, 1H, J = 7.7 Hz), 4.18 (br-
d, 1H, J = 11.1 Hz), 3.26–3.95 (m, 103H), 2.82 (br-s, 1H), 2.50 (br-
s, 1H), 1.55–1.64 (m, 4H), 1.36 (br-s, 4H); 13C NMR (100 MHz,
D2O) d 103.6, 103.3, 85.1, 85.0 ꢃ 2, 76.5, 74.2, 74.1, 74.0 ꢃ 2,
70.4, 69.3, 68.9, 61.6, 61.5, 20.9; IR (solid): 3308, 2897, 1982,
1811, 1656, 1373, 1043, 891, 649, 526 cmꢀ1; HRMS (ESI-TOF) calcd
for C108H186NO86 [M+H]+ m/z = 2873.0212, found: 2873.0244.
-glucopyranoside (2)
To a stirred solution of the protected hexadecasaccharide 38
(50.7 mg, 9.34 mol) in NH3/THF/EtOH (8.50 mL/1.00 mL/
l
0.500 mL) was added a large amount of lithium (100 mg) at
ꢀ78 °C. After being stirred under reflux for 1.5 h, the reaction mix-
ture was added methanol (1.00 mL). After being stirred at room
temperature for 12 h, the reaction mixture was concentrated in va-
cuo. The residue was purified by size exclusion column chromatog-
raphy on Sephadex LH-20 eluted with water and further purified
by revese-phase column chromatography (Bond Elut-C18) with
80:20 water:methanol to give hexadecasaccharide 2 (20.2 mg,
4.1.32. 6-Aminohexyl 3-O-(3-O-(3-O-(3-O-(3-O-(3-O-(3-O-(b-D-
glucopyranosyl)-b-
glucopyranosyl)-b-
glucopyranosyl)-b-
D
D
D
-glucopyranosyl)-b-
D
-glucopyranosyl)-b-
-glucopyranosyl)-b-
D-
-glucopyranosyl)-b-
D
D-
-glucopyranoside (6)
To
a
stirred solution of the protected octasaccharide 34
mol) in NH3/THF/EtOH (8.50 mL/1.00 mL/0.500
7.45
l
mol, 80%). ½a 2D3
ꢂ
ꢀ22.5 (c = 0.130, H2O); 1H NMR (400 MHz,
(28.5 mg, 10.6
l
D2O) d 4.71–4.77 (m, 14H), 4.58 (d, 1H, J = 7.7 Hz), 4.45 (d, 1H,
J = 7.7 Hz), 3.88–3.92 (m, 17H), 3.67–3.78 (m, 32H), 3.32–3.56
(m, 49H), 2.96 (t, 2H, J = 7.7 Hz), 1.57–1.63 (m, 4H), 1.38 (br-s,
4H); 13C NMR (100 MHz, D2O) d 103.4, 85.1, 76.5, 74.1, 70.4, 69.0,
61.5; IR (solid): 3341, 2898, 2327, 2155, 1597, 1458, 1313, 1154,
mL) was added a large amount of lithium (100 mg) at ꢀ78 °C. After
being stirred under reflux for 1.5 h, the reaction mixture was
added methanol (1.00 mL). After being stirred at room tempera-
ture for 12 h, the reaction mixture was concentrated in vacuo.
The residue was purified by size exclusion column chromatogra-
phy on Sephadex LH-20 eluted with water and further purified
1072, 1042, 806, 670, 526 cmꢀ1
102H176NO81 [M+H]+ m/z = 2710.9684, found: 2710.9724.
; HRMS (ESI-TOF) calcd for
C

H. Tanaka et al. / Bioorg. Med. Chem. 20 (2012) 3898–3914
3913
4.2. Biological experiment
9-19 pulse train, and probe temperature was set to 5 °C. For STD-
NMR experiments, either 128 scans (cryogenic probe) or 1024
scans (normal probe) with 4 repetition loops were required to ob-
tain good signal to noise ratio, and protein signals were suppressed
using T2rho filter. 1H and 13C chemical shifts indicated with parts
per million (ppm) were calibrated based on outer standards of
the chemical shift of 4,4-dimethyl-4-silapentane-1-sulfonic acid
(DSS) at 0 ppm. Assignments of 1H and 13C NMR signals of
b(1,3)Glc 16-mer 2 were obtained by 1H–1H DQF-COSY, 1H–1H
4.2.1. Binding assay to soluble Dectin-1
Binding ability of various oligosaccharides to dectin-1 was as-
sessed by competitive enzyme linked immunosorbent assay (ELI-
SA). Briefly, (1,6)-monoglucosyl branched (1,3)-b-
Schizophyllum commune, SPG (1 g/ml), dissolved in phosphate
buffer pH 6.9, was coated on ELISA plate (NUNC) by overnight incu-
bation at 4 °C. The unbound SPG was washed with phosphate-buf-
fered saline (PBS) containing 0.05% Tween20 (PBST), and the plate
was blocked with PBS containing 0.5% BSA (BPBS) by 2 h incuba-
tion at room temperature. Various oligosaccharides or SPG samples
D-glucan from
l
NOESY, 1H-13
C C HSQC-TOCSY experiments
HSQC and 1H–13
measured at 25 °C. NMR data were processed with XWIN-NMR
(ver.3.5) and the spectra were displayed using XWIN-PLOT
(ver.3.5).
were diluted with BPBS to prepare 0 to 500
lg/ml and mixed with
soluble Dectin-1-Fc (1 g/ml) for 30 min before adding to the SPG-
l
coated ELISA plate. The plate containing soluble Dectin-1-Fc (sDec-
tin-1) was incubated for 1 h at room temperature, washed with
PBST, and further incubated with 2000-fold diluted peroxidase-
conjugated anti-HA IgG (SantaCruz). The binding of Dectin-1 to so-
lid-phase SPG was monitored by reaction of peroxidase substrate
TMB (KPL Inc., MD) and color development was stopped with
1 m phosphoric acid. The absorbance at 450 nm was measured
by microplate reader, MTP450 (Corona electric, Japan) The data
are presented as means for duplicate samples.
4.3.1. Expression of Dection-1
Mouse Dectin-1 CTLD (Gly113–Leu244) was produced as inclu-
sion bodies in Escherichia coli, and the protein was refolded as de-
scribed following. We constructed pET28a-Dectin-1 CTLD vector,
which composed of an N-terminal hexahistidine-tag and the
Dectin-1 CTLD coding sequence with a thrombin cleavage site
(Novagen). The plasmid was then transformed into E. coli
BL21(DE3)codonplus. Cells were cultured in LB medium and har-
vested after 6 h induction with isopropylthio-b-D-galactopyrano-
side at 37 °C, and then sonicated in PBS with BugBuster
(Novagen). The resultant inclusion bodies were solubilized with
8 M urea including 50 mM Tris–HCl (pH 8.0) and 50 mM NaCl.
The solubilized protein was refolded by dilution into 200 mM
4.2.2. Luciferase-assisted NF-B assay
On the day prior to transfection, 293T cells were plated (1 ꢃ 104
cells/well) in 96-well plates cultured in DMEM containing 10% FBS.
The cells incubated for 20 h at 37 °C under 5% CO2 atmospheres.
Then, transfection was performed in the 96-well plates using Lipo-
fectamine LTX (Invitrogen) and PLUS Reagent (Invitrogen) with
Plasmid DNA mixtures for Dectin-1 (p3x-FLAG-CMV 14/Dectin-
1A, p3x-FLAG-CMV 14/CARD9, pBud-CE4.1/Bcl10, pGL4.32[luc2P/
Tris–HCl (pH 8.0), 0.4 M L-arginine, 5.0 mM reduced glutathione
and 0.5 mM oxidized glutathione at 4 °C for 16 h. After refolding,
the protein solution was passed through a Ni-Sepharose 6 Fast Flow
column (GE Healthcare) and desired protein was eluted with
500 mM imidazole in PBS. Yielded protein was treated with throm-
bin to remove the hexahistidine-tag moiety, and then concentrated
by ultrafiltration using Amicon Ultra 10 K (Millipore). Finally, the
protein was dissolved in appropriate buffer for NMR analysis.
NF-
jB-RE/Hygro], and pGL4.74[hRluc/TK]) or for TLR4 (p3xFLAG-
CMV9/TLR4, pBud-CE4.1/M
D-2, pGL4.32[luc2P/NF- B-RE/Hygro],
j
and pGL4.74[hRluc/TK]). The transfection mixture was added drop-
wise to the cells and incubated for 18 h in DMEM containing 10%
FBS. At 18 h after transfection, the cells were stimulated with the
oligosaccharides, SPG, or combination of the oligosaccharides and
LPS for 6 h. The cells were then lysed with Passive Lysis Buffer (Pro-
mega). Luciferase activity was measured using the Dual-Luciferase
Reporter Assay System (Promega). The luciferase assay reagents
were added to 20 lL of the lysate with an infector, and the results
were read immediately with Microplate Luminometer (Berthold
Technologies GmbH, KG, Bad Wildbad, Germany). Luciferase activ-
ity was expressed as the ratio of the NF-jB luciferase (firefly) activ-
Acknowledgments
We sincerely thank Dr. Yukishige Ito (RIKEN, ERATO JST) and Dr.
Osamu Kanie (ERATO JST) for use of the NMR spectrometer. We
thank Ms. Akemi Ikeda for expression and purification of Dectin-
1.This work was supported by Grant-in-Aid for Scientific Research
(B) (Grant-in-aid No. 23310150) and Research (C) (Grant-in-aid No.
20550149) from the Ministry of Education, Culture, Sports, Science,
and Technology. We thank EIWEISS Chemical Corporation for gen-
erous gift of EDCI and Central Glass Co., Ltd. for their gift of triflu-
oromethanesufonic acid anhydride.
ity to the RL-TK (renilla) activity. The data are presented as
means standard deviation for triplicate samples.
4.3. NMR experiments
Supplementary data
NMR spectra were recorded with 500 MHz and 600 MHz spec-
trometers (DRX-500 and DRX-600, BrukerBioSpin) equipped with
either a triple resonance inverse cryogenic (cryo-TXI) probe or a
triple resonance inverse (TXI) probe. Extracellular C-type lectin-
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.04.017.
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