Lactotriaose-containing carbosilane dendrimers: Syntheses and lectin-binding activities

Article abstract of DOI:10.1016/j.bmc.2006.12.030

Carbosilane dendrimers periphery-functionalized with lactotriaose (GlcNAcβ1-3Galβ1-4Glc) with valencies of three, four, six, and twelve were prepared for use in a lectin-binding assay. A lactotriaose derivative was prepared from d-glucosamine and d-lactos

Full text of DOI:10.1016/j.bmc.2006.12.030

Bioorganic & Medicinal Chemistry 15 (2007) 1606–1614
Lactotriaose-containing carbosilane dendrimers: Syntheses
and lectin-binding activities
Akihiro Yamada,a Ken Hatano,a Tetsuo Koyama,a Koji Matsuoka,a
Naonori Takahashi,b,c Kazuya I. P. J. Hidari,b,c Takashi Suzuki,b,c
Yasuo Suzukic,d and Daiyo Terunumaa,*
aArea for Molecular Function, Division of Material Science, Graduate School of Science and Engineering, Saitama University,
255 Shimo-ohkubo, Sakura-ku, Saitama 338-8570, Japan
bDepartment of Biochemistry, University of Shizuoka, School of Pharmaceutical Sciences 52-1, Yada, Shizuoka-shi,
Shizuoka 422-8526, Japan
cCore Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation,
and COE Program in the 21st Century, Japan
dDepartment of Biomedical Sciences, College of Life and Health Sciences, Chubu University, 1200 Matsumoto-cho,
Kasugai-shi, Aichi 487-8501, Japan
Received 4 November 2006; revised 13 December 2006; accepted 14 December 2006
Available online 20 December 2006
Abstract—Carbosilane dendrimers periphery-functionalized with lactotriaose (GlcNAcb1–3Galb1–4Glc) with valencies of three,
four, six, and twelve were prepared for use in a lectin-binding assay. A lactotriaose derivative was prepared from D-glucosamine
and D-lactose derivatives. The N-Troc-protected glucosamine glycosyl donor and 30-O-unprotected lactose glycosyl acceptor were
condensed in the presence of silver trifluoromethanesulfonate and methylsulfenyl bromide to provide corresponding trisaccharide
with new b-1-3 linkages in 92% yield. The protection group of the trisaccharide was transformed into an acetyl group. The 4-pen-
tenyl glycoside was prepared from the acetate via glycosyl bromide. The alkene was converted into acetyl sulfide by addition of thio-
acetic acid under radical conditions. The lactotriaose unit was linked with carbosilane dendrimers to afford acetyl-protected
glycodendrimers. De-O-acetylation of the dendrimers was carried out in the presence of sodium methoxide and then aq NaOH
to give the desired lactotriaose clusters using a carbosilane dendrimer backbone. Their biological activities toward WGA were eval-
uated by fluorescence methods. The binding constants of free lactotriaose and trivalent, tetravalent, hexavalent, and dodecavalent
glycodendrimers to WGA were determined to be 1.1 · 103, 4.4 · 104, 5.1 · 104, 2.8 · 106, and 1.3 · 106 Mꢀ1, respectively. The hexa-
valent glycodendrimer showed a 2500-fold larger binding effect than that of free lactotriaose.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
also reported syntheses of some glycodendrimers having
globotriaose,4 galabiose,5 sialyllactose,6 mannobiose,7
and functional saccharides8 in which carbosilane dendri-
mers were employed as the scaffolds of carbohydrate,
and we have described the biological activities of some
of these glycodendrimers.5,9 The hexavalent glycoden-
drimer periphery-functionalized with globotriaose
(Gala1–4Galb1–4Glcb1–) neutralized Vero toxin-pro-
ducing Escherichia coli O157:H7 with high affinity in
in vivo experiments using mice.9a
It is known that carbohydrate substructures of cell sur-
face glycoconjugates constitute important binding sites
for a variety of pathogen infections.1 Multivalent carbo-
hydrate–protein interactions are often observed in bio-
logical systems and they appear to enhance affinity.
This event due to multivalency has also been called the
glycoside cluster effect.2 Artificial multivalent carbohy-
drate ligands have been synthesized, and some of them
have shown remarkable enhancement effects.3 We have
Recently, syntheses of lacto-N-neotetraose (Galb1–
4GlcNAcb1–3Galb1–4Glc) clusters targeting dengue
virus have been reported.10 Dengue virus is involved in
dengue fever and dengue hemorrhagic fever. The inci-
dence of these viral infections is increasing in many
Keywords: WGA; Glycocluster; Carbosilane dendrimer; Lactotriaose.
*
Corresponding author. Tel./fax: +81 48 858 3532; e-mail:
teru@fms.saitama-u.ac.jp
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.12.030

A. Yamada et al. / Bioorg. Med. Chem. 15 (2007) 1606–1614
1607
OAc
RO OBn
countries located in tropical and subtropical areas.
However, no effective vaccines or anti-dengue agents
have yet been developed. Suzuki et al. discovered that
dengue virus preferentially adheres to lacto-N-neotetra-
ose located on the surfaces of eukaryotic cells. Synthe-
sized hexavalent lacto-N-neotetraose cluster inhibited
dengue virus infection of BHK-21 cells, thus demon-
strating the potential therapeutic usefulness of lacto-N-
neotetraose cluster compounds as dengue virus inhibitors.11
OBn
OBn
O
AcO
AcO
O
BnO
SEt
+
O
HO
O
NHTroc
OBn
OBn
2 R = H
3 R = Ac
1
a or b
AcO OR1
OR1
OR1
OAc
O
O
R1O
AcO
AcO
O
O
O
OR1
NHR2
OR1
4 R1 = Bn, R2 = Troc
5 R1 = Bn, R2 = Ac
6 R1 = R2 = Ac
f
c
It would be interesting to investigate why hexavalent
clusters are so effective between different types of donors
and acceptors. The biological activities of many artificial
glycoclusters have been evaluated by using lectins. Lec-
tins are thought to be ideal models for studying carbo-
hydrate–protein interactions with glycoclusters. To
investigate the binding activities of lactotriaose clusters,
we selected the wheat germ agglutinin (WGA), which
specifically recognizes N-acetyl glucosamine. It is known
that fluorescence methods can be used for binding
experiments with WGA and saccharide derivatives.12
HO
O
OH
OH
OH
d
O
e
O
HO
O
HO
HO
OH
O
OH
NHAc
OH
7
AcO OAc
OAc
OAc
O
O AcO
O
O
AcO
O
AcO
O
OAc
OAc
NHAc
8
g
AcO OAc
OAc
OAc
O
O
AcO
O
AcO
AcO
O
SAc
O
O
OAc
NHAc
OAc
Herein, we describe the syntheses of lactotriaose dendri-
mers and the specific binding properties of families of
lactotriaose dendrimers with WGA.
9
Scheme 1. Reagents and conditions: (a) 2, AgOTf, MeCN, CH2Cl2,
ꢀ78 ꢁC, 10 min, then MeSBr, ClCH2CH2Cl, ꢀ78 ꢁC, 15 h, then Ac2O,
pyridine, 2 h (43%); (b) 3, AgOTf, MeCN, CH2Cl2, ꢀ60 ꢁC, 20 min,
then MeSBr, ClCH2CH2Cl, ꢀ60 to ꢀ30 ꢁC, 1 h (92%); (c) Zn, AcOH,
30 min, then Ac2O, pyridine 7.5 h (97%); (d) H2, Pd(OH)2/C, MeOH,
16 h, then Ac2O, DMAP, pyridine, 7 h (quant); (e) NaOMe, MeOH,
2 h, then aqueous NaOH, 4 h (82%); (f) HBr/AcOH, 16 h, then 4-
Furthermore, chemical synthesis of a lacto-N-neotetra-
ose residue and clusters requires many reaction steps.
It is important to improve the synthetic problems for
the practical use of a carbosilane dendrimer having lac-
to-N-neotetraose. A lactotriaose (GlcNAcb1–3Galb1–
4Glc) cluster is one of the precursors for the efficient
synthesis of a lacto-N-neotetraose cluster. Since the syn-
thesis of a lactotriaose cluster is easier than that of a lac-
to-N-neotetraose cluster, and the trisaccharide can be
easily converted into the tetrasaccharide by using enzy-
matic glycosylation, Zanini and Roy13 reported success-
ful enzymatic conversion of GlcNAc to LacNAc on a
synthetic dendrimer. Moreover, Narvor and co-
workers14 reported enzymatic synthesis of lacto-N-
neotetraose using dendrimeric polyethylene glycol.
Lacto-N-neotetraose was obtained by using b-(1–4)-ga-
lactosyl transferase with a chemo-enzymatically synthe-
sized lactotriaose cluster using dendrimeric polyethylene
glycol. Therefore, carbosilane dendrimers having lacto-
triaose are expected to be candidates for improving the
synthetic problems of the dengue virus inhibitor, having
lacto-N-neotetraose.
˚
pentene-1-ol, Ag2CO3, MS 4 A, I2, CH2Cl2, 2 days (50%); (g) AcSH,
AIBN, 1,4-dioxane, 80 ꢁC, 3 h (87%).
O-glycosylated tetrasaccharide was obtained as
a
by-product. Consequently, 30-O-unprotected lactose
derivative (3)18 was chosen as another glycosyl acceptor.
The compound 3 could be readily obtained from com-
pound 2 by treatment with methyl orthoacetate and then
with acetic acid. Glycosylation of 1 with 30-O-unprotect-
ed derivative 3 under promotion of MeSBr and AgOTf
in CH2Cl2 for 1 h at ꢀ60 to ꢀ30 ꢁC furnished trisaccha-
ride 4 in 92% yield; no stereoisomeric glycosides could
be isolated from the reaction mixture. The b-glucosa-
1
mine linkage was confirmed by the H NMR signal at
d 4.67 ppm (J1 ;2 ¼ 8:7 Hz, H-100) and the 13C NMR sig-
00 00
nals at d 102.42, 101.91, and 100.92 ppm (C-1, C-10, and
C-100, respectively). Elemental analysis and FAB-MS
spectrum ([M+H]+ 1386.7) also support the trisaccha-
ride structure. The N-Troc group of 4 was transformed
into an acetamido group by zinc powder in acetic acid,
followed by N-acetylation, to give N-Ac-protected gly-
coside (5)19 in 97% yield. Compound 5 was de-O-benzy-
lated by hydrogenolysis in methanol, and the crude
product was O-acetylated by acetic anhydride and a cat-
alytic amount of 4-dimethylaminopyridine in pyridine to
furnish acetate (6)19 (mixture of a and b anomers) in
quantitative yield. De-O-acetylation of 6 with sodium
methoxide in methanol and then saponification yielded
lactotriaose (7)20 in 82% yield after gel filtration. The
4-pentenyl glycoside (8) was prepared via glycosyl bro-
mide by treatment with silver carbonate in 50% yield.
Treatment of 8 with thioacetic acid and AIBN gave
5-acetylthiopentyl glycoside (9) in 87% yield. The struc-
2. Results and discussion
2.1. Syntheses of glycodendrimers
The known N-Troc-protected thioglycoside (1)15 was
prepared from glucosamine hydrochloride. Glycosyla-
tion of the known 30,40-O-unprotected acceptor (2)16
with donor 1 under promotion of methylsulfenyl bro-
mide (MeSBr)17 and silver trifluoromethanesulfonate
(AgOTf) in CH2Cl2 for 15 h at ꢀ78 ꢁC, followed by
O-acetylation, furnished trisaccharide (4) in 43% yield,
and about 28% of the 30,40-O-acetylated glycosyl accep-
tor was recovered (Scheme 1). A trace amount of 30,40-

1608
A. Yamada et al. / Bioorg. Med. Chem. 15 (2007) 1606–1614
1
ture of 9 was confirmed by H and 13C NMR spectra,
FAB-MS spectrum, and elemental analysis. The signals
at 2.83 (t, 2H, J = 7.3 Hz, CH2SAc) and 2.31 (s, 3H,
2.2. Interaction of glycodendrimers with WGA
The versatility of lactotriaose cluster compounds was
investigated by determining the binding specificity of
these glycoclusters with WGA. Fluorescence methods
have been used extensively to study the specific interac-
tion of WGA with sugars12 and glycopolymers.21 It is
known that the values of binding constants of WGA with
chitin oligomers [(GlcpNAc)n] increase with increase in
the length of these oligomers. Privat et al.12b reported that
changes in tryptophan fluorescence by addition of a chitin
oligomer depend on the ligand size and that oligomers
produced a 10-nm shift toward shorter wavelength and
46% enhancement of fluorescence intensity. The binding
constants of chitin oligomers to WGA were determined
to be 6.9 · 102 (GlcpNAc), 4.5 · 103 [(GlcpNAc)2],
2.0 · 104 [(GlcpNAc)3], and 2.3 · 104 Mꢀ1 [(GlcpNAc)4].
Nishimura et al.21a reported that a synthesized glycopoly-
mer carrying a GlcNAc residue effectively bound to WGA
and that the polymer produce a 6-nm shift toward a short-
er wavelength and 43% enhancement of fluorescence
intensity.
1
CH2SAc) ppm in the H NMR spectrum of 9 prove
the formation of acetylthio function.
Carbosilane dendrimers 10 and 11 were synthesized by a
previously described method.4b,c Lactotriaose derivative
9 was attached to tri- (10), tetra- (11), hexa- (12), and
dodecavalent dendritic carbosilane scaffolds (13)
(Scheme 2). Compound 9 (1.4 equiv per bromine func-
tionality) was coupled in good yields to the respective
(poly)bromide dendrimers by treatment with sodium
methoxide in a mixture of methanol and DMF. The
crude product was O-acetylated by acetic anhydride with
a catalytic amount of 4-dimethylaminopyridine in pyri-
dine. The resultant products were purified by a recycling
preparative HPLC to give acetylated glycodendrimers 14
(66%), 15 (45%), 16 (61%), and 17 (73%). All acetylated
glycodendrimers were examined by means of 1H and 13
C
NMR spectra. Finally, de-O-acetylation of the acetylat-
ed glycodendrimers with sodium methoxide in methanol
and then saponification gave lactotriaose-coated
carbosilane dendrimers trivalent 18 (68%), tetravalent
19 (81%), hexavalent 20 (54%), and dodecavalent 21
(89%). These glycodendrimers were identified by 1H
and 13C NMR spectra. Further evidence of the synthe-
sized glycodendrimers was obtained by high-resolution
mass spectroscopy (Table 1). The results showed good
agreement with the calculated values for the expected
structures. The molecular weight of the acetylated and
de-O-acetylated dodecavalent dendrimers (17 and 21)
was too great to measure with high-resolution mass
spectra.
Synthesized carbosilane dendrimers having lactotriaose,
in contrast to glycopolymers, have a monodispersed and
well-defined structure. Therefore, tests of binding activ-
ities of the dendrimers with WGA are expected to give
clearer results.
Figure 1 shows the emission spectra of WGA and its
complexs with monovalent saccharide 7 (Fig. 1A)
and dodecavalent dendrimer 21 (Fig. 1B) at pH 7.8.
The monovalent 7 showed no enhancing effect on
fluorescence intensity in the range of micromolar
R
R
R
R
Si
Si
R
R
R
R
R
R
R
R
R
R
Me
Si
Me
R
Si
Si
Si
R
R
10 R = Br
R
Si
Si
14 R = R1, R2 = Ac
18 R = R1, R2 = H
Si
R
R
R
12 R = Br
R
16 R = R1, R2 = Ac
20 R = R1, R2 = H
R
13 R = Br
17 R = R1, R2 = Ac
21 R = R1, R2 = H
Si
R
R
R2O OR2
11 R = Br
OR2
OR2
15 R = R1, R2 = Ac
19 R = R1, R2 = H
O
R2O
O
R2O
O
R1
=
O
S
R2O
O
O
NHAc
OR2
OR2
R = R1, R2 = Ac
R = R1, R2 = H
10
14
15
16
17
18
19
20
21
11
12
13
a
b
9
+
Scheme 2. Reagents and conditions: (a) NaOMe, MeOH, DMF, 3 h, then Ac2O, DMAP, pyridine, 50 ꢁC, 3 h (66% for 14, 45% for 15, 61% for 16,
and 73% for 17); (b) NaOMe, MeOH, then aqueous NaOH (68% for 18, 81% for 19, 54% for 20, and 89% for 21).

A. Yamada et al. / Bioorg. Med. Chem. 15 (2007) 1606–1614
Table 1. HRMS (ESI) of the glycodendrimers
1609
Compound
Formula
144H209N3O75S3Si+2Na+
m/z (calculated)
m/z (found)
Acetylated-trivalent (14)
Acetylated-tetravalent (15)
Acetylated-hexavalent (16)
Acetylated-dodecavalent (17)
Trivalent (18)
C
1675.0674
1454.1550
1672.0773
13136.8a
1107.9248
1413.5477
1465.5832
2164.9
1675.0668
1454.1552
1672.0805
13135.5b
1107.9281
1413.5525
1465.5858
2166.5
C184H272N4O100S4Si+3Na+
C284H426N6O150S6Si3+4Na+
C564H840N12O300S12Si5+Na+
C90H155N3O48S3Si+2Na+
Tetravalent (19)
Hexavalent (20)
Dodecavalent (21)
C
112H200N4O64S4Si+2Na+
C176H318N6O96S6Si3+3Na+
C
348H624N12O192S12Si5+4Na+
a The value is average mass.
b The value is from MALDI-TOFMS.
Table 2. Ka values of the glycodendrimers with WGA
a
Ka (Mꢀ1
)
Relative potency
0.4
A
Compound
Monovalent (7)
Trivalent (18)
1.1 · 103
4.4 · 104
5.1 · 104
2.8 · 106
1.3 · 106
1
40
0.3
0.2
Tetravalent (19)
Hexavalent (20)
Dodecavalent (21)
46
/F
2500
1200
0.1
0
a Ka values are calculated from Steck–Wallack plot analyses.
0
0.5
1
1.5
2
[S] (mM)
concentrations. However, in the range of millimolar
concentrations of 7, maximum fluorescence intensity
was enhanced by 30% and the emission maximum
was shifted from 349 to 345 nm (Fig. 1A). In contrast,
when lectin was saturated with dodecavalent 21, max-
imum fluorescence intensity was enhanced by 40% and
the emission maximum was shifted from 350 to 344 nm
(Fig. 1B). Three other dendrimers also showed
enhancement of fluorescence intensities and 8-nm
shifts of the emission maximum. The intensity was
enhanced by 33% (18), 30% (19), and 44% (29). The
Ka values of these clusters and monomeric lactotriaose
to WGA were determined by Steck–Wallack plot anal-
ysis (Table 2). The Ka value of trivalent glycocluster 18
was similar to that of tetravalent 19. The 18 and 19
were 40 times and 46 times more potent, respectively,
than lactotriaose monomer 7. The hexavalent cluster
20 showed the maximum Ka value (2.8 · 106 Mꢀ1) in
the clusters, the Ka value being 2500 times larger than
that of monovalent 7. Interestingly, dodecavalent clus-
ter 21 showed less potency than hexavalent 20, which
could be attributed to the over-crowding of the poorly
accessible surface saccharide moieties of the dendrimer
21. These results clearly indicate that lactotriaoses sup-
ported on the hexavalent glycodendrimer are suitable
for binding to WGA.
310
360
410
460
Wavelength
B
0.5
0.4
0.3
/F
0.2
0.1
0
0
5
10
15
20
[S] (μM)
3. Conclusion
310
360
410
460
High stereo-selective synthesis of a lactotriaose deriva-
tive in high yield was accomplished by using the 30-O-
unprotected lactose derivative 3 and N-Troc-protected
glucosamine derivative 1. A series of new carbosilane
dendrimers periphery-functionalized with lactotriaose
was successfully synthesized. The hexavalent glycoden-
drimer showed a 2500-fold larger binding effect than
that of free lactotriaose, though the dodecavalent one
exhibited only a 1200-fold larger binding effect.
Wavelength
Figure 1. Changes in fluorescence spectrum of WGA (0.69 lM, 3.0 mL
of Tris–HCl buffer-containing 1.25 M NaCl and 25 mM CaCl2, pH
7.8, 5.0 ꢁC) upon addition of (A) 40-lL aliquots of the monovalent
saccharide 7 (11.6 mM) and (B) 10-lL aliquots of the dodecavalent
dendrimer 21 (0.32 mM). DF is change in the fluorescence intensity at
fluorescence maximum wavelength of a solution containing the lectin
obtained by exciting at 295 nm with a total ligand concentration [S],
and F0 is the fluorescence intensity of lectin alone.

1610
A. Yamada et al. / Bioorg. Med. Chem. 15 (2007) 1606–1614
J1 ;2 ¼ 7:7 Hz, H-10), 4.46 (d, 1H, J1,2 = 7.6 Hz, H-1),
4.39–4.45 (m, 2H, CHaPh, CHbPh), 4.27 (d, 1H,
J = 11.8 Hz, CHaPh), 4.20 (d, 2H, J = 3.4 Hz, H-600ab),
4.06 (t, 1H, J = 9.4 Hz, H-4), 3.80 (dd, 1H, J = 2.8 Hz,
J = 10.4 Hz, H-6b), 3.66–3.71 (m, 2H, H-6a, H-30),
3.45–3.61 (m, 6H, H-2, H-3, H-20, H-50, H-200, H-500),
3.31–3.35 (m, 3H, H-5, H-60ab), 2.06 (s, 3H, OAc),
2.03 (s, 3H, OAc), 2.01 (s, 3H, OAc), 1.96 (s, 3H,
OAc). 13C NMR (CDCl3) d 170.71, 170.50, 169.78,
169.24, 153.79, 138.97, 138.52, 138.13, 137.96, 137.40,
128.65, 128.37, 128.32, 128.30, 128.22, 128.08, 127.99,
127.90, 127.85, 127.83, 127.68, 127.62, 127.57, 127.50,
127.30, 126.85, 102.42 (C-1), 101.91 (C-10), 100.92 (C-
100), 95.53, 82.63, 81.59, 80.89, 75.83, 75.26, 74.97,
74.84, 74.20, 73.53, 73.42, 72.51, 71.82, 71.66, 70.87,
69.37, 68.47, 67.94, 67.84, 61.85, 55.98, 20.70, 20.67,
20.56, 20.52; Anal. Calcd for C71H78Cl3NO21: C, 61.45;
H, 5.67; N, 1.01. Found: C, 61.33; H, 5.59; N, 0.94. FAB-
MS: [M+H]+ 1386.7.
0
0
4. Experimental
4.1. General methods
1H and 13C NMR spectra were recorded on a Bruker
DRX 400, at 400 MHz for proton and at 100 MHz for
carbons, respectively. Proton chemical shifts are given
in ppm with use of tetramethylsilane (0 ppm) or residual
solvent peaks as internal standard. NMR signals were
assigned by 1H, 13C, HH, and HC COSY measurements.
FAB mass spectra were obtained with a JEOL JMS-
HX110A spectrometer. Optical rotations were recorded
on a JASCO DIP-1000 digital polarimeter at ambient
temperature, using a 10-cm micro cell. Recycling pre-
parative HPLC was performed with a LC-908 or LC-
918W (Japan Analytical Industry Co., Ltd) connected
to an RI detector RI-5.
4.1.1. Benzyl O-[3,4,6,-tri-O-acetyl-2-deoxy-2-(2,2,2,-tri-
chloroethoxycarbonylamino)- b-D-glucopyranosyl]-(1 !
3)-O-(2,6-di-O-benzyl-4-O-acetyl-b-D-galactopyranosyl)-
(1 ! 4)-2,3,6-tri-O-benzyl-b-D-glucopyranoside (4).
4.1.1.1. Prepared from 1 and 2. A solution of AgOTf
(135.1 mg, 0.526 mmol) in MeCN (0.9 mL) was added to
a mixture of glycosyl donor 115 (123.0 mg, 0.234 mmol)
and acceptor 216 (139.1 mg, 0.158 mmol) in CH2Cl2
(1.75 mL) at ꢀ78 ꢁC under Ar. After 10 min, a 4 M solu-
tion of methylsulfenyl bromide in 1,2-dichloroethane
(98.8 lL)17 was added during 5 min. The reaction mix-
ture was stirred for 15 h at same temperature, diisopro-
pylamine (1.0 mL) was added, and the mixture was
stirred for 30 min. After concentration of the reaction
mixture, CHCl3 was added to the residue. The mixture
was filtered and concentrated, and the residue was treat-
ed with pyridine (4 mL) and Ac2O (2 mL) for 2 h. The
resulting solution was concentrated, and the residue
was purified by a recycling preparative HPLC (column,
JAIGEL-1H and 2H; solvent, chloroform) to give com-
pound 4 (93.4 mg, 43%).
4.1.2. Benzyl O-(2-acetoamido-3,4,6,-tri-O-acetyl-2-de-
oxy-b-D-glucopyranosyl)-(1 ! 3)-O-(2,6-di-O-benzyl-4-
O-acetyl-b-D-galactopyranosyl)-(1 ! 4)-2,3,6-tri-O-ben-
zyl-b-D-glucopyranoside (5). Compound
4
(5.03 g,
3.62 mmol) was dissolved in AcOH (50 mL), and Zinc
powder (10.25 g) was added. The mixture was stirred
for 30 min, filtered through Celite, and concentrated.
The residue was treated with pyridine (15 mL) and
Ac2O (7 mL) at room temperature for 7.5 h. The mix-
ture was co-concentrated with toluene. The residue
was dissolved in EtOAc, washed with 1 M HCl aq, sat-
urated NaHCO3 solution, and brine. The organic layer
was dried (MgSO4) and concentrated. The residue was
purified by column chromatography on silica gel (1:1
EtOAc/hexane then EtOAc) to give compound 519
(4.39 g, 97%).
4.1.3. O-(2-Acetoamido-3,4,6,-tri-O-acetyl-2-deoxy-b-D-
glucopyranosyl)-(1 ! 3)-O-(2,4,6-tri-O-acetyl-b-D-galac-
topyranosyl)-(1 ! 4)-2,3,6-tri-O-acetyl-b-D-glucopyrano-
syl acetate (6). A mixture of compound 5 (4.12 g,
3.28 mmol) and 20% Pd(OH)2/C (1.0 g) in MeOH
(15 mL) was stirred under hydrogen gas for 16 h at
room temperature and then filtered through Celite.
The filtrate was concentrated. The residue was treated
with pyridine (30 mL), Ac2O (20 mL), and 4-dimethyla-
minopyridine (28 mg) at room temperature for 7 h. The
mixture was co-concentrated with toluene. The residue
was dissolved in EtOAc, washed with 1 M HCl aq, sat-
urated NaHCO3 solution, and brine. The organic layer
was dried (MgSO4) and concentrated. The residue was
purified by column chromatography on silica gel
(EtOAc) to give compound 619 (3.16 g, quant).
4.1.1.2. Prepared from 1 and 3. A solution of AgOTf
(16.64 g, 64.8 mmol) in MeCN (90 mL) was added to a
mixture of glycosyl donor 1 (16.40 g, 31.2 mmol)
and acceptor 318 (19.16 g, 20.7 mmol) in CH2Cl2
(190 mL) at ꢀ60 ꢁC under Ar. After 20 min, a 4 M solu-
tion of methylsulfenyl bromide in 1,2-dichloroethane
(13.1 mL) was added during 40 min. The temperature
was gradually raised to ꢀ30 ꢁC during 1 h, diisopropyl-
amine (50 mL) was added, and the mixture was stirred
for 15 min at ꢀ30 ꢁC. After concentration of the reaction
mixture, CHCl3 was added to the residue. The mixture
was filtered and concentrated, and the residue was puri-
fied by column chromatography on silica gel (3:7 then 2:3
26
EtOAc/hexane) to give compound 4 (26.47 g, 92%). ½aꢁD
ꢀ9.2ꢁ (c 1.1, CHCl3). 1H NMR (CDCl3) d 7.42–7.18 (m,
30H, aromatic), 5.41 (d, 1H, J = 3.5 Hz, H-40), 5.00 (t,
1H, J = 9.6 Hz, H-400), 4.97 (d, 1 H, J = 10.4 Hz,
CHbPh), 4.93 (d, 1 H, J = 12.3 Hz, CHbPh), 4.90 (d, 1
H, J = 11.2 Hz, CHbPh), 4.87 (d, 1 H, J = 13.5 Hz,
CHaPh), 4.69–4.78 (m, 4H, H-300, CHaPh, CHbPh · 2),
4.1.4. O-(2-Acetamido-2-deoxy-b-D-glucopyranosyl)-(1 !
3)-O-(b-D-galactopyranosyl)-(1 ! 4)-D-glucopyranose (7).
A solution of 6 (567.8 mg, 0.588 mmol) in MeOH
(5 mL) was treated with NaOMe (31.7 mg, 0.587 mmol)
at room temperature for 2 h. The mixture was concentrat-
ed, and 0.1 M NaOH aq (4 mL) was then added to the
residue. After 4 h, the solution was neutralized by Amber-
lite IR120B (H+) resin. The resin was filtered off and fil-
trate was concentrated to dryness. Purification of the
4.67 (d, 1H, J1 ;2 ¼ 8:7 Hz, H-100), 4.62–4.68 (m, 3H,
00 00
OCH2CCl3, CHaPh), 4.58 (d, 1H, J = 12.0 Hz, CHaPh),
4.49 (d, 1H, J = 11.6 Hz, NH), 4.48 (d, 1H,

A. Yamada et al. / Bioorg. Med. Chem. 15 (2007) 1606–1614
1611
5.44–5.49 (m, 2 H, H-300, NH), 5.31 (d, 1H, J3 ;4 ¼ 3:3
Hz, H-40), 5.15 (t, 1H, J = 9.4 Hz, H-3), 4.97–5.05 (m,
3H, H-20, H-100, H-400), 4.86 (dd, 1H, J1,2 = 8.0 Hz,
J2,3 = 9.5 Hz, H-2), 4.42–4.46 (m, 2H, H-1, H-6b),
4.33–4.38 (m, 2H, H-10, H-60b), 4.10 (dd, 1H,
J = 5.4 Hz, J = 11.9 Hz, H-6a), 4.00–4.07 (m, 3H, H-
60a, H-600ab), 3.70–3.84 (m, 4 H, H-4, H-30, H-50, one
of OCH2), 3.64–3.68 (m, 1H, H-500), 3.58–3.59 (m, 1H,
H-5), 3.41–3.47 (m, 1H, one of OCH2), 3.24–3.31 (m,
1H, H-200), 2.83 (t, 2H, J = 7.3 Hz, SCH2), 2.31 (s, 3H,
SAc), 2.11 (s, 3H, Ac), 2.10 (s, 3H, Ac), 2.09 (s, 3H,
Ac), 2.08 (s, 3H, Ac), 2.07 (s, 3H, Ac), 2.03 (s, 3H,
Ac), 2.01 (s, 3H, Ac), 2.00 (s, 3H, Ac), 1.95 (s, 3H,
Ac), 1.90 (s, 3H, Ac), 1.52–1.57 (m, 4H,
OCH2CH2CH2CH2CH2S), 1.32–1.42 (m, 2H, OCH2-
CH2CH2CH2CH2S). 13C NMR (CDCl3) d 195.76,
170.58, 170.44, 170.39, 170.27, 169.74, 169.52, 169.43,
169.33, 169.01, 100.58, 100.39, 99.44, 75.97, 75.63,
72.55, 72.46, 71.53, 71.45, 71.06, 70.79, 69.58, 68.73,
68.58, 62.04, 61.53, 61.02, 55.92, 39.18, 35.01, 30.48,
29.01, 28.76, 28.71, 24.86, 23.13, 20.73, 20.60, 20.55,
20.51; Anal. Calcd for C43H61NO25: C, 50.60; H, 6.13;
N, 1.31. Found: C, 50.29; H, 6.09; N, 1.29. FABMS:
[M+H]+ 1068.5; [M+Na]+ 1090.5.
0
0
crude product by gel permeation chromatography (Sepha-
dex G50, 5% HOAc aq eluent) gave 720 (263.3 mg, 82%)
as white powder after lyophilization.
4.1.5. 4-Pentenyl (2-acetoamido-3,4,6,-tri-O-acetyl-2-
deoxy-b-D-glucopyranosyl)-(1 ! 3)-O-(2,4,6-tri-O-ace-
tyl-b-D-galactopyranosyl)-(1 ! 4)-2,3,6-tri-O-acetyl-b-
D-glucopyranoside (8). To a suspension of compound 6
(6.64 g, 6.87 mmol), AcOH (30 mL) was added 33%
HBr/AcOH (3.66 mL, 20.62 mmol) at 0 ꢁC. The mixture
was stirred for 16 h at room temperature under dark-
ness. The mixture was poured into ice-water and extract-
ed with CHCl3. The solution was washed with water,
saturated NaHCO3 solution, and brine. The organic
layer was dried (MgSO4) and concentrated. The crude
˚
product was dissolved in CH2Cl2 (66 mL), and 4 A
molecular sieves powder (6.78 g) and 4-pentene-1-ol
(3.55 mL, 34.37 mmol) were added successively. The
mixture was stirred for 1 h at room temperature.
Ag2CO3 (4.75 g, 17.23 mmol) and I2 (39.4 mg) were add-
ed to the mixture at 0 ꢁC. After stirring under the dark
at room temperature for 2 days, the mixture was filtered
through Celite and concentrated. The residue was puri-
fied by column chromatography on silica gel (9:1
EtOAc/toluene) to give compound 8 (3.39 g, 50%).
26
1
½aꢁD +13.9ꢁ (c 1.3, CHCl3). H NMR (CDCl3) d 5.72–
5.83 (m, 1H, –CH = CH2), 5.48 (dd, 1H, J = 9.3 Hz,
J = 10.5 Hz, H-300), 5.40 (d, 1H, J = 5.4 Hz, NH), 5.33
4.1.7. Acetylated trivalent glycodendrimer (14). A mix-
ture of 10 (37.5 mg, 79.6 lmol) and 9 (360.2 mg,
337.3 lmol) was dissolved in a mixture of DMF
(0.4 mL) and MeOH (0.3 mL), and the solution was
treated with 28% NaOMe methanolic solution (82 lL,
337.3 lmol) at room temperature for 3 h. AcOH
(0.5 mL) was then added to the mixture, and the result-
ing solution was evaporated under reduced pressure.
The residue was treated with pyridine (10 mL), AcOH
(5 mL), and 4-dimethylaminopyridine (10.1 mg) at
50 ꢁC for 3 h. The resulting solution was poured into
ice-water and extracted with EtOAc. The organic solu-
tion was washed with 1 M HCl aq, saturated NaHCO3
aq, and brine. Organic phase was dried (MgSO4) and
concentrated. Purification by recycling preparative
(d, 1H, J3 ;4 ¼ 3:4 Hz, H-40), 5.17 (t, 1H, J = 9.4 Hz,
H-3), 4.95–5.07 (m, 5H, H-20, H-100, H-400, –CH@CH2),
4.89 (dd, 1H, J1,2 = 8.0 Hz, J2,3 = 9.6 Hz, H-2), 4.45
(dd, 1H, J5,6b = 1.7 Hz, J6a,6b = 11.6 Hz, H-6b), 4.44
(d, 1H, J1,2 = 7.6 Hz, H-1), 4.38 (dd, 1H, J = 2.5 Hz,
0
0
J = 12.3 Hz, H-60b), 4.35 (d, 1H, J1 ;2 ¼ 8:0 Hz, H-10),
4.12 (dd, 1H, J5,6b = 5.4 Hz, J6a,6b = 11.9 Hz, H-6a),
4.04–4.08 (m, 3H, H-60a, H-600ab), 3.81–3.87 (m, 1H,
one of OCH2), 3.72–3.80 (m, 3H, H-4, H-30, H-50),
3.64–3.68 (m, 1H, H-500), 3.57–3.61 (m, 1H, H-5),
3.45–3.50 (m, 1H, one of OCH2), 3.25–3.31 (m, 1H,
H-200), 1.91–2.36 (m, 32H, Ac, OCH2CH2CH2
CH@CH2), 1.62–1.70 (m, 2H, OCH2CH2CH2
CH@CH2); 13C NMR (CDCl3) d 170.65, 170.49,
170.45, 170.32, 169.81, 169.56, 169.49, 169.39, 169.68,
137.71 (–CH@CH2), 115.00 (–CH@CH2), 100.64
(C-10), 100.54 (C-1), 99.49 (C-100), 75.96, 75.71, 72.60,
72.53, 71.61, 71.56, 71.10, 70.90, 69.22, 68.80, 68.63,
62.11, 61.56, 61.06, 56.51, 29.73, 28.50, 23.21, 20.79,
20.67, 20.61, 20.57; Anal. Calcd for C43H61NO25: C,
52.07; H, 6.20; N, 1.41. Found: C, 51.87; H, 6.24; N,
1.41. FABMS: [M+H]+ 992.7; [M+Na]+ 1014.7.
0
0
HPLC (column, JAIGEL-2.5H and 3H; solvent, chloro-
form) afforded 14 (172.9 mg, 66%). ½aꢁD +10.7ꢁ (c 0.8,
22
CHCl3). 1H NMR (CDCl3) d 7.43–7.45, 7.32–7.33
(2m, 5H, SiPh), 5.44–5.49 (m, 6H, H-300, NH), 5.32 (s,
3H, H-40), 5.15 (t, 3H, J = 9.4 Hz, H-3), 4.97–5.05 (m,
9H, H-20, H-100, H-400), 4.86 (dd, 3H, J1,2 = 8.1 Hz,
J2,3 = 9.3 Hz, H-2), 4.42–4.46 (m, 6H, H-1, H-6b),
4.33–4.38 (m, 6H, H-10, H-60b), 4.04–4.11 (m, 12H, H-
6a, H-60a, H-600ab), 3.65–3.82 (m, 15H, H-4, H-30, H-
50, H-500, one of OCH2), 3.55–3.61 (m, 3H, H-5), 3.39–
3.46 (m, 3H, one of OCH2), 3.24–3.31 (m, 3H, H-200),
2.48 (t, 6H, J = 7.1 Hz, SCH2), 2.42 (t, 6H, J = 7.1 Hz,
SCH2), 1.90–2.11 (m, 90H, Ac), 1.49–1.60 (m, 18H,
OCH2CH2CH2CH2CH2S), 1.33–1.40 (m, 6H, SiCH2-
CH2CH2S), 0.87–0.91 (m, 6H, SiCH2). 13C NMR
(CDCl3) d 170.50, 170.36, 170.30, 170.25, 169.67,
169.48, 169.35, 169.24, 168.92, 136.07, 133.79, 128.94,
127.68, 100.55, 100.37, 99.41, 76.11, 75.59, 72.49,
72.40, 71.40, 71.03, 70.66, 69.68, 68.62, 68.53, 62.01,
61.52, 60.98, 55.80, 35.67, 31.77, 29.14, 28.83, 24.92,
23.84, 23.11, 20.70, 20.51, 11.67; HRMS(ESI) Anal.
Calcd for C144H209N3O75S3Si[M+2Na]2+: 1675.0674.
Found: 1675.0668.
4.1.6. 5-Acetylthiopentyl (2-acetoamido-3,4,6,-tri-O-ace-
tyl-2-deoxy-b-D-glucopyranosyl)-(1 ! 3)-O-(2,4,6-tri-O-
acetyl-b-D-galactopyranosyl)-(1 ! 4)-2,3,6-tri-O-acetyl-
b-D-glucopyranoside (9). A solution of compound 8
(479.9 mg, 0.484 mmol) in 1,4-dioxane (0.5 mL) was
treated with thioacetic acid (0.69 mL, 9.676 mmol) and
AIBN (158.8 mg, 0.968 mmol) at 80 ꢁC for 3 h under
Ar. Cyclohexene (98 lL, 0.968 mmol) was added with
stirring for 10 min. The resulting solution was purified
by column chromatography on silica gel (1:1 EtOAc/
hexane then EtOAc) to yield compound 9 (449.7 mg,
29
1
87%). ½aꢁD +9.9ꢁ (c 1.2, CHCl3). H NMR (CDCl3) d

1612
A. Yamada et al. / Bioorg. Med. Chem. 15 (2007) 1606–1614
4.1.8. Acetylated tetravalent glycodendrimer (15). A cou-
pling reaction between 11 (27.7 mg, 53.7 lmol) and 9
3.66–3.69 (m, 12H, H-500), 3.59–3.63 (m, 12H, H-5),
3.42–3.48 (m, 12H, one of OCH2), 3.29–3.33 (m, 12H,
H-200), 2.45–2.50 (m, 48H, SCH2), 1.91–2.12 (m, 360H,
Ac), 1.50–1.62 (m, 72H, OCH2CH2CH2CH2CH2S),
1.36–1.42 (m, 24H, SiCH2CH2CH2S), 1.19–1.25 (m,
8H, SiCH2CH2CH2Si), 0.49–0.62 (m, 20H, SiCH2).
13C NMR (CDCl3) d 170.51, 170.36, 169.67, 169.49,
169.34, 169.22, 168.99, 100.61, 100.41, 99.50, 75.66,
72.46, 71.40, 71.10, 70.62, 69.72, 68.54, 62.06, 61.55,
61.01, 55.95, 55.73, 35.91, 31.96, 29.25, 28.91, 24.99,
24.12, 23.20, 20.79, 20.54, 20.44, 18.26, 17.34, 11.90;
MALD-TOFMS Anal. Calcd for C564H840N12O300S12-
Si5[M+Na]+: 13136.8 (average mass). Found: 13135.5.
(333.1 mg, 311.9 lmol) was carried out in the same man-
22
ner as described for 14 to give 15 (103.0 mg, 45%). ½aꢁD
1
+10.1ꢁ (c 1.1, CHCl3). H NMR (CDCl3) d 5.46–5.50
(m, 8H, H-300, NH), 5.34 (s, 4H, H-40), 5.17 (t, 4H,
J = 9.3 Hz, H-3), 4.99–5.07 (m, 12H, H-20, H-100, H-
400), 4.88 (dd, 4H, J1,2 = 8.1 Hz, J2,3 = 9.4 Hz, H-2),
4.43–4.49 (m, 8H, H-1, H-6b), 4.35–4.40 (m, 8H, H-10,
H-60b), 4.05–4.13 (m, 16H, H-6a, H-60a, H-600ab),
3.72–3.86 (m, 16H, H-4, H-30, H-50, one of OCH2),
3.66–3.68 (m, 4H, H-500), 3.58–3.62 (m, 4 H, H-5),
3.42–3.48 (m, 4H, one of OCH2), 3.26–3.32 (m, 4H,
H-200), 2.45–2.50 (m, 16H, SCH2), 1.91–2.13 (m, 120H,
Ac), 1.55–1.62 (m, 24H, OCH2CH2CH2CH2CH2S),
1.38–1.44 (m, 8H, SiCH2CH2CH2S), 0.59–0.64 (m, 8H,
SiCH2). 13C NMR (CDCl3) d 170.61, 170.45, 170.39,
170.30, 169.75, 169.52, 169.45, 169.34, 169.03, 100.62,
100.48, 99.49, 76.04, 75.65, 72.60, 72.49, 71.57, 71.49,
71.09, 70.82, 69.79, 68.73, 68.58, 62.08, 61.52, 61.03,
55.98, 35.94, 32.01, 29.27, 28.96, 25.03, 24.11, 23.20,
20.80, 20.63, 20.61, 20.58, 20.54, 11.86; HRMS(ESI)
4.1.11. Trivalent glycodendrimer (18). A solution of 14
(65.9 mg, 19.9 lmol) in MeOH (5 mL) was treated with
28% NaOMe methanolic solution (20 lL) at room tem-
perature for 12 h. The mixture was concentrated, and
0.1 M NaOH aq (5 mL) was then added to the residue.
After overnight, the solution was neutralized by Amber-
lite IR120B (H+) resin. The resin was filtered off and fil-
trate was concentrated to dryness. The crude product
was purified by recycling preparative HPLC (column,
JAIGEL GS-320 and JAIGEL GS-220; solvent, 5% aq
Anal. Calcd for C184H272N4O100S4Si[M+3Na]3+
1454.1550. Found: 1454.1552.
:
HOAc) to give 18 (29.5 mg, 68%) as white powder after
23
4.1.9. Acetylated hexavalent glycodendrimer (16). A cou-
pling reaction between 12 (36.6 mg, 39.3 lmol) and 9
lyophilization. ½aꢁD ꢀ2.3ꢁ (c 0.9, H2O). 1H NMR (D2O)
d 7.46, 7.29 (2 br s, 5H, Si–Ph), 4.45 (d, 3H, J = 7.6 Hz),
4.40 (d, 3H, J = 7.6 Hz), 4.15 (s, 3 H), 3.55–3.91 (m),
3.46–3.47 (m, 6H), 3.30–3.34 (m, 3H), 2.46 (br s, 12H,
SCH2), 2.05 (s, 9H, NHAc), 1.55–1.62 (m, 18 H, SCH2
CH2CH2CH2CH2O), 1.40 (br s, 6H, SiCH2CH2CH2S),
0.89 (br s, 6H, SiCH2). 13C NMR (D2O) d 175.44,
137.13, 134.66, 129.90, 128.68, 103.62, 103.41, 102.98,
82.70, 82.36, 79.09, 76.30, 75.51, 75.34, 75.17, 74.22,
73.48, 70.84, 70.61, 70.33, 68.93, 61.60, 61.14, 60.88,
56.34, 36.10, 32.28, 29.99, 29.54, 25.61, 24.56, 23.01,
12.21; HRMS(ESI) Anal. Calcd for C90H155N3O48S3-
Si[M+2Na]2+: 1107.9248. Found: 1107.9281.
(351.7 mg, 329.3 lmol) was carried out in the same man-
33
ner as described for 14 to give 16 (157.8 mg, 61%). ½aꢁD
+8.8ꢁ (c 1.2, CHCl3). 1H NMR (CDCl3) d 5.45–5.50 (m,
12H, H-300, NH), 5.33 (s, 6H, H-40), 5.16 (t, 6H,
J = 9.3 Hz, H-3), 4.98–5.06 (m, 18H, H-20, H-100, H-
400), 4.87 (dd, 6H, J1,2 = 8.2 Hz, J2,3 = 9.1 Hz, H-2),
4.43–4.47 (m, 12H, H-1, H-6b), 4.34–4.38 (m, 12H, H-
10, H-60b), 4.04–4.13 (m, 24H, H-6a, H-60a, H-600ab),
3.72–3.81 (m, 24H, H-4, H-30, H-50, one of OCH2),
3.65–3.67 (m, 6 H, H-500), 3.57–3.62 (m, 6H, H-5),
3.41–3.47 (m, 6H, one of OCH2), 3.25–3.31 (m, 6H,
H-200), 2.45–2.50 (m, 24 H, SCH2), 1.90–2.12 (m,
180H, Ac), 1.49–1.60 (m, 36H, OCH2CH2CH2
CH2CH2S), 1.36–1.44 (m, 12H, SiCH2CH2CH2S),
1.22–1.30 (m, 4H, SiCH2CH2CH2Si), 0.51–0.62 (m,
20H, SiCH2), ꢀ0.51 (s, 6H, SiMe). 13C NMR (CDCl3)
d 170.55, 170.40, 170.33, 170.26, 169.70, 169.48,
169.39, 169.29, 168.97, 100.59, 100.43, 99.46, 76.07,
75.62, 72.55, 72.44, 71.51, 71.06, 70.34, 69.74, 68.67,
68.54, 62.03, 61.50, 60.99, 55.90, 35.93, 31.93, 29.23,
28.92, 24.99, 24.14, 23.16, 20.76, 20.57, 20.22, 18.18,
16.94, 11.94, ꢀ3.37; HRMS(ESI) Anal. Calcd for
4.1.12. Tetravalent glycodendrimer (19). Reaction condi-
tions were as described for 18, with 15 (94.0 mg,
21.9 lmol). The crude product was purified by gel per-
meation chromatography (Sephadex G50, 5% HOAc
aq eluent) to give 19 (49.5 mg, 81%) as white powder
after lyophilization. ½aꢁD ꢀ0.4ꢁ (c 0.8, H2O). H NMR
(D2O) d 4.44 (br s, 8H), 4.15 (s, 4H), 3.55–3.93 (m),
3.46–3.50 (m, 8H), 3.29–3.35 (m, 4H), 2.59 (br s, 16H,
SCH2), 2.05 (s, 12H, NHAc), 1.66 (br s, 24H,
SCH2CH2CH2CH2CH2O), 1.49 (br s, 8H, SiCH2
CH2CH2S), 0.74 (br s, 8H, SiCH2). 13C NMR (D2O) d
175.49, 103.63, 103.43, 102.96, 82.68, 79.12, 76.33,
75.53, 75.39, 75.18, 74.24, 73.49, 70.91, 70.64, 70.34,
68.96, 61.62, 61.16, 60.86, 56.35, 36.30, 32.35, 30.04,
29.56, 25.64, 24.80, 22.99, 12.42; HRMS(ESI) Anal.
Calcd for C112H200N4O64S4Si[M+2Na]2+: 1413.5477.
Found: 1413.5525.
27
1
C284H426N6O150S6Si3[M+4Na]4+
1672.0805.
:
1672.0773. Found:
4.1.10. Acetylated dodecavalent glycodendrimer (17). A
coupling reaction between 13 (30.7 mg, 17.3 lmol) and
9 (312.5 mg, 291.0 lmol) was carried out in the same
manner as described for 14 to give 17 (165.7 mg, 73%).
21
1
½aꢁD +13.2ꢁ (c 0.7, CHCl3). H NMR (CDCl3) d 5.72
(br s, 12H, NH), 5.47 (t, 12H, H-300), 5.34 (s, 12H, H-
40), 5.17 (t, 12H, J = 9.1 Hz, H-3), 4.99–5.07 (m, 36 H,
H-20, H-100, H-400), 4.87 (t, 12H, J = 8.6 Hz, H-2), 4.45–
4.48 (m, 24H, H-1, H-6b), 4.36–4.38 (m, 24H, H-10,
H-60b), 4.05–4.14 (m, 48H, H-6a, H-60a, H-600ab),
3.73–3.85 (m, 48H, H-4, H-30, H-50, one of OCH2),
4.1.13. Hexavalent glycodendrimer (20). Reaction condi-
tions were as described for 18, with 16 (128.0 mg,
19.4 lmol). The crude product was purified by recycling
preparative HPLC (column, JAIGEL GS-320 and JAI-
GEL GS-220; solvent, 5% aq HOAc) to give 20
(45.2 mg, 54%) as white powder after lyophilization.

A. Yamada et al. / Bioorg. Med. Chem. 15 (2007) 1606–1614
1613
19
1
tials of Glycobiology; Varki, A., Cummings, R., Esko, J.,
Freeze, H., Hart, G., Marth, J., Eds.; Cold Spring Harbor
Laboratory: Spring Harbor, NY, 1999; pp 429–440.
2. (a) Lee, Y. C.; Townsend, M. R.; Hardy, M. R.;
Lo¨nngren, J.; Arnarp, J.; Haraldsson, M.; Lo¨nn, H.
J. Biol. Chem. 1983, 258, 199–202; (b) Lee, Y. C. FASEB
J. 1992, 6, 3193–3200.
3. (a) Lundquist, J. J.; Toone, E. J. Chem. Rev. 2002, 102,
555–578; (b) Wang, Q.; Dordick, J. S.; Linhardt, R. J.
Chem. Mater. 2002, 14, 3232–3244.
4. (a) Matsuoka, K.; Terabatake, M.; Esumi, Y.; Terunuma,
D.; Kuzuhara, H. Tetrahedron Lett. 1999, 40, 7839–7842;
(b) Matsuoka, K.; Terabatake, M.; Umino, A.; Esumi, Y.;
Hatano, K.; Terunuma, D.; Kuzuhara, H. Biomacromol-
ecules 2006, 7, 2274–2283; (c) Matsuoka, K.; Terabatake,
M.; Esumi, Y.; Hatano, K.; Terunuma, D.; Kuzuhara, H.
Biomacromolecules 2006, 7, 2284–2290.
5. Yamada, A.; Hatano, K.; Matsuoka, K.; Koyama, T.;
Esumi, Y.; Koshino, H.; Hino, K.; Nishikawa, K.; Natori,
Y.; Terunuma, D. Tetradedron 2006, 62, 5074–5083.
6. Matsuoka, K.; Oka, H.; Koyama, T.; Esumi, Y.; Teru-
numa, D. Tetrahedron Lett. 2001, 42, 3327–3330.
7. Mori, T.; Hatano, K.; Matsuoka, K.; Esumi, Y.; Toone,
E. J.; Terunuma, D. Tetrahedron 2005, 61, 2751–2760.
8. (a) Matsuoka, K.; Terabatake, M.; Saito, Y.; Hagihara,
C.; Esumi, Y.; Terunuma, D.; Kuzuhara, H. Bull. Chem.
Soc. Jpn. 1998, 71, 2709–2713; (b) Matsuoka, K.; Saito,
Y.; Terunuma, D.; Kuzuhara, H. Kobunshironbunsyu
2000, 57, 691–695; (c) Matsuoka, K.; Ohtawa, T.; Hinou,
H.; Koyama, T.; Esumi, Y.; Nishimura, S.; Hatano, K.;
Terunuma, D. Tetrahedron Lett. 2003, 44, 3617–3620; (d)
Matsuoka, K.; Kurosawa, H.; Esumi, Y.; Terunuma, D.;
Kuzuhara, H. Carbohydr. Res. 2000, 329, 765–772.
9. (a) Kiyotaka, N.; Matsuoka, K.; Kita, E.; Okabe, N.;
Mizuguchi, M.; Hino, K.; Miyazawa, S.; Yamasaki, C.;
Aoki, J.; Takashima, S.; Yamakawa, Y.; Nishijima, M.;
Terunuma, D.; Kuzuhara, H.; Natori, T. Proc. Natl. Acad.
Sci. U.S.A. 2002, 99, 7669–7674; (b) Nishikawa, K.;
Matsuoka, K.; Watanabe, M.; Igai, K.; Hino, K.; Hatano,
K.; Yamada, A.; Abe, N.; Terunuma, D.; Kuzuhara, H.;
Natori, Y. J. Infect. Dis. 2005, 191, 2097–2105.
10. Yamada, A.; Hatano, K.; Koyama, T.; Matsuoka, K.;
Esumi, Y.; Terunuma, D. Carbohydr. Res. 2006, 341, 467–
473.
11. Aoki, C.; Hidari, K. I. P. J.; Itonori, S.; Yamada, A.;
Takahashi, N.; Kasama, T.; Hasebe, F.; Islam, M. A.;
Hatano, K.; Matsuoka, K.; Taki, T.; Guo, C.-T.; Takah-
ashi, T.; Sakano, Y.; Suzuki, T.; Miyamoto, D.; Sugishita,
M.; Terunuma, D.; Morita, K.; Suzuki, Y. J. Biochem.
2006, 139, 607–614.
½aꢁD ꢀ1.7ꢁ (c 0.7, H2O). H NMR (D2O) d 4.45 (br s,
12H), 4.16 (s, 6H), 3.58–3.92 (m), 3.46–3.48 (m, 12H),
3.34 (br s, 6H), 2.58 (br s, 24H, SCH2), 2.06 (s, 18 H,
NHAc), 1.65 (br s, 36H, SCH2CH2CH2CH2CH2O),
1.49 (br s, 16H, SiCH2CH2CH2SiCH2CH2CH2S), 0.71
(br s, 20H, SiCH2), 0.00 (s, 6H, SiMe). 13C NMR
(D2O) d 175.44, 103.65, 103.43, 103.03, 82.72, 79.15,
78.89, 76.33, 75.54, 75.39, 75.20, 74.25, 73.51, 70.84,
70.64, 70.36, 70.19, 68.96, 61.63, 61.17, 60.92, 56.37,
36.41, 32.44, 30.11, 29.64, 25.70, 24.86, 23.04, 20.93,
19.26, 18.04, 12.51, ꢀ1.71; HRMS(ESI) Anal. Calcd
for C176H318N6O96S6Si3[M+3Na]3+: 1465.5832. Found:
1465.5858.
4.1.14. Dodecavalent glycodendrimer (21). Reaction condi-
tions were as described for 18, with 17 (142.6 mg,
10.9 lmol). The crude product was purified by gel perme-
ation chromatography (Sephadex G50, 5% HOAc aq elu-
ent) to give 21 (83.1 mg, 89%) as white powder after
lyophilization. ½aꢁD ꢀ0.3ꢁ (c 1.0, H2O). H NMR (H2O)
d 4.46 (br s, 24H), 4.16 (s, 12H), 3.59–3.92 (m), 3.46–
3.48 (m, 24H), 3.34 (br s, 12H), 2.61 (br s, 48H, SCH2),
2.05 (s, 36H, NHAc), 1.67 (br s, 72H, SCH2CH2CH2
CH2CH2O), 1.50 (br s, 32H, SiCH2CH2CH2SiCH2
CH2CH2S), 0.74 (br s, 40H, SiCH2). 13C NMR (D2O) d
175.41, 103.58, 103.38, 102.955, 82.66, 79.08, 76.27,
75.48, 75.33, 75.14, 74.19, 73.45, 70.82, 70.58, 70.30,
68.90, 61.57, 61.11, 60.85, 56.30, 36.37, 32.42, 30.02,
29.55, 25.63, 24.84, 22.98, 19.43, 18.31, 12.56; HRMS(E-
SI) Anal. Calcd for C348H624N12O192S12Si5[M+4Na]4+:
2164.8624. Found: 2166.5.
23
1
4.2. Fluorescence measurements
Wheat germ agglutinin (lectin from Triticum vulgaris, lot
No. 054K8925) was purchased from Sigma. Emission
spectra of WGA induced by excitation at 295 nm are
uncorrected and were recorded with SHIMADZU RF-
500PC spectrometer. The solutions were contained in
1 cm quarts cuvettes, mounted in thermostated holders,
and the measurements were carried out at 5 ꢁC in order
to remove the effect of nonspecific binding on the spec-
tra. The concentration of WGA was estimated to be
0.69 lM by using the absorption coefficient at 280 nm.22
12. (a) Lotan, R.; Sharon, N. Biochem. Biophys. Res. Com-
mun. 1973, 55, 1340–1346; (b) Privat, c.-P.; Delmotte, F.;
Mialonier, G.; Bouchard, P.; Monsigny, M. Eur.
J. Biochem. 1974, 47, 5–14; (c) Landschoot, A. V.;
Loontiens, F. G.; Clegg, R. M.; Sharon, N.; Bruyne, K.
D. Eur. J. Biochem. 1977, 79, 275–283.
Acknowledgments
Authors are grateful to Dr. Takemichi Nakamura (The
Institute of Physical and Chemical Research) for mea-
suring the high-resolution mass spectroscopy. This work
was supported by a Health and Labour Sciences Re-
search Grant for Research on Advanced Medical Tech-
nology (14-N-015) from the Ministry of Health, Labour,
and Welfare of Japan.
13. Zanini, D.; Roy, R. Bioconjugate Chem. 1997, 8, 187–192.
´
14. Renaudie, L.; Daniellou, R.; Auge, C.; Narvor, C. L.
Carbohydr. Res. 2004, 339, 693–698.
15. Ellervik, U.; Magnusson, G. Carbohydr. Res. 1996, 280,
251–260.
16. Ogawa, T.; Sugimoto, M. Carbohydr. Res. 1985, 135, C5–
C9.
17. Dasgupta, F.; Garegg, P. J. Carbohydr. Res. 1988, 177,
C13–C17.
References and notes
1. (a) Varki, A. Glycobiology 1993, 3, 97–130; (b) Angata, T.;
Varki, A. Chem. Rev. 2002, 102, 439–470; (c) Karlsson,
K.-A. Annu. Rev. Biochem. 1989, 58, 309–350; (d) Esko, J.
D. Microbial Carbohydrate-Binding Proteins. In Essen-
18. Manzoni, L.; Lay, L.; Schmidt, R. R. J. Carbohydr. Chem.
1998, 17, 739–758.
19. Ito, Y.; Sato, S.; Mori, M.; Ogawa, T. J. Carbohydr.
Chem. 1988, 7, 359–376.

1614
A. Yamada et al. / Bioorg. Med. Chem. 15 (2007) 1606–1614
20. Paulsen, H.; Hadamczyk, D.; Kutschker, W.; Bunsch, A.
¨
Liebigs Ann. Chem. 1985, 129–141.
21. (a)Nishimura, S.-I.; Furuike, T.; Matsuoka, K.;Maruyama,
K.; Nagata, K.; Kurita, K.; Nishi, N.; Tokura, S. Macro-
molecules 1994, 27, 4876–4880; (b) Takasu, A.; Horikoshi,
S.; Hirabayashi, T. Biomacromolecules 2005, 6, 2334–2342.
22. Thomas, M. W.; Walborg, E. F., Jr.; Jirgensons, B. Arch.
Biochem. Biophys. 1977, 178, 625–630.