Syntheses and Vero toxin-binding activities of carbosilane dendrimers periphery-functionalized with galabiose

Article abstract of DOI:10.1016/j.tet.2006.03.042

Carbosilane dendrimers bearing galabiose (Galα1-4Gal) with three, four, and six galabiose units at the periphery of the dendrimers were synthesized for use as artificial inhibitors against Shiga toxins (Stxs) produced by Escherichia coli O157:H7. The gala

Full text of DOI:10.1016/j.tet.2006.03.042

Tetrahedron 62 (2006) 5074–5083
Syntheses and Vero toxin-binding activities of carbosilane
dendrimers periphery-functionalized with galabiose
Akihiro Yamada,a Ken Hatano,a Koji Matsuoka,a Tetsuo Koyama,a Yasuaki Esumi,b
Hiroyuki Koshino,b Kumiko Hino,c Kiyotaka Nishikawa,c,d Yasuhiro Natoric
and Daiyo Terunumaa,
*
aDepartment of Functional Materials Science, Faculty of Engineering, Saitama University, 255 Shimo-ohkubo,
Sakura-ku, Saitama 338-8570, Japan
bThe Institute of Physical and Chemical Research, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
cDepartment of Clinical Pharmacology, Research Institute, International Medical Center of Japan,
1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan
dPRESTO, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama, Japan
Received 19 January 2006; revised 14 March 2006; accepted 15 March 2006
Available online 5 April 2006
Abstract—Carbosilane dendrimers bearing galabiose (Gala1–4Gal) with three, four, and six galabiose units at the periphery of the den-
drimers were synthesized for use as artificial inhibitors against Shiga toxins (Stxs) produced by Escherichia coli O157:H7. The galabiose
unit, prepared from penta-O-acetyl-b-D-galactopyranose, was linked with carbosilane dendrimers of three shapes to afford acetyl-protected
glycodendrimers in good yields. De-O-acetylation of the clusters was carried out in the presence of NaOMe and then aq NaOH to give the
desired three shapes of galabiose-coated carbosilane dendrimers. Their biological activities toward Stxs were evaluated by kinetic analysis,
binding assays, and cytotoxic assays.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
consisting a single catalytically active A subunit and a pen-
tamer of B subunits.6 The B subunit is responsible for
binding to globotriaose (Gala1–4Galb1–4Glc).7 Successful
preparation of carbosilane dendrimers periphery-functional-
ized with globotriaose for the purpose of neutralizing Stxs
has been reported.4a It was found that the dendrimers carry-
ing globotriaose, named Dumbbell(1)6 and Dumbbell(2)18
(Fig. 1), completely inhibited Stxs in vivo.5a
Protein–carbohydrate interactions play important roles in
many biological phenomena such as processes of cell adhe-
sion with proteins of pathogens.1 Although the interactions
between single carbohydrate ligands and protein receptors
are usually weak, it is known that clustering of carbohydrate
ligands induces enhancement of the interaction due to the
glycoside cluster effect.2 Artificial clustering carbohydrate
models,2,3 i.e., glycopolymers and glycodendrimers, have
been utilized extensively for the enrichment of protein–
carbohydrate interaction. Recently, a large number of neo-
glycoconjugates carrying a globotriaose cluster have been
synthesized by several groups,4 and their activities have
been evaluated.5
On the other hand, galabiose (Gala1–4Gal), the constitutive
part of globotriaose, is also known to have the ability to bind
to Stxs.7 Enhancement of the binding ability by clustering
of galabiose would be advantageous compared to clustering
of globotriaose from the viewpoint of practical use for
a Stxs neutralizer, because preparation of the former disac-
charide cluster is easier than that of the latter trisaccharide
one.
Shiga toxins (Stxs: Stx1 and Stx2) produced by Escherichia
coli O157:H7, known as Vero toxins, cause diarrhea and he-
molytic uremic syndrome (HUS). Stxs are multimeric toxins
Magnusson8a and Pieters8b independently reported the prep-
aration of galabiose clusters using dendrimers and the results
of evaluation of their potency for inhibiting Streptococcus
suis. We describe herein the syntheses and Stxs binding
activities of dendritic galabiose clusters using a carbosilane
dendrimer as a glycocluster backbone.
* Corresponding author. Tel./fax: +81 48 858 3532; e-mail: teru@fms.
saitama-u.ac.jp
0040–4020/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2006.03.042

A. Yamada et al. / Tetrahedron 62 (2006) 5074–5083
5075
R
R
R
R
R
R
Si
R
R
R
R
R
R
Si
Si
Me
Si
Me
Me
Si
Me
Si
Si
Si
R
R
Si
Si
R
R
Si
Si
R
R
R
R
Dumbbell(1)6
Dumbbell(2)18
R
R
R
R
O
R
R
OH
OH
HO
R =
Si
R
HO
O
OH
OH
O
O
HO
O
S
O
HO
Fan(0)3
OH
OH
Figure 1. Structure of globotriaose cluster compounds.
2. Results and discussion
bromide. The carbosilane dendrimers 6, 7, and 8 were
obtained in 18%, 41%, and 15% yields, respectively. The
structures of these bromide dendrimers were confirmed by
1H, 13C NMR, and FABMS spectra.
2.1. Syntheses of glycoclusters
In the course of our studies on the synthesis of an Stxs inhib-
itor,4a the preparation and characterization of new carbo-
silane dendrimers of three shapes having galabiose were
carried out. One essential point for the molecular design of
a carbosilane dendrimer bearing peripheral galabiose is ad-
justment of the molecular size to that of Stxs adhesion-point
On the other hand, galabiose derivatives were synthesized
by a procedure closely analogous to that used for the syn-
thesis of a globotriaose derivative (Scheme 2).4a Reductive
opening of the 4,6-O-benzylidene ring of 4-pentenyl 2,3-di-
O-benzyl-4,6-O-benzylidene-b-D-galactopylanoside (9)11
gave 4-pentenyl 2,3,6-tri-O-benzyl-b-D-galactopyranoside
(10) in 90% yield. Stereoselective glycosylation of 10
and 2,3,4,6-tetra-O-benzyl-a-D-galactopyranosyl chloride12
was carried out in the presence of AgOTf as a promoter in
ether at ꢀ20 ꢁC to afford galabioside (11) in 71% yield.
˚
distance, ca. 46 A (The information is obtained from crystal-
lographic analysis.), because the size of galabiose is smaller
than that of the already reported Dumbbell(1)6 for globo-
triaose (Fig. 1). Thus, a carbosilane dendrimer larger than
that of Dumbbell(1)6 was designed. Another essential point
is that the carbosilane dendrimer derivative must have suffi-
cient water solubility. The water solubility of the glycoclus-
ter is influenced by the balance between hydrophobicity of
the carbosilane dendrimer and hydrophilicity of the saccha-
ride used. Either an increase of hydrophobicity by using
a longer-armed carbosilane dendrimer or a decrease of
hydrophilicity by using a disaccharide results in low water
solubility of the galabiose clusters.
1
The a-galactose linkage was confirmed by the H NMR
signal at d 5.02 ppm (J1 ,2 ¼1.2 Hz, H-1 ) and the 13C NMR
0
0
0
Br
Me
Me
Si
Me
Br
Si
Me Si
Me
Si
Me
At first, new carbosilane dendrimers shown in Figure 2 were
designed. The carbosilane dendrimers were prepared in
a manner similar to that described in the literature (Scheme
1).9 The hydrosilylation reaction of commercially available
triallylmethylsilane (1) with chlorodimethylsilane catalyzed
by H2PtCl6 followed by alkylation with allyl grignard re-
agent gave tri-branched dendrimer 2 in 31% yield. In the re-
action mixture, a small amount of probably olefin-rearranged
Me
Br
6
Br
Br
Br
Br
Me
Si
Me
Me
Si
Me
Me
Si
Me
Me Si
Si
Me
1
7
by-products was detected by H NMR spectra, 5.93–6.08
(m), 5.58–5.63 (m), 1.82 (d, J¼1.6, 6.2 Hz) ppm. The by-
products were carefully removed by distillation. Both tetra-
branched dendrimer (4) and hexa-branched dendrimer (5)
were also synthesized from bis(allyldimethylsilylpropyl)di-
methylsilane (3)10 using the corresponding chlorosilanes.
The u-brominated carbosilane dendrimers 6–8 were pre-
pared by: (i) hydroxylation of terminal olefin, (ii) mesyla-
tion, and then (iii) bromination using excess sodium
Br
Br
Me
Si
Me
Me
Si
Me
Me
Si
Me
Br
Si
Si
Br
Br
Br
8
Figure 2. Structure of new carbosilane dendrimers.

5076
A. Yamada et al. / Tetrahedron 62 (2006) 5074–5083
Br
Br
Me
Si
Me
Me
Si
Me
Me
Me
a
d, e, f
Br
Si
Me
Me Si
Si
Me
Me Si
Me Si
Si
Me
Si
Me
Me
Me
1
2
6
Me
Si
Me
Me
Si
Me
Me
Si
Me
d, e, f
b
c
Me Si
Si
Me
Me
Si
Me
Si
Me
Si
7
Me
Me
Me
4
3
Me
Si
Me
Si
Me
Si
d, e, f
Si
Si
8
Me
Me
Me
5
Br
Br
Me
Br
Br
Me
Si
Me
Me
Si
Me
Me
Si
Me
Me
Si
Me
Me
Si
Me
Me
Si
Me
Me Si
Si
Br
Si
Si
Br
Br
Br
Br
Br
7
8
Scheme 1. Reagents and conditions: (a) HSiMe2Cl, Speier cat., THF, 50 ꢁC, then allyl grignard, reflux (31%); (b) HSiMeCl2, Speier cat., THF, 40 ꢁC, then allyl
grignard, reflux (81%); (c) HSiCl3, Speier cat., THF, rt-reflux, then allyl grignard, reflux (37%); (d) cyclohexylborane, THF, then 3 M NaOH aq, 30% H2O2 aq,
60 ꢁC; (e) MsCl, Py, ꢀ20 ꢁC; (f) NaBr, DMF, 80 ꢁC.
signals at d 103.87 and 100.37 ppm (C-1 and C-10, respec-
tively). Elemental analysis and FABMS spectrum ([MꢀH]+
1039.0) also support the disaccharide structure. Debenzyla-
tion of 11 without affecting the terminal double bond of the
aglycon was conducted through Birch reduction. Removal
of the benzyl groups from 11 followed by acetylation
afforded acetyl-protected galabioside (12) in 38% yield
(Scheme 2), and about 60% of the starting compound 11
was recovered. Acetylthio function was introduced into
12 carrying an olefin terminal by treatment with AcSH
and AIBN to give 13 in 93% yield after purification by
silica gel column chromatography. The structure of 13 was
confirmed by 1H and 13C NMR spectra, FABMS spectrum,
and elemental analysis. The signals at 2.86 (t, 2H,
J¼7.2 Hz, CH2SAc) and 2.32 (s, 3H, CH2SAc) ppm in
1
the H NMR spectrum of 13 prove the formation of ace-
tylthio function.
Finally, acetylated glycocluster Fan(1)3-Ga2OAc (14) was
synthesized by coupling of the dendrimer 6 with the ace-
tylthio-terminated galabiose 13 (2.1 equiv for each terminal
branch) in the presence of NaOMe (1.1 equiv for sugar
OBn
OBn
Ph
O
OH
OBn
BnO
O
O
BnO
b
O
a
O
OBn
O
O
BnO
O
OBn
O
BnO
O
BnO
OBn
OBn
9
10
OAc
OAc
OAc
OAc
O
O
AcO
AcO
AcO
AcO
c
d
O
O
OAc
OAc
O
O
O
SAc
O
AcO
AcO
OAc
OAc
12
13
Scheme 2. Reagents and conditions: (a) AlCl3, Me3N–BH3, MS4A, THF, rt (90%); (b) 2,3,4,6-tetra-O-benzyl-b-D-galactopyranosyl chloride, AgOTf, MS4A,
Et2O, ꢀ20 ꢁC (71%); (c) Na, liq. NH3, dimethoxyethane, ꢀ72 ꢁC, then Ac2O, Py, rt (38%); (d) AcSH, AIBN, 1,4-dioxane, 80 ꢁC (93%).

A. Yamada et al. / Tetrahedron 62 (2006) 5074–5083
5077
R
R
R
Me
Si
Me
Me
Si
Me
Me
Si
Me
R
Me Si
Si
Me
Me
Me
Si
Me
R
15 (R1 = Ac)
18 (R1 = H)
Si
R
Me Si
Me
R
R
Si
Me
Me
Me
Si
Me
Me
Si
Me
Me
Si
Me
R
R
Si
Si
R
14 (R1 = Ac)
17 (R1 = H)
R
R
16 (R1 = Ac)
19 (R1 = H)
OR
1 OR1
O
R1O
R1O
R =
O
OR1
O
O
S
R1O
OR1
R1 = Ac
14
R1 = H
17
6
7
8
a
b
+
15
16
13
18
19
Scheme 3. (a) NaOMe, DMF, MeOH, rt, then Ac2O, Py, rt (86% for 14, 72% for 15, and 75% for 16); (b) NaOMe, MeOH, rt, then dil NaOH aq, rt (91% for 17,
99% for 18, 98% for 19).
Table 1. HRMS (ESI) of galabiose clusters
Compounds
Formula
m/z (Calculated)
m/z (Found)
Fan(1)3-Ga2OAc (14)
Dumbbell(2)4-Ga2OAc (15)
Dumbbell(2)6-Ga2OAc (16)
Fan(1)3-Ga2 (17)
Dumbbell(2)4-Ga2 (18)
Dumbbell(2)6-Ga2 (19)
C118H192O54S3Si4+Na+
C156H252O72S4Si5+2Na+
C222H348O108S6Si5+2Na+
C76H150O33S3Si4+Na+
C100H196O44S4Si5+Na+
C138H264O66S6Si5+2Na+
2704.04148
1795.67912
2559.93525
1821.81962
2392.07263
1677.71339
2704.04324
1795.68283
2559.92997
1821.81874
2392.07023
1677.71763
moiety) (Scheme 3).13 Purification by recycling preparative
HPLC afforded pure 14 in 86% yield. Dumbbell(2)4-
Ga2OAc (15) and Dumbbell(2)6-Ga2OAc (16) were
prepared from the corresponding dendrimers in a manner
similar to that described above in 72% and 75% yields,
respectively. De-O-acetylation by NaOMe in MeOH and
successive saponification of 14, 15, and 16 gave target
compounds Fan(1)3-Ga2 (17), Dumbbell(2)4-Ga2 (18),
and Dumbbell(2)6-Ga2 (19) in 91%, 99%, and 98% yields,
respectively. The galabiose clusters 18 and 19 were soluble
in an aqueous solution. Fortunately, the level of water solu-
bility was adequate for biological assay systems, although
that of 17 was low. All the synthesized acetyl- and de-acet-
ylated clusters were fully characterized by high-resolution
mass spectrometry (Table 1). The results showed good
agreement with the calculated values for the expected struc-
tures.
(KD) of these clusters to the Stx1 and Stx2 B subunits were
determined by Scatchard plot analysis. Compound 19
showed KD values of 1.3 and 1.6 mM for Stx1 and Stx2,
respectively (Table 2). Previously reported KD values of
hexavalent globotriaose cluster Dumbbell(1)6 were 0.11
and 0.21 mM for Stx1 B subunit and Stx2 B subunit, respec-
tively.5a,14 Therefore, the potency of 19 is one-tenth of that of
Dumbbell(1)6. However, the KD values of 17 are similar to
those of trivalent globotriaose cluster Fan(0)3 (structure of
Fan(0)3 is shown in Fig. 1). The potency of the clusters 17,
Table 2. Kinetic analysis of dendrimers having oligosaccharide toward Stxs
Dendrimers
Stx1 B subunit
Stx2 B subunit
KD, mM
KD, mM
Fan(1)3-Ga2 (17)
Dumbbell(2)4-Ga2 (18)
61.1
10.5
1.3
53.9
10.1
1.6
Dumbbell(2)6-Ga2 (19)
Fan(0)3a
2.2. Inhibition studies of the glycoclusters
64.8
0.11
124
0.21
Dumbbell(1)6a
The synthesized galabiose cluster compounds were assessed
by using a BIAcore instrument. The dissociation constants
a
See Refs. 5a and 14.

5078
A. Yamada et al. / Tetrahedron 62 (2006) 5074–5083
A
B
100
80
60
40
20
0
100
80
60
40
20
0
Stx1
Stx2
10-3 10-2 10-1
1
10
102
103
10-1
1
10
102
103
104
100
100
80
60
40
20
0
80
60
40
20
0
10-3 10-2 10-1
Concentration (
1
10
g/ml)
102 103
10-1
1
10
Concentration (
102
g/ml)
103
104
µ
µ
Figure 3. Inhibitory effects of carbosilane dendrimers having galabiose on the biological activities of Stxs in Vero cells. (A) Results of 125I-Stxs binding assay.
(B) Results of cytotoxicity assay using Vero cells. The symbols :, C, and A indicate Fan(1)3-Ga2 (17), Dumbbell(2)4-Ga2 (18), and Dumbbell(2)6-Ga2 (19),
respectively.
18, and 19 was further evaluated by 125I-labeled Stxs (125I-
Stxs) binding assay and cytotoxic assay. It was found that
the clusters 17, 18, and 19 inhibited the binding of 125I-
Stx1 and 125I-Stx2 to Vero cells (Fig. 3A). The IC50 values
in the 125I-Stxs binding assay are shown in Table 3. The min-
imum IC50 value was 13.6 mM, which is larger than that of
Dumbbell(1)6.5a On the other hand, 17, 18, and 19 showed
very weak inhibitory effects (Fig. 3B) in the cytotoxic assay.
Only the IC50 value of 19 for Stx2 was determined (362 mM).
Interestingly, the inhibitory potency in these analyses of 19 is
lower than that of Dumbbell(1)6, although the potency of 17
is similar to that of Fan(0)3. The intra-sugar distances be-
4. Experimental
4.1. General methods
1H and 13C NMR spectra were recorded on a Bruker AM 400
and DRX 400, at 400 and 400 MHz for proton and at 100 and
100 MHz for carbons, respectively. Proton chemical shifts
are given in parts per million with the use of tetramethyl-
silane (0 ppm) or residual solvent peaks as internal standard.
NMR signals were assigned by 1H, 13C, HH, and HC COSY
measurements. FAB and ESI mass spectra were obtained
with a JEOL JMS-HX110A spectrometer and a JEOL
JMS-T100LC spectrometer, respectively. Optical rotations
were recorded on a JASCO DIP-1000 digital polarimeter
at ambient temperature, using a 10-cm micro cell. Recycling
preparative HPLC was performed with a LC-908 or LC-
918W (Japan Analytical Industry Co., Ltd) connected to
an RI detector RI-5. The computer generated low energy
conformations were calculated using the CaChe software
(Fujitsu Inc.).
˚
tween each branches of 17, about 29 A, are similar to those
˚
of globotriaose clusters, about 27 A. In contrast, the intra-
˚
sugar distances of 19, about 15 A, are shorter than those
of globotriaose clusters. These results suggest that a long
intra-sugar distance in dendritic galabiose is preferable for
high binding activity of Stxs.
Table 3. Determination of the IC50 values in 125I-Stxs binding assay
4.1.1. Tris[3-(allyldimethylsilyl)propyl]methylsilane (2).
A solution of chlorodimethylsilane (16.8 g, 177.6 mmol)
in THF (3 mL) was added dropwise to a mixture of 1
(1.97 g, 11.8 mmol) and Speier catalyst (six drops) in THF
(3 mL) under Ar, and the reaction mixture was stirred at
50 ꢁC for 24 h. Chlorodimethylsilane and THF were elimi-
nated by heating the mixture. The residue was dissolved in
THF (25 mL) and a solution of allyl grignard reagent
(88.8 mmol) in diethyl ether (45 mL) was added to the reac-
tion solution at 0 ꢁC. The mixture was refluxed overnight
and then 1 M HCl aq was added dropwise to the resulting
mixture. The mixture was extracted with diethyl ether and
washed with water and brine. The organic layer was dried
(Na2SO4) and the filtrate concentrated. Distillation of the
residue gave compound 2 (1.73 g, 31%). Bp 220 ꢁC/4 torr.
1H NMR (CDCl3) d 5.75–5.81 (m, 3H, –CH]CH2), 4.80–
4.86 (m, 6H, –CH]CH2), 1.52 (d, 6H, J¼16.8 Hz,
SiCH2CH]CH2), 1.32–1.34 (m, 6H, SiCH2CH2CH2Si),
0.53–0.61 (m, 12H, SiCH2), 0.01 (s, 21H, Si–CH3); 13C
NMR (CDCl3) d 135.28 (–CH]CH2), 112.54 (–CH]
CH2), 23.53, 19.72, 19.01, 18.38, ꢀ3.88, ꢀ4.97.
Dendrimers
Stx1 B subunit
Stx2 B subunit
IC50, mM
IC50, mM
Fan(1)3-Ga2 (17)
Dumbbell(2)4-Ga2 (18)
Dumbbell(2)6-Ga2 (19)
18.9
23.6
14.2
17.8
23.6
13.6
Fan(0)3a
21.4
0.08
>50
Dumbbell(1)6a
0.50
a
See Ref. 5a.
3. Conclusion
A series of new carbosilane dendrimers periphery-function-
alized with galabiose was synthesized. The synthesized
dendrimers having galabiose were evaluated by biological
potency of Stx1 and Stx2 using kinetic analysis, binding as-
says, and cytotoxic assays. The results show that about 29 A
intra-sugar distance between each branches is required for
increasing binding activity toward Stxs.
˚

A. Yamada et al. / Tetrahedron 62 (2006) 5074–5083
5079
4.1.2. Bis(diallylmethylsilylpropyldimethylsilylpropyl)-
dimethylsilane (4). A solution of dichloromethylsilane
(3.38 g, 29.3 mmol) in THF (10 mL) was added dropwise
to a mixture of 3 (2.00 g, 5.87 mmol) and Speier catalyst
(five drops) in THF (10 mL) under Ar, and the reaction mix-
ture was stirred at 40 ꢁC for 39 h. The reaction mixture was
then processed in the same way described for compound 2.
The residue was chromatographed on silica gel with hexane
as eluent to give 4 (2.82 g, 81%). 1H NMR (CDCl3) d 5.73–
5.84 (m, 4H, –CH]CH2), 4.81–4.90 (m, 8H, –CH]CH2),
1.55 (d, 8H, J¼8.2 Hz, SiCH2CH]CH2), 1.26–1.41 (m,
8H, SiCH2CH2CH2Si), 0.49–0.64 (m, 16H, SiCH2), ꢀ0.01
(s, 6H, SiCH3), ꢀ0.05 (s, 18H, SiCH3); 13C NMR (CDCl3)
d 134.86 (–CH]CH2), 112.99 (–CH]CH2), 21.48, 20.13,
20.09, 18.43, 18.19, 17.80, ꢀ3.21, ꢀ5.74.
added dropwise to the solution. The mixture was extracted
with CHCl3, and washed with 5% H2SO4 aq (v/v), 5%
NaHCO3 aq (w/w) and brine, dried (Na2SO4), and concen-
trated. The residue was purified by column chromatography
on silica gel (1:100 MeOH/CHCl3) to yield the title
compound (1.86 g, 98%). 1H NMR (CDCl3) d 4.15 (t,
6H, J¼6.9 Hz, CH2O), 2.98 (s, 9H, SO2CH3), 1.67–1.75
(m, 6H, CH2CH2O), 1.27–1.31 (m, 6H, SiCH2CH2CH2Si),
0.48–0.56 (m, 18H, SiCH2), ꢀ0.04 (s, 18H,
CH2Si(CH3)2CH2), ꢀ0.09 (s, 3H, SiCH3); 13C NMR
(CDCl3) d 72.54, 37.28, 23.89, 19.73, 18.66, 18.21, 10.62,
ꢀ3.54, ꢀ5.05.
4.1.4.3. Synthesis of 6. NaBr (4.89 g, 45.5 mmol) was
added to a solution of above mesylated compound (2.39 g,
3.17 mmol) in DMF (17 mL) under Ar. The mixture was
heated at 80 ꢁC for 3 h. The resulting mixture was diluted
with water and toluene. The solution was extracted with tol-
uene, washed with brine, dried (Na2SO4), and concentrated.
The residue was purified by column chromatography on
silica gel (1:99 EtOAc/hexane) to yield compound 6
4.1.3. Bis(triallylsilylpropyldimethylsilylpropyl)di-
methylsilane (5). A solution of trichlorosilane (2.38 g,
17.6 mmol) in THF (3 mL) was added dropwise to a mixture
of 3 (2.00 g, 5.87 mmol) and Speier catalyst (six drops) in
THF (4 mL) under Ar, and the reaction mixture was stirred
at room temperature overnight and then refluxed for 2 h.
The reaction mixture was then processed in the same way de-
1
(1.78 g, 79%). H NMR (CDCl3) d 3.38 (t, 6H, J¼7.1 Hz,
CH2Br), 1.81–1.85 (m, 6H, CH2CH2Br), 1.29–1.31 (m,
6H, SiCH2CH2CH2Si), 0.52–0.62 (m, 18H, SiCH2), ꢀ0.02
(s, 18H, CH2Si(CH3)2CH2), ꢀ0.08 (s, 3H, SiCH3); 13C
NMR (CDCl3) d 37.18 (CH2Br), 28.01 (CH2CH2Br), 19.90,
18.97, 13.39, 14.64, ꢀ3.37, ꢀ4.96. FABMS (negative):
[M+Br]ꢀ 786.8.
1
scribed for compound 4 to give 5 (1.40 g, 37%). H NMR
(CDCl3) d 5.73–5.82 (m, 6H, –CH]CH2), 4.85–4.91 (m,
12H, –CH]CH2), 1.58 (d, 12H, J¼8.1 Hz, SiCH2CH]
CH2), 1.25–1.40 (m, 8H, SiCH2CH2CH2Si), 0.63–0.67 (m,
4H, CH2CH2SiCH2CH]CH2), 0.52–0.57 (m, 12H, SiCH2),
ꢀ0.05 (s, 18H, SiCH3); 13C NMR (CDCl3) d 134.50
(–CH]CH2), 113.44 (–CH]CH2), 20.23, 20.14, 20.07,
19.72, 18.42, 18.10, 16.28, ꢀ3.20, ꢀ3.23.
4.1.5. Bis(bis(3-bromopropyl)methylsilylpropyldi-
methylsilylpropyl)dimethylsilane (7).
4.1.5.1. Synthesis of bis(bis(3-hydroxypropyl)methyl-
silylpropyldimethylsilylpropyl)dimethylsilane. Reaction
conditions and workup were as described in Section 4.1.4.1,
with compound 4 (2.00 g, 3.37 mmol). The residue was
purified by reprecipitation from hexane then a mixture of
1:5 EtOAc/hexane again to yield title compound (1.34 g,
60%). 1H NMR (DMSO-d6) d 4.40 (t, 4H, J¼5.2 Hz,
–OH), 3.28–3.33 (m, 8H, CH2OH), 1.26–1.41 (m, 16H,
SiCH2CH2), 0.47–0.53 (m, 16H, SiCH2), 0.39–0.43 (m,
8H, SiCH2), ꢀ0.09 and ꢀ0.10 (each s, 24H, SiCH3); 13C
NMR (DMSO-d6) d 64.13, 27.19, 19.82, 19.70, 19.68,
18.27, 18.209.53, ꢀ2.91, ꢀ4.90.
4.1.4. Tris(3-bromopropyldimethylsilylpropyl)methyl-
silane (6).
4.1.4.1. Synthesis of tris(3-hydroxypropyldimethyl-
silylpropyl)methylsilane.
A solution of 2 (0.98 g,
2.1 mmol) in THF (4 mL) was added to a solution of cyclo-
hexylborane, which was prepared from borane/THF complex
(4.7 mL of 1 M solution) and cyclohexene (0.39 g,
4.7 mmol) in THF at 0 ꢁC under Ar. After stirring at room
temperature for 2 h, MeOH (3 mL) was added to the reaction
mixture. Aqueous NaOH (5 mL of 3 M solution) and H2O2
(2 mL of 30% solution) were added to the resulting solution
at 0 ꢁC, subsequently. The reaction mixture was stirred at
60 ꢁC for 1 h. The resulting mixture was extracted with
THF; the solution was washed with brine, dried (Na2SO4),
and concentrated. The residue was purified by reprecipitation
from cold hexane (ꢀ30 ꢁC) to yield the title compound
4.1.5.2. Synthesis of bis(bis(3-methylsulfonyloxy-
propyl)methylsilylpropyldimethylsilylpropyl)dimethylsi-
lane. Reaction conditions and workup were as described in
Section 4.1.4.2, with above hydoxylated compound (1.29 g,
1.94 mmol). Title compound (1.86 g, 98%) was obtained. 1H
NMR d 4.15 (t, 8H, J¼6.8 Hz, CH2O), 2.99 (s, 12H,
SO2CH3), 1.69–1.73 (m, 8H, SiCH2CH2CH2O), 1.27–1.31
(m, 8H, SiCH2CH2CH2Si), 0.50–0.61 (m, 24H, SiCH2),
ꢀ0.02 and ꢀ0.07 (each s, 24H, SiCH3); 13C NMR
(CDCl3) d 72.29, 37.31, 23.81, 20.14, 20.03, 19.99, 18.32,
18.21, 18.01, 9.10, ꢀ3.27, ꢀ5.52.
1
(0.79 g, 73%). H NMR (CDCl3) d 3.58 (t, 6H, J¼6.8 Hz,
CH2OH), 1.64 (br s, 3H, OH), 1.50–1.58 (m, 6H,
CH2CH2OH), 1.27–1.35 (m, 6H, SiCH2CH2CH2Si), 0.52–
0.60 (m, 12H, SiCH2CH2CH2Si), 0.45–0.50 (m, 6H,
SiCH2CH2CH2Br), ꢀ0.04 (s, 18H, CH2Si(CH3)2CH2),
ꢀ0.09 (s, 3H, SiCH3); 13C NMR (CDCl3) d 65.83, 27.17,
19.99, 18.78, 18.43, 10.89, ꢀ3.37, ꢀ4.93.
4.1.4.2. Synthesis of tris(3-methylsulfonyloxypropyl-
dimethylsilylpropyl)methylsilane. Methanesulfonyl chlo-
ride (1.8 mL, 32.2 mmol) was added dropwise to a solution
of the above hydroxylated compound (1.29 g, 1.94 mmol)
in pyridine (7 mL) at ꢀ20 ꢁC under Ar, and the reaction
mixture was stirred at same temperature for 3 h. The solution
was diluted with CHCl3 (10 mL), and water (10 mL) was
4.1.5.3. Synthesis of 7. Reaction conditions and workup
were as described in Section 4.1.4.3, with above mesylated
compound (1.81 g, 1.85 mmol). The compound 7 (1.40 g,
83%) was obtained. 1H NMR d 3.38 (t, 8H, J¼7.0 Hz,
CH2Br), 1.79–1.86 (m, 8H, CH2CH2Br), 1.29–1.33 (m,
8H, SiCH2CH2CH2Si), 0.52–0.65 (m, 24H, SiCH2), ꢀ0.02
and ꢀ0.05 (each s, 24H, SiCH3); 13C NMR (CDCl3)

5080
A. Yamada et al. / Tetrahedron 62 (2006) 5074–5083
d 37.05, 27.83, 20.18, 20.11, 20.06, 18.40, 18.28, 18.23,
12.99, ꢀ3.19, ꢀ5.26. FABMS (negative): [M+Br]ꢀ 995.0.
J1,2¼7.7 Hz, H-1), 4.02 (d, 1H, J3,4¼3.2 Hz, H-4), 3.93–
3.98 (m, 1H, one of OCH2CH2CH2CH]CH2), 3.80 (dd,
1H, J¼6.0 Hz, J¼9.9 Hz, H-6a), 3.72 (dd, 1H, J¼6.0 Hz, J¼
9.9 Hz, H-6b), 3.64 (dd, 1H, J1,2¼7.8 Hz, J2,3¼9.2 Hz, H-2),
3.53–3.57 (m, 2H, H-5 and one of OCH2CH2CH2CH]CH2),
3.49 (dd, 1H, J2,3¼9.4 Hz, J3,4¼3.4 Hz, H-3), 2.13–2.19
(m, 2H, OCH2CH2CH2CH]CH2), 1.69–1.80 (m, 2H,
OCH2CH2CH2CH]CH2), 1.60 (br s, 2H, –OH and H2O);
13C NMR (CDCl3) d 138.55, 138.05, 137.96, 137.85,
128.39, 128.37, 128.25, 128.05, 127.81, 127.77, 127.72,
127.69, 127.56, 114.80 (–C]CH2), 103.63 (C-1), 80.55,
78.91, 75.14, 73.65, 73.10, 72.34, 69.17, 66.83 (C-4),
30.17, 28.91; Anal. Calcd for C32H38O6+0.5H2O: C, 72.84;
H, 7.45. Found: C, 73.08; H, 7.35. FABMS (positive):
[M+Na]+ 541.3.
4.1.6. Bis(tris(3-bromopropyl)silylpropyldimethylsilyl-
propyl)dimethylsilane (8).
4.1.6.1. Synthesis of bis(tris(3-hydroxypropyl)silyl-
propyldimethylsilylpropyl)dimethylsilane. Reaction con-
ditions and workup were as described in Section 4.1.4.1, with
compound 5 (1.38 g, 2.14 mmol). The residue was purified
by reprecipitation from a mixture of 1:5 EtOAc/hexane to
1
yield the title compound (1.35 g, 84%). H NMR (DMSO-
d6) d 4.40 (t, 6H, J¼5.2 Hz, –OH), 3.28–3.37 (q, 12H,
J¼6.6 Hz, CH2OH), 1.26–1.60 (m, 20H, SiCH2CH2CH2),
0.50–0.56 (m, 16H, CH2SiCH2CH2CH2OH), 0.40–0.44 (m,
12H, SiCH2), ꢀ0.08 (s, 18H, SiCH3); 13C NMR (DMSO-
d6) d 64.20, 27.21, 19.94, 19.69, 19.65, 18.24, 18.21,
17.00, 8.15, ꢀ2.89.
4.1.8. 4-Pentenyl (2,3,4,6-tetra-O-benzyl-a-D-galacto-
pyranosyl)-(1/4)-2,3,6-tri-O-benzyl-b-D-galactopyra-
noside (11). A mixture of 2,3,4,6-tetra-O-benzyl-a-D-
galactopyranosyl chloride (17.4 g, 31.0 mmol), compound
4.1.6.2. Synthesis of bis(tris(3-methylsulfonyloxypro-
pyl)silylpropyldimethylsilylpropyl)dimethylsilane. Reac-
tion conditions and workup were as described in Section
4.1.4.2, with the above hydoxylated compound (1.03 g,
1.37 mmol). Purification by chromatography on silica gel
(1:50 MeOH/CHCl3) yielded the title compound (1.62 g,
˚
10 (7.58 g, 14.6 mmol), and molecular sieves (4 A powder,
7.6 g) in diethyl ether (300 mL) was stirred at room temper-
ature for 1 h under Ar. Silver triflate (12.0 g, 47.0 mmol) was
added to the mixture at ꢀ20 ꢁC under darkness and reaction
mixture was stirred at same temperature for 2 h. The insolu-
ble solids were separated by filtration through a Cerite bed,
the filtrate was diluted with CHCl3 and washed with ice-
water, saturated NaHCO3 solution, and brine. The organic
layer was dried (MgSO4) and concentrated. The residue was
purified by column chromatography on silica gel (3:97 then
5:95 EtOAc/toluene) to yield compound 11 (10.8 g, 71%).
1
96%). H NMR d 4.17 (t, 12H, J¼6.4 Hz, CH2O), 3.02 (s,
18H, SO2CH3), 1.70–1.76 (m, 12H, CH2CH2O), 1.28–1.31
(m, 8H, SiCH2CH2CH2Si), 0.52–0.67 (m, 28H, SiCH2),
ꢀ0.05 (s, 18H, SiCH3); 13C NMR (CDCl3) d 72.11, 37.34,
23.75, 20.31, 20.02, 19.98, 18.31, 18.23, 16.56, 7.57,
ꢀ3.26, ꢀ3.29.
1
4.1.6.3. Synthesis of 8. Reaction conditions and workup
were as described in Section 4.1.4.3, with above mesylated
compound (2.08 g, 1.70 mmol). Purification was effected
by column chromatography on silica gel (5:95 EtOAc/
hexane) to yield compound 8 (0.98 g, 51%). 1H NMR
(CDCl3) d 3.39 (t, 12H, J¼6.8 Hz, CH2Br), 1.79–1.85 (m,
12H, CH2CH2Br), 1.30–1.32 (m, 8H, SiCH2CH2CH2Si),
0.52–0.69 (m, 28H, SiCH2), ꢀ0.05 (s, 18H, SiCH3); 13C
NMR (CDCl3) d 30.99, 27.67, 20.32, 20.12, 20.05, 18.40,
18.28, 16.79, 11.44, ꢀ3.19. FABMS (negative): [M+Br]ꢀ
1208.8.
[a]3D1 +41.0 (c 1.2, CHCl3); H NMR (CDCl3) d 7.16–7.38
(m, 35H, aromatics), 5.76–5.86 (m, 1H, –CH]CH2), 5.02
0
0
0
(d, 1H, J1 ,2 ¼1.3 Hz, H-1 ), 4.87–4.99 (m, 5H, –CH]CH2,
CH2Ph), 4.77–4.80 (m, 4H, CH2Ph), 4.68 (d, 1H, J¼
11.9 Hz, CH2Ph), 4.55 (d, 1H, J¼3.1 Hz, CH2Ph), 4.52 (d,
1H, J¼4.6 Hz, CH2Ph), 4.12–4.45 (m, 1H, H-50), 4.31 (d,
1H, J1,2¼7.6 Hz, H-1), 4.26 (d, 1H, J¼11.8 Hz, CH2Ph),
4.21 (d, 1H, J¼11.8 Hz, CH2Ph), 4.10–4.17 (m, 5H, H-20,
H-30, H-40 and CH2Ph), 4.02 (d, 1H, J3,4¼2.4 Hz, H-4),
3.91–3.98 (m, 2H, H-6a and one of OCH2CH2CH2CH]
CH2), 3.67 (dd, 1H, J1,2¼7.6 Hz, J2,3¼9.9 Hz, H-2), 3.51–
3.57 (m, 4H, H-5, H-6b, H-60a and one of OCH2CH2CH2
CH]CH2), 3.38 (dd, 1H, J2,3¼9.9 Hz, J3,4¼2.8 Hz, H-3),
3.24 (dd, 1H, J¼4.7 Hz, J¼8.3 Hz, H-60b), 2.15–2.19 (m,
2H, OCH2CH2CH2CH]CH2), 1.76–1.78 (m, 2H, OCH2
CH2CH2CH]CH2); 13C NMR (CDCl3) d 138.90, 138.74,
138.67, 138.60, 138.06, 137.99, 128.25, 128.19, 128.16,
128.14, 128.10, 128.02, 127.96, 127.86, 127.72, 127.50,
127.47, 127.35, 127.24, 114.79, 103.87 (C-1), 100.37
(C-10), 80.86, 78.91, 78.86, 76.51, 75.02, 74.80, 74.76,
74.64, 73.58, 73.51, 73.07, 72.95, 72.27, 72.17, 69.57,
69.26, 69.19, 67.97, 67.87, 30.12, 28.89; Anal. Calcd for
C66H72O11: C, 76.13; H, 6.97. Found: C, 75.99; H, 7.01.
FABMS (positive): [MꢀH]+ 1039.0. [M+Na]+ 1062.9.
4.1.7. 4-Pentenyl 2,3,6-tri-O-benzyl-b-D-galactopyrano-
side (10) one half hydrate. A mixture of compound 9
˚
(5.03 g, 9.75 mmol) and molecular sieves (4 A powder,
5.0 g) in THF (80 mL) was stirred at room temperature for
2 h under Ar, trimethylamine boron complex (4.98 g,
68.2 mmol) was added. Aluminum chloride (9.10 g,
68.2 mmol) was added to the reaction mixture in some por-
tion at 0 ꢁC. After stirring at room temperature for 4 h, the re-
sulting mixture was filtered through Celite and filtrate was
diluted with CHCl3. The solution was poured into ice-water
and extracted with CHCl3. The organic solution was washed
with water, saturated NaHCO3 solution, and brine. The
organic layer was dried (MgSO4) and concentrated. The
residue was purified by column chromatography on silica gel
(1:4 EtOAc/hexane) to give compound 10 in hydrated form
(4.54 g, 90%). [a]D31 +7.6 (c 1.0, CHCl3); 1H NMR (CDCl3)
d 7.28–7.37 (m, 15H, aromatics), 5.76–5.87 (m, 1H,
–CH]CH2), 4.94–5.03 (m, 2H, –CH]CH2), 4.91 (d, 1H,
J¼11.2 Hz, CH2Ph), 4.73 (d, 1H, J¼11.6 Hz, CH2Ph), 4.72
(s, 2H, CH2Ph), 4.59 (s, 2H, CH2Ph), 4.35 (d, 1H,
4.1.9. 4-Pentenyl (2,3,4,6-tetra-O-acetyl-a-D-galacto-
pyranosyl)-(1/4)-2,3,6-tri-O-acetyl-b-D-galactopyrano-
side (12). A solution of compound 11 (5.16 g, 4.92 mmol) in
dimethoxyethane (30 mL) was added dropwise to sodium
(4.56 g, 198 mmol) in liq. NH3 (150 mL) at ꢀ72 ꢁC. After
stirring at same temperature for 45 min, NH4Cl (10.6 g,
198 mmol) in limited amounts was added to the reaction

A. Yamada et al. / Tetrahedron 62 (2006) 5074–5083
5081
mixture. The mixture was allowed to warm at room temper-
ature and stirred overnight, and then evaporated to dryness.
The residue was treated with pyridine (33.5 mL, 420 mmol)
and Ac2O (19.7 mL, 210 mmol) at room temperature for
19 h. The mixture was poured into ice-water and extracted
with CHCl3. The solution was washed with 1 M HCl aq,
saturated 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) to give compound 12 (1.32 g, 38%) and
3.26 g of the starting compound. [a]2D9 +73.5 (c 1.4,
solution was treated with NaOMe (11.6 mg, 215 mmol) at
room temperature for 21 h. Acetic acid (0.25 mL) was then
added to the mixture, the resulting solution was evaporated
under reduced pressure. The residue was suspended in a mix-
ture of pyridine (6 mL) and acetic acid (3 mL). The suspen-
sion was stirred for 5 h at room temperature, after resulting
solution was poured into ice-water and extracted with
EtOAc. The organic solution was washed with 1 M HCl aq,
saturated NaHCO3 aq, and brine. Organic phase was dried
(MgSO4) and concentrated. Purification by recycling prepar-
ative HPLC (column, JAIGEL-1H and 2H; solvent, chloro-
form) afforded Fan(1)3-Ga2OAc 14 (75.6 mg, 86%). [a]D33
CHCl3); 1H NMR (CDCl3)
d
5.75–5.85 (m, 1H,
0
1
0
0
–CH]CH2), 5.57 (d, 1H, J3 ,4 ¼3.1 0Hz, H-4 ), 5.39 (dd,
+59.0 (c 2.0, CHCl3); H NMR (CDCl3) d 5.57 (d, 3H,
J3 ,4 ¼2.8 Hz, H-40), 5.39 (dd, 3H, J2 ,3 ¼11.0 Hz,
0
0
0
0
0
0
0
0
1H, J2 ,3 ¼10.9 Hz, J3 ,4 ¼3.1 Hz, H-3 ), 5.16–5.21 (m, 2H,
H-2 and H-20), 4.96–5.04 (m, 3H, H-10, –CH]CH2), 4.82
(dd, 1H, J2,3¼10.7 Hz, J3,4¼2.4 Hz, H-3), 4.54 (t, 1H,
J¼6.9 Hz, H-50), 4.44–4.48 (m, 2H, H-1 and H-6b), 4.06–
4.25 (m, 4H, H-4, H-6a and H-60ab), 3.87–3.93 (m, 1H,
one of OCH2CH2CH2CH]CH2), 3.78 (t, 1H, J¼6.6 Hz,
H-5), 3.47–3.53 (m, 1H, one of OCH2CH2CH2CH]CH2),
1.99–2.13 (m, 23H, –OAc, OCH2CH2CH2CH]CH2),
1.64–1.76 (m, 2H, OCH2CH2CH2CH]CH2); 13C NMR
(CDCl3) d 170.70, 170.55, 170.45, 170.41, 170.10, 169.72,
169.06, 137.90 (–CH]CH2), 114.95 (–CH]CH2), 101.17
(C-1), 99.30 (C-10), 72.75 (C-3), 71.79 (C-5), 69.12, 68.76,
68.56, 67.81 (C-40), 67.35 (C-30), 66.99 (C-50), 61.94 (C-
6), 60.46, 29.84, 28.55, 20.92, 20.73, 20.66, 20.64, 20.59;
Anal. Calcd for C31H44O18: C, 52.84; H, 6.29. Found: C,
52.66; H, 6.23. FABMS (positive): [M+Na]+ 726.9.
0
0
0
0
J3 ,4 ¼3.3 Hz, H-3 ), 5.15–5.21 (m, 6H, H-2 and H-2 ), 5.00
0
0
0
(d, 3H, J1 ,2 ¼3.6 Hz, H-1 ), 4.81 (dd, 3H, J2,3¼10.8 Hz,
J3,4¼2.7 Hz, H-3), 4.53 (t, 3H, J¼6.7 Hz, H-50), 4.43–4.48
(m, 6H, H-1 and H-6b), 4.06–4.20 (m, 12H, H-4, H-6a and
H-60ab), 3.88–3.90 (m, 3H, one of OCH2CH2), 3.77 (t, 3H,
J¼6.6 Hz, H-5), 3.46–3.48 (m, 3H, one of OCH2CH2–),
2.50 (t, 12H, J¼7.5 Hz, CH2S), 1.99–2.13 (m, 63H, OAc),
1.53–1.61 (m, 18H, OCH2CH2CH2CH2CH2SCH2CH2CH2
Si), 1.43–1.47 (m, 6H, OCH2CH2CH2CH2CH2S), 1.26–
1.32 (m, 6H, SiCH2CH2CH2Si), 0.51–0.60 (m, 18H,
SiCH2), ꢀ0.04 (s, 18H, SiCH3), ꢀ0.09 (s, 3H, SiCH3); 13
C
NMR (CDCl3) d 170.68, 170.53, 170.43, 170.39, 170.08,
169.71, 169.06, 101.13 (C-1), 99.31 (C-10), 72.71, 71.79,
69.71, 68.71, 68.54, 67.80, 67.33, 66.98, 61.92, 60.45,
35.94, 31.98, 29.41, 29.03, 25.16, 24.38, 20.90, 20.72,
20.66, 20.62, 20.58, 19.98, 18.74, 18.37, 15.13, ꢀ3.39,
ꢀ5.01; HRMS (ESI) Anal. Calcd for C118H192O54S3Si4
[M+Na]+: 2704.04148. Found: 2704.04324.
4.1.10. 5-Acetylthiopentyl (2,3,4,6-tetra-O-acetyl-a-D-
galactopyranosyl)-(1/4)-2,3,6-tri-O-acetyl-b-D-galacto-
pyranoside (13). A solution of compound 12 (1.41 g,
2.00 mmol) in 1,4-dioxane (1 mL) was treated with thioace-
tic acid (3.0 ml, 42.6 mmol) and AIBN (0.679 g, 4.26 mmol)
at 80 ꢁC for 2 h under Ar. Cyclohexene (432 mL, 4.26 mmol)
was added with stirring for 10 min. The resulting solution
was purified by column chromatography on silica gel (1:1
EtOAc/hexane) to yield compound 13 (1.46 g, 93%). [a]D32
4.1.12. Dumbbell(2)4-Ga2OAc (15). A coupling reaction
between 7 (60.8 mg, 66.3 mmol) and 13 (312.5 mg,
400.0 mmol) was carried out in the same manner as de-
scribed in synthesis of 14 to give Dumbbell(2)4-Ga2OAc
15 (169.6 mg, 72%) after purification by recycling prepara-
tive HPLC (column, JAIGEL-2H and 2.5H; solvent, chloro-
+66.1 (c 1.1, CHCl3); 1H NMR (CDCl3) d 5.57 (d, 1H, J3 ,4
¼
form). [a]3D4 +61.1 (c 1.3, CHCl3); 1H NMR (CDCl3) d 5.57
0
0
2.6 Hz, H-40), 5.39 (dd, 1H, J2 ,3 ¼11.0 Hz, J3 ,4 ¼3.3 Hz, H-
(d, 4H, J3 ,4 ¼2.4 Hz, H-4 ), 5.39 (dd, 4H, J2 ,3 ¼11.0 Hz,
0
0
0
0
0
0
0
0
0
30), 5.15–5.21 (m, 2H, H-2 and H-20), 5.00 (d, 1H, J1 ,2
¼
J3 ,4 ¼3.2 Hz, H-3 ), 5.15–5.21 (m, 8H, H-2 and H-2 ),
0
0
0
0
0
0
3.6 Hz, H-10), 4.81 (dd, 1H, J2,3¼10.8 Hz, J3,4¼2.7 Hz,
H-3), 4.53 (t, 1H, J¼6.6 Hz, H-50), 4.43–4.47 (m, 2H, H-1
and H-6b), 4.06–4.19 (m, 4H, H-4, H-6a and H-60ab),
3.85–3.91 (m, 1H, one of OCH2CH2), 3.78 (t, 1H, J¼
6.6 Hz, H-5), 3.45–3.51 (m, 1H, one of OCH2CH2–), 2.86
(t, 2H, J¼7.2 Hz, CH2SAc), 2.32 (s, 3H, SAc), 2.13 (s, 3H,
Ac), 2.10 (s, 3H, Ac), 2.08 (s, 3H, Ac), 2.07 (s, 3H, Ac),
2.06 (s, 3H, Ac), 2.04 (s, 3H, Ac), 1.99 (s, 3H, Ac), 1.55–
1.66 (m, 4H, OCH2CH2CH2CH2CH2S), 1.39–1.48 (m, 2H,
OCH2CH2CH2CH2CH2S); 13C NMR (CDCl3) d 195.83
(SC]O), 170.68, 170.53, 170.43, 170.39, 170.08, 169.71,
169.05, 101.09 (C-1), 99.31 (C-10), 72.72 (C-3), 71.80
(C-5), 69.51 (OCH2CH2), 68.69, 68.54, 67.80, 67.33,
66.98, 61.93, 60.44, 30.57, 29.16, 28.89, 28.83, 25.03,
20.90, 20.72, 20.63, 20.58. Anal. Calcd for C33H48O19S:
C, 50.76; H, 6.20. Found: C, 50.47; H, 6.10. FABMS (posi-
tive): [M+H]+ 781.2. [M+Na]+ 803.2.
5.00 (d, 4H, J1 ,2 ¼3.6 Hz, H-1 ), 4.81 (dd, 4H, J2,3
¼
0
0
0
10.8 Hz, J3,4¼2.7 Hz, H-3), 4.52 (t, 4H, J¼7.3 Hz, H-50),
4.43–4.48 (m, 8H, H-1 and H-6b), 4.06–4.20 (m, 16H, H-
4, H-6a and H-60ab), 3.88–3.90 (m, 4H, one of OCH2CH2),
3.78 (t, 4H, J¼6.6 Hz, H-5), 3.46–3.48 (m, 4H, one of
OCH2CH2–), 2.50 (t, 16H, J¼6.4 Hz, CH2S), 1.98–2.22
(m, 84H, OAc), 1.53–1.61 (m, 24H, OCH2CH2CH2CH2CH2
SCH2CH2CH2Si), 1.43–1.46 (m, 8H, OCH2CH2CH2CH2
CH2S), 1.27–1.33 (m, 8H, SiCH2CH2CH2Si), 0.52–0.61
(m, 28H, SiCH2), ꢀ0.05 and ꢀ0.06 (s, 24H, SiCH3); 13C
NMR (CDCl3) d 170.70, 170.54, 170.44, 170.40, 170.10,
169.72, 169.06, 101.15 (C-1), 99.34 (C-10), 72.73, 71.80,
69.72, 68.72, 68.55, 67.80, 67.34, 66.99, 61.92, 60.46,
36.00, 32.04, 29.41, 29.05, 25.17, 24.33, 20.93, 20.75,
20.68, 20.65, 20.60, 20.21, 20.10, 18.36, 13.58, ꢀ3.22,
ꢀ5.28; HRMS (ESI) Anal. Calcd for C156H252O72S4Si5
[M+2Na]2+/2: 1795.67912. Found: 1795.68283.
4.1.11. Fan(1)3-Ga2OAc (14). A mixture of 6 (22.1 mg,
31.0 mmol) and 13 (152.6 mg, 195.0 mmol) was dissolved
in a mixture of DMF (0.25 mL) and MeOH (0.25 mL), the
4.1.13. Dumbbell(2)6-Ga2OAc (16). A coupling reaction
between 8 (49.3 mg, 43.6 mmol) and 13 (316.5 mg,
405.0 mmol) was carried out in the same manner as

5082
A. Yamada et al. / Tetrahedron 62 (2006) 5074–5083
described in synthesis of 14 to give Dumbbell(2)6-Ga2OAc
16 (166.2 mg, 75%) after purification by recycling prepara-
tive HPLC (column, JAIGEL-2.5H and 3H; solvent, chloro-
69.00, 60.82, 59.70, 35.93, 32.00, 29.61, 29.22, 25.25,
24.44, 20.36, 18.64, 13.51, ꢀ2.55, ꢀ4.55; HRMS (ESI)
Anal. Calcd for C100H196O44S4Si5 [M+Na]+: 2392.07263.
Found: 2392.07023.
form). [a]3D5 +61.1 (c 1.1, CHCl3); 1H NMR (CDCl3) d 5.57
0
0
0
0
0
(d, 6H, J3 ,4 ¼2.4 Hz, H-4 ), 5.38 (dd, 6H, J2 ,3 ¼11.0 Hz,
0
0
0
0
J3 ,4 ¼3.3 Hz, H-3 ), 5.15–5.21 (m, 12H, H-2 and H-2 ),
4.1.16. Dumbbell(2)6-Ga2 (19). A mixture of Dumb-
bell(2)6-Ga2OAc 16 (35.2 mg, 7.0 mmol) and 1 M NaOMe
methanolic solution (1 mL) was stirred at room temperature
for 3 h, 0.25 M NaOH aq (3 mL) was then added to the reac-
tion mixture. The solution was stirred overnight at room
temperature. Workup and purification were as described
in synthesis of Fan(1)3-Ga2 17. Dumbbell(2)6-Ga2 19
(22.4 mg, 98%) was obtained as white powder after lyophi-
lization. [a]3D1 +56.7 (c 1.0, H2O); 1H NMR (D2O) d 4.97 (s,
12H), 4.32 (br s, 12H), 4.00 (br s, 12H), 3.51–3.86 (m), 2.53
(br s, 24H, CH2S), 1.19–1.61 (m, 98H), 0.57 (br s, 28H,
SiCH2), ꢀ0.03 (br s, 18H, SiCH3); 13C NMR (D2O)
d 103.29, 100.34, 76.84, 74.70, 72.64, 70.92, 70.25, 69.31,
69.06, 68.81, 60.67, 59.61, 35.81, 31.88, 29.49, 29.09,
25.11, 24.30, 20.30, 20.13, 18.59, 11.90, ꢀ2.60; HRMS
(ESI) Anal. Calcd for C138H264O66S6Si5 [M+2Na]2+/2:
1677.71339. Found: 1677.71763.
0
0
0
5.00 (d, 6H, J1 ,2 ¼3.6 Hz, H-1 ), 4.81 (dd, 6H, J2,3
¼
10.7 Hz, J3,4¼2.6 Hz, H-3), 4.53 (t, 6H, J¼6.7 Hz, H-50),
4.43–4.48 (m, 12H, H-1 and H-6b), 4.07–4.20 (m, 24H, H-4,
H-6a and H-60ab), 3.88–3.91 (m, 6H, one of OCH2CH2),
3.78 (t, 6H, J¼6.6 Hz, H-5), 3.46–3.50 (m, 6H, one of
OCH2CH2–), 2.50 (t, 24H, J¼6.8 Hz, CH2S), 1.99–2.23
(m, 126H, OAc), 1.58–1.62 (m, 36H, OCH2CH2CH2CH2
CH2SCH2CH2CH2Si), 1.42–1.46 (m, 12H, OCH2CH2CH2
CH2CH2S), 1.25–1.31 (m, 8H, SiCH2CH2CH2Si), 0.54–
0.61 (m, 28H, SiCH2), ꢀ0.05 (s, 18H, SiCH3); 13C NMR
(CDCl3) d 170.60, 170.46, 170.35, 170.30, 170.02, 169.64,
168.99, 101.05 (C-1), 99.24 (C-10), 72.62, 71.70, 69.64,
68.63, 68.45, 67.71, 67.25, 66.89, 61.83, 60.38, 35.97,
34.04, 32.00, 29.96, 29.30, 29.10, 28.96, 27.75, 25.24,
25.06, 24.18, 23.13, 20.84, 20.66, 20.59, 20.56, 20.51,
20.27, 20.02, 18.39, 18.25, 16.96, 12.01, 11.40, ꢀ3.32;
HRMS (ESI) Anal. Calcd for C222H348O108S6Si5
[M+2Na]2+/2: 2559.93525. Found: 2559.92997.
4.2. Kinetic analysis of dendrimers carrying galabiose
binding to immobilized B subunits
4.1.14. Fan(1)3-Ga2 (17). A solution of Fan(1)3-Ga2OAc
14 (103.6 mg, 29.2 mmol) in MeOH (1 mL) was treated
with 28% NaOMe methanolic solution (20 mL) at room tem-
perature for 1 h, DMF (7 mL) and 0.1 M NaOH aq (6 mL)
were then added to the reaction mixture. After overnight,
the solution was neutralized by Amberlite IR120B (H+)
resin. The resin was filtered off and filtrate was concentrated
to dryness. Purification of the crude product was carried out
by gel permeation chromatography (Sephadex G50, 5%
HOAc aq eluent) to give Fan(1)3-Ga2 17 (47.8 mg, 91%)
as white powder after lyophilization. [a]3D1 +52.8 (c 1.1,
The dendrimer binding to immobilized recombinant histi-
dine-tagged Stx1 B (1B-His) and Stx2 subunits (2B-His)
was quantified using a BIAcore instrument (BIAcore,
Uppsala, Sweden) as described previously.5b Ni2+ was fixed
on a nitrilotriacetic acid sensor chip (BIAcore), and re-
combinant 1B-His or 2B-His (10 mg/mL) was injected into
the system, where it was immobilized on the chip. Various
concentrations of the dendrimers were injected (time 0)
over the immobilized 1B-His or 2B-His at flow rate of
20 mL/min to reach plateau at 25 ꢁC. The binding kinetics
were analyzed by Scatchard plot using the software BIAE-
VALUATION 3.0 (BIAcore).
1
DMSO); H NMR (CD3OD) d 4.97 (s, 3H), 4.27–4.32 (m,
6H), 44.06–4.12 (m, 3H), 4.00 (d, 3H, J¼2.5 Hz), 3.78–
3.92 (m), 3.68–3.76 (m), 3.47–3.63 (m), 2.51 (t, 12H,
J¼7.2 Hz, CH2S), 1.49–1.67 (m, 18H, OCH2CH2CH2CH2
CH2SCH2CH2CH2Si), 1.43–1.47 (m, 6H, OCH2CH2CH2
CH2CH2S), 1.37–1.40 (m, 6H, SiCH2CH2CH2Si), 0.57–
0.65 (m, 18H, SiCH2), ꢀ0.03 (s, 18H, SiCH3), ꢀ0.06 (s,
3H, SiCH3); 13C NMR (CD3OD) d 105.16, 102.51, 78.96,
76.08, 74.70, 72.82, 72.60, 71.38, 71.16, 71.05, 70.80,
62.63, 60.89, 36.80, 32.85, 30.76, 30.49, 26.38, 25.58,
21.04, 19.83, 19.79, 15.86, 14.46, ꢀ2.97, ꢀ4.67; HRMS
(ESI) Anal. Calcd for C76H150O33S3Si4 [M+Na]+:
1821.81962. Found: 1821.81874.
4.3. 125I-Stx binding assay
125I-Stx binding assay was performed as described pre-
viously.5a Vero cells were treated with 125I-Stx1 or 125I-
Stx2 (1 mg/mL) in the absence or presence of the desired
amount of a given compound for 30 min at 4 ꢁC. After exten-
sive washing, the cells were dissolved in lysis solution
(0.1 M NaOH, 0.5% SDS). Recovered radioactivity was
measured by a g-counter (Packard).
4.4. Cytotoxicity assay
4.1.15. Dumbbell(2)4-Ga2 (18). A mixture of Dumb-
bell(2)4-Ga2OAc 15 (50.1 mg, 14.0 mmol) and 1 M NaOMe
methanolic solution (1 mL) was stirred at room temperature
for 4 h, 0.25 M NaOH aq (3 mL) was then added to the reac-
tion mixture. The solution was stirred for 14 h at room
temperature. Workup and purification were as described in
synthesis of Fan(1)3-Ga2 17. Dumbbell(2)4-Ga2 18
(31.6 mg, 99%) was obtained as white powder after lyophi-
lization. [a]2D8 +47.6 (c 1.1, H2O); 1H NMR (D2O) d 5.02 (br
s, 4H), 4.36 (br s, 4H), 4.04 (br s), 3.55–3.90 (m), 2.55 (br s,
16H, CH2S), 1.26–1.62 (m, 40H), 0.60 (br s, 24H, SiCH2),
ꢀ0.01 (br s, 24H, SiCH3); 13C NMR (D2O) d 103.47,
110.49, 76.97, 74.82, 72.80, 71.04, 70.32, 69.47, 69.21,
Subconfluent Vero cells in a 96-well plate were treated with
Stx1 or Stx2 (10 pg/mL) in the absence or presence of the de-
sired amount of a given compound for 72 h. The relative
number of living cells was determined by using a WST-1
Cell Counting Kit (Wako Pure Industries).
Acknowledgements
This work was supported by a Health and Labour Sciences
Research Grant for Research on Advanced Medical Tech-
nology (14-N-O15) from the Ministry of Health, Labour,
and Welfare of Japan.

A. Yamada et al. / Tetrahedron 62 (2006) 5074–5083
5083
8834–8836; (b) Lindberg, A. A.; Brown, J. E.; Stromberg, N.;
¨
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