Convenient use of non-malodorous thioglycosyl donors for the assembly of multivalent globo- and isoglobosyl trisaccharides.

Article abstract of DOI:10.1016/S0008-6215(02)00093-9

New thioglycosyl donors (o-methoxycarbonylphenyl 2,3,4,6-tetra-O-benzyl-1-thio-beta-D-galactopyranoside and its 6-O-acetyl analogue) were designed and used for the synthesis of glycoconjugate polymers carrying Gb(3) [Gal(alpha1-->4)Gal(beta1-->4)Glc] and

Full text of DOI:10.1016/S0008-6215(02)00093-9

Carbohydrate Research 337 (2002) 983–989
www.elsevier.com/locate/carres
Convenient use of non-malodorous thioglycosyl donors for the
assembly of multivalent globo- and isoglobosyl trisaccharides
Hirofumi Dohi,a Yoshihiro Nishida,a,* Tae Takeda,b Kazukiyo Kobayashia,*
aDepartment of Molecular Design and Engineering, Graduate School of Engineering, Nagoya Uni6ersity, Furo-cho, Chikusa-ku,
Nagoya 464-8603, Japan
bDepartment of Infectious Disease Research, National Children’s Medical Research Center, Taishido, Setagaya, Tokyo 154, Japan
Received 28 December 2001; accepted 25 March 2002
Abstract
New thioglycosyl donors (o-methoxycarbonylphenyl 2,3,4,6-tetra-O-benzyl-1-thio-b-
D
-galactopyranoside and its 6-O-acetyl
analogue) were designed and used for the synthesis of glycoconjugate polymers carrying Gb3 [Gal(a1“4)Gal(b1“4)Glc] and
isoGb3 [Gal(a1“3)Gal(b1“4)Glc] clusters as side chains. These donors scarcely evolved the unpleasant odor of thiophenols and
showed a high a-anomeric selectivity in the galactosylation of p-nitrophenyl b-lactoside derivatives, although in moderate yields.
The derived trisaccharides were converted to multivalent carbohydrate ligands and were subjected to a biological assay with Shiga
toxins. The multivalent Gb3 ligand was highly active in inhibiting the toxicity, while the isoGb3 ligand showed no activity,
indicating that Stx-I discriminates between the carbohydrate structures. © 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Multivalent oligosaccharides; Stereo- and regioselective glycosylations; Glycoconjugate polymers; Gal(a1“3)Gal epitope; Pk antigenic
trisaccharide; Shiga toxins
1. Introduction
Because of their biological significance, Gb3 and
isoGb3 trisaccharides and other related a-galactoside
epitopes have resulted in a number of synthetic5,6 and
biomedical7 studies. We already reported the synthesis
of Gb3 and a multivalent model and showed that these
ligands displayed an inhibitory activity against Shiga
Cell surface oligosaccharides with
a
terminal
Gal(a1“3)Gal or Gal(a1“4)Gal linkage are known
as ligands of various bacterial toxins (Scheme 1).1 For
example, a Pk antigenic globotrioside [Gb3: Gal(a1“
4)Gal(b1“4)Glc] is recognized by Shiga toxins (Stx-I
and Stx-II) produced by Escherichia. coli O-157:H-7,
which cause a serious hemolytic uremic syndrome in
humans.2 The isomeric isoglobotrioside [isoGb3:
Gal(a1“3)Gal(b1“4)Glc] is the ligand of Toxin A
produced by Clostridium difficile, and the toxin plays a
major role in causing antibiotic-associated diarrhea.3 It
is also well known that the analogous Gal(a1“
3)Gal(b1“4)GlcNAc linkage is the key determinant of
animal antigens responsible for hyperacute rejection
upon xenotransplantation.4
* Corresponding authors. Tel.: +81-52-7892553/2488; fax:
+81-52-7892528.
Scheme 1. Structures of Pk antigenic trisaccharide and
Gal(a1“3)Gal epitope.
E-mail
addresses:
nishida@mol.nagoya-u.ac.jp
(Y.
Nishida), kobayash@mol.nagoya-u.ac.jp (K. Kobayashi).
0008-6215/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(02)00093-9

984
H. Dohi et al. / Carbohydrate Research 337 (2002) 983–989
expect possible applications in the biomedical field.
This paper reports a convenient synthesis of p-
aminophenyl (pAP) Gb3 glycoside 1 and isoGb3 gly-
coside 2 as precursors of multivalent Gb3 and isoGb3
ligands (Scheme 2). The synthesis was conducted effi-
ciently by using the novel thiogalactosyl donors 3 and
4, which were designed as non-malodorous thioglycosyl
donors.
2. Results and discussion
Design of non-malodorous thiogalactosyl donors and
its application to h-anomeric selecti6e galactosylation.—
The synthetic methods,5,6 already reported for glycosyl
ceramides, could not be applied conveniently to the
synthesis of glycoconjugate polymers because of their
lack of a suitable functional group for the grafting and
polymerization step. Thus, we adopted our previous
pathway8 starting from p-nitrophenyl b-lactoside lead-
ing to glycosyl acceptors 5 and 6 for the synthesis of 1
and 2 (Scheme 3). At first, a conventional glycosylation
step between 5 and glycosyl fluoride 7 was examined8
(Table 1). Since glycosyl donors with an electron-with-
drawing group at O-6 have been reported to promote
an a-anomeric specificity,10 donor 7 was derivatized
with a 6-O-acetyl group. Glycosylation of fluoride 7
with glycosyl acceptor 5 using tin(II) chloride and silver
perchlorate as activators11 proceeded with the expected
a-anomeric selectivity to give the desired product 8
(38% isolated yield, a/b a19:1, 1H NMR analysis).
Despite the fact that the yield of this reaction could be
increased by further optimization, we decided to try
another route towards the desired trisaccharide 8.
Next, we designed thioglycosyl donors 312 and 4 for
the construction of the a-(1“3) galactosyl linkages and
tested their reactivity. It was expected that the electron-
withdrawing o-methoxycarbonyl group might decrease
the reactivity of thiophenyl donors to improve the
stereo- and regioselectivity. The donors were obtained
from an SN2 reaction of methyl thiosalicylate with
Scheme 2. Structures of the target molecules 1 and 2, and
novel thiogalactosyl donors 3 and 4.
Scheme 3. Synthesis of target molecules 1 and 2. Reaction
conditions: (a) (i) NaOMe, MeOH, rt, 6 h, 93%; (ii) Pd(OH)2/
C, HCl, H2, MeOH, rt, 6 h, 92%; (b) NaOMe, MeOH, rt, 6
h, 97%; (ii) Pd(OH)2/C, HCl, H2, MeOH, rt, 6 h, 97%.
toxin-I (CD50 10−8ꢀ10−9 M).8 A variety of multiva-
lent Gb3 structures have been also designed by other
groups which showed a potent activity against Shiga
toxins.9 These studies clearly demonstrate the signifi-
cance of carbohydrate clusters as effective ligands of
bacterial toxins and other binding proteins and let
Table 1
Glycosylation of 5 and 6 with various glycosyl donors
Run
Acceptor
Donor equiv a
Activator equiv b
Temp (°C)
Time (h)
Yield c (%)
1
2
3
4
5
5
5
6
7, 1.5
3, 1.5
4, 1.5
3, 2.0
AgClO4 (1.5), SnCl2 (1.5)
NIS (2.0), TfOH (ca.)
NIS (2.0), TfOH (ca.)
NIS (2.0), TfOH (ca.)
0“rt
−10
−10
−10
12
2
1
8, 38
9, 60 d
8, 65
3
10, 79
a Against acceptor.
b Against donor.
c Isolated yield.
d 4%-O-Galactosylated byproduct was generated (17% isolated yield, a/b 82:18).

H. Dohi et al. / Carbohydrate Research 337 (2002) 983–989
985
1
yield, a/b ratio a19:1, H NMR analysis), while the
donor 3 without the 6-O-acetyl group tended to show
higher reactivity but lower regioselectivity (9 60%,
a(1“4) isomer in 17% yield). The utility of these new
donors could be verified in the reactions with the less
reactive acceptor 6 carrying a bulky pivaloyl group in
the vicinity. The reaction between 3 and 6 gave the
desired globotrioside 10 in high yield (79%, a/b a19:1)
by using a mixture of ethyl ether and dichloromethane
as solvent.
Scheme 4. Synthesis of novel thioglycosyl donors 3 and 4.
Reaction conditions: (c) methyl thiosalicylate, K2CO3, DMF,
rt, 2 h, 90%; (d) NaOMe, MeOH, rt, 3 h, 99%; (e) NaH,
BnBr, DMF, 16 h, 91%; (f) TBDPSCl, Et3N, DMAP, Py, rt,
8 h, 85%; (g) BnBr, NaH, DMF, rt, 12 h, 92%; (h) TBAF,
THF, rt, 12 h; (i) Ac2O, Py, rt, 7 h, 94% (2 steps).
The Gb3 and isoGb3 derivatives 10 and 8, thus
derived, were quantitatively converted to the target
compounds 1 and 2, respectively, via O-deacetylation
with sodium methoxide in methanol followed by cata-
lytic hydrogenation (palladium hydroxide in methanol)
to reduce the pNP group and cleave the benzyl groups.
Syntheses of multi6alent Gb3 and isoGb3 ligands and
their inhibiting acti6ity towards Shiga toxins.—Globo-
syl- and isoglobosyl amines 1 and 2 were converted to
the artificial glycoconjugate polymers 17 and 19 via
N-acryloylation at the terminal amino group13 followed
by radical copolymerization with acrylamide (feed mo-
lar ratio 1:4) in the presence of 2,2-azobis(2-
amidinopropane) dihydrochloride (AAPD)14 (Scheme
5). After dialysis in water (Mw 8000 cut-off) and freeze-
drying, 17 and 19 bearing respective Gb3 and isoGb3
clusters at the side chain were obtained as water-soluble
colorless powders (molar ratio carbohydrate–acrylam-
1
ide ca. 1:8 as determined by H NMR spectroscopy).
The polymeric ligands were subjected to a biological
assay involving neutralization of the toxicity of Shiga
Scheme 5. Synthesis of glycoconjugate polymers 17 and 19.
Reaction conditions: (j) acryloyl chloride, Et3N, CH2Cl2,
MeOH, 0 °C“rt, 1.5 h, 92%; (k) acrylamide, 2,2-azobis(2-
amidinopropane) dihydrochloride, H2O, 60 °C, 12 h, 70%; (l)
acryloyl chloride, Et3N, CH2Cl2, MeOH, 0 °C“rt, 1.5 h,
93%; (m) acrylamide, 2,2-azobis(2-amidinopropane) dihy-
drochloride, H2O, 60 °C, 12 h, 76%.
per-O-acetyl galactosyl bromide and isolated as crys-
talline solids (Scheme 4). Though the thiosalicylate
smells weakly like a blend of methyl salicylate and
thiophenol, the reaction does not emit the unpleasant
odor of mercapto alcohol and phenol. Glycosylation
between 5 and 4 gave the desired product 8 in satisfac-
tory yield and a-anomeric selectivity (65% isolated
Fig. 1. Neutralization effect of copolymer 17 and 19 against
Stx-I and Stx-II. Each symbol indicates as follow: ꢀ, Gb3
copolymer 17 and Stx-I assay; ꢁ, copolymer 17 and Stx-II
assay; ꢂ, isoGb3 copolymer 19 and Stx-I assay; ꢃ, copoly-
mer 19 and Stx-II assay.

986
H. Dohi et al. / Carbohydrate Research 337 (2002) 983–989
1
toxins against human ACHN cells (Fig. 1). Under the
conditions that each of Stx-I and Stx-II gave fatal
damage to the cells, the Gb3 multivalent model 17
showed a highly neutralizing activity against Stx-I
(CD50 10−8ꢀ10−9 M), but no activity against Stx-II.
This result accorded with our previous results, which
showed a critical difference in the carbohydrate recog-
nition between the two toxins. On the other hand, the
isoGb3 multivalent model 19 did not show any activity
against both toxins. This means that the strong activity
of 17 against Stx-I can be ascribed to a highly specific
interaction between the Gb3 cluster and the toxin. The
toxin strictly discriminates between Gb3 and isoGb3
structures.
Until recently, most conventional oligosaccharide
syntheses have targeted an oligosaccharide of its repeti-
tive sequence. It is now recognized that biological activ-
ities of cell surface oligosaccharides are associated with
multivalent binding to receptor proteins that are com-
posed of multiple binding subunits. For example, the
Shiga toxins are composed of five B-subunits for the
tight binding to the Gb3 cluster on the cell surface.15
Anthrax toxins consist of heptameric structures for
internalization into the host cells, though the contribu-
tion of carbohydrate–protein interactions is not clear.16
This means that a single oligosaccharide sequence can-
not bind these toxins effectively, while the multivalent
model can. The potential use of these glycosides has
been demonstrated by synthesis and biological evalua-
tion of multivalent Gb3 and isoGb3 models.
workstation. Unless otherwise stated, H NMR spectra
were recorded at 25 °C in CDCl3 using an internal
Me4Si standard at 0 ppm. Optical rotations were deter-
mined with JASCO DIP-1000 digital polarimeter using
a water-jacketed 100-mm cell at 25 °C. IR spectra were
recorded on a JASCO FT/IR-230 Fourier transform
infrared spectrometer in the form of a KBr disc. Size-
exclusion chromatography was performed with
a
JASCO 800 high-performance liquid chromatograph on
Shodex B804+B805 columns using PBS as eluent.
o-Methoxycarbonylphenyl 6-O-(tert-butyldiphenylsi-
lyl)-1-thio-i- -galactopyranoside (13).—To a solution
D
of alcohol 12 (2.00 g, 6.05 mmol), Et3N (2.48 mL, 18.2
mmol), and DMAP (79 mg, 0.650 mmol) in pyridine
(50 mL) was added tert-butylchlorodiphenylsilane (2.33
mL, 9.08 mmol) gradually at 0 °C, and the reaction
mixture was stirred at rt for 8 h. The reaction mixture
was quenched by addition of 1 N HCl and extracted
with CHCl3. The combined organic layer was washed
with 1 N HCl, satd NaHCO3, and water, dried over
MgSO4, and concentrated under diminished pressure.
The residue was purified by chromatography (silica gel,
1:1 toluene–EtOAc) to give silyl ether 13 (2.92 g, 85%)
as a colorless crystals: mp 112–114 °C; [h]2D4 −13.2° (c
1.0, CHCl3); IR (disc); w 3412 (OH), 1723 and 1251
cm−1 (CO2Me); H NMR (CDCl3): l 7.13–7.86 (m, 14
1
H, ArH), 5.02 (d, 1 H, J1,2 8.0 Hz, H-1), 4.05 (dd, 1 H,
J1,2 8.0, J2,3 9.0 Hz, H-2), 4.07 (d, 1 H, J3,4 2.6 Hz,
H-4), 3.90 (s, 3 H, CO2Me), 3.63–3.75 (m, 4 H, H-3,
H-5, and H-6R,S), 1.03 (s, 9 H, t-Bu); FAB−MS: m/z
567 [M−1]−: Anal. Calcd for C30H36O7SSi: C, 63.35;
H, 6.38. Found: C, 63.36; H, 6.36.
In summary, we have accomplished an efficient syn-
thesis of p-aminophenyl Gb3 and isoGb3 glycosides
using novel thioglycosyl donors. These glycosides could
be easily converted into their multivalent models via
radical copolymerization. The synthetic pathway, as
well as the biological data presented in this paper, may
extend the scope for a practical use of cell surface
oligosaccharides.
o-Methoxycarbonylphenyl
2,3,4-tri-O-benzyl-6-O-
-galactopyranoside
(tert-butyldiphenylsilyl)-1-thio-i-
D
(14).—To a solution of the silyl ether 13 (2.50 g, 4.40
mmol) in DMF (100 mL) was added NaH (475 mg,
19.8 mmol) and BnBr (2.35 mL, 19.8 mmol) slowly at
0 °C, and the reaction mixture was stirred at rt for 12 h.
The reaction mixture was quenched by addition of
MeOH, and to the mixture was added EtOAc. The
organic layer was washed with brine, dried over
MgSO4, and concentrated under diminished pressure.
The residue was purified by chromatography (silica gel,
10:1 toluene–EtOAc) to afford the benzyl ether 14
(3.40 g, 92%) as a white powder: [h]2D4 −24.7° (c 1.0,
CHCl3); IR (disc); w 1722 and 1250 (CO2Me), 1294 and
3. Experimental
General methods.—Reagents of the highest commer-
cial quality were purchased and used without further
purification. Anhydrous solvents were purchased from
Kanto Chemical Co., Inc. All reactions, except poly-
merization, were carried out under a dry nitrogen at-
mosphere, and monitored by thin-layer chromato-
graphy (TLC) on E. Merck aluminium roll Silica Gel
60-F254 with visualization with UV light and ethanoic
phosphomolybdic acid or p-anisaldehyde solution. E.
Merck Silica Gel 60 (particle size 0.040–0.063 mm) was
employed for column chromatography using toluene–
EtOAc and CHCl3–MeOH as eluents.
1132 cm−1 (OBn); H NMR (CDCl3): l 7.08–7.83 (m,
1
29 H, ArH), 4.66 (d, 1 H, J1,2 9.5 Hz, H-1), 4.63–4.99
(m, 6 H, ArCH2O), 4.03 (dd, 1 H, J1,2 9.5, J2,3 9.0 Hz
H-2), 3.99 (bd, J3,4 2.5 Hz, H-4), 3.84 (s, 3 H, CO2Me),
3.85 (dd, 1 H, H-6S), 3.78 (dd, 1 H, H-6R), 3.59 (dd, 1
H, J2,3 9.0, J3,4 2.5 Hz, H-3), 3.48 (dd, 1 H, H-5), 1.05
(s, 9 H, t-Bu); FAB−MS: m/z 838 [M−1]−: Anal.
Calcd for C51H54O7SSi: C, 73.00; H, 6.49. Found: C,
73.10; H, 6.47.
1H NMR (500 MHz) spectra were recorded with a
Varian Inova 500 spectrometer equipped with Sun

H. Dohi et al. / Carbohydrate Research 337 (2002) 983–989
987
mixture was stirred at rt for 6 h. To the mixture was
added TfOH (10 mL) at −10 °C, and the reaction
mixture was stirred at the same temperature for 2 h.
The reaction mixture was filtered through a pad of
Celite, and the filtrate was washed with 5% aq Na2S2O3,
satd NaHCO3 and water, dried over MgSO4, and con-
centrated under diminished pressure. The residue was
purified by chromatography (silica gel, 5:1 toluene–
EtOAc) to furnish the trisaccharide 8 as a white powder
(533 mg, 65%): [h]2D4 −62.6° (c 1.0, CHCl3); IR (film);
w 1747 and 1228 (Ac), 1519 cm−1 (NO2); 1H NMR
(CDCl3): l 8.20 and 7.04 (2×d, 4 H, pNP), 7.15–7.44
(m, 15 H, ArH), 5.15 (d, 1 H, J1,2 7.5 Hz, H-1Glc),
4.60–4.94 (d×6, 6 H, ArCH2O), 4.72 (d, 1 H, J1%,2% 1.6
Hz, H-1%Gal), 4.32 (d, 1 H, J1,2 8.5 Hz, H-1Gal), 3.75
(bs, 1 H, GalH-4), 3.61 (dd, 1 H, J2,3 9.5, J3,4 3.5 Hz,
GalH-3), 1.97–2.14 (6×s, 18 H, Ac); FAB−MS: m/z
1147 [M−1]−: Anal. Calcd for C57H65NO24: C, 59.63;
H, 5.71; N, 1.22. Found: C, 59.57; H, 5.72; N, 1.22.
o-Methoxycarbonylphenyl
6-O-acetyl-2,3,4-tri-O-
benzyl-1-thio-i- -galactopyranoside (4).—To a solu-
D
tion of benzyl ether 14 (3.00 g, 3.58 mmol) in THF (100
mL) was added TBAF (7.15 mL, 7.15 mmol, 1 M in
THF) and the reaction mixture was stirred at rt for 12
h. The reaction mixture was concentrated under dimin-
ished pressure, and the residue was diluted with CHCl3.
The organic layer was washed with brine, dried over
MgSO4, and concentrated under diminished pressure.
The residue containing alcohol 15 was dissolved in
pyridine (50 mL), and to the stirred mixture was added
Ac2O (25 mL). After 7 h, the reaction mixture was
quenched by addition of EtOH, and the mixture was
azeotropically repeatedly dried with toluene. The
residue was dissolved in CHCl3, and the organic layer
was washed with 1 N HCl, satd NaHCO3, and brine,
dried over MgSO4, and concentrated under diminished
pressure. The residue was purified by chromatography
(silica gel, 5:1 toluene–EtOAc) to give acetate 4 (2.16 g,
94%, 2 steps) as a white powder; [h]2D4 −39.6° (c 1.0,
CHCl3); IR (film); w 1743 and 1230 (Ac), 1720 and 1250
(CO2Me), 1301 and 1136 cm−1 (OBn); 1H NMR
(CDCl3): l 7.18–7.87 (m, 19 H, ArH), 4.65–5.01 (m, 6
H, ArCH2O), 4.74 (d, 1 H, J1,2 8.0 Hz, H-1), 4.26 (dd,
1 H, H-6S), 4.10 (dd, 1 H, H-6S), 4.06 (dd, 1 H, J1,2 8.0,
J2,3 9.0 Hz, H-2), 3.88 (bs, 1 H, H-4), 3.87 (s, 3 H,
CO2Me), 3.67 (ddd, 1 H, H-5), 3.64 (dd, 1 H, J2,3 9.0,
J3,4 3.0 Hz, H-3), 2.01 (s, 3 H, Ac); FAB−MS: m/z 641
[M−1]−: Anal. Calcd for C37H38O8S: C, 69.14; H,
5.96. Found: C, 69.01; H, 5.95.
p-Aminophenyl (h-
D
-galactopyranosyl)-(1“3)-(i-
D-
galactopyranosyl)-(1“4)-i-
D
-glucopyranoside (2).—To
a solution of trisaccharide 8 (60 mg, 50 mmol) in
MeOH (3 mL) was added NaOMe (10 mg), and the
reaction mixture was stirred at rt for 6 h. The reaction
mixture was quenched by addition of Amberlyst 15E-
ion exchange resin, filtered, and concentrated under
diminished pressure. The residue was purified by chro-
matography (silica gel, 30:1 CHCl3–MeOH) to afford a
deacetylated compound as a white powder (29 mg,
97%). The powder (60 mg, 50 mmol) was dissolved in
MeOH (5 mL), and the mixture was added to 10 N HCl
(10 mL, 0.10 mmol) and 10% Pd(OH)2/C. The reaction
mixture was stirred vigorously under H2 atmosphere at
rt for 6 h. The reaction mixture was filtered through a
pad of Celite, and the filtrate was concentrated under
diminished pressure to give amine 2 (29 mg) as a brown
amorphous solid which was used for the next step
without further purification: [h]2D4 −16.9° (c 1.0, wa-
ter); IR (film); w 3419 (OH), 1228 and 1076 cm−1
p-Nitrophenyl
galactopyranosyl)-(1“3)-(2,6-di-O-acetyl-i-
pyranosyl)-(1“4)-2,3,6-tri-O-acetyl-i-
(6-O-acetyl-2,3,4-tri-O-benzyl-h-
-galacto-
-glucopyranos-
D-
D
D
ide (8)
,
Method A. A suspension of flame-dried MS 4 A,
AgClO4 (218 mg, 1.05 mmol), and SnCl2 (199 mg, 1.05
mmol) in dry CH2Cl2 (6 mL) was stirred at rt for 16 h.
The stirred mixture was added to a solution of diol 5
(215 mg, 0.350 mmol) in dry CH2Cl2 (2 mL) at 0 °C,
and the mixture was stirred at rt for 6 h. The mixture
was added to a solution of fluoride 7 (400 mg, 0.702
mmol) in dry CH2Cl2 (2 mL) gradually at 0 °C, and the
reaction mixture was stirred at the same temperature
for 12 h. The reaction mixture was filtered through a
pad of Celite, and the filtrate was washed with satd
NaHCO3 and water, dried over MgSO4, and concen-
trated under diminished pressure. The residue was
purified by chromatography (silica gel, 5:1 toluene–
EtOAc) to furnish trisaccharide 8 (124 mg, 38%) as a
white powder.
1
(NH2); H NMR (D2O): l 6.83 and 6.64 (2×d, 4 H,
pAP), 4.82 (d, 1 H, J1,2 8.1 Hz, H-1Glc), 4.77 (d, 1 H,
J1%,2% 3.2 Hz, H-1%Gal), 4.35 (d, 1 H, J1,2 7.7 Hz,
H-1Gal).
p-N-Acryloylamidophenyl
(h-
D
-galactopyranosyl)-
-glucopyra-
(1“3)-(i- -galactopyranosyl)-(1“4)-i-
D
D
noside (18).—To a solution of amine 2 (25 mg, 0.04
mmol) and Et3N (22 mL, 0.120 mmol) in MeOH (3 mL)
was added a solution of acryloyl chloride (4 mL, 0.05
mmol) in CH2Cl2 (0.5 mL) slowly at 0 °C, and the
reaction mixture was stirred at rt for 1.5 h. The reaction
mixture was concentrated under diminished pressure
and the residue was purified by chromatography
(TSKgel HW-40S, water) to furnish monomer 18 (31
mg, 93%) as a white powder: [h]2D4 −5.8° (c 1.0, water);
IR (disc); w 3298 (OH), 1660 (amide), 1033 and 827
,
Method B. A suspension of flame-dried MS 4 A and
NIS (322 mg, 1.43 mmol) in dry CH2Cl2 (6 mL) was
stirred for 16 h. The stirred mixture was added to a
solution of diol 5 (292 mg, 0.476 mmol) and acetate 4
(459 mg, 0.714 mmol) in dry CH2Cl2 (4 mL), and the

988
H. Dohi et al. / Carbohydrate Research 337 (2002) 983–989
1
cm−1 (olefin); H NMR (D2O): l 7.32 and 7.02 (2×
d, 4 H, pAP), 6.28 (dd, 1 H, H-olefin), 6.20 (dd, 1 H,
H-trans), 5.75 (dd, 1 H, H-cis), 5.01 (d, 1 H, J1,2 7.8
Hz, H-1Glc), 4.99 (d, 1 H, J1%,2% 3.6 Hz, H-1%Gal),
4.42 (d, 1 H, J1,2 7.7 Hz, H-1Gal), 4.07 (bt, 1 H,
H-5Gal); FAB−MS: m/z 648 [M−1]−: Anal. Calcd
for C27H39NO17: C, 49.92; H, 6.05; N, 2.16. Found:
C, 49.90; H, 6.05; N, 2.16.
References
1. Vaughan H. A.; Loveland B. E.; Sandrin M. S. Trans-
plantation 1994, 58, 879–882.
2. (a) Lingwood C. A.; Law H.; Richardson S.; Petric M.;
Brunton J. L.; Graandis S. D.; Karmali M. J. Biol. Chem.
1987, 262, 8834–8839;
(b) Lindberg A. A.; Brown J. E.; Stro¨mberg N.; Westling-
Ryd M.; Schultz J. E.; Karlsson K. A. J. Biol. Chem.
1987, 262, 1779–1785;
Glycoconjugate polymer (19).—To a solution of
monomer 18 (30 mg, 0.046 mmol) and acrylamide (13
mg, 0.184 mmol) in degassed water (0.1 mL) was
added 2,2-azobis(2-amidinopropane) dihydrochloride
(1 mol% of monomers, 2.3 mmol in degassed water).
The reaction mixture was frozen and degassed repeat-
edly under reduced pressure. The polymerization tube
was sealed under reduced pressure, and incubated at
60 °C for 12 h. The reaction mixture was dialyzed in
water (Mw 8000 cut off) for 3 days, and lyophilized
to give 19 (34 mg, 76%) as a white powder: Mn 3.2×
105 (SEC analysis, pullulan standard); mol fraction of
sugar unit=0.12 (as determined by 1H NMR spec-
trum); IR (disc); w 3248 (OH), 1662 cm−1 (amide);
1H NMR (D2O, 80 °C): l 7.02–7.50 (br, ArH), 5.10
(bs, H-1Glc), 4.92 (bs, H-1%Gal), 4.31 (bs, H-1Gal),
3.61–4.23 (m, sugar-H), 2.39–2.61 (br, methyne of
main chain), 1.66–2.03 (br, methylene of main chain).
In the same manner as described above, polymer 17
was prepared from monomer 16. 17: Mn 4.3×105
(SEC analysis, pullulan standard); mol fraction of
sugar unit=0.13 as determined by 1H NMR spec-
trum); IR (disc); w 3225 (OH), 1661 cm−1 (amide);
1H NMR (D2O, 80 °C): l 7.05–7.48 (br, ArH), 5.22
(bs, H-1Glc), 5.05 (bs, H-1%Gal), 4.40 (bs, H-1Gal),
3.56–4.31 (m, sugar-H), 2.32–2.65 (br, methyne of
main chain), 1.72–1.93 (br, methylene of main chain).
Inhibitory acti6ity of polymers against Stx-I and
Stx-II.—A mixture of each Stx (250 pg/mL in PBS)
and the diluted polymer solution (10−2ꢀ10−14 mg/
mL) was preincubated in a well at 37 °C for 1 h. To
the well were added ACHN cells (65 mL of 8×
(105×cells/mL) in Eagle’s medium containing 10%
FCS), and the mixture was incubated for 3 days at
37 °C. The mixture was treated with 0.014% neutral
red solution (100 mL) for 1 h at 37 °C, washed with
PBS (2×200 mL), and then mixed with 0.5 N HCl–
35% EtOH (100 mL). The absorbance at 540 nm was
measured at different concentrations.
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Acknowledgements
This work was carried out with the support of the
Japan Society for the Promotion of Science Research
for a research fellowship for young scientists (DC for
H. Dohi).
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