Synthesis of the carbohydrate moiety from the parasite Echinococcus multilocularis and their antigenicity against human sera

Article abstract of DOI:10.1016/j.ejmech.2011.02.030

Stereocontrolled syntheses of biotin-labeled oligosaccharide portions with a Galβ1-3GalNAc core of the Em2 glycoprotein antigen obtained from the parasite Echinococcus multilocularis have been accomplished. Trisaccharide Galβ1-3(GlcNAcβ1-6)GalNAcα1-R (G),

Full text of DOI:10.1016/j.ejmech.2011.02.030

European Journal of Medicinal Chemistry 46 (2011) 1768e1778
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of the carbohydrate moiety from the parasite Echinococcus multilocularis
and their antigenicity against human sera
,
Akihiko Koizumi a, Kimiaki Yamano b, Frank Schweizer c d, Tadahiro Takeda a,
,
Fumiyuki Kiuchi a, Noriyasu Hada a
*
a Faculty of Pharmacy, Keio University, 1-5-30 Shibakoen, Minato-ku, Tokyo 105-8512, Japan
b Hokkaido Institute of Public Health, Kita-19, Nishi-12, Kita-ku, Sapporo 060-0819, Japan
c Department of Chemistry, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada
d Department of Medical Microbiology, University of Manitoba, Winnipeg, Manitoba R3E 3P4, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
Stereocontrolled syntheses of biotin-labeled oligosaccharide portions with a Galb1-3GalNAc core of the
Received 18 November 2010
Received in revised form
10 February 2011
Accepted 14 February 2011
Available online 22 February 2011
Em2 glycoprotein antigen obtained from the parasite Echinococcus multilocularis have been accom-
plished. Trisaccharide Gal 1-3(GlcNAc 1-6)GalNAc 1-R (G), tetrasaccharide Gal 1-3(Gal 1-4GlcNAc 1-
6)GalNAc 1-R (J) and pentasaccharide Gal 1-3(Gal 1-4Gal 1-4GlcNAc 1-6)GalNAc 1-R (K)
(R ¼ biotinylated probe) were synthesized by block synthesis by the use of 5-(methoxycarbonyl)pentyl
2,3,4,6-tetra-O-acetyl- -galactopyranosyl-(1/3)-2-azido-4-O-benzyl-2-deoxy- -galactopyranoside
as a common glycosyl acceptor. Moreover, linear trisaccharide Gal 1-4Gal 1-3GalNAc 1-R (H) and
branched tetrasaccharide Gal 1-4Gal 1-3(GlcNAc 1-6)GalNAc 1-R (I) were synthesized by stepwise
b
b
a
b
b
b
a
b
a
b
b
a
b
-
D
a-D
a
b
a
Keywords:
Glycoprotein
Echinococcus multilocularis
Host-parasite interactions
Stereocontrolled synthesis
Antigenicity
a
b
b
a
condensation. We examined the antigenicity of these five oligosaccharides by an enzyme linked
immunosorbent assay (ELISA). Our results demonstrate that biotinylated oligosaccharides H, I and K
show good serodiagnostic potential to detect infections caused by the parasite E. multilocularis. Among
them the linear sequence Gala1-4Galb1-3GalNAca1-R in oligosaccharide (H) appears to show the highest
sensitivity (95%). Moreover, our study clarified the dominant carbohydrate epitope of Em2 antigen.
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
selective antigens that permit serological detection of echinococ-
cosis in patients [4,5]. Persat et al. reported in 1991 that sera from
Alveolar echinococcosis (AE) in humans is a severe chronic
helminthic disease caused by infection with the metacestode stage
of Echinococcus multilocularis, a cestode of the genus Echinococcus,
which is limited to the northern hemisphere such as Alps in Europe
and Hokkaido in Japan. The adult parasites mainly reside in the
intestines of foxes, wolves and dogs where they produce eggs. The
eggs released in feces are usually ingested by rodents, the inter-
mediate hosts of the parasite, or accidentally by humans [1,2].
Metacestodes are fluid-filled vesicles which are surrounded by an
acellular, carbohydrate-rich surface structure that protects the
parasite from immunological and physiological reactions of the
host. In order to study the molecular features and selectivity of
the immune response of metacestodes, an access to potential
oligosaccharide epitopes expressed on the extracellular surface of
E. multilocularis is required [3]. Moreover, there is demand for
AE patients recognized a neutral glycosphingolipid fraction
extracted from metacestodes of E. multilocularis [6]. Structure
determination of this glycosphingolipid fraction revealed that it
belonged to the neogala-series (Galb1-6Gal). In addition, it was
further shown that neogala-based glycosphingolipids had signifi-
cant serodiagnostic potential in differentiating alveolar from cystic
echinococcosis (CE) [7]. Based on this information, we previously
synthesized four kinds of glycosphingolipids, [Galb1-6(Fuca1-3)
Gal 1-6Gal 1-6] and its precursors [8], and reported their serodi-
b
b
agnostic potential [9]. In 1985, Gottstein isolated two protein
fractions (Em1 and Em2) from an extract of E. multilocularis
metacestode tissue and showed that Em2 was species specific for
E. multilocularis [10].
Hülsmeier et al. reported that the structure of Em2 antigen was
a novel mucin-type glycoprotein and studied the carbohydrate
moieties in more detail [11]. However, they were unable to deter-
mine the exact linkage connectivity of the carbohydrate sequence
in this novel mucin-type glycoprotein. Based on their estimated
structures, we synthesized six oligosaccharides (AeF) with
* Corresponding author. Tel.: þ81 3 5400 2666; fax: þ81 3 5400 2556.
E-mail address: hada-nr@pha.keio.ac.jp (N. Hada).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.02.030

A. Koizumi et al. / European Journal of Medicinal Chemistry 46 (2011) 1768e1778
1769
GlcNAcβ1
-
6
GlcNAcβ1
-
GalNAcα1-R
Galβ1-4Galβ1-3GalNAcα1-R
6
3
GalNAcα1-R
-
3
-
Galβ1
A
Galβ1-4Galβ1
B
C
Galα1-4Galβ1-4GlcNAcβ1
-
Galβ1-4Galβ1-4GlcNAcβ1
-
Galβ1-4GlcNAcβ1
-
6
6
6
GalNAcα1-R
GalNAcα1-R
GalNAcα1-R
3
-
3
-
3
-
F
Galβ1
R = OCH2CH2Si(CH3)3
Fig. 1. Structures of the oligosaccharide derivatives, AeF from the parasite Echinococcus multilocularis synthesized in the previous report.
Galβ1
D
E
Galβ1
trimethylsilylethyl (TMSEt) group as an aglycon [12] (Fig. 1).
However, these oligosaccharides did not react with sera of patients
suffering from echinococcosis (data not shown) in an ELISA assay.
Recently, Díaz et al. reported that the extracellular matrix of
a related cestode Echinococcus granulosus contains a novel mucin-
6-hydroxyhexanoate using a combination of tetrabutylammonium
iodide (TBAI) and iodine (I2) as activator [12]. Although the prep-
aration of 2-(trimethylsilyl)ethyl glycoside from glycosylnitrate 1
gave an anomeric ratio of 3:1 (
anomeric ratio of 5-(methoxycarbonyl)pentyl glycoside 2 was 10:1
, from 1H NMR). Zemplén deacetylation followed by protection
of the 4- and 6-hydroxy groups as a benzylidine group provided 3. It
was possible to separate the major -anomer from the minor
a:b) in our previous paper [12], the
type O-glycan capping motif consisting of Galp
the non-reducing end [13]. This information suggests that oligo-
saccharides containing Galp 1-4Gal capping motifs, such as struc-
a
1-4Gal linkages at
(a:b
b
a
b-
tures B, C and E, are not likely to be involved in the immune
response. Moreover, in our previous investigation, we used TMSEt
group as an aglycon, which seemed to have poor adherence ability
to the microplate rendering from the use in ELISA assays. In order to
enhance the adherence ability of the oligosaccharide probes to the
plate, we decided to take advantage of the strong affinity of biotin
to avidin. Biotinylated compounds have been widely used for
investigating various biological responses [14e17]. We expected
that biotinylated oligosaccharide probes may show improved
adherence to streptavidin-coated microtitre plates that could find
application as a potential serodiagnostic tool to detect AE infections
in humans. We describe here the syntheses of biotinylated glycan
portions of the glycoprotein antigen of E. multilocularis, GeK
(Fig. 2), among which H, I and K contain the postulated novel
glycoside at this stage. Glycosylation [19] of acceptor 3 with the
trichloroacetimidate donor 4 [20] was carried out in the presence of
trimethylsilyl trifluoromethanesulfonate (TMSOTf) and molecular
sieves AW-300 in CH2Cl2 to afford desired b-glycoside 5 in 74% yield
as a single anomer. Reductive ring opening of the benzylidene
acetal 5 was achieved by treatment with dichlorophenylborane
(PhBCl2) and triethylsilane (Et3SiH) in CH2Cl2 [21] to produce
disaccharide acceptor 6 as a single regioisomer in 79% yield.
Glycosylation of imidate donor 7 [22] with acceptor 6 was
achieved in the presence of TMSOTf to afford trisaccharide 8 in 81%
yield (Scheme 2). Subsequently, reduction of the azido- and 2,2,2-
trichloroethoxycarbonyl (Troc) groups of 8 with Zn-AcOH followed
by debenzylation with catalytic hydrogenolysis over 10% Pd/C in
THF and acetylation afforded 9 in 62% yield. After deacylation by
Zemplén-based method, compound 10 was converted into an
ethylenediamine monoamide [23] by exposure to ethylenediamine
followed by conjugation to N-hydroxy succinimide ester of
biotin (biotin-NHS) to afford target biotinylated trisaccharide G
after column chromatographic purification on Sephadex LH-20
(Scheme 2).
capping motif Galpa1-4Gal at the non-reducing end, and their
antigenicity against sera of AE patients.
2. Chemistry
2.1. Syntheses of the target oligosaccharides G, H, I, J and K
The synthesis of tetrasaccharide J containing core-II type
structure was achieved by glycosylation of disaccharide donor 11
with previously prepared acceptor 6 (Scheme 3). Thioglycoside
donor 11 was prepared according to a previously established
synthetic methodology [12]. Glycosylation of donor 11 with
acceptor 6 in the presence of NIS, TfOH and MS AW-300 in CH2Cl2
afforded desired tetrasaccharide 12 in quantitative yield. The
nature of the new glycosidic linkage was determined from the
coupling constant of the anomeric proton (H-1 of GlcN,
As a temporary protecting group, 5-(methoxycarbonyl)pentyl
group was chosen to ensure future conjugation to biotin for ELISA
test for all target compounds. The synthetic routes for target
compounds GeK are outlined in Schemes 2e6. Initially, disaccha-
ride acceptor 6, which serves as a common acceptor for the
syntheses of compound G, J and K, was prepared (Scheme 1).
Compound 2 was obtained by in situ conversion of glycosylnitrate
1
[18] into glycosyl iodide followed by coupling to methyl
GlcNAcβ1
-
6
GlcNAcβ1
-
GalNAcα1-R
Galα1-4Galβ1-3GalNAcα1-R
6
3
GalNAcα1-R
-
3
-
Galβ1
G
Galα1-4Galβ1
H
I
Galα1-4Galβ1-4GlcNAcβ1
-
Galβ1-4GlcNAcβ1
-
6
6
GalNAcα1-R
GalNAcα1-R
3
-
3
-
K
Galβ1
J
Galβ1
O
H
N
S
N
H
R=
O
O
H
H
HN
NH
O
Fig. 2. Structures of the target oligosaccharide derivatives, GeK.

1770
A. Koizumi et al. / European Journal of Medicinal Chemistry 46 (2011) 1768e1778
Ph
O
OAc
AcO
OAc
O
AcO
AcO
O
1) NaI
CH3CN
1) NaOMe
O
MeOH
O
AcO
2)
methyl 6-hydroxyhexanoate
2)
BDA
OMe
N3
HO
N3
O
ONO2
I2
CSA
N3
3
1
O
OMe
2
TBAB
O
CH3CN
MS AW-300
CH2Cl2
O
90% (2 steps)
49% (2 steps)
Ph
PhBCl2
OH
OBz
OBz
O
O
BzO
Et3SiH
BnO
O
BzO
BzO
OBz
O
TMSOTf
O
BzO
MS AW-300
CH2Cl2
O
O
MS AW-300
O
+ 3
CCl3
BzO
CH2Cl2
74%
O
BzO
OBz
N3
O
79%
OBz
OMe
N3
OBz
O
OMe
6
O
NH
O
4
5
O
Scheme 1. Preparation of disaccharide acceptor.
d
¼ 4.55 ppm, JH1,
¼ 7.9 Hz). Removal of the Troc-protecting
acceptor 6 afforded desired b-glycoside 16 in 99% yield. Depro-
H2
group using ZneCu in the presence of acetic anhydride and acetic
acid in THF, followed by catalytic hydrogenation over 10% PdeC in
THF and subsequent N- and O-acetylation provided tetrasaccharide
13. After deacetylation of 13, 5-(methoxycarbonyl)pentyl glycoside
14 was converted into the ethylenediamine monoamide by expo-
sure to ethylenediamine and conjugated to biotin to afford tetra-
saccharide-biotin conjugate J in 68% yield (Scheme 3).
Branched pentasaccharide K was synthesized by glycosylation
of thioglycoside donor 15 [12] with acceptor 6 (Scheme 4). NIS/
TfOH-promoted activation of donor 15 and coupling to disaccharide
tection and biotinylation were performed as described for
compound J to provide target pentasaccharide K (Scheme 4).
Oligosaccharides H and I were prepared by stepwise condensation
as described in our previous paper [12] (Schemes 5 and 6). Both
oligosaccharides H and I are derived from a common trisaccharide
24.
Common intermediate 24 was synthesized from the acceptor 3
and known thioglycoside donor 19 [12]. Glycosylation of acceptor 3
with 19 in the presence of NIS/TfOH provided disaccharide 20 in
75% yield. Selective removal of the chloroacetyl group in 20 with
R2
AcO
AcO
O
OAc
O
TMSOTf
O
MS AW-300
AcO
OBz
AcO
+ 6
CCl3
R3O
AcO
BzO
BzO
CH2Cl2
81%
O
TrocHN
O
O
O
NH
R1
7
OBz
OMe
O
O
8 R1 = N3, R2 = NHTroc, R3 = Bn
9 R1 = R2 = NHAc, R3 = Ac
1) Zn-Cu, THF-Ac2O-AcOH
2) Pd(OH)2-C, H2, THF
3) Ac2O, Pyr.
62% (3 steps)
NHAc
HO
HO
O
O
HO
HO
OH
NaOMe
MeOH
HO
O
O
O
HO
AcHN
OH
OMe
O
O
10
NHAc
HO
HO
O
O
1)
2)
ethylenediamine
HO
OH
HO
HO
HO
Biotin-NHS
DIPEA
O
O
DMF
O
G
H
N
O
S
AcHN
OH
47% (3 steps)
O
N
H
O
H
H
HN
NH
O
Scheme 2. Synthesis of trisaccharide G.

A. Koizumi et al. / European Journal of Medicinal Chemistry 46 (2011) 1768e1778
1771
OAc
AcO
R2
O
NIS
BzO
O
b
TfOH
O
OAc
AcO
AcO
AcO
O
MS AW-300
OAc
BzO
R3O
OBz
NHTroc
SDMP
O
BzO
O
R3O
CH2Cl2
+
6
O
OAc
quant.
O
O
BnO
a
O
BzO
11
R1
OBz
OMe
O
O
12 R1 = N3, R2 = NHTroc, R3 = Bn
13 R1 = R2 = NHAc, R3 = Ac
1) Zn-Cu, THF-Ac2O-AcOH
2) Pd(OH)2-C, H2, THF
3) Ac2O, Pyr.
OH
HO
NHAc
O
HO
O
b
O
HO
O
62% (3 steps)
OH
HO
HO
OH
NaOMe
MeOH
92%
HO
O
O
a
O
HO
AcHN
OH
OMe
O
14
O
OH
HO
NHAc
O
HO
1)
2)
O
ethylenediamine
b
O
14
HO
O
OH
HO
Biotin-NHS
DIPEA
OH
DMF
HO
HO
O
O
68% (2 steps)
a
O
HO
H
N
O
S
AcHN
OH
O
N
H
O
J
H
H
HN
NH
O
Scheme 3. Synthesis of tetrasaccharide J.
OAc
AcO
O
OAc
AcO
c
R3O
R3O
O
O
OR3
b
BnO
R2
NIS
BnO
O
OBn
O
BzO
O
O
TfOH
O
b
R3O
NHTroc
SDMP
MS AW-300
O
OBz
BzO
R3O
OBz
a
+
6
O
BnO
CH2Cl2
O
R3O
OBz
BzO
BnO
99%
O
O
a
O
BzO
15
R1
OBz
OMe
O
O
16
R
1 = N3, R2 = NHTroc, R3 = Bn
1) Zn-Cu, THF-Ac2O-AcOH
2) Pd(OH)2-C, H2, THF
3) Ac2O, Pyr.
17 R1
= R2 = NHAc, R3 = Ac
61% (3 steps)
OH
HO
HO
OH
HO
O
c
O
c
HO
HO
OH
HO
O
OH
O
NHAc
O
OH
1) ethylenediamine
NHAc
O
NaOMe
MeOH
HO
O
b
O
HO
O
HO
b
O
2)
Biotin-NHS
DIPEA
O
HO
O
HO
OH
OH
HO
OH
DMF
HO
O
HO
80%
HO
O
HO
O
O
42% (2 steps)
a
O
O
HO
a
AcHN
HO
OH
OMe
O
H
N
O
AcHN
O
OH
O
S
N
H
O
K
H
HN
H
18
NH
O
Scheme 4. Synthesis of pentasaccharide K.

1772
A. Koizumi et al. / European Journal of Medicinal Chemistry 46 (2011) 1768e1778
Ph
NIS
OBn
O
O
ClAcO
BnO
TfOH
OBn
O
RO
MS AW-300
O
O
SPh
+
3
CH2Cl2
75%
O
OBz
BnO
N3
OBz
OMe
19
O
20 R = ClAc
21 R = H
O
thiourea, EtOH-Pyr.
81%
OR2
R1O
O
Si
b
R5O
NIS
O
TfOH
R5O
OR4
O
OR5
MS AW-300
CH2Cl2
R3O
+ 21
SPh
O
O
O
O
BnO
a
R5O
OBn
O
94%
R7
OR6
22
OMe
O
O
23 R1 = R2 = DTBS, R3 = R4 = CHPh, R5 = Bn, R6 = Bz, R7 = N3
24 R1 = R2 = Ac, R3 = R4 = CHPh, R5 = Bn, R6 = Bz, R7 = N3
25 R1 = R2 = R3 = R4 = Ac, R5 = Bn, R6 = Bz, R7 = N3
26 R1 = R2 = R3 = R4 = Ac, R5 = Bn, R6 = Bz, R7 = NHAc
27 R1 = R2 = R3 = R4 = R5 = R6 = H, R7 = NHAc
1) HF-Pyr., THF
2) Ac2O, Pyr.; 73% (2 steps)
1) 80%AcOH
2) Ac2O, Pyr.; 98% (2 steps)
Zn-Cu, THF-Ac2O-AcOH; 88%
1) Pd(OH)2-C, H2, THF, 2) Ac2O, Pyr.
3) NaOMe, MeOH; 88% (3 steps)
OH
OH
O
b
HO
1) ethylenediamine
OH
OH
OH
OH
O
2)
Biotin-NHS
DIPEA
O
AcHN
O
OH
a
DMF
O
HO
H
N
O
71% (2 steps)
S
O
N
H
O
H
H
H
HN
NH
O
Scheme 5. Synthesis of trisaccharide H.
thiourea [24] produced disaccharide acceptor 21 which was used
directly for the next glycosylation step. In order to prepare the
Gala1-4Gal sequence with high a-stereoselectivity, we selected
4,6-O-di-tert-butylsilylene (DTBS)- protected galactose donor 22
followed by O-acetylation and deacetylation to produce depro-
tected target trisaccharide 27 in 88% yield. Compound 27 was then
used for ligation to biotin using the common methodology to give
target trisaccharide H (Scheme 5).
[25]. Previous studies have indicated that DTBS-protected galactose
To prepare the branched tetrasaccharide I, it was necessary to
donors induce high
a
-selectivity in glycosylation reactions [26].
introduce a (1/6)-linked N-acetyl glucosamine moiety into
NIS/TfOH-promoted activation of thioglycoside donor 22 and
coupling to disaccharide acceptor 21 generated trisaccharide 23 in
trisaccharide 24 (Scheme 6). In order to accomplish this, a reductive
and regioselective ring opening of the benzylidene acetal group in
24 was necessary. This was achieved by exposure of 24 to Et3SiH
and PhBCl2 in CH2Cl2 to afford 28. Subsequently, trisaccharide
acceptor 28 was glycosylated with glycosyl imidate 7 to generate
tetrasaccharide 29 in 93% yield. The same deprotection and bio-
tinylation reactions described for oligosaccharide J afforded target
tetrasaccharide I in 52% yield (Scheme 6).
94% yield. The newly formed a-glycosidic linkage was confirmed by
1H NMR spectroscopy. The anomeric proton of the galactose moiety
(b) in 23 appeared at 4.95 ppm as a doublet with a protoneproton
coupling constant of 3.3 Hz (H-1 of Gal(b),
d
¼ 4.95 ppm, JH1b,
¼ 3.3 Hz). Selective removal of the DTBS group in 23 was ach-
H2b
ieved with HF/pyridine followed by acetylation with acetic anhy-
dride in pyridine to afford 24 in 73% yield. Global deprotection was
performed by a combination of protection/deprotection steps. At
first the benzylidene acetal was removed by acidic hydrolysis fol-
lowed by O-acetylation using acetic anhydride in pyridine to afford
25 in 88% yield. Secondly, the azido-group was converted to an
acetamido moiety by reduction with Zn/Cu AcOH in the presence of
acetic anhydride. Finally, the obtained N-acylated product 26 was
debenzylated by catalytic hydrogenolysis using Pearlman’s catalyst
3. Antigenicity of oligosaccharides by ELISA
The reactivity of the five oligosaccharides G, H, I, J and K to
alveolar echinococcosis (AE) patient sera was examined using
microplates coated with streptavidin. The antibody response of the
AE patient group differed from that of the normal healthy (NH)
group (Fig. 3). AE patient group tended to show higher ELISA values

A. Koizumi et al. / European Journal of Medicinal Chemistry 46 (2011) 1768e1778
1773
OAc
AcO
OAc
AcO
BnO
O
O
Ph
b
b
PhBCl2
BnO
BnO
Et3SiH
BnO
O
OBn
O
OH
BnO
O
O
OBn
O
MS AW-300
CH2Cl2
85%
O
O
O
O
O
a
a
BnO
BnO
N3
OBz
N3
OMe
OBz
24
OMe
O
O
O
28
O
NHTroc
OAc
AcO
BnO
AcO
AcO
O
OAc
O
O
1) Zn-Cu, THF-Ac2O-AcOH
2) Pd(OH)2-C, H2, THF
O
TMSOTf
b
AcO
OBn
O
AcO
AcO
MS AW-300
BnO
28
+
3) Ac2O, Pyr.
BnO
O
O
O
CCl3
TrocHN
7
CH2Cl2
93%
4)NaOMe, MeOH
54% (4 steps)
O
NH
a
BnO
N3
OBz
29
OMe
O
O
NHAc
OH
HO
HO
HO
O
NHAc
OH
O
HO
HO
HO
HO
O
O
b
1)
2)
ethylenediamine
HO
OH
O
HO
O
b
HO
HO
OH
Biotin-NHS
DIPEA
HO
O
O
HO
HO
O
O
O
DMF
a
HO
O
O
H
O
52% (2 steps)
AcHN
a
S
OH
I
N
N
H
O
O
HO
O
AcHN
OH
OMe
O
H
H
30
O
HN
NH
O
Scheme 6. Synthesis of tetrasaccharide I.
than NH group against oligosaccharide probes H, I and K, which
have Gal 1-4Gal sequence. On the contrary, very little response
was observed to G and J, which lack the capping Gal 1-4Gal
probe K induced a higher response compared to F in the ELISA assay
(results not shown). This is most likely the result of the significantly
improved adherence of the biotinylated probe K to the streptavi-
din-coated microtitre plate leading to higher sensitivity and
reproducibility in this serodiagnostic assay.
a
a
sequence. Compound H showed the most strong response to the AE
group resulting in clear differentiation between the AE and NH
groups. The absorbance (A) values of AE group (n ¼ 60) were
significantly higher than those in the NH group (n ¼ 60) (P < 0.001,
Student’s t-test). For the NH group shown in Fig. 3, the average A
value against H was 0.24 ꢀ 0.09; hence the cut off value that
separated positive and negative was set to 0.42 calculated from the
average A þ (2 ꢁ standard deviation) for the 60 NH sera. Fifty-seven
sera from the AE group showed A values above 0.42 (sensitivity:
95%); in contrast, all NH sera showed A values below 0.42 (speci-
ficity: 100%). This represents a higher sensitivity than the Em2
antigen (89.3%) previously reported by Gottstein [5]. Although the
carbohydrate sequence of K and F are identical, the biotinylated
4. Conclusions
We have prepared oligosaccharide-biotin conjugates GeK in
order to study the antigenicity of putative carbohydrate sequences
at the metacestode stage of the parasite E. multilocularis. Antige-
nicity of these compounds was examined by ELISA. The glyco-
conjugates H, I and K showed good serodiagnostic potential for
alveolar echinococcosis. Among these compounds, the linear
sequence Gal
a1-4Galb1-3GalNAca1-R in oligosaccharide (H)
exhibited the highest sensitivity (95%) in the serodiagnostic assay.
Moreover, we clarified the dominant epitope of Em2 antigen and
confirmed that the unusual capping sequence Galpa1-4Gal is an
3.00
2.50
2.00
1.50
1.00
0.50
0.00
important contributor for selective antibody/antigen recognition.
The synthetic biotinylated oligosaccharide probe H, will serve as
a tool for diagnosis of AE patients by an ELISA assay.
5. Experimental section
5.1. General procedures
Optical rotations were measured with a Jasco P-1020 digital
polarimeter. 1H and 13C NMR spectra were recorded with a Varian
500 FT NMR spectrometer. Me4Si and acetone were used as internal
standards for CDCl3 and D2O, respectively. MALDI-TOFMS was
recorded on an AB Sciex Voyager RP mass spectrometer. High-
resolution mass spectra were recorded on a JEOL JMS-700 under
Fig. 3. ELISA reaction between human sera and the five oligosaccharides (GeK) used.
(þ): alveolar echinococcosis (AE) patient group (ꢂ):normal healthy (NH) group. The
line of H show the cut off value (0.42).

1774
A. Koizumi et al. / European Journal of Medicinal Chemistry 46 (2011) 1768e1778
0
0
0
0
0
FAB conditions. TLC was performed on Silica Gel 60 F254 (E. Merck)
with detection by quenching of UV fluorescence and by charring
with 10% H2SO4. Column chromatography was carried out on Silica
Gel 60 (E. Merck).
J3 , 4 ¼ 3.4 Hz, H-3 ), 5.49 (s, 1H, PhCH), 5.17 (d, 1H, J1 , 2 ¼ 8.0 Hz, H-
10), 4.93 (d, 1H, J1, 2 ¼ 3.3 Hz, H-1), 4.77 (dd, 1H, J5 , 6 a ¼ 8.8 Hz, J6 a,
0
0
0
0
0
¼ 13.0 Hz, H-6 a), 4.47 (d, 1H, J3, 4 ¼ 3.2 Hz, H-4) 4.44e4.40 (m,
6 b
2H, H-50, H-60b), 4.15e4.12 (m, 2H, H-3, H-6a), 3.76e3.74 (m, 2H, H-
3, H-6b), 3.69e3.64 (m, 4H, OCH2CH2 a, OCH3), 3.49 (s, 1H, H-5),
3.47e3.43 (m, 1H, OCH2CH2 b), 2.32 (t, 2H, J ¼ 7.3 Hz), 1.69e1.59 (m,
5.2. 5-(Methoxycarbonyl)pentyl 3,4,6-tri-O-acetyl-2-azido-2-
deoxy-
D
-galactopyranoside (2)
4H), 1.42e1.36(m, 2H). 13C NMR (125 MHz, CDCl3):
d174.9, 166.0,
165.6, 165.2, 137.7, 133.6, 133.5, 133.3, 130.0, 129.8, 129.73, 129.72,
129.45, 129.44, 129.03, 128.97, 128.79, 128.71, 128.67, 128.57, 128.3,
128.2, 128.1, 126.1, 103.0 (C-10), 100.6 (CHPh) 98.6 (C-1), 76.4 (C-3),
76.0 (C-4), 72.0 (C-30), 71.5 (C-50), 69.5 (C-20), 69.0 (C-6), 68.3
(OCH2CH2), 68.2 (C-40), 63.0 (C-5), 62.5 (C-60), 58.5 (C-2), 51.5
(OCH3), 33.9, 29.0, 25.7, 24.6.
To a solution of 1 (870 mg, 2.31 mmol) in CH3CN (10.0 mL) was
added NaI (2.42 g, 18.6 mmol). The reaction mixture was stirred for
1 h at room temperature, then extracted with Et2O, washed with aq.
Na2S2O3, dried (MgSO4), and concentrated to give glycosyl iodide
intermediate. To a solution of methyl 6-hydroxyhexanoate (1.01 g,
6.93 mmol), tetrabutylammonium iodide (TBAI) (1.71 g, 4.62 mmol)
and MS AW300 (1.0 g) in CH2Cl2 (5.0 mL) were stirred under an
atmosphere of argon for 2 h at room temperature, then solution of
the glycosyl iodide in CH2Cl2 (5.0 mL) and iodine (I2) (879 mg
3.47 mmol) were added and stirring was continued for 88 h.
The reaction mixture was diluted with CHCl3, washed with satu-
rated aqueous Na2S2O3, dried (MgSO4), and concentrated. The
product was purified by silica gel column chromatography (20:1
HRFABMS: Calcd for C54H53N3O16Na: m/z 1022.3324 Found: m/z
1022.3276 [M þ Na]þ.
5.5. 5-(Methoxycarbonyl)pentyl 2,3,4,6-tetra-O-benzoyl-b-D-
galactopyranosyl-(1/3)-2-azido-4-O-benzyl-2-deoxy-
galactopyranoside (6)
a-D-
To a solution of 5 (207 mg, 0.21 mmol)) in dry CH2Cl2 (4.0 mL)
tolueneeacetone) as eluent to give 2 (
a
:
b
ratio 10:1) (524 mg, 49%,
was added MS AW-300 (200 mg), and the mixture was stirred for
two steps). MALDI-TOFMS: Calcd for C19H29N3O10Na: m/z 482.2
2 h at room temperature, then cooled to ꢂ78 ꢃC Et3SiH (100
mL,
Found: m/z 482.7 [M þ Na]þ.
0.62 mmol) and PhBCl2 (91.6 mL 0.70 mmol) were added, and the
mixture was stirred for 5 min, then neutralized with Et3N and
added to MeOH. The precipitates were filtrated off and washed
with CHCl3. The combined filtrate and washings were successively
washed with water, dried (MgSO4), and concentrated. The product
was purified by silica gel column chromatography (1:1 hexane-
5.3. 5-(Methoxycarbonyl)pentyl 2-azido-4,6-O-benzylidene-2-
deoxy-a-D-galactopyranoside (3)
To a solution of 2 (524 mg, 1.14 mmol) in MeOH (10 mL) was
added NaOMe (31 mg, 0.51 mmol) and the mixture was stirred for
30 min, then neutralized with Amberlite IR 120 [Hþ]. The mixture
was filtered off and concentrated. To a solution of the residue
(367 mg) in CH3CN (10 mL) were added benzaldehyde dimethyla-
cetal (0.34 mL, 2.28 mmol) and camphor-10-sulfonic acid (132 mg,
0.51 mmol) at room temperature. The reaction mixture was stirred
for 3 h, then neutralized with Et3N. After evaporation, column
chromatography of the residue on silica gel (2:1 hexane-AcOEt)
ethyl acetate) to give 6 (164 mg, 79％). [
a
]
D þ 104 (c 1.0, CHCl3). 1H
0
NMR (500 MHz, CDCl3):
d
8.03e7.21 (m, 30H, Ar), 6.04 (d, 1H, J3 ,
¼ 3.0 Hz, H-40), 5.98 (dd, 1H, J1 , 2 ¼ 7.8 Hz, J2 , 3 ¼ 10.5 Hz, H-20),
40
0
0
0
0
5.65 (dd, 1H, J2 , 3 ¼ 10.3 Hz, J3 , 4 ¼ 3.3 Hz, H-30), 5.20 (d, 1H, J1 ,
0
0
0
0
0
¼ 7.9 Hz H-10), 5.19 and 4.75 (each d, 2H, Jgem ¼ 11.2 Hz, benzyl
20
0
methylene), 4.85 (d, 1H, J1,
¼ 3.6 Hz, H-1), 4.76 (d, 1H, J6 a,
2
¼ 11.4 Hz, H-60a), 4.50 (dd, 1H, J5 , 6 a ¼ 5.7 Hz, J6 a, 6 b ¼ 11.2 Hz,
0
0
0
0
0
6 b
H-60b), 4.45 (t, 1H, J5 , 6 a ¼ 6.7 Hz, H-50), 4.16e4.12 (m, 2H, H-4, H-
3), 3.72e3.60 (m, 7H, H-2, H-5, H-6a, OCH2CH2 a, OCH3), 3.46e3.37
(m, 2H, H-6a, OCH2CH2 a), 2.32 (t, 2H, J ¼ 7.3 Hz), 1.67e1.56 (m, 4H),
0
0
gave 3 (433 mg, 90%). [
a
]
D þ 126 (c 1.0, CHCl3). 1H NMR (500 MHz,
CDCl3):
d
7.50e7.37 (m, 5H, Ar), 5.58 (s, 1H, PhCH), 4.99 (d, 1H, J1,
¼ 3.4 Hz H-1), 4.31e4.26 (m, 2H, H-4, H-6a), 4.18 (dd., 1H, J2,
1.41e1.35(m, 2H). 13C NMR (125 MHz, CDCl3):
d174.9, 166.0, 165.6,
2
3
¼ 10.6 Hz, J3, 4 ¼ 3.5 Hz, H-3), 4.11e4.08 (m, 1H, H-6b), 3.75e3.70
165.34, 165.28, 138.2, 133.6, 133.5, 133.3, 133.2, 129.80, 129.78,
129.74, 129.70, 129.3, 129.2, 128.9, 128.8, 128.7, 128.6, 128.32,
128.28, 128.1, 103.0 (C-10), 98.2 (C-1), 78.6 (C-3), 76.8 (C-4), 75.0
(CH2Ph), 71.7 (C-30), 71.5 (C-50), 70.6 (C-5), 69.7 (C-20), 68.1
(OCH2CH2, C-40), 62.3 (C-6), 62.1 (C-60), 59.4 (C-2), 51.5 (OCH3), 33.9,
28.9, 25.7, 24.6. HRFABMS: Calcd for C54H55N3O16Na: m/z
1024.3480 Found: m/z 1024.3456 [M þ Na]þ.
(m, 2H, OCH2CH2 a, H-5), 3.67 (s, 3H, OCH3), 3.55e3.48 (m, 2H, H-2,
OCH2CH2 b), 2.33 (t, 2H, J ¼ 7.4 Hz), 1.69e1.60 (m, 4H,), 1.45e1.39
(m, 2H). 13C NMR (125 MHz, CDCl3):
d 174.1, 137.3, 129.3, 128.3,
126.2, 101.3 (PhCH), 98.7 (C-1), 75.6 (C-4), 69.3 (C-6), 68.4
(OCH2CH2), 67.3 (C-3), 62.8 (C-5), 60.7 (C-2), 51.5 (OCH3), 33.9, 29.1,
25.7, 24.6. HRFABMS: Calcd for C20H28N3O7: m/z 422.1927 Found:
m/z 422.1974 [M þ H]þ.
5.6. 5-(Methoxycarbonyl)pentyl 2,3,4,6-tetra-O-benzoyl-
galactopyranosyl-(1/3)-[3,4,6-tri-O-acetyl-2-deoxy-2-(2,2,2-
trichloroethoxycarbonylamino)- -glucopyranosyl-(1/6)]-2-
azido-4-O-benzyl-2-deoxy- -galactopyranoside (8)
b-D-
5.4. 5-(Methoxycarbonyl)pentyl 2,3,4,6-tetra-O-benzoyl-b-D-
galactopyranosyl-(1/3)-2-azido-4,6-O-benzylidene-2-deoxy-
galactopyranoside (5)
a-D
-
b-D
a-D
To a solution of 3 (138 mg, 0.33 mmol) and 4 (364 mg,
0.49 mmol) in dry CH2Cl2 (3.0 mL) was added MS AW-300
(500 mg), and the mixture was stirred for 2 h at room temperature,
To a solution of 6 (160 mg, 0.16 mmol) and 7 (150 mg,
0.24 mmol) in dry CH2Cl2 (2 mL) was added MS AW-300 (300 mg),
and the mixture was stirred for 2 h at room temperature, then
then cooled to ꢂ20 ꢃC. TMSOTf (17.8
mL, 98.2
m
mol) was added, and
cooled to ꢂ20 ꢃC. TMSOTf (8.7
mL, 48.0 mmol) was added, and the
the mixture was stirred for 1 h at 0 ꢃC, then neutralized with Et3N.
The precipitates were filtrated off and washed with CHCl3. The
combined filtrate and washings were successively washed with
water, dried (MgSO4), and concentrated. The product was purified
by silica gel column chromatography (17:1 toluene-EtOAc) to give 5
mixture was stirred for 1 h at ꢂ20 ꢃC, then neutralized with Et3N.
The precipitates were filtrated off and washed with CHCl3. The
combined filtrate and washings were successively washed with
water, dried (MgSO4), and concentrated. The product was purified
by silica gel column chromatography (5:1 toluene-EtOAc) to give 8
(241 mg, 74％). [
d
a
]
D þ 127 (c 1.0, CHCl3). 1H NMR (500 MHz, CDCl3):
(190 mg, 81％). [
CDCl3):
a
]
þ 65.0 (c 1.0, CHCl3). 1H NMR (500 MHz,
D
8.09e7.16 (m, 25H, Ar), 6.00 (d, 1H,0J3 , 4 ¼ 3.4 Hz, H-40), 5.92 (dd,
d
5.20 (d, 1H, J1, ¼ 8.0 Hz, H-1 of Gal), 4.77 (d, 1H, J1,
0
0
2
1H, J1 , 2 ¼ 7.8 Hz, J2 , 3 ¼ 10.4 Hz, H-2 ), 5.60 (dd, 1H, J2 , 3 ¼ 10.4 Hz,
¼ 3.4 Hz, H-1 of GalN), 4.76 (d, 1H, J1, 2 ¼ 8.1 Hz, H-1 of GlcN). 13
C
0
0
0
0
0
0
2

A. Koizumi et al. / European Journal of Medicinal Chemistry 46 (2011) 1768e1778
1775
NMR (125 MHz, CDCl3):
d
102.3 (C-1 of Gal), 100.7 (C-1 of GlcN),
saturated aqueous Na2S2O3 and water, dried (MgSO4), and
concentrated. The product was purified by silica gel column chro-
matography (1:1 hexane-EtOAc) to give 12 (187 mg, quant.).
98.1 (C-1 of GalN). HRFABMS: Calcd for C69H74Cl3N4O25: m/z
1463.3708 Found: m/z 1463.3666 m/z [M þ H]þ.
[a
]
D þ 41.3 (c 1.0, CHCl3). 1H NMR (500 MHz, CDCl3):
d 5.19 (d, 1H, J1,
5.7. 5-(Methoxycarbonyl)pentyl 2,3,4,6-tetra-O-benzoyl-
galactopyranosyl-(1/3)- [2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-
-glucopyranosyl-(1/6)]-2-acetamido-4-O-acetyl-2-deoxy-
galactopyranoside (9)
b
-D-
¼ 8.0 Hz, H-1 of Gal a), 4.79 (d, 1H, J1, 2 ¼ 3.3 Hz, H-1 of GalN), 4.65
2
(d, 1H, J1, 2 ¼ 8.3 Hz, H-1 of GlcN), 4.45 (d, 1H, J1, 2 ¼ 7.2 Hz, H-1 of
b
-D
a-D
-
Gal b). 13C NMR (125 MHz, CDCl3):
d
104.2 (C-1 of Gal a), 101.4 (C-1
of GlcN), 100.1 (C-1 of Gal b), 98.1 (C-1 of GalN). MALDI-TOFMS:
Calcd for C91H95Cl3N4O32Na: m/z 1883.5 Found: m/z 1883.6
[M þ Na]þ.
To a solution of 8 (190 mg, 0.13 mmol) in THF-AcOH-Ac2O (3:2:1,
6.0 mL) was added ZneCu (760 mg). The mixture was stirred for
30 min at room temperature. After completion of the reaction, the
solid was filtered off. The filtrate was concentrated and purified by
silica gel column chromatography (3:2 tolueneeacetone) to give an
acetamido compound. To a solution of this compound (135 mg) in
THF (2.0 mL) was hydrogenolysed in the presence of Pd(OH)2/C
(100 mg) for 8 h at room temperature. The mixture was filtered and
concentrated, and the residue was acetylated with acetic anhydride
(3.0 mL) in pyridine (5.0 mL). After the reaction was quenched with
MeOH, toluene was added and co-evaporated several times. The
product was purified by silica gel column chromatography (1:1
5.10. 5-(Methoxycarbonyl)pentyl 2,3,4,6-tetra-O-benzoyl-
galactopyranosyl-(1/3)-[2,3,4,6-tetra-O-acetyl-
galactopyranosyl-(1/4)-2-acetamido-6-O-acetyl-3-O-benzoyl-2-
deoxy]- -glucopyranosyl-(1/6)]-2-acetamido-4-O-acetyl-2-
deoxy- -galactopyranoside (13)
b-D-
b-D-
b-D
a-D
Compound 13 was prepared from 12 (389 mg, 0.21 mmol) as
described for preparation of 9. The product was purified by silica gel
column chromatography (2:1 tolueneeacetone) to give 13 (214 mg,
62%). [
a
]
D þ 59.1 (c 1.0, CHCl3). 1H NMR (500 MHz, CDCl3):
d 5.01 (d,
tolueneeacetone) to give 9 (104 mg, 62％). [
CHCl3). 1H NMR (500 MHz, CDCl3):
5.00 (d, 1H, J1, 2 ¼ 7.8 Hz, H-1 of
Gal), 4.81 (d,1H, J1, 2 ¼ 3.5 Hz, H-1 of GalN), 4.62 (d,1H, J1, 2 ¼ 8.3 Hz,
H-1 of GlcN). 13C NMR (125 MHz, CDCl3):
101.3 (C-1 of Gal), 101.0
a
]
þ 77.3 (c 1.0,
1H, J1, 2 ¼ 7.7 Hz, H-1 of Gal a), 4.81 (d, 1H, J1, 2 ¼ 3.3 Hz, H-1 of
D
d
GalN), 4.55 (d, 1H, J1, ¼ 7.9 Hz, H-1 of GlcN), 4.53 (d, 1H, J1,
2
¼ 8.0 Hz, H-1 of Gal b). 13C NMR (125 MHz, CDCl3):
d 101.31 (C-1 of
2
d
Gal a), 101.25 (C-1 of GlcN), 100.9 (C-1 of Gal b), 97.1 (C-1 of GalN).
MALDI-TOFMS: Calcd for C82H92N2O34Na: m/z 1671.5 Found: m/z
1671.5 [M þ Na]þ.
(C-1 of GlcN), 97.0 (C-1 of GalN). HRFABMS: Calcd for C65H75N2O26
:
m/z 1299.4608 Found: m/z 1299.4567 [M þ H]þ.
5.8. Biotinylated trisaccharide (G)
5.11. 5-(Methoxycarbonyl)pentyl
b-D-galactopyranosyl-(1/3)-[b-
D-galactopyranosyl-(1/4)-2-acetamido-2-deoxy-b-D-
To a solution of 9 (100 mg, 77.0
mmol) in MeOH (5 mL) was
glucopyranosyl-(1/6)]-2-acetamido-2-deoxy-
a-D-
added NaOMe (100 mg, 4.32 mmol) and the mixture was stiþrred at
50 ꢃC for 19 h, then neutralized with Amberlite IR 120 [H ]. The
mixture was filtered off and concentrated. The product was purified
by Sephadex LH-20 column chromatography in H2O to give 10. The
residue was dissolved in anhydrous ethylenediamine (10 mL) and
heated at 70 ꢃC for 44 h. The mixture was concentrated with
toluene and the product was purified by Sephadex LH-20 column
chromatography in H2O to give an amine intermediate. The amine
galactopyranoside (14)
To a solution of 13 (210 mg, 77.0 mmol) in MeOH (15 mL) was
added NaOMe (100 mg) at room temperature and the mixture was
stirred at 50 ꢃC for 12 h, then neutralized with Amberlite IR 120
[Hþ]. The mixture was filtered off and concentrated. The product
was purified by Sephadex LH-20 column chromatography in H2O to
give 14 (102 mg, 92%). [
D2O):
a
]
þ 41.2 (c 1.0, H2O). 1H NMR (500 MHz,
D
(42.3 mg, 56.9
mmol) was dissolved in DMF (6.0 mL), and the pH
d
4.67 (d, 1H, J1, ¼ 3.4 Hz, H-1 of GalN), 4.36 (d, 1H, J1,
2
was adjusted to 8e9 using DIPEA. Biotine-NHS (23.3 mg, 56.9
mmol)
¼ 8.3 Hz, H-1 of GlcN), 4.282 (d, 1H, J1, 2 ¼ 7.8 Hz, H-1 of Gal b),
2
was added and the reaction stirred for 12 h at room temperature.
Toluene was added to and evaporated from the residue several
times. The product was purified by Sephadex LH-20 column chro-
4.276 (d, 1H, J1, 2 ¼ 7.8 Hz, H-1 of Gal a). 13C NMR (125 MHz, D2O):
d
104.3 (C-1 of Gal a), 102.5 (C-1 of Gal b), 101.0 (C-1 of GlcN), 96.4
(C-1 of GalN). HRFABMS: Calcd for C35H60N2O23Na: m/z 899.3485
matography in H2O to give G (35.0 mg, 47%). [
1H NMR (500 MHz, D2O):
4.67 (d, 1H, J1, 2 ¼ 3.9 Hz, H-1 of GalN),
4.34 (d, 1H, J1, 2 ¼ 8.4 Hz, H-1 of GlcN), 4.28 (d, 1H, J1, 2 ¼ 7.8 Hz, H-1
of Gal). 13C NMR (125 MHz, D2O):
104.2 (C-1 of Gal), 101.1 (C-1 of
a]
D þ 56.7 (c 1.0, H2O).
Found: m/z 899.3520 [M þ Na]þ.
d
5.12. Biotinylated tetrasaccharide (J)
d
GlcN), 96.3 (C-1 of GalN). HRFABMS: Calcd for C40H69N6O19SNa: m/z
Compound 14 (102 mg, 0.12 mmol) was dissolved in neat
anhydrous ethylenediamine (10 mL) and heated at 70 ꢃC for 48 h.
The mixture was concentrated with toluene and the product was
purified by Sephadex LH-20 column chromatography in H2O to give
991.4158 Found: m/z 991.4184 [M þ Na]þ.
5.9. 5-(Methoxycarbonyl)pentyl 2,3,4,6-tetra-O-benzoyl-
galactopyranosyl-(1/3)-[2,3,4,6-tetra-O-acetyl-
galactopyranosyl-(1/4)-3-O-benzoyl-6-O-benzyl-2-deoxy-2-
(2,2,2-trichloroethoxycarbonylamino)- -glucopyranosyl-(1/6)]-
2-azido-4-O-benzyl-2-deoxy- -galactopyranoside (12)
b-D-
b-
D-
an amine intermediate. The amine (84.5 mg, 93.4 mmol) was dis-
solved in DMF (10 mL), and the pH was adjusted to 8e9 using
DIPEA. Biotine-NHS (38.3 mg 0.11 mmol) was added and the
reaction stirred for 12 h at room temperature. Toluene was added to
and evaporated from the residue several times. The product was
purified by Sephadex LH-20 column chromatography in H2O to give
b-D
a-D
To a solution of 6 (100 mg, 99.8
mmol) and 11 (150 mg,
0.150 mmol) in dry CH2Cl2 (2 mL) was added powdered MS AW-300
(250 mg), and the mixture was stirred under Ar atmosphere for 2 h
at room temperature, then cooled to ꢂ40 ꢃC. NIS (67.5 mg,
J (88.7 mg, 68%). [
a
]
D þ 47.2 (c 1.0, H2O). 1H NMR (500 MHz, D2O):
d
4.67 (d, 1H, J1, 2 ¼ 3.7 Hz, H-1 of GalN), 4.36 (d, 1H, J1, 2 ¼ 8.2 Hz, H-1
of GlcN), 4.284 (d, 1H, J1, 2 ¼ 7.8 Hz, H-1 of Gal b), 4.278 (d, 1H, J1,
0.30 mmol) and TfOH (2.7
m
L, 30.0
m
mol) were added to the
¼ 7.8 Hz, H-1 of Gal a). 13C NMR (125 MHz, D2O):
d 104.3 (C-1 of
2
mixture, which was stirred for 1 h at ꢂ50 ꢃC, then neutralized with
Et3N. The precipitates were filtered off and washed with CHCl3. The
combined filtrate and washings were successively washed with
Gal a), 102.5 (C-1 of Gal b), 101.0 (C-1 of GlcN), 96.4 (C-1 of GalN).
HRFABMS: Calcd for C46H73N6O24SNa: m/z 1153.4686 Found: m/z
1153.4659 [M þ Na]þ.

1776
A. Koizumi et al. / European Journal of Medicinal Chemistry 46 (2011) 1768e1778
5.13. 5-(Methoxycarbonyl)pentyl 2,3,4,6-tetra-O-benzoyl-
galactopyranosyl-(1/3)-[4,6-di-O-acetyl-2,3-di-O-benzyl-
galactopyranosyl-(1/4)-2-O-benzoyl-3,6-di-O-benzyl-
galactopyranosyl-(1/4)-3-O-benzoyl-6-O-benzyl-2-deoxy-2-
(2,2,2-trichloroethoxycarbonylamino)- -glucopyranosyl-(1/6)]-
2-azido-4-O-benzyl-2-deoxy- -galactopyranoside (16)
b
-
-
D
-
-
5.17. 5-(Methoxycarbonyl)pentyl 2-O-benzoyl-3,6-di-O-benzyl-4-
O-chloroacetyl- -galactopyranosyl-(1/3)-2-azido-4,6-O-
benzylidene-2-deoxy- -galactopyranoside (20)
a
D
b-D
b
-
D
-
a-D
b-
D
Compound 20 was prepared from 3 (123 mg, 0.292 mmol) and
19 (222 mg, 0.350 mmol) as described for preparation of 12. The
product was purified by silica gel column chromatography (14:1
a-D
Compound 16 was prepared from 6 (60.0 mg, 59.9
m
mol) and 15
toluene-ethyl acetate) to give 20 (206 mg, 75%). [
a
]
þ 99.4 (c 1.0,
D
(102 mg, 65.8 mol) as described for preparation of 12. The product
m
CHCl3). 1H NMR (500 MHz, CDCl3):
d
8.01e7.06 (m, 20H, Ar), 5.68 (d,
0
0
0
0
was purified by silica gel column chromatography (16:9 hexane-
1H, J3 , 4 ¼ 3.3 Hz, H-4 ), 5.46e5.42 (m, 2H, H-2 , PhCH), 4.91 (d, 1H,
D þ 71.1 (c 1.0, CHCl3). 1H
J1, 2 ¼ 3.5 Hz, H-1), 4.82 (d, 1H, J1 , 2 ¼ 8.1 Hz, H-10), 4.65 and 4.42
(each d, 2H, Jgem ¼ 12.7 Hz, benzyl methylene), 4.54 and 4.49 (each
d, 2H, Jgem ¼ 11.7 Hz, benzyl methylene), 4.37 (d, 1H, J3, 4 ¼ 3.1 Hz,
H-4), 4.19 and 4.11 (each d, 2H, Jgem ¼ 15.2 Hz, COCH2Cl), 4.16 (dd,
1H, J5, 6a ¼ 12.5 Hz, J6a, 6b ¼ 1.5 Hz, H-6a), 4.06 (dd,1H, J2, 3 ¼ 10.8 Hz,
J3, 4 ¼ 3.3 Hz, H-3), 3.90e3.87 (m, 2H, H-5, H-6b), 3.70e3.62 (m, 7H,
0
0
ethyl acetate) to give 16 (143 mg, 99%). [
NMR (500 MHz, CDCl3):
a
]
d
5.15 (d,1H, J1, 2 ¼ 8.1 Hz, H-1 of Gal a), 4.97
(d, 1H, J1, 2 ¼ 3.4 Hz, H-1 of Gal c), 4.75 (d, 1H, J1, 2 ¼ 3.7 Hz, H-1 of
GalN), 4.61 (d, 1H, J1, ¼ 7.8 Hz, H-1 of Gal b), 4.52 (d, 1H, J1,
2
¼ 8.3 Hz, H-1 of GlcN),. 13C NMR (125 MHz, CDCl3):
d 102.9 (C-1 of
2
Gal a), 101.4 (C-1 of GlcN), 101.1 (C-1 of Gal b), 100.4 (C-1 of Gal c),
98.0 (C-1 of GalN). MALDI-TOFMS: Calcd for C128H129Cl3N4O36Na:
m/z 2425.7 Found: m/z 2425.7 [M þ Na]þ.
0
0
0
H-2, H-3 , H-6 a, OCH2CH2 a, OCH3), 3.59e3.56 (m, 2H, H-5 , H-6’b),
3.45e3.41 (m, 1H, OCH2CH2 b), 2.30 (t, 2H, J ¼ 7.5 Hz), 1.66e1.57 (m,
4H), 1.40e1.33(m, 2H). 13C NMR (125 MHz, CDCl3):
d174.9, 167.0,
165.2, 137.7, 137.4, 136.9, 133.0, 129.90, 129.87, 129.0, 128.8, 128.6,
128.3, 128.2, 128.11, 128.06, 128.0, 127.8, 126.1, 102.5(C-10), 100.5
(CHPh) 98.7 (C-1), 76.2 (C-30), 75.8 (C-4), 75.4 (C-3), 73.8 (CH2Ph),
72.1 (C-5), 71.1 (CH2Ph), 70.7 (C-20), 69.1 (C-6), 68.3 (C-40), 68.1
(OCH2CH2), 67.9 (C-60), 63.0 (C-50), 58.5 (C-2), 51.5(OCH3), 40.1
(CH2Cl), 33.9, 29.0, 25.6, 24.6. HRFABMS: Calcd for
C49H54ClN3O14Na: m/z 966.3192 Found: m/z 966.3234 [M þ Na]þ.
5.14. 5-(Methoxycarbonyl)pentyl 2,3,4,6-tetra-O-benzoyl-
galactopyranosyl-(1/3)-[2,3,4,6-tetra-O-acetyl-
galactopyranosyl-(1/4)-3,6-di-O-acetyl-2-O-benzoyl-
b
-D-
a-D-
b
-D
-
galactopyranosyl-(1/4)-2-acetamido-6-O-acetyl-3-O-benzoyl-2-
deoxy-
b
-D
-glucopyranosyl-(1/6)]-2-acetamido-4-O-acetyl-2-
deoxy-a-D-galactopyranoside (17)
Compound 17 was prepared from 16 (281 mg, 0.117 mmol) as
described for preparation of 9. The product was purified by silica gel
column chromatography (3:2 tolueneeacetone) to give 23 (142 mg,
5.18. 5-(Methoxycarbonyl)pentyl 2-O-benzoyl-3,6-di-O-benzyl-b-
D
-galactopyranosyl-(1/3)-2-azido-4,6-O-benzylidene-2-deoxy-
a-
61%). [
a
]
D þ 95.1 (c 1.0, CHCl3). 1H NMR (500 MHz, CDCl3):
d 4.97 (d,
D-galactopyranoside (21)
1H, J1, 2 ¼ 7.9 Hz, H-1 of Gal a), 4.92 (d, 1H, J1, 2 ¼ 3.5 Hz, H-1 of Gal
To a solution of 20 (125 mg 0.13 mmol) in EtOH-pyridine (3:1,
c), 4.78 (d, 1H, J1, 2 ¼ 3.5 Hz, H-1 of GalN), 4.69 (d, 1H, J1, 2 ¼ 7.7 Hz,
H-1 of Gal b) 4.45 (d, 1H, J1, ¼ 7.8 Hz, H-1 of GlcN),. 13C NMR
4 mL) was added thiourea (30 mg 0.40 mmol), and the mixture was
stirred for 2 h at 80 ꢃC. The mixture was diluted with CHCl3, washed
with saturated aqueous NaHCO3 and water, dried (MgSO4), and
concentrated. The product was purified by silica gel column chro-
matography (1:1 hexane-ethyl acetate) to give 21 (93.4 mg, 81%).
2
(125 MHz, CDCl3): d 101.3 (C-1 of Gal b), 101.19 (C-1 of GlcN), 101.15
(C-1 of Gal a), 99.3 (C-1 of Gal c), 97.0 (C-1 of GalN). MALDI-TOFMS:
Calcd for C99H110N2O42Na: m/z 2021.6 Found: m/z 2021.6 [M þ Na]þ.
[
a
]
þ 89.2 (c 1.0, CHCl3). 1H NMR (500 MHz, CDCl3):
d
8.04e7.12
D
5.15. 5-(Methoxycarbonyl)pentyl
-galactopyranosyl-(1/4)- -galactopyranosyl-(1/4)-2-
acetamido-2-deoxy- -glucopyranosyl-(1/6)]-2-acetamido-2-
deoxy- -galactopyranoside (18)
b
-
D
-galactopyranosyl-(1/3)-[
a-
0
0
0
0
0
(m, 20H, Ar), 5.92 (dd,1H, J1 , 2 ¼ 8.0 Hz, J2 , 3 ¼ 9.6 Hz, H-2 ), 5.44 (s,
D
b-D
0
0
1H, PhCH), 4.91 (d, 1H, J1, 2 ¼ 3.5 Hz, H-1), 4.80 (d, 1H, J1 , 2 ¼ 8.0 Hz
H-10), 4.66 and 4.51 (each d, 2H, Jgem ¼ 12.4 Hz, benzyl methylene),
4.61 and 4.56 (each d, 2H, Jgem ¼ 11.7 Hz, benzyl methylene), 4.41 (d,
b-D
a-D
1H, J3,
¼ 3.1 Hz, H-4), 4.15e4.08 (m, 3H, H-3, H-6a, H-40),
4
Compound 18 was prepared from 17 (142 mg, 77
mmol) as
3.86e3.83 (m, 3H, H-6b, H-60a, H-60b), 4.16 (dd, 1H, J5, 6a ¼ 5.8 Hz, J5,
described for preparation of 14. The product was purified by
Sephadex LH-20 column chromatography in H2O to give 18 (59 mg,
¼ 6.8 Hz, H-5), 3.68e3.63 (m, 6H, H-2, H-30, OCH2CH2 a, OCH3),
6b
3.57 (s, 1H, H-50), 3.45e3.41 (m, 1H, OCH2CH2 b), 2.30 (t, 2H,
D þ 61.8 (c 1.0, H2O). 1H NMR (500 MHz, D2O):
d 4.76 (d, 1H,
J ¼ 7.5 Hz), 1.66e1.57 (m, 4H), 1.40e1.35(m, 2H). 13C NMR (125 MHz,
80%). [
a
]
J1, 2 ¼ 3.9 Hz, H-1 of Gal c), 4.67 (d, 1H, J1, 2 ¼ 3.7 Hz, H-1 of GalN),
4.36 (d, 1H, J1, 2 ¼ 8.7 Hz, H-1 of GlcN), 4.34 (d, 1H, J1, 2 ¼ 7.8 Hz, H-1
of Gal b), 4.28 (d, 1H, J1, 2 ¼ 7.8 Hz, H-1 of Gal a). 13C NMR (125 MHz,
CDCl3): d174.9, 165.3, 138.0, 137.7, 137.1, 129.8, 128.7, 128.5, 128.4,
128.2, 128.00, 127.96, 127.8, 127.7, 126.2, 102.5 (C-10), 100.5 (CHPh)
98.7 (C-1), 78.4 (C-30), 75.8 (C-4), 75.0 (C-3), 73.7 (CH2Ph), 73.6 (C-
5), 71.3 (CH2Ph), 71.0 (C-20), 69.4 (C-60), 69.0 (C-6), 68.2 (OCH2CH2),
66.2 (C-40), 63.1 (C-50), 58.5 (C-2), 51.5 (OCH3), 33.9, 29.0, 25.6, 24.6.
HRFABMS: Calcd for C47H53N3O13Na: m/z 890.3476 Found: m/z
890.3494 [M þ Na]þ.
D2O): d 104.3 (C-1 of Gal a), 102.8 (C-1 of Gal b), 101.0 (C-1 of GlcN),
99.8 (C-1 of Gal c), 96.4 (C-1 of GalN). HRFABMS: Calcd for
C41H70N2O28Na: m/z 1061.4013 Found: m/z 1061.4044 [M þ Na]þ.
5.16. Biotinylated pentasaccharide (K)
5.19. 5-(Methoxycarbonyl)pentyl 2,3-di-O-benzyl-4,6-O-di-tert-
Compound K was prepared from 18 (102 mg, 0.116 mmol) as
butylsilylene-
benzyl- -galactopyranosyl-(1/3)-2-azido-4,6-O-benzylidene-2-
deoxy- -galactopyranoside (23)
a-D-galactopyranosyl-(1/4)-2-O-benzoyl-3,6-di-O-
described for preparation of J, yielding 31 mg (42%). [
a
]
D þ 68.7 (c
b-D
a-D
0.8, H2O). 1H NMR (500 MHz, D2O):
d
4.76 (d,1H, J1, 2 ¼ 3.9 Hz, H-1 of
Gal c), 4.67 (d, 1H, J1, ¼ 3.7 Hz, H-1 of GalN), 4.36 (d, 1H, J1,
2
¼ 8.7 Hz, H-1 of GlcN), 4.34 (d, 1H, J1, 2 ¼ 7.8 Hz, H-1 of Gal b), 4.28
Compound 23 was prepared from 21 (90.0 mg, 0.10 mmol) and
22 (92.5 mg, 0.16 mmol) as described for preparation of 12. The
product was purified by silica gel column chromatography (14:1
2
(d, 1H, J1, 2 ¼ 7.8 Hz, H-1 of Gal a). 13C NMR (125 MHz, D2O):
d 104.3
(C-1 of Gal a), 102.8 (C-1 of Gal b), 101.0 (C-1 of GlcN), 99.8 (C-1 of
Gal c), 96.4 (C-1 of GalN). HRFABMS: Calcd for C52H88N6O29SNa: m/z
1315.5214 Found: m/z 1315.5194 [M þ Na]þ.
hexane-ethyl acetate) to give 23 (132 mg, 94%). [
a
]
þ 114 (c 1.0,
D
CHCl3). 1H NMR (500 MHz, CDCl3):
d
4.95 (d, 1H, J1, 2 ¼ 3.3 Hz, H-1

A. Koizumi et al. / European Journal of Medicinal Chemistry 46 (2011) 1768e1778
1777
of Gal b), 4.90 (d, 1H, J1, 2 ¼ 3.0 Hz, H-1 of GalN), 4.79 (d, 1H, J1,
5.23. 5-(Methoxycarbonyl)pentyl
-galactopyranosyl-(1/3)-2-acetamido-2-deoxy-
galactopyranoside (27)
a
-
D
-galactopyranosyl-(1/4)-
b
-
¼ 7.8 Hz, H-1 of Gal a). 13C NMR (125 MHz, CDCl3):
d
102.7 (C-1 of
D
a-D-
2
Gal a), 101.0 (C-1 of Gal b), 98.7 (C-1 of GalN). HRFABMS: Calcd
for C75H91N3O18SiNa: m/z 1372.5965 Found: m/z 1372.5992
[M þ Na]þ.
Compound 26 (184 mg, 0.141 mmol) in THF (5.0 mL) was
hydrogenolysed in the presence of Pd(OH)2/C (150 mg) for 4 h at
room temperature, and the mixture was filtered and concentrated.
The residue was acetylated with acetic anhydride (3.0 mL) in
pyridine (5.0 mL). The reaction mixture was poured into ice-water
and extracted with CHCl3. After the reaction was quenched with
MeOH, toluene was added and co-evaporated several times. To
a solution of the residue (153 mg) in MeOH (6 mL) was added
NaOMe (50 mg) at room temperature and the mixture was þstirred
at 40 ꢃC for 4 h, then neutralized with Amberlite IR 120 [H ]. The
mixture was filtered off and concentrated. The product was purified
by Sephadex LH-20 column chromatography in H2O to give 27
5.20. 5-(Methoxycarbonyl)pentyl 4,6-di-O-acetyl-2,3-di-O-benzyl-
a-D-galactopyranosyl-(1/4)-2-O-benzoyl-3,6-di-O-benzyl-b-D-
galactopyranosyl-(1/3)-2-azido-4,6-O-benzylidene-2-deoxy-
a-D-
galactopyranoside (24)
A solution of 23 (746 mg 0.55 mmol) in THF (6 mL) was added
HF-Pyr. (3.5 mL) at ꢂ10 ꢃC and then was stirred for 4 h. The reaction
mixture was added to water, extracted with ethyl acetate, and the
organic layer was washed with saturated aqueous NaHCO3 and
water, dried (MgSO4), and concentrated. The residue was treated
with Ac2O (6 mL) in pyridine (10 mL). The reaction mixture was
poured into ice-water and extracted with CHCl3. The extract was
washed sequentially with 5% HCl, saturated aqueous NaHCO3 and
water, dried (MgSO4), and concentrated. The product was purified
by silica gel column chromatography (1:1 hexane-EtOAc) to give 24
(84.0 mg, 88%). [
a
]
þ 127 (c 1.0, H2O). 1H NMR (500 MHz, D2O):
d
D
4.77 (d, 1H, J1, 2 ¼ 3.9 Hz, H-1 of Gal b), 4.69 (d, 1H, J1, 2 ¼ 3.6 Hz, H-1
of GalN), 4.36 (d, 1H, J1, 2 ¼ 7.5 Hz, H-1 of Gal a). 13C NMR (125 MHz,
D2O):
d 104.5 (C-1 of Gal a), 100.0 (C-1 of Gal b), 96.6 (C-1 of GalN).
HRFABMS: Calcd for C27H47NO18Na: m/z 696.2691 Found: m/z
696.2707 [M þ Na]þ.
(522 mg, 73％). [
CDCl3):
a
]
þ 123 (c 1.0, CHCl3). 1H NMR (500 MHz,
D
d
5.06 (d, 1H, J1, 2 ¼ 3.7 Hz, H-1 of Gal b), 4.90 (d, 1H, J1,
¼ 3.8 Hz, H-1 of GalN), 4.80 (d, 1H, J1, 2 ¼ 8.5 Hz, H-1 of Gal a). 13
C
5.24. Biotinylated trisaccharide (H)
2
NMR (125 MHz, CDCl3):
d 103.0 (C-1 of Gal a), 100.9 (C-1 of Gal b),
Compound H was prepared from 27 (84.0 mg, 0.125 mmol) as
98.7 (C-1 of GalN). HRFABMS: Calcd for C71H79N3O20Na: m/z
1316.5155 Found: m/z 1316.5110 [M þ Na]þ.
described for preparation of J, yielding 82.3 mg (71%). [
a
]
D þ 119 (c
1.0, H2O). 1H NMR (500 MHz, D2O):
d
4.77 (d,1H, J1, 2 ¼ 4.0 Hz, H-1 of
Gal b), 4.69 (d, 1H, J1, ¼ 3.8 Hz, H-1 of GalN), 4.35 (d, 1H, J1,
2
5.21. 5-(Methoxycarbonyl)pentyl 4,6-di-O-acetyl-2,3-di-O-benzyl-
¼ 7.7 Hz, H-1 of Gal a). 13C NMR (125 MHz, D2O):
d 104.5 (C-1 of
2
a-D
-galactopyranosyl-(1/4)-2-O-benzoyl-3,6-di-O-benzyl-
b-
D-
Gal a), 100.0 (C-1 of Gal b), 96.6 (C-1 of GalN). HRFABMS: Calcd for
galactopyranosyl-(1/3)-4,6-di-O-acetyl-2-azido-2-deoxy-
galactopyranoside (25)
a-D
-
C38H65N5O19SNa: m/z 950.3892 Found: m/z 950.3934 [M þ Na]þ.
5.25. 5-(Methoxycarbonyl)pentyl 4,6-di-O-acetyl-2,3-di-O-benzyl-
a-D-galactopyranosyl-(1/4)-2-O-benzoyl-3,6-di-O-benzyl-b-D-
A solution of 24 (213 mg 0.165 mmol) in 80% AcOH was stirred at
70 ꢃC for 2 h, then was diluted with toluene and concentrated. The
residue was acetylated with acetic anhydride (3.0 mL) in pyridine
(5.0 mL). After the reaction was quenched with MeOH, toluene was
added and co-evaporated several times. The product was purified
by silica gel column chromatography (3:2 hexane-ethyl acetate) to
galactopyranosyl-(1/3)-2-azido-4-O-benzyl-2-deoxy-
a-D-
galactopyranoside (28)
Compound 28 was prepared from 24 (270 mg, 0.21 mmol) as
described for preparation of 6. The product was purified by silica gel
column chromatography (1:1 hexane-ethyl acetate) to give 28
give 25 (208 mg, 98％). [
a
]
D þ 121 (c 1.0, CHCl3). 1H NMR (500 MHz,
þ 91.3 (c 1.0, CHCl3). 1H NMR (500 MHz,
CDCl3):
d
5.07 (d, 1H, J1, 2 ¼ 3.4 Hz, H-1 of Gal b), 4.85 (d, 1H, J1,
(229 mg, 85%). [
CDCl3):
a
]
D
¼ 3.6 Hz, H-1 of GalN), 4.70 (d, 1H, J1, 2 ¼ 7.8 Hz, H-1 of Gal a). 13
C
2
d
5.10 (d, 1H, J1, 2 ¼ 3.5 Hz, H-1 of Gal b), 4.82 (d, 1H, J1,
¼ 7.7 Hz, H-1 of Gal a), 4.80 (d, 1H, J1, 2 ¼ 3.6 Hz, H-1 of GalN). 13
C
NMR (125 MHz, CDCl3):
d 102.0 (C-1 of Gal a), 100.5 (C-1 of Gal b),
2
98.0 (C-1 of GalN). HRFABMS: Calcd for C68H79N3O22Na: m/z
NMR (125 MHz, CDCl3):
d 103.1 (C-1 of Gal a), 100.8 (C-1 of Gal b),
1312.5053 Found: m/z 1312.5048 [M þ Na]þ.
98.3 (C-1 of GalN). HRFABMS: Calcd for C71H81N3O20Na: m/z
1318.5311 Found: m/z 1318.5349 [M þ Na]þ.
5.22. 5-(Methoxycarbonyl)pentyl 4,6-di-O-acetyl-2,3-di-O-benzyl-
a
-
D
-galactopyranosyl-(1/4)-2-O-benzoyl-3,6-di-O-benzyl-
galactopyranosyl-(1/3)-2-acetamido-4,6-di-O-acetyl-2-deoxy-
-galactopyranoside (26)
b
-
D
-
5.26. 5-(Methoxycarbonyl)pentyl 4,6-di-O-acetyl-2,3-di-O-benzyl-
a-
a
-
D
-galactopyranosyl-(1/4)-2-O-benzoyl-3,6-di-O-benzyl-
galactopyranosyl-(1/3)-[3,4,6-tri-O-acetyl-2-deoxy-2-(2,2,2-
trichloroethoxycarbonylamino)- -glucopyranosyl-(1/6)]-2-
azido-4-O-benzyl-2-deoxy- -galactopyranoside (29)
b
-
D
-
D
b-D
To a solution of 25 (208 mg, 0.161 mmol) in THF-AcOH-Ac2O
a-D
(3:2:1, 10 mL) was added ZneCu (800 mg). The mixture was stirred
for 30 min at room temperature. After completion of the reaction,
the mixture was filtered through Celite. The filtrate was concen-
trated with toluene and purified by silica gel column chromatog-
Compound 29 was prepared from 28 (110 mg, 84.8
m
mol) and 7
(106 mg, 0.17 mmol) as described for preparation of 5. The product
was purified by silica gel column chromatography (3:2 hexane-
raphy (5:1 tolueneeacetone) to give 25 (184 mg, 88%). [
a
]
D þ 124 (c
ethyl acetate) to give 29 (139 mg, 93%). [
NMR (500 MHz, CDCl3):
a
]
D þ 61.2 (c 1.0, CHCl3). 1H
1.0, CHCl3)
d
5.05 (d, 1H, J1, 2 ¼ 3.7 Hz, H-1 of Gal b),
1H NMR (500 MHz, CDCl3):
d
5.06 (d, 1H, J1, 2 ¼ 3.6 Hz, H-1 of Gal
b), 4.90 (d, 1H, J1, 2 ¼ 3.9 Hz, H-1 of GalN), 4.56 (d, 1H, J1, 2 ¼ 7.7 Hz,
H-1 of Gal a). 13C NMR (125 MHz, CDCl3):
100.8 (C-1 of Gal a),
4.81 (d, 1H, J1, 2 ¼ 7.9 Hz, H-1 of Gal a), 4.74 (d, 1H, J1, 2 ¼ 3.6 Hz, H-1
of GalN), 4.69 (d, 1H, J1, 2 ¼ 8.6 Hz, H-1 of GlcN). 13C NMR (125 MHz,
d
CDCl3): d 103.2 (C-1 of Gal a), 101.0 (C-1 of GlcN), 100.7 (C-1 of Gal
100.6 (C-1 of Gal b), 97.1 (C-1 of GalN). HRFABMS: Calcd for
b), 98.2 (C-1 of GalN). MALDI-TOFMS: Calcd for C86H99Cl3N4O29Na:
C70H83NO23Na: m/z 1328.5254 Found: m/z 1328.5278 [M þ Na]þ.
m/z 1779.5 Found: m/z 1778.8 [M þ Na]þ.

1778
A. Koizumi et al. / European Journal of Medicinal Chemistry 46 (2011) 1768e1778
5.27. 5-(Methoxycarbonyl)pentyl
a
-
D
-galactopyranosyl-(1/4)-
b-
at 405 nm on a microplate reader (Model 680; BIORAD, Hercules,
California, USA).
D-galactopyranosyl-(1/3)- [2-acetamido-2-deoxy-b-D-
glucopyranosyl-(1/6)]-2-acetamido-2-deoxy-
a-D-
galactopyranoside (30)
5.31. Data analysis
To a solution of 29 (267 mg, 0.152 mmol) in THF-AcOH-Ac2O
(3:2:1, 12 mL) was added ZneCu (1 g). The mixture was stirred for
30 min at room temperature. After completion of the reaction, the
solid was filtered off. The filtrate was concentrated and purified by
silica gel column chromatography (1:1 toluene: acetone) to give an
acetamido intermediate. This compound (183 mg) in THF (5.0 mL)
was hydrogenolysed in the presence of Pd(OH)2/C (150 mg) for 5 h
at room temperature, then the mixture was filtered and concen-
trated. The residue was acetylated with acetic anhydride (3.0 mL) in
pyridine (5.0 mL). After the reaction was quenched with MeOH,
toluene was added and co-evaporated several times. To a solution
of per-acylated compound (133 mg) in MeOH (5 mL) was added
NaOMe (50 mg) and the mixture was stirred at 40 ꢃC for 7 h, then
neutralized with Amberlite IR 120 [Hþ]. The mixture was filtered off
and concentrated. The product was purified by Sephadex LH-20
column chromatography in H2O to give 30 (71.9 mg, 54%).
The cut off value that separated positive and negative was
defined as the average A plus two standard deviations for the 60 NH
sera. The significant difference between the two groups was
analyzed by the Student’s t-test.
Acknowledgments
This work was supported by a Grant-in-Aid for Scientific
Research (No. 22590014) from the Ministry of Education, Culture,
Sports, Science and Technology of Japan (MEXT), and High-Tech
Research Center project of the MEXT. We gratefully acknowledge
financial support in the form of the Sasagawa Scientific Research
Grant and the Uehara Memorial Foundation. The authors are
grateful to Ms. J. Hada for providing HR-FAB MS measurements
data.
[
a]
þ 87.3 (c 1.0, H2O). 1H NMR (500 MHz, D2O):
d 4.77 (d, 1H, J1,
D
References
¼ 3.9 Hz, H-1 of Gal b), 4.67 (d, 1H, J1, 2 ¼ 3.7 Hz, H-1 of GalN), 4.35
2
(d, 1H, J1, 2 ¼ 7.6 Hz, H-1 of Gal a), 4.34 (d, 1H, J1, 2 ¼ 8.6 Hz, H-1 of
GlcN). 13C NMR (125 MHz, D2O):
d
104.5 (C-1 of Gal a), 101.1 (C-1 of
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GlcN), 100.0 (C-1 of Gal b), 96.4 (C-1 of GalN). HRFABMS: Calcd for
C35H60N2O23Na: m/z 899.3485 Found: m/z 899.3530 [M þ Na]þ.
5.28. Biotinylated tetrasaccharide (I)
Compound I was prepared from 30 (71.9 mg, 82.0
described for preparation of J, yielding 48.3 mg (52%). [
D þ 73.1 (c
1.0, H2O). 1H NMR (500 MHz, D2O):
4.76 (d,1H, J1, 2 ¼ 4.0 Hz, H-1 of
mmol) as
a
]
d
Gal b), 4.66 (d, 1H, J1, ¼ 3.6 Hz, H-1 of GalN), 4.35 (d, 1H, J1,
2
¼ 7.7 Hz, H-1 of Gal a), 4.34 (d, 1H, J1, 2 ¼ 8.4 Hz, H-1 of GlcN). 13
C
2
NMR (125 MHz, D2O):
d 104.5 (C-1 of Gal a), 101.1 (C-1 of GlcN),
100.0 (C-1 of Gal b), 96.4 (C-1 of GalN). HRFABMS: Calcd for
C46H73N6O24SNa: m/z 1153.4686 Found: m/z 1153.4681 [M þ Na]þ.
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5.29. Serum samples
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Serum samples examined by ELISA were obtained from 60
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patients who were confirmed to have AE and 60 NH individuals.
5.30. ELISA protocol
ELISA was performed using as previously described [27,28] with
some modifications. The oligosaccharides in H2O (13 pmol per well)
were added to the wells of flat-bottomed microplates (Streptavidin
C96, No. 236001; Nunc, Roskilde, Denmark) coated with strepta-
vidin and these plates were incubated for 1 h at 37 ꢃC. After the
coating solution was discarded, the microplates were washed with
0.05% Tween-PBS (250
with 0.05% Tween-PBS (200
wells and incubated overnight at 4 ꢃC. After washing with 0.05%
Tween-PBS, 200 L of anti-human IgG/HRP (P0214; DakoCytoma-
mL per well). Serum samples diluted 1:250
mL per well) were then added to the
m
tion, Denmark; 1:1000 in 0.05% Tween-PBS) was added, and the
microplates were incubated for 1 h at 37 ꢃC. After further washing,
bound antibodies were detected by the addition of ABTS peroxidase
substrate solution (KPL, Gaithersburg, MD, USA, 200 mL per well).
After incubation period of 8 min at 37 ꢃC, the reaction was stopped
by the addition of 1% SDS, and the absorbance (A) values were read