Synthetic studies on the carbohydrate moiety of the antigen from the parasite Echinococcus multilocularis

Article abstract of DOI:10.1016/j.carres.2009.02.019

Stereocontrolled syntheses of branched tri-, tetra-, and pentasaccharides displaying a Galβ1→3GalNAc core in the glycan portion of the glycoprotein antigen from the parasite Echinococcus multilocularis have been accomplished. Trisaccharide Galβ1→3(GlcNAcβ

Full text of DOI:10.1016/j.carres.2009.02.019

Carbohydrate Research 344 (2009) 856–868
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Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthetic studies on the carbohydrate moiety of the antigen from the parasite
Echinococcus multilocularis
a
b
c
Akihiko Koizumi a, Noriyasu Hada a, , Asuka Kaburaki , Kimiaki Yamano , Frank Schweizer ,
*
Tadahiro Takeda 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, Canada R3T 2N2
a r t i c l e i n f o
a b s t r a c t
Article history:
Stereocontrolled syntheses of branched tri-, tetra-, and pentasaccharides displaying a Galb1?3GalNAc
core in the glycan portion of the glycoprotein antigen from the parasite Echinococcus multilocularis
Received 2 December 2008
Received in revised form 19 February 2009
Accepted 19 February 2009
Available online 24 February 2009
have been accomplished. Trisaccharide Galb1?3(GlcNAcb1?6)GalNAc
Galb1?3(Galb1?4GlcNAcb1?6)GalNAc 1-OR (D), and pentasaccharides Galb1?3(Galb1?4Galb1?4Gl
cNAcb1?6)GalNAc 1-OR (E) and Gal b1?3(Gal 1?4Galb1?4GlcNAcb1?6)GalNAc 1-OR (F) (R = 2-
(trimethylsilyl)ethyl) were synthesized by block synthesis. The disaccharide 2-(trimethylsilyl)ethyl
2,3,4,6-tetra-O-acetyl-b- -galactopyranosyl-(1?3)-2-azido-4-O-benzyl-2-deoxy- -galactopyranoside
served as a common glycosyl acceptor in the synthesis of the branched oligosaccharides. Moreover, linear
trisaccharide Galb1?4Galb1?3GalNAc 1-OR (B) and branched tetrasaccharide Galb1?4Galb1?3(Glc-
NAcb1?6)GalNAc 1-OR (C) were synthesized by stepwise condensation.
Ó 2009 Elsevier Ltd. All rights reserved.
a1-OR (A), tetrasaccharide
a
a
a
a
Keywords:
Glycoprotein
Echinococcus multilocularis
Host–parasite interactions
Stereocontrolled syntheses
D
a-D
a
a
1. Introduction
trematode Fasciola sp (III) contain glycolipids, while those of the
nematodes Dirofilaria immitis (II) and Toxocara sp. (II) contain gly-
coproteins. In contrast, glycolipids and glycoproteins were identi-
fied in the extracellular matrix of cestode Echinococcus sp. (I) and
nematode Caenorhabditis elegans (II).2a Glycosphingolipids found
in mammals are classified into different types on the basis of their
basic carbohydrate structure; these types include the globo, lacto,
ganglio, neolacto, and isoglobo series.4 However, to the best of our
knowledge, the carbohydrate structure of parasitic helminth
including schisto, arthro, spirometo, and neogala series represents
a different type of glycolipid core structure.5 Due to these differ-
ences in the glycan structure, we have been interested in the rela-
tionships between the structure and biological activity of
glycolipids from invertebrate species and have synthesized oligo-
saccharides from various helminths.6 Recently, we have developed
Infection by parasitic protozoans and helminthes constitutes a
serious health problem all over the world, but no vaccines exist
against major human parasitic diseases such as malaria, African
trypanosomiasis, leishmaniasis, schistosomiasis, and alveolar echi-
nococcosis.1 Recent studies on a number of parasites revealed that
immune responses to parasites in infected animals and humans are
directed toward glycoconjugate glycan determinants on the cell
surface where they play important roles in host–parasite interac-
tions.2 Moreover, evidence is mounting that carbohydrate antigens,
rather than protein antigens, dominate the immune responses to
many helminthic parasites.3 Because of the prospect of developing
conjugate vaccines and new diagnostics for parasite infections,
parasite-derived glycans are compelling vaccine targets. In this
context it is of particular interest that the glycan portion of para-
site-derived glycoprotein-based antigens commonly possesses un-
ique glycan sequences and sorts that distinguish them from
mammalian glycans.2a Parasitic helminthes are divided into three
major groups: the cestodes (tapeworms) I; the nematodes (round-
worms) II; and the trematodes (flukes) III. Among the major para-
sites it has been shown that the extracellular matrixes of cestode
Spirometra erinacei (I), the nematode Ascaris suum (II), and the
a
strong interest in the structure and synthesis of glyco-
sphingolipids from Echinococcus multilocularis. E. multilocularis is
a parasite, which belongs to the class cestoda of the Phylum Platy-
helminthes and causes alveolar echinococcosis (AE), a severe dis-
ease that can be fatal in the absence of efficient treatment. AE is
endemic in the Alps and in Hokkaido (Japan), and it is known as
a severe parasitic zoonosis caused by ingestion of the parasite’s
eggs. The adult parasites mainly reside in the intestine of the red
fox, Vulpes vulpes schrencki, where they produce eggs. These eggs
are released in feces and are usually ingested by rodents, the inter-
mediate hosts of the parasite, or accidentally by humans. However,
* Corresponding authors. Tel.: +81 3 5400 2666; fax: +81 3 5400 2556 (N.H.).
E-mail address: hada-nr@pha.keio.ac.jp (N. Hada).
0008-6215/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2009.02.019

A. Koizumi et al. / Carbohydrate Research 344 (2009) 856–868
857
no perfect antigen exists for the serological detection of patients
with echinococcosis. Persat et al. reported7 in 1991 that sera from
AE patients recognized a neutral glycosphingolipid fraction ex-
tracted from metacestodes of E. multilocularis. Structure determina-
tion of this glycosphingolipid fraction revealed that it belonged to
the neogala series (Galb1?6Gal). In addition, it was further shown
that neogala-based glycosphingolipids show significant serodiag-
nostic potential in differentiating alveolar echinococcosis from cys-
tic echinococcosis (CE).8 Based on this information, we previously
conjugation to proteins, lipids, and other macromolecular devices.
Trisaccharides A and tetrasaccharide D have been previously pre-
pared;11b,11e however, oligosaccharides B, C, E, and F are novel
compounds.
2.1. Syntheses of compounds A, D, E, and F
The synthetic strategy for the synthesis of oligosaccharides is
shown in Scheme 1. The disaccharide, 2-(trimethylsilyl)ethyl
synthesized four kinds of glycosphingolipids [Galb1?6(Fuc
a
1?3)
2,3,4,6-tetra-O-acetyl-b-
D-galactopyranosyl-(1?3)-2-azido-4-O-
Galb1?6Galb1?6] and their precursors,6a,6b and described their
benzyl-2-deoxy-b- -galactopyranoside (6) was chosen as the
D
serodiagnostic potential.9
acceptor, while suitably protected mono-, di-, and trisaccharide-
based thioglycosides (8, 13, 22, and 28) were selected as glycosyl
donors.
Attachment of carbohydrates to protein occurs via three major
types of linkages: (a) an N-glycosidic linkage between the reducing
terminal sugar and the amide group of asparagines (N-glycans) (b)
O-glycosidic linkage between the sugar and the hydroxyl group of
an amino acid, most commonly serine and threonine (O-glycans),
and (c) via ethanolamine phosphate groups between the C-termi-
nal residue of the protein and an oligosaccharide attached to phos-
phatidylinositiol (GPI anchor). O-Glycans often possess
characteristic cores, the most frequent of which are core-I type
[Galb1?3GalNAc] and core-II type [Galb1?3(GlcNAcb1?6)Gal-
NAc]. Köhler and co-workers recently isolated novel mucin-type
glycoforms from the metacestode of E. multilocularis.10 These gly-
coforms contained mucin-type core-I type and core-II type struc-
tures that were further diversified by addition of GlcNAc or Gal
residues. Although several approaches to core-II type structures
have been reported,11 none of them is suitable for conjugation to
lipids and proteins. We report here on the synthesis of the glycan
portions of the glycoprotein antigen of E. multilocularis in order
to elucidate the interactions between oligosaccharides and sera
of AE by enzyme-linked immunosorbent assay (ELISA).
2.1.1. Synthesis of trisaccharide A
The preparation of disaccharide acceptor 6 is shown in Scheme
2. Compound 2 was obtained by in situ conversion of glycosylni-
trate 114 into the glycosyl iodide, followed by coupling to 2-(tri-
methylsilyl)ethanol using a combination of tetrabutylammonium
iodide (TBAI) and iodine (I2) as activator. Activation of the glycosyl
iodide using only TBAI15 or iodine16 resulted in lower yields and re-
duced
tection of the 4- and 6-hydroxy group as the benzylidine acetal,
provided an anomeric mixture of 3a–b ( :b ratio 3:1) that was sep-
arable into the
- and b-glycosides at this stage. Glycosylation17 of
a-stereoselectivity. Zemplén deacetylation, followed by pro-
a
a
3a with the imidate donor 418 was carried out in the presence of
trimethylsilyl trifluoromethanesulfonate (TMSOTf) and AW-300
molecular sieves in CH2Cl2 to afford the desired b-glycoside 5 in
90% yield. Reductive ring opening of the benzylidene acetal 5
was achieved by treatment with dichlorophenylborane (PhBCl2)
19
and triethylsilane (Et3SiH) in CH2Cl2 to produce disaccharide
acceptor 6 as a single regioisomer in 73% yield.
Initially, we studied the preparation of the type-II trisaccharide
motif by regioselective glycosylation of acceptor 2-(trimethyl-
2. Results and discussion
The exact anomeric linkages of the carbohydrate sequence of
the glycoforms derived from E. multilocularis are currently un-
known. Previous studies have indicated that the E. multilocularis
antigen contains mucin-type core-I type [Galb1?3GalNAc] and
core-II type [Galb1?3(GlcNAcb1?6)GalNAc] and branched core-I
and core-II structures attached to both serine and threonine.
silyl)ethyl 2,3,4,6-tetra-O-acetyl-b-
D-galactopyranosyl-(1?3)-2-
azido-2-deoxy- -galactopyranoside with slight excess of
a-D
thioglycoside donors (data not shown). This, however, resulted in
the formation of regioisomeric mixtures. As a result, we employed
acceptor 6 for further studies.
In order to complete the synthesis of trisaccharide A, we
originally studied the glycosylation of acceptor 6 with phenyl
Although
a-anomeric linkages in cores I and II cannot be ruled
out, it was suggested that the b-linkage is more likely.12 Based
on this information we were interested in developing an approach
that would permit the synthesis of the branched oligosaccharides
A, C, D, E, and F exhibiting core-I and core-II structures and of
the linear oligosaccharide B with only a core-I structure. Branched
oligosaccharides A, C, and D differ in the number of sugar residues
attached to core-II, while pentasaccharides E and F vary at the ano-
meric linkage of the terminal Gal residue (Fig. 1). The presence of
oligosaccharide sequences A–F in E. multilocularis glycoprotein
antigen was previously suggested on the basis of mass spectromet-
ric O-glycan profiling and sequencing.10
In order to study the structure–activity relationships of the car-
bohydrate moiety in the antigenic glycoprotein, access to oligosac-
charide sequences A–F is required. In particular, we were
interested in developing a block synthesis approach that would en-
able the synthesis of oligosaccharides A and C–F from common
suitably protected di- and trisaccharide acceptors and various
mono-, di-, and trisaccharide donors. Due to the fact that the
nature of the O-glycosidic linkage of the oligosaccharides to the
polypeptide is unknown, a synthetic approach giving access to
3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-1-thio-b-D-glucopyran-
oside donor using N-iodosuccinimide (NIS)–trifluoromethanesulfo-
nic acid (TfOH)20 as promoter. However, this resulted in poor yields
(26%) of the corresponding trisaccharide. Significantly improved
yields were obtained by replacement of the bulky phthaloyl group
by 2-trichloroethoxycarbonyl (Troc). Donor 8 was prepared from
common compound 721 using standard Lewis acid-catalyzed con-
ditions. Glycosylation of donor 8 with acceptor 6 was achieved in
the presence of NIS and TfOH to afford trisaccharide 9 in 47% yield.
Subsequently, the azido group and Troc group were converted to
an acetamido moiety by reduction with Zn–AcOH, followed by
debenzylation and catalytic hydrogenolysis over 10% Pd/C in
MeOH and acetylation to yield 10 in 37% yield. Finally, Zemplén-
based deacetylation and column chromatography on Sephadex
LH-20 furnished target trisaccharide A (Scheme 3).
2.1.2. Synthesis of tetrasaccharide D
The synthesis of tetrasaccharide D containing core-II type struc-
ture was achieved by glycosylation of disaccharide donor 13 with
previously prepared acceptor 6 (Scheme 4). Disaccharide donor
13 was prepared from 8 in a five-step process. At first, compound
8 was deacetylated, protected as 4,6-benzylidene, and benzoylated
to provide compound 11 in 65% yield. Regioselective acid-cata-
lyzed reductive ring opening of the benzylidene acetal using
both
a- and b-glycosidic linkages was selected. This was realized
by preparing oligosaccharide analogs bearing a non-participating
azido group at C-2 in the GalNAc portion. The temporary 2-(tri-
methylsilyl)ethyl-13 protecting group was chosen to ensure future

858
A. Koizumi et al. / Carbohydrate Research 344 (2009) 856–868
NHAc
OH
HO
HO
HO
HO
O
O
O
O
HO
O
OH
O
OH
HO
OH
O
HO
HO
HO
O
OH
O
HO
O
AcHN
TMS
TMS
OH
AcHN
TMS
OH
O
O
B
A
OH
O
HO
HO
NHAc
NHAc
O
OH HO
HO
HO
O
OH
O
HO
HO
HO
O
O
O
HO
HO
OH
HO
OH
HO
O
O
O
O
O
O
OH
O
HO
HO
AcHN
TMS
AcHN
OH
C
OH
O
O
OH
O
HO
HO
D
OH
O
HO
HO
HO
OH
O
O
O
OH
NHAc
NHAc
HO
O
HO
O
OH
O
HO
O
HO
O
O
O
HO
HO
OH
HO
OH
HO
OH HO
OH HO
O
O
O
O
O
O
HO
HO
TMS
TMS
OH
OH
AcHN
AcHN
O
O
E
F
Figure 1. Structure of the target oligosaccharide derivatives, A–F from the parasite Echinococcus multilocularis.
OAc
AcO
AcO
OAc
O
NHTroc
SDMP
O
BzO
O
AcO
AcO
SDMP
NHTroc
O
OAc
BnO
8
13
D
A
OAc
O
AcO
AcO
BnO OH
O
O
N3
OAc
TMS
F
O
6
E
OAc
OAc
O
AcO
BnO
AcO
AcO
O
OBn
O
O
NHTroc
SDMP
BnO
BzO
O
BnO
OAc
BnO
OBn
O
O
NHTroc
SDMP
O
BzO
O
BnO
OBz
BnO
O
22
OBz
28
Scheme 1. Synthetic plan of the target oligosaccharides, A, D, E, and F.
(1)
(2)
NaI
CH3CN
(1)
NaOMe
MeOH
TMSEtOH
I2, TBAI
MS 4Å
Ph
O
(2) BDA
OAc
O
OAc
O
O
AcO
AcO
AcO
AcO
CSA
CH2Cl2
O
CH3CN
TMS
HO
ONO2
N3
1
O
N3
N3
88% (2steps)
TMS
59% (2steps)
O
2
3a
3b
(β anomer)
+
OAc
O
AcO
AcO
O
CCl3
NH
Ph
4OAc
Et3SiH
PhBCl2
TMSOTf
O
MS AW-300
CH2Cl2
O
Å
MS 4
OAc
O
AcO
OAc BnO OH
AcO
AcO
CH2Cl2
73%
O
O
O
O
O
AcO
90%
N
3O
OAc
TMS
N
TMS
OAc
3O
5
6
Scheme 2. Preparation of disaccharide acceptor.

A. Koizumi et al. / Carbohydrate Research 344 (2009) 856–868
859
R4
O
6
R1O
2,6-dimethylthiophenol
NIS, TfOH
MS AW-300
CH2Cl2
R1O
O
BF3-OEt2
CH2Cl2
AcO
AcO
R1O
AcO
O
O
AcO
OR1
R1O
R1O
R2O
SDMP
NHTroc
AcO
AcO
OAc
77%
47%
O
R3
O
OR1
NHTroc
O
8
7
TMS
R
1 = Ac, R2 = Bn, R3 = N3, R4 = NHTroc
O
(1) Zn, AcOH-Ac2O-THF
(2) H2, Pd/C , MeOH
(3) Ac2O, Pyr.
9
NHAc
10 R1 = R2 = Ac, R3 = R4 = NHAc
HO
HO
HO
37% (3steps)
O
O
NaOMe, MeOH
95%
10
OH HO
HO
HO
O
O
O
TMS
AcHN
OH
O
A
Scheme 3. Synthesis of trisaccharide A.
(1) NaOMe
MeOH
NaBH3CN
BDA
MS 3
Å
(2)
OBn
CSA
HCl-Et2O
THF
O
Ph
O
CH3CN
O
HO
BzO
O
8
SDMP
SDMP
BzO
3)BzCl
92%
NHTroc
NHTroc
Pyr.
12
11
65% (3steps)
OR2
R2O
R5
6
4
R1O
R3O
O
NIS, TfOH
TMSOTf
MS AW-300
CH2Cl2
b
MS AW-300 R2O
CH2Cl2
OAc
O
AcO
AcO
O
O
NHTroc
SDMP
O
OR2
BzO
O
OR2 R3O
O
R2O
O
70%
86%
BnO
OAc
O
a
O
13
R2O
OR2
TMS
R4
O
(1) Zn, AcOH-Ac2O-THF 14 R1 = Bz, R2 = Ac, R3 = Bn, R4 = N3, R5 = NHTroc
(2) H2, Pd/C , MeOH
(3) Ac2O, Pyr.
R1 = Bz, R2 = R3 = Ac, R4 = R5 = NHAc
15
OH
O
HO
44% (3steps)
NHAc
HO
b
HO
O
O
O
OH
HO
NaOMe, MeOH
97%
HO
15
OH HO
O
O
a
O
HO
AcHN
OH
TMS
O
D
Scheme 4. Synthesis of tetrasaccharide D.
sodium cyanoborohydride in acidified ether afforded acceptor 12
in excellent yield (92%). Finally TMSOTf-catalyzed glycosylation
of 12 with imidate donor 4 in the presence of AW-300 MS in CH2Cl2
produced the desired disaccharide 13 as the sole product in 86%
yield. The b-glycosidic linkage was assigned on the basis of homo-
synthesis of trisaccharide 22 started from known phenylthio
galactopyranoside 16.22 Regioselective reductive ring opening of
the benzylidene acetal provided 17 in 78% yield, which was chlo-
roacetylated by exposure to chloroacetyl chloride in pyridine to
produce 18. Thioglycoside 18 was converted to glycosyl fluoride
19 using N,N-diethylaminosulfur trifluoride23 in CH2Cl2. Donor
19 was coupled to monosaccharide acceptor 12 by activation
nuclear coupling constants (H-10, d 4.49, JH1 ,H2 7.9 Hz). With disac-
charide donor 13 in hand, we then studied the glycosylation of
donor 13 with acceptor 6. We were pleased to see that NIS–
TfOH-promoted glycosylation of 13 with 6 in the presence of
AW-300 MS in CH2Cl2 afforded the desired tetrasaccharide 14 in
70% yield. The nature of the new glycosidic linkage was deter-
mined by measurements of the homonuclear coupling constants
(H-1 of GlcNAc, d 4.60, JH1,H2 7.9 Hz). Removal of the Troc-protect-
ing group using Zn in a mixture containing Ac2O and HOAc, fol-
lowed by catalytic hydrogenation over 10% Pd–C in MeOH, and
acetylation provided 15. Finally, deacylation of 15 with NaOMe
in MeOH afforded the desired target tetrasaccharide D in 97% yield
(Scheme 4).
0
0
24
with AgOTf and Cp2HfCl2 to afford disaccharide 20 in 78%
yield. Selective removal of the chloroacetyl group in 20 was
achieved by treatment with thiourea in a solvent mixture con-
taining pyridine and ethanol25 to produce acceptor 21 in 73%
yield. Accepor 21 served as precursor to prepare trisaccharide
donor 22. This was achieved by TMSOTf-promoted activation of
imidate donor 4 and coupling to 21 to provide desired trisaccha-
ride donor 22 in 73% yield. With donor 22 in hand we then stud-
ied the formation of pentasaccharide E. NIS–TfOH-promoted
activation of donor 22 and coupling to disaccharide acceptor 6
afforded the desired b-glycoside 23 in 57% yield. Deprotection
was performed as previously described for compound D. That
is, sequential de(trichloroethoxycarbonyl)ation and N-acetylation,
followed by debenzylation and O-acetylation, provided pentasac-
charide 24 in 34% yield. Finally, Zemplén-based deacetylation
2.1.3. Synthesis of pentasaccharide E
Branched pentasaccharide E was synthesized by glycosylation
of thioglycoside donor 22 with acceptor 6 (Scheme 5). The

860
A. Koizumi et al. / Carbohydrate Research 344 (2009) 856–868
NaBH3CN
MS 3Å
Ph
ClAcCl
ClAcO
BnO
OBn
Pyr.
OBn
HCl-Et2O
THF
HO
O
O
CH2Cl2
O
O
O
SPh
86%
BnO
78%
X
OBz
18 X = SPh (b)
SPh
BnO
OBz
OBz
17
DAST
16
CH2Cl2
19 X = F (
:
= 2.2:1)
α β
86%
12
AgOTf
4
OAc
Cp2HfCl2
MS AW-300
CH2Cl2
TMSOTf
MS AW-300
CH2Cl2
AcO
AcO
OBn
RO
O
OBn
NHTroc
SDMP
O
NHTroc
SDMP
b
O
BzO
O
BzO
O
O
OAc
a
BnO
O
73%
78%
BnO
O
OBz
20
BnO
BnO
OBz
22
R = ClAc
thiourea
Pyr., EtOH
21 R = H
73%
OR2
R2O
OH
HO
O
O
6
OR3
O
OH
O
c
R5
c
O
R2O
O
NHAc
HO
NIS, TfOH
MS AW-300
CH2Cl2
R1O
OR2
HO
O
OH
b
b
R3O
NaOMe, MeOH
95%
HO
O
O
O
OR3
O
R3O
HO
OR1
OH
O
24
OR2
O
OH
O
HO
R2O
HO
57%
O
O
a
O
O
a
R2O
E
HO
AcHN
R4
TMS
OH
OR2
TMS
O
O
23 R1 = Bz, R2 = Ac, R3 = Bn, R4 = N3, R5 = NHTroc
24
(1) Zn, Ac2O-AcOH-THF
(2) H2, Pd/C , MeOH
(3) Ac2O, Pyr.
R1 = Bz, R2 = R3 = Ac, R4 = R5 = NHAc
34% (3steps)
Scheme 5. Synthesis of pentasaccharide E.
and column chromatography on Sephadex LH-20 furnished tar-
get pentasaccharide E in 95% yield (Scheme 5).
blocks 4, 18, 3a, and 8. Both oligosaccharides B and C are derived
from the common trisaccharide 32.
2.1.4. Synthesis of pentasaccharide F
2.2.1. Synthesis of common intermediate 32 and conversion
into trisaccharide B and tetrasaccharide C
The synthesis of branched pentasaccharide F required access
to trisaccharide donor 28 containing a Gal
sequence. In order to prepare the Gal 1?4Gal sequence with high
-stereoselectivity, we selected 4,6-O-di-tert-butylsilylene (DTBS)-
protected galactose donor 26. Previous studies have indicated that
a
1?4Galb1?4GlcNAc
Common intermediate 32 was synthesized by glycosylation of
acceptor 3a with donor 18 in the presence of NIS–TfOH to provide
disaccharide 30 in 79% yield. The b-glycosidic linkage was con-
firmed by 1H NMR spectroscopy. The anomeric proton of the newly
formed interglycosidic linkage appeared as a doublet with a homo-
a
a
DTBS-protected galactose donors induce high
a-selectivity in
glycosylation reactions.26 Donor 26 was prepared from known
phenylthioglycoside donor 2527 by exposure to DAST and N-bro-
mosuccinimide (NBS)28 at ꢀ20 °C to form glycosyl fluoride 26 in
78% yield. Coupling of glycosyl fluoride 26 to previously prepared
disaccharide acceptor 21 was achieved by activation with AgOTf–
nuclear coupling constant of 8.6 Hz (H-10, d 4.52, JH1 ,H2 8.6 Hz).
Selective removal of the chloroacetyl group in 30 with thiourea
gave disaccharide acceptor 31, which was used directly for the next
glycosylation step. TMSOTf-promoted activation of imidate donor
4 and coupling to acceptor 31 generated trisaccharide 32 in 74%
yield. The newly formed b-glycosidic linkage was confirmed by
1H NMR spectroscopy. The anomeric proton of the galactose moi-
ety in 32 appeared at d 5.01 as a doublet with a homonuclear pro-
ton–proton coupling constant of 7.9 Hz (H-1 of Gal b, d 5.01,
JH1b,H2b 7.9 Hz). Catalytic hydrogenation followed by acetylation
in pyridine produced trisaccharide 33 that was deblocked using so-
dium methoxide in methanol to produce target compound B in
quantitative yield (Scheme 8).
0
0
Cp2HfCl2 to produce trisaccharide 27 with complete
a-selectivity
in excellent yield (91%). The -interglycosidic linkage in 27 was
a
confirmed by 1H NMR spectroscopy. The anomeric proton of gal-
actose moiety (b) appeared as a doublet with a homonuclear cou-
pling constant of 3.1 Hz. Selective removal of the DTBS group in 27
was achieved with HF–pyridine, and subsequent acetylation with
Ac2O in pyridine provided 28 in 93% yield. NIS–TfOH-promoted
glycosylation of donor 28 and coupling to acceptor 6 afforded
pentasaccharide derivative 29 in 46% yield. Deblocking as de-
scribed above for oligosaccharides A and D provided target penta-
saccharide F (Scheme 6).
To prepare the branched tetrasaccharide C, it was necessary
to introduce the (1?6)-linked N-acetyl glucosamine moiety into
trisaccharide 32. In order to do so a reductive and regioselective
ring opening of the benzylidene acetal group in 32 was neces-
sary. This was achieved by exposure of 32 to Et3SiH and PhBCl2
in CH2Cl2 to afford trisaccharide acceptor 34. Subsequently,
acceptor 34 was glycosylated with thioglycoside donor 8 to gen-
erate protected tetrasaccharide 35 in 81% yield. Deblocking using
reaction conditions that were the same as those described for
oligosaccharides A and D afforded target tetrasaccharide C in
91% yield (Scheme 9).
2.2. Syntheses of compounds B and C
Initial attempts to synthesize trisaccharide B by coupling of a
disaccharide donor with acceptor 3a failed because of the steric
hindrance and low activities of the glycosyl donor. We therefore
decided to prepare oligosaccharides B and C by stepwise condensa-
tion as outlined in Scheme 7. This required access to building

A. Koizumi et al. / Carbohydrate Research 344 (2009) 856–868
861
OR
RO
21
O
Si
AgOTf
b
Si
BnO
Cp2HfCl2
MS AW-300
CH2Cl2
DAST
NBS
O
BnO
O
OBn
O
O
O
NHTroc
O
O
CH2Cl2
O
a
BzO
SDMP
BnO
BnO
O
BnO
SPh
O
BnO
26
OBz
78%
91%
OBn
BnO
25
F
27
R = DTBS
1) HF-Pyr., THF
2) Ac2O, Pyr.
28
R = Ac
93%
OH
O
HO
OAc
O
AcO
c
HO
c
(1) Zn, Ac2O/AcOH/THF
(2) H2, Pd/C , MeOH
(3) Ac2O, Pyr.
6
BnO
HO
OH
O
NIS, TfOH
MS AW-300
CH2Cl2
BnO
O
NHAc
OBn
O
O
NHTroc
HO
O
b
(4) NaOMe, MeOH
BnO
O
HO
O
b
O
BnO
O
O
HO
OH
BnO
OBz
21% (4steps)
OH
O
46%
HO
HO
BnO
OAc
O
AcO
O
a
O
O
HO
a
O
OAc
AcO
29
AcHN
OH
TMS
N3
TMS
O
F
O
Scheme 6. Synthesis of pentasaccharide F.
JMN A500 FT NMR spectrometer with Me4Si as the internal stan-
dard for solutions in CDCl3. MALDI-TOFMS was recorded on a
Perseptive Voyager RP mass spectrometer. High-resolution mass
spectra were recorded on a JEOL JMS-700 under 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).
C
OAc
O
AcO
AcO
AcO
O
AcO
OH
O
OBn BnO
SDMP
O
AcO
O
NHTroc
OAc
BnO
O
N3
8
OBz
TMS
34
O
4.2. 2-(Trimethylsilyl)ethyl 3,4,6-tri-O-acetyl-2-azido-2-deoxy-
D
-galactopyranoside (2)
Ph
OAc
O
AcO
AcO
O
OBn
O
O
O
To a solution of 1 (1.00 g, 2.66 mmol) in CH3CN (5.0 mL) was
B
O
OAc
added NaI (2.78 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 a solution
of the residue in CH2Cl2 (5.0 mL) at room temperature were
added 2-(trimethylsilyl)ethanol (TMSEtOH) (1.88 mL, 13.3
mmol), tetrabutylammonium iodide (TBAI) (196 mg, 0.53 mmol)
and 4 Å MS (1.0 g). The reaction mixture was stirred under an
atmosphere of argon for 2 h at room temperature, then iodine
(I2) (2.04 g, 7.98 mmol) was added and stirring was continued
for 16 h. After that time, the reaction mixture was extracted
with CHCl3, washed with satd aq Na2S2O3, dried (MgSO4), and
concentrated. The product was purified by silica gel column
chromatography using 6:1 hexane–AcOEt as eluent to give 2
(678 mg, 59%, two steps). MALDI-TOFMS: calcd for C17H29N3O8-
SiNa: ([M+Na]+) 454.2, found 454.0.
O
BnO
N3
OBz
TMS
32
O
Ph
O
OAc
O
AcO
AcO
OBn
ClAcO
BnO
O
O
SPh
O
O
CCl3
NH
OAc
OBz
HO
4
N3
18
TMS
O
3a
Scheme 7. Synthetic plan of the target oligosaccharides, B and C.
3. Conclusions
4.3. 2-(Trimethylsilyl)ethyl 2-azido-4,6-O-benzylidene-2-
deoxy-D-galactopyranoside (3)
In summary, a systematic and integrated approach for the syn-
thesis of six oligosaccharides A–F found in the metacestode of the
parasite, E. multilocularis was developed. It is expected that these
compounds will find use in future studies designed to reveal the
relationships between the structure and biological activity for spe-
cific antibody detection in patients with alveolar echinococcosis.
To a solution of 2 (3.73 g, 8.64 mmol) in MeOH (40 mL) was
added NaOMe (233 mg, 4.32 mmol), and the mixture was stirred
for 30 min, and then neutralized with Amberlite IR 120 [H+]. The
mixture was filtered and concentrated. To a solution of the res-
idue (2.64 g) in CH3CN (5.0 mL) were added benzaldehyde
dimethylacetal (2.58 mL, 17.3 mmol) and camphor-10-sulfonic
acid (1.00 g, 4.32 mmol) at room temperature. The reaction mix-
ture was stirred for 5 h, and then neutralized with Et3N. After
evaporation, column chromatography of the residue on silica
gel (6:1?1:1 hexane–AcOEt) gave 3a (2.25 g, 66%) and 3b
4. Experimental
4.1. General methods
Optical rotations were measured with a Jasco P-1020 digital
polarimeter. 1H NMR and 13C NMR spectra were recorded with a

862
A. Koizumi et al. / Carbohydrate Research 344 (2009) 856–868
Ph
OR2
O
4
R2O
R2O
18
O
TMSOTf
MS AW-300
CH2Cl2
NIS, TfOH
MS AW-300
CH2Cl2
O
OR4
O
OBn
O
R4O
O
RO
b
OR3
OR2
R3O
O
O
N
O
O
OBz
a
BnO
3a
TMS
74%
3O
OR1
R5
79%
TMS
O
thiourea
30 R=ClAc
31 R=H
32 R1 = Bz, R2 = Ac, R3 = Bn, R4 = CHPh, R5 = N3
Pyr., EtOH
(1) H2, Pd(OH)2/C , MeOH
(2) Ac2O, Pyr.
89%
33 R1 = Bz, R2 = R3 = R4 = Ac, R5 = NHAc
64% (2steps)
OH
HO
HO
O
b
O
OH
HO
OH
NaOMe, MeOH
quant.
OH
O
O
a
33
O
HO
OH
TMS
AcHN
O
B
Scheme 8. Synthesis of trisaccharide B.
Et3SiH
OAc
OAc
AcO
Ph
AcO
AcO
PhBCl2
O
O
O
MS 4Å
OH
O
O
OBn
BnO
OBn
O
AcO
O
CH2Cl2
OAc
OAc
O
O
O
74%
O
OBz
O
OBz
34
BnO
BnO
TMS
TMS
N3
N3
O
O
32
8
R5
O
NHAc
NIS, TfOH
MS AW-300
CH2Cl2
HO
HO
R2O
R2O
R2O
OH
OR2
O
O
HO
HO
R2O
R2O
O
O
O
HO
O
NaOMe, MeOH
91%
O
OR3
O
OH
36
R3O
O
HO
O
81%
OR2
OH
O
O
HO
R3O
O
TMS
R4
AcHN
OR1
O
OH
TMS
O
C
R1 = Bz, R2 = Ac, R3 = Bn, R4 = N3, R5 = NHTroc
(1) Zn, Ac2O/AcOH/THF
(2) H2, Pd/C , MeOH
(3) Ac2O, Pyr.
35
36
R
1 = Bz, R2 = R3 = Ac, R4 = R5 = NHAc
36% (3steps)
Scheme 9. Synthesis of tetrasaccharide C.
(725 mg, 22%). Compound 3a ½a D24
ꢁ
+142 (c 1.0, CHCl3). 1H NMR
1H, J1 ,2 7.9 Hz, J2 ,3 10.4 Hz, H-20), 4.99 (d, 1H, J1,2 3.7 Hz, H-
0
0
0
0
(500 MHz, CDCl3): d 7.51–7.16 (m, 5H, Ar), 5.58 (s, 1H, PhCH),
5.03 (d, 1H, J1,2 3.1 Hz, H-1), 4.30–4.27 (m, 2H, H-4, H-6a),
4.18 (br, 1H, H-3), 4.10 (m, 1H, H-6b), 3.81 (m, 1H, SiCH2CH2),
3.76 (s, 1H, H-5), 3.63–3.57 (m, 2H, H-2, SiCH2CH2), 2.46 (br.
d, 1H, OH), 1.08–0.93 (m, 2H, SiCH2), 0.04 (s, 9H, Si(CH3)3). 13C
NMR (CDCl3): d 137.4, 129.3, 128.3, 126.2, 101.3(PhCH), 98.1(C-
1), 75.6(C-4), 69.3(C-6), 67.7(C-3), 66.2(SiCH2CH2), 62.8(C-5),
60.8(C-2), 18.0(SiCH2), –1.42(Si(CH3)3). HRFABMS: calcd for
C18H28NO5Si: ([M+H]+) 394.1798, found 394.1765.
1), 4.96 (dd, 1H, J2 ,3 10.4 Hz, J3 ,4 3.7 Hz, H-30), 4.72 (d, 1H,
0
0
0
0
J1 ,2 7.9 Hz, H-10), 4.29 (d, 1H, H-4), 4.19 (d, 1H, J5,612.2 Hz, H-
0
0
6a), 4.14 (m, 1H, H-60a), 4.07–4.03 (m, 2H, H-60b, H-3), 3.99 (d,
1H, J5,612.2 Hz, H-6b), 3.86 (t, 1H, J5 ,6 a J5 ,6 b 6.7 Hz, H-50), 3.78
(dd, 1H, J1,2 3.7 Hz, J2,3 11.0 Hz, H-2), 3.74 (m, 1H, SiCH2CH2),
3.62 (s, 1H, H-5), 3.56 (m, 1H, SiCH2CH2), 2.09–1.91 (s ꢂ 4,
12H, COCH3),1.01–0.89 (m, 2H, SiCH2), ꢀ0.03 (s, 9H, Si(CH3)3).
13C NMR (125 MHz, CDCl3): d 170.3, 170.1, 169.4, 137.7, 128.8,
128.1, 126.1, 102.4 (C-10), 100.6 (CHPh) 97.9 (C-1), 75.9 (C-3),
75.8 (C-4), 71.0 (C-30), 70.8 (C-50), 69.2 (C-6), 68.7 (C-200), 67.0
(C-40), 66.0 (SiCH2CH), 63.1 (C-5), 61.4 (C-60), 59.0 (C-2), 20.7
(COCH3), 20.5 (COCH3), 18.1 (SiCH2), ꢀ1.47 (Si(CH3)3). HRFABMS:
calcd for C32H45N3O14SiNa: ([M+Na]+) 746.2569, found 746.2579.
0
0
0
0
4.4. 2-(Trimethylsilyl)ethyl 2,3,4,6-tetra-O-acetyl-b-
galactopyranosyl-(1?3)-2-azido-4,6-O-benzylidene-2-deoxy-
-galactopyranoside (5)
D-
a-
D
To a solution of 3a (200 mg, 0.51 mmol) and 4 (376 mg, 0.77
4.5. 2-(Trimethylsilyl)ethyl 2,3,4,6-tetra-O-acetyl-b-D-
mmol) in dry CH2Cl2 (2.0 mL) was added AW-300 MS (600 mg),
and the mixture was stirred for 2 h at room temperature, and
galactopyranosyl-(1?3)-2-azido-4-O-benzyl-2-deoxy-a-D-
galactopyranoside (6)
then cooled to ꢀ20 °C. TMSOTf (18.4
lL, 0.15 mmol) was added,
and the mixture was stirred for 1 h at 0 °C, and then neutralized
with Et3N. The solids were filtered 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 using 2:1 hex-
To a solution of 5 (123 mg, 0.51 mmol) in dry CH2Cl2 (2.0
mL) was added 4Å MS (500 mg), and the mixture was stirred
for 2 h at room temperature, and then cooled to ꢀ78 °C. Et3SiH
(82.4
lL, 0.51 mmol) and PhBCl2 (75.2 mL, 0.58 mmol) were
added, and the mixture was stirred for 5 min, and then neutral-
ized with Et3N in MeOH. The solids were filtered off and washed
with CHCl3. The combined filtrate and washings were succes-
sively washed with water, dried (MgSO4), and concentrated.
ane–EtOAc as eluent to give 5 (330 mg, 90%). ½a D24
ꢁ
+75.8 (c
1.0, CHCl3). 1H NMR (500 MHz, CDCl3): d 7.47–7.20 (m, 5H,
Ar), 5.49 (s, 1H, PhCH), 5.34 (d, 1H, J3 ,4 3.7 Hz H-40), 5.22 (dd,
0
0

A. Koizumi et al. / Carbohydrate Research 344 (2009) 856–868
863
The product was purified by silica gel column chromatography
using 15:1 toluene–acetone as eluent to give 6 (90.0 mg, 73%).
+32.3 (c 1.0, CHCl3), 1H NMR (500 MHz, CDCl3): d 7.42–
7.18 (m, 5H, Ar), 5.45 (d, 1H, J3’,4’ 2.4 Hz H-40), 5.33 (dd, 1H,
4.8. 2-(Trimethylsilyl)ethyl 2,3,4,6-tetra-O-acetyl-b-
galactopyranosyl-(1?3)-[2-acetamido-3,4,6-tri-O-acetyl-2-
deoxy-b- -glucopyranosyl-(1?6)]-2-acetamido-4-O-acetyl-2-
deoxy- -galactopyranoside (10)
D-
½
a 2D4
ꢁ
D
a
-D
J1 ,2 7.9 Hz, J2 ,3 10.4 Hz, H-20), 5.08 (dd, 1H, J2 ,3 10.4 Hz, J3 ,4
0
0
0
0
0
0
0
0
3.7 Hz, H-30), 4.97 (d, 1H, J1,2 4.3 Hz, H-1), 4.96 (d, 1H, Jgem
To a solution of 9 (102 mg, 85.7 lmol) in 3:2:1 AcOH–Ac2O–THF
11.0 Hz, benzyl methylene a), 4.80 (d, 1H, J1 ,2 7.9 Hz, H-10),
4.72 (d, 1H, Jgem 11.6 Hz, benzyl methylene b), 4.23 (m, 1H, H-
60a), 4.16 (m, 1H, H-60b), 4.08 (dd, 1H, J2,3 10.4 Hz, J3,4 2.4 Hz,
H-3), 3.99–3.97 (m, 2H, H-4, H-50), 3.80–3.74 (m, 3H, H-2, H-5,
SiCH2CH2), 3.66 (m, 1H, H-6a), 3.54 (m, 1H, SiCH2CH2), 3.47 (br
d, 1H, H-6b), 2.15-2.00 (s ꢂ 4, 12H, COCH3 ꢂ 4), 1.07–0.91 (m,
2H, SiCH2), 0.02 (s, 9H, Si(CH3)3). 13C NMR (125 MHz, CDCl3): d
170.3, 170.1, 169.6, 138.1, 129.0, 128.5, 128.2, 102.6 (C-10),
97.6 (C-1), 78.5 (C-3), 75.5 (C-4), 74.3 (PhCH2), 71.0 (C-50), 70.8
(C-5), 70.5 (C-30), 68.9 (C-20), 67.0 (C-40), 65.9 (CH2CH2Si), 62.0
(C-6), 61.2 (C-60), 59.9 (C-2), 20.7 (COCH3), 20.6 (COCH3), 20.5
(COCH3), 18.1 (SiCH2), ꢀ1.5(Si(CH3)3). HRFABMS: calcd for
C32H47N3O14SiNa: ([M+Na]+) 748.2725, found 748.2700.
(3.0 mL) was added Zn powder (1.0 g). The mixture was stirred for
1 h at room temperature. After completion of the reaction, the mix-
ture was filtered through Celite. The filtrate was concentrated and
purified by silica gel column chromatography (2:1 toluene–ace-
tone) to give an acetamido residue. A solution of this compound
0
0
(50 mg, 46.7 lmol) in MeOH (1.0 mL) was hydrogenolyzed in the
presence of 10% Pd–C (60 mg) for 1 h at room temperature, then
filtered, and concentrated. The residue was acetylated with Ac2O
(3.0 mL) in pyridine (5.0 mL). The reaction mixture was poured
into ice-water and extracted with CHCl3. The extract was washed
sequentially with 5% HCl, aq NaHCO3, and water, dried (MgSO4),
and concentrated. The product was purified by silica gel column
chromatography using 2:1 toluene–acetone as eluent to give 10
(32.0 mg, 37%). ½a D24
ꢁ
+32.5 (c 0.8, CHCl3). 1H NMR (500 MHz,
4.6. 2,6-Dimethylphenyl 3,4,6-tri-O-acetyl-2-deoxy-2-(2,2,2-
trichloroethoxycarbonylamino)-1-thio-b-D-glucopyranoside (8)
CDCl3): d 4.84 (d, 1H, J1,2 3.7 Hz, H-1 of GalNAc), 4.67 (d, 1H, J1,2
8.6 Hz, H-1 of GlcNAc), 4.57 (d, 1H, J1,2 7.9 Hz, H-1 of Gal). 13C
NMR (125 MHz, CDCl3): d 100.6 (C-1 of GlcNAc), 100.5 (C-1 of
Gal), 97.1 (C-1 of GalNAc). MALDI-TOFMS: calcd for C43H66N2O24Si-
Na: ([M+Na]+) 1045.4, found 1045.5.
To a solution of 7 (4.87 g, 9.32 mmol) in CH2Cl2 (50 mL)
cooled at 0 °C were added 2,6-dimethylthiophenol (2.48 mL,
18.6 mmol) and BF3ꢃEt2O (1.52 mL, 12.1 mmol). The mixture
was stirred for 14 h at 40 °C. After completion of the reaction,
the mixture was neutralized with Et3N and concentrated. The
residue was purified by silica gel column chromatography using
4.9. 2-(Trimethylsilyl)ethyl b-
acetamido-2-deoxy-b- -glucopyranosyl-(1?6)]-2-acetamido-2-
deoxy- -galactopyranoside (A)
D-galactopyranosyl-(1?3)-[2-
D
a
-D
3:1 hexane–AcOEt as eluent to give 8 (4.30 g, 77%). [a] +12.0 (c
D
1.0, CHCl3). 1H NMR (500 MHz, CDCl3): d 7.26–7.09 (m, 3H, Ar),
5.39 (d, 1H, J2,NH 9.2 Hz, NH), 5.20 (t, 1H, J2,3 J3,4 9.8 Hz, H-3),
5.05 (t, 1H, J3,4 J4,5 9.8 Hz, H-4), 4.77 (s, 2H, Troc CH2), 4.52 (d,
1H, J1,2 10.4 Hz, H-1), 4.15 (m, 1H, H-6a), 4.03 (m, 1H, H-6b),
3.87 (q, 1H, J1,2 10.4 Hz, J2,3 9.8 Hz, H-2), 3.53–3.50 (m, 1H, H-
5), 2.53 (s, 6H, CH3 ꢂ 2), 2.04–1.95 (s ꢂ 3, 9H, COCH3 ꢂ 3). 13C
NMR (125 MHz, CDCl3): d 144.1, 129.3, 128.2, 89.0 (C-1), 75.4
(C-5), 74.7 (Troc CH2), 73.2 (C-3), 68.7 (C-4), 62.4 (C-6), 56.0
(C-2), 22.3 (CH3 ꢂ 2, COCH3), 20.6 (COCH3), 20.4 (COCH3). MAL-
DI-TOFMS: calcd for C23H28Cl3NO9SNa: ([M+Na]+) 622.0, found
623.0. HRFABMS: calcd for C23H28Cl3NO9SNa: ([M+Na]+)
622.0448, found 622.0486.
To a solution of 10 (32.0 mg, 31.3 lmol) in MeOH (2.0 mL) was
added NaOMe (10.0 mg) at room temperature, and the mixture
was stirred for 15 h, and 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 MeOH
to give A (20.5 mg, 95%).½a D24
ꢁ
+33.6 (c 0.5, H2O). 1H NMR (500 MHz,
D2O): d 4.76 (d, 1H, J1,2 3.7 Hz, H-1 of GalNAc), 4.40 (d, 1H, J1,2 8.6
Hz, H-1 of GlcNAc), 4.32 (d, 1H, J1,2 7.9 Hz, H-1 of Gal). 13C NMR
(125 MHz, D2O): d 102.8 (C-1 of GlcNAc), 101.7 (C-1 of Gal), 96.8
(C-1 of GalNAc). MALDI-TOFMS: calcd for C27H50N2O16SiNa:
([M+Na]+) 709.3, found 709.9. HRFABMS: calcd for C27H50N2O16Si-
Na: ([M+Na]+) 709.2828, found 709.2847.
4.7. 2-(Trimethylsilyl)ethyl 2,3,4,6-tetra-O-acetyl-b-
galactopyranosyl-(1?3)-[3,4,6-tri-O-acetyl-2-deoxy-2-(2,2,2-
trichloroethoxycarbonylamino)-b- -glucopyranosyl-(1?6)]-2-
azido-4-O-benzyl-2-deoxy- -galactopyranoside (9)
D
-
4.10. 2,6-Dimethylphenyl 3-O-benzoyl-4,6-O-benzylidene-2-
deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-1-thio-b-D-
glucopyranoside (11)
D
a
-D
To a solution of 8 (2.80 g, 4.66 mmol) in MeOH (30 mL) was
added NaOMe (126 mg, 2.33 mmol), and the mixture was stirred
for 1 h, and then neutralized with Amberlite IR 120 [H+]. The
mixture was filtered and concentrated. To a solution of the res-
idue in CH3CN (47 mL) were added benzaldehyde dimethylacetal
(1.39 mL, 9.32 mmol) and camphor-10-sulfonic acid (1.00 g 2.33
mmol) at room temperature. The reaction mixture was stirred
for 16 h at 40 °C, and then neutralized with Et3N. After evapora-
tion, the residue was recrystallized with 1,4-dioxane–n-hexane
to give the benzylidene derivative. To a solution of this com-
pound in pyridine (47 mL) was added benzoyl chloride (1.1
mL, 9.32 mmol), and the mixture was stirred for 16 h at room
temperature. The reaction mixture was poured into ice-water
and extracted with CHCl3. The extract was washed sequentially
with 5% HCl, satd aq NaHCO3, and water, dried (MgSO4), and
concentrated. The product was purified by silica gel column
chromatography using 1:1 hexane–EtOAc as eluent to give 11
To a solution of 6 (83.8 mg, 0.115 mmol) and 8 (104 mg,
0.173 mmol) in dry CH2Cl2 (1 mL) was added powdered AW-
300 MS (200 mg), and the mixture was stirred under an Ar
atmosphere for 2 h at room temperature, and then cooled to
ꢀ50 °C. NIS (117 mg, 0.52 mmol) and TfOH (3.0
lL, 17.3 lmol)
were added to the mixture, which was stirred for 2 h at ꢀ50
°C, and then neutralized with Et3N. The solids were filtered
off and washed with CHCl3. The combined filtrate and washings
were successively washed with satd aq Na2S2O3 and water,
dried (MgSO4), and concentrated. The product was purified by
silica gel column chromatography using 1:1 hexane–EtOAc as
eluent to give 9 (64.0 mg, 47%). ½a D24
ꢁ
+20.8 (c 1.2, CHCl3). 1H
NMR (500 MHz, CDCl3): d 4.82 (d, 1H, J1,2 3.7 Hz, H-1 of Gal-
NAc), 4.75 (d, 1H, J1,2 7.9 Hz, H-1 of GlcNAc), 4.39 (br. d, 1H,
J1,2 7.9 Hz, H-1 of Gal). 13C NMR (125 MHz, CDCl3): d 102.3
(C-1 of GlcNAc), 100.5 (C-1 of Gal), 97.5 (C-1 of GalNAc). MAL-
DI-TOFMS: calcd for C47H65Cl3N4O23SiNa: ([M+Na]+) 1209.3,
found 1210.0.
(2.03 g, 65%). ½a D24
ꢁ
+0.98 (c 1.0, CHCl3). 1H NMR (500 MHz,
CDCl3): d 8.07–7.02 (m, 13H, Ar), 6.07 (d, 1H, J2,NH 9.8 Hz, NH),

864
A. Koizumi et al. / Carbohydrate Research 344 (2009) 856–868
5.65 (t, 1H, J2,3 J3,4 9.8 Hz, H-3), 5.42 (s, 1H, PhCH), 4.67 (s, 2H,
Troc CH2), 4.62 (d, 1H, J1,2 10.4 Hz, H-1), 4.18 (q, 1H, H-4), 3.94
(m, 1H, H-6a), 3.86 (t, 1H, J3,4 J4,5 9.8 Hz, H-4), 3.67(t, 1H, J5,6a
J6a,6b 10.4 Hz, H-6b), 3.61(m, 1H, H-5), 2.52 (s, 6H, CH3 ꢂ 2).
13C NMR (125 MHz, CDCl3): d 154.6, 144.1, 136.8, 133.5, 129.3,
128.8, 128.5, 128.3, 127.9, 125.8, 100.8 (PhCH), 89.9 (C-1), 78.6
(C-4), 74.5 (Troc CH2), 73.4 (C-3), 70.2 (C-5), 68.1 (C-6), 56.3
(C-2), 22.4 (CH3 ꢂ 2). HRFABMS: calcd for C31H31Cl3NO7S:
([M+H]+) 666.0887, found 666.0909.
1020.2, found 1020.8. HRFABMS: calcd for C45H50Cl3NO16SNa:
([M+Na]+) 1020.1814, found 1020.1863.
4.13. 2-(Trimethylsilyl)ethyl 2,3,4,6-tetra-O-acetyl-b-
galactopyranosyl-(1?3)-[2,3,4,6-tetra-O-acetyl-b-
galactopyranosyl-(1?4)-3-O-benzoyl-6-O-benzyl-2-deoxy-2-
(2,2,2-trichloroethoxycarbonylamino)-b- -glucopyranosyl-
(1?6)]-2-azido-4-O-benzyl-2-deoxy- -galactopyranoside (14)
D-
D
-
D
a-D
Compound 14 was prepared from 6 (53.6 mg, 73.8
l
mol) and 13
mol) as described for preparation of 9, yielding 82
+8.8 (c 1.0, CHCl3). 1H NMR (500 MHz, CDCl3): d 4.89
4.11. 2,6-Dimethylphenyl 3-O-benzoyl-6-O-benzyl-2-deoxy-2-
(88.4 mg, 88.6 l
(2,2,2-trichloroethoxycarbonylamino)-1-thio-b-
D
-
mg (70%). ½a 2D4
ꢁ
glucopyranoside (12)
(d, 1H, J1,2 3.7 Hz, H-1 of GalNAc), 4.79 (d, 1H, J1,2 7.9 Hz, H-1 of Gal
a), 4.60 (d, 1H, J1,2 7.9 Hz, H-1 of GlcNAc), 4.42 (d, 1H, J1,2 7.9 Hz, H-
1 of Gal b). 13C NMR (125 MHz, CDCl3): d 102.3 (C-1 of Gal a), 101.3
(C-1 of GlcNAc), 99.9 (C-1 of Gal), 97.5 (C-1 of GalNAc). MALDI-
TOFMS: calcd for C69H87Cl3N4O30SiNa: ([M+Na]+) 1607.4, found
1607.7.
To a solution of 11 (2.03 g, 3.04 mmol) and NaBH3CN (1.72 g, 27.4
mmol) in dry THF (30 mL) was added powdered 3 Å MS (2 g), and the
mixturewas stirredfor 2 h at roomtemperature, and thencooledto 0
°C. HCl–Et2O was added until the solution was acidic (pH paper, gas
evolution). After 30 min, the reaction mixture was poured into
ice-water and extracted with CHCl3. The extract was washed
sequentially with satd aq NaHCO3 and water, dried (Na2SO4), and
concentrated. The product was purified by silica gel column chroma-
tography using 20:1 toluene–acetone as eluent to give 12 (1.86 g,
4.14. 2-(Trimethylsilyl)ethyl 2,3,4,6-tetra-O-acetyl-b-
galactopyranosyl-(1?3)-[2,3,4,6-tetra-O-acetyl-b-
galactopyranosyl-(1?4)-2-acetamido-6-O-acetyl-3-O-benzoyl-
2-deoxy-b- -glucopyranosyl-(1?6)]-2-acetamido-4-O-acetyl-2-
deoxy- -galactopyranoside (15)
D-
D
-
D
92%). ½a 2D4
ꢁ
+17.6 (c 1.0, CHCl3). 1H NMR (500 MHz, CDCl3): d 8.02–
a-D
7.06 (m, 13H, Ar), 5.75 (d, 1H, J2,NH 9.8 Hz, NH), 5.34 (t, 1H, J2,3 J3,4
9.8 Hz, H-3), 4.69 and 4.63 (each d, 2H, Jgem 12.2 Hz, PhCH2), 4.53
(d, 1H, J1,2 10.4 Hz, H-1), 4.51 and 4.47 (each d, 2H, Jgem 11.6 Hz, Troc
CH2), 4.08 (q, 1H, H-2), 3.90 (br, 1H, H-4), 3.69 (m, 2H, H-6a, H-6b),
3.44–3.40 (m, 1H, H-5), 3.07, (br d, 1H, OH), 2.53 (s, 6H, CH3 ꢂ 2).
13C NMR (125 MHz, CDCl3): d 167.4, 154.5, 144.0, 137.6, 133.6,
131.1, 130.0, 129.1, 129.0, 128.44, 128.41, 127.8, 127.6, 125.3,
95.3, 89.3 (C-1), 77.9 (C-5), 76.7 (C-3), 74.5 (PhCH2), 73.7 (Troc
CH2), 70.7 (C-4), 70.3 (C-6), 55.7 (C-2), 22.4(CH3 ꢂ 2). HRFABMS:
calcd for C31H33Cl3NO7S: ([M+H]+) 668.1043, found 668.1080.
Compound 15 was prepared from 14 (132 mg, 83.2 mmol) as
described for preparation of 10, yielding 50.1 mg (44%). ½a D24
ꢁ
+19.3 (c 1.0, CHCl3). 1H NMR (500 MHz, CDCl3): d 4.72 (d, 1H, J1,2
3.7 Hz, H-1 of GalNAc), 4.79 (d, 1H, J1,2 7.9 Hz, H-1 of Gal a), 4.60
(d, 1H, J1,2 7.9 Hz, H-1 of GlcNAc), 4.42 (d, 1H, J1,2 7.9 Hz, H-1 of
Gal b). 13C NMR (125 MHz, CDCl3): d 102.3 (C-1 of Gal a), 101.3
(C-1 of GlcNAc), 99.9 (C-1 of Gal b), 97.5 (C-1 of GalNAc). MALDI-
TOFMS: calcd for C60H84N2O32SiNa: ([M+Na]+) 1395.5, found
1395.8.
4.12. 2,6-Dimethylphenyl 2,3,4,6-tetra-O-acetyl-b-
D
-
4.15. 2-(Trimethylsilyl)ethyl b-
D-galactopyranosyl-(1?3)-[b-D-
galactopyranosyl-(1?4)-3-O-benzoyl-6-O-benzyl-2-deoxy-2-
galactopyranosyl-(1?4)-2-acetamido-2-deoxy-b-
D
-
(2,2,2-trichloroethoxycarbonylamino)-1-thio-b-
D
-
glucopyranosyl-(1?6)]-2-acetamido-2-deoxy-a-D-
glucopyranoside (13)
galactopyranoside (D)
To a solution of 4 (147 mg, 0.30 mmol) and 12 (100 mg, 0.15
mmol) in dry CH2Cl2 (1.0 mL) was added AW-300 MS (300 mg),
and the mixture was stirred for 2 h at room temperature, then cooled
Compound D was prepared from 15 (50.1 mg, 36.5
l
mol) as de-
scribed for preparation of A, yielding 30.0 mg (97%). ½a D24
ꢁ
+41.6 (c
0.5, H2O). 1H NMR (500 MHz, D2O): d 4.72 (d, 1H, J1,2 3.7 Hz, H-1
of GalNAc), 4.38 (d, 1H, J1,2 8.5 Hz, H-1 of GlcNAc), 4.281 (d, 1H,
J1,2 7.9 Hz, H-1 of Gal a), 4.276 (d, 1H, J1,2 7.9 Hz, H-1 of Gal b).
13C NMR (125 MHz, D2O): d 104.1 (C-1 of GlcNAc), 102.5 (C-1 of
Gal a), 101.0 (C-1 of Gal b), 96.2 (C-1 of GalNAc). MALDI-TOFMS:
calcd for C33H60N2O21SiNa: ([M+Na]+) 871.3, found 871.7.
HRFABMS: calcd for C33H60N2O21SiNa: ([M+Na]+) 871.3356, found
871.3398.
to ꢀ20 °C. TMSOTf (5.4
lL, 29.8 lmol) was added, and the mixture
was stirred for 1 h at ꢀ20 °C, and then neutralized with Et3N. The sol-
ids 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 using 4:1 hexane–EtOAc as eluent to give 13 (128
mg, 86%). ½a 2D4
ꢁ
–14.3 (c 1.0, CHCl3). 1H NMR (500 MHz, CDCl3): d
8.05–7.10 (m, 13H, Ar), 5.47 (d, 1H, J2,NH 9.8 Hz, NH), 5.31 (t, 1H,
J2,3 J3,4 9.8 Hz, H-3), 5.13 (d, 1H, J3 ,4 3.7 Hz, H-40), 4.93 (dd, 1H, J1 ,2
4.16. Phenyl 2-O-benzoyl-3,6-di-O-benzyl-1-thio-b-D-
galactopyranoside (17)
0
0
0
0
7.9 Hz, J2 ,3 10.4 Hz, H-20), 4.77 (dd, 1H, J2 ,3 10.4 Hz, J3 ,4 3.7 Hz, H-
0
0
0
0
0
0
30), 4.73 and 4.50 (each d, 2H, Jgem 11.6 Hz, Troc CH2), 4.72 and 4.62
(each d, 2H, Jgem 12.2 Hz, PhCH2), 4.49 (d, 1H, J1 ,2 7.9 Hz, H-100),
4.47 (d, 1H, J1,2 10.4 Hz, H-1), 4.16 (t, 1H, J3,4 J4,5 9.8 Hz, H-4), 4.08
(q, 1H, H-2), 3.68 (m, 1H, H-6a), 3.61–3.58 (m, 2H, H-6b, H-60a),
3.52 (m, 1H, H-50), 3.47(m, 1H, H-60b), 3.33(m, 1H, H-5), 2.56 (s,
6H, CH3 ꢂ 2), 2.00-1.87 (s ꢂ 4, 12H, COCH3 ꢂ 4). 13C NMR (125
MHz, CDCl3): d 170.1, 168.9, 166.2, 154.4, 144.0, 137.8, 133.4,
129.9, 129.1, 128.5, 128.3, 128.2, 128.0, 100.1(C-10), 89.4(C-1),
78.7(C-5), 74.5 (PhCH2), 74.3 (C-4), 74.2 (C-3), 73.9 (TrocCH2), 70.9
(C-30), 70.5 (C-5), 69.2 (C-20), 67.7 (C-6), 66.5 (C-40), 60.6 (C-60),
56,1 (C-2), 22.4 (Ph(CH3)2), 20.6 (COCH3 ꢂ 2), 20.5 (COCH3), 20.4
(COCH3). MALDI-TOFMS: calcd for C45H50Cl3NNaO16S: ([M+Na]+)
Compound 17 was prepared from 16 (850 mg, 1.53 mmol) as
0
0
described for preparation of 12, yielding 666 mg (78%). ½a D24
ꢁ
+33.6 (c 0.5, CHCl3). 1H NMR (500 MHz, CDCl3): d 8.02–7.12 (m,
20H, Ar), 5.50 (t, 1H, J1,2 J2,3 9.8 Hz, H-2), 4.75 (d, 1H, H-1), 4.65
and 4.52 (each d, 2H, Jgem 12.2 Hz, PhCH2), 4.59 (s, 2H, PhCH2),
4.17 (br.d, 1H, H-4), 3.87–3.81 (m, 2H, H-6a, H-6b), 3.71 (t, 1H,
H-5), 3.66 (dd, 1H, H-3). 13C NMR (125 MHz, CDCl3): d 138.0,
133.3, 133.1, 132.2, 129.9, 128.8, 128.4, 128.0, 127.9, 127.8,
127.6, 86.6 (C-1), 79.3 (C-3), 77.5 (C-5), 73.7, 71.3 (each PhCH2),
69.6 (C-6), 69.3 (C-2), 66.4 (C-4). MALDI-TOFMS: calcd for
C33H32O6SNa: ([M+Na]+) 579.2, found 579.4.

A. Koizumi et al. / Carbohydrate Research 344 (2009) 856–868
865
4.17. Phenyl 2-O-benzoyl-3,6-di-O-benzyl-4-O-chloroacetyl-1-
thio-b- -galactopyranoside (18)
(8mL) was stirred underan atmosphereof argon for 2 h at room tem-
perature. After cooling to ꢀ20 °C, successively Cp2HfCl2 (460 mg,
1.79 mmol) and AgOTf (340 mg, 0.90 mmol) were added, and stirring
was continued at ꢀ20 °C for 2 h. The mixture was then neutralized
with Et3N. The reaction mixture was filtered, and the filtrate was
washed with CHCl3 and brine, dried (MgSO4), and concentrated.
The product was purified by silica gel column chromatography using
D
To a solution of 17 (1.92 g, 3.45 mmol) in 15:1 CH2Cl2–pyridine
(32 mL) was added chloroacetyl chloride (550 L, 6.90 mmol), and
l
the mixture was stirred for 1 h at 0 °C. Toluene was added and
evaporated, and the residue was extracted with CHCl3. The extract
was washed with 5% HCl, aq NaHCO3, and water, dried (MgSO4),
and concentrated. The product was purified by silica gel column
chromatography using 7:1 hexane–EtOAc as the eluent to give 18
60:1 toluene–EtOAc as the eluent to give 20 (276 mg, 78%). ½a D24
ꢁ
+12.4 (c 1.0, CHCl3). 1H NMR (500 MHz, CDCl3): d 8.01–7.02 (m,
33H, Ar), 5.52 (d, 1H, J3 ,4 3.7 Hz H-40), 5.43 (d, 1H, J2,NH 9.8 Hz,
0
0
(1.88 g, 86%). ½a D24
ꢁ
+47.8 (c 1.0, CHCl3). 1H NMR (500 MHz, CDCl3):
NH), 5.24 (t, 1H, J2,3 J3,4 9.8 Hz, H-3), 5.08 (dd, 1H, J1 ,2 7.9 Hz, J2 ,3
0
0
0
0
d 7.99–7.04 (m, 20H, Ar), 5.73 (d, 1H, J3,4 3.7 Hz, H-4), 5.36 (t, 1H,
J2,3 9.8 Hz, H-2), 4.77(d, 1H, J1,2 10.4 Hz, H-1), 4.64 and 4.41 (each d,
2H, Jgem 12.8 Hz, PhCH2), 4.58 and 4.46 (each d, 2H, Jgem 11.6 Hz,
PhCH2), 4.11 and 4.00 (each d, 2H, Jgem 14.6 Hz, COCH2Cl), 3.86 (t,
1H, H-5), 3.70 (dd, 1H, J2,3 9.8 Hz, J3,4 3.1 Hz, H-3), 3.67–3.64 (m,
1H, H-6a), 3.57-3.54 (m, 1H, H-6b). 13C NMR (125 MHz, CDCl3): d
166.9, 165.1, 137.3, 136.9, 133.2, 132.7, 132.5, 129.9, 129.7,
128.8, 128.5, 128.33, 128.26, 128.15, 128.06, 128.02, 127.9, 127.8,
86.8 (C-1), 77.2 (C-3), 75.8 (C-5), 73.7 (PhCH2), 71.1 (PhCH2), 69.4
(C-2), 68.2 (C-4), 67.5 (C-6), 40.8 (COCH2Cl). MALDI-TOFMS: calcd
for C35H33ClO7SNa: ([M+Na]+) 655.2, found 655.5. HRFABMS: calcd
for C35H33ClO7SNa: ([M+Na]+) 655.1534, found 655.1520.
10.4 Hz, H-20), 4.70 and 4.57 (each d, 2H, Jgem 12.2 Hz, PhCH2), 4.57
0
0
and 4.31 (each d, 2H, Jgem 12.8 Hz, PhCH2), 4.52 (d, 1H, J1 ,2 8.6 Hz,
H-10), 4.50 and 4.36 (each d, 2H, Jgem 12.2 Hz, PhCH2), 4.47 (d, 1H,
J1,2 10.4 Hz, H-1), 4.22 (t, 2H, Jgem 11.6 Hz, Troc CH2), 4.09–4.03 (m,
2H, H-4, H-2), 3.94 and 3.64 (each d, 2H, Jgem 14.6 Hz, COCH2Cl),
3.44–3.41 (m, 2H, H-6a, H-60a), 3.32(d, 1H, J3,4 11.0 Hz, H-6a), 3.14
(m, 1H, H-5), 3.07 (m, 1H, H-60b), 2.84 (t, 1H, H-5), 2.49 (s, 6H,
CH3 ꢂ 2),. 13C NMR (125 MHz, CDCl3): d 166.7, 166.0, 164.6, 154.4,
144.0. 138.0, 137.1, 130.9, 130.0, 129.7, 129.5, 129.0, 128.5, 128.3,
128.2, 128.1, 128.02, 127.97, 127.90, 127.8, 127.7, 125.3, 100.5 (C-
10), 89.2 (C-1), 78.3 (C-5), 76.0 (C-6), 74.6 (C-3), 74.4 (C-2, PhCH2),
73.5 (Troc CH2), 73.4 (PhCH2), 71.3 (C-20), 71.2 (C-60), 70.8 (PhCH2),
67.8 (C-6), 67.2 (C-40), 66.0 (C-4), 56.1 (CH2Cl), 22.3 (Ph(CH3)2). MAL-
DI-TOFMS: calcd for C60H59Cl4NO14SNa: ([M+Na]+) 1212.2, found
1212.2. HRFABMS: calcd for C60H59Cl4NO14SNa: ([M+Na]+)
1212.2308, found 1212.2349.
4.18. 2-O-Benzoyl-3,6-di-O-benzyl-4-O-chloroacetyl-D-
galactopyranosyl fluoride (19)
To a solution of 18 (1.88 g, 0,126 mmol) in CH2Cl2 (30 mL) was
added DAST (1.19 mL) at 0 °C, and the mixture was stirred at 40 °C
for 16 h. The reaction mixture was diluted with CHCl3 and ex-
tracted with satd aq NaHCO3, and brine. The organic layer was
dried (MgSO4), filtered, and concentrated. The residue was purified
by chromatography on silica gel (9:1?8:1 hexane–EtOAc) to give
4.20. 2,6-Dimethylphenyl 2-O-benzoyl-3,6-di-O-benzyl-b-
galactopyranosyl-(1?4)-3-O-benzoyl-6-O-benzyl-2-deoxy-2-
(2,2,2-trichloroethoxycarbonylamino)-1-thio-b-D-
glucopyranoside (21)
D-
19
a
(990 mg, 60%) and 19b (422 mg, 25.6%) as pale-yellow syrups.
To a solution of 20 (514 mg, 0.431 mmol) in 5:1 EtOH–pyridine
(12 mL) was added thiourea (98.4 mg, 1.29 mmol), and the mixture
was stirred for 2 h at 80 °C. The mixture was diluted with CHCl3,
washed with satd aq NaHCO3, and water, dried (MgSO4), and con-
centrated. The product was purified by silica gel column chroma-
tography using 15:1 toluene–acetone as eluent to give 21 (350
Compound 19
a
: ½a 2D4
ꢁ
+113 (c 1.0, CHCl3). 1H NMR (500 MHz,
CDCl3): d 8.03–7.20 (m, 15H, Ar), 5.85 (dd, 1H, J1,2 2.4 Hz, J1,F
53.1 Hz, H-1), 5.83 (d, 1H, J3,4 2.4 Hz, H-4), 5.33 (ddd, 1H, J1,2 2.1
Hz, J2,3 10.4 Hz, J2,F 24.4 Hz, H-2), 4.72 and 4.51 (each d, 2H, Jgem
11.6 Hz, PhCH2), 4.58 and 4.47 (each d, 2H, Jgem 11.6 Hz, PhCH2),
4.34 (t, 1H, H-5), 4.10 and 4.02 (each d, 2H, Jgem 11.6 Hz, COCH2Cl),
4.09 (dd, 1H, J2,3 10.4 Hz, J3,4 3.1 Hz, H-3), 3.62–3.51 (m, 2H, H-6a,
H-6b). 13C NMR (125 MHz, CDCl3): d 166.7, 165.7, 137.2, 137.0,
133.5, 130.0, 129.2, 128.5, 128.4, 128.2, 128.0, 127.9, 104.8 (d, JC-
mg, 73%). ½a 2D4
ꢁ
+10.4 (c 1.0, CHCl3). 1H NMR (500 MHz, CDCl3): d
8.00–7.00 (m, 33H, Ar), 5.54 (d, 1H, J2,NH 9.8 Hz, NH), 5.31–5.25
(m, 2H, H-3, H-20), 4.67 and 4.58 (each d, 2H, Jgem 12.2 Hz, PhCH2),
0
0
4.58 and 4.38 (each d, 2H, Jgem 12.8Hz, PhCH2), 4.53 (d, 1H, J1 ,2 7.9
Hz, H-10), 4.48 and 4.25 (each d, 2H, Jgem 12.2Hz, PhCH2), 4.43 (d,
1H, J1,2 10.4 Hz, H-1), 4.35 and 4.30 (each d, 2H, Jgem 12.6 Hz, Troc
226 Hz, C-1), 73.7 (PhCH2), 72.1 (C-3), 71.5 (PhCH2), 69.8 (C-
1,F
5), 69.3 (d, JC-2,F 23 Hz, C-2), 68.6 (C-4), 67.0 (C-6). HRFABMS: calcd
for C29H29ClFO7Na: ([M+Na]+) 543.1586, found 543.1562.
CH2), 4.11–4.05 (m, 2H, H-4, H-2), 4.00 (d, 1H, J3 ,4 3.0 Hz H-40),
3.50–3.47 (m, 1H, H-6a), 3.40 (dd, 1H, J2,3 9.8 Hz, J3,4 3.7 Hz, H-
3), 3.32(d, 1H, J3,4 9.8 Hz, H-6a), 3.29–3.18 (m, 3H, H-5, H-50, H-
60a), 3.12 (m, 1H, H-60b), 2.49 (s, 6H, CH3 ꢂ 2),. 13C NMR (125
MHz, CDCl3): d 166.6, 164.8, 154.5, 144.0, 138.2, 138.0, 137.2,
133.0, 130.3, 130.0, 129.7, 129.0, 128.4, 128.3, 128.2, 128.1,
127.7, 127.62, 127.55, 100.7(C-10), 89.3(C-1), 78.3(C-5), 78.1(C-
30), 74.8(C-2, C-20), 74.4(PhCH2), 73.4(PhCH2), 73.3(Troc CH2),
72.7(C-50), 71.5(C-3), 70.5(PhCH2), 68.0(C-6), 67.3(C-60), 64.7(C-
4,C-40), 22.4(Ph(CH3)2). MALDI-TOFMS: calcd for C58H58Cl3NO13S-
Na: ([M+Na]+) 1136.3, found 1136.5. HRFABMS: calcd for
C58H58Cl3NO13SNa: ([M+Na]+) 1136.2592, found 1136.2632.
0
0
Compound 19b: ½a D24
ꢁ
+55.0 (c 1.0, CHCl3). 1H NMR (500MHz,
CDCl3): d 7.98–7.09 (m, 15H, Ar), 5.72 (s, 1H, H-4), 5.48–5.42 (m,
1H, H-2), 5.27 (dd, 1H, J1,2 7.3 Hz, J1,F 52.5 Hz, H-1), 4.67 and
4.44 (each d, 2H, Jgem 12.2 Hz, PhCH2), 4.59 and 4.47 (each d, 2H,
Jgem=12.8 Hz, PhCH2), 4.15 and 4.04 (each d, 2H, Jgem 11.6 Hz,
COCH2Cl), 3.92 (t, 1H, H-5), 3.70 (dd, 1H, J2,3 9.8 Hz, J3,4 3.1 Hz,
H-3), 3.68–3.58 (m, 2H, H-6a, H-6b). 13C NMR (125 MHz, CDCl3):
d 166.8, 165.0, 137.1, 136.7, 133.4, 129.9, 129.3, 128.6, 128.4,
128.2, 128.13, 128.05, 127.9, 107.3 (d, JC-1,F 219 Hz, C-1), 75.2 (d,
JC-3,F 10.4 Hz, C-3), 73.8 (PhCH2), 72.2 (C-5), 71.3 (PhCH2), 70.8(d,
JC-2,F 22.8 Hz, C-2), 67.3(C-4), 66.9(C-6), 40.7. HRFABMS: calcd for
C29H29ClFO7Na: ([M+Na]+) 543.1586, found 543.1616.
4.21. 2,6-Dimethylphenyl 2,3,4,6-tetra-O-acetyl-b-
pyranosyl-(1?4)-2-O-benzoyl-3,6-di-O-benzyl-b-
galactopyranosyl-(1?4)-3-O-benzoyl-6-O-benzyl-2-deoxy-2-
(2,2,2-trichloroethoxycarbonylamino)-1-thio-b-D-
glucopyranoside (22)
D-galacto-
D-
4.19. 2,6-Dimethylphenyl 2-O-benzoyl-3,6-di-O-benzyl-4-O-
chloroacetyl-b-
benzyl-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-1-
thio-b- -glucopyranoside (20)
D-galactopyranosyl-(1?4)-3-O-benzoyl-6-O-
D
A solution of compound 12 (200 mg, 0.30 mmol) and 19 (325 mg,
0.60 mmol) containing activated AW-300 MS (500 mg) in dry CH2Cl2
Compound 22 was prepared from 21 (150 mg, 0.13 mmol) and 4
(528 mg, 0.54 mmol) as described for preparation of 13, yielding

866
A. Koizumi et al. / Carbohydrate Research 344 (2009) 856–868
142 mg (73%). ½a 2D4
ꢁ
ꢀ1.11 (c 1.4, CHCl3). 1H NMR (500 MHz, CDCl3):
hexane–EtOAc) to give 26 (167 mg, 78%). ½a D24
ꢁ
+41.7 (c 1.1, CHCl3).
d 4.88 (d, 1H, J1,2 7.9Hz, H-1 of Gal b), 4.45 (d, 1H, J1,2 7.9Hz, H-1 of
Gal a), 4.27 (d, 1H, J1,2 11.0Hz, H-1 of GlcNAc). 13C NMR (125 MHz,
CDCl3): d 100.9 (C-1 of Gal a), 100.3 (C-1 of Gal b), 89.3 (C-1 of Glc-
NAc). MALDI-TOFMS: calcd for C72H76Cl3NO22SNa: ([M+Na]+)
1466.4, found 1466.8.
1H NMR (500 MHz, CDCl3): d 7.43–7.25 (m, 10H, Ar), 5.54 (dd, 1H,
J1,2 3.1 Hz, J1,F 53.7 Hz, H-1), 4.92–4.71 (m, 4H, PhCH2 ꢂ 2), 4.55 (d,
1H, J3,4 2.4 Hz, H-4), 4.22–4.14 (m, 2H, H-6a, H-6b), 3.99 (ddd, 1H,
J1,2 2.4 Hz, J2,3 9.8 Hz, J2,F 26.2 Hz, H-2), 3.79 (s, 1H, H-5), 1.06 (s, 9H,
Si(CH3)3), 0.97 (s, 9H, Si(CH3)3). 13C NMR (125 MHz, CDCl3): d 138.5,
138.0, 128.4, 128.3, 127.9, 127.7, 106.9 (d, JC-1,F 226 Hz, C-1), 77.3
(C-3), 74.0 (PhCH2), 73.7 (d, JC-2,F 23 Hz, C-2), 71.1 (PhCH2), 70.5
(C-4), 69.6 (C-2), 66.7 (C-6), 27.6 (Si(CH3)3), 27.2 (Si(CH3)3). MALDI-
TOFMS: calcd for C28H39FO5SiNa: ([M+Na]+) 525.2, found 525.4.
4.22. 2-(Trimethylsilyl)ethyl 2,3,4,6-tetra-O-acetyl-b-
pyranosyl-(1?3)-[2,3,4,6-tetra-O-acetyl-b- -galactopyranosyl-
(1?4)-2-O-benzoyl-3,6-di-O-benzyl-b- -galactopyranosyl-
(1?4)-3-O-benzoyl-6-O-benzyl-2-deoxy-2-(2,2,2-
trichloroethoxycarbonylamino)-b- -glucopyranosyl-(1?6)]-2-
azido-4-O-benzyl-2-deoxy- -galactopyranoside (23)
D-galacto-
D
D
D
4.26. 2,6-Dimethylphenyl 2,3-di-O-benzyl-4,6-di-tert-
a
-D
butylsilylene-
a-D-galactopyranosyl-(1?4)-2-O-benzoyl-3,6-di-
O-benzyl-b- -galactopyranosyl-(1?4)-3-O-benzoyl-6-O-benzyl-
D
Compound 23 was prepared from 6 (64.8 mg, 89.3
l
mol) and 22
mol) as described for preparation of 14, yielding 104
+17.2 (c 1.1, CHCl3). 1H NMR (500 MHz, CDCl3): d 4.95
2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-1-thio-b-D-
glucopyranoside (27)
(142 mg, 98.2
l
mg (57%). ½a 2D4
ꢁ
(d, 1H, J1,2 7.3 Hz, H-1 of Gal c), 4.84 (d, 1H, J1,2 4.9 Hz, H-1 of GalNAc),
4.74 (d, 1H, J1,2 7.3 Hz, H-1 of Gal a), 4.45 (d, 1H, J1,2 7.3 Hz, H-1 of Glc-
NAc), 4.42(d, 1H, J1,2 5.5Hz, H-1 of Gal b). 13C NMR(125MHz, CDCl3):
d 102.3 (C-1 of Gal b), 101.1 (C-1 of Gal a), 100.6 (C-1 of GlcNAc),
100.2 (C-1 of Gal c), 95.5 (C-1 of GalNAc). MALDI-TOFMS: calcd for
C96H113Cl3N4O36SiNa: ([M+Na]+) 2053.6, found 2053.8.
Compound 27 was prepared from 21 (200 mg, 0.18 mmol) and
26 (135 mg, 0.27 mmol) as described for preparation of 20, yielding
260 mg (91%). ½a 2D4
ꢁ
+41.7 (c 1.0, CHCl3). 1H NMR (500 MHz, CDCl3):
d 4.83 (d, 1H, J1,2 3.1 Hz, H-1 of Gal b), 4.72 (d, 1H, J1,2 7.3 Hz, H-1 of
Gal a), 4.37 (d, 1H, J1,2 10.4 Hz, H-1 of GlcNAc). 13C NMR (125 MHz,
CDCl3): d 101.6 (C-1 of Gal a), 100.7 (C-1 of Gal b), 89.5 (C-1 of Glc-
NAc). MALDI-TOFMS: calcd for C86H96Cl3NO18SSiNa: ([M+Na]+)
1618.5, found1618.4.
4.23. 2-(Trimethylsilyl)ethyl 2,3,4,6-tetra-O-acetyl-b-
galactopyranosyl-(1?3)-[2,3,4,6-tetra-O-acetyl-b-
galactopyranosyl-(1?4)-3,6-di-O-acetyl-2-O-benzoyl-b-
galactopyranosyl-(1?4)-2-acetamido-6-O-acetyl-3-O-benzoyl-
2-deoxy-b- -glucopyranosyl-(1?6)]-2-acetamido-4-O-acetyl-2-
deoxy- -galactopyranoside (24)
D-
D
-
D-
4.27. 2,6-Dimethylphenyl 4,6-di-O-acetyl-2,3-di-O-benzyl-
galactopyranosyl-(1?4)-2-O-benzoyl-3,6-di-O-benzyl-b-
galactopyranosyl-(1?4)-3-O-benzoyl-6-O-benzyl-2-deoxy-2-
a-D-
D-
D
a-D
(2,2,2-trichloroethoxycarbonylamino)-1-thio-b-D-
glucopyranoside (28)
Compound 24 was prepared from 23 (251 mg, 0.123 mmol) as
described for preparation of 10, yielding 73.3 mg (34%). ½a D24
ꢁ
A solution of 27 (260 mg, 0.163 mmol) in THF (5 mL) was trea-
ted with HF–pyridine (500 L) at 0 °C and then stirred for 2 h. The
+25.9 (c 1.0, CHCl3). 1H NMR (500 MHz, CDCl3): d 4.82 (d, 1H, J1,2
3.7 Hz, H-1 of GalNAc), 4.59 (d, 1H, J1,2 7.9 Hz, H-1 of Gal c), 4.54
(d, 1H, J1,2 7.9Hz, H-1 of GlcNAc), 4.43 (d, 1H, J1,2 7.3 Hz, H-1 of
Gal a), 4.36 (d, 1H, J1,2 7.9 Hz, H-1 of Gal b). 13C NMR (125 MHz,
CDCl3): d 102.3 (C-1 of Gal b), 101.1 (C-1 of Gal a), 100.6 (C-1 of
GlcNAc), 100.2 (C-1 of Gal c), 95.5 (C-1 of GalNAc). MALDI-TOFMS:
calcd for C77H102N2O40SiNa: ([M+Na]+) 1745.5, found 1745.3.
l
reaction mixture was added to water, extracted with EtOAc, and
the organic layer was washed with satd aq NaHCO3 and water,
dried (MgSO4), and concentrated. The residue was treated with
Ac2O (3 mL) in pyridine (5 mL). The reaction mixture was poured
into ice-water and extracted with CHCl3. The extract was washed
sequentially with 5% HCl, satd aq NaHCO3, and water, dried
(MgSO4), and concentrated. The product was purified by silica gel
column chromatography using 7:1 hexane–EtOAc as eluent to give
4.24. 2-(Trimethylsilyl)ethyl b-
galactopyranosyl-(1?4)-b- -galactopyranosyl-(1?4)-2-
acetamido-2-deoxy-b- -glucopyranosyl-(1?6)]-2-acetamido-2-
deoxy- -galactopyranoside (E)
D-galactopyranosyl-(1?3)-[b-D-
D
28 (234 mg, 93%). ½a D24
ꢁ
+39.1 (c 1.0, CHCl3). 1H NMR (500 MHz,
D
CDCl3): d 4.98 (d, 1H, J1,2 3.7 Hz, H-1 of Gal b), 4.60 (d, 1H, J1,2
7.3 Hz, H-1 of Gal a), 4.36 (d, 1H, J1,2 10.4 Hz, H-1 of GlcNAc). 13C
NMR (125 MHz, CDCl3): d 101. (C-1 of Gal a), 100.4 (C-1 of Gal
b), 95.2 (C-1 of GlcNAc). MALDI-TOFMS: calcd for C82H84Cl3NO20S-
Na: ([M+Na]+)1562.4, found 1562.5.
a-D
Compound E was prepared from 24 (69.7 mg, 40.4
l
mol) as de-
scribed for preparation of A, yielding 38.5 mg (95%). ½a D24
ꢁ
+34.6 (c
1.0, H2O). 1H NMR (500 MHz, D2O): d 4.72 (d, 1H, J1,2 3.7 Hz, H-1
of GalNAc), 4.41 (d, 1H, J1,2 7.9 Hz, H-1 of Gal c), 4.37 (d, 1H, J1,2
8.5 Hz, H-1 of GlcNAc) 4.31 (d, 1H, J1,2 7.9 Hz, H-1 of Gal a), 4.28
(d, 1H, J1,2 7.9 Hz, H-1 of Gal b). 13C NMR (125 MHz, D2O): d 104.1
(C-1 of Gal c), 103.8 (C-1 of Gal b), 102.5 (C-1 of Gal a), 101.0 (C-1
of GlcNAc), 98.8 (C-1 of GalNAc). MALDI-TOFMS: calcd for
C39H70N2O26SiNa: ([M+Na]+) 1033.4, found 1034.0. HRFABMS: calcd
for C39H70N2O26SiNa: ([M+Na]+) 1033.3884, found 1033.3920.
4.28. 2-(Trimethylsilyl)ethyl 2,3,4,6-tetra-O-acetyl-b-
galactopyranosyl-(1?3)-[4,6-di-O-acetyl-2,3-di-O-benzyl-
galactopyranosyl-(1?4)-2-O-benzoyl-3,6-di-O-benzyl-b-
galactopyranosyl-(1?4)-3-O-benzoyl-6-O-benzyl-2-deoxy-2-
(2,2,2-trichloroethoxycarbonylamino)-b- -glucopyranosyl-
(1?6)]-2-azido-4-O-benzyl-2-deoxy- -galactopyranoside (29)
D-
a-D-
-
D
D
a-D
Compound 29 was prepared from 6 (42.3 mg, 58.3
lmol) and 28
4.25. 2,3-Di-O-benzyl-4,6-di-tert-butylsilylene-
galactopyranosyl fluoride (26)
a
-
D
-
(108 mg, 70.0 lmol) as described for preparation of 14, yielding
56.4 mg (46%). ½a D24
ꢁ
+43.6 (c 1.0, CHCl3).
1H NMR (500 MHz, CDCl3): d 4.91 (d, 1H, J1,2 3.1 Hz, H-1 of Gal
c), 4.79 (d, 1H, J1,2 4.3 Hz, H-1 of GalNAc), 4.68 (d, 1H, J1,2 8.5 Hz, H-
1 of Gal b), 4.51 (d, 1H, J1,2 7.9 Hz, H-1 of GlcNAc), 4.37 (br d, 1H, J1,2
7.3 Hz, H-1 of Gal a). 13C NMR (125 MHz, CDCl3): d 102.3(C-1 of
Gal b), 101.2 (C-1 of GlcNAc), 101.12 (C-1 of Gal a), 100.4 (C-1
of Gal c), 97.4 (C-1 of GalNAc). MALDI-TOFMS: calcd for
C106H121Cl3N4NaO34Si: ([M+Na]+) 2149.7, found 2150.7.
To a solution of 25 (253 mg, 0.43 mmol) in CH2Cl2 (4 mL) were
added DAST (84 L, 0.64 mmol) and NBS (98.8 mg, 0.56 mmol) at
l
ꢀ15 °C, and the mixture was stirred for 1.5 h. The reaction mixture
was diluted with CHCl3 and extracted with satd aq NaHCO3 and brine.
The organic layer was dried (MgSO4), filtered, and concentrated.
The residue was purified by chromatography on silica gel (20:1

A. Koizumi et al. / Carbohydrate Research 344 (2009) 856–868
867
4.29. 2-(Trimethylsilyl)ethyl b-
galactopyranosyl-(1?4)-b- -galactopyranosyl-(1?4)-2-
acetamido-2-deoxy-b- -glucopyranosyl-(1?6)]-2-acetamido-2-
deoxy- -galactopyranoside (F)
D
-galactopyranosyl-(1?3)-[
a
-
D
-
reaction mixture was poured into ice-water and extracted with
CHCl3. The extract was washed sequentially with 5% HCl, satd aq
NaHCO3, and water, dried (MgSO4), and concentrated. The product
was purified by silica gel column chromatography using 3:1 tolu-
D
D
a-D
ene–acetone as eluent to give 33 (78.3 mg, 64%). ½a D24
ꢁ
+67.5 (c
Compound F was prepared from 29 (122 mg, 57.3
l
mol) as de-
1.0, CHCl3). 1H NMR (500 MHz, CDCl3): d 4.96 (d, 1H, J1,2 3.7 Hz,
H-1 of GalNAc), 4.74 (d, 1H, J1,2 7.9 Hz, H-1 of Gal a), 4.47 (d, 1H,
J1,2 7.9 Hz, H-1 of Gal b). 13C NMR (125 MHz, CDCl3): d 101.6 (C-
1 of Gal b), 99.7 (C-1 of Gal a), 96.6 (C-1 of GalNAc). MALDI-TOFMS:
calcd for C43H66N2O24SiNa: ([M+Na]+) 1108.4, found 1108.6.
scribed for preparation of A, yielding 12.1 mg (21% four steps). ½a D24
ꢁ
+41.9 (c 0.3, H2O). 1H NMR (500 MHz, D2O): d 4.76 (d, 1H, J1,2 3.7
Hz, H-1 of Gal c), 4.72 (d, 1H, J1,2 3.7, H-1 of GalNAc), 4.38 (d, 1H,
J1,2 7. 9Hz, H-1 of GlcNAc) 4.34 (d, 1H, J1,2 7.3 Hz, H-1 of Gal b),
4.28 (d, 1H, J1,2 7.9 Hz, H-1 of Gal a). 13C NMR (125 MHz, D2O): d
104.2 (C-1 of Gal b), 102.8 (C-1 of Gal a), 101.0 (C-1 of GlcNAc),
99.9 (C-1 of Gal c), 96.2 (C-1 of GalNAc). MALDI-TOFMS: calcd for
C39H70N2O26SiNa: ([M+Na]+) 1033.4, found 1034.2. HRFABMS: calcd
for C39H70N2O26SiNa: ([M+Na]+) 1033.3884, found 1033.3907.
4.34. 2-(Trimethylsilyl)ethyl b-D-galactopyranosyl-(1?4)-b-D-
galactopyranosyl-(1?3)-2-acetamido-2-deoxy-a-D-
galactopyranoside (B)
Compound B was prepared from 33 (73.8 mg, 67.9 lmol) as de-
4.30. 2-(Trimethylsilyl)ethyl 2-O-benzoyl-3,6-di-O-benzyl-4-O-
scribed for preparation of A, yielding 48.6 mg (quant.). ½a D24
ꢁ
+65.2
chloroacetyl-b-
benzylidene-2-deoxy-
D
-galactopyranosyl-(1?3)-2-azido-4,6-O-
-galactopyranoside (30)
(c 1.0, H2O). 1H NMR (500 MHz, D2O): d 4.73 (d, 1H, J1,2 3.7 Hz,
H-1 of GalNAc), 4.40 (d, 1H, J1,2 7.3 Hz, H-1 of Gal b), 4.32 (d, 1H,
J1,2 7.9 Hz, H-1 of Gal a). 13C NMR (125 MHz, D2O): d 104.2 (C-1
of Gal a), 103.8 (C-1 of Gal b), 96.1 (C-1 of GalNAc). MALDI-TOFMS:
calcd for C25H47NO16SiNa: ([M+Na]+) 668.3, found 668.6.
HRFABMS: calcd for C25H47NO16SiNa: ([M+Na]+) 668.2562, found
668.2602.
a-D
Compound 30 was prepared from 3a (106 mg, 0.249 mmol) and
18 (256 mg, 0.408 mmol) as described for preparation of 9, yielding
195 mg (79%). [a]
D +103 (c 1.0, CHCl3). 1H NMR (500 MHz, CDCl3): d
4.89 (d, 1H, J1,2 3.7 Hz, H-1 of GalNAc), 4.76 (d, 1H, J1,2 7.9 Hz, H-1
of Gal). 13C NMR (125 MHz, CDCl3): d 102.3 (C-1 of Gal), 97.9 (C-1
of GalNAc). MALDI-TOFMS: calcd for C47H54ClN3O12SiNa:
([M+Na]+) 938.3, found: 939.0. HRFABMS: calcd for C47H54ClN3O12-
SiNa: ([M+Na]+) 938.3063, found 938.3097.
4.35. 2-(Trimethylsilyl)ethyl 2,3,4,6-tetra-O-acetyl-b-
D-
galactopyranosyl-(1?4)-2-O-benzoyl-3,6-di-O-benzyl-b-
D-
galactopyranosyl-(1?3)]-2-azido-4-O-benzyl-2-deoxy-a-D-
galactopyranoside (34)
4.31. 2-(Trimethylsilyl)ethyl 2-O-benzoyl-3,6-di-O-benzyl-b-
galactopyranosyl-(1?3)-2-azido-4,6-O-benzylidene-2-deoxy-
-galactopyranoside (31)
D
-
a
-
Compound 34 was prepared from 32 (189 mg, 0.16 mmol) as
D
described for preparation of 6, yielding 140 mg (74%). ½a D24
ꢁ
+21.3
(c 1.0, CHCl3). 1H NMR (500 MHz, CDCl3): d 4.89 (d, 1H, J1,2
7.9Hz, H-1 of Gal b), 4.83 (d, 1H, J1,2 3.7Hz, H-1 of GalNAc), 4.79
(d, 1H, J1,2 7.9Hz, H-1 of Gal a). 13C NMR (125 MHz, CDCl3): d
102.1 (C-1 of Gal a), 100.4 (C-1 of Gal b), 97.9 (C-1 of GalNAc).
MALDI-TOFMS: calcd for C59H73N3O20SiNa: ([M+Na]+) 1194.5,
found 1195.3. HRFABMS: calcd for C59H73N3O20SiNa: ([M+Na]+)
1194.4454, found 1194.4517.
Compound 31 was prepared from 30 (364 mg, 0.40 mmol) as
described for preparation of 21, yielding 297 mg (89%). ½a D24
ꢁ
+81.6 (c 1.0, CHCl3). 1H NMR (500 MHz, CDCl3): d 4.88 (d, 1H, J1,2
3.7 Hz, H-1 of GalNAc), 4.72 (d, 1H, J1,2 7.9 Hz, H-1 of Gal). 13C
NMR (125 MHz, CDCl3): d 102.3 (C-1 of Gal), 97.9 (C-1 of GalNAc).
MALDI-TOFMS: calcd for C45H53N3O11SiNa: ([M+Na]+) 862.3, found
863.1. HRFABMS: calcd for C45H53N3O11SiNa: ([M+Na]+) 862.3347,
found 862.3387.
4.36. 2-(Trimethylsilyl)ethyl 2,3,4,6-tetra-O-acetyl-b-
galactopyranosyl-(1?4)-2-O-benzoyl-3,6-di-O-benzyl-b-
galactopyranosyl-(1?3)-[3,4,6-tri-O-acetyl-2-deoxy-2-(2,2,2-
trichloroethoxycarbonylamino)-b- -glucopyranosyl-(1?6)-]-2-
azido-4-O-benzyl-2-deoxy- -galactopyranoside (35)
D-
D-
4.32. 2-(Trimethylsilyl)ethyl 2,3,4,6-tetra-O-acetyl-b-
D-galac-
topyranosyl-(1?4)-2-O-benzoyl-3,6-di-O-benzyl-b- -galacto-
D
D
pyranosyl-(1?3)-2-azido-4,6-O-benzylidene-2-deoxy-
a-
D
-
a-D
galactopyranoside (32)
Compound 35 was prepared from 34 (76.3 mg, 65.1 lmol) and 8
Compound 32 was prepared from 31 (131 mg, 0.156 mmol) and
4 (308 mg, 0.624 mmol) as described for preparation of 13, yielding
(78.2 mg, 0.130 mmol) as described for preparation of 14, yielding
85.6 mg (81%). ½a D24
ꢁ
+19.9 (c 1.0, CHCl3). 1H NMR (500 MHz, CDCl3):
135 mg (74%). ½a 2D4
ꢁ
+62.1 (c 1.0, CHCl3). 1H NMR (500 MHz, CDCl3):
d 4.96 (d, 1H, J1,2 3.7 Hz, H-1 of GalNAc), 4.86 (d, 1H, J1,2 7.9 Hz, H-1
of Gal a), 4.77 (d, 1H, J1,2 7.9 Hz, H-1 of GlcNAc), 4.60 (br d, 1H, J1,2
12.8 Hz, H-1 of Gal b). 13C NMR (125 MHz, CDCl3): d 103.0 (C-1 of
Gal a), 101.2 (C-1 of GlcNAc), 100.2 (C-1 of Gal b), 97.6 (C-1 of Gal-
NAc). MALDI-TOFMS: calcd for C74H91Cl3N4O29SiNa: ([M+Na]+)
1655.5, found 1655.2.
d 5.01 (d, 1H, J1,2 7.9 Hz, H-1 of Gal b), 4.93 (d, 1H, J1,2 3.7 Hz, H-1 of
GalNAc), 4.77 (d, 1H, J1,2 = 7.9 Hz, H-1 of Gal a). 13C NMR (125 MHz,
CDCl3): d 102.1 (C-1 of Gal a), 100.4 (C-1 of Gal b), 97.9 (C-1 of Gal-
NAc). MALDI-TOFMS: calcd for C59H71N4O20SiNa: ([M+Na]+)
1192.4, found 1192.4. HRFABMS: calcd for C59H71N3O20SiNa:
([M+Na]+) 1192.4298, found 1192.4354.
4.37. 2-(Trimethylsilyl)ethyl 2,3,4,6-tetra-O-acetyl-b-
galactopyranosyl-(1?4)-3,6-di-O-acetyl-2-O-benzoyl-b-
galactopyranosyl-(1?3)-[2-acetamido-3,4,6-tri-O-acetyl-2-
deoxy-b- -glucopyranosyl-(1?6)-]-2-acetamido-4-O-acetyl-2-
deoxy- -galactopyranoside (36)
D-
4.33. 2-(Trimethylsilyl)ethyl 2,3,4,6-tetra-O-acetyl-b-
D
-galacto-
D-
pyranosyl-(1?4)-3,6-di-O-acetyl-2-O-benzoyl-b- -galacto-
D
pyranosyl-(1?3)-2-acetamido-4,6-di-O-acetyl-2-deoxy-
galactopyranoside (33)
a-D-
D
a-D
A solution of 32 (132 mg, 0.113 mmol) in MeOH (3.0 mL) was
hydrogenolyzed in the presence of 10% Pd(OH)2–C (100 mg) for
20 h at room temperature, then filtered, and concentrated. The res-
idue was acetylated with Ac2O (3.0 mL) in pyridine (5.0 mL). The
Compound 36 was prepared from 35 (119 mg, 72.9
l
mol) as de-
+36.0 (c
scribed for preparation of 10, yielding 36.4 mg (36%). ½a D24
ꢁ
1.0, CHCl3). 1H NMR (500 MHz, CDCl3): d 4.90 (d, 1H, J1,2 3.7 Hz, H-1
of GalNAc), 4.75 (d, 1H, J1,2 7.9 Hz, H-1 of Gal b), 4.63 (d, 1H, J1,2 7.9

868
A. Koizumi et al. / Carbohydrate Research 344 (2009) 856–868
Hz, H-1 of GlcNAc), 4.48 (d, 1H, J1,2 7.9 Hz, H-1 of Gal a). 13C NMR
(125 MHz, CDCl3): d 101.6 (C-1 of Gal a), 100.7 (C-1 of GlcNAc),
99.6 (C-1 of Gal b), 96.6 (C-1 of GalNAc). MALDI-TOFMS: calcd
for C60H84N2O32SiNa: ([M+Na]+) 1395.5, found 1396.1.
3. (a) Hokke, C. H.; Yazdnbakhsh, M. Parasite Immunol. 2005, 27, 257–264; (b)
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Nakatani, F. Biochim. Biophys. Acta 1994, 1215, 163–169.
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Sugita, M.; Takeda, T. Chem. Pharm. Bull. 2002, 50, 600–604; (d) Hada, N.;
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4.38. 2-(Trimethylsilyl)ethyl b-
galactopyranosyl-(1?3)-[2-acetamido-2-deoxy-b-
glucopyranosyl-(1?6)-]-2-acetamido-2-deoxy-a-D-
galactopyranoside (C)
D-galactopyranosyl-(1?4)-b-D-
D-
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Compound C was prepared from 36 (36.4 mg,
l
mol) as de-
scribed for preparation of A, yielding 20.4 mg (91%). ½a D24
ꢁ
+52.0 (c
0.5, H2O). 1H NMR (500 MHz, D2O): d 4.72 (d, 1H, J1,2 3.7Hz, H-1
of GalNAc), 4.40 (d, 1H, J1,2 7.3 Hz, H-1 of Gal b), 4.35 (d, 1H, J1,2
8.5 Hz, H-1 of GlcNAc), 4.30 (d, 1H, J1,2 7.9 Hz, H-1 of Gal a). 13C
NMR (125 MHz, D2O): d 104.2 (C-1 of GlcNAc), 103.8 (C-1 of Gal
b), 101.1 (C-1 of Gal a), 96.2 (C-1 of GalNAc). MALDI-TOFMS: calcd
for C33H60N2O21SiNa: ([M+Na]+) 871.3, found: 871.8. HRFABMS:
calcd for C33H66N2O21SiNa: ([M+Na]+) 871.3356, found 871.3376.
Acknowledgments
This work was supported by a Grant-in-Aid for Scientific Re-
search (No. 19590011) from the Ministry of Education, Culture,
Sports, Science, and Technology of Japan (MEXT), and High-Tech
Research Center project of the MEXT. The authors are grateful to
Ms. J. Hada for providing HR-FAB MS data.
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Supplementary data
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Supplementary data (Copies of the NMR spectra for all new
compounds are provided) associated with this article can be found,
in the online version, at doi:10.1016/j.carres.2009.02.019.
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