Design and synthesis of a novel ganglioside ligand for influenza a viruses

Article abstract of DOI:10.3390/molecules17089590

A novel ganglioside bearing Neuα2-3Gal and Neuα2-6Gal structures as distal sequences was designed as a ligand for influenza A viruses. The efficient synthesis of the designed ganglioside was accomplished by employing the cassette coupling approach as a ke

Full text of DOI:10.3390/molecules17089590

Molecules 2012, 17, 9590-9620; doi:10.3390/molecules17089590
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Design and Synthesis of a Novel Ganglioside Ligand for
Influenza A Viruses †
Tomohiro Nohara 1, Akihiro Imamura 1,2,*, Maho Yamaguchi 3, Kazuya I. P. J. Hidari 3,
Takashi Suzuki 3, Tatsuya Komori 1, Hiromune Ando 1,2, Hideharu Ishida 1 and Makoto Kiso 1,2,
*
1
Department of Applied Bioorganic Chemistry, Gifu University, 1-1 Yanagido, Gifu-shi,
Gifu 501-1193, Japan; E-Mails: noharah18@yahoo.co.jp (T.N.); komorih17@yahoo.co.jp (T.K.);
hando@gifu-u.ac.jp (H.A.); ishida@gifu-u.ac.jp (H.I.)
2
3
Institute for Integrated Cell-Material Sciences, Kyoto University, 69 Konoe-cho, Yoshida,
Sakyo-ku, Kyoto 606-8501, Japan
Department of Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada,
Suruga-ku, Shizuoka-shi, Shizuoka 422-8526, Japan; E-Mails: d11107@u-shizuoka-ken.ac.jp (M.Y.);
hidari@u-shizuoka-ken.ac.jp (K.I.P.J.H.); suzukit@u-shizuoka-ken.ac.jp (T.S.)
†
Synthetic studies on sialoglycoconjugates, Part 157.
* Authors to whom correspondence should be addressed; E-Mails: aimamura@gifu-u.ac.jp (A.I.);
kiso@gifu-u.ac.jp (M.K.); Tel.: +81-58-293-3453 (A.I.); Fax: +81-58-293-2918 (A.I.).
Received: 9 July 2012; in revised form: 6 August 2012 / Accepted: 8 August 2012 /
Published: 10 August 2012
Abstract: A novel ganglioside bearing Neu2-3Gal and Neu2-6Gal structures as distal
sequences was designed as a ligand for influenza A viruses. The efficient synthesis of the
designed ganglioside was accomplished by employing the cassette coupling approach as a
key reaction, which was executed between the non-reducing end of the oligosaccharide and
the cyclic glucosylceramide moiety. Examination of its binding activity to influenza A
viruses revealed that the new ligand is recognized by Neu2-3 and 2-6 type viruses.
Keywords: ganglioside; sialic acid; total synthesis; glycosylation; influenza virus

Molecules 2012, 17
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1. Introduction
Influenza viruses cause a substantial number of deaths during annual epidemics and occasional
pandemics [1,2]. Based on the antigenicity of their internal proteins the viruses are divided into three
types, A, B, and C, of which either type A or B viruses cause seasonal influenza in humans. When the
viruses bind to the host cell, hemagglutinin (HA) on their cell surface plays a significant role in the
infection process. The HA protein recognizes sialoglycoconjugates expressed on the plasma membrane
of the host cell, for example, sialoglycoproteins and gangliosides (sialoglycosphingolipids), as
cellular ligands. Furthermore, HA can also recognize specific linkages between sialic acid
(Neu5Ac/Gc) and lactosamine (LacNAc: Gal1-4GlcNAc) residues, which are found at the terminal
end of glycoconjugates [3,4]. The structure and distribution of sialoglycans are crucial for viruses to
determine their host animals, and two major linkage types, that is, Neu5Ac2-3LacNAc and
Neu5Ac2-6LacNAc, are essential for viral transmission. Human and swine viruses predominantly
recognize the Neu5Ac2-6LacNAc sequence, while avian and equine viruses bind preferentially to the
Neu5Ac2-3Gal (including Neu5Ac2-3LacNAc) moiety. Swine are considered as intermediate hosts
between humans and birds since they possess an abundance of both Neu5Ac2-3LacNAc and
Neu5Ac2-6LacNAc structures as receptor carbohydrate determinants. The simultaneous infection of
an intermediate host, such as swine, with avian and human viruses could lead to genetic recombination
between the viruses, resulting in the generation of a new pathogenic virus that could potentially cause
severe pandemics. However, the exact natural ligand for influenza A viruses in an intermediate host,
such as pigs, remains unclear. Therefore, in this study, we focused on identifying a new carbohydrate
ligand that was not only highly recognized by influenza A viruses but also functions as a natural
receptor for viral HA. For this purpose, a ganglioside bearing both the Neu5Ac2-3 and Neu5Ac2-
6LacNAc sequences was designed (Figure 1). It was hypothesized that the designed ganglioside 1
could be recognized by human- and avian-derived viruses because it contains two types of sialoglycan
in a single molecule. We report the chemical synthesis of ganglioside 1 and its binding activity to
influenza A viruses.
2. Results and Discussion
2.1. Chemical Synthesis
It was envisaged that the efficient synthesis of 1 could be achieved using the cassette approach
between the non-reducing end of the oligosaccharide and the glucosylceramide, which was recently
developed by our group [5–10]. Furthermore, it was thought that the construction of the non-reducing
end of the heptasaccharide moiety, which includes two types of sialoside, Neu5Ac2-3/2-6Gal, should
be executed through a convergent synthetic approach. For the convergent synthesis of a relatively large
oligosaccharide such as 1, the design of the building blocks often affects the efficiency of the total
synthesis as well as the overall yield. Our preliminary experiment on the synthesis of a ganglioside
similar to 1 gave a significant finding that monosaccharyl (GlcN) units are more useful as glycosyl
donors than oligosaccharyl (Neu5Ac2-3/2-6Gal1-4GlcN) donors for the formation of branched
structure at the 3- and 6-positions of the inner galactose residue (data not shown). Therefore, target 1

Molecules 2012, 17
9592
was divided into four major components, from which each building block (Units A–D) was designed
(Figure 1).
Figure 1. Structures of the target ganglioside 1 and the designed building blocks (Units A–D).
OAc
AcO
CO2Me
O
AcHN
AcO
O
NHTroc
O
O
AcO
BnO
ClAcO
BnO
OAc
BzO
O
TCAI
AcO
OBn
O
OBz
O
LevO
BnO
Unit B
O
OSE
NHTroc
OBz
OH
Unit C
HO
CO2H
O
O
AcHN
HO
HO
HO
NHAc
O
O
O
HO
HO
O
HO
OH
OH
HO
CO2H
O
HO
OH
OH
O
OH
OH
O
HO
O
O
AcHN
HO
O
C13H27
C17H35
O
O
NHAc
HO
O
O
OH
HO
OH
HN
HO
OH
O
1
O
O
O
OAc
O
O
AcO
CO2Me
O
O
OBz
O
HO
PMBO
O
TCAI
O
C13H27
C17H35
AcHN
AcO
OBz
HN
OAc
AcO OBz
Unit A
Unit D
The synthetic method for the terminal Neu5Ac2-3Gal unit A has been already established by our
group. The coupling of the 5-N-Troc-protected sialyl donor 2 and galactosyl acceptor 3, carrying a
p-methoxyphenyl (MP) group at the anomeric position, generated the Neu5Troc2-3Gal disaccharide
in good yield. The isolation of -sialoside from the reaction mixture was easily accomplished by
recrystallization [11]. The obtained disaccharide was readily converted into the corresponding
trichloroacetimidate donor as Unit A [12]. Similarly, the other terminal Neu5Ac2-6Gal unit (B) was
prepared efficiently according to the synthetic procedure for Unit A (Scheme 1). The sialylation of the
diol galactosyl acceptor 5 was performed in the presence of NIS and TfOH [13,14] in a mixed solvent
system, propionitrile–dichloromethane (5:1), at −30 °C [15,16]. This mixed solvent system was used
because of the poor solubility of the acceptor 5 in acetonitrile. In addition, temperatures lower than
−30 °C led to a significant decrease in the yield, possibly because of the observed precipitation of 5
during the reaction. As a result of optimization, the desired -glycoside 6 was obtained in 69% yield
along with a 14% yield of the -isomer. Purification of the -glycoside 6 by silica gel column
chromatography was troublesome compared with that of the regioisomer, Neu5Troc2-3GalMP,
which has benzyl groups on the O-2 and O-6 positions of its galactose residue, which can be isolated
easily by recrystallization from an EtOAc/n-hexane system [12]. The selective deprotection of the Troc
group with Zn–Cu [17] (giving 7) and the subsequent concomitant acetylation of the liberated amine

Molecules 2012, 17
9593
and the hydroxyl group at the C-4 position of the galactose residue afforded compound 8 in excellent
yield. Hydrogenolysis of the benzyl groups over Pearlman’s catalyst in 1,4-dioxane followed by
benzoylation generated compound 10 in 94% yield over two steps. The exposure of 10 to CAN and
H2O [18] (giving 11) and the subsequent introduction of a trichloroacetimidate group [19] afforded the
Neu5Ac2-6Gal donor 12 (Unit B) in 63% yield over two steps.
Scheme 1. Efficient synthesis of Units A and B from the N-Troc-protected sialyl donor 2.
HO
OBn
O
OAc
HO
OMP
AcO
CO2Me
SPh
OAc
AcO
OBn
CO2Me
O
BzO
3
O
OC(NH)CCl3
TrocHN
O
AcHN
OAc
AcO
O
OBz
Ref. 11,12
AcO
OAc
AcO
2
4
(Unit A)
HO
BnO
OH
O
AcO
OAc
OMP
6: R1 = Troc, R2 = H, R3 = Bn, R4 = OMP, R5 = H
7: R1 = H, R2 = H, R3 = Bn, R4 = OMP, R5 = H
8: R1 = R2 = Ac, R3 = Bn, R4 = OMP, R5 = H
9: R1 = R2 = Ac, R3 = H, R4 = OMP, R5 = H
10: R1 = R2 = Ac, R3 = Bz, R4 = OMP, R5 = H
11: R1 = R2 = Ac, R3 = Bz, R4, R5 = H, OH
12 (Unit B):
R1HN
AcO
a
b
c
d
e
OBn
5
CO2Me
97% (2 steps)
94% (2 steps)
b
O
NIS
TfOH
MS3Å
OAc
O
R2O
EtCN–CH2Cl2
(5:1)
O
R3O
R4 63% (2 steps)
a
f
–30 °C
69%
R3O
R5
R1 = R2 = Ac, R3 = Bz, R4, R5 = H, OC(NH)CCl3
Reagents and Conditions: (a) Zn–Cu, AcOH–CH2Cl2 (3:2), 40 °C; (b) Ac2O, DMAP, Py, r.t.;
(c) H2, Pd(OH)2/C, 1,4-dioxane, r.t.; (d) Bz2O, DMAP, Py, r.t.; (e) CAN, MeCN–PhMe–H2O
(6:5:3), r.t.; (f) CCl3CN, DBU, CH2Cl2, 0 °C.
The inner core trisaccharide structure, Unit C, was prepared starting from the 2-N-Troc protected
glucosamine derivative 13 (Schemes 2 and 3). First, the glucosaminyl donors 16 and 17 were prepared
as shown in Scheme 2. Removal of the acetyl groups from 13 and the subsequent formation of cyclic
benzylidene acetal between O-4 and O-6 afforded 14 in good yield. The following benzylation step in
the presence of a Troc group was conducted under reductive conditions. Optimization of this reductive
benzylation with benzaldehyde, TESOTf, and triethylsilane [20] revealed that the use of toluene as a
solvent could increase the yield. The successive reductive opening of the benzylidene group by
treatment with BF3 etherate and triethylsilane [21] gave 15 in 78% yield over two steps from 14. The
obtained alcohol 15 was transformed into two types of glucosaminyl donors, namely, 16 and 17, via
the introduction of a levulinoyl (Lev) and monochloroacetyl (ClAc) group to the hydroxyl group at
C-4, respectively.

Molecules 2012, 17
Scheme 2. Preparation of glucosaminyl donors 16 and 17 towards Unit C.
9594
1) PhCHO, TESOTf
TESH
OAc
O
THF-PhMe (1:4)
–20 °C
OBn
O
1) NaOMe, MeOH, r.t.
Ph
O
AcO
AcO
O
HO
BnO
O
SPh
SPh
SPh
HO
2) PhCH(OMe)2, CSA
THF-MeCN (1:4)
r.t. 80% (2 steps)
2) BF3•OEt2, TESH
CH2Cl2
0 °C, 78% (2 steps)
NHTroc
NHTroc
NHTroc
13
14
15
LevOH
EDC•HCl
DMAP
ClAc2O
DMAP
OBn
O
OBn
O
LevO
BnO
ClAcO
BnO
15
15
SPh
SPh
NHTroc
CH2Cl2
r.t., 93%
THF
r.t., 90%
NHTroc
16
17
Scheme 3. Assembly of the inner core fragment, Unit C.
Ph
Ph
Ph
Ph
Zn–Cu
AcOH
Bz2O
DMAP
O
O
O
O
O
O
O
O
TrocCl
O
O
O
O
TrocO
TrocO
HO
HO
OSE
OSE
OSE
OSE
CH2Cl2
r.t.
Py-CH2Cl2
(1:4)
Py
r.t.
OH
OH
OBz
OBz
84%
–40 °C
81%
quant.
18
19
20
21
Ph
NIS
TfOH
O
O
HO
80% AcOH aq.
OH
O
OBn
O
OBn
O
16
+
21
O
LevO
LevO
a
b
O
O
OSE
OSE
50 °C
99%
MS4Å
CH2Cl2
0 °C
BnO
BnO
OBz
OBz
NHTroc
NHTroc
22
23
81%
OBn
OBn
17
NIS
TfOH
O
O
HO
ClAcO
BnO
b
O
O
a
Ac2O
DMAP
BnO
O
DABCO
AcO
O
TrocHN
TrocHN
AcO
HO
OBn
O
OBn
O
O
THF
r.t.
96%
EtOH
50 °C
98%
MS4Å
CH2Cl2
0 °C
OSE
O
OBz
O
LevO
BnO
LevO
BnO
O
c
O
OSE
OSE
OBz
OBz
NHTroc
NHTroc
49%
24
27
28
(Unit C)
The glucosaminyl donors 16 and 17 were then incorporated into the galactosyl acceptor 21, which
was prepared readily from the known galactose derivative 18 [22] via three steps. Selective protection
of the hydroxyl group at C-3 by the Troc group was carried out under basic conditions with TrocCl at a
low temperature (−40 °C) to afford 19 in 81% yield. Benzoylation under standard conditions (giving 20)
and the selective removal of the Troc group with Zn–Cu furnished the galactosyl acceptor 21 in good
yield. The obtained 21 was then subjected to glycosylation with donor 16 in the presence of NIS and
TfOH in CH2Cl2 at 0 °C, affording the disaccharide 22 in 81% yield. The stereochemical assignment
was confirmed by 1H-NMR, where the J1,2 value of 7.6 Hz for H-1 indicated the -configuration of the
glucosamine residue. Next, hydrolysis of the benzylidene group with 80% AcOH aq at 50 °C afforded

Molecules 2012, 17
9595
the diol 23 at an almost quantitative yield. A second round of glucosaminidation was conducted
between 17 and 23 under the same conditions as those of the initial glucosaminidation between 16 and
21. As a result, the desired trisaccharide 24 was obtained as Unit C in a moderate yield of 49%. In this
reaction, a non-negligible amount of the tetrasaccharide 25, in which both hydroxyl groups were
glucosaminylated, was observed as a byproduct (Figure 2). An attempt at using a lower temperature to
increase the selectivity failed due to the poor solubility of acceptor 23 in CH2Cl2. Furthermore,
changing the other factors for glycosylation, for example, the leaving group (using trichloroacetimidate)
and how the donor was added, did not improve the yield of 24. It is of importance that the generation
of the tetrasaccharide 25 during the reaction was faster than the complete consumption of the acceptor
23. In addition, the trisaccharide 26, which was glucosaminylated at C-4 of the galactose residue, was
not detected among the by-products. These findings suggested that the newly formed trisaccharyl
alcohol 24 was preferred to the disaccharyl acceptor 23 as a glycosyl acceptor. This phenomenon
might be explained by the poor solubility of the disaccharyl alcohol 23 in CH2Cl2 compared with the
trisaccharyl alcohol 24 (Figure 2). Next, the acetylation of 24 with acetic anhydride and DMAP in
THF [23] was carried out to protect the free hydroxyl group, affording 27 in 96% yield (Scheme 3).
The monochloroacetyl group on 27 was then unblocked using DABCO in ethanol [24] with an
excellent yield, providing the inner core trisaccharide acceptor 28, which was ready for the next
glycosylation step.
Figure 2. Explanation for the poor regioselectivity observed during glucosaminylation.
Lower solubility in CH2Cl2
Higher solubility in CH2Cl2
OBn
O
ClAcO
BnO
O6-selective
glucosaminylation
O
HO
OH
O
OBn
O
TrocHN
LevO
BnO
HO
O
OSE
OBz
OBn
O
O
LevO
NHTroc
O
OSE
OBz
BnO
NHTroc
23
24
OBn
O
SPh
NHTroc
Preferential ?
ClAcO
BnO
O4-selective
glucosaminylation
Undesired second
glucosaminylation
on O4
17
TrocHN
BnO
ClAcO
BnO
BnO
OBn
O
O
O
ClAcO
BnO
O
O
ClAcO
O
BnO
TrocHN
OBn
O
TrocHN
O
OH
O
BnO
LevO
BnO
O
LevO
BnO
O
O
OSE
OBz
OSE
OBz
NHTroc
NHTroc
26
25
(Not detected)

Molecules 2012, 17
9596
As depicted in Scheme 4, the coupling of the trisaccharide acceptor 28 with the Neu5Ac2-6Gal
donor 12 promoted by TMSOTf was conducted in CH2Cl2 at room temperature, affording the
pentasaccharide 29 in 74% yield. During this glycosylation step, the generation of several by-products
containing trichloroacetamide glycoside, which is occasionally formed as a by-product during
glycosylation using trichloroacetimidate donors, made the purification process an arduous task.
Column chromatography on silica gel followed by gel filtration was found to be useful for purification.
Next, the conversion of the Troc carbamate at C-2 of both glucosamine residues into acetamide was
achieved by treatment of alloyed zinc with copper in AcOH and followed by acetylation, giving the
acetamide compound 31 in 61% yield over two steps. Finally, cleavage of the levulinoyl group by
using hydrazine monoacetate in THF [25] released the 4-OH to provide the pentasaccharide acceptor
32 in 92% yield.
Scheme 4. Coupling of Units A and C followed by transformation of the corresponding acceptor.
OAc
AcO
CO2Me
e
O
AcHN
AcO
O
29: R1 = Lev, R2 = Troc
30: R1 = Lev, R2 = H
31: R1 = Lev, R2 = Ac
32: R1 = H, R2 = Ac
R2HN
AcO
a
b
c
OAc
BzO
TMSOTf
61% (2 steps)
92%
O
BnO
O
12 + 28
c
O
a
d
O
MS3Å
CH2Cl2
r.t.
BnO
BzO
AcO
OBn
R1O
BnO
O
O
b
O
74%
OSE
NHR2
OBz
Reagents and Conditions: (a) Zn–Cu, AcOH–(CH2Cl2)2 (3:2), 50 °C; (b) Ac2O, DMAP, Py, r.t.;
(c) NH2NH2·AcOH, THF, r.t.
Scheme 5 shows the assembly of the non-reducing end heptasaccharide moiety. The Neu5Ac2-
3Gal donor 4 was coupled with 32 in the presence of TMSOTf in CH2Cl2 at room temperature to
provide the heptasaccharide 33 in 62% yield. During this glycosylation step, the generation of the
trichloroacetamide glycoside and the dimer of donor 4, which was formed by the nucleophilic attack of
the hydrolyzed donor on the oxocarbenium species derived from the donor, as by-products, made the
purification of the desired product 33 laborious. The structure of isolated 33 was elucidated based on
1
13
its MS, H, and C-NMR spectra. For instance, the -configuration of the newly formed glycosidic
linkage was evident from the coupling constant of the anomeric proton at  5.01 (J1,2 = 7.5 Hz). Next,
cleavage of the benzyl groups by hydrogenolysis (giving 34) followed by acetylation with
conventional conditions afforded 35 in 89% yield over two steps. Selective exposure of the anomeric
hydroxyl group was easily achieved by treatment with trifluoroacetic acid in CH2Cl2 to yield 36. This
was then converted to the corresponding trichloroacetimidate donor 37 in 95% yield over two steps
from 35, which was then ready for cassette coupling with the glucosylceramide block 38 (Unit D).

Molecules 2012, 17
Scheme 5. Synthesis of the non-reducing end heptasaccharide part.
9597
OAc
AcO
CO2Me
e
O
AcHN
AcO
O
AcO
AcHN
OAc
O
BnO
O
c
O
a
d
BzO
O
BnO
BzO
HO
AcO
OBn
O
O
b
O
OSE
BnO
OBz
NHAc
32
TMSOTf
MS3Å
CH2Cl2
r.t.
O
4
AcO
O
O
O
62%
N
CCl3
BzO
H
OAc
e
AcO
trichloroacetamide glycoside
CO2Me
O
AcHN
AcO
O
AcO
AcHN
OAc
R1O
O
O
c
O
d
BzO
O
R1O
OAc
g
BzO
BzO
AcO
AcO
OR1
CO2Me
O
O
R2
O
a
O
f
O
AcHN
b
O
R1O
O
BzO
AcO
R3
OAc
NHAc
OBz
AcO
33: R1 = Bn, R2 = OSE, R3 = H
34: R1 = H, R2 = OSE, R3 = H
35: R1 = Ac, R2 = OSE, R3 = H
36: R1 = Ac, R2, R3 = H, OH
a
b
c
d
89% (2 steps)
95% (2 steps)
37: R1 = Ac, R2, R3 = H, OC(NH)CCl3
Reagents and Conditions: (a) H2, Pd(OH)2/C, 1,4-dioxane, r.t.; (b) Ac2O, DMAP, Py, r.t.; (c) TFA,
CH2Cl2, 0 °C; (d) CCl3CN, DBU, CH2Cl2, 0 °C.
We previously addressed the development of the cassette coupling approach between a non-reducing
end oligosaccharide and a glucosylceramide (GlcCer) moiety for the synthesis of various glycolipids,
particularly gangliosides [5–10]. This approach resulted in a solution to the inevitable low yield of
sugar and ceramide fragments. Hitherto, we developed two types of GlcCer units: one is a cyclic type
GlcCer tethered by succinic ester between the sugar and lipid portions [5,7,8], while the other is an
acyclic type GlcCer [6,9,10]. In this study, we chose the cyclic type due to its ease of preparation. The
reported cyclic GlcCer acceptor 38 [7] was subjected to glycosylation with the oligosaccharide donor
37 in the presence of TMSOTf in CHCl3 at room temperature, affording the fully protected ganglioside
39 in a moderate yield of 49%. In this reaction, chloroform was employed as solvent instead of the
conventional dichloromethane because of the somewhat poor solubility of 38 in CH2Cl2. Following the
same protocol as that used for the other glycosylation reactions, the structure of 39 was elucidated.
Next, cleavage of the p-methoxybenzyl (PMB) group by TFA in CH2Cl2 at 0 °C provided 40 in 90%

Molecules 2012, 17
9598
yield. Finally, global deprotection under Zemplén conditions followed by saponification generated the
target ganglioside 1 in good yield (Scheme 6).
Scheme 6. Final coupling by the cassette approach followed by global deprotection.
OAc
AcO
CO2Me
O
AcHN
AcO
O
AcO
AcHN
O
OAc
BzO
O
AcO
O
AcO
O
OAc
BzO
AcO
AcO
CO2Me
O
OAc
BzO
O
O
O
O
AcHN
O
AcO
O
OBz
O
CCl3
NH
BzO
AcO
OAc
NHAc
AcO
37
O
O
O
O
TMSOTf
MS5Å
CHCl3
r.t.
O
HO
PMBO
O
C13H27
C17H35
OBz
HN
49%
38
(Unit D)
O
OAc
e
AcO
CO2Me
O
AcHN
AcO
O
AcO
AcHN
OAc
BzO
O
AcO
O
c
O
d
O
O
AcO
OAc
BzO
AcO
AcO
HN
O
C17H35
C13H27
CO2Me
OBz
OAc
O
BzO
O
PMBO
O
h
O
O
a
g
O
f
O
AcHN
b
O
O
O
O
AcO
O
OBz
OBz
AcO
O
OAc
NHAc
39
AcO
TFA
CH2Cl2
0 °C
90%
OAc
AcO
CO2Me
O
AcHN
AcO
O
AcO
AcHN
O
OAc
O
AcO
O
AcO
O
O
O
BzO
OAc
BzO
AcO
AcO
HN
O
C
17H35
C13H27
CO2Me
O
OBz
OAc
BzO
O
OBz
HO
O
O
O
O
O
AcHN
O
O
O
O
AcO
O
AcO
OAc
NHAc
40
OBz
AcO
NaOMe
MeOH
THF
then H2O
40 °C
61%
1

Molecules 2012, 17
9599
2.2. Binding Assay
The synthesized ganglioside 1 (termed GSC-734) was then assessed for its binding activity to
influenza viruses (Figure 3). The binding assay showed that GSC-734 was recognized by Neu5Ac2-3
and 2-6 type viruses. Moreover, the binding activity of the Neu5Ac2-6 type virus (H3N2) was almost
identical to that of the previously reported 2-6 sialylparagloboside [26]. This observation suggests
that the branched structure of the sugar part does not potently influence its binding activity to the viruses.
Figure 3. Binding activity of the synthetic ligand (GSC-734) for influenza A viruses.
3. Experimental
3.1. General Methods for Chemical Synthesis
All reactions were carried out under a positive pressure of argon, unless otherwise noted. All
chemicals were purchased from commercial suppliers and used without further purification, unless
otherwise noted. Molecular sieves were purchased from Wako Chemicals Inc. (Osaka, Japan) and
dried at 300 °C for 2 h in a muffle furnace prior to use. Solvents as reaction media were dried over
molecular sieves and used without purification. TLC analysis was performed on Merck TLC (silica gel
60F254 on glass plate, Darmstadt, Germany). Compound detection was either by exposure to UV light
(2536 Å) or by soak in a solution of 10% H2SO4 in ethanol followed by heating. Silica gel (80 mesh
and 300 mesh) manufactured by Fuji Silysia Co. (Kasugai, Japan) was used for flash column
chromatography. Quantity of silica gel was usually estimated as 100 to 150-fold weight of sample to
be charged. Solvent systems in chromatography were specified in v/v. Evaporation and concentration
1
were carried out in vacuo. H-NMR and 13C-NMR spectra were recorded with JEOL ECA
1
400/500/600 spectrometers. Chemical shifts in H-NMR spectra are expressed in ppm (δ) relative to
the signal of Me4Si, adjusted to δ 0.00 ppm. Data are presented as follow: Chemical shift, multiplicity
(s = singlet, d = doublet, t = triplet, dd = double of doublet, dt = double of triplet, m = multiplet and/or
multiple resonances), integration, coupling constant in Hertz (Hz), position of the corresponding
proton. COSY methods were used to confirm the NMR peak assignments. MALDI-TOF mass spectra
were run in a Bruker Autoflex (Billerica, MA, USA) and CHCA was used as the matrix. High-
resolution mass (ESI-TOF MS) spectra were run in a Bruker micrOTOF. Optical rotations were
measured with a ‘Horiba SEPA-300’ high-sensitive polarimeter (Kyoto, Japan).

Molecules 2012, 17
9600
OAc
AcO
CO2Me
b
O
TrocHN
AcO
O
HO
OAc
O
a
BnO
OMP
OBn
4-Methoxyphenyl (methyl 4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-(2,2,2-trichloroethoxycarbamoyl)-D-
glycero--D-galacto-2-nonulopyranosylonate)-(26)-2,3-di-O-benzyl--D-galactopyranoside (6). To
a mixture of 2 (691 mg, 0.964 mmol) and 5 (300 mg, 0.643 mmol) in EtCN/CH2Cl2 (5:1, 9.6 mL) was
added 3 Å molecular sieves (991 mg) at r.t. After stirring for 1 h and then cooling to −30 °C, NIS (324 mg,
1.44 mmol) and TfOH (12.7 μL, 0.144 mmol) were added to the mixture. After stirring for 45 min at
the same temperature as the reaction was monitored by TLC (1:3 EtOAc–toluene, twice development),
the reaction was quenched by the addition of triethylamine. The precipitate was filtered through Celite.
The filtrate was evaporated to remove EtCN and then diluted with CHCl3, washed with satd aq
Na2S2O3 and brine. The organic layer was subsequently dried over Na2SO4, concentrated and the
residue was purified by silica gel column chromatography (1:5 EtOAc–toluene) to give 6 (473 mg,
69%) along with its -isomer (96 mg, 14%). []D −13.9° (c 0.4, CHCl3); 1H-NMR (500 MHz, CDCl3)
 7.38–6.80 (m, 14 H, Ar), 5.39 (m, 1 H, H-8b), 5.36 (dd, 1 H, J6,7 = 1.7 Hz, H-7b), 5.00 (d, 1 H,
Jgem = 10.2 Hz, OCH2), 4.99 (m, 1 H, H-4b), 4.89 (d, 1 H, Jgem = 12.2 Hz, OCH2), 4.86 (d, 1 H,
J5,NH = 9.7 Hz, NH), 4.84 (d, 1 H, J1,2 = 8.0 Hz, H-1a), 4.83 (d, 1 H, Jgem = 10.2 Hz, OCH2), 4.76 (2 d,
2 H, Jgem = 12.0 Hz, OCH2), 4.47 (d, 1 H, Jgem = 12.2 Hz, OCH2), 4.32 (dd, 1 H, H-9b), 4.19 (dd, 1 H,
J6,7 = 1.7 Hz, H-6b), 4.10 (dd, 1 H, H-9'b), 4.07 (near d, 1 H, H-4a), 3.94–3.89 (m, 2 H, H-2a, H-5a),
3.81–3.75 (m, 7 H, H-6a, 2 OMe), 3.65–3.61 (m, 2 H, H-6'a, H-5b), 3.57 (dd, 1 H, H-3a), 2.67–2.63
(m, 2 H, H-3beq, OH), 2.12–1.99 (m, 12 H, 4 Ac), 1.91 (t, 1 H, H-3bax); 13C-NMR (100 MHz, CDCl3)
 193.2, 191.4, 170.7, 170.3, 170.1, 169.9, 167.9, 155.2, 154.0, 151.7, 138.4, 137.9, 129.0, 128.4,
128.3, 128.2, 128.1, 127.8, 127.7, 127.6, 125.3, 118.6, 114.4, 102.9, 98.7, 95.3, 80.6, 78.7, 77.2, 75.3,
74.5, 72.6, 72.3, 72.2, 68.6, 68.4, 67.5, 66.0, 63.1, 62.3, 55.6, 53.0, 51.6, 37.5, 29.7, 29.5, 21.4, 21.0,
20.8, 20.7. m/z (MALDI): found [M+Na]+ 1094.32, C48H56Cl3NO20 calcd for [M+Na]+ 1094.32.
OAc
AcO
COOMe
b
O
AcO
O
AcHN
AcO
AcO
O
a
BnO
OMP
OBn
4-Methoxyphenyl (methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero--D-galacto-2-
nonulopyranosylonate)-(26)-4-O-acetyl-2,3-di-O-benzyl--D-galactopyranoside (8). To a solution of
6 (3.77 g, 3.51 mol) in AcOH/CH2Cl2 (3:2, 70 mL) was added Zn/Cu couple (18.9 g) at r.t. The
reaction mixture was heated to 40 °C and was stirred for 45 min at the same temperature as the
reaction was monitored by TLC (4:1 toluene–EtOAc). The precipitate was filtered through Celite and
the filtrate was co-evaporated with toluene. The obtained residue was exposed to high vacuum for 6 h.
The crude residue was dissolved in pyridine (35 mL) and acetic anhydride (1.32 L, 14.0 mmol),
DMAP (4.3 mg, 35.1 mol) were then added to the mixture at 0 °C. After stirring for 3 d at r.t as the
reaction was monitored by TLC (2:1 toluene–EtOAc), the reaction mixture was evaporated. The

Molecules 2012, 17
9601
residue was diluted with CHCl3, washed with 2 M HCl, H2O, satd aq NaHCO3 and brine, dried over
Na2SO4, and concentrated. The obtained residue was purified by silica gel column chromatography
(4:1 toluene–EtOAc) to give 8 (3.35 g, 97%). []D −11.3° (c 1.0, CHCl3); 1H-NMR (500 MHz, CDCl3)
δ 7.37–7.24 (m, 10 H, 2 Ph), 6.97 (2 d, 4 H, Ar), 5.95 (d, 1 H, JNH,5 = 6.3 Hz, NH), 5.62 (d, 1 H,
J3,4 = 2.3 Hz, H-4a), 5.41 (m, 1 H, H-8b), 5.32 (d, 1 H, H-7b), 4.96–4.81 (m, 5 H, H-1a, H-4b,
3OCH2), 4.68 (d, 1 H, OCH2), 4.34 (d, 1 H, H-9b), 4.14–4.05 (m, 3 H, H-5b, H-6b, H-9'b), 3.90–3.69
(m, 11 H, H-2a, H-3a, H-5a, H-6a, H-6'a, 2 OMe), 2.62 (m, 1 H, Jgem = 11.5 Hz, H-3beq), 2.16–1.87
13
(m, 19 H, 6 Ac, H-3bax); C-NMR (100 MHz, CDCl3)  170.4, 170.2, 170.0, 169.8, 169.7, 169.5,
167.7, 154.9, 151.2, 138.2, 137.5, 128.7, 128.0, 127.9, 127.7, 127.3, 127.2, 118.0, 114.1, 102.2, 98.4,
79.1, 78.3, 75.0, 72.4, 71.8, 71.3, 68.7, 68.3, 67.0, 65.9, 62.7, 62.3, 55.2, 52.5, 48.6, 37.5, 22.7, 20.7,
20.5, 20.4, 20.4, 20.3. m/z (MALDI): found [M+Na]+ 1004.17, C49H59NO20 calcd for [M+Na]+ 1004.35.
OAc
AcO
COOMe
b
O
AcO
O
AcHN
AcO
AcO
O
a
BzO
OMP
OBz
4-Methoxyphenyl (methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero--D-galacto-2-
nonulopyranosylonate)-(26)-4-O-acetyl-2,3-di-O-benzoyl--D-galactopyranoside (10). To a solution
of 8 (3.34 g, 3.41 mol) in 1,4-dioxane (34 mL) was added Pd(OH)2/C (3.34 g). After stirring for
45 min at r.t. under a hydrogen atmosphere as the reaction was monitored by TLC (15:1 CHCl3–MeOH),
the mixture was filtered through Celite. The filtrate was concentrated and the obtained crude residue
was roughly purified by silica gel column chromatography. The obtained product was exposed to high
vacuum for 24 h. The residue was then dissolved in pyridine (34 mL). Benzoic anhydride (3.09 g,
13.6 mmol) and DMAP (20.8 mg, 0.171 mol) were added to the mixture at 0 °C. After stirring for 9 h
at r.t. as the reaction was monitored by TLC (15:1 CHCl3–MeOH), the reaction was quenched by the
addition of MeOH at 0 °C. The mixture was co-evaporated with toluene and the residue was then
diluted with CHCl3, and washed with 2 M HCl, H2O, satd aq NaHCO3 and brine. The organic layer
was subsequently dried over Na2SO4, and concentrated. The resulting residue was purified by silica gel
column chromatography (1:1 toluene–EtOAc) to give 10 (3.22 g, 94%). []D +40.3° (c 1.0, CHCl3);
1H-NMR (500 MHz, CDCl3)  8.00–7.37 (m, 10 H, 2 Ph), 7.06–6.76 (2 d, Ar), 5.91 (near t, 1 H,
J1,2 = 7.8 Hz, J2,3 = 10.6 Hz, H-2a), 5.78 (d, 1 H, J3,4 = 3.4 Hz, H-4a), 5.60 (dd, 1 H, H-3a), 5.50 (m,
1 H, J7,8 = 6.8 Hz, H-8b), 5.33–5.27 (m, 2 H, H-1a, H-7b), 5.15 (d, 1 H, NH), 4.87 (m, 1 H, J3eq,4 = 4.6 Hz,
H-4b), 4.41 (dd, 1 H, Jgem = 12.4 Hz, H-9), 4.30 (near t, 1 H, J5,6 = 6.9 Hz, H-5a), 4.17–4.02 (m, 3 H,
H-5b, H-6b, H-9'b), 3.89–3.73 (m, 7 H, 2 OMe, H-6a), 3.58 (near t, 1 H, Jgem = 10.1 Hz, H-6'a), 2.55
(dd, 1 H, Jgem = 12.4 Hz, H-3beq), 2.25–1.89 (m, 19 H, 6 Ac, H-3bax); 13C-NMR (100 MHz, CDCl3) 
170.5, 170.4, 170.0, 169.7, 169.5, 167.7, 165.1, 165.0, 155.1, 151.0, 133.0, 129.4, 129.3, 129.1, 128.8,
128.1, 128.1, 118.2, 114.1, 100.1, 98.9, 72.5, 71.7, 71.6, 69.4, 68.6, 67.9, 67.0, 62.9, 55.2, 52.7, 48.7,
37.6, 22.8, 20.8, 20.5, 20.4, 20.3. m/z (MALDI): found [M+Na]+ 1032.21, C49H55NO22 calcd for
[M+Na]+ 1032.31.

Molecules 2012, 17
9602
OAc
AcO
COOMe
b
O
AcO
O
AcHN
AcO
AcO
O
a
BzO
BzO
OC(NH)CCl3
(Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero--D-galacto-2-nonulopyranosylonate)-
(26)-4-O-acetyl-2,3-di-O-benzoyl-D-galactopyranosyl trichloroacetimidate (12). To a solution of 10
(3.22 g, 3.19 mmol) in MeCN/PhMe/H2O (32 mL, 6:5:3) was added diammonium cerium (IV) nitrate
(CAN; 17.5 g, 31.9 mmol) at r.t. The mixture was stirred for 2 h at r.t., as the proceeding of the
reaction was monitored by TLC (10:1 CHCl3–MeOH). The reaction mixture was diluted with CHCl3
and washed with H2O, satd aq NaHCO3 and brine. The organic layer was subsequently dried over
Na2SO4 and concentrated. The resulting residue was purified by silica gel column chromatography
(70:1 CHCl3–MeOH) to give 11. The obtained hemiacetal compound 11 (2.27 g, 2.51 mmol) was
dissolved in CH2Cl2 (50 mL). To the mixture was added CCl3CN (2.5 mL, 25.1 mmol), DBU (449 L,
3.01 mmol) at 0 °C. After stirring for 6 h at 0 °C as the reaction was monitored by TLC (15:1
CHCl3–MeOH), the reaction mixture was evaporated. The crude residue was purified by silica gel
1
column chromatography (70:1 CHCl3–MeOH) to give 12 (2.10 g, 63% over 2 steps). 12: H-NMR
(500 MHz, CDCl3)  8.61 (s, 1 H, C=NH), 7.95–7.28 (m, 10 H, 2 Ph), 6.80 (d, 1 H, J1,2 = 3.4 Hz,
H-1a), 5.90–5.82 (m, 2 H, H-2a, H-3a), 5.79 (m, 1 H, J3,4 = 2.8 Hz, H-4a), 5.38–5.30 (m, 3 H, H-7b,
H-8b, NHb), 4.87–4.86 (m, 1 H, H-4b), 4.51 (br t, 1 H, H-5a), 4.28 (dd, 1 H, Jgem = 12.6 Hz, J8,9 = 2.9 Hz,
H-9b), 4.11 (dd, 1 H, J8,9´ = 5.9 Hz, H-9'b), 4.06–3.97 (m, 3 H, H-6a, H-5b, H-6b), 3.78 (s, 3 H,
COOMe), 3.42 (dd, 1 H, J5,6' = 2.6 Hz, Jgem = 10.6 Hz, H-6'a), 3.55 (dd, 1 H, Jgem = 11.6 Hz, J3eq,4 = 3.6 Hz,
H-3beq), 2.35–1.87 (m, 19 H, 6 Ac, H-3bax).
Ph
O
O
O
SPh
NHTroc
HO
Phenyl 4,6-O-benzylidene-2-deoxy-1-thio-2-(2,2,2-trichloroethoxycarbamoyl)--D-glucopyranoside (14).
To a solution of 13 (8.43 g, 14.7 mmol) in MeOH (84 mL) was added NaOMe (28% solution in
MeOH, 39.7 mg, 0.735 mmol) at 0 °C. After stirring for 2.5 h at room temperature as the reaction was
monitored by TLC (10:1 CHCl3–MeOH), the reaction was neutralized with Dowex (H+) resin. The
resin was filtered through cotton and the filtrate was then evaporated. The residue was exposed to high
vacuum for 12 h. The obtained crude mixture was then dissolved in THF/MeCN (1:4, 135 mL). To the
mixture were added benzaldehyde dimethyl acetal (4.39 mL, 29.4 mmol) and (±)-camphor-10-sulfonic
acid (CSA) (512 mg, 2.21 mmol) at 0 °C. After stirring for 1 h at room temperature as the reaction was
monitored by TLC (20:1 CHCl3–MeOH), the reaction was quenched by the addition of triethylamine.
The reaction mixture was concentrated and then subjected to crystallization from hot acetone/n-hexane
to give 14 (6.27 g, 80%) as a white crystal. The physical data of 14 was identical to those reported in
the literature [27].

Molecules 2012, 17
9603
OBn
O
HO
SPh
BnO
NHTroc
Phenyl 3,6-di-O-benzyl-2-deoxy-1-thio-2-(2,2,2-trichloroethoxycarbamoyl)--D-glucopyranoside (15).
To a solution of 14 (4.89 g, 9.15 mmol) in THF/toluene (1:4, 9.0 mL) was added TESOTf (4.1 mL,
18.3 mmol) at −20 °C. After stirring for 45 min at −20 °C, benzaldehyde (4.7 mL, 45.7 mmol) and
triethylsilane (2.2 mL, 13.7 mmol) were added to the mixture. After stirring for 2 h at −20 °C as the
reaction was monitored by TLC (1:4 EtOAc–toluene), the reaction was quenched by satd aq Na2CO3.
Dilution of the mixture with EtOAc provided a solution, which was then washed with satd aq Na2CO3
and brine. The organic layer was subsequently dried over Na2SO4 and concentrated. The obtained
residue was exposed to high vacuum for 24 h. The resulting residue was dissolved in CH2Cl2 (183 mL)
and cooled to 0 °C. BF3·OEt2 (4.7 mL, 18.3 mmol) and triethylsilane (14.6 mL, 91.5 mmol) were
added to the solution at 0 °C and the mixture was then stirred for 1 h at 0 °C as the reaction was
monitored by TLC (1:4 EtOAc–toluene). The reaction was quenched by the addition of satd aq
Na2CO3 at 0 °C and then diluted with CHCl3, and washed with satd aq Na2CO3 and brine. The organic
layer was subsequently dried over Na2SO4 and concentrated. The resulting residue was purified by
silica gel column chromatography (200:1 CHCl3–MeOH) to give 15 (4.50 g, 78%). []D −24.2° (c 0.3,
CHCl3); 1H-NMR (400 MHz, CDCl3)  7.49–7.21 (m, 15 H, 3 Ph), 5.17 (d, 1 H, J2,NH = 8.2 Hz, NH),
4.91 (d, 1 H, , J1,2 = 10.1 Hz, H-1), 4.75 (s, 4 H, 2 OCH2), 4.56 (2 d, 2 H, OCH2), 3.77–3.67 (m, 4 H,
H-3, H-5, H-6, H-6'), 3.52–3.42 (m, 2 H, H-2, H-4), 2.85 (s, 1 H, OH); 13C-NMR (100 MHz, CDCl3) 
153.8, 137.9, 137.7, 132.7, 132.3, 128.9, 128.5, 128.4, 128.1, 128.0, 127.8, 127.7, 95.4, 86.0, 81.9,
78.0, 74.5, 74.4, 73.7, 72.6, 70.4, 56.0. m/z (MALDI): found [M+Na]+ 648.05, C29H30Cl3NO6S calcd
for [M+Na]+ 648.08.
OBn
O
LevO
BnO
SPh
NHTroc
Phenyl 3,6-di-O-benzyl -2-deoxy-4-O-levulinoyl-1-thio-2-(2,2,2-trichloroethoxycarbamoyl)--D-
glucopyranoside (16). To a solution of 15 (200 mg, 0.314 mmol) in CH2Cl2 (3.1 mL) were added
levulinic acid (55 mg, 0.472 mmol), EDC·HCl (90 mg, 0.472 mmol), and DMAP (4.2 mg, 3.44 mol).
After stirring for 4 h at r.t. as the reaction was monitored by TLC (2:3 EtOAc–n-hexane), the reaction
mixture was diluted with CHCl3, and washed with 2 M HCl and brine. The organic layer was
subsequently dried over Na2SO4, and concentrated, and the obtained residue was purified by silica gel
column chromatography (1:4 EtOAc–n-hexane) to give 16 (215 mg, 93%). []D +3.7° (c 0.9, CHCl3);
1H-NMR (600 MHz, acetone-d6)  7.53–7.24 m, 16 H, 3 Ph, NH 5.06 (d, 1 H, J1,2 = 10.3 Hz, H-1),
5.03 (t, 1 H, J3,4 = 9.6 Hz, J4,5 = 9.7 Hz, H-4), 4.83 (2 d, 2 H, Jgem = 12.4 Hz, OCH2), 4.72 (2 d, 2 H,
Jgem = 11.0 Hz, OCH2), 4.52 (2 d, 2 H, Jgem = 11.7 Hz, OCH2), 3.99 (t, 1 H, J2,3 = 9.7 Hz, H-3), 3.79–3.75
(m, 2 H, H-2, H-5), 3.65 (dd, 1 H, Jgem = 11.0 Hz, J5,6 = 2.8 Hz, H-6), 3.65 (dd, 1 H, J5,6' = 6.9 Hz, H-6'),
2.73–2.40 (m, 4 H, CH2CH2C(=O)O), 2.09 (s, 3 H, C(=O)CH3); 13C-NMR (150 MHz, CDCl3)  166.1,
153.7, 137.6, 137.5, 132.6, 132.1, 129.0, 128.5, 128.3, 128.0, 128.0, 127.9, 127.7, 95.4, 85.2, 79.0,
76.8, 74.5, 74.4, 73.6, 73.2, 69.5, 56.7, 40.5. m/z (MALDI): found [M+Na]+ 746.09, C34H36Cl3NO8S
calcd for [M+Na]+ 746.11.

Molecules 2012, 17
9604
OBn
O
ClAcO
BnO
SPh
NHTroc
Phenyl 3,6-di-O-benzyl-4-O-chloroacetyl-2-deoxy-1-thio-2-(2,2,2-trichloroethoxycarbamoyl)--
D-glucopyranoside (17). To a solution of 15 (200 mg, 0.314 mmol) in THF (3.1 mL) were added
monochloroacetic anhydride (81 mg, 0.472 mmol) and DMAP (0.5 mg, 4.09 mol) at 0 °C. After
stirring for 7 h at r.t. as the reaction was monitored by TLC (2:3 EtOAc–n-hexane), THF was
evaporated and the residue was then diluted with CHCl3, and washed with 2 M HCl, H2O, satd aq
NaHCO3, and brine. The organic layer was subsequently dried over Na2SO4, and concentrated. The
residue was purified by silica gel column chromatography (1:8 EtOAc–n-hexane) to give 17 (200 mg,
90%). []D −2.6° (c 1.0, CHCl3); 1H-NMR (600 MHz, CDCl3)  7.51–7.21 (m, 15 H, 3 Ph), 5.37 (d, 1 H,
J
NH,2 = 8.2 Hz, NH), 5.12 (d, 1 H, J1,2 = 10.3 Hz, H-1), 5.07 (near t, 1 H, J3,4 = 8.9 Hz, J4,5 = 9.6 Hz,
H-4), 4.75 (2 d, 2 H, Jgem = 11.8 Hz, OCH2), 4.61 (2 d, 2 H, Jgem =11.7 Hz, OCH2), 4.48 (m, 2 H,
Jgem = 11.7 Hz, OCH2), 4.11 (near t, 1 H, J2,3 = 9.6 Hz, J3,4 = 8.9 Hz, H-3), 3.68–3.55 (m, 5 H, H-5, H-6,
H-6', CH2Cl), 3.36 (br dd, 1 H, J2,3 = 9.6 Hz, H-2); 13C-NMR (125 MHz, CDCl3)  166.1, 153.7,
137.7, 137.6, 132.8, 132.0, 129.1, 128.5, 128.4, 128.2, 128.0, 127.9, 127.8, 95.4, 85.2, 79.0, 77.6, 74.7,
74.5, 73.7, 73.4, 69.7, 56.9, 40.5. m/z (MALDI): found [M+Na]+ 724.05, C31H31Cl4NO7S calcd for
[M+Na]+ 724.05.
Ph
O
O
O
TrocO
OSE
OH
2-(Trimethylsilyl)ethyl 4,6-O-benzylidene-3-O-(2,2,2-trichloroethoxycarbonyl)--D-galactopyranoside
(19). To a solution of 18 (49.5 mg, 0.134 mmol) in pyridine/CH2Cl2 (1:4, 1.3 mL) was added
trichloroethyl chloroformate (20 L, 0.148 mmol) at −40 °C. After stirring for 3 h at the same
temperature as the reaction was monitored by TLC (30:1 CHCl3–MeOH), solvents were removed by
co-evaporation with toluene, and then the residue was diluted with CHCl3, washed with 2 M HCl, H2O,
satd aq NaHCO3, and brine. The organic layer was subsequently dried over Na2SO4, and concentrated.
The resulting residue was purified by silica gel column chromatography (1:5 EtOAc–n-hexane) to give
19 (59.1 mg, 81%). []D +41.7° (c 1.0, CHCl3); 1H-NMR (400 MHz, CDCl3) δ 7.48–7.30 (m, 5 H, Ph),
5.49 (s, 1 H, PhCH<), 4.82–4.72 (m, 3 H, H-3, OCH2CCl3), 4.45 (d, 1 H, J3,4 = 3.7 Hz, H-4), 4.34
(d, 1 H, J1,2 = 7.7 Hz, H-1), 4.33 (d, 1 H, Jgem = 13.8 Hz, H-6), 4.08–4.01 (m, 3 H, H-2, H-6',
OCH2CH2SiMe3), 3.60–3.53 (m, 1 H, OCH2CH2SiMe3), 3.48 (s, 1 H, H-5), 2.51 (br s, 1 H, OH),
13
1.03–0.97 (m, 2 H, OCH2CH2SiMe3), 0.00 (s, 9 H, OCH2CH2SiMe3); C-NMR (100 MHz, CDCl3) 
153.6, 137.3, 129.0, 128.1, 126.2, 102.3, 100.9, 94.2, 78.2, 77.2, 76.9, 73.0, 69.0, 68.5, 67.5, 66.2,
18.1, −1.4. m/z (MALDI): found [M+Na]+ 565.11, C16H16Cl3O7 calcd for [M+Na]+ 565.06.

Molecules 2012, 17
9605
Ph
O
O
O
TrocO
OSE
OBz
2-(Trimethylsilyl)ethyl 2-O-benzoyl-4,6-O-benzylidene-3-O-(2,2,2-trichloroethoxycarbonyl)--D-
galactopyranoside (20). To a solution of 19 (2.39 g, 4.39 mmol) in THF (1.3 mL) were added benzoic
anhydride (1.49 g, 6.59 mmol) and DMAP (268 mg, 2.20 mol) at 0 °C. After stirring for 8.5 h at r.t.
as the reaction was monitored by TLC (30:1 CHCl3–MeOH), THF was evaporated and the residue was
then diluted with CHCl3, washed with 2 M HCl, H2O, satd aq NaHCO3, and brine. The organic layer
was subsequently dried over Na2SO4, concentrated. The residue was purified by silica gel column
chromatography (20:1 toluene–EtOAc) to give 20 (2.90 g, quant.). []D +38.8° (c 1.2, CHCl3); 1H-NMR
(400 MHz, CDCl3) δ 8.11–7.43 (m, 10 H, 2 Ph), 5.79–7.75 (near t, 1 H, J2,3 = 10.6 Hz, J1,2 = 7.8 Hz,
H-2), 5.64 (s, 1 H, PhCH<), 5.22 (dd, 1 H, J3,4 = 3.7 Hz, H-3), 4.81–4.78 (2 d, 2 H, OCH2CCl3, H-1),
4.68 (d, 1 H, Jgem = 11.9 Hz, OCH2CCl3), 4.58 (d, 1 H, H-4), 4.67–4.50 (dd, 1 H, Jgem = 12.4 Hz,
J5,6 = 1.8 Hz, H-6), 4.23–4.19 (dd, 1 H, J5,6´ = 1.8 Hz, H-6'), 4.15–4.08 (m, 1 H, OCH2CH2SiMe3),
3.68–3.62 (m, 2 H, H-5, OCH2CH2SiMe3), 1.03–0.91 (m, 2 H, OCH2CH2SiMe3), 0.00 (s, 9 H,
OCH2CH2SiMe3); 13C-NMR (100 MHz, CDCl3) δ 164.8, 153.7, 137.2, 133.1, 129.8, 129.7, 129.1,
128.3, 128.2, 126.4, 101.1, 100.5, 94.1, 76.8, 76.6, 73.4, 69.2, 68.9, 67.0, 66.2, 17.9, −1.5. m/z
(MALDI): found [M+Na]+ 669.09, C28H33Cl3O9Si calcd for [M+Na]+ 669.09.
Ph
O
O
O
HO
OSE
OBz
2-(Trimethylsilyl)ethyl 2-O-benzoyl-4,6-O-benzylidene--D-galactopyranoside (21). To a solution of
20 (2.18 g, 3.36 mol) in AcOH/CH2Cl2 (3:2, 33.6 mL) was added Zn/Cu couple (6.00 g) at r.t. The
reaction mixture was stirred for 1 h at r.t. as the reaction was monitored by TLC (30:1 CHCl3–MeOH).
The precipitate was filtered through Celite and the filtrate was co-evaporated with toluene. The residue
was diluted with CHCl3 and washed with satd aq NaHCO3 and brine, dried over Na2SO4, and concentrated.
The obtained residue was purified by silica gel column chromatography (4:1 EtOAc–n-hexane) to give
21 (1.33 g, 84%). []D −3.8° (c 1.1, CHCl3); 1H-NMR (400 MHz, CDCl3)  8.15–7.45 (m, 10 H, 2 Ph),
5.65 (s, 1 H, PhCH<), 5.43 (near t, 1 H, J2,3 = 10.3 Hz, J1,2 = 8.3 Hz, H-2), 4.68 (d, 1 H, J1,2 = 8.3 Hz,
H-1), 4.45 (d, 1 H, Jgem = 12.3 Hz, H-6), 4.33 (d, 1 H, J3,4 = 4.2 Hz, H-4), 4.18 (dd, 1 H, Jgem = 12.4 Hz,
H-6'), 4.12 (m, 1 H, OCH2CH2SiMe3), 3.97 (m, 1 H, J3,OH = 11.0 Hz, J2,3 = 10.3 Hz, H-3), 3.68–3.60
(m, 2 H, H-5, OCH2CH2SiMe3), 2.72 (d, 1 H, OH), 0.98 (m, 2 H, CH2CH2SiMe3), 0.00 (s, 3 H,
CH2CH2SiMe3); 13C-NMR (100 MHz, CDCl3)  166.2, 137.4, 132.9, 130.0, 129.8, 129.2, 128.2, 126.4,
101.4, 100.3, 75.6, 72.9, 71.9, 69.0, 67.0, 66.5, 17.9, −1.5. m/z (MALDI): found [M+Na]+ 495.12,
C25H32O7Si calcd for [M+Na]+ 495.18.

Molecules 2012, 17
9606
Ph
O
O
OBn
O
O
LevO
BnO
a
b
O
OSE
NHTroc
OBz
2-(Trimethylsilyl)ethyl 3,6-di-O-benzyl-2-deoxy-4-O-levulinoyl-2-(2,2,2-trichloroethoxycarbamoyl)--
D-glucopyranosyl-(13)-2-O-benzoyl-4,6-O-benzylidene--D-galactopyranoside (22). To a mixture of
16 (230 mg, 0.317 mmol) and 21 (100 mg, 0.212 mmol) in CH2Cl2 (3.2 mL) was added 4 Å molecular
sieves AW-300 (400 mg) at r.t. After stirring for 1 h, the mixture was cooled to 0 °C. NIS (143 mg,
0.636 mmol) and TfOH (2.8 L, 31.7 mol) were then added to the mixture at 0 °C. After stirring for
2 h at the same temperature as the reaction was monitored by TLC (1:4 EtOAc–toluene), the reaction
was quenched by the addition of triethylamine. The solution was diluted with CHCl3 and filtered
through Celite. The filtrate was then washed with satd aq Na2S2O3 and brine. The organic layer was
subsequently dried over Na2SO4, and concentrated. The resulting residue was purified by silica gel
column chromatography (1:4 EtOAc–toluene, then 200:1 CHCl3–MeOH) to give 22 (186 mg, 81%).
1
[]D +17.4° (c 0.8, CHCl3); H-NMR (600 MHz, CDCl3)  8.07–7.13 (m, 20 H, 4 Ph), 5.55 (near t,
1 H, J2,3 = 10.3 Hz, J1,2 = 7.6 Hz, H-2a), 5.49 (s, 1 H, PhCH<), 5.26 (d, 1 H, JNH,2 = 6.2 Hz, NH), 5.08
(d, 1 H, J1,2 = 7.6 Hz, H-1b), 4.92 (near t, 1 H, J3,4 = 9.9 Hz, J4,5 = 9.4 Hz, H-4b), 4.53 (d, 1 H, H-1a),
4.55–4.51 (m, 2 H, 2 OCH2), 4.46–4.41 (m, 3 H, 3 OCH2), 4.40 (d, 1 H, J3,4 = 3.4 Hz, H-4a), 4.26 (d, 1 H,
Jgem = 12.4 Hz, H-6'a), 4.15 (t, 1 H, J3,4 = 9.9 Hz, H-3b), 4.00–3.95 (m, 2 H, H-3a, OCH2CH2SiMe3),
3.83 (d, 1 H, Jgem = 12.4 Hz, H-6a), 3.66 (br m, 1 H, H-5b), 3.55 (m, 3 H, H-6b, H-6'b, OCH2CH2SiMe3),
3.35 (s, 1 H, H-5a), 3.28 (br d, 1 H, OCH2), 3.17 (br dd, 1 H, H-2b), 2.61–2.36 (m, 4 H, CH2CH2C(=O)O),
2.10 (s, 3 H, C(=O)CH3), 0.86–0.76 (m, 2 H, CH2CH2SiMe3), −0.12 (m, 9 H, CH2CH2SiMe3);
13C-NMR (125 MHz, CDCl3)  206.2, 171.6, 164.9, 153.4, 138.1, 138.0, 137.8, 133.0, 130.2, 129.9,
128.9, 128.4, 128.3, 128.3, 128.1, 127.7, 127.6, 127.6, 127.5, 126.5, 101.1, 100.8, 100.2, 95.4, 78.7,
77.5, 76.2, 74.2, 73.4, 73.3, 73.2, 71.6, 70.5, 69.9, 68.8, 66.7, 66.6, 58.2, 37.7, 29.7, 27.8, 17.9, −1.6.
m/z (MALDI): found [M+Na]+ 1108.51, C53H62Cl3NO15Si calcd for [M+Na]+ 1108.29.
OH
OBn
O
OH
O
LevO
BnO
a
b
O
OSE
NHTroc
OBz
2-(Trimethylsilyl)ethyl 3,6-di-O-benzyl-2-deoxy-4-O-levulinoyl-2-(2,2,2-trichloroethoxycarbamoyl)--
D-glucopyranosyl-(13)-2-O-benzoyl--D-galactopyranoside (23). Compound 22 (42 mg, 38.3 mol)
was dissolved in 80% AcOH aq (1.5 mL) and the solution was stirred for 4 h at 50 °C as the reaction
was monitored by TLC (15:1 CHCl3–MeOH). After the reaction mixture was co-evaporated with
toluene, the crude residue was purified by silica gel column chromatography (50:1 CHCl3–MeOH) to
1
give 23 (38 mg, 99%). []D −1.43° (c 0.7, CHCl3); H-NMR (400 MHz, acetone-d6)  8.34–7.32 (m,
15 H, 3 Ph), 7.03 (d, 1 H, JNH,2 = 9.2 Hz, NH), 5.60 (near t, 1 H, J2,3 = 9.7 Hz, J1,2 = 8.0 Hz, H-2a),
5.13 (d, 1 H, J1,2 = 8.6 Hz, H-1b), 5.09 (near t, J3,4 = 9.9 Hz, J4,5 = 8.3 Hz, H-4b), 4.77–4.64 (m, 5 H,
H-1a, 4 OCH2), 4.48 (d, 1 H, J3,4 = 3.4 Hz, H-4a), 4.51 (d, 1 H, OCH2), 4.23 (dd, 1 H, H-3a), 4.13–4.08
(m, 2 H, H-3b, OCH2), 3.96–3.63 (m, 9 H, 2 OCH2, H-2b, H-5b, H-6b, H-6'b, H-5a, H-6a, H-6'a), 2.98
(s, 2 H, 2 OH), 2.80–2.49 (m, 4 H, CH2CH2C(=O)O), 2.20 (s, 3 H, C(=O)CH3), 1.01–0.83 (m, 2 H,
CH2CH2SiMe3), 0.00 (s, 9 H, CH2CH2SiMe3); 13C-NMR (100 MHz, acetone-d6)  206.5, 172.5, 165.6,

Molecules 2012, 17
9607
154.8, 139.5, 133.7, 131.7, 130.7, 129.2, 129.0, 128.8, 128.4, 128.3, 128.2, 128.0, 102.7, 101.7, 96.8,
82.4, 80.2, 75.9, 74.6, 74.1, 74.0, 73.9, 72.1, 72.0, 70.4, 69.5, 67.0, 62.6, 58.3, 38.1, 29.6, 28.6, 18.6,
−1.4. m/z (MALDI): found [M+Na]+ 1020.26, C46H58Cl3NO15Si calcd for [M+Na]+ 1020.25.
OBn
O
ClAcO
BnO
b
O
TrocHN
HO
OBn
O
O
LevO
BnO
a
c
O
OSE
NHTroc
OBz
2-(Trimethylsilyl)ethyl 3,6-di-O-benzyl-2-deoxy-4-O-levulinoyl-2-(2,2,2-trichloroethoxycarbamoyl)--
D-glucopyranosyl-(13)-[3,6-di-O-benzyl-4-O-chloroacetyl-2-deoxy-2-(2,2,2-trichloroethoxycarbamoyl)-
-D-glucopyranosyl-(16)]-2-O-benzoyl--D-galactopyranoside (24). To a mixture of 17 (694 mg,
0.987 mmol) and 23 (658 mg, 0.658 mmol) in CH2Cl2 (6.6 mL) was added 4 Å molecular sieves
(1.40 g) at r.t. After stirring for 1 h, the mixture was cooled to 0 °C. NIS (296 mg, 1.32 mmol) and
TfOH (8.7 L, 98.7 mol) were then added to the mixture at 0 °C. After stirring for 2 h at r.t. as the
reaction was monitored by TLC (30:1 CHCl3–MeOH), the reaction was quenched by the addition of
triethylamine. The solution was diluted with CHCl3 and filtered through Celite. The filtrate was then
washed with satd aq Na2S2O3 and brine. The organic layer was subsequently dried over Na2SO4, and
concentrated. The residue was purified by silica gel column chromatography (100:1 CHCl3–MeOH) to
give 24 (511 mg, 49%). []D −19.4° (c 1.8, CHCl3); 1H-NMR (400 MHz, DMSO-d6)  8.20–7.32 (m,
27 H, 5 Ph, NHb, NHc), 5.38 (t, 1 H, J2,3 = 8.3 Hz, J1,2 = 8.7 Hz, H-2a), 5.14 (t, 1 H, J4,5 = 9.6 Hz,
H-4c), 5.06 (d, 1 H, Jgem = 12.4 Hz, OCH2), 5.01 (t, 1 H, J3,4 = 9.6 Hz, J4,5 = 9.1 Hz, H-4b), 4.90 (d,
1 H, J1,2 = 8.2 Hz, H-1c), 4.75 (d, 1 H, J1,2 = 8.3 Hz, H-1b), 4.66 (d, 1 H, H-1a), 4.91–4.61 (m, 10 H,
OH, 9 OCH2), 4.49 (2 d, 2 H, OCH2), 4.24 (br s, 1 H, H-4a), 4.04–3.52 (m, 18 H, H-3a, H-5a, H-6a,
H-6'a, H-2b, H-3b, H-5b, H-6b, H-6'b, H-2c, H-3c, H-5c, H-6c, H-6'c, OCH2CH2SiMe3, CH2Cl),
2.83–2.80 (m, 2 H, CH2CH2C(=O)O), 2.56 (m, 2 H, CH2CH2C(=O)O), 2.26 (s, 3 H, C(=O)CH3),
13
0.93–0.78 (m, 2 H, CH2CH2SiMe3), 0.00 (s, 9 H, CH2CH2SiMe3); C-NMR (100 MHz, DMSO-d6) 
206.8, 171.5, 166.5, 164.7, 154.4, 153.8, 138.4, 138.3, 138.2, 133.2, 130.3, 129.6, 128.6, 128.4, 128.3,
128.2, 127.7, 127.7, 127.6, 101.9, 101.1, 100.2, 96.4, 96.0, 80.7, 79.4, 78.9, 73.7, 73.6, 73.3, 72.8,
72.7, 72.6, 72.5, 72.2, 72.0, 70.9, 70.7, 69.6, 69.0, 68.7, 68.6, 66.1, 57.0, 41.1, 37.4, 29.7, 27.8, 17.6,
−1.3. m/z (MALDI): found [M+Na]+ 1611.27, C71H83Cl7N2O22Si calcd for [M+Na]+ 1611.29.
OBn
O
ClAcO
BnO
b
O
TrocHN
AcO
OBn
O
O
a
LevO
BnO
c
O
OSE
NHTroc
OBz
2-(Trimethylsilyl)ethyl 3,6-di-O-benzyl-2-deoxy-4-O-levulinoyl-2-(2,2,2-trichloroethoxycarbamoyl)--
D-glucopyranosyl-(13)-[3,6-di-O-benzyl-4-O-chloroacetyl-2-deoxy-2-(2,2,2-trichloroethoxycarbamoyl)-
-D-glucopyranosyl-(16)]-4-O-acetyl-2-O-benzoyl--D-galactopyranoside (27). To a solution of 24
(759 mg, 0.476 mmol) in THF (4.8 mL) were added acetic anhydride (94 L, 0.956 mmol) and DMAP
(5.8 mg, 47.6 mol) at 0 °C. The reaction mixture was stirred for 1.5 h at r.t. as the reaction was

Molecules 2012, 17
9608
monitored by TLC (15:1 CHCl3–MeOH). After THF was evaporated, the obtained crude residue was
purified by silica gel column chromatography (100:1 CHCl3–MeOH) to give 27 (745 mg, 96%). []D
1
−5.5° (c 1.8, CHCl3); H-NMR (400 MHz, DMSO-d6)  8.20–7.32 (m, 27 H, 5 Ph, NHb, NHc), 5.58
(br s, 1 H, H-4a), 5.34 (near t, 1 H, J2,3 = 9.7 Hz, J1,2 = 8.2 Hz, H-2a), 5.16–5.07 (m, 3 H, H-4c, H-4b,
OCH2), 4.86–4.61 (m, 10 H, H-1b, H-1c, H-1a, 7 OCH2), 4.58–4.39 (m, 3 H, 3 OCH2), 4.31 (br dd,
1 H, H-3a), 4.18 (br dd, 1 H, H-6a), 4.06–3.48 (m, 17 H, H-5a, H-6'a, H-2b, H-3b, H-5b, H-6b, H-6'b,
H-2c, H-3c, H-5c, H-6c, H-6'c, OCH2, OCH2CH2SiMe3, CH2Cl), 2.80 (m, 2 H, CH2CH2C(=O)O), 2.54
(m, 2 H, CH2CH2C(=O)O), 2.25–2.22 (2 s, 6 H, 2 C(=O)CH3), 0.96–0.79 (m, 2 H, CH2CH2SiMe3),
13
0.00 (s, 9 H, CH2CH2SiMe3); C-NMR (100 MHz, DMSO-d6)  206.7, 179.5, 171.3, 170.0, 166.5,
164.5, 154.4, 153.8, 138.5, 138.3, 138.3, 138.2, 133.3, 130.1, 129.6, 128.7, 128.4, 128.3, 128.3, 128.1,
127.7, 127.6, 127.6, 127.5, 127.2, 101.3, 100.9, 100.0, 96.3, 96.0, 79.3, 79.0, 77.6, 73.6, 73.1, 72.7,
72.4, 72.3, 72.0, 71.1, 70.3, 70.2, 69.0, 68.8, 68.4, 66.4, 57.0, 41.1, 37.4, 29.7, 27.8, 20.8, 17.6, −1.3.
m/z (MALDI): found [M+Na]+ 1653.57, C73H85Cl7N2O23Si calcd for [M+Na]+ 1653.30.
OBn
O
HO
b
O
BnO
TrocHN
AcO
OBn
O
O
LevO
BnO
c
a
O
OSE
NHTroc
OBz
2-(Trimethylsilyl)ethyl 3,6-di-O-benzyl-2-deoxy-4-O-levulinoyl-2-(2,2,2-trichloroethoxycarbamoyl)-
-D-glucopyranosyl-(13)-[3,6-di-O-benzyl-2-deoxy-2-(2,2,2-trichloroethoxycarbamoyl)--D-
glucopyranosyl-(16)]-4-O-acetyl-2-O-benzoyl--D-galactopyranoside (28). To a solution of 27 (712 mg,
0.435 mmol) in EtOH (4.4 mL) was added DABCO (732 mg, 6.52 mmol) at 0 °C. After stirring for 1 h
at 50 °C as the reaction was monitored by TLC (30:1 CHCl3–MeOH), EtOH was evaporated. The
obtained crude residue was purified by silica gel column chromatography (100:1 CHCl3–MeOH) to
give 28 (665 mg, 98%). []D −1.8° (c 1.5, CHCl3); 1H-NMR (500 MHz, CDCl3)  8.19–7.27 (m, 25 H,
5 Ph), 5.63 (d, 1 H, J3,4 = 3.4 Hz, H-4a), 5.52 (near t, 1 H, J2,3 = 10.3 Hz, J1,2 = 8.0 Hz, H-2a), 5.32 (br
d, 1 H, NHb), 5.18 (d, 1 H, JNH,2 = 6.9 Hz, NHc), 5.07 (d, 1 H, J1,2 = 9.2 Hz, H-1c), 5.05 (near t, 1 H,
J3,4 = 9.2 Hz, H-4c), 4.85 (d, 1 H, J1,2 = 8.6 Hz, H-1b), 4.91–4.80 (m, 3 H, 3 OCH2), 4.71 (d, 1 H,
OCH2), 4.63 (d, 1 H, H-1a), 4.69–4.59 (m, 6 H, 6 OCH2), 4.50 (d, 1 H, OCH2), 4.16 (br t, 1 H,
J2,3 = 10.3 Hz, H-3c), 4.09 (m, 1 H, OCH2CH2SiMe3), 4.07 (dd, 1 H, H-3a), 4.01 (dd, 1 H, J5,6 = 4.1 Hz,
J
gem = 12.8 Hz, H-6a), 3.89–3.56 (m, 11 H, H-5a, H-6'a, H-3b, H-5b, H-6b, H-6'b, H-5c, H-6c, H-6'c,
OCH2, OCH2CH2SiMe3), 3.44 (br dd, 1 H, H-2b), 3.23 (br dd, 1 H, H-2c), 2.96 (s, 1 H, OH), 2.72–2.37
(m, 4 H, CH2CH2C(=O)O), 2.22 (m, 6 H, 2 C(=O)CH3), 1.01–0.86 (m, 2 H, CH2CH2SiMe3), 0.00 (s,
13
9 H, CH2CH2SiMe3); C-NMR (100 MHz, CDCl3)  206.1, 171.4, 169.9, 164.8, 154.0, 153.3, 138.2,
138.1, 137.7, 137.5, 133.2, 129.9, 128.5, 128.4, 128.3, 128.0, 127.9, 127.8, 127.7, 127.6, 127.6, 100.7,
100.2, 95.6, 95.4, 80.5, 77.5, 74.3, 74.3, 73.9, 73.7, 73.4, 73.4, 73.2, 73.1, 71.5, 71.4, 70.5, 70.1, 69.6,
67.4, 57.9, 57.3, 37.6, 29.6, 27.8, 20.8, 17.9, −1.5. m/z (MALDI): found [M+Na]+ 1577.28,
C71H84Cl6N2O22Si calcd for [M+Na]+ 1577.33.

Molecules 2012, 17
9609
OAc
AcO
COOMe
O
Oe
AcHN
AcO
AcO
TrocHN
AcO
O
BnO
b
d
BzO
O
O
O
OBz
OBn
OBn AcO
O
c
aO
OBz
LevO
BnO
O
OSE
NHTroc
2-(Trimethylsilyl)ethyl (methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero--D-galacto-2-
nonulopyranosylonate)-(26)-4-O-acetyl-2,3-di-O-benzoyl--D-galactopyranosyl-(14)-3,6-di-O-
benzyl-2-deoxy-2-(2,2,2-trichloroethoxycarbamoyl)--D-glucopyranosyl-(16)-[3,6-di-O-benzyl-2-
deoxy-4-O-levulinoyl-2-(2,2,2-trichloroethoxycarbamoyl)--D-glucopyranosyl-(13)]-4-O-acetyl-2-
O-benzoyl--D-galactopyranoside (29). To a mixture of 12 (80 mg, 76.5 mol) and 28 (70 mg,
45.0 mol) in CH2Cl2 (0.9 mL) was added 3 Å molecular sieves (250 mg) at r.t. After stirring for 1 h,
the mixture was cooled to 0 °C. TMSOTf (1.4 L, 7.65 mol) was then added to the mixture at 0 °C.
After stirring for 25 h at r.t., TMSOTf (1.4 L, 7.65 mol) was added to the mixture. After the stirring
was continued for 3 h at r.t. as the reaction was monitored by TLC (30:1 CHCl3–MeOH), the reaction
was quenched by the addition of triethylamine. The mixture was diluted with CHCl3 and filtered
through Celite. The filtrate was then washed with satd aq NaHCO3 and brine. The organic layer was
subsequently dried over Na2SO4, and concentrated. The resulting residue was purified by silica gel
column chromatography (90:1 CHCl3–MeOH) followed by gel filtration column chromatography (LH-20)
1
using MeOH as eluent, giving 29 (81 mg, 74%). []D −2.9° (c 1.4, CHCl3); H-NMR (600 MHz,
CDCl3)  8.05–7.14 (m, 35 H, 7 Ph), 5.61 (d, 1 H, J3,4 = 3.3 Hz, H-4d), 5.59 (near t, 1 H, J1,2 = 8.3 Hz,
H-2d), 5.45 (d, 1 H, J3,4 = 3.4 Hz, H-4a), 5.40–5.36 (m, 1 H, H-8e), 5.36 (t, 1 H, J1,2 = 8.2 Hz, H-2a),
5.34 (m, 1 H, H-7e), 5.30 (dd, 1 H, J2,3 = 10.2 Hz, H-3d), 5.23 (2 br d, 2 H, NHe, NHb), 5.02 (d, 1 H,
J2,NH = 6.9 Hz, NHc), 5.12 (d, 1 H, Jgem = 11.0 Hz, OCH2), 4.94 (d, 1 H, H-1d), 4.94–4.86 (m, 2 H, H-1c,
H-4c), 4.84 (m, 1 H, J3eq,4 = 4.2 Hz, H-4e), 4.73–4.68 (m, 3 H, 3 OCH2), 4.58–4.30 (m, 10 H, H-1a,
H-1b, H-9e, 7 OCH2), 4.14–3.71 (m, 15 H, H-3a, H-6'a, H-3b, H-4b, H-3c, H-4c, H-5d, H-6d, H-6'd,
H-5e, H-6e, OCH2, OCH3), 3.58–3.37 (m, 10 H, H-6a, H-2b, H-5b, H-6b, H-6'b, H-5c, H-6c, H-6'c,
2 OCH2), 3.28–3.26 (m, 1 H, H-5a), 3.08 (br dd, 1 H, H-2c), 2.60–2.51 (m, 3 H, H-3eeq,
CH2CH2C(=O)O), 2.38–2.24 (m, 2 H, CH2CH2C(=O)O), 2.16–1.91 (m, 25 H, 8 C(=O)CH3, H-3eax),
13
0.85–0.69 (m, 2 H, CH2CH2SiMe3), −0.14 (s, 9 H, CH2CH2SiMe3); C-NMR (100 MHz, CDCl3) 
206.0, 171.4, 170.9, 170.7, 170.3, 170.1, 169.8, 169.6, 167.8, 165.3, 165.2, 164.8, 153.9, 153.3, 138.6,
138.1, 138.0, 137.7, 133.3, 133.1, 129.8, 129.6, 129.5, 129.1, 128.5, 128.4, 128.4, 128.3, 128.3, 128.2,
127.9, 127.8, 127.7, 127.6, 127.6, 127.4, 100.6, 100.1, 99.8, 99.0, 95.4, 77.4, 76.8, 76.1, 74.4, 74.3,
73.9, 73.7, 73.3, 73.3, 73.1, 72.8, 71.7, 71.5, 71.4, 70.2, 70.1, 69.6, 68.7, 68.4, 68.1, 67.3, 62.4, 57.9,
57.0, 52.9, 49.4, 37.7, 37.6, 29.6, 27.8, 23.1, 21.0, 20.8, 20.5, 17.8, −1.5. m/z (MALDI): found
[M+Na]+ 2463.09, C113H131Cl6N3O42Si calcd for [M+Na]+ 2462.60.

Molecules 2012, 17
9610
OAc
AcO
COOMe
O
AcO
e
O
AcHN
AcO
AcHN
AcO
O
BnO
b
d
BzO
O
O
O
OBz
OBn
OBn AcO
O
O
O
LevO
BnO
c
a
OSE
NHAc
OBz
2-(Trimethylsilyl)ethyl (methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero--D-galacto-2-
nonulopyranosylonate)-(26)-4-O-acetyl-2,3-di-O-benzoyl--D-galactopyranosyl-(14)-2-acetamido-
3,6-di-O-benzyl-2-deoxy--D-glucopyranosyl-(16)-[2-acetamido-3,6-di-O-benzyl-2-deoxy-4-
O-levulinoyl--D-glucopyranosyl-(13)]-4-O-acetyl-2-O-benzoyl--D-galactopyranoside (31). To a
solution of 29 (220 mg, 89.9 mol) in CH2Cl2/AcOH (2:3, 3.5 mL) was added Zn/Cu couple (2.20 g)
at r.t. The reaction mixture was heated to 50 °C and was stirred for 2.5 h at the same temperature as the
reaction was monitored by TLC (15:1 CHCl3–MeOH). The precipitate was filtered through Celite and
the filtrate was co-evaporated with toluene. The obtained residue was exposed to high vacuum for 6 h.
The crude residue was dissolved in pyridine (3.6 mL) and acetic anhydride (68 L, 0.719 mmol),
DMAP (2.5 mg, 47.6 mol) were then added to the mixture at 0 °C. After stirring for 12 h at r.t as the
reaction was monitored by TLC (15:1 CHCl3–MeOH), the reaction mixture was evaporated. The
obtained residue was diluted with CHCl3 and washed with 2 M HCl, satd aq NaHCO3, and brine,
dried over Na2SO4, and concentrated. The resulting residue was purified by silica gel column
chromatography (40:1 CHCl3–MeOH) to give 31 (120 mg, 61%). []D −1.9° (c 0.5, CHCl3); 1H-NMR
(600 MHz, CDCl3)  8.02–7.13 (m, 35 H, 7 Ph), 5.93 (d, 1 H, J2,NH = 8.3 Hz, NHb), 5.63 (d, 1 H,
J3,4 = 3.4 Hz, H-4d), 5.56 (near t, 1 H, J1,2 = 7.5 Hz, J2,3 = 8.2 Hz, H-2d), 5.41–5.31 (m, 5 H, H-2a, H-4a,
H-3d, H-7e, H-8e), 5.26 (d, 1 H, J2,NH = 7.6 Hz, NHc), 5.13 (d, 1 H, J5,NH = 7.5 Hz, NHe), 4.95 (d, 1 H,
J1,2 = 8.2 Hz, H-1c), 4.90–4.83 (m, 4 H, H-4c, H-1d, H-4e, OCH2), 4.70 (d, 1 H, Jgem = 11.7 Hz,
OCH2), 4.58 (d, 1 H, J1,2 = 6.9 Hz, H-1c), 4.44 (d, 1 H, J1,2 = 8.3 Hz, H-1a), 4.54–4.31 (m, 7 H, H-9e,
3 OCH2), 4.13 (t, 1 H, J2,3 = 9.6 Hz, H-3c), 4.10–4.02 (m, 4 H, H-6a, H-4b, H-5e, H-9e), 3.97–3.75 (m,
9 H, H-3a, H-6'a, H-3b, H-6d, H-6e, OCH2CH2SiMe3, OCH3), 3.67–3.35 (m, 11 H, H-5a, H-2b, H-5b,
H-6b, H-6'b, H-5c, H-6c, H-6'c, H-5d, H-6'd, OCH2CH2SiMe3), 3.07 (dd, 1 H, H-2c), 2.54–2.51
(m, 3 H, H-3eeq, CH2CH2C(=O)O), 2.35–2.27 (m, 2 H, CH2CH2C(=O)O), 2.15–1.89 (m, 28 H,
9 C(=O)CH3, H-3eax), 0.88–0.69 (m, 2 H, OCH2CH2SiMe3), −0.16 (s, 9 H, OCH2CH2SiMe3); 13C (125
MHz, CDCl3)  206.1, 171.5, 170.9, 170.7, 170.5, 170.3, 170.1, 169.9, 169.7, 167.8, 165.3, 165.1,
138.9, 138.2, 138.1, 133.4, 133.3, 133.2, 129.8, 129.7, 129.6, 129.1, 128.6, 128.5, 128.5, 128.4, 128.3,
127.9, 127.9, 127.8, 127.7, 127.6, 127.5, 127.4, 100.6, 100.2, 99.9, 99.7, 99.0, 78.0, 77.4, 74.6, 73.6,
73.4, 73.3, 73.2, 73.1, 72.7, 71.8, 71.8, 71.6, 71.5, 70.2, 70.1, 69.7, 68.8, 68.8, 68.1, 67.6, 67.4, 67.2,
62.7, 62.5, 57.6, 52.9, 49.5, 37.8, 37.7, 29.7, 27.9, 23.4, 23.2, 22.7, 21.0, 20.8, 20.7, 20.6, 17.8, −1.5.
m/z (MALDI): found [M+Na]+ 2198.83, C111H133N3O40Si calcd for [M+Na]+ 2198.81.

Molecules 2012, 17
9611
OAc
AcO
COOMe
e
O
O
AcO
AcHN
AcO
AcHN
AcO
O
BnO
b
d
BzO
O
O
O
OBn
OBz
OBn AcO
O
O
O
HO
BnO
c
a
OSE
NHAc
OBz
2-(Trimethylsilyl)ethyl (methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero--D-galacto-2-
nonulopyranosylonate)-(26)-4-O-acetyl-2,3-di-O-benzoyl--D-galactopyranosyl-(14)-2-acetamido-
3,6-di-O-benzyl-2-deoxy--D-glucopyranosyl-(16)-[2-acetamido-3,6-di-O-benzyl-2-deoxy--D-
glucopyranosyl-(13)]-4-O-acetyl-2-O-benzoyl--D-galactopyranoside (32). To a solution of 31
(119 mg, 54.7 mol) in THF (2.0 mL) was added hydrazine monoacetate (4.9 mg, 54.7 mol) at 0 °C.
After stirring for 1 h at r.t. as the reaction was monitored by TLC (30:1 CHCl3–MeOH), THF was
evaporated. The residue was diluted with CHCl3 and washed with 2 M HCl, satd aq NaHCO3, and
brine, dried over Na2SO4, and concentrated. The obtained residue was purified by silica gel column
chromatography (40:1 CHCl3–MeOH) to give 32 (104 mg, 92%). []D −5.2° (c 1.9, CHCl3); 1H-NMR
(500 MHz, CDCl3)  8.02–7.20 (m, 35 H, 7 Ph), 6.01 (d, 1 H, JNH,2 = 8.6 Hz, NHb), 5.64 (d, 1 H,
J3,4 = 3.5 Hz, H-4d), 5.58 (near t, 1 H, J1,2 = 8.0 Hz, J2,3 = 10.3 Hz, H-2d), 5.43–5.32 (m, 6 H, H-2a, H-4a,
H-3d, H-7e, H-8e, NHc), 5.21 (d, 1 H, JNH,5 = 7.4 Hz, NHe), 4.89–4.81 (m, 3 H, H-1d, H-4e, OCH2),
4.82 (d, 1 H, J1,2 = 9.7 Hz, H-1c), 4.72–4.65 (2 d, 2 H, Jgem = 11.5 Hz, 2 OCH2), 4.60–4.45 (m, 5 H,
H-1b, 4 OCH2), 4.44 (d, 1 H, J1,2 = 8.0 Hz, H-1a), 4.37–4.32 (m, 2 H, OCH2, H-9e), 4.12–4.04 (m, 4 H,
H-6a, H-6d, H-6e, H-9'e), 3.98–3.62 (m, 14 H, H-3a, H-5a, H-6'a, H-2b, H-3b, H-6b, H-6'b, H-3c, H-5d,
H-6'd, OCH3, OCH2CH2SiMe3), 3.54 (t, 1 H, J3,4 = J4,5 = 12.0 Hz, H-4c), 3.49–3.36 (m, 6 H, H-5c,
H-6c, H-6'c, H-4b, H-5b, OCH2CH2SiMe3), 3.16 (dd, 1 H, H-2c), 2.96 (s, 1 H, OH), 2.53 (dd, 1 H,
Jgem = 12.4 Hz, J3eq,4 = 4.6 Hz, H-3eeq), 2.18–1.87 (m, 25 H, 8 C(=O)CH3, H-3eax), 0.88–0.69 (m,
2 H, OCH2CH2SiMe3), −0.16 (s, 9 H, OCH2CH2SiMe3); 13C-NMR (125 MHz, CDCl3)  170.8, 170.6,
170.4, 170.2, 170.1, 169.9, 169.6, 169.5, 167.7, 165.6, 165.2, 165.0, 138.7, 138.4, 138.0, 137.7, 133.4,
133.2, 133.1, 129.7, 129.7, 129.6, 129.5, 129.0, 128.4, 128.4, 128.3, 128.3, 128.2, 127.8, 127.8, 127.7,
127.6, 127.5, 127.3, 100.4, 100.2, 100.1, 99.6, 98.9, 79.8, 77.9, 75.2, 74.5, 73.8, 73.5, 73.5, 73.2, 73.1,
73.0, 72.8, 72.6, 71.7, 71.6, 71.4, 70.7, 70.1, 70.0, 68.7, 68.1, 67.5, 67.3, 67.2, 62.5, 62.4, 56.4, 54.0,
52.8, 49.2, 37.7, 29.6, 23.3, 23.1, 22.8, 20.9, 20.7, 20.6, 20.5, 17.7, −1.6. m/z (MALDI): found
[M+Na]+ 2100.96, C106H127N3O38Si calcd for [M+Na]+ 2100.78.
OAc
AcO
COOMe
e
O
AcO
O
AcHN
AcO
AcHN
AcO
O
BnO
b
d
BzO
O
O
O
OAc
OBz
OBn
AcO
COOMe
O
OBn AcO
OBz
O
O
O
O
g
O
a
O
f
c
OSE
AcHN
AcO
BnO
NHAc
OBz
AcO
OBz
AcO

Molecules 2012, 17
9612
2-(Trimethylsilyl)ethyl (methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero--D-galacto-2-
nonulopyranosylonate)-(23)-4-O-acetyl-2,6-di-O-benzoyl--D-galactopyranosyl-(14)-2-acetamido-
3,6-di-O-benzyl-2-deoxy--D-glucopyranosyl-(13)-[(methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-
dideoxy-D-glycero--D-galacto-2-nonulopyranosylonate)-(26)-4-O-acetyl-2,3-di-O-benzoyl--D-
galactopyranosyl-(14)-2-acetamido-3,6-di-O-benzyl-2-deoxy--D-glucopyranosyl-(16)]-4-
O-acetyl-2-O-benzoyl--D-galactopyranoside (33). To a mixture of 4 (103 mg, 98.7 mol) and 32
(93 mg, 44.5 mol) in CH2Cl2 (1.8 mL) was added 3 Å molecular sieves (300 mg) at r.t. After stirring
for 1 h, the mixture was cooled to 0 °C. TMSOTf (1.8 L, 9.94 mol) was then added to the mixture at
0 °C. The reaction was stirred for 2 h at r.t. as the reaction was monitored by TLC (15:1:1 CHCl3–
MeOH–EtOAc). Another portion of TMSOTf (1.8 L, 9.94 mol) was added to the mixture at 0 °C.
After the stirring was continued for 4 h at r.t, the reaction was quenched by the addition of
triethylamine. The reaction mixture was diluted with CHCl3 and filtered through Celite. The filtrate
was then washed with satd aq NaHCO3 and brine. The organic layer was subsequently dried over
Na2SO4, and concentrated. The resulting residue was purified by silica gel column chromatography
1
(40:1 CHCl3–MeOH) to give 33 (82 mg, 62%). []D −5.1° (c 0.8, CHCl3); H-NMR (600 MHz,
CDCl3)  8.22–7.12 (m, 45 H, 9 Ph), 5.92 (d, 1 H, NHb), 5.65 (m, 1 H, H-4g), 5.63 (d, 1 H, J3,4 = 3.4 Hz,
H-4d), 5.55 (near t, 1 H, J1,2 = 8.2 Hz, J2,3 = 10.2 Hz, H-2d), 5.40 (m, 1 H, H-8e), 5.37 (dd, 1 H, H-3d),
5.33–5.13 (m, 7 H, H-2a, H-4a, H-7e, H-2f, H-7g, NHc, NHe), 5.06 (d, 1 H, J3,4 = 3.4 Hz, H-4f), 5.01
(d, 1 H, J1,2 = 7.5 Hz, H-1f), 5.00 (d, 1 H, J5,NH = 7.5 Hz, NHg), 4.87–4.82 (m, 5 H, H-1d, H-4e, H-4g,
2 OCH2), 4.75 (dd, 1 H, J2,3 = 10.2 Hz, H-3f), 4.71 (d, 1 H, Jgem = 11.6 Hz, OCH2), 4.68 (d, 1 H,
J1,2 = 7.5 Hz, H-1c), 4.68–4.47 (m, 4 H, H-1b, 3 OCH2), 4.42 (d, 1 H, J1,2 = 10.7 Hz, H-1a), 4.28 (m, 4 H,
H-9e, H-9g, 2 OCH2), 4.11–4.00 (m, 7 H, H-4b, H-4c, H-6c, H-6'c, H-5e, H-6e, H-9'e), 3.97–3.55 (m,
23 H, H-3a, H-5a, H-6a, H-6'a, H-2b, H-3b, H-6b, H-6'b, H-3c, H-6d, H-6'd, H-6f, H-6'f, H-5g, H-6g,
H-9'g, 2 OCH3, OCH2CH2SiMe3), 3.49–3.40 (m, 3 H, H-5b, H-5c, OCH2CH2SiMe3), 3.31–3.28 (m,
2 H, H-5c, H-5f), 3.14 (br dd, 1 H, H-2c), 2.53 (dd, 1 H, Jgem = 11.3 Hz, J3eq,4 = 4.8 Hz, H-3geq), 2.49
(m, 1 H, Jgem = 11.7 Hz, J3eq,4 = 4.8 Hz, H-3eeq), 2.15–1.74 (m, 43 H, 14 Ac, H-3eax), 1.70 (t, 1 H,
H-3gax), 1.51 (s, 3 H, Ac), 0.88–0.68 (m, 2 H, OCH2CH2SiMe3), −0.17 (s, 9 H, OCH2CH2SiMe3);
13C-NMR (150 MHz, CDCl3)  170.9, 170.7, 170.7, 170.3, 170.2, 170.1, 170.1, 170.0, 169.8, 169.7,
169.6, 168.0, 167.8, 165.7, 165.5, 165.3, 165.2, 164.9, 138.8, 138.8, 138.7, 138.1, 133.4, 133.3, 133.2,
133.1, 133.0, 130.3, 129.9, 129.8, 129.7, 129.7, 129.6, 129.5, 129.0, 128.6, 128.5, 128.4, 128.2, 128.1,
128.0, 127.9, 129.6, 129.5, 129.0, 128.6, 128.5, 128.4, 128.2, 128.1, 128.0, 127.9, 127.8, 127.5, 127.4,
127.2, 127.2, 127.0, 100.5, 100.3, 99.6, 99.5, 99.0, 96.8, 78.1, 77.6, 77.5, 75.2, 75.0, 74.6, 74.5, 73.6,
73.3, 73.0, 72.7, 72.6, 71.8, 71.7, 71.4, 71.3, 70.4, 70.2, 70.1, 69.4, 68.7, 68.1, 68.0, 67.5, 67.3, 67.2,
67.1, 66.6, 62.4, 61.3, 53.0, 52.9, 49.4, 48.8, 37.8, 37.3, 29.7, 23.3, 23.2, 23.1, 22.6, 21.3, 21.0, 20.8,
20.8, 20.7, 20.6, 20.6, 20.3, 17.7, −1.5. m/z (MALDI): found [M+Na]+ 2986.28, C148H174N4O58Si calcd
for [M+Na]+ 2986.05.

Molecules 2012, 17
9613
OAc
AcO
COOMe
e
O
AcO
O
AcHN
AcO
AcHN
AcO
O
AcO
b
d
BzO
COOMe
O
O
O
OAc
OAc
OBz
AcO
OAc AcO
OBz
O
O
NHAc
O
g
f
O
O
O
a
c
OSE
AcHN
AcO
AcO
O
OBz
OBz
AcO
AcO
2-(Trimethylsilyl)ethyl (methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero--D-galacto-2-
nonulopyranosylonate)-(23)-4-O-acetyl-2,6-di-O-benzoyl--D-galactopyranosyl-(14)-2-acetamido-
3,6-di-O-acetyl-2-deoxy--D-glucopyranosyl-(13)-[(methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-
dideoxy-D-glycero--D-galacto-2-nonulopyranosylonate)-(26)-4-O-acetyl-2,3-di-O-benzoyl--D-
galactopyranosyl-(14)-2-acetamido-3,6-di-O-acetyl-2-deoxy--D-glucopyranosyl-(16)]-4-O-
acetyl-2-O-benzoyl--D-galactopyranoside (35). To a solution of 33 (42.0 mg, 14.2 mol) in
1,4-dioxane (0.6 mL) was added Pd(OH)2/C (210 mg). After stirring for 4 h at r.t. under a hydrogen
atmosphere as the reaction was monitored by TLC (10:1 CHCl3–MeOH), the mixture was filtered
through Celite. The filtrate was concentrated and the obtained crude residue was roughly purified by
silica gel column chromatography (10:1 CHCl3–MeOH). The obtained product was exposed to high
vacuum for 24 h. The residue was then dissolved in pyridine (1.4 mL). Acetic anhydride (11 L,
0.114 mmol) and DMAP (1.0 mg, 8.18 mol) were added to the mixture at 0 °C. After stirring for 72 h
at r.t. as the reaction was monitored by TLC (10:1 CHCl3–MeOH), the reaction was quenched by the
addition of MeOH at 0 °C. The mixture was co-evaporated with toluene and the residue was then
diluted with CHCl3, and washed with 2 M HCl, H2O, satd aq NaHCO3 and brine. The organic layer
was subsequently dried over Na2SO4, and concentrated. The resulting residue was purified by silica gel
column chromatography (40:1 CHCl3–MeOH) to give 35 (35 mg, 89%). []D +5.1° (c 0.7, CHCl3);
1H-NMR (500 MHz, CDCl3)  8.17–7.27 (m, 25 H, 5 Ph), 5.92 (d, 1 H, JNH,2 = 9.2 Hz, NHb), 5.68 (d,
1 H, J3,4 = 3.5 Hz, H-4d), 5.77 (m, 1 H, H-8g), 5.55 (near t, 2 H, J1,2 = 8.0 Hz, J2,3 = 10.3 Hz, H-2d),
5.44 (m, 1 H, H-3d), 5.38 (m, 1 H, H-8e), 5.32–5.16 (m, 7 H, H-2a, H-3a, H-3b, H-7e, H-2f, H-7g,
NHe), 5.09 (d, 1 H, J3,4 = 2.8 Hz, H-4f), 4.96 (d, 1 H, JNH,5 = 9.8 Hz, NHg), 4.91–4.80 (m, 6 H, NHc,
H-3c, H-1d, H-4e, H-1f, H-4g), 4.74–4.71 (dd, 1 H, J2,3 = 9.7 Hz, H-3f), 4.47 (d, 1 H, J1,2 = 8.0 Hz,
H-1b), 4.43 (d, 1 H, J1,2 = 8.0 Hz, H-1a), 4.41–4.28 (m, 6 H, H-6a, H-1c, H-6d, H-9e, H-6f, H-9g),
4.17–3.93 (m, 10 H, H-6'a, H-6b, H-6'b, H-6'd, H-5e, H-6e, H-9'e, H-6'f, H-9'g, OCH2CH2SiMe3),
3.88–3.68 (m, 15 H, H-3a, H-2b, H-4b, H-5b, H-2c, H-4c, H-6c, H-6'c, H-5g, 2 OCH3), 3.55–3.53 (m,
2 H, H-5d, H-6g), 3.48–3.42 (m, 3 H, H-5a, H-5f, OCH2CH2SiMe3), 3.27–3.25 (m, 1 H, J5,4 = 9.7 Hz,
H-5c), 2.55 (dd, 1 H, Jgem = 12.6 Hz, J3eq,4 = 4.6 Hz, H-3geq), 2.51 (dd, 1 H, Jgem = 12.6 Hz, J3eq,4 = 4.6 Hz,
H-3eeq), 2.20–1.78 (m, 52 H, 17 Ac, H-3eax), 1.62 (t, 1 H, H-3gax), 1.54 (s, 6 H, 2 Ac), 0.88–0.72 (m,
2 H, OCH2CH2SiMe3), −0.15 (s, 9 H, OCH2CH2SiMe3); 13C-NMR (150 MHz, CDCl3)  170.9, 170.7,
170.7, 170.5, 170.4, 170.4, 170.2, 170.1, 170.1, 170.0, 169.8, 169.8, 169.7, 168.0, 167.7, 165.6, 165.3,
165.0, 164.9, 164.8, 133.5, 133.3, 133.2, 130.3, 129.8, 129.7, 129.5, 129.0, 128.9, 128.7, 128.5, 128.5,
128.4, 128.4, 101.2, 100.7, 100.6, 100.5, 100.4, 99.2, 96.8, 75.6, 74.6, 72.9, 72.7, 72.5, 72.4, 71.9,
71.7, 71.1, 70.9, 70.0, 69.8, 69.6, 69.4, 68.6, 67.8, 67.4, 67.1, 66.2, 62.9, 62.4, 62.2, 62.0, 61.0, 60.9,
53.9, 53.0, 49.4, 48.8, 37.8, 37.2, 23.2, 23.1, 22.7, 21.3, 21.0, 20.9, 20.8, 20.7, 20.6, 20.5, 20.4, 17.8,
−1.5. m/z (MALDI): found [M+Na]+ 2793.65, C128H158N4O62Si calcd for [M+Na]+ 2793.90.

Molecules 2012, 17
9614
OAc
AcO
COOMe
e
O
AcO
O
AcHN
AcO
AcHN
AcO
O
AcO
b
d
BzO
COOMe
O
O
O
OAc
OAc
OBz
AcO
OAc AcO
OBz
O
O
NHAc
O
g
f
O
O
O
a
c
AcHN
AcO
AcO
O
OBz
BzO
AcO
O
CCl3
NH
AcO
(Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero--D-galacto-2-nonulopyranosylonate)-
(23)-4-O-acetyl-2,6-di-O-benzoyl--D-galactopyranosyl-(14)-2-acetamido-3,6-di-O-acetyl-2-
deoxy--D-glucopyranosyl-(13)-[(methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-
-D-galacto-2-nonulopyranosylonate)-(26)-4-O-acetyl-2,3-di-O-benzoyl--D-galactopyranosyl-
(14)-2-acetamido-3,6-di-O-acetyl-2-deoxy--D-glucopyranosyl-(16)]-4-O-acetyl-2-O-benzoyl-D-
galactopyranosyl trichloroacetimidate (37). To a solution of 35 (28 mg, 10.2 mol) in CH2Cl2
(0.75 mL) was added TFA (0.25 mL) at 0 °C. After stirring for 2 h at 0 °C as the reaction was
monitored by TLC (15:1:1 CHCl3–MeOH–EtOAc), the reaction mixture was co-evaporated with
toluene and then roughly purified by silica gel column chromatography (20:1 CHCl3–MeOH). The
obtained product was exposed to high vacuum for 24 h and then dissolved in CH2Cl2 (1.0 mL).
CCl3CN (10.2 L, 0.102 mmol) and DBU (1.8 L, 12.2 mol) were added to the mixture at 0 °C.
After stirring for 45 min at 0 °C as the reaction was monitored by TLC (10:1 CHCl3–MeOH), the
reaction mixture was evaporated. The obtained crude residue was purified by silica gel column
chromatography (30:1 CHCl3–MeOH) to give 37 (27 mg, 95%, : = 3:1). 37: 1H-NMR (600 MHz,
CDCl3)  8.60 (s, 1 H, C(=NH)), 8.19–7.27 (m, 25 H, 5 Ph), 6.48 (d, 1 H, J1,2 = 4.1 Hz, H-1a), 5.81 (d,
1 H, JNH,2 = 9.0 Hz, NHc), 5.68 (d, 1 H, J3,4 = 3.5 Hz, H-4d), 5.60 (m, 1 H, H-8g), 5.55 (near t, 1 H,
J1,2 = 8.3 Hz, J2,3 = 10.3 Hz, H-2d), 5.48 (dd, 1 H, J2,3 = 10.3 Hz, H-2a), 5.43–5.42 (m, 2 H, H-3a, H-4a),
5.38 (m, 1 H, H-8e), 5.32 (dd, 1 H, H-7e), 5.24 (m, 2 H, H-3a, H-7g), 5.20 (near t, 1 H, J1,2 = 8.3 Hz,
J
2,3 = 9.6 Hz, H-2f), 5.16 (t, 1 H, J2,3 = J3,4 = 9.0 Hz, H-3b), 5.13 (d, 1 H, J5,NH = 9.6 Hz, NHe), 4.92 (d,
1 H, J3,4 = 2.3 Hz, H-4f), 4.90–4.80 (m, 8 H, H-3c, H-1d, H-4e, H-1f, H-3d, H-4g, NHc, NHg), 4.73
(dd, 1 H, H-3f), 4.55 (d, 1 H, J1,2 = 7.6 Hz, H-1b), 4.45 (d, 1 H, J1,2 = 7.4 Hz, H-1c), 4.41–4.01 (m, 13 H,
H-3a, H-6a, H-6'a, H-5e, H-6e, H-9e, H-9'e, H-6d, H-6'd, H-6f, H-6'f, H-9g, H-9'g), 3.88–3.70 (m, 15
H, H-2b, H-4b, H-6b, H-6'b, H-2c, H-4c, H-6c, H-6'c, H-5e, 2 OCH3), 3.55–3.36 (m, 5 H, H-5a, H-5b,
H-5c, H-5d, H-5f), 2.55 (dd, 1 H, Jgem = 13.1 Hz, J3eq,4 = 4.8 Hz, H-3eeq ), 2.51 (dd, 1 H, Jgem = 11.6 Hz,
J
3eq,4 = 4.8 Hz, H-3geq), 2.20–1.78 (m, 52 H, 17 Ac, H-3eax), 1.63–1.39 (m, 7 H, 2 Ac, H-3gax).
OAc
AcO
COOMe
e
O
AcO
O
AcHN
AcO
AcHN
AcO
O
AcO
b
d
BzO
COOMe
O
O
O
OAc
O
O
OAc
OBz
AcO
OAc AcO
OBz
OBz
HN
O
C17H35
C13H27
MPMO
O
O
O
NHAc
g
h
f
O
O
O
a
c
O
O
AcHN
AcO
AcO
O
O
O
OBz
BzO
AcO
AcO
O

Molecules 2012, 17
9615
(2S,3R,4E)-1-O-({(Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero--D-galacto-2-
nonulopyranosylonate)-(23)-4-O-acetyl-2,6-di-O-benzoyl--D-galactopyranosyl-(14)-2-acetamido-
3,6-di-O-acetyl-2-deoxy--D-glucopyranosyl-(13)-[(methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-
dideoxy-D-glycero--D-galacto-2-nonulopyranosylonate)-(26)-4-O-acetyl-2,3-di-O-benzoyl--D-
galactopyranosyl-(14)-2-acetamido-3,6-di-O-acetyl-2-deoxy--D-glucopyranosyl-(16)]-4-O-acetyl-
2-O-benzoyl--D-galactopyranosyl}-(14)-2-O-benzoyl-3-O-p-methoxybenzyl--D-glucopyranosyl)-
2-octadecanamido-3,6´-succinyl-octadec-4-ene-1,3-diol (39). To a mixture of 37 (38 mg, 13.4 mol)
and 38 (14 mg, 13.4 mol) in CH2Cl2 (1.3 mL) was added 5 Å molecular sieves (100 mg) at r.t. After
stirring for 1 h, the mixture was cooled to 0 °C. TMSOTf (0.3 L, 1.34 mol) was then added to the
mixture at 0 °C. After stirring for 1 h at r.t. as the reaction was monitored by TLC (15:1:5 CHCl3–
MeOH–EtOAc), the reaction was quenched by the addition of triethylamine. The reaction mixture was
diluted with CHCl3 and filtered through Celite. The filtrate was then washed with satd aq NaHCO3 and
brine. The organic layer was subsequently dried over Na2SO4, and concentrated. The residue was
purified by silica gel column chromatography (40:1:5 CHCl3–MeOH–EtOAc) to give 39 (24 mg,
49%). []D −10.3° (c 2.0, CHCl3); 1H-NMR (500 MHz, CD3CN)  8.13–7.39 (m, 30 H, 6 Ph), 7.04 (d,
2 H, Ar), 6.66 (d, 2 H, Ar), 6.14 (d, 1 H, J5,NH = 9.7 Hz, NHe), 6.09 (d, 1 H, J2,NH = 9.2 Hz, NHCer),
6.00 (d, 1 H, J5,NH = 9.7 Hz, NHg), 5.95 (d, 1 H, J2,NH = 9.1 Hz, NHb), 5.79 (d, 1 H, J2,NH = 9.8 Hz,
NHc), 5.63 (dt, 1 H, J5,6 = 6.9 Hz, J = 14.9 Hz, J = 6.9 Hz, H-5Cer), 5.54 (d, 1 H, J3,4 = 3.5 Hz, H-4d),
5.47 (m, 3 H, H-3d, H-7e, H-8e), 5.37 (near t, 1 H, J1,2 = 7.5 Hz, J2,3 = 10.3 Hz, H-2d), 5.36 (m, 1 H,
H-8g), 5.27 (d, 1 H, J3,4 = 3.5 Hz, H-4a), 5.25–5.17 (m, 4 H, H-2a, H-4f, H-7g, H-4Cer), 5.11 (br t,
1 H, J3,4 = J2,3 = 7.5 Hz, H-3Cer), 5.03 (near t, 1 H, J1,2 = 8.0 Hz, J2,3 = 10.3 Hz, H-2f), 4.94 (t, 1 H,
J1,2 = J2,3 = 8.3 Hz, H-2h), 4.88 (t, 1 H, J2,3 = J3,4 = 9.3 Hz, H-3b), 4.76 (m, 8 H, H-1c, H-3c, H-1d,
H-4e, H-1f, H-3f, H-4g, ArCH2), 4.68 (d, 1 H, J1,2 = 8.1 Hz, H-1a), 4.54 (d, 1 H, J1,2 = 8.5 Hz, H-1b),
4.47 (d, 1 H, H-1h), 4.46 (d, 1 H, Jgem = 10.9 Hz, ArCH2), 4.35 (dd, 1 H, J8,9 = 2.9 Hz, Jgem = 12.6 Hz,
H-9e), 4.30–4.21 (m, 4 H, H-6b, H-6'b, H-6d, H-9g), 4.11–3.89 (m, 13 H, H-3a, H-6'a, H-6c, H-6'c, H-6'd,
H-5e, H-6e, H-9'e, H-6f, H-9g, H-6h, H-6'h, H-2Cer), 3.78–3.53 (m, 21 H, H-5a, H-4b, H-2c, H-4c,
H-5d, H-6'a, H-6'f, H-5g, H-6g, H-3h, H-4h, H-1Cer, 3 OCH3), 3.51 (m, 1 H, H-2b), 3.40–3.32 (m, 3 H,
H-5b, H-5h, H-1'Cer), 3.18–3.12 (m, 2 H, H-5c, H-5f), 2.56 (dd, 1 H, J3eq,4 = 4.6 Hz, Jgem = 12.6 Hz,
H-3eeq), 2.48–2.34 (m, 5 H, H-3geq, 2 C(=O)CH2), 2.21–1.61 (m, 62 H, H-3eax, H-6Cer, H-6'Cer,
C(=O)CH2Cer, 19 Ac), 1.55–1.08 (m, 53 H, H-3gax, 26 –CH2–), 0.87 (t, 6 H, 2 –CH3Cer); C-NMR
13
(150 MHz, CDCl3)  172.6, 171.0, 170.9, 170.8, 170.7, 170.6, 170.5, 170.4, 170.3, 170.2, 170.1,
170.0, 169.7, 169.7, 169.5, 168.0, 167.7, 165.6, 165.2, 165.2, 165.1, 165.0, 164.5, 159.1, 138.6, 133.8,
133.3, 133.1, 130.7, 130.3, 129.8, 129.7, 129.6, 129.5, 129.5, 129.5, 129.0, 128.9, 128.8, 128.6, 128.6,
128.5, 128.4, 128.3, 124.8, 113.9, 101.6, 101.3, 100.8, 100.6, 100.3, 99.7, 99.3, 96.8, 91.8, 80.8, 79.7,
76.3, 75.9, 74.7, 74.3, 73.2, 72.9, 72.7, 72.6, 72.5, 71.9, 71.8, 71.7, 71.7, 71.0, 70.9, 70.1, 69.8, 69.7,
69.4, 68.6, 67.8, 67.4, 67.1, 66.3, 63.2, 63.0, 62.6, 62.1, 61.9, 61.1, 60.9, 55.1, 53.6, 53.1, 53.0, 49.9,
49.4, 48.8, 37.9, 37.2, 37.1, 36.6, 32.7, 32.2, 31.9, 30.0, 29.7, 29.6, 29.5, 29.5, 29.3, 29.3, 29.2, 28.8,
27.1, 25.5, 23.6, 23.2, 23.1, 22.7, 21.3, 21.0, 20.9, 20.8, 20.7, 20.7, 20.6, 20.5, 20.5, 19.7, 14.1. m/z
(MALDI): found [M+Na]+ 3709.15, C184H239N5O73 calcd for [M+Na]+ 3709.50.

Molecules 2012, 17
9616
OAc
e
AcO
CO2Me
O
AcHN
AcO
O
AcO
AcHN
OAc
O
AcO
O
c
O
a
d
O
O
BzO
O
AcO
OAc
g
BzO
AcO
AcO
HN
O
C17H35
C13H27
CO2Me
OBz
OAc
BzO
O
HO
O
O
h
O
O
O
f
O
AcHN
b
O
O
O
O
AcO
O
OBz
OBz
AcO
OAc
NHAc
AcO
(2S,3R,4E)-1-O-({(Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero--D-galacto-2-
nonulopyranosylonate)-(23)-4-O-acetyl-2,6-di-O-benzoyl--D-galactopyranosyl-(14)-2-acetamido-
3,6-di-O-acetyl-2-deoxy--D-glucopyranosyl-(13)-[(methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-
dideoxy-D-glycero--D-galacto-2-nonulopyranosylonate)-(26)-4-O-acetyl-2,3-di-O-benzoyl--D-
galactopyranosyl-(14)-2-acetamido-3,6-di-O-acetyl-2-deoxy--D-glucopyranosyl-(16)]-4-O-acetyl-
2-O-benzoyl--D-galactopyranosyl}-(14)-2-O-benzoyl--D-glucopyranosyl)-2-octadecanamido-3,6'-
succinyl-octadec-4-ene-1,3-diol (40). To a solution of 39 (9.0 mg, 2.47 mol) in CH2Cl2 (0.60 mL)
was added TFA (0.30 mL) at 0 °C. After stirring for 30 min at 0 °C as the reaction was monitored by
TLC (10:1 CHCl3–MeOH), the reaction was quenched by the addition of satd aq NaHCO3. The
mixture was diluted with CHCl3 and washed with brine. The organic layer was subsequently dried over
Na2SO4, and concentrated. The residue was purified by silica gel column chromatography (20:1
1
CHCl3–MeOH) to give 40 (8.0 mg, 91%). []D −6.3° (c 0.8, CHCl3); H-NMR (600 MHz, CDCl3) 
8.17–7.27 (m, 30 H, 6 Ph), 6.68 (d, 1 H, J2,NH = 8.2 Hz, NHCer), 6.21 (d, 1 H, J2,NH = 9.0 Hz, NHb),
5.85 (near dt, 1 H, J5,6 = 6.9 Hz, J = 14.4 Hz, J = 7.5 Hz, H-5Cer), 5.70 (d, 1 H, J3,4 = 3.4 Hz, H-4d),
5.59 (m, 1 H, H-8g), 5.57 (near t, 1 H, J1,2 = 8.3 Hz, J2,3 = 10.3 Hz, H-2d), 5.45 (dd, 1 H, H-3d), 5.41–5.38
(m, 1 H, H-8e), 5.35–5.32 (m, 2 H, H-2a, H-7e), 5.27–5.17 (m, 7 H, H-4a, H-3b, H-2f, H-7g, NHe,
H-3Cer, H-4Cer), 5.09 (d, 1 H, J3,4 = 2.7 Hz, H-4f), 5.06 (near t, 1 H, J1,2 = 4.8 Hz, J2,3 = 5.5 Hz, H-2h),
4.94 (d, 1 H, J5,NH =10.3 Hz, NHg), 4.91–4.81 (m, 5 H, H-3c, H-1d, H-4e, H-4g, NHc), 4.74–4.70 (m,
2 H, H-1a, H-3f), 4.62 (d, 1 H, H-1h), 4.45–4.34 (m, 6 H, H-1b, H-1c, H-9e, H-6f, H-9g, H-6h), 4.27–4.23
(m, 3 H, H-2cer, H-6a, H-6d), 4.17–3.98 (m, 11 H, H-6'a, H-6c, H-6'c, H-6d, H-5e, H-6e, H-9'e, H-9'g,
H-5h, H-6h, H-6'h), 3.88–3.65 (m, 17 H, H-3a, H-2b, H-4b, H-6b, H-6'b, H-2c, H-4c, H-5g, H-3h, H-4h,
H-1Cer, 2 OCH3), 3.60–3.53 (m, 3 H, H-5d, H-6g, H-1Cer), 3.50–3.44 (m, 2 H, H-5b, H-5f), 3.29–3.23
(m, 2 H, H-5a, H-5c), 2.56–2.39 (m, 6 H, H-3eeq, H-3geq, 2 C(=O)CH2), 2.20–1.78 (m, 56 H, 17 Ac,
H-3eax, H-6Cer, H-6'Cer, C(=O)CH2Cer), 1.62 (t, 1 H, Jgem = J3ax,4 = 12.6 Hz, H-3gax), 1.54 (s, 3 H, Ac),
1.51–1.46 (m, 2 H, C(=O)CH2CH2), 1.42 (s, 3 H, Ac), 1.38–1.20 (m, 50 H, 25 –CH2–), 0.88 (m, 6 H,
2 –CH3Cer); 13C-NMR (150 MHz, CDCl3)  172.9, 171.0, 170.9, 170.8, 170.8, 170.7, 170.6, 170.5,
170.5, 170.3, 170.3, 170.3, 170.1, 170.1, 170.0, 169.9, 169.8, 169.7, 169.7, 168.0, 167.8, 165.7, 165.4,
165.3, 165.2, 165.0, 164.7, 138.5, 134.0, 133.5, 133.3, 133.2, 130.3, 129.8, 129.7, 129.7, 129.6, 129.5,
129.4, 129.0, 129.0, 128.7, 128.6, 128.5, 128.4, 128.4, 128.4, 125.0, 101.7, 101.2, 100.7, 99.8, 99.6,
99.3, 98.5, 96.8. 82.0, 77.5, 75.2, 74.5, 74.2, 73.8, 73.5, 72.9, 72.7, 72.6, 72.5, 72.0, 71.9, 71.7, 71.2,
71.0, 70.9, 70.7, 70.1, 69.9, 69.6, 69.4, 68.6, 67.8, 67.6, 67.4, 67.2, 67.1, 66.6, 66.3, 62.9, 69.4, 68.6,
67.8, 67.6, 67.4, 67.2, 67.1, 66.6, 66.3, 62.9, 62.5, 62.4, 62.0, 61.1, 60.9, 54.2, 53.9, 53.0, 53.0, 50.1,
49.5, 48.8, 37.8, 37.2, 36.4, 32.2, 31.9, 30.4, 29.7, 29.7, 29.7, 29.6, 29.5, 29.5, 29.3, 29.2, 28.8, 25.6,
23.6, 23.2, 23.1, 22.7, 22.5, 21.4, 21.1, 20.8, 20.8, 20.7, 20.6, 20.5, 20.4, 14.1. m/z (MALDI): found

Molecules 2012, 17
9617
[M+Na]+ 3589.60, C176H231N5O72 calcd for [M+Na]+ 3589.45. HRMS (ESI): found [1/2M+Na]+
1806.2182, C176H231N5O72 calcd for [1/2M+Na]+ 1806.2181.
OH
HO
CO2H
e
O
O
AcHN
HO
HO
HO
HO
NHAc
O
O
O
HO
c
d
O
HO
OH
OH
HO
CO2H
O
HO
OH
OH
O
OH
O
OH
O
HO
h
g
O
O
f
a
AcHN
b
O
C13H27
C17H35
O
O
HO
O
OH
HO
HO
OH
HN
NHAc
HO
OH
O
{3-O-[(5-Acetamido-3,5-dideoxy-D-glycero--D-galacto-2-nonulopyranosylonic
acid)--D-
galactopyranosyl-(14)-2-acetamido-2-deoxy--D-glucopyranosyl]-6-O-[(5-acetamido-3,5-dideoxy-
D-glycero--D-galacto-2-nonulopyranosylonic acid)--D-galactopyranosyl-(14)-2-acetamido-2-
deoxy--D-glucopyranosyl]--D-galactopyranosyl}-(14)--D-glucopyranosyl-(11)-(2S,3R,4E)-2-
octadecanamido-4-octadecene-1,3-diol (1). To a solution of 40 (8.0 mg, 2.24 mol) in MeOH/THF
(1:1, 1.0 mL) was added NaOMe (28% solution in MeOH, 12.1 g, 0.672 mol) at 0 °C. After stirring
for 7 d at 40 °C as the reaction was monitored by TLC (6:4:1 CHCl3–MeOH–0.2% aq CaCl2) and
MALDI-TOF MS, water (0.1 mL) was added to the reaction mixture. After stirring for 2 d at 40 °C,
the reaction was neutralized with Dowex (H+) resin. The resin was filtered through cotton and the
filtrate was then evaporated. The residue was purified by gel filtration column chromatography (LH-20)
using MeOH as eluent followed by silica gel column chromatography (6:4:1 CHCl3–MeOH–H2O) to
give 1 (3.0 mg, 61%). []D −78.8° (c 0.4, MeOH); 1H-NMR (600 MHz, 1:1 CD3OD–D2O)  5.73 (m,
1 H, H-5Cer), 5.46 (m, 1 H, H-4Cer), 2.86 (dd, 1 H, H-3geq), 2.78 (dd, 1 H, H-3eeq), 2.20 (t, 2 H,
NHCOCH2), 2.04 (s, 14 H, H-6Cer, H-6'Cer, 4 NAc), 1.73 (m, 2 H, H-3gax, H-3eax), 1.58 (m, 2 H,
NHCOCH2CH2), 1.30 (m, 50 H, 25 –CH2–), 0.92 (t, 6 H, 2 –CH3); 13C-NMR (125 MHz, 5:6:0.5
CD3OD–D2O–CDCl3)  174.2, 174.1, 173.7, 173.3, 134.1, 128.6, 102.9, 102.3, 102.0, 101.8, 99.9,
79.9, 79.0, 77.8, 76.5, 75.1, 74.6, 74.1, 74.0, 73.5, 73.1, 72.5, 72.2, 72.0, 71.7, 71.6, 71.1, 70.9, 70.6, 70.2,
69.3, 68.7, 68.2, 67.9, 67.8, 67.5, 67.3, 66.8, 62.4, 62.2, 60.4, 59.7, 54.6, 54.5, 52.4, 51.6, 51.5, 48.1, 46.7,
39.7, 39.4, 35.6, 31.6, 31.1, 31.0, 29.0, 28.8, 28.7, 28.6, 28.6, 28.5, 28.4, 25.3, 21.8, 21.6, 21.4, 21.2, 21.1,
12.9. HRMS (ESI): found [M+Na]− 2225.0943, C98H171N5O49 calcd for [M+Na]– 2225.0940.
3.2. Materials and Methods for Binding Assay
3.2.1. Virus Preparation
A/Memphis/1/71 (H3N2) and A/Puerto Rico/8/34 (H1N1) were used in this study. The virus was
propagated in 11 days old embryonated hen’s eggs. The virus was purified by ultra-centrifugation and
stored at −80 °C before use.
3.2.2. Solid-Phase Binding Assay
Virus binding to sialylglycolipids was determined according to a method described previously [28].
Synthetic and authentic gangliosides were 2-fold serially diluted in 100% ethanol from 0.625 to

Molecules 2012, 17
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2.5 pmol/μL. Ten μL of each diluted ganglioside was placed into a well of a polystyrene
Universal-BINDTM microplates (flat-bottom, Product# 2503, Corning, Tokyo, Japan) and incubated for
approximately 1 h at 37 °C until the ethanol had completely evaporated. gangliosides were covalently
immobilized on the surface of plates by exposure for 1 min under ultraviolet irradiation (254 nm)
according to the manufacture’s instruction. Each well was blocked for 1 h at room temperature with
PBS containing 2% bovine serum albumin. After 3 washes with PBS, the plates were incubated
in solutions containing viruses in PBS (128 HA unit/50 μL/well) overnight at 4 °C. After 5 washes
with ice-cold PBS, the plates were incubated in a substrate solution containing 40 µM
2'-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid in PBS (50 μL/well) at 37 °C for 60 min to
detect virus neuraminidase (NA) activity associated with bound viruses. The reactions were terminated
by addition of 500 mM carbonate buffer (pH 10.2) (50 μL/well). Fluorescence intensity of the
4-methylumbelliferone released by viral NAs was measured at 355 nm (excitation) and 460 nm
(emission). Triplicate measurements were performed in each assay. The direct virus binding activity
was calculated as follows: Virus-binding score = [(average of NA activity of triplicate ganglioside-
immobilized wells) − (average of NA activity of triplicate ethanol-treated wells)]/[NA activity of
applied virus (128 HA units/50 μL)]. The virus-binding score was expressed as mean score ± SD.
4. Conclusions
We efficiently synthesized a novel ganglioside designed as a ligand for influenza A viruses
employing the cassette coupling approach between the heptasaccharyl sugar part and the cyclic
glucosylceramide moiety. The present study revealed that the cassette approach can be applied to the
synthesis of lacto-series gangliosides as well as ganglio-series gangliosides. This success will expand
the applicability of the cassette approach to the synthesis of other glycolipids. In addition, we
examined the binding activity of the synthesized ganglioside ligand to influenza A viruses. It was
found that the synthetic ligand is recognized by Neu2-3 and 2-6 type viruses, suggesting that a glycan
structure containing both the Neu2-3Gal and Neu2-6Gal sequences in a single molecule could exist
as a natural ligand for influenza A viruses. To identify an actual natural ligand for influenza viruses,
the synthesis of a series of gangliosides with both the Neu2-3Gal and Neu2-6Gal sequences in a
single molecule is currently underway.
Acknowledgments
This work was financially supported by the Ministry of Education, Culture, Sports, Sciences, and
Technology (MEXT) of Japan (WPI program and Grant-in-Aid for Scientific Research to M.K.,
No.17101007 and 22380067).
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