Synthesis of ganglioside analogs containing fluorescently labeled GalNAc for single-molecule imaging

Article abstract of DOI:10.1080/07328303.2019.1609019

Gangliosides, glycosphingolipids containing sialic acid(s) in the glycan moieties, as major constituents of functional membrane domains such as lipid rafts participate in various biological processes. Previously, we developed fluorescent ganglioside analogs useful for single-molecule imaging of lipid raft formations. In this paper, we describe the development of a, 6-N₃-GalNTCAc donor and its application to the synthesis of fluorescent gangliosides containing fluorescent GalNAc residue(s) at the glycan terminus, namely, fluorescent analogs of GalNAc-GD1a and asialo-GM2.

Full text of DOI:10.1080/07328303.2019.1609019

Journal of Carbohydrate Chemistry
ISSN: 0732-8303 (Print) 1532-2327 (Online) Journal homepage: https://www.tandfonline.com/loi/lcar20
Synthesis of ganglioside analogs containing
fluorescently labeled GalNAc for single-molecule
imaging
Machi Koikeda, Naoko Komura, Hide-Nori Tanaka, Akihiro Imamura,
Hideharu Ishida, Makoto Kiso & Hiromune Ando
To cite this article: Machi Koikeda, Naoko Komura, Hide-Nori Tanaka, Akihiro Imamura, Hideharu
Ishida, Makoto Kiso & Hiromune Ando (2019): Synthesis of ganglioside analogs containing
fluorescently labeled GalNAc for single-molecule imaging, Journal of Carbohydrate Chemistry, DOI:
10.1080/07328303.2019.1609019
To link to this article: https://doi.org/10.1080/07328303.2019.1609019
Published online: 27 May 2019.
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JOURNAL OF CARBOHYDRATE CHEMISTRY
https://doi.org/10.1080/07328303.2019.1609019
Synthesis of ganglioside analogs containing
fluorescently labeled GalNAc for
single-molecule imaging
Machi Koikedaa, Naoko Komurab , Hide-Nori Tanakab, Akihiro Imamuraa,
Hideharu Ishidaa,b, Makoto Kisoa,c, and Hiromune Andob,c
aCenter for Highly Advanced Integration of Nano and Life Sciences (G-CHAIN), Gifu University,
b
c
Gifu, Japan, Department of Applied Bioorganic Chemistry, Gifu University, Gifu, Japan; Institute
for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Kyoto, Japan
ABSTRACT
ARTICLE HISTORY
Received 4 February 2019
Accepted 26 March 2019
Gangliosides, glycosphingolipids containing sialic acid(s) in the
glycan moieties, as major constituents of functional mem-
brane domains such as lipid rafts participate in various bio-
logical processes. Previously, we developed fluorescent
ganglioside analogs useful for single-molecule imaging of lipid
raft formations. In this paper, we describe the development of
a, 6-N3-GalNTCAc donor and its application to the synthesis of
fluorescent gangliosides containing fluorescent GalNAc resi-
due(s) at the glycan terminus, namely, fluorescent analogs of
GalNAc-GD1a and asialo-GM2.
KEYWORDS
Lipid raft; single-molecule
imaging; ganglioside;
GalNAc-GD1a; asialo-GM2
GRAPHICAL ABSTRACT
Introduction
Gangliosides are sialic acid (Neu)-containing glycosphingolipids. Hundreds
of different ganglioside species are widely distributed throughout the
CONTACT Naoko Komura
Sciences (G-CHAIN), Gifu University, Gifu, Japan; Hiromune Ando
komura@gifu-u.ac.jp
Center for Highly Advanced Integration of Nano and Life
hando@gifu-u.ac.jp Center for Highly
Advanced Integration of Nano and Life Sciences (G-CHAIN), Gifu University, Gifu, Japan; Institute for Integrated
Cell-Material Sciences (WPI-iCeMS), Kyoto University, Kyoto, Japan
ß 2019 Taylor & Francis Group, LLC

2
M. KOIKEDA ET AL.
nervous system and participate in various biological processes, such as syn-
aptic transmission, neuronal differentiation, and neuronal cell adhesion.[1]
Gangliosides are present in cell plasma membranes (PMs) and function
through both vertical interactions with proteins in the PMs of other cells
(trans interactions) and lateral interactions with proteins in the PM of the
same cell (cis interactions). The trans interactions of gangliosides have been
studied thoroughly using X-ray crystallography and NMR spectroscopy,
revealing the importance of the sialic acid residues.[2,3] However, the cis
interactions of gangliosides remain poorly understood at the molecular
level owing to the complexity of the processes involved, including transient
segregation with other lipids and proteins into the membrane domains
referred to as lipid rafts.[4,5]
Single-molecule imaging of fluorescently labeled membrane molecules is
a powerful technique for elucidating their dynamic behavior, lateral interac-
tions, and formation of lipid rafts.[6] Endogenous gangliosides in PMs have
been indirectly detected using fluorescently tagged antibodies and ganglio-
side-binding proteins such as cholera toxin, which specifically binds to
GM1. However, these biomolecules are generally multivalent and thus
crosslink the gangliosides, altering their characteristics and behavior in the
PMs. We previously developed fluorescent ganglioside analogs useful for
single-molecule imaging. The fluorescent nature of these analogs permitted
visualization of their dynamic behavior, transient lateral associations with
lipid-raft-associated proteins and glycosylphosphatidylinositol-anchored
proteins (CD59, ꢀ40 ms lifetime), and lipid raft formation at the single-
molecule level in the PMs.[7]
Our previous research into ganglioside probes revealed that the fluores-
cent dye must be introduced at the terminus of the glycan moiety of the
ganglioside to maximally retain the biophysical and biochemical properties
of the native ganglioside.[7,8] To synthesize the glycan-labeled gangliosides,
we developed amino-modified saccharide units that permitted the site-
selective conjugation of the unprotected gangliosides with a fluorescent dye,
namely, 9-NHTFAc-sialyl a(2,3)Gal acceptor for GM3, 6-NHTFAc-Gal
donor for GM1, and 6-N3-GalN donor for GM2. From a synthetic perspec-
tive, the development of amine-modified GalN units was challenging due
to the limited availability of suitable protecting groups for the amine moi-
ety, although this was considered valuable because the chemistry of the
GalN unit would be extendable to the synthesis of fluorescent glycans con-
taining GlcNAc. Previously, we briefly reported the chemical synthesis of a
fluorescent analog of ganglioside GM2 using the 6-N3-GalN donor as the
key intermediate.[7,8] Herein, we describe in detail the synthesis of the 6-
N3-GalN donor, in addition to the syntheses of new fluorescent analogs of
[9]
ꢀ
a ganglioside related to Guillain–Barre syndrome, GalNAc-GD1a
and

JOURNAL OF CARBOHYDRATE CHEMISTRY
3
asialo-GM2, which are expected to be useful for evaluating the role of the
sialic acid residues of gangliosides in raft-related interactions.
Results and discussion
In our previous study,[7] we found that ganglioside analogs labeled at the
glycan terminus exhibited behavior similar to that of native gangliosides in
PMs and could be used for single-molecule imaging, whereas other analogs,
such as those labeled at the central region of the glycan moiety or at the
lipid moiety, displayed altered biophysical properties in PMs. We also dem-
onstrated the necessity of using a highly hydrophilic fluorescent dye for the
analogs to act in the same way as the native gangliosides in PMs. Based on
these results, to synthesize a fluorescent analog of GM2, we introduced the
fluorescent dye at an outermost position of the glycan moiety, the C6 pos-
ition of GalNAc, and confirmed that the fluorescent GM2 analog exhibited
similar biophysical properties to native GM2. Similarly, for GalNAc-GD1a
and asialo-GM2, we selected the C6 position of GalNAc for labeling with a
fluorescent dye (ATTO594) via an amide bond, affording the target mole-
cules, 594-G6-GalNAc-GD1a (1) and 594-G6-asialo-GM2 (2) (Figure 1).
Retrosynthetic analysis of the GalNAc-GD1a probe was based on the
synthesis of the natural ganglioside GalNAc-GD1a previously reported by
our group (Scheme 1).[10] According to our previous report of fluorescent
gangliosides, we elected to introduce the chemically unstable fluorescent
dye in the final step of the synthesis. Therefore, the 6-OH group of the ter-
minal GalNAc was replaced with an amino group to allow for incorpor-
ation of the fluorescent dye via an amide linkage in the final step. Thus,
the target molecule was expected to be accessible from the corresponding
aminoganglioside 3. Compound 3 was then disconnected at the b-(1,4)-
glycosidic linkage in the Gal-Glc sequence to provide the hexasaccharide
Figure 1. Structures of GM2 and fluorescent analogs of GalNAc-GD1a and asialo-GM2.

4
M. KOIKEDA ET AL.
Scheme 1. Retrosynthetic analyses of the fluorescent analogs 1 and 2.
donor 4, in which the amino group of the GalNAc residue was protected
with a base-labile trifluoroacetyl (TFAc) group, and the Glc-Cer cassette
5.[11–13] Disconnection of the hexasaccharide component 4, which is the
tandem sequence of a trisaccharide (referred to as GM2 core), gave the
GM2 core donor 6 and the previously reported GM2 core acceptor 8.[10]
For the synthesis of 6, we designed a GalN donor with the appropriate
chemistry for the b-selective glycosylation with 9[14] and subsequent con-
version into 6-NHTFAc-GalNAc. Owing to the difficulty of the direct
replacement of a hydroxyl group with NHTFAc, a stepwise introduction of
the NHTFAc group involving the installation and reduction of an azide
group followed by trifluoroacetylation was conceived. To achieve this
design, we envisaged that a trichloroacetamide (NHTCAc) group at the C2
position could be exploited as both a neighboring group and a precursor of
the desired acetamide group. Thus, the 6-azido-GalNTCAc donor could be
used for the glycosylation of 9, which could be followed by simultaneous
selective reduction of the azide and trichloroacetamide groups and subse-
quent trifluoroacetylation, affording a suitably protected amino-containing
GM2 core intermediate. A similar synthetic route was conceived for the

JOURNAL OF CARBOHYDRATE CHEMISTRY
5
asialo-GM2 analog 2, which started from the coupling of the 6-azido-
GalNTCAc donor with Gal acceptor 10.[15]
First, we focused on developing a 6-azido-GalNTCAc donor that allowed
for efficient glycosylation and subsequent selective conversion into the
desired 6-NHTFAc-GalNAc residue. We examined possible protecting
group strategies for the C3 and C4 hydroxyl groups suitable for b-glycosy-
lation (Scheme 2). The N-TCAc derivative 14[16] was converted into triol
15, which was subsequently transformed into 16 by regioselective acetona-
tion of the 3,4-diol. Tosylation of the C6 hydroxyl group of 16 followed by
treatment with NaN3 and 18-crown-6 in N,N-dimethylacetamide (DMA) at
80 ꢁC afforded 6-azido-GalNTCAc donor 18. Subsequent acidic hydrolysis
of the acetonide and acetylation furnished 3,4-di-O-acetyl-GalNTCAc
donor 20. To exploit the arming effect of an ether protecting group,[17,18]
we also examined the synthesis of a 4-O-benzyl-GalNTCAc donor. In this
case, triol 15 was converted into 6-OH derivative 23 via 4,6-benzylidena-
tion[19], acetylation, and reductive ring cleavage of the benzylidene group
using TESH and PhBCl2. Next, a tosyl group was introduced at the 6-OH
position to afford 24, and subsequent treatment with N,N,N0,N0-tetrame-
thylguanidinium azide (TMGN3)[20] at 80 ꢁC delivered 4-O-Bn donor 25 in
a moderate yield (44%), which was accompanied by degradation of the
starting material and desired product. Although the synthesis of 25 was
also attempted via triflate 26, the reaction of 23 with Tf2O and 2,6-lutidine
provided 6-SPh oxazoline 27 as the main product. Compound 27 might be
produced immediately from 26 via intramolecular replacement of the
Scheme 2. Synthesis of GalN donors. (a) PhSH, BF3ꢂEt2O/CH2Cl2, rt, 65%; (b) NaOMe/MeOH, rt;
(c) CSA/2,2-dimethoxypropane, rt; (d) MeOH-H2O, reflux, 93% (3 steps); (e) TsCl/Pyr, rt, 92%; (f)
NaN3, 18-crown-6/DMA, 80 ꢁC, 44%; (g) 80% AcOH aq., 50 ꢁC; (h) Ac2O, DMAP/Pyr, rt, quant. (2
steps); (i) BDA, CSA/MeCN-THF, rt, 94% (2 steps); (j) Ac2O, DMAP/Pyr, rt, 94%; (k) TESH, PhBCl2/
CH2Cl2, MS4Å, ꢃ20 ꢁC; (l) TsCl/Pyr, rt, 94% (2 steps); (m) TMGN3/DMA, 80 ꢁC, 44%; (n) Tf2O, 2,6-
lutidine/CH2Cl2, ꢃ20 ꢁC, 27 (75%).

6
M. KOIKEDA ET AL.
Scheme 3. Synthesis of GalN donors. (a) H2NNH2ꢂAcOH/DMF, rt, 78%; (b) TBDPSCl, imidazole/
DMF, 60 ꢁC, 95%; (c) NaOMe/MeOH, rt; (d) BDA, p-TsOH/MeCN, rt; (e) Ac2O/Pyr, rt; (f) TESH,
PhBCl2/CH2Cl2, MS4Å, ꢃ20 ꢁC, 87% (4 steps); (g) Tf2O, 2,6-lutidine/CH2Cl2, ꢃ20 ꢁC; (h) NaN3, 18-
crown-6/DMA, 60 ꢁC, 87% (2 steps); (i) TBAF, AcOH/THF, rt, 98%; (j) CCl3CN, DBU/CH2Cl2, 0 ꢁC,
73%; (k) PhSH, TMSOTf/CH2Cl2, MS AW-300, 0 ꢁC, 69%.
triflate at the C6 position by the nucleophilic sulfur atom at the C1 pos-
ition, facilitated by the nucleophilic participation of the NHTCAc group, to
form the oxazoline as depicted in Scheme 2.
Following these results, triflate 34 bearing a TBDPS ether moiety at the
C1 position was synthesized and examined for azidation at the C6 position
(Scheme 3). The 1-O-TBDPS derivative 33, which was prepared from
GalNTCAc tetraacetate 13, was treated with Tf2O and 2,6-lutidine to afford
the 6-OTf derivative 34. Next, triflate 34 was reacted with NaN3 at 60 ꢁC to
afford 35 in an 81% yield over two steps. Azide 35 was readily converted
into trichloroacetimidate 37 via TBDPS deprotection using TBAF in the
presence of AcOH followed by treatment with CCl3CN and DBU at 0 ꢁC.
Trichloroacetimidate 37 was further converted into thioglycoside 25 by
reaction with PhSH in the presence of TMSOTf.
Next, 6-azido-GalNTCAc donors 18, 20, 25, and 37 were used to glyco-
sylate an equimolar amount of sialyl a(2,3)Gal (Neu-Gal) acceptor 9 at
0 ꢁC in CH2Cl2 (Table 1). Attempted activation of 3,4-O-isopropylidene-
protected donor 18 using N-iodosuccinimide (NIS) and TfOH was unsuc-
cessful, unexpectedly generating oxazoline 41 as the predominant product
(94%, entry 1). Reaction of 4-O-Ac-protected donor 20 under the same
conditions afforded the corresponding trisaccharide 39 in a 42% yield and
the oxazoline derivative 42 in a 57% yield (entry 2). The coupling yield
increased to 76% upon using 1.5 equiv of donor 20 (entry 3). In contrast,
the glycosylation reaction of 4-O-Bn GalNTCAc donor 25 (1.0 equiv) pref-
erentially provided trisaccharide 40 in a 73% yield (entry 4), which
increased to a 84% yield upon using 1.5 equiv of donor (entry 5). A com-
parable yield was obtained for trichloroacetimidate donor 37 in the pres-
ence of TMSOTf as promoter (entry 6). The glycosidation of 20 (entry 2,
80 min) proceeded more slowly than that of 25 (entry 4, 30 min). These
results indicated that the C4 protecting group strongly affected the reactiv-
ity and coupling yields of 6-azido-GalNTCAc donors. The b-galactosamini-
dation reactions proceeded via the 1,3-oxazolenium intermediate, and thus
competed with oxazoline formation. The 4-O-Bn group in 25 might stabil-
ize the oxonium cation intermediate, decreasing the likelihood of forming

JOURNAL OF CARBOHYDRATE CHEMISTRY
7
a
Table 1. Results of galactosaminidation to produce the GM2 core trisaccharide.
Conversion yield.
the 1,3-oxazolenium intermediate. Then, b-glycosylation might be directed
by electrostatic interaction between the TCAc group and the anomeric cen-
ter in the vicinity of the a face.
For the synthesis of asialo-GM2, equimolar amounts of 4-O-Bn thiogly-
coside 25 and Gal acceptor 10 were coupled to afford disaccharide 44 in
an 84% yield (Scheme 4). The higher yield of this reaction compared with
the reaction to form trisaccharide 40 (73%, Table 1, entry 4) may be attrib-
utable to reduced steric hindrance at the 4-OH group of the Gal moiety
owing to the smaller substituent at the C3 position.
Next, the GM2 core 40 and the asialo-GM2 core 44 were converted into
the corresponding glycosyl donors 6 and 7, respectively (Scheme 5). For
this step, we first addressed the simultaneous conversion of TCAc and
azide groups in the GalN residues into acetyl (Ac) and amino groups,
respectively. Treatment with Zn in AcOH was found to be the most effi-
cient method for reducing trisaccharide 40 to 2-NHAc-6-NH2 derivative 45
(87%). Next, the amino group in 45 was protected with the trifluoroacetyl
group. The protecting groups in GM2 trisaccharide 46 were manipulated
through hydrogenolysis of the three benzyl groups and subsequent

8
M. KOIKEDA ET AL.
Scheme 4. Synthesis of the disaccharide moiety of asialo-GM2.
Scheme 5. (A) Synthesis of GM2 core donor. (a) H2, Pd(OH)2-C/EtOH, rt, 96%; (b) Bz2O, DMAP/
Pyr, rt, 91%; (c) CAN, H2O/MeCN-toluene, 0 ꢁC, 91%; (d) CCl3CN, DBU/CH2Cl2, 0 ꢁC, 79%. (B)
Synthesis of asialo-GM2 core donor. (a) NaOMe/MeOH-CH2Cl2, rt; (b) H2, Pd(OH)2-C/1,4-dioxane,
rt; (c) Bz2O, DMAP/Pyr, rt, 89% (3 steps); (d) CAN, H2O/MeCN-toluene, 0 ꢁC, 82%; (e) CCl3CN,
DBU/CH2Cl2, 0 ꢁC, 92%.
benzoylation to afford 48 (Scheme 5A). The cleavage of the MP group in
48 using cerium(IV) ammonium nitrate in an aqueous solvent mixture
afforded the hemiacetal, which was transformed into the 6-NHTFAc-con-
taining GM2 core trichloroacetimidate 6. Similar procedures were used to
efficiently convert disaccharide donor 44 into donor 7 for asialo-GM2
(Scheme 5B).
To our delight, the glycosylation of GM2 acceptor 8 with 6-NHTFAc-
containing GM2 core donor 6, promoted by TMSOTf in CH2Cl2 at 0 ꢁC,
afforded successfully GalNAc-GD1a hexasaccharide 56 in a 72% yield
(Scheme 6). Subsequent hydrogenolysis of the benzyl groups followed by
benzoylation delivered 58 in a high yield. To permit the final coupling with
Glc-Cer cassette 5, 58 was converted into trichloroacetimidate donor 4 in a
similar manner as that with the GM2 donor 6.
With glycosyl donors 4 and 7, we constructed the glycolipid frameworks
of GalNAc-GD1a and asialo-GM2. Thus, Glc-Cer acceptor 5 (1.5 equiv)
were glycosylated with the two donors in the presence of TMSOTf in
CH2Cl2, furnishing the protected GalNAc-GD1a 60 (71%) and asialo-GM2
61 (75%) in high yields (Table 2).

JOURNAL OF CARBOHYDRATE CHEMISTRY
9
Scheme 6. Synthesis of GalNAc-GD1a core donor. (a) H2, Pd(OH)2-C/1,4-dioxane, rt, 92%; (b)
Bz2O, DMAP/Pyr, rt, 95%; (c) CAN, H2O/MeCN-toluene, 0 ꢁC, 72%; (d) CCl3CN, DBU/CH2Cl2,
0 ꢁC, 82%.
Table 2. Synthesis of ganglioside frameworks.
Finally, 60 and 61 were fully deprotected to afford the C6-amino deriva-
tives 3 and 12, respectively, which were then reacted with the activated
ester of ATTO594 in DMF/H2O, delivering the target molecules of 594-G6-
GalNAc-GD1a (1) and 594-G6-asialo-GM2 (2) (Scheme 7).
Conclusion
We have developed a 6-N3-GalNTCAc donor and applied it to the synthe-
sis of fluorescent analogs of gangliosides GalNAc-GD1a and asialo-GM2.
The combination of the 6-N3 and C2-NHTCAc groups allowed single-step
conversion into the 6-NHTFAc-GalNAc residue following the assembly of
the GM2 core trisaccharide and asialo-GM2 disaccharide. The glycosylation
reactions of GalN donors revealed the influence of the 4-OH protecting
group on the coupling efficiency. Furthermore, the use of the 6-NHTFAc-
GalNAc-containing saccharide units permitted fluorescent analogs of
GalNAc-GD1a and asialo-GM2 to be successfully realized for the first time.

10
M. KOIKEDA ET AL.
Scheme 7. Global deprotection and fluorescent labeling.
Biophysical experiments using these fluorescent probes are currently under
way. These fluorescent analogs are expected to prove valuable for elucidat-
ing raft-associated neuropathies.
Experimental section
General procedures
1H- and 13C-NMR spectra were recorded using Bruker Avance III 500 and
Avance III 800 spectrometers. Chemical shifts are expressed in ppm (d)
relative to Me4Si as an internal standard. High-resolution mass spectrom-
etry (HRMS) was performed using a Bruker Daltonics micrOTOF (ESI-
TOF) spectrometer. Specific rotations were determined using a Horiba
SEPA-300 high-sensitivity polarimeter. Molecular sieves were purchased
from FUJIFILM Wako Pure Chemical Corporation and dried at 300 ꢁC for
2 h in a muffle furnace prior to use. Dry reaction solvents (CH2Cl2, toluene,
THF, CH3CN, DMF, and pyridine) were purchased from Kanto Chemical
Co. Inc. and used without further purification. Thin-layer chromatography
(TLC) was performed on Merck TLC plates (silica gel 60 F254 on glass
plates), and analytes were detected by either UV illumination (253.6 nm) or
soaking in an ethanolic solution of 10% H2SO4 followed by heating. Flash
column chromatography was performed using silica gel (80 mesh and 300
mesh) manufactured by Fuji Silysia Co. The quantity of silica gel was typic-
ally estimated as 150–200 times the sample weight to be charged. The chro-
matographic solvent systems are specified by volume. Evaporation and
condensation steps were performed under vacuum. ATTO594 N-succini-
midyl ester was purchased from ATTO-Tec. The sugar units are numbered
using letters from a to g; GalNAc-GD1a: GalNAc (a), Gal (b), and NeuAc

JOURNAL OF CARBOHYDRATE CHEMISTRY
11
(c) in terminal GM2 core, GalNAc (e), Gal (f), and NeuAc (g) in inner
GM2 core, Glc (d); asialo-GM1: GalNAc (a), Gal (b), and Glc (d) (e.g., see
Table 2).
594-G6-GalNAc-GD1a (1)
Compound 3 (4.1 mg, 2.9 mmol) and ATTO594 N-succinimidyl ester (4.2 mg,
3.0 mmol) were dissolved in DMF/H2O (180 mL/17mL), and triethylamine
(5.6 mL, 40 mmol) was added at room temperature. After the reaction had
reached completion, as confirmed by TLC (CHCl3/MeOH/H2O ¼ 5:6:2), the
reaction mixture was purified by column chromatography on Sephadex LH-
20, using CHCl3/MeOH (1:1) followed by preparative TLC (CHCl3/MeOH/
1
H2O ¼ 5:5:1) to afford 594-G6-GalNAc-GD1a (1) (4.0 mg, 70%): H NMR
(500 MHz, CD3OD) d 7.75–7.38 (m, 4 H, ArATTO594), 7.33–5.88 (m, 6 H,
ArATTO594), 5.68 (dt, 1 H, J5,6a ¼ J5,6b ¼ 6.7 Hz, J4,5 ¼ 15.3Hz, H-5Cer), 5.44
(dd, 1 H, J3,4 ¼ 7.7 Hz, H-4Cer), 4.89-3.11 (m, 63H, H-1a, H-2a, H-3a, H-4a,
H-5a, H-6aa, H-6ba, H-1b, H-2b, H-3b, H-4b, H-5b, H-6ab, H-6bb, H-4c, H-5c,
H-6c, H-7c, H-8c, H-9ac, H-9bc, H-1d, H-2d, H-3d, H-4d, H-5d, H-6ad, H-6bd,
H-1e, H-2e, H-3e, H-4e, H-5e, H-6ae, H-6be, H-1f, H-2f, H-3f, H-4f, H-5f, H-
6af, H-6bf, H-4g, H-5g, H-6g, H-7g, H-8g, H-9ag, H-9bg, H-1aCer, H-1bCer, H-
2
Cer, H-3Cer, 3 NCH2ATTO594, 2 CH2SO3ATTO594), 2.76–2.64 (m, 5 H, H-3eqc,
H-3eqg, NMeATTO594), 2.18–2.15 (m, 2 H, NHCOCH2Cer), 2.01–1.99 (m,
14H, H-6aCer, H-6bCer, 4 Ac), 1.90-1.83 (m, 4 H, H-3axc, H-3axg,
NCH2CH2CH2ATTO594), 1.69-1.49 (m, 14 H, NHCOCH2CH2Cer, 4 MeATTO594),
1.39–1.29 (m, 58H, 25 CH2Cer, NCH2CH2ATTO594, 2 NCH2CH3ATTO594),
0.91–0.88 (m, 6 H, 2 MeCer); 13C NMR (200 MHz, CD3OD) d 175.9, 175.6,
175.6, 175.2, 175.1, 175.0, 174.7, 174.6, 171.0, 169.8, 161.5, 159.2, 154.9,
154.2, 154.1, 138.5, 138.5, 137.6, 135.1, 132.2, 131.6, 131.4, 131.2, 130.8,
129.5, 129.4, 128.8, 126.1, 126.0, 124.2, 122.8, 122.8, 115.0, 115.0, 106.5,
104.9, 104.5, 104.4, 104.2, 103.5, 103.3, 97.1, 83.0, 81.0, 79.6, 78.8, 76.5, 76.4,
76.3, 76.1, 75.8, 75.6, 75.3, 75.2, 75.1, 74.9, 74.2, 74.0, 73.5, 73.4, 73.0, 71.0,
71.0, 70.4, 70.0, 69.9, 69.7, 69.7, 69.6, 65.3, 64.9, 63.0, 61.8, 61.7, 61.7, 54.7,
54.2, 54.1, 53.9, 53.9, 53.8, 52.5, 49.8, 49.5, 49.4, 49.3, 47.8, 41.4, 41.4, 40.9,
38.8, 38.7, 38.6, 37.4, 33.9, 33.5, 33.1, 33.1, 32.7, 30.9, 30.9, 30.8, 30.8, 30.7,
30.7, 30.5, 30.5, 30.5, 30.4, 29.5, 29.4, 29.3, 29.3, 27.2, 23.8, 23.8, 23.6, 22.6,
14.5, 13.8, 13.7; HRMS (ESI) m/z: found [M–3H]3– 941.1016,
C133H207N9O52S2 calcd for [M–3H]3– 941.1018.
594-G6-asialo-GM2 (2)
Compound 12 (3.2 mg, 2.9 mmol) and ATTO594 N-succinimidyl ester
(6.2 mg, 4.5 mmol) were dissolved in DMF/H2O (270 mL/27 mL), and

12
M. KOIKEDA ET AL.
triethylamine (12.2 mL, 87.5 mmol) was added in two portions over 2.5 h at
room temperature. After the reaction had reached completion, as con-
firmed by TLC (CHCl3/MeOH/H2O ¼ 5:4:1), the reaction mixture was
purified by column chromatography on Sephadex LH-20, using CHCl3/
MeOH (1:1) followed by preparative TLC (CHCl3/MeOH/H2O ¼ 5:2:0.1) to
1
afford 594-G6-asialo-GM2 (2) (2.8 mg, 51%): H NMR (800 MHz, CD3OD)
d 7.73-5.89 (m, 10 H, ArATTO594), 5.70 (m, 1 H, H-5Cer), 5.44 (m, 1 H, H-
4Cer), 4.59-3.21 (m, 35 H, H-1a, H-2a, H-3a, H-4a, H-5a, H-6aa, H-6ba, H-
1b, H-2b, H-3b, H-4b, H-5b, H-6ab, H-6bb, H-1d, H-2d, H-3d, H-4d, H-5d,
ATTO594
H-6ad, H-6bd, H-1aCer
2CH2SO3
,
H-1bCer
,
H-2Cer
,
H-3Cer
,
3NCH2
,
ATTO594)
, 2.73-2.64 (2 s, 3 H, NMeATTO594), 2.17-2.03 (m, 7 H, H-
Cer
6aCer, H-6bCer, NHCOCH2Cer, Ac), 1.76–1.29 (m, 74 H, 26CH2
,
,
ATTO594
ATTO594
ATTO594
NCH2CH2
,
NCH2CH2CH2
,
2NCH2CH3
4 MeATTO594), 0.91–0.89 (m, 6 H, 2 MeCer); 13C NMR (200 MHz, CD3OD) d
175.9, 175.4, 175.3, 175.1, 175.0, 171.5, 171.2, 159.2, 155.1, 154.9, 154.2,
154.1, 138.4, 138.3, 137.5, 137.4, 135.1, 132.1, 132.0, 131.9, 131.7, 131.4,
131.2, 131.0, 130.8, 130.3, 129.4, 128.8, 126.1, 126.0, 124.4, 124.1, 124.1,
122.9, 122.9, 115.1, 115.0, 114.9, 105.1, 104.4, 104.3, 104.1, 97.0, 80.8, 80.8,
78.2, 78.0, 76.4, 76.2, 76.1, 74.9, 74.8, 74.4, 74.4, 74.3, 72.9, 72.6, 72.5, 69.8,
69.8, 61.8, 61.7, 61.7, 55.1, 55.1, 54.6, 54.2, 53.8, 53.8, 47.3, 41.3, 41.3, 38.7,
37.4, 33.6, 33.5, 33.1, 33.1, 32.6, 30.9, 30.9, 30.8, 30.8, 30.8, 30.7, 30.7, 30.5,
30.5, 30.5, 30.4, 29.5, 29.4, 29.3, 27.2, 23.9, 23.8, 23.1, 14.5, 13.8, 13.7, 13.7;
HRMS (ESI) m/z: found [M–H]– 1877.9969, C97H149N6O26S2 calcd for
[M–H]– 1877.9968.
4-Methoxyphenyl (3-O-acetyl-6-azido-4-O-benzyl-2,6-dideoxy-2-
trichloroacetamido-b-D-galactopyranosyl)-(1fi4)-[(methyl 5-acetamido-
4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-a-D-galacto-2-
nonulopyranosylonate)-(2fi3)]-2,6-di-O-benzyl-b-D-galactopyranoside (40)
To a solution of 9 (81.9 mg, 87.1 lmol) and 25 (75.7 mg, 132 lmol) in
CH2Cl2 (4.4 mL) were added 4 Å molecular sieves (224 mg) at room tem-
perature under an Ar atmosphere, and the resulting mixture was stirred for
30 min. NIS (51.3 mg, 228 lmol) and TfOH (2.1 lL, 24 lmol) were then
added to the mixture at 0 ꢁC. After stirring for 1 h at 0 ꢁC, as the reaction
was monitored by TLC (n-hexane/AcOEt ¼ 4:1, developed twice), the solu-
tion was filtered through a Celite pad and the pad was washed with CHCl3.
The combined filtrate and washings were extracted with CHCl3, washed
with saturated NaHCO3, Na2S2O3, and brine, dried over Na2SO4, and con-
centrated. The residue was purified by column chromatography on silica
gel, using toluene/acetone (5:1) to afford 40 (102 mg, 84%): [a]D ꢃ17.0ꢁ (c
1
1.1, CHCl3); H NMR (500 MHz, CDCl3) d 7.45–6.78 (m, 20 H, NH-2a,

JOURNAL OF CARBOHYDRATE CHEMISTRY
13
Ar), 5.24-5.18 (m, 5 H, H-3a, H-4c, H-7c, H-8c, NH-5c), 5.16 (d, 1 H,
J1,2 ¼ 8.6 Hz, H-1a), 5.04–4.92 (2d, 2 H, CH2Ph), 4.87 (d, 1 H, J1,2 ¼ 7.7 Hz,
H-1b), 4.64–4.50 (m, 5 H, H-2a, CH2Ph), 4.14 (m, 1 H, H-5a), 4.10 (d, 1 H,
J3,4 ¼ 2.1 Hz, H-4a), 4.06–4.00 (m, 3 H, H-3b, H-9ac, H-9bc), 3.94 (s, 3 H,
COOMe), 3.93–3.79 (m, 6 H, H-2b, H-4b, H-6ab, H-6bb, H-5c, H-6c), 3.77
(s, 3 H, OMe), 3.69 (m, 1 H, H-5b), 3.51 (dd, 1 H, J5,6a ¼ 7.3 Hz,
Jgem ¼ 12.7 Hz, H-6aa), 3.08 (dd, 1 H, J5,6b ¼ 5.3 Hz, H-6ba), 2.29 (dd, 1 H,
J3ax,4 ¼ 11.5 Hz, Jgem ¼ 14.3 Hz, H-3axc), 2.14–2.09 (m, 7 H, H-3eqc, 2 Ac),
2.01-1.88 (4 s, 12 H, 4 Ac); 13C NMR (125 MHz, CDCl3) d 170.8, 170.6,
170.4, 169.7, 169.4, 168.6, 162.0, 155.4, 151.4, 138.5, 138.2, 137.9, 128.6,
128.4, 128.4, 128.3, 128.1, 128.0, 127.8, 127.7, 127.6, 118.7, 114.6, 103.3,
100.9, 99.5, 92.9, 77.5, 77.3, 77.2, 76.9, 75.3, 75.3, 75.0, 74.6, 74.4, 74.3,
73.9, 73.2, 72.0, 70.3, 69.3, 67.7, 66.6, 62.0, 55.7, 53.5, 53.0, 51.1, 49.4, 35.7,
23.3, 21.1, 21.1, 20.9, 20.8, 20.7; HRMS (ESI) m/z found [M þ Na]þ
1424.3684, C64H74Cl3N5O24 calcd for [M þ Na]þ 1424.3682.
4-Methoxyphenyl (3-O-acetyl-6-azido-4-O-benzyl-2,6-dideoxy-2-
trichloroacetamido-b-D-galactopyranosyl)-(1fi4)-2,3,6-tri-O-benzyl-b-D-
galactopyranoside (44)
To a solution of 10 (62.1 mg, 111 lmol) and 25 (63.8 mg, 111 lmol) in
CH2Cl2 (4.4 mL) were added 4 Å molecular sieves (225 mg) at room tem-
perature under an Ar atmosphere, and the resulting mixture was stirred for
30 min. NIS (37.6 mg, 167 lmol) and TfOH (1.5 lL, 17 lmol) were then
added to the mixture at 0 ꢁC. After stirring for 2 h at 0 ꢁC, as the reaction
was monitored by TLC (toluene/AcOEt ¼ 9:1, developed twice), the solu-
tion was filtered through a Celite pad and the pad was washed with CHCl3.
The combined filtrate and washings were extracted with CHCl3, washed
with saturated NaHCO3, Na2S2O3, and brine, dried over Na2SO4, and con-
centrated. The residue was purified by column chromatography on silica
gel, using toluene/AcOEt (19:2) to afford 44 (95.6 mg, 84%): [a]D ꢃ2.4ꢁ (c
1
1.0, CHCl3); H NMR (500 MHz, CDCl3) d 7.40–6.74 (m, 24 H, Ar), 6.94
(d, 1 H, J2,NH ¼ 8.0 Hz, NH-2a), 5.05–5.00 (m, 2 H, H-3a, CH2Ph), 4.89 (d,
1 H, J1,2 ¼ 8.6 Hz, H-1a), 4.86–4.84 (m, 2 H, CH2Ph), 4.80 (d, 1 H,
J1,2 ¼ 7.7 Hz, H-1b), 4.76–4.49 (m, 5 H, CH2Ph), 4.35 (m, 1 H, H-2a), 4.00
(d, 1 H, J3,4 ¼ 2.9 Hz, H-4b), 3.88 (dd, 1 H, J2,3 ¼ 9.5 Hz, H-2b), 3.80–3.72
(m, 6 H, H-4a, H-6ab, H-6bb, OMe), 3.66 (m, 1 H, H-5b), 3.62 (dd, 1 H,
J3,4 ¼ 3.3 Hz, H-3b), 3.50 (m, 1 H, H-5a), 3.43 (dd, 1 H, J5,6a ¼ 7.2 Hz,
Jgem ¼ 12.4 Hz, H-6aa), 3.13 (dd, 1 H, J5,6b ¼ 9.5 Hz, H-6ba), 2.04 (s, 3 H,
Ac); 13C NMR (125 MHz, CDCl3) d 170.6, 161.8, 155.3, 151.5, 138.7, 138.5,
137.8, 137.6, 129.0, 128.7, 128.5, 128.5, 128.4, 128.4, 128.3, 127.9, 127.8,
127.7, 118.6, 114.6, 103.1, 101.1, 92.5, 81.5, 79.8, 75.4, 75.1, 74.8, 74.5, 74.0,

14
M. KOIKEDA ET AL.
73.9, 73.8, 73.7, 70.0, 55.7, 53.0, 50.9, 20.9; HRMS (ESI) m/z: found
[M þ Na]þ 1041.2618, C51H53Cl3N4O12 calcd for [M þ Na]þ 1041.2618.
4-Methoxyphenyl (2-acetamido-3-O-acetyl-4-O-benzoyl-2,6-dideoxy-6-
trifluoroacetamido-b-D-galactopyranosyl)-(1fi4)-{[(methyl 5-acetamido-
4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-a-D-galacto-2-
nonulopyranosylonate)-(2fi3)]-2,6-di-O-benzoyl-b-D-galactopyranosyl}-(1fi3)-
(2-acetamido-4,6-di-O-acetyl-2-deoxy-b-D-galactopyranosyl)-(1fi4)-[(methyl
5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-a-D-galacto-2-
nonulopyranosylonate)-(2fi3)]-2,6-di-O-benzyl-b-D-galactopyranoside (56)
To a solution of 6 (323 mg, 254 lmol) and 8 (254 mg, 175 lmol) in CH2Cl2
(8.8 mL) were added 4 Å molecular sieves (AW-300, 1.71 g) at room tem-
perature under an Ar atmosphere, and the resulting mixture was stirred for
1 h. TMSOTf (3.8 lL, 21 lmol) was then added to the mixture at 0 ꢁC.
After stirring for 2 h at 0 ꢁC, as the reaction was monitored by TLC (tolu-
ene/acetone ¼ 1:1), the reaction mixture was filtered through a Celite pad
and the pad was washed with CHCl3. The combined filtrate and washings
were extracted with CHCl3, washed with saturated Na2CO3 and brine, dried
over Na2SO4, and concentrated. The residue was purified by column chro-
matography on Sephadex LH-20, using CHCl3/MeOH (1:1) followed by
flash column chromatography on silica gel, using CHCl3/MeOH (20:1) to
1
afford 56 (318 mg, 72%): [a]D þ8.9ꢁ (c 1.4, CHCl3); H NMR (500 MHz,
CD3CN) d 8.15–6.79 (m, 30 H, NH-6a, Ar), 6.43 (d, 1 H, J2,NH ¼ 9.1 Hz,
NH-2a), 6.10–6.07 (m, 3 H, NH-5c, NH-2e, NH-5g), 5.51 (d, 1 H,
J3,4 ¼ 2.8 Hz, H-4a), 5.44 (d, 1 H, J3,4 ¼ 2.9 Hz, H-4e), 5.31 (dd, 1 H,
J2,3 ¼ 11.2 Hz, H-3a), 5.24–5.22 (m, 2 H, H-1b, H-7Neu), 5.18–5.11 (m, 3 H,
H-2b, H-8c, H-8g), 5.01 (m, 1 H, H-4Neu), 4.98 (d, 1 H, J1,2 ¼ 8.6 Hz, H-1a),
4.95–4.88 (m, 2 H, H-1f, H-4Neu), 4.90 (dd, 1 H, J6,7 ¼ 3.0 Hz, J7,8 ¼ 7.7 Hz,
H-7Neu), 4.77 (d, 1 H, CH2Ph), 4.65 (d, 1 H, J1,2 ¼ 8.6 Hz, H-1e), 4.62 (dd,
1 H, J5,6a ¼ 5.3 Hz, Jgem ¼ 11.5 Hz, H-6ab), 4.57–4.53 (m, 2 H, H-3b, CH2Ph),
4.51–4.43 (m, 3 H, H-6bb, CH2Ph), 4.36 (t, 1 H, J5,6a ¼ J5,6b ¼ 6.7 Hz, H-5a),
4.30–4.24 (m, 2 H, H-2a, H-3f), 4.14 (dd, 1 H, J2,3 ¼ 12.3 Hz, H-3e),
4.08–3.72 (m, 16 H, H-4b, H-5b, H-5c, H-6c, H-9ac, H-9bc, H-2e, H-5e, H-
6ae, H-6be, H-4f, H-5f, H-5g, H-6g, H-9ag, H-9bg), 3.78–3.77 (2 s, 6 H, 2
COOMe), 3.72 (s, 3 H, OMe), 3.63–3.55 (m, 2 H, H-6af, H-6bf), 3.46–3.35
(m, 3 H, H-6aa, H-6ba, H-2f), 2.40 (dd, 1 H, J3eq,4 ¼ 4.9 Hz, Jgem ¼ 13.5 Hz,
H-3eqNeu), 2.11–1.17 (m, 48 H, H-3eqNeu, H-3axc, H-3axg, 15 Ac); 13C
NMR (125 MHz, CD3CN) d 171.6, 171.4, 171.3, 171.2, 171.1, 171.0, 170.9,
170.9, 170.8, 170.6, 170.6, 170.5, 170.4, 169.6, 169.4, 167.2, 166.8, 165.1,
158.4, 158.1, 157.8, 157.5, 156.3, 152.5, 139.9, 139.8, 134.7, 134.3, 134.3,
131.2, 130.9, 130.7, 130.7, 130.6, 130.2, 129.9, 129.7, 129.6, 129.2, 129.1,

JOURNAL OF CARBOHYDRATE CHEMISTRY
15
128.5, 128.4, 119.1, 118.6, 115.7, 115.5, 103.3, 103.1, 102.7, 100.5, 100.3,
79.4, 79.1, 78.9, 75.9, 75.8, 74.3, 74.2, 73.6, 72.8, 72.6, 72.5, 72.2, 72.0, 71.3,
71.3, 71.0, 70.7, 70.6, 70.5, 69.3, 69.2, 68.7, 67.6, 65.0, 64.3, 62.8, 62.6, 56.2,
54.0, 53.9, 52.3, 51.4, 49.4, 48.9, 40.4, 36.2, 35.8, 32.6, 30.0, 23.3, 23.1, 23.1,
21.5, 21.5, 21.2, 21.0, 21.0, 21.0, 20.9, 14.4; HRMS (ESI) m/z: found
[M þ Na]þ 2536.8154, C118H138F3N5O52 calcd for [M þ Na]þ 2536.8152.
(2-Acetamido-3-O-acetyl-4-O-benzoyl-2,6-dideoxy-6-trifluoroacetamido-b-D-
galacto-pyranosyl)-(1fi4)-{[(methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-
dideoxy-D-glycero-a-D-galacto-2-nonulopyranosylonate)-(2fi3)]-2,6-di-O-
benzoyl-b-D-galacto-pyranosyl}-(1fi3)-(2-acetamido-4,6-di-O-acetyl-2-deoxy-
b-D-galactopyranosyl)-(1fi4)-{[(methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-
dideoxy-D-glycero-a-D-galacto-2-nonulopyranosylonate)-(2fi3)]-2,6-di-O-
benzoyl-b-D-galactopyranosyl}-(1fi4)-(2-O-benzoyl-3,6-di-O-p-methoxybenzyl-
b-D-glucopyranosyl)-(1fi1)-(2S,3R,4E)-3-O-benzoyl-2-octadecanamido-4-
octadecene-1,3-diol (60)
To a solution of 5 (69.9 mg, 59.4 lmol) and 4 (102 mg, 39.4 lmol) in
CH2Cl2 (2.5 mL) were added 4 Å molecular sieves (AW-300, 487 mg) at
room temperature under an Ar atmosphere, and the resulting mixture was
stirred for 1 h. TMSOTf (0.8 lL, 4 lmol) was then added to the mixture at
0 ꢁC. After stirring for 5 h at 0 ꢁC, as the reaction was monitored by TLC
(CHCl3/MeOH ¼ 15:1, developed twice), triethylamine was added. The
reaction mixture was filtered through a Celite pad and the pad was washed
with CHCl3. The combined filtrate and washings were extracted with
CHCl3, washed with saturated Na2CO3 and brine, dried over Na2SO4, and
concentrated. The residue was purified by flash column chromatography
on silica gel, using CHCl3/MeOH (30:1) to afford 60 (101 mg, 71%): [a]D
1
þ25.7ꢁ (c 1.5, CHCl3); H NMR (500 MHz, CDCl3) d 8.15–6.60 (m, 44 H,
NH-6a, Ar), 6.22 (d, 1 H, J2,NH ¼ 7.6 Hz, NH-2a), 5.76 (dt, 1 H,
J5,6a ¼ J5,6b ¼ 6.8 Hz, J4,5 ¼ 15.2 Hz, H-5Cer), 5.70–5.68 (m, 2 H, H-3a, NH-
2Cer), 5.64 (m, 1 H, H-8Neu), 5.52 (d, 1 H, J2,NH ¼ 6.5 Hz, NH-2e), 5.49–5.44
(m, 3 H, H-4e, H-3Cer, H-7Neu), 5.41–5.31 (m, 3 H, H-2b, H-4Cer, H-8Neu),
5.27–5.24 (m, 2 H, H-2f, H-7Neu), 5.18–5.11 (m, 4 H, H-1a, H-4a, H-2d, H-
1e), 5.07 (d, 1 H, J1,2 ¼ 7.8 Hz, H-1f), 5.07 (m, 1 H, NH-5Neu), 5.00 (dd, 1 H,
J3,4 ¼ 3.4 Hz, J2,3 ¼ 10.8 Hz, H-3e), 4.96–4.90 (m, 2 H, H-4Neu, NH-5Neu),
4.82 (d, 1 H, J1,2 ¼ 7.7 Hz, H-1b), 4.79–4.73 (m, 2 H, H-4Neu, CH2Ar), 4.69
(dd, 1 H, J5,6a ¼ 5.3 Hz, Jgem ¼ 11.5 Hz, H-6ab), 4.59 (dd, 1 H, J5,6a ¼ 6.4 Hz,
Jgem ¼ 11.0 Hz, H-6af), 4.53 (d, 1 H, CH2Ar), 4.43–4.39 (m, 2 H, H-6bb, H-
3f), 4.36–4.28 (m, 2 H, H-3b, H-2Cer), 4.35 (d, 1 H, J1,2 ¼ 7.8 Hz, H-1d), 4.24
(near dd, 1 H, H-9Neu), 4.17–4.13 (m, 2 H, H-5a, H-9Neu), 4.10-3.66 (m,
20 H, H-2a, H-4b, H-5b, H-5c, H-6c, H-3d, H-4d, H-5e, H-6ae, H-6be, H-4f,

16
M. KOIKEDA ET AL.
H-5f, H-6bf, H-5g, H-6g, H-9Neu, H-9Neu, H-1aCer, CH2Ar), 3.81–3.73 (2 s,
6 H, 2 COOMe), 3.69–3.56 (2 s, 6 H, 2 OMe), 3.51 (d, 1 H, Jgem ¼ 9.8 Hz,
H-6ad), 3.48-3.41 (m, 2 H, H-5d, H-1bCer), 3.39-3.32 (m, 2 H, H-6aa, H-
6bd), 3.26 (m, 1 H, H-6ba), 3.15 (m, 1 H, H-2e), 2.76 (dd, 1 H,
J3eq,4 ¼ 4.0 Hz, Jgem ¼ 12.8 Hz, H-3eqNeu), 2.36 (dd, 1 H, J3eq,4 ¼ 4.5 Hz,
Jgem ¼ 13.2 Hz, H-3eqNeu), 2.17-1.44 (15 s, 45 H, 15 Ac), 1.96-1.90 (m, 3 H,
H-6aCer, H-6bCer, H-3axNeu), 1.73-1.65 (m, 3 H, H-3axNeu, NHCOCH2Cer),
1.33-1.14 (m, 50 H, 25 CH2Cer), 1.06 (m, 2 H, NHCOCH2CH2Cer), 0.89-0.83
(m, 6 H, 2 MeCer); 13C NMR (125 MHz, CDCl3) d 172.7, 172.0, 171.2,
170.9, 170.8, 170.7, 170.6, 170.5, 170.5, 170.4, 170.3, 170.1, 169.8, 168.4,
168.2, 167.0, 166.7, 165.6, 165.3, 165.0, 164.4, 159.0, 158.9, 157.7, 157.4,
137.1, 133.9, 133.5, 133.4, 133.3, 133.0, 132.8, 130.5, 130.4, 130.4, 130.3,
130.2, 130.1, 130.0, 130.0, 129.9, 129.8, 129.7, 129.6, 129.6, 129.1, 128.9,
128.6, 128.4, 128.3, 124.9, 117.0, 114.7, 113.6, 113.6, 101.2, 100.9, 100.8,
100.7, 98.6, 98.5, 97.7, 80.9, 76.8, 75.9, 75.2, 74.7, 74.0, 73.5, 73.4, 72.9,
72.8, 72.3, 72.0, 71.9, 71.5, 71.1, 70.6, 70.3, 70.1, 69.6, 69.1, 69.0, 68.9, 67.5,
67.2, 67.2, 66.7, 66.5, 64.3, 63.5, 63.2, 62.6, 62.2, 55.3, 55.2, 55.0, 53.3, 52.7,
52.4, 50.6, 49.3, 39.9, 36.9, 36.5, 36.4, 32.4, 32.0, 30.1, 29.8, 29.8, 29.7, 29.6,
29.6, 29.5, 29.4, 29.4, 29.3, 29.1, 25.6, 23.4, 23.3, 23.2, 23.2, 22.8, 21.4, 21.3,
20.9, 20.9, 20.8, 20.8, 20.5, 20.3, 20.2, 14.2; HRMS (ESI) m/z: found
[M þ 2Na]2þ
1819.7374,
C183H231F3N6O64
calcd
for
[M þ 2Na]2þ 1819.7371.
(2-Acetamido-3,4-di-O-benzoyl-2,6-dideoxy-6-trifluoroacetamido-b-D-galacto-
pyranosyl)-(1fi4)-(2,3,6-tri-O-benzoyl-b-D-galactopyranosyl)-(1fi4)-(2-O-
benzoyl-3,6-di-O-p-methoxybenzyl-b-D-glucopyranosyl)-(1fi1)-(2S,3R,4E)-3-O-
benzoyl-2-octadecanamido-4-octadecene-1,3-diol (61)
To a solution of 5 (74.7 mg, 63.5 lmol) and 7 (48.4 mg, 41.9 lmol) in
CH2Cl2 (2.2 mL) were added 4 Å molecular sieves (AW-300, 146 mg) at
room temperature under an Ar atmosphere, and the resulting mixture was
stirred for 1 h. TMSOTf (0.8 lL, 4 lmol) was then added to the mixture at
0 ꢁC. After stirring for 75 min at 0 ꢁC, as the reaction was monitored by
TLC (toluene/acetone ¼ 4:1), triethylamine was added. The reaction mixture
was filtered through a Celite pad and the pad was washed with CHCl3. The
combined filtrate and washings were extracted with CHCl3, washed with
saturated Na2CO3 and brine, dried over Na2SO4, and concentrated. The
residue was purified by flash column chromatography on silica gel, using
toluene/acetone (7:2) to afford 61 (67.6 mg, 75%): [a]D þ43.5ꢁ (c 1.0,
1
CHCl3); H NMR (500 MHz, CDCl3) d 8.07-6.56 (m, 44 H, NH-6a, Ar),
5.79 (dt, 1 H, J5,6a ¼ J5,6b ¼ 6.8 Hz, J4,5 ¼ 15.2 Hz, H-5Cer), 5.71-5.68 (m, 2 H,
H-2b, NH-2Cer), 5.55 (d, 1 H, J3,4 ¼ 3.0 Hz, H-4a), 5.50 (t, 1 H,

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J2,3 ¼ J3,4 ¼ 7.1 Hz, H-3Cer), 5.42 (dd, 1 H, H-4Cer), 5.33 (dd, 1 H,
J3,4 ¼ 3.3 Hz, J2,3 ¼ 11.3 Hz, H-3a), 5.30 (d, 1 H, J2,NH ¼ 5.3 Hz, NH-2a),
5.23-5.17 (m, 2 H, H-3b, H-2d), 4.94 (d, 1 H, CH2Ar), 4.90 (d, 1 H,
J1,2 ¼ 8.0 Hz, H-1b), 4.83 (m, 1 H, H-6ab), 4.82 (d, 1 H, J1,2 ¼ 8.3 Hz, H-1a),
4.65–4.54 (2 d, 2 H, CH2Ar), 4.50 (d, 1 H, J3,4 ¼ 2.2 Hz, H-4b), 4.45 (m, 1 H,
H-2a), 4.38 (d, 1 H, J1,2 ¼ 8.0 Hz, H-1d), 4.37 (m, 1 H, H-2Cer), 4.21-4.14 (m,
3 H, H-6bb, H-4d, CH2Ar), 4.04 (dd, 1 H, J1a,2 ¼ 3.0 Hz, Jgem ¼ 9.9 Hz, H-
1aCer), 3.84 (t, 1 H, J5,6a ¼ J5,6b ¼ 5.8 Hz, H-5b), 3.80-3.76 (m, 2 H, H-5a, H-
3d), 3.72 (s, 3 H, OMe), 3.64 (dd, 1 H, J5,6a ¼ 3.3 Hz, Jgem ¼ 11.2 Hz, H-6ad),
3.50 (dd, 1 H, J1b,2 ¼ 3.9 Hz, H-1bCer), 3.46 (s, 3 H, OMe), 3.46–3.35 (m,
2 H, H-6aa, H-6bd), 3.30–3.24 (m, 2 H, H-6ab, H-5d), 2.01 (s, 3 H, Ac),
1.98–1.94 (m, 2 H, H-6aCer, H-6bCer), 1.73 (t, 2 H, NHCOCH2Cer), 1.43–1.07
(m, 52 H, 26 CH2Cer), 0.89–0.86 (m, 6 H, 2 MeCer); 13C NMR (125 MHz,
CDCl3) d 172.8, 170.5, 166.9, 166.6, 166.1, 165.9, 165.4, 165.3, 165.3, 159.6,
158.9, 158.0, 157.7, 137.3, 134.2, 133.9, 133.5, 133.3, 132.9, 130.7, 130.5,
130.2, 130.2, 129.9, 129.8, 129.8, 129.7, 129.6, 129.3, 129.0, 128.9, 128.8,
128.7, 128.7, 128.6, 128.6, 128.5, 128.4, 125.0, 117.0, 114.7, 114.1, 113.5,
101.3, 101.2, 100.4, 79.8, 75.2, 74.7, 74.6, 74.6, 73.7, 73.5, 73.4, 72.4, 71.2,
70.6, 70.1, 68.9, 67.5, 64.1, 55.3, 54.9, 51.9, 50.6, 40.1, 36.5, 32.4, 32.0, 29.8,
29.8, 29.7, 29.7, 29.6, 29.5, 29.5, 29.4, 29.3, 29.1, 25.6, 23.4, 22.8, 14.2;
HRMS (ESI) m/z: found [M þ Na]þ 2179.0145, C123H148F3N3O27 calcd for
[M þ Na]þ 2179.0145.
Funding
This work was supported in part by JSPS KAKENHI Grant Numbers JP15H04495 and
JP18H03942 (H. A.); JST CREST Grant Number JPMJCR18H2 (H. A.); and by Mizutani
Foundation for Glycoscience (H. A.). The iCeMS is supported by a World Premier
International Research Center Initiative (WPI), Ministry of Education, Culture, Sports,
Science and Technology (MEXT), Japan.
ORCID
Naoko Komura
Hiromune Ando
http://orcid.org/0000-0002-8104-750X
http://orcid.org/0000-0002-0551-0830
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