Extending the glucosyl ceramide cassette approach: Application in the total synthesis of ganglioside GalNAc-GM1b

Article abstract of DOI:10.3390/molecules181215153

The development of a novel cyclic glucosyl ceramide cassette acceptor for efficient glycolipid syntheses was investigated. p-Methoxybenzyl (PMB) groups were selected as protecting groups at C2 and C3 of the glucose residue with the aim of improving the functionality of the cassette acceptor. The choice of the PMB group resulted in a loss of β-selectivity, which was corrected by using an appropriate tether to control the spatial arrangement and the nitrile solvent effect. To investigate the effect of linker structure on the β-selectivity of intramolecular glycosylation, several linkers for tethering the glucose and ceramide moiety were designed and prepared, namely, succinyl, glutaryl, dimethylmalonyl, and phthaloyl esters. The succinyl ester linker was the best for accessing the cassette form. The newly designed glucosyl ceramide cassette acceptor was then applied in the total synthesis of ganglioside GalNAc-GM1b.

Full text of DOI:10.3390/molecules181215153

Molecules 2013, 18, 15153-15181; doi:10.3390/molecules181215153
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Extending the Glucosyl Ceramide Cassette Approach:
Application in the Total Synthesis of Ganglioside GalNAc-GM1b
Miku Konishi 1,2, Akihiro Imamura 1,*, Kohki Fujikawa 1,2, Hiromune Ando 1,2
Hideharu Ishida 1,* and Makoto Kiso 1,2
,
1
Department of Applied Bio-Organic Chemistry, Faculty of Applied Biological Sciences,
Gifu University, 1-1 Yanagido, Gifu-shi, Gifu 501-1193, Japan;
E-Mails: konishi@gifu-u.ac.jp (M.K.); kouki.fujikawa@riken.jp (K.F.);
hando@gifu-u.ac.jp (H.A.); kiso@gifu-u.ac.jp (M.K.)
2
Institute for Integrated Cell-Material Sciences, Kyoto University, 69 Konoe-cho, Yoshida,
Sakyo-ku, Kyoto 606-8501, Japan
* Authors to whom correspondence should be addressed; E-Mails: aimamura@gifu-u.ac.jp (A.I.);
ishida@gifu-u.ac.jp (H.I.); Tel.: +81-58-293-3453 (A.I.); Fax: +81-58-293-2918 (H.I.).
Received: 7 November 2013; in revised form: 1 December 2013 / Accepted: 2 December 2013 /
Published: 10 December 2013
Abstract: The development of a novel cyclic glucosyl ceramide cassette acceptor for
efficient glycolipid syntheses was investigated. p-Methoxybenzyl (PMB) groups were
selected as protecting groups at C2 and C3 of the glucose residue with the aim of
improving the functionality of the cassette acceptor. The choice of the PMB group resulted
in a loss of β-selectivity, which was corrected by using an appropriate tether to control the
spatial arrangement and the nitrile solvent effect. To investigate the effect of linker
structure on the β-selectivity of intramolecular glycosylation, several linkers for tethering
the glucose and ceramide moiety were designed and prepared, namely, succinyl, glutaryl,
dimethylmalonyl, and phthaloyl esters. The succinyl ester linker was the best for accessing
the cassette form. The newly designed glucosyl ceramide cassette acceptor was then
applied in the total synthesis of ganglioside GalNAc-GM1b.
Keywords: ganglioside; GalNAc-GM1b; total synthesis; cassette approach

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1. Introduction
Gangliosides, which are glycosphingolipids that contain one or more sialic acid residues, are
components of all animal cell membranes and participate in many biological events, such as cell–cell
interaction, signal transduction, immunological reaction, and neuronal differentiation [1–3]. Found in
high abundance in the nervous system, several neuronal gangliosides have been linked with
neurological disorders including Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease [4].
Moreover, autoimmune neuropathy such as Guillain–Barré syndrome arises from the production of
anti-ganglioside antibodies [5,6]. The growing body of research regarding the physiological and
pathological implications of gangliosides has given rise to immense interest, not only among
biologists, but also among synthetic chemists. Many synthetic organic chemists have contributed to
developing methodology for the total synthesis of natural gangliosides and encountered several notable
synthetic challenges, including regio- and stereo-selective sialylation and the introduction of the
ceramide moiety into the oligosaccharide chain. Reliable methods for α-sialylation have been
developed and used in numerous syntheses of natural gangliosides and analogues [7–9]; however,
linking the flexible ceramide moiety to a large glycan remains a challenging undertaking. The typical
procedure for connecting the lipid and glycan units is first to prepare the entire oligosaccharide framework
and then to link it either to 2-azide sphingosine, which serves as a ceramide precursor, or to the ceramide
moiety directly. This general procedure has proved effective for small gangliosides such as GM4 and
GM3 [10,11]. In the synthesis of complex gangliosides, however, the oligosaccharide donor generally
couples to the lipid acceptor in low yields. Our group has recently developed the glucosyl ceramide
(GlcCer) cassette approach in order to overcome these synthetic challenges; our procedure involves
coupling of glucose and ceramide (forming GlcCer) early in the total synthesis. This methodology has
been used for efficiently synthesizing a series of natural gangliosides including GQ1b [12], GM3 [13],
GalNAc-GD1a [14], X2 [15], and LLG-3 [16] in satisfactory overall yields. Having established a
robust method for synthesizing gangliosides, we have shifted our attention to efficiently preparing
GlcCer cassette acceptors. Two types of GlcCer cassette acceptors have been developed to date: an
acyclic type [12,15,16] and a cyclic type [13,14]. Of these two types, acyclic cassettes are more
reactive, but cyclic cassettes are easier to prepare. Against this background, we set out to develop a
highly reactive cyclic GlcCer cassette acceptor. Here we describe the development of a novel cyclic
GlcCer cassette acceptor and its application in the total synthesis of ganglioside GalNAc-GM1b.
2. Results and Discussion
2.1. Synthesis of a Novel Cyclic GlcCer Cassette Acceptor
2.1.1. Design of a Novel Cyclic GlcCer Cassette Acceptor
The structure of the previously used cyclic GlcCer (1) is shown in Figure 1. We speculated that
the low reactivity of the 4-OH group of the glucose residue was due to the presence of the
electron-withdrawing acetyl group at the C3 position. Thus, installing an electron-donating protecting
group at the neighboring C3 position was expected to enhance the nucleophilicity of 4-OH.
Furthermore, to retain a route for accessing the cassette, the same protecting groups should be installed

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on O2 and O3 of the glucose. Based on the above considerations, the p-methoxybenzyl (PMB) group
was chosen as a protecting group because it can serve as an electron-donating group and be selectively
removed under mild acidic conditions. A point of concern, however, was that the non-participating
PMB group at the C2 position would cause a loss of stereoselectivity for the β-product in the
intramolecular glycosylation. Therefore, we envisioned controlling stereoselectivity by means of a
tethered structure; in particular, we anticipated that nucleophilic attack by the primary alcohol of the
ceramide on the anomeric center of the glucose could be restricted to a single face and that the
β-anomer could be selectively prepared. The four linkers evaluated in this study are shown in Figure 1:
(1) succinyl ester, which is used in 1; (2) glutaryl ester, which has a more flexible longer chain;
(3) dimethylmalonyl ester, which has a bulkier shorter chain; and (4) phthaloyl ester, which has a more
rigid chain.
Figure 1. Structure of our previously reported cyclic GlcCer cassette acceptor (left).
Structure of newly designed cyclic GlcCer cassette acceptor (right).
Previously reported cyclic GlcCer cassette
Newly designed cyclic GlcCer cassette
O
Linker
O
O
O
O
O
O
O
HO
AcO
O
HO
PMBO
O
C13H27
C17H35
O
C13H27
C17H35
OAc
HN
HN
OPMB
O
1
O
O
O
O
O
O
O
O
Linkers evaluated in this study
2.1.2. Preparation of Cyclic GlcCer Cassette Acceptors with Various Linkers
As shown in Scheme 1, 2,3-di-O-p-methoxybenzyl-protected glucose derivative 4 was efficiently
prepared from phenylthio-β-D-glucopyranoside 2 in three steps.
Scheme 1. Synthesis of the 2,3-di-O-PMB-protected glucose derivative.
Installation of anisylidene protecting groups at the C4 and C6 positions of 2 followed by
introduction of PMB protecting groups at the C2 and C3 positions afforded fully protected glucose

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derivative 3 in excellent yield. Hydrolysis of the anisylidene group under acidic conditions gave diol 4,
which was ready for linking to the ceramide moiety.
Scheme 2 shows the procedure for linking glucose derivative 4 to the ceramide moiety. The 3-OH
ceramide derivative 5 [13] was treated with succinic anhydride, glutaric anhydride, dimethylmalonyl
chloride, or phthalic anhydride under optimized conditions to form the corresponding carboxylic acid
derivatives 6, 7, 8, or 9 in almost quantitative yield, except for compound 8.
Scheme 2. Tethering between the glucose residue and ceramide derivative by various
types of dicarboxylate linkers.
Reagents and conditions: (a) 4, EDC·HCl, DMAP, CH2Cl2, 0 °C → r.t.; (b) TBAF, AcOH, THF,
0 °C → r.t.
The preparation of 8 was hampered by an undesired main reaction that formed an isobutyrate
product via decarboxylation. Succinic acid derivative 6 [13] was linked to glucose 4 in the presence of
EDC·HCl and DMAP in CH2Cl2 at 0 °C, giving tethered product 10 in 70% yield. The
tert-butyldimethylsilyl (TBS) group on 10 was removed by TBAF treatment to afford 11 in 95% yield.
By the same procedure, compound 7 was linked to 4 to give 12, along with a by-product in which the
ceramide moiety was tethered to C4 of the glucose. Since these regioisomers were difficult to separate
by column chromatography, the mixture was directly subjected to the next reaction without isolating
the products. Upon removal of the TBS group, 13 was obtained in 47% yield over the two operations.
Next, we attempted to form the dimethylmalonyl diester. After several attempts, we found that
hetero-diester of dimethylmalonic acid was difficult to form and the best yield of the coupled product

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14 was moderate (51%). Conversion of 14 into 15 proceeded smoothly in excellent yield. Lastly,
phthalic acid derivative 9 was reacted with glucose 4 under the same conditions, providing the desired
diester 16 in poor yield (32%). In this reaction, an unexpected phthalate product formed in which the
C6 and C4 positions of the glucose residue were tethered. Subsequent removal of the TBS group
furnished 17 in 90% yield.
2.1.3. Intramolecular Glycosylation towards Novel Cyclic GlcCer Cassette Acceptors
The alcohols prepared in Scheme 2 were subjected to the intramolecular glycosylation to evaluate
the β-selectivity of the reaction (Table 1). Intramolecular glycosylation of 11, which contained the
succinyl linker, was promoted by dimethyl(methylthio)sulfonium triflate [17,18] in CH2Cl2 at 0 °C.
The reaction proceeded smoothly and afforded intramolecularly glycosylated 18 in 67% yield with
poor anomeric selectivity (α/β = 1:1.7, entry 1).
Table 1. Investigation into the effect of various linkers on intramolecular glycosylation.
Linker
Linker
O
O
DMTST
(3.0 eq.)
O
O
O
HO
PMBO
O
C13H27
C17H35
HO
PMBO
O
SPh HO
C13H27
C17H35
0 °C
HN
PMBO
HN
PMBO
O
O
11, 13, 15, 17
18, 19, 20, 21
Entry
Compd.
11
Linker
Condition a
Product
18
% Yield b
α/β Ratio
1/1.7
O
1
2
3
4
5
6
7
A
B
A
B
A
B
A
67
77
34
40
75
76
53
11
18
1/8.2
O
O
O
13
19
1/2.0
13
19
1/7.7
O
O
15
20
1/2.4
15
20
1/9.1
17
21
1/2.0
8
17
B
21
71
1/5.2
O
O
a
Condition A: CH2Cl2, molecular sieve 4 Å; Condition B: CH3CN–CH2Cl2 (2:1), molecular sieve 3 Å;
b Isolated yield. DMTST: dimethyl(methylthio)sulfonium trifluoromethanesulfonate.
Acetonitrile, which generally promotes β-selective glycosylation, was examined as the main
solvent: as expected, the nitrile solvent effect gave improved β-selectivity (α/β = 1:8.2, entry 2). Note
that the desired β-product could be purified by recrystallization in the case of 18 only. Similarly,
intramolecular glycosylation of glutaryl ester-tethered 13 was performed (entries 3 and 4). The longer
more flexible linker compared with the one in 11 reduced both the yield and the stereoselectivity of the
glycosylation (77%, α/β = 1:8.2 vs. 40%, α/β = 1:7.7). Compound 15, which had the shortest linker,
was used to examine whether the chain length of the linker would affect the stereochemical outcome of
the intramolecular glycosylation. The β-selectivity for intramolecular glycosylation of 15 was only
slightly better than that of 11. Also, the α- and β-anomers were difficult to separate. Considering that a

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flexible linker appeared to hinder β-selectivity, we turned our attention to the compound with a more
rigid linker, namely, compound 17, which contained a rigid phthaloyl linker that could suppress free
rotation around the tether (entries 7 and 8). As a result, a slight shift toward α-selectivity was observed
(entry 8, α/β = 1:5.2).
Contemplating the above results, we considered that the succinyl linker might be the best for
accessing the desired novel cyclic GlcCer cassette acceptor. Next, the novel GlcCer cassette acceptor
18β was utilized for the total synthesis of ganglioside GalNAc-GM1b to investigate its applicability to
glycolipid synthesis.
2.2. Total Synthesis of Ganglioisde GalNAc-GM1b
2.2.1. Assembly of the Non-Reducing End Pentasaccharide Donor
Ganglioside GalNAc-GM1b was first isolated from Tay–Sachs brain in 1981 [19] and from murine
T lymphocytes in 1989 [20], and has been suggested to play important roles in the mammalian immune
system. Furthermore, immunoglobulin M monoclonal antibody against GalNAc-GM1b has been
isolated from patients with Guillain–Barré syndrome [21–23]. Having been implicated in these
intractable diseases, GalNAc-GM1b has elicited much interest. The chemical total synthesis of
GalNAc-GM1b was achieved first by Ogawa and co-workers in 1990 [24], who adopted the standard
procedure for introducing the ceramide moiety into the glycan. Although their construction of the
glycan sequence was elegant, the final coupling of the ceramide acceptor and hexasaccharide donor
was accomplished in with a low yield of 15%. Thus, we decided to apply our novel cyclic GlcCer
cassette to the total synthesis of GalNAc-GM1b in order to extend the generality of the GlcCer cassette
approach (Figure 2).
Figure 2. Structure of ganglioside GalNAc-GM1b and key disconnections for total synthesis.
GM2-core trisaccharide
Inner disaccharide
HO
OH
O
Glucosyl ceramide
HO
O
HO
OH
O
OH
O
AcHN
O
HO
OH
HO
O
O
O
OH
OH
O
OH
OH
AcHN
AcHN
HO
CO2H
HO
O
C13H27
C17H35
O
O
HO
HN
OH
OH
O
GalNAc-GM1b
The non-reducing end glycan sequence of GalNAc-GM1b was efficiently prepared as shown in
Scheme 3. Glycosylation of known galactosyl acceptor 22 [25] with galactosaminyl donor 23 [26] was
carried out in the presence of NIS and TfOH [27,28] in CH2Cl2 at 0 °C, giving disaccharide 24 in 86%
yield. Reductive removal of the Troc group by treatment with zinc gave 25 in excellent yield. Then,
under optimized acidic conditions (AcOH/1,4-dioxane: 1:4; 60 °C), acetyl migration from the C3
position of the galactosamine residue to the liberated amine was achieved in good yield (inner
disaccharide acceptor 26, 81%) [14]. For efficient migration of the acetyl group, the selected solvent

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and AcOH/solvent ratio were important. Also, undesired migration of an acetyl group from C4 to C3
of the galactosamine was observed, giving in 8% yield a disaccharide with an unprotected 4-OH group
in the galactosamine residue. The GM2-core trisaccharide donor 28 was prepared from 23 and the
sialylα(2,3)galactose unit 27 according to a previously reported procedure [26]. The coupling of 28
and 26 was promoted by a catalytic amount of TMSOTf in CH2Cl2 at 0 °C to give pentasaccharide 29
in 75% yield. The benzyl groups in 29 were replaced with benzoyl groups by hydrogenation and
subsequent benzoylation, affording 30 in good yield. Selective removal of the p-methoxyphenyl (MP)
group with CAN [29] followed by introduction of the trichloroacetimidate leaving group [30] gave the
non-reducing end glycan donor 31 in 91% yield over two steps.
Scheme 3. Synthesis of the non-reducing end glycan sequence of GalNAc-GM1b.
2.2.2. Final Glycosylation by the GlcCer Cassette Approach and Global Deprotection
First, the novel cyclic GlcCer cassette acceptor 18β was glycosylated with 31 in the presence of
TMSOTf in CHCl3 at room temperature, giving the fully protected GalNAc-GM1b framework in a
meager 26% yield. In this glycosylation, most of the donor was hydrolyzed to form the corresponding
hemiacetal compound and ca. 67% of the acceptor were recovered. Contrary to our expectations, the
acceptor equipped with the electron-donating PMB groups at the C2 and C3 positions of the glucose
residue did not serve as a good cassette. Although we cannot explain this low yield with certainty, we
speculate that the functionality at C2 might significantly lower the nucleophilicity of the 4-OH group
because similar 2-O-benzoyl-protected cyclic GlcCer acceptor, which only differed by the protecting
group at O-2 compared to 18β, served as a good acceptor in our previous experiment [14]. Next, the

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previously reported GlcCer cassette 1 [14] was used as an alternative for the final glycosylation. When
1.0 eq. of 1 was used as the glycosyl acceptor, the desired GalNAc-GM1b framework (33) was
obtained in 31% yield. Increasing the equivalent amount of the acceptor increased the coupling yield to
60%. Finally, global deprotection to remove the acetyl, benzoyl, and succinyl groups was performed
by treatment with NaOMe in MeOH/THF (1:1) followed by addition of water, furnishing the target
ganglioside GalNAc-GM1b in 88% yield (Scheme 4).
Scheme 4. Final glycosylation using the GlcCer cassette approach and global deprotection.
O
O
O
O
O
HO
RO
O
C13H27
C17H35
OR
HN
O
18: R = PMB (1.0 eq.)
1: R = Ac (1.0 or 2.0 or 3.0 eq.)
31
(1.0 eq.)
TMSOTf
MS4Å
CHCl3
r. t.
AcO
AcO
AcO
OAc
O
O
AcO
O
OBz
O
OAc
O
O
O
AcHN
AcO
O
O
O
OBz
O
OR
HN
O
C17H35
C13H27
AcHN
AcHN
OBz
RO
CO2Me
O
BzO
O
O
OAc
O
AcO
OBz
O
32: R = PMB (26%)
33: R = Ac (31% [based on 1.0 eq. of 1], 48% [2.0 eq.], 60% [3.0 eq.])
NaOMe
MeOH–THF
(1:1)
r. t.; H2O
88%
GalNAc-GM1b
3. Experimental
General Methods
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. (Miyazaki, 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 plates
(silica gel 60F254 on glass plate). 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)

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manufactured by Fuji Silysia Co. was used for flash column chromatography. Quantity of silica gel was
usually estimated as 200 to 400-fold weight of sample to be charged. Solvent systems in
chromatography were specified in v/v. Evaporation and concentration were carried out in vacuo.
13
1H-NMR and C-NMR spectra were recorded with JEOL ECA 400/500/600 and Bruker UltraShield
Plus 500 spectrometers. Chemical shifts in 1H-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, td = triple doublet, 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. 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.
MP
O
O
O
SPh
OPMB
PMBO
Phenyl 4,6-O-anisylidene-2,3-di-O-p-methoxybenzyl-1-thio-β-D-glucopyranoside (3). To the solution
of 2 (20.0 g, 73.5 mmol) in the mixed solvent (CH3CN–DMF 735 mL:200 mL) were added
p-anisaldehyde dimethyl acetal (25.0 mL, 147 mmol) and (±)-camphor-10-sulfonic acid (CSA)
(680 mg, 2.94 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 quenched by the addition of triethylamine.
The reaction mixture was concentrated and diluted with EtOAc, of which solution was then added to
separatory funnel. After addition of distilled water to the solution, the desired 4,6-O-anisylidenated
product was appeared as a pure crystalline material (26.0 g, 91%), the physical data of which was
identical to those reported in the literature [31]. To a solution of the 4,6-O-anisylidenated product
obtained (2.00 g, 5.13 mmol) in DMF (25.7 mL) was added sodium hydride (492 mg, 20.5 mmol) at 0 °C.
After stirring for 1 h at 0 °C, p-methoxybenzyl chloride (2.8 mL, 20.5 mmol) was added to the
mixture. After stirring for 17 h at rt as the reaction was monitored by TLC (1:2.5 EtOAc–n-hexane),
the reaction was quenched by MeOH at 0 °C. Dilution of the mixture with EtOAc provided a solution,
which was then 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
1
chromatography (1:3 EtOAc–n-hexane) to give 3 (2.94 g, 91%). [α]D +3.3° (c 0.3, CHCl3); H-NMR
(500 MHz, CDCl3) δ 7.54–6.82 (m, 17H, 4Ar), 5.54 (s, 1H, ArCH), 4.85 (d, 1H, Jgem = 10.9 Hz,
ArCH2), 4.78–4.70 (m, 4H, ArCH2, H-1), 4.35 (dd, 1H, J5,6 = 5.2 Hz, Jgem = 10.3 Hz, H-6), 3.81–3.76
(m, 11H, 3OCH3, H-3, H-6'), 3.66 (t, 1H, J3,4 = J4,5 = 9.5 Hz, H-4), 3.45 (m, 2H, H-2, H-5); 13C-NMR
(125 MHz, CDCl3) δ 160.0, 159.4, 159.3, 133.1, 132.2, 130.5, 130.2, 129.9, 129.8, 129.0, 127.8,
127.3, 113.8, 113.6, 101.1, 88.3, 82.7, 81.4, 80.1, 75.5, 74.9, 70.2, 68.6, 55.3, 55.2. HRMS (ESI) m/z:
found [M+Na]+ 653.2180, C36H38O8S calcd for [M+Na]+ 653.2180.
OH
O
HO
PMBO
SPh
OPMB

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Phenyl 2,3-di-O-p-methoxybenzyl-1-thio-β-D-glucopyranoside (4). Compound 3 (2.60 g, 4.13 mmol)
was dissolved in 80% AcOH aq (41.3 mL) and the solution was stirred for 1.5 h at 50 °C as the
reaction was monitored by TLC (2:1 EtOAc–n-hexane). The reaction mixture was diluted with EtOAc
and carefully washed with ice-cooled 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 (1:1 EtOAc–n-hexane) to give 4 (2.11 g, quant.). [α]D −18.0° (c 0.4, CHCl3);
1H-NMR (500 MHz, CDCl3) δ 7.52–6.87 (m, 13H, 3Ar), 4.90 (d, 1H, Jgem = 11.3 Hz, ArCH2), 4.88 (d,
1H, Jgem = 11.0 Hz, ArCH2), 4.70 (m, 2H, ArCH2, H-1), 4.64 (d, 1H, ArCH2), 3.86 (m, 1H, H-6), 3.80
(2 s, 6H, 2OCH3), 3.73 (m, 1H, H-6'), 3.55–3.43 (m, 3H, H-4, H-3, H-2), 3.32 (m, 1H, H-5), 2.28 (d,
1H, J4,OH = 2.5 Hz, OH), 2.08 (t, 1H, J6,OH = J6',OH = 6.6 Hz, OH); 13C-NMR (125 MHz, CDCl3)
δ 159.5, 159.5, 133.6, 131.7, 130.4, 130.0, 129.9, 129.7, 129.6, 129.0, 127.6, 114.1, 113.9, 87.7, 85.6,
80.6, 79.1, 75.0, 75.0, 70.4, 62.8, 55.3, 55.3. HRMS (ESI) m/z: found [M+Na]+ 535.1758, C28H32O7S
calcd for [M+Na]+ 535.1761.
O
O
HO
O
C13H27
C17H35
TBSO
HN
O
(2S,3R,4E)-1-O-tert-Butyldimethylsilyl-3-O-(4-hydroxycarbonylbutanoate)-2-octadecanamido-4-octa-
decene-1,3-diol (7). To a solution of 5 (25 mg, 36.8 µmol) in CH2Cl2 (368 µL) were added glutaric
anhydride (21 mg, 184 µmol) and DBU (11 µL, 73.6 µmol) at 0 °C. After stirring for 1 h at rt as the
reaction was monitored by TLC (4:1 diethylether–n-hexane), the reaction was quenched by the
addition of MeOH at 0 °C. The reaction mixture was evaporated. The crude residue obtained was
purified by silica gel column chromatography (1:3 diethylether–n-hexane) to give 7 (28 mg, 96%).
1
[α]D +4.7° (c 0.3, CHCl3); H-NMR (500 MHz, CDCl3) δ 5.76 (m, 2H, H-5, NH), 5.42 (dd, 1H,
J3,4 = 7.2 Hz, J4,5 = 15.3 Hz, H-4), 5.32 (t, 1H, J2,3 = 7.2 Hz, H-3), 4.28 (m, 1H, H-2), 3.72 (dd, 1H,
J
1,2 = 3.1 Hz, Jgem = 10.3 Hz, H-1), 3.59 (dd, 1H, J1',2 = 4.4 Hz, H-1'), 2.46–2.36 (m, 4H, 2C(=O)CH2),
2.17 (m, 2H, C(=O)CH2Cer), 2.01 (m, 2H, H-6, H-6'), 1.95 (m, 2H, -CH2-), 1.59 (m, 2H,
C(=O)CH2CH2), 1.25 (m, 50H, 25-CH2-Cer), 0.88 (m, 15H, t-Bu, 2-CH3Cer), 0.05-0.04 (2 s, 6H,
13
Si(CH3)2); C-NMR (125 MHz, CDCl3) δ 177.3, 173.3, 173.1, 171.1, 136.9, 124.6, 73.7, 61.6, 51.8,
51.6, 37.0, 33.4, 33.1, 33.0, 32.9, 32.4, 31.9, 30.0, 29.7, 29.7, 29.5, 29.5, 29.4, 29.4, 29.3, 29.2, 29.0,
25.8, 25.7, 22.7, 20.1, 19.9, 18.2, 14.1, −5.6, −5.6. HRMS (ESI) m/z: found [M+Na]+ 816.6507,
C47H91NO6Si calcd for [M+Na]+ 816.6508.
O
O
HO
O
O
C13H27
C17H35
TBSO
HN

Molecules 2013, 18
15163
(2S,3R,4E)-1-O-tert-Butyldimethylsilyl-3-O-(2-hydroxycarbonylisobutanoate)-2-octadecanamido-4-
octadecene-1,3-diol (8). To a solution of 5 (47 mg, 69.2 µmol) in CH2Cl2 (1.4 mL) were added
dimethylmalonyl dichloride (92 µL, 692 µmol) and triethylamine (96 µL, 692 µmol) at 0 °C. After
stirring for 3 h at 0 °C as the reaction was monitored by TLC (1:1 diethylether–n-hexane), the reaction
was diluted with CHCl3. The solution was then washed with H2O and brine. The organic layer was
subsequently dried over Na2SO4, and concentrated. The resulting residue was purified by silica gel
column chromatography (1:4 diethylether–n-hexane) to give 8 (17 mg, 31%). [α]D +4.4° (c 0.8,
CHCl3); 1H-NMR (500 MHz, CDCl3) δ 6.44 (d, 1H, J2,NH = 9.0 Hz, NH), 5.77 (m, 1H, H-5), 5.41 (dd,
1H, J3,4 = 7.4 Hz, J4,5 = 15.3 Hz, H-4), 5.32 (t, 1H, J2,3 = 7.4 Hz, H-3), 4.20 (m, 1H, H-2), 3.76 (dd,
1H, J1,2 = 2.7 Hz, Jgem = 10.3 Hz, H-1), 3.62 (dd, 1H, J1',2 = 4.2 Hz, H-1'), 2.28 (m, 2H, C(=O)CH2),
2.00 (m, 2H, H-6, H-6'), 1.60 (m, 2H, C(=O)CH2CH2), 1.49 (s, 6H, CH3CCH3), 1.25 (m, 50H,
25-CH2-), 0.89 (m, 15H, t-Bu, 2-CH3Cer), 0.05 (2 s, 6H, Si(CH3)2). LRMS (ESI) m/z: found [M−H]−
792.6438, C47H91NO6Si calcd for [M−H]− 792.6543.
HO
O
O
O
C13H27
C17H35
TBSO
HN
O
(2S,3R,4E)-1-O-tert-Butyldimethylsilyl-3-O-(o-hydroxycarbonylbenzoate)-2-octadecanamido-4-
octadecene-1,3-diol (9). To a solution of 5 (1.00 g, 1.47 mmol) in pyridine (7.4 mL) were added
phthalic anhydride (327 mg, 2.21 mmol), DMAP (18 mg, 147 µmol) and triethylamine (612 µL,
4.41 mmol) at 0 °C. After stirring for 23 h at 40 °C as the reaction was monitored by TLC
(4:1 diethylether–n-hexane), the solvent was removed by co-evaporation with toluene, and then the
residue was diluted with CHCl3, washed with H2O. The organic layer was subsequently dried over
Na2SO4, and concentrated. The resulting residue was purified by silica gel column chromatography
(1:2 diethylether–n-hexane) to give 9 (1.22 g, quant.). [α]D +58.3° (c 0.3, CHCl3); 1H-NMR (600 MHz,
CDCl3) δ 7.80–7.52 (m, 4H, Ar), 6.07 (d, 1H, J2,NH = 10.3 Hz, NH), 5.81 (m, 1H, H-5), 5.71 (t, 1H,
J2,3 = J3,4 = 6.9 Hz, H-3), 5.54 (dd, 1H, J4,5 = 15.5 Hz, H-4), 4.44 (m, 1H, H-2), 3.76 (dd, 1H,
J1,2 = 4.1 Hz, Jgem = 10.3 Hz, H-1), 3.64 (dd, 1H, J1',2 = 5.5 Hz, H-1'), 2.27 (m, 2H, C(=O)CH2), 2.03
(m, 2H, H-6, H-6'), 1.63 (m, 2H, C(=O)CH2CH2), 1.33 (m, 50H, 25-CH2-), 0.88 (m, 15H, t-Bu,
13
2-CH3), 0.05 (2 s, 6H, Si(CH3)2); C-NMR (150 MHz, CDCl3) δ 174.7, 169.8, 166.4, 136.9, 132.7,
131.4, 131.2, 130.8, 129.5, 128.8, 123.7, 75.3, 62.1, 52.3, 37.0, 32.4, 31.9, 29.7, 29.7, 29.5, 29.4, 29.4,
29.3, 29.3, 29.0, 25.8, 25.7, 22.7, 18.1, 14.1, −5.5, −5.6. HRMS (ESI) m/z: found [M+Na]+ 850.6351,
C51H91NO5Si calcd for [M+Na]+ 850.6351.
O
O
C13H27
O
C17H35
O
TBSO
SPh
HN
O
HO
PMBO
O
a
PMBO

Molecules 2013, 18
15164
Phenyl 6-O-{3-[(2S,3R,4E)-1-O-tert-butyldimethylsilyl-2-octadecanamido-4-octadecene-3-yloxy-1,3-
diol]carbonylpropanoyl}-2,3-di-O-p-methoxybenzyl-1-thio-β-D-glucopyranoside (10). To a solution of
4 (124 mg, 242 µmol) in CH2Cl2 (4.8 mL) were added 6 (200 mg, 242 µmol), EDC·HCl (51 mg, 266
µmol) and DMAP (3.0 mg, 24.2 µmol) at 0 °C. After stirring for 2.5 h at rt as the reaction was
monitored by TLC (1:1 EtOAc–n-hexane), the mixture was diluted with CHCl3. The solution was then
washed with H2O. 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 10
1
(215 mg, 70%). [α]D −7.7° (c 0.3, CHCl3); H-NMR (500 MHz, CDCl3) δ 7.56–7.24 (m, 5H, Ph),
7.54–6.85 (m, 8H, 2Ar), 5.74 (m, 2H, H-5Cer, NHCer), 5.43 (dd, 1H, J3,4 = 7.2 Hz, J4,5 = 15.2 Hz,
H-4Cer), 5.36 (t, 1H, J2,3 = 7.2 Hz, H-3Cer), 4.84 (d, 1H, Jgem = 11.0 Hz, ArCH2), 4.83 (d, 1H,
Jgem = 10.0 Hz, ArCH2), 4.77 (d, 1H, ArCH2), 4.68 (d, 1H, ArCH2), 4.65 (d, 1H, J1,2 = 9.2 Hz, H-1a),
4.39 (dd, 1H, J5,6 = 4.6 Hz, Jgem = 12.0 Hz, H-6a), 4.33 (dd, 1H, J5,6' = 2.0 Hz, H-6'a), 4.23 (m, 1H,
H-2Cer), 3.80–3.79 (2 s, 6H, 2 OCH3), 3.69 (dd, 1H, J1,2 = 9.7 Hz, Jgem = 10.4 Hz, H-1Cer), 3.57 (m, 2H,
H-1'Cer, H-4a), 3.50 (t, 1H, J2,3 = J3,4 = 9.2 Hz, H-3a), 3.44 (m, 3H, H-2a, H-5a, OHa), 2.66–2.60 (m,
4H, 2C(=O)CH2), 2.15 (m, 2H, C(=O)CH2Cer), 2.01 (m, 2H, H-6Cer, H-6'Cer), 1.57 (m, 2H,
C(=O)CH2CH2), 1.30 (m, 50H, 25-CH2-), 0.88 (m, 15H, t-Bu, 2-CH3Cer), 0.04 (2 s, 6H, Si(CH3)2);
13C-NMR (125 MHz, CDCl3) δ 172.8, 172.5, 170.7, 159.4, 159.3, 136.7, 133.7, 131.9, 130.6, 130.2,
129.9, 129.6, 128.8, 127.5, 124.6, 113.9, 113.8, 87.7, 85.6, 80.2, 77.5, 75.2, 75.0, 74.0, 69.8, 63.6,
61.6, 55.2, 55.2, 51.9, 36.9, 32.3, 31.9, 30.0, 29.6, 29.5, 29.5, 29.5, 29.4, 29.3, 29.2, 29.0, 29.0, 25.8,
25.8, 22.6, 18.1, 14.2, 14.1, −5.5, −5.6. HRMS (ESI) m/z: found [M+Na]+ 1296.8116, C74H119NO12SSi
calcd for [M+Na]+ 1296.8114.
O
O
C13H27
O
C17H35
O
HN
HO
O
HO
PMBO
O
a
SPh
PMBO
Phenyl
6-O-{3-[(2S,3R,4E)-2-octadecanamido-4-octadecene-3-yloxy-1,3-diol]carbonylpropanoyl}-
2,3-di-O-p-methoxybenzyl-1-thio-β-D-glucopyranoside (11). To a solution of 10 (183 mg, 144 µmol) in
THF (1.4 mL) were added AcOH (26 µL, 432 µmol) and TBAF (432 µL, 432 µmol) at 0 °C. After
stirring for 2 h at rt as the reaction was monitored by TLC (1:1 EtOAc–n-hexane), the mixture was
diluted with CHCl3. The solution was then washed with satd. aq. NaHCO3. The organic layer was
subsequently dried over Na2SO4 and concentrated. The resulting residue was purified by silica gel
column chromatography (1:1.5 EtOAc–n-hexane) to give 11 (159 mg, 95%). [α]D −12.0° (c 0.3,
1
CHCl3); H-NMR (500 MHz, CDCl3) δ 7.55–7.25 (m, 5H, Ph), 7.35–6.86 (m, 8H, 2Ar), 6.05 (d, 1H,
J2,NH = 9.0 Hz, NHCer), 5.76 (m, 1H, H-5Cer), 5.42 (dd, 1H, J3,4 = 7.3 Hz, J4,5 = 15.2 Hz, H-4Cer), 5.35
(t, 1H, J2,3 = 7.3 Hz, H-3Cer), 4.85 (d, 1H, Jgem = 10.9 Hz, ArCH2), 4.84 (d, 1H, Jgem = 10.4 Hz,
ArCH2), 4.72 (d, 1H, ArCH2), 4.68 (d, 1H, ArCH2), 4.66 (d, 1H, J1,2 = 9.6 Hz, H-1a), 4.45 (dd, 1H,
J5,6 = 4.6 Hz, Jgem = 11.9 Hz, H-6a), 4.29 (dd, 1H, J5,6' = 1.6 Hz, H-6'a), 4.15 (m, 1H, H-2Cer),
3.80–3.79 (2 s, 6H, 2OCH3), 3.66 (dd, 1H, J1,2 = 4.2 Hz, Jgem = 11.6 Hz, H-1Cer), 3.60 (near dd, 1H,
H-1'Cer), 3.54–3.42 (m, 4H, H-3a, H-5a, H-4a, H-2a), 3.27 (br s, 1H, OHa), 2.83 (br s, 1H, OHCer),

Molecules 2013, 18
15165
2.72–2.62 (m, 4H, 2C(=O)CH2), 2.16 (m, 2H, C(=O)CH2Cer), 2.11 (m, 2H, H-6Cer, H-6'Cer), 1.58 (m,
13
2H, C(=O)CH2CH2), 1.30 (m, 50H, 25-CH2-), 0.88 (m, 6H, 2-CH3Cer); C-NMR (125 MHz, CDCl3)
δ 173.6, 172.7, 171.5, 159.4, 137.2, 133.7, 131.8, 130.4, 130.1, 129.9, 129.7, 128.9, 127.5, 124.5,
114.0, 113.8, 87.8, 85.6, 80.2, 77.3, 75.3, 75.0, 74.6, 69.6, 63.7, 61.6, 55.3, 55.2, 52.9, 36.7, 32.3, 31.9,
29.7, 29.6, 29.6, 29.5, 29.5, 29.4, 29.3, 29.3, 29.2, 29.1, 28.9, 25.7, 22.7, 14.1. HRMS (ESI) m/z: found
[M+Na]+ 1182.7250, C68H105NO12S calcd for [M+Na]+ 1182.7250.
O
O
C13H27
O
C17H35
O
HN
HO
O
O
HO
PMBO
a
SPh
PMBO
Phenyl 6-O-{4-[(2S,3R,4E)-2-octadecanamido-4-octadecene-3-yloxy-1,3-diol]carbonylbutanoyl}-2,3-
di-O-p-methoxybenzyl-1-thio-β-D-glucopyranoside (12). To a solution of 4 (90 mg, 175 µmol) in
CH2Cl2 (3.5 mL) were added 7 (139 mg, 175 µmol), EDC·HCl (37 mg, 193 µmol) and DMAP
(2.1 mg, 17.5 µmol) at 0 °C. After stirring for 1.5 h at rt as the reaction was monitored by TLC
(1:1 EtOAc–n-hexane), the mixture was diluted with CHCl3. The solution was then washed with H2O.
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 the crude mixture mainly
containing 12. The crude mixture was exposed to high vacuum for 15 h and then dissolved in THF
(1.8 mL). AcOH (31 µL, 525 µmol) and TBAF (525 µL, 525 µmol) were added to the mixture at 0 °C.
After stirring for 4 h at rt as the reaction was monitored by TLC (1:1 EtOAc–n-hexane), the mixture
was diluted with CHCl3. The solution was then washed with satd aq NaHCO3. The organic layer was
subsequently dried over Na2SO4 and concentrated. The resulting residue was purified by silica gel
column chromatography (1:2 EtOAc–n-hexane) to give 13 (96 mg, 47% in two steps). [α]D −22.5°
1
(c 1.0, CHCl3); H-NMR (500 MHz, CDCl3) δ 7.55–7.25 (m, 5H, Ph), 7.36–6.87 (m, 8H, 2Ar), 6.00
(d, 1H, J2,NH = 8.6 Hz, NHCer), 5.76 (m, 1H, H-5Cer), 5.44 (dd, 1H, J3,4 = 7.4 Hz, J4,5 = 15.4 Hz,
H-4Cer), 5.28 (t, 1H, J2,3 = 7.4 Hz, H-3Cer), 4.87 (d, 1H, Jgem = 11.1 Hz, ArCH2), 4.85 (d, 1H,
Jgem = 9.9 Hz, ArCH2), 4.69 (d, 1H, ArCH2), 4.68 (d, 1H, ArCH2), 4.65 (d, 1H, J1,2 = 9.4 Hz, H-1a),
4.34 (near s, 2H, H-6a, H-6'a), 4.12 (m, 1H, H-2Cer), 3.81–3.80 (2 s, 6H, 2OCH3), 3.66 (dd, 1H,
J1,2 = 3.8 Hz, Jgem = 11.7 Hz, H-1Cer), 3.60 (dd, 1H, J1',2 = 2.7 Hz, H-1'Cer), 3.50–3.42 (m, 4H, H-2a,
H-3a, H-4a, H-5a), 2.85 (br s, 1H, OHCer), 2.75 (s, 1H, OHa), 2.43–2.39 (m, 4H, 2C(=O)CH2),
2.16 (m, 2H, C(=O)CH2Cer), 2.01 (m, 2H, H-6Cer, H-6'Cer), 1.96 (m, 2H, -CH2-), 1.58 (m, 2H,
C(=O)CH2CH2), 1.29 (m, 50H, 25-CH2-Cer), 0.88 (m, 6H, 2-CH3Cer); 13C-NMR (125 MHz, CDCl3)
δ 173.4, 173.1, 172.7, 159.5, 159.4, 137.4, 133.7, 131.9, 130.4, 130.0, 129.9, 129.7, 128.9, 127.6,
124.7, 114.1, 113.9, 87.7, 85.5, 80.3, 77.6, 75.2, 75.0, 74.3, 69.9, 63.6, 61.7, 55.3, 55.2, 53.1, 36.8,
33.4, 33.0, 32.3, 31.9, 30.0, 29.6, 29.6, 29.5, 29.5, 29.4, 29.3, 29.3, 29.2, 28.9, 25.7, 22.7, 20.0, 14.1.
HRMS (ESI) m/z: found [M+Na]+ 1196.7404, C69H107NO12S calcd for [M+Na]+ 1196.7406.

Molecules 2013, 18
15166
O
O
O
C13H27
C17H35
O
HN
TBSO
O
HO
PMBO
O
a
SPh
PMBO
Phenyl 6-O-{2-[(2S,3R,4E)-1-O-tert-butyldimethylsilyl-2-octadecanamido-4-octadecene-3-yloxy-1,3-
diol]carbonylisobutanoyl}-2,3-di-O-p-methoxybenzyl-1-thio-β-D-glucopyranoside (14). To a solution
of 4 (55 mg, 107 µmol) in CH2Cl2 (2.1 mL) were added 8 (85 mg, 107 µmol), EDC·HCl
(23 mg, 118 µmol) and DMAP (1.3 mg, 10.7 µmol) at 0 °C. After stirring for 5 h at rt as the reaction
was monitored by TLC (2:1 diethylether–n-hexane), the mixture was diluted with CHCl3. The solution
was then washed with H2O. 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
14 (70 mg, 51%). 1H-NMR (500 MHz, CDCl3) δ 7.54–6.85 (m, 13H, 3Ar), 6.70 (d, 1H, J2,NH = 9.0 Hz,
NHCer), 5.75 (m, 1H, H-5Cer), 5.37 (m, 2H, H-4Cer, H-3Cer), 4.84 (d, 1H, Jgem = 11.0 Hz, ArCH2), 4.82
(d, 1H, Jgem = 10.0 Hz, ArCH2), 4.72 (d, 1H, ArCH2), 4.66 (d, 1H, ArCH2), 4.63 (d, 1H, J1,2 = 9.6 Hz,
H-1a), 4.40 (dd, 1H, J5,6 = 4.0 Hz, Jgem = 11.9 Hz, H-6a), 4.34 (near dd, 1H, H-6'a), 4.15 (m, 1H,
H-2Cer), 3.81–3.80 (2 s, 6H, 2OCH3), 3.68 (dd, 1H, J1,2 = 3.3 Hz, Jgem = 10.4 Hz, H-1Cer), 3.57 (dd, 1H,
J1',2 = 4.3 Hz, H-1'Cer), 3.50–3.47 (m, 3H, H-3a, H-4a, H-5a), 3.40 (m, 1H, H-2a), 2.87 (br s, 1H, OHa),
2.28 (m, 2H, C(=O)CH2), 1.99 (m, 2H, H-6Cer, H-6'Cer), 1.59 (m, 2H, C(=O)CH2CH2), 1.45 (s, 6H,
CH3CCH3), 1.25 (m, 50H, 25-CH2-), 0.88 (m, 15H, t-Bu, 2-CH3Cer), 0.05 (2 s, 6H, Si(CH3)2).
O
O
O
C13H27
C17H35
O
HN
HO
O
HO
PMBO
O
a
SPh
PMBO
Phenyl 6-O-{2-[(2S,3R,4E)-2-octadecanamido-4-octadecene-3-yloxy-1,3-diol]carbonylisobutanoyl}-
2,3-di-O-p-methoxybenzyl-1-thio-β-D-glucopyranoside (15). To a solution of 14 (62 mg, 48.1 µmol) in
THF (481 µL) were added AcOH (8.6 µL, 144 µmol) and TBAF (144 µL, 144 µmol) at 0 °C. After
stirring for 3.5 h at rt as the reaction was monitored by TLC (1:2 EtOAc–n-hexane), the mixture was
diluted with CHCl3. The solution was then washed with satd aq NaHCO3. The organic layer was
subsequently dried over Na2SO4 and concentrated. The resulting residue was purified by silica gel
column chromatography (1:3 EtOAc–n-hexane) to give 15 (51 mg, 91%). 1H-NMR (500 MHz, CDCl3)
δ 7.54–6.86 (m, 13H, 3Ar), 6.71 (d, 1H, J2,NH = 8.6 Hz, NHCer), 5.75 (m, 1H, H-5Cer), 5.41 (dd, 1H,
J3,4 = 7.4 Hz, J4,5 = 15.4 Hz, H-4Cer), 5.29 (t, 1H, J2,3 = 7.4 Hz, H-3Cer), 4.83 (d, 1H, Jgem = 10.8 Hz,
ArCH2), 4,82 (d, 1H, Jgem = 9.9 Hz, ArCH2), 4.72 (d, 1H, ArCH2), 4.66 (d, 1H, ArCH2), 4.64 (d, 1H,
J1,2 = 9.2 Hz, H-1a), 4.39 (near d, 2H, H-6a, H-6'a), 4.08 (m, 1H, H-2Cer), 3.81–3.80 (2 s, 6H, 2OCH3),
3.55–3.46 (m, 5H, H-1Cer, H-1'Cer, H-3a, H-4a, H-5a), 3.40 (t, 1H, J2,3 = 9.2 Hz, H-2a), 3.32 (d, 1H,
J4,OH = 3.4 Hz, OHa), 2.83 (br s, 1H, OHCer), 2.30 (m, 2H, C(=O)CH2), 2.01 (m, 2H, H-6Cer, H-6'Cer),

Molecules 2013, 18
15167
1.60 (m, 2H, C(=O)CH2CH2), 1.45 (2 s, 6H, CH3CCH3), 1.25 (m, 50H, 25-CH2-), 0.88 (m, 6H,
2-CH3Cer). HRMS (ESI) m/z: found [M+Na]+ 1196.7487, C69H107NO12S calcd for [M+Na]+ 1196.7406.
O
O
C13H27
C17H35
O O
O
HO
PMBO
a
SPh
TBSO
HN
PMBO
O
Phenyl 6-O-{[(2S,3R,4E)-1-O-tert-butyldimethylsilyl-2-octadecanamido-4-octadecene-3-yloxy-1,3-
diol]carbonylbenzoyl}-2,3-di-O-p-methoxybenzyl-1-thio-β-D-glucopyranoside (16). To a solution of 4
(87 mg, 169 µmol) in CH2Cl2 (3.4 mL) were added 9 (140 mg, 169 µmol), EDC·HCl (36 mg,
186 µmol) and DMAP (2.1 mg, 16.9 µmol) at 0 °C. After stirring for 5.5 h at rt as the reaction was
monitored by TLC (1:1 EtOAc–n-hexane), the mixture was diluted with CHCl3. The solution was then
washed with H2O. The organic layer was subsequently dried over Na2SO4 and concentrated. The
resulting residue was purified by silica gel column chromatography (1:3 EtOAc–n-hexane) to give 16
1
(71 mg, 32%). [α]D +4.5° (c 1.0, CHCl3); H-NMR (500 MHz, CDCl3) δ 7.77–6.83 (m, 17H, 4Ar),
5.93 (d, 1H, J2,NH = 9.6 Hz, NHCer), 5.77 (m, 1H, H-5Cer), 5.61 (t, 1H, J2,3 = J3,4 = 6.9 Hz, H-3Cer), 5.54
(dd, 1H, J4,5 = 15.4 Hz, H-4Cer), 4.82 (s, 2H, ArCH2), 4.79 (d, 1H, Jgem = 9.9 Hz, ArCH2), 4.68 (m, 3H,
ArCH2, H-1a, H-6a), 4.57 (dd, 1H, J5,6' = 1.9 Hz, Jgem = 12.0 Hz, H-6'a), 4.39 (m, 1H, H-2Cer), 4.11
(br s, 1H, OHa), 3.81–3.79 (2 s, 6H, 2OCH3), 3.71 (m, 2H, H-1Cer, H-4a), 3.63 (dd, 1H, J1',2 = 5.1 Hz,
Jgem = 10.5 Hz, H-1'Cer), 3.57 (m, 2H, H-3a, H-5a), 3.45 (t, 1H, J1,2 = J2,3 = 9.2 Hz, H-2a), 2.15 (m, 2H,
C(=O)CH2), 2.03 (m, 2H, H-6Cer, H-6'Cer), 1.55 (m, 2H, C(=O)CH2CH2), 1.25 (m, 50H, 25-CH2-), 0.88
(m, 15H, t-Bu, 2-CH3Cer), 0.04–0.03 (2 s, 6H, Si(CH3)2); 13C-NMR (125 MHz, CDCl3) δ 173.1, 166.9,
166.8, 159.4, 159.3, 136.2, 133.5, 133.1, 132.2, 131.8, 131.5, 131.0, 130.8, 130.7, 130.3, 129.8, 129.6,
129.2, 128.8, 127.5, 124.4, 113.9, 113.9, 113.8, 87.4, 85.8, 79.9, 77.8, 77.6, 75.8, 75.3, 75.0, 69.9,
64.3, 62.0, 55.3, 55.2, 52.0, 36.9, 32.4, 31.9, 29.7, 29.7, 29.5, 29.4, 29.4, 29.3, 29.2, 29.1, 25.8, 25.7,
22.7, 18.2, 14.1, −5.4, −5.6. HRMS (ESI) m/z: found [M+Na]+ 1344.8116, C78H119NO12SSi calcd for
[M+Na]+ 1344.8114.
O
O
C13H27
C17H35
O O
SPh
O
HO
PMBO
a
HN
HO
PMBO
O
Phenyl 6-O-{[(2S,3R,4E)-2-octadecanamido-4-octadecene-3-yloxy-1,3-diol]carbonylbenzoyl}-2,3-di-
O-p-methoxybenzyl-1-thio-β-D-glucopyranoside (17). To a solution of 16 (190 mg, 144 µmol) in THF
(1.4 mL) were added AcOH (26 µL, 432 µmol) and TBAF (432 µL, 432 µmol) at 0 °C. After stirring
for 4 h at rt as the reaction was monitored by TLC (1:1 EtOAc–n-hexane), the mixture was diluted
with CHCl3. The solution was then washed with satd aq NaHCO3. The organic layer was subsequently
dried over Na2SO4 and concentrated. The resulting residue was purified by silica gel column

Molecules 2013, 18
15168
chromatography (1:2 EtOAc–n-hexane) to give 17 (156 mg, 90%). [α]D +3.3° (c 0.3, CHCl3); 1H-NMR
(500 MHz, CDCl3) δ 7.85–6.84 (m, 17H, 4Ar), 6.41 (d, 1H, J2,NH = 9.2 Hz, NHCer), 5.82 (m, 1H, H-5Cer),
5.61 (t, 1H, J2,3 = J3,4 = 6.6 Hz, H-3Cer), 5.47 (dd, 1H, J4,5 = 15.4 Hz, H-4Cer), 4.87–4.80 (m, 3H,
ArCH2, H-6a), 4.73 (d, 1H, Jgem = 10.7 Hz, ArCH2), 4.68 (d, 1H, J1,2 = 9.2 Hz, H-1a), 4.65 (d, 1H,
Jgem = 10.0 Hz, ArCH2), 4.48 (near d, 1H, H-6'a), 4.27 (m, 1H, H-2Cer), 3.80–3.78 (2 s, 6H, 2OCH3),
3.74 (br d, 1H, H-1Cer), 3.69 (s, 1H, OHa), 3.57–3.51 (m, 4H, H-4a, H-5a, H-3a, H-1'Cer), 3.42 (t, 1H,
J2,3 = 9.2 Hz, H-2a), 2.85 (br s, 1H, OHCer), 2.21 (m, 2H, C(=O)CH2), 2.03 (m, 2H, H-6Cer, H-6'Cer),
13
1.62 (m, 2H, C(=O)CH2CH2), 1.30 (m, 50H, 25-CH2-), 0.88 (m, 6H, 2-CH3Cer); C-NMR (125 MHz,
CDCl3) δ 173.6, 167.3, 167.1, 159.4, 137.3, 133.5, 132.8, 132.0, 131.1, 130.4, 130.3, 130.1, 129.9,
129.8, 129.8, 129.7, 129.0, 128.9, 127.5, 124.3, 114.0, 113.9, 113.9, 87.6, 85.8, 80.0, 77.6, 77.4, 76.2, 75.5,
75.0, 69.2, 64.1, 61.6, 55.3, 55.2, 52.5, 36.7, 32.3, 31.9, 29.6, 29.6, 29.5, 29.5, 29.3, 29.2, 28.9, 25.7, 22.7,
14.1. HRMS (ESI) m/z: found [M+Na]+ 1230.7250, C72H105NO12S calcd for [M+Na]+ 1230.7250.
O
O
O
O
O
HO
PMBO
a
O
C13H27
C17H35
HN
PMBO
O
(2,3-Di-O-p-methoxybenzyl-β-D-glucopyranosyl)-(1'→1)-(2S,3R,4E)-2-octadecanamido-4-octadecene-
1,3-diol-3,6'-succinate (18β). Condition A: To a mixture of 11 (32 mg, 27.6 µmol) in CH2Cl2 (5.5 mL)
was added 4 Å molecular sieves (64 mg) at rt. After stirring for 1 h, the mixture was cooled to 0 °C.
DMTST (45 mg, 82.8 µmol) was then added to the mixture at 0 °C. After stirring for
2 h at 0 °C as the reaction was monitored by TLC (1:2 acetone–n-hexane), the solution was diluted
with CHCl3 and filtered through Celite. The filtrate was then washed with satd aq NaHCO3 and H2O.
The organic layer was subsequently dried over Na2SO4, and concentrated. The residue was purified by
silica gel column chromatography (1:5 acetone–n-hexane) to give 18 (19 mg, 67%, α:β = 1:1.7).
Condition B: To a mixture of 11 (47 mg, 40.5 µmol) in acetonitrile/CH2Cl2 (2:1 8.1 mL) was added
3 Å molecular sieves (95 mg) at rt. After stirring for 1 h, the mixture was cooled to 0 °C. DMTST
(65 mg, 121 µmol) was then added to the mixture at 0 °C. After stirring for 2 h at 0 °C as the reaction
was monitored by TLC (1:2 acetone–n-hexane), the solution was diluted with CHCl3 and filtered
through Celite. The filtrate was then washed with satd aq NaHCO3 and H2O. The organic layer was
subsequently dried over Na2SO4, and concentrated. The residue was purified by silica gel column
chromatography (1:5 acetone–n-hexane) to give 18 (33 mg, 77%, α:β = 1:8.2). 18β: [α]D −10.4° (c 0.5,
1
CHCl3); H-NMR (500 MHz, CDCl3) δ 7.27–6.86 (m, 8H, 2Ar), 5.93 (d, 1H, J2,NH = 9.2 Hz, NHCer),
5.77 (m, 1H, H-5Cer), 5.55 (t, 1H, J2,3 = J3,4 = 6.3 Hz, H-3Cer), 5.30 (dd, 1H, J4,5 = 15.5 Hz, H-4Cer),
4.87 (d, 1H, Jgem = 11.4 Hz, ArCH2), 4.78 (d, 1H, Jgem = 10.8 Hz, ArCH2), 4.67–4.59 (m, 2H, H-6a,
ArCH2), 4.55 (d, 1H, ArCH2), 4.40 (m, 1H, H-2Cer), 4.34 (d, 1H, J1,2 = 7.0 Hz, H-1a), 4.09 (dd, 1H,
J5,6' = 2.1 Hz, Jgem = 11.6 Hz, H-6'a), 3.97 (dd, 1H, J1,2 = 4.72 Hz, Jgem = 10.3 Hz, H-1Cer), 3.80 (s, 6H,
2OCH3), 3.77 (near d, 1H, H-1'Cer), 3.44 (td, 1H, J4,5 = 2.1 Hz, H-5a), 3.38–3.29 (m, 3H, H-2a, H-3a,
H-4a), 2.79–2.61 (m, 4H, 2C(=O)CH2), 2.19 (d, 1H, J4,OH = 1.8 Hz, OHa), 2.17 (m, 2H, C(=O)CH2Cer),
1.99 (m, 2H, H-6Cer, H-6'Cer), 1.61 (m, 2H, C(=O)CH2CH2), 1.25 (m, 50H, 25-CH2-), 0.88 (m, 6H,

Molecules 2013, 18
15169
2-CH3Cer); 13C-NMR (125 MHz, CDCl3) δ 173.1, 172.6, 169.9, 159.4, 159.3, 136.5, 130.4, 130.1,
129.8, 129.6, 124.1, 114.0, 113.8, 102.8, 83.2, 81.0, 74.7, 74.1, 73.7, 73.4, 70.8, 65.0, 63.7, 55.2, 50.4,
36.8, 32.2, 31.9, 29.7, 29.7, 29.7, 29.6, 29.6, 29.5, 29.5, 29.4, 29.3, 29.2, 29.0, 28.8, 25.7, 22.6, 14.1.
HRMS (ESI) m/z: found [M+Na]+ 1072.7060, C62H99NO12 calcd for [M+Na]+ 1072.7059.
O
O
O
O
O
HO
PMBO
a
C13H27
C17H35
O
HN
PMBO
O
(2,3-Di-O-p-methoxybenzyl-D-glucopyranosyl)-(1'→1)-(2S,3R,4E)-2-octadecanamido-4-octadecene-
1,3-diol-3,6´-glutarate (19). Condition A: To a mixture of 13 (27 mg, 23.0 µmol) in CH2Cl2 (4.6 mL)
was added 4 Å molecular sieves (55 mg) at rt. After stirring for 1 h, the mixture was cooled to 0 °C.
DMTST (37 mg, 69.0 µmol) was then added to the mixture at 0 °C. After stirring for
3 h at 0 °C as the reaction was monitored by TLC (1:2 acetone–n-hexane), the solution was diluted
with CHCl3 and filtered through Celite. The filtrate was then washed with satd aq NaHCO3 and H2O.
The organic layer was subsequently dried over Na2SO4, and concentrated. The residue was purified by
silica gel column chromatography (1:5 acetone–n-hexane) to give 19 (8.3 mg, 34%, α:β = 1:2.0).
Condition B: To a mixture of 13 (29 mg, 24.7 µmol) in acetonitrile/CH2Cl2 (2:1 4.9 mL) was added
3 Å molecular sieves (60 mg) at rt. After stirring for 1 h, the mixture was cooled to 0 °C. DMTST
(40 mg, 74.1 µmol) was then added to the mixture at 0 °C. After stirring for 20 h at 0 °C as the
reaction was monitored by TLC (1:2 acetone–n-hexane), the solution was diluted with CHCl3 and
filtered through Celite. The filtrate was then washed with satd aq NaHCO3 and H2O. The organic layer
was subsequently dried over Na2SO4, and concentrated. The residue was purified by silica gel column
1
chromatography (1:5 acetone–n-hexane) to give 19 (10.4 mg, 40%, α:β = 1:7.7). 19β: H-NMR
(500 MHz, CDCl3) δ 7.29–6.86 (m, 8H, 2Ar), 5.79 (m, 1H, H-5Cer), 5.73 (d, 1H, J2,NH = 8.7 Hz,
NHCer), 5.32 (m, 2H, H-4Cer, H-3Cer), 4.89 (d, 1H, Jgem = 11.3 Hz, ArCH2), 4.79 (d, 1H, Jgem = 10.9 Hz,
ArCH2), 4.68 (d, 1H, ArCH2), 4.58 (m, 2H, H-6a, ArCH2), 4.37 (d, 1H, J1,2 = 7.3 Hz, H-1a), 4.27
(m, 1H, H-2Cer), 4.02 (dd, 1H, J5,6´ = 4.5 Hz, Jgem = 11.8 Hz, H-6´a), 3.96 (dd, 1H, J1,2 = 4.6 Hz,
J
gem = 9.9 Hz, H-1Cer), 3.80 (2 s, 6H, 2OCH3), 3.69 (dd, 1H, J1',2 = 3.0 Hz, H-1'Cer), 3.44 (m, 2H, H-4a,
H-5a), 3.37 (m, 2H, H-2a, H-3a), 2.57–2.29 (m, 4H, 2C(=O)CH2), 2.13 (s, 1H, OHa), 2.09–1.85 (m,
6H, C(=O)CH2Cer, H-6Cer, H-6'Cer, -CH2-), 1.54 (m, 2H, C(=O)CH2CH2), 1.23 (m, 50H, 25-CH2-Cer),
0.88 (m, 6H, 2-CH3Cer); 13C-NMR (125 MHz, CDCl3) δ 172.9, 172.7, 171.3, 159.5, 159.4, 137.2,
130.4, 130.3, 129.6, 129.5, 125.1, 114.1, 114.0, 113.9, 102.7, 83.2, 81.7, 77.6, 74.8, 74.5, 72.6, 72.4,
69.9, 67.0, 62.5, 55.3, 55.2, 50.9, 36.8, 33.0, 32.5, 32.3, 31.9, 30.0, 29.7, 29.6, 29.6, 29.5, 29.4, 29.4,
29.2, 28.9, 25.7, 22.7, 19.6, 14.1. HRMS (ESI) m/z: found [M+Na]+ 1086.7216, C63H101NO12 calcd for
[M+Na]+ 1086.7216.

Molecules 2013, 18
15170
O
O
O
O
O
O
HO
PMBO
a
O
C13H27
C17H35
HN
PMBO
(2,3-Di-O-p-methoxybenzyl-D-glucopyranosyl)-(1'→1)-(2S,3R,4E)-2-octadecanamido-4-octadecene-
1,3-diol-3,6´-(2,2-dimethylmalonate) (20). Condition A: To a mixture of 15 (24 mg,
20.4 µmol) in CH2Cl2 (4.1 mL) was added 4 Å molecular sieves (50 mg) at rt. After stirring for 1 h, the
mixture was cooled to 0 °C. DMTST (33 mg, 61.2 µmol) was then added to the mixture at 0 °C. After
stirring for 2 h at 0 °C as the reaction was monitored by TLC (1:2 acetone–n-hexane), the solution was
diluted with CHCl3 and filtered through Celite. The filtrate was then washed with satd aq NaHCO3 and
H2O. The organic layer was subsequently dried over Na2SO4, and concentrated. The residue was
purified by silica gel column chromatography (1:5 acetone–n-hexane) to give 20 (16 mg, 75%,
α:β = 1:2.4). Condition B: To a mixture of 15 (26 mg, 22.2 µmol) in acetonitrile/CH2Cl2 (2:1 4.5 mL)
was added 3 Å molecular sieves (55 mg) at rt. After stirring for 1 h, the mixture was cooled to 0 °C.
DMTST (36 mg, 66.6 µmol) was then added to the mixture at 0 °C. After stirring for 2 h at 0 °C as the
reaction was monitored by TLC (1:2 acetone–n-hexane), the solution was diluted with CHCl3 and
filtered through Celite. The filtrate was then washed with satd aq NaHCO3 and H2O. The organic layer
was subsequently dried over Na2SO4, and concentrated. The residue was purified by silica gel column
chromatography (1:5 acetone–n-hexane) to give 20 (18 mg, 76%, α:β = 1:9.1). 20β: 1H-NMR (500 MHz,
CDCl3) δ 7.24–6.85 (m, 8H, 2Ar), 6.55 (d, 1H, J2,NH = 7.0 Hz, NHCer), 5.75 (m, 1H, H-5Cer), 5.59 (t,
1H, J2,3 = J3,4 = 6.4 Hz, H-3Cer), 5.33 (dd, 1H, J4,5 = 15.4 Hz, H-4Cer), 4.81 (d, 1H, Jgem = 11.5 Hz,
ArCH2), 4.66 (d, 1H, Jgem = 11.0 Hz, ArCH2), 4.59 (dd, 1H, J5,6 = 5.2 Hz, Jgem = 11.7 Hz, H-6a), 4.55
(d, 1H, ArCH2), 4.54 (d, 1H, ArCH2), 4.44 (m, 2H, H-1a, H-6'a), 4.56 (dd, 1H, Jgem = 11.9 Hz,
J1,2 = 2.1 Hz, H-1Cer), 3.89 (m, 1H, H-2Cer), 3.59 (m, 2H, H-1'Cer, H-5a), 3.48 (t, 1H, J3,4 = J4,5 = 7.3 Hz,
H-4a), 3.44 (t, 1H, J1,2 = J2,3 = 7.3 Hz, H-2a), 3.36 (t, 1H, H-3a), 2.44 (s, 1H, OHa), 2.30 (m, 2H,
C(=O)CH2), 2.00 (m, 2H, H-6Cer, H-6'Cer), 1.60 (m, 2H, C(=O)CH2CH2), 1.41–1.40 (2 s, 6H,
CH3CCH3), 1.25 (m, 50H, 25-CH2-), 0.88 (m, 6H, 2-CH3Cer); C-NMR (125 MHz, CDCl3) δ 175.0,
13
172.4, 170.7, 159.5, 159.4, 136.2, 130.3, 130.1, 129.7, 129.7, 129.6, 129.5, 129.0, 127.6, 127.1, 124.6,
114.2, 114.0, 114.0, 113.9, 103.6, 82.2, 80.5, 74.1, 73.3, 72.8, 72.2, 70.4, 69.8, 62.7, 55.3, 54.6, 50.8,
50.7, 34.5, 32.3, 29.6, 29.5, 29.5, 29.3, 29.3, 29.2, 29.2, 29.0, 28.9, 25.0, 23.7, 22.7, 21.8, 14.1. HRMS
(ESI) m/z: found [M+Na]+ 1086.7217, C63H101NO12 calcd for [M+Na]+ 1086.7216.
O
O
O
O
O
O
HO
PMBO
a
C13H27
C17H35
HN
PMBO
O
(2,3-Di-O-p-methoxybenzyl-D-glucopyranosyl)-(1'→1)-(2S,3R,4E)-2-octadecanamido-4-octadecene-
1,3-diol-3,6'-phthalate (21). Condition A: To a mixture of 17 (50 mg, 41.4 µmol) in CH2Cl2 (8.3 mL)

Molecules 2013, 18
15171
was added 4 Å molecular sieves (100 mg) at rt. After stirring for 1 h, the mixture was cooled to 0 °C.
DMTST (67 mg, 124 µmol) was then added to the mixture at 0 °C. After stirring for
1 h at 0 °C as the reaction was monitored by TLC (1:2 acetone–n-hexane), the solution was diluted
with CHCl3 and filtered through Celite. The filtrate was then washed with satd aq NaHCO3 and H2O.
The organic layer was subsequently dried over Na2SO4, and concentrated. The residue was purified by
silica gel column chromatography (1:5 acetone–n-hexane) to give 21 (24 mg, 53%, α:β = 1:2.0).
Condition B: To a mixture of 17 (45 mg, 37.3 µmol) in acetonitrile/CH2Cl2 (2:1 11.3 mL) was added
3 Å molecular sieves (90 mg) at rt. After stirring for 1 h, the mixture was cooled to 0 °C. DMTST
(60 mg, 112 µmol) was then added to the mixture at 0 °C. After stirring for 1 h at 0 °C as the reaction
was monitored by TLC (1:2 acetone–n-hexane), the solution was diluted with CHCl3 and filtered
through Celite. The filtrate was then washed with satd aq NaHCO3 and H2O. The organic layer was
subsequently dried over Na2SO4, and concentrated. The residue was purified by silica gel column
chromatography (1:5 acetone–n-hexane) to give 21 (29 mg, 71%, α:β = 1:5.2). 21β: 1H-NMR (500 MHz,
CDCl3) δ 7.75–6.86 (m, 12H, 3Ar), 6.18 (br d, 1H, J2,NH = 9.8 Hz, NHCer), 5.88 (m, 1H, H-5Cer), 5.67
(m, 1H, H-3Cer), 5.41 (dd, 1H, J3,4 = 7.8 Hz, J4,5 = 15.3 Hz, H-4Cer), 4.90 (d, 1H, Jgem = 11.4 Hz,
ArCH2), 4.81 (d, 1H, Jgem = 10.7 Hz, ArCH2), 4.64 (d, 1H, ArCH2), 4.56 (d, 1H, ArCH2), 4.53–4.47
(m, 3H, H-6a, H-6'a, H-2Cer), 4.33 (d, 1H, J1,2 = 7.4 Hz, H-1a), 3.97 (dd, 1H, J1,2 = 4.4 Hz, Jgem = 10.3 Hz,
H-1Cer), 3.90 (near dd, 1H, J1',2 = 5.4 Hz, H-1'Cer), 3.82–3.78 (m, 7H, H-4a, 2OCH3), 3.50 (m, 1H,
H-5a), 3.43–3.36 (m, 2H, H-2a, H-3a), 2.22 (m, 2H, C(=O)CH2), 2.16 (s, 1H, OHa), 2.00 (m, 2H,
H-6Cer, H-6'Cer), 1.64 (m, 2H, C(=O)CH2CH2), 1.25 (m, 50H, 25-CH2-), 0.88 (m, 6H, 2-CH3Cer);
13C-NMR (125 MHz, CDCl3) δ 172.8, 159.5, 159.4, 131.5, 130.9, 130.4, 130.2, 129.8, 129.7, 129.6,
129.0, 124.4, 124.1, 114.1, 114.1, 114.0, 113.9, 103.1, 83.4, 81.3, 77.6, 75.1, 74.8, 74.3, 70.9, 55.3,
51.8, 50.2, 37.0, 32.3, 31.9, 29.7, 29.7, 29.7, 29.6, 29.6, 29.5, 29.5, 29.4, 29.2, 28.8, 25.9, 25.6, 22.7,
14.1. HRMS (ESI) m/z: found [M+Na]+ 1120.7060, C66H99NO12 calcd for [M+Na]+ 1120.7059.
AcO
OAc
O
c
AcO
O
OBn
O
TrocHN
BnO
b
OMP
OBn
4-Methoxyphenyl 3,4,6-tri-O-acetyl-2-deoxy-2-(2,2,2-trichloroethoxycarbamoyl)-β-D-galactopyranosyl-
(1→4)-2,3,6-tri-O-benzyl-β-D-galactopyranoside (24). To a mixture of 22 (80 mg, 144 µmol) and 23
(82 mg, 144 µmol) in CH2Cl2 (1.4 mL) was added 4 Å molecular sieves (320 mg) at rt. After stirring
for 1 h, the mixture was cooled to 0 °C. NIS (49 mg, 216 µmol) and TfOH (1.9 µL, 21.6 µmol) were
then added to the mixture at 0 °C. After stirring for 2 h at 0 °C as the reaction was monitored by TLC
(4:1 toluene–EtOAc), the reaction was quenched by the addition of satd aq NaHCO3. 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 (10:1 toluene–EtOAc) to give 24 (126 mg, 86%).
[α]D −5.6° (c 0.8, CHCl3); 1H-NMR (600 MHz, CDCl3) δ 7.39–7.26 (m, 15H, 3 Ph), 7.04–6.79 (m, 4H,
Ar), 5.65 (d, 1H, J2,NH = 6.9 Hz, NHc), 5.27 (d, 1H, J3,4 = 2.8 Hz, H-4c), 5.04 (d, 1H, Jgem = 11.0 Hz,

Molecules 2013, 18
15172
PhCH2), 4.96 (d, 1H, Jgem = 11.0 Hz, PhCH2), 4.91 (d, 1H, Jgem = 12.4 Hz, PhCH2), 4.84 (d, 2H,
J1,2 = 7.6 Hz, H-1b, PhCH2), 4.70 (m, 2H, H-3c, H-1c), 4.64 (d, 1H, PhCH2), 4.55 (q, 2H, Jgem = 11.7 Hz,
OCH2CCl3), 4.41 (d, 1H, PhCH2), 4.10 (dd, 1H, J5,6 = 7.6 Hz, Jgem = 11.0 Hz, H-6c), 4.06 (d, 1H,
J3,4 = 2.8 Hz, H-4b), 4.04 (dd, 1H, J5,6' = 6.2 Hz, H-6'c), 3.92 (m, 2H, H-2b, H-2c), 3.80 (dd, 1H,
J5,6 = 5.8 Hz, Jgem = 10.0 Hz, H-6b), 3.77 (s, 3H, OCH3), 3.76–3.71 (m, 2H, H-6'b, H-5c), 3.66 (m, 2H,
H-3b, H-5b), 2.14–1.94 (3 s, 9H, 3Ac); 13C-NMR (150 MHz, CDCl3) δ 170.3, 170.2, 155.3, 154.3,
151.4, 138.2, 138.1, 137.2, 129.0, 128.7, 128.5, 128.4, 128.2, 128.0, 127.8, 127.7, 127.6, 125.3, 118.6,
114.5, 102.9, 101.9, 95.8, 81.6, 79.6, 75.7, 75.3, 74.6, 74.3, 73.6, 73.5, 71.7, 70.9, 69.1, 66.5, 61.1,
55.6, 52.7, 20.6, 20.5. HRMS (ESI) m/z: found [M+Na]+ 1040.2402, C49H54Cl3NO16 calcd for
[M+Na]+ 1040.2400.
AcO
OAc
O
c
AcO
O
OBn
O
H2N
b
BnO
OMP
OBn
4-Methoxyphenyl 3,4,6-tri-O-acetyl-2-amino-2-deoxy-β-D-galactopyranosyl-(1→4)-2,3,6-tri-O-benzyl-
β-D-galactopyranoside (25). To a solution of 24 (100 mg, 98.3 µmol) in acetonitrile/AcOH (4:1, 3.3 mL)
was added Zn (500 mg) at rt. After stirring for 30 min at rt as the reaction was monitored by TLC (1:1
toluene–EtOAc), the solution was diluted with EtOAc and filtered through Celite. The filtrate was then
washed with satd aq Na2CO3 and brine. The organic layer was subsequently dried over Na2SO4, and
concentrated. The residue was purified by silica gel column chromatography (1.5:1 toluene–EtOAc) to
give 25 (81 mg, 98%). [α]D −12.3° (c 0.4, CHCl3); 1H-NMR (600 MHz, CDCl3) δ 7.36–7.24 (m, 15H,
3Ph), 7.06–6.78 (m, 4H, Ar), 5.29 (d, 1H, J3,4 = 2.1 Hz, H-4c), 5.01 (d, 1H, Jgem = 11.0 Hz, PhCH2),
4.87 (d, 1H, J1,2 = 7.6 Hz, H-1b), 4.83 (d, 1H, Jgem = 11.7 Hz, PhCH2), 4.82 (d, 1H, Jgem = 11.6 Hz,
PhCH2), 4.69 (d, 1H, PhCH2), 4.66 (dd, 1H, J2,3 = 10.7 Hz, H-3c), 4.55 (s, 2H, PhCH2), 4.49 (d, 1H,
J1,2 = 8.2 Hz, H-1c), 4.08 (dd, 1H, J5,6 = 7.6 Hz, Jgem = 11.0 Hz, H-6c), 4.04 (d, 1H, J3,4 = 2.8 Hz, H-
4b), 4.02 (dd, 1H, J5,6' = 6.2 Hz, H-6'c), 3.94 (dd, 1H, J1,2 = 7.6 Hz, J2,3 = 9.6 Hz, H-2b), 3.80 (dd, 1H,
J5,6 = 4.8 Hz, Jgem = 10.3 Hz, H-6b), 3.76 (s, 3H, OCH3), 3.76–3.73 (m, 2H, H-6'b, H-5c), 3.66 (m, 1H,
H-5b), 3.59 (dd, 1H, H-3b), 3.15 (dd, 1H, H-2c), 2.09–2.01 (3 s, 9H, 3Ac); 13C-NMR (150 MHz,
CDCl3) δ 170.4, 170.3, 170.2, 155.2, 151.5, 138.2, 138.1, 137.9, 129.0, 128.6, 128.4, 128.3, 128.2,
128.0, 127.8, 127.6, 127.5, 125.3, 118.3, 114.5, 105.1, 102.9, 81.0, 78.8, 75.2, 74.8, 74.1, 73.9, 73.6,
73.6, 70.5, 69.8, 66.3, 61.5, 55.6, 51.9, 21.4, 20.8, 20.7, 20.6. HRMS (ESI) m/z: found [M+Na]+
866.3358, C46H53NO14 calcd for [M+Na]+ 866.3358.
AcO
OAc
O
c
HO
O
OBn
O
AcHN
BnO
b
OMP
OBn

Molecules 2013, 18
15173
4-Methoxyphenyl 2-acetamido-4,6-di-O-acetyl-2-deoxy-β-D-galactopyranosyl-(1→4)-2,3,6-tri-O-benzyl-
β-D-galactopyranoside (26). Compound 25 (270 mg, 320 µmol) was dissolved in 1,4-dioxane/AcOH
(4:1, 32 mL) and the solution was stirred for 50 h at 60 °C as the reaction was monitored by TLC
(1:1.5 toluene–EtOAc). Dilution of the mixture with CHCl3 provided a solution, which was then
washed with satd aq Na2CO3. The organic layer was subsequently dried over Na2SO4, and
concentrated. The residue was purified by silica gel column chromatography (2:1 toluene–EtOAc) to
give 26 (218 mg, 81%). [α]D −3.5° (c 0.6, CHCl3); 1H-NMR (600 MHz, CDCl3) δ 7.38–7.24 (m, 15H,
3Ph), 7.17 (d, 1H, J2,NH = 3.4 Hz, NHc), 7.05–6.78 (m, 4H, Ar), 5.91 (d, 1H, J3,OH = 1.4 Hz, OHc), 5.27
(d, 1H, J3,4 = 3.4 Hz, H-4c), 5.12 (d, 1H, Jgem = 11.0 Hz, PhCH2), 4.88 (d, 1H, J1,2 = 7.6 Hz, H-1b), 4.87
(d, 1H, Jgem = 9.6 Hz, PhCH2), 4.73 (d, 2H, PhCH2), 4.56 (q, 2H, Jgem = 11.7 Hz, PhCH2), 4.46 (d, 1H,
J1,2 = 8.2 Hz, H-1c), 4.15 (dd, 1H, J5,6 = 6.5 Hz, Jgem = 11.3 Hz, H-6c), 4.05 (m, 2H,
H-6'c, H-4b), 3.89–3.83 (m, 3H, H-2b, H-2c, H-6b), 3.77 (s, 3H, OCH3), 3.76–3.69 (m, 4H, H-6'b,
13
H-3b, H-5b, H-5c), 3.53 (br d, 1H, H-3c), 2.14–1.61 (3 s, 9H, 3Ac); C-NMR (150 MHz, CDCl3)
δ 173.9, 170.5, 170.3, 155.4, 151.2, 138.1, 138.0, 136.4, 129.1, 129.0, 128.8, 128.5, 128.4, 128.2,
128.0, 127.9, 127.7, 127.5, 118.5, 114.5, 102.9, 102.7, 81.6, 79.9, 75.7, 75.3, 74.3, 73.6, 71.3, 69.3,
67.9, 61.9, 55.8, 55.6, 29.7, 22.3, 20.8, 20.7. HRMS (ESI) m/z: found [M+Na]+ 866.3358, C46H53NO14
calcd for [M+Na]+ 866.3358.
AcO
AcO
AcO
OAc
f
O
O
AcO
O
OBz
O
OAc
O
AcHN
O
AcO
d
c
O
O
e
OBn
O
AcHN
AcHN
BnO
OBz
CO2Me
b
OMP
OAc
AcO
OBn
4-Methoxyphenyl
2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-galactopyranosyl-(1→4)-{[methyl
5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosylonate]-
(2→3)}-2,6-di-O-benzoyl-β-D-galactopyranosyl-(1→3)-2-acetamido-4,6-di-O-acetyl-2-deoxy-β-D-
galactopyranosyl-(1→4)-2,3,6-tri-O-benzyl-β-D-galactopyranoside (29). To a mixture of 28 (180 mg,
135 µmol) and 26 (114 mg, 135 µmol) in CH2Cl2 (4.5 mL) was added 4 Å molecular sieves (300 mg)
at rt. After stirring for 1 h, the mixture was cooled to 0 °C. TMSOTf (2.4 µL, 13.5 µmol) was then
added to the mixture at 0 °C. After stirring for 2 h at 0 °C as the reaction was monitored by TLC (1:1
CHCl3–acetone), the reaction was quenched by the addition of satd aq NaHCO3. The solution 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 CHCl3–MeOH) to give 29 (203 mg, 75%).
1
[α]D −7.7° (c 0.2, CHCl3); H-NMR (600 MHz, DMSO-d6) δ 8.00–7.19 (m, 25H, 5Ph), 7.57 (d, 1H,
J5,NH = 8.9 Hz, NHe), 7.20 (d, 1H, J2,NH = 8.2 Hz, NHc), 6.96–6.78 (m, 4H, Ar), 6.78 (d, 1H,
J2,NH = 6.9 Hz, NHf), 5.32 (d, 1H, J3,4 = 3.5 Hz, H-4c), 5.29–5.25 (m, 2H, H-8e, H-3f), 5.23 (d, 1H,
J3,4 = 2.7 Hz, H-4f), 5.14–5.09 (m, 2H, H-2d, H-7e), 4.90 (d, 1H, J1,2 = 7.6 Hz, H-1d), 4.85 (d, 2H,
J1,2 = 7.6 Hz, H-1b, H-1f), 4.78 (m, 1H, H-4e), 4.74 (d, 1H, Jgem = 11.7 Hz, PhCH2), 4.68 (m, 2H,
H-1c, PhCH2), 4.59 (m, 2H, H-3d, PhCH2), 4.52 (d, 1H, Jgem = 12.4 Hz, PhCH2), 4.47 (m, 2H, H-6d,

Molecules 2013, 18
15174
PhCH2), 4.41 (d, 1H, Jgem = 12.4 Hz, PhCH2), 4.28 (m, 1H, H-3c), 4.25 (dd, 1H, J5,6' = 5.5 Hz,
Jgem = 11.0 Hz, H-6'd), 4.08–4.00 (m, 3H, H-6c, H-6f, H-9e), 3.98–3.91 (m, 4H, H-6'c, H-6'f, H-9'e,
H-5d), 3.86–3.72 (m, 8H, H-4d, H-5c, H-5f, H-2c, H-2f, H-6e, H-6b, H-5e), 3.75–3.68 (2 s, 6H,
2OCH3), 3.66 (near t, 1H, H-2b), 3.59–3.53 (m, 4H, H-3b, H-4b, H-5b, H-6'b), 2.30 (dd, 1H,
J3eq,4 = 4.8 Hz, Jgem = 13.1 Hz, H-3eeq), 1.80 (near t, 1H, H-3eax), 2.08–1.64 (12 s, 36H, 12 Ac);
13C-NMR (150 MHz, CDCl3) δ 171.3, 170.7, 170.6, 170.5, 170.4, 170.4, 170.2, 169.9, 169.8, 168.1,
166.0, 164.3, 155.1, 151.5, 138.4, 137.9, 133.2, 133.1, 130.0, 129.5, 128.6, 128.5, 128.4, 128.3, 128.0,
127.7, 127.5, 118.3, 114.4, 102.8, 100.7, 100.5, 100.0, 98.3, 80.7, 79.2, 75.3, 74.9, 74.1, 74.0, 73.8,
73.5, 73.2, 72.1, 72.0, 70.8, 70.3, 70.1, 70.0, 69.3, 68.7, 67.4, 66.9, 66.5, 63.7, 62.7, 62.1, 61.4, 55.6,
54.2, 53.1, 51.8, 49.2, 36.1, 31.9, 29.7, 29.3, 23.4, 23.1, 22.7, 21.1, 20.8, 20.7, 20.7, 20.7, 20.5, 20.4,
14.1. HRMS (ESI) m/z: found [M+Na]+ 2038.7055, C100H117N3O41 calcd for [M+Na]+ 2038.7055.
AcO
AcO
AcO
OAc
O
f
O
AcO
O
OBz
O
OAc
O
AcHN
O
AcO
d
c
O
O
e
OBz
O
AcHN
BzO
AcHN
OBz
CO2Me
b
OMP
OAc
AcO
OBz
4-Methoxyphenyl
2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-galactopyranosyl-(1→4)-{[methyl
5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosylonate]-
(2→3)}-2,6-di-O-benzoyl-β-D-galactopyranosyl-(1→3)-2-acetamido-4,6-di-O-acetyl-2-deoxy-β-D-
galactopyranosyl-(1→4)-2,3,6-tri-O-benzoyl-β-D-galactopyranoside (30). To a solution of 29
(215 mg, 107 µmol) in EtOH (10.7 mL) was added Pd(OH)2/C (20%, 215 mg). After stirring for 16 h
at rt 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 crude residue obtained was
exposed to high vacuum for 3 h. The resulting residue was then dissolved in pyridine (535 µL).
Benzoic anhydride (145 mg, 642 µmol) and DMAP (7.8 mg, 64.2 µmol) were added to the mixture at
0 °C. After stirring for 40 min at rt 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 and satd aq NaHCO3.
The organic layer was subsequently dried over Na2SO4, and concentrated. The resulting residue was
purified by silica gel column chromatography (25:1 CHCl3–MeOH) to give 30 (180 mg, 82%).
1
[α]D +6.3° (c 0.4, CHCl3); H-NMR (600 MHz, DMSO-d6) δ 8.01–7.35 (m, 26H, 5Ph, NHe), 7.11
(br d, 1H, J2,NH = 5.5 Hz, NHc), 7.01 (d, 1H, J2,NH = 8.2 Hz, NHf), 6.85–6.62 (m, 4H, Ar), 5.60 (t, 1H,
J1,2 = J2,3 = 7.6 Hz, H-2b), 5.52 (dd, 1H, J3,4 = 2.8 Hz, H-3b), 5.39 (d, 1H, H-1b), 5.30 (d, 1H,
J3,4 = 2.7 Hz, H-4c), 5.25 (m, 3H, H-3f, H-8e, H-4f), 5.12 (m, 2H, H-2d, H-7e), 4.87 (m, 2H, H-1d,
H-1f), 4.81 (m, 1H, H-4e), 4.74 (d, 1H, J1,2 = 8.3 Hz, H-1c), 4.59 (d, 1H, J2,3 = 10.3 Hz, H-3d),
4.49–4.41 (m, 5H, H-6b, H-4b, H-6'b, H-6d, H-5b), 4.29 (br dd, 1H, J2,3 = 10.3 Hz, H-3c), 4.24 (dd,
1H, J5,6' = 5.5 Hz, Jgem = 11.0 Hz, H-6'd), 4.06 (m, 2H, H-6f, H-9e), 3.98–3.93 (m, 3H, H-6'f, H-9'e,
H-6c), 3.90–3.72 (m, 7H, H-4d, H-5d, H-5f, H-2f, H-6e, H-5e, H-6'c), 3.75 (s, 3H, OCH3), 3.67 (m,
1H, H-5c), 3.62 (s, 3H, OCH3), 3.60 (m, 1H, H-2c), 2.26 (near dd, 1H, H-3eeq), 2.09–1.75 (m, 37H,
H-3eax, 12Ac); 13C-NMR (150 MHz, CDCl3) δ 171.1, 170.6, 170.5, 170.5, 170.5, 170.3, 170.2, 169.9,

Molecules 2013, 18
15175
169.7, 168.1, 166.3, 166.1, 166.0, 165.3, 164.3, 155.5, 151.2, 133.6, 133.4, 133.1, 132.9, 130.1, 130.0,
130.0, 129.8, 129.7, 129.6, 129.5, 129.4, 128.6, 128.5, 128.4, 128.3, 118.7, 114.3, 100.9, 100.8, 100.4,
98.2, 97.8, 74.7, 74.0, 73.4, 72.7, 72.0, 71.9, 71.2, 70.6, 70.1, 70.0, 69.7, 69.4, 68.8, 67.3, 67.0, 66.5,
63.7, 63.5, 62.8, 62.3, 61.4, 55.5, 53.1, 52.1, 52.0, 49.3, 36.3, 29.7, 23.4, 23.2, 21.1, 20.9, 20.8, 20.8,
20.7, 20.5, 20.4. HRMS (ESI) m/z: found [M+Na]+ 2080.6434, C100H111N3O44 calcd for [M+Na]+
2080.6433.
AcO
AcO
AcO
OAc
O
f
O
AcO
O
OBz
O
OAc
O
AcHN
O
AcO
d
c
O
O
e
OBz
O
AcHN
BzO
AcHN
OBz
CO2Me
b
OAc
AcO
BzO
O
CCl3
NH
2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-galactopyranosyl-(1→4)-{[methyl 5-acetamido-4,7,8,9-
tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosylonate]-(2→3)}-2,6-di-O-
benzoyl-β-D-galactopyranosyl-(1→3)-2-acetamido-4,6-di-O-acetyl-2-deoxy-β-D-galactopyranosyl-
(1→4)-2,3,6-tri-O-benzoyl-α-D-galactopyranosyl trichloroacetimidate (31). To a solution of 30
(115 mg, 55.9 µmol) in acetonitrile/toluene/H2O (6:5:3, 1.1 mL) was added CAN (245 mg, 447 µmol)
at 0 °C. After stirring for 2 h at 0 °C as the reaction was monitored by TLC (10:1 CHCl3–MeOH), the
mixture was diluted with CHCl3. The solution was then washed with H2O, satd aq NaHCO3 and brine.
The organic layer was subsequently dried over Na2SO4, and concentrated. The residue was roughly
purified by silica gel column chromatography (30:1 CHCl3–MeOH). The product obtained was
exposed to high vacuum for 20 h and then dissolved in CH2Cl2 (502 µL). CCl3CN (101 µL, 1.00
mmol) and DBU (9.0 µL, 60.2 µmol) were added to the mixture at 0 °C. After stirring for 30 min at rt
as the reaction was monitored by TLC (15:1 CHCl3–MeOH), the reaction mixture was evaporated. The
crude residue obtained was purified by silica gel column chromatography (40:1 CHCl3–MeOH) to give
1
31 (94 mg, 81%). [α]D +14.0° (c 0.6, CHCl3); H-NMR (600 MHz, DMSO-d6) δ 9.65 (br s, 1H,
C(=NH)), 8.01–7.37 (m, 26H, 5Ph, NHe), 7.21 (br s, 1H, NHc), 6.98 (d, 1H, J2,NH = 8.2 Hz, NHf), 6.57
(br s, 1H, H-1b), 5.77 (br d, 1H, H-2b), 5.64 (near dd, 1H, H-3b), 5.31 (s, 1H, H-4c), 5.23 (m, 3H, H-
3f, H-8e, H-4f), 5.10 (m, 2H, H-2d, H-7e), 4.89 (d, 1H, J1,2 = 7.6 Hz, H-1d), 4.86–4.81 (m, 3H, H-1f,
H-1c, H-4e), 4.62 (m, 2H, H-4b, H-5b), 4.56 (br d, 1H, H-3d), 4.48 (m, 2H, H-6b, H-6d), 4.38 (near
dd, 1H, H-6'b), 4.31 (br d, 1H, H-3c), 4.25 (near dd, 1H, H-6'd), 4.06 (m, 2H, H-6f, H-9e), 3.98–3.84
(m, 8H, H-6'f, H-9'e, H-6c, H-4d, H-5d, H-5f, H-2f, H-6'c), 3.81–3.69 (m, 6H, H-6e, OCH3, H-5e, H-
13
5c), 3.62 (m, 1H, H-2c), 2.26 (near dd, 1H, H-3eeq), 2.09–1.66 (m, 37H, H-3eax, 12Ac); C-NMR
(150 MHz, CDCl3) δ 171.0, 170.8, 170.4, 170.4, 170.4, 170.3, 170.2, 169.8, 169.7, 166.1, 165.9,
165.3, 164.2, 160.4, 133.5, 133.3, 133.1, 133.0, 129.9, 129.8, 129.7, 129.6, 129.5, 128.7, 128.4, 128.4,
128.3, 128.2, 100.7, 100.5, 98.2, 98.0, 93.4, 90.8, 74.8, 73.5, 73.1, 71.9, 71.9, 71.4, 71.3, 70.9, 70.4,
70.1, 70.0, 69.5, 68.7, 67.2, 66.9, 66.4, 63.8, 63.5, 62.8, 62.2, 61.4, 55.3, 53.0, 51.2, 49.1, 36.1, 29.6,
23.3, 23.1, 22.9, 22.6, 21.0, 20.8, 20.8, 20.7, 20.6, 20.4, 20.3, 14.1. HRMS (ESI) m/z: found [M+Na]+
2117.5111, C95H105Cl3N4O43 calcd for [M+Na]+ 2117.5110.

Molecules 2013, 18
15176
AcO
AcO
AcO
OAc
O
f
O
AcO
O
OBz
O
OAc
O
O
O
AcHN
O
AcO
d
c
O
O
e
OBz
O PMBO
OPMB HN
O
O
C17H35
C13H27
AcHN
AcHN
BzO
OBz
CO2Me
a
b
O
O
OAc
AcO
OBz
O
O
4-O-{2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-galactopyranosyl-(1→4)-[(methyl 5-acetamido-
4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosylonate)-(2→3)]-2,6-di-O-
benzoyl-β-D-galactopyranosyl-(1→3)-2-acetamido-4,6-di-O-acetyl-2-deoxy-β-D-galactopyranosyl-
(1→4)-2,3,6-tri-O-benzoyl-β-D-galactopyranosyl-(1→4)}-2,3-di-O-p-methoxybenzyl-β-D-glucopyranosyl-
(1'→1)-(2S,3R,4E)-2-octadecanamido-4-octadecene-1,3-diol-3,6'-succinate (32). To a mixture of
31 (59 mg, 28.2 µmol) and 18β (30 mg, 28.2 µmol) in CHCl3 (940 µL) was added 4 Å molecular
sieves (100 mg) at rt. After stirring for 1 h, the mixture was cooled to 0 °C. TMSOTf (0.5 µL,
2.82 µmol) was then added to the mixture at 0 °C. After stirring for 1.5 h at rt as the reaction was
monitored by TLC (1.5:1 acetone–n-hexane), the reaction was quenched by the addition of satd aq
NaHCO3. The solution 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 (1:1 acetone–n-hexane) to
1
give 32 (22 mg, 26%). [α]D +7.8° (c 0.4, CHCl3); H-NMR (500 MHz, CDCl3) δ 8.08–6.80 (m, 33H,
7Ar), 6.77 (d, 1H, J2,NH = 7.9 Hz, NHc), 5.73 (m, 2H, J2,NH = 8.9 Hz, H-5Cer, NHCer), 5.58–5.53 (m,
2H, H-3b, H-2d), 5.47–5.35 (m, 3H, NHf, H-3Cer, H-8e), 5.33 (d, 1H, J3,4 = 3.1 Hz, H-4c), 5.31–5.20
(m, 4H, H-4f, H-2b, H-4Cer, H-7e), 5.14 (d, 1H, J5,NH = 9.9 Hz, NHe), 5.09 (d, 1H, J1,2 = 8.1 Hz, H-1f),
5.05 (d, 1H, J1,2 = 8.4 Hz, H-1c), 4.99 (m, 1H, H-4e), 4.96 (d, 1H, J1,2 = 7.8 Hz, H-1d), 4.84–4.73 (m,
3H, 2 ArCH2, H-3f), 4.71 (d, 1H, J1,2 = 7.7 Hz, H-1b), 4.66 (d, 1H, Jgem = 10.8 Hz, ArCH2), 4.61 (m,
2H, H-6b, H-6d), 4.52 (d, 1H, ArCH2), 4.47 (d, 1H, J3,4 = 1.6 Hz, H-4d), 4.33–4.19 (m, 5H, H-6'b,
H-2Cer, H-1a, H-6f, H-6c), 4.14–4.03 (m, 5H, H-6'd, H-6'f, H-3c, H-9e, H-9'e), 4.00–3.87 (m, 6H, H-
4b, H-6'c, H-6a, H-2c, H-3d, H-5e), 3.85–3.72 (m, 8H, H-6'a, OCH3, H-5b, H-6e, H-5d, H-1Cer), 3.66–
3.58 (m, 3H, H-1'Cer, H-3a, H-4a), 3.54 (m, 1H, H-5a), 3.48 (m, 2H, H-5c, H-5f), 3.33 (t, 1H, J1,2 = J2,3
=
7.2 Hz, H-2a), 3.16 (m, 1H, H-2f), 2.58–2.45 (m, 4H, 2C(=O)CH2), 2.29 (dd, 1H, J3eq,4 = 4.8 Hz, Jgem
=
13.4 Hz, H-3eeq), 2.18–1.55 (m, 43H, C(=O)CH2CH2Cer, H-3eax, H-6Cer, H-6'Cer, 12Ac), 1.26 (m, 50H,
13
25-CH2-), 0.88 (m, 6H, 2-CH3Cer); C-NMR (125 MHz, CDCl3) δ 172.8, 171.8, 171.2, 171.0, 170.6,
170.5, 170.4, 170.4, 170.2, 169.9, 169.8, 168.1, 166.2, 166.0, 165.6, 164.3, 159.3, 159.0, 137.1, 133.6,
133.3, 133.1, 130.5, 130.0, 130.0, 129.8, 129.8, 129.7, 129.6, 129.6, 129.6, 129.0, 128.8, 128.5, 128.4,
128.4, 128.3, 128.2, 125.6, 124.3, 120.2, 113.8, 113.7, 101.8, 100.9, 100.6, 98.3, 97.8, 81.8, 80.7, 78.5,
77.6, 75.0, 73.9, 73.7, 73.5, 73.3, 72.7, 72.2, 72.0, 71.2, 70.6, 70.4, 70.4, 70.1, 70.0, 69.5, 68.7, 67.3,
66.9, 66.5, 63.5, 62.8, 62.7, 62.2, 61.4, 55.3, 55.2, 55.1, 53.8, 53.1, 51.9, 49.2, 43.0, 38.7, 36.6, 36.2,
32.3, 31.9, 31.7, 30.3, 29.7, 29.7, 29.7, 29.6, 29.6, 29.5, 29.4, 29.3, 29.3, 29.1, 29.0, 28.9, 28.8, 25.9,
25.6, 23.9, 23.8, 23.4, 23.1, 23.0, 22.9, 22.8, 22.7, 22.6, 21.1, 20.8, 20.8, 20.8, 20.6, 20.4, 20.4, 14.1,
14.1, 14.0, 11.0, 10.9. HRMS (ESI) m/z: found [1/2M+Na]+ 1514.6482, C155H202N4O54 calcd for
[1/2M+Na]+ 1514.6484.

Molecules 2013, 18
15177
AcO
AcO
AcO
OAc
f O
O
AcO
O
OBz
O
OAc
O
O
O
AcHN
AcO
d
c
O
O
e
O
OBz
O
OAc
HN
O
C17H35
C13H27
AcHN
BzO
AcHN
OBz
AcO
CO2Me
a
O
O
b
O
O
OAc
AcO
OBz
O
4-O-{2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-galactopyranosyl-(1→4)-[(methyl
5-acetamido-
4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosylonate)-(2→3)]-2,6-di-O-
benzoyl-β-D-galactopyranosyl-(1→3)-2-acetamido-4,6-di-O-acetyl-2-deoxy-β-D-galactopyranosyl-
(1→4)-2,3,6-tri-O-benzoyl-β-D-galactopyranosyl-(1→4)}-2,3-di-O-acetyl-β-D-glucopyranosyl-
(1'→1)-(2S,3R,4E)-2-octadecanamido-4-octadecene-1,3-diol-3,6'-succinate (33). To a mixture of 31
(53 mg, 25.3 µmol) and 1 (23 mg, 25.3 µmol) in CHCl3 (843 µL) was added 4 Å molecular sieves (120 mg)
at rt. After stirring for 1 h, the mixture was cooled to 0 °C. TMSOTf (0.5 µL, 2.53 µmol) was then
added to the mixture at 0 °C. After stirring for 2.5 h at rt as the reaction was monitored by TLC (4:3
acetone–n-hexane), the reaction was quenched by the addition of satd aq NaHCO3. The solution 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 (1:1 acetone–n-hexane) to give 33 (22 mg, 31%). The
yields of 33 based on the use of 2.0 eq. and 3.0 eq. of 1 were 48% and 60%, respectively. [α]D +8.0° (c
1
0.5, CHCl3); H-NMR (600 MHz, CDCl3) δ 8.11–7.31 (m, 25H, 5Ph), 6.31 (d, 1H, J2,NH = 8.2 Hz,
NHc), 5.98 (d, 1H, J2,NH = 6.2 Hz, NHf), 5.77 (m, 1H, H-5Cer), 5.59 (d, 1H, J2,NH = 9.6 Hz, NHCer),
5.52 (m, 2H, H-3b, H-2d), 5.38 (m, 1H, H-8e), 5.34–5.29 (m, 4H, H-4c, H-4f, H-2b, H-3Cer),
5.26–5.21 (m, 2H, H-4Cer, H-7e), 5.18–5.14 (m, 2H, H-3a, H-1f), 5.10 (d, 1H, J5,NH = 9.7 Hz, NHe),
5.04 (d, 1H, J1,2 = 8.3 Hz, H-1c), 5.00 (m, 1H, H-4e), 4.88 (dd, 1H, J3,4 = 3.4 Hz, J2,3 = 11.0 Hz, H-3f),
4.83 (t, 1H, J1,2 = J2,3 = 7.2 Hz, H-2a), 4.75 (d, 1H, J1,2 = 7.6 Hz, H-1b), 4.69 (d, 1H, J1,2 = 7.6 Hz,
H-1d), 4.62 (m, 2H, H-6b, H-6d), 4.52 (d, 1H, J3,4 = 2.8 Hz, H-4d), 4.34 (m, 3H, H-1a, H-6'b, H-6'd),
4.25 (m, 2H, H-2Cer, H-6f), 4.19 (near dd, 1H, H-6c), 4.20–4.05 (m, 4H, H-6´f, H-3c, H-6´c, H-9e),
4.02–3.88 (m, 6H, H-9'e, H-4b, H-6a, H-2c, H-3d, H-5e), 3.85–3.75 (m, 8H, H-1Cer, OCH3, H-1'Cer,
H-5b, H-5d, H-6e), 3.70 (m, 2H, H-4a, H-6'a), 3.60 (m, 2H, H-5c, H-5f), 3.50 (near t, 1H, H-5a), 3.12
(m, 1H, H-2f), 2.59–2.40 (m, 4H, 2C(=O)CH2), 2.29 (dd, 1H, Jgem = 13.0 Hz, J3eq,4 = 4.8 Hz, H-3eeq),
2.19–1.55 (m, 49H, C(=O)CH2CH2Cer, H-3eax, H-6Cer, H-6'Cer, 14Ac), 1.25 (m, 50H, 25-CH2-), 0.88
13
(m, 6H, 2-CH3Cer); C-NMR (150 MHz, CDCl3) δ 172.7, 171.3, 171.2, 171.1, 170.5, 170.4, 170.3,
170.3, 169.9, 169.7, 169.3, 168.1, 166.1, 166.0, 165.9, 164.9, 164.3, 133.6, 133.3, 133.2, 130.4, 130.1,
130.0, 129.8, 129.7, 129.5, 129.4, 129.0, 128.8, 128.7, 128.6, 128.5, 128.4, 128.2, 124.7, 101.0, 100.7,
100.4, 99.2, 98.3, 97.5, 76.5, 75.2, 74.0, 73.6, 73.3, 72.4, 72.1, 72.0, 72.0, 71.7, 71.3, 70.5, 70.4, 70.0,
69.6, 68.7, 67.3, 67.0, 66.5, 63.5, 63.0, 62.8, 62.2, 61.4, 53.1, 51.8, 50.0, 49.3, 36.8, 36.1, 32.3, 31.9,
29.7, 29.7, 29.5, 29.5, 29.4, 29.3, 29.3, 28.8, 25.6, 23.4, 23.2, 23.2, 23.1, 22.9, 22.8, 22.7, 22.6, 22.2,
22.0, 21.8, 21.1, 20.9, 20.8, 20.7, 20.6, 20.5, 20.4, 14.1. HRMS (ESI) m/z: found [1/2M+Na]+
1436.6014, C143H190N4O54 calcd for [1/2M+Na]+ 1436.6015.

Molecules 2013, 18
15178
HO
HO
OH
O
f
O
HO
OH
O
OH
O
AcHN
O
HO
HO
c
d
O
O
O
e
OH
OH
O
OH
O
OH
AcHN
HO
AcHN
HO
CO2H
a
b
O
C13H27
C17H35
O
OH
HO
HN
OH
OH
O
GalNAc-GM1b: 2-acetamido-2-deoxy-β-D-galactopyranosyl-(1→4)-[5-acetamido-3,5-dideoxy-D-glycero-
α-D-galacto-2-nonulopyranosylonic acid-(2→3)]-β-D-galactopyranosyl-(1→3)-2-acetamido-2-deoxy-β-D-
galactopyranosyl-(1→4)-β-D-galactopyranosyl-(1→4)-β-D-glucopyranosyl-(1'→1)-(2S,3R,4E)-2-
octadecanamido-4-octadecene-1,3-diol. To a solution of 33 (15.0 mg, 5.31 µmol) in MeOH/THF (1:1,
532 µL) was added NaOMe (28% solution in MeOH, 102 µg, 0.531 µmol) at 0 °C. After stirring for
6 d at rt as the reaction was monitored by TLC (5:4:1 CHCl3–MeOH–10 mM aq ZnCl2), water (10 µL)
was added to the mixture. After stirring for 8 d at rt, 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 CHCl3–MeOH as eluent followed by
silica gel column chromatography (5:4:0.5 CHCl3–MeOH–H2O) to give the target GalNAc-GM1b
1
(8.2 mg, 88%). [α]D +12.5° (c 0.2, 1:1 CHCl3–MeOH); H-NMR (600 MHz, 1:1 CDCl3–CD3OD) δ
5.70 (m, 1H, H-5Cer), 5.45 (dd, 1H, J3,4 = 7.6 Hz, J4,5 = 15.1 Hz, H-4Cer), 2.73 (br d, 1H, H-3eeq), 2.18
(m, 2H, C(=O)CH2), 2.05–2.01 (m, 11H, 3Ac, H-6Cer, H-6'Cer), 1.85 (br t, 1H, H-3eax), 1.59 (m, 2H,
C(=O)CH2CH2), 1.37–1.19 (m, 50H, 25-CH2-), 0.89 (m, 6H, 2-CH3); 13C-NMR (150 MHz, 1:1
CDCl3–CD3OD) δ 174.8, 174.6, 173.7, 173.4, 134.4, 129.7, 129.5, 128.0, 104.4, 103.8, 103.1, 102.0,
79.0, 76.2, 75.2, 75.0, 74.7, 74.5, 73.8, 73.6, 73.5, 72.1, 71.9, 71.3, 69.6, 69.5, 68.7, 68.6, 68.2, 64.6,
62.0, 61.5, 60.4, 60.2, 53.3, 53.1, 52.6, 51.8, 47.7, 36.4, 32.4, 32.0, 29.7, 29.6, 29.6, 29.6, 29.4, 29.3,
26.1, 22.7, 22.7, 22.0, 13.8. HRMS (ESI) m/z: found [M−H]− 1747.9487, C81H144N4O36 calcd for
[M−H]− 1747.9488.
4. Conclusions
In this study, we investigated the development of a GlcCer cassette acceptor that was both readily
accessible and highly reactive. We designed and prepared a novel cassette acceptor bearing
electron-donating PMB groups at C2 and C3 of the glucose residue. Various types of linkers and their
effect on the stereoselectivity of intramolecular glycosylation were examined. Although varying the
linker did not significantly increase β-selectivity, the use of a nitrile solvent gave predominantly the
desired β-product. Considering the accessibility of the acceptor, we opted for the succinyl linker. In the
experiment on coupling the cassette acceptor and oligosaccharide donor, we found that the use of
PMB groups as protecting groups at C2 and C3 positions of the glucose residue did not enhance the
reactivity as a GlcCer cassette acceptor. This interesting finding should provide useful information for
the future design of glycosyl acceptors. Furthermore, we extended the generality of the GlcCer cassette
approach by applying it to the efficient total synthesis of the ganglioside GalNAc-GM1b. Our

Molecules 2013, 18
15179
laboratory is now conducting further studies to evaluate the scope and limitations of the GlcCer
cassette approach.
Acknowledgments
The iCeMS is supported by World Premier International Research Center Initiative (WPI), MEXT,
Japan. This work was financially supported in part by MEXT of Japan (a Grant-in-Aid for Scientific
Research (B) No. 22380067 to M.K. and a Grant-in-Aid for Young Scientists (A) No. 23688014 and
Grant-in-Aid on Innovative Areas No. 24110505, Deciphering sugar chain-based signals regulating
integrative neuronal functions, to H.A.). We thank Kiyoko Ito (Gifu University) for providing
technical assistance.
Conflicts of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are not available from the authors.
© 2013 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).