Synthesis of a GM3 ganglioside analogue carrying a phytoceramide moiety by intramolecular glycosylation as a key step

Article abstract of DOI:10.1016/j.carres.2008.05.007

A novel analogue of ganglioside GM3, in which sphingosine was replaced with a phytosphingosine moiety, was synthesized by intramolecular glycosylation as a key step. Glucose, a reducing terminal of the saccharide, and phytoceramide were first tethered by succinic acid and the derivative used for the subsequent glycosidic bond formation. The obtained glycosyl phytoceramide was further glycosylated with the sialyl galactose residue to afford a fully protected GM3 derivative, which was converted into the desired, final compound by using conventional deprotection procedures.

Full text of DOI:10.1016/j.carres.2008.05.007

Carbohydrate Research 343 (2008) 2729–2734
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Carbohydrate Research
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Synthesis of a GM3 ganglioside analogue carrying a phytoceramide moiety
by intramolecular glycosylation as a key step q
a,b
Kohki Fujikawa a, Akihiro Imamura b, Hideharu Ishida a, , Makoto Kiso
*
a Department of Applied Bio-organic Chemistry, Faculty of Applied Biological Sciences, Gifu University, Gifu 501-1193, Japan
b CREST, Japan Science and Technology Agency (JST), Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel analogue of ganglioside GM3, in which sphingosine was replaced with a phytosphingosine moi-
ety, was synthesized by intramolecular glycosylation as a key step. Glucose, a reducing terminal of the
saccharide, and phytoceramide were first tethered by succinic acid and the derivative used for the sub-
sequent glycosidic bond formation. The obtained glycosyl phytoceramide was further glycosylated with
the sialyl galactose residue to afford a fully protected GM3 derivative, which was converted into the
desired, final compound by using conventional deprotection procedures.
Received 16 February 2008
Received in revised form 23 April 2008
Accepted 4 May 2008
Available online 13 May 2008
Keywords:
Ó 2008 Published by Elsevier Ltd.
Ganglioside GM3
Intramolecular glycosylation
Phytosphingosine
1. Introduction
2. Results and discussion
Gangliosides, sialic acid-containing glycosphingolipids, are com-
monly present in cell-surface membranes. These molecules are con-
sidered to be involved in many biological processes such as cell
growth, cell differentiation, cell adhesion, microbial and viral infec-
tions, immune response, oncogenesis, and many other receptor-
mediated reactions.1–3 Since gangliosides have been found to play
important functional roles, synthetic gangliosides are of critical
importance to investigate structure–activity relationships. There-
fore, numerous reports on the synthesis of gangliosides have been
published, most of them employ a typical synthetic strategy to
introduce the ceramide moiety4 or an azido derivative of sphingo-
sine5 as an equivalent of sphingosine, after having completed the
construction of the oligosaccharides. However, reduction in yield
during the introduction of the ceramide moiety or multiple steps
required for the conversion of the azido derivative to a ceramide
moiety having an oligosaccharide unit makes the synthesis of gly-
colipids in large quantities a formidable task. Intramolecular glyco-
sylation can be a promising approaches to overcome this problem
because of enhanced reactivity and stereo-/regiospecificity6 as al-
The retro-synthetic scheme of the target compound is shown
in Chart 1, where a sialyl galactosyl donor 11, which has already
been employed successfully in the synthesis of a series of ganglio-
sides,8–12 and glycosyl phytoceramide acceptor 10, which was syn-
thesized by intramolecular glycosylation and also serves as a
glycosyl acceptor for the first time in this study, have been
employed as the key building blocks. The glucose derivative 3
was synthesized from the known 4,6-O-benzylidene protected glu-
cose derivative 1.13 First, 1 was benzolyated at O-2 to give 2, then
removal of the benzylidene acetal from 2 gave the desired glucose
derivative 3 (Scheme 1).
The phytoceramide derivative 6 was synthesized from phyto-
ceramide 4,14 which was previously prepared from the commer-
cially available phytosphingosine. Regioselective silylation of the
primary hydroxyl with tert-butyldiphenylsilyl chloride gave 5,
then subsequent treatment with succinic anhydride and acetic
anhydride gave 6 in 40% yield accompanied by 7 (40%) as by-
product.
Linking of glucose derivative 3 with phytoceramide 6 was
accomplished in the presence of 2,4,6-trichlorobenzoyl chloride,
triethylamine and 4-(dimethylamino)pyridine15 to give the desired
compound 8 in 76% yield (Scheme 2). In intramolecular glycosyla-
tion, succinyl and malonyl spacers are well investigated and are
known to provide good results in terms of yield and/or stereocon-
trol.16 The tert-butyldiphenylsilyl group was removed by treat-
ment with acetic acid and tetrabutylammonium fluoride to yield
9 in 77% yield.
ready demonstrated in
a
-glycosylation and b-mannosylation.7 As
a part of our continuous interest in the synthesis of glycolipids,
we describe herein the efficient synthesis of a GM3 analogue by
employing intramolecular glycosylation as a key step.
q
Part 145 in the series: Synthetic studies on sialoglycoconjugates.
* Corresponding author. Tel./fax: +81 58 293 2918.
E-mail address: ishida@gifu-u.ac.jp (H. Ishida).
0008-6215/$ - see front matter Ó 2008 Published by Elsevier Ltd.
doi:10.1016/j.carres.2008.05.007

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K. Fujikawa et al. / Carbohydrate Research 343 (2008) 2729–2734
OH
OH
OH
O
HO
OH
OH
O
C14H29
HO
O
AcHN
HO
O
O
O
OH
OH
C17H35
COOH
OH
OH
HN
14
GM3 derivative
O
O
OBz
OBz
O
AcO
OAc
O
O
AcHN
AcO
O
O
O
O
+
C17H35
HN
BzO
O
CCl3
NH
COOMe
O
HO
MPMO
OAc
O
C14H29
OBz
OAc
11
10
OAc
C14H29
OH
O
TBDPSO
H35C17
O
HO
MPMO
SPh
O
NH
OBz
OH
O
O
3
6
Chart 1. Retro-synthetic scheme for ganglioside GM3 derivative.
O
O
Ph
Ph
a
b
O
O
O
O
SPh
SPh
3
MPMO
MPMO
OH
OBz
1
2
OH
OH
OH
C14H29
C14H29
HO
c
TBDPSO
d,e
C14H29
OH
OH
HO
HN C17H35
HN C17H35
NH2OH
O
O
phytosphingosine
4
5
O
O
HO
O
C14H29
+
6
TBDPSO
O
OH
O
HN
40%
(2 steps)
O
C17H35
O
7
40%
Scheme 1. Reagents and conditions: (a) BzCl, py, rt, 80%; (b) 85% aq AcOH, 40 °C, 86%; (c) TBDPSCl, DMAP, Et3N, CH2Cl2, rt, 76%; (d) succinic anhydride, DMAP, py, rt to 40 °C;
(e) Ac2O, DMAP, py, rt, 40% in two steps.
The pre-arranged glycolipid 9 was then treated with N-iodosuc-
cinimide and trifluoromethanesulfonic acid (NIS–TfOH)17 in
dichloromethane to give the desired glucosyl ceramide 10 in 85%
yield as an intramolecular glycosylation product. When the 2-hy-
droxy group was protected with a non-participating MPM group,
this glycosylation reaction gave an anomeric mixture of the

K. Fujikawa et al. / Carbohydrate Research 343 (2008) 2729–2734
2731
O
O
O
C17H35
O
HN
a
O
+
3
6
O
OTBDPS
HO
H
29C14
SPh
MPMO
OBz
OAc
8
O
O
O
C17H35
OH
c
b
HN
O
O
10
O
HO
MPMO
SPh H29C14
OBz
OAc
9
Scheme 2. Reagents and conditions: (a) 2,4,6-trichlorobenzoyl chloride, Et3N, DMAP, CH2Cl2, rt, 76%; (b) TBAF, AcOH, THF, 0 °C, 77%; (c) NIS, TfOH, CH2Cl2, 0 °C, 85%.
glycoside unless nitriles were used as a solvent (data not shown).
The anomeric signal of 10 at d 4.47 (d, J1,2 7.5 Hz, H-1 of Glc)
confirmed the b-glycosidic linkage.
3. Experimental
3.1. General methods
The obtained glycosyl ceramide was then further glycosylated
to give the fully protected GM3 derivative 12 (Scheme 3). Glycosyl-
ation of 10 with the trichloroacetimidate18 derivative of sialyl
galactose (11) was carried out by use of trimethylsilyl trifluoro-
methanesulfonic acid (TMSOTf) to give the desired GM3 derivative
12 in 70% yield. A proton doublet in the 1H NMR spectrum of 12 at
d 5.10 (d, J1,2 8.2 Hz, H-1 of Gal) showed that the sialyl galactose
unit was incorporated into the glycolipid as a b-glycoside. These
results suggest that the glycosyl acceptor 10 could be applied as
versatile building unit for the synthesis of more complicated gan-
gliosides such as GM1 and GM2.
Finally, all the protecting groups were removed by conventional
deprotection procedures, such as treatment with trifluoroacetic
acid to remove the MPM group (?13) sodium methoxide deacety-
lation to remove acetyl and benzoyl groups, and a final saponifica-
tion of the methyl ester to afford the target compound 14 in
quantitative yield.
1H and 13C NMR spectra were measured using Varian INOVA
400 and 500 equipments. Chemical shifts are expressed in ppm
(d) relative to the signal of Me4Si adjusted to d 0 ppm. High-resolu-
tion mass spectra were recorded in positive ion mode on a Bruker
micro TOF (ESI-TOFMS). Molecular sieves were purchased from
Wako Chemicals Inc. and dried at 300 °C for 2 h in muffle furnace
prior to use. Solvents as reaction media were dried over molecular
sieves and used without purification. TLC analysis was performed
on Merck TLC (Silica Gel 60F254 on glass plate). Silica gel (80 mesh
and 300 mesh) manufactured by Fuji Silysia Co. was used for flash
column chromatography. Amount of silica gel was usually esti-
mated as 200–400-fold weight of sample to be charged. Solvent
systems for chromatography are specified in v/v. Evaporation and
concentration were carried out under diminished pressure. Specific
rotations were determined with a Horiba SEPA-300 high sensitive
polarimeter.
In conclusion, the present study demonstrates the usefulness of
the intramolecular glycosylation of newly developed glucose-cera-
mide derivatives and its application in the synthesis of a ganglio-
side GM3 analogue carrying a phytoceramide moiety in place of
mammalian ceramide. The key intermediate glucosylceramide
could be used as an acceptor in glycosylation reactions for the effi-
cient synthesis of higher gangliosides and their analogues. Further
study on this subject is currently underway.
3.2. Phenyl 2-O-benzoyl-4,6-O-benzylidene-3-O-4-
methoxybenzyl-1-thio-b-D-glucopyranoside (2)
To a soln of 1 (171 mg, 0.356 mmol) in pyridine (3.5 mL) were
added benzoyl chloride (62.0 L, 0.534 mmol) and DMAP
l
(4.35 mg, 0.036 mmol), and the mixture was stirred for 4 h at room
temperature. After complete consumption of the starting material
a
+
10
11
OAc
OR1
BzO
OBz
O
AcO
R2O
O
C14H29
AcHN
AcO
O
O
O
O
O
HN
C17H35
BzO
CO2Me
R1 = Bz, R2 = MPM
R1 = Bz, R2 = H
OAc
12
13
O
O
OAc
b
O
O
c,d
14
GM3 derivative
Scheme 3. Reagents and conditions: (a) TMSOTf, CH2Cl2, 0 °C, 70%; (b) TFA, CH2Cl2, rt; (c) NaOMe, MeOH, rt; (d) NaOMe, MeOH and water, rt, quantitative in three steps.

2732
K. Fujikawa et al. / Carbohydrate Research 343 (2008) 2729–2734
(TLC 50:1 toluene–MeOH), MeOH was added to the reaction mix-
ture at 0 °C and the soln was co-evaporated with toluene. The mix-
ture was diluted with CHCl3 and washed with 2 M HCl, water, satd
NaHCO3 and brine, dried over Na2SO4 and concentrated. The resi-
due was chromatographed on a column of silica gel (CHCl3) to give
Ac2O (2.5 mL, 26.5 mmol) was added at 0 °C. After stirring at
40 °C for 12 h (TLC monitoring: 40:1:1 CHCl3–MeOH–5% aq CaCl2),
MeOH was added to the mixture at 0 °C. The reaction mixture was
co-evaporated with toluene and extracted with EtOAc. The organic
layer was washed with 2 M HCl, water, satd NaHCO3 and brine,
dried (Na2SO4) and concentrated. The residue was chromato-
graphed on a column of silica gel (1:6 EtOAc–hexane) to give 6
2 (166 mg, 80%) as an amorphous product: [
a
]
D
+47.3 (c 1.0,
CHCl3); 1H NMR (400 MHz, CDCl3): d 7.20–8.01 (m, 15H, 3Ph),
6.55–7.10 (2d, 4H, MeOPh), 5.60 (s, 1H, PhCH), 5.26 (t, 1H,
J1,2 = J2,3 9.9 Hz, H-2), 4.84 (d, 1H, H-1), 4.73 (d, 1H, Jgem 11.7 Hz,
PhCH2), 4.59 (d, 1H, PhCH2), 4.41 (t, 1H, H-4), 3.68–3.89 (m, 3H,
H-6, H-60, H-3), 3.68 (s, 3H, OMe), 3.56 (m, 1H, H-5); 13C NMR
(100 MHz, CDCl3): d 164.9, 159.1, 137.2, 133.1, 132.9, 132.2,
129.9, 129.8, 129.7, 129.0, 128.9, 128.3, 128.2, 128.1, 126.0,
113.5, 101.2, 87.0, 81.4, 78.7, 73.8, 72.0, 70.6, 68.6, 55.0; HRESIMS:
calcd for C34H32O7S: 607.1761 [M+Na]+, found: m/z 607.1773.
(138 mg, 40%) as an amorphous solid: [a] +60.0 (c 0.1, CHCl3);
D
1H NMR (400 MHz, CDCl3): d 7.35–7.65 (m, 10H, 2Ph), 5.97 (d,
1H, J2,NH 9.5 Hz, NH), 5.33 (dd, 1H, J2,3 9.2 Hz, J3,4 2.4 Hz, H-3),
4.95 (m, 1H, H-4), 4.25 (m, 1H, H-2), 3.66 (m, 2H, H-1,10), 2.54–
2.67 (m, 4H, –OCOCH2CH2COO–), 2.15 (m, 2H, H-6, H-60), 1.92 (s,
3H, OAc), 1.60 (m, 2H, H-5,50), 1.00–1.70 (m, 54H, –CH2–), 0.90
(t, 6H, 2Me); 13C NMR (100 MHz, CDCl3): d 177.0, 172.8, 172.0,
169.8, 135.6, 135.5, 132.9, 132.6, 129.9, 127.8, 127.8, 73.9, 71.4,
62.3, 49.2, 36.8, 31.9, 29.7, 29.6, 29.52, 29.47, 29.4, 29.34, 29.30,
29.28, 29.1, 28.9, 27.8, 26.8, 25.6, 25.5, 22.7, 20.7, 19.2, 14.1; HRE-
SIMS: calcd for C58H97NO8Si: 986.6876 [M+Na]+, found: m/z =
986.6843.
3.3. Phenyl 2-O-benzoyl-3-O-4-methoxybenzyl-1-thio-b-D-
glucopyranoside (3)
To a soln of 2 (200 mg, 0.330 mmol) in AcOH (15 mL) was added
water (3.0 mL). After stirring at 40 °C for 12 h (TLC monitoring 1:1
toluene–EtOAc), the mixture was diluted with CHCl3, and washed
with water, satd NaHCO3 and brine, dried (Na2SO4) and concen-
trated. The residue was chromatographed on a column of silica
gel (3:1 toluene–EtOAc) to give 3 (145 mg, 86%) as an amorphous
3.6. Phenyl 6-O-[{(2S,3S,4R)-3-O-acetyl-1-O-tert-
butyldiphenylsilyl-2-octadecanoylamino-octadecane-4-
yloxy}carbonylpropanoyl]-2-O-benzoyl-3-O-p-methoxybenzyl-
1-thio-b-D-glucopyranoside (8)
mass: [
a]
+0.4 (c 1.0, CHCl3); 1H NMR (400 MHz, CDCl3): d 7.20–
To a soln of 6 (204 mg, 0.212 mmol) in CH2Cl2 (2.1 mL) were
added 2,4,6-trichlorobenzoyl chloride (50.0 L, 0.318 mmol),
DMAP (39.0 mg, 0.318 mmol), Et3N (44.4 L, 0.318 mmol) and 3
D
8.10 (m, 10H, 2Ph), 6.60–7.15 (2d, 4H, MeOPh), 5.23 (t, 1H,
J1,2 = J2,3 9.9 Hz, H-2), 4.83 (d, 1H, H-1), 4.63 (d, 1H, Jgem 11.7 Hz,
PhCH2), 4.59 (d, 1H, PhCH2), 3.90 (m, 1H, H-60), 3.83 (m, 1H, H-6),
3.74 (m, 1H, H-4), 3.70 (m, 1H, H-3), 3.65 (s, 3H, OMe), 3.44 (m,
1H, H-5), 3.42 (d, 1H, J4,4-OH 3.6 Hz, 4-OH), 2.77 (m, 1H, 6-OH);
13C NMR (100 MHz, CDCl3): d 165.2, 159.1, 133.2, 132.8, 132.0,
129.8, 129.6, 129.6, 128.8, 128.3, 127.8, 113.7, 86.3, 83.1, 79.5,
74.3, 72.2, 70.2, 62.3, 55.0; HRESIMS: calcd for C27H28O7S:
519.1448 [M+Na]+, found: m/z 519.1454.
l
l
(105 mg, 0.212 mmol). The mixture was stirred for 2 h at room
temperature. After complete consumption of the starting material
(TLC: 2:1 toluene–EtOAc), the mixture was diluted with CHCl3 and
washed with satd NaHCO3, water and brine, dried (Na2SO4) and
concentrated. The residue was chromatographed on a column of
silica gel (1:6 EtOAc–hexane) to give 8 (229 mg, 76%) as an amor-
phous solid: [a]
+0.4 (c 1.9, CHCl3); 1H NMR (500 MHz, CDCl3): d
D
7.20–8.10 (m, 20H, 4Ph), 6.62–7.15 (2d, 4H, MeOPh), 5.95 (d, 1H,
J2,NH 9.5 Hz, NH), 5.35 (dd, 1H, J2,3 9.6 Hz, J3,4 2.4 Hz, H-3Cer), 5.19
(t, 1H, J1,2 = J2,3 9.9 Hz, H-2Glc), 4.95 (m, 1H, H-4Cer), 4.76 (d, 1H,
3.4. (2S,3S,4R)-1-O-tert-butyldiphenylsilyl-2-
octadecanoylamino-octadecane (5)
H-1Glc), 4.66 (d, 1H, Jgem 11.0 Hz, PhCH2), 4.63 (d, 1H, PhCH2),
0
0
To a soln of 4 (500 mg, 0.857 mmol) and Et3N (10 mL) in CH2Cl2
4.46 (dd, 1H, J5,6 2.4 Hz, Jgem 12.0 Hz, H-6 Glc), 4.39 (dd, 1H, J5;6
(8.6 mL) were added TBDPSCl (260
lL, 1.03 mmol) and DMAP
4.0 Hz, H-60Glc), 4.22 (m, 1H, H-2Cer), 3.72 (m, 1H, J4,4-OH 2.8 Hz,
(209 mg, 1.71 mmol). After stirring at room temperature for 20 h
(TLC monitoring 50:1 CHCl3–MeOH), MeOH was added to the reac-
tion mixture at 0 °C. After concentration, the residue was chro-
matographed on a column of silica gel (100:1 CHCl3–MeOH) to
H-4Glc), 3.71 (s, 3H, OMe), 3.63 (m, 1H, H-3Glc), 3.53 (m, 1H, H-
5
Glc), 3.10 (d, 1H, 4-OHGlc), 2.60–2.66 (m, 4H, –OCOCH2CH2COO–),
2.10 (m, 2H, H-6Cer, H-60Cer), 1.95 (s, 3H, OAc), 1.52 (m, 2H, H-
5
Cer, H-50Cer), 1.00–1.60 (m, 54H, –CH2–), 0.90 (t, 6H, 2Me); 13C
give 5 (533 mg, 76%) as an amorphous product: [
a
]
+1.3 (c 1.0,
NMR (125 MHz, CDCl3): d 172.7, 172.2, 171.9, 170.5, 165.1, 159.3,
135.7, 135.5, 133.2, 132.8, 132.6, 129.9, 129.8, 129.7, 128.7,
128.4, 127.9, 127.84, 127.79, 113.8, 86.3, 82.9, 73.9, 72.1, 71.6,
69.8, 63.0, 62.3, 55.1, 49.2, 36.8, 31.9, 29.70, 29.66, 29.6, 29.5,
29.39, 29.35, 29.1, 27.9, 26.8, 25.7, 22.7, 20.7, 19.2, 14.1; HRESIMS:
calcd for C85H123NO14SSi: 1464.8289 [M+Na]+, found: m/z
1464.8357.
D
CHCl3); 1H NMR (400 MHz, CDCl3): d 7.30–7.80 (m, 10H, 2Ph),
0
6.15 (d, 1H, J2,NH 8.4 Hz, NH), 4.17 (m, 1H, J1,2 3.6 Hz, J1 ;2 4.4 Hz,
H-2), 4.05 (dd, 1H, Jgem 10.6 Hz, H-1), 3.79 (dd, 1H, H-10), 3.58 (m,
2H, J3,3-OH 6.8 Hz, J4,4-OH 7.6 Hz, H-3, H-4), 3.46 (d, 1H, 4-OH),
2.80 (d, 1H, 3-OH), 2.08 (m, 2H, HNC(@O)CH2–), 1.70 (m, 2H, H-
6, H-60), 1.50 (m, 2H, HNC(@O)CH2CH2–), 1.45 (m, 2H, H-5, H-50),
1.00–1.40 (m, 50H, –CH2–), 0.85 (t, 6H, 2Me); 13C NMR
(100 MHz, CDCl3): d 173.1, 135.5, 135.4, 134.8, 132.5, 132.2,
130.0, 127.9, 127.5, 75.6, 73.3, 63.8, 51.3, 36.7, 33.3, 31.9, 29.7,
29.6, 29.5, 29.34, 29.33, 29.28, 26.9, 26.5, 25.9, 25.6, 22.7, 19.1,
14.1; HRESIMS: calcd for C52H91NO4Si: 844.6615 [M+Na]+, found:
m/z 844.6623.
3.7. Phenyl 6-O-[{(2S,3S,4R)-3-O-acetyl-2-octadecanolyamino-
octadecane-4-yloxy}carbonylpropanoyl]-2-O-benzoyl-3-O-4-
methoxybenzyl-1-thio-b-D-glucopyranoside (9)
To a soln of 8 (48.8 mg, 0.034 mmol) in THF (338
added AcOH (6.0 L, 0.101 mmol) and tetrabutylammonium fluo-
ride (102 L, 0.102 mmol). After stirring at 0 °C for 12 h (TLC mon-
lL) were
l
3.5. (2S,3S,4R)-3-O-acetyl-1-O-tert-butyldiphenylsilyl-4-O-
l
succinoyl-2-octadecanoylamino-octadecane (6)
itoring: 1:2 EtOAc–hexane), the mixture was diluted with CHCl3,
and washed with satd NaHCO3 and brine, dried (Na2SO4) and con-
centrated. The residue was chromatographed on a column of silica
gel (1:5 toluene–EtOAc) to give 9 (30 mg, 77%) as an amorphous
solid: [
8.05 (m, 10H, 2Ph), 6.65–7.19 (2d, 4H, MeOPh), 6.23 (d, 1H,
To a soln of 5 (300 mg, 0.365 mmol) in pyridine (1.8 mL) was
added succinic anhydride (40.2 mg, 0.402 mmol) at 0 °C. The mix-
ture was stirred for 24 h at 40 °C. After complete consumption of
the starting material (TLC: 40:1:1 CHCl3–MeOH–5% aq CaCl2),
a]
+7.4 (c 0.6, CHCl3); 1H NMR (500 MHz, CDCl3): d 7.20–
D

K. Fujikawa et al. / Carbohydrate Research 343 (2008) 2729–2734
2733
J2,NH = 9.0 Hz, NH), 5.21 (t, 1H, J1,2 = J2,3 10.0 Hz, H-2Glc), 5.01–5.06
(m, 2H, H-3Cer, H-4Cer), 4.78 (d, 1H, H-1Glc), 4.63 (2d, 2H, Jgem
H-8Neu), 5.55 (t, 1H, J1,2 8.2 Hz, J2,3 8.2 Hz, H-2Gal), 5.36 (d, 1H, H-
4
1
Gal), 5.24 (dd, 1H, J6,7 2.8 Hz, J7,8 9.2 Hz, H-7Neu), 5.10 (d, 1H, H-
Gal), 5.08 (m, 1H, H-4Cer), 5.02 (d, 1H, J1,2 7.8 Hz, H-2Glc), 4.91–
11.5 Hz, PhCH2), 4.48 (dd, 1H, J5;6 4.0 Hz, Jgem 12.0 Hz, H-60Glc),
0
4.39 (dd, 1H, J5,6 2.0 Hz, H-6Glc), 4.19 (m, 1H, J2,NH 9.0 Hz, H-2Cer),
3.65–3.74 (m, 6H, H-10Cer, H-3Glc, H-4Glc, OMe), 3.54–3.59 (m, 2H,
H-1Cer, H-5Glc), 2.60–2.75 (m, 4H, –OCOCH2CH2COO–), 2.20 (m,
2H, H-6Cer, H-60Cer), 2.15 (s, 3H, OAc), 1.60 (m, 2H, H-5Cer, H-50Cer),
1.20–1.79 (m, 54H, –CH2–), 0.89 (t, 6H, 2Me); 13C NMR
(100 MHz, CDCl3): d 173.3, 172.6, 171.9, 171.5, 165.2, 159.3,
133.2, 132.8, 132.5, 129.83, 129.79, 129.7, 129.0, 128.7, 128.4,
128.2, 127.8, 125.3, 113.7, 86.4, 82.9, 77.8, 74.5, 73.4, 73.2, 72.0,
69.7, 63.3, 61.4, 55.1, 49.6, 36.7, 31.9, 29.7, 29.6, 29.5, 29.34,
29.28, 29.1, 28.6, 25.6, 25.5, 22.7, 20.9, 14.1; HRESIMS: calcd for
C69H105NO14S: 1226.7148 [M+Na]+, found: m/z 1226.7180.
4.98 (m, 3H, H-3Cer, NHNeu, H-3Gal), 4.81 (m, 1H, H-4Neu), 4.66–
4.87 (2d, 2H, Jgem 11.0 Hz, PhCH2), 4.42 (dd, 1H, Jgem 12.4 Hz, H-
90Neu), 4.35 (d, 1H, H-1Glc), 4.14–4.37 (m, 6H, H-5Gal, H-6Gal, H-60Gal
,
H-6Glc, H-60Glc, H-2Cer), 4.05 (dd, 1H, J8,9 6.0 Hz, Jgem 12.4 Hz, H-9Neu),
3.71–3.82 (m, 8H, H-1Cer, H-10Cer, H-3Glc, H-4Glc, H-5Neu, OMe), 3.61
(dd, 1H, H-6Neu), 3.57 (s, 3H, OMe), 3.50 (m, 1H, H-5Glc), 2.36–
2.67 (m, 4H, –OCOCH2CH2COO–), 2.48 (dd, 1H, J3eq.,4 4.6 Hz, H-
3eq.Neu), 1.64 (t, 1H, Jgem 12.4 Hz, H-3ax.Neu), 1.52–2.18 (6s, 18H,
OAc), 1.80 (m, 2H, H-6Cer, H-60Cer), 1.40 (m, 2H, H-5Cer, H-50Cer),
1.10–1.50 (m, 54H, –CH2–), 0.87 (t, 6H, 2Me); 13C NMR
(100 MHz, CDCl3): d 172.8, 171.0, 170.9, 170.8, 170.7, 170.6,
170.3, 170.1, 168.2, 165.7, 165.6, 165.1, 158.8, 133.3, 133.1,
130.4, 130.2, 129.9, 129.8, 129.7, 129.6, 129.4, 128.6, 128.5,
128.3, 113.4, 101.3, 98.8, 96.9, 79.5, 78.7, 77.7, 74.5, 74.4, 73.4,
73.1, 73.1, 72.0, 71.6, 71.4, 70.9, 69.4, 68.2, 67.6, 66.6, 63.3, 62.4,
61.7, 55.0, 53.2, 48.8, 46.6, 37.4, 36.3, 31.9, 30.8, 30.0, 29.7, 29.6,
29.6, 29.5, 29.4, 29.2, 25.3, 25.2, 23.2, 22.7, 21.5, 20.9, 20.8, 20.7,
3.8. 2-O-Benzoyl-3-O-4-methoxybenzyl-b-D-glucopyranosyl-
(1?1)-(2S,3S,4R)-3-O-acetyl-2-octadecanoylamino-octadecane-
4,6-succinate (10)
To a soln of 9 (43 mg, 0.036 mmol) in CH2Cl2 (1.2 mL) was
added MS4Å (40 mg). After stirring for 1 h, NIS (16.0 mg,
20.4, 14.1; HRESIMS: calcd for C110H148N2O34
:
2063.9811
0.071 mmol) and TfOH (0.6
lL, 0.0079 mmol) were added to the
[M+Na]+, found: m/z 2063.9823.
suspension at 0 °C. The mixture was stirred for 5 h at 0 °C. After
completion of the reaction (TLC 1:1 toluene–EtOAc), the mixture
was filtered through Celite. The combined filtrate and washings
were extracted with CHCl3, washed with satd NaHCO3, satd
Na2S2O3 and brine, dried (Na2SO4) and concentrated. The residue
was chromatographed on a column of silica gel (3:1 toluene–
3.10. O-(5-Acetamido-3,5-dideoxy-
nonulopyranosylonic acid)-(2?3)-(O-b-
(1?4)-O-b- -glucopyranosyl-(1?1)-(2S,3S,4R)-2-
D
-glycero-
a-D-galacto-2-
D-galactopyranosyl)-
D
octadecanoylamino-octadecane-1,3,4-triol (14)
EtOAc) to give 10 (33 mg, 85%) as an amorphous solid: [
a
]
+3.7
To a soln of 12 (38 mg, 0.0184 mmol) in CH2Cl2 (800 lL) was
added trifluoroacetic acid (370 lL). The mixture was stirred for
7 h at room temperature. After complete consumption of the start-
ing material (TLC 6:1 EtOAc–hexane), Et3N was added at 0 °C. The
reaction mixture was co-evaporated with toluene. After concentra-
tion, the residue was dissolved in MeOH (1.0 mL) and NaOMe
D
(c 1.1, CHCl3); 1H NMR (500 MHz, CDCl3): d 7.40–8.05 (m, 5H,
Ph), 6.75–7.12 (2d, 4H, MeOPh), 6.02 (d, 1H, J2,NH 9.0 Hz, NH),
5.18 (m, 2H, J1,2 7.5 Hz, H-4Cer, H-2Glc), 5.10 (t, 1H, H-3Cer), 4.66
(d, 1H, Jgem 11.5 Hz, PhCH2), 4.55 (d, 1H, PhCH2), 4.47 (d, 1H, H-
1
Glc), 4.39 (d, 1H, J1,2 5.5 Hz, H-2Cer), 4.37–4.41 (m, 2H, H-6Glc, H-
60Glc), 3.81 (dd, 1H, Jgem 11.5 Hz, H-1Cer), 3.73 (s, 3H, OMe), 3.61–
(7.2 mM soln in MeOH, 100 lL, 5.2 lmol) was added at 0 °C. After
3.69 (m, 3H, H-10Cer, H-3Glc, H-5Glc), 3.49 (dt, 1H, J4,4-OH 2.0 Hz, H-
stirring at room temperature for 26 h (TLC monitoring: 10:1:1
BuOH–MeOH–water), water was added to the mixture at 0 °C.
The mixture was stirred at room temperature for 10 h (TLC moni-
toring: 10:1:1 BuOH–MeOH–water). The reaction mixture was
neutralized with Dowex (H+) and filtered. The combined filtrate
and washings were concentrated. Column chromatography
(MeOH) of the residue on Sephadex LH-20 (200 g) gave 14
4
Glc), 2.50–2.80 (m, 4H, –OCOCH2CH2COO–), 2.50 (d, 1H, 4-OHGlc),
2.10 (s, 3H, OAc), 2.00 (m, 2H, H-6Cer, H-60Cer), 1.50 (m, 2H, H-5Cer
,
H-50Cer), 1.10–1.60 (m, 54H, –CH2–), 0.84 (t, 6H, 2Me); 13C NMR
(100 MHz, CDCl3): d 172.9, 171.4, 171.2, 170.8, 165.0, 159.3,
133.3, 129.8, 129.7, 129.6, 129.5, 128.5, 114.0, 113.9, 100.1, 81.5,
74.7, 74.3, 73.8, 73.7, 72.7, 70.4, 63.8, 55.1, 47.4, 37.4, 37.1, 36.5,
33.5, 32.7, 31.9, 30.2, 30.0, 29.7, 29.64, 29.55, 29.52, 29.48, 29.4,
29.34, 29.32, 29.2, 27.4, 27.1, 25.4, 25.0, 24.4, 22.7, 21.0, 19.7,
14.1; HRESIMS: calcd for C63H99NO14: 1116.6963 [M+Na]+, found:
m/z 1116.6923.
(22.1 mg, quantitative) as an amorphous solid: [
a
]
+8.0 (c 1.0,
D
MeOH); 1H NMR (600 MHz, CD3OD): d 4.42 (d, 1H, J1,2 7.6 Hz, H-
1
Gal), 4.31 (d, 1H, J1,2 7.6 Hz, H-1Glc), 2.85 (dd, 1H, Jgem 11.7 Hz, H-
3eq.Neu), 2.20 (m, 2H, H-6Cer, H-60Cer), 2.00 (s, 3H, NAc), 1.73 (t,
1H, H-3ax.Neu), 1.50 (m, 2H, H-5Cer, H-50Cer), 1.10–1.70 (m, 54H, –
CH2–), 0.90 (t, 6H, 2Me); 13C NMR (150 MHz, CD3OD): d 176.0,
175.5, 175.0, 105.1, 104.5, 101.1, 80.9, 77.7, 77.1, 76.5, 76.2, 75.1,
75.0, 74.8, 73.1, 73.0, 70.9, 70.2, 70.1, 69.4, 69.0, 64.6, 62.7, 61.8,
54.0, 51.9, 49.9, 49.6, 42.1, 37.3, 33.1, 32.3, 30.9, 30.9, 30.8, 30.8,
30.7, 30.6, 30.5, 30.5, 30.4, 27.1, 23.7, 22.6, 14.4; HRESIMS: calcd
for C59H110N2O22: 1221.7442 [M+Na]+, found: m/z 1221.7450.
3.9. (Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
glycero- -galacto-2-nonulopyranosylonate)-(2?3)-(2,4,6-tri-
O-benzoyl-b- -galactopyranosyl)-(1?4)-2-O-benzoyl-3-O-4-
methoxybenzyl-b- -glucopyranosyl-(1?1)-(2S,3S,4R)-3-O-
acetyl-2-octadecanoylamino-octadecane-4,6-succinate (12)
D-
a-D
D
D
To
0.137 mmol) in CH2Cl2 (1.1 mL) were added molecular sieves
(AW-300) (200 mg). After stirring for 1 h, TMSOTf (1.0 L,
a soln of 10 (63 mg, 0.0578 mmol) and 11 (152 mg,
Acknowledgements
l
0.00548 mmol) was added to the suspension at 0 °C. The progress
of the reaction was monitored by TLC (1:3 toluene–EtOAc). After
stirring for 4 h at 0 °C, the reaction mixture was filtered through
a pad of Celite. The combined filtrate and washings were extracted
with CHCl3, and washed with satd NaHCO3, and brine, dried
(Na2SO4) and concentrated. The residue was chromatographed on
a column of silica gel (1:2 toluene–EtOAc) to give 12 (83 mg,
This work was partly supported by the Ministry of Education,
Culture, Sports, Science, and Technology (MEXT) of Japan (Grant-
in-Aid for Scientific Research to M. Kiso, No. 17101007). We thank
Dr. Hirofumi Dohi for helpful discussions and Ms. Kiyoko Ito for
technical assistance.
References
70%) as an amorphous solid: [a]
+24.4 (c 0.7, CHCl3); 1H NMR
D
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