Ganglioside GM3 derivatives with truncated ceramide moiety: Facial synthesis and inhibitory activity against KB cell growth

Article abstract of DOI:10.1080/07328300600803518

An expeditious sialylation reaction with phenylthioglycoside 4 as a sialyl donor and MeSOTf as a promoter was developed. These conditions are very useful for synthesizing ganglioside GM 3 (1), its C8-ceramide analog 2, and 3-deoxy analog 3 of 2 in an efficient manner. The GM 3 analog 2, whose hydrophilicity is increased by shortening the ceramide moiety, exhibits increased growth inhibiton of KB cells. The 3-hydoxy group of ceramide does not influence its activity against KB cells. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/07328300600803518

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Ganglioside GM3 Derivatives with Truncated
Ceramide Moiety: Facial Synthesis and
Inhibitory Activity against KB Cell Growth
Dr. Michio Kurosu a & Isao Kitagawa b
a Department of Microbiology, Immunology and Pathology, Colorado State
University, Fort Collins, USA
b Graduate School of Pharmaceutical Sciences, Osaka University, Suita,
Osaka, Japan
Published online: 28 Oct 2011.
To cite this article: Dr. Michio Kurosu & Isao Kitagawa (2006): Ganglioside GM3 Derivatives with Truncated
Ceramide Moiety: Facial Synthesis and Inhibitory Activity against KB Cell Growth, Journal of Carbohydrate
Chemistry, 25:5, 427-439
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Journal of Carbohydrate Chemistry, 25:427–439, 2006
Copyright # Taylor & Francis Group, LLC
ISSN: 0732-8303 print 1532-2327 online
DOI: 10.1080/07328300600803518
Ganglioside GM3
Derivatives with Truncated
Ceramide Moiety: Facial
Synthesis and Inhibitory
Activity against KB
Cell Growth
Michio Kurosu
Department of Microbiology, Immunology and Pathology, Colorado State University,
Fort Collins, USA
Isao Kitagawa
Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka, Japan
An expeditious sialylation reaction with phenylthioglycoside 4 as a sialyl donor and
MeSOTf as a promoter was developed. These conditions are very useful for synthesizing
ganglioside GM3 (1), its C8-ceramide analog 2, and 3-deoxy analog 3 of 2 in an efficient
manner. The GM3 analog 2, whose hydrophilicity is increased by shortening the
ceramide moiety, exhibits increased growth inhibiton of KB cells. The 3-hydoxy group
of ceramide does not influence its activity against KB cells.
Keywords Ganglioside GM3, truncated ceramide, inhibitory activity against KB cell
growth
INTRODUCTION
Gangliosides are sialic acid-containing glycosphingolipids that are present on
all mammalian plasma membranes, where they participate in recognition and
signaling activity.[1] Ganglioside GM3 (1, herein referred to as GM3), a simple
Received December 15, 2006; accepted March 01, 2006.
Address correspondence to Dr. Michio Kurosu, Department of Microbiology, Immu-
nology and Pathology, Colorado State University, Fort Collins CO, 80523-1682, USA.
E-mail: michio.kurosu@colostate.edu
427

M. Kurosu and I. Kitagawa
428
and widely distributed glycosphingolipid, is known as (1) a negative regulator of
insulin signaling, which makes it a potential therapeutic target in type II
diabetes,[2] (2) a modulator of cell growth through inhibition of EGF receptor-
associated tyrosine kinase,[3] (3) a tumor-associated antigen in humans,[4] (4)
a modulator of EGF receptor phosphorylation, (5) a regulator of protein
kinase A (PKA) activity, and (6) an inducer of CD4 internalization in human
lymphocytes.[5] GM3 also interacts with HIV surface envelope glycoproteins
and modulates Ca2þ flux in triad membranes.[6] Although large-scale isolation
of GM3 from hybridoma cells has been developed, the purification typically
produces as a mixture of the ceramide moiety (N-acylsphingosine) with
C16 : 0-, C18 : 0-, C20 : 0-, C22 : 0-, and C24 : 1-fatty acids.[7] Interestingly, the
ceramide itself plays an important role in the regulation of several biological
processes of the cell. Some diseases are related to ceramide accumulation and
subsequent apoptosis induction.[8] Because glycolipids isolated from eukaryotes
and prokaryotes generally exhibit both amphiphilic and amphiphatic nature,[9]
it is important to (1) improve the physiochemical properties of glycolipids that
facilitate the penetration of a biological membrane and (2) design the mimic of
lipid moieties that can be recognized by glycosylases. As a part of our studies
on the synthesis of bioactive glycolipids and on the development of inhibitors
of glycosyltransferases and fatty acid biosyntheses, we wish to address the
function of the ceramide of glycolipids.[10] For these investigations we first
selected GM3 because of its significant biological activity (vide infra) and
because of its structural simplicity compared to the other higher gangliosides.
The ceramides are biosynthesized from L-serine and the C3-OH group, and
the configuration is important for their function as an acceptor for glycosyltrans-
ferases.[11] However, C3-OH group may not be an essential functional group for
gangliosides to exhibit their biological activities.[12] It is also important to under-
stand the minimum chain-length of the lipids required for enzymatic recognitions
and biological activities. In order to gain information about these questions, we
need to synthesize GM3 analogs containing shorter carbon-chain ceramides.
We now wish to report an expeditious sialylation of the lactose derivative 5 and
syntheses of GM3 (1, as a control), C8-GM3 2,[13] and its 3-deoxy analog 3
(Fig. 1), and a preliminary biological evaluation of these analogs against KB cells.
Figure 1: Structures of ganglioside GM3 and its analogues.

Ganglioside GM3 Derivatives
429
Syntheses of GM3 Analogs
A number of elegant syntheses of gangliosides have been reported to
date.[14] One of challenges in the syntheses of gangliosides is how efficiently
one can achieve the sialylation selectively at the desired position of the
acceptor. In order to achieve efficient a-selective sialylation reactions, it is
very important to find an appropriate combination between leaving group of
the sialyl donor and its promoter.[14c,d] Some sialylation conditions require
long reaction times at controlled temperatures and do not afford exclusively
the desired a-glycoside in good yield. On the contrary, enzymatic sialylations
have an advantage over chemical methods because of exclusive regio- and
stereoselectivity with unprotected acceptors.[15] However, chemical synthesis
of structurally less complicated GM3 derivatives is more feasible. We selected
phenythio glycoside 4 (a 1 : 6.6 mixture of a- and b-anomers) as a silayl
donor,[16] which could be synthesized in 85% overall yield in three steps from
sialic acid. We observed that MeSOTf [17] generated from AgOTf and MeSBr
was very effective to activate 4 in propionitrile at 2788C or in CH3CN. (Import-
ance of using a nitrile solvent in a-selective sialylation was described in a
number of literatures (see ref. 12)) at 2458C in which the sialylations with
benzyl alcohol and 2-trimethylsilylethanol were completed within 1 min to
afford the corresponding sialyl glycoside as a 6 ꢀ 7 : 1 mixture of a- and b-gly-
cosides in quantitative yields. Gratifyingly, when these conditions were applied
to partially protected TBDMS lactoside 5, which was synthesized by Hasega-
wa’s procedures,[14d,18] the a-sialyl lactoside 6 was obtained within 5 min in
85% yield without a detectable amount of undesired b-glycoside. (The structure
of 6 was confirmed after conversion of 6 into the imidate 16 whose physical data
was identical to that reported in the literature. Hasegawa also reported better
a-selectivity of sialylations with the partially protected lactoside than those
with the primary alcohols (see ref. 14d)) The same reaction was demonstrated
on a gram-scale and we confirmed reproducibility of selectivity and reaction
yield. a-Sialyl lactoside 6 was protected with Ac2O and pyridine to give the
per-protected trisaccharide 7 in quantitative yield. We stored 7 as a building
block for the systematic syntheses of GM3 analogs (Sch. 1).[19]
The synthesis of ceramide analogs began with the corresponding sphin-
gosine analogs. By using 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline
Scheme 1:

M. Kurosu and I. Kitagawa
430
(EEDQ), D-erythro-C18-sphingosine (8) was condensed with stearic acid to
provide the ceramide 9 in 80% yield. The C3-hydroxyl group of the
ceramide 9 was protected as its TBDPS ether in three steps (1, tritylation
of primary alcohol; 2, TBDPS silylation; and 3, deprotection of trityl group
.
using PhSH in the presence of BF3 OEt2) to afford 10 in 70% overall
yield. (The corresponding C3-benzoyl and acetyl-protected ceramide were
not useful in the glycosylations (16 ! 17 and 18); the coupling between 16
and the acylated ceramides gave the desired products in less than 30%
yields. It was speculated that C3-acyl carbonyl deactivates the reactivity of
the C1-alcohol by the formation of an eight-membered Lewis acid complex)
In a similar manner C8-sphingosine 11[20] was converted to its TBDPS
ether 13. (R,E)-2-aminooct-4-en-1-ol (14) was synthesized from D-glyceralde-
hyde derivative in eight steps in 40% overall yield and coupled with octanoic
acid using EEDQ (Sch. 2).[20]
With the protected ceramide 10 (a natural form) and its analogs 13 and 15
in our hands, the fully protected trisaccharide 7 was then converted to the
imidate 16 in two steps: selective desialylation of the anomeric position, and
imidate formation.[21] The coupling reactions between the sialyl-lactosylimi-
date 16 and the ceramide analogs 10, 13, and 15 smoothly underwent at 08C
under Schmidt’s conditions to give exclusively b-glycosides 17, 18, and 19 in
good yields of 70%, 75%, and 80%, respectively. Global deprotections of these
coupling products were achieved by the treatment of aq. HF (for 17 and 18)
followed by saponifications to provide 1, 2, and 3 in excellent yields. Thus,
we could synthesize the GM3 ceramide analog in a convergent manner as
illustrated in Scheme 3.
Scheme 2:

Ganglioside GM3 Derivatives
431
Scheme 3:
Activity of GM3 Ceramide Analogs on KB
Cell Proliferation
As a preliminary study on the biological activity of GM3 analogs, we first
examined the growth-inhibitory property of the synthesized GM3 ceramide
analogs 2 and 3 against KB cells in vitro. We observed a pronounced growth
inhibitory effect of 2 and 3 against KB cells compared to the natural form 1;
the analogs 2 and 3 inhibited the growth of KB cells at 0.5 nmol/mL; however,
1 did not inhibit growth at the same concentrations. The analogs 2 and 3 are
about 100-fold more potent than 1 in inhibiting the growth of KB cells (Figure 2).
DISCUSSION AND CONCLUSION
We have demonstrated expeditious a-sialylation with phenylthioglycoside 4 as
a sialyl donor and methylthiotrilate as a promoter. Introduction of the ceramide
Figure 2: A. Effect of GM3 derivatives 2 and 3 on growth of KB cells. Cells were treated at
50 nmol/mL. B. Dose response of 2 and 3 on growth of KB cells at various concentrations.

M. Kurosu and I. Kitagawa
432
analogs to the trisaccharide donor was also achieved in very good yield by using
ceramide 13 possessing a TBDPS-protecting group at C3 position. The shorter
chain length of ceramide portion of GM3 significantly improved its biological
activity as demonstrated by the inhibition of KB cell proliferation. The C3-
hydroxy group of the ceramide moiety did not contribute to the inhibitory
activity against KB cells.
In studies of the immunosuppressive activity of gangliosides it is reported
that ceramide containing a shorter fatty acyl chain influences its immuno-
suppressive activity. Ceramides with chain lengths of 16 and 18 carbons
were 6- to 10-fold more active than those with 22 and 24 carbons.[22] Thus,
the subtle difference in the chain length of fatty acid of ganglioside makes a
dramatic difference in immunosuppressive activity. The structures of the
carbohydrate moiety in certain glycolipids are identical between normal and
tumor cells. However, structural diversity in the ceramide moiety has been
observed. Therefore, synthesis of diverse ceramide analogs and their biologi-
cal evaluations at the molecular level have been of great interest.[23] As a
result, it was realized that certain ceramide analogs have important func-
tional roles such as cell adhesion and apoptosis.[24] However, there is lack of
data of biological function for ceramide structures conjugated with oligosac-
charides. No rationale has been found for the optimization of ceramide struc-
ture of each biologically significant glycolipid. Our study described here and
others[13,22,24a] strongly suggest that biological properties of gangliosides
would be altered by the change in length of ceramide chains. We are currently
validating the effects of ceramide-chain truncated GM3 on other cancer cell
lines as well as enzymatic recognition of the truncated ceramides and their
glycoconjugates.
EXPERIMENTAL
Methyl (phenyl 5-acetoamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-D-
galacto-2-nonulopyranoside)onate (4). To a stirred solution of 5-aceta-
mido-2,4,7,8,9-penta-O-acetyl-3,5-dideoxy-D-glycero-D-galacto-2-nonulosonic
acid methyl ester[25] (5.0 g, 9.57 mmol) and PhSH 3.93 mL, 38.3 mmol) in
.
CH2Cl2 (95 mL) at 08C was added BF3 OEt2 (10.9 g, 76.6 mmol). The
reaction mixture was warmed to r.t. After being stirred for 12 h, the reaction
mixture was poured into cold aq. NaHCO3 solution. The water phase was
extracted with CH2Cl2. The combined extracts were dried over Na2SO4
and concentrated in vacuo. Purification by silica gel chromatography
(toluene:acetone ¼ 15 : 1) gave 4 (5.1 g, 92%) as a white solid: [a]D ¼ 294.0
1
(c 1.2, CHCl3); b-glycoside; H NMR (500 MHz, CDCl3) 3.26 ppm (3H, s), 2.26
(1H, dd, J ¼ 12.5, 4.8 Hz), 2.08, 2.04, 1.96, 1.90, 1.85 (3H each, s), 2.00 (1H,
dd, J ¼ 13.9, 11.4 Hz); a-glycoside; 3.57 (3H, s), 2.83 (1H, dd, J ¼ 12.5,
4.5 Hz), 2.15, 2.14, 2.05, 2.02, 1.87 (3H each, s), 2.00 (1H, dd, J ¼ 12.5,

Ganglioside GM3 Derivatives
433
10.5 Hz); 13C NMR (125 MHz, CDCl3) 86.0 ppm, 85.9 (anomeric carbons); IR
(CHCl3) 3065 cm21
,
2957, 2936, 1745, 1602. FAB-HRMS calcd for
C26H33NO12S [M þ Hþ]: 454.17.2244. Found: 454.2246. The anomeric ratio
was determined to be 1 : 6.6 (a/b) from the integration value of C3-equatorial
1
protons in H NMR.
General procedure for sialylation with 4. MeSBr was prepared from
Me2S2 (200 mL, 2.72 mmol) and Br2 (133 mL, 2.59 mmol) in ClCH2CH2Cl
(3 mL) at r.t. This solution could be stored in the dark for over 1 month
without loss of activity. Thiophenyl glycoside 4 (2.5 eq), the glycosyl
˚
acceptor, AgOTf (3 eq), and MS 3A in EtCN (0.1 ꢀ 0.3 M) were stirred at
r.t. for 1 h. The reaction mixture was cooled to 2788C and MeSBr (ca.
1.55 M, 2.8 eq) was added. After 1 ꢀ 15 min, the reaction mixture was
diluted with CH2Cl2 and filtered through celite. The organic phase was
washed with sat. aq. NaHCO3, dried over Na2SO4, and concentrated in
vacuo. The crude product was purified by silica gel chromatography
(toluene:acetone ¼ 10 : 1).
tert-Butyldimetylsilyl O-(methyl 5-acetoamido-4,7,8,9-tetra-O-acetyl-
3,5-dideoxy-dideoxy-D-glycero-a-D-galacto-2-nonulopyranosylonate)-
(2 ! 3)-O-(6-O-benzoyl-b-D-galactopyranosyl)-(1 ! 4)-2,6-di-O-benzoyl-
b-D-glucopyranoside (6): 85% yield as a white solid: [a]D ¼ þ45.3 (c 0.8,
1
CHCl3); H NMR (500 MHz, CDCl3) 8.1–7.2 ppm (15H, m), 3.79 (3H, s), 2.57
(1H, dd, J ¼ 12.5, 4.6 Hz), 2.14, 2.06, 2.04, 1.95 (3H each s), 1.90 (3H, s), 0.89
(9H, s); 13C NMR (125 MHz, CDCl3) 104.3 ppm, 97.5, 90.9 (anomeric
carbons); IR (KBr) 3470 cm21, 3065, 2957, 2932, 1745, 1602. FAB-HRMS
calcd for C59H76NO26Si [M þ Naþ]: 1264.4244. Found: 1264.4237.
tert-Butyldimetylsilyl O-(methyl 5-acetoamido-4,7,8,9-tetra-O-acetyl-3,5-
dideoxy-D-glycero-a-D-galacto-2-nonulopyranosylonate)-(2 ! 3)-O-(2,4-
di-O-acetyl-6-O-benzoyl-b-D-galactopyranosyl)-(1 ! 4)-3-O-acetyl-2,6-
di-O-benzoyl-b-D-glucopyranoside (7). To a stirred solution of 6 (2.0 g,
1.61 mmol) in pyridine (5 mL) was added Ac2O (5 mL). After 12 h, all volatiles
were evaporated under high vacuum to give the crude product. Purification by
silica gel chromatography (toluene: acetone ¼ 15 : 1) gave 7 (2.2 g, 100%) as a
white solid: [a]D ¼ þ42.8 (c 0.6, CHCl3); 1H NMR (500 MHz, CDCl3) 8.1–
7.4 ppm (15H, m), 5.78 (1H, t, J ¼ 9.9 Hz), 4.58 (1H, dd, J ¼ 9.9, 3.3 Hz), 4.19
(1H, dd, J ¼ 11.6, 7.6 Hz), 4.30 (1H, m), 3.68 (3H, s), 2.58 (1H, dd, J ¼ 11.9,
4.28 Hz), 2.21, 2.12, 2.04, 2.01, 1.98, 1.95 (3H each s), 1.82 (3H, s), 1.69
(1H, t, J ¼ 11.9), 0.89 (9H, s), 0.01 (3H, s), 0.00 (3H, s); 13C NMR (125 MHz,
CDCl3) 101.4 ppm, 96.7, 90.5 (anomeric carbons); IR (KBr) 3470 cm21, 3378,
3067, 2859, 1748, 1603. FAB-HRMS calcd for C65H81NO29Si [M þ Naþ]:
1390.4561. Found: 1390.4618.

M. Kurosu and I. Kitagawa
434
O-(Methyl 5-acetoamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-
a-D-galacto-2-nonulopyranosyl-onate)-(2 ! 3)-O-(2,4-di-O-acetyl-6-O-
benzoyl-b-D-galactopyranosyl)-(1 ! 4)-3-O-acetyl-2,6-di-O-benzoyl-b-
D-glucopyranosyl)trichloroacertymidate (16). To a stirred solution of 7
(113.9 mg, 0.0833 mmol) in THF (2.8 mL) was added AcOH (194 mL,
0.28 mmol, 3.4 eq.) and TBAF (1.0 M, 183 mL, 2.2 eq). After 48 h, the reaction
was quenched with MeOH (1 mL). All volatiles were evaporated in vacuo. Puri-
fication by silica gel chromatography (toluene:acetone ¼ 5 : 1) gave O-(methyl
5-acetoamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-a-D-galacto-2-non-
ulopyranosylonate)-(2 ! 3)-O-(2,4-di-O-acetyl-6-O-benzoyl-b-D-galactopyranosyl)-
(1 ! 4)-3-O-acetyl-2,6-di-O-benzoyl-b-D-glucopyranose (101.3 mg, 97%) as a
white solid. This was dissolved in CH2Cl2 (1 mL). Into the solution Cl3CCN
(156 mL, 1.3 eq) and DBU (38 mL, 0.026 mmol, 0.5 eq) were added at 08C.
After 1 h, all volatiles were evaporated in vacuo to provide the crude imidate.
Purification by silica gel chromatography (toluene: acetone ¼ 6 : 1) gave 16[18]
(68.3 mg, 95%) as a white solid: [a]D ¼ þ33.0 (c 0.9, CHCl3); 1H NMR
(500 MHz, CDCl3) 8.55 ppm (1H, s), 8.1–7.3 (15H, m), 6.66 (1H, d,
J ¼ 9.9 Hz), 5.86 (1H, t, J ¼ 9.9 Hz), 5.6–4.9 (m), 4.60 (1H, dd, J ¼ 9.9,
3.3 Hz), 4.4–3.9 (m), 3.71 (3H, s), 3.58 (1H, dd, J ¼ 10.6, 2.3 Hz), 2.59 (1H,
dd, J ¼ 13.6, 4.28 Hz), 2.21, 2.13, 2.10, 2.00, 1.97, 1.83 (3H each s); IR (KBr)
3327 cm21, 2961, 1746, 1678, 1602. Anal. Calcd for C61H67N2O29Cl3: C,
52.39; H, 4.83; N, 2.00. Found: C, 52.42; H, 4.85; N, 1.90.
(2S,3R,4E)-3-O-tert-Butyldiphenylsilyl-2-octadecanamide-octadec-4-
1
ene-1,3-diol (10): [a]D ¼ 211.6 (c 2.3, CHCl3); H NMR (500 MHz, CDCl3)
7.7–7.3 ppm (10H, m), 5.94 (1H, br.d), 5.42–5.32 (1H, m), 4.35–4.32 (1H, m),
3.91–3.81 (2H, m), 3.64–3.57 (1H, m), 2.00–1.93 (2H, m), 1.90–1.86 (2H, m),
1.53–1.48 (2H, m), 1.45–1.20 (50H, m), 1.07 (9H, s), 0.88 (6H, t-like); IR
(KBr) 3300 cm21, 2924, 2855, 1645. FAB-HRMS calcd for C52H89NO3Si
[M þ Naþ]: 826.6500. Found: 826.6499.
(2S,3R,4E)-3-O-tert-Butyldiphenylsilyl-2-octanamide-oct-4-ene-1,3-diol
1
(13): [a]D ¼ 230.6 (c 1.0, CHCl3); H NMR (500 MHz, CDCl3) 7.7–7.3 ppm
(10H, m), 5.94 (1H, br.d), 5.67 (1H, m), 4.53 (1H, m), 3.91–3.81 (2H, m),
3.60–3.57 (1H, m), 2.00–1.93 (2H, m), 1.90–1.86 (2H, m), 1.53–1.20 (12H,
m), 1.07 (9H, s), 0.96 (6H, t-like); IR (KBr) 3305 cm21, 2924, 2855, 1648.
FAB-HRMS calcd for C32H49NO3Si [M þ Naþ]: 548.3392. Found: 548.3389.
(2R,4E)-2-Octamido-oct-4-en-1-ol (15): [a]D ¼ 23.5 (c 0.5, CHCl3); 1H NMR
(500 MHz, CDCl3) 8.19 ppm (1H, br.d), 5.4–5.7 (2H, m), 4.5 (1H, m), 3.9 (2H,
m), 2.7–2.5 (2H, m), 2.37 (2H, t, J ¼ 7.25 Hz), 1.4–1.1 (12H, m), 1.53–1.48
(2H, m), 1.4–1.1 (12H, m), 0.82 (6H, m); IR (KBr) 3300 cm21, 2924, 2854,
1644. FAB-HRMS calcd for C16H31NO2 [M þ Naþ]: 292.4236. Found: 292.4231.

Ganglioside GM3 Derivatives
435
O-(Methyl 5-acetoamido-4,7,8,9-tetra-O-acetyl-3,5-di-deoxy-D-glyc-
ero-a-D-galacto-2-nonulopyranosyl-onate)-(2 ! 3)-O-(2,4-di-O-acetyl-
6-O-benzoyl-b-D-galactopyranosyl)-(1 ! 4)-3-O-acetyl-2,6-di-O-benzoyl-
b-D-glucopyranosyl)-(1 ! 1)-(2S,3R,4E)-3-O-tert-butyldiphenylsilyl-2-
octadecanamide-octadec-4-ene-1,3-diol (17). To a stirred suspension of 16
(20.9 mg, 0.026 mmol, 1.3 eq), 10 (28 mg, 0.020 mmol), and MS 3A (100 mg),
ClCH2CH2Cl (1 mL) was stirred for 1 h at r.t. and cooled to 220 ꢀ 2258C.
.
Into the reaction mixture BF3 OEt2 (11.3 mg, 0.080 mmol, 4 eq) was added.
The reaction mixture was stirred for 12 h at 08C and diluted with CH2Cl2
and filtered through celite. The organic phase was washed with sat. aq.
NaHCO3, dried over Na2SO4, and concentrated in vacuo. The crude product
was purified by silica gel chromatography (toluene:acetone ¼ 5 : 1) to give 17
(28.5 mg, 70%) as a white solid: [a]D ¼ 22.0 (c 0.5, CHCl3); 1H NMR
(500 MHz, CDCl3) 8.2–7.3 ppm (25H, m), 5.6–3.6 (21H, m), 3.71 (3H, s), 2.58
(1H, dd, J ¼ 13.4, 4.8 Hz), 2.20, 2.12, 2.04, 2.02, 2.01, 2.00, 1.92, 1.84 (3H
each s), 1.2 (52H, m), 0.87 (6H, s), 0.86 (9H, s); IR (KBr) 3312 cm21, 3070,
2926, 1748, 1685, 1602. FAB-HRMS calcd for C111H154NO31Si [M þ Naþ]:
2062.0202. Found: 2062.0327.
O-(Methyl 5-acetoamido-4,7,8,9-tetra-O-acetyl-3,5-di-deoxy-D-glycero-a-
D-galacto-2-nonulopyranosyl-onate)-(2 ! 3)-O-(2,4-di-O-acetyl-6-O-
benzoyl-b-D-galactopyranosyl)-(1 ! 4)-3-O-acetyl-2,6-di-O-benzoyl-b-
D-glucopyranosyl)-(1 ! 1)-(2S, 3R, 4E)-3-O-tert-butyldiphenylsilyl-2-octa
namide-oct-4-ene-1,3-diol (18). To a stirred suspension of 16 (21.0 mg,
0.026 mmol, 1.3 eq), 13 (10.5 mg, 0.020 mmol), and MS 3A (100 mg),
ClCH2CH2Cl (1 mL) was stirred for 1 h at r.t. and cooled to 220 ꢀ 2258C.
.
Into the reaction mixture BF3 OEt2 (11.3 mg, 0.080 mmol, 4 eq) was added.
The reaction mixture was stirred for 12 h at 08C and diluted with CH2Cl2 and
filtered through celite. The organic phase was washed with sat. aq. NaHCO3,
dried over Na2SO4, and concentrated in vacuo. The crude product was
purified by silica gel chromatography (toluene:acetone ¼ 5 : 1) to give 18
(26.4 mg, 75%) as a white solid: [a]D ¼ 210 (c 0.1, CHCl3); 1H NMR
(500 MHz, CDCl3) 8.1–7.2 ppm (25H, m), 5.5–3.5 (21H, m), 3.71 (3H, s), 2.58
(1H, dd, J ¼ 13.4, 4.8 Hz), 2.21, 2.13, 2.05, 2.02, 2.01, 2.00, 1.92, 1.84 (3H
each s), 1.9–1.3 (12H, m), 0.90 (6H, s), 0.86 (9H, s); IR (KBr) 3313 cm21, 3077,
2928, 1749, 1680, 1602. FAB-HRMS calcd for C91H113NO32Si [M þ Naþ]:
1783.1192. Found: 1783.1203.
O-(Sodium 5-acetoamido-3,5-di-deoxy-D-glycero-a-D-galacto-2-nonulo-
pyranosyl-onate)-(2 ! 3)-b-D-galactopyranosyl)-(1 ! 4)-3-O-b-D-gluco-
pyranosyl)-(1 ! 1)-(2S,3R,4E)-2-octadecanamide-octadec-4-ene-1,3-
diol (ganglioside GM3, 1). To a stirred solution of 17 (18 mg, 0.0088 mmol) in
THF (1 mL) was added an HF solution (46% HF/CH3CN ¼ 3/7, 0.5 mL). After

M. Kurosu and I. Kitagawa
436
16 h at r.t. the reaction mixture was poured into aq. NaHCO3. The water phase
was extracted with CHCl3. The combined organic phase was dried over
Na2SO4 and evaporated in vacuo. This was dissolved in absolute MeOH
(0.5 mL) and NaOMe (3.8 mg, 0.0352 mmol, 8eq) was added. After 12 h at
r.t., water (0.3 mL) was added. The reaction mixture was stirred for an
additional 4 h and all volatile were evaporated under high vaccum to give
the crude product. This was purified by Sephadex LH-20 (CHCl3:MeOH:H2-
O ¼ 24 : 24 : 1) to provide 1[26] (10.2 mg, 95%) as a white solid: [a]D ¼ þ4.0 (c
0.5, CHCl3:MeOH¼1 : 1); 1H NMR (500 MHz, d6-DMSO:D2O ¼ 1 : 1) 5.55
(1H, dt, J ¼ 15.3, 6.8 Hz), 5.37 (1H, dd, J ¼ 15.3, 6.8 Hz), 4.19 (1H, d,
J ¼ 7.3 Hz), 4.14 (1H, d, J ¼ 7.9 Hz), 2.73 (1H, dd, J ¼ 11.6, 4.3 Hz), 2.04
(2H, t, J ¼ 7.9 Hz), 1.9 (2H, m), 1.5–1.3 (13H, m), 0.84 (6H, t, J ¼ 7.3 Hz);
13C NMR (125 MHz, d6-DMSO:D2O ¼ 1 : 1) 173.7 ppm, 173.5, 172.0, 132.7,
131.5, 104.2, 100.0, 93.7; IR (KBr) 3312 cm21, 3070, 2926, 1748, 1685, 1602.
FAB-HRMS calcd for C39H67N2O21Na [M þ Naþ2 ]: 1225.7161. Found:
1225.7203.
O-(Sodium 5-acetoamido-3,5-di-deoxy-D-glycero-a-D-galacto-2-nonulo-
pyranosyl-onate)-(2 ! 3)-b-D-galacto-pyranosyl)-(1 ! 4)-3-O-b-D-glu-
copyranosyl)-(1 ! 1)-(2S,3R,4E)-2-octanamide-oct-4-ene-1,3-diol (2):
[a]D ¼ þ19.6 (c 1.1, CHCl3); 1H NMR (500 MHz, d6-DMSO:D2O ¼ 1 : 1)
5.55 (1H, dt, J ¼ 15.3, 6.8 Hz), 5.37 (1H, dd, J ¼ 15.3, 6.8 Hz), 4.19 (1H,
d, J ¼ 7.3 Hz), 4.14 (1H, d, J ¼ 7.9 Hz), 2.73 (1H, dd, J ¼ 11.6, 4.3 Hz),
2.04 (2H, t, J ¼ 7.9 Hz), 1.9 (2H, m), 1.5–1.3 (13H, m), 0.84 (6H, t,
J ¼ 7.3 Hz); 13C NMR (125 MHz, d6-DMSO:D2O ¼ 1 : 1) 173.6 ppm, 173.4,
172.1, 132.7, 131.5, 104.2, 100.7, 93.6; IR (KBr) 3327 cm21, 2926, 1749,
1624. FAB-HRMS calcd for C39H67N2O21Na [M þ Naþ2 ]: 945.4032. Found:
945.4048.
O-(Sodium 5-acetoamido-3,5-di-deoxy-D-glycero-a-D-galacto-2-nonulo-
pyranosyl-onate)-(2 ! 3)-b-D-galacto-pyranosyl)-(1 ! 4)-3-O-b-D-gluco-
pyranosyl)-(1 ! 1)-(2R,4E)-2-octanamide-oct-4-ene-1-ol (3): [a]D ¼ þ5.1
1
(c 0.3, CHCl3); H NMR (500 MHz, d6-DMSO:D2O ¼ 50 : 1) 5.5–5.3 (2H, m),
4.19 (1H, d, J ¼ 7.9 Hz), 4.15 (1H, d, J ¼ 7.9 Hz), 2.74 (1H, dd, J ¼ 11.6,
4.3 Hz), 2.04 (2H, t, J ¼ 7.9 Hz), 1.9 (2H, m), 1.5–1.1 (13H, m), 0.84 (6H,
t-like); IR (KBr) 3320 cm21, 2926, 1734, 1626. FAB-HRMS calcd for
C39H67N2O20Na [M þ Naþ2 ]: 929.4032. Found: 929.4048.
ACKNOWLEDGEMENTS
This work was supported by Colorado State University and Osaka University.

Ganglioside GM3 Derivatives
437
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