Synthesis of ganglioside GD3 and its comparison with bovine GD3 with regard to oligodendrocyte apoptosis mitochondrial damage

Article abstract of DOI:10.1002/1521-3765(20010518)7:10<2178::AID-CHEM2178>3.0.CO;2-E

2,3-Dehydroneuraminic acid derivative 5 was transformed in five efficient steps into sialyl donor 2, which has a phenylthio group on the β-side of the 3-position for anchimeric assistance and a diethyl phosphite residue as leaving group at the anomeric ca

Full text of DOI:10.1002/1521-3765(20010518)7:10<2178::AID-CHEM2178>3.0.CO;2-E

FULL PAPER
Synthesis of Ganglioside GD3 and its Comparison with Bovine GD3 with
Regard to Oligodendrocyte Apoptosis Mitochondrial Damage
Julio C. Castro-Palomino,[a] Bernadett Simon,[b] Oliver Speer,[c] Marcel Leist,*[b] and
Richard R. Schmidt*[a]
Abstract: 2,3-Dehydroneuraminic acid
derivative 5 was transformed in five
efficient steps into sialyl donor 2, which
has a phenylthio group on the b-side of
the 3-position for anchimeric assistance
and a diethyl phosphite residue as leav-
ing group at the anomeric carbon. The
known GM3 intermediate 10 was trans-
formed into the 4b,4c,8c-O-unprotected
acceptor 3, which was then allowed to
react with 2 by using TMSOTf as
catalyst and acetonitrile as solvent to
afford the desired tetrasaccharide 12,
which has an a(2 ± 8)-linkage between
two neuraminic acid residues. Removal
of the phenylthio group gave intermedi-
ate 13, which was transformed into O-
tetraosyl trichloroacetimidate 16 as gly-
cosyl donor. Application of the azido-
sphingosine glycosylation procedure fur-
nished GD3 (1) in high overall yield.
Comparison of synthetic GD3 with bo-
vine-brain-derived GD3 showed that
there were similar effects in GD3-trig-
gered uncoupling of mitochondrial res-
piration and in induction of apoptosis in
oligodendrocytes.
Keywords: anchimeric assistance ´
apoptosis
´ gangliosides ´ glyco-
lipids ´ synthesis design
However, these investigations were hampered by the varying
biological activity and undefined nature of different lots of
commercially available natural GD3 isolates. Therefore, we
initiated a chemical synthesis of GD3 and compared the
proapoptotic effect of the synthetic ganglioside with those of
purified bovine-brain fractions.
Introduction
Gangliosides have attracted a lot of attention because of their
manifold biological roles.[1] Disialoganglioside GD3
(Scheme 1, 1), and particularly its 9d-O-acetyl derivative,
were found to be human melanoma-associated antigens.[2, 3]
Recently, endogenously formed GD3 has been implicated in
intracellular signalling with proapoptotic function.[4] More-
over, extracellular GD3 was reported to induce apoptotic cell
death in a variety of cell types, most likely by triggering
permeability transition. Under neuroinflammatory condi-
tions, GD3 may be formed by microglial cells and is found
at increased concentrations in cerebral liquor. Thus, the
ganglioside may contribute to selective oligodendrocyte loss
in conditions such as multiple sclerosis, and examination of
the cell pathways triggered by GD3 may reveal new targets
for pharmacological intervention in degenerative diseases.[7]
Results and Discussion
Synthesis of GD3: The chemical synthesis of GD3 has to
overcome the difficult formation of the a(2 ± 8)-linkage
between two N-acetyl neuraminic acid (Neu5Ac) residues.[8]
This task has been successfully addressed by a few research
groups.[8±14] The rather low reactivity observed for the
8-hydroxy group of variously protected neuraminic acid
acceptors and the tendency of the sialyl donors towards b-
linkage formation and/or towards competing 2,3-dehydro-
neuraminic acid generation led to the conclusion that for
successful a(2 ± 8)-linkage formation the activated sialyl
donor requires anchimeric assistance in order to shield the
b-face and to provide stabilisation of the incipient carbonium
ion intermediate. Hence, phenylthio[9±11] and the phenylthio-
carbonyloxy[8, 13, 14] groups were introduced on the b-side.
Benzyl and acetyl groups were chosen for hydroxy-group
protection, and bromo, chloro, phosphite and ethylthio groups
were selected as leaving groups at the 2-position. We report
here on the usefulness of sialyl donor 2 (Scheme 1), which
possesses an anchimerically assisting 3-phenylthio group on
[a] Prof. Dr. R. R. Schmidt, J. C. Castro-Palomino
Fachbereich Chemie, Universität Konstanz
Fach M725, 78457 Konstanz (Germany)
Fax : (‡49)7531-88-3135
E-mail: richard.schmidt@uni-konstanz.de
[b] Priv.-Doz. Dr. M. Leist, B. Simon
Fachbereich Biologie, Universität Konstanz
Fach X911, 78457 Konstanz (Germany)
E-mail: marcel.leist@uni-konstanz.de
[c] O. Speer
Institute of Cell Biology, ETH-Hoenggerberg
8093 Zürich (Switzerland)
2178
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Chem. Eur. J. 2001, 7, No. 10

2178±2184
Scheme 1.
the b-side and diethyl phosphite as leaving group, which
requires only catalytic amounts of acid for activation. Thus,
we combined the anchimeric assistance as introduced by
Ogawa et al.[9] and the activation of the anomeric leaving
group by acid catalysis as introduced by us.[15] The protective-
group pattern of 2 was selected in order to permit trans-
formation of the product into an 8-O-acceptor for further
chain extension with 2.
The retrosynthesis in Scheme 1 shows that besides 2
(disconnection at ꢀ1 ), a GM3 trisaccharide building block 3
is required which should be available from a lactose acceptor
and a conventional sialyl donor (disconnection at ꢀ2 ) follow-
ing literature precedents.[15] The disconnection at ꢀ3 liberates
the ceramide moiety which can be attached through the
azidosphingosine glycosylation procedure[16] which calls for
the known 3-O-benzoyl-azidosphingosine[17] and stearic acid.
For the synthesis of sialyl donor 2, the known 2,3-
dehydroneuraminic acid ester 5[18] (Scheme 2) was treated
with acetone in the presence of trifluoromethanesulfonic acid
with sodium thiophenolate in THF gave, with inversion of
configuration, the 3-phenylthio derivative 9; the structural
1
assignment of which was confirmed by the H NMR data:
J(3,4) ˆ 10.5 Hz. Reaction of 9 with diethyl chlorophosphite
in the presence of Hünigꢁs base furnished the desired sialyl
donor 2; the structural assignment was confirmed by the
1H NMR data: J(3,4) ˆ J(4,5) ˆ 10.2 Hz.
The transformation of the known GM3 trisaccharide 10[15]
into the required acceptor 3 could be readily carried out
(Scheme 3). Selective removal of the O-acetyl protecting
Scheme 3.
group of 10 was performed in methanol at 208C in the
presence of a catalytic amount of 1,7-diazabicyclo[5.4.0]un-
dec-7-ene (DBU) to afford compound 11 in almost quantita-
tive yield. Treatment of 11 with tert-butyldimethylsilyl chlor-
ide (TBS-Cl) in the presence of imidazole led to regioselective
4c,9c-O-silylation, thus providing 3 in 72% yield. Because of
the generally observed low reactivity of the 4-hydroxy group
of galactose and the 7-hydroxy group of Neu5Ac residues in
sialylation reactions, protection of the 4b- and 7c-hydroxy
groups was not required.
Scheme 2.
Sialylation of acceptor 3 with donor 2 was performed in
acetonitrile[15, 19] at 258C in the presence of 0.1 equivalent of
TMSOTf as catalyst giving the desired tetrasaccharide 12 in
54% yield (Scheme 4). Removal of the 3d-phenylthio group
was achieved by treatment with tributyltin hydride and
activation with azoisobutironitrile (AIBN) to afford com-
pound 13. Reaction with tetrabutylammonium fluoride
(TBAF) in THF at 208C in the presence of acetic acid led
(TfOH) as catalyst to furnish exclusively the 8,9-O-isopropyl-
idene derivative 6.[14] Benzylation of 6 with benzyl bromide
and sodium hydride as base in DMF afforded the 4-O-benzyl-
protected derivative 7 in high yield. For the a-side-selective
bromine addition, 7 was first treated with acetic anhydride in
pyridine and then with N-bromo-succinimide in acetonitrile at
608C to afford the desired 3-bromo derivative 8. Treatment
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M. Leist, R. R. Schmidt et al.
FULL PAPER
Scheme 4.
and then RP-18 column chromatography afforded pure GD3
(1) which had optical rotation and NMR data in accordance
with those previously reported.[10, 12, 22]
to the desilylated compound 14. Hydrogenolysis in methanol
with palladium on carbon as catalyst and TfOH as promoter
led to O-debenzylation with concomitant removal of the
8d,9d-O-isopropylidene group. Reaction of the crude product
with acetic anhydride in pyridine furnished O-acyl-protected
tetrasaccharide 15. Regioselective 1a-O-deacetylation of 15
with hydrazinium acetate[20] and ensuing treatment with
trichloroacetonitrile in the presence of DBU as base afforded
trichloroacetimidate 16. Only the a-isomer was isolated in
83% yield.
For the attachment of the ceramide residue, the azidos-
phingosine glycosylation procedure was employed.[16] To this
end, the known 3-O-benzoyl-azidosphingosine 4[17] was treat-
ed with tetrasaccharide donor 16 in the presence of borontri-
fluoride diethyl ether as promoter; this afforded the desired b-
linked glycoside 17 in high yield (1H NMR: J(1a,2a) ˆ 8.8 Hz).
Transformation of the azido group into the amino group was
performed by treatment with hydrogen sulfide in aqueous
pyridine. The crude product was immediately treated with
stearic acid and water soluble carbodiimide (WSC) as
condensing agent to afford fully O-acylated GD3 (18) in
GD3-triggered uncoupling of mitochondrial respiration:
Gangliosides, especially disialoganglioside GD3, have recent-
ly been implicated in the signalling of apoptosis.[4] Although
the mechanisms involved have not been completely elucidat-
ed yet, the triggering of mitochondrial permeability transition
(PT) appears to be a key event.[5] PT is characterised by a loss
of the permeability barrier of the inner mitochondrial
membrane to molecules >1500 Da and it can be specifically
inhibited by the cyclophilin ligand cyclosporine A.[23] PT-
dependent effects of GD3 on mitochondrial function, such as
the uncoupling of respiration have been demonstrated
directly with the isolated organelles.[5, 24] We used this well-
characterised in vitro system for an initial comparison of
synthetic and bovine-brain (BB) GD3. The uncoupling-
induced increase in mitochondrial respiration after exposure
of rat-liver mitochondria to synthetic and BB GD3 was
measured in an oxygraph. Synthetic and BB GD3 showed a
similar concentration dependency with respect to the uncou-
pling of mitochondria (Figure 1a), while the control ganglio-
Â
74% yield. De-O-acylation was performed under Zemplen
conditions[21] and the methyl ester was cleaved with potassium
‡
hydroxide. Ion exchange with Amberlite IR-120 (H form)
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Synthesis of GD3
2178±2184
cyclosporine A did not prevent increased respiration after
exposure to the nonspecific protonophore uncoupler FCCP
within the same system (not shown).
Comparison of GD3 preparations with respect to induction of
apoptosis in oligodendrocytes: GD3 has been shown to trigger
apoptotic death in various cell types. We chose oligodendro-
cytes, the most sensitive cell population in the brain,[25] to
compare the effects of synthetic and BB GD3. Both prepa-
rations induced a similar degeneration of cellular processes
later followed by apoptotic chromatin condensation (Fig-
ure 1b). Also, both preparations triggered a defined sequence
of degenerative events different from a potential nonspecific
detergent effect. This is exemplified by the intactness of the
plasma membrane (retention of calcein) 24 h after exposure
to GD3, a time point when the chromatin shows features of
apoptotic condensation (Figure 1c). Specificity is further
indicated by the finding that the related ganglioside GD1b
did not cause death in oligodendrocytes. An exact concen-
tration-response comparison of the two preparations showed
that slightly higher concentrations of synthetic GD3 were
required for a given apoptotic effect (Figure 1b). This may be
due the different composition of fatty acid residues in
synthetic and BB GD3. While all fatty acid residues of
synthetic GD3 have the same length (C18), a mixture of fatty
acids forms the lipophilic moiety of BB GD3. The formation
of micelles is favoured for this reason in the synthetic GD3.
The potential re-formation of micelles after sonication of
GD3 and the start of the incubation in the medium had more
pronounced effects in the cell culture experiments that
extended over 24 h than in the experiments with isolated
mitochondria that were terminated after 30 min.
Figure 1. Biological studies. A: Isolated rat-liver mitochondria were
incubated in an oxygraph chamber. Initial respiration was set to 100%.
Increasing respiration due to the uncoupling effect of GD3 was measured.
The specificity of the effect was controlled by blocking with cyclosporine A
(CsA, 2mm) or adding of a noneffective control ganglioside (GD1b). Data
are means of two experiments with SD < 10%. B: Murine oligodendrocytes
were incubated for 24 h with different concentrations of either synthetic
GD3 or GD3 extracted from bovine brains. Cells were then fixed and
stained for the oligodendrocyte marker protein cyclic nucleotide 2',3'-
phosphodiesterase (CNPase). Nuclei were stained with the DNA dye
H-33342. Apoptotic oligodendrocytes were scored by counting CNPase-
positive cells that showed alterations in nuclear morphology, as indicated
by chromatin condensation. Data are averaged from six experiments in
three different cell preparations. C: Murine oligodendrocytes were
incubated for 24 h with 400mm of either synthetic GD3 or bovine brain
extracted GD3. Cells were then stained with 1mm calcein-acetoxymethy-
lester (AM), a dye that fluoresces when it is accumulated in vital cells.
Phase contrast images and the corresponding images from the calcein
staining are shown.
Conclusion
Sialyl donor 2 can be readily obtained from the known 2,3-
dehydroneuraminic acid derivative 5. Donor 2 is highly
reactive. With the GM3-derived acceptor 3, it afforded the
desired a(2 ± 8)-linkage between two neuraminic acid residues
in good yield, thus finally affording, via trisaccharide 12, GD3
in good overall yield (8 steps, 13%). Intermediate 12 should
also be an ideal precursor for further chain extension with
sialyl donor 2.[26] It is clearly shown that damage of oligoden-
drocytes from mouse brain and respiration of mitochondria
from rat liver cells are dependent on the concentration of the
GD3 thus obtained.
Experimental Section
General techniques: Solvents were purified according to standard proce-
dures. Flash chromatography was performed on Baker silica gel60 (0.040 ±
side GD1b did not show this effect. Uncoupling of mitochon-
dria may be due to contaminating lipophilic protonophores
present in either GD3 preparation. Such an effect would not
be subject to inhibition by cyclosporine A. However, the
complete prevention of GD3-induced mitochondrial changes
in the presence of cyclosporine A indicates a specific effect of
both synthetic and BB GD3, mediated by PT. Conversely,
0.063 mm) at
a pressure of 0.4 bar. Thin-layer chromatography was
performed on Merck silica gel plastic plates 60F254; compounds were
visualised by treatment with a solution of (NH4)6Mo7O24 ´ 4H2O (20 g) and
Ce(SO4)2 (0.4 g) in 10% sulfuric acid (400 mL) and heating at 1508C.
Optical rotations were measured on a Perkin ± Elmer241 polarimeter in a
1 dm cell at 228C. NMR measurements were recorded at 228C on a
BrukerAC250 Cryospec or a BrukerDRX600 apparatus. TMS or the
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M. Leist, R. R. Schmidt et al.
FULL PAPER
resonance of the deuterated solvent was used as an internal standard;
solvents: CDCl3, d ˆ 7.24; CD3OD, d ˆ 3.31.
Glucopyranoside 10: Compound 10 was synthesised following a published
procedure. The physical data are in accordance with published values.[15]
Glucopyranoside 11: A solution of the known trisaccharide 10 (2 g,
1.46 mmol) in dry methanol (30 mL) was cooled to 208C and DBU
(30 mL) was added. The solution was stirred for 4 h at 208C, then
Methyl 5-Acetamido-2,3,5-trideoxy-d-glycero-d-galacto-non-2-enopyra-
nosonate (5): Compound
procedure.[18]
5 was synthesised following a published
‡
neutralised (H -Amberlite), filtered and concentrated. Column chroma-
Methyl
5-Acetamido-2,3,5-trideoxy-8,9-O-isopropylidene-d-glycero-d-
tography of the residue (chloroform/acetone 8:1) afforded 11. Yield: 1.6 g,
galacto-non-2-enopyranosonate (6): Triflic acid (30 mL) was added to a
suspension of 5 (5 g, 164 mmol) in dry acetone (30 mL) and the mixture was
stirred for 3 h at room temperature until a clear solution was obtained. The
solution was then neutralised with Et3N and concentrated to give 6 (5.2 g,
quant.). The physical data are in accordance with published values.[14]
94%; [a]D
ˆ
8 (c ˆ 0.5, CHCl3); 1H NMR (250 MHz, CD3OD): d ˆ 1.15
(s, 3H; OPiv), 1.96 (s, 3H; NCOMe), 2.10 (dd, 1H, J(gem) ˆ 13.0 Hz,
J(3ax,4) ˆ 12.3 Hz; 3cax-H), 2.58 (dd, 1H, J(gem) ˆ 13.0 Hz, J(3eq,4) ˆ
4.6 Hz; 3ceq-H), 3.33 (m, 1H; H5a), 3.36 (s, 3H; COOMe), 3.57 ± 3.72 (m,
6H; H3a, H5b, H9c, H9'c, H8c, H7c), 3.77 ± 3.87 (m, 2H; H6a, H2b), 3.95 ±
4.15 (m, 8H; H6'a, H6b, H6'b, H4c, H5c, H6c, NH, H4b), 4.30 ± 4.46 (m,
4H; H4a, H3b, CH2Ph), 4.48 (d, 1H, J(1a,2a) ˆ 8.0 Hz; H1a), 4.48 ± 5.02
(m, 9H; 4CH2Ph, H1b), 5.05 (dd, 1H, J(1a,2a) ˆ 8.0 Hz, J(2a,3a) ˆ 8.0 Hz;
H2a), 7.10 ± 7.69 (m, 30H; 6Ph); elemental analysis calcd (%) for
C64H79NO20 (1181.2): C 65.01, H 6.68, N 1.18; found C 65.11, H 6.67, N 1.14.
Methyl 5-Acetamido-4-O-benzyl-2,3,5-trideoxy-8,9-O-isopropylidene-d-
glycero-d-galacto-non-2-enopyranosonate (7):
A solution of 6 (2 g,
5.8 mmol) and benzyl bromide (0.9 mL, 7.5 mmol) in DMF (10 mL)was
cooled to 08C. Sodium hydride (185 mg, 7.6 mmol) was then added over a
period of 30 min at 08C. The reaction was stirred for another 10 min at 08C,
then methanol (0.1 mL) was added, and the mixture was evaporated in
vacuo. The residue was dissolved in dichloromethane (15 mL), washed with
water (2 Â 5 mL), dried (MgSO4), filtered and concentrated. The desired
compound 7 crystallised from hexane/ethyl acetate (15:1). Yield: 2.5 g,
83%; m.p. 1448C; [a]D ˆ ‡10 (c ˆ 0.5, CHCl3); 1H NMR (600 MHz,
CDCl3): d ˆ 1.27, 1.30 (2s, 6H; 2Me-isopropyl), 1.92 (s, 3H; NCOMe), 3.45
(dd, 1H, J(6,7) ˆ 2.88 Hz, J(7,8) ˆ 8.16 Hz; 7H), 3.72 (s, 3H; COOMe),
3.98 (m, 2H; H6, H9), 4.11 (m, 2H; H9', H4), 4.18 (m, 1H; H5), 4.26 (m,
1H; H8), 4.52 (m, 2H; CH2Ph), 5.25 (d, 1H, J(NH,5) ˆ 9.5 Hz; NH), 6.02
(d, 1H, J(3,4) ˆ 2.88 Hz; H3), 7.29 (m, 5H, Ph); elemental analysis calcd
(%) for C22H29NO8 (435.6): C 60.68, H 6.66, N 3.21; found C 60.71, H 6.67, N
3.14.
Glucopyranoside (3): tert-Butyldimethylsilyl chloride (0.7 g, 3.74 mmol)
and imidazole (0.31 g, 3.82 mmol) were added to a solution of 11 (2 g,
1.7 mmol) in dry dichloromethane (20 mL). The mixture was stirred for 8 h
at room temperature, then filtered and concentrated. Column chromatog-
raphy (toluene/acetone 3:1) of the residue gave 3 as a white foam. Yield:
1.81 g, 72%; [a]D
ˆ
15 (c ˆ 0.5, CHCl3); 1H NMR (250 MHz, CDCl3): d ˆ
0.10, 0.11, 0.13, 0.14 (4s, 12H; 4SiMe), 0.86, 0.89 (2s, 6H; 2tBu), 1.16 (s,
3H; OPiv), 1.96 (s, 3H; NCOMe), 2.10 (dd, 1H, J(gem) ˆ 13.0 Hz,
J(3ax,4) ˆ 12.3 Hz; 3cax-H), 2.58 (dd, 1H, J(gem) ˆ 13.0 Hz, J(3eq,4) ˆ
4.6 Hz; 3ceq-H), 3.33 (m, 1H; H5a), 3.36 (s, 3H; COOMe), 3.57 ± 3.72 (m,
6H; H3a, H5b, H9c, H9'c, H8c, H7c), 3.77 ± 3.87 (m, 2H; H6a, H2b), 3.95 ±
4.15 (m, 8H; H6'a, H6b, H6'b, H4c, H5c, H6c, NH, H4b), 4.30 ± 4.46 (m,
4H; H4a, H3b, CH2Ph), 4.48 (d, 1H, J(1a,2a) ˆ 8.0 Hz; H1a), 4.48 ± 5.02
(m, 9H; 4 CH2Ph, H1b), 5.00 (dd, 1H, J(1a,2a) ˆ 8.0 Hz, J(2a,3a) ˆ 8.0 Hz;
H2a), 7.10 ± 7.69 (m, 30H; 6Ph); elemental analysis calcd (%) for
C76H107NO20Si (1409.1): C 64.72, H 7.59, N 0.99; found C 64.61, H 7.57, N
0.95.
Methyl 5-Acetamido-4-O-benzyl-3-bromo-3,5-dideoxy-8,9-O-isopropyl-
idene-b-d-erythro-l-manno-2-nonulopyranosonate (8): Acetic anhydride
(5 mL) was added to a solution of 7 (2 g, 3.5 mmol) in pyridine (10 mL).
The solution was stirred for 2 h, then concentrated. A solution of the
residue in acetonitrile/water (6:1, 14 mL) was heated at 608C. N-
Bromosuccinimide (0.8 g, 3.7 mmol) was then added, and the solution
was stirred at 608C for 10 min, then allowed to reach room temperature.
Solvents were evaporated and the residue was purified by column
chromatography (toluene/acetone 4:1) to afford 8. Yield: 1.85 g, 71%;
[a]D ˆ ‡24 (c ˆ 1, CHCl3); 1H NMR (600 MHz, CDCl3): d ˆ 1.34, 1.35 (2s,
6H; 2Me-isopropyl), 1.98 (s, 3H; NCOMe), 2.18 (s, 3H; OCOMe), 3,51 (m,
1H; H9), 3.75 (s, 3H; COOMe), 4.08 (m, 1H; H9'), 4.52 (m, 2H; CH2Ph),
4.71 (m, 2H; H6, H3), 5.28 (dd, 1H, J(6,7) ˆ 2.88 Hz, J(7,8) ˆ 8.16 Hz; H7),
5.70 (brs, 1H; OH), 5.82 (d, 1H, J(NH,5) ˆ 9.8 Hz; NH), 7.44 (m, 5H, Ph);
elemental analysis calcd (%) for C24H32BrNO10 (574.4): C 50.17, H 5.57, N
2.44; found C 50.11, H 5.46, N 2.34.
Glucopyranoside 12: A solution of trisaccharide acceptor 3 (500 mg,
0.34 mmol) and the phosphite donor
2 (370 mg, 0.5 mmol) in dry
acetonitrile (15 mL) was cooled to 258C. Trimethylsilyl trifluorometha-
nesulfonate (9 mL, 0.05 mmol) was then added and the solution was stirred
for 2 h at 258C, then allowed to reach room temperature. The solution
was neutralised with Et3N and concentrated. Column chromatography of
the residue (toluene/acetone 3.5:1) afforded 12. Yield: 410 mg, 54%;
[a]D
ˆ
11 (c ˆ 1, CHCl3); 1H NMR (600 MHz, CDCl3): d ˆ 0.10, 0.11,
0.13, 0.14 (4s, 12H; 4 SiMe), 0.86, 0.89 (2s, 18H; 2tBu), 1.16 (s, 3H; OPiv),
1.32, 1.33 (2s, 6H; 2Me-isopropyl), 1.86,1.96 (2s, 6H; 2NCOMe), 2.05 (dd,
1H, J(gem) ˆ 13.0 Hz, J(3ax,4) ˆ 12.3 Hz; 3cax-H), 2.10 (s, 3H; OCOMe),
2.58 (dd, 1H, J(gem) ˆ 13.0 Hz, J(3eq,4) ˆ 4.6 Hz; 3ceq-H), 3.33 (m, 1H;
H5a), 3.36 (s, 3H; COOMe), 3.54 (s, 3H; COOMe), 3.57 ± 3.72 (m, 7H;
H3a, H5b, H9c, H9'c, H8c, H7c, H3d), 3.77 ± 3.87 (m, 4H; H6a, H2b, H9d,
H9'd), 3.95 ± 4.15 (m, 10H; H6'a, H6b, H6'b, H4c, H5c, H6c, NH, H4b, H4d,
H5d), 4.30 ± 4.46 (m, 5H; H4a, H3b, CH2Ph, H6d), 4.48 (d, 1H, J(1a,2a) ˆ
8.0 Hz; H1a), 4.48 ± 5.02 (m, 11H; 5CH2Ph, H1b), 5.10 (dd, 1H, J(1a,2a) ˆ
8.0 Hz, J(2a,3a) ˆ 8.0 Hz; H2a), 5.34 (dd, 1H, J(6,7) ˆ 2.12 Hz, J(7,8) ˆ
8.10 Hz; H7d), 5.42 (m, 2H, 2NH), 7.10 ± 7.69 (m, 40H; 8Ph); elemental
analysis calcd (%) for C106H143N2O29SSi (1995.2): C 63.75, H 7.16, N 1.40;
found C 63.71, H 7.23, N 1.44.
Methyl 5-Acetamido-4-O-benzyl-3,5-dideoxy-8,9-O-isopropylidene-3-phe-
nylthio-b-d-erythro-l-gluco-2-nonulopyranosonate (9): Sodium thiophe-
nolate (0.5 g, 3.7 mmol) was added to a solution of 8 (2 g, 3.7 mmol) in dry
THF (15 mL), and the mixture was stirred for 20 min, then concentrated.
Column chromatography of the residue (toluene/acetone 3:1) afforded 9.
Yield: 2.3 g, 92%; [a]D ˆ ‡14 (c ˆ 1, CHCl3); 1H NMR (600 MHz, CDCl3):
d ˆ 1.32, 1.33 (2s, 6H; 2Me-isopropyl), 1.82 (s, 3H; NCOMe), 2.10 (s, 3H;
OCOMe), 3.60 (s, 3H; COOMe), 3.63 (d, 1H; J(3,4) ˆ 10.5 Hz, H3), 3.80 ±
4.28 (m, 4H; H9, H9', H4, H5), 4.42 (dd, 1H, J(5,6) ˆ 8.2 Hz, J(6,7) ˆ
2.1 Hz; H6), 4.78 (m, 2H; CH2Ph), 5.05 (brs, 1H; OH), 5.34 (dd, 1H,
J(6,7) ˆ 2.12 Hz, J(7,8) ˆ 8.10 Hz; H7), 5.80 (d, 1H, J(NH,5) ˆ 9.8 Hz;
NH), 7.10 ± 7.50 (m, 10H, 2Ph); elemental analysis calcd (%) for
C30H37NO10S (603.8): C 59.68, H 6.13, N 2.32; found C 59.76, H 6.18, N 2.34.
Glucopyranoside 13: Tributylstannane (0.18 mL, 0.7 mmol) and AIBN
(20 mg) were added to a solution of 12 (400 mg, 0.2 mmol) in toluene
(10 mL) under an argon atmosphere. The mixture was heated at 1208C for
3 h, still under an argon atmosphere, then concentrated. Column chroma-
tography of the residue (toluene/acetone 3:1) afforded 13. Yield: 274 mg,
Methyl 5-Acetamido-4-O-benzyl-3,5-dideoxy-8,9-O-isopropylidene-3-phe-
nylthio-b-d-erythro-l-gluco-2-nonulopyranosonate-diethylphosphite (2):
Ethyldiisopropylamine (0.8 mL, 4.6 mmol) and diethylchlorophosphite
(0.6 mL, 3.9 mmol) were added to a solution of 9 (2 g, 3.31 mmol) in dry
acetonitrile (15 mL). The solution was stirred for 30 min at room temper-
ature, then concentrated. Column chromatography of the residue on silica
gel (toluene/acetone 4:1) gave 2 as a pale yellow syrup. Yield: 2.1 g, 92%;
[a]D ˆ ‡22 (c ˆ 1, CHCl3); 1H NMR (250 MHz, CDCl3): d ˆ 1.28 ± 1.41 (m,
12H; 2Me-isopropyl, 2Me), 1.82 (s, 3H; NCOMe), 2.12 (s, 3H; OCOMe),
3.52 (d, 1H; J(3,4) ˆ 10.5 Hz, H3), 3.58 (s, 3H; COOMe) 3.78 ± 4.20 (m,
7H; 2CH2, H9, H9', H5), 4.32(dd, 1H, J(3,4) ˆ 10.5 Hz, J(4,5) ˆ 10.5 Hz,
H4), 4.42 (dd, 1H, J(5,6) ˆ 8.2 Hz, J(6,7) ˆ 2.1 Hz; H6), 4.76 (m, 2H;
CH2Ph), 5.42 (m, 2H; NH, H7), 7.10 ± 7.50 (m, 10H, 2Ph).
74%; [a]D
ˆ
22 (c ˆ 1, CHCl3); 1H NMR (600 MHz, CDCl3): d ˆ 0.10,
0.11, 0.13, 0.14 (4s, 12H; 4SiMe), 0.86, 0.89 (2s, 18H; 2tBu), 1.18 (s, 3H;
OPiv), 1.32, 1.33 (2s, 6H; 2Me-isopropyl), 1.86,1.96 (2s, 6H; 2NCOMe),
2.05 (m, 2H; 3cax-H, 3dax-H), 2.12 (s, 3H; OCOMe), 2.56 (m, 2H; 3ceq-H,
3deq-H), 3.35 (m, 1H; H5a), 3.37 (s, 3H; COOMe), 3.55 (s, 3H; COOMe),
3.57 ± 3.72 (m, 6H; H3a, H5b, H9c, H9'c, H8c, H7c), 3.77 ± 3.87 (m, 4H;
H6a, H2b, H9d, H9'd), 3.95 ± 4.15 (m, 10H; H6'a, H6b, H6'b, H4c, H5c,
H6c, NH, H4b, H4d, H5d), 4.30 ± 4.46 (m, 5H; H4a, H3b, CH2Ph, H6d),
4.48 (d, 1H, J(1a,2a) ˆ 8.0 Hz; H1a), 4.48 ± 5.02 (m, 11H; 5CH2Ph, H1b),
5.10 (dd, 1H, J(1a,2a) ˆ 8.0 Hz, J(2a,3a) ˆ 8.0 Hz; H2a), 5.34 (dd, 1H,
J(6,7) ˆ 2.12 Hz, J(7,8) ˆ 8.10 Hz; H7d), 5.42 (m, 2H, 2NH), 7.10 ± 7.69 (m,
2182
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Synthesis of GD3
2178±2184
35H; 7Ph); elemental analysis calcd (%) for C100H138N2O29Si (1886.1): C
63.62, H 7.31, N 1.48; found C 63.51, H 7.32, N 1.45.
Diol 18: Hydrogen sulfide was bubbled through a stirred solution of 17
(102 mg, 0.06 mmol) in aqueous 83% pyridine (5 mL) for 48 h at 08C. The
reaction was monitored by TLC. After completion of the reaction, the
mixture was concentrated, and the residue was stirred with octadecanoic
acid (39 mg, 0.12 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodi-
imide hydrochloride (39 mg, 0.18 mmol) in dry dichloromethane (5 mL) for
12 h at room temperature. Column chromatography of the residue
(toluene/acetone 2:1) on silica gel gave 18 as an amorphous mass. Yield:
126 mg, 74%; [a]D ˆ ‡8 (c ˆ 0.8, CHCl3); 1H NMR (600 MHz, CDCl3):
d ˆ 0.88 (m, 6H; 2MeCH2), 1.18 (s, 9H; OPiv), 1.24 (m, 54H; 27CH2), 1.86±
2.20 (m, 46H; 3cax-H, 3dax-H, CH2, 14COMe), 2.68 (m, 3H; 3ceq-H, 3deq-H,
CH2CON), 3.35 (m, 2H; H5a, H4b), 3.38(m, 1H, H2d), 3.60 ± 4.46 (m, 24H;
H4a, H5a, H6a, H6'a, H3b, H5b, H6b, H6'b, H5c, H6c, H9c, H9'c, H5d,
H6d, H9d, H9'd, 2COOMe, OCH2R), 4.50 ± 4.80 (m, 4H; H1b, H4c, H4d,
H8c), 4.93 (d, 1H, J(1,2) ˆ 8.8 Hz; H1a), 4.96 ± 5.24 (m, 2H; H2a, H2b,
NH), 5.33 ± 5.54 (m, 4H; H3a, H7c, H4d, H7d, H8d, CHOBz, CH ˆ CHR),
5.86 (m, 1H; CH ˆ CHR), 7.40 ± 8.00 (m, 5H; OBz); elemental analysis
calcd (%) for C108H163N3O43 (2189.1): C 59.20, H 7.44, N 1.92; found C 59.11,
H 7.43, N 1.85.
Glucopyranoside 14: Acetic acid (10 mL) and TBAF (1m solution in THF,
0.4 mL) were added to a solution of 13 (200 mg, 0.1mmol) in dry THF
(3mL) at 208C. The reaction was stirred for 6 h at 208C, then acetic acid
(1 mL) was added, and the solution was concentrated. Column chromatography
of the residue (toluene/acetone 1:1) afforded 14 as a white foam. Yield:
125 mg, 74%; [a]D
ˆ
18 (c ˆ 1, CHCl3); 1H NMR (600 MHz, CDCl3): d ˆ
1.18 (s, 3H; OPiv), 1.32, 1.33 (2s, 6H; 2Me-isopropyl), 1.86, 1.96 (2s, 6H;
2NCOMe), 2.05 (m, 2H; 3cax-H, 3dax-H), 2.12 (s, 3H; OCOMe), 2.56 (m,
2H; 3ceq-H, 3deq-H), 3.35 (m, 1H; H5a), 3.37 (s, 3H; COOMe), 3.55 (s, 3H;
COOMe), 3.57 ± 3.72 (m, 6H; H3a, H5b, H9c, H9'c, H8c, H7c), 3.77 ± 3.87
(m, 4H; H6a, H2b, H9d, H9'd), 3.95 ± 4.15 (m, 10H; H6'a, H6b, H6'b, H4c,
H5c, H6c, NH, H4b, H4d, H5d), 4.30 ± 4.46 (m, 5H; H4a, H3b, CH2Ph,
H6d), 4.48 (d, 1H, J(1a,2a) ˆ 8.0 Hz; H1a), 4.48 ± 5.02 (m, 11H; 5CH2Ph,
H1b), 5.10 (dd, 1H, J(1a,2a) ˆ 8.0 Hz, J(2a,3a) ˆ 8.0 Hz; H2a), 5.34 (dd,
1H, J(6,7) ˆ 2.12 Hz, J(7,8) ˆ 8.10 Hz; H7d), 5.48 (m, 2H, 2NH), 7.10 ± 7.69
(m, 35H; 7Ph); elemental analysis calcd (%) for C88H108N2O29S (1656.2): C
63.76, H 6.52, N 1.69; found C 63.72, H 6.25, N 1.64.
Ganglioside GD3 (1): Sodium methoxide (20 mg) was added to a solution
of 18 (120 mg) in methanol (5 mL) and the mixture was stirred for 24 h at
room temperature. A solution of potassium hydroxide (0.2m, 1 mL) in
methanol was then added and the solution was stirred for another 24 h at
Glucopyranose 15: A solution of 14 (160 mg, 0.1 mmol) in methanol
(10 mL) and triflic acid (2 mL) was hydrogenated in the presence of 10%
PdC (50 mg) for 12 h at room temperature, then filtered and concentrated.
The residue was treated with acetic anhydride (1 mL), pyridine (3 mL) and
4-dimethylaminopyridine (20 mg) for 24 h at room temperature. The
solvents were evaporated and the residue was purified by column
chromatography on silica gel (toluene/acetone 5:2) to afford 15. Yield:
‡
room temperature, neutralised with Amberlite IR-120 (H ) resin and
filtered, the resin was washed with chloroform/methanol (1:1), and the
combined filtrate and washings were concentrated. Column chromatog-
raphy (methanol/water 1:1 ± 6:1) of the residue on RP-18 column gave 1 as
123 mg, 88%; [a]D
ˆ
24 (c ˆ 1, CHCl3); 1H NMR (600 MHz, CDCl3): d ˆ
an amorphous mass. Yield: 82 mg, 96%; [a]D
ˆ
3 (c ˆ 1, CHCl3 MeOH,
1.25 (s, 3H; OPiv), 1.86± 2.20 (m, 47H; 3cax-H, 3dax-H, 15COMe), 2.60 (m,
2H; 3ceq-H, 3deq-H), 3.35 (m, 2H; H5a, H4b), 3.70 ± 4.46 (m, 22H; H4a,
H5a, H6a, H6'a, H3b, H5b, H6b, H6'b, H5c, H6c, H9c, H9'c, H5d, H6d,
H9d, H9'd, 2COOMe), 4.50 ± 4.80 (m, 4H; H1b, H4c, H4d, H8c), 4.93 ±
5.24(m, 4H; H2aa, H2ab, H3aa, H2b, NH), 5.33 ± 5.54(m, 4H; H3aa, H7c,
H4d, H7d, H8d), 5.67(d, 1H, J(1,2) ˆ 8.2 Hz; H1ab), 6.26(d, 1H, J(1,2) ˆ
3.7 Hz; H1aa); elemental analysis calcd (%) for C67H92N2O41 (1580.3): C
50.88, H 5.82, N 1.77; found C 50.81, H 5.73, N 1.74.
1:1); [a]D
ˆ
2.6 (c ˆ CHCl3 MeOH;[12] 1H NMR (600 MHz, MeOH,
408C): d ˆ 0.79 (m, 6H; 2CH3CH2), 1.18 (s, 52H; 26CH2), 1.93, 1.95 (2s,
6H; 2NCOMe), 2.08 (t, 2H; COCH2), 2.48 (m, 2H; H3cax, H3dax), 2.85 (m,
2H; H3ceq, H3deq), 4.22 (d, 1H, J(1,2) ˆ 8.4 Hz; H1a), 4.34 (d, 1H, J(1,2) ˆ
ˆ
ˆ
7.9 Hz; H1b), 5.35 (m, 1H; CH CHR), 5.75 (m, 1H; CH CHR). The
1H NMR data are in accordance with those published previously.[10, 12, 22]
Mitochondrial respiration: Mitochondria were isolated from three-month-
old rats.[27] Livers were homogenised in ice-cold isolation buffer A [250mm
sucrose, 10mm Hepes (N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic
acid), pH 7.4, 1mm glutathione (GSH), 1mm ethyleneglycol bis(2-amino-
ethylether)tetraacetic acid (EGTA), 1% bovine serum albumin (BSA)].
The homogenate was centrifuged for 10 min at 700 Â g and the supernatant
was recentrifuged for 10 min at 1000 Â g. After resuspending the pellets in
isolation buffer B (125mm KCl, 10mm Hepes, pH 7.4, 1mm GSH, 0.1mm
EGTA), they were centrifuged for 10 min at 700 Â g. The supernatant was
recentrifuged for 10 min at 1000 Â g and the resultant pellet was used as
mitochondrial fraction. Isolated rat-liver mitochondria (protein concen-
tration 0.4 mgmL 1) were incubated in a medium containing 125mm KCl,
10mm Hepes, 1mm, GSH, 2mm rotenone, 5mm Mg2‡-phosphate, 5mm
succinate, pH 7.2. Oxygen consumption was measured at 258C in oxygraphs
(Geiger and Para, Innsbruck, Austria) equipped with thermostatic control
and magnetic stirrers. GD3 from bovine brain (Sigma, Deisenhofen,
Germany) or synthetic GD3 was added to mitochondria after intense
sonication in a buffer. The increase of respiration due to uncoupling effects
of GD3 was measured as described.[5] Mitochondrial function was
controlled at the end of every experiment by adding 100nm carbonyl-
cyanide-p(trifluoromethoxy)phenyl hydrazone (FCCP) to stimulate max-
imal respiration rate. The initial oxygen consumption of mitochondria
energised with succinate (31‡55.6 nmol O2 per min per mg of protein) was
used as the 100% reference value.
Trichloroacetimidate 16: A solution of 14 (1 g, 0.75 mmol) and hydrazin-
ium acetate (101 mg, 1.10 mmol) in dry DMF (8 mL) was stirred for
20 min at 408C, then diluted with EtOAc (50 mL), washed with water, with
saturated aqueous NaHCO3 solution and then again with water, dried
(MgSO4) and concentrated. A solution of the residue, trichloroacetonitrile
(1 mL, 10 mmol) and DBU (0.15 mL, 1 mmol) in dichloromethane (10 mL)
was stirred for 45 min at room temperature, then concentrated. The residue
was eluted from a column of silica gel (toluene/acetone 2:1 containing
0.1% of Et3N) to afforded 16 as a white foam. Yield: 0.95 g, 83%; [a]D
ˆ
1
‡8 (c ˆ 0.8, CHCl3); H NMR (600 MHz, CDCl3): d ˆ 1.30 (s, 3H; OPiv),
1.86± 2.20 (m, 44H; 3cax-H, 3dax-H, 14COMe), 2.60 (m, 2H; 3ceq-H, 3deq
-
H), 3.35 (m, 2H; H5a, H4b), 3.70 ± 4.46 (m, 22H; H4a, H5a, H6a, H6'a,
H3b, H5b, H6b, H6'b, H5c, H6c, H9c, H9'c, H5d, H6d, H9d, H9'd,
2COOMe), 4.50 ± 4.80 (m, 4H; H1b, H4c, H4d, H8c), 4.93 ± 5.24 (m, 3H;
H2a, H2b, NH), 5.33 ± 5.54 (m, 4H; H3a, H7c, H4d, H7d, H8d), 6.43 (d, 1H,
J(1,2) ˆ 3.2 Hz, H1a), 8.63(s, 1H, NH).
(2S,3R,4E)-2-Azido-3-O-benzoyl-4-octadecene-1,3-diol (4): Compound 4
was synthesised following a published procedure.[17]
Diol 17: A solution of 4 (68 mg, 0.16 mmol) and borontrifluoride etherate
(20 mL, 0.16 mmol) in dry dichloromethane (2 mL) was cooled to 08C. A
solution of the imidate 16 (118 mg, 0.08 mmol) in dry dichloromethane
(1 mL) was added dropwise to this solution under a nitrogen atmosphere.
After 1 h the solution was neutralised with triethylamine and evaporated in
vacuo. The residue was purified by flash chromatography (toluene/acetone
2:1) to afford 17. Yield: 118 mg, 82%; [a]D ˆ ‡13 (c ˆ 0.8, CHCl3);
1H NMR (600 MHz, CDCl3): d ˆ 0.86 (t, 3H; MeCH2), 1.16 (s, 9H; OPiv),
1.24 (m, 22H; 11CH2), 1.86± 2.20 (m, 46H; 3cax-H, 3dax-H, CH2, 14COMe),
2.68 (m, 2H; 3ceq-H, 3deq-H), 3.35 (m, 2H; H5a, H4b), 3.38(m, 1H, H2d),
3.60 ± 4.46 (m, 24H; H4a, H5a, H6a, H6'a, H3b, H5b, H6b, H6'b, H5c, H6c,
H9c, H9'c, H5d, H6d, H9d, H9'd, 2COOMe, OCH2R), 4.50 ± 4.80 (m, 4H;
H1b, H4c, H4d, H8c), 4.94 (d, 1H, J(1,2) ˆ 8.8 Hz; H1a), 4.96 ± 5.24 (m,
3H; H2a, H2b, NH), 5.33 ± 5.54 (m, 4H; H3a, H7c, H4d, H7d, H8d,
CHOBz, CH ˆ CHR), 5.84 (m, 1H; CH ˆ CHR), 7.40 ± 8.00 (m, 5H; OBz);
elemental analysis calcd (%) for C90H127N5O42 (1949.2): C 55.41, H 6.51, N
3.59; found C 55.32, H 6.63, N 3.64.
Oligodendrocyte culture: Mouse oligodendrocytes were isolated from a
primary mixed brain culture[28] prepared from the brains of BALB/c murine
embryos at day 15 after gestation. Oligodendrocyte precursors were shaken
off from the astrocyte monolayer at 15 to 20 days after preparation and
maintained in Dulbeccos Modified Eagle Medium (Life Technologies,
Grand Island, NY) supplemented with 10 ngmL 1 biotin, 100 mgmL 1 BSA
and 1% foetal calf serum. Fresh medium was mixed 1:1 with medium
preconditioned by astrocytes for 24 h. Four days after seeding, oligoden-
drocytes were stimulated with GD3. After 24 h, cells were fixed with
paraformaldehyde (PFA, 4%, dissolved in phosphate buffered saline) and
immunostained for the oligodendrocyte marker 2'3' cyclic nucleotide
phosphodiesterase (CNPase). , Cells were briefly permeabilised with 0.1%
Triton X-100, incubated for 45 min with a murine monoclonal antibody
Chem. Eur. J. 2001, 7, No. 10
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0947-6539/01/0710-2183 $ 17.50+.50/0
2183

M. Leist, R. R. Schmidt et al.
FULL PAPER
against CNPase (1:150, Sigma, Deisenhofen, Germany) that had been
incubated for 30 min with a goat-antimouse antibody coupled to Alexa-488
(Molecular Probes, Eugene, OR), stained for DNA with H-33342 (Roche
Biochemicals, Mannheim, Germany) and mounted in Aquapolymount
(Polysciences, Warrington, PA, USA). The number of oligodendrocytes
with changed nuclear morphology was scored by counting six microscopic
fields for each experimental condition by using a fluorescent microscope
(DM IRBIL, Leica Mikroskopie und Systeme GmbH, Wetzlar, Germany).
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Acknowledgements
This work was supported by the Deutsche Forschungsgemeinschaft and the
research focus ªEndogenous tissue destruction We686/18º. We are grateful
for the technical help by Elvira Gawlitta-Gorka.
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Received: September 11, 2000
Revised version: January 29, 2001 [F2725]
2184
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
0947-6539/01/0710-2184 $ 17.50+.50/0
Chem. Eur. J. 2001, 7, No. 10