Synthesis of the glycosphingolipid β-galactosyl ceramide and analogues via olefin cross metathesis

Full text of DOI:10.1021/jo051069y

SCHEME 1a
Synthesis of the Glycosphingolipid
â-Galactosyl Ceramide and Analogues via
Olefin Cross Metathesis
Anand Narain Rai and Amit Basu*
Department of Chemistry, Brown University, Box H,
Providence, Rhode Island 02912
abasu@brown.edu
Received May 28, 2005
a Conditions: (a) (i) tBuPh2SiCl, imidazole, 61%; (ii) p-MeO-
benzyl trichloroacetimidate, La(OTf)3, 81%. (b) (i) NaN3, Bu4NCl,
DMF, 90 °C; (ii) tBuPh2SiCl, imidazole, 67% over two steps. (c) (i)
Zn, NH4Cl, MeOH, 83%; (ii) Fmoc-Cl, Hunig’s base, CH2Cl2, 76%.
The preparation of the glycosphingolipid galactosyl ceramide
from an orthogonally protected five-carbon building block
is described. The main chain of the lipid is installed via a
highly stereoselective olefin cross metathesis reaction. The
methodology permits the facile preparation of glycolipids
which vary in the length of the main carbon chain.
cosphingolipids. We recently reported a cross metathesis
route for the preparation of the sphingolipid 4,5-unsatur-
ated lipid chain and demonstrated an application of this
methodology for the synthesis of ceramide.7,8 In this
paper we present a cross metathesis approach for the
synthesis of â-GalCer and its analogues.9
The centerpiece of the cross metathesis synthetic route
is the orthogonally protected building block 5 (Scheme
1). Our previously reported preparation of 5 used diethyl
tartrate as the starting material.7 While the use of
tartrate permits access to both enantiomers of sphin-
golipids, we now report a significantly improved method
for the preparation of 5 starting from the mesylate diol
2. The synthesis of 2 has been reported by Bundle and
co-workers in a patent application.10 Following these
procedures, we prepared 2 on a multigram scale without
The glycosphingolipid â-galactosyl ceramide (â-GalCer)
is abundant in the myelin sheath of both the central and
peripheral nervous system. â-GalCer isolated from my-
elin consists of a variety of lipids, which vary in chain
length, olefination, and hydroxylation state of the hy-
drocarbons in the main and acyl chains.1 These lipids are
also collectively referred to as cerebroside. A carbohy-
drate-carbohydrate interaction between cerebroside and
cerebroside sulfate, the 3-sulfate derivatives of cerebro-
side, mediates the compaction of the myelin sheath.2
Defective formation and/or compaction of myelin is
implicated in a variety of pathologies, including multiple
sclerosis, Krabbe’s and metachromatic leukodystrophies
(MLD), and congenital hypomyelination.3 Additional roles
for â-GalCer include its function as a ligand for the HIV-1
viral glycoprotein gp120, mediating viral entry into
epithelial cells.4 â-GalCer has been suggested as a
possible ligand for the adhesion of Helicobacter pylori to
cells in the gastric system.5
(6) (a) Santacroce, P. V.; Basu, A. Angew. Chem., Int Ed. 2003, 42,
95-98. (b) Santacroce, P. V.; Basu, A. Glycoconjugate J. 2004, 21, 89-
95.
(7) Rai, A. N.; Basu, A. Org. Lett. 2004, 6, 2861-2863.
(8) For other approaches to sphingolipids involving olefin cross
metathesis, see: (a) Hasegawa, H.; Yamamoto, T.; Hatano, S.; Hakogi,
T.; Katsumura, S. Chem. Lett. 2004, 33, 1592-1593. (b) Singh, O. V.;
Kampf, D. J.; Han, H. Tetrahedron Lett. 2004, 45, 7239-7242. (c)
Torssell, S.; Somfai, P. Org. Biomol. Chem. 2004, 2, 1643-1646.
(9) For other applications of olefin cross metathesis for the prepara-
tion of O-linked as well as C-linked glycoconjugates, see the following.
O-Linked: (a) Biswas, K.; Coltart, D. M.; Danishefsky, S. J. Tetrahe-
dron Lett. 2002, 43, 6107-6110. (b) Kirschning, A.; Chen, G.-W.
Tetrahedron Lett. 1999, 40, 4665-4668. (c) Berkowitz, D. B.; Maiti,
G.; Charette, B. D.; Dreis, C. D.; MacDonald, R. G. Org. Lett. 2004, 6,
4921-4924. (d) Roy, R.; Dominique, R.; Das, S. K. J. Org. Chem. 1999,
64, 5408-5412. (e) Plettenburg, O.; Mui, C.; Bodmer-Narkevitch, V.;
Wong, C.-H. Adv. Synth. Catal. 2002, 344, 622-626. (f) Barrett, A. G.
M.; Beall, J. C.; Braddock, D. C.; Flack, K.; Gibson, V. C.; Salter, M.
M. J. Org. Chem. 2000, 65, 6508-6514. C-Linked: (g) Huwe, C. M.;
Woltering, T. J.; Jiricek, J.; Weitz-Schmidt, G.; Wong, C.-H. Bioorg.
Med. Chem. 1999, 7, 773-788. (h) Chen, G.; Schmieg, J.; Tsuji, M.;
Franck, R. W. Org. Lett. 2004, 6, 4077-4080. (i) Vernall, A. J.; Abell,
A. D. Org. Biomol. Chem. 2004, 2, 2555-2557. (j) Godin, G.; Compain,
P.; Martin, O. R. Org. Lett. 2003, 5, 3269-3272. (k) Nolen, E. G.;
Kurish, A. J.; Wong, K. A.; Orlando, M. D. Tetrahedron Lett. 2003, 44,
2449-2453. (l) Postema, M. H. D.; Piper, J. L. Tetrahedron Lett. 2002,
43, 7095-7099. (m) McGarvey, G. J.; Benedum, T. E.; Schmidtmann,
F. W. Org. Lett. 2002, 4, 3591-3594. (n) Dondoni, A.; Giovannini, P.
P.; Marra, A. J. Chem. Soc., Perkin Trans. 1 2001, 2380-2388. (o) Liu,
S.; Ben, R. N. Org. Lett. 2005, 7, 2385-2388.
Our interest in â-GalCer stems from our ongoing
studies of glycolipid carbohydrate-carbohydrate interac-
tions.6 As part of this work, we required a modular
synthetic approach toward â-GalCer and related gly-
(1) (a) Stoffel, W.; Bosio, A. Curr. Opin. Neurobiol. 1997, 7, 654-
661. (b) Curatolo, W. Biochim. Biophys. Acta 1987, 906, 137-160.
(2) Boggs, J. M.; Wang, H. M.; Gao, W.; Arvantis, D. N.; Gong, Y.
P.; Min, W. X. Glycoconjugate J. 2004, 21, 97-110.
(3) (a) Kolter, T.; Sandhoff, K. Angew. Chem., Int. Ed. 1999, 38,
1532-1568. (b) Ohler, B.; Revenko, I.; Husted, C. J. Struct. Biol. 2001,
133, 1-9.
(4) (a) Villard, R.; Hammache, D.; Delapierre, G.; Fotiadu, F.; Buono,
G.; Fantini, J. ChemBioChem 2002, 3, 517-525. (b) Nolting, B.; Yu,
J. J.; Liu, G.; Cho, S. J.; Kauzlarich, S.; Gervay-Hague, J. Langmuir
2003, 19, 6465-6473.
(5) Tang, W.; Seino, K.; Ito, M.; Konishi, T.; Senda, H.; Makuuchi,
M.; Kojima, N.; Mizuochi, T. FEBS Lett. 2001, 504, 31-35.
(10) Bundle, D. R.; Ling, C. C.; Zhang, P. In WO 03/101937 A1, 2003.
10.1021/jo051069y CCC: $30.25 © 2005 American Chemical Society
Published on Web 08/25/2005
8228
J. Org. Chem. 2005, 70, 8228-8230

SCHEME 2a
SCHEME 3a
a Conditions: (a) (i) Piperidine/DMF; (ii) palmitoyl chloride,
Et3N, DMAP, CH2Cl2, 66% over two steps. (b) 1-Pentadecene,
Grubbs second-generation catalyst, 72%. (c) (i) Piperidine/DMF;
(ii) palmitoyl chloride, Et3N, DMAP, CH2Cl2, 70% over two steps.
(d) (i) CF3CO2H/CH2Cl2/H2O; (ii) NaOMe/MeOH, 77% over two
steps.
a Conditions: (a) HF‚pyridine, 88%. (b) BF3‚Et2O, 4 Å MS, 71%.
(c) Alkene, Grubbs second-generation catalyst.
chromatographic purification. The primary alcohol of the
diol was selectively protected as a bulky silyl ether,
followed by lanthanum triflate-promoted etherification
of the secondary alcohol to provide 3.11 Subsequent azide
displacement of the sulfonate proceeded efficiently, but
the desired product 4 was always accompanied by some
material that had lost the silyl ether under the reaction
conditions. The crude reaction mixture was resubjected
to silylation conditions to provide 4 in 67% overall yield.
We had found that olefin cross metathesis reactions of
the azide-containing building block 4 suffered from poor
yields and undesirable side reactions,7 so the azide was
reduced and the resulting amine was protected as the
Fmoc carbamate. The orthogonally protected alkene 5
was obtained in 20% overall yield from the diol 2 and
could be prepared in multigram quantities.
The silyl ether was removed from 5 by treatment with
HF‚pyridine to afford the alcohol 6 in 88% yield. This
alcohol was efficiently glycosylated with the tetrapivaloyl
trichloroacetimidate 7 derived from galactose using BF3‚
Et2O as the promoter to provide the desired â-linked
glycoside 8 in 71% yield. It is worth commenting that
the glycosylation yield and anomeric selectivity obtained
with the N-Fmoc-protected acceptor 5 compare favorably
with that obtained when â-azido glycosyl acceptors were
used.12
observed in the cross metathesis reactions of 5, the trans-
alkene was formed with very high selectivity. In most
1
cases, only the trans isomer was observed by H NMR.
While R-olefins underwent facile cross metathesis to
afford the protected glycolipids in good yield, the presence
of adjacent heteroatoms was problematic. The ethylene
glycol derivative 9e failed to provide any cross-coupled
product, despite variations in solvent, temperature, stoi-
chiometry, and ruthenium carbene catalyst. One possible
reason for the failure of 9e to couple may be the formation
of nonproductive chelated metallacarbene intermedi-
ates.15
Completion of the synthesis of â-GalCer was achieved
by deprotecting the Fmoc group from 10c, followed by
acylation with palmitoyl chloride to provide 12. Alterna-
tively, 12 could be obtained from 8 by initial Fmoc group
removal and palmitoylation, followed by metathesis as
the last step in the sequence. Subsequent global depro-
tection of the PMB ether and pivaloyl esters provided
1
â-galactosyl ceramide 13. The H NMR spectrum of the
final product was in good agreement with that reported
by Schmidt.12b
Alkene 5 is a versatile building block for (glyco)-
sphingolipid synthesis. The majority of glycosphingolipid
syntheses install the alkene-containing chain first, fol-
lowed by glycosylation and acylation.12c Using building
block 5, one can install the three functionalities (sugar,
acyl group, main chain) in a wider variety of sequences
such as those shown in Scheme 3. Additionally, the
terminal alkene in 5 provides a useful chemical handle
for alternative functionalization chemistries and biocon-
With the glycoside in hand, we evaluated its compe-
tence as a coupling partner in the olefin cross metathesis
reaction (Scheme 2). The cross coupling could be effected
with a variety of alkenes in high yields using the
commercially available “Grubbs second-generation” ru-
thenium carbene catalyst.13,14 As we had previously
(11) Rai, A. N.; Basu, A. Tetrahedron Lett. 2003, 44, 2267-2269.
(12) (a) Rich, J. R.; Bundle, D. R. Org. Lett. 2004, 6, 897-900. (b)
Zimmermann, P.; Bommer, R.; Ba¨r, T.; Schmidt, R. R. J. Carbohydr.
Chem. 1988, 7, 435-452. (c) Vankar, Y. D.; Schmidt, R. R. Chem. Soc.
Rev. 2000, 29, 201-216. (d) Compostella, F.; Franchini, L.; De Libero,
G.; Palmisano, G.; Ronchetti, F.; Panza, L. Tetrahedron 2002, 58,
8703-8708.
(14) Excess lipid alkene was used in these experiments to obtain
products in reasonable yields and time frames. Although these
reactions generate mixtures of the lipid alkene homodimer and
monomer, we have not observed dimerization of the unreacted sugar
alkene, which can be recovered.
(15) See: Hoveyda, A. H.; Ve´zina, M. Org. Lett. 2005, 7, 2113.
McNaughton, B. R.; Buchholz, K. M.; Camaan˜o-Moure, A.; Miller, B.
L. Org. Lett. 2005, 7, 733 and references contained therein.
(13) Scholl, M.; Ding, S.; Lee, C. W.; Grubbs, R. H. Org. Lett. 1999,
1, 953-956.
J. Org. Chem, Vol. 70, No. 20, 2005 8229

jugation.12a This flexibility will facilitate the synthesis
of glycolipid derivatives and provide a powerful tool for
the elucidation of structure-function relationships in this
class of biomolecules.16
61.0, 66.6, 66.8, 68.0, 69.1, 70.5, 70.9, 71.0, 79.4, 101.2, 113.7,
119.9, 125.05, 125.1, 127.0, 127.3, 127.6, 129.3, 130.4, 137.1,
141.2, 141.3, 143.9, 144.0, 155.8, 159.1, 176.8, 176.9, 177.2, 177.8.
HRMS (FAB): calcd for C67H97NaNO14 1162.6806, found
1162.6830
12: A solution of 10c (40 mg, 0.035 mmol) and 20% piperidine
in DMF (3 mL) was stirred for 1 h. The excess piperidine and
DMF was removed by flowing N2 through the reaction mixture
for 3 h to provide the crude amine. The crude amine was taken
up in dichloromethane (3 mL), and triethylamine (0.03 mL, 0.2
mmol), DMAP (2 mg, 0.016 mmol), and palmitoyl chloride (0.2
mL, 0.07 mmol) were added. The reaction mixture was stirred
at room temperature for 3 h. The reaction mixture was diluted
with dichloromethane (10 mL) and washed successively with 1
N HCl (5 mL), water (5 mL), and brine (5 mL). The dichlo-
romethane layer was dried (Na2SO4) and concentrated to give
the crude, which was purified by two successive column chro-
matography runs (SiO2, ethyl acetate/hexanes, 1/10) to provide
Experimental Section
8: To a cold (-10 °C) solution of 6 (50 mg, 0.11 mmol) and 74
(181 mg, 0.28 mmol) in dichloromethane (5 mL) was added BF3‚
OEt (0.28 mL, 0.022 mmol, 0.78 M solution in CH2Cl2). The
reaction mixture was stirred for 2 h. To the reaction mixture
was added saturated sodium bicarbonate (aq). The mixture was
stirred for 5 min and diluted with dichloromethane (20 mL). The
dichloromethane layer was dried (Na2SO4) and concentrated to
give the crude, which was purified by by column chromatography
(SiO2, ethyl acetate/hexanes, 1/12) to provide 80 mg (71%, 0.083
mmol) of 8 as a white solid. 1H NMR (300 MHz, CDCl3): δ 1.12
(s, 9H), 1.13 (s, 9H), 1.17 (s, 9H), 1.24 (s, 9H), 3.59 (d, J ) 7.9
Hz, 1H), 3.77 (s, 3H), 3.89-3.90 (m, 2H), 3.96 (t, J ) 6.9 Hz,
1H), 4.05 (dd, J ) 7.2, 10.9 Hz, 1H), 4.14 (dd, J ) 5.7, 11 Hz,
1H), 4.18-4.23 (m, 2H), 4.25-4.28 (m, 1H), 4.39 (ab, J ) 10.9
Hz, 2H), 4.49 (d, J ) 10 Hz, 1H), 4.51 (d, J ) 10 Hz, 1H), 4.97
(d, J ) 8 Hz, 1H), 5.12 (dd, J ) 3.2, 10.4 Hz, 1H), 5.23 (dd, J )
7.8, 10.3 Hz, 1H), 5.30 (d, J ) 9.8 Hz, 1H), 5.31 (d, J ) 17.9 Hz,
1H), 5.415 (d, J ) 2.8 Hz, 1H), 5.78 (ddd, J ) 7.2, 9.9, 17 Hz,
1H), 6.86 (d, J ) 8.6 Hz, 2H), 7.26 (m, 2H), 7.30 (td, J ) 1.1, 7.4
Hz, 2H), 7.39 (t, J ) 7.5 Hz, 2H), 7.55 (d, J ) 7.6, 1H), 7.58 (d,
J ) 11 Hz, 1H), 7.76 (d, J ) 7.6 Hz, 2H). 13C NMR (100 MHz,
CDCl3): δ 27.1, 27.14, 38.7, 38.76, 38.8, 39.1, 47.2, 53.6, 55.2,
61.1, 66.6, 67.8, 69.0, 70.85, 70.9, 71.0, 79.7, 101.1, 113.8, 119.6,
119.9, 125.0, 127.0, 127.6, 129.4, 130.2, 135.7, 141.3, 143.9, 155.9,
159.2, 176.8, 176.9, 177.3, 177.8. HRMS (FAB): calcd for C54H71-
NaNO14 980.4772, found 980.4791.
1
28 mg (70%, 0.024 mmol) of 12 as a waxy solid. H NMR (400
MHz, CDCl3): δ 0.88 (t, Jave ) 6.8 Hz, 6H), 1.12 (s, 9H), 1.159
(s, 9H), 1.16 (s, 9H), 1.23-1.38 (m, 48 H), 1.25 (s, 9H), 1.99-
2.13 (m, 4H), 3.59 (dd, J ) 3.4, 9.1 Hz, 1H), 3.79 (s, 3H), 3.82 (t,
J ) 8 Hz, 1H), 3.94 (t, J ) 7.1 Hz, 1H), 4.04 (dd, J ) 7.4, 10.9
Hz, 1H), 4.12 (dd, J ) 6.7, 10.9 Hz, 1H), 4.14-4.18 (m, 1H), 4.20
(dd, J ) 4.0, 8.9 Hz, 1H), 4.36 (ab, J ) 11 Hz, 2H), 4.50 (d, J )
7.6 Hz, 1H), 5.11 (dd, J ) 3.2, 10.4 Hz, 1H), 5.19 (dd, J ) 7.6,
10.4 Hz, 1H), 5.35 (dd, J ) 8.4, 15.5 Hz, 1H), 5.405 (d, J ) 2.9
Hz, 1H), 5.57 (d, J ) 8.8 Hz, 1H), 5.67 (dt, J ) 6.9, 15.3 Hz,
1H), 6.87 (d, J ) 8.6 Hz, 2H), 7.22 (d, J ) 8.6 Hz, 2H). 13C NMR
(100 MHz, CDCl3): δ 14.5, 23.1, 26.1, 27.5, 27.6, 29.7, 29.8, 30.0,
30.1, 32.4, 32.7, 37.4, 39.1, 39.2, 39.5, 52.0, 55.7, 61.4, 67.0, 68.4,
69.6, 70.7, 71.3, 71.4, 79.8, 101.6, 114.2, 127.9, 129.7, 131.0,
137.2, 159.5, 172.8, 177.2, 177.3, 177.6, 178.2. HRMS (FAB)
calcd for C68H118NO13 1156.8603, found 1156.8570.
13: To a cold (0 °C) solution of 12 (22 mg, 0.019 mmol) in
dichloromethane was added 20% TFA (2 mL, solution in dichlo-
romethane). The reaction mixture was stirred for 12 h. To the
reaction mixture was added solid sodium bicarbonate with
vigorous stirring. The resulting mixture was filtered and con-
centrated. The crude reaction mixture was directly used in next
step without any further purification. The crude mixture was
taken in methanol (3 mL) and added to a solution of sodium
methoxide (0.19 mmol) in methanol. The reaction mixture was
stirred for 12 h. To the reaction mixture was added Amberlite
(IR-120, H+), and the reaction mixture was stirred until the
solution was neutral. The reaction mixture was filtered and
concentrated to give the crude, which was purified by column
chromatography (SiO2, hexanes/ethyl acetate, 9/1) to generate
10.3 mg (77%, 0.015 mmol) of 134 as a waxy solid.
10c: To a solution of 8 (55 mg, 0.057 mmol) and pentadecene-1
(0.078 mL, 0.29 mmol) in dichloromethane (3 mL) was added
Grubbs second-generation catalyst (13.6 mg, 0.016 mmol). The
reaction mixture was heated to reflux for 24 h. To the reaction
mixture was added pentadecene-1 (0.078 mL, 0.29 mmol), and
the reaction mixture was refluxed for an additional 12 h. The
reaction mixture was concentrated and purified by column
chromatography (SiO2, ethyl acetate/hexanes, 1/12) to provide
1
53 mg (81%, 0.046 mmol) of 10c as white waxy solid. H NMR
(400 MHz, CDCl3): δ 0.88 (t, Jave ) 6.8 Hz, 3H), 1.12 (s, 9H),
1.14 (s, 9H), 1.17(s, 9H), 1.24 (s, 9H), 1.25-1.33 (m, 22H), 2.0
(m, 2H), 3.61 (dd, J ) 1.4, 8.6 Hz, 1H), 3.78 (s, 3H), 3.82-3.90
(m, 2H), 3.96 (m, 1H), 4.07 (dd, J ) 7.4, 10.9 Hz, 1H), 4.15 (dd,
J ) 6.6, 10.8 Hz, 1H), 4.17-4.20 (m, 1H), 4.20-4.24 (m, 1H),
4.25 (d, J ) 9 Hz, 1H), 4.39 (ab, J ) 11 Hz, 2H), 4.4 (dd, J )
6.7, 6.8 Hz, 1H), 4.96 (d, J ) 8.6 Hz, 1H), 5.12 (dd, J ) 3.2, 10.4
Hz, 1H), 5.23 (dd, J ) 7.8, 10.3 Hz, 1H), 5.37 (dd, J ) 8, 15 Hz,
1H), 5.415 (d, J ) 3.1 Hz, 1H), 5.73 (dt, J ) 6.6, 15.4 Hz, 1H),
6.86 (d, J ) 8.5 Hz, 2H), 7.24 (d, J ) 8.5 Hz, 2H), 7.30 (t, J )
7.4 Hz, 2H), 7.39 (t, J ) 7.5 Hz, 2H), 7.55 (d, J ) 7.5 Hz, 1H),
7.59 (d, J ) 7.5 Hz, 1H), 7.76 (d, J ) 7.6 Hz, 2H). 13C NMR
(100 MHz, CDCl3): δ 14.1, 22.7, 27.07, 27.14, 27.2, 29.2, 29.4,
29.5, 29.7, 31.9, 32.3, 38.7, 38.75, 38.8, 39.1, 47.2, 53.8, 55.2,
Acknowledgment. This work was supported by an
NSF-CAREER award (CHE-0134908) to A.B.
Supporting Information Available: Experimental pro-
tocols for compounds 3-5, 10a,b,d, and 11 and characteriza-
tion data for all new compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
(16) (a) Chang, Y. T.; Choi, J.; Ding, S.; Prieschl, E. E.; Baumruker,
T.; Lee, J.-M.; Chung, S.-K.; Schultz, P. G. J. Am. Chem. Soc. 2002,
124, 1856-1857. (b) Lingwood, C. A.; Mylvaganam, M. Methods
Enzymol. 2003, 363, 264-283.
JO051069Y
8230 J. Org. Chem., Vol. 70, No. 20, 2005