Synthesis of Ceramide Mimics with a Pseudo Cyclic Framework

Article abstract of DOI:10.1055/s-2003-42094

We have designed and synthesized ceramide mimics with a pseudo cyclic framework that can be converted to 'dimeric' sphingomyelins and glycosphingolipids for potential components of artificial rafts. These molecules are characterized by the presence of (i) two hydrophilic head groups, (ii) a covalently bonded hydrocarbon chain, and (iii) two untethered alkyl chains. Self-assembling of a ceramide mimic into nanorods is briefly discussed.

Full text of DOI:10.1055/s-2003-42094

LETTER
2163
Synthesis of Ceramide Mimics with a Pseudo Cyclic Framework
S
ynthesis of Ceram
i
ide Mi
k
mics
w
ith
a
o
Pseudo Cycl
k
ic
F
ramewor
a
k
zu Suzuki, Michiko Mori, Motonari Shibakami*
National Institute for Materials and Chemical Process, Institute of Advanced Industrial Science and Technology (AIST), Central 5th,
1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan
Fax +81(29)8614547; E-mail: moto.shibakami@aist.go.jp
Received 8 August 2003
nique. Recently we have started a synthetic program aim-
Abstract: We have designed and synthesized ceramide mimics
ing at preparing ‘dimeric’ ceramide mimics with a pseudo
with a pseudo cyclic framework that can be converted to ‘dimeric’
cyclic framework that can be converted to ‘dimeric’
sphingomyelins and glycosphingolipids. Under appro-
sphingomyelins and glycosphingolipids for potential components
of artificial rafts. These molecules are characterized by the presence
of (i) two hydrophilic head groups, (ii) a covalently bonded hydro- priate conditions, rafts that contain such dimers should
carbon chain, and (iii) two untethered alkyl chains. Self-assembling
of a ceramide mimic into nanorods is briefly discussed.
be relatively long-lived for detection. A long-range goal
that we have been trying to reach has been to construct
Key words: ceramide, lipid microdomain, pseudo cyclic frame-
work, raft, sphingolipids, self-assembly, regioselectivity
‘visual’ artificial rafts that can be available for clarifying
the size and molecular-level structure of rafts.
OH
OH
In this paper we describe the design and synthesis of cer-
amide mimics that contain a pseudo cyclic framework.1
Such amphiphiles could be converted into sphingolipid
mimics that are designed to span the inner and outer leaf-
let, or adopt a ‘U-shaped’ conformation (Figure 1).2
CH3(CH2)m
HO
(CH2)mCH3
(CH2)n
OH
HN
NH
O
O
1a (m=4, n=14 )
1b (m=12, n=30)
Figure 2 Structure of ceramide mimics with a pseudo cyclic frame-
work.
Specific ceramide mimics that were chosen as a synthetic
target are shown in Figure 2. The design principle that we
have used for the construction of the mimics incorporates
structural elements that are found in the naturally occur-
ring sphingolipids. Specifically, it includes (i) two hydro-
philic head groups composed of amide and hydroxyl
groups, (ii) a covalently bonded hydrocarbon chain, and
a
b
Figure 1 Possible conformations of ceramide mimics with a pseudo
cyclic framework: a) a spanning conformation; and b) a ‘U-shaped’ (iii) two untethered alkyl chains. The flexibility of unteth-
conformation.
ered alkyl chains may produce ‘local’ liquid-crystalline
phase when the lipids assemble in membranes, and there-
by allow the resulting lipid microdomains to accommo-
date membrane proteins.7 A ceramide mimic 1a,
containing fourteen methylene units in a covalently bond-
ed chain was expected to adopt a ‘U-shaped’ conforma-
tion in a leaflet, since the maximum length of 1a (17 Å) is
too short to span the membrane (ca 30 Å). A mimic 1b that
bears thirty methylene units was expected to span the hy-
drocarbon region of a phospholipid bilayer, or adopt a ‘U-
shaped’ conformation.
Our motivation for this work originally stems from the hy-
pothesis that lipid microdomains, referred to as ‘rafts’,3
that are composed of, at least, sphingolipids, i.e., sphingo-
myelins and glycosphingolipids, and cholesterol, play an
important role in cell-cell communication, signal trans-
duction,4 and immunorecognition.5 Despite numerous in-
vestigations, the postulate of ‘raft’ still remains to be a
concept awaiting definite proof.6 Part of the difficulty for
unmasking rafts has been that they are in dynamic equili-
brium. Thus, we hypothesized that if sphingolipids that
exist in a lipid microdomain are ‘dimerized’ by connect-
ing two alkyl chains, thereby prolonging raft’s lifetime,
one may ‘snap a picture’ of rafts with an appropriate tech-
Our construction of the ceramide mimics hinges on the
use of a covalently bonded alkyl chain. That is, two D-
erythro-sphingosine units are coupled with dicarboxylic
acid. Hexadecanedioic acid for the preparation of 1a is
commercially available, while dotriacontanedioic acid for
1b was synthesized by efficient use of diacetylenic cou-
pling reaction.
SYNLETT 2003, No. 14, pp 2163–2166
0
6
.1
1
.2
0
0
3
Advanced online publication: 15.10.2003
DOI: 10.1055/s-2003-42094; Art ID: U16203ST
© Georg Thieme Verlag Stuttgart · New York

2164
H. Suzuki et al.
LETTER
a
b
c
OH
OH
OTHP
OTHP
C13H27
C
13H27
2
5
3
4
d
e
(CH2)14OH
(CH2)13CO2H
f
(CH2)13CO2H
2
6
7
8
g
h
MeO2C(CH2)30CO2Me
HO2C(CH2)30CO2H
9
10
Scheme 1 Reagents and conditions: (a) DHP, p-TSA (cat.), r.t., 24 h, quant.; (b) C13H27Br, n-BuLi, –30 °C, 1 h; (c) p-TSA (cat.), MeOH, r.t.,
quant. from 3; (d) NaH, 1,3-DAP, 70 °C, 68%; (e) PDC, DMF, 69% (f) Cu(OAc)2, pyridine, reflux, 10 min; (g) 10% Pd/C, H2, CHCl3–MeOH,
r.t., overnight; (h) 1 M NaOH (aq), THF, r.t., 74% from 7. 1,3-DAP = 1,3-diaminopropane, PDC = pyridinium dichromate,
DHP = dihydropyran, p-TSA = p-toluenesulfonic acid, THP = tetrahydropyranyl.
Scheme 1 summarizes the synthetic route, which has been chromatography (silica). Subsequent deprotection of the
employed in the preparation of dotriacontanedioic acid. In hydroxyl group and the amino moiety (p-TSA), reduction
essence, the primary hydroxyl group of propargyl alcohol of the triple bond to trans-double bond with Red-Al®,11c
2 was protected with THP. Subsequent reaction of 3 and deprotection of the amino moiety (TFA) afforded
with 1-bromotridecane, deprotection of the hydroxyl sphingosine (15a and 15b). Alternatively, we examined
group, to give 5, and acetylenic zipper reaction8 of 5 with the availability of the route in which reduction of the triple
sodium hydride and 1,3-diaminopropane provided termi- bond precedes deprotection. This procedure, however, re-
nal acetylenic alcohol 6. After oxidation of 6 with PDC sulted in a lower yield. N-acylation of sphingosine (15a)
(to give 7), coupling9 of 7 in the presence of Cu(OAc)2 with N-succinimidyl ester of hexadecanedioic acid afford-
yielded 8. Catalytic reduction of diacetylenic unit with Pd/ ed 1a (74%).12,13 In contrast, the use of coupling reagents
C afforded the methyl ester of the desired compound 9. such as 1,3-dicyclohexylcarbodiimide and 1-(3-dimethyl-
We assume that such esterification was catalyzed by aminopropyl)-3-ethylcarbodiimide hydrochloride in place
hydrochloric acid that derived from chloroform. Finally, of the N-succinimidyl ester provided the desired com-
hydrolysis of 9 with sodium hydroxide afforded 10.
pound in a lower yield. Compound 1b13 was obtained by
using acid chloride prepared from dotriacontanedioic acid
and oxalyl chloride (9.4%). Relatively lower yield is at-
tributable to the lower solubility of 1b in organic solvents
when subjected to column chromatography.
The synthetic route that was used to prepare the sphin-
gosine moieties is summarized in Scheme 2. This proce-
dure is reminiscent of Liotta’s and Herold’s approach to
sphingosine. In brief, reaction of aldehyde 11, which was
prepared according to the scheme previously reported,10 Since the resulting ceramide mimics include two hydroxyl
with lithiated 1-heptyne and 1-pentadecyne afforded 12a groups, the protection of the secondary hydroxyl group is
and 12b, respectively. This reaction provided both the al- needed for the introduction of phosphoric choline or sugar
cohol as a mixture of erythro- and threo-isomers,11 and into the primary hydroxyl group. To establish such protec-
the desired isomer (erythro) was purified by column tion procedure, some combinations of protecting groups
O
OH
OH
a
b
O
H
O
HO
NBoc
NBoc
(CH2)mCH3
NHBoc
(CH2)mCH3
12a (m=4)
12b (m=12)
13a (m=4)
13b (m=12)
11
OH
e
c
1a , 1b
HO
(CH2)mCH3
NHX
14a (m=4, X=Boc), 14b (m=12, X=Boc)
15a (m=4, X=H), 15b (m=12, X=H)
d
Scheme 2 Reagents and conditions; (a) 1-Heptyne, n-BuLi, THF, from –40 °C to –20 °C within 1 h, D-erythro (12a: 72%, 12b: 70%): L-threo
(7.5%, 5.0%); (b) p-TSA, MeOH, r.t., 24 h, (13a: 95%, 13b: 90%) (c) Red-Al®, Et2O, r.t., 24 h, (14a: 67%, 14b: 70%); (d) TFA, 30 min, (15a:
98%, 15b: 90%); (e) hexadecanedioic acid succinimide ester, Et3N, THF, r.t., 24 h, (1a: 74%), ClCO(CH2)30COCl, pyridine, CH2Cl2(1b: 9.4%).
Boc = t-butoxycarbonyl, Red-Al® = sodium bis(2-methoxyethoxy)aluminum hydride.
Synlett 2003, No. 14, 2163–2166 © Thieme Stuttgart · New York

LETTER
Synthesis of Ceramide Mimics with a Pseudo Cyclic Framework
2165
OR2
OTBDPS
c
R1O
(CH2)4CH3
HO
(CH2)4CH3
HN
HN
(CH2)14
(CH2)14
2
2
O
O
1a (R1, R2) = (H, H)
16 (R1, R2) = (Bz, H)
17 (R1, R2) = (Bz, TBDPS)
a
18
b
Scheme 3 Reagents and conditions: (a) BzCl, DMAP, pyridine, –40 °C, 65%; (b) TBDPSCl, imidazole, 60 °C, 85%; (c) K2CO3,
MeOH–THF–H2O, 4 °C, 60%. BzCl = benzoyl chloride, DMAP = 4-dimethylamino pyridine, TBDPSCl = tert-butyldiphenylsilyl chloride.
have been examined by use of 1a as a starting material. work, and that one of the mimics forms organogel. Efforts
Scheme 3 summarizes the established synthetic route. In aimed at preparing sphingolipids with a pseudo cyclic
essence, regioselective benzoylation14 and silylation of framework and constructing artificial rafts are continuing
the primary and secondary hydroxyl groups, respectively, in our laboratory.
to give 17, followed by debenzoylation afforded the de-
sired product (18) in 50% for two steps.
Acknowledgment
Our working hypothesis for this work has been that
‘dimeric’ lipids potentially associate with each other in
transient rafts, thereby prolonging raft’s lifetime. Other
We are grateful to T. Watanabe. (the Technical Center, AIST) for
technical assistance. This research was supported by Industrial
Technology Research Grant Program from New Energy and Indu-
structural elements that may contribute to the prolonga-
tion include amide and hydroxyl groups. One may imag-
ine that intramolecular hydrogen bonding through these
groups, together with dimerization, would provide robust
microdomains in phospholipid bilayers. In order to test for
the presence of cooperative forces for forming domains,
we have chosen to examine self-assembling of 1a in or-
ganic solvents as an alternative method, since no method
of detection of microdomains has not been established.
Specific protocols that were used for self-assembling
were similar to those for recrystallization of organic com-
pounds. Briefly, 1a (30 mg) was dissolved in ethyl acetate
(1 mL) at 60 °C. After the clear solution was sonicated for
1–2 min by use of mild (bath-type) sonicator at ambient
temperature, white gel was obtained. Examination of
the gel by scanning electron microscopy revealed the for-
mation of nanorods that construct three-dimensional
networks (Figure 3).
strial Technology Development Organization (NEDO) of Japan.
Reference
(1) Several lipids with a pseudo cyclic framework are reported
in the literature: (a) Lecollinet, G.; Gulik, A.; Mackenzie,
G.; Goodby, J. W.; Benvegnu, T.; Plusquellec, D. Chem.–
Eur. J. 2002, 8, 585. (b) Eguchi, T.; Arakawa, K.;
Kakinuma, K.; Rapp, G.; Ghosh, S.; Nakatani, Y.; Ourisson,
G. Chem.–Eur. J. 2000, 6, 3351. (c) Wang, G.;
Hollingsworth, R. I. J. Org. Chem. 1999, 64, 4140.
(d) Lecollinet, G.; Auzély-Velty, R.; Danel, M.; Benvegnu,
T.; Mackenzie, G.; Goodby, J. W.; Plusquellec, D. J. Org.
Chem. 1999, 64, 3139. (e) Auzély-Velty, R.; Benvegnu, T.;
Plusquellec, D.; Mackenzie, G.; Haley, J. A.; Goodby, J. W.
Angew. Chem. Int. Ed. 1998, 37, 2511. (f) Kim, J.-M.;
Thompson, D. Langmuir 1992, 8, 637.
(2) Delfino, J. M.; Schreiber, S. L.; Richards, F. M. J. Am.
Chem. Soc. 1993, 115, 3458.
(3) Simons, K.; Ikonen, E. Nature 1997, 387, 569.
(4) For recent reviews on cell-cell communication and signal
transduction mediated by sphingolipids, see: (a) Kolesnick,
R. J. Clinical Inv. 2002, 110, 3. (b) Hannun, Y. A.; Luberto,
C.; Argraves, K. M. Biochemistry 2001, 40, 4893.
(c) Kolter, T.; Sandhoff, K. Angew. Chem. Int. Ed. 1999, 38,
1532. (d) Brown, D. A.; London, E. Annu. Rev. Cell Dev.
Biol. 1998, 14, 111; and references therein.
The results reported herein indicate that a facile synthetic
route for the ceramide mimics with a pseudo cyclic frame-
(5) (a) Sharma, K.; Shi, Y. F. Cell Res. 1999, 9, 1.
(b) Nakamura, S.; Kozutsumi, Y.; Sun, Y. D.; Miyake, Y.;
Fujita, T.; Kawasaki, T. J. Biol. Chem. 1996, 271, 1255.
(6) (a) Kenworthy, A. K.; Petranova, N.; Edidin, M. Mol. Biol.
Cell. 2000, 11, 1645. (b) Friedrichson, T.; Kurzchlia, T. V.
Nature (London) 1998, 394, 802. (c) Varma, R.; Mayor, S.
Nature (London) 1998, 394, 798. (d) Kenworthy, A.;
Edidin, M. J. Cell Biol. 1988, 142, 69.
(7) Menger, F. M.; Wong, Y.-L. J. Org. Chem. 1996, 61, 7382.
(8) Djura, P.; Faulkner, J. D. J. Org. Chem. 1980, 45, 735.
(9) Hebert, N.; Beck, A.; Lennox, B. R.; Just, G. J. Org. Chem.
1992, 57, 1777.
(10) (a) Garner, P.; Park, J. M. Org. Synth. 1991, 70, 18.
(b) Garner, P.; Park, J. M. J. Org. Chem. 1987, 52, 2361.
Figure 3 Electron micrograph of the organogel of 1a.
Synlett 2003, No. 14, 2163–2166 © Thieme Stuttgart · New York

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H. Suzuki et al.
LETTER
(11) (a) Williams, L.; Zhang, Z.; Shao, F.; Carroll, P. J.; Joullie,
M. M. Tetrahedron 1996, 52, 11673. (b) Nimkar, S.;
Menaldino, D.; Merrill, A. H.; Liotta, D. Tetrahedron Lett.
1988, 29, 3037. (c) Herold, P. Helv. Chem. Acta 1988, 71,
354.
(12) Synthesis of 1a: To a solution of 15a (19 mg, 0.101 mmol)
and Et3N (10.2 mg, 0.101 mmol) in dry THF (1.0 mL) was
added N-succinimidyl ester of hexadecanedioic acid (15 mg,
0.054 mmol) at r.t. After 24 h, the reaction mixture was
concentrated under reduced pressure to give a residue. The
residue was purified by column chromatography [silica,
EtOAc–MeOH–n-hexane (100:10:100 to 100:5:0)] to give
1a (23 mg, 74%).
(13) All new compounds gave satisfactory analytical and spectral
data. Compound 1a: colorless solid, [a]D29 –18.76 (c 0.50,
MeOH). 1H NMR (500 MHz, CDCl3): d = 6.28 (2 H, d,
J = 7.7 Hz), 5.79 (2 H, dtd, J = 15.3, 6.7, 1 Hz), 5.53 (2 H,
ddt, J = 15.3, 6.4, 1 Hz), 4.32 (2 H, br s), 3.95 (2 H, br d,
J = 11.9 Hz), 3.91 (2 H, m), 3.71 (2 H, m), 2.85 (4 H, br s),
2.23 (4 H, t, J = 7.4 Hz), 2.06 (4 H, dt, J = 7.5, 6.7 Hz), 1.64
(4 H, tt, J = 7.4, 7.4 Hz), 1.38 (4 H, tt, J = 7.4, 7.4 Hz), 1.25–
1.30 (28 H, m), 0.89 (6 H, t, J = 7.0 Hz) ppm. 13C NMR (125
MHz, CDCl3): d = 174.0, 136.4, 128.8, 74.7, 62.5, 54.5,
36.8, 32.2, 31.4, 29.3, 29.2, 29.2, 29.2, 29.2, 28.8, 25.7, 22.5,
14.0 ppm. LRMS (ESI, m/z): 647 (M + Na). Compound 1b:
colorless amorphous solid, 1H NMR (500 MHz, CDCl3): d =
6.32 (2 H, m), 5.76 (2 H, dt, J = 15.3, 6.7 Hz), 5.50 (2 H, dd,
J = 15.3, 6.8 Hz), 4.27 (2 H, m), 3.90 (2 H, m), 3.85 (2 H, m),
3.67 (2 H, m), 2.84 (4 H, br s), 2.25 (4 H, t, J = 7.3 Hz), 2.05
(4 H, m), 1.62 (4 H, m), 1.25–1.45 (96 H, m), 0.86 (6 H, t,
J = 7.0 Hz) ppm. LRMS (ESI, m/z): 1097 (M + Na).
Synthesis of 1b: To a solution of dotriacontanedioic acid
(93.5 mg, 183 mmol) in CH2Cl2 (3.7 mL) was added oxalyl
chloride (7.3 mL, 84 mmol) at r.t. After 12 h, the reaction
mixture was concentrated under reduced pressure. Then, the
resulting residue was added to a mixture that was composed
of 15b (137 mg, 457 mmol), pyridine (3.7 mL) and CH2Cl2
(7.3 mL) at r.t. After stirring for 12 h, the reaction was
quenched by the addition of 1.5 mL of H2O. The mixture was
then extracted with CHCl3 (100 mL). The organic layer was
washed with H2O (5mL × 2) and brine (5mL), and
concentrated under reduced pressure to yield a residue. The
residue was purified by column chromatography [silica,
CHCl3–EtOAc–MeOH (10/10/1 to 5/5/1, v/v/v)] to give 1b
(18.4 mg, 9.4%).
(14) Regioselective Benzoylation of 1a: Compound 1a (170 mg,
0.272 mmol) and DMAP (33 mg, 0.272 mmol) were
dissolved in pyridine (3 mL) and stirred at –40 °C for 1 h in
an atmosphere of nitrogen gas. After addition of benzoyl
chloride (0.070 mL, 0.598 mmol), the reaction mixture was
then stirred at –40 °C for 4 h. A sat. NH4Cl aqueous solution
was added to quench the reaction, and the mixture was then
extracted with CHCl3 (15 mL × 4). The organic layer was
washed with brine (20 mL), dried over anhyd Na2SO4, and
concentrated under reduced pressure to give a residue. The
residue was purified by column chromatography [silica, n-
hexane/EtOAc/CHCl3 (6:6:1)] to give 16a (158 mg, 70%).
Synlett 2003, No. 14, 2163–2166 © Thieme Stuttgart · New York