Synthesis of ether-linkage-based cyclic glycerophospholipid carrying methylene moiety that improves membrane fluidity

Article abstract of DOI:10.1055/s-0028-1087935

A cyclic glycerophospholipid, in which two glycerols are connected via alkyl chains to form a 64-membered macrocycle, was synthesized. The structural features that differentiate this from our analogous lipids previously reported are two methylene moieties

Full text of DOI:10.1055/s-0028-1087935

LETTER
759
Synthesis of Ether-Linkage-Based Cyclic Glycerophospholipid Carrying
Methylene Moiety that Improves Membrane Fluidity
S
ynthesis of Cycli
u
c
G
lyceropho
t
spholi
apid ka Ono, Masato Namekata, Rie Goto, Makoto Nakamura, Motonari Shibakami*
Institute of Biological Resources and Functions, National 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 10 October 2008
membrane systems that work as a model of living cells or
functional micro/nanostructures.
Abstract: A cyclic glycerophospholipid, in which two glycerols are
connected via alkyl chains to form a 64-membered macrocycle, was
synthesized. The structural features that differentiate this from our
analogous lipids previously reported are two methylene moieties in
the alkyl chains, which were incorporated to improve membrane
fluidity. Fluorescence recovery after photobleaching revealed that
the membranes made from the methylene-containing cyclic lipid
demonstrated fluidity at room temperature unlike those of a linear
alkyl-chain lipid of equal ring size.
The feasibility of obtaining large quantities of ether cyclic
lipids from natural sources presently appears dubious;
only trace amounts can be obtained by standard culturing
methods. Thus, we need to develop synthetic methods for
the natural cyclic lipids or those mimics. We previously
reported the preparation of ether lipids 1 (Figure 1), which
mimic natural cyclic lipids.10,11 These molecules consist
of two glycerol components that are attached to a phos-
phorylcholine residue at each end and are connected via
two long hydrocarbon chains to form a macrocycle (n = 8,
10, 12, and 14 where the total length of each chain is 20,
24, 28, and 32 carbons). Diyne moieties at the middle of
the chains have been incorporated to make the molecule
more rigid and keep the molecular conformation a straight
I-shape. By doing so, self-assembly of the lipid molecules
into monolayer membrane can be expected.11,12 A series
of lipids 1 with different chain lengths were prepared so
that the thickness of the resulting vesicles would match a
variety of functional membrane proteins that would be in-
corporated. We found that synthetic cyclic lipids were
successfully formed into monolayer structures such as
vesicles.11,12 We also found that the membranes of lipid 1
(n = 8), which has a maximum thickness of 4.8 nm and a
fluidic character at room temperature, reconstituted bacte-
riorhodopsin (a membrane protein found in the natural
purple membrane of Halobacterium salinarum with a
thickness of 4.9 nm).13 However, we anticipated that ves-
Key words: lipids, macrocycles, ether lipid, phospholipid, cyclic
lipid, FRAP
Phospholipids are a major component of membranes in
living cells. Some of these have been utilized to construct
artificial membranes mimicking biomembranes and other
micro/nanostructures that have beneficial functions.1–5
Physical lability in the membranes sometimes arises as a
problem in the design of these assemblies when their
functions are utilized and examined.6,7 We focused our at-
tention on ether-linkage-based cyclic lipids as potent ma-
terials that could overcome this weakness. These are
naturally found in some species of archaea distributed
over extreme environments such as those with high tem-
peratures.8,9 Thus, a membrane that is composed of such
lipids is expected that it would be much more stable than
one composed of ester-based, two-tailed phospholipids.
With this idea in mind, we are continuing to make
progress with our research on ether lipids to establish
O
O
( )n
( )n
O
P
O
O
O
N+
N+
( )n
( )n
O
O
O
P
O
O–
1: n = 8, 10, 12, 14
O
O
O
O
O
( )7
( )7
( )4
( )4
( )4
( )7
( )7
O
O
N+
N+
( )4
O
P
O
O
P
O
O–
O–
2
Figure 1
SYNLETT 2009, No. 5, pp 0759–0762
x
x.
x
x.
2
0
0
9
Advanced online publication: 24.02.2009
DOI: 10.1055/s-0028-1087935; Art ID: U11108ST
© Georg Thieme Verlag Stuttgart · New York

760
Y. Ono et al.
LETTER
O
O
OH
( )4
a
b
c
+
TBDMSO
TBDMSO
Br
RO
TBDMSO
( )7
( )4
( )4
( )7
( )7
( )4
( )7
4
3
5: R = TBDMS
6: R = H
7: R = Ms
d
e
Scheme 1 Reagents and conditions: (a) Mg, THF, reflux, then hept-6-ynal, 0 °C, 91%; (b) cat. TPAP, NMO, CH2Cl2, r.t., 89%; (c) MePh3P+I–,
NaH, DMSO, r.t., 63%; (d) concd HCl, EtOH, r.t., 87%; (e) MsCl, Et3N, CH2Cl2, 0 °C, 91%.
icles made from lipids with longer chain lengths (1, n = 10, are tough works that require a complicated reaction se-
12, 14) would be too rigid for reconstituting membrane quence.16 The alkylidene group can be introduced more
proteins.11 That is, the vesicles demonstrated gel-to- easily (by using a practical method such as the Wittig
liquid-crystalline phase transition above room tempera- reaction, for example) because it does not require a stereo-
ture (in water). This property is detrimental, because pro- controlled or stereospecific reaction to introduce it. In oth-
teins exhibit functions in the liquid-crystalline phase but er words, when the ‘horn’ moiety is introduced into the
not in the gel phase, and they normally degenerate at high- chain, a chemical conversion that leads to an additional
er temperatures.
asymmetric carbon into the molecule and makes it dia-
stereomeric or racemic is detrimental (e.g., the Grignard
reaction makes the carbonyl carbon racemic, yielding a
mixture of stereoisomers). The structure of the ether lipid
2 synthesized in this work is outlined in Figure 1. We also
report its membrane properties.
To resolve this drawback with lipids 1 (n = 10), we intro-
duced a ‘horn’ onto the chains connecting the glycerols.
We expected that it would work as a steric hindrance to
the aggregation of lipid molecules and weaken the inter-
action between molecules when the lipid membrane
formed, so that the phase-transition temperature could be First, half the hydrocarbon chains that connected the two
reduced. We chose a methylene group as the ‘horn’. In glycerols was synthesized. The procedure is outlined in
natural membrane lipids incorporated olefin and iso- Scheme 1. 7-Bromoheptan-1-ol with the hydroxy group
prenoid moieties have been found to work as a functional being protected as TBDMS ether was joined with hept-6-
group that drops the phase-transition temperature of a lip- ynal by using the Grignard reaction in THF. The alcohol
id membrane.14,15 The alkylidene group we introduced 3 thus obtained was subjected to oxidation with TPAP/
this time is a novel type of such functionality that may NMO to give ketone 4. Then, the ‘horn’ moiety was intro-
have the ability to drop the phase-transition temperature, duced by using the Wittig reaction. Thus, the action of 4
which gives high mobility to the membrane. Preparing with methylene triphenylphosphine generated from
and introducing a natural or related optically active iso- methyltriphenylphosphonium iodide and NaH in DMSO
prenoid moiety into an alkyl chain as its partial structure successfully gave olefin 5. To perform further transfor-
O
O
O
a
b
( )7
( )4
( )7
( )4
( )4
( )7
7
O
O
O
8
9
O
O
O
O
O
O
O
O
O
e
c
( )7
( )4
( )4
( )4
( )7
( )7
( )7
( )4
( )4
( )7
OH
HO
( )7
( )4
TrO
OTr
10: R = H
RO
RO
OR
d
12
11: R = Tr
O
O
O
f
( )7
( )4
( )4
( )4
( )4
( )7
O
( )7
( )7
OR
13: R = Tr
14: R = H
g
O
O
O
( )7
( )7
( )4
( )4
( )4
( )4
( )7
( )7
h
O
O
O
N+
N+
O
P
O
O
P
O
2
O–
O–
Scheme 2 Reagents and conditions: (a) 1,2-isopropylidene-sn-glycerol, NaH, DMSO, r.t., 91%; (b) CuCl, TMEDA, O2, acetone, reflux, 97%;
(c) TsOH, MeOH, r.t., 71%; (d) TrCl, DMAP, Et3N, CH2Cl2, r.t., 46%; (e) 7, NaH, DMSO, 60 °C, 51%; (f) Cu(OAc)2, pyridine, reflux, 61%;
(g) TsOH, MeOH, r.t., 85%, (h) bromoethyl dichlorophosphate, Et3N, benzene, r.t., then 30% aq Me3N, i-PrOH–MeCN–CHCl3, 60 °C, 50%.
Synlett 2009, No. 5, 759–762 © Thieme Stuttgart · New York

LETTER
Synthesis of Cyclic Glycerophospholipid
761
(5) Martin, S. F.; Josey, J. A.; Wong, Y.-L.; Dean, D. W. J. Org.
Chem. 1994, 59, 4805; and references cited therein.
(6) Kornberg, R. D.; McConnell, H. M. Biochemistry 1971, 10,
1111.
(7) Nicolussi, A.; Massari, S.; Colonna, R. Biochemistry 1982,
21, 2134.
(8) Tornabebe, T. G.; Langworthy, T. A. Science 1979, 203, 51.
(9) De Rosa, M.; Gambacorta, A.; Gliozzi, A. Microbiol. Rev.
1986, 50, 70.
(10) Miyawaki, K.; Takagi, T.; Shibakami, M. Synlett 2002,
1326.
(11) Nakamura, M.; Goto, R.; Tadokoro, T.; Shibakami, M.
J. Colloid Interface Sci. 2007, 310, 630.
(12) Miyawaki, K.; Goto, R.; Shibakami, M. Chem. Lett. 2003,
32, 1170.
(13) Shibakami, M.; Tsuihiji, H.; Miyoshi, S.; Nakamura, M.;
Goto, R.; Mitaku, S.; Sonoyama, M. Biosci. Biotechnol.
Biochem. 2008, 72, 1623.
(14) Lewis, R. N. A. H.; McEthaney, R. N. In The Structure of
Biological Membranes, 2nd ed.; Yeagle, P. L., Ed.; CRC
Press: Boca Raton, 2004, 66.
mations, the TBDMS protecting group on 5 was replaced
by the Ms group to yield compound 7, which is the key
counterpart in the preparation of the required cyclic lipids.
Next, by using the Williamson reaction, the half-chain
part 7 was joined with a glycerol in which two of the three
hydroxy groups were protected as acetonide (1,2-isopro-
pylidene-sn-glycerol, Scheme 2). By using glycerol ace-
tonide, the entire molecule became chiral. We consider
that the lipid molecules needed to be chiral for the sake of
mimicry with natural membrane lipids, mainly from the
viewpoint of interaction with protein molecules. Then,
terminal alkyne 8 was coupled to give diyne 9 by using a
CuCl catalyst under Hay conditions in hot acetone.17 Be-
fore the second chain moiety was introduced, the ace-
tonide protective groups of 9 were replaced by Tr groups;
the acetonides were hydrolyzed by using an acid catalyst,
and the terminal primary hydroxy groups in 1018 in
Scheme 2 were selectively masked by the Tr groups. The
reaction of 1119 in Scheme 2 with NaH and then 7 in warm
DMSO yielded 12, which was cyclized to 13 under highly
diluted Eglinton coupling conditions in the presence of
excess Cu(OAc)2 in refluxing pyridine.20,21
(15) (a) Koga, Y.; Morii, H. Biosci., Biotechnol., Biochem. 2005,
69, 2019. (b) Blöcher, D.; Gutermann, R.; Henkel, B.; Ring,
K. Biochim. Biophys. Acta 1984, 778, 74.
(16) For example, see: Eguchi, T.; Arakawa, K.; Terachi, T.;
Kakinuma, K. J. Org. Chem. 1997, 62, 1924.
(17) Selected Physical Data of 9
Finally, the Tr protecting groups on 13 were removed and
the hydroxy groups were converted into phosphoryl-
choline residue by successive action with bromoethyl
dichlorophosphate and trimethylamine. Isolation with gel
permeation chromatography using CHCl3–MeOH as an
eluent gave the required ‘horned’ cyclic lipid 2 in pure
form.22
1H NMR (400 MHz, CDCl3): d = 1.30 (12 H, br s), 1.36 (6
H, s), 1.42 (6 H, s), 1.52 (8 H, quint, J = 5.4 Hz), 1.96–2.02
(8 H, m), 1.58 (8 H, br m), 2.26 (8 H, br m), 3.39–3.53 (8 H,
m), 3.72 (2 H, dd, J = 8.2, 6.4 Hz), 4.05 (2 H, dd, J = 8.2, 6.4
Hz), 4.26 (2 H, quint, J = 6.0 Hz), 4.69 (4 H, br s). ESI-MS
(TOF): m/z = 694 [M + Na+], 672 [M + H+].
(18) Selected Physical Data of 10
1H NMR (400 MHz, CDCl3): d = 1.32 (12 H, br s), 1.42 (4
H, quint, J = 7.2 Hz), 1.53 (8 H, quint, J = 3.4 Hz), 1.59 (4
H, quint, J = 7.0 Hz), 1.98–2.02 (8 H, br s), 2.28 (4 H, br m),
3.45–3.56 (8 H, m), 3.66 (2 H, dd, J = 11.4, 5.0 Hz), 3.73 (2
H, dd, J = 11.5, 4.1 Hz), 3.87 (2 H, quint, J = 4.7 Hz), 4.71
(4 H, br s). ESI-MS (TOF): m/z = 614 [M + Na+], 591 [M +
H+].
The membrane fluidity composed of the ‘horned’ cyclic
lipid 2 was proved by fluorescence recovery after pho-
tobleaching (FRAP). After bleaching with irradiation, the
recovery of fluorescence was observed in the defined re-
gion of a monolayer membrane of 2 developed on a glass
plate. This meant that the bleached molecules were ex-
changed with unbleached molecules on the membrane,
thus revealing the mobility of lipid molecules that com-
posed the membrane. However, the membranes of the
previously synthesized straight chain analogue 1 (n = 10)
demonstrated gel-like characteristics at room tempera-
ture.11,23 Altogether, we think that the ‘horn’ worked well
as an effective steric hindrance to improve membrane
fluidity.
(19) Selected Physical Data of 11
1H NMR (400 MHz, CDCl3): d = 1.28 (12 H, br s), 1.40 (4
H, quint, J = 7.1 Hz), 1.51 (8 H, quint, J = 4.2 Hz), 1.96–
1.99 (8 H, br m), 2.25 (4 H, br m), 2.42 (2 H, d, J = 4.6 Hz),
3.19 (4 H, qd, J = 7.6, 5.4 Hz), 3.40–3.54 (8 H, m), 3.94 (2
H, sext, J = 5.1 Hz), 4.69 (4 H, br m), 7.22 (6 H, t, J = 7.3
Hz), 7.29 (12 H, t, J = 7.3 Hz), 7.42 (12 H, d, J = 7.3 Hz).
ESI-MS (TOF): m/z = 1098 [M + Na+].
(20) Procedure for the Preparation of 13
To a solution of Cu(OAc)2 (314 mg, 1.73 mmol) in refluxing
pyridine (100 mL), a solution of 12 (257 mg, 0.173 mmol) in
pyridine (10 mL) was added dropwise with a syringe pump
over 5.5 h. The solution gradually turned from blue to dark
green. After completion of the addition the solution was
heated at the same temperature for 1 h. The mixture was
cooled to r.t. and evaporated to give a residue, which was
purified on a silica gel column chromatography eluted with
hexane–EtOAc (10:1) to give 13 as a yellow oil; yield 157
mg (61%).
Acknowledgment
We are grateful to the AIST Upbringing of Talent in the Nanobio-
technology Course and the Promotion Budget for Science and Tech-
nology from the Ministry of Education, Culture, Sports, Science
and Technology for their support in this research.
1H NMR (400 MHz, CDCl3): d = 1.28 (24 H, br s), 1.39 (8
H, br m), 1.51–1.57 (16 H, br m), 1.96–2.01 (16 H, m), 2.28
(br s, 8 H), 3.13–3.20 (4 H, br m), 3.39 (4 H, br t, J = 6.9 Hz),
3.45–3.58 (10 H, m), 4.68 (8 H, br s), 7.20 (6 H, t, J = 7.1
Hz), 7.27 (12 H, t, J = 7.3 Hz), 7.45 (12 H, d, J = 7.8 Hz).
ESI-MS (TOF): m/z = 1505 [M + Na+].
References and Notes
(1) Gennis, R. B. Biomembranes: Molecular Structure and
Function; Springer: New York, 1989.
(2) Eibl, H. Chem. Phys. Lipids 1980, 26, 405.
(3) Beck, A.; Heissler, D.; Duportail, G. Tetrahedron 1991, 47,
1459.
(4) Paltauf, F.; Hermetter, A. Prog. Lipid Res. 1994, 33, 239.
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Y. Ono et al.
LETTER
(22) Purification of 2 was conduced on an LC-908 instrument
(21) The sequence from 7 and glycerol moiety to 12 was altered
from the one we previously reported for preparing a straight-
chain analogue 1.10,11 At the step of the connection of chain
moiety and protected glycerol in the previous study, we used
1-trityl-3-p-methoxybenzoyl-sn-glycerol, which was
prepared by starting from 1,2-isopropylidene-sn-glycerol in
three steps. Through this modification, several protection–
deprotection steps could be omitted. The order in which the
chains was introduced into the glycerol moiety was also
changed. This change would cause an increase in the steric
hindrance around the secondary hydroxy groups during the
Williamson reaction sequence; however, the reaction
proceeded without difficulties even though the yield of 12
was somewhat lower.
(Japan Analytical Industries) equipped with a JAIGEL-
GS310 column [eluent, CHCl3–MeOH = 1:2 (v/v)].
Colorless gummy oil. 1H NMR (400 MHz, CDCl3–
CD3OD = 2:1): d = 1.30 (28 H, br s), 1.41 (12 H, br s), 1.52
(20 H, br s), 1.97–2.01 (16 H, t, J = 7.6 Hz), 2.27 (8 H, br m),
3.23 (18 H, s), 3.38–3.49 (6 H, m), 3.55–3.61 (12 H, m), 3.88
(4 H, br s), 4.25 (4 H, br s), 4.70 (8 H, br s). ESI-MS (TOF):
m/z = 1350 [M + Na+], 1328 [M + H+].
(23) Schubert, T.; Seitz, P. C.; Schneck, E.; Nakamura, M.;
Shibakami, M.; Funari, S. S.; Konovalov, O.; Tanaka, M.
J. Phys. Chem. B 2008, 112, 10041.
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