Giant vesicles from 72-membered macrocyclic archaeal phospholipid analogues: Initiation of vesicle formation by molecular recognition between membrane components

Article abstract of DOI:10.1002/1521-3765(20000915)6:18<3351::AID-CHEM3351>3.0.CO;2-4

Stereochemically pure archaeal acyclic bola-amphiphilic diphosphates 4 and 5, with the basic structure of the phospholipids found in Sulfolobus, have been synthesized for the first time. The self-assembly properties have been compared with those of the ne

Full text of DOI:10.1002/1521-3765(20000915)6:18<3351::AID-CHEM3351>3.0.CO;2-4

FULL PAPER
Giant Vesicles from 72-Membered Macrocyclic Archñal Phospholipid
Analogues: Initiation of Vesicle Formation by Molecular Recognition
between Membrane Components
Tadashi Eguchi,*[a] Kenji Arakawa,[b] Katsumi Kakinuma,[b] Gert Rapp,[c] Sangita Ghosh,[d]
Yoichi Nakatani,*[d] and Guy Ourisson*[d]
Abstract: Stereochemically pure arch-
ñal acyclic bola-amphiphilic diphosphates
4 and 5, with the basic structure of the
phospholipids found in Sulfolobus, have
been synthesized for the first time. The
self-assembly properties have been com-
pared with those of the nearly identical
72-membered macrocyclic tetraether
phosphates 3a and 3b, analogues of the
major phospholipid components of Sul-
folobus, Thermoplasma, and methano-
genic Archña, which were also synthe-
sized. Phase contrast and fluorescence
microscopies have shown that the dipo-
lar lipids 1 and 2 spontaneously formed
vesicles. Whereas the macrocyclic dipo-
lar phosphates 3 spontaneously formed
vesicles (phase contrast and fluores-
cence microscopies), the bolaform phos-
phate 4 gave only a lamellar structure
(synchrotron diffraction pattern: repeat
distance of about 4.25 nm but with only a
few layers). However, upon addition of
the unphosphorylated precursors phyta-
nol, phytol, or geranylgeraniol to the
acyclic lipids 4 and 5, giant vesicles were
rapidly formed. Addition of n-hexadeca-
nol or cholesterol did not lead to vesicle
formation. Therefore it was concluded
that this vesicle formation occurs only
when the added molecule is closely
compatible with the constituents of the
lipid layer and can be inserted into
the double layer. Aslight mismatch
(cholesterol or n-hexadecanol/polyprenyl
chains) is therefore enough to block the
insertion process presumably required
for vesicle formation.
Keywords: amphiphiles ´ Archña ´
macrocycles ´ molecular recognition
´ vesicles
lipids, but of the iso- and anteiso-acyl type). Another striking
feature of some archñal lipids is the presence of 36- (2; Yˆ
PO3(CH2)2NMe3) or 72-membered rings (3a, 3b; X ˆ PO3H2),
or of bolaform lipids (4, 5; X ˆ PO3H2).[1]
Introduction
Archña, the third major kingdom of living organisms, possess
membrane lipids structurally unique in that their polar
headgroups are linked to polyprenyl chains (unsaturated or
saturated) by ether bonds, in contrast to the ester bonds and n-
acyl chains of Eucarya (Procarya often contain also branched
We have undertaken to study systematically the effect of
these structural peculiarities on the self-assembly properties
of natural archñal lipids and analogues, such as the formation
of vesicles and their permeability. We have previously shown
that the archñal 36-membered macrocyclic diether phospha-
tidylcholine 2 (Yˆ PO3(CH2)2NMe3) gave vesicles which
were much less water-permeable than those composed of
acyclic n-diacyl or n-dialkyl phosphocholines.[2]
[a] T. Eguchi
Department of Chemistry and Materials Science
Tokyo Institute of Technology
O-okayama, Meguro-ku, Tokyo (Japan)
Fax : (‡81)3-57-34-26-31
E-mail: eguchi@chem.titech.ac.jp
The 72-membered macrocyclic tetraether phosphocholine
lipids are major membrane lipids of the highly thermophilic
Sulfolobus and Thermoplasma, and of some methanogenic
Archña.[1a] They carry two nonequivalent polar heads, based
on glycerol (or more complex polyols[3]); two of the carbon
atoms of glycerol are linked by ether bonds to C40 octaprenyl
chains, themselves linked distally to the second headgroup.
The diversity of these phospholipids comes from the nature of
the headgroups and from the presence of zero to four
cyclopentane rings per polyprenyl chain. It was also shown
recently that the 72-membered lipids are present as a mixture
of regioisomers 3a and 3b (with various complex polar
[b] K. Arakawa, K. Kakinuma
Department of Chemistry, Tokyo Institute of Technology
O-okayama, Meguro-ku, Tokyo (Japan)
[c] G. Rapp
MPI-KGF, c/o HASYLAB at DESY
Notkestrasse 85, 22603 Hamburg (Germany)
[d] Y. Nakatani, G. Ourisson, S. Ghosh
Â
Laboratoire de Chimie Organique des Substances Naturelles, associe
au CNRS, Centre de Neurochimie Universite Louis Pasteur
Â
5 rue Blaise Pascal, 67084 Strasbourg (France)
Fax : (‡33)3-88-60-76-20
E-mail: nakatani@chimie.u-strasbg.fr
ourisson@chimie.u-strasng.fr
Chem. Eur. J. 2000, 6, No. 18
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3351

T. Eguchi, Y. Nakatani, G. Ourisson et al.
FULL PAPER
hypothesized that these poly-
prenyl phosphates may have
been the most primitive phos-
pholipids. As diphytanylglycer-
yl phosphate 1 easily formed
vesicles,[11] we now study the
phosphates 3ab (X ˆ PO3H2,
1:1 mixture [molar] of 3a and
3b), 4 (X ˆ PO3H2), and 5 (X ˆ
PO3H2).
Results
Synthesis
In order to compare the mem-
brane properties of the 72-
membered macrocyclic diphos-
phates 3ab (X ˆ PO3H2) and
their acyclic counterparts
4
(X ˆ PO3H2) and
5
(X ˆ
headgroups).[4] These lipids can be expected, by virtue of their
obvious amphiphilicity, to form organized systems in which
the very long chain is extended across the membrane,[5]
leading to molecular assemblies structurally similar to the
classical bilayers of eucaryotic biomembranes but linked by
PO3H2), we have synthesized these lipids according to
Scheme 1.
72-Membered macrocyclic diphosphates 3ab: We have re-
cently developed a convenient synthesis of the 72-membered
macrocyclic diol 3ab (X ˆ H, mixture of 1:1 molar ratio of 3a
and 3b).[12] In the presence of 4-dimethylaminopyridine
(DMAP) and pyridine, a mixture of the diols 3a and 3b
(X ˆ H)[13] was treated with diphenylphosphoryl chloride[14] in
benzene to give the corresponding bisphospho-triesters. This
mixture was subjected to catalytic hydrogenolysis in the
presence of platinum (from PtO2) to afford the desired
bisphosphoric acid derivatives 3a,b (X ˆ PO3H2, 72% yield).
À
C C bonds in mid-membrane. Indeed, formation of multi-
lamellar liposomes has been reported with the native lipid
mixture isolated from Sulfolobus acidocaldarius,[6] whereas
some close derivatives of 3 form predominantly nonlamellar
assemblies such as cubic phases.[7] Menger and Chen have
synthesized a fully demethylated 72-membered macrocyclic
archñal lipid analogue based on n-alkyl chains, rather than
polyprenyl, which did not form vesicular systems.[8] Some
models of natural acyclic bola-amphiphilic lipids have also
been synthesized and their phase properties have been
reported.[9]
Acyclic bola-amphiphilic diphosphates 4 and 5: The bola-
amphiphilic phosphate 4, structurally analogous to the macro-
cyclic lipid 3ab but not macrocyclic, has also been found in
Sulfolobus solfataricus.[20] In this lipid, each of the polar
headgroups is linked to two chains, a C-40 bisphytanyl and a
C-20 phytanyl one. It was synthesized as follows.
We have previously shown that phosphates instead of
phosphatidylcholines or other complex headgroups are effi-
cient for membrane formation with polyprenyl derivatives,
both with two chains[10a] or with only one,[10b] and we have
Phytanol (6) was oxidized under Swern conditions to give in
82% yield aldehyde 7, which was treated with sn-1-O-
benzylglycerol 8[15] in the presence of toluene-p-sulfonic acid
and MgSO4 to give quantitatively the acetal 9 as a diastereo-
meric mixture. The acetal 9 was treated with diisobutylalu-
minum hydride (DIBAL-H) to give a mixture of positionally
isomeric monoalkylated benzylglycerol derivatives, which
were easily separated by silica ± gel column chromatography
to afford the sn-2-O-alkylated benzylglycerol 10 and the sn-3-
O-alkylated benzylglycerol 11 in 48 and 50% yields, respec-
tively.[16] The sn-2-O-alkylated glycerol 10 was further alky-
lated via its sodium alkoxide with 17[17] to afford 2,3-O-
disubstituted sn-1-O-benzylglycerol 12 in 85% yield. Subse-
quent deprotection of the TBS group, Swern oxidation, and
Wittig olefination afforded the half-size diether 15. This was
dimerized by metathesis using the ruthenium-alkylidene
Abstract in French: Les diphosphates acycliques bola-amphi-
Â
Â
 Â
philiques de Sulfolobus (une archeobacterie)ont e te pour la
Á
Â
Â
 Â
Â
 Â
premiere fois synthetises stereoselectivement. Les proprietes
 Â
Â
d'auto-organisation de ces lipides ont ete comparees avec celles
Â
de diphosphates macrocycliques possedant un squelette voisin.
Le microscopie en contraste de phase et en fluorescence montre
Â
que les lipides dipolaires macrocycliques 3 donnent spontane-
Â
Â
ment des vesicules geantes, tandis que les lipides acycliques 4 et
Â
Ã
Â
5 ne forment pas de vesicules par eux-memes. L'etude par
diffraction de rayons X (Synchrotron DESY)montre que le
Á
Á
diphosphate 4 donne un systeme de couches paralleles distantes
d'environ 4,25 nm. Par addition de phytanol, de phytol ou de
Â
Â
Â
Â
 Â
Â
geranylgeraniol, des vesicules geantes sont ete formees rapide-
Á
Â
ment a partir de 4 et de 5. L'addition de n-hexadecanol ou de
Â
Â
cholesterol n'induit pas la formation de vesicules.
ˆ
complex, [RuCl2( CHPh)(PCy3)2] developed by Grubbs
3352
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Chem. Eur. J. 2000, 6, No. 18

Giant Vesicles
3351±3358
ˆ
Scheme 1. Synthesis of the dipolar lipids 3ab (X ˆ PO3H2), 4 (X ˆ PO3H2) and 5 (X ˆ PO3H2). Reagents: a) (PhO)2P( O)Cl, DMAP/Py/benzene; b) H2,
PtO2/HOAc; c) Swern oxidation; d) compound 7, p-TsOH, MgSO4/CH2Cl2; e) DIBAL-H/toluene; f) NaH, compound 17/DMSO; g) TBAF/THF;
ˆ
ˆ
h) Ph3P CH2/THF; i) [RuCl2( CHPh)(PCy3)2]/CH2Cl2; j) H2, 10%Pd-C/EtOAc/HOAc. TBAF ˆ tetrabutylammonium fluoride.
Scheme 2. Synthesis of the dipolar lipid 5 (X ˆ PO3H2). Reagents see Scheme 1.
et al.[18] The reaction proceeded smoothly to yield the
unsaturated tetraether 16 in 68% yield. Reduction of the
double bond and deprotection of the benzyl groups of 16 by
catalytic hydrogenation afforded 4 (X ˆ OH) quantitatively.
Finally, phosphorylation as described above gave the acyclic
tetraether core lipid 4 (X ˆ PO3H2) in 62% yield.
addition of Nile Red, a neutral lipophilic fluorescent probe,
one can visualize more clearly the image of the membrane
around the vesicles in the fluorescence mode. When the 72-
membered macrocyclic phosphates 3ab (X ˆ PO3H2), with the
same core structures as the mixture of lipids found in
thermophilic Archña, were dispersed in a neutral buffer, they
easily formed giant vesicles (50 ± 60 mm) (Figure 1).
Lipid 4 (X ˆ PO3H2), when placed on a microscope slide
and covered by a buffer (Tris ± HCl or Glycine ± NaOH), did
not form vesicles. Mild sonication, or heating to 458C, did not
give any sign of formation of vesicles. However, to our
surprise, formation of giant vesicles started very rapidly when
The acyclic tetraether core lipid 5 (X ˆ PO3H2) was
synthesized from 11 in the same manner (Scheme 2).
Optical microscopy: ªGiantº vesicles, with a diameter of
10 mm or more, can be observed in situ, in water, with an
optical microscope working in the Nomarsky mode.[19] By
Chem. Eur. J. 2000, 6, No. 18
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3353

T. Eguchi, Y. Nakatani, G. Ourisson et al.
FULL PAPER
philic lipid 5 did not give giant
vesicles when pure, but only
after addition of phytanol.
The insertion of phytanol, phy-
tol, or geranylgeraniol (which
themselves, when pure, did not
form vesicles) into the mem-
brane of vesicles of 4 is of course
made possible by the nearly
identical lipophilic parts of these
molecules. The additives may
favor the formation of vesicles
mainly by reduction of the elec-
Figure 1. Phase contrast image (left) and fluorescent microscopic image (right) of giant vesicles from the 72-
membered macrocyclic dipolar phosphates 3ab (X ˆ PO3H2).
trostatic interactions between
the ionized headgroups, and
we treated with the same buffer a film of 4 premixed with a 2 ±
3 molar excess of phytanol (6), phytol (23) or geranylgeraniol
(24). These vesicles grew in size with time, apparently by
capture of further material from unorganized reservoirs as
also act as space-filling partners, facilitating the curvature of
the system to form vesicles. Two similar cases of vesicle
formation induced by additives have been reported.[7c, 21]
Synchrotron X-ray diffraction:
We and others[11] have shown
that the simplest phosphate 1
easily forms giant vesicles, how-
ever, the diffraction pattern
unexpectedly showed well-ex-
pressed hexagonal structures
over the entire temperature
range measured (from 58C to
878C), with a distance between
cylinders of 6.23 nm at 208C
and 5.76 nm at 878C. This ap-
parent contradiction, may be
due to two differences: the
synchrotron study was run at a
103 higher concentration of lip-
id, in pure water (pH 7) instead
shown in Figure 2. n-Hexadecanol (25) or cholesterol (26), of
similar polarity and dimensions as 6, 23, and 24 (as judged
from scale molecular models), but nevertheless of different
shapes, were also tested as additives, and found not to induce
vesicle formation. In addition, the regioisomeric bola-amphi-
of a pH 7.8 ± 8.4 buffer. This may be checked when we can
obtain beam time.
The diffraction pattern of phosphate 4 and water, without
added phytanol, showed only, by a small and broad reflection,
a lamellar structure with a repeat distance of 4.25 nm at 48C
and 4.18 at 508C, but with only
a few layers. This interlamellar
distance is compatible with the
thickness expected for a system
made of molecules such as 4,
fully extended.
Discussion
These studies suggest several
remarks. First in the context of
our hypothesis of the primitive-
ness of membranes of polyter-
penyl phosphates, our observa-
tions may have some signifi-
cance: assuming polyprenols to
Figure 2. a) Phase contrast image of a giant vesicle from the lipid 4/phytanol system (1/2 molar ratio). The vesicle
starts growing almost immediately from the lipid droplet at the center of the image. b) Same frame photographed
after one day, showing that the size of the vesicle has increased. The droplet at the center of the image appears to
be the source of the new material required for the growth of the vesicle. Bar: 10mm.
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Giant Vesicles
3351±3358
ˆ
bisphosphoric acid 3ab (X ˆ P( O)(OH)2) (27 mg, 72%) as a hygroscopic
be formed prebiotically, it would not be necessary to
phosphorylate them fully to induce vesicle formation, but
only to about 1/4 ± 1/3. It may even have been advantageous to
have had, at an early stage of evolution of Archña, an
incomplete conversion to the phosphates of the precursors of
the present complex membrane constituents. In fact, it would
appear warranted to study more critically the mixture of polar
lipids from an archñal organism, to check whether or not they
contain non-phosphorylated polyprenols alongside the highly
polar phospholipids already characterized. It may also be
possible that these mixtures contain phospholipids still with
simple phosphate headgroups, as we had postulated earlier for
ªprimitiveº microorganisms.[10]
The factors governing the spontaneous formation of
vesicles or cubic phases from archñal lipids and water, are
not clear,[7] and it seems that so far spontaneous vesicle
formation has only been described from the native mixture
(headgroups and chains) of polar lipids from Sulfolobus
acidocaldarius.[6] To the best of our knowledge, this is
therefore the first report of the formation of vesicles from a
defined stereochemically pure archñal polar lipid mixture,
which differs from the natural ones only by its phosphate
headgroups (instead of phosphocholines, for example).
Finally, it will also be interesting to refine the observation
made here of a very high selectivity of ªrecognitionº of the
nonpolar part of vesicle-forming phospholipids, and of
membrane-improving additives.
1
wax. H NMR (400 MHz, CDCl3/CD3OD ˆ 8:1): d ˆ 0.84 ± 0.89 (m, 48H),
1.00 ± 1.70 (m, 104H), 3.45 ± 3.70 (m, 14H), 3.99 (br, 4H); 13C NMR
(100 MHz, CDCl3/CD3OD ˆ 8:1): d ˆ 19.52, 19.63, 19.72, 24.27, 24.35,
29.59, 29.74, 32.63, 32.68, 32.91, 34.11, 36.47, 36.74, 37.26, 65.23, 68.81,
69.98, 70.25, 77.52 (d, J ˆ 7.4 Hz); 31P NMR (162 MHz, CDCl3/CD3OD ˆ
8:1): d ˆ 0.79; elemental analysis (%) calcd for C86H174O12P2: C 70.63, H
11.99; found: C 70.90, H 12.21.
(3R,7R,11R)-3,7,11,15-Tetramethylhexadecanal (7): To a stirred solution of
oxalyl chloride (29.0 mL, 2m in CH2Cl2, 58.0 mmol) in CH2Cl2 (40 mL) was
slowly added DMSO (5.50 mL, 77.5 mmol) at À788C. The mixture was
stirred for 35 min. Asolution of 6 (5.78 g, 19.4 mmol) in CH2Cl2 (44 mL)
was added dropwise over 5 min. The mixture was stirred for 20 min,
warmed to À258C, and stirring was continued for 2.5 h. The mixture was
cooled to À788C, and Et3N (25.0 mL, 179 mmol) was added dropwise over
5 min. The mixture was gradually warmed to room temperature, and a
saturated aqueous solution of NH4Cl was added. The mixture was extracted
with ethyl acetate. The organic phase was washed with brine, dried
(Na2SO4), filtered, and concentrated to dryness. The residue was chromato-
graphed over silica gel with hexane/EtOAc (7:1) to give aldehyde 7 (4.73 g,
82%) as an oil. [a]2D6 ‡8 (c 0.83, CHCl3); 1H NMR (400 MHz): d ˆ 0.85 (d,
J ˆ 6.6 Hz, 6H), 0.87 (d, J ˆ 6.8 Hz, 6H), 0.96 (d, J ˆ 6.6 Hz, 3H), 1.00 ± 1.43
(m, 20H), 1.52 (m, 1H), 2.05 (m, 1H), 2.22 (ddd, J ˆ 2.7, 7.8 and 16.1 Hz,
1H), 2.40 (ddd, J ˆ 2.0, 2.8 and 16.0 Hz, 1H), 9.76 (t, J ˆ 2.2 Hz, 1H);
13C NMR (100 MHz): d ˆ 19.72, 19.74, 20.02, 22.62, 22.71, 24.37, 24.44,
24.78, 27.97, 28.20, 32.73, 32.77, 37.06, 37.25, 37.26, 37.34, 37.41, 39.35, 51.07,
203.21; IR (neat): nÄ ˆ 1379, 1464, 1730, 2870, 2927, 2954 cmÀ1; elemental
analysis (%) calcd for C20H40O: C 81.01, H 13.60; found: C 80.91, H 13.72.
(4R)-4-Benzyloxymethyl-2-[(2R,6R,10R)-2,6,10,14-tetramethyl
penta-
decanyl]-1,3-dioxolane (9): Amixture of 7 (4.21 g, 14.2 mmol), 1-O-
benzyl-sn-glycerol (8)[15] (2.85 g, 15.6 mmol), toluene-p-sulfonic acid
(130 mg, 0.755 mmol), and MgSO4 (2.23 g, 18.5 mmol) in CH2Cl2 (70 mL)
was stirred for 4.5 h at room temperature. The mixture was filtered, EtOAc
and saturated aqueous NaHCO3 were added to the filtrate, and the organic
phase was separated. The aqueous phase was extracted with EtOAc. The
organic phase was washed with brine, dried (Na2SO4), filtered, and
concentrated to dryness. The residue was chromatographed over silica
gel with hexane/EtOAc (20:1) to give an oily 9 as a 7:3 mixture of
diastereomers (6.52 g, quant.). 1H NMR (300 MHz): d ˆ 0.83 ± 0.88 (m,
12H), 0.93 (d, J ˆ 6.6 Hz, 3H), 0.97 ± 1.74 (m, 25H), 3.41 ± 3.59 (m, 2H),
3.64 (dd, J ˆ 6.8 and 8.0 Hz, 0.3H), 3.79 (dd, J ˆ 5.1 and 8.3 Hz, 0.7H), 3.90
(dd, J ˆ 7.1 and 8.1 Hz, 0.7H), 4.12 (dd, J ˆ 6.8 and 8.3 Hz, 0.3H), 4.18 ± 4.30
(m, 1H), 4.57 (s, 0.6H), 4.58 (s, 1.4H), 4.94 (t, J ˆ 4.9 Hz, 0.7H), 5.03 (dd,
J ˆ 4.4 and 5.6 Hz, 0.3H), 7.25 ± 7.34 (m, 5H); 13C NMR (75 MHz): d ˆ
19.76, 19.90, 19.98, 22.62, 22.71, 24.18, 24.45, 24.78, 27.96, 29.27, 32.78, 37.26,
37.27, 37.37, 37.44, 37.63, 37.72, 39.36, 41.07, 41.19, 67.45, 70.53, 71.11, 73.48,
74.37, 74.64, 103.80, 104.35, 127.66, 127.71, 127.73, 128.39, 137.96; IR (neat):
Experimental Section
Synthesis: All reactions, except for catalytic hydrogenations, were carried
out in an inert (Ar or N2) atmosphere. IR spectra were taken on a Horiba
1
FT-710 Fourier transform infrared spectrometer. H NMR and 13C NMR
spectra were recorded on a JEOL LA-300 and/or a LA-400 spectrometers.
31P NMR spectra were recorded on a JEOL LA-400 spectrometer.
Deuteriochloroform (99.8 atom% enriched, Merck) was used as the
NMR solvent, unless otherwise indicated. 1H NMR and 13C NMR chemical
shifts are reported in d values based on internal TMS (dHˆ0), or solvent
signal (CDCl3 dCˆ77.0) as reference. Phosphoric acid was used (dPˆ0) as an
external standard for 31P NMR spectroscopy. Column chromatography was
carried out on Kieselgel 60 (70 ± 230 mesh or 230 ± 400 mesh, Merck). THF
was distilled from sodium/benzophenone ketyl prior to use. Triethylamine
was distilled from potassium hydroxide. DMSO, CH2Cl2, benzene, and
toluene were distilled from calcium hydride.
nÄ ˆ 698, 735, 758, 1030, 1103, 1128, 1377, 1462, 2868, 2925, 2952 cmÀ1
;
elemental analysis (%) calcd for C30H52O3: C 78.21, H 11.38; found: C 78.41,
H 11.67.
1-O-Benzyl-2-O-[(3R,7R,11R)-3,7,11,15-tetramethylhexadecanyl]-sn-glyc-
erol (10) and 1-O-benzyl-3-O-[(3R,7R,11R)-3,7,11,15-tetramethyl hexa-
decanyl]-sn-glycerol (11): Asolution of DIBAL-H (42.0 mL, 1 m in toluene,
42.0 mmol) was added to a solution of 9 (6.38 g, 13.8 mmol) in toluene
(30 mL) at 08C. The mixture was stirred for 5 min at the same temperature,
and then at room temperature for 16 h. The reaction was quenched by
addition of saturated aqueous NH4Cl at 08C. After 10 min, diehtyl ether
and HCl (2n) were added. The diethyl ether layer was separated and the
aqueous phase was extracted three times with ether. The organic phase was
washed with saturated aqueous NaHCO3 and brine, dried (Na2SO4),
filtered and concentrated to dryness. The residue was purified by flash
chromatography over silica gel with hexane/EtOAc (15:1 ± 7:1) to give a
more polar product 10 (3.08 g, 48%) and a less polar product 11 (3.19 g,
50%). Compound 10: [a]2D5 À8 (c ˆ 1.07, CHCl3); 1H NMR (400 MHz): d ˆ
0.84 (d, J ˆ 6.6 Hz, 6H), 0.86 (d, J ˆ 6.8 Hz, 6H), 0.87 (d, J ˆ 6.6 Hz, 3H),
1.01 ± 1.75 (m, 24H), 2.13 (br, 1H), 3.52 ± 3.75 (m, 7H), 4.54 (s, 2H), 7.25 ±
7.37 (m, 5H); 13C NMR (100 MHz): d ˆ 19.67, 19.73, 19.74, 22.60, 22.70,
24.33, 24.45, 24.77, 27.95, 29.81, 32.77, 37.05, 37.27, 37.33, 37.37, 37.43, 37.47,
39.35, 62.87, 68.67, 70.00, 73.50, 78.47, 127.61, 127.68, 128.39, 138.00; IR
(2S,7R,11R,15S,19S,22S,26S,30R,34R,38S,43R,47R,51S,55S,58S,62S,66R,
70R)-2,38-Bis(phosphoryloxymethyl)-7,11,15,19,22,26,30,34,43,47,51,55,58,
62,66,70-hexadecamethyl-1,4,37,40-tetraoxacyclodoheptacontane and (2S,
7R,11R,15S,19S,22S,26S,30R,34R,38S,43R,47R,51S,55S,58S,62S,66R,70R)-
2,39-bis(phosphoryloxymethyl)-7,11,15,19,22,26,30,34,43,47,51,55,58,62,66,
70-hexadecamethyl-1,4,37,40-tetraoxacyclodoheptacontane [3ab (X ˆ
PO3H2)]: Diphenylphosphoryl chloride (145 mL, 0.70 mmol) was added
dropwise to a mixture of 3ab (X ˆ H)[13] (35 mg, 27 mmol) and DMAP
(14 mg, 0.12 mmol) in pyridine (0.5 mL) and benzene (2 mL) at room
temperature, and the mixture was stirred at room temperature for 12 h.
Aqueous HCl (2m, 1 mL) was added and the mixture was extracted with
EtOAc. The organic layer was washed with saturated NaHCO3 and brine,
dried (Na2SO4), filtered, and concentrated to dryness. The residue was
chromatographed over silica gel with benzene/EtOAc (25:1) to give the
bisphosphotriester (46 mg, 95%) as an oil. Amixture of bisphosphotriester
(43 mg, 25 mmol) and PtO2 (18 mg) in acetic acid (6 mL) was stirred at
room temperature under a hydrogen atmosphere for 19 h. The catalyst was
filtered through a pad of Celite and washed with CHCl3/CH3OH (2:1 v/v).
The filtrate and washings were concentrated to dryness. The residue was
purified over SephadexꢁLH-20 with CHCl3/CH3OH (2:1 v/v) to give the
(neat): nÄ ˆ 698, 735, 820, 1099, 1377, 1462, 2868, 2924, 2954, 3454 cmÀ1
;
elemental analysis (%) calcd for C30H54O3: C 77.87, H 11.76; found: C 77.67,
Chem. Eur. J. 2000, 6, No. 18
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2000
0947-6539/00/0618-3355 $ 17.50+.50/0
3355

T. Eguchi, Y. Nakatani, G. Ourisson et al.
FULL PAPER
H 11.98. Compound 11: [a]D24 À0.4 (c ˆ 1.15, CHCl3); 1H NMR (400 MHz):
d ˆ 0.84 (d, J ˆ 6.6 Hz, 6H), 0.86 (d, J ˆ 6.6 Hz, 6H), 0.87 (d, J ˆ 6.6 Hz,
3H), 1.00 ± 1.80 (m, 24H), 2.48 (br, 1H), 3.44 ± 3.57 (m, 6H), 3.98 (m, 1H),
4.56 (s, 2H), 7.26 ± 7.37 (m, 5H); 13C NMR (100 MHz): d ˆ 19.70, 19.74,
19.76, 22.62, 22.71, 24.35, 24.46, 24.78, 27.97, 29.90, 32.79, 36.57, 37.28, 37.34,
37.39, 37.44, 37.49, 39.37, 69.53, 69.98, 71.40, 71.82, 73.44, 127.70, 128.41,
138.03; IR (neat): nÄ ˆ 698, 735, 750, 1115, 1377, 1462, 2868, 2925, 2952,
3448 cmÀ1; elemental analysis (%) calcd for C30H54O3: C 77.87, H 11.76;
found: C 77.76, H 12.06.
To
a
suspension of methyltriphenylphosphonium bromide (310 mg,
0.868 mmol) in THF (7 mL) was added nBuLi (550 mL, 1.50m in hexane,
0.825 mmol) at À788C, and the mixture was stirred at the same temper-
ature for 20 min and at room temperature for 20 min. The mixture was
recooled to À788C and a solution of aldehyde 14 (324 mg, 0.428 mmol) in
THF (5 mL) was added dropwise over 5 min. The mixture was stirred at
À788C for 15 min and at À258C for 1 h. Saturated aqueous NH4Cl solution
(2 mL) was added, and the mixture was extracted with EtOAc. The organic
phase was washed with brine, dried (Na2SO4), filtered, and concentrated to
dryness. The residue was chromatographed over silica gel with hexane/
EtOAc (50:1 ± 30:1) to give olefin 15 (324 mg, quant.) as an oil. [a]2D7 ‡5
(c ˆ 0.967, CHCl3); 1H NMR (400 MHz): d ˆ 0.83 ± 0.87 (m, 24H), 0.98 (d,
J ˆ 6.6 Hz, 3H), 1.00 ± 1.70 (m, 47H), 2.11 (m, 1H), 3.42 ± 3.66 (m, 9H), 4.55
(s, 2H), 4.89 (ddd, J ˆ 1.7, 2.0 and 10 Hz, 1H), 4.94 (ddd, J ˆ 1.2, 1.7 and
17 Hz, 1H), 5.69 (ddd, J ˆ 7.6, 10 and 17 Hz, 1H), 7.26 ± 7.34 (m, 5H);
13C NMR (100 MHz): d ˆ 19.69, 19.72, 19.75, 19.76, 20.23, 22.62, 22.72,
24.37, 24.49, 24.63, 24.79, 27.98, 29.83, 29.92, 32.76, 32.80, 32.82, 36.65, 36.99,
37.12, 37.30, 37.40, 37.47, 37.54, 37.75, 39.38, 68.89, 69.98, 70.35, 70.82, 73.36,
77.97, 112.19, 127.49, 127.58, 128.30, 138.46, 145.01; IR (neat): nÄ ˆ 696, 733,
908, 1115, 1377, 1462, 1641, 2866, 2925, 2952 cmÀ1; elemental analysis (%)
calcd for C51H94O3: C 81.10, H 12.54; found: C 81.24, H 12.79.
1-O-Benzyl-2-O-[(3R,7R,11R)-3,7,11,15-tetramethylhexadecanyl]-3-O-
[(3R,7R,11S,15S)-16-tert-butyldimethylsilyloxy-3,7,11,15-tetramethylhexa-
decanyl]-sn-glycerol (12): To prewashed NaH (71.7 mg, 1.79 mmol) in
DMSO (2 mL) was added a solution of 10 (261 mg, 0.564 mmol) in DMSO
(3.5 mL). The mixture was stirred for 30 min at room temperature and a
solution of the mesylate 17[17] (264 mg, 0.520 mmol) in DMSO (2.5 mL) was
added. The mixture was stirred at room temperature for 1 h and at 408C for
23 h. After cooling to 08C, water (5 mL) was added to the mixture. The
mixture was extracted with EtOAc. The organic phase was washed with
brine, dried (Na2SO4), filtered, and concentrated to dryness. The residue
was chromatographed over silica gel with hexane/EtOAc (20:1) to give 12
(385 mg, 85%) as an oil. [a]D28 ‡1.5 (c ˆ 1.77, CHCl3); 1H NMR (400 MHz):
d ˆ 0.04 (s, 6H), 0.84 (d, J ˆ 6.6 Hz, 12H), 0.87 (d, J ˆ 6.6 Hz, 15H), 0.89 (s,
9H), 1.00 ± 1.70 (m, 48H), 3.34 (dd, J ˆ 6.8 and 9.8 Hz, 1H), 3.43 ± 3.66 (m,
10H), 4.56 (s, 2H), 7.26 ± 7.34 (m, 5H); 13C NMR (100 MHz): d ˆ À5.35,
16.81, 18.35, 19.68, 19.72, 19.75, 22.63, 22.72, 24.36, 24.39, 24.47, 24.79, 25.96,
27.97, 29.81, 29.89, 32.76, 32.79, 32.80, 33.51, 35.76, 36.64, 37.09, 37.29, 37.40,
37.45, 37.53, 39.36, 68.42, 68.87, 69.97, 70.31, 70.78, 73.35, 77.94, 127.48, 127.57,
128.29, 138.42; IR (neat): nÄ ˆ 837, 1101, 1252, 1377, 1462, 2858, 2927,
2954 cmÀ1; elemental analysis (%) calcd for C56H108O4Si: C 77.00, H 12.46;
found: C 76.71, H 12.68.
3,3'-O-[(3R,7R,11S,15S,18S,22S,26R,30R)-3,7,11,15,18,22,26,30-Octame-
thyldotriacont-16-ene-1,32-diyl]-1,1'-di-O-benzyl-2,2'-di-O-[(3R,7R,11R)-
3,7,11,15-tetramethylhexadecanyl]-sn-diglycerol (16): Amixture of olefin
ˆ
15 (195 mg, 0.258 mmol) and [RuCl2( CHPh)(PCy3)2] (42.3 mg, 51.4 mmol)
in CH2Cl2 (5.1 mL) was refluxed for 33 h. After concentration of the
mixture, the resulting residue was purified by flash chromatography over
silica gel with hexane/EtOAc (30:1) to afford 16 (131 mg, 68%) and
unreacted 15 (51 mg, 26%). Compound 16: [a]2D7 ‡8 (c ˆ 0.515, CHCl3);
1H NMR (400 MHz): d ˆ 0.82 ± 0.87 (m, 48H), 0.94 (d, J ˆ 6.6 Hz, 6H),
1.00 ± 1.70 (m, 94H), 2.02 (br, 2H), 3.42 ± 3.66 (m, 18H), 4.55 (s, 4H), 5.17
(dd, J ˆ 2.2 and 4.6 Hz, 2H), 7.24 ± 7.34 (m, 10H); 13C NMR (100 MHz):
d ˆ 19.69, 19.71, 19.75, 19.77, 21.18, 22.63, 22.72, 24.36, 24.48, 24.51, 24.74,
24.79, 27.97, 29.82, 29.92, 32.76, 32.80, 32.81, 32.84, 36.65, 36.71, 37.11, 37.30,
37.40, 37.46, 37.54, 39.37, 68.88, 69.97, 70.34, 70.81, 73.36, 77.96, 127.48, 127.56,
128.29, 134.57, 138.44; IR (neat): nÄ ˆ 696, 733, 1115, 1377, 1462, 2866, 2925,
2952 cmÀ1; elemental analysis (%) calcd for C100H184O6: C 81.02, H 12.51;
found: C 80.99, H 12.77.
1-O-Benzyl-2-O-[(3R,7R,11R)-3,7,11,15-tetramethylhexadecanyl]-3-O-
[(3R,7R,11S,15S)-16-hydroxy-3,7,11,15-tetramethylhexadecanyl]-sn-glyc-
erol (13): Asolution of tetrabutylammonium fluoride (1.00 mL, 1 m in THF,
1.00 mmol) was added to a solution of 12 (385 mg, 0.440 mmol) in THF
(10 mL). The mixture was stirred for 8 h at room temperature and
concentrated in vacuo. The residue was chromatographed over silica gel
with hexane/EtOAc (7:1 ± 5:1) to give alcohol 13 (333 mg, quant.) as an oil.
[a]2D7 ‡0.5 (c ˆ 0.767, CHCl3); 1H NMR (400 MHz): d ˆ 0.83 ± 0.93 (m,
24H), 0.92 (d, J ˆ 6.8 Hz, 3H), 1.00 ± 1.70 (m, 48H), 3.39 ± 3.66 (m, 11H),
4.55 (s, 2H), 7.26 ± 7.34 (m, 5H); 13C NMR (100 MHz): d ˆ 16.63, 19.68,
19.72, 19.76, 22.62, 22.71, 24.36, 24.40, 24.44, 24.47, 24.79, 27.97, 29.83, 29.91,
32.76, 32.79, 32.80, 33.49, 35.78, 36.64, 37.11, 37.29, 37.30, 37.36, 37.38, 37.40,
37.46, 37.53, 39.37, 68.39, 68.89, 69.97, 70.34, 70.81, 73.36, 77.95, 127.48, 127.57,
3,3'-O-[(3R,7R,11S,15S,18S,22S,26R,30R)-3,7,11,15,18,22,26,30-Octame-
thyldotriacontane-1,32-diyl]-2,2'-di-O-[(3R,7R,11R)-3,7,11,15-tetrame-
thylhexadecanyl]-sn-diglycerol [4 (X ˆ H)]: Amixture of 16 (131 mg,
88.2 mmol) and 10% Pd-C (106 mg) in EtOAc (40 mL) was stirred for 18 h
under an atmospheric pressure of hydrogen at room temperature. The
mixture was filtered through a pad of Celite and washed with EtOAc. The
filtrate and washings were concentrated to dryness. The residue was
chromatographed over silica gel with hexane/EtOAc (7:1) to give 4 (X ˆ H)
(115 mg, quant.) as an oil. [a]2D8 ‡9 (c ˆ 0.743, CHCl3); 1H NMR
(400 MHz): d ˆ 0.84 ± 0.89 (m, 54H), 1.00 ± 1.70 (m, 100H), 2.21 (br, 2H),
3.45 ± 3.74 (m, 18H); 13C NMR (100 MHz): d ˆ 19.67, 19.69, 19.75, 22.62,
22.72, 24.36, 24.46, 24.79, 27.96, 29.83, 29.88, 32.79, 33.05, 34.32, 36.58, 37.07,
37.28, 37.35, 37.39, 37.42, 37.44, 37.49, 37.57, 39.36, 63.07, 68.63, 70.15, 70.94,
128.29, 138.43; IR (neat): nÄ ˆ 1113, 1377, 1462, 2868, 2925, 2952, 3423 cmÀ1
;
elemental analysis (%) calcd for C50H94O4: C 79.09, H 12.48; found: C
78.80, H 12.46.
1-O-Benzyl-2-O-[(3R,7R,11R)-3,7,11,15-tetramethylhexadecanyl]-3-O-
[(3R,7R,11S,15S)-15-formyl-3,7,11,15-tetramethylpentadecanyl]-sn-glycer-
ol (14): To a stirred solution of oxalyl chloride (1.00 mL, 2m in CH2Cl2,
2.00 mmol) in CH2Cl2 (6 mL) was slowly added DMSO (200 mL,
2.82 mmol) at À788C. The mixture was stirred for 30 min. Asolution of
13 (333 mg, 0.439 mmol) in CH2Cl2 (5 mL) was added dropwise over
10 min. The mixture was stirred for 25 min, warmed to À258C, and stirring
was continued for 4 h. The mixture was recooled to À788C, Et3N (1.00 mL,
7.17 mmol) was added dropwise, and the mixture was gradually warmed to
room temperature. Saturated aqueous NH4Cl solution (3 mL) was added,
and the mixture was extracted with EtOAc. The organic phase was washed
with brine, dried (Na2SO4), filtered, and concentrated to dryness. The
residue was chromatographed over silica gel with hexane/EtOAc (20:1) to
give aldehyde 14 (325 mg, 98%) as an oil. [a]2D7 ‡8 (c ˆ 0.667, CHCl3);
1H NMR (400 MHz): d ˆ 0.83 ± 0.87 (m, 24H), 1.09 (d, J ˆ 7.1 Hz, 3H),
1.00 ± 1.75 (m, 48H), 2.34 (m, 1H), 3.42 ± 3.66 (m, 9H), 4.55 (s, 2H), 7.26 ±
7.34 (m, 5H), 9.61 (d, J ˆ 2.2 Hz, 1H); 13C NMR (100 MHz): d ˆ 13.37,
19.65, 19.69, 19.72, 19.75, 19.77, 22.63, 22.72, 24.37, 24.42, 24.45, 24.48, 24.79,
27.98, 29.83, 29.92, 30.89, 32.66, 32.80, 32.82, 36.65, 37.01, 37.12, 37.30, 37.34,
37.39, 37.41, 37.47, 37.54, 39.38, 46.35, 68.89, 69.98, 70.35, 70.82, 73.36, 77.97,
127.49, 127.57, 128.30, 138.46, 205.36; IR (neat): nÄ ˆ 696, 735, 1115, 1377,
1462, 1730, 2860, 2925, 2952 cmÀ1; elemental analysis (%) calcd for
C50H92O4: C 79.30, H 12.25; found: C 79.02, H 11.98.
78.30; IR (neat): nÄ ˆ 1049, 1117, 1377, 1462, 2868, 2925, 2952, 3448 cmÀ1
;
elemental analysis (%) calcd for C86H174O6: C 79.19, H 13.45; found: C
79.13, H 13.71.
3,3'-O-[(3R,7R,11S,15S,18S,22S,26R,30R)-3,7,11,15,18,22,26,30-Octame-
thyldotriacontane-1,32-diyl]-2,2'-di-O-[(3R,7R,11R)-3,7,11,15-tetrame-
thylhexadecanyl]-sn-diglycero-1,1'-bisphosphoric acid [4 (X ˆ PO3H2)]:
Diphenylphosphoryl chloride (100 mL, 0.48 mmol) was added dropwise to
a
mixture of diol
4
(X ˆ H) (26 mg, 20 mmol) and DMAP (13 mg,
0.11 mmol) in pyridine (0.5 mL) and benzene (2 mL) at room temperature,
and the mixture was stirred at room temperature for 6 h. Aqueous HCl
(2m, 1 mL) was added and the mixture was extracted with EtOAc. The
organic layer was washed with saturated NaHCO3 and brine, dried
(Na2SO4), filtered, and concentrated to dryness. The residue was chromato-
graphed over silica gel with benzene/EtOAc (25:1) to give bisphospho-
triester (34 mg, 97%) as an oil. Amixture of bisphosphotriester (31 mg,
18 mmol) and PtO2 (17 mg) in acetic acid (5 mL) was stirred at room
temperature under a hydrogen atmosphere for 10 h. The catalyst was
filtered through a pad of Celite and washed with CHCl3/CH3OH (2:1 v/v).
The filtrate and washings were combined and concentrated to dryness. The
residue was purified over SephadexꢁLH-20 with CHCl3/CH3OH (2:1 v/v)
1-O-Benzyl-2-O-[(3R,7R,11R)-3,7,11,15-tetramethylhexadecanyl]-3-O-
[(3R,7R,11S,15S)-3,7,11,15-tetramethylheptadec-16-enyl]-sn-glycerol (15):
3356
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2000
0947-6539/00/0618-3356 $ 17.50+.50/0
Chem. Eur. J. 2000, 6, No. 18

Giant Vesicles
3351±3358
1H NMR (400 MHz): d ˆ 0.82 ± 0.87 (m, 48H), 0.94 (d, J ˆ 6.6 Hz, 6H),
1.00 ± 1.70 (m, 94H), 2.03 (br, 2H), 3.42 ± 3.66 (m, 18H), 4.55 (s, 4H), 5.18
(dd, J ˆ 2.2 and 4.6 Hz, 2H), 7.25 ± 7.33 (m, 10H); 13C NMR (100 MHz):
d ˆ 19.69, 19.72, 19.76, 19.77, 21.18, 22.63, 22.72, 24.38, 24.48, 24.52, 24.74,
24.80, 27.98, 29.84, 29.92, 32.77, 32.80, 32.82, 32.85, 36.66, 36.71, 37.12, 37.30,
37.39, 37.42, 37.44, 37.46, 37.54, 37.56, 39.38, 68.89, 69.97, 70.36, 70.81, 73.36,
77.98, 127.48, 127.57, 128.29, 134.58, 138.46; IR (neat): nÄ ˆ 696, 733, 1115,
1377, 1462, 2866, 2925, 2952 cmÀ1; elemental analysis (%) calcd for
C100H184O6: C 81.02, H 12.51; found: C 81.31, H 12.58.
ˆ
to give bisphosphoric acid
4
(X ˆ P( O)(OH)2) (17 mg, 64%) as
a
hygroscopic wax. 1H NMR (400 MHz, CDCl3/CD3OD (8:1)): d ˆ 0.84 ±
0.89 (m, 54H), 1.00 ± 1.70 (m, 100H), 3.45 ± 3.70 (m, 14H), 3.98 (br, 4H);
13C NMR (100 MHz, CDCl3/CD3OD ˆ 8:1): d ˆ 19.42, 19.45, 19.54, 19.63,
22.41, 22.51, 24.22, 24.33, 24.63, 27.80, 29.52, 29.65, 29.78, 32.67, 32.89, 34.10,
36.47, 36.75, 37.13, 37.26, 37.28, 37.31, 37.41, 39.51, 65.04 (d, J ˆ 5.8 Hz), 68.81,
70.01, 70.24, 77.56 (d, J ˆ 7.4 Hz); 31P NMR (162 MHz, CDCl3/CD3OD ˆ
8:1): d ˆ 2.64; elemental analysis (%) calcd for C86H176O12P2: C 70.54, H
12.11; found: C 70.83, H 12.21.
2,2'-O-[(3R,7R,11S,15S,18S,22S,26R,30R)-3,7,11,15,18,22,26,30-Octame-
thyldotriacontane-1,32-diyl]-3,3'-di-O-[(3R,7R,11R)-3,7,11,15-tetrame-
thylhexadecanyl]-sn-diglycerol [5 (X ˆ H)]: Compound 22 (100 mg,
0.0677 mmol) was treated in the same manner as described for the
preparation of 4 (X ˆ H) to give 5 (X ˆ H) (87 mg, 98%) as an oil. [a]2D7 ‡9
(c ˆ 0.533, CHCl3); 1H NMR (400 MHz): d ˆ 0.84 ± 0.89 (m, 54H), 1.00 ±
1.70 (m, 100H), 2.19 (t, J ˆ 6.1 Hz, 2H), 3.44 ± 3.75 (m, 18H); 13C NMR
(100 MHz): d ˆ 19.67, 19.70, 19.72, 19.75, 22.62, 22.71, 24.36, 24.47, 24.79,
27.97, 29.86, 29.89, 32.80, 33.06, 33.14, 34.33, 34.43, 36.59, 37.08, 37.29, 37.35,
37.38, 37.40, 37.44, 37.49, 37.52, 37.58, 39.37, 63.10, 68.65, 70.16, 70.95, 78.30;
IR (neat): nÄ ˆ 1049, 1115, 1377, 1462, 2868, 2925, 2952, 3458 cmÀ1; elemental
analysis (%) calcd for C86H174O6: C 79.19, H 13.45; found: C 79.18, H 13.61.
1-O-Benzyl-2-O-[(3R,7R,11S,15S)-16-tert-butyldimethylsilyloxy-3,7,11,15-
tetramethylhexadecanyl]-3-O-[(3R,7R,11R)-3,7,11,15-tetramethylhexade-
canyl]-sn-glycerol (18): Compound 11 (571 mg, 1.23 mmol) was treated in
the same manner as described for the preparation of 12 to give 18 (656 mg,
61%) as an oil. [a]2D8 ‡0.1 (c ˆ 0.887, CHCl3); 1H NMR (400 MHz): d ˆ 0.04
(s, 6H), 0.84 (d, J ˆ 6.6 Hz, 12H), 0.87 (d, J ˆ 6.6 Hz, 15H), 0.89 (s, 9H),
1.00 ± 1.70 (m, 48H), 3.34 (dd, J ˆ 6.8 and 9.8 Hz, 1H), 3.43 ± 3.66 (m, 10H),
4.56 (s, 2H), 7.26 ± 7.34 (m, 5H); 13C NMR (100 MHz): d ˆ À5.34, 16.82,
18.37, 19.69, 19.72, 19.76, 22.63, 22.72, 24.38, 24.41, 24.49, 24.80, 25.97, 27.98,
29.85, 29.92, 32.78, 32.80, 32.83, 33.54, 35.78, 36.66, 37.13, 37.30, 37.40, 37.43,
37.47, 37.54, 37.56, 39.38, 68.44, 68.91, 69.99, 70.38, 70.83, 73.37, 77.98, 127.49,
127.58, 128.30, 138.47; IR (neat): nÄ ˆ 775, 837, 1101, 1252, 1377, 1462, 2858,
2927, 2952 cmÀ1; elemental analysis (%) calcd for C56H108O4Si: C 77.00, H
12.46; found: C 76.74, H 12.76.
2,2'-O-[(3R,7R,11S,15S,18S,22S,26R,30R)-3,7,11,15,18,22,26,30-Octameth-
yldotriacontane-1,32-diyl]-3,3'-di-O-[(3R,7R,11R)-3,7,11,15-tetramethyl-
ˆ
hexadecanyl]-sn-diglycero-1,1'-bisphosphoric
acid
[5
(X ˆ P( O)-
1-O-Benzyl-2-O-[(3R,7R,11S,15S)-16-hydroxy-3,7,11,15-tetramethylhexa-
decanyl]-3-O-[(3R,7R,11R)-3,7,11,15-tetramethylhexadecanyl]-sn-glycer-
ol (19): Compound 18 (545 mg, 0.623 mmol) was treated in the same
manner as described for the preparation of 13 to give alcohol 19 (423 mg,
89%) as an oil. [a]2D7 ‡0.1 (c ˆ 1.01, CHCl3); 1H NMR (400 MHz): d ˆ
0.83 ± 0.87 (m, 24H), 0.92 (d, J ˆ 6.6 Hz, 3H), 1.00 ± 1.70 (m, 48H), 3.39 ±
3.66 (m, 11H), 4.55 (s, 2H), 7.26 ± 7.34 (m, 5H); 13C NMR (100 MHz): d ˆ
16.64, 19.69, 19.72, 19.77, 22.63, 22.72, 24.37, 24.40, 24.44, 24.48, 24.80, 27.98,
29.83, 29.91, 32.76, 32.80, 32.82, 33.50, 35.79, 36.65, 37.11, 37.30, 37.31, 37.39,
37.41, 37.46, 37.53, 37.55, 39.37, 68.40, 68.90, 69.98, 70.34, 70.81, 73.36, 77.96,
127.49, 127.58, 128.30, 138.44; IR (neat): nÄ ˆ 1113, 1377, 1462, 2868, 2925,
2952, 3444 cmÀ1; elemental analysis (%) calcd for C50H94O4: C 79.09, H
12.48; found: C 78.81, H 12.50.
(OH)2)]: Compound 5 (X ˆ H) (25 mg, 19 mmol) was treated in the same
manner as described for the preparation of 4 to give bisphosphotriester
(29 mg, 86%), part of which (25 mg, 14 mmol) was further treated to afford
ˆ
5
(X ˆ P( O)(OH)2) (9 mg, 43%) as
a
hygroscopic wax. 1H NMR
(400 MHz, CDCl3/CD3OD (2:1)): d ˆ 0.84 ± 0.89 (m, 54H), 1.00 ± 1.70 (m,
100H), 3.45 ± 3.70 (m, 14H), 3.98 (br, 4H); 13C NMR (100 MHz, CDCl3/
CD3OD (2:1)): d ˆ 19.22, 19.32, 19.38, 22.17, 22.27, 24.05, 24.13, 24.45, 27.63,
29.33, 29.44, 29.57, 32.47, 32.71, 33.94, 36.27, 36.58, 36.93, 37.09, 37.21, 39.03,
65.07 (br), 68.68, 69.83, 70.07, 77.30 (br); 31P NMR (162 MHz, CDCl3/
CD3OD ˆ 2:1): d ˆ 0.57; elemental analysis (%) calcd for C86H176O12P2: C
70.54, H 12.11; found: C 70.24, H 11.94.
Microscopy: The self-organization of the amphiphiles 1, 3ab, 4, and 5 in
water was studied by phase contrast and fluorescence microscopy. Giant
vesicles were prepared from the lipids following reported procedures.[2, 22]
The lipid (0.5 mg) was hydrated in Tris ± HCl buffer (3.2 mL, 0.05m) at
pH 7.8 or glycine ± NaOH buffer (0.05m) at pH 8.4 (conditions leading to
about 50% dianion). For lipid 4, a 2 ± 3 molar excess of neat phytanol (6),
phytol (23), or geranylgeraniol (24) was added to the lipid film and
hydrated as usual. Fluorescence microscopy was used to confirm vesicle
formation: the lipid film was prepared with a CHCl3/MeOH solution of
Nile Red and, after hydration of the film, the plate was observed in a
fluorescence microscope.
1-O-Benzyl-2-O-[(3R,7R,11S,15S)-15-formyl-3,7,11,15-tetramethylpenta-
decanyl]-3-O-[(3R,7R,11R)-3,7,11,15-tetramethylhexadecanyl]-sn-glycer-
ol (20): Compound 19 (371 mg, 0.488 mmol) was treated in the same
manner as described for 14 to give aldehyde 20 (299 mg, 80%) as an oil.
[a]2D8 ‡7 (c ˆ 0.880, CHCl3); 1H NMR (400 MHz): d ˆ 0.83 ± 0.87 (m, 24H),
1.09 (d, J ˆ 7.1 Hz, 3H), 1.00 ± 1.75 (m, 48H), 2.34 (ddd, J ˆ 1.7, 6.8 and
13.4 Hz, 1H), 3.42 ± 3.66 (m, 9H), 4.55 (s, 2H), 7.26 ± 7.34 (m, 5H), 9.61 (d,
J ˆ 2.0 Hz, 1H); 13C NMR (100 MHz): d ˆ 13.33, 19.63, 19.66, 19.70, 19.73,
22.60, 22.69, 24.34, 24.39, 24.42, 24.45, 24.77, 27.93, 29.79, 29.86, 30.85, 32.62,
32.76, 32.78, 36.61, 36.97, 37.07, 37.26, 37.31, 37.35, 37.37, 37.42, 37.49, 37.51,
39.34, 46.31, 68.84, 69.93, 70.29, 70.76, 73.31, 77.92, 127.45, 127.54, 128.26,
138.39, 205.29; IR (neat): nÄ ˆ 665, 696, 735, 1115, 1377, 1462, 1730, 2860,
2925, 2952 cmÀ1; elemental analysis (%) calcd for C50H92O4: C 79.30, H
12.25; found: C 79.34, H 12.23.
Phase contrast and fluorescence microscopies were carried out with an
inverted microscope (Axiovert 135, Carl Zeiss) equipped with a charge-
coupled device camera (C2400 ± 75i, Hamamatsu Photonics). The images
were stored and processed on a 7500/100 PowerMacintosh connected to an
image processor (Argus-20, Hamamatsu Photonics).
1-O-Benzyl-2-O-[(3R,7R,11S,15S)-3,7,11,15-tetramethylheptadec-16-en-
yl]-3-O-[(3R,7R,11R)-3,7,11,15-tetramethylhexadecanyl]-sn-glycerol (21):
Compound 20 (203 mg, 0.268 mmol) was treated in the same manner as
described for 15 to give olefin 21 (183 mg, 90%) as an oil. [a]2D8 ‡5 (c ˆ
0.750, CHCl3); 1H NMR (400 MHz): d ˆ 0.83 ± 0.87 (m, 24H), 0.98 (d, J ˆ
6.6, 3H), 1.00 ± 1.70 (m, 47H), 2.11 (m, 1H), 3.42 ± 3.66 (m, 9H), 4.55 (s,
2H), 4.89 (ddd, J ˆ 1.7, 2.0 and 10 Hz, 1H), 4.94 (ddd, J ˆ 1.2, 1.7 and 17 Hz,
1H), 5.69 (ddd, J ˆ 7.6, 10 and 17 Hz, 1H), 7.26 ± 7.34 (m, 5H); 13C NMR
(100 MHz): d ˆ 19.69, 19.72, 19.75, 19.76, 20.23, 22.63, 22.72, 24.37, 24.49,
24.63, 24.80, 27.98, 29.83, 29.92, 32.76, 32.80, 32.82, 36.65, 36.99, 37.12, 37.30,
37.40, 37.48, 37.54, 37.76, 39.38, 68.89, 69.98, 70.35, 70.82, 73.36, 77.97, 112.20,
127.49, 127.58, 128.30, 138.46, 145.01; IR (neat): nÄ ˆ 665, 696, 733, 908, 1115,
1377, 1462, 1639, 2866, 2925, 2952 cmÀ1; elemental analysis (%) calcd for
C51H94O3: C 81.10, H 12.54; found: C 80.80, H 12.77.
X-ray diffraction: The samples of lipids 1 or 4 (2 mg) were suspended in
Millipore ªpureº water (50 mL, pH 7.0) and transferred into thin-walled
glass tubes and mounted on a Peltier-controlled sample holder with X-ray
transparent windows. Diffraction patterns were recorded on the A2 double
focusing monochromator-mirror camera[23] at Hasylab, at the Deutsches
Elektronen Synchrotron (DESY) in Hamburg, on the storage ring Doris.
The wavelength was fixed at 0.15 nm. We used a data acquisition system
allowing simultaneous recording of reflections at different angular
regimes.[24, 25] Afast solenoid-driven lead shutter controlled by the data
acquisition system was used to prevent irradiation of the sample when no
diffraction data were collected. For further details on the X-ray diffraction
technique and data analysis, see refs.[26, 27]
Acknowledgements
2,2'-O-[(3R,7R,11S,15S,18S,22S,26R,30R)-3,7,11,15,18,22,26,30-Octame-
thyldotriacont-16-ene-1,32-diyl]-1,1'-di-O-benzyl-3,3'-di-O-[(3R,7R,11R)-
3,7,11,15-tetramethylhexadecanyl]-sn-diglycerol (22): Compound 21
(85.0 mg, 0.113 mmol) was treated in the same manner as described for
the preparation of 16 to give E-olefin 22 (51 mg, 61%) and unreacted 21
(30 mg, 36%) as an oil. Compound 22: [a]2D4 ‡9 (c ˆ 0.510, CHCl3);
This work was supported in part by the Japanese Ministry of Education
(Monbusho) and by COST. We are grateful for support granted by CNRS
to S.Ghosh. We thank Dr.O. Dannenmuller and Miss N. Huther for their
technical assistance.
Chem. Eur. J. 2000, 6, No. 18
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2000
0947-6539/00/0618-3357 $ 17.50+.50/0
3357

T. Eguchi, Y. Nakatani, G. Ourisson et al.
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Received: March 15, 2000 [F2368]
3358
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