Membrane properties of sodium 2- and 6-(poly)prenyl-substituted polyprenyl phosphates

Article abstract of DOI:10.1039/b101802g

The amphiphilic 2- and 6-(poly)prenyl-substituted polyprenyl phosphates and their isomers, spontaneously form vesicles. Vesicle formation depends on the pH of the buffer and the structural features of the hydrophobic chains such as their lengths and the double bond positions. The compactness of the vesicles was estimated by measuring their water permeability, evaluating the intramolecular attractive van der Waals forces between the highly branched chains and measuring the spin-lattice relaxation times in ¹³C NMR spectra.

Full text of DOI:10.1039/b101802g

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Membrane properties of sodium 2- and 6-(poly)prenyl-substituted
polyprenyl phosphates
Saho Takajo,a Hajime Nagano,*a Olivier Dannenmuller,b Sangita Ghosh,b
Anne Marie Albrecht,c Yoichi Nakatani*b and Guy Ourissonb
a Department of Chemistry, Faculty of Science, Ochanomizu University, Otsuka, Bunkyo-ku,
T okyo 112-8610, Japan. E-mail: nagano=cc.ocha.ac.jp; Fax: ]81-3-5978-5715
b L aboratoire de Chimie Organique des Substances Naturelles associe au CNRS, Centre de
Neurochimie, Universite L ouis Pasteur, 5 rue Blaise Pascal, F-67084 Strasbourg, France.
E-mail: nakatani=chimie.u-strasbg.fr; Fax: ]33 3 88 60 76 20
c Ecole Europeenne de Chimie, Polymeres et Materiaux, L aboratoire de Physico-Chimie
Bio-inorganique associe au CNRS, Institut de Chimie, 1 rue Blaise Pascal, F-67070 Strasbourg,
France
Received (in Montpellier, France) 26th February 2001, Accepted 11th April 2001
First published as an Advance Article on the web 19th June 2001
The amphiphilic 2- and 6-(poly)prenyl-substituted polyprenyl phosphates and their isomers, spontaneously form
vesicles. Vesicle formation depends on the pH of the bu†er and the structural features of the hydrophobic chains
such as their lengths and the double bond positions. The compactness of the vesicles was estimated by measuring
their water permeability, evaluating the intramolecular attractive van der Waals forces between the highly branched
chains and measuring the spinÈlattice relaxation times in 13C NMR spectra.
biomembranes or shorter9 (Fig. 2). We now report the pH
1
Introduction
dependence of the spontaneous formation of vesicles from
these amphiphilic molecules (observed by optical microscopy)
and their water permeability as an index of the compactness
of their membranes. We also estimated the intramolecular
attractive van der Waals forces between the hydrophobic
chains of the alcohols corresponding to these phosphates and
their mobility on the basis of 13C spin lattice relaxation time
measurements.
All known living organisms are cellular and the inside of each
cell is separated from the outside by a thin water-insoluble
membrane formed by the self-assembly of amphiphilic mol-
ecules. Except for Archaea, cell membranes of extant
organisms are built of ester phospholipids with n-acyl chains
(C ÈC ), or often, in bacteria, with branched chains.
However, polyprenyl phosphates could have been involved in
14 18
the prebiotic formation of the most primitive membranes.1,2
Terpenoids are ubiquitous and very varied in biological mem-
branes of all the living organisms, either as structural constitu-
ents (polyprenyl phospholipid ethers in Archaea) or as
mechanical reinforcers (hopanoids and carotenoids in Pro-
carya; sterols in bacteria).3 Furthermore, in contrast to n-acyl
chains, terpenoids, normally biosynthesized by a series of
enzymatic reactions, can be obtained under extremely simple
conditions such as acid-catalyzed reactions, from simple mol-
ecules.1,4
2
Materials and methods
2.1 Di†erential interference contrast microscopy, phase
contrast microscopy and image analysis
Inverted optical microscope (Axiovert 135, 63]/1.40 Plan
Achromat Oil DIC objective, ]2.5 insertion lens, Carl Zeiss),
light sources: Hg (HBO 100, bandpass 450È490 blue Ðlter,
bandpass 546È558 green Ðlter, Carl Zeiss) and halogen lamp
(12 V, 100 W, Carl Zeiss), charge-coupled device (CCD)
camera (C 2400-75 H, Hamamatsu Photonics), image pro-
cessor (Argus 20, Hamamatsu Photonics), S-VHS video
recorder (SVO-9500 MDP, Sony), Trinitron color video
monitor (PVM-1443 MD, Sony), microcomputer (Power
MacIntosh 7500/100, Apple), video printer (VP-1800 EPM,
Sony) and digital image recorder (Focus). Improvement of the
optical microscopy image quality is achieved by increasing the
ratio optical signal : background noise, by use of appropriate
softwares (M.I.H. Image 1.55 and Photoshop 4.0).
We have shown that monopolyprenyl and dipolyprenyl
phosphates (1 and 2) indeed form vesicles, provided the poly-
prenyl chains contain at least three prenyl units, as in 1 (n \ 1
or 2) and 2 (m ] n P 1).2 Furthermore, we have postulated
that the highly branched isoprenoid alkanes and alkenes 3
(C , C , C and C ), which are distributed widely and
20
25
30
35
abundantly in many sediments,5 may have been derived from
the corresponding polyprenylated polyprenyl amphiphiles 5
present in biomembranes in primitive organisms. The recent
isolation of the branched isoprenoid hydrocarbons 3 and of
their isomers 4 from diatomaceous algae6 also suggests that
the corresponding alcohols must still exist on Earth7,8 at least
as biosynthetic intermediates (Fig. 1).
Preparation and observation of vesicles. A typical procedure
was as follows. A phosphate (3 mg) was dissolved in 300 ll of
2 : 1 (v/v) chloroformÈmethanol. An aliquot (15 ll) of the solu-
tion was dropped on a coverglass (0.17 mm thick). After 10
min of drying at room temperature, the lamellar solid remain-
ing on the slide was brought into focus and 500 ll of a bu†er
at 25 ¡C were added (pH 3.1, 4.0 and 4.5: sodium acetateÈ
We have recently synthesized a series of 2- and 6-(poly)
prenyl-substituted polyprenyl phosphates and their isomers as
their sodium salts 6È22, possessing a hydrophobic portion of
about 20 A in length, one half of the thickness of all known
DOI: 10.1039/b101802g
New J. Chem., 2001, 25, 917È929
917
This journal is ( The Royal Society of Chemistry and the Centre National de la Recherche ScientiÐque 2001

View Article Online
Fig. 1 Structures of 1È5.
acetic acid. pH 7.0: Na HPO ÈNaH PO ; pH 7.86:
2
4
2
4
Tris É HCl pH 8.5 and 8.85: borate). Vesicles were observed to
grow from the edges of the solid and to detach after estab-
lishing their optimal size. Unilamellar and multilamellar vesi-
cles and tubules of various shapes and sizes ranging from 0.2
to 15 lm were observed. The vesicles moved at speeds of 0.15È
345 nm s~1. The membranes appear to be thick because of
the di†raction-limited resolution of the optical system
(interference contrast).
Fig. 2 Structures of synthesized 2- and 6-(poly)prenyl-substituted
polyprenyl phosphates 6È22.
mounted under liquid nitrogen in a Gatan 626 Cryoholder,
transferred to the electron microscope and observed at
[170 ¡C. Observations were made in a CM 12 Philips cryo-
transmission electron microscope operating at 100 kV. The
microscope was equipped with an additional anti-
contamination device. Images were not taken under low-dose
conditions, as pure lipid solutions are not as sensitive to beam
irradiation as proteins. Images were recorded on Kodak SO
163 Ðlm and developed under standard conditions.
2.2 Metal shadowing cryo-transmission electron microscopy
A typical procedure for the preparation and observation of
vesicles was as follows. A phosphate (5 mg) was dissolved in 1
ml of 2 : 1 (v/v) chloroformÈmethanol. After evaporation of
the solvents, the Ðlm was dried under vacuum (ca. 0.1 Torr)
overnight. The lipidic Ðlm was then hydrated by addition of 1
ml of a 100 lM sodium acetateÈacetic acid bu†er (pH 4.5) and
the vesicular suspension was sonicated for 2 min at room tem-
perature. 700 Mesh copper grids were dipped into the lipid
stock solution (5 mg ml~1) and plunged into liquid ethane
cooled with liquid nitrogen. Obtained at the temperature of
[100 ¡C and under high vacuum (pressure: ca. 10~8 Torr),
the digital prints of the micrometrically cut liposome prep-
aration result from a double metallic deposit of Pt/C Ðrst
(incidence angle 45¡; thickness 0.6È1.0 nm) and, thereafter, of
C (incidence angle 90¡; thickness 6È10 nm). Grids were Ðnally
2.3 Conformational analysis
The conformational analysis of 26È33 was performed using
the CONFLEX program on a CACheTM system. CACheTM is
a registered trademark of Oxford Molecular Inc.
2.4 NMR spectroscopy
1H NMR spectra were recorded on a JEOL GSX-270 (270
MHz) or GSX-400 (400 MHz) spectrometer with CDCl as
3
918
New J. Chem., 2001, 25, 917È929

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the solvent and tetramethylsilane as an internal standard. J
values are given in Hz. 13C NMR spectra were recorded on
Disodium 6-[(Z)-1,5-Dimethyl-4-hexenylidene]-(2E,8E)-
3,9,13-Trimethyl-2,8,12-tetradecatrienyl phosphate 14. Oil. 1H
NMR d 5.35 (1H, m, 2-H), 5.03 (3H, m, 3 ] CH2), 4.33 (2H, m,
the spectrometers operating at 67.8 or 100.4 MHz with CDCl
3
as the solvent and internal standard (d 77.00). A spectral width
1-H), 2.67 (2H, d, J 5.3, 1@-H), 1.98 (12H, m, 6 ] CH CH2),
2
of 5 kHz using 33 K data points was applied to the 13C mea-
1.66 (3H, s, Me), 1.64 (6H, s, 2 ] Me), 1.61 (6H, s, 2 ] Me),
surements of carbonÈcarbon vicinal spin coupling constants.
1.58 (3H, s, Me) and 1.56 (3H, s, Me) 13C NMR d 140.61,
134.40, 131.90, 131.00, 128.98, 124.55, 124.28, 123.59, 120.71,
62.08, 39.88, 38.64, 34.72, 31.60, 30.88, 27.28, 26.82, 25.76,
18.12, 17.74 and 16.20. 31P NMR d 3.47 (br s). Negative
FAB-MS (glycerol) m/z 437 [M [ (2 ] Na`) ] H`, 100%)].
Found: m/z 437.2793 [M [ (2 ] Na`) ] H`]. Calc. for
The measurements of spinÈlattice relaxation times T were
1
performed using proton-decoupled 13C spectra by the Fourier
transform technique on the spectrometers and
a pulse
sequence (pÈqÈ%) at 25 ¡C. 31P NMR spectra with complete
proton decoupling were recorded on the JEOL GSX-270
spectrometer operating at 109.4 MHz in CDCl (external
C
H
O P: 437.2820.
3
standard: phosphoric acid in D O). Mass spectra (EI, 70 eV
2
25 42
4
and negative FAB) were obtained on a JEOL JMS-700 mass
6-[(Z)-1,5-Dimethyl-4-hexenylidene]-(2E,8E,12E)-3,9,13,17-
spectrometer. Accurate mass measurements were made on the
mass spectrometer.
tetramethyl-2,8,12,16-octadecatetraen-1-ol 61. Oil. 1H NMR d
5.41 (1H, t, J 6.9, 2-H), 5.10 (3H, m, 3 ] CH2), 5.03 (1H, t-like,
J 7.3, CH2), 4.15 (2H, d, J 6.0, 1-H), 2.73 (2H, d, J 7.3, 1@-H),
2.04 (16H, m, 8 ] CH CH2), 1.69 (3H, s, Me), 1.68 (6H, s,
2
2.5 Syntheses
2 ] Me), 1.65 (6H, s, 2 ] Me) and 1.60 (9H, s, 3 ] Me). 13C
NMR d 140.17, 134.81, 134.71, 131.85, 131.20, 131.13, 129.06,
124.42, 124.30, 124.08, 123.49, 123.00, 59.43, 39.87, 39.77, 38.41,
34.60, 31.16, 30.82, 27.26, 26.83, 26.71, 25.78, 25.75, 18.19,
17.75, 17.70, 16.42, 16.24 and 16.09. MS (EI) m/z 426 (M`,
Triethyl phosphonoacetate-1-13C and triethyl phospho-
noacetate-2-13C were purchased from Aldrich. Precoated
Merck Kieselgel 60 F254 and Wakogel C-300 were used for
thin layer chromatography (TLC) and Ñash column chroma-
tography, respectively. Synthesis of 13C-labeled compounds 44
and 54 was performed according to Schemes 1 and 2.
5%), 408 (M` [ H O, 4) , 339 (9), 271 (12), 203 (22), 147 (22),
2
109 (26), 81 (42) and 69 (100). Found: m/z 426.3906 (M`).
Calc. for C
H
O: 426.3862.
30 50
Disodium (2E)-3,7-dimethyl-2-(3-methyl-2-butenyl)-2,6-
octadienyl phosphate 6. Oil. 1H NMR d 5.08 (1H, m, CH2),
5.01 (1H, t, J 6.4, CH2), 4.31 (2H, d, J 5.0, 1-H), 2.80 (2H, d, J
Disodium 6-[(Z)-1,5-dimethyl-4-hexenylidene]-(2E,8E,12E)-
3,9,13,17-tetramethyl-2,8,12,16-octadeca tetraenyl phosphate
16. Oil. 1H NMR d 5.37 (1H, br t, J 6.3, 2-H), 5.08 (2H, m,
2 ] CH2), 4.98 (2H, m, 2 ] CH2), 4.35 (2H, br s, 1-H), 2.68
6.4, 1@-H), 1.99 (4H, m, 2 ] CH CH2), 1.71 (3H, s, Me), 1.66
2
(3H, s, Me), 1.62 (3H, s, Me) and 1.58 (3H, s, Me). 13C NMR d
(2H, d, J 4.0, 1@-H), 1.99 (16H, m, 8 ] CH CH2), 1.66 (6H, s,
2
135.42, 131.26, 129.19, 124.55, 123.30, 64.86, 34.90, 29.74, 29.13,
26.83, 25.69, 18.03, 17.84 and 17.59. 31P NMR d 5.13 (s). Nega-
tive FAB-MS (glycerol): m/z 301 [M [ (2 ] Na`) ] H`,
100%]. Found: m/z 301.1596 [M [ (2 ] Na`) ] H`]. Calc.
2 ] Me), 1.65 (3H, s, Me), 1.62 (3H, s, Me), 1.61 (3H, s, Me),
1.58 (3H, s, 2 ] Me) and 1.57 (3H, s, Me). 13C NMR d 140.96,
134.82, 134.57, 131.98, 131.10, 129.15, 124.63, 124.43, 124.23,
123.60, 120.65, 62.17, 39.86, 39.73, 38.56, 34.64, 31.50, 30.77,
27.20, 26.78, 25.67, 18.02, 17.64, 17.59, 16.21, 16.13 and 15.97.
31P NMR d 4.29 (br s).Negative FAB-MS (glycerol) m/z 505
[M [ (2 ] Na`) ] H`, 77%] and 183 (100). Found: m/z
for C
H
O P: 301.1568.
15 26
4
2-[(E)-1,5-Dimethyl-4-hexenylidene]-(4E,8E)-5,9,13-
trimethyl-4,8,12-tetradecatrien-1-ol 56. Oil. IR (neat) 3327 and
997 cm~1. 1H NMR d 5.09 (4H, m, 4 ] CH2), 4.09 (2H, s, J
505.3425 [M [ (2 ] Na`) ] H`]. Calc. for
505.3447.
C
H
O P:
30 50
4
4.9, 1-H), 2.87 (2H, d,
J 7.3, 1@-H), 2.08 (12H, m, 6
] CH CH2), 1.76 (3H, s, Me), 1.68 (9H, s, 3 ] Me), 1.61 (3H,
Ethyl [2-13C]-(2E/Z,6E)-3,7,11-trimethyl-2,6,10-dodeca-
trienoates 35. A solution of triethyl phosphonoacetate-2-13C
(99 atom% 13C, 90 mg, 0.40 mmol) in dry THF (0.5 ml) cooled
to 0 ¡C was added dropwise to a suspension of sodium
hydride (60% in mineral oil, 32 mg, 0.80 mmol) in dry THF
(2.0 ml) under nitrogen. The mixture was stirred at room tem-
perature for 1 h and cooled to 0 ¡C. Geranylacetone 34 (155
mg, 0.80 mmol) was added dropwise and the mixture stirred at
this temperature for 3 h. Water was added and the aqueous
solution extracted three times with diethyl ether. The com-
bined ether solutions were washed with water and saturated
brine, and dried over anhydrous sodium sulfate. Evaporation
gave a crude product which was chromatographed on silica
gel (5.3 g; hexaneÈdiethyl ether, 40 : 1) to give the a,b-unsatu-
rated ester 35 as a mixture of diastereomers (115 mg, 89%,
2
s, Me), 1.59 (6H, s, 2 ] Me) and 1.15 (1H, br t, J 4.9, OH). MS
(EI) m/z 358 (M`, 14%), 340 (M`ÈH O, 24), 297 (28), 271 (23),
2
221 (21), 203 (71), 161 (52), 147 (67), 81 (66) and 69 (100).
Found: m/z 358.3205 (M`). Calc. for C
H
O: 358.3235.
25 42
2-[(E)-1,5-Dimethyl-4-hexenylidene]-(4E)-5,9-dimethyl-4,8-
decadien-1-ol 57. Oil. IR (neat) 3329 and 998 cm~1. 1H NMR
d 5.11È5.02 (3H, m, 3 ] CH2), 4.08 (2H, s, J 5.0, 1-H), 2.85
(2H, d, J 7.3, 1@-H), 2.06 (8H, m, 4 ] CH CH2), 1.75 (3H, s,
2
Me), 1.67 (9H, s, 3 ] Me), 1.59 (3H, s, Me), 1.58 (3H, s, Me)
and 1.24 (1H, br s, OH). MS (EI) m/z 290 (M`, 15%), 272
(M`ÈH O, 11), 259 (M`ÈCH OH, 16), 229 (18), 221 (25), 203
2
2
(65), 161 (34), 147 (63), 109 (55), 81 (49) and 69 (100). Found:
m/z 290.2645 (M`). Calc. for C
H
O: 290.2610.
2E : 2Z \ 4.4 : 1) as an oil. 35E: 1H NMR d 5.66 (1H, d, 1J
20 34
CH
159, 13CH2), 5.08 (2H, m, 2 ] 2CH), 4.14 (2H, q, J 7.1,
6-[(Z)-1,5-Dimethyl-4-hexenylidene]-(2E,8E)-3,9,13-
CH CH ), 2.16 (7H, m, Me and 2 ] 2CCH ), 2.00 (4H, m,
2
3
2
trimethyl-2,8,12-tetradecatrien-1-ol 60. Oil. 1H NMR d 5.41
(1H, t-like, J 6.9, 2-H), 5.11 (2H, m, 2 ] CH2), 5.02 (1H, t-like,
J 7.5, CH2), 4.14 (2H, d, J 6.9, 1-H), 2.73 (2H, d, J 7.3, 1@-H),
2 ] 2CCH ), 1.68 (3H, s, Me), 1.60 (6H, s, 2 ] Me) and 1.28
2
(3H, t, J 7.1, CH CH ); 13C NMR d 115.90 (s, 13C-enriched
2
3
2CH); MS (EI) m/z 265 (M`, 20%), 220 (M` [ OEt, 28), 129
2.04 (12H, m, 6 ] CH CH2), 1.69 (3H, s, Me), 1.68 (3H, s, Me),
(93) and 69 (100); Found m/z 265.2079 (M`); Calc. for
2
1.67 (3H, s, Me), 1.66 (6H, s, 2 ] Me), 1.60 (3H, s, Me), 1.59
C 13CH O 265.2123. 35Z: 1H NMR d 5.65 (1H, dq, J 159,
16
28
2
(3H, s, Me) and 1.18 (1H, br s, OH). MS (EI) m/z 358 (M`,
1.2, 13CHCO ), 5.09 (2H, m, 2 ] 2CH), 4.14 (2H, q, J 7.2,
2
24%), 340 (M`ÈH O), 289 (58), 271 (34), 203 (56), 189 (34), 161
CH CH ), 2.65 (2H, td, J 7.8, 4.5, 2CCH ), 2.16 (2H, m,
2
2
3
2
(38), 147 (57), 109 (62), 93 (58), 81 (59) and 69 (100). Found:
2CCH ), 2.06 (2H, q, J 7.3, 2CCH ), 1.98 (2H, t, J 7.3,
2
2
m/z 358.3198 (M`). Calc. for C
H
O: 358.3235.
2CCH ), 1.89 (3H, dd, J 6.1, 1.2, Me), 1.68 (3H, s, Me), 1.60
25 42
2
New J. Chem., 2001, 25, 917È929
919

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(6H, s, 2 ] Me) and 1.27 (3H, t, J 7.3, CH CH ); 13C NMR d
enriched CH2O) and 122.21 (d, 3J 0.62, 13C-enriched CH2);
2
3
CC
116.23 (s, 13C-enriched 2CH).
1H non-decoupled 13C NMR d 191.32 (d, J
172, 13C-
CH
enriched CH2O) and 122.21 (dt, J 153, 4.6, 13C-enriched
CH
[2-13C]-(2E,6E)-3,7,11-Trimethyl-2,6,10-dodecatrien-1-ol
36. To a solution of the ester 35 (E major; 105 mg, 0.40 mmol)
in anhydrous diethyl ether (9.9 ml) was added a solution of
diisobutylaluminium hydride in hexane (0.95 M; 1.3 ml, 1.2
mmol) at 0 ¡C and the solution stirred at this temperature for
50 min. An aqueous solution of sodium hydroxide (10%) and
diethyl ether were added. The organic layer was washed suc-
cessively with aqueous NaOH (10%), water (until neutral) and
brine, and dried over anhydrous sodium sulfate. Chromatog-
raphy on silica gel (10 g; hexaneÈdiethyl ether, 4 : 1) gave the
alcohol 36 (82 mg, 92%) as an oil.
CH2); MS (EI) m/z 426 (M`, 2), 408 (M` [ H O, 1), 81 (43)
2
and 69 (100); Found m/z 426.3813; Calc. for C 13C H
O
28
2 48
426.3772.
43: 1H NMR d 10.04 (1H, d J 171, 13CH2O), 5.09 (4H,
CH
m, 4 ] CH2), 5.07 (1H, d, J 154, 13CH2), 2.98 (2H, m, 1@-H),
CH
2.60 (2H, t, J 7.5, CH ), 2.29 (2H, q, J 7.5, CH ), 2.05 (12H, m,
2
2
6 ] CH ), 1.97 (3H, s, Me), 1.68 (9H, s, 3 ] Me), 1.60 (9H, s,
2
3 ] Me) and 1.58 (3H, s, Me); 1H decoupled 13C NMR d
190.37 (d, 3J 0.61, 13C-enriched CH2O) and 121.62 (d, 3J
CC
CC
0.61, 13C-enriched CH2); 1H non-decoupled 13C NMR d
190.37 (d, J 171, 13C-enriched CH2O) and 121.62 (dt, J
CH
CH
154, 4.6, 13C-enriched CH2); MS (EI) m/z 426 (M`, 2), 408
[2-13C]-(2E,6E)-1-Bromo-3,7,11-trimethyl-2,6,10-dodeca-
triene 37. To a solution of the alcohol 36 (82 mg, 0.37 mmol)
in anhydrous diethyl ether (1.2 ml) was added pyridine (9.8
mg, 0.12 mmol) under nitrogen. The mixture was cooled to
0 ¡C and phosphorus tribromide (33 mg, 0.12 mmol) added
dropwise. After 20 min, saturated aqueous sodium hydro-
gencarbonate was added, and the crude product extracted
with diethyl ether. The organic layer was washed with saturat-
ed aqueous sodium hydrogencarbonate, water and brine, and
the solvent evaporated to give the bromide 37 (88 mg, 84%
yield) as a pale yellow oil. The bromide (E major) was used for
the following reaction without further puriÐcation.
(M` [ H O, 1), 139 (14), 109 (11), 81 (21) and 69 (100); Found
2
m/z 426.3797; Calc. for C 13C H O 426.3772.
28
2 48
[1, 4-13C ]-(4E,8E)-5,9,13-Trimethyl-2-[(E)-1,5,9-
2
trimethyl-4,8-decadienylidene]-4,8,12-tetradecatrien-1-ol 44
and [1, 4-13C ]-(4E,8E)-5,9,13-trimethyl-2-[(Z)-1,5,9,13-
2
tetramethyl-4,8,12-tetradecatrienylidene]-4,8,12-tetradeca-
trien-1-ol 45. Following the procedures described previously,9a
the aldehydes 42 and 43 were reduced with diisobutylalumin-
ium hydride to give the alcohols 44 and 45, respectively.
44: 1H NMR d 5.11 (2H, m, 2 ] CH2), 5.09 (2H, m,
2 ] CH2), 5.07 (1H, dt, 1J 152, 6.8, 13CH2), 4.09 (2H, d,
CH
1J 142, 13CH OH), 2.88 (2H, dd, 2J 5.6, 3J 6.8, 1@-CH ),
Ethyl [1-13C]-(2E/Z,6E)-3,7,11-Trimethyl-2,6,10-dodeca-
trienoates 38. Following the procedures described for the
preparation of 35, the compounds were prepared from triethyl
phosphonoacetate-1-13C (99 atom% 13C; 90 mg, 0.4 mmol)
and geranylacetone 34 (116 mg, 0.8 mmol) in 57% yield and in
a ratio of E : Z \ 7 : 1. 38E: 13C NMR d 166.90 (s, 13C-
CH
2
CH
HH
2
2.09 (4H, m, 2 ] CH ), 2.08 (4H, m, 2 ] CH ), 2.04 (4H, m,
2
2
2 ] CH ), 1.99 (4H, m, 2 ] CH ), 1.77 (3H, s, Me), 1.68 (9H, s,
2
2
3 ] Me) and 1.60 (12H, s, 4 ] Me); 1H decoupled 13C NMR
d 122.92 (d, 3J 0.77, 13C-enriched CH2) and 62.51 (d, 3J
CC
CC
0.77, 13C-enriched CH OH); 1H non-decoupled 13C NMR d
2
122.92 (d, 1J 152, 13C-enriched CH2) and 62.51 (d, 1J 142,
enriched C2O). 38Z: 13C NMR (CDCl ) d 166.35 (s, 13C-
CH
CH
13C-enriched CH OH); MS (EI) m/z 428 (M`, 24), 410 (M`
3
enriched C2O).
2
[ H O, 13), 273 (64), 149 (45), 137 (59), 122 (53), 109 (60), 81
2
(85) and 69 (100); Found: m/z 428.3948; Calc. for
[1-13C]-(2E,6E)-3,7,11-Trimethyl-2,6,10-dodecatrien-1-ol
39. Following the procedures described for the preparation of
36, the compound (E major) was prepared from 38 in 86%
yield.
C
13C H O 428.3929.
28
2
50
45: 1H NMR d 5.15 (1H, t-like, J 7.6, CH2), 5.09 (3H, m,
3 ] CH2), 5.07 (1H, dt, 1J 151, 6.6, 13CH2), 4.06 (2H, dd,
CH
1J 142, 3J 5.3, 13CH OH), 2.86 (2H, dd, 2J 5.6, 3J
CH
HH
2
CH
HH
6.6, 1@-CH ), 2.04 (16H, m, 8 ] CH ), 1.73 (3H, s, Me), 1.68
N-{ [1-13C]-(2E,6E)-3,7,11-Trimethyl-2,6,10-dodeca-
2
2
(9H, s, 3 ] Me) and 1.60 (12H, s, 4 ] Me); 1H decoupled 13C
trienylidene}cyclohexylamine 41. A mixture of activated MnO
NMR d 122.53 (d, 3J 0.70, 13C-enriched CH2) and 61.99 (d,
2
(332 mg) and the alcohol 39 (41 mg, 0.18 mmol) in hexane (2.3
CC
3J
0.70, 13C-enriched CH OH); 1H non-decoupled 13C
ml) was stirred at room temperature for 3 h. After Ðltration,
the solvent was evaporated to give the corresponding alde-
hyde (30 mg, 73% yield) as a pale yellow oil. To a solution of
the aldehyde (41 mg, 0.13 mmol) in benzene (0.8 ml) cooled to
0 ¡C were added cyclohexylamine (28 mg, 0.28 mmol) and pot-
assium carbonate (6 mg, 0.04 mmol). The mixture was stirred
at 0 ¡C for 1 h and then at room temperature for 1 h. After
Ðltration the solvent was evaporated to give quantitatively the
imine 41.
CC
2
NMR d 122.53 (d, 1J 151, 13C-enriched CH2) and 62.00 (d,
CH
1J 142, 13C-enriched CH OH); MS (EI) m/z 428 (M`, 11),
CH
2
410 (M` [ H O, 5) and 69 (100); Found m/z 428.3929; Calc.
2
for C 13C H O 426.3929.
28
2
50
Ethyl (4E)-2-{ [1-13C]acetyl}-5,9-dimethyl-4,8-decadienoate
47. To a suspension of sodium hydride (60% in mineral oil; 51
mg, 1.27 mmol) in anhydrous THF (9.3 ml) cooled to 0 ¡C was
added a solution of ethyl [3-13C]acetoacetate (99 atom% 13C,
849 mg, 3.91 mmol) in anhydrous THF (1.0 ml) under argon.
The mixture was stirred at room temperature for 30 min and
then cooled to 0 ¡C. A solution of geranyl bromide (0.85 g, 3.9
mmol) in anhydrous THF (6 ml) was added and the mixture
stirred at room temperature for 3 h. Quenching with water
and the usual work-up followed by Ñash chromatography on
silica gel gave 47 (163 mg, 62% yield) as an oil and ethyl (4E)-
2-([1-13C]acetyl)-2-[(2E)-3,7-dimethyl-6-octenyl]-5,9-dimethyl-
4,8-decadienoate (oil; 97 mg, 33% yield). Spectral data for 47:
1H NMR d 5.05 (1H, m, CH2), 5.04 (1H, m, CH2), 4.18 (2H, q,
[1, 4-13C ]-(4E,8E)-5,9,13-Trimethyl-2-[(E)-1,5,9-
2
trimethyl-4,8-decadienylidene]-4,8,12-tetradecatrienal 42 and
[1,4-13C ]-(4E,8E)-5,9,13-trimethyl-2-[(Z)-1,5,9,13-tetra-
2
methyl-4,8,12-tetradecatrienylidene]-4,8,12-tetradecatrienal
43. Following the procedures reported previously,9a the imine
41 (0.13 mmol) was treated with lithium diisopropylamide and
then with the bromide 37 (88 mg, 0.31 mmol) to give 42 (5.6
mg, 10% yield; including c-substituted isomer) and 43 (43 mg,
76% yield). Acid-catalyzed isomerization of 43 gave 42.9c
42: 1H NMR d 10.15 (1H, d J 172, 13CH2O), 5.08 (4H,
CH
m, 4 ] CH2), 4.92 (1H, dt, J 153, 5.8, 13CH2), 2.99 (2H, d, J
J
7.0, OCH CH ), 3.44 (1H, dq, 3J
7.5, 2J
5.8,
6.1,
CH
2
3
HH
CH
CH
4.6, 1@-H), 2.28 (2H, m, CH ), 2.19 (2H, m, CH ), 2.19 (3H, s,
13C(2O)CH), 2.55 (2H, m, CH ), 2.22 (3H, d, 2J
2
2
2
Me), 2.05 (6H, m, 3 ] CH ), 1.97 (6H, m, 3 ] CH ), 1.68 (9H,
13C(2O)Me), 2.04 (2H, q, J 7.3, CH ), 1.97 (2H, t, J 7.3, CH ),
2
2
2
2
s, 3 ] Me), 1.61 (3H, s, Me), 1.60 (6H, s, 2 ] Me) and 1.57 (3H,
1.67 (3H, s, Me), 1.63 (3H, s, Me), 1.59 (3H, s, Me) and 1.27
s, Me); 1H decoupled 13C NMR d 191.32 (d, 3J 0.62, 13C-
(3H, t, J 7.0, Me); 13C NMR d 203.18 (s, 13C-enriched C2O).
CC
920
New J. Chem., 2001, 25, 917È929

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[2-13C]-(5E)-6,10-Dimethyl-5,9-undecadien-2-one 48. To a
solution of the b-keto ester 47 (163 mg, 0.61 mmol) in ethanol
(9 ml) was added an aqueous solution of sodium hydroxide
(1.6 M; 16 ml). The mixture was stirred at 50 ¡C for 2 h and
then acidiÐed with acetic acid. After evaporation of the solvent
the residue was extracted with diethyl ether. The crude
product was chromatographed on silica gel (30 g; hexaneÈ
ethyl acetate, 20 : 1) to give the methyl ketone 48 (96 mg,
81%), an oil. 1H NMR d 5.08 (2H, m, 2 ] CH2), 2.46 (2H, dt,
3J 7.3, 2J 5.5, 13C(2O)CH ), 2.27 (2H, m, CH ), 2.14 (3H,
and 69 (100); Found m/z 428.3959 (M`); Calc. for
C
13C H O 428.3929.
50
55: 1H NMR d 5.11 (2H, m, 2 ] CH2), 5.09 (2H, m,
28
2
2 ] CH2), 5.07 (1H, m, CH2), 4.07 (2H, d, 3J 5.3, 2J 3.3,
HH CH
1-H), 2.85 (2H, dd, 3J 6.3, 2J 5.9, 1@-H), 2.04È1.97 (16H, m,
HH CH
8 ] CH ), 1.72 (3H, d, 3J 5.0, Me), 1.68 (9H, s, 3 ] Me) and
2
CH
1.60 (12H, s, 4 ] Me); 1H-decoupled 13C NMR d 135.73 (d,
3J 2.8, 13C-enriched 3@-C) and 132.26 (d, 3J 2.8, 13C-
CC
CC
enriched 2-C); MS (EI) m/z 428 (M`, 32%), 410 (M` [ H O,
2
12), 273 (39), 137 (40), 123 (37), 109 (36), 81 (58) and 69 (100);
HH
CH
2
2
d, 2J 5.8, 13C(2O)Me), 2.05 (2H, m, CH ), 1.97 (2H, m, CH ),
Found m/z 428.3929 (M`); Calc. for C 13C H O 428.3929.
CH
2
2
28
2 50
1.68 (3H, s, Me), 1.61 (3H, s, Me) and 1.60 (3H, s, Me). 13C
NMR d 208.94 (s, 13C-enriched C2O).
3
Results and discussion
Ethyl [3-13C]-(2E,6E)-3,7,11-Trimethyl-2,6,10-dodeca-
trienoate 49. Following the procedures described for the prep-
aration of 35, the a,b-unsaturated ester 49 was prepared from
triethyl phosphonoacetate (1.56 g, 6.98 mmol) and the ketone
48 (136 mg, 0.70 mmol) as a mixture of E and Z diastereomers
in 75% yield.
3.1 Vesicle formation
The spontaneous formation of vesicles (liposomes) depends on
the relative sizes of the hydrophilic and hydrophobic parts in
the molecule. For bilayer-forming lipids the critical packing
parameter v/a l (a \ the optimal area per molecule at the
0 c
0
lipidÈwater interface; l \ chain length; v \ hydrocarbon
c
[3-13C]-(2E,6E)-3,7,11-Trimethyl-2,6,10-dodecatrien-1-ol
50. Following the procedures for the preparation of 36, the
a,b-unsaturated ester 49 was reduced with diisobutylalumin-
ium hydride to give 50 in 93% yield together with the corre-
sponding 2,3-epoxy alcohol (6%). Spectral data of 50: 1H
NMR d 5.42 (1H, t-like, J 7.1, 2-H), 5.11 (1H, m, CH2), 5.09
volume) must lie between 1/2 and 1.10 The lipids with two
chains adopting cylindrical shapes would be accommodated
in the bilayer phase. The surface area a depends on the net
charge of the polar region. Charge repulsion increases the
0
e†ective area per molecule in the polar region and therefore
the e†ective area of the phosphate group increases in the
order : diacid \ monoanion \ dianion. In order to reveal the
relationships between the pH values of bu†ers and the sizes of
lipophilic polyprenyl chains in the spontaneous vesicle forma-
tion of the phosphates 6È20, we examined the vesicle forma-
tion in bu†ers of pH range 3.1È8.85, following the procedures
described in the Experimental section. Vesicle formation was
observed by di†erential interference contrast microscopy at
25.0 ¡C. Unilamellar vesicles of diameter larger than 0.2 lm
were observed, besides multilamellar structures and tubules. A
summary of the results is shown in Table 1, from which we
can draw the following conclusions.
(1H, m, CH2), 4.16 (2H, d, J 7.1, 1-CH ), 2.05 (8H, m, 4
2
] CH ), 1.70 (3H, d, 2J , 4.9, Me), 1.68 (3H, s, Me) and 1.60
2
CH
(3H, s, Me); 13C NMR d 139.78 (s, 13C-enriched C2O).
[3-13C]-(2E,6E)-1-Bromo-3,7,11-trimethyl-2,6,10-dodeca-
triene 51. Following the procedures described for the prep-
aration of 37, the alcohol 50 was treated with phosphorus tri-
bromide to give the bromide 51 in 78% yield.
(2E,6E)-3,7,11-Trimethyl-2,6,10-dodecatrienal 52. Following
the procedures described for the preparation of the imine 41,
the alcohol 36 was oxidized with activated MnO to give 52 in
2-Prenylgeranyl phosphate 6 formed vesicles spontaneously
in bu†ers of pH 3.1, 4.0 and 4.5. Fig. 3 shows an optical
micrograph of the vesicles of 6 prepared at pH 3.1. In con-
trast, it has been reported that the single-chain farnesyl phos-
2
92% yield as an oil, 1H NMR d 10.00 (1H, dd, 2J 24.4, 3J
CH
HH
8.1, CH2O), 5.88 (1H, dd, 1J 158, 3J 8.1, CH2), 5.07 (2H,
CH
m, 2 ] CH2), 2.23 (4H, m, 2 ] CH ), 2.17 (3H, dd, 3J 4.3,
CH
HH
2
4J 1.2 H, Me), 2.04 (4H, m, 2 ] CH ), 1.68 (3H, s, Me) and
phate 1 (n \ 1, C ) forms only very small vesicles which were
HH
2
15
1.60 (6H, s, 2 ] Me). 13C NMR d 127.32 (s, 13C-enriched C-2).
detected by electron microscopy, but no giant vesicle was seen
MS (EI) m/z 221 (M`, 27%), 203 (M` [ H O, 15), 178 (27),
by optical microscopy.2c Furthermore, prenylgeranyl phos-
2
150 (23), 136 (67), 123 (49), 109 (51), 85 (92), 81 (63) and 69
phate 2, with one C and one C chain (m \ 0, n \ 1), has
5
10
(100). Found: m/z 221.1832 (M`). Calc. for C 13CH O:
been shown to give a clear solution in water.2a,b Poten-
tiometric measurements with phytyl phosphate 24 and related
compounds have shown their dissociation constants to be:
pK 3.2È3.3 and pK 6.6È6.7.2c Under acidic conditions, the
14
24
221.1861.
[2,5-13C ]-(4E,8E)-5,9,13-Trimethyl-2-[(E)-1,5,9-
2
a1
a2
trimethyl-4,8-decadienylidene]-4,8,12-tetradecatrien-1-ol 54
phosphate 6 would be present as diacid or monoanionic
and [2,5-13C ]-(4E,8E)-5,9,13-trimethyl-2-[(Z)-1,5,9,13-
2
tetramethyl-4,8,12-tetradecatrienylidene]-4,8,12-tetradeca-
trien-1-ol 55. The aldehyde 52 was then transformed into 54
and 55 following the procedures reported above. It was
treated with cyclohexylamine to give 53. Allylation of the car-
banion of 53 with the bromide 51, followed by acid-catalyzed
hydrolysis, gave (2E)- and (2Z)-2-farnesylfarnesals. Acid-
catalyzed isomerization of the (2Z)-aldehyde gave the (2E)
isomer in pure form. Reduction of the (2E)- and (2Z)-2-far-
nesylfarnesals with diisobutylaluminium hydride gave 54 and
55, respectively.
54: 1H NMR d 5.11 (2H, m, 2 ] CH2), 5.09 (2H, m,
2 ] CH2), 5.07 (1H, m, CH2), 4.10 (2H, d, 2J 3.3, 1-H), 2.88
CH
(2H, dd, 3J
6.3, 2J
5.9, 1@-H), 2.09È1.99 (16H, m, 8
HH
CH
CH
] CH ), 1.77 (3H, d, 3J 4.9, Me), 1.68 (9H, s, 3 ] Me) and
2
1.60 (12H, s, 4 ] Me); 1H-decoupled 13C NMR d 136.10 (d,
3J 2.8, 13C-enriched 3@-C) and 131.78 (d, 3J 2.8, 13C-
CC
CC
enriched 2-C); MS (EI) m/z 428 (M`, 11%), 410 (M` [ H O,
Fig. 3 Phase contrast microscope images of the giant vesicles of
phosphate 6 at pH 3.1.
2
6), 273 (41), 149 (35), 137 (44), 123(45), 109 (56), 95 (60), 81 (86)
New J. Chem., 2001, 25, 917È929
921

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Table 1 Spontaneous vesicle formation of sodium polyprenyl phosphates 6È20 at various pH valuesa
pH Value of bu†erb
Number of C atoms
Phosphate
(Main and side chains)
3.1
4.0
4.5
7.0
7.86
8.5
8.85
6
7
15(10 ] 5)
25(15 ] 10)
30(20 ] 10)
35(20 ] 15)
35(20 ] 15)
20(10 ] 10)
20(15 ] 5)
20(15 ] 5)
25(15 ] 10)
25(15 ] 10)
30(15 ] 15)
30(15 ] 15)
30(20 ] 10)
30(20 ] 10)
30(20 ] 10)
]
[
[
[
[
[
]
[
]
]
[
]
[
]
[
[
]
]
[
[
]
]
]
]
]
]
]
[
]
]
]
]
]
]
8
]
]
]
9
[
[
[
[
]
]
]
10
11
12
13
14
15
16
17
18
19
20
]
]
[
[
[
]
]
]
]
]
[
]
]
]
[
]
[
[
]
]
a Observed by di†erential interference contrast microscopy at 25.0 ^ 0.1 ¡C. b pH 3.1 and 4.5; acetate bu†er; pH 7.0, phosphate bu†er; pH 7.86;
Tris É HCl bu†er; pH 8.5 and 8.85; borate bu†er. ]; Vesicle formation; [; no vesicle formation; blank; not tested.
forms, with smaller polar heads than those of the dianion
form. In the bu†ers of pH 7.0 and 8.5 the dianion of phos-
phate 6 did not form vesicles, due to the higher hydro- : lipo-
philicity ratio.10
mixing in pH 7.0 phosphate bu†er (10~4 M), were also
observed by negative staining (3% Ac UO ) transmission elec-
2
2
tron microscopy (Fig. 5).
In the vesicle formation with 6-(poly)prenyl-substituted
polyprenyl phosphates 11È19 a similar trend was observed in
the relationship between the pH values of bu†ers and the sizes
of the lipophilic polyprenyl chains.
Both the phosphate 11 (disodium 6-isopropenyl-3,9-
dimethyl-2,8-decadienyl phosphate) and the slightly larger
homolog 13 (disodium 6-isopropyl-3,11-dimethyl-7-methyl-
ene-2,10-dodecadienyl phosphate) formed vesicles in the pH
range 4.5È8.5 (Fig. 6), but neither in a highly acidic bu†er (pH
3.1) nor in a basic bu†er (pH 8.85 for 13).
Both phosphates 14 and 15 formed vesicles in a wide range
of pH (3.1È8.5). However, the phosphate 12 and its isomer 13
showed di†erent behaviours in vesicle formation. The former,
possessing a prenyl chain attached to the sp2 carbon atom (C-
6), formed vesicles at pH 3.1, but not the latter possessing a
prenyl chain at the sp3 carbon atom (C-6). A similar trend was
observed for the isoprenologs 16 and 17 at pH 4.5, i.e. the
former formed vesicles at this pH but not the latter.
The higher isoprenologs 7 and 8, which possess C and
15
C
main chains and a C side chain at C-2, did not form
20
10
vesicles under acidic conditions, but did so at pH 7.0 and
higher. Further increase of the size of the lipophilic polyprenyl
chains, as in the case of 2-farnesyl-substituted geranylgeranyl
phosphates 9 and 10, inhibited vesicle formation in the bu†ers
of pH 7.86 and lower: their hydro- : lipo-philicity ratios are
inappropriate for vesicle formation. The vesicle formation of
the isomeric phosphates 9 and 10 indicates that the double
bond positions are indi†erent (vide infra).
The spherical structure of the vesicles prepared by sonica-
tion of phosphate 8 in pH 4.5 acetate bu†er (10~4 M) was
conÐrmed by metal shadowing cryo-transmission electron
microscopy (Fig. 4; the white parts of the images are due to
the side-shadowing). The vesicles of 8, prepared by vortex
Fig. 4 Cryo-transmission electron microscope shadowed images of
the vesicles of phosphate 8 prepared by sonication in pH 4.5 acetate
bu†er (10~4 M). The black part of the images corresponds to the
carbon shadowing (90¡) and the white to the platinum shadowing
(45¡). The bar represents 0.5 lm.
Fig. 5 Negatively stained (3% Ac UO ) electron microscope images
2
2
of the vesicles of phosphate 8 prepared by vortex mixing in pH 7.0
phosphate bu†er (10~4 M). The bar represents 0.5 lm.
922
New J. Chem., 2001, 25, 917È929

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Fig. 7 Structures of 23, 24 and 25.
Fig. 6 Phase contrast micrograph of giant vesicles of phosphate 11.
The large lipophilic portion of the phosphate 19 was inap-
propriate for vesicle formation under acidic conditions and
required the larger polar head-group of the dianion to obtain
an adequate hydrophilic : lipophilic ratio.
The phosphate 20, which was derived as a by-product from
the synthesis of 8, but was not a candidate primitive bio-
membrane constituent,9a formed vesicles at pH higher than
7.0.
Thus, Table 1 shows that the 6-polyprenyl-substituted poly-
prenyl phosphates 11È19 form vesicles in a wide range of pH,
unlike the 2-polyprenyl-substituted polyprenyl phosphates
6È10. We shall discuss the di†erences in vesicle formation and
their compactness in the following sections.
We also attempted the preparation of vesicles of 21 and 22,
but as predicted for their 2Z geometry no vesicle formation
was observed by optical microscopy (see: the global minimum
conformation in Fig. 12).
constant similar to those of disodium (7R,11R)-phytyl phos-
phate 24 and disodium geranylgeranyl phosphate (1, n \ 2).
These unexpected results indicate that the vesicles of the
unsaturated polyprenyl phosphates are more impermeable to
water, by a factor of 100, than those of the saturated poly-
prenyl phosphates. However, it has been shown that phos-
phate 24 does not form stable monolayers in alkaline
solutions and phosphate 1 (n \ 2) does not form monolayers
even under acidic conditions.12 The decomposition of the
unstable vesicles during mixing (ca. 3 ms) and/or swelling may
be the cause of the extremely small rate constant in this case.
Closed vesicles should be capable of holding inside their
cavity hydrophilic molecules unable to cross the lipid layer.2
However, the entrapment of calcein, a hydrophilic dye, in the
vesicles of 14 failed, probably because the membranes were
unstable and the dye therefore leaked out rapidly through the
loosely packed membranes.13 We abandoned further attempts
to measure the permeability of the other phosphates.
3.2 Water permeability of the vesicles
In order to evaluate the rigidity of the vesicles of phosphates 1
(n \ 2), 11, 20 and 23È25 (Fig. 7), osmotic swelling of the uni-
lamellar vesicles was measured by use of the stopped-Ñow/
light-scattering method at pH 7.86 and at 33 ¡C.2b,11
Following the procedures described previously,2b the vesicles
of these phospates were prepared and we measured their
water permeability. The results are summarized in Table 2
together with those previously reported.2b The phosphate 20
showed an average Ðrst-order rate constant similar to those of
1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) 2511
and difarnesyl phosphate 2 (m \ n \ 2). It was also observed
3.3 Calculations of intramolecular attractive van der Waals
forces
Among the major forces holding the membrane molecules
together are van der Waals forces between the hydrocarbon
chains of adjacent lipid molecules. The stability of the vesicles
mentioned above should also depend on both the rigidity of
each unsaturated hydrophobic chain and on the intramolecu-
lar attractive van der Waals forces between the chains. It has
been shown that unsaturated chains are more mobile in solu-
tion than their saturated counterparts.14 To evaluate the sta-
bility of the vesicles of 20, we estimated the intramolecular
attractive van der Waals forces between the two chains a and
b in the alkene 27 and docosane 26. The latter was chosen as a
substitute for the saturated counterpart of 27 (Fig. 8).
that, when the chain length increases from
C
[2
15
(m \ n \ 2)] to C [2 (m \ n \ 3)], the water permeability
20
decreases. This could be due to the increase in the attractive
cooperative van der Waals forces. Phosphate 11 showed a rate
Table 2 Shrinkage experiments on vesicles of phosphates 1, 2, 11, 20 and 23È25
Phosphate
d
a/nm
SDb/nm
kc/s~1
SDb
Reference
av
86
11
27
21
28
19
26
2.0 ] 10~2
0.1 ] 10~2
0.8
0.1
d
d
d
d
e
f
d
e
e
e
20
106
91
39.8
3.5
23
24
94
88
6.5 ] 10~2
36.1
3.5 ] 10~1
2.6 ] 10~2
0.5 ] 10~2
25(DMPC)
25(DMPC)
1 (n \ 2)
2 (m \ n \ 1)
2 (m \ n \ 2)
2 (m \ n \ 3)
0.9
200
90.3
26
0.2 ] 10~2
150È200
115È130
133È140
26È35
35È36
20.5È31
0.3È0.6
0.2È0.4
12.0È13.2
a Average diameter. b Standard deviation. c Average rate constant. d This work. e Ref. 10. f Ref. 2(b).
New J. Chem., 2001, 25, 917È929
923

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mol~1 obtained as the sum of the two energy di†erences was
slightly higher than that of docosane 26. The attractive van
der Waals force decreased as the enthalpy of the conformer
increased. Recently Gung et al. have reported an ab initio MO
study of 1,5-hexadiene at the MP2/6-31G* level which shows
that, unlike n-butane, there is little energy di†erence between
the anti and the gauche conformations.18 This indicates that
the energies for the gauche conformations of 27, 29 and 30
were overestimated. The attractive van der Waals forces are
possibly weaker than those predicted using MM2 calculations.
Exhaustive conformation searches of 29 using the CAChe/
ConÑex system gave 568 structures within 5.0 kcal mol~1 of
the global minimum energy structure. The intramolecular
attractive van der Waals forces for the global minimum con-
formations 29A (Fig. 9) and for octadecane were estimated to
be 1.99 and 3.87 kcal mol~1, respectively. The weaker intra-
molecular attractive force of 29 compared to that of octade-
cane shows a loosely packed state of the membranes of the
2-polyprenyl-substituted polyprenyl phosphates 6 and 8. The
larger attractive forces of 27A (5.27 kcal mol~1) and 30A (3.58
kcal mol~1 obtained by the calculations shown in Fig. 10)
imply that the interaction between the two alkenyl chains
attached to the same sp2 carbon atom is more e†ective. The
vesicles of the phosphates 7 and 10 were less stable than those
of the phosphate 20. In the compounds 7 and 10 the polar
head group is attached to the mobile unsaturated side chain
E, whereas in the phosphate 20 the polar head group is
attached directly to the rigid unit A composed of Ðve carbon
atoms (Fig. 11). The compact and rigid structure of 20 implies
that the phosphate has a lower molecular volume and greater
intermolecular attractive van der Waals interaction (as a
result of a higher surface : volume ratio)19 than those of 2-
polyprenylpolyprenyl phosphates such as 6 and 8 (D). In these
analyses the e†ects of solvent and of bu†er components were
Fig. 8 Optimized structures of 26, 27 and 28, their relative energies
(MM2) and the intramolecular van der Waals forces of 26 and 27.
Recently Goodman has reported that the Ðrst alkane for
which the hairpin conformation (see: conformer of docosane
26 in Fig. 8) is lower in energy than the extended one is octa-
decane, according to MM2 force Ðeld calculations.15 The
hairpin conformation is preferable because intramolecular
attractive van der Waals forces outweigh the energetic cost of
twisting the carbon chain away from the preferred extended
anti conformation to the gauche conformation. A chain can be
reversed with a combination of one anti and four gauche con-
formations. Following GoodmanÏs protocol we calculated the
attractive van der Waals forces of docosane 26, whose carbon
chain lengths (chains a and b) are comparable to those of 27.
The attractive van der Waals force of 4.76 kcal mol~1 can be
obtained as the sum of the energy cost of twisting (\four
times the energy di†erence between anti and gauche) and the
di†erence in energy between extended and hairpin conformers.
Molecular mechanics and ab initio calculations have been
employed to examine the phase conformation of prenyl deriv-
atives in solution.16 Exhaustive conformational searches were
performed by varying all the dihedral angles (CÈCÈCÈC and
C2CÈCÈC) between [120 and 120¡ in two steps using the
CAChe/ConÑex system (optimized with molecular mechanics
calculations, MM2).17 The conformation searches of 27@ gave
2140 structures within 5.0 kcal mol~1 of the global minimum
energy structure. The di†erence in energy between the folded
conformer 27A (global minimum conformer) and the extended
conformer 27B was found to be 3.78 kcal mol~1. The con-
former 27B is transformed from 27A by twisting a single bond
in the chain a from gauche to anti (indicated by an arrow). The
di†erence in energy between the gauche 28A and anti 28C con-
formers of 3,7-dimethyl-2,6-octadiene was calculated to be
1.49 kcal mol~1. The attractive van der Waals force 5.27 kcal
Fig. 9 Optimized structures of 29A and 29, their relative energies
(MM2) and the intramolecular van der Waals force of 29.
924
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Fig. 10 Optimized structures of 30, 31 and 32, their relative energies (MM2) and the intramolecular van der Waals force of 30.
ignored, but exclusion from water may be expected to com-
press and rigidify the folded hydrophobic system, favoring the
smallest possible surface area.15
at pH 4.5 (see Table 1) suggest that the contribution of the
hydrophobic region of the unit C is more important than that
of unit B. As was discussed above, the unit C (corresponding
to 30: 3.58 kcal mol~1) has a larger attractive van der Waals
interaction than B (corresponding to 29A: 1.99 kcal mol~1).
Therefore, for the formation of vesicles with phosphates 13 or
17, an increase of their polar head region is needed. This could
be obtained by increasing pH.
The fact that 6-polyprenyl-substituted polyprenyl phos-
phates form vesicles in a wide range of pH, unlike 2-
polyprenyl-substituted polyprenyl phosphates (Table 1),
indicates that the units B and C (11È19) are preferable to D
and E (6È10) for vesicle formation. In the units D and E the
polar head group and the junction of two chains are neighbor-
ing, whereas B and C possess a prenyl unit, as a spacer,
between the polar head group and the junction. The vesicles
of phosphate 20 possessing a rigid spacer (unit A) can be more
tightly packed than those of 6-polyprenyl-substituted poly-
prenyl phosphates 11È19.
The dihedral angles C1ÈC2ÈC3ÈC4 and C3ÈC4ÈC5ÈC6 in a
1,5-hexadiene system are governed by a 1,3-allylic strain. To
minimize this steric interaction these dihedral angles are
expected to be 90¡. In the chain b of the global minimum con-
formation 29A, a less preferable CÈC eclipsed form is present.
This may be due to maximization of the attractive force
between the chains a and b. The fact that unsaturated poly-
prenols are mobile unlike alkanes can be ascribed to the small
rotational barrier of the CÈC bond14 and the presence of
enormous structures within a small amount of energy of the
global minimum energy structure.18 Exhaustive conformation
searches of 33 using the CAChe/ConÑex system gave 2810
structures within 5.0 kcal mol~1 of the global minimum
energy structure. Most of them, including the global minimum
conformation (Fig. 12), were folded to maximize the attractive
van der Waals interactions between the polyprenyl chains.
The hydroxymethyl group is located inside the U-shaped
polyprenyl chains. The conformational analysis indicates that
the (2Z)-2-polyprenyl-substituted geranylgeranyl phosphates
21 and 22 can not form vesicles even if they adopt either the
folded conformations similar to those of 33 or extended con-
formations. In fact, no vesicle formation was observed for 21
and 22.
The di†erences in vesicle formation between the 6-prenyl-
substituted farnesyl phosphates 12 and 13 at pH 3.1 and
between 6-farnesyl-substituted farnesyl phosphates 16 and 17
3.4 Structural parameters obtained from NMR spectral
analysis
In order to reveal the conformation of the 1,4-pentadiene part
of 2-polyprenyl-substituted polyprenols we needed to measure
the dihedral angles C1ÈC2ÈC1@ÈC2@ and C2ÈC1@ÈC2@ÈC3@.20
The numbering systems used to identify the carbon atoms are
shown in Fig. 13. The Ðrst angle can be obtained from the
Fig. 11 ClassiÐcation of substituted polyprenyl phosphates into Ðve
types of structures: A (compound 20), B (compounds 12, 14, 16, 19), C
(compounds 11, 13, 15, 17, 18), D (compounds 6, 8, 9), E (compounds
7, 10).
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phosphate 46 was attempted in CDCl , but the signals were
3
too broad to obtain the coupling constant, which is very
small. The averaged value of the dihedral angles calculated for
the 30 structures (67% total population) of 2-geranylfarnesol
within 1.57 kcal mol~1 of the global minimum structure is
74¡. In these polyprenols the 1,3-allylic strain may be one of
the major factors governing their conformation.
For the fragment including terminal oleÐn C2ÈC1@ÈC2@2C3@
the Karplus relationship is not obeyed, due to hypercon-
jugative interaction at dihedral angles near 90¡.22 Instead of
the dihedral angle, we can use the angle H1@ÈC1@ÈC2@ÈH2@ for
the conformational analysis. The vicinal spin coupling con-
stants (J \ 6.9È7.3 Hz)9a,c between the 1@-H and 2@-H of the 2-
and 6-polyprenyl-substituted polyprenols and their tetrabutyl-
ammonium phosphates are typical of free rotation.20,23b
However, the vicinal coupling constants for the phosphates 6,
8, 9, 12, 14 and 16 were found to decrease with increasing
chain length, i.e. the J values of 12, 6, 14, 8 and 16 were 6.6,
6.4, 5.3, 4.4 and 4.0 Hz, respectively. Phosphates 12 and 6
Fig. 12 Global minimum conformation of (2Z)-2-geranylfarnesol 33.
spin coupling constant between C1 and C2@ using the Karplus
relation. We have synthesized [1,2@-13C ]-2-farnesylfarnesol 44
2
(Scheme 1).21 In the small scale synthesis we avoided using
volatile prenylacetone as starting material and instead chose
having a small prenyl group may rotate freely (see also the T
1
the less volatile geranylacetone 34. The 3J coupling constant
maps in Fig. 14). The smaller J values for 14, 8 and 16, which
CC
between the labeled carbon atoms is 0.77 Hz. The Karplus
have a larger substituent at C-2 or C-6 such as a geranyl or
farnesyl group, can be ascribed to some hindered internal
rotation due to the increase in the intramolecular attractive
van der Waals forces. The vicinal spin coupling constants
relation showed the dihedral angle C1ÈC2ÈC1@ÈC2@ to be near
90¡. In this conformation the 1,3-allylic strain may be mini-
mized. The measurement of the 3J coupling constant of the
CC
Scheme 1 Reagents: (a) (EtO) P(2O)13CH CO Et, NaH; (b) AliBu H; (c) PBr , pyridine; (d) (EtO) P(2O)CH 13CO Et, NaH; (e) active MnO ;
2
2
2
2
3
2
2
2
2
(f) cyclohexylamine, K CO ; (g) LiNiPr , bromide 37; (h) CF CO H, THF for the isomerization of Z-aldehyde 43 to E-aldehyde 42.
2
3
2
3
2
926
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Fig. 13 Structures and numbering systems of 56È63.
Scheme 2 Reagents: (a) NaH, geranyl bromide; (b) aq. NaOH then
HCl; (c) (EtO) P(2O)CH CO Et, NaH; (d) AliBu H; (e) PBr , pyri-
2
2
2
2
3
dine; (f) activated MnO ; (g) cyclohexylamine, K CO ; (h) LiNiPr ,
chemical shift (d 133.36) is slightly larger than those of termin-
al disubstituted oleÐnic carbons, i.e. C-7 in geraniol 62 and
C-11 in farnesol 63 ordinarily resonating at d ca. 131. C-4: the
methylene carbon Ñanking the disubstituted oleÐnic carbon is
observed at a higher Ðeld (ca. 5 ppm) than the corresponding
methylene carbons in 62 and 63 due to the c e†ect of the side
chain cis to C-4. C-5, C-5@ and C-9@: the signals observed in
common at d ca. 26.8 in the spectra of 56È58 were assigned to
the carbon atoms at C-5, and the other signals resonating at a
slightly higher Ðeld were assigned to C-5@ of 56 and 57. For
farnesol 63, the methylenic carbon atom C-5 resonates at a
slightly higher Ðeld than the terminal methylenic carbon atom
2
2
3
2
bromide 51; (h) CF CO H, THF for the isomerization of Z- to E-
3
2
aldehyde.
between the 1@-H and 2@-H of the (2Z)-phosphate 21 and the
corresponding alcohol are 4.4 and 7.1 Hz, respectively.9a
Viterbo and co-workers have reported the hindered internal
rotation of the central C11ÈC12 bond in squalene (J \ 3.8
Hz).23b
The assignment of the 13C NMR spectra of polyprenols
56È61 was carried out independently from the T measure-
1
ment as much as possible, with the aid of the chemical shift
values of 13C-labeled compounds 44 and 54 (Schemes 1 and 2)
and the known assignment of geraniol 62 and farnesol 63 (Fig.
13 and Table 3). 2-Polyprenylgeraniols 56 and 57 and 6-
geranyl- and 6-farnesyl-geraniols 60 and 61 were freshly pre-
pared following the procedures reported previously.9a,c
Firstly, the assignment of the 13C NMR spectra of 56È58
was performed as follows. The signals observed in common in
the spectra of 56È58 were assigned to the carbon atoms at
C1ÈC12, 3-Me, 7-Me, 11-Me and C-1@. The four signals having
nearly identical chemical shifts with those of the labeled
carbons in 44 and 54 were assigned to C-1, C-2, C-2@ and C-3@.
C-1@: the signals showing almost the same chemical shifts with
that of C-1@ in 59 were assigned to C-1@ (vide infra). C-3: the
C-9. Based on this trend and with the aid of their T values,
the assignment of C-5@ and C-9@ in 56 was performed. C-6,
1
C-6@ and C-10@: for farnesol 63, the terminal monosubstituted
oleÐnic carbon atom C-10@ resonates at a slightly lower Ðeld
than the inner oleÐnic carbon atom C-7. The signal at d
124.19 of 56, which appears at a slightly lower Ðeld than those
of the two other monosubstituted oleÐnic carbon atoms, is
therefore assigned to the terminal oleÐnic carbon C-10@. The
T
value of C-10@ being larger than those of the other two
1
carbons supported the assignment.22 C-6 and C-6@ were tenta-
tively assigned. C-7 and C-11@: the resonances at d 131.30 and
130.88 cannot be assigned unequivocally owing to their
similar chemical shifts. The resonance (d 130.88) possessing
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signal observed at d 17.57 was assigned to the terminal cis-
methyl carbon atoms, 7-Me and 11@-Me (see: 11-Me of far-
nesol 63). The di†erentiation of 3-Me and 7@-Me was difficult
(see also: 3-Me and 7-Me of 63) and the assignment was
achieved with the aid of their T values. The remainder reson-
1
ating at a lower Ðeld was assigned to 3@-Me.
The assignment of the 13C NMR spectra of 59 was per-
formed as follows. The carbon atoms C-1, C-2, C-3, C-4, C-9,
3-Me and 11-Me were assigned with the aid of the known
assignment of geraniol 62 and farnesol 63. The assignment of
C-2, C-10, C-1@ and C-2@ was performed on the basis of their
selective proton-decoupled spectra. Thus, the methylenic
carbon C-5, whose chemical shift is slightly di†erent, was
assigned unequivocally. C-6 and C-7: the assignment was per-
formed by comparing the chemical shifts with those of the
tetraalkyl-substituted alkenes.24 C-8: see the assignment of
C-4 in 56. C-11 and C-3@: the signals observed in common in
the spectra of 59È61 were assigned to the carbon atoms at
C-11. For 60 and 61, see Experimental section. The chemical
shift depended on the concentration of 59 and the d value
(131.22) of C-11 observed at lower concentration was slightly
larger than that shown in Table 3.9c Based on the trend that
the terminal methyl resonates at lower Ðeld than the inner
methyl, we assigned 7-Me and 3@-Me.
Fig. 14 SpinÈlattice relaxation time NT (in s) maps of the 13C
3.5 Spin–lattice relaxation data
1
atoms of 2-farnesylgeraniol 56 and 6-prenylfarnesol 59. Solutions of
56 (0.65 M) and 59 (0.82 M) in CDCl were used for the measure-
13C SpinÈlattice relaxation times T can be a parameter of the
Ñexibility of the 2- and 6-polyprenyl-substituted polyprenyl
3
1
ments. N in NT means the number of protons attached directly to a
1
carbon atom.a,b The averaged NT values for C-8 and C-12@ and 7-Me
phosphates, although being qualitative.25 We have measured
1
and C11@-Me.c The averaged NT value for C-8 and C-12@.
1
the T values of the 2-farnesyl-geraniol 56 and 6-prenyl-far-
1
nesol 59 (Fig. 14).9c In the former the tetrasubstituted double
larger T must be assigned to the more mobile carbon, C-
bond is rooted and the mobility of the main and side chains
increased with increasing distance from C-2 and C-3, respec-
tively. The relatively rigid structure of the central tetra-
substituted double bond part of the 6-prenylfarnesol 59 was
indicated by the spinÈlattice relaxation times. The vesicle for-
1
11@.23 C-4@ and C-8@: for farnesol 63, the methylenic carbon
atom C-4 resonates at a slightly lower Ðeld than the methyl-
enic carbon atom C-8. Based on the trend the assignment of
C-4@ and C-8@ was performed. Methyl groups: the overlapping
Table 3 13C NMR Chemical shifts of polyprenols 44, 54, 56È59, 62 and 63
Chemical shift values, d
Carbon
56a
57
58
59b
62
63
44/54
2
3
6
7
10
11
131.81
133.36
123.86c
131.30
131.83
133.76
124.07d
131.60
132.06
133.82
124.04
131.77
123.13
139.17
131.69
128.75
124.25
130.84
123.44
130.64
123.40
138.20
123.72
131.11
123.8
139.5
123.5
135.3
124.4
131.2
131.78
2@
122.94
135.44
124.00c
134.80
124.19
130.88
62.07
34.67
26.87
25.62c
122.99
135.78
124.07d
131.45
122.99
131.60
122.92
136.10
3@
6@
7@
10@
11@
1
62.36
34.59
26.77
25.59d
62.49
34.84
26.93
25.87
58.90
38.26
31.07
34.39
27.04
25.55d
30.77
25.55d
58.52
39.26
26.12
25.25
59.3
39.7
26.4
39.6
26.8
25.6
62.51
4
5
8
9
12
1
29.21
39.73
26.53
39.66
26.71
25.62d
16.04
17.57d
29.28
39.66
26.48
25.59d
29.54
25.77
4@
5@
8@
9@
12@
3-Me
7-Me
11-Me
3@-Me
7@-Me
11@-Me
15.95
17.56
17.70
17.89
16.13
17.92
17.64
17.43
15.80
17.23
15.9
16.2
17.6
17.97
15.95
17.57d
17.95
17.51
18.12
a Obtained from a 0.65 M solution. b Obtained from a 0.82 M solution. c The assignment may be interchanged. d The peaks are overlapping.
928
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S. J. Rowland, Org. Geochem., 1997, 27, 497; E. J. Wraige, L.
Johns, S. T. Belt, G. Masse, J.-M. Robert and S. J. Rowland,
Phytochemistry, 1998, 51, 69; E. J. Wraige, S. T. Belt, G. Masse,
J.-M. Robert and S. J. Rowland, Org. Geochem., 1998, 28, 855;
J. S. S. Damste, W. I. C. Rijpstra, S. Schouten, H. Peletier, M. J.
E. van der Maarel and W. W. C. Gieskes, Org. Geochem., 1999,
30, 95; L. Johns, E. J. Wraige, S. T. Belt, C. A Lewis, G. Masse, J.
M. Robert and S. J. Rowland, Org. Geochem., 1999, 30, 1471; J. S.
S. Damste, S. Schouten, W. C. Rijpstra, E. C. Hopmans, H. Pele-
tier, W. W. C. Gieskes and J. A. J. Geenevasen, Org. Geochem.,
1999, 30, 1581; S. T. Belt, G. Allard, G. Masse, J.-M. Robert and
S. Rowland, Chem. Commun., 2000, 501; S. J. Rowland, D. A.
Yon, C. A. Lewis and J. R. Maxwell, Org. Geochem., 1985, 8, 207;
C. Porte, D. Barcelo, T. M. Tavares, V. C. Rocha and J. Albaiges,
Arch. Environ. Contam. T oxicol., 1990, 19, 263; S. J. Rowland,
S. T. Belt, E. J. Wraige, G. Masse, C. Roussakis and J.-M. Robert,
Phytochemistry, 2001, 56, 597.
For highly branched polyprenols and related alcohols isolated
from higher plants, see: E. Lemmich, Phytochemistry, 1979, 18,
1195; H. Nagano, M. Tori, M. Shiota and J. de Pascual Teresa,
Bull. Chem. Soc. Jpn., 1984, 57, 2971; M. Bruno, L. Lamartina, F.
Lentini, C. Pascual and G. Savona, T etrahedron L ett., 1984, 25,
4287; L. Mangoni, D. Merola, P. Monaco, M. Parrilli and L.
Previtera, T etrahedron L ett., 1984, 25, 2597; C. Porte, D. Barcelo,
T. M. Tavares, V. C. Rocha and J. Albaiges, Arch. Environ.
Contam. T oxicol., 1990, 19, 263; J.-L. Giner, J. D. Berkowitz and
T. Andersson, J. Nat. Prod., 2000, 63, 267; S. Faure, J. D. Con-
nolly, C. O. Fakunle and O. Piva, T etrahedron, 2000, 56, 9647;
H. R. Nawaz, A. Malik and M. S. Ali, Phytochemistry, 2001, 56,
99.
mation of 6-polyprenyl-substituted polyprenyl phosphates 12,
14 and 16 in the bu†ers with a wide range of pH may partly
be attributed to the rigid structure.
These results from T measurements show that the poly-
1
prenols 56 and 59 are more mobile in solution than their satu-
rated counterparts such as phytol.23c,26 The fact that the
vesicles from unsaturated polyprenyl phosphates are less
stable than those from their saturated counterparts can be
interpreted based on the di†erence in the rigidity of the hydro-
phobic chains. To estimate experimentally the degree of con-
tribution of the intramolecular attractive interaction to vesicle
stabilization, we would require further systematic investiga-
tion on the spinÈlattice relaxation times T employing solu-
1
tions of polyprenols and their phosphates with various chain
lengths in polar and non-polar solvents.
7
Conclusion
The spontaneous vesicle formation of polyprenylated poly-
prenyl phosphates 6È20 and the structural properties of these
amphiphiles and the corresponding alcohols have been report-
ed. We now include these amphiphiles 6È19 as possible primi-
tive biomembrane constituents to the hypothetical family tree
of the chemical evolution of terpenoids as biomembrane con-
stituents and reinforcers.1b
8
9
For highly branched polyprenyl acetate isolated from higher
plants, see: M. J. Gieselmann, D. S. Moreno, J. Fargerlund, H.
Tashiro and W. L. Roelofs, J. Chem. Ecol., 1979, 5, 27.
(a) H. Nagano and K. Kudo, Bull. Chem. Soc. Jpn., 1996, 69,
2071; (b) H. Nagano, T. Nagasawa and M. Sakuma, Bull. Chem.
Soc. Jpn., 1997, 70, 1969; (c) H. Nagano, E. Nakanishi, S. Takajo,
M. Sakuma and K. Kudo, T etrahedron, 1999, 55, 2591.
Acknowledgements
We are grateful for support granted by CNRS to S. G.,
Association ““Amis des SciencesÏÏ to O. D. We thank Mr N.
Midani for his technical assistance, Dr M. Tsuji, Nisshin
Flour Milling, Dr T. Takigawa, Kuraray and Professor T.
Eguchi, Tokyo Institute of Technology for their generous gifts
of polyprenols.
10 H. Goto and E. Osawa, J. Am. Chem. Soc., 1989, 111, 8950; H.
Goto and E. Osawa, J. Chem. Soc., Perkin T rans. 2, 1993, 187;
JCPE, P040 and P021, OCPE, no. 59, developed by H. Goto and
E. Osawa.
11 J. N. Israelachvili, Intermolecular and Surface Forces, Academic
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