Polymerization of the inverted hexagonal phase

Article abstract of DOI:10.1021/ja970052x

The hydration of polar natural and synthetic lipids yields a variety of lipid phases including various inverted cubic phases and the inverted hexagonal (H(II)) phase. The H(II) phase can be considered as aqueous columns encased with a monolayer of lipids and arranged in a hexagonal pattern. The polar head groups are well-ordered at the water interface, whereas the lipid tails are disordered to fill the volume between the tubes of water. A particularly interesting characteristic of the H(II) phase is the large temperature effect on the basis vector length d of the hexagonal lattice. Previous studies indicate that polymerization of the lipid region of the H(II) phase might reduce the sensitivity of the basis vector to temperature. A phosphoethanolamine (PE) was designed and synthesized with dienoyl groups in each lipid tail in an attempt to cross-link the lipids around and along the water core of the H(II) phase. The synthesis of the the PE was accomplished by acylation of 3-(4-methoxybenzyl)-sn-glycerol with 2,4,13- (E,E,Z)-docosatrienoic acid, followed by deprotection, then phosphorylation with dichloro-[[N-[(2,2,2- trichloroethoxy)carbonyl]-2-aminolethyl]phosphinic acid to give the Troc-PE, which was converted to the PE with activated zinc and acetic acid. The hydrated PE (1/1 weight lipid/water) formed the H(II) phase over an extended temperature range. Polymerization to high conversion was accomplished at 60 °C with the aid of redox initiators. Polymerization was followed in-situ using X-ray diffraction over a period of 48 h. The scattering, which weakened over the course of the reaction, remained consistent with a hexagonal phase. Temperature cycling of the polymerized H(II) phase showed an unaltered pattern on decreasing temperature while maintaining the same lattice parameter, unlike that of the unpolymerized phase where the value increased with decreasing temperature. Thus it is possible to fix the dimensions of the H(II) phase by cross-linking polymerization of appropriately designed reactive lipids.

Full text of DOI:10.1021/ja970052x

4866
J. Am. Chem. Soc. 1997, 119, 4866-4873
Polymerization of the Inverted Hexagonal Phase
Warunee Srisiri,† Thomas M. Sisson,† David F. O’Brien,*,† K. M. McGrath,‡
Yuqi Han,‡ and Sol M. Gruner‡
Contribution from C.S. MarVel Laboratories, Department of Chemistry, The UniVersity of
Arizona, Tucson, Arizona 85721, and Joseph Henry Laboratories, Department of Physics,
Princeton UniVersity, Princeton, New Jersey 08544
ReceiVed January 6, 1997X
Abstract: The hydration of polar natural and synthetic lipids yields a variety of lipid phases including various inverted
cubic phases and the inverted hexagonal (HII) phase. The HII phase can be considered as aqueous columns encased
with a monolayer of lipids and arranged in a hexagonal pattern. The polar head groups are well-ordered at the water
interface, whereas the lipid tails are disordered to fill the volume between the tubes of water. A particularly interesting
characteristic of the HII phase is the large temperature effect on the basis vector length d of the hexagonal lattice.
Previous studies indicate that polymerization of the lipid region of the HII phase might reduce the sensitivity of the
basis vector to temperature. A phosphoethanolamine (PE) was designed and synthesized with dienoyl groups in
each lipid tail in an attempt to cross-link the lipids around and along the water core of the HII phase. The synthesis
of the the PE was accomplished by acylation of 3-(4-methoxybenzyl)-sn-glycerol with 2,4,13-(E,E,Z)-docosatrienoic
acid, followed by deprotection, then phosphorylation with dichloro-[[N-[(2,2,2-trichloroethoxy)carbonyl]-2-amino]-
ethyl]phosphinic acid to give the Troc-PE, which was converted to the PE with activated zinc and acetic acid. The
hydrated PE (1/1 weight lipid/water) formed the HII phase over an extended temperature range. Polymerization to
high conversion was accomplished at 60 °C with the aid of redox initiators. Polymerization was followed in-situ
using X-ray diffraction over a period of 48 h. The scattering, which weakened over the course of the reaction,
remained consistent with a hexagonal phase. Temperature cycling of the polymerized HII phase showed an unaltered
pattern on decreasing temperature while maintaining the same lattice parameter, unlike that of the unpolymerized
phase where the value increased with decreasing temperature. Thus it is possible to fix the dimensions of the HII
phase by cross-linking polymerization of appropriately designed reactive lipids.
Introduction
The hydration of polar natural and synthetic lipids yields a
variety of lipid phases depending on the concentration, tem-
perature, and pressure. At high concentrations certain lipids
form quite complex lyotropic liquid crystals. These include
various inverted cubic phases and the inverted hexagonal (HII)
phase.1-3 The most commonly studied of these phases is the
HII, which is readily formed from lipids that exhibit a large
spontaneous curvature under certain conditions. Curvature is
a consequence of an imbalance of forces across a lipid layer.4
When the repulsive lateral pressure in the lipid tail region is
greater than the repulsive forces acting at the head group, a
lipid layer assumes a negative curvature as shown in Figure 1.
Lipids with cis-double bonds or branching substitutents in the
tails and relatively small, poorly hydrated head groups form
the HII phase at moderate to low temperatures.5
Figure 1. Schematic representation of the inverted hexagonal phase.
The HII phase can be considered as aqueous columns arranged
in a hexagonal pattern (Figure 1). Each water channel is encased
within a monolayer of lipid. The polar head groups are well-
ordered at the water interface, whereas the lipid tails are
disordered to fill the volume between the columns of water. A
particularly interesting characteristic of the HII phase is the large
temperature effect on the basis vector length d of the hexagonal
lattice. Tate and Gruner showed that reduction in the radius of
the water channel accounts for most of the change in this lattice
vector.5 It may therefore be possible to polymerize of the lipid
region in a manner to lock-in the column diameter rendering
the unit cell of the HII phase insensitive to changes in
temperature.
Previously reported methods to polymerize and stabilize
lamellar assemblies, i.e. bilayer vesicles, offer approaches to
the polymerization of nonlamellar assemblies. Since the first
reports of the polymerization of hydrated bilayers in the early
1980s,6-9 a wide variety of polymerizable groups have been
successfully employed. The various strategies for the polym-
† The University of Arizona.
‡ Princeton University.
X Abstract published in AdVance ACS Abstracts, May 1, 1997.
(1) Lindblom, G.; Rilfors, L. Biochim. Biophys. Acta 1989, 988, 221-
256.
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6638-6640.
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(3) Tate, M. W.; Eikenberry, E. F.; Turner, D. C.; Shyamsunder, E.;
Gruner, S. M. Chem. Phys. Lipids 1991, 57, 147-164.
(4) Gruner, S. M. Proc. Natl. Acad. Sci. 1985, 82, 3665-3669.
(5) Tate, M. W.; Gruner, S. M. Biochemistry 1989, 28, 4245-4253.
(7) Hub, H.; Hupfer, B.; Koch, H.; Ringsdorf, H. Angew. Chem., Int.
Ed. Engl. 1980, 19, 938-940.
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Biophys. Acta 1980, 602, 57-69.
S0002-7863(97)00052-8 CCC: $14.00 © 1997 American Chemical Society

Polymerization of the InVerted Hexagonal Phase
J. Am. Chem. Soc., Vol. 119, No. 21, 1997 4867
Results and Discussion
erization of lamellar phases have been reviewed.10-13
A
particularly useful method relies on the design of suitable
polymerizable amphiphiles, which upon hydration form as-
semblies that can then be polymerized in place. Several reactive
lipids, e.g. acryloyl, dienoyl, and sorbyl, may be polymerized
by radical chain processes in either the Lâ or LR phases, whereas
the topotactic polymerization of diacetylenes limits this chem-
istry to the solid-analogous (Lâ) phase.9 Lipids in the LR phase
exhibit rapid lateral diffusion,14 in a manner that facilitates the
polymerization process.15 A series of systematic studies have
provided new insights into the effect of the two-dimensional
nature of the lipid bilayer on the rate and degree of polymer-
ization in the LR phase.16-19 In recent years the effects of
polymerization on the partial phase diagram of some hydrated
lipids have been reported.20-23
On the basis of the current understanding of the polymeri-
zation of the lamellar phase and our success in extending these
methods to polymerization of a bicontinuous cubic phase,22 we
began an examination of the polymerization of the HII phase.
Various strategies for the design of polymerizable lipids were
considered at the outset of these studies. First, the reactive group
could be located on either or both lipid tail(s) near the lipid
backbone, i.e. the glycerol unit in the case of phospholipids.
Second, the reactive group could be placed at the end of the
lipid tails, although this may be less desirable because polym-
erization of the disordered terminal end of the lipid tails may
perturb the HII phase. Third, the reactive group could be
attached to the lipid head group as a hydrophilic substitutent.
The first approach appeared to be the most promising because
covalent linkage of lipids near the backbone would have less
effect on the important forces that act at the head group and
tails of the lipids. Furthermore the use of a diene group
conjugated with the acyl chain carbonyl does not interfere with
the biocompatibility of the lipid-water interface. Here we
report the design and synthesis of a PE with dienoyl groups in
each lipid tail in a manner that is expected to yield a cross-
linked polymer around and along the water core of the HII phase.
Studies of the polymerization of bis-substituted dienoyl lipids,
e.g. phosphatidylcholines (PC), in the lamellar phase show the
formation of cross-linked bilayer membranes.24 In addition the
formation and polymerization of the HII phase was accomplished
in a manner that preserves the lipid assembly as well as leads
to the formation of individually polymerized unit cells, i.e. tubes
of lipid surrounding the water core.
(9) O’Brien, D. F.; Whitesides, T. H.; Klingbiel, R. T. J. Polym. Sci:
Polym. Lett. Ed. 1981, 19, 95-101.
(10) Ringsdorf, H.; Schlarb, B.; Venzmer, J. Angew. Chem., Int. Ed. Engl.
1988, 27, 113-158.
(11) O’Brien, D. F.; Ramaswami, V. Encycl. Polym. Sci. Eng. 1989, 17,
108-135.
(12) O’Brien, D. F. Trends Polym. Sci. 1994, 2, 183-188.
(13) Singh, A.; Schnur, J. M. In Phospholipids Handbook; Cevc, G.,
Ed.; Marcel Dekker: New York, 1993; pp 233-291.
(14) Fahey, P. F.; Webb, W. W. Biochemistry 1978, 17, 3046-3053.
(15) Ko¨lchens, S.; Lamparski, H.; O’Brien, D. F. Macromolecules 1993,
26, 398-400.
(16) Sells, T. D.; O’Brien, D. F. Macromolecules 1994, 27, 226-233.
(17) Lei, J.; O’Brien, D. F. Macromolecules 1994, 27, 1381-1388.
(18) Lamparski, H. G.; O’Brien, D. F. Macromolecules 1995, 28, 1786-
1794.
(19) Sisson, T. M.; Lamparski, H. G.; Ko¨lchens, S.; Elayadi, A.; O’Brien,
D. F. Macromolecules 1996, 29, 8321-8329.
(20) Barry, J. A.; Lamparski, H.; Shyamsunder, E.; Osterberg, F.; Cerne,
J.; Brown, M. F.; O’Brien, D. F. Biochemistry 1992, 31, 10114-10120.
(21) Stro¨m, P.; Anderson, D. M. Langmuir 1992, 8, 691-709.
(22) Lee, Y.-S.; Yang, J.-Z.; Sisson, T. M.; Frankel, D. A.; Gleeson, J.
T.; Aksay, E.; Keller, S. L.; Gruner, S. M.; O’Brien, D. F. J. Am. Chem.
Soc. 1995, 117, 5573-5578.
(23) McGrath, K. M.; Drummond, C. J. Colloid Polym. Sci. 1996, 274,
316-333.
(24) Tsuchida, E.; Hasegawa, E.; Kimura, N.; Hatashita, M.; Makino,
C. Macromolecules 1992, 25, 207-212.
[1,2-Bis[2,4,13-(E,E,Z)-docosatrienoyl]-sn-glycerol]-3-phos-
phoethanolamine (1) that contains a polymerizable dienoyl group
in each hydrophobic chain was designed and synthesized. A
cis-double bond was incorporated into each hydrophobic tail
to facilitate the formation of HII phase at suitable temperatures.
Polymerization of the diene substituted lipids can be ac-
complished with the aid of either thermal or redox initiators or
by direct photopolymerization.24 In this study, redox-initiated
radical polymerizations were employed, as they can be per-
formed at reasonable rates over a wide range of temperatures.
Synthesis of Lipid 1. The synthesis of PE lipids has been
studied extensively in the past decade, and several methods have
been reported.25-27 However, methods for the chemical syn-
thesis of polymerizable PE lipids have only recently been
described.28 Our preparative approach started with the synthesis
of a 1,2-bis[2,4,13-(E,E,Z)-docosatrienoyl]glycerol (2) followed
by phosphorylation. Acylation of 3-(4-methoxybenzyl)-sn-
glycerol29,30 with 2,4,13-(E,E,Z)-docosatrienoic acid (3a) and
then deprotection of the 4-methoxybenzyl ether group via Lewis
acid catalyzed hydrolysis at low temperature afforded 2 (Scheme
1).
The polymerizable fatty acid 3 was accessible in three steps
from commercially available oleoyl alcohol (Scheme 2). The
alcohol was oxidized to the corresponding aldehyde 4 using
pyridinium dichromate (PDC) in CH2Cl2.31 The Wittig-Horner
reaction of 4 and trimethyl 4-phosphonocrotonate gave methyl
dienoate 5, which upon base-catalyzed hydrolysis using 1.5 mol
equiv of KOH in methanol afforded acid 3. This acid was
obtained as a mixture between (E,E)- and (E,Z)-isomers as
determined by 1H NMR spectroscopy. We utilized urea
inclusion complexation to separate the (E,E)-acid 3a from its
(E,Z)-isomer.28 This purification was undertaken to prepare a
single lipid in order to minimize the complexity of the phase
and polymerization studies. Urea is known to form a crystalline
inclusion complex with certain compounds. The hydrogen
bonded urea molecules in methanol orient in a helical crystal
lattice in such a way as to leave a narrow cylindrical channel
with a diameter of 5.3 Å, and compounds with small cross-
section diameters can reside within these clathrate channels.32,33
The optimum ratio of the host urea to the guest fatty acid was
predetermined to be 18. The mixed-isomer fatty acid sample
was added to a methanolic solution of urea. The more linear
(E,E)-dienoyl acid 3a preferentially formed an inclusion com-
plex with the urea and precipitated. The bent (E,Z)-isomer
apparently did not fit within the urea clathrate channel.
Filtration followed by extraction of the inclusion complex with
ether produced dienoic acid that was predominantly (E,E)-isomer
based on the absence of the characteristic vinyl proton of (E,Z)-
isomer at 7.55-7.68 ppm in the 1H NMR spectrum (Figure 2).34
The selection of the protecting group for glycerol is crucial
for a successful synthesis. The deprotection step cannot interfere
with the dienoyl polymerizable group, and it must not facilitate
(25) Eibl, H. Angew. Chem., Int. Ed. Engl. 1984, 23, 257-328.
(26) Bittman, R. Phospholipid Handbook 1993, 141-232.
(27) Martin, S. F.; Josey, J. A. Tetrahedron Lett. 1988, 29, 3631-3634.
(28) Srisiri, W.; Lee, Y.-S.; O’Brien, D. F. Tetrahedron Lett. 1995, 36,
8945-8948.
(29) DeMedeiros, E. F.; Herbert, J. M.; Taylor, R. J. K. J. Chem. Soc.,
Perkin Trans. 1 1991, 2725-2730.
(30) Hebert, N.; Beck, A.; Lennox, R. B.; Just, G. J. Org. Chem. 1992,
57, 1777-1783.
(31) Corey, E. J.; Schmidt, G. Tetrahedron Lett. 1979, 5, 399-402.
(32) Smith, A. E. Acta Crystallogr. 1952, 5, 224-226.
(33) Farina, N. In Inclusion Compounds; Atwood, J. L. Ed.; Academic
Press: New York, 1984; Vol. 2, pp 69-98.
(34) Srisiri, W.; Lamparski, H.; O’Brien, D. F. J. Org. Chem. 1996, 61,
5911-5915.

4868 J. Am. Chem. Soc., Vol. 119, No. 21, 1997
Srisiri et al.
Scheme 1a
Scheme 2a
a (a) PDC, CH2Cl2. (b) NaH, trimethyl phosphonocrotonate, THF.
(c) KOH, MeOH. (d) Urea inclusion.
the deprotection proceeded without interfering with the poly-
merizable dienoyl group and isomerization of 1,2-diacylglycerol
to its 1,3-isomer.28,35
The final step was phosphorylation of 2 with a N-protected
phosphorylating agent. 2,2,2-Trichloroethoxylamide (Troc-
amide) was employed as an amine protecting group since it
could be readily removed via reduction with activated zinc under
nonhydrolytic conditions thereby avoiding reduction of the
carbon-carbon double bonds.36 Treatment of 2 with phos-
phorylating agent dichloro-[[N-[(2,2,2-trichloroethoxy)carbonyl]-
2-amino]ethyl]phosphinic acid36 afforded Troc-PE 7 in 70%
yield. The Troc-protecting group was removed by stirring 7
overnight with activated zinc in a mixture of glacial acetic acid
and ether at room temperature (rt) to yield the desired poly-
merizable PE 1 as a white solid in 80% yield.
Unsaturated PEs such as those found in biological membranes
are particularly sensitive to oxidation.37 Lipid 1 suffers from a
similar sensitivity. Samples of hydrated 1 in the presence of
oxygen, without initiator, began to noticeably deteriorate at
temperatures of 60 °C or higher as judged by an increase in
yellow color. If the lipid samples were handled under argon
they were significantly more stable. Consequently the experi-
ments described here were performed under argon to minimize
lipid degradation. The nature of the oxygen-dependent degrada-
tion of lipid 1 remains to be determined.
Phase Study of Lipid 1. Variable-temperature 31P NMR was
used to examine the phase of lipid 1 at high concentration. A
1:1 mixture of 1 and MilliQ water was incubated at 5 °C under
Ar for 48 h after hydration yielding an opaque solid. The
proton-decoupled 31P-NMR spectrum of 1 at 5 °C showed the
characteristic line shape of a well-ordered hexagonal assembly
with a baseline width of 26 ppm (Figure 3a). The sample
temperature of 1 was then increased rapidly to 60 °C and another
spectrum acquired after the temperature stabilized for ca. 5 min.
The spectrum at 60 °C had the same line shape as that acquired
at 5 °C (Figure 3b). The apparent slight narrowing of the width
(25 ppm) is probably due to increased rotational and translational
motions of the lipids at the higher temperature.
a (a) DCC, DMAP, CHCl3. (b) Me2BBr, CH2Cl2, -78 °C. (c)
Pyridine. (d) Zn/HOAc.
the isomerization from 1,2-diacylglycerol to 1,3-diacylglycerol.
The 4-methoxybenzyl ether meets these criteria.29,30 Herbert
et al. reported that its deprotection via Lewis acid Me2BBr
catalyzed hydrolysis at very low temperature (-78 °C) for a
short period of time (10-15 min) proceeded in quantitative yield
without any isomerization of 1,2-diacylglycerol.30
The acylation of 3-(4-methoxybenzyl)-sn-glycerol with 2,4,-
13-(E,E,Z)-docosatrienoic acid (3a) using dicyclohexylcarbo-
diimide (DCC) and 4-(dimethylamino)pyridine (DMAP) in
CH2Cl2 gave 1,2-bis(dienoyl)-3-(4-methoxybenzyl)-sn-glycerol
6. When an excess of fatty acid was employed, 6 was obtained
in less than 20% yield and accompanied by acid anhydride and
acid-DCC complex side products. The yield of 6 was
improved to 50% (based on fatty acid) when excess protected
glycerol (1.5 mol equiv) was used. The deprotection of the
4-methoxybenzyl ether group was accomplished by treating it
with excess dimethylboron bromide in dry CH2Cl2 at -78 °C
for 15 min. The reaction was quenched by diluting with ether
and washing with water until the aqueous extracts were neutral.
The purity of the product was determined by thin layer
chromatography and 1H NMR spectroscopy, which showed that
X-ray diffraction characterization of the phase behavior of a
1:1 mixture of 1 and MilliQ water revealed the formation of
(35) Ponpipom, M. M.; Bugianesi, R. L. J. Lipid Res. 1980, 21, 136-
139.
(36) Pfeiffer, F. R.; Cohen, S. R.; Weisbach, J. A. J. Org. Chem. 1969,
34, 2795-2796.
(37) Evstigneeva, R. In Phospholipids Handbook; Cevc, G. Ed.; Marcel
Dekker, Inc: New York, 1993; pp 323-334.

Polymerization of the InVerted Hexagonal Phase
J. Am. Chem. Soc., Vol. 119, No. 21, 1997 4869
Figure 2. 1H-NMR spectra of (a) mixture of (E,E)- and (E,Z)-dienoic acid 3, (b) (E,Z)-dienoic acid, and (c) (E,E)-dienoic acid 3a.
Figure 4. Variation in unit cell parameters measured via X-ray
diffraction as a function of temperature along the isopleth 1/1 lipid to
MilliQ water. Squares ) double diamond bicontinuous cubic phase,
triangles ) HII phase, and circles ) polymerized HII phase. Closed
symbols indicate increasing temperature run and open decreasing
temperature run.
phases. Wide-angle X-ray diffraction showed a single diffuse
ring centered at 4.5 Å for all temperatures, consistent with the
phases having fluid hydrocarbon chains. At 0 °C the two phases
coexist. The dominant signal is due to the HII phase, although
it is difficult to quantitate the contribution of the bicontinuous
cubic phase. The absence of an isotropic component in the
31P-NMR spectrum of a sample of lipid 1/water with a weight
Figure 3. 31P-NMR spectra of lipid 1 and water (1:1 by wt) at (a) 5
°C and (b) 60 °C.
ratio of 1 at 5 °C indicates that the amount of the cubic phase
is quite small. An attempt to produce a pure double diamond
two liquid crystalline phases, an inverse hexagonal and a
bicontinuous cubic (consistent with the Pn3hm space group) over
the temperature range 0-80 °C. Figure 4 shows the variation
with temperature in the unit cell parameters for each of these
(Pn3hm) bicontinuous cubic phase by continued heat cycling and
incubation about the temperature range of the transition from
lamellar to nonlamellar (ca. -10 °C) was unsuccessful.38
A
pure lamellar phase with a lattice parameter of approximately

4870 J. Am. Chem. Soc., Vol. 119, No. 21, 1997
Srisiri et al.
a
Figure 6. Absorption spectra of methanol extractions of samples of 1
before (a) and after (b) polymerization with potassium persulfate/L-
cysteine as described in the Experimental Section.
erization was performed at 60 °C in the absence of oxygen,
with a ratio of monomer to initiator of 8. This redox system
generates hydroxyl radicals which are free to diffuse into the
nonlamellar assemblies where they react with the polymerizable
dienoyl groups. Redox polymerization of similar dienoyl lipids
in lamellar assemblies afforded polymers having a degree of
polymerization of 30.24 The solubility of 1 in organic solvents
was tested before and after polymerization. The unpolymerized
lipid 1 was soluble in several organic solvents at room
temperature, whereas the poly-1 was insoluble in organic
solvents including 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP).
This dramatic change in solubility confirmed that the polymer
obtained was cross-linked.19 The conversion to polymer was
determined by methanol extraction of a known weight of freeze-
dried lipid and subsequent observation of the intensity of the
dienoyl group at 265 nm. The UV spectrum showed that the
conversion was greater than 90% (Figure 6).
b
Hydrogen peroxide (30% in water) was employed as a redox
initiator for polymerization of the HII phase at a lipid/water
weight ratio of 1. After addition of the same weight deoxy-
genated H2O2 solution to 1, the sample was centrifuged at rt
and kept at 5 °C for 1 day to allow the sample to equilibrate.
At this time, the UV spectrum of the lipid showed no decrease
in the absorbance of the dienoyl peak at 265 nm; therefore, little
or no polymerization took place during this initial incubation
period. The sample was then incubated at 60 °C under Ar(g)
for 2 days to perform the polymerization. The UV spectrum
of the methanol-extracted polymerized lipid showed that the
conversion to polymer was ca. 90%. The polymers obtained
were insoluble in organic solvents including HFIP, the same as
those obtained at low concentration. A control experiment with
DOPE and the redox chemistry showed that the cis-double bonds
in each acyl chain did not react under these conditions.
Phase Study of Polymerized Lipid 1. Lipid 1 was hydrated
with MilliQ water at a concentration of 100 mg/mL. An opaque
solid in excess water was observed over the temperature range
5-90 °C. The proton-decoupled 31P-NMR specrtum of 1 at
25 °C showed the characteristic line shape of an inverted
hexagonal assembly in excess water with a width of 26 ppm.
The line shape is indicative of a multidomain hexagonal
assembly which is expected upon hydration in excess water.
The excess water at the edge of the assembly somewhat distorts
the line shape (Figure 7a). The characteristic 31P-NMR line
shape for an inverted hexagonal assembly was retained after
redox polymerization, but with a slightly broader width of 27-
30 ppm (Figure 7b). Addition resonances within the baseline
were observed on the sides of the hexagonal line shape.
Figure 5. Azimuthal integration of the two-dimensional X-ray
diffraction patterns upon heating of a hydrated 1:1 lipid to MilliQ water
mixture: (a) 25 °C, coexistence of double diamond (112.6 Å) and
inverse hexagonal (76.1 Å) phases; (b) 60 °C, hexagonal phase (72.6
Å). Tick marks indicate the expected positions of the appropriate
corresponding lattice.
55 Å was observed below -20 °C. Further characterization of
the low-temperature behavior was complicated by the partial
freezing of sample water. At temperatures above 40 °C the HII
phase was found in a pure state. The measured lattice
parameters for the hexagonal and the cubic phases decreased
on heating in a reversible manner, varying between 81.1 Å at
0 °C and 69.3 Å at 80 °C and 121.2 Å at 0 °C and 108.2 Å at
40 °C, respectively. Figure 5 shows the corresponding azi-
muthal integrations of the two-dimensional X-ray diffraction
patterns in each of the regions along this isopleth.
Polymerization of the HII Phase. Polymerization studies
of the HII phase were performed at both low and high
concentrations. Redox-initiated radical polymerization using
K2S2O8/L-cysteine (1/1) as the initiator was employed for the
HII assemblies at low concentration (100 mg/mL). The polym-
(38) Shyamsunder, E.; Gruner, S. M.; Tate, M. W.; Turner, D. C.; So,
P. T. C.; Tilcock, C. P. S. Biochemistry 1988, 27, 2332-2336.

Polymerization of the InVerted Hexagonal Phase
J. Am. Chem. Soc., Vol. 119, No. 21, 1997 4871
a
b
Figure 7. 31P-NMR spectra of hydrated lipid 1 (100 mg/mL) before
(a) and after (b) polymerization as described in the Experimental
Section.
Presumably this is due to the decrease in rotational and
translational motions of the lipids upon polymerization of the
assembly unit cells.
A sample of lipid 1 in aqueous hydrogen peroxide in the HII
phase at 60 °C (being the only phase along this isopleth) was
polymerized using thermal initiation of the hydrogen peroxide.
Polymerization was followed in-situ using X-ray diffraction over
a period of 48 h. The pattern was observed to weaken over the
course of the reaction, but still indexed to a hexagonal phase.
After 48 h the pattern no longer evolved. At this point a
temperature cycle showed that the pattern remained unaltered
on decreasing temperature while maintaining the same lattice
parameter (70.6 Å as compared with 72.6 Å for the unpoly-
merized sample at 60 °C), unlike that of the unpolymerized
phase where the value increased with decreasing temperature.
The sample remained stable without further alteration. The
spectrum characteristic of the double diamond phase was not
observed to form. Figure 8 shows the diffraction patterns of
the polymerized HII phase at 60 and 20 °C. The diffraction
suggests that the hexagonal phase is more poorly ordered after
than before polymerization. The predominant diffraction is
consistent with a hexagonal phase, even though the asymmetry
of the (1,0) peaks suggests that the sample may contain some
small fraction of material in either a different phase or different
lattice spacing.
Figure 8. Azimuthal integration of the two-dimensional X-ray
diffraction patterns of polymerized HII phase following 48 h incubation
at 60 °C in a 30% H2O2 solution such that lipid to total solvent is 1:1:
(a) 60 °C, with hexagonal lattice parameter equal to 70.6 Å and (b)
corresponding pattern after the temperature is decreased to 20 °C, lattice
parameter 71.0 Å. Note weakening of the pattern in comparison to that
of the unpolymerized sample at 60 °C and the lack of formation of the
double diamond phase and constancy of the pattern on reducing
temperature as compared with a similar sequence for the unpolymerized
system. Tick marks indicate the expected positions of the corresponding
hexagonal lattice.
the design of lipids for the polymerization of the HII phase. The
synthetic route employed for the preparation of 1 was straight-
forward with generally high yields for each step. This procedure
allows the preparation of research quantities of the polymerizable
lipid in high purity, and it can be readily adapted for the
preparation of lipids with other substitution patterns in the fatty
acid tails. The critical design features of 1 include the
following: a relatively poorly hydrated PE head group to
decrease the head group size and permit the membrane contact
required for lipid reorganization during the lamellar to non-
lamellar transition; cis-double bonds in each lipid tail to enhance
chain disorder;39 and long lipid tails (22 carbons) to lower the
lamellar to nonlamellar phase transition temperature.40 The
Conclusions
Phosphoethanolamines are the most thoroughly studied lipids
that readily form the HII phase.2 These lipids when hydrated
usually show a thermotropic transition from the gel (Lâ) phase
to the liquid-crystalline (LR) phase (designated Tm) and a higher
temperature, lower enthalpy transition from the LR to the HII
phase. This literature provides an excellent starting point for

4872 J. Am. Chem. Soc., Vol. 119, No. 21, 1997
Srisiri et al.
relative significance of these features in nonpolymerizable lipids
was examined by Lewis et al.40 We are currently exploring
the effect of lipid tail structure on the phase behavior of
polymerizable PEs.
tetrahydrofuran at 0 °C. After 1 h, oleyl aldehyde (2.0 g, 7.5
mmol) in 15 mL of THF was added dropwise at 0 °C. The
reaction was then allowed to warm to rt and monitored by TLC
using hexane/EtOAc (95/5) as the mobile phase. After the
reaction was completed, excess NaH was destroyed by slow
addition of cold water. After evaporation of THF, the residue
was diluted by diethyl ether and extracted several times with
water and brine. The organic layer was dried with anhydrous
MgSO4, then concentrated. The crude product was purified by
column chromatography using hexane/EtOAc (95/5) to give
methyl ester 5 in 75% yield. 1H-NMR (CDCl3): 7.32-7.22
(m, 1H), 6.17-6.12 (m, 2H), 5.82-5.76 (d, J ) 15.38 Hz, 1H),
5.37-5.33 (m, 2H), 3.74 (s, 3H), 2.18-2.00 (m, 6H), 1.42-
1.27 (b, 22H), 0.91-0.85 (t, J ) 6.52 Hz, 3H) ppm.
2,4,13-(Z)-Docosatrienoic Acid (3). The methanolic solution
of methyl ester 5 (2.0 g, 5.7 mmol in 100 mL) was treated with
1.5 mol equiv of 85% aqueous solution of KOH. The mixture
was refluxed gently until the reaction was finished (about 5 h)
as determined by TLC using hexane/ethyl acetate (95/5) as the
mobile phase. The methanolic solution was concentrated and
then diluted with ether. After the solution was acidified to pH
3 with dilute HCl solution, it was extracted several times with
water. The organic layer was dried with anhydrous MgSO4 and
then concentrated, affording the crude dienoic acid 3 in 85%
yield.
Although the molecular design was successful for this study,
the coexistence of a bicontinuous cubic and HII phases over
part of the temperature range investigated was somewhat
surprising. Whether this is a consequence of the water to lipid
ratio (1:1 by weight), which is an excess of water required for
the HII phase, or is intrinsic to this lipid remains to be
determined. The water to lipid ratio was selected to facilitate
the addition of the initiator after the formation of the HII phase.
The initiator chemistry yields radicals that can diffuse from the
water into the lipid. Other polymerization protocols that could
be employed without excess water are also being examined.
The successful polymerization of the HII phase of 1 at 60 °C
effectively fixed the diameter of the hexagonal unit cell at 70.6
Å even after the sample was cooled to 20 °C. In the absence
of polymerization the diameter would have increased to 77 Å.
This observation is consistent with the previous observation of
Tate and Gruner that the change of the lattice vector with
temperature is primarily due to variation in the water channel
size.5 Furthermore it suggests the dimensions of the unit cell
of a given polymerizable lipid in the HII phase can be varied
by temperature prior to polymerization and then fixed by
polymerization at a desired temperature. It has not escaped our
notice that this characteristic provides a means to prepare lipid
tubes of selected dimension that have an aqueous interior and
a hydrophobic exterior. This is a consequence of the polym-
erization of the unit cell of the HII phase without polymerization
of the whole assembly. We have already demonstrated that the
polymerized HII phase of 1 can be dispersed by selected organic
solvents to yield supramolecular polymers. The nature of these
polymers is currently under study and will be reported in due
course.
2,4,13-(E,E,Z)-Docosatrienoic Acid (3a). A well-stirred
solution of urea (5.0 g, 83 mmol) in methanol (100 mL) was
treated with a solution of acid 3 (2.0 g, 5.7 mmol) in methanol
(100 mL). The solution was then kept at 0 °C overnight. The
needle-like crystals were filtered, washed many times with
methanol, and then dried under vacuum. These crystals were
dissolved in ether and washed several times with dilute HCl
solution and water. The organic layer was combined and dried
with anhydrous MgSO4. After concentration, the crude acid
was purified by recrystallization from hexane at -30 °C, giving
the ester 3a as colorless needles in 80% yield. 1H-NMR
(CDCl3): 7.39-7.31 (m, 1H), 6.25-6.14 (m, 2H), 5.90-5.72
(d, J ) 15.22 Hz, 1H), 5.37-5.28 (m, 2H), 2.34-2.01 (m, 6H),
1.44-1.31 (b, 22H), 0.92-0.86 (t, J ) 6.77 Hz, 3H) ppm.
1,2-Bis[2,4,13-(E,E,Z)-docosatrienoyl]-3-(4-methoxybenzyl)-
sn-glycerol (6). 4-Methoxybenzyl-protected glycerol (0.3 g,
1.4 mmol), acid 3a (0.6 g, 1.8 mmol), and DMAP (0.2 g, 1.8
mmol) were dissolved in 20 mL of chloroform, and then 0.4 g
(1.8 mmol) of DCC in 10 mL of chloroform was added. After
being stirred at rt overnight, the urea was filtered and the filtrate
was concentrated. The crude product was purified by column
chromatography using hexane/EtOAc (9/1) to give the protected
glyceride 6 in 50% yield. 1H-NMR (CDCl3): 7.19-7.15 (m,
4H), 6.81-6.77 (d, J ) 8.69 Hz, 2H), 6.10-6.05 (m, 4H), 5.76-
5.65 (dd, J ) 12.65, 15.20, 2H), 5.37-5.20 (m, 2H), 5.25-
5.23 (m, 1H), 4.42-4.40 (d, J ) 13.62 Hz, 2H), 4.31-4.25
(m, 2H), 3.72 (s, 3H), 3.55-3.53 (d, J ) 5.14 Hz, 2H), 2.30-
2.00 (m, 12H), 1.50-1.30 (b, 44H), 0.83-0.78 (m, 6H) ppm.
1,2-Bis[2,4,13-(E,E,Z)-docosatrienoyl]-sn-glycerol (2). A
solution of the protected glycerol 6 (0.1 g, 0.1 mmol) in dry
dichloromethane at -78 °C was treated with dimethylboron
bromide (0.1 mL, 1.2 mmol) by addition through a syringe under
argon. The reaction mixture was stirred for 15 min, while the
dry-ice bath was removed. The reaction was then diluted with
ether and quenched with water slowly. The organic layer was
washed many times with water until the aqueous part was
neutral. After evaporation of the solvent, the crude glyceride
2 was purified by column chromatography using hexane/EtOAc
(8/1) and then (1/1) (80% yield). 1H-NMR (CDCl3): 7.35-
7.23 (m, 2H), 6.18-6.15 (m, 4H), 5.84-5.76 (m, 2H), 5.35-
Experimental Section
Methods and Materials. All chemicals were obtained from
Aldrich Chemical Corp., except trimethyl 4-phosphonocrotonate,
which was purchased from Lancaster Synthesis Inc. Solvents
were dried and distilled prior to use. Compounds that contained
UV-sensitive groups were handled under yellow light. The
reactions were monitored by TLC visualized by UV light and/
or phosphomolybdic acid reagent. NMR spectra were recorded
on a Bruker AM-250 magnetic resonance spectrometer in
chloroform-d with TMS as an internal reference.
Synthesis of 1. 9-(Z)-Octadecenal (4). Pyridinium dichro-
mate (PDC, 4.2 g, 11.1 mmol) was added to a solution of oleyl
alcohol (2.0 g, 7.4 mmol) in 30 mL of dichloromethane. The
reaction was stirred at rt for 18 h and then filtered through silica
gel to remove the used PDC. The filtrate was concentrated,
and the crude product was purified by column chromatography
(hexane/EtOAc, 95/5) (75% yield). 1H-NMR (CDCl3): 9.77
(m, 1H), 5.34-5.32 (m, 2H), 2.43-2.39 (m, 4H), 2.02-2.00
(m, 4H), 1.63-1.60 (2H), 1.30-1.27 (b, 18H), 0.90-0.85 (t, J
) 6.09 Hz, 3H) ppm.
Methyl 2,4,13-(Z)-Docosatrienoate (5). A solution of tri-
methyl 4-phosphonocrotonate (2.0 g, 9.6 mmol) in 10 mL of
tetrahydrofuran was added slowly to a suspension of sodium
hydride (60% dispersion in mineral oil, 0.4 g) in 5 mL of
(39) Cullis, P. R.; de Kruijff, B.; Hope, M. J.; Verkleij, A. J.; Nayar, R.;
Farren, S. B.; Tilcock, C. P. S.; Madden, T. D.; Bally, M. B. In Membrane
Fluidity in Biology; Aloia, R. C., Ed.; Academic Press: New York, 1983;
Vol. 1, pp 39-81.
(40) Lewis, R. N. A. H.; Mannock, D. A.; McElhaney, R. N.; Turner,
D. C.; Gruner, S. M. Biochemistry 1989, 28, 541-548.

Polymerization of the InVerted Hexagonal Phase
J. Am. Chem. Soc., Vol. 119, No. 21, 1997 4873
5.19 (m, 4H), 5.19-5.15 (m, 1H), 4.62-4.37 (d, J ) 5.08 Hz,
2H), 3.81-3.77 (m, 2H), 2.48 (b, 1H), 2.20-2.13 (m, 12H),
1.45-1.20 (b, 44H), 0.91-0.85 (m, 6H) ppm.
position of the lipid over time and with increase in temperature.
Capillaries were made by injection of a solution of the lipid in
cyclohexane which was then lyophilized, and the capillaries were
weighed to determine the amount of lipid and the corresponding
amount of deionized water was added. Capillaries were then
flame sealed. Samples were made and kept in the dark.
X-ray diffraction data were obtained using a Rigaku RU-
200 rotating anode X-ray diffractometer equipped with a
microfocus cup. The generated Cu KR X-rays were focused
via bent mirror optics. Two-dimensional X-ray images were
collected with the Princeton PM area detector.41 The digital
powder diffraction images were azimuthally integrated along
an arc of (15° from the meridional axis to generate plots of
scattered intensity versus Q ) 4π sin d/1.54 Å, where 2d is the
angle between the incident and scattered beam directions.
Thermal polymerization was affected by replacing the deion-
ized water with a 30% solution of H2O2, which was used as the
source of hydroxyl radicals at elevated temperatures, which
diffuse into the hydrocarbon region to react with the dienoyl
moieties. Samples were incubated at 60 °C for 2 days.
Polymerization of Hydrated Lipid 1. Potassium Persul-
fate/L-Cysteine (1/1) Initiator. Lipid 1 (6 mg) was hydrated
with an argon-flushed K2S2O8 buffer solution (60 µL) through
several freeze-thaw-vortex cycles and then incubated at 60
°C. Potassium persulfate (300 mg, 1.1 mmol) and L-cysteine
(174 mg, 1.1 mmol) were diluted to 10 mL with deoxygenated
MilliQ water. A volume of this solution was added to the
thermally incubated lipid sample to give [M]/[I] ) 8. After
polymerization under Ar(g) at 60 °C for 2 days, the sample
was freeze-dried. The methanol extract of a known weight lipid
was then characterized using UV spectroscopy. The absorbance
of dienoyl peak at 265 nm was measured for the calculation of
percent conversion to polymer. The residue of sample after
extraction with methanol was tested for its solubility in organic
solvents including chloroform, dichloromethane, tetrahydrofuran,
and HFIP.
H2O2 Initiator. Aqueous hydrogen peroxide solution (30%
H2O2 in water, 6 µL) was flushed with argon then added to 6
mg of lipid 1. The sample was centrifuged at rt and equilibrated
at 5 °C for 1 day. The polymerization was then performed at
60 °C under Ar (g) for 2 days, after which the sample was
freeze-dried. The isolated polymer was then characterized as
described above.
DOPE Control. A sample of DOPE (60 mg) was hydrated
with an argon-flushed K2SO4 buffer solution (2 mL) and
incubated at 60 °C for a day to form the HII phase. Potassium
persulfate solution (200 mM) and L-cysteine solution (200 mM)
were then added to the hydrated lipid to give [M]/[I] ) 8. After
incubation of the reaction mixture for 2 days at 60 °C, the
sample was freeze-dried and then partly dissolved in chloroform.
The chloroform extract was concentrated, and its 1H-NMR
spectrum was taken in chloroform-d. The ratio of integration
area between vinyl and terminal methyl protons on the lipid
chains was to 2:3, the same as that obtained for DOPE prior to
the reaction. The chloroform-insoluble residue was totally
soluble in water, precluding the presence of lipid polymers in
this fraction.
N-[(2,2,2-Trichloroethoxy)carbonyl][1,2-bis[2,4,13-(E,E,Z)-
docosatrienoyl]-sn-glycerol]-3-phosphoethanolamine 7. The
solution of 2 (50 mg, 0.07 mmol) and pyridine (0.03 mL) in
chloroform (5 mL) was transferred slowly into the solution of
dichloro-[[N-[(2,2,2-trichloroethoxy)carbonyl]-2-amino]ethyl]-
phosphinic acid (42 mg, 0.12 mmol) in benzene (2 mL) under
Ar(g) at 0 °C. The reaction was allowed to warm to rt and
stirred overnight. After dilution with ether, it was extracted
with water, dilute HCl solution, saturated NaHCO3 solution,
and then brine. The organic layer was dried with anhydrous
MgSO4 and concentrated. The crude product was purified by
column chromatography using hexane/EtOAc (1/1) and then
chloroform/methanol/water (65/25/4) (70% yield). 1H-NMR
(CDCl3): 7.59-7.25 (m, 2H), 6.78-6.53 (b, 1H), 6.21-6.19
(m, 4H), 5.83-5.75 (m, 2H), 5.39-5.25 (m, 5H), 4.76 (s, 2H),
4.49-3.95 (m, 6H), 3.49-3.45 (b, 2H), 2.37-2.00 (m, 12H),
1.42-1.03 (b, 44H), 0.89-0.83 (m, 6H) ppm.
[1,2-Bis[2,4,13-(E,E,Z)-docosatrienoyl]-sn-glycerol]-3-phos-
phoethanolamine (1). Zinc dust (0.2 g, 3.2 mmol) was
carefully added to the solution of 7 (0.4 g, 0.4 mmol) in glacial
acetic acid (6 mL) and diethyl ether (3 mL). The suspension
was stirred under Ar(g) at rt overnight. The mixture was then
diluted with ether, and zinc metal was filtered off. The filtrate
was washed many times with water, saturated NaHCO3 solution,
and then brine. The organic layer was dried with anhydrous
MgSO4 and concentrated. The crude product was purified by
column chromatography using chloroform/methanol/water (65/
25/4). Bis-PE lipid 1 was obtained as a white solid in 80%
yield. 1H-NMR (CDCl3): 8.40-8.12 (b, 3H), 7.69-7.15 (m,
2H), 6.13-6.04 (m, 4H), 5.91-5.70 (m, 2H), 5.37-5.20 (b,
5H), 4.34-3.78 (b, 6H), 3.27-3.15 (m, 2H), 2.30-1.97 (m,
12H), 1.36-1.18 (b, 44H), 0.86-0.82 (m, 6H) ppm. FAB-
MS: 849.4 (M+), 707.6 (M+ - [OPO2CH2CH2NMe2]), 360.3
(707.6-dienoyl acid). Elemental analysis. Calcd: C, 69.40; H,
10.15; N, 1.65; O, 15.12; P, 3.66. Found: C, 69.73; H, 9.85.
Phase Behavior of Hydrated Lipid 1. 31P NMR.
A
hydrated PE lipid sample was sealed in a 5 mm NMR tube.
The sample temperature was controlled to (0.2 °C. The proton-
decoupled 31P-NMR spectra were acquired on a Varian-Unity
300 spectrometer operating at 121.4 MHz (7.0 T). A phase
cycled pulse sequence (90°-τ1-180°-τ2-acquisition) was used
with a 90° pulse of 14 µs, the delay τ1 of 100 µs, the time
before acquisition τ2 of 60 µs, and the delay between sequences
of 0.5 s. At low lipid concentration, the free induction decays
consisted of 80 000-90 000 acquisitions and were multiplied
by a line broadening of 100 Hz after Fourier transform. At
high lipid concentration, the free induction decays consisted of
5000 acquisitions and were multiplied by a line broadening of
50 Hz after Fourier transform.
The polymerized sample was handled in the same manner
except that the NMR tube and sample were flushed with argon
to provide an oxygen free environment. Polymerization was
initiated by the addition of a 13 µL aliquot of potassium
persulfate/sodium bisulfite (1/1) initiator (see below) to the NMR
sample. After polymerization the NMR spectrum was obtained
as above.
Acknowledgment. The authors thank the NSF-CTS (D.F.O.),
the Army Research Office, NSF, and DOE (S.M.G.) for support
of this research. We thank Professor Go¨ran Lindblom for
helpful discussions.
X-ray Diffraction. Phase behavior of 1 was determined
along a single isopleth over the temperature range -40 to 80
°C at a concentration of 1:1 water to lipid. All samples were
made directly into X-ray transparent capillaries under an argon
atmosphere: failure to either store the lipid under argon or make
the liquid crystalline samples under argon resulted in decom-
JA970052X
(41) Gruner, S. M.; Milch, J. R.; Reynolds, G. T. ReV. Sci. Instrum. 1982,
53, 1770-1778.