Self-Assembly in a Bipolar Phosphocholine-Water System: The Formation of Nanofibers and Hydrogels

Article abstract of DOI:10.1002/anie.200351731

The bolaamphiphile PC-C32-PC with two phosphocholine headgroups and one long C₃₂ alkyl chain (see the space-filling model, left) forms nanofibrils in aqueous solution at room temperature solely by hydrophobic interactions. The gels that result were studied by electron microscopy (image on the right), differential scanning calorimetry, and FT-IR spectroscopy. At higher temperatures the fibers "melt" and small aggregates form.

Full text of DOI:10.1002/anie.200351731

Angewandte
Chemie
Nanomaterials
Self-Assembly in a Bipolar Phosphocholine–
Water System: The Formation of Nanofibers and
Hydrogels
Karen Köhler, Günter Förster, Anton Hauser,
Bodo Dobner, Ulrich F. Heiser, Friederike Ziethe,
Walter Richter, Frank Steiniger, Markus Drechsler,
Heiko Stettin, and Alfred Blume*
Bipolar lipids with only one long alkyl chain and two polar
headgroups at the ends are model compounds for the
chemically more complicated bola lipids from Archea bac-
teria, for instance, the bis(diphytanyl) diglycerol tetraethers.[1]
In Irlbachia alata and Anthocleista djalonensis, the bola lipid
docosan-1,22-diyl-bis-[2-(trimethylammonio)ethylphosphate]
(irlbacholine) has a C22 alkyl chain that connects two polar
phosphocholine groups and has been used in plant medicine.
It was found to be effective against the fungus Trichophyton
rubrum, which affects the skin, and other fungal infections.[2]
In the course of studies on the possibility of using similar
bola lipids with one long alkyl chain as membrane stabilizers,
a bola phospholipid with a C32 alkyl chain was synthesized,
namely the lipid dotriacontan-1,32-diyl-bis-[2-(trimethylam-
monio)ethylphosphate] (PC-C32-PC, 1; Figure 1).[3] During
the initial characterization of 1 we observed unusual aggre-
gation behavior, which necessitated further physico-chemical
investigations. To obtain sufficient quantities of 1 a more
efficient synthetic route to long-chain bola compounds was
necessary.
Figure 1. Structure of 1 with a bulky, polar phosphocholine headgroup
at each end of the C32 hydrocarbon chain.
hydrolysis. The importance of the new reaction is underlined
by the possibility of functionalizing the 1- and 32-positions.
The phosphocholine moieties were introduced by the reaction
of dotriacontan-1,32-diol with 2-bromoethyl phosphoric acid
dichloride in chloroform. This classic phosphorylation
reagent was more effective than more modern variants. The
trimethylammonium groups in 1 were introduced by estab-
lished procedures.[4]
Because of the long alkyl chain and the large polar
headgroups at both chain ends of 1, it forms novel types of
aggregate structures not observed previously. We investigated
the self-assembly of the bipolar compound in aqueous
solution using differential scanning calorimetry, electron
microscopy, X-ray scattering, viscosity measurements, and
FT-IR spectroscopy.
We prepared lipid 1 in gram quantities by a newly
developed simple and effective procedure. The construction
of the chain with 32carbon atoms is possible in a one-step
synthesis by a copper-catalyzed coupling of undec-10-en-1-
ylmagnesium bromide with 1,10-dibromodecane in a molar
ratio of 2:1 (see Experimental Section). Dotriacontan-1,31-
diene is then converted into dotriacontan-1,32-diol by hydro-
boration with disiamyl borane and subsequent oxidative
In highly dilute aqueous solution (c < 0.5 wt%), a very
viscous, almost transparent gel is observed. The 0.1-wt%
sample shown in Figure 2has a water/ 1 molar ratio of ca.
45000:1; we see that an enormous
amount of water can be “immobilized”
by the gel structure. At temperatures
[*] Dipl.-Chem. K. Köhler, Dr. G. Förster, Dr. A. Hauser, Prof. Dr. A. Blume
MLU Halle-Wittenberg
Institut für Physikalische Chemie
Mühlpforte 1, 06108 Halle/Saale (Germany)
Fax : (+49)345-5527157
above 508C or through mechanical stress
the gel character disappears and a fluid
E-mail: blume@chemie.uni-halle.de
solution is obtained; the viscosity drops
Prof. Dr. B. Dobner, Dr. U. F. Heiser, Dr. F. Ziethe
MLU Halle-Wittenberg
Institut für Pharmazeutische Chemie
by about five orders of magnitude. Pre-
liminary viscosity measurements at room
temperature show an indication of a
yield stress (t0*) of roughly 7.2Pa for a
Wolfgang-Langenbeck-Strasse 4, 06120 Halle/Saale (Germany)
Dr. W. Richter, F. Steiniger
FSU Jena, Institut für Ultrastrukturforschung
Ziegelmühlenweg 1, 07740 Jena (Germany)
0.8-wt% solution (Figure 3). Tempera-
ture-dependent viscosity measurements
indicate a viscosity of ca. 5 ” 103 Pas at
Dr. M. Drechsler
FSU Jena, Institut für Pharmazie
Philosophenweg 14, 07743 Jena (Germany)
room temperature which drops to values
Figure 2. Hydrogel
below 5 ” 10À4 Pas at temperatures
of a solution of 1
above 708C, that is, values close to that
Dr. H. Stettin
(0.1 wt%)in water
of pure water at the same temperature.
The rheological behavior found for this ture.
Physica Messtechnik GmbH
Helmuth-Hirth-Strasse 6, 73760 Ostfildern (Germany)
at room tempera-
Angew. Chem. Int. Ed. 2004, 43, 245 –247
DOI: 10.1002/anie.200351731
ꢀ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
245

Communications
self-assembly would result in a helical structure with a
thickness of roughly one molecular length.
The formation of gels in aqueous solution induced by
organic molecules is well known, particularly in the polymer
field. However, the type of gels formed by aggregating low-
molecular-weight molecules can be quite different because
the gel formation is connected to a self-assembly process. Gel
formation has been observed for other bola compounds, such
as nucleotide bolaamphiphiles and compounds containing
bisurea dicarboxylic acid groups.[6] In these cases, the gel
formation arises mainly through polar interactions, especially
hydrogen bonds. As a consequence, the fibers formed are
much thicker. In gels of 1, however, intermolecular polar
interactions do not play a role, and the aggregation process
seems to be driven exclusively by hydrophobic effects, that is,
the minimization of the hydrophobic surface area of the long
alkyl chains.
Figure 3. Rheological measurements on an aqueous solution of 1 with
constant shear stress (controlled stress oscillation). The characteristic
relation between the storage and loss moduli G’>G’’ indicate the
region of linear strain response; the shear stress amplitude g0
Freeze-fracture electron microscopy was used to inves-
tigate the lipid self-assembly in more concentrated samples in
the presence of glycerol (Figure 5). In a sample quick-frozen
*
increases linearly up to the yield stress of t0 =7.2 Pa. T=238C,
w=10 Hz, c=0.8 wt%.
system is complicated and in some way related to the behavior
of other association colloids such as wormlike micelles.[5]
.
The gel state at room temperature can be explained by the
existence of a dense three-dimensional network of fibrils,
which can be found even in highly dilute aqueous suspensions
(0.03 wt%) by cryo-transmission electron microscopy. All the
fibrils are approximately 6–7 nm thick (Figure 4). This
Figure 5. Freeze-fracture electron microscopic picture of a solution of
Figure 4. Cryo-TEM image of an aqueous suspension of 1 (0.03 wt%).
A nanofiber with helical structure is marked with an arrow.
1 (2 wt%)in H O/glycerol (4:1). Top: Sample was kept for one day at
2
238C and then quick-frozen. Bottom: Sample was heated to 808C and
then quick-frozen.
corresponds roughly to the length of 1. The fibrils appear to
have a helical structure, but the resolution of cryo-electron
micrographs is notoriously poor, and an unequivocal descrip-
tion of the structure is not possible. Since 1 is not chiral there
must be just as many right-handed as left-handed helices.
Indeed, in some cryo-transmission electron micrographs
fibrils with opposite handedness seem to be present. Because
the fibers are so small in diameter, it is not possible to obtain
quantitative statistics. The formation of helices could be
explained by the following: two or more molecules are
arranged side by side in one direction, but because of the
bulky headgroups the chains cannot adopt a completely
parallel packing in the direction perpendicular to the first one.
To optimize the hydrophobic interaction the long axes of
adjacent molecules twist relative to each other. This type of
from room temperature to À1968C the fracture faces show
oriented fibrils 7 nm thick sticking out of the surface at a
certain angle. In samples cooled from 458C this structure is
still present, but when the samples are quenched from starting
temperatures of 508C or even 808C the fibrillar structure has
disappeared and only aggregates or smaller particles are seen
with diameters in the range of 3–10 nm. We also investigated
solutions quenched from 808C using dynamic light scattering
(DLS). We found that the sample consists of both small
monodisperse particles (radius 2–5 nm) as well as larger
aggregates. When the samples were kept at room temperature
for longer times, the viscosity increased strongly, again
indicating the reformation of the gel, that is, the formation
of nanofibrils.
246
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Angewandte
Chemie
decane (6 g, 20 mmol) in dry THF (20 mL) was added in one portion.
To detect the exact temperature for the transition of the
nanofibrils into smaller aggregates DSC measurements were
performed. Three endothermic peaks were seen upon heating
and a strong hysteresis in the transition behavior was
observed. After the first heating a defined annealing protocol
had to be applied to reproduce the initial data. For all
measured concentrations a main transition with the largest
latent heat is found at Tꢀ 488C along with a peculiar jump in
the heat capacity. This could be a consequence of changes in
the interactions of the chain region with water, that is, an
increase in the wetting of hydrophobic surfaces. At this
particular temperature the breakdown of the gel was also
observed.
After the mixture had been stirred for 2min, dilithium tetrachloro-
cuprate (3.5 mL, 1m solution in THF) was added dropwise at 08C.
Within some minutes the reaction mixture became turbid. After
20 min at 08C the mixture was diluted with 20 mL of dry THF, and
stirring was continued for a further 3 h at the same temperature. For
the workup of the reaction 100 mL of ether were added and the
resulting mixture was poured into a cold saturated solution of
ammonium chloride. The organic layer was separated and the
aqueous phase was extracted with ether (50 mL). The combined
organic phases were washed with water, dried over sodium sulfate,
and concentrated to dryness under reduced pressure. For purification
the residue was dissolved in 100 mL of ether/THF (1:1). After
addition of 70 mL of acetone, the precipitate was filtered off. This
precipitation procedure was repeated to give the pure product as a
waxy white solid. Yield: 7.8 g (87%), m.p. 59–618C (uncorrected);
ESI-MS: 446 [M+]; 1H NMR: (CDCl3, 400 MHz, 298 K): d = 1.18–
The largest heat for the main transition was observed
when the sample had been kept at 28C for eight hours and the
gel had fully developed. At room temperature apparently
metastable states (smaller aggregates) can occur which only
slowly convert into the equilibrium aggregation state (nano-
fibers). In the heating curves a further small endothermic
transition at Tꢀ 408C is seen, and at T= 738C there is
another very broad peak. In contrast to the other transitions
the high-temperature peak does not change its position and
shape under various experimental conditions (e.g. different
heating rates or longer equilibration at low temperatures).
To find out if conformational changes in the chains occur
at the different transition temperatures, FT-IR measurements
were carried out. The symmetric and antisymmetric CH2
stretching vibrations are reliable indicators of the order of
the hydrocarbon chains of alkyl compounds. In FT-IR spectra
recorded between 28C and 858C, two frequency increases of
the stretching modes at T= 478C and 728C indicate a
stepwise formation of gauche conformers. These temper-
atures correspond to the temperature of the two upper peaks
observed in the DSC experiment (Tꢀ 488C and 728C).
It can be concluded that the bola architecture of 1 reduces
the ability for formation of lamellar phases. In dilute aqueous
solution at room temperature fibrils of 1 with rigid chain
packing are formed solely by hydrophobic interactions and
not by hydrogen bonds. The fibers can extend over several
micrometers. As a consequence, the solution becomes gel-
like. At higher concentrations the fibers seem to be arranged
in a parallel fashion and form sheets consisting of parallel
fibrils. The stability of the fibers is not very great, and changes
in length may be induced by mechanical stress. At a defined
main transition temperature of 488C the long chains of 1
become more disordered, and as a consequence the aggregate
size and morphology change dramatically. The reformation of
the fibrils at low temperature after cooling is time and
concentration dependent.
=
1.37 (m, 52H, -CH2-), 1.99–2.05 (m, 4H, CH-CH2-), 4.89–4.99 (m,
=
=
4H, CH2 ), 5.75–5.85 ppm (m, 2H, CH-); Elemental analysis calcd
for C32H62: C 86.10, H 13.89; found: C 86.01, H 13.96.
The rest of the synthesis of 1 can be found in references [3,4].
Received: April 23, 2003
Revised: July 28, 2003 [Z51731]
Keywords: amphiphiles · hydrogels · nanomaterials ·
.
phospholipids
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Experimental Section
Dotriacontan-1,31-diene: A solution of 11-bromoundec-1-ene (14 g,
60 mmol) in dry ether (50 mL) was added dropwise to stirred
magnesium turnings (2.1 g, 85 mmol) under an argon atmosphere.
After the exothermic reaction had subsided, the mixture was heated
at reflux for 2h. The excess magnesium was removed, and the
Grignard solution was concentrated under reduced pressure at 108C.
Dry THF (180 mL) was added to the oily residue at a rate such that
the temperature remained below 08C. A solution of 1,10-dibromo-
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