Amino-functionalized single-chain bolalipids: Synthesis and aggregation behavior of new basic building blocks

Article abstract of DOI:10.1016/j.bpc.2010.01.013

Herein, we report the synthesis of two novel, amino-functionalized single-chain bolalipids and, based on those, a general synthetic approach for the insertion of various carboxylic acids into the bolalipid headgroups, e.g. α-lipoic acid for one-dimensional fixation of gold nanoparticles, sorbic acid for polymerization experiments, or lysine for the use in gene delivery systems. The temperature- and pH-dependent self-assembly of amino-functionalized bolalipids into nanofibers and micelles was investigated by differential scanning calorimetry (DSC), transmission electron microscopy (TEM) and dynamic light scattering (DLS). Rheological measurements were used to describe the macroscopic behavior of the formed temperature switchable hydrogels that can be fine-tuned for drug delivery applications. We showed that the viscoelastic properties of the hydrogel strongly depend on ionic interactions between bolalipid headgroups as well as on the ability to form hydrogen bonds.

Full text of DOI:10.1016/j.bpc.2010.01.013

Biophysical Chemistry 150 (2010) 136–143
Contents lists available at ScienceDirect
Biophysical Chemistry
journal homepage: http://www.elsevier.com/locate/biophyschem
Amino-functionalized single-chain bolalipids: Synthesis and aggregation behavior of
☆
new basic building blocks
b
c
a
b,d,
Simon Drescher a, , Gesche Graf , Gerd Hause , Bodo Dobner , Annette Meister
⁎
⁎
a
Institute of Pharmacy, Biochemical Pharmacy, MLU Halle-Wittenberg, Wolfgang-Langenbeck-Str. 4, 06120 Halle (Saale), Germany
Institute of Chemistry, Physical Chemistry, MLU Halle-Wittenberg, Von-Danckelmann-Platz 4, 06120 Halle (Saale), Germany
Biocenter, MLU Halle-Wittenberg, Weinbergweg 23, 06120 Halle (Saale), Germany
HALOmem, Biophysical Chemistry of Membranes, MLU Halle-Wittenberg, Kurt-Mothes-Str. 3, 06120 Halle (Saale), Germany
b
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 January 2010
Received in revised form 28 January 2010
Accepted 29 January 2010
Available online 4 February 2010
Herein, we report the synthesis of two novel, amino-functionalized single-chain bolalipids and, based on
those, general synthetic approach for the insertion of various carboxylic acids into the bolalipid
a
headgroups, e.g. α-lipoic acid for one-dimensional fixation of gold nanoparticles, sorbic acid for
polymerization experiments, or lysine for the use in gene delivery systems. The temperature- and pH-
dependent self-assembly of amino-functionalized bolalipids into nanofibers and micelles was investigated by
differential scanning calorimetry (DSC), transmission electron microscopy (TEM) and dynamic light
scattering (DLS). Rheological measurements were used to describe the macroscopic behavior of the formed
temperature switchable hydrogels that can be fine-tuned for drug delivery applications. We showed that the
viscoelastic properties of the hydrogel strongly depend on ionic interactions between bolalipid headgroups
as well as on the ability to form hydrogen bonds.
Keywords:
Bolalipids
Self-assembly
Nanofibers
Synthetic methods
Bipolar amphiphiles
Hydrogel
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
nanoparticles of self-assembled bolalipids, which are uncommon for
the natural compounds [5,6].
Over the last 30 years, representatives in the class of bipolar lipids
(bolalipids) emerged as basic structures for wide-spread modifica-
tions enabling versatile applications in the field of bioscience. As
substantially reviewed, those bolalipids, originating in the membrane
lipids of certain species of Archaea, connect two hydrophilic head-
groups via a lipophilic spacer mainly consisting of one or two alkyl
chains [1]. The membrane-spanning nature and, hence, membrane-
stabilizing properties of these bolalipids are responsible for the
outstanding stability of the Archaea, and those qualities make them
attractive candidates for the use in vesicular drug delivery systems [2]
or for the stabilization of supported biosensor devices [3]. With regard
to their challenging synthesis, numerous attempts were made to
simplify their chemical structure resulting in the preparation of
single-chain bolalipids [4]. Unexpectedly, this class of simplified
bipolar lipids forms novel aggregate structures, e.g. nanofibers and
Recently, we reported the synthesis and aggregation behavior of
symmetrical, single-chain polymethylene-1,ω-bis(phosphocholines)
with alkyl chain lengths of 22 to 32 carbon atoms and two
phosphocholine headgroups bilaterally attached [7]. These simplified
archaeal model lipids self-assemble in water at room temperature into
well defined, long and flexible nanofibers, forming a dense network,
which gels water very efficiently — even at very low concentrations of
0.3 mg/ml [8,9]. Within the fiber, bolalipid molecules are arranged
side by side, but twisted relative to each other due to the bulky
headgroups. These packing constraints lead to a helical superstructure
of the nanofibers [10]. Since the phosphocholine headgroups are not
able to form intermolecular hydrogen bonds, the self-assembly
process is exclusively driven by van-der-Waals interactions between
the long alkyl chains and, thus, it is strongly related to temperature:
Above a certain transition temperature, which depends on the lengths
of the alkyl spacer, the nanofibers reversibly transform into smaller
aggregates like spherical micelles or discs and the gel character of the
suspensions is completely lost [7].
☆
Dedicated to Prof. Alfred Blume on the occasion of his 65th birthday.
⁎
Corresponding authors. Drescher is to be contacted at the Institute of Pharmacy,
An additional stabilization can be achieved by replacing the
phosphocholine with phosphodimethylethanolamine headgroups,
which introduces further hydrogen bonds [11,12]. The presence of
dissociable H-atoms induces the formation of pH-sensitive bolaform
hydrogelators, which might be of importance for drug delivery
systems. Protonable moieties can also be realized by amino-
Biochemical Pharmacy, Martin-Luther-University Halle-Wittenberg, Wolfgang-Lan-
genbeck-Str. 4, 06120 Halle (Saale), Germany. Tel.: +49 345 55 25077; fax: +49 345 55
27018. Meister, HALOmem, Biophysical Chemistry of Membranes, Martin-Luther-
University Halle-Wittenberg, Kurt-Mothes-Str. 3, 06120 Halle (Saale), Germany.
Tel.: +49 345 55 24935; fax: +49 345 55 27408.
E-mail addresses: simon.drescher@pharmazie.uni-halle.de (S. Drescher),
annette.meister@chemie.uni-halle.de (A. Meister).
0301-4622/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.bpc.2010.01.013

S. Drescher et al. / Biophysical Chemistry 150 (2010) 136–143
137
functionalized headgroups. However, the first attempt to synthesize
an amino-functionalized single-chain bipolar lipid using phosphory-
lation of dotriacontane-1,32-diol and a subsequent quarternisation
with N,N-dimethylethylenediamine failed due to the limited purifi-
cation feasibility of the final reaction mixture [13].
In the work presented here, we seized on this last concept: We
developed the synthesis of two novel amino group containing
bolalipids using both a one-side protected N,N-dimethylethylenedia-
mine for the quarternisation reaction and the click-chemistry in
combination with a 2-azidoethylamine acting as linker. These amino-
functionalized bolalipids represent new basic building blocks for
tailor-made bolalipids carrying various functional groups. Both
building blocks and a selection of modified bolalipids as well as
their potential applications are illustrated in Fig. 1.
and pH-dependent self-assembly of AEPC-C32-AEPC and AETPC-C32-
AETPC in water was investigated in order to test the stability range of the
formed aggregates and to adjust the viscoelastic properties of the
formed hydrogels. Therefore, we studied pure and mixed bolalipid
suspensions with the unmodified dotriacontane-1,32-diyl-bis[2-(tri-
methylammonio)ethylphosphate] (PC-C32-PC) by means of differential
scanning calorimetry (DSC), transmission electron microscopy (TEM)
and dynamic light scattering (DLS). The macroscopic behavior of the
formed hydrogels was described by rheological measurements.
2. Materials and methods
2.1. Materials and synthesis
The presence of amino-functionalized headgroups allows the
implementation of 1) α-lipoic acid for the one-dimensional fixation
of gold nanoparticles on bolaform nanofibers [10] with a possible
quantum dot behavior [14] and 2) sorbic acid for the polymerization
of the headgroup region in order to stabilize the structure of the fibers.
Moreover, 3) basic amino acids like lysine can be used enabling the
fixation of DNA and/or the stabilization of the DNA-transporting
liposomes which might be employed in gene delivery systems [15].
Further headgroup modifications are under investigation. They are
mentioned here to show the diversity of feasible functionalization
reactions. For instance, 1) the insertion of a fluorescence label [16]
could yield information about the dynamics of the bolalipids within
the self-assembled nanofibers. The coupling of 2) low-molecular
weight peptides might be of interest for applications addressing
biological sensing [17], and 3) the binding of polyethoxy moieties
should result in nanofibers with hydophilized surfaces.
Sodium chloride, sodium carbonate and sodium hydrogen car-
bonate (p.a. grade) were purchased from Carl Roth GmbH&Co
(Karlsruhe, Germany). Substances for syntheses were purchased
from Sigma Aldrich Co. and were used without further purification.
β-Bromoethylphosphoric acid dichloride was prepared according to the
literature [18], and dotriacontane-1,32-diol as well as PC-C32-PC were
synthesized according to the procedures described previously [7]. All
organic solvents used were purified and dried. The purity of all
compounds was checked by thin-layer chromatography (TLC; obtained
from Merck). The purification of the final bolalipids was carried out by
middle pressure liquid chromatography (MPLC; Büchi) or conventional
column chromatography using silica gel (Merck, 0.032–0.060 mm).
Detailed analytical data concerning synthesized substances can be
found in the Supplementary material.
2.1.1. Protection of amino groups via carbonodithioimidic acid dimethyl
ester
A solution of 2-(dimethylamino)ethylamine (1) (8.82 g, 0.1 mol)
and triethylamine (TEA, 20.2 g, 0.2 mol) in dry CHCl3 (75 ml) was
stirred at room temperature. Carbon disulfide (8.36 g, 0.11 mol)
In this work we present the synthesis of the amino-functionalized
bolalipids AEPC-C32-AEPC and AETPC-C32-AETPC. Based on those, we
propose a general synthetic approach for the insertion of various
carboxylic acids in the headgroup of these bolalipids. The temperature-
Fig. 1. Potential applications of amino-functionalized bolalipids with different headgroup modifications: Arrow 1 points to amino-functionalized bolalipids, arrow 2 shows
headgroup modifications with carboxylic acids described in the manuscript, and arrow 3 indicates potential modifications that are currently under investigation.

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S. Drescher et al. / Biophysical Chemistry 150 (2010) 136–143
dissolved in dry CHCl3 (20 ml) was added in such a rate that the
temperature remained below 50 °C and stirring was continued for
further 1 h. Afterwards, methyl iodide (15.6 g, 0.11 mol) was added
slowly and the mixture was heated at reflux for 3 h. After cooling down
to room temperature, the organic layer was washed with water
(2×150 ml) and concentrated to dryness under reduced pressure. The
residue was dissolved again in Et2O (250 ml), washed with water
(2×50 ml), dried over sodium sulfate, and concentrated to dryness
under reduced pressure. The obtained light yellow crystals of [2-
(dimethylamino)ethyl]carbamodithioic acid methyl ester (2) (12.33 g,
69.2 mmol, 69% yield) was dried in vacuo over phosphorus pentoxide.
For the second methylation, 2 was dissolved in dry acetone (50 ml)
under argon atmosphere, a solution of sodium bis(trimethylsilyl)amide
in THF (1 M, 70 ml) was added slowly, and the mixture was stirred for
2 h. Afterwards, methyl iodide (9.94 g, 70 mmol) was added and the
resulted mixture was heated at reflux for further 2 h. For work-up the
solvent was evaporated and the residue was dissolved in Et2O (100 ml),
washed with water (2×30 ml), dried over sodium sulfate concentrated
to dryness under reduced pressure to give the [2-(dimethylamino)-
ethyl]carbonodithioimidic acid dimethyl ester (3) as oily substance in
90% yield (11.98 g, 62.3 mmol) and 62% over-all yield.
cooled down to 0 °C. Et2O (5 ml) and subsequent solid KOH (1.6 g)
were added. After vigorous stirring for 30 min, the organic phase was
separated and the aqueous layer was extracted with Et2O (3×25 ml).
The combined organic layers were dried over sodium sulfate, filtered,
and the solvent was removed carefully under reduced pressure
affording the azide 8 as a light yellow liquid which was used without
1
further purification. H NMR spectra were found to be identical with
the one described in the literature [19].
2.1.5. General procedure for synthesis of amides from 2-azidoethylamine
Equimolar amounts of 2-azidoethylamine 8 (86 mg, 1 mmol), the
appropriate carboxylic acid, (benzotriazol-1-yloxy)tris(dimethyla-
mino)phosphonium hexafluorophosphate (BOP, 442 mg, 1 mmol),
and 1.5fold excess of TEA (150 mg, 1.5 mmol) were dissolved in dry
CH2Cl2 (10 ml) and the mixture was stirred at room temperature.
After 24 h, the solvent was removed under reduced pressure and the
residue was purified by MPLC using gradient technique and heptane/
CHCl3 or CHCl3/MeOH, respectively, as eluent affording the carboxylic
acid N-(2-azidoethyl)amides 9a–9c in 80–91% yield.
2.1.6. Coupling of carboxylic acid N-(2-azidoethyl)amides with propynyl
modified bolalipid by click-reaction
2.1.2. Phosphorylation and quarternisation
The N-(2-azidoethyl)carboxylic acid amides 9a–9c as well as the
azide 8 were used for subsequent, copper-catalyzed click-reaction with
the propynyl modified phosphocholine dotriacontane-1,32-diyl-bis[2-
(N,N-dimethyl-N-propynylammonio)ethylphosphate] (10) described
previously [13]. Propynylcholine 10 (10 mg, 11.6 µmol) and the
appropriate azide 8 or 9a–9c, respectively, (30 µmol) were suspended
in water/EtOH (10 ml, 2/1, v/v). Sodium ascorbate (8 µmol in aqueous
solution) followed by copper(II) acetate monohydrate (4 µmol in
aqueous solution) were added and the mixture was stirred for 24 h at
room temperature. After evaporation of the solvent, the residue was
purified by column chromatography using gradient technique and
CHCl3/MeOH/water as eluent affording functionalized bolalipids 11a–
11c and the amino group containing bolalipid 11d (AETPC-C32-AETPC).
β-Bromoethylphosphoric acid dichloride [18] (1.93 g, 8 mmol) was
poured into dry CHCl3 (20 ml) under cooling with ice/water. A mixture
of dry TEA (1.42 g, 14 mmol) and dry CHCl3 (20 ml) was added slowly
and stirring was continued for 30 min at 0 °C. Dotriacontane-1,32-diol
(4) [7] (0.48 g, 1 mmol) was added as solid substance in one portion.
The suspension was heated to 60 °C until diol 4 was dissolved, then
rapidly cooled to room temperature. Stirring was continued for further
24 h at this temperature. After TLC control (CHCl3/Et2O, 8/2, v/v)
showed complete conversion of diol 4, crushed ice (40 ml) was added
and the mixture was stirred for a further 2 h. The organic layer was
separated and the aqueous phase was diluted with cold saturated
solution of NaCl (50 ml) and then extracted with CHCl3 (3×50 ml).
The combined organic phases were concentrated to dryness under
reduced pressure, the oily residue was dissolved in THF/water (9/1, v/v,
30 ml) and stirred for 1 h. Then, the solvent was evaporated and the oily
residue was transferred into a mixture of dry CHCl3 (30 ml), dry
acetonitrile (30 ml) and dry EtOH (10 ml) under argon atmosphere. The
amine 3 (7.7 g, 40 mmol) was added and the mixture was kept in a
closed tube at 45 °C for 72 h. Afterwards, the mixture was concentrated
by evaporation of the solvent and the residue was purified by MPLC
using gradient technique and CHCl3/MeOH/water as eluent to give the
dotriacontane-1,32-diyl-bis{2-[N-(2-{[bis(methylthio)methylen]
amino}ethyl)-N,N-dimethylammonio]ethylphosphate} (5) as white
solid (42% yield) which was dried in vacuo over phosphorus pentoxide
at room temperature for at least two days.
2.1.7. Cleavage of BOC-protecting groups
For cleavage of the BOC-protecting groups, the bolalipid 11c (10 mg,
5.9 µmol) and equimolar amounts of trifluoroacetic acid (TFA) were
dissolved in dry CH2Cl2 (10 ml) and stirred for at least 48 h at room
temperature. After evaporation of the solvent, the residue was purified
by MPLC using gradient technique and CHCl3/MeOH/water as eluent.
2.2. Sample preparation
The appropriate amount of the pure bolalipid was suspended in
water (pH 6) or 10 mM carbonate buffer at pH 10 for DSC and TEM
measurements. Binary bolalipid mixtures were prepared from lipid
stock solution in CHCl3/MeOH (2/1, v/v) as solvent by mixing of
appropriate volumes of the stock solution. The organic solvent was
then rapidly removed in a stream of nitrogen at elevated tempera-
tures. The resulting bolalipid films were kept in an evacuated flask for
24 h to remove residual traces of solvent. The aqueous suspensions
were then prepared by adding a certain volume of deionised water
and 1 M NaCl solution, respectively. Homogenous suspensions were
obtained by heating to 90 °C and vortexing the sample.
2.1.3. Deprotection of amino groups—synthesis of the amino-functionalized
bolalipid
The bolalipid 5 (26.8 mg, 24.8 µmol) was suspended in water
(10 ml) under slightly warming, 0.2 ml HCl (2 M) was added and the
mixture was stirred at room temperature. After 24 h, the mixture was
alkalized with a solution of potassium carbonate (pH∼10) and
evaporated under reduced pressure until a small amount of a clear
colorless solution is left. Addition of dry acetone resulted in the
precipitation of dotriacontane-1,32-diyl-bis{2-[N-(2-aminoethyl)-N,
N-dimethylammonio]ethylphosphate} (AEPC-C32-AEPC, 6) as white
solid. The product was collected by centrifugation and was dried in
vacuo over phosphorus pentoxide at room temperature.
2.3. DSC
DSC measurements were performed using a MicroCal VP-DSC
differential scanning calorimeter (MicroCal Inc. Northampton, MA,
USA). Before the measurements, the sample suspension (1 mg/ml) and
the water or buffer reference were degassed under vacuum while stirring.
A heating rate of 20 K/h was used, and the measurements were performed
in the temperature interval from 2 to 95 °C. To check the reproducibility,
three consecutive scans were recorded for each sample. The buffer–buffer
2.1.4. Synthesis of 2-azidoethylamine
2-Bromoethylamine hydrobromide 7 (1.0 g, 4.9 mmol) was added
to a solution of sodium azide (0.95 g, 14.6 mmol) in water (5 ml). The
stirred solution was heated to 80 °C for at least 24 h before it was

S. Drescher et al. / Biophysical Chemistry 150 (2010) 136–143
139
baseline was subtracted from the thermograms of the samples, and the
DSC scans were evaluated using MicroCal Origin 7.0 software.
1,ω-diols and subsequent quarternisation with amines [7]. As we
described recently, the use of 2-(dimethylamino)ethylamine (1)
during the quarternisation reaction resulted in a mixture of secondary
and quarternary amines [13]. A separation and purification of single
compounds was not possible due to the very similar chromatographic
properties of the products obtained. To avoid the problem of two
reaction centers within the amine 1 the primary amino group had to
be protected as imine (Schiffs base) using various aldehydes or
ketones. Considering that an increase of the amine size led to lower
yields during the quarternisation [13] common protecting groups for
primary amino moieties such as tert.-butoxycarbonyl (Boc) [21],
benzyloxycarbonyl (Cbz) [22] or 9-fluorenylmethyloxycarbonyl
(Fmoc) [23] should be evaded. The first attempt employing acetone
as protecting reagent failed because of polymerization and decom-
position of the reaction mixture. A second attempt dealt with carbon
disulfide in combination with a two-step alkylation with methyl
iodide. In contrast to the procedures described by SAUTER et al. [24]
potassium carbonate used for the second deprotonation of [2-
(dimethylamino)ethyl]carbamodithioic acid methyl ester (2) had to
be replaced by sodium bis(trimethylsilyl)amide in order to obtain
acceptable yields (62% over-all yield) of the [2-(dimethylamino)-
ethyl]carbonodithioimidic acid dimethyl ester (3). The one-side
protected amine 3 was then used for the quarternisation of the
phosphorylated dotriacontane-1,32-diol (4). For the foregoing phos-
phorylation reaction we used β-bromoethylphosphoric acid dichlor-
ide in combination with TEA as basic additive owing to the higher
efficiency of this reagent compared to 2-chloro-1,3,2-dioxopho-
spholane or phosphites which were also used by other groups
performing bolalipid synthesis [25,26]. This two-step procedure
resulted in the formation of dotriacontane-1,32-diyl-bis{2-[N-(2-
{[bis(methylthio)methylen]amino}ethyl)-N,N-dimethylammonio]
ethylphosphate} (5) in moderate yields (42%) with regard to the diol
4. For the cleavage of the imidic protecting group the bolalipid 5 was
suspended in diluted hydrochloric acid and stirred of 24 h affording
the amino-functionalized bolalipid dotriacontan-1,32-diyl-bis{2-[N-
(2-aminoethyl)-N,N-dimethylammonio]ethylphosphate} (AEPC-C32-
AEPC, 6) in nearly quantitative yields. The complete synthesis of
AEPC-C32-AEPC (6) is summarized in Scheme 1.
2.4. TEM
The negatively stained samples were prepared at 5 °C by spreading
5 µl of the dispersion onto a Cu grid coated with a formvar-film. After
1 min, excess liquid was blotted off with filter paper and 5 µl of 1%
aqueous uranyl acetate solution were placed onto the grid and
drained off after 1 min. The dried specimens were examined with a
Zeiss EM 900 transmission electron microscope.
2.5. DLS
The DLS experiments were performed with an ALV-NIBS-HPPS
particle sizer (ALV-Laser Vertriebsgesellschaft mbH, Langen, Ger-
many). The device was equipped with a 3 mW HeNe laser with a
wavelength of 632.8 nm. Because of the principle of noninvasive back
scattering the scattering angle was 173°. All samples (1 mg/ml) were
freshly prepared and then filtered through a membrane filter of
0.2 µm pore size (at 80 °C) into quartz cuvettes (HELLMA GmbH & Co.
KG, Muehlheim, Germany). Before starting the measurement, each
sample was equilibrated for 60 min. Three individual measurements
were performed for each system to test the reproducibility. The
experimental data were analyzed with the aid of the ALV-5000/E
software based on the modified CONTIN method [20], with the
temperature correction of the viscosity being taken into account.
2.6. Rheology
Rheological properties of 1 mg/ml bolalipid suspensions in water
were studied using a MCR301 rheometer (Anton Paar, Germany)
controlled by the software Rheoplus 3.0. For the oscillatory measure-
ments we used a cone and plate shear geometry (2°/50 mm) with a
thermostatting unit (Peltier system: −40 to 200 °C).
3. Results and discussion
3.1. Synthetic methods
The deprotonation of the primary amino groups of AEPC-C32-AEPC
(6) could be realized by addition of an aqueous solution of potassium
carbonate. The influence of the degree of protonation on the
temperature-dependent aggregation behavior of AEPC-C32-AEPC (6)
is explained in Section 3.2.
For the synthesis of symmetrical, amino-functionalized single-
chain bolalipids we established two different strategies. The first
approach uses the well-established bis-phosphorylation of long-chain
Scheme 1. Synthesis of the amino-functionalized bolalipid 6 (AEPC-C32-AEPC) via phosphorylation and quarternisation reaction.

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S. Drescher et al. / Biophysical Chemistry 150 (2010) 136–143
Scheme 2. Synthesis of headgroup modified bolalipids 11a–11d using click-reaction in combination with 2-azidoethylamides.
For the second synthetic approach of amino-functionalized single-
(AETPC-C32-AETPC, 11d). The AETPC-C32-AETPC represents the
second amino-functionalized bolalipid introduced in this work. It's
temperature-dependent aggregation behavior is described in Sec-
tion 3.2. The physico-chemical characterization of the headgroup
modified bolalipids 11a–11c is currently under investigation.
For the synthesis of the lysine modified bolalipid 12 the BOC-
protecting groups were cleaved using trifluoroacetic acid (TFA).
Therefore, the bolalipid 11c was suspended in CH2Cl2 with equimolar
amounts of TFA and stirred for at least 48 h at room temperature
resulting in the dotriacontane-1,32-diyl-bis(2-{N-[(1-{2-[(2,6-di-
amino-1-oxohex-1-yl)amino]ethyl}-1,2,3-triazol-4-yl)methyl]-N,N-
dimethylammonio}ethylphosphate) (12) (see Scheme 3).
chain bolalipids we used a click-reaction [27,28] of propynes with
azides whose feasibility was shown previously [13]. The advantages of
this copper(I) catalyzed Huisgen 1,3-cyclo addition are 1) the modular
and broad applicability, 2) simple reaction procedures at ambient
temperatures, and 3) high yields avoiding the formation of by-
products. Nevertheless, the reaction might be limited to the marginal
commercial availability of functionalized azides. To overcome this
restriction, the use of 2-azidoethylamine as linker was proposed.
Therefore, 2-bromoethylamine hydrobromide (7) was converted into
the amine 8 using sodium azide proceeding as described by MAYER
et al. [19] and, afterwards, this amine 8 was coupled with various
carboxylic acids deploying (benzotriazol-1-yloxy)tris(dimethyla-
mino)phosphonium hexafluorophosphate (BOP) as the coupling
reagent. This reaction procedure led to the formation of N-(2-
azidoethyl)amides of 1,2-dithiolan-3-pentanoic acid (α-lipoic acid,
9a), hexa-2(E),4(E)-dienoic acid (sorbic acid, 9b), and 2,6-bis(tert.-
butoxycarbonylamino)hexanioc acid (di-BOC-lysine, 9c). The purifi-
cation of the final products was carried out using MPLC with a
gradient technique and heptane/CHCl3 or CHCl3/MeOH, respectively,
as eluent affording the azidoamides 9a–9c in 80 – 91% isolated yields
(see Scheme 2).
The azidoamides 9a–9c were used for subsequent copper(I)
catalyzed click-reaction with the propynyl modified bolalipid dotria-
contane-1,32-diyl-bis[2-(N,N-dimethyl-N-propynylammonio)ethyl-
phosphate] (10) and catalytic amounts of copper(I) salt which was
prepared in situ by reduction of copper(II) acetate with sodium
ascorbate and water/EtOH (2/1, v/v) as solvent [13,28]. After 24 h,
thin-layer chromatography (TLC) indicated the complete conversion
of the propynylcholine 10 into headgroup modified bolalipids
containing α-lipoic acid (11a), sorbic acid (11b), and di-BOC-lysine
(11c), respectively (see Scheme 2). In addition, the unmodified 2-
azidoethylamine (8) was also used for click-reaction resulting in the
formation of dotriacontane-1,32-diyl-bis[2-(N-{[1-(2-aminoethyl)-
1,2,3-triazol-4-yl]methyl}-N,N-dimethylammonio)ethylphosphate]
3.2. Temperature-dependent aggregation at different pH values
The self-assembly of symmetric single-chain bolalipids in water is
driven by hydrophobic, electrostatic, and hydrogen bonding interac-
tions. In addition, the ratio between the cross-sectional area of the
chain and the headgroup of the bolalipid strongly determines the
shape of the formed aggregates. In the case of the amino-functiona-
lized bolalipid AEPC-C32-AEPC each headgroup is composed of a
phosphate group and an N-(aminoethyl)-N,N-dimethylammonium
group with a permanent positive charge at the quarternary nitrogen
(the N,N-dimethylammonium part) and a dissociable proton at the
primary nitrogen (the aminoethyl part). Therefore, at intermediate
pH the bolalipid molecule should be positively charged due to the
protonation of the aminoethyl group, whereas at higher pH the
dissociation of this proton should lead to a lipid with zwitterionic
headgroups (see Fig. 2).
At very low pH-values the headgroup should be doubly positively
charged due to the protonation of the phosphate and the aminoethyl
group. Recently, we determined the pKa values of a similar bolalipid
with phosphodimethylammonium groups by titration in water [11].
The measurements yielded an apparent pKa value for the phosphate
group of 3.3, and one of 6.5 for the dimethylammonium group.
Scheme 3. Synthesis of the lysine modified bolalipid 12.

S. Drescher et al. / Biophysical Chemistry 150 (2010) 136–143
141
In contrast, the fibers composed of AETPC-C32-AETPC are up to
300 nm long and they arrange in a parallel fashion. At pH 10, both
bolalipids self-assemble into long and flexible fibers with a length of
more than 1 µm.
To clarify the temperature-dependent stability of these nanofibers,
we performed DLS measurements at temperatures that were chosen
according to the transition temperatures detected by DSC measure-
ments. Fig. 4 shows the DSC curves for suspensions of both amino-
functionalized bolalipids at pH 6 and 10, respectively. All heating
curves display two endothermic transitions. The fibers visualized by
TEM are present up to the first transition temperature Tm1. Above this
temperature, DLS measurements indicate the presence of spherical
aggregates with a diameter of about 4 to 7.6 nm (see Supplementary
material). In Table 1, the DSC transition temperatures Tm1 and Tm2 are
summarized together with the radii of micelles that were determined
by DLS at temperatures between Tm1 and Tm2 as well as above Tm2.
The transition temperatures Tm1 of AEPC-C32-AEPC and AETPC-C32-
AETPC are more than 15 K lower for the suspensions at pH 6 than for
pH 10. In addition, all transitions are very broad, which indicates that
they are less cooperative.
All heating curves show a very broad second transition (Tm2),
which occurs within the stability range of the micellar aggregates (see
Table 1). The cooling curves indicate almost no hysteresis for the
suspensions at pH 6 but a strong hysteresis for pH 10. These distinct
differences for both bolalipids at pH 6 and pH 10 should be due to
differences in the protonation state of the headgroups. At pH 6, the
positively charged headgroups cause repulsive interactions between
neighboring headgroups and, as a consequence, the Tm values are
significantly lower.
Fig. 2. Headgroup protonation state of the bolalipid AEPC-C32-AEPC at different pH
values.
According to these results and based on pKa values of basic amino
acids [29], the investigations with AEPC-C32-AEPC and AETPC-C32-
AETPC were carried out either in water at pH 6, where the headgroups
are positively charged, or in carbonate buffered solutions at pH 10,
where the bolalipids has zwitterionic headgroups.
The temperature-dependent aggregation behavior of AEPC-C32-
AEPC and AETPC-C32-AETPC was investigated by transmission
electron microscopy (TEM), differential scanning calorimetry (DSC)
and dynamic light scattering (DLS). Fig. 3 shows TEM images of
stained (uranyl acetate) suspensions of both bolalipids at pH 6 and 10
that were prepared at 5 °C. The suspensions at pH 6 indicate the
formation of short fiber aggregates with a diameter of about 5 nm that
corresponds roughly to the molecular length of the bolalipids. AEPC-
C32-AEPC forms a few long fibers (1 µm) but mainly short fiber
segments with up to 50 nm length are visible.
We were interested in the gelling properties of symmetric long-
chain bolalipids that originate from entangled fiber aggregates. For
some of these bolalipids the formation of hydrogels has been
Fig. 3. TEM images of stained (uranyl acetate) suspensions (0.1 mg/ml) of amino-functionalized bolalipids. Top row: AEPC-C32-AEPC at pH 6 (left) and pH 10 (right), Bottom row:
AETPC-C32-AETPC at pH 6 (left) and pH 10 (right). The bar corresponds to 100 nm.

142
S. Drescher et al. / Biophysical Chemistry 150 (2010) 136–143
gelator at room temperature due to the formation of a dense network
of cross-linked fibers [9]. However, a 1:10 mixture of AEPC-C32-AEPC
and PC-C32-PC in the presence of NaCl (1 M) shows the formation of a
viscous, transparent gel, which is stable up to temperatures of about
48 °C. The gel state is due to the existence of a fiber network. Salt was
added to screen the positive headgroup charges of AEPC-C32-AEPC at
pH 6 and to prevent repulsive interactions between neighboring
headgroups. DSC measurements indicate the transformation of fibers
into spherical micelles at 48 °C and they show that 1 M NaCl has only a
marginal effect onto the transition temperatures (see Supplementary
material). Rheograms show at 2, 20, and 60 °C the typical signature of
a viscoelastic fluid (not shown). At low frequencies, both storage and
loss moduli are rather independent of frequency, and at 2 and 20 °C G′
is about an order of magnitude larger than G″. Similar values for G′ and
G″ were found for PC-C32-PC at 2 and 20 °C. Above the first transition
temperature at 48 °C, the shear modulus G′ breaks down (see Fig. 5A)
and the gel character is lost due to the transformation of fibers into
spherical micelles.
After continuous heating of the samples up to 80 °C and
subsequent cooling to 2 °C, the moduli are significantly lower than
before the temperature treatment. However, at 2 °C both moduli
show a distinct increase during the next 10 h. Whereas the pure PC-
Fig. 4. DSC heating curves of AEPC-C32-AEPC (A) and AETPC-C32-AETPC (B) at pH 6 and
10, respectively. Cooling curves are presented with dashed lines.
described recently [9,30–33]. The fibers become cross-linked by
noncovalent interactions so that they are able to trap water molecules.
As a consequence, the hydrogels are often thermally reversible and
the gel properties can easily be controlled by changing the
temperature, pH, or ionic strength of the suspension.
The bolalipids AEPC-C32-AEPC and AETPC-C32-AETPC in dilute
aqueous suspension (1 mg/ml) show no pronounced gelling proper-
ties. This can be explained by the absence of a fiber network and the
presence of short fiber segments at pH 6. In contrast, the phosphocho-
line analogue PC-C32-PC exhibits excellent properties as a hydro-
Table 1
DSC transition temperatures (Tm1 and Tm2) of aqueous suspensions (1 mg/ml) of AEPC-
C32-AEPC and AETPC-C32-AETPC, as well as radii of micelles determined by DLS at
temperatures between Tm1 and Tm2 and above Tm2 at pH 6 and 10, respectively.
Bolalipid
pH
DSC
DLS
Tm [°C]
T [°C]
Radius [nm]
AEPC-C32-AEPC
6
10
6
Tm
Tm
Tm
Tm
Tm
Tm
Tm
Tm
1
2
1
2
1
2
1
2
18.2
57.0
44.0
70.0
24.6
56.0
41.6
59.0
30
75
55
80
35
75
50
75
2.0 0.1
1.8 0.1
3.8 0.1
3.2 0.1
3.1 0.1
2.7 0.1
3.3 0.1
3.1 0.1
Fig. 5. (A) Temperature dependence of G′ and G″ of aqueous suspensions (1 mg/ml) of a
1:10 AEPC-C32-AEPC/PC-C32-PC mixture in the presence of 1 M NaCl recorded at a
deformation γ of 1% and a frequency ω of 1 rad/s. Moduli for pure PC-C32-PC are added
for comparison. The heating rate was 20 K/h. (B) Time dependence of G′ and G″ at 2 °C
after heating the sample to 80 °C and subsequent cooling to 2 °C. The heating and
cooling rate was 20 K/h.
AETPC-C32-AETPC
10

S. Drescher et al. / Biophysical Chemistry 150 (2010) 136–143
143
line-water system: the formation of nanofibers and hydrogels, Angew. Chem. Int. Ed.
43 (2004) 245–247.
C32-PC suspension reaches the initial G′ and G″ values, the AEPC-C32-
AEPC/PC-C32-PC mixture (1:10) shows a large increase of both
moduli of more than one order of magnitude (see Fig. 5B). This is
probably due to the formation of intermolecular hydrogen bonds
between headgroups of AEPC-C32-AEPC. In contrast, PC-C32-PC is not
able to form intermolecular hydrogen bonds. These results clearly
indicate that a tailor-made modification of the bolalipid headgroup
is a promising way to enhance the gelling properties of bolalipid
hydrogelators.
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In summary, we developed the synthesis of two novel, amino-
functionalized bolalipids (AEPC-C32-AEPC and AETPC-C32-AETPC)
using either a one-side protected diamine for the quarternisation
reaction of the phosphorylated dotriacontane-1,32-diol or the click-
chemistry in combination with a 2-azidoethylamine. Especially the
second way offers the possibility to fix versatile carboxylic acids, e.g. α-
lipoic acid, sorbic acid, or lysine, leading to different tools for the use in
biochemical and biophysical sensing. Actually, the insertion of a
fluorescence label is under investigation. This label will be used to get
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self-assembled nanofibers. The insertion of the additional amino-
moiety within the bolalipid headgroup gives rise to a pH-dependent
adjustment of the self-assembly properties ranging from short fiber
segments at pH 6 to long and flexible fibers at pH 10. Furthermore, the
addition of AEPC-C32-AEPC to a hydrogel composed of unmodified PC-
C32-PC can enhance the gelling properties of bolaform hydrogelators.
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Acknowledgements
The authors wish to thank Prof. Andrea Sinz and Dr. Christian
Ihling for the high resolution mass spectrometry analysis. This work
was supported by grants from the Deutsche Forschungsgemeinschaft
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