Highly Asymmetrical Glycerol Diether Bolalipids: Synthesis and Temperature-Dependent Aggregation Behavior

Article abstract of DOI:10.1021/acs.langmuir.5b02951

In the present work, we describe the synthesis and temperature-dependent aggregation behavior of two examples of a new class of highly asymmetrical glycerol diether bolaphospholipids. The bolalipids contain a long alkyl chain (C32) bound to glycerol in the sn-3 position, carrying a hydroxyl group at the ω position. The C16 alkyl chain in the sn-2 position either possesses a racemic methyl branch at the 10 position of the short alkyl chain (lipid II) or does not (lipid I). The sn-1 position of the glycerol is linked to a zwitterionic phosphocholine moiety. The temperature-dependent aggregation behavior of both bolalipids was studied using differential scanning calorimetry (DSC), Fourier-transform infrared (FTIR) spectroscopy, and X-ray scattering. Aggregate structures were visualized by transmission electron microscopy (TEM). We show that both bolalipids self-assemble into large lamellar sheetlike aggregates. Closed lipid vesicles or other aggregate structures such as tubes or nanofibers, as usually found for diglycerol tetraether lipids, were not observed. Within the lamellae the bolalipid molecules are arranged in an antiparallel (interdigitated) orientation. Lipid I, without an additional methyl moiety in the short alkyl chain, shows a lamellar phase with high crystallinity up to a temperature of 34 °C, which was not observed before for other phospholipids.

Full text of DOI:10.1021/acs.langmuir.5b02951

Article
pubs.acs.org/Langmuir
Highly Asymmetrical Glycerol Diether Bolalipids: Synthesis and
Temperature-Dependent Aggregation Behavior
Thomas Markowski,† Simon Drescher,*,† Gunter Forster,‡ Bob-Dan Lechner,‡ Annette Meister,§
̈
̈
Alfred Blume,‡ and Bodo Dobner*,†
†Institute of Pharmacy, Martin Luther University (MLU) Halle-Wittenberg, Wolfgang-Langenbeck-Str. 4, 06120 Halle (Saale),
Germany
‡Institute of Chemistry, MLU Halle-Wittenberg, von-Danckelmann-Platz 4, 06120 Halle (Saale), Germany
§Center for Structure and Dynamics of Proteins (MZP), MLU Halle-Wittenberg, Biocenter, Weinbergweg 22, 06120 Halle (Saale),
Germany
S
* Supporting Information
ABSTRACT: In the present work, we describe the synthesis
and temperature-dependent aggregation behavior of two
examples of a new class of highly asymmetrical glycerol
diether bolaphospholipids. The bolalipids contain a long alkyl
chain (C32) bound to glycerol in the sn-3 position, carrying a
hydroxyl group at the ω position. The C16 alkyl chain in the
sn-2 position either possesses a racemic methyl branch at the
10 position of the short alkyl chain (lipid II) or does not (lipid
I). The sn-1 position of the glycerol is linked to a zwitterionic
phosphocholine moiety. The temperature-dependent aggrega-
tion behavior of both bolalipids was studied using differential
scanning calorimetry (DSC), Fourier-transform infrared
(FTIR) spectroscopy, and X-ray scattering. Aggregate
structures were visualized by transmission electron microscopy (TEM). We show that both bolalipids self-assemble into large
lamellar sheetlike aggregates. Closed lipid vesicles or other aggregate structures such as tubes or nanofibers, as usually found for
diglycerol tetraether lipids, were not observed. Within the lamellae the bolalipid molecules are arranged in an antiparallel
(interdigitated) orientation. Lipid I, without an additional methyl moiety in the short alkyl chain, shows a lamellar phase with
high crystallinity up to a temperature of 34 °C, which was not observed before for other phospholipids.
However, the isolation of these lipids from natural sources is
extremely expensive and leads to mixtures with respect to a
variety of alkyl chain structures found in the natural extracts.
On the other hand a total synthesis as described by Kakinuma
et al.20−23 is complex and time-consuming and thus is not
useful for the production of large batches. Therefore, several
attempts have been made24−27 to synthesize simpler archaeal
model compounds having only one membrane-spanning alkyl
chain with glycerol moieties bound to both ends of the alkyl
chain. The original second transmembrane chain is replaced by
two shorter alkyl chains, in many cases a phytanyl chain, bound
to each of the glycerol moieties.
We have shown before that tetraether bolalipids containing
two phosphocholine (PC) headgroups and methyl branches at
“sensitive” places in the 10 and 10′ positions of the membrane-
spanning alkyl chain and/or in the 10 position of the short
chain can mimic the behavior of natural lipids in terms of
INTRODUCTION
■
Archaea, which exist under very harsh living conditions,
represent a third kingdom within the organisms. Due to their
living environment they are quite different from prokaryotes
and eukaryotes,1−5 which in turn also concern the chemical
structure of the membrane lipids of those archaea. In detail, the
hydrophobic alkyl chains are connected by ether linkages in the
inverse sn-2,3 configuration to the glycerol backbone, and these
alkyl chains could contain a variable number of cyclopentane
rings as well as several methyl branches in an isoprenoid
substitution pattern.6,7 These archaeal membrane lipids can be
divided into archaeol- and caldarchaeol-type lipids. The latter
ones have two membrane-spanning (transmembrane) alkyl
chains forming a macrocycle with two glycerol residues and two
polar headgroups at both ends and are found in some
methanogens and thermoacidophiles.8 These bipolar lipids,
which are also called bolalipids or bolaamphiphiles,9 are of great
interest for applications in biotechnology, pharmaceuticals, and
materials science.10−15 Liposomes composed of such bipolar
lipids are sometimes called archaeosomes16 and are suitable as
long-circulating vehicles for drug-delivery purposes.17−19
Received: August 8, 2015
Revised: September 13, 2015
© XXXX American Chemical Society
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liposome formation and phase transitions.28 Recently, we found
that the absolute configuration of the methyl moieties, which is
the R configuration in nature, has only a marginal impact on the
phase behavior.29 Both classes of archaeal model compounds,
either with optically pure or racemic methyl branches within
the alkyl chains, are able to form stable liposomes.28,29
However, these bolalipids are symmetric compounds with
respect to the polar rests (PC headgroups).
In this study we focused on the synthesis and physical−
chemical characterization of asymmetrical bipolar lipids. Beside
various bolalipids resulting from the esterification of the single-
chain dotriacontane-1,32-diol30 with different phosphoric acid
ester residues,31 we now synthesize an asymmetric bipolar,
glycerol-containing diether lipid with a large difference in the
polarity of both headgroups. The term “asymmetry” in
connection with bolalipids is used to illustrate that the two
different headgroups bound to the membrane-spanning alkyl
chain have different sizes, polarities, and/or ability to
(de)protonate. Recently, we showed that the headgroup
asymmetry strongly influences the aggregation behavior of
single-chain bolalipids.32,33
small elongated micelles at pH 5 or lamellar structures with an
interdigitated orientation of the molecules in a monolayer
arrangement at pH 10.32
In our study we wanted to address the question of whether
the additional methyl group in position 10 of the sn-2 chain has
an influence on the temperature-dependent aggregation
behavior in an aqueous suspension. The systems were
investigated using transmission electron microscopy (TEM),
differential scanning calorimetry (DSC), Fourier transform
infrared (FTIR) spectroscopy, and X-ray scattering.
EXPERIMENTAL SECTION
■
Syntheses. The synthesis procedures and the analytical data of the
substances are described in detail in the Supporting Information (SI).
Methods. Sample Preparation. Appropriate amounts of the
bolalipid were suspended in H2O (Milli-Q) and D2O (Sigma-Aldrich).
Homogeneous suspensions were obtained by heating to 90 °C and
vortex mixing.
Transmission Electron Microscopy (TEM). The samples were
prepared by spreading 5 μL of the bolalipid suspension (c = 0.03 mg
mL−1) onto a copper grid coated with a Formvar film. After 1 min,
excess liquid was blotted off with filter paper and 5 μL of a 1% aqueous
uranyl acetate solution was placed on the grid and drained off after 1
min. All specimens were examined with a Zeiss EM 900 transmission
electron microscope (Carl Zeiss Microscopy GmbH, Oberkochen,
Germany).
The new bolalipids contain a membrane-spanning C32 alkyl
chain that is bound with one end to the sn-3 position of a
glycerol unit whereas the other end carries only a hydroxyl
group. The sn-2 position of the glycerol moiety is etherified
with either a hexadecyl (lipid I) or a racemic 10-
methylhexadecyl (lipid II) residue (Figure 1). In the sn-1
Differential Scanning Calorimetry (DSC). DSC measurements
were performed using a MicroCal VP-DSC differential scanning
calorimeter (MicroCal Inc. Northampton, MA, USA). Before the
measurements, the sample suspension and the water reference were
degassed under vacuum while stirring. A heating rate of 20 K h−1 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 water−water
baseline was subtracted from the thermogram of the sample, and the
DSC scans were evaluated using MicroCal Origin 8.0 software.
Fourier Transform Infrared Spectroscopy (FTIR). Infrared spectra
were collected on a Bruker Vector 22 Fourier transform spectrometer
with a DTGS detector operating at 2 cm−1 resolution. The sample
suspension (c = 100 mg mL−1 in D2O) was placed between two CaF2
windows, separated by a 6 μm spacer. IR spectra were recorded in
steps of 2 K in the temperature range from 9 to 89 °C. After an
equilibration time of 8 min, 64 scans were recorded and accumulated.
The corresponding spectra of the solvent (D2O) were subtracted from
the sample spectra using the OPUS software supplied by Bruker.
X-ray Scattering. Powder diffraction experiments were carried out
using monochromatic Cu Kα1 radiation (λ = 0.154051 nm from a
Ge(111) monochromator (Seifert X-ray/GE Inc. Freiberg) and a
curved linear position-sensitive detector (range: 2Θ = 0−40°). The
bolalipid samples (50 wt % H2O) were sealed in glass capillaries
(diameter 1.5 mm). The scattering was corrected with respect to a
capillary filled with H2O (Inorm = Isample/Icap). The X-ray patterns were
combined in a single contour diagram to continuously present the
scattering intensity from the SAXS to the WAXS region (2Θ = 0−44°,
s = 0−4.7 nm−1) between −36 and 105 °C in steps of 2 K. The heating
rate was 1/15 K min−1 (5 min of equilibration, 10 min of exposition
for each pattern) for the applied temperature protocol.
Figure 1. Chemical structure of asymmetrical model lipids I (R = H)
and II (R = CH3) (top) and CPK models of lipid I (middle) and lipid
II (bottom).
position of the glycerol the bolalipid carries a zwitterionic PC
moiety. Thus, the two polar groups are very different in size,
which leads to a cone-shaped molecular structure. The
aggregation in water could lead to structures with different
orientations of the molecule: either parallel, i.e., only
headgroups of the same type are arranged at one site on the
lamella, or antiparallel. A parallel orientation could trigger the
formation of rods, nanotapes,34 nanotubes,35−37 or even
liposomes with different curvatures of the inner and outer
surfaces of the aggregate. An antiparallel (interdigitated)
orientation of the bolalipid could result in the formation of
large lamellae or sheetlike structures composed of a monolayer
with a thickness essentially determined by the length of the
molecule.36,38 Recently, we investigated the aggregation
behavior of DMAPPC-C32-OH in water;32 an asymmetrical
single-chain bolalipid bearing a large 2-(dimethylaminopropyl)-
PC (DMAPPC) headgroup and a small hydroxy group at both
ends of a C32-alkyl chain. This bolalipid self-assembles,
depending on the pH value and hence protonation state, into
RESULTS AND DISCUSSION
■
Synthesis of Bolalipids. The main challenges for a
straightforward preparation of glycerol-containing di- as well
as tetraether model lipids are on the one hand an O-alkylation
reaction giving high yields and on the other hand a versatile
protection group strategy for the selective introduction of
different alkyl groups in the glycerol moiety. For the synthesis
of asymmetrical model lipids I and II (Figure 1) we used
commercially available (S)-1,2-O-isopropylideneglycerol (3) as
B
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a
Scheme 1. Synthesis Pathway for the Preparation of Asymmetrical Bipolar Lipids I and II
a
Reactions and conditions: (i) PPh3, Br2, CH2Cl2/CHCl3, r.t. (ii) PPTS, MeOH/CHCl3, reflux and then MsCl, CHCl3, TEA, DMAP. (iii) KH,
toluene, reflux and then 2a or 2b, reflux. (iv) PPTS, MeOH/CHCl3, reflux. (v) trityl chloride, pyridine/CHCl3, r.t. (vi) KH, toluene, reflux and then
1-bromohexadecane or (10R,S)-1-bromo-10-methylhexadecane, reflux. (vii) BF3-Et2O, CH2Cl2/MeOH, 0 °C. (viii) Cl2P(O)O(CH2)2Br, TEA,
CHCl3, 45 °C and then THF−water. r.t. (ix) CHCl3, CH3CN, EtOH, N(CH3)3, 45 °C. (x) H2, Pd/C (10%), EtOH, 5 atm, 50 °C.
the starting material. The first O-alkylation was performed with
benzyl-blocked 32-bromodotriacontan-1-ol (benzyl 32-bromo-
dotriacontyl ether, 2a), which was synthesized from the
appropriate orthogonally protected dotriacontane-1,32-diol31
(1) with the use of triphenylphosphoranediyl dibromide
following the procedure described by Schwarz et al.39 (Scheme
1). Then, compound 3 was deprotonated with potassium
hydride in toluene and subsequently alkylated with bromide 2a,
resulting in the formation of 3-O-[32-(benzyloxy)dotriacontyl]-
1,2-O-isopropylidene-sn-glycerol (4) in good yield (70%). This
yield is in line with yields from other O-alkylation reactions at
the glycerol moiety described previously.28,29 Higher yields (up
to 86%) could possibly be obtained using a method described
by Kakinuma and co-workers.22 They have worked in dry
DMSO, used sodium hydride as a deprotonating agent, and the
corresponding methanesulfonate (mesylate) instead of bromide
2a. Adopting this method, we first synthesized mesylate 2b
from compound 1 in two steps: cleavage of the tetrahydropyr-
anyl (THP) group and reaction with methanesulfonyl chloride
(MsCl). However, the subsequent alkylation of compound 1
with 2b using sodium hydride did not succeed due to the very
low solubility of 2b in dry DMSO. Performing the same
alkylation reaction in toluene with the use of potassium hydride
resulted in a 44% yield of 4 (Scheme 1). Thus, the use of KH in
toluene and an appropriate bromide (instead of the mesylate)
is, in our hands, the method of choice for O-alkylation reactions
at glycerol moieties.
performed in CHCl3/pyridine (1/1 v/v) at room temperature,
leading to compound 6 in a very good yield of 96%. Due to the
orthogonal cleavage of the trityl protection group in the
presence of a benzyl moiety, we used this tritylation instead of
the insertion of a second benzyl group.29 The subsequent
second O-alkylation of glycerol 6 was performed under nearly
the same conditions described above using potassium hydride
in toluene and 1-bromohexadecane or (10RS)-1-bromo-10-
methylhexadecane.29 The yields of resulting 2,3-O,O-dialkyl-1-
O-trityl-sn-glycerols 7a and 7b (53−65%) are in line with the
yields obtained previously in other O-alkylation reactions of
trityl-blocked 2,3-O,O-dialkyl-sn-glycerols,28 and both are
somewhat lower compared to those of the O-alkylations of
benzyl-blocked 2,3-O,O-dialkyl-sn-glycerols29 due to steric
hindrance. For the selective cleavage of the trityl blocking
group, BF3 × Et2O in MeOH was used in accordance with the
procedure described by Hermetter and Paltauf,40 which
resulted in the formation of 2,3-O,O-dialkyl-sn-glycerols 8a
and 8b in acceptable yields of 73−75%. The introduction of the
phosphocholine (PC) headgroup was carried out by the
method described by Eibl et al.41 using the 2-bromoethylphos-
phoric acid dichloride42 as phosphorylating reagent followed by
the quarternization with trimethylamine in 54−56% yield with
respect to compounds 8. Finally, the remaining benzyl blocking
group was removed from compounds 9a and 9b using a
hydrogenation reaction on palladium/carbon (10%) in EtOH
at 5 bar and 50 °C, and asymmetrical bolalipids I and II were
obtained in 84−93% yield after purification and with respect to
compound 9 or in 9.7−14.0% overall yield with respect to
starting material 3.
The following steps in the synthesis of asymmetrical
bolalipids I and II are well-established procedures. The
isopropylidene (IP) blocking group of 4 was cleaved using
pyridinium p-toluenesulfonate (PPTS) in a mixture of MeOH
and CHCl3 (5/1 v/v) due to the low solubility of acetal 4 in
pure MeOH. The sn-1 position of glycerol 5 was subsequently
blocked by tritylation. The reaction with trityl chloride was
The synthesis strategy presented herein allows the
preparation of asymmetrical glycerol diether bolalipids with a
variable alkyl chain pattern and different headgroups. Moreover,
the free hydroxy moiety offers the possibility of further
C
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modifications of the bolalipid, e.g., the introduction of a second,
pH-sensitive headgroup or the attachment of fluorescent or
EPR spin probes, sugar moieties, or peptide fragments (RGD
fragment) to force cell adhesion.
structures (liposomes) should display a characteristic folding
or disruption of these aggregates as shown for samples of our
symmetrical, diglycerol tetraether lipids described previously
(see also Figure S1).28,29 The aggregation behavior of lipids I
and II is comparable to the aggregation behavior observed for
asymmetrical single-chain and glycerol-free bolalipids including
a hydroxy group at one end and a PC33 or a DMAPPC32
headgroup at the other end of the single alkyl chain. The
formation of these large lamellar aggregates of lipids I and II
also explains why the extrusion through a 100 nm membrane
filter failed and the filter is clogged.
Temperature-Dependent Aggregation Behavior in an
Aqueous Suspension. TEM. As a first observation, neither
bolalipid (lipids I and II) is water-soluble. A turbid and
opalescent suspension in water (c = 1 mg mL−1) can be formed
by repeated heating and vortex mixing. An attempt to prepare
liposomes of both bolalipids by sonication and extrusion
through a 100 nm polycarbonate membrane filter failed.
Dynamic light scattering (DLS) of the suspension after 1 h of
storage showed the presence of particles with large hydro-
dynamic radii and a high polydispersity index (PdI; data not
shown). In addition, the corresponding correlation functions of
the size distributions revealed the presence of very large and
polydisperse aggregates. This indicates that lipids I and II are
not able to form stable liposomes with a 100 nm radius. To
visualize the structure of the aggregates formed in an aqueous
suspension (c = 0.03 mg mL−1), TEM images from negatively
stained samples prepared at 22 °C were obtained (Figure 2).
DSC and FTIR. The DSC heating curve of lipid I in water (c
= 1 mg mL−1) shows two endothermic transitions (Figure 3,
Figure 3. DSC heating (red lines) and cooling (blue lines) curves of
aqueous suspensions (c = 1 mg mL−1) of lipids I (solid lines) and II
(dashed lines). The heating rate was 20 K h−1. The curves are shifted
vertically for clarity.
red solid line) at 31.8 and at 94.0 °C, where a very small
increase in heat capacity can already be observed at about 85
°C. Both transitions are very cooperative, as indicated by the
narrow shape of the peak. The corresponding cooling curve
reveals the same peak pattern with almost no hysteresis: the
first peak is observed at 93.5 °C with a small additional peak at
91.5 °C and the second one at 31.6 °C (Figure 3, blue solid
line).
In contrast to lipid I, the DSC heating curve of lipid II, which
contains a methyl branch within the short C16 alkyl chain,
shows only one endothermic transition at 63.7 °C, which is a
bit less cooperative compared to both transitions of lipid I
(Figure 3, red dashed line). The corresponding cooling curve
shows again nearly no hysteresis; the transition peak is
observed at 62.9 °C, including a shoulder at 61.8 °C (Figure
3, blue dashed line).
Figure 2. TEM images of aqueous suspensions (c = 0.03 mg mL−1) of
lipid I (A, B) and lipid II (C, D) at different magnifications. The
samples were prepared at 22 °C and stained with uranyl acetate. The
white arrows point to large lamellar aggregates whereas the black
arrows point to smaller, round structures.
Compared to symmetrical diglycerol tetraether lipids, which
consist of two PC headgroups and a variable number of methyl
branches within the alkyl chain,28,29 the transition temperatures
(Tm) of both lipids (I and II) are much higher and the
transitions themselves are more cooperative. On the other
hand, Tm of lipids I and II are somewhat comparable to Tm
values of other asymmetrical but single-chained bolalipids, e.g.,
The TEM image of unbranched lipid I shows the formation
of large lamellar sheets with a size of several hundred
nanometers (white arrows in Figure 2A,B), which are
sometimes folded over (probably due to the drying process
during EM sample preparation). In addition, some smaller,
almost circular lamellar sheets with a diameter of 100−200 nm
are observed (black arrows). The TEM image of lipid II also
shows large lamellar aggregates, about 1 μm in size, as well as
round structures with a diameter of 200−500 nm (Figure
2C,D). All observed structures seem to be to sheetlike
structures rather than liposomes since negatively stained
TEM images of samples containing collapsed vesicular
PC-C32-OH43 (Tm = 51.2 °C), PC-C17pPhC17-OH33 (Tm =
53.4 °C), and DMAPPC-C32-OH32 (Tm1 = 36.0 °C and Tm
=
2
78.5 °C at pH 10). This is the first indication that the
headgroup asymmetry of bolalipids, independent of the number
of alkyl chains the bolalipid consists of, i.e., single chain or
double chain, leads to a better/denser packing of the lipid
D
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Figure 4. Wavenumber (heating, red; cooling, blue) of the symmetric methylene stretching vibrational band (A, C) and the methylene scissoring
vibrational band (B, D) as a function of temperature for suspensions of lipid I (A, B) and lipid II (C, D; c = 100 mg mL−1 in D2O).
molecules within the aggregate structure and hence higher Tm
values.
≈ 31 °C (Figure S2). The frequencies of both methylene
stretching vibrational bands remain very low, indicating that the
alkyl chains of lipid I are still in an all-trans conformation, i.e.,
no “melting” of the alkyl chain occurs at temperatures below 88
°C. Unfortunately, with our experimental setup we could not
obtain sample temperatures covering the range of the second
transition observed at 94.0 °C where the chains are obviously
“melting” and a liquid-crystalline lamellar phase is formed.
For lipid II the frequency of both methylene stretching
vibrational bands are located at low temperatures at 2848.7 and
2916.7 cm−1 for νsCH2 (Figure 4C) and νasCH2 (Figure S3).
These values are higher compared to CH2 stretching
frequencies of lipid I at low temperatures, indicating a structure
with less-ordered chains compared to lipid I. This is caused by
the additional methyl group, which leads to an increased space
requirement preventing a perfect parallel packing of the chains.
However, despite the perturbation of the packing by the methyl
group, both frequency values are indicative of an all-trans
conformation of the alkyl chain. When the sample is heated, the
wavenumber of both bands increase to 2852.1 (νsCH2) and
2921.9 (νasCH2) cm−1 at 87.6 °C, indicating that the chains are
in the “fluid” phase at this temperature. The “melting” of the
chains occurs at Tm of lipid II (around 63 °C) as can be seen by
the stepwise increase in the frequency at this temperature.
The cooling curves of the frequency of the νsCH2 and
νasCH2 bands of both bolalipids (Figures 4A,C, S2, and S3)
show only very small hysteresis. This hysteresis and the fact
that the end values of the cooling curves did not reach the
starting values of the heating curves are characteristic of a
To obtain more information on the nature of the
thermotropic transitions observed in the DSC scans of lipids
I and II, we performed temperature-dependent FTIR experi-
ments on suspensions of both bolalipids. The wavenumbers of
the symmetric (νsCH2) and the antisymmetric (νasCH2)
methylene stretching vibrational bands provide information
about the conformational order of the alkyl chain.44,45 The
results of the FTIR experiments are presented in Figure 4 and
in the SI in Figures S2−S4.
For unbranched lipid I, the frequencies of the bands at low
temperature are at 2847.0 cm−1 for νsCH2 (Figure 4A) and
2914.8 cm−1 for νasCH2 (Figure S2). These frequency values
are very low compared to the CH2 stretching frequencies of
other asymmetrical single-chain bolalipids, e.g., PC-
C17pPhC17-OH33 (2849.8 and 2917.0 cm−1) and DMAPPC-
C32-PC32 (2848.4 and 2917.7 cm−1), indicating that the alkyl
chains of lipid I are in an all-trans conformation and are well-
ordered in a quasi-crystalline state. With increasing temperature
the wavenumber of both bands remains nearly constant up to
the first endothermic transition Tm ≈ 31 °C. Above Tm, the
wavenumber of the νsCH2 band jumps to 2848.0 cm−1 and
increases further to 2849.0 cm−1 at a temperature of 87.7 °C,
just below the second endothermic transition. This increase is
more pronounced at temperatures above 80 °C, which could be
related to the second transition observed in the DSC. The
wavenumber of the νasCH2 band shows only a continuous
increase with temperature with only a slight discontinuity at Tm
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delayed reorganization of the ordered structures in the lamellar
assemblies of both bolalipids.
For a closer look at the nature of the transitions, the
methylene scissoring vibrational band (δCH2) was analyzed.
The temperature-dependent position of the δCH2 vibrational
band for lipid I is depicted in Figures 4B and S4. It shows a
characteristic splitting of the CH2 deformation band at low
temperatures (1472.7 and 1462.4 cm−1), indicative of an
orthorhombic and highly ordered (quasi-crystalline) packing of
the alkyl chain.45 With increasing temperature, this splitting
changes at the first Tm (≈ 33 °C), and a peak at 1466.4 cm−1
appears, including a shoulder at higher wavenumbers (Figure
S4). Upon further heating, this shoulder vanishes and the
splitting disappears within the temperature range of 34−43 °C.
The frequency of the remaining single δCH2 vibrational band
located at 1466.9 cm−1 (at 51.1 °C) stays almost constant up to
high temperatures. The first change in the splitting of the
methylene scissoring band is due to a change in packing from
an ordered orthorhombic Lβ′c (Go) to an ordinary ortho-
rhombic Lβ′ (Gd) phase. This was also observed before for long-
chain monopolar phosphocholines at very low temperatures.46
The single δCH2 vibrational band at about 1467 cm−1 above 51
°C is indicative of a hexagonal packing of chains. Such a change
in packing modes (from orthorhombic to hexagonal) was also
described for another single-chain bolalipid (DMAPPC-C32-
OH),32 although a change between different orthorhombic
phases was not observed for that bolalipid.
Figure 5. X-ray contour diagram of a 50 wt % lipid I sample in water.
The scattering intensities are shown in the upper part as a function of
the reciprocal lattice spacing (ordinate) and temperature (abscissa). In
the lower part the temperature course during the experiment is shown
as a ramp. The two arrows pointing down indicate the onset of
freezing and melting of interlamellar excess water. In the temperature
range between these arrows, additional intense ice reflections are seen.
The arrows pointing upward indicate the transition temperatures of
the bolalipid in the up and down scans. The intensities in the diagram
have been scaled differently in the different temperature ranges, visible
by the changes in gray scaling of the background intensity. Long
spacings (different orders of the repeat distance, white dashes) and
respective short spacings (fingerprint scattering due to the aliphatic
chain packing, yellow dashes) belong together at selected temperatures
I−V: (I) d = 6.80 nm (s = 0.147 nm−1, 17 °C), (II) d = 6.67 nm (s =
0.150 nm−1, 32 °C), (III) d = 5.88 nm (s = 0.170 nm−1, − 36 °C),
(IV) d = 5.88 nm (s = 0.170 nm−1, 98 °C), and (V) d = 5.00 nm (s =
0.200 nm−1, 104 °C). Two light vertical lines are drawn at 0 °C.
In contrast, the frequency of the δCH2 vibrational band of
lipid II is located at 1467.5 cm−1 at ambient temperature
(Figure 4D). This clearly indicates that for lipid II the chains
are not ordered in an orthorhombic lattice but show hexagonal
chain packing due to the additional methyl group in the short
alkyl chain. Upon heating, the wavenumber of the δCH2
vibrational band decreases with a pronounced jump at Tm
(about 64 °C), indicating the chain melting transition.
X-ray. To gain information on the molecular packing and
structural organization of the lipids within their lyotropic
aggregates, aqueous suspensions of each lipid (50 wt % H2O)
were investigated using temperature-dependent X-ray diffrac-
tion (Figures 5, 6, and S5−S9 and Tables S1 and S2).
For lipid I up to six sharp equidistant Bragg reflections in the
small-angle scattering (SAXS) region of the diffraction patterns
can be observed throughout the whole temperature range (−36
to 105 °C), which is indicative of an underlying lamellar
packing (Figures 5 and S5). Thus, lipid I forms lamellar
aggregates over the whole investigated temperature range. At
ambient temperature a lamellar repeat distance (membrane
thickness plus thickness of the hydration layer plus interlamellar
excess water layer thickness) of d = 6.80 nm (s1 = 0.147 nm−1)
was determined. Four sharp reflections were detected in the
SAXS region, indicating highly ordered alkyl chains. The
observed value is typical of bolalipid monolayers.32,33,47,48
Figure 6. X-ray contour diagram of a 50 wt % lipid II sample in water.
For details on the contour plot, see Figure 5, I−V: (I) d = 7.41 nm (s =
0.135 nm−1, 20 °C), (II) d = 7.41 nm (s = 0.135 nm−1, − 11 °C), (III)
d = 6.17 nm (s = 0.162 nm−1, − 30 °C), (IV) d = 7.41 nm (s = 0.135
nm−1, 70 °C), and (V) d = 8.41 nm (s = 0.120 nm−1, 87 °C).
Other asymmetrical bolalipids, e.g., DMAPPC-C32-PC,32
a
single-chain bolalipid with unmodified alkyl chains, or PC-
C17pPhC17-OH,33 a single-chain phenylene-modified bolali-
pid, show comparable layer thicknesses with d = 6.2 and 6.66
nm, respectively. Moreover, an antiparallel packing of the bola
molecules in a monolayer with interdigitated alkyl chains is
obvious, since other artificial asymmetrical bolalipids32,33 and
also bolalipids that are derived from archaea49 can form
interdigitated monolayer membranes. The wide-angle reflec-
tions are also indicative of highly ordered chains, which has also
been found before for phospholipid bilayers with all-trans alkyl
chains.50−52 To explain the observed d value an aggregation of
lipid I in the form of a double layer with a U-shaped
conformation of the C32 chain of the bola molecule can in
principle be assumed. However, this U shape is energetically
unfavorable since it requires the existence of at least four gauche
conformers in the C32 chain for a complete U-turn. FTIR
measurements reveal an all-trans conformation of the alkyl
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chain and an orthorhombic packing of the chains (see above).
Therefore, this U-shaped arrangement can be excluded.
After heating to 80 °C and cooling again to room
temperature, the SAXS reflections become more intense and
move to higher s values, indicating a dehydration of the
headgroup region with a concomitant reduction in water layer
thickness.
The sample was then frozen and heated again. Below 0 °C,
sharp ice reflections appear in the WAXS region of the
scattering patterns and there is a slight upshift of the five sharp
reflections from the alkyl chain packing (Lβ′c subcell) in the
fingerprint region, indicating a slight shortening of the cell
parameters. The ice reflections are a consequence of the
freezing of interlamellar excess water. Thus, the repeat distance
of the lamellae is decreased by 0.92 nm to d = 5.88 nm (s1 =
0.170 nm−1 at −36 °C).57 When the system is heated again, the
ice reflections vanish at 0 °C, and the previous SAXS scattering
and hence the repeat distance and degree of hydration are
restored.
In the wide-angle scattering (WAXS) region, five sharp
reflections are observed, indicating a high order of densely
packed alkyl chains. We could index the reflections with
orthorhombic subcell Lβ′c of symmetry Pbnm (Miller indices
are given on the right side of the contour plot in Figure 5) and
lattice parameters a = 0.495 nm and b = 0.744 nm.53,54 From
the lattice parameters, the average cross-sectional area per alkyl
chain of Σ = 0.1842 nm2 can be calculated, which is in the range
of typical Σ values of crystallized saturated olefins, for instance,
crystalline dotriacontane (C32H66) with a Σ value of 0.1841
nm2.55 The cross-sectional area of the acyl chains of DPPC in
the Lβ′ phase (at 33 °C) is much higher with Σ = 0.20 nm2.56
Therefore, the alkyl chains of lipid I are almost as densely
packed as the crystalline C32 alkane. The X-ray results thus
support the finding obtained by FTIR that lipid I forms a
monolayer of antiparallel (fully interdigitated) molecules with
an orthorhombic packing where the molecules can possibly be
tilted. If one assume a molecular length of lipid I of about 5.4
nm, full interdigitation of alkyl chains, and a tilt angle of
roughly 20° with respect to the layer normal, then one can
calculate a layer thickness of about 5.0 nm (Figure S10). Thus,
an interlamellar water layer thickness of 1.8 nm can be
calculated. This is adequate to ensure full hydration of the PC
headgroups. A partial interdigitation of lipid molecules, where
the hydroxy moiety of one bola molecule came in contact with
the methyl group from the end of the short alkyl chain of
another lipid molecule, is not possible since a layer thickness of
7.6 nm is calculated in that case (Figure S11).
At around 90 °C, the positions of the SAXS reflections
resulting in a decreased d value to d = 5.88 nm (s1 = 0.170 nm−1
at 98 °C) was reproducible. Thus, the more dehydrated
lamellar system seems to be the equilibrium state.
The transition into a liquid-crystalline phase (Lα) seems to
occur at a higher temperature. Only when reaching a
temperature of 102 °C a change in the reflection is observed
in both the SAXS and WAXS regions. Above this temperature,
the equidistant SAXS reflections show very poor intensities and
their position is shifted to higher s values; therefore, the
lamellar repeat distance shifts to d = 5.00 nm (s1 = 0.200 nm−1
at 104 °C). In the WAXS region no sharp reflection but only a
broad halo is visible, indicating a high-temperature phase with
fluid alkyl chains.
Upon heating to 32 °C, a change in the reflections in the
In contrast to lipid I, the diffraction patterns of lipid II,
bearing a methyl branch within the short alkyl chain, look
fundamentally different. At ambient temperature below the
transition observed in DSC and FTIR, five sharp equidistant
Bragg reflections are present in the SAXS region, indicating a
lamellar packing with a repeat distance of d = 7.41 nm (s1 =
0.135 nm−1 at 20 °C; see Figure 6 and S7 and Table S2). The d
value is 0.6 nm higher than for lipid I. Assuming a similar
antiparallel packing of the molecules in the lamellae (fully
interdigitated), either the thickness of the interlamellar excess
water layer would be much larger or no chain tilt is present in
the lamellae. The WAXS region reveals only one sharp peak at
s110 = 2.351 nm−1. However, closer inspection reveals that the
sharp peak is superimposed by a very broad peak at s020 = 2.358
nm−1 (Figure S8). This indicates an underlying Lβ packing
mode with alkyl chains oriented parallel to the layer normal
(without chain tilt). Obviously, the additional methyl moiety in
the short alkyl chain of lipid II does not allow for the very
dense (quasi-crystalline) packing of the alkyl chains as found
for lipid I below 32 °C.
When the system is cooled below 0 °C, sharp ice reflections
appear in the WAXS region, as is the case for lipid I. The
lamellar repeat distance of lipid II decreases by 1.24 nm to d =
6.17 nm (s1 = 0.162 nm−1, at −30 °C), much more than for
lipid I, due to the freezing out of excess water from the
interlamellar space. Still, the lamellae of lipid II show higher d
values than we observed for lipid I. Besides the sharp reflection
in the WAXS region at s110 = 2.389 nm−1, a second peak at s020
= 2.545 nm−1 can be detected (Figure S8). This suggests an Lβ′
packing mode and hence the alkyl chains tilted with respect to
the layer normal below 0 °C.
WAXS region occurs and only two distinct reflections (s110
=
2.386 nm−1 and s020 = 2.541 nm−1, see Figures 5 and S6) are
visible for temperatures above this first DSC transition up to a
temperature of about 45 °C. The two reflections can be indexed
as (110) and (020) reflections, respectively, indicating a
herringbone chain packing mode with subcell symmetry
Pbnm.54 Obviously, lipid I now forms a gel phase (Lβ′),56
which is typical of bolalipid monolayers and phospholipid
bilayers. It is likely that the alkyl chains of lipid I have a chain
tilt with respect to the monolayer normal as postulated for the
low-temperature phase because the positions of the SAXS
reflections and thus the repeat distance of the lamellae are
unchanged (Table S1).
Upon further heating, the two WAXS reflections move closer
together, and at about 45 °C, only one reflection is visible in the
diffraction pattern (Figure 5). This indicates that in the
temperature range between 32 and 45 °C the packing changes
from herringbone to a hexagonal alkyl chain packing. A closer
inspection reveals that the sharp WAXS peak is likely to be
superimposed on a very broad peak (Figure S6), which would
indicate an Lβ chain packing mode without chain tilt.56
However, the repeat distance d of the lamellar packing does
not change significantly in this temperature range so that this
change in tilt is not very likely. The IR spectra taken in this
temperature range agree with these findings as the splitting of
the δCH2 vibrational band is no longer detectable above 45 °C
(see above and Figure S4).
The hexagonal packing is present over a wide temperature
range, but the positions of the WAXS reflections shift to lower s
values with increasing temperature, indicating a temperature-
induced expansion of the Lβ subcell lattice.
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Above 64 °C up to a temperature of ca. 80 °C one broad
halo at s = 2.178 nm−1 superimposed on a sharp peak at s =
2.328 nm−1 is present, suggesting the coexistence of a fluid and
an ordered phase (Figure S9). The higher-order reflections in
the SAXS show very low intensities, also pointing to a
reduction of the lamellar packing order by the partial formation
of fluid domains. However, the DSC data show only one broad
transition at 64 °C, and also in the IR spectra the νCH2
vibrational bands shift to higher wavenumbers at the same
temperature, indicating the transition from all-trans to fluid
alkyl chains (see above). No two-phase region is observed in
these experiments. A possible explanation for these apparent
discrepancies might lie in the different hydration conditions: In
the DSC as well as the IR experiments the samples had a much
larger quantity of excess water than in the sample used for X-ray
experiments. If 50 wt % water would not be sufficient for the
complete hydration and formation of excess water, then a
broader two-phase region shifted to higher temperature could
be expected. The effect of decreased hydration on the phase
transition has been observed before for phospholipids, such as
dipalmitoylphosphatidylcholine (DPPC).58
Above a temperature of 80 °C only the halo is present in the
WAXS region indicating that a fluid phase is present. In the
SAXS region, the reflections show very poor intensities and
their position is shifted to lower s values, hence the lamellar
repeat distance is shifted up to d = 8.41 nm (s = 0.120 nm−1 at
87 °C). The transition processes are reversible, and the same
scattering patterns appear when the sample is cooled at about
the same temperatures as observed for heating.
The stereochemistry of the additional methyl branch in the
short alkyl chain of lipid II should have no or only a marginal
effect on the lyotropic behavior of the bolalipid. As we have
shown for diglycerol tetraether model lipids bearing non, two,
or four methyl moieties at distinct positions of the alkyl chain,
the stereospecificity of those methyl branches is not of
substantial significance for the properties of archaeal model
lipids: the optically pure28 and also the racemic29 model lipids
showed comparable lyotropic behavior (transition temper-
atures) and aggregation behavior (formation of liposomes).
layer normal. This high crystallinity at ambient temperatures
was not observed before for other phospholipids. With
increasing temperature, changes in the chain packing mode
occur: above 31 °C the ordered orthorhombic Lβ′c (Go) phase
transforms into an ordinary orthorhombic Lβ′ (Gd) phase also
with tilted alkyl chains, which further changes into a hexagonal
phase without chain tilt (Lβ) at temperatures above 48 °C.
However, the alkyl chains are still in the all-trans conformation
up to high temperatures of about 102 °C. Above this
temperature a melting of the alkyl chains into liquid-crystalline
(Lα) phases occur.
Lipid II also self-assembles in an aqueous suspension into
large lamellar sheets with a fully interdigitated arrangement of
the bola molecules within the monolayer. The thickness of the
lamellae is 7.4 nm and therefore larger compared to the
thickness of 6.8 nm observed for lipid I. The lipid molecules are
oriented parallel to the layer normal and are not as densely
packed as observed before for lipid I due to the additional
methyl group within the short alkyl chain of lipid II, which
leads to an increased space requirement of the hydrophobic
part of the bolalipid. Concomitant with this increased volume
requirement, more water seems to be necessary for complete
hydration of the headgroups. This is evident in the X-ray
sample, where a two-phase region above 64 °C up to 80 °C is
observed, which is not seen in the samples used for DSC and IR
that had a much higher water content. Above 80 °C, a liquid-
crystalline (Lα) phase is present.
The results show that a delicate balance between the steric
requirements of headgroups (asymmetry) and the alkyl chains
as well as the shape of the whole molecule and intermolecular
interactions within these long-chain compounds determines the
aggregation behavior of the bolalipids. The addition of one
small methyl branch in the alkyl chains changes this aggregation
behavior dramatically. The question of whether this novel class
of bolalipids is miscible with conventional phospholipids, e.g.,
dipalmitoylphosphatidylcholine (DPPC), which can lead to
stabilized liposomes applicable for drug-delivery purposes, is
part of ongoing research.
ASSOCIATED CONTENT
* Supporting Information
■
CONCLUSIONS
S
■
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.lang-
muir.5b02951.
Two examples from a new class of highly asymmetrical glycerol
diether bolalipids were synthesized using different high-yielding
O-alkylation reactions as well as a versatile protection-group
strategy. The preparation methods presented are also applicable
for other alkyl chain lengths and/or variable headgroup
structures. Moreover, the free hydroxyl moiety of both
bolalipids allows the introduction of any further modification.
The aggregation behavior of both bolalipids (lipid I without
the methyl branch and lipid II bearing one methyl group within
the short alkyl chain) in the aqueous suspension as a function
of temperature showed that both bolalipids form large sheetlike
lamellar aggregates. This is in contrast to diglycerol tetraether
lipids, either naturally occurring in archaea or artificial ones,
which self-assemble into liposomes with curved lamellae. In the
case of lipid I, the thickness of the lamellae is 6.8 nm, indicating
a monolayer of bola molecules with fully interdigitated alkyl
chains. The alkyl chains are in an all-trans conformation, very
densely packed and in a quasi-crystalline state as indicated by a
splitting of the δCH2 vibrational band (FTIR) and by the value
for the cross-sectional area per alkyl chain of Σ = 0.18 nm2 (X-
ray), which is comparable to Σ values of crystallized saturated
olefins. The molecules seem to be tilted with respect to the
Synthesis procedures, analytical data of prepared
compounds, and further TEM, FTIR, and X-ray
measurements (PDF)
AUTHOR INFORMATION
Corresponding Authors
*E-mail: simon.drescher@pharmazie.uni-halle.de. Phone: +49-
345-5525196. Fax: +49-345-5527026.
■
*E-mail: bodo.dobner@pharmazie.uni-halle.de. Phone: +49-
345-5525120. Fax: +49-345-5527018.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Dedicated to Prof. Gerald Brezesinski on the occasion of his
65th birthday. This work was financially supported by grants for
postgraduate studies of the German Federal State Sachsen-
Anhalt (to T.M.) and by grants from the Deutsche
H
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of 72-Membered Tetraether Model Compounds. Bull. Chem. Soc. Jpn.
1997, 70, 2545−2554.
Forschungsgemeinschaft (DFG), project DR 1024/1-1 (to
S.D.), and within the Forschergruppe FOR 1145 (to B.-D.L.
and A.B.). The support of Dr. Gerd Hause (Biocenter, Martin
Luther University Halle-Wittenberg) by providing us access to
the electron microscope facility is greatly appreciated.
(21) Eguchi, T.; Arakawa, K.; Terachi, T.; Kakinuma, K. Total
Synthesis of Archaeal 36-Membered Macrocyclic Diether Lipid. J. Org.
Chem. 1997, 62, 1924−1933.
(22) Eguchi, T.; Ibaragi, K.; Kakinuma, K. Total Synthesis of
Archaeal 72-Membered Macrocyclic Tetraether Lipids. J. Org. Chem.
1998, 63, 2689−2698.
(23) Arakawa, K.; Eguchi, T.; Kakinuma, K. An Olefin Metathesis
Approach to 36- and 72-Membered Archaeal Macrocyclic Membrane
Lipids. J. Org. Chem. 1998, 63, 4741−4745.
(24) Benvegnu, T.; Brard, M.; Plusquellec, D. Archaeabacteria bipolar
lipid analogues: structure, synthesis and lyotropic properties. Curr.
Opin. Colloid Interface Sci. 2004, 8, 469−479.
REFERENCES
■
(1) Woese, C. R.; fox, G. F. Phylogenetic structure of the prokaryotic
domain: The primary kingdoms. Proc. Natl. Acad. Sci. U. S. A. 1977, 74,
5088−5090.
(2) Koch, R.; Zablowski, P.; Spreinat, A.; Antranikian, G. Extremely
thermostable amylolytic enzyme from the archaebacterium Pyrococcus
furiosus. FEMS Microbiol. Lett. 1990, 71, 21−26.
(25) Brard, M.; Laine,
́
C.; Ret
́ ́
hore, G.; Laurent, I.; Neveu, C.;
(3) Woese, C. R.; Magrum, L. J.; Fox, G. E. Archaebacteria. J. Mol.
Evol. 1978, 11, 245−252.
Lemieg
̀
re, L.; Benvegnu, T. Synthesis of Archaeal Bipolar Lipid
Analogues: A Way to Versatile Drug/Gene Delivery Systems. J. Org.
Chem. 2007, 72, 8267−8279.
(26) Meister, A.; Blume, A. Self-assembly of bipolar amphiphiles.
Curr. Opin. Colloid Interface Sci. 2007, 12, 138−147.
(4) Baumeister, W.; Lembcke, G. Structural features of archaebacte-
rial cell envelopes. J. Bioenerg. Biomembr. 1992, 24, 567−575.
(5) Lewalter, K.; Muller, V. Bioenergetics of archaea: Ancient energy
̈
conserving mechanisms developed in the early history of life. Biochim.
Biophys. Acta, Bioenerg. 2006, 1757, 437−445.
̀
(27) Jacquemet, A.; Lemiegre, L.; Lambert, O.; Benvegnu, T. How
the Stereochemistry of a Central Cyclopentyl Ring Influences the Self-
Assembling Properties of Archaeal Lipid Analogues: Synthesis and
CryoTEM Observations. J. Org. Chem. 2011, 76, 9738−9747.
(28) Markowski, T.; Drescher, S.; Meister, A.; Hause, G.; Blume, A.;
Dobner, B. Synthesis of Optically Pure Diglycerol Tetraether Model
Lipids with Non-Natural Branching Pattern. Eur. J. Org. Chem. 2011,
2011, 5894−5904.
(6) Langworthy, T. A. Long-chain diglycerol tetraethers from
Thermoplasma acidophilum. Biochim. Biophys. Acta, Lipids Lipid
Metab. 1977, 487, 37−50.
(7) De Rosa, M.; Esposito, E.; Gambacorta, A.; Nicolaus, B.; Bu’Lock,
J. D. Effects of temperature on ether lipid composition of Caldariella
acidophila. Phytochemistry 1980, 19, 827−831.
(8) Nishihara, M.; Morii, H.; Koga, Y. Structure Determination of a
Quartet of Novel Tetraether Lipids from Methanobacterium
thermoautotrophicum. J. Biochem. 1987, 101, 1007−1015.
(9) Fuhrhop, J.-H.; Wang, T. Bolaamphiphiles. Chem. Rev. 2004, 104,
2901−2937.
(29) Markowski, T.; Drescher, S.; Meister, A.; Blume, A.; Dobner, B.
Structure-property relationships in a series of diglycerol tetraether
model lipids and their lyotropic assemblies: the effect of branching
topology and chirality. Org. Biomol. Chem. 2014, 12, 3649−3662.
(30) Drescher, S.; Meister, A.; Blume, A.; Karlsson, G.; Almgren, M.;
Dobner, B. General synthesis and aggregation behaviour of a series of
single-chain 1,ω-bis(phosphocholines). Chem. - Eur. J. 2007, 13,
5300−5307.
(10) Cornell, B. A.; Braach-Maksvytis, V. B. L.; King, L. G.; Osmann,
P. D. J.; Raguse, B.; L, W.; Pace, R. J. A biosensor that uses ion-channel
switches. Nature 1997, 387, 580−583.
(11) Bakowsky, U.; Rothe, U.; Antonopoulos, E.; Martini, T.;
Henkel, L.; Freisleben, H. J. Monomolecular organization of the main
tetraether lipid from Thermoplasma acidophilum at the water-air
interface. Chem. Phys. Lipids 2000, 105, 31−42.
(31) Drescher, S.; Dobner, B. Synthesis of novel asymmetrical single-
chain phosphoglycol-based bolaamphiphiles. Synth. Commun. 2014,
44, 564−573.
(32) Graf, G.; Drescher, S.; Meister, A.; Garamus, V. M.; Dobner, B.;
Blume, A. Tuning the Aggregation Behaviour of Single-Chain
Bolaamphiphiles in Aqueous Suspension by Changes in Headgroup
Asymmetry. Soft Matter 2013, 9, 9562−9571.
́ ́
(12) Benvegnu, T.; Rethore, G.; Brard, M.; Richter, W.; Plusquellec,
D. Archaeosomes based on novel synthetic tetraether-type lipids for
the development of oral delivery systems. Chem. Commun. 2005,
5536−5538.
́
(33) Drescher, S.; Lechner, B.-D.; Garamus, V. M.; Almasy, L.;
(13) Brown, D. A.; Venegas, B.; Cooke, P. H.; English, V.; Chong, P.
L.-G. Bipolar tetraether archaeosomes exhibit unusual stability against
autoclaving as studied by dynamic light scattering and electron
microscopy. Chem. Phys. Lipids 2009, 159, 95−103.
Meister, A.; Blume, A. The headgroup (a)symmetry strongly
determines the aggregation behavior of single-chain phenylene-
modified bolalipids and their miscibility with classical phospholipids.
Langmuir 2014, 30, 9273−9284.
́
(14) Jain, N.; Arntz, Y.; Goldschmidt, V. r.; Duportail, G.; Mely, Y.;
(34) Kameta, N.; Masuda, M.; Minamikawa, H.; Shimizu, T. Self-
Assembly and Thermal Phase Transition Behavior of Unsymmetrical
Bolaamphiphiles Having Glucose- and Amino-Hydrophilic Head-
groups. Langmuir 2007, 23, 4634−4641.
(35) Fuhrhop, J.-H.; Spiroski, D.; Boettcher, C. Molecular monolayer
rods and tubules made of α-(L-lysine),ω-(amino) bolaamphiphiles. J.
Am. Chem. Soc. 1993, 115, 1600−1601.
(36) Masuda, M.; Shimizu, T. Lipid nanotubes and microtubes:
experimental evidence for unsymmetrical monolayer membrane
formation from unsymmetrical bolaamphiphiles. Langmuir 2004, 20,
5969−5977.
(37) Kameta, N.; Masuda, M.; Minamikawa, H.; Mishima, Y.;
Yamashita, I.; Shimizu, T. Functionalizable Organic Nanochannels
Based on Lipid Nanotubes: Encapsulation and nanofluidic Behavior of
Biomacromolecules. Chem. Mater. 2007, 19, 3553−3560.
(38) Masuda, M.; Shimizu, T. Multilayer structure of an unsym-
metrical monolayer lipid membrane with a ’head-to-tail’ interface.
Chem. Commun. 2001, 2442−2443.
(39) Schwarz, M.; Oliver, J. E.; Sonnet, P. E. Synthesis of 3,11-
dimethyl-2-nonacosanone, a sex pheromone of the German cockroach.
J. Org. Chem. 1975, 40, 2410−2411.
Klymchenko, A. S. New Unsymmetrical Bolaamphiphiles: Synthesis,
Assembly with DNA, and Application for Gene Delivery. Bioconjugate
Chem. 2010, 21, 2110−2118.
(15) Nuraje, N.; Bai, H.; Su, K. Bolaamphiphilic molecules: Assembly
and applications. Prog. Polym. Sci. 2013, 38, 302−343.
(16) Krishnan, L.; Deschatelets, L.; Stark, F. C.; Gurnani, K.; Sprott,
G. D. Archaeosome Adjuvant Overcomes Tolerance to Tumor-
Associated Melanoma Antigens Inducing Protective CD8+ T Cell
Responses. Clin. Dev. Immunol. 2010, 2010, 1−13.
(17) Sprott, G. D.; Tolson, D. L.; Patel, G. B. Archaeosomes as novel
antigen delivery systems. FEMS Microbiol. Lett. 1997, 154, 17−22.
(18) Patel, G. B.; Agnew, B. J.; Deschatelets, L.; Fleming, L. P.;
Sprott, G. D. In vitro assessment of archaeosome stability for
developing oral delivery systems. Int. J. Pharm. 2000, 194, 39−49.
(19) Patel, G. P.; Ponce, A.; Zhou, H.; Chen, W. Safety of
Intranasally Administered Archaeal Lipid Mucosal Vaccine Adjuvant
and Delivery (AMVAD) Vaccine in Mice. Int. J. Toxicol. 2008, 27,
329−339.
(20) Eguchi, T.; Kano, H.; Arakawa, K.; Kakinuma, K. Synthetic
Studies of Archaeal Macrocyclic Tetraether Lipids: Practical Synthesis
I
DOI: 10.1021/acs.langmuir.5b02951
Langmuir XXXX, XXX, XXX−XXX

Langmuir
Article
(40) Hermetter, A.; Paltauf, F. A new method for the detritylation of
1,2-diradyl-3-O-tritylglycerols. Chem. Phys. Lipids 1981, 29, 191−195.
(41) Eibl, H.; Nicksch, A. 1,3-Propanedeol phosphatides. Ger. Offen.
DE 2345057 A1 19750327, 1975.
(42) Hirt, G.; Berchtold, R. Synthesis of phosphatides. A new
synthesis of lecithin. Pharm. Acta Helv. 1958, 33, 349−356.
(43) Zimmermann, I. Struktur- und Phasenverhalten von Neuen
Lipiden mit Molekularen Modifizierungen in Hydrophilen Bereich.
Ph.D. Thesis, Martin Luther University Halle-Wittenberg, Halle
(Saale), 1997.
(44) Mantsch, H. H.; McElhaney, R. N. Phospholipid phase
transitions in model and biological membranes as studied by infrared
spectroscopy. Chem. Phys. Lipids 1991, 57, 213−226.
(45) Mendelsohn, R.; Moore, D. J. Vibrational spectroscopic studies
of lipid domains in biomembranes and model systems. Chem. Phys.
Lipids 1998, 96, 141−157.
(46) Snyder, R. G.; Liang, G. L.; Strauss, H. L.; Mendelsohn, R. IR
Spectroscopic Study of the Structure and Phase Behavior of Long-
Chain Diacylphosphatidylcholines in the Gel State. Biophys. J. 1996,
71, 3186−3198.
(47) Meister, A.; Drescher, S.; Karlsson, G.; Hause, G.; Baumeister,
U.; Hempel, G.; Garamus, V. M.; Dobner, B.; Blume, A. Formation of
square lamellae by self-assembly of long-chain bolaphospholipids in
water. Soft Matter 2010, 6, 1317−1324.
(48) Pohle, W.; Selle, C.; Rettig, W.; Heiser, U.; Dobner, B.;
Wartewig, S. Phase transitions and hydrogen bonding in a bipolar
phosphocholine evidenced by calorimetry and vibrational spectrosco-
py. Arch. Biochem. Biophys. 2001, 396, 151−161.
(49) Gliozzi, A. In Handbook of Nonmedical Applications of Liposomes;
Barenholz, Y., Lasic, D. D., Eds.; CRC Press: Boca Raton, FL, 1996;
Vol. II, pp 329−348.
(50) Janiak, M. J.; Small, D. M.; Shipley, G. G. Nature of the Thermal
Pretransition of Synthetic Phospholipids: Dimyristoyl- and Dipalmi-
toyllecithin. Biochemistry 1976, 15, 4575−4580.
(51) Tristram-Nagle, S.; Nagle, J. F. Lipid bilayers: thermodynamice,
structure, uctuations, and interactions. Chem. Phys. Lipids 2004, 127,
3−14.
(52) Tristram-Nagle, S.; Liu, Y.; Legleiter, J.; Nagle, J. F. Structure of
Gel Phase DMPC Determined by X-Ray Diffraction. Biophys. J. 2002,
83, 3324−3335.
(53) Forster, G.; Meister, A.; Blume, A. Chain packing modes in
̈
crystalline surfactant and lipid bilayers. Curr. Opin. Colloid Interface Sci.
2001, 6, 294−302.
(54) Forster, G.; Meister, A.; Blume, A. Van der Waals energy
̈
contour map describing the orthorhombic hydrocarbon chain packing
of symmetry Pbnm. Phys. Chem. Chem. Phys. 2000, 2, 4503−4508.
(55) Vand, V. Density and Unit Cell of n-Hexatriacontane. Acta
Crystallogr. 1953, 6, 797−798.
(56) Tardieu, A.; Luzzati, V.; Reman, F. C. Structure and
Polymorphism of the Hydrocarbon Chains of Lipids: A AStudy of
Lecithin-Water Phases. J. Mol. Biol. 1973, 75, 711−733.
(57) Forster, G.; Brezesinski, G. Lyotropic stress in lipid-water model
̈
systems. Structural changes induced by freezing of the solvent. Liq.
Cryst. 1989, 5, 1659−1668.
(58) Grabielle-Madelmont, C.; Perron, R. Calorimetric studies on
phospholipidwater systems: I. DL-Dipalmitoylphosphatidylcholine
(DPPC)water system. J. Colloid Interface Sci. 1983, 95, 471−482.
J
DOI: 10.1021/acs.langmuir.5b02951
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