Membrane properties of amacrocyclic tetraether bisphosphatidylcholine lipid: Effect of a single membrane-spanning polymethylene cross-linkage between two head groups of ditetradecylphosphatidylcholine membrane

Article abstract of DOI:10.1016/j.bbamem.2021.183569

The plasma membranes of archaea are abundant in macrocyclic tetraether lipids that contain a single or double long transmembrane hydrocarbon chains connecting the two glycerol backbones at both ends. In this study, a novel amacrocyclic bisphosphatidylcholine lipid bearing a single membrane-spanning octacosamethylene chain, 1,1’-O-octacosamethylene-2,2′-di-O-tetradecyl-bis-(sn-glycero)-3,3′-diphosphocholine (AC-(di-O-C14PC)₂), was synthesized to elucidate effects of the interlayer cross-linkage on membrane properties based on comparison with its corresponding diether phosphatidylcholine, 1,2-di-O-tetradecyl-sn-glycero-3-phosphocholine (DTPC), that forms bilayer membrane. Several physicochemical techniques demonstrated that while AC-(di-O-C14PC)₂ monolayer, which adopts a particularly high-ordered structure in the gel phase, shows remarkably high thermotropic transition temperature compared to DTPC bilayer, the fluidity of both phospholipids above the transition temperature is comparable. Nonetheless, the fluorescent dye leakage from inside the AC-(di-O-C14PC)₂ vesicles in the fluid phase is highly suppressed. The origin of the membrane properties characteristic of AC-(di-O-C14PC)₂ monolayer is discussed in terms of the single long transmembrane hydrophobic linkage and the diffusional motion of the lipid molecules.

Full text of DOI:10.1016/j.bbamem.2021.183569

BBA - Biomembranes 1863 (2021) 183569
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BBA - Biomembranes
journal homepage: www.elsevier.com/locate/bbamem
Membrane properties of amacrocyclic tetraether bisphosphatidylcholine
lipid: Effect of a single membrane-spanning polymethylene cross-linkage
between two head groups of ditetradecylphosphatidylcholine membrane
,
,
Naoyuki Tsuchidaa, Toshiyuki Takagib *, Hiroshi Takahashic *, Toshitada Yoshiharaa,
,d,e,**
Seiji Tobitaa, Masashi Sonoyamaa
a Division of Molecular Science, Faculty of Science and Technology, Gunma University, Kiryu, Gunma 376-8515, Japan
b Cellular and Molecular Biotechnology Research Institute, AIST, Tsukuba, Ibaraki 305-8565, Japan
c Division of Pure and Applied Science, Faculty of Science and Technology, Gunma University, Maebashi, Gunma 371-8510, Japan
d Gunma University Initiative for Advanced Research (GIAR), Gunma University, Kiryu, Gunma 376-8515, Japan
e Gunma University Center for Food Science and Wellness (GUCFW), Gunma University, Kiryu, Gunma 376-8515, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords:
The plasma membranes of archaea are abundant in macrocyclic tetraether lipids that contain a single or double
long transmembrane hydrocarbon chains connecting the two glycerol backbones at both ends. In this study, a
novel amacrocyclic bisphosphatidylcholine lipid bearing a single membrane-spanning octacosamethylene chain,
1,1’-O-octacosamethylene-2,2′-di-O-tetradecyl-bis-(sn-glycero)-3,3′-diphosphocholine (AC-(di-O-C14PC)2), was
synthesized to elucidate effects of the interlayer cross-linkage on membrane properties based on comparison with
its corresponding diether phosphatidylcholine, 1,2-di-O-tetradecyl-sn-glycero-3-phosphocholine (DTPC), that
forms bilayer membrane. Several physicochemical techniques demonstrated that while AC-(di-O-C14PC)2
monolayer, which adopts a particularly high-ordered structure in the gel phase, shows remarkably high ther-
motropic transition temperature compared to DTPC bilayer, the fluidity of both phospholipids above the tran-
sition temperature is comparable. Nonetheless, the fluorescent dye leakage from inside the AC-(di-O-C14PC)2
vesicles in the fluid phase is highly suppressed. The origin of the membrane properties characteristic of AC-(di-O-
C14PC)2 monolayer is discussed in terms of the single long transmembrane hydrophobic linkage and the
diffusional motion of the lipid molecules.
Amacrocyclic phospholipid
Ether-linked phospholipid
Thermotropic transition
Membrane fluidity
Leakage assay
1. Introduction
phospholipids with distinctive chemical structures. The principal dif-
ference is that archaeal phospholipids are built on a backbone of sn-
Biomembrane, a common fundamental structure in the cells of living
organisms, is mainly composed of a phospholipid membrane and
embedded integral or peripheral membrane proteins. Structural and
physical properties of phospholipid membranes significantly vary be-
tween the domains of organisms [1–6]. In eukaryotes and bacteria, sn-
glycerol-3-phosphate-based ester-linked phospholipid molecules
together assemble into a bilayer membrane of an inner leaflet and an
outer leaflet. On the other hand, archaea, which thrive in various harsh
environments, e.g., hot springs and salt lakes, typically utilize
glycerol-1-phosphate bearing ether-linkages with a hydrocarbon chain.
An additional striking structural feature reported for some archaeal
lipids is that two double-chained ether-linked phospholipid molecules,
which generally form bilayer membrane in the aqueous media, are cross-
linked through a single or double interlayer bridge of a phytanyl chain to
yield amacrocyclic or macrocyclic tetraether phospholipids, respectively
[1–6]. Typically, cyclopentene rings of varying proportions are included
in the phytanyl chains [1–6]. Such archaea-specific amacrocyclic and
macrocyclic tetraether phospholipids have a single or double long
Abbreviations: AC-(di-O-C14PC)2, amacrocyclic 1,1’-O-octacosamethylene-2,2′-di-O-tetradecyl-bis-(sn-glycero)-3,3′-diphosphocholine; 6-CF, 6-carboxyfluorescein;
di-O-C14PC, 1,2-di-O-tetradecyl-sn-glycero-3-phosphocholine; DPH, diphenylhexatriene; Laurdan, 2-dimethylamino-6-lauroylnaphthalene; WAXD, wide-angle X-ray
diffraction.
* Corresponding authors.
** Correspondence to: Masashi Sonoyama, Division of Molecular Science, Faculty of Science and Technology, Gunma University, Kiryu, Gunma 376-8515, Japan.
E-mail addresses: t.takagi@aist.go.jp (T. Takagi), hirotakahashi@gunma-u.ac.jp (H. Takahashi), sonoyama@gunma-u.ac.jp (M. Sonoyama).
https://doi.org/10.1016/j.bbamem.2021.183569
Received 10 November 2020; Received in revised form 14 January 2021; Accepted 27 January 2021
Available online 5 February 2021
0005-2736/© 2021 Published by Elsevier B.V.

N. Tsuchida et al.
BBA - Biomembranes 1863 (2021) 183569
phytanyl chain with cyclopentene rings and the two polar head groups at
its ends via the ether-linkage with the glycerol moiety. Therefore, it
turns out that in the aqueous media, the archaea-specific amacrocyclic
and macrocyclic tetraether phospholipids spontaneously form vesicles
composed of a “eukaryotic naturally-occurring phospholipid bilayer-like
monolayer” membrane. Thus, the transmembrane cross-linkage, the
methyl branches, and the cyclopentene rings are the three structural
segments characteristic of archaea-specific amacrocyclic and macrocy-
clic tetraether phospholipids.
purchased from Avanti Polar Lipids, Inc. Laurdan (6-Dodecanoyl-2-
dimethylaminonaphthalene) and DPH (Diphenylhexatriene) were from
Molecular Probes, Inc. 6-CF (6-Carboxyfluorescein) was obtained from
Tokyo Chemical Industry Co., Ltd. All other chemical reagents used were
of research-grade. For efficient preparations of samples for several
physicochemical measurements, stock solutions of the phospholipids
and the fluorescent dyes except for 6-CF were prepared by dissolving
them into chloroform. Methanol was used for the stock solution of 6-CF.
In addition to be important targets in the various fields of basic
research [7–22], such archaea-specific amacrocyclic and macrocyclic
tetraether phospholipids have been attracting much attention as prom-
ising materials for pharmaceutical and biomedical applications [23–34].
The highly advantageous properties of such archaea-specific phospho-
lipids are high thermal and mechanical stability and low permeability of
membranes. To reveal structural features responsible for the significant
archaea phospholipid-specific membrane properties, not only native
[15–18,30–34] but also synthetic [7–11,13–14,19–29] amacrocyclic
and macrocyclic tetraether phospholipids have been extensively inves-
tigated with the use of various biophysical techniques. However, the
amacrocyclic and macrocyclic tetraether phospholipids studied in the
previous studies are assembled of some of the archaea-specific structural
segments such as the covalently cross-linked alkyl chain, the methyl
branch, and the cyclopentene ring. Therefore, it is not straightforward to
separately clarify effects of the membrane-spanning cross-linkage.
In the present study, a novel amacrocyclic tetraether phospholipid
bearing a single transmembrane polymethylene chain that connects two
phosphatidylcholine (PC) groups, 1,1’-O-octacosamethylene-2,2′-di-O-
tetradecyl-bis-(sn-glycero)-3,3′-diphosphocholine (Fig. 1, AC-(di-O-
C14PC)2), is developed to elucidate effects of a simple membrane-
spanning cross-linkage on membrane properties. The amacrocyclic tet-
raether phospholipid used corresponds to a dimer of a diether phos-
pholipid 1,2-di-O-tetradecyl-sn-glycero-3-phosphocholine (di-O-C14PC)
connected through the cross-linkage between the two PC groups in both
inner and outer leaflets of the di-O-C14PC bilayer membrane. Therefore,
based on detailed comparisons between AC-(di-O-C14PC)2 monolayer
membrane and di-O-C14PC bilayer membrane, it is expected that the
effects of a single transmembrane cross-linkage on physical properties
are unraveled. The comparative analyses between di-O-C14PC bilayer
vesicle and AC-(di-O-C14PC)2 monolayer vesicle demonstrated that
compared to the di-O-C14PC vesicle, the leakage of fluorescent com-
pounds inside the AC-(di-O-C14PC)2 vesicle is highly suppressed even in
the liquid crystalline phase despite high fluidity comparable to the di-O-
C14PC bilayer.
2.2. Organic synthesis of AC-(di-O-C14PC)2
AC-(di-O-C14PC)2 (1) was synthesized by the following procedures
(Scheme 1). Propargyl alcohol protected as THP ether was reacted with
1-bromoundecane by treated with n-BuLi in THF-HMPA, and the THP
ether was removed to give 2-tetradecyn-1-ol. The isomerization of the
alcohol bearing internal triple bond was treated with NaH in 1,3-prop-
andiamine to yield 13-tetradecyn-1-ol having terminal triple bond.
The alcohol was mesylated to obtain compound 2 [35–36].
Compound 3 was prepared from (S)-(+)-2,2-dimethyl-1,3-dioxolane-
4-methanol according to the method described in the literature [37].
Compound 3 was reacted with 1-bromotetradecane in the presence of
NaH, the trityl group was deprotected by treated with cat. pTsOH,
compound 2 was reacted, and the PMB group was removed by DDQ
oxidation to provide compound 4 [8,38]. Diol compound having diac-
etylene group obtained by homo-coupling reaction of compound 4 in the
presence of Cu(OAc)2 in pyridine was reduced by treated with Pd(OH)2/
C in H2 atmosphere to obtain compound 5, and the two hydroxyl groups
were converted to phosphocholine groups to get compound 1 [39–40].
2-Bromoethyl phosphorodichloride was prepared from phosphoryl
dichloride according to the method of W. J. Hansen et al. [41]. Com-
pound 5 (1.17 g/ 1.21 mmol) was treated with 2-bromoethyl phos-
phorodichloride (0.90 g/ 3.73 mmol) in benzene containing
triethylamine (0.85 mL/6.10 mmol) at room temperature for 18 h, and
then the reaction mixture was evaporated. The residue was extracted
with CHCl3 after being treated with water at room temperature for 4 h.
The solvent was removed under reduce pressure to afford the phos-
phoryl intermediate. Trimethylamine 30% solution was added to the
intermediate in a mixture of [CH3CN]/[iPrOH]/[CHCl3] (5/5/3). The
reaction mixture was stirred for 18 h at 60 ◦C. After the removal of the
solvent under reduce pressure, the residue was purified by column
chromatography (SiO2, [CHCl3]:[MeOH]:[H2O] = 65:35:4 to 65:35:8),
Sephadex LH20 (MeOH), and ODS (MeOH)), to give compound 1 (1.08
g/68%).
1: MS (FAB+) m/z: 1298 (M + H)+. HRMS (FAB+) calcd for
C72H151N2O12P2 (M + H)+ 1298.0737: Found 1298.0747. 1H NMR
(CD3OD-CDCl3, TMS): 4.21–4.34 (m, 4H), 3.87 (t, J = 5.5 Hz, 4H),
3.65–3.75 (m, 4H), 3.50–3.64 (m, 8H), 3.49–3.37 (m, 6H), 3.29 (s, 18H),
1.46–1.61 (m, 8H), 1.20–1.38 (m, 92H), 0.88 (t, J = 6.8 Hz, 6H).
2. Materials and methods
2.1. Materials
All the reagents for organic synthesis of AC-(di-O-C14PC)2 were
commercially available and were used without further purification. Di-
O-C14PC (1,2-Di-o-ditetradecyl-sn-glycero-3-phosphocholine) was
2.3. DSC measurements
A comparison of thermotropic behaviors of phospholipid suspension
Fig. 1. Chemical structures of (A) di-O-C14PC and (B) AC-(di-O-C14PC)2. For comparison, di-O-C14PC is depicted as a piece of bilayer membrane.
2

N. Tsuchida et al.
BBA - Biomembranes 1863 (2021) 183569
Scheme 1. Synthesis of Amacrocyclic Bisphosphatidylcholine lipid 1
of AC-(di-O-C14PC)2 and di-O-C14PC was performed by using DSC
measurements. For the measurements, dried films of AC-(di-O-C14PC)2
and di-O-C14PC were obtained by evaporating the stock solutions under
a stream of dry nitrogen and were kept in vacuum for 16 h to remove
residual solvents. Multilamellar liposome samples were prepared by
suspending the dried phospholipid films in Milli-Q water and sonicating
at 55 ◦C for 1 h. The concentrations of AC-(di-O-C14PC)2 and di-O-
C14PC were 50 and 100 mM, respectively. The thermotropic transition
of the phospholipid suspensions was investigated with a SEIKO DSC
6100-Exstar6000 differential scanning calorimeter (Chiba, Japan). The
heating rate was 1.0 K minꢀ 1. The melting transition of indium was used
as a reference for calibration for the instrument.
For measurements of fluorescent dyes of DPH in the phospholipid
membrane, the stock solutions of AC-(di-O-C14PC)2 and di-O-C14PC
were mixed with that of the fluorescent dye at the molar ratio of 250:1
and 500:1, respectively, and films of the phospholipid/fluorescent dye
mixture were prepared by evaporating the solvent at 40 ◦C and then
drying under the reduced pressure for 3 h. The dried films were sus-
pended with Milli-Q water and vortexed at 60 ◦C to obtain the suspen-
sions samples for the steady-state fluorescence measurements. The final
phospholipid concentrations of the samples are 0.25 and 0.5 mM for AC-
(di-O-C14PC)2 and di-O-C14PC, respectively.
An Edinburgh FS 900 CDT fluorometer (Edinburgh Analytical In-
struments) was utilized for fluorescence depolarization measurements of
DPH in the phospholipid membrane. The excitation wavelength was
360 nm, and the fluorescence spectra in the wavelength region of
420–440 nm were obtained in the temperature range of 20–70 ◦C. The
anisotropy (r) values were obtained from intensity measurements of
vertically and horizontally polarized emissions with vertically polarized
excitation (IVV and IVH) and a grating factor G that derives from in-
tensities of vertically and horizontally polarized emissions with hori-
zontally polarized excitation (IHV and IHH). The temperature control was
performed with the use of a temperature-controlled water bath (Thermo
Scientific NESLAB RTE-7 Circulating Bath).
2.4. WAXD measurements
X-ray diffraction measurements were performed at the bending-
magnet beamline BL-6A [42] and BL-10C [43] of the Photon Factory
(PF) using synchrotron radiation beams from the PF storage ring at High
Energy Accelerator Research Organization, KEK (Tsukuba, Japan). The
wavelength of X-ray beam was 0.15 nm (BL-6A) or 0.10 nm (BL-10C).
Typical detector-to-sample distances were ~300 mm and ~500 mm for
BL-6A and BL-10C, respectively. The distance was determined by
analyzing the diffraction patterns of standard sample, silver behenate
[44]. The temperature of samples was controlled by a modified differ-
ential scanning calorimeter (FP 84, Mettler-Toledo International Inc.)
[45]. For X-ray diffraction studies, the samples were pelleted by
centrifugation at 18,000g for 30 min at 2 ◦C with a temperature-
controlled centrifuge (MX-150, TOMY SEIKO Co. Ltd.). Two-
dimensional X-ray diffraction patterns were recorded with an X-ray
photon counting pixel array detector PILATUS100K or PILATUS1M
(DECTRIS, Switzerland) [42]. Typical exposure time was 20 s.
2.6. Laurdan fluorescence measurements
A polarity-sensitive fluorescent Laurdan was used to reveal the po-
larity around the hydrophilic/hydrophobic interface region of the
phospholipid membrane [52–54]. A film was obtained by evaporation of
the mixture of a phospholipid and Laurdan stock solutions with a
phospholipid/Laurdan molar ratio of 500:1 under a stream of oxygen-
free dry nitrogen. For Laurdan fluorescence measurements, the film of
phospholipid and Laurdan was hydrated with Milli-Q water at around
55 ◦C for about 10 min and was vortexed after the incubation at the final
lipid concentration of about 0.5 mM.
By using a FIT2D software [46–47], circular integrations were car-
ried out to transform into one-dimensional intensity data from the two-
dimensional diffraction image data. In this paper, we used the reciprocal
spacing (S), S = 1/d = 2 sinθ/λ (where d is the real spacing, 2θ is the
scattering angle, and λ is the wavelength of X-ray) for the horizontal axis
to display X-ray diffraction data.
A Hitachi F-4500 fluorescence spectrophotometer was employed for
measurements of steady-state fluorescence emission spectra in the re-
gion of 380 to 650 nm. The excitation wavelength was 361 nm. The
temperature was controlled with an accuracy of ~0.01 ◦C by using a
Julabo F25 recirculating chiller. The fluorescence spectral shift was
quantitatively evaluated by calculating the generalized polarization
(GP) function proposed by Parasassi et al. [52–54].
2.5. DPH fluorescence depolarization measurements
Steady-state fluorescence depolarization measurements of DPH in
the phospholipid membrane [48–51] was employed to probe differences
in the membrane fluidity between AC-(di-O-C14PC)2 and di-O-C14PC.
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N. Tsuchida et al.
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2.7. Fluorescent dye leakage assay
The dye leakage assay based on an anionic fluorophore 6-CF self-
quenching properties [15,55–56] was tested to investigate differences
in the permeability of the phospholipid liposomes between AC-(di-O-
C14PC)2 and di-O-C14PC. A previous study by Komatsu and Chong [15]
demonstrated that the tight and rigid lipid packing is responsible for the
leakage rate of 6-CF. For the preparation of 6-CF-loaded liposomes,
phospholipid/dye film samples were prepared by evaporating the
mixture of the stock solution of AC-(di-O-C14PC)2/di-O-C14PC and 6-CF
at the molar ratio of 10/5 to 1, respectively, at 40 ◦C, under a stream of
oxygen-free dry nitrogen and were dried under a reduced-pressure for 3
h. Furthermore, dried films were suspended with 10 mM HEPES buffer
(pH 7.4) above the phase transition temperatures. After the overnight
incubation for hydration at room temperature, the obtained suspension
samples were subject to the freeze-thaw cycles several times and
centrifugation (4000 g, 15 min) twice at 4 ◦C to obtain 6-CF-loaded
unilamellar vesicle and remove residual unloaded fluorescent dyes.
The final phospholipid/dye suspension samples used for the dye leakage
assay were obtained by suspending the pellet with 10 mM HEPES buffer
(pH 7.4) at the final phospholipid concentrations of 2.25 mM for AC-(di-
O-C14PC)2 and 4.5 mM for di-O-C14PC, respectively.
Fig. 3. Wide-angel X-ray diffraction patterns of (A) di-O-C14PC and (B) AC-(di-
O-C14PC)2. Peaks are offset vertically for clarity.
Fluorescence of liposomes loaded with 6-CF was monitored at 520
nm (excitation at 495 nm) with a Hitachi F-4500 spectrofluorometer.
The extent of 6-CF efflux was calculated as (Ft-F0)/(F100-F0), where F0
and Ft represent the initial fluorescence intensity and the fluorescence
intensity at the time t, respectively, and F100 is the fluorescence intensity
after complete disruption of liposomes by addition of 0.1% Triton X-100.
The temperature was controlled by using a Julabo F25 recirculating
chiller.
Kꢀ 1 molꢀ 1, respectively. The thermodynamic values are also very
similar to those in the previous work [57].
For AC-(di-O-C14PC)2, on the other hand, an endothermic peak ap-
pears at 53.4 ± 0.1 ◦C, which is as high as ~27 ◦C compared to di-O-
C14PC. In order to investigate what is responsible for the endothermic
peak, WAXD measurements were performed for the AC-(di-O-C14PC)2
membrane in the temperature range of 20–60 ◦C. The WAXD pattern of
AC-(di-O-C14PC)2 at 20 ◦C is composed of a remarkably sharp peak at S
= 2.31 nmꢀ 1 with a broad one centered at S = 2.45 nmꢀ 1 (Fig. 3B). Upon
heating to 50–55 ◦C, the characteristic WAXD peak dramatically
changes to a broad featureless one, indicating that the endothermic peak
at 53.4 ± 0.1 ◦C in the DSC thermogram (Fig. 2B) is due to the chain-
melting transition. The transition enthalpy and entropy of the AC-(di-
O-C14PC)2 membrane were estimated from the DSC data and are shown
in Table 1. Because AC-(di-O-C14PC)2 molecules assemble into a
monolayer membrane, the thermodynamic values per molecule ob-
tained for the AC-(di-O-C14PC)2 membrane are approximately doubled
compared to those obtained for bilayer membranes of usual phospho-
lipids (e.g., dimyristoylphosphatidylcholine). Therefore, halves of the
thermodynamic values for the AC-(di-O-C14PC)2 membrane are also
listed as converted values for comparison. The converted ΔH and en-
tropy ΔS for AC-(di-O-C14PC)2 are 30.8 ± 0.8 kJ molꢀ 1 and 94.2 ± 2.7 J
3. Results and discussion
3.1. Thermotropic characterization
DSC measurements were carried out for phospholipid suspensions to
reveal differences in thermotropic behaviors of phospholipid membrane
between AC-(di-O-C14PC)2 and di-O-C14PC. As shown in Fig. 2, a
remarkably sharp endothermic peak is observed for di-O-C14PC at 26.4
± 0.1 ◦C, which is in good agreement with the previous report [57].
According to the previous work [57], the DSC peak is attributable to the
gel-to-liquid crystalline phase transition (Pβ’ → Lα), which is also sup-
ported by the temperature-dependent changes of the WAXD patterns of
di-O-C14PC shown in Fig. 3A. The transition enthalpy ΔH and entropy
ΔS obtained from the DSC data are 22.4 ± 0.9 kJ molꢀ 1 and 74.7 ± 3.1 J
K
ꢀ 1 molꢀ 1, respectively, that are higher than di-O-C14PC.
Of particular note is that the marked sharp peak at S = 2.31 nmꢀ 1
appears for AC-(di-O-C14PC)2 in the gel phase. The full width at half
maximum for the sharp peak of AC-(di-O-C14PC)2 at 20 ◦C is roughly
estimated to be ~0.016 nmꢀ 1 from the diffraction peak, which is
approximately an order of magnitude smaller than that for the central
peak at S = 2.34 nmꢀ 1 for di-O-C14PC at 20 ◦C (~0.14 nmꢀ 1, Fig. 3A).
The remarkable difference in the width of the dominant endothermic
peak at 20 ◦C strongly suggests that in the AC-(di-O-C14PC)2 membrane,
Table 1
Thermodynamic properties for the phase transition of di-O-C14PC and AC-(di-O-
C14PC)2. Parenthesized values of ΔH and ΔS for AC-(di-O-C14PC)2 are the
values reduced to half for comparison with di-O-C14PC.
Lipid
Tm
ΔH
ΔS
(◦C)
(kJ molꢀ 1
)
(J Kꢀ 1 molꢀ 1
)
di-O-C14PC
26.6 ± 0.1
53.4 ± 0.1
22.4 ± 0.9
61.5 ± 1.5
(30.8 ± 0.8)
74.7 ± 3.1
188.3 ± 5.5
(94.2 ± 2.7)
Fig. 2. Differential scanning calorimetric curves of (A) di-O-C14PC and (B) AC-
(di-O-C14PC)2. Thermograms are offset vertically for clarity.
AC-(di-O-C14PC)2
4

N. Tsuchida et al.
BBA - Biomembranes 1863 (2021) 183569
a much more highly ordered structure is formed in the hydrophobic
region compared to di-O-C14PC. It is reasonable to conclude that the
highly ordered structure formed in the AC-(di-O-C14PC)2 membrane is
attributable to a single membrane-spanning linkage of a long methylene
chain of (CH2)28 between the phosphatidylcholine groups in the inner
and the outer sides of the membrane and contributes to the above-
mentioned significant difference of ~27 ◦C in Tm between AC-(di-O-
C14PC)2 and di-O-C14PC. Preliminary small-angle X-ray diffraction
analyses of AC-(di-O-C14PC)2 membrane at 2 ◦C demonstrated that the
membrane thickness is ~3.8 nm. The value of the thickness is very
similar to that of the AC-(di-O-C14PC)2 monolayer membrane expected
from its chemical structure, strongly suggesting that AC-(di-O-C14PC)2
membrane in the gel phase does not adopt the interdigitated structure,
unlike di-O-C14PC bilayer (Tsuchida et al., unpublished results).
Therefore, it is plausible that the long transmembrane hydrocarbon
segments are in the all-trans planar zigzag conformation, which could
induce the ordering of the AC-(di-O-C14PC)2 membrane. The tentative
picture of the AC-(di-O-C14PC)2 will be addressed by vibrational spec-
troscopy in the near future. On the other hand, the less ordered structure
and lower Tm of di-O-C14PC bilayer membrane may be attributable to
weaker interlayer interaction, compared to the covalently bonded long
hydrophobic segment of AC-(di-O-C14PC)2.
as the DMPC membrane in the fluid phase [48–49], showing that fluidity
of phospholipid membrane is mostly independent of whether the hy-
drophobic chains are linked to the glycerol moiety through the ester or
the ether bond. Moreover, the r-value of the AC-(di-O-C14PC)2 mem-
brane changes in a very similar temperature-dependent manner; the
anisotropy value, which is almost constant at ~0.32 in the gel phase,
sharply decreases at the temperature of the endothermic peak and
gradually diminishes to ~0.14 with further temperature elevation.
These experimental results demonstrate that the monolayer membrane
of the amacrocyclic tetraether phosphocholine AC-(di-O-C14PC)2 un-
dergoes the thermotropic transition from the gel phase to the liquid
crystalline phase, and the fluidity of AC-(di-O-C14PC)2 after the tran-
sition is comparable to that of the bilayer membrane of the corre-
sponding double-chained diether phosphocholine di-O-C14PC.
As an essential membrane property closely related to membrane
fluidity, the polarity around the hydrophilic/hydrophobic membrane
interface region, which strongly reflects water penetration induced by
the phase transition, was also probed by a polarity-sensitive fluorescent
Laurdan. The GP parameters obtained from fluorescence spectral
changes for Laurdan in the AC-(di-O-C14PC)2 and di-O-C14PC mem-
brane are shown in Fig. 5. The GP values for di-O-C14PC are mostly
constant at ca. 0.3 in the temperature range of the gel phase, which is
significantly small compared to those for DMPC [58]. The significant
differences in the GP values between di-O-C14PC and DMPC demon-
strate that the polarity around the interface region of the di-O-C14PC
bilayer is higher than that of DMPC. The origin of the polarity difference
in the hydrophobic/hydrophilic interface is attributable to the chemical
structural variations of the ester or the ether bond and/or the accessi-
bility of water molecules [59], which would result in some perturbation
to the molecular interaction between phospholipids. It should be noted
that between di-O-C14PC and DMPC in the gel phase, the Laurdan
fluorescence revealed very different properties in the hydrophilic/hy-
drophobic membrane interface, while no significant variations in the
transmembrane region were demonstrated by DPH fluorescence as
mentioned above. These experimental results strongly suggest that the
double-chained ester-linked and ether-linked PCs bearing the hydro-
carbon segment with the same chain length exhibit similar structural
and physical properties for the hydrophobic core region, but different
3.2. Membrane fluidity
The membrane fluidity was probed by steady-state fluorescence de-
polarization measurements of DPH in the temperature range of 20–70
◦C. In Fig. 4, thermal changes of the anisotropy (r) values for DPH in the
AC-(di-O-C14PC)2 membrane are shown with those in the di-O-C14PC
membrane for comparison. The r-value for di-O-C14PC in the tempera-
ture range of the gel phase is almost constant at ~0.34, which is very
similar to those for 1,2-dimyristoyl-sn-glycero-3-phosphocholine
(DMPC) in the gel phase, the ester-linked phosphatidylcholine corre-
sponding to di-O-C14PC, indicating that in the gel phase, rotational
depolarization of DPH molecules is significantly limited in the di-O-
C14PC membrane like in the DMPC membrane [48–49]. Upon temper-
ature elevation above Tm, however, the value of the fluorescence
anisotropy dramatically decreases to ~0.15, which is at the same level
Fig. 5. Generalized polarization (GP) values calculated form Laurdan fluores-
cence intensities at 440 and 490 in di-O-C14PC (red circles) and AC-(di-O-
C14PC)2 (blue squares) as a function of temperature.
Fig. 4. Anisotropy values of DPH in di-O-C14PC (red circles) and AC-(di-O-
C14PC)2 (blue squares) as a function of temperature.
5

N. Tsuchida et al.
BBA - Biomembranes 1863 (2021) 183569
polarity-related properties for the hydrophilic/hydrophobic interface.
Furthermore, the GP values for AC-(di-O-C14PC)2 in the gel phase are
very similar to those for di-O-C14PC. This indicates that irrespective of
the single transmembrane alkyl linkage between the two glycer-
ophosphate head groups, which induces more ordered chain packing
inside the phospholipid membrane as indicated by the WAXD mea-
surements, there are no significant differences in the polarity of the
hydrophilic/hydrophobic membrane interface region between AC-(di-
O-C14PC)2 and di-O-C14PC.
In the temperature range of the phase transition, however, both di-O-
C14PC and AC-(di-O-C14PC)2 showed a remarkable drop in the GP
values, followed by a gradual decrease to ca. -0.4 at higher tempera-
tures. The dramatic falls of GP to ca. -0.4 indicate that the polarity of the
hydrophilic/hydrophobic interface region of the AC-(di-O-C14PC)2
membrane becomes higher, which is attributable to enhancement of
membrane fluidity by the gel-to-liquid crystalline phase transition. Of
note is that compared to DMPC, the GP values for both AC-(di-O-
C14PC)2 and di-O-C14PC in the fluid phase are much smaller, that is, the
interface region of the AC-(di-O-C14PC)2 and di-O-C14PC membrane
exhibit highly polar character, which is in the very similar situation to
the gel phase as mentioned before.
3.3. Fluorescent dye leakage assay
Dye leakage assay based on 6-CF self-quenching properties was
employed to probe the entrapping capability of water-soluble substances
inside phospholipid liposomes, which is directly related to the perme-
ability of the phospholipid membrane. A previous study on the mem-
brane permeability of macrocyclic bipolar tetraether lipids from
Thermoacidophilic Archaebacterium Sulfolobus acidocaldarius revealed
that the 6-CF low leakage rate is attributable to the tight and rigid lipid
packing and the negative charges on the membrane surface [15]. The
extents of 6-CF leakage for AC-(di-O-C14PC)2 and di-O-C14PC were
investigated at 10, 35, and 60 ◦C to reveal differences between the phase
behaviors of phospholipid membrane as well as the two kinds of ether-
linked phospholipids. The results are shown in Fig. 5.
Fig. 6. Time courses of 6-CF leakage from liposome of AC-(di-O-C14PC)2 (blue)
and di-O-C14PC (red) at (A) 10 ◦C, (B) 35 ◦C and (C) 60 ◦C.
At 10 ◦C in the gel phase for both AC-(di-O-C14PC)2 and di-O-C14PC,
6-CF leakage is highly suppressed at the same level irrespective of the
chemical structural difference between the two kinds of phospholipids,
which is due to the limited lateral and rotational diffusion of phospho-
lipid molecules. However, upon temperature elevation to 35 ◦C, the
extent of the fluorescent dye leakage remarkably increases to nearly a
half for di-O-C14PC in the liquid crystalline phase. In contrast, AC-(di-O-
C14PC)2 shows no significant changes at the level of less than 10% even
after the temperature elevation, which is readily explained by the
experimental results that the AC-(di-O-C14PC)2 membrane adopts the
gel phase at the temperature.
revealed by comparison with the di-O-C14PC bilayer are summarized as
follows: (a) formation of significantly highly ordered structure in the gel
phase, (b) dramatic temperature elevation of the thermotropic transi-
tion, (c) fluidity in the liquid crystalline phase comparable to the di-O-
C14PC bilayer and (d) remarkable leakage suppression of substances
entrapped inside vesicles in the fluid phase. The very high transition
temperature of the AC-(di-O-C14PC)2 monolayer membrane can be
discussed in connection with the remarkably high order in its membrane
structure. As stated in Section 3.1, the unusual sharp WAXD peak
observed for AC-(di-O-C14PC)2 demonstrates that the highly ordered
chain structure is formed in the transmembrane region, which is unique
as phospholipid membrane bearing bulky polar head group like PC. It is
reasonable to infer that the most plausible origin for the extraordinarily
high structural order of AC-(di-O-C14PC)2 monolayer membrane is the
transmembrane linkage via the extended octacosamethylene group be-
tween the head groups on the opposite sides with each other. A single
layer of AC-(di-O-C14PC)2 formed through the covalent interlayer
linkage between di-O-C14PC molecules behaves in a unified manner,
which leads to the high structural order in the transmembrane region.
On the other hand, two independent layers of di-O-C14PC formed by the
noncovalent intermolecular interaction in the lateral direction assemble
to common lipid bilayer membranes through the vertical noncovalent
interlayer interaction, which results in the less ordered di-O-C14PC
membrane.
As shown in Fig. 6, a further increase to 60 ◦C in temperature leads to
a tremendous loss of 6-CF up to ~40% inside the AC-(di-O-C14PC)2
vesicle, which is attributable to the membrane fluidity induced by the
phase transition from the gel phase to the liquid crystalline phase. It is of
significant note that the extent of the 6-CF leakage for the AC-(di-O-
C14PC)2 membrane is highly suppressed compared to the di-O-C14PC
membrane, where more than twice fluorescent dye molecules leak from
the vesicle. These experimental results strongly suggest that even in the
fluid phase, AC-(di-O-C14PC)2 phospholipid molecules do not diffuse
freely, unlike di-O-C14PC, but interact with each other to some extent.
The peculiar membrane properties of AC-(di-O-C14PC)2 in the liquid
crystalline phase, which are reasonably ascribed to a single membrane-
spanning linkage between the inner and the outer leaflets, are discussed
in the next section.
3.4. Effect of a single membrane-spanning linkage on membrane
properties
As stated in Sections 3.1 and 3.2, highly ordered AC-(di-O-C14PC)2
monolayer membrane undergoes the thermotropic transition from the
gel phase to the liquid crystalline phase like ordinary phospholipid
Remarkable physical properties of the AC-(di-O-C14PC)2 monolayer
6

N. Tsuchida et al.
BBA - Biomembranes 1863 (2021) 183569
bilayer membrane, although the transition temperature is remarkably
higher than its corresponding bilayer membrane of di-O-C14PC. Of
significant note is that fluorescence-probed measurements with DPH
and Laurdan demonstrated that the AC-(di-O-C14PC)2 monolayer shows
fluidic characters comparable to the di-O-C14PC bilayer membrane. The
fluidic property of phospholipid membrane fundamentally derives from
spontaneous lateral and rotational diffusion of phospholipid molecules.
In the bilayer membrane, while the lateral and rotational movements of
phospholipid molecules are confined in either the inner or the outer
leaflet, the spontaneous transverse diffusion between the different
leaflets, i.e., the flip-flop, is restricted in a significant way in the absence
of phospholipid-transporting membrane proteins. On the other hand,
the AC-(di-O-C14PC)2 membrane comprises a single layer of the large
transmembrane molecules. Although the diffusional behavior of the AC-
(di-O-C14PC)2 molecules in the membrane is unknown, it can be
deduced from the 6-CF leakage assay experimental results.
induced some unique membrane properties that are not observed for
typical phospholipid bilayer membranes. In particular, the high ability
for entrapping water-soluble substances inside phospholipid vesicles
even in the fluidic phase is a significant feature of the amacrocyclic
tetraether bisphosphatidylcholine lipid, which is presumably the
essential membrane property of archaea that can survive in the extreme
environment.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
It should be noted that in the liquid crystalline phase, the leakage of
water-soluble fluorescent 6-CF molecules inside AC-(di-O-C14PC)2
monolayer vesicles is much more highly suppressed compared to di-O-
C14PC bilayer vesicles, even though DPH and Laurdan fluorescence
measurements demonstrated that AC-(di-O-C14PC)2 molecules have
high fluidic property comparable to di-O-C14PC. It is reasonably
mentioned that the lateral diffusion of phospholipid molecules plays a
central role in the leakage of the water-soluble dye molecules 6-CF from
inside vesicles because temporal fractures in the membrane are required
for the water-soluble molecules to transect the hydrophobic lipid layer.
Therefore, while the lateral diffusion of AC-(di-O-C14PC)2 molecules is
controlled at the moderately low level even in the liquid crystalline
phase, which results in the remarkable suppression of the 6-CF leakage,
their rotational diffusion is sufficiently activated above the transition
temperature, which is the essential origin of the fluidic monolayer
membrane comparable to the di-O-C14PC bilayer. The characteristic
membrane properties of AC-(di-O-C14PC)2 are attributable to the
extended transmembrane bridge via the octacosamethylene group be-
tween the polar head groups on the opposite sides with each other. Our
ongoing X-ray diffraction studies on the membrane structure of AC-(di-
O-C14PC)2 strongly suggest that the decrease in the membrane thickness
by the thermotropic phase transition is highly suppressed compared to
common bilayer phospholipid membranes, which may indicate that the
transmembrane cross-linkage props the monolayer membrane even in
the liquid crystalline phase (Tsuchida et al., unpublished results).
As mentioned above, the present study demonstrated that the single
interlayer cross-linkage via the linear polymethylene chain induces the
highly ordered membrane structure in the gel phase and the remarkable
temperature elevation of the thermotropic transition to 53.4 ◦C
compared to the corresponding bilayer membrane of di-O-C14PC.
Supposing that double interlayer cross-linkages via the linear poly-
methylene chain are introduced to provide macrocyclic tetraether
phospholipid molecules, which is the general backbone structure of
naturally occurring archaeal bipolar tetraether lipid, it is inferred that
the phase transition temperature of the macrocyclic tetraether phos-
pholipid membrane is further elevated from 53.4 ◦C. Such a high ther-
motropic transition temperature leads to the highly rigid phospholipid
membrane even at substantially high temperatures. Too much high ri-
gidity would not be suitable for various biological functions on the
membrane. As reported in the previous works [15–18,30–34], long
transmembrane cross-linkages of native archaeal macrocyclic tetraether
lipids contain the phytanyl chain and the cyclopentane ring moieties in
some cases, which would moderately loosen the molecular packing,
decrease in the phase transition temperature and yield well-suited
functional fields for membrane proteins.
This work was supported in part by Grants-in-Aid for Scientific
Research (KAKENHI: 15K05558 and 20K06573 to M.S., 18K06568 to T.
T., and 19K03762 to H.T.) from the Japan Society for the Promotion of
Science (JSPS). X-ray diffraction experiments were carried out under the
approval of the Photon Factory Program Advisory Committee (Proposals
No. 20013G675 and 2018G095). The authors wish to thank Professors
N. Shimizu and N. Igarashi and the SAXS beamline staff of Photon
Factory for their kind assistance.
References
[1] M. Kates, The phytanyl ether-linked polar lipids and isoprenoid neutral lipids of
extremely halophilic bacteria, Prog. Chem. Fats Other Lipids 15 (1977) 301–342,
https://doi.org/10.1016/0079-6832(77)90011-8.
[2] M. De Rosa, A. Gambacorta, A. Gliozzi, Structure, biosynthesis, and
physicochemical properties of archaebacterial lipids, Microbiol. Rev. 50 (1986)
70–80.
[3] Y. Koga, M. Nishihara, H. Morii, M. Akagawa-Matsushita, Ether polar lipids of
methanogenic bacteria: structures, comparative aspects, and biosynthesis,
Microbiol. Rev. 57 (1993) 164–182.
[4] Y. Koga, H. Morii, M. Akagawa-Matsushita, M. Ohga, Correlation of polar lipid
composition with 16s rrna phylogeny in methanogens. Further analysis of lipid
component parts, Biosci. Biotech. Biochem. 62 (1998) 230–236, https://doi.org/
10.1271/bbb.62.230.
[5] Y. Koga, H. Morii, Recent advances in structural research on ether lipids from
archaea including comparative and physiological aspects, Biosci. Biotechnol.
Biochem. 69 (2005) 2019–2034.
[6] A. Caforio, A.J.M. Driessen, Archaeal phospholipids: structural properties and
biosynthesis, Biochim. Biophys. Acta 1862 (2017) 1325–1339, https://doi.org/
10.1016/j.bbalip.2016.12.006.
[7] R.A. Moss, T. Fujita, Y. Okumura, Dynamics of a bolaamphiphilic lipid in a bilayer
liposome, Langmuir 7 (1991) 2415–2418, https://doi.org/10.1021/la00059a001.
[8] J.M. Kim, D.H. Thompson, Tetraether bolaform amphiphiles as models of
archaebacterial membrane lipids: synthesis, differential scanning calorimetry, and
monolayer studies, Langmuir 8 (1992) 637–644, https://doi.org/10.1021/
la00038a056.
[9] D.H. Thompson, K.F. Wong, R. Humphry-Baker, J.J. Wheeler, J.M. Kim, S.
B. Rananavare, Tetraether bolaform amphiphiles as models of archaebacterial
membrane lipids: Raman spectroscopy, phosphorus-31 NMR, x-ray scattering, and
electron microscopy, J. Am. Chem. Soc. 114 (1992) 9035–9042, https://doi.org/
10.1021/ja00049a040.
[10] A.P. Patwardhan, D.H. Thompson, Novel flexible and rigid tetraether amacrocyclic
and macrocyclic bisphosphocholines: Synthesis and monolayer properties,
Langmuir 16 (2000) 10340–10350, https://doi.org/10.1021/la001104q.
[11] K. Yamauchi, K. Doi, M. Kinoshita, F. Kii, H. Fukuda, Archaebacterial lipid models:
highly salt-tolerant membranes from 1,2-diphytanylglycero-3-phosphocholine,
Biochim. Biophys. Acta 1110 (1992) 171–177, https://doi.org/10.1016/0005-
2736(92)90355-P.
[12] Q. Fan, A. Relini, D. Cassinadri, A. Gambacorta, A. Gliozzi, Stability against
temperature and external agents of vesicles composed of archaeal bolaform lipids
and egg PC, Biochim. Biophys. Acta 1240 (1992) 83–88, https://doi.org/10.1016/
0005-2736(95)00157-X.
[13] T. Eguchi, K. Ibaragi, K. Kakinuma, Total synthesis of archaeal 72-membered
macrocyclic tetraether lipids, J. Org. Chem. 63 (1998) 2689–2698, https://doi.
org/10.1021/jo972328p.
[14] K. Arakawa, H. Kano, T. Eguchi, Y. Nishiyama, K. Kakinuma, Significance of the
72-membered macrocyclic structure found in archaeal membrane lipids: model
studies of the macrocyclic tetraether diphospholipids by calorimetric, 31P NMR,
and electron microscopic analyses, Bull. Chem. Soc. Jpn. 72 (1999) 1575–1581,
https://doi.org/10.1246/bcsj.72.1575.
4. Conclusions
The monolayer formation of phospholipid membrane by introducing
the long membrane-spanning linkage via the polymethylene chain
7

N. Tsuchida et al.
BBA - Biomembranes 1863 (2021) 183569
[15] H. Komatsu, P.L.-G. Chong, Low permeability of liposomal membranes composed
of bipolar tetraether lipids from thermoacidophilic archaebacterium Sulfolobus
acidocaldarius, Biochem. 37 (1998) 107–115, https://doi.org/10.1021/bi972163e.
[16] J.L. Gabriel, P.L.-G. Chong, Molecular modeling of archaebacterial bipolar
tetraether lipid membranes, Chem. Phys. Lipids 105 (2000) 193–200, https://doi.
org/10.1016/S0009-3084(00)00126-2.
[38] A.P. Patwardhan, D.H. Thompson, Novel flexible and rigid tetraether amacrocyclic
and macrocyclic bisphosphocholines: synthesis and monolayer properties,
Langmuir 16 (2000) 10340–10350.
[39] N. Hebert, A. Beck, R.B. Lennox, G. Just, A new reagent for the removal of the 4-
methoxybenzyl ether: application to the synthesis of unusual macrocyclic and
bolaform phosphatidylcholines, J. Org. Chem. 57 (1992) 1777–1783, https://doi.
org/10.1021/jo00032a033.
[17] P.L.-G. Chong, R. Ravindra, M. Khurana, V. English, R. Winter, Pressure
perturbation and differential scanning calorimetric studies of bipolar tetraether
liposomes derived from the thermoacidophilic archaeon Sulfolobus acidocaldarius,
Biophys. J. 89 (2005) 1841–1849, https://doi.org/10.1529/biophysj.105.063933.
[18] P.L.-G. Chong, Archaebacterial bipolar tetraether lipids: physico-chemical and
membrane properties, Chem. Phys. Lipids 163 (2010) 253–265, https://doi.org/
10.1016/j.chemphyslip.2009.12.006.
[40] T. Takagi, K. Takai, T. Baba, T. Kanamori, Synthesis of phospholipids containing
perfluorooctyl group and their interfacial properties, J. Fluor. Chem. 128 (2007)
133–138, https://doi.org/10.1016/j.jfluchem.2006.10.020.
[41] W.J. Hansen, R. Murari, Y. Wedmid, W.J. Baumann, An improved procedure for
the synthesis of choline phospholipids via 2-bromoethyl dichlorophosphate, Lipids
17 (1982) 453–459, https://doi.org/10.1007/BF02535226.
[19] T. Koyanagi, G. Leriche, D. Onofrei, G.P. Holland, M. Mayer, J. Yang, Cyclohexane
rings reduce membrane permeability to small ions in archaea-inspired tetraether
lipids, Angew. Chem. Int. Ed. 55 (2016) 1890–1893, https://doi.org/10.1002/
anie.201510445.
[42] N. Shimizu, T. Mori, N. Igarashi, H. Ohta, Y. Nagatani, T. Kosuge, K. Ito,
Refurbishing of small-angle X-ray scattering beamline, BL-6A at the Photon
Factory, J. Phys. Conf. Ser. 425 (2013) 202008, https://doi.org/10.1088/1742-
6596/425/20/202008.
[20] T.B.H. Schroeder, G. Leriche, T. Koyanagi, M.A. Johnson, K.N. Haengel, O.
M. Eggenberger, C.L. Wang, Y.H. Kim, K. Diraviyam, D. Sept, J. Yang, M. Mayer,
Effects of lipid tethering in extremophile-inspired membranes on H(+)/OH(-) flux
at room temperature, Biophys. J. 110 (2016) 2430–2440, https://doi.org/
10.1016/j.bpj.2016.04.044.
[43] N. Igarashi, Y. Watanabe, Y. Shinohara, Y. Inoko, G. Matsuba, H. Okuda, T. Mori,
K. Ito, Upgrade of the small angle X-ray scattering beamlines at the photon factory,
J. Phys. Conf. Ser. 272 (2011), 012026 https://doi.org/10.1088/1742-6596/272/
1/012026.
[44] T.C. Huang, H. Toraya, T.N. Blanton, Y. Wu, X-ray powder diffraction analysis of
silver behenate, a possible low-angle diffraction standard, J. Appl. Crystallogr. 26
(1993) 180–184, https://doi.org/10.1107/S0021889892009762.
[45] H. Takahashi, S. Matuoka, Y. Amemiya, I. Hatta, Simultaneous differential
scanning calorimetry and time-resolved X-ray diffraction of lipid-water system. I.
Relationship between chain melting and endothermic heat at the main transition of
a dipalmitoylphosphatidylcholine-water system, Chem. Phys. Lipids 76 (1995)
115–121, https://doi.org/10.1016/0009-3084(94)02428-8.
[21] V. Paolucci, G. Leriche, T. Koyanagi, J. Yang, Evaluation of tetraether lipid-based
liposomal carriers for encapsulation and retention of nucleoside-based drugs,
Bioorg. Med. Chem. Lett. 27 (2017) 4319–4322, https://doi.org/10.1016/j.
bmcl.2017.08.032.
[22] G. Leriche, D. Stengel, D. Onofrei, T. Koyanagi, G.P. Holland, J. Yang, Fusion of
bipolar tetraether lipid membranes without enhanced leakage of small molecules,
Sci. Rep. 9 (2019) 19359, https://doi.org/10.1038/s41598-019-55494-z.
[23] T. Benvegnu, M. Brard, D. Plusquellec, Archaeabacteria bipolar lipid analogues:
structure, synthesis and lyotropic properties, Curr. Opin. Colloid Interface Sci. 8
(2004) 469–479, https://doi.org/10.1016/j.cocis.2004.01.005.
¨
[46] E.F. Eikenberry, Ch. Bronnimann, G. Hülsen, H. Toyokawa, R. Horisberger,
B. Schmitt, C. Schulze-Briese, T. Tomizaki, PILATUS: a two-dimensional X-ray
detector for macromolecular crystallography, Nucl. Instrum. Methods Phys. Res. A
501 (2003) 260–266, https://doi.org/10.1016/S0168-9002(02)02044-2.
[47] A.P. Hammersley, S.O. Svensson, M. Hanfland, A.N. Fitch, D. Hausermann, Two-
dimensional detector software: from real detector to idealised image or two-theta
scan, High Pressure Res. 14 (1996) 235–248, https://doi.org/10.1080/
08957959608201408.
[24] M. Brard, W. Richter, T. Benvegnu, D. Plusquellec, Synthesis and supramolecular
assemblies of bipolar archaeal glycolipid analogues containing a cis-1,3-
disubstituted cyclopentane ring, J. Am. Chem. Soc. 126 (2004) 10003–10012,
https://doi.org/10.1021/ja049805n.
´
´
[25] T. Benvegnu, G. Rethore, M. Brard, W. Richter, D. Plusquellec, Archaeosomes
based on novel synthetic tetraether-type lipids for the development of oral delivery
systems, Chem. Commun. 44 (2005) 5536–5538, https://doi.org/10.1039/
B511440C.
[48] J.R. Lakowicz, F.G. Prendergast, D. Hogen, Differential polarized phase
fluorometric investigations of diphenylhexatriene in lipid bilayers quantitation of
hindered depolarizing rotations, Biochem. 18 (1979) 508–519, https://doi.org/
10.1021/bi00570a02.
´
´
´
`
[26] M. Brard, C. Laine, G. Rethore, I. Laurent, C. Neveu, L. Lemiegre, T. Benvegnu,
Synthesis of archaeal bipolar lipid analogues: a way to versatile drug/gene delivery
systems, J. Org. Chem. 72 (2007) 8267–8279, https://doi.org/10.1021/jo071181r.
[49] J.R. Lakowicz, F.G. Prendergast, D. Hogen, Fluorescence anisotropy measurements
under oxygen quenching conditions as a method to quantify the depolarizing
rotations of fluorophores. Application to diphenylhexatriene in isotropic solvents
and in lipid bilayers, Biochem. 18 (1979) 520–527, https://doi.org/10.1021/
bi00570a022.
`
[27] T. Benvegnu, L. Lemiegre, S. Cammas-Marion, Archaeal lipids: innovative
materials for biotechnological applications, Eur. J. Org. Chem. 28 (2008)
4725–4744, https://doi.org/10.1002/ejoc.200800452.
`
[28] A. Jacquemet, J. Barbeau, L. Lemiegre, T. Benvegnu, Archaeal tetraether bipolar
[50] M. Adler, T.R. Tritton, Fluorescence depolarization measurements on oriented
membranes, Biophys. J. 53 (1988) 989–1005, https://doi.org/10.1016/S0006-
3495(88)83179-5.
lipids: structures, functions and applications, Biochimie 91 (2009) 711–717,
https://doi.org/10.1016/j.biochi.2009.01.006.
`
[29] A. Jiblaoui, J. Barbeau, T. Vives, P. Cormier, V. Glippa, B. Cosson, T. Benvegnu,
[51] N.C.S. Mykytczuk, J.T. Trevors, L.G. Leduc, G.D. Ferroni, Fluorescence polarization
in studies of bacterial cytoplasmic membrane fluidity under environmental stress,
Prog. Biophys. Mol. Biol. 95 (2007) 60–82, https://doi.org/10.1016/j.
pbiomolbio.2007.05.001.
Folate-conjugated stealth archaeosomes for the targeted delivery of novel
antitumoral peptides, RSC Adv. 6 (2016) 75234–75241, https://doi.org/10.1039/
C6RA15713K.
[30] J. Parmentier, B. Thewes, F. Gropp, G. Fricker, Oral peptide delivery by tetraether
lipid liposomes, Int. J. Pharm. 415 (2011) 150–157, https://doi.org/10.1016/j.
ijpharm.2011.05.066.
[52] T. Parasassi, G. De Stasio, A. d’Ubaldo, E. Gratton, Phase fluctuation in
phospholipid membranes revealed by Laurdan fluorescence, Biophys. J. 57 (1990)
1179–1186, https://doi.org/10.1016/S0006-3495(90)82637-0.
[31] J. Parmentier, M.M.M. Becker, U. Heintz, G. Fricker, Stability of liposomes
containing bio-enhancers and tetraether lipids in simulated gastro-intestinal fluids,
Int. J. Pharm. 405 (2011) 210–217, https://doi.org/10.1016/j.
ijpharm.2010.12.005.
[53] T. Parasassi, G. De Stasio, G. Ravagnan, R.M. Rusch, E. Gratton, Quantitation of
lipid phases in phospholipid vesicles by the generalized polarization of Laurdan
fluorescence, Biophys. J. 60 (1991) 179–189, https://doi.org/10.1016/S0006-
3495(91)82041-0.
¨
[32] J. Parmentier, G. Hofhaus, S. Thomas, L.C. Cuesta, F. Gropp, R. Schroder,
[54] T. Parasassi, E.K. Krasnowska, L. Bagatolli, E. Gratton, Laurdan and prodan as
polarity-sensitive fluorescent membrane probes, J. Fluoresc. 8 (1998) 365–373,
https://doi.org/10.1023/A:1020528716621.
K. Hartmann, G. Fricker, Improved oral bioavailability of human growth hormone
by a combination of liposomes containing bio-enhancers and tetraether lipids and
omeprazole, J. Pharm. Sci. 103 (2014) 3985–3993, https://doi.org/10.1002/
jps.24215.
[55] J.N. Weinstein, S. Yoshikami, P. Henkart, R. Blumenthal, W.A. Hagins, Liposome-
cell interaction: transfer and intracellular release of a trapped fluorescent marker,
Science 195 (1977) 489, https://doi.org/10.1126/science.835007.
[56] J. Barbet, P. Machy, A. Truneh, L.D. Leserman, Weak acid-induced release of
liposome-encapsulated carboxyfluorescein, Biochim. Biophys. Acta 772 (1984)
347–356, https://doi.org/10.1016/0005-2736(84)90152-4.
[33] P. Uhl, S. Pantze, P. Storck, J. Parmentier, D. Witzigmann, G. Hofhaus, J. Huwyler,
W. Mier, G. Fricker, Oral delivery of vancomycin by tetraether lipid liposomes, Eur.
J. Pharm. Sci. 108 (2017) 111–118, https://doi.org/10.1016/j.ejps.2017.07.013.
´
[34] G. Mahmoud, J. Jedelska, B. Strehlow, U. Bakowsky, Bipolar tetraether lipids
derived from thermoacidophilic archaeon Sulfolobus acidocaldarius for membrane
stabilization of chlorin e6 based liposomes for photodynamic therapy, Eur. J.
Pharm. Biopharm. 95 (2015) 88–98, https://doi.org/10.1016/j.ejpb.2015.04.009.
[35] S.R. Macaulay, Isomerization of internal triple bonds of alkyn-1-ols with sodium
hydride in 1,3-diaminopropane, J. Org. Chem. 45 (1980) 734–735, https://doi.
org/10.1021/jo01292a042.
[57] H. Matsuki, E. Miyazaki, F. Sakano, N. Tamai, S. Kaneshina, Thermotropic and
barotropic phase transitions in bilayer membranes of ether-linked phospholipids
with varying alkyl chain lengths, Biochim. Biophys. Acta 1768 (2007) 479–489,
https://doi.org/10.1016/j.bbamem.2006.10.005.
[58] M. Yoshino, H. Takahashi, T. Takagi, T. Baba, K. Morita, H. Amii, T. Kanamori,
M. Sonoyama, Effect of partial fluorination in the myristoyl groups on thermal and
interfacial properties of dimyristoylphosphatidylcholine, Chem. Lett. 41 (2012)
1495–1497, https://doi.org/10.1246/cl.2012.1495.
[36] J.F. Carvalho, G.D. Prestwich, Synthesis of ω-tritiated and ω-fluorinated analogs of
the trail pheromone of subterranean termites, J. Org. Chem. 49 (1984) 1251–1258,
https://doi.org/10.1021/jo00181a023.
[59] L.A. Bagatoll, T. Parasassi, G.D. Fidelio, E. Gratton, A Model for the interaction of
6-lauroyl-2-(N,N-dimethylamino)naphthalene with lipid environments:
implications for spectral properties, Photochem. Photobiol. 70 (1999) 557–564,
https://doi.org/10.1111/j.1751-1097.1999.tb08251.x.
[37] K. Miyawaki, T. Takagi, M. Shibakami, Efficient Synthesis of parallel
cyclobolaphiles having two diacetylenes: Mimetics of archaeal membrane lipids,
Synlett 2002 (2002) 1326–1328, https://doi.org/10.1055/s-2002-32987.
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