Highly Asymmetrical Glycerol Diether Bolalipids: Synthesis and

Publication Date (Web): September 14, 2015. Copyright © 2015 American Chemical Society. *E-mail: [email protected]. Phone: +49-34...
0 downloads 4 Views 4MB Size
Article pubs.acs.org/Langmuir

Highly Asymmetrical Glycerol Diether Bolalipids: Synthesis and Temperature-Dependent Aggregation Behavior Thomas Markowski,† Simon Drescher,*,† Günter Förster,‡ 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 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.



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 © XXXX American Chemical Society

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 membranespanning alkyl chain and/or in the 10 position of the short chain can mimic the behavior of natural lipids in terms of Received: August 8, 2015 Revised: September 13, 2015

A

DOI: 10.1021/acs.langmuir.5b02951 Langmuir XXXX, XXX, XXX−XXX

Article

Langmuir 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 singlechain 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 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 10methylhexadecyl (lipid II) residue (Figure 1). In the sn-1

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). 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

DOI: 10.1021/acs.langmuir.5b02951 Langmuir XXXX, XXX, XXX−XXX

Article

Langmuir Scheme 1. Synthesis Pathway for the Preparation of Asymmetrical Bipolar Lipids I and IIa

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-bromodotriacontyl 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 tetrahydropyranyl (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. 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

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-10methylhexadecane.29 The yields of resulting 2,3-O,O-dialkyl-1O-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-bromoethylphosphoric 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 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

DOI: 10.1021/acs.langmuir.5b02951 Langmuir XXXX, XXX, XXX−XXX

Article

Langmuir

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. DSC and FTIR. The DSC heating curve of lipid I in water (c = 1 mg mL−1) shows two endothermic transitions (Figure 3,

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. 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 hydrodynamic 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).

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). 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., PC-C32-OH43 (Tm = 51.2 °C), PC-C17pPhC17-OH33 (Tm = 53.4 °C), and DMAPPC-C32-OH32 (Tm1 = 36.0 °C and Tm2 = 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

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.

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 D

DOI: 10.1021/acs.langmuir.5b02951 Langmuir XXXX, XXX, XXX−XXX

Article

Langmuir

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).

≈ 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

molecules within the aggregate structure and hence higher Tm values. 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 experiments 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., PCC17pPhC17-OH33 (2849.8 and 2917.0 cm−1) and DMAPPCC32-PC32 (2848.4 and 2917.7 cm−1), indicating that the alkyl chains of lipid I are in an all-trans conformation and are wellordered 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 E

DOI: 10.1021/acs.langmuir.5b02951 Langmuir XXXX, XXX, XXX−XXX

Article

Langmuir 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 orthorhombic Lβ′ (Gd) phase. This was also observed before for longchain 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-C32OH),32 although a change between different orthorhombic phases was not observed for that bolalipid. 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 diffraction (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 Other asymmetrical bolalipids, e.g., DMAPPC-C32-PC,32 a single-chain bolalipid with unmodified alkyl chains, or PCC17pPhC17-OH,33 a single-chain phenylene-modified bolalipid, 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 reflections are also indicative of highly ordered chains, which has also been found before for phospholipid bilayers with all-trans alkyl

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.

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).

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 F

DOI: 10.1021/acs.langmuir.5b02951 Langmuir XXXX, XXX, XXX−XXX

Article

Langmuir

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. 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. 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.

chain and an orthorhombic packing of the chains (see above). Therefore, this U-shaped arrangement can be excluded. 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). Upon heating to 32 °C, a change in the reflections in the 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 temperatureinduced expansion of the Lβ subcell lattice. G

DOI: 10.1021/acs.langmuir.5b02951 Langmuir XXXX, XXX, XXX−XXX

Article

Langmuir 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 temperatures) 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 liquidcrystalline (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.

CONCLUSIONS 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 (Xray), which is comparable to Σ values of crystallized saturated olefins. The molecules seem to be tilted with respect to the

S Supporting Information *





ASSOCIATED CONTENT

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.5b02951. Synthesis procedures, analytical data of prepared compounds, and further TEM, FTIR, and X-ray measurements (PDF)



AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: +49345-5525196. Fax: +49-345-5527026. *E-mail: [email protected]. Phone: +49345-5525120. 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 SachsenAnhalt (to T.M.) and by grants from the Deutsche H

DOI: 10.1021/acs.langmuir.5b02951 Langmuir XXXX, XXX, XXX−XXX

Article

Langmuir

of 72-Membered Tetraether Model Compounds. Bull. Chem. Soc. Jpn. 1997, 70, 2545−2554. (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. (25) Brard, M.; Lainé, C.; Réthoré, G.; Laurent, I.; Neveu, C.; Lemiègre, 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. (27) Jacquemet, A.; Lemiègre, L.; Lambert, O.; Benvegnu, T. How the Stereochemistry of a Central Cyclopentyl Ring Influences the SelfAssembling 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. (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. (31) Drescher, S.; Dobner, B. Synthesis of novel asymmetrical singlechain 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. (33) Drescher, S.; Lechner, B.-D.; Garamus, V. M.; Almásy, L.; Meister, A.; Blume, A. The headgroup (a)symmetry strongly determines the aggregation behavior of single-chain phenylenemodified bolalipids and their miscibility with classical phospholipids. Langmuir 2014, 30, 9273−9284. (34) Kameta, N.; Masuda, M.; Minamikawa, H.; Shimizu, T. SelfAssembly and Thermal Phase Transition Behavior of Unsymmetrical Bolaamphiphiles Having Glucose- and Amino-Hydrophilic Headgroups. 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 unsymmetrical 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 3,11dimethyl-2-nonacosanone, a sex pheromone of the German cockroach. J. Org. Chem. 1975, 40, 2410−2411.

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.



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. (3) Woese, C. R.; Magrum, L. J.; Fox, G. E. Archaebacteria. J. Mol. Evol. 1978, 11, 245−252. (4) Baumeister, W.; Lembcke, G. Structural features of archaebacterial cell envelopes. J. Bioenerg. Biomembr. 1992, 24, 567−575. (5) Lewalter, K.; Müller, V. Bioenergetics of archaea: Ancient energy conserving mechanisms developed in the early history of life. Biochim. Biophys. Acta, Bioenerg. 2006, 1757, 437−445. (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. (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. (12) Benvegnu, T.; Réthoré, 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. (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. (14) Jain, N.; Arntz, Y.; Goldschmidt, V. r.; Duportail, G.; Mély, Y.; 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 TumorAssociated 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

Article

Langmuir (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 LongChain 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 spectroscopy. 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 Dipalmitoyllecithin. 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) Förster, G.; Meister, A.; Blume, A. Chain packing modes in crystalline surfactant and lipid bilayers. Curr. Opin. Colloid Interface Sci. 2001, 6, 294−302. (54) Förster, 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) Förster, 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 Langmuir XXXX, XXX, XXX−XXX