New Lipid Family That Forms Inverted Cubic Phases in Equilibrium

Sep 6, 2008 - Ibaraki 305-8565, Japan, and New Business DeVelopment DiVision, Kuraray Company, Ltd. 1-1-3,. Otemachi, Chiyoda-ku, Tokyo 100-8115, ...
0 downloads 0 Views 907KB Size
Download PDF
12286

J. Phys. Chem. B 2008, 112, 12286–12296

New Lipid Family That Forms Inverted Cubic Phases in Equilibrium with Excess Water: Molecular Structure-Aqueous Phase Structure Relationship for Lipids with 5,9,13,17-Tetramethyloctadecyl and 5,9,13,17-Tetramethyloctadecanoyl Chains Jun Yamashita,†,§ Manzo Shiono,‡,| and Masakatsu Hato*,† Nanotechnology Research Institute, AIST, Tsukuba Central-5, Higashi 1-1-1, Tsukuba, Ibaraki 305-8565, Japan, and New Business DeVelopment DiVision, Kuraray Company, Ltd. 1-1-3, Otemachi, Chiyoda-ku, Tokyo 100-8115, Japan ReceiVed: April 7, 2008; ReVised Manuscript ReceiVed: July 2, 2008

With a view to discovering a new family of lipids that form inverted cubic phases, the aqueous phase behavior of a series of lipids with isoprenoid-type hydrophobic chains has been examined over a temperature range from -40 to 65 °C by using optical microscopy, DSC (differential scanning calorimetry), and SAXS (smallangle X-ray scattering) techniques. The lipids examined are those with 5,9,13,17-tetramethyloctadecyl and 5,9,13,17-tetramethyloctadecanoyl chains linked to a series of headgroups, that is, erythritol, pentaerythritol, xylose, and glucose. All of the lipid/water systems displayed a “water + liquid crystalline phase” two-phase coexistence state when sufficiently diluted. The aqueous phase structures of the most diluted liquid crystalline phases in equilibrium with excess water depend both on the lipid molecular structure and on the temperature. Given an isoprenoid chain, the preferred phase consistently follows a phase sequence of an HII (an inverted hexagonal phase) to a QII (an inverted bicontinuous cubic phase) to an LR (a lamellar phase) as A* (crosssection area of the headgroup) increases. For a given lipid/water system, the phase sequence observed as the temperature increases is LR to QII to HII. The present study allowed us to find four cubic phase-forming lipid species, PEOC18+4 [mono-O-(5,9,13,17-tetramethyloctadecyl)pentaerythritol], β-XylOC18+4 [1-O-(5,9,13,17tetramethyloctadecyl)-β-D-xylopyranoside], EROCOC17+4 [1-O-(5,9,13,17-tetramethyloctadecanoyl)erythritol], and PEOCOC17+4 [mono-O-(5,9,13,17-tetramethyloctadecanoyl)pentaerythritol]. The values of TK (hydrated solid-liquid crystalline phase transition temperature) of the cubic phase-forming lipids are all below 0 °C. Quantitative analyses of the lipid molecular structure-aqueous phase structure relationship in terms of the experimentally evaluated “surfactant parameter” allow us to rationally select an optimum combination of hydrophilic/hydrophobic part of a lipid molecule that will form a desired phase in a desired temperature range. Introduction Inverted bicontinuous cubic phases (QII) in lipid/water systems, in particular, those that are stable in equilibrium with excess water, have unique features favoring their growing interest in biophysical and pharmaceutical fields, such as carriers for drug delivery systems and cosmetics, and matrixes for membrane protein crystallization.1-6 While recent progress in understanding of the inverted cubic phases has been significant, the cubic phases currently available present problems that have hampered the actual applications. First, the cubic phases often have a limited range of temperature over which they can be used. In particular, due to high values of TK of QII-forming lipids, the conventional cubic phases are generally unstable at low temperatures in a range 0-4 °C; they transform into a solid phase when handled and/or stored at the low temperature * Corresponding author. Phone: +81-45-503-9214. Fax: +81-45-5039201. E-mail: [email protected]. Present address: System and Structural Biology Center (SSBC), Yokohama Institute, RIKEN, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa-230-0045, Japan. † Nanotechnology Research Institute, AIST. ‡ Kuraray Co. § Present address: CytoPathfinder, Inc., Kiba KI Bldg. 4F, 6-4-2 Kiba, Koto-ku, Tokyo 135-0042, Japan. | Present address: Department for Research on the Future Key Technology, Japan Science and Technology Agency, 4-1-7, Kudan-Kita, Chiyodaku, Tokyo 102-0073, Japan.

conditions.7-11 This poses a problem particularly in their pharmaceutical or biological applications, where low temperature operations are often prerequisite for manipulating temperature-sensitive actives. Second, as different types of lipid species that can form QII phases are limited, engineering of the cubic phase to realize required performance is not yet as easy as might be wished. The challenge therefore is to develop a new lipid library that consists of a series of QII-forming lipids with varying hydrophobic chain lengths and headgroups that can form QII phase, which is stable over a technically relevant temperature range, say from 0 to 50 °C. Lipids with isoprenoid-type hydrophobic chains (hereafter referred to as an isoprenoid-chained lipid) are a unique class of lipids and have been synthesized and their physical properties have been investigated.12-21 We have recently proposed polyolbased lipids with single and double isoprenoid-type hydrophobic chains and demonstrated that they can form a range of liquid crystalline phases, for example, HII, LR, and inverted cubic phases with Pn3m/Ia3d/Fd3m symmetry.21-27 Moreover, the values of TK of isoprenoid-chained lipids are in most cases below 0 °C even when the hydrophobic chain length is extended to 16 carbon atoms long, indicating that the isoprenoid-chained lipids are lipids of choice for our present purpose. This expectation has prompted us to perform a more thorough survey of the aqueous phase behavior of the isoprenoid-chained lipids.

10.1021/jp8029874 CCC: $40.75  2008 American Chemical Society Published on Web 09/06/2008

New Lipid Family That Forms Inverted Cubic Phases

Figure 1. Chemical structure of the isoprenoid-chained lipids examined: (1) 1-O-(5,9,13,17-tetramethyloctadecyl)erythritol (EROC18+4), (2) mono-O-(5,9,13,17-tetramethyloctadecyl)pentaerythritol (PEOC18+4), (3) 1-O-(5,9,13,17-tetramethyloctadecyl)-β-D-xylopyranoside (β-XylOC18+4), (4) 1-O-(5,9,13,17-tetramethyloctadecyl)-β-D-glucopyranoside (β-GlcOC18+4), (5) 1-O-(5,9,13,17-tetramethyloctadecanoyl)erythritol (EROCOC17+4), and (6) mono-O-(5,9,13,17-tetramethyloctadecanoyl)pentaerythritol (PEOCOC17+4).

We have therefore synthesized isoprenoid-chained lipids with the hydrophobic chain lengths in a range C12-C18, which are linked to a range of sugars or sugar alcohols as the hydrophilic group to control the spontaneous curvature of the lipid monolayer. The work reported here is part of our current project aimed at discovering a new family of inverted cubic phases that are stable over technically relevant temperatures. In this Article, we describe how the aqueous phase structures are controlled by modulating the hydrophilic headgroup of the isoprenoid-chained lipids with C18-C17 chains (chains of 18 and 17 carbon atoms long), that is, with 5,9,13,17-tetramethyloctadecyl and 5,9,13,17-tetramethyloctadecanoyl chains. The headgroups are systematically altered from erythritol (ER), to pentaerythritol (PE), to xylose (Xyl), and to glucose (Glc) in order of increasing values of A*. The values of A*, which are estimated from the CPK molecular model, are about 0.2, 0.3, 0.3, and 0.4 nm2 for ER, PE, Xyl, and Glc-headgroup, respectively. Because all of the lipid/water systems displayed a “water + liquid crystalline phase” two-phase state at high dilution, we here focus on the identity and the temperature dependence of the most diluted liquid crystalline phase (a liquid crystalline phase that is in equilibrium with excess water) over a temperature range from -40 to 65 °C. Experimental Section Materials. The chemical structures of lipids examined are shown in Figure 1, that is, four different ether-lipids with a 5,9,13,17-tetramethyloctadecyl chain and two ester-lipids with a 5,9,13,17-tetramethyloctadecanoyl chain. We expressed an isopsrenoid chain as Cp+q, where p and q stand for the number of carbon atoms in the main chain and the number of methyl branches, respectively. For example, 5,9,13,17-tetramethyloctadecyl chain is expressed as C18+4, explicitly indicating that the hydrophobic chain is of 18 carbon atoms long and there are 4 methyl branches on the C18 main chain. Thus, 1-O-(5,9,13,17tetramethyloctadecyl)erythritol is expressed as EROC18+4, indicating that the erythritol headgroup is linked to the C18+4

J. Phys. Chem. B, Vol. 112, No. 39, 2008 12287 hydrophobic chain via an ether linkage. Similarly, 5,9,13,17tetramethyloctadecanoyl chain is expressed as C17+4, indicating that the hydrophobic chain is of 17 carbon atoms long with 4 methyl branches on it. Mono-O-(5,9,13,17-tetramethyloctadecanoyl)pentaerythritol is therefore expressed as PEOCOC17+4, indicating that the pentaerythritol headgroup is linked to the C17+4 hydrophobic chain via an ester linkage. The lipids were synthesized by conventional procedures.24,28,29 As a detailed description of each lipid including the synthetic procedures is given in the Appendix, only the purity of the lipids is given here. The purity of lipids with PE-headgroup (PEOC18+4 and PEOCOC17+4) as estimated by HPLC was at least 99.4%. The anomeric purity of β-XylOC18+4 and β-GlcOC18+4 estimated by 1H NMR and gas chromatography of its acetate form was at least 96%. The purity of lipids with ER-headgroup, EROC18+4, consisted of 77.8% of 1-O-(5,9,13,17-tetramethyloctadecyl)erythritol and 22.2% of 2-O-(5,9,13,17-tetramethyloctadecyl)erythritol. EROCOC17+4 consisted of 91.6% of 1-O(5,9,13,17-tetramethyloctadecanoyl)erythritol and 8.4% of 2-O(5,9,13,17-tetramethyloctadecanoyl)erythritol. Water was purified as follows: tap water was first treated by a water purification system designed by our institute (reverse osmosis, ion-exchange, filtration through a 0.22 µm filter) and finally treated by an Elga water purification unit just before experiments. Methods for Phase Identification and Thermal Stability of the Liquid Crystalline Phases. Water Penetration Scan and Small-Angle X-ray Scattering (SAXS). An outline of the phase behavior of each lipid/water system was first examined by a water penetration scan30 at three different temperatures, 1, 20, and 60 °C. To ensure the thermal equilibrium, a lipid, water, glass slides, and cover glasses used in the penetration experiment were initially equilibrated at each experimental temperature before the penetration scan was initiated. The optical textures of the lipid/water system were observed by an Olympus BX51 polarizing microscope,31 equipped with a Mettler FP82HTtemperature control stage. The optical images were captured by an Olympus C-4040 digital camera. For lipid/water systems that displayed an optically isotropic phase in equilibrium with excess water, which we considered to represent a QII phase, the phase identity was further examined by SAXS measurements. The SAXS measurements were performed with Ni-filtered Cu KR radiation [λ(wavelength) ) 0.154 nm] generated by a Rigaku RU-200 X-ray generator (40 kV, 100 mA) with a double pinhole collimator (0.5 mm φ × 0.3 mm φ). The sample temperature was controlled with a Mettler FP82HT temperature control-stage within an accuracy of (0.5 °C. Exposure time was 0.5-1 h at a sample-to-film distance of 195 mm, which was calibrated using B-form of stearic acid and 4.385 nm as the lattice constant.32 For several samples, the SAXS measurements using a Rigaku FR-D X-ray generator (50 kV, 60 mA) were also performed with an exposure time of 5 min and a sample-to-film distance of 500 mm. The diffraction pattern was recorded with an imaging plate (Fuji Photo films, HR-IIIN) in a flat camera. The images were digitized by a Rigaku imaging plate reader and were analyzed by a Rigaku R-AXIS system. The indexing of the SAXS peaks of cubic phases can be assessed by plotting the reciprocal spacing (1/dhkl) of the reflections verses (h2 + k2 + l2), where h, k, and l are the Miller indices. For a given cubic structure, such a plot passes through the origin and is linear with a slope of 1/ac, where ac is the cubic cell lattice constant, for example, inset in Figure 3c. Sample Preparation and Procedures of SAXS Measurement. Samples for SAXS and DSC measurements were prepared by using a modified micro syringe-based mixing device.27,33 To

12288 J. Phys. Chem. B, Vol. 112, No. 39, 2008

Yamashita et al. Berlin, 1.5 mm φ in outer diameter) by using a sample transfer device27 and was immediately flame-sealed. The flame-sealed sample was then incubated at 1 °C for 2-5 days before the SAXS measurement was initiated at 1 °C. We prepared two specimens from each lipid/water mixture and confirmed that the two specimens gave identical SAXS diffraction at 1 °C. Specimens that gave different diffractions were discarded, and new samples were prepared. In parallel experiments, we incubated the samples at -60 °C for 2-5 days before being incubated at 1 °C for 15 h. We confirmed that the 1 °C sample prepared by this deep freezing protocol gave a 1 °C SAXS profile identical to that observed by the present protocol. We therefore considered the phases at 1 °C obtained in this work to represent an equilibrium phase. After the SAXS measurement at 1 °C was completed, the SAXS measurement was continued with increasing temperature at a 5-10 °C interval until 65 °C was reached. The lipid/water samples were incubated for 15-24 h at each temperature before the SAXS measurements were performed. The actual composition of each sample was determined gravimetrically. Differential Scanning Calorimetry (DSC). The values of TK for the lipid/water systems were estimated by a Seiko SSC/ 560U differential scanning calorimeter, where effort has gone into developing a hydrated lipid solid by incubating the samples at -60 °C for 3 h before a heating run was initiated from -40 °C at a rate of 0.2-0.3 °C/min. The surfactant/water mixture was sealed in a silver DSC capsule. Results

Figure 2. (a) Water penetration scans for the ether-type isoprenoidchained lipid/water systems at 1, 20, and 60 °C. (1) EROC18+4/water system, (2) PEOC18+4/water system, (3) β-XylOC18+4/water system, (4) β-GlcOC18+4/water system. (b) Water penetration scans for the estertype isoprenoid-chained lipid/water systems at 1, 20, and 60 °C. (1) EROCOC17+4/water system, (2) PEOCOC17+4/water system.

avoid supercooling in the phase transformation involving cubic phases,34 we employed the following sample preparation procedure. Known weights of lipid and water, which gave a liquid crystalline phase + excess water coexistence phase, were loaded separately into each microsyringe at room temperature, 20-25 °C; that is, water was loaded in one syringe, and the lipid was loaded in the other syringe. The two microsyringes were then connected with a stainless steal coupler, and the whole device was incubated at 1 °C for at least 15 h before homogenization of the lipid and water at 1 °C was initiated. To avoid freezing of water, 1 °C was taken as the lowest temperature at which the stability of a cubic phase was examined. Because the values of TK of the lipid/water systems are below 0 °C (see below), and no transition was observed between the 0 and 1 °C temperature range, we consider the QII phase, which proved to be stable at 1 °C, is stable in a temperature range 0-1 °C as well. Below 0 °C, freezing of water causes dehydration of the lipid phase leading to the formation of a lamellar phase, LR (0 °C > T > TK). The hydrated surfactant was then transferred into a quartz-capillary (Glas,

In Figure 2, the water penetration scans for each lipid performed at 1, 20, and 60 °C are presented with increasing lipid concentration from left to right. Figures 3 and 4 report typical examples of SAXS profiles and DSC thermograms for the QII-forming lipid/water systems, respectively. Tables 1-4 summarize the numerical values of temperature-dependent SAXS data for the most diluted liquid crystalline phase for the QII-forming lipid/water systems. Temperature-Dependent Phase Behavior of the EtherLipid/Water Systems. EROC18+4/Water System. Water penetration scan for the EROC18+4/water system at 1, 20, and 60 °C indicated that the most diluted liquid crystalline phase in contact with excess water is birefringent (Figure 2a-1). The optical textures appeared to be typical of a hexagonal phase, which consists of long straight cylinders parallel to each other and packed in a two-dimensional hexagonal lattice. By analogy with other lipid/water systems, low hydration capacity of the mesophase suggests that the structure of this phase is of type II, where the interior of the cylinders is occupied by the polar moiety. The SAXS data corroborate the above speculation; the SAXS profile of 50 wt % EROC18+4 at 1 and 25 °C gave three peaks in a ratio 1:1/3:1/2, (6.11, 3.53, 3.06 nm at 1 °C and 5.62, 3.27, 2.83 nm at 25 °C). Thus, the most diluted liquid crystalline phase of the EROC18+4/water system is an HII phase over a temperature range 1-60 °C. PEOC18+4/Water System. Water penetration scans for the PEOC18+4/water system at 1 and 20 °C gave two optically isotropic regions, water (W) and an isotropic lipid region (QII), which we considered to represent a QII phase. At 60 °C, the most diluted liquid crystalline region in contact with excess water displayed a birefringent texture, suggesting formation of an HII phase. This suggests that the PEOC18+4/water system undergoes a QII to an HII phase transition as temperature increases (Figure 2a-2). It is noted that the QII/water or HII/ water interfaces were very irregular as compared to those of

New Lipid Family That Forms Inverted Cubic Phases

J. Phys. Chem. B, Vol. 112, No. 39, 2008 12289

Figure 3. Temperature evolution of SAXS profiles for the QII-forming lipid/water systems: (a) 59.3 wt % PEOC18+4/water system at 1, 25, and 35 °C, (b) 61.5 wt % β-XylOC18+4/water system at 1, 25, and 65 °C, (c) 52.8 wt % EROCOC17+4/water system at 4 °C. Inset shows the (1/dhkl) verses (h2 + k2 + l2) plot, (d) 51.1 wt % PEOCOC17+4/water system at 4 and 45 °C. The SAXS profiles in (a) and (b) were captured by a Rigaku RU-200 X-ray generator system, while those in (c) and (d) were captured by a Rigaku FR-D X-ray generator system. The arrows on traces indicate the location of the diffraction peaks. q ) (4π sin θ)/λ is the scattering vector, where 2θ is the scattering angle.

the other lipid/water systems examined. This is due to the fact that anhydrous PEOC18+4 is an isotropic liquid of low viscosity. Upon contact with the lipid, water readily penetrated into the lipid region forming many narrow channel-like structures, thereby leading to the irregular interface as shown in the pictures. The SAXS profiles observed for a 59.3 wt % PEOC18+4 support the above conclusion (Figure 3a, Table 1). In a lower temperature range 1-30 °C, the SAXS displayed six peaks that correspond to a cubic phase with the crystallographic space group Pn3m. In a higher temperature range 35-65 °C, the Pn3m cubic phase gave way to an inverted hexagonal phase HII. Figure 4a reports a heating thermogram of a 66.2 wt % PEOC18+4/ water system over the temperature range from -40 to 10 °C. An endothermic peak associated with melting of hydrated PEOC18+4 occurred at -33 °C (∆H ) 13.3 kJ/mol). The large endothermic peak at 0 °C is due to melting of ice. In summary, the value of TK for the PEOC18+4/water system is -33 °C, and the most diluted liquid crystalline phase of the system is temperature dependent, a Pn3m cubic phase in the lower temperature range 1-30 °C and an HII phase above 35 °C. β-XylOC18+4/Water System. Water penetration scan for the β-XylOC18+4/water system at 1 and 20 °C gave four distinct regions, that is, water (W), an isotropic region (QII), a birefringent region (LR), and a weakly birefringent anhydrous lipid region (L) as the water content decreases from left to right (Figure 2a-3). We consider the isotropic region in contact with water as being an inverted cubic phase, QII. At 60 °C, a most diluted lipidic phase in contact with water became birefringent under crossed polarizer configuration (an inset). The fanlike

optical texture observed at 60 °C appeared typical of a hexagonal phase. Thus, the β-XylOC18+4/water system exhibits a phase sequence QII to HII as the temperature increases. The temperature-dependent SAXS data (Figure 3b and Table 2) support the above speculation. In a lower temperature range 1-45 °C, the SAXS profile observed for a 61.5 wt % β-XylOC18+4 displayed six peaks that correspond to a cubic phase with the crystallographic space group Pn3m. Above 55 °C, the Pn3m cubic phase completely gave way to an inverted hexagonal phase, HII. Figure 4b reports a heating thermogram of a 60 wt % β-XylOC18+4/water system over the temperature range from -40 to 10 °C, displaying an exothermic peak at about -24 °C and two endothermic peaks at -13 and at 0 °C, respectively. Although we did not investigate the exact nature of the exothermic transition at -24 °C, it may be ascribable to a transition from a “less ordered solid” to an “ordered solid” or “hydrated crystalline phase” of β-XylOC18+4, which then melts endothermically at -13 °C (∆H ) 14.5 kJ/mol). The large endothermic peak at 0 °C is due to melting of ice. In summary, the value of TK for the β-XylOC18+4/water system is -13 °C, and the most diluted liquid crystalline phase of the β-XylOC18+4/water system is a Pn3m cubic phase in the temperature range 1-45 °C, and an HII phase at higher temperatures above 55 °C. β-GlcOC18+4/Water System. Water penetration scan for the β-GlcOC18+4/water system performed at 1, 20, and 60 °C gave practically identical profiles; myelin figures formed at the lipid/ water interface (Figure 2a-4), indicating that a lamellar phase is the preferred phase for the β-GlcOC18+4/water system. Thus,

12290 J. Phys. Chem. B, Vol. 112, No. 39, 2008

Yamashita et al. TABLE 2: SAXS Diffraction Peaks Observed for the Most Diluted Liquid Crystalline Phases of the β-XylOC18+4/Water System (61.5 wt % β-XylOC18+4) That Are in Equilibrium with Excess Watera temperature (°C)

d-spacing (nm)

phase identity

1 10 25 35 45 55 60 65

7.30, 6.00, 5.11, 4.21, 3.65, 3.51 7.30, 6.03, 5.23, 4.23, 3.69. 3.49 7.07, 5.84, 4.94, 4.09, 3.52, 3.36 6.80, 5.55, 4.79, 3.90, 3.39, 3.22 6.42, 5.32, 4.59, 3.74, 3.29, 3.06 5.80, 3.34, 2.88 5.69, 3.29, 2.85 5.62, 3.26, 2.81

QII(Pn3m) QII(Pn3m) QII(Pn3m) QII(Pn3m) QII(Pn3m) HII HII HII

a

ac (nm) 10.4 10.4 10.0 9.6 9.2

ac: Lattice constant of a cubic phase.

TABLE 3: SAXS Diffraction Peaks Observed for the Most Diluted Liquid Crystalline Phases of the EROCOC17+4/ Water System (52.8 wt % EROCOC17+4) That Are in Equilibrium with Excess Watera temperature (°C)

d-spacing (nm)

phase identity

1 10 25 45 60 65

8.01, 6.70, 5.62, 4.69, 4.02, 3.87 8.15, 6.75, 5.66, 4.71, 4.13, 3.90 8.15, 6.80, 5.80, 4.74, 4.09, 3.90 7.30, 5.99, 5.08, 4.21, 3.65, 3.51 6.51, 5.32, 4.66, 3.77, 3.29, 3.11 5.39, 3.10, 2.69

QII(Pn3m) QII(Pn3m) QII(Pn3m) QII(Pn3m) QII(Pn3m) HII

a

ac (nm) 11.5 11.6 11.6 10.3 9.3

ac: Lattice constant of a cubic phase.

TABLE 4: SAXS Diffraction Peaks Observed for the Most Diluted Liquid Crystalline Phases of the PEOCOC17+4/Water System (51.1 wt % PEOCOC17+4) That Are in Equilibrium with Excess Watera Figure 4. DSC heating thermograms for the lipid/water systems: (a) 66.2 wt % PEOC18+4/water system (a heating rate at 0.3 °C/min), (b) 60 wt % β-XylOC18+4/water system (a heating rate at 0.2 °C/min), and (c) 61.7 wt % PEOCOC17+4/water system (a heating rate at 0.3 °C/ min).

temperature (°C)

d-spacing (nm)

phase identity

ac (nm)

1 15 20 25 30 40 45 60 65

4.76, 2.38 4.69, 2.38 4.69, 2.34 4.65, 2.32 4.63, 2.33 7.80, 6.42, 5.55, 4.50, 3.90, 3.71 7.60, 6.24, 5.29, 4.41, 3.82, 3.63 6.85, 5.62, 4.81, 3.97, 3.45, 3.26 6.70, 5.39, 4.66, 3.83, 3.38, 3.16

LR LR LR LR LR QII(Pn3m) QII(Pn3m) QII(Pn3m) QII(Pn3m)

11.1 10.8 9.7 9.4

TABLE 1: SAXS Diffraction Peaks Observed for the Most Diluted Liquid Crystalline Phases of the PEOC18+4/Water System (59.3 wt % PEOC18+4) That Are in Equilibrium with Excess Watera temperature (°C)

d-spacing (nm)

phase identity

1 25 30 35 45 60 65

6.96, 5.69, 4.86, 4.02, 3.49, 3.33 5.95, 4.86, 4.23, 3.43, 2.98, 2.81 5.80, 4.74, 4.13, 3.34, 2.90, 2.75 5.44, 3.12, 2.71 5.24, 3.02, 2.62 4.98, 2.89, 2.50 4.94, 2.85, 2.46

QII(Pn3m) QII(Pn3m) QII(Pn3m) HII HII HII HII

a

ac(nm) 9.86 8.42 8.22

ac: Lattice constant of a cubic phase.

the most diluted liquid crystalline phase of the β-GlcOC18+4/ water system in equilibrium with excess water is a lamellar phase, LR, at least over a temperature range 1-60 °C. Temperature-Dependent Phase Behavior of the EsterLipid/Water Systems. EROCOC17+4/Water System. Water penetration scan for the EROCOC17+4/water system at 1 and 20 °C gave four distinct regions, that is, water (W), a stiff isotropic region (QII), a birefringent region (LR), and an anhydrous lipid region (L) as the water content decreases from left to right. The water penetration scan at 60 °C gave three distinct regions, water (W), an isotropic region (QII), and a

a

ac: Lattice constant of a cubic phase.

strongly birefringent anhydrous lipid region (L) as the water content decreases (Figure 2b-1). Thus, the isotropic region (QII) of the EROCOC17+4/water system, which we consider to represent an inverted cubic phase, persists at least over the temperature range 1-60 °C. The SAXS profiles observed for a 52.8 wt % EROCOC17+4 corroborate this speculation; the SAXS data gave at least six peaks that correspond to a cubic phase with a crystallographic space group Pn3m over the temperature range 1-60 °C, and above 65 °C, the Pn3m cubic phase gives way to an HII phase (Table 3). An SAXS profile of a 52.8 wt % EROCOC17+4 at 4 °C is reported in Figure 3c displaying eight peaks that correspond to a Pn3m cubic phase with a lattice constant of 11.5 nm. The DSC thermogram of a 63 wt % EROCOC17+4/water system measured over a temperature range from -40 to 15 °C gave only a single endothermic peak associated with melting of ice (data not shown). No thermal event associated with the

New Lipid Family That Forms Inverted Cubic Phases

J. Phys. Chem. B, Vol. 112, No. 39, 2008 12291 TABLE 5: Phase Sequence, TK, an Lr to QII Phase Transition Temperature (TLQ), a QII to HII Phase Transition Temperature (TQH), the Surfactant Parameter of the Lipid (Whc/{as(dhc)dhc}), and the Area Averaged Mean Curvature of the Lipid Monolayer (〈Hhc〉) for the Fully Hydrated Most Diluted Liquid Crystalline Phases at 25 °C lipid Ether-Lipids EROC18+4 PEOC18+4 β-XylOC18+4 β-GlcOC18+4

Figure 5. Temperature-dependent phase sequence of the most diluted liquid crystalline phases of the ether-type isoprenoid-chained lipid/water systems (A) and of the ester-type isoprenoid-chained lipid/water systems (B). Light blue: an inverted hexagonal phase, HII. Violet: a cubic phase QII. Green: a lamellar phase LR.

melting of hydrated solid EROCOC17+4 was observed, indicating the system remained in a liquid crystalline state even after the 3 h incubation at -60 °C. This may arise either from kinetically hindered crystallization of hydrated EROCOOC17+4 or from a TK value (if any) of lower than -40 °C. Although we were unable to identify the TK value, we speculate that the value of TK for the EROCOOC17+4/water system is most presumably below 0 °C. In summary, the most diluted liquid crystalline phase of the EROCOC17+4/water system is a Pn3m cubic phase over a temperature range 1-60 °C, and an HII phase above 65 °C. PEOCOC17+4/Water System. At 1 and 20 °C, the water penetration scans for the PEOCOC17+4/water system are dominated by myelin figures, which are growing at the water/lipid interface, indicating that a lamellar phase LR is the preferred phase at 1-20 °C (Figure 2b-2). At 60 °C, the lipidic phase in contact with water becomes isotropic, indicating that the preferred phase of the PEOCOC17+4/water system is temperature dependent. The corresponding SAXS data corroborate the above observation (Figure 3d and Table 4). According to the SAXS data, the LR phase is stable in the lower temperature range 1-30 °C. Above 40 °C, the LR phase gives way to a cubic phase with the crystallographic space group Pn3m. The DSC thermogram for 61.7 wt % PEOCOC17+4 reported in Figure 4c displays an endothermic peak at -17 °C (∆H ) 20.4 kJ/mol), which is associated with melting of hydrated PEOCOC17+4. In summary, the value of TK of the PEOCOC17+4/ water system is -17 °C, and the most diluted liquid crystalline phase of the PEOCOC17+4/water system is an LR phase at lower temperatures, 0-30 °C, and a Pn3m cubic phase at higher temperatures, 40-65 °C. It is finally noted that the system transforms into an HII phase at higher temperatures above ∼73 °C (data not shown). Thus, a phase sequence observed in this system as the temperature increases is LR to QII to HII. Discussion Lipid Molecular Structure-Aqueous Phase Structure Relationship. Outline. The temperature-dependent phase sequence of the most diluted liquid crystalline phase of each lipid/ water system is summarized in Figure 5, where the lipid/water systems are grouped into two categories, the ether-lipid/water (Figure 5A) and the ester-lipid/water systems (Figure 5B). The

TK TLQ phase sequencea (°C) (°C) HII QII-HII QII-HII LR

0 °C), while an HII phase is formed above ∼1.2 (T > 0 °C). Second, temperature span of a QII phase stability range increases as the “surfactant parameter” decreases, displaying a maximum temperature span at or close to the QII-LR phase boundary. These relations allow us to rationally select an optimum combination of the hydrophilic/ hydrophobic parts of a lipid molecule that will form a desired phase in a desired temperature range. Let us finally consider the case of EROC18+4 and βGlcOC18+4, which predominantly prefer an HII and an LR phase, respectively. Because their hydrophobic part is identical to that of PEOC18+4 and β-XylOC18+4, a “surfactant parameter” assigned for their “hypothetical QII phase” may be estimated by assuming that as(dhc) is proportional to A*. A “surfactant parameter” of EROC18+4 thus estimated was about 1.7 (0.3/0.2 times that of PEOC22 or β-XylOC18+4), which is well above 1.2, predicting that TQH of EROC18+4 is below 0 °C, so the

New Lipid Family That Forms Inverted Cubic Phases EROC18+4/water system forms an HII phase (>0 °C), in accord with the present results. A surfactant parameter of a “hypothetical QII phase” of β-GlcOC18+4 similarly estimated was about 0.85, which is well below 1.05, predicting that the β-GlcOC18+4/ water system will form an LR phase above 0 °C, again in accord with the present experimental result. Conclusion We have investigated the lipid molecular structure-aqueous phase structure relationship for the series of isoprenoidchained lipids with 5,9,13,17-tetramethyloctadecyl and 5,9,13,17-tetramethyloctadecanoyl chains. The systematic phase sequences observed in this study as functions of molecular structure and of temperature are as follows: (1) for a given hydrophobic chain type, the preferred phases of the lipid/water system consistently shift from HII to QII to LR phase as the headgroup size increases, and (2) given a lipid/water system, the phase sequence observed as the temperature increases is LR to QII to HII. Quantitative analyses of the lipid molecular structure-aqueous phase structure relationship in terms of the experimentally evaluated “surfactant parameter” of the lipid/water systems indicate that both TQH and TLQ are depressed as the “surfactant parameter” Vhc/(as(dhc)dhc) of the lipid/water system increases, providing us with a useful measure to rationally select a combination of the hydrophilic/hydrophobic parts of a lipid molecule that will form a desired phase in a desired temperature range. Finally, the values of TK of the cubic phase-forming lipids are all below 0 °C, further demonstrating that isoprenoidchained lipids are invaluable in designing a range of inverted phases in the presence of excess water. The lipid molecular structure-aqueous phase structure relationship for the isoprenoid-chained lipids with shorter chains series will be published in future work. Acknowledgment. This work was financially supported by the Special Coordination Funds for Promoting Science and Technology of Ministry of Education, Culture, Sports, Science and Technology, MEXT. Thanks are also due to Professor M. Sato at Yokohama City University for allowing us to use the FR-D X-ray generator and Professor K. Aramaki at Yokohama National University for allowing us to perform SAXS measurements of the EROC18+4/water system. Appendix Estimation of Whc/(as(dhc)dhc) and 〈Hhc〉 Cubic phases have been proposed to be composed of surfactant bilayer units forming a bicontinuous threedimensional network, which separates two water-channel systems.37 It is well recognized that infinite periodic minimal surfaces, IPMS, afford reasonable structural models of bicontinuous cubic phases; for example, the diamond surface models the bilayer geometry of mesophases of the Pn3m symmetry, and the gyroid surface describes well that of the space group Ia3d.38,39 The polar/apolar dividing surfaces of the lipid bilayer in a QII phase were approximated by two displaced “parallel” surfaces, a constant distance dhc away from each point of a base (minimal) surface where the terminal methyl groups are located.40,41 Based on the general formula for the area element and the mean curvature on a displaced surface (e.g., eqs 10 and 11 in ref 41), the “surfactant parameter” and the mean curvature were estimated from the following equations:

J. Phys. Chem. B, Vol. 112, No. 39, 2008 12293

φhca3c Vhc ) as(dhc)dhc 2(a2 + 2πχu d2 )d c

〈Hhc 〉 )

E hc

(A1)

hc

-2πχEudhc

(A2)

a2c + 2πχEud2hc

where ac, φhc, and  are an experimentally observed lattice constant, the volume fraction of the hydrophobic chain part, and a dimensionless constant that represents the surface area per unit cell with a lattice constant of unity, for example, 1.919 for a Pn3m cubic phase.40-42 χuE is the Euler characteristic per unit cell, for example, -2 for a Pn3m cubic phase. dhc was estimated using an equation that relates φhc as a function of dhc (eq 7 in ref 40):

( )

φhc ) 2

( )

dhc dhc 4 + πχEu ac 3 ac

3

(A3)

φhc was estimated from the experimental data using the equation

φhc )

nLVhc nL(Vhc + Vhead) + nWVw

(A4)

where Vhc, Vhead, and Vw are the molar volumes of the hydrophobic chain part, the headgroup part, and water, respectively. nw and nL are the numbers of moles of water and the lipid in the system, respectively. The values of Vhc were estimated, assuming group additivity of the molecular volume, from the densities of 5,9,13-trimethyltetradecanol, 3,7,11,15tetramethylhexadecanol, 5,9,13,17-tetramethyloctadecanol, 5,9,13trimethyltetradecanoic acid, and 5,9,13,17-tetramethyloctadecanoic acid, which were measured by a Mettler DMA 602 densitometer over the temperature range 4-60 °C. The molecular volume of the headgroup, Vhead, was estimated from the literature value of the density of erythritol (1.4 × 103 kg/m3),43 pentaerythritol (1.4 × 103 kg/m3),44 and xlylose (1.5 × 103 kg/ m3),45 respectively. The density of water was taken from the literature value.46 For an HII phase, the mean curvature 〈Hhc〉 was estimated from the equation

〈Hhc 〉 )

1 2rp

(A5)

√

where rp[)L (√3⁄2π(1-φhc))] is a radius of the polar core (water + headgroup part of the lipid) and L is the basis vector length. The surfactant parameter for an HII phase was estimated from

√3L2φhc Vhc ) as(dhc)dhc 4πr d eq

(A6)

p hc

As the lipid chain length in the HII phase is not homogeneous,

max eq we assumed that dhc ) d eq hc. d hc[)√(3√3⁄2π) (rp + d hc ) - rp] is an equivalent chain length for an equivalent cylinder of polar core radius rp, in which the total lipid volume is equal to that in the HII phase.47 d max ) (L/√3 - rp) is the maximum hc hydrocarbon chain length in the HII phase. The area, which the lipid occupies at the polar/apolar interface a(dhc), is given by a(dhc) ) (4πrpVhc)/(√3L2φhc). For an LR phase, the surfactant parameter and the mean curvature were taken to be one and zero from the flat bilayer geometry.

12294 J. Phys. Chem. B, Vol. 112, No. 39, 2008 Synthesis of the Lipids (1) 1-O-(5,9,13,17-Tetramethyloctadecyl)erythritol (EROC18+4). The solution of 5,9,13,17-tetramethyloctadecanol (30 g, 0.09 mol) and pyridine (8.72 g, 0.11mol) in 200 mL of anhydrous dichloromethane was dropwise added into 100 mL of an anhydrous dichloromethane solution of p-toluenesulfonylchloride (19.3 g, 0.10 mol) at 0 °C under nitrogen atmosphere. The reaction mixture was stirred at ambient temperature overnight, washed with 200 mL of water, 20 mL of 2 N HCl, and 200 mL of saturated aqueous sodium-bicarbonate successively, and dried over anhydrous magnesium sulfate. The solution was filtered and evaporated under reduced pressure to afford 42 g of crude 5,9,13,17-tetramethyloctadecyl p-toluenesulfonate. A powder of 60% NaH in mineral oil (3.7 g, 0.09 mol) was added by small portion into a solution of erythritol (22.6 g, 0.19 mol) in 200 mL of anhydrous N,N-dimethylformamide (DMF) at 0 °C. After the complete addition of NaH, the reaction mixture was stirred for 1 h at ambient temperature and warmed to 50 °C. 5,9,13,17-Tetramethyloctadecyl p-toluenesulfonate (21 g) and 55 mL of DMF were added to the reaction mixture, which was further stirred at 80 °C for 4 h. The reaction mixture was evaporated in vacuo, and twice extracted with 500 mL of diethylether. The ether extract was washed with a saturated aqueous sodium chloride, dried over anhydrous magnesium sulfate, filtered, and evaporated. The resulting syrup was purified by silica gel column chromatography (dichloromethane to methanol/dichloromethane) to afford 2.2 g of 1-O-(5,9,13,17tetramethyloctadecyl)erythritol. According to HPLC analysis [acetonitrile:water (4:1) CAPCELLPAK SG-120 (5 µm)], the product consisted of 77.8% of 1-O-(5,9,13,17-tetramethyloctadecyl)erythritol and 22.2% of 2-O-(5,9,13,17-tetramethyloctadecyl)erythritol. 1H NMR (270 MHz CDCl , TMS): δ 0.83-0.88 (m, 15H), 3 1.0-1.6 (m, 28H), 2.40 (br. s, 1H), 2.71 (br. s, 1H), 2.84 (br. s, 1H), 3.39-3.7 (m, 4H), 3.7-3.85 (m, 4H). (2) Mono-O-(5,9,13,17-tetramethyloctadecyl)pentaerythritol (PEOC18+4). Under an atmosphere of nitrogen, 240 mL of an anhydrous dichloromethane solution containing 5,9,13,17tetramethyloctadecanol (30 g, 0.09 mol) and pyridine (8.72 g, 0.11 mol) was added dropwise into 100 mL of an anhydrous dichloromethane solution of p-toluenesulfonylchloride (19.3 g, 0.10 mol). After being stirred overnight at ambient temperature, the reaction mixture was washed successively with 200 mL of water, 20 mL of 2 N HCl, and 200 mL of saturated aqueous sodium hydrogen carbonate. The organic phase was dried over anhydrous magnesium sulfate and concentrated under reduced pressure after filtration to afford 42 g of 5,9,13,17-tetramethyloctadecyl p-toluenesulfonate. Under atmosphere of nitrogen, pentaerythritol (25 g, 0.18 mol) was dissolved in 200 mL of anhydrous DMF at elevated temperature. The powder of 3.7 g of 60% NaH in mineral oil was added to the pentaerythritol solution under ice cooling. The reaction mixture was then stirred for 1 h at ambient temperature. Twenty-one grams of 5,9,13,17tetramethyloctadecyl p-toluenesulfonate and 55 mL of DMF were added into the above reaction mixture at 50 °C, and the resulting mixture was stirred for 4 h at 80 °C. The solvent was removed under reduced pressure, and the resulting residue was extracted twice with 500 mL of diethylether. The ether extract was washed twice with saturated aqueous sodium chloride and dried with anhydrous magnesium sulfate. After filtration, the ether solution was evaporated, and the residue was purified by silica gel column chromatography (dichloromethane to methanol/dichloromethane) to afford 7.3 g

Yamashita et al. of mono-O-(5,9,13,17-tetramethyloctadecyl)pentaerythritol as a viscous transparent liquid. According to HPLC analysis [acetonitrile:water (4:1) CAPCELLPAK SG-120(5 µm)], the purity of the product was at least 99.5%. 1H NMR (270 MHz CDCl , TMS): δ 0.83-0.88 (m, 15H), 3 1.0-1.6 (m, 28H), 2.88 (br. s, 3H), 3.39-3.52 (m, 4H), 3.71 (d, J ) 3.9 Hz, 6H). (3) 1-O-(5,9,13,17-Tetramethyloctadecyl)-β-D-xylopyranoside (β-XylOC18+4). Under argon atmosphere, 318 mg of β-xylose tetraacetate was dissolved in 6 mL of dichloromethane and cooled to 0 °C. The dichloromethane solution of 0.12 mL of SnCl4 was added dropwise into the above β-xylose tetraacetate solution. The reaction mixture was cooled to -10 °C after stirring for 20 min at ambient temperature. Under cooling (-10 °C), 5,9,13,17-tetramethyloctadecanol (326.6 mg, 1.00 mmol) in 1 mL of dichloromethane was added dropwise to the above mixture. After stirring for 4 h, sodium hydrogen carbonate solution was added to the reaction mixture. Next, the reaction mixture was extracted three times with dichloromethane, washed with water, and dried over anhydrous sodium sulfate. After evaporation of the solvent, the resulting residue was purified by silica gel chromatography to obtain 93 mg of 1-O-(5,9,13,17tetramethyloctadecyl)-β-D-xylopyranoside triacetate. Under argon atmosphere, 54 mg of sodium methoxide was added into an anhydrous methanol solution of 1-O-(5,9,13,17-tetramethyloctadecyl)-β-D-xylopyranoside triacetate (584.8 mg in 5 mL of methanol). The reaction mixture was stirred overnight at room temperature, and 1 mL of 1 N HCl was added under cooling (0 °C). The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane to methanol/dichloromethane) to afford 413 mg of 1-O-(5,9,13,17-tetramethyloctadecyl)-β-D-xylopyranoside as a waxlike solid. The anomeric purity of 1-O-(5,9,13,17tetramethyloctadecyl)-β-D-xylopyranoside estimated by 1H NMR and by gas chromatography of its acetate form after treatment of acetic anhydride-pyridine at 60 °C for 2 h was at least 96%. 1H NMR (300 MHz CDCl , TMS): δ 0.84, 0.86 (d, J ) 6.4 3 Hz, J ) 6.8 Hz, 15H), 1.0-1.7 (m, 31H), 3.2-3.7 (m, 5H), 3.82 (dd, J ) 16 Hz, 7.7 Hz, 1H), 3.94 (dd, J ) 11.6 Hz, 5 Hz, 1H), 4.25 (d, J ) 7.1 Hz, 1H). (4) 1-O-(5,9,13,17-Tetramethyloctadecyl)-β-D-glucopyranoside (β-GlcOC18+4). Under argon atmosphere, 180 mg of βglucose pentaacetate was dissolved in 5 mL of dichloromethane and cooled to 0 °C. Next, 6.3 mL of a dichloromethane solution of 0.12 mL of SnCl4 was added dropwise into the above β-glucose pentaacetate solution and stirred for 20 min at ambient temperature. The reaction mixture was then cooled and stirred for 10 min at -10 °C. Under cooling (-10 °C), 5,9,13,17tetramethyloctadecanol (327 mg, 1.00 mmol) dissolved in 1 mL of dichloromethane was added dropwise and stirred for 4 h. Next, aqueous sodium hydrogen carbonate solution was added to the reaction mixture. Afterward, the reaction mixture was extracted three times with dichloromethane, washed with saturated aqueous sodium chloride, and dried over anhydrous sodium sulfate. After the solvent was evaporated under reduced pressure, the residue was purified by silica gel column chromatography (dichloromethane to methanol/dichloromethane) to afford 79 mg of 1-O-(5,9,13,17-tetramethyloctadecyl)-β-Dglucopyranoside tetraacetate. Under argon atmosphere, a methanol solution of 1-O-(5,9,13,17-tetramethyloctadecyl)-β-Dglucopyranoside tetraacetate (131 mg in 2 mL of methanol) was added dropwise into a methanol solution of sodium methoxide (1.62 mg in 5 mL of methanol) and stirred for 8 h at room temperature. Under cooling (0 °C), 1 mL of 1 N HCl was added.

New Lipid Family That Forms Inverted Cubic Phases The reaction mixture was extracted three times with dichloromethane, washed once with saturated aqueous sodium chloride, and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane to methanol/dichloromethane) to afford 83 mg of 1-O-(5,9,13,17tetramethyloctadecyl)-β-D-glucopyranoside as a waxlike solid. The purity estimated by 1H NMR was at least 96%. 1H NMR (300 MHz CDCl , TMS): δ 0.84, 0.86 (d, J ) 6.4 3 Hz, J ) 6.6 Hz, 15H), 1.0-1.4 (m, 28H), 1.4-1.7 (m, 4H), 3.3 (dd, J ) 16 Hz, 8 Hz, 2H), 3.44-3.6 (m, 3H), 3.7-3.8 (m, 3H), 4.29 (d, J ) 7.9 Hz, 1H). (5) 1-O-(5,9,13,17-Tetramethyloctadecanoyl)erythritol(EROCOC17+4). Under nitrogen atmosphere and at ambient temperature, thionylchloride (5.2 g, 0.044 mol) was added dropwise into 30 mL of an anhydrous dichloromethane solution of 5,9,13,17-tetramethyloctadecanoic acid (10 g, 0.029 mol) and a drop of pyridine. The reaction mixture was refluxed for 1 h and evaporated in vacuo to obtain 10.5 g of 5,9,13,17tetramethyloctadecanoylchloride. Under nitrogen atmosphere, erythritol (2.56 g, 0.0209 mol) was mixed with a solution of pyridine (2.21 g, 0.0279 mol) in 70 mL of anhydrous DMF at elevated temperature. The resulting solution was cooled to ambient temperature. Five grams of 5,9,13,17-tetramethyloctadecanoylchloride dissolved in 10 mL of dichloromethane was added dropwise into the solution. Next, the reaction mixture was stirred for 1 h at ambient temperature. The reaction mixture was then diluted with 100 mL of dichloromethane, washed three times with saturated aqueous sodium chloride, and dried over anhydrous sodium sulfate. The product was concentrated under reduced pressure and purified by silica gel column chromatography (dichloromethane to methanol/dichloromethane) to obtain 2.83 g of 1-O-(5,9,13,17tetramethyloctadecanoyl)erythritol as a transparent liquid of high viscosity. According to HPLC analysis [acetonitrile:water (4: 1) CAPCELLPAK SG-120 (5 µm)], the product consisted of 91.6% of 1-O-(5,9,13,17-tetramethyloctadecanoyl)erythritol and 8.4% of 2-O-(5,9,13,17-tetramethyloctadecanoyl)erythritol. 1H NMR (270 MHz CDCl , TMS): δ 0.8-0.9 (m, 15H), 3 1.0-1.7 (m, 26H), 2.11 (br. s, 1H), 2.33 (t, J ) 7.9 Hz, 2H), 2.66 (br. S, 1H), 2.75 (br. s, 1H), 3.6-3.9 (m, 4H), 4.29-4.36 (m, 2H). (6) Mono-O-(5,9,13,17-tetramethyloctadecanoyl)pentaerythritol (PEOCOC17+4). Under an atmosphere of nitrogen and at ambient temperature, thionylchloride (1.14 g, 9.58 mmol) was added dropwise into 22 mL of anhydrous dichloromethane solution containing 5,9,13,17-tetramethyloctadecanoic acid (2.0 g, 5.9 mmol) and a drop of pyridine. After the reaction mixture was refluxed for 1 h followed by evaporation of the solvent under reduced pressure, 2.0 g of 5,9,13,17-tetramethyloctadecanoylchloride was obtained. Under nitrogen atmosphere, pentaerythritol (0.88 g, 6.46 mmol), pyridine (0.69 g, 8.7 mmol), and 25 mL of anhydrous 1,3-dimethyl-2-imidazolidinone were mixed at an elevated temperature. After cooling, 1.32 g of 5,9,13,17-tetramethyloctadecanoylchloride in a 5 mL of dichloromethane was added dropwise into the pentaerythritol solution at ambient temperature. The reaction mixture was stirred for 1 h at ambient temperature, diluted with 100 mL of dichloromethane, and washed five times with saturated aqueous sodium chloride. The organic layer was dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane to methanol/dichloromethane) to obtain 0.64 g of mono-O-

J. Phys. Chem. B, Vol. 112, No. 39, 2008 12295 (5,9,13,17-tetramethyloctadecyl)pentaerythritol as a viscous transparent liquid. According to HPLC analysis [acetonitrile: water (4:1) CAPCELLPAK SG-120 (5 µm)], the purity of the product was at least 99.4%. 1H NMR (270 MHz CDCl , TMS): δ 0.7-0.9 (m, 12H), 0.95 (d, 3 J ) 7 Hz, 3H), 1.0-1.6 (m, 22H), 1.9 (br. s, 1H), 2.15 (dd, J ) 14 Hz, 9 Hz), 2.38 (dd, J ) 14 Hz, 7 Hz, 1H), 3.17 (br. s, 2H), 3.62 (s, 6H), 4.16 (s, 2H). References and Notes (1) (a) Larsson, K. J. Phys Chem. 1989, 93, 7304–7314. (b) Larsson, K. Curr. Opin. Colloid Interface Sci. 2001, 6, 268–276. (2) Ganem-Quintanar, A.; Quintanar-Guerrero, D.; Buri, P. Drug DeV. Ind. Pharm. 2000, 26, 809–820. (3) Shah, J. C.; Sadhale, Y.; Chilukuri, D. M. AdV. Drug DeliVery ReV. 2001, 47, 229–250. (4) Drummond, C. J.; Fong, C. Curr. Opin. Colloid Interface Sci. 2000, 4, 449–456. (5) Rummel, G.; Hardmeyer, A.; Widmer, C.; Chiu, M. L.; Nollert, P.; Locher, K. P.; Pedruzzi, I.; Landau, E. M.; Rosenbush, J. J. Struct. Biol. 1998, 121, 82–91. (6) Bicontinuous Liquid Crystals, Surfactant Science Series; Lynch, M. J.; Spicer, P. T., Eds.; Taylor & Francis: Boca Raton, FL, 2005; Vol. 127. (7) Briggs, J.; Caffrey, M. Biophys. J. 1994, 66, 573–587. (8) Briggs, J.; Caffrey, M. Biophys. J. 1994, 67, 1594–1602. (9) Lutton, E. S. J. Am. Oil Chem. Soc. 1965, 42, 1068–1070. (10) Qui, H.; Caffrey, M. J. Phys. Chem. B 1998, 102, 4819–4829. (11) Misquitta, Y.; Cherezov, V.; Havas, F.; Patterson, S.; Mohan, J. M.; Wells, A. J.; Hart, D. J.; Caffrey, M. J. Struct. Biol. 2004, 148, 169–175. (12) Ghosh, G.; Lee, S. J.; Ito, K.; Akiyoshi, K.; Sunamoto, J.; Nakatani, Y.; Ourisson, G. Chem. Commun. 2000, 4, 267–268. (13) Yamauchi, K.; Sakamoto, Y.; Moriya, A.; Yamada, K.; Hosokawa, T.; Higuchi, T.; Kinoshita, M. J. Am. Chem. Soc. 1990, 112, 3188–3191. (14) Stewart, L. C.; Kates, M. Chem. Phys. Lipids 1989, 50, 23–42. (15) Blo¨cher, D.; Six, L.; Gutermann, R.; Henkel, B.; Ring, K. Biochim. Biophys. Acta 1985, 818, 333–342. (16) Menger, F. M.; Chen, X. Y.; Brocchini, S.; Hopkins, H. P.; Hamilton, D. J. Am. Chem. Soc. 1993, 115, 6600–6608. (17) Fong, C.; Wells, D.; Krodkiewska, I.; Booth, J.; Hartley, P. G. J. Phys. Chem. B 2007, 111, 1384–1392. (18) Fong, C.; Wells, D.; Krodkiewska, I.; Hartley, P. G.; Drummond, C. J. Chem. Mater. 2006, 18, 594–597. (19) Milkereit, G.; Garamus, V. M.; Yamashita, J.; Hato, M.; Morr, M.; Vill, V. J. Phys. Chem. B 2005, 109, 1599–1608. (20) Barauskas, J.; Landh, T. Langmuir 2003, 19, 9562–9565. (21) Hato, M.; Minamikawa, H.; Tamada, K.; Baba, T.; Tanabe, Y. AdV. Colloid Interface Sci. 1999, 80, 233–270, and references cited therein. (22) Hato, M.; Minamikawa, H.; Salkar, R. A.; Matsutani, S. Langmuir 2002, 18, 3425–3429. (23) Hato, M.; Minamikawa, H.; Salkar, R. A.; Matsutani, S. Prog. Colloid Polym. Sci. 2004, 123, 56–60. (24) Salkar, R. J.; Minamikawa, H.; Hato, M. Chem. Phys. Lipids 2004, 127, 65–75. (25) Minamikawa, H.; Hato, M. Chem. Phys. Lipids 2005, 134, 151–160. (26) Yamashita, I.; Kawabata, Y.; Kato, T.; Hato, M.; Minamikawa, H. Colloids Surf., A 2004, 250, 485–490. (27) Hato, M.; Yamashita, I.; Kato, T.; Abe, Y. Langmuir 2004, 20, 11366–11373. (28) Japan laid-open patent S59-170085. (29) Banoub, J.; Bundle, D. Can. J. Chem. 1979, 57, 2091–2097, and references cited therein. (30) Laughlin, R. G. AdV. Colloid Interface Sci. 1992, 41, 57–79. (31) Rosevear, F. B. J. Am. Oil Chem. Soc. 1954, 31, 628–639. (32) von Sydow, E. Acta Crystallogr. 1955, 8, 557–560. (33) Cheng, A.; Hummel, B.; Qui, H.; Caffrey, M. Chem. Phys. Lipids 1998, 95, 11–21. (34) Fontell, K. Colloid Polym. Sci. 1990, 268, 264–285. (35) The sign of the curvature is taken to be positive when the lipid headgroup surface bends toward the water. (36) Israelachvili, J. N. Intermolecular and Surface Forces; Academic Press: San Diego, CA, 1985; pp 246-264. (37) Lindblom, G.; Larsson, K.; Johansson, L.; Fontell, K.; Forsen, S. J. Am. Chem. Soc. 1979, 101, 5465–5470. (38) Longley, W.; McIntosh, T. J. Nature 1983, 303, 612–614. (39) Hyde, S. T.; Andersson, S.; Ericsson, B.; Larsson, K. Z. Kristallogr. 1984, 168, 213–219. (40) Anderson, D.; Wennerstrom, H.; Olsson, U. J. Phys. Chem. 1989, 93, 4243–4253. (41) Anderson, D. M.; Davis, H. T.; Scriven, L. E. J. Chem. Phys. 1989, 91, 3246–3251.

12296 J. Phys. Chem. B, Vol. 112, No. 39, 2008 (42) By using the Gauss-Bonnet theorem, integration of eq 10 in ref 41 over the unit cell gives the area at the polar/apolar interface as 2(ac2 + 2 2πχuEdhc ). As the number of lipid molecules in the unit cell is φhcac3/Vhc, the area per lipid at the polar/apolar interface, as(dhc), is given by aS(dhc) ) (2(a2c + 2πχuEd2hc)Vhc)/φhca3c , leading to eq A1. Equation A2 can be derived in a similar manner by integrating eq 11 in ref 41 over the unit cell. (43) Schroeder, H. Chem. Ber. 12 1879, 562. (44) Eilerman, D.; Rudman, R. Acta Crystallogr., Sect. B 1979, 35, 2458.

Yamashita et al. (45) The Merck Index, 10th ed.; Merck & Co. Inc.: Rahway, NJ, 1983. (46) Handbook of Chemistry and Physics, 54th ed.; CRC Press: Cleveland, OH, 1974. (47) Marsh, D. Biophys. J. 1996, 70, 2248–2255.

JP8029874