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Chemically modified lipids — a suitable tool to study molecular interactions in model systems
Colloids and Surfaces A: Physicochemical and Engineering Aspects 183– 185 (2001) 391– 401 www.elsevier.nl/locate/colsurfa
Chemically modified lipids — a suitable tool to study molecular interactions in model systems F. Bringezu a,*, G. Brezesinski b a
Department of Chemical Engineering, Eng. II, Uni6ersity of California Santa Barbara, Santa Barbara, CA 93106 -50580, USA b Max Planck Institute of Colloids and Interfaces, Am Muehlenberg 1, D-14476 Golm/Potsdam, Germany
Abstract The polymorphism of triple-chain glycerophosphoethanolamines differing in the degree of methylation of the head group has been investigated in both monolayers at the air/water interface and in multilayers of the lipid/water dispersions by means of film balance measurements, Grazing incidence X-ray diffraction (GIXD), differential scanning calorimetry (DSC) and simultaneous small- and wide-angle X-ray diffraction, respectively. Despite the larger area requirements of the three chains compared to the smaller head group, the change in the degree of methylation affects the thermodynamical data and structures formed in mono- and multilamellar systems. Increasing methylation leads to larger tilt angles of the hydrocarbon chains in monolayers at low lateral pressures and shifts the pressure for the tilting transition from NN tilted centered rectangular to untilted hexagonal phases towards larger values. In the lipid/water dispersions, the change in head group methylation is connected with a change of the phase sequence. For the phosphatidylethanolamines (PE) and the monomethyl derivative a stabilization of lamellar liquid-crystalline phases by interdigitation of adjacent head groups was found, whereas the introduction of a second methyl group results in the appearance of an HII phase. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Glycerophosphoethanolamines; X-ray diffraction; Methylation
1. Introduction Due to their importance as major components of biological membranes, the self-organization of lipids in both monolayers at the air/water interface and multilayers in aqueous dispersions has gained great scientific interest. One approach of
* Corresponding author. Tel.: + 1-805-8934325; fax: + 1805-8934731. Present address: Institute of Biophysics and Xray Research, Schmiedlstr. 6, Graz, A-8042, Austria. E-mail address: [email protected] (F. Bringezu).
systematic studies in those systems uses chemically modified lipids providing information about competitive interactions between hydrophilic and hydrophobic moieties of the amphiphiles. The main goal of such experiments is to get a better understanding of the relation between chemical structure and lipid polymorphism [1–4]. The modifications have been made in different parts of the molecule. Recently, the influence of branches in the acyl chains on the lipid polymorphism has gained our interest because long-chain branches enable a systematic change of the area requirements of the two molecule parts without changing
0927-7757/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 7 - 7 7 5 7 ( 0 1 ) 0 0 5 2 2 - 2
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the ‘chemical character’ of the environment [5,6]. On the other hand, chemically modified phospholipids are interesting substrates for phospholipases. It is known that the substrate properties have a strong influence on the activity of phospholipases [7,8]. The study of phospholipids with defined chemical modifications should help to understand the hydrolysis mechanism and to find potential inhibitors [9,10]. Although much is known about the phase behavior of multiple-chain phosphatidylethanolamines (PE) and phosphatidylcholines (PC), relatively few studies on the N-methylated intermediates have been published [11,12]. In previous studies on monolayers of triple-chain lipids, it was shown that despite the large hydrophobic area specific interactions between the head groups must be taken into account [13,14]. The present work is focused on the phase behavior of triple chain phospholipids in monolayers and aqueous dispersions. The degree of the head group methylation has been changed leading to new substances with differences in polarity of the head group, ability to form a hydrogen-bonding network and in the specific area requirements which influence the competitive interaction between hydrophobic and hydrophilic parts of these molecules. The systems were investigated by means of differential scanning calorimetry (DSC), small- and wide-angle X-ray diffraction and Grazing incidence X-ray diffraction (GIXD).
2. Experimental
2.1. Materials For the synthesis of the branched-chain PEs, 2-alkyl branched fatty acids have been prepared
according to [15] via the alkyl malonic acid esters. The structures of the acids were confirmed by mass spectrometry. The melting points were found to be in agreement with the literature data [16,17]. The fatty acids were transformed into their anhydrides using dicyclohexylcarbodiimide (Merck). The acylation of 1-O-benzyl-2-O-hexadecyl-glycerol in presence of 4,4%-dimethylaminopyridine as a catalyst revealed the corresponding racemic 1-O-benzyl-2-O-hexadecyl-3acyl-glycerols. From these derivatives, the benzyl-blocking group was removed and the resulting primary hydroxy-function was then phosphorylated using 2-bromoethyl-phosphoric acid dichloride [18]. The conversion of the bromo-esters using ammonia, monomethyl- and dimethylammonia saturated ethanol at room temperature revealed the final products (see Fig. 1 for chemical structures), which have been purified by column chromatography using methanol/chloroform/water/ammonia gradients for elution. Compounds 1 –3 were characterized by mass spectrometry, high performance liquid chromatography, and microanalysis. Selected analytical data: rac. 1(2-hexadecyloctadecanoyl)-2-O-hexadecylglycerophosphoethanolamine (1): 1(2C16-18:0)-2H-PE; C55H112N1O7P1; M= 930.47 g mol − 1, [M]+ = 931.1; FP = 192–193°C; micro analysis: (% calc./ found) N: 1.51/1.57, C: 71.00/71.22, H: 12.13/ 12.19 rac. 1(2-hexadecyloctadecanoyl)-2-O-hexadecylglycerophospho-N-methyl-ethanolamine (2): 1(2C16-18:0)-2H-PE(Me); C56H114N1O7P1; M= 944.50 g mol − 1, [M]+ = 944.9; FP = 78–80°C; micro analysis: (% calc./found) N: 1.48/1.44, C: 71.21/71.01, H: 11.86/12.04
Fig. 1. Chemical structure of the triple-chain lipids investigated (x =3, compound 1; x = 2, compound 2; x = 1, compound 3).
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rac. 1(2-hexadecyloctadecanoyl)-2-O-hexadecylglycerophospho-N,N-dimethyl-ethanolamine (3): C57H116N1O7P1; 1(2C16-18:0)-2H-PE(Me)2; −1 + M = 958.52 g mol , [M] =958.2; FP = 106.5– 107°C; micro analysis: (% calc./ found) N: 1.46/1.42, C: 71.73/70.83, H: 12.20/ 12.04
2.2. Methods The thermotropic phase behavior in the water saturated two-phase region (60 wt.% water) was characterized by DSC using a DSC-2 (PerkinElmer, Norfolk, CT). For sample preparation, all lipids were dried in a vacuum oven for 2 h at 50°C before weighing. The samples (3– 5 mg) were transferred into aluminum pans and mixed with water before closing. Multilamellar dispersions were formed during the equilibration above the main-transition temperature (Tm) for 1 h. An empty pan was used as reference. The data were recorded through an interface to a PC. After polynomial baseline fit the transition enthalpies and main-transition temperatures have been determined. The amount of strongly bound water was quantitatively calculated from the ice peak resulting from the melting of frozen excess water not bound to the head groups [19]. X-ray measurements of the lipid-water dispersions were carried out at the X13 double focusing monochromator-mirror camera of the EMBL outstation at DESY, Hamburg, Germany, using a monochromatic X-ray beam (u =0.15 nm). The diffracted intensities in the small- and wide-angle regions were simultaneously recorded using a data acquisition system described in [20]. The beamline setup has been described in detail elsewhere [21]. The reciprocal spacings s= 1/d were calibrated by the diffraction pattern of dry rat-tail collagen with a long spacing of 65 nm (SAXS) and p-bromobenzoic acid (WAXS). For sample preparation, weighed amounts of the lipid were dispersed in ultra-pure water (60% w/w). The dispersions were vortexed and then transferred into glass capillaries (d= 1 mm) (Hilgenberg, Malsfeld, Ger-
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many), which were placed in the temperature controlled sample holder. Initially static exposures have been undertaken in order to gain information about the structures formed after sample preparation. The experiments were continued with temperature scans (1°C min − 1) between 5 and 95°C. Static exposures were taken below and above the main-transition and compared between the cycles to ensure the sample quality and exclude possible radiation damage. The monolayers were spread from an 1 mM p.a. grade chloroform solution onto an ultra pure water subphase. The water was purified using a Millipore desktop unit. The pressure-area isotherms were recorded with a Wilhelmy film balance. For the Synchrotron measurements the trough is located in a He-filled, sealed container. The X-ray experiments were performed using the liquid-surface diffractometer on the undulator beamline BW1 at HASYLAB, DESY, Hamburg (Germany). The experimental set-up and evaluation procedures have been described in detail elsewhere [22,23]. The Synchrotron beam was made monochromatic by Bragg reflection on a beryllium (002) crystal and was adjusted to strike the monolayer on the water surface at an angle of incidence hi = 0.85hc, where hc is the critical angle of total external reflection. The intensity of the diffracted radiation is detected by a position sensitive detector (PSD) (OED-100-M, Braun, Garching, Germany). The resolution of the horizontal scattering angle 2Uhor is given by a Soller collimator located in front of the PSD. The scattering vector Q has an in-plane component Qxy :(4y/ u) sin(2Uhor/2) and an out-of-plane component Qz : (2y/u) sin hf, where u is the X-ray wave length and hf the vertical scattering angle. The accumulated position-resolved scans were corrected for polarization, footprint-area, and powder averaging (Lorentz factor). Model peaks taken to be Lorentzian in the in-plane direction and Gaussian in the out-of-plane direction were fitted to the corrected intensities. The lattice spacings are obtained from the in-plane diffraction. The lattice parameters can be calculated from the lattice spacings and the unit cell area Axy is calculated from the lattice parameters. The Bragg rods
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Fig. 2. Surface pressure –molecular area isotherms of compound 2 at temperatures indicated. The insert shows both the transition enthalpy DH(p), calculated from the area change DA during the first order transition, and the transition pressure (yc) ( ) as function of temperature.
additionally yield the chain tilt angle t and the tilt azimuth.
3. Results
3.1. Lipid monolayers At the air/water interface, compounds 1–3 form stable monolayers. Fig. 2 displays the temperature dependency of compound 2 recorded between 10 and 40°C. Below 30°C the isotherm is fully condensed, whereas at 30°C a plateau indicates a first order transition between a liquid-expanded (LE) and a condensed state (LC). Both, the transition pressure (yc) and the area change DA involved in the transition show the typical linear temperature dependency (see insert Fig. 2). The linear extrapolation of yc towards zero yields the temperature T0 above which the LE phase
occurs (T0 = 26.5°C). The transition enthalpy DH can be calculated by a Clausius–Clapeyron equation using the area change DA [24]. The extrapolation of DH towards zero yields the critical temperature Tc above which the monolayer cannot be compressed into a condensed state (Tc = 51.3°C). GIXD measurements were applied in order to investigate the structures of the condensed phases. Fig. 3 (left) shows the intensity profile of the diffracted beam for compound 2 at 15°C. At low lateral pressures, two low order diffraction peaks (the degenerated (1, 91) above the horizon and the non-degenerated (0, 2) at the horizon) of a centered rectangular unit cell are observed. Such an intensity distribution indicates tilted hydrocarbon chains with a tilt direction towards the nearest neighbor (NN) along the short axis of the unit cell (see inserts). Increasing pressure leads to a phase transition towards a hexagonal phase exhibiting only one diffraction peak with a Bragg rod maximum at the horizon, hence the chains are vertically oriented. This tilting transition can also be seen in the isotherm (Fig. 3 (right)) which shows a change in slope at about 17 mN m − 1. Compounds 1 and 3 show similar scattering profiles with the same type of phase transition. The calculated lattice parameters are presented in Table 1. The in-plane area (Axy ) calculated from the X-ray results as a function of the lateral pressure is also presented in Fig. 3 (right). The Axy values are in good agreement with the measured isotherm, hence the monolayer contains only a small number of defects. The condensed isotherms show a linear dependency between lateral pressure and molecular area: Axy = K1 − K2y, where Axy = A0/cos(t) with A0 as the cross-sectional area of the chains and t as the chain tilt angle. Therefore, the extrapolation of the plot of 1/cos(t)= f(y) to zero tilt gives the value of the tilting transition pressure. Fig. 4 shows this relation for compounds 1–3. The PE (1) shows a transition pressure of approximately 14.0 mN m − 1. The successive methylation of the head group results in a shift towards larger values. For compound 2, a value of 18.0 mN m − 1
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was obtained, whereas compound 3 gives 20.5 mN m − 1. The slope of the linear function increases with increasing head-group methylation. Therefore, the possible differences in the chain tilt are more pronounced at low lateral pressures. The extrapolation towards zero pressure yields a chain tilt of approximately 13.6° for the PE (1), 15.5° for compound 2 and 16.9° for compound 3. Despite the large hydrophobic area that should determine the structure of the condensed phases, interactions between the head-groups must be obviously taken into account. The extended hydrophobic area should provide sufficient space for the head-groups. However, the data indicate that the chemical structure of the head-groups has a pronounced influence on the chain structure investigated by GIXD. On the one hand, the systematic introduction of methyl units increases the size of
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the head-groups continuously, but in any case, the three hydrophobic chains occupy a much larger area. On the other hand, specific interactions between adjacent lipid molecules must be taken into account, which should be responsible for the variation found in the chain lattice (tilt angles) and thermodynamical data (transition pressures). Compared to the hexagonal phase of upright oriented chains, the centered rectangular unit cell found at low lateral pressures is distorted. The direction of the distortion d coincides with the NN tilt direction. Investigations on monolayers of single-chain molecules (for review see [25]) revealed two sources of the unit cell distortion: the chain tilt and backbone ordering. According to the Landau theory [26], the lattice distortion d should be linearly dependent on sin2(t): d= K sin2(t)+ d0. Extrapolation towards zero tilt al-
Fig. 3. Left: Contour plots of the corrected X-ray intensities vs. the in-plane and the out-of-plane scattering vector components Qxy and Qz at different lateral pressures (indicated) at 15°C for compound 2. The inserts show the lattice and the tilt directions of the hydrocarbon chains. Right: Isotherm ( –) and molecular area ( ) calculated from the GIXD data for compound 2 at 15°C.
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Table 1 Lattice parameters calculated from the GIXD measurements Compound
T (°C)
y (mN m−1)
a (nm)
b (nm)
Axy (nm2)
t (°)
1
15
2
15
3
15
0 4 14.5 37 4 11 13 18 25 37 4 11 18 25 37
0.496 0.493 0.485 0.482 0.494 0.492 0.488 0.485 0.484 0.483 0.502 0.493 0.487 0.485 0.484
0.491 0.489 0.485 0.482 0.490 0.488 0.489 0.485 0.484 0.483 0.494 0.490 0.487 0.485 0.484
0.210 0.208 0.204 0.201 0.209 0.208 0.206 0.204 0.203 0.202 0.214 0.209 0.206 0.204 0.203
13.5 12.4 0 0 11.5 10.1 9.0 0 0 0 15.2 11.9 10.1 0 0
lows the separation of the two contributions. In Fig. 5, d is shown as a function of sin2(t). For all samples, the expected linear correlation has been approved. The introduction of methyl-groups on going from 1 to 2 and 3 induces a pronounced change in the slope and offset d0. The d0 values of compounds 2 and 3 deviate slightly from zero, whereas d0 of compound 1 is practically zero. This indicates that there is a small contribution of backbone ordering to the distortion of 2 and 3.
3.2. Lipid dispersions
sharp reflection in the SAXS and a symmetric gel peak in the WAXS are obtained corresponding to spacings of 6.12 and 0.427 nm, respectively (Fig. 6 bottom, left). The absence of higher order reflections for lamellar gel phases of PEs has also been observed previously in investigations of triplechain PEs with shorter acyl chains [27] and for the double-chain DMPE [28]. The sharp first order reflection and the absence of higher order peaks indicate a poor correlation between the layers but a good ordering within one layer. The gel peak is symmetric suggesting an orientation of the chains
The lipid/water dispersions have been characterized in the water saturated two-phase region (60 wt.% water). In the DSC measurements, compound 1 shows a main transition temperature of 48.2°C. With increasing degree of methylation, a shift towards higher Tm-values is observed (2: 50.5°C; 3: 50.9°C, for additional data see Table 2). Besides the main-transition, no further transitions have been obtained up to 95°C. For all compounds was shown that the main transition is reversible, however, a large hysteresis of about 13°C for the PE (1) and of about 10°C for compounds 2 and 3 was obtained. The X-ray diffraction measurements were performed between 10 and 95°C. Fig. 6 summarizes the results obtained for compound 1. Only one
Fig. 4. 1/cos(t) as function of the lateral pressure y for compounds 1 ( ), 2 () and 3 ( ) at 15°C. t is the tilt angle of the hydrocarbon chains.
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Fig. 5. The unit cell distortion d vs. sin2(t) for compounds 1 ( ), 2 () and 3 ( ) at 15°C. t is the tilt angle of the hydrocarbon chains.
normal to the bilayer plane. Therefore, it can be assumed that below Tm compound 1 exhibits a lamellar Lb phase. The cross-sectional area of the chains amounts to 0.209 nm2. Between 25 and 49°C, the spacings decrease with increasing temperature starting from 6.19 to 6.06 nm. Above the main transition, the diffraction profile is changed. The WAXS displays a broad scattering from molten chains with a maximum position at 2.27 nm − 1 that corresponds to a cross-sectional area of about 0.223 nm2. In the SAXS, a broad reflection with low intensity occurs. The peak width is six times larger than that observed below Tm. The temperature dependency of the d-values remains almost linear with a negative slope of about 0.0076 nm °C − 1. For compound 2 a comparable scattering profile was obtained. Below Tm, the maximum
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position of the first order reflection in the SAXS is shifted towards lower values (0.147 nm − 1 at 45°C). Thus, the introduction of the methyl-group in the hydrophilic area of the molecule results in remarkably increased d-spacings by approximately 0.8 nm. At 45°C, spacings of about 6.8 nm are obtained. The gel peak (WAXS) is again symmetric and has a high intensity as observed for compound 1, indicating non-tilted chains in an Lb gel phase. Above Tm, an La-phase with spacings between 5.38 nm (53°C) and 4.89 nm (93°C) occurs (Fig. 7). The dimethyl derivative 3 displays a changed phase sequence. Fig. 8 shows the d-spacings as function of temperature and typical diffraction patterns obtained below and above Tm. First exposures taken below Tm revealed two reflections in the SAXS corresponding to a lamellar gel phase with almost constant spacings of about 7 nm. At 15°C, a sharp reflection at 2.4 nm in the WAXS indicates a non-tilted b-conformation of the chains. On going to higher temperatures, this peak shifts towards lower values (2.34 nm at 49°C). Therefore, the cross-sectional area increases from 0.200 nm2 (15°C) to 0.211 nm2 (49°C). Above the phase transition, three reflections of the typical hexagonal diffraction pattern (a=2d/ 3 = 6.35 nm at 54°C) are obtained. The WAXS displays the diffuse scattering from molten chains. Since the head group is small compared to the large hydrophobic area an inverse hexagonal phase (HII) must be assumed. Increasing temperature leads to a shift of the peak positions towards lower values leading to a lattice constant of 6.04 nm at a high temperature of 93°C.
4. Discussion Table 2 Main phase transition temperatures (Tm), enthalpies (DH) and entropies (DS) for aqueous dispersions (60 wt.%) of compounds 1–3 Compound
Tm (°C)
DH (kJ mol−1)
DS (Jmol−1 K−1)
1 2 3
48.2 50.5 50.9
49.3 57.9 52.5
153 179 156
The Tm-values of double-chain phospholipids decrease with increasing degree of head group methylation [11,12]. In contrast, triple-chain lipids with a slightly shorter main-chain revealed an increase in the Tm values on going from the PE to the PC head group [13]. The same behavior was found in this work for the triple-chain lipids with a longer main-chain. For compounds 1 and 2, that exhibit the same phase sequence, the transi-
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Fig. 6. Top: Long spacings (d) of the lamellar gel (Lb) and lamellar liquid-crystalline (La) phases obtained for compound 1 as a function of temperature. Bottom: Typical small- and wide-angle (insert) X-ray diffraction patterns at 42.2°C (left) and 53.1°C (right).
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Fig. 7. Long spacings (d) of the lamellar gel (Lb) and lamellar liquid-crystalline (La) phases obtained for compound 2 as a function of temperature.
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tion enthalpy also increases with increasing methylation of the head group. Three chains connected to the glycerol backbone require more space compared to the small PE head group. Therefore, from the molecular shape concept one would expect the existence of an inverse hexagonal phase above the main transition. However, up to 95°C only a lamellar liquid-crystalline phase was obtained. Both compounds 1 and 2 show a broad reflection of weak intensity in the La phase, but sharp reflections in the gel-state. The comparison of the area requirements of the hydrophobic and hydrophilic parts leads to the assumption of interactions between the head groups from adja-
Fig. 8. Top: Long spacings (d) of the lamellar gel (Lb) and lattice constants (a) of the hexagonal phases obtained for compound 3 as a function of temperature. Bottom: Typical small- and wide-angle (insert) X-ray diffraction patterns at 45.3°C (left) and 55.1°C (right) are given.
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cent layers. A model of interdigitated head groups must be assumed at least for compound 1 which has a much smaller d-value in the gel phase. The head groups interdigitate and form a hydrogen-bonding network that remains upon heating above the main transition. This model has been proposed previously based on investigations of triple chain PEs with an equal but shorter acyl chain length of n = 16 and is confirmed by the results obtained on compound 1. Increasing flexibility and larger area requirements of the molten chains result in a breakdown of the lamellar structure into fragments. This argumentation is supported by the small coherence length observed in these lamellar systems. Assuming an exponential decay of the coherence length as known from liquid crystals, values of about 60 nm above Tm and at least 500 nm in the gel state can be estimated. This interdigitation should be responsible for the inverse behavior of Tm as a function of methylation compared to the double-chain lipids. In the case of compound 1, the system is not so flexible due to the stronger hydrogen bonds formed between the head groups what leads to the slightly lower transition temperature and enthalpy compared to 2. In the case of compound 2, it is also possible that the head group interdigitation occurs at the same time as the chain melting. This model is supported by the larger d-value in the gel phase, the comparison with the Tc value in monolayers (see below) and the occurrence of an La-phase. Two methyl groups are necessary for the appearance of the expected hexagonal phase. With increasing head-group methylation, the size of the hydrophilic part is increased. Additionally, the possibility to form a hydrogen-bonding network between adjacent head groups is restricted (compound 2) or impossible (compound 3). Therefore, in the case of compound 3 an interdigitation of the head groups is less favorable. The larger d-spacings in the gel phase also point to the formation of bilayers with non-interdigitated head groups. At this point, it should be noted that in case of the PC head group hexagonal phases have been obtained for triple chain analogs [26]. Therefore, it must be assumed that
in the case of PEs and monomethyl-PEs with large hydrophobic areas lamellar phases are stabilized by specific head-group interactions. The analysis of the isotherm measurements for compound 2 resulted in a critical temperature that is in the range of the main transition temperature of the lipid water dispersion. For compound 1 previous investigations revealed a Tc value of 64°C, which is much larger than Tm obtained in the DSC experiments. These findings suggest that the arrangement in condensed monolayers and gel phase bilayers is similar in the case of compound 2 and completely different in the case of compound 1 (interdigitated head groups). The transition pressure to the non-tilted phase increases continuously with increasing degree of methylation indicating that the increased head group size has also an influence on the chain packing in the condensed monolayers.
Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft (Emmy-Noether-Program grant: BR 1826/2-1 and BR 1378/6-1). We gratefully acknowledge the help with the settingup of the X-ray experiments by Kristian Kjaer and Gert Rapp and thank HASYLAB for providing very generous access to the experimental facilities.
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Chemically modified lipids — a suitable tool to study molecular interactions in model systems F. Bringezu a,*, G. Brezesinski b a
Department of Chemical Engineering, Eng. II, Uni6ersity of California Santa Barbara, Santa Barbara, CA 93106 -50580, USA b Max Planck Institute of Colloids and Interfaces, Am Muehlenberg 1, D-14476 Golm/Potsdam, Germany
Abstract The polymorphism of triple-chain glycerophosphoethanolamines differing in the degree of methylation of the head group has been investigated in both monolayers at the air/water interface and in multilayers of the lipid/water dispersions by means of film balance measurements, Grazing incidence X-ray diffraction (GIXD), differential scanning calorimetry (DSC) and simultaneous small- and wide-angle X-ray diffraction, respectively. Despite the larger area requirements of the three chains compared to the smaller head group, the change in the degree of methylation affects the thermodynamical data and structures formed in mono- and multilamellar systems. Increasing methylation leads to larger tilt angles of the hydrocarbon chains in monolayers at low lateral pressures and shifts the pressure for the tilting transition from NN tilted centered rectangular to untilted hexagonal phases towards larger values. In the lipid/water dispersions, the change in head group methylation is connected with a change of the phase sequence. For the phosphatidylethanolamines (PE) and the monomethyl derivative a stabilization of lamellar liquid-crystalline phases by interdigitation of adjacent head groups was found, whereas the introduction of a second methyl group results in the appearance of an HII phase. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Glycerophosphoethanolamines; X-ray diffraction; Methylation
1. Introduction Due to their importance as major components of biological membranes, the self-organization of lipids in both monolayers at the air/water interface and multilayers in aqueous dispersions has gained great scientific interest. One approach of
* Corresponding author. Tel.: + 1-805-8934325; fax: + 1805-8934731. Present address: Institute of Biophysics and Xray Research, Schmiedlstr. 6, Graz, A-8042, Austria. E-mail address: [email protected] (F. Bringezu).
systematic studies in those systems uses chemically modified lipids providing information about competitive interactions between hydrophilic and hydrophobic moieties of the amphiphiles. The main goal of such experiments is to get a better understanding of the relation between chemical structure and lipid polymorphism [1–4]. The modifications have been made in different parts of the molecule. Recently, the influence of branches in the acyl chains on the lipid polymorphism has gained our interest because long-chain branches enable a systematic change of the area requirements of the two molecule parts without changing
0927-7757/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 7 - 7 7 5 7 ( 0 1 ) 0 0 5 2 2 - 2
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the ‘chemical character’ of the environment [5,6]. On the other hand, chemically modified phospholipids are interesting substrates for phospholipases. It is known that the substrate properties have a strong influence on the activity of phospholipases [7,8]. The study of phospholipids with defined chemical modifications should help to understand the hydrolysis mechanism and to find potential inhibitors [9,10]. Although much is known about the phase behavior of multiple-chain phosphatidylethanolamines (PE) and phosphatidylcholines (PC), relatively few studies on the N-methylated intermediates have been published [11,12]. In previous studies on monolayers of triple-chain lipids, it was shown that despite the large hydrophobic area specific interactions between the head groups must be taken into account [13,14]. The present work is focused on the phase behavior of triple chain phospholipids in monolayers and aqueous dispersions. The degree of the head group methylation has been changed leading to new substances with differences in polarity of the head group, ability to form a hydrogen-bonding network and in the specific area requirements which influence the competitive interaction between hydrophobic and hydrophilic parts of these molecules. The systems were investigated by means of differential scanning calorimetry (DSC), small- and wide-angle X-ray diffraction and Grazing incidence X-ray diffraction (GIXD).
2. Experimental
2.1. Materials For the synthesis of the branched-chain PEs, 2-alkyl branched fatty acids have been prepared
according to [15] via the alkyl malonic acid esters. The structures of the acids were confirmed by mass spectrometry. The melting points were found to be in agreement with the literature data [16,17]. The fatty acids were transformed into their anhydrides using dicyclohexylcarbodiimide (Merck). The acylation of 1-O-benzyl-2-O-hexadecyl-glycerol in presence of 4,4%-dimethylaminopyridine as a catalyst revealed the corresponding racemic 1-O-benzyl-2-O-hexadecyl-3acyl-glycerols. From these derivatives, the benzyl-blocking group was removed and the resulting primary hydroxy-function was then phosphorylated using 2-bromoethyl-phosphoric acid dichloride [18]. The conversion of the bromo-esters using ammonia, monomethyl- and dimethylammonia saturated ethanol at room temperature revealed the final products (see Fig. 1 for chemical structures), which have been purified by column chromatography using methanol/chloroform/water/ammonia gradients for elution. Compounds 1 –3 were characterized by mass spectrometry, high performance liquid chromatography, and microanalysis. Selected analytical data: rac. 1(2-hexadecyloctadecanoyl)-2-O-hexadecylglycerophosphoethanolamine (1): 1(2C16-18:0)-2H-PE; C55H112N1O7P1; M= 930.47 g mol − 1, [M]+ = 931.1; FP = 192–193°C; micro analysis: (% calc./ found) N: 1.51/1.57, C: 71.00/71.22, H: 12.13/ 12.19 rac. 1(2-hexadecyloctadecanoyl)-2-O-hexadecylglycerophospho-N-methyl-ethanolamine (2): 1(2C16-18:0)-2H-PE(Me); C56H114N1O7P1; M= 944.50 g mol − 1, [M]+ = 944.9; FP = 78–80°C; micro analysis: (% calc./found) N: 1.48/1.44, C: 71.21/71.01, H: 11.86/12.04
Fig. 1. Chemical structure of the triple-chain lipids investigated (x =3, compound 1; x = 2, compound 2; x = 1, compound 3).
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rac. 1(2-hexadecyloctadecanoyl)-2-O-hexadecylglycerophospho-N,N-dimethyl-ethanolamine (3): C57H116N1O7P1; 1(2C16-18:0)-2H-PE(Me)2; −1 + M = 958.52 g mol , [M] =958.2; FP = 106.5– 107°C; micro analysis: (% calc./ found) N: 1.46/1.42, C: 71.73/70.83, H: 12.20/ 12.04
2.2. Methods The thermotropic phase behavior in the water saturated two-phase region (60 wt.% water) was characterized by DSC using a DSC-2 (PerkinElmer, Norfolk, CT). For sample preparation, all lipids were dried in a vacuum oven for 2 h at 50°C before weighing. The samples (3– 5 mg) were transferred into aluminum pans and mixed with water before closing. Multilamellar dispersions were formed during the equilibration above the main-transition temperature (Tm) for 1 h. An empty pan was used as reference. The data were recorded through an interface to a PC. After polynomial baseline fit the transition enthalpies and main-transition temperatures have been determined. The amount of strongly bound water was quantitatively calculated from the ice peak resulting from the melting of frozen excess water not bound to the head groups [19]. X-ray measurements of the lipid-water dispersions were carried out at the X13 double focusing monochromator-mirror camera of the EMBL outstation at DESY, Hamburg, Germany, using a monochromatic X-ray beam (u =0.15 nm). The diffracted intensities in the small- and wide-angle regions were simultaneously recorded using a data acquisition system described in [20]. The beamline setup has been described in detail elsewhere [21]. The reciprocal spacings s= 1/d were calibrated by the diffraction pattern of dry rat-tail collagen with a long spacing of 65 nm (SAXS) and p-bromobenzoic acid (WAXS). For sample preparation, weighed amounts of the lipid were dispersed in ultra-pure water (60% w/w). The dispersions were vortexed and then transferred into glass capillaries (d= 1 mm) (Hilgenberg, Malsfeld, Ger-
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many), which were placed in the temperature controlled sample holder. Initially static exposures have been undertaken in order to gain information about the structures formed after sample preparation. The experiments were continued with temperature scans (1°C min − 1) between 5 and 95°C. Static exposures were taken below and above the main-transition and compared between the cycles to ensure the sample quality and exclude possible radiation damage. The monolayers were spread from an 1 mM p.a. grade chloroform solution onto an ultra pure water subphase. The water was purified using a Millipore desktop unit. The pressure-area isotherms were recorded with a Wilhelmy film balance. For the Synchrotron measurements the trough is located in a He-filled, sealed container. The X-ray experiments were performed using the liquid-surface diffractometer on the undulator beamline BW1 at HASYLAB, DESY, Hamburg (Germany). The experimental set-up and evaluation procedures have been described in detail elsewhere [22,23]. The Synchrotron beam was made monochromatic by Bragg reflection on a beryllium (002) crystal and was adjusted to strike the monolayer on the water surface at an angle of incidence hi = 0.85hc, where hc is the critical angle of total external reflection. The intensity of the diffracted radiation is detected by a position sensitive detector (PSD) (OED-100-M, Braun, Garching, Germany). The resolution of the horizontal scattering angle 2Uhor is given by a Soller collimator located in front of the PSD. The scattering vector Q has an in-plane component Qxy :(4y/ u) sin(2Uhor/2) and an out-of-plane component Qz : (2y/u) sin hf, where u is the X-ray wave length and hf the vertical scattering angle. The accumulated position-resolved scans were corrected for polarization, footprint-area, and powder averaging (Lorentz factor). Model peaks taken to be Lorentzian in the in-plane direction and Gaussian in the out-of-plane direction were fitted to the corrected intensities. The lattice spacings are obtained from the in-plane diffraction. The lattice parameters can be calculated from the lattice spacings and the unit cell area Axy is calculated from the lattice parameters. The Bragg rods
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Fig. 2. Surface pressure –molecular area isotherms of compound 2 at temperatures indicated. The insert shows both the transition enthalpy DH(p), calculated from the area change DA during the first order transition, and the transition pressure (yc) ( ) as function of temperature.
additionally yield the chain tilt angle t and the tilt azimuth.
3. Results
3.1. Lipid monolayers At the air/water interface, compounds 1–3 form stable monolayers. Fig. 2 displays the temperature dependency of compound 2 recorded between 10 and 40°C. Below 30°C the isotherm is fully condensed, whereas at 30°C a plateau indicates a first order transition between a liquid-expanded (LE) and a condensed state (LC). Both, the transition pressure (yc) and the area change DA involved in the transition show the typical linear temperature dependency (see insert Fig. 2). The linear extrapolation of yc towards zero yields the temperature T0 above which the LE phase
occurs (T0 = 26.5°C). The transition enthalpy DH can be calculated by a Clausius–Clapeyron equation using the area change DA [24]. The extrapolation of DH towards zero yields the critical temperature Tc above which the monolayer cannot be compressed into a condensed state (Tc = 51.3°C). GIXD measurements were applied in order to investigate the structures of the condensed phases. Fig. 3 (left) shows the intensity profile of the diffracted beam for compound 2 at 15°C. At low lateral pressures, two low order diffraction peaks (the degenerated (1, 91) above the horizon and the non-degenerated (0, 2) at the horizon) of a centered rectangular unit cell are observed. Such an intensity distribution indicates tilted hydrocarbon chains with a tilt direction towards the nearest neighbor (NN) along the short axis of the unit cell (see inserts). Increasing pressure leads to a phase transition towards a hexagonal phase exhibiting only one diffraction peak with a Bragg rod maximum at the horizon, hence the chains are vertically oriented. This tilting transition can also be seen in the isotherm (Fig. 3 (right)) which shows a change in slope at about 17 mN m − 1. Compounds 1 and 3 show similar scattering profiles with the same type of phase transition. The calculated lattice parameters are presented in Table 1. The in-plane area (Axy ) calculated from the X-ray results as a function of the lateral pressure is also presented in Fig. 3 (right). The Axy values are in good agreement with the measured isotherm, hence the monolayer contains only a small number of defects. The condensed isotherms show a linear dependency between lateral pressure and molecular area: Axy = K1 − K2y, where Axy = A0/cos(t) with A0 as the cross-sectional area of the chains and t as the chain tilt angle. Therefore, the extrapolation of the plot of 1/cos(t)= f(y) to zero tilt gives the value of the tilting transition pressure. Fig. 4 shows this relation for compounds 1–3. The PE (1) shows a transition pressure of approximately 14.0 mN m − 1. The successive methylation of the head group results in a shift towards larger values. For compound 2, a value of 18.0 mN m − 1
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was obtained, whereas compound 3 gives 20.5 mN m − 1. The slope of the linear function increases with increasing head-group methylation. Therefore, the possible differences in the chain tilt are more pronounced at low lateral pressures. The extrapolation towards zero pressure yields a chain tilt of approximately 13.6° for the PE (1), 15.5° for compound 2 and 16.9° for compound 3. Despite the large hydrophobic area that should determine the structure of the condensed phases, interactions between the head-groups must be obviously taken into account. The extended hydrophobic area should provide sufficient space for the head-groups. However, the data indicate that the chemical structure of the head-groups has a pronounced influence on the chain structure investigated by GIXD. On the one hand, the systematic introduction of methyl units increases the size of
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the head-groups continuously, but in any case, the three hydrophobic chains occupy a much larger area. On the other hand, specific interactions between adjacent lipid molecules must be taken into account, which should be responsible for the variation found in the chain lattice (tilt angles) and thermodynamical data (transition pressures). Compared to the hexagonal phase of upright oriented chains, the centered rectangular unit cell found at low lateral pressures is distorted. The direction of the distortion d coincides with the NN tilt direction. Investigations on monolayers of single-chain molecules (for review see [25]) revealed two sources of the unit cell distortion: the chain tilt and backbone ordering. According to the Landau theory [26], the lattice distortion d should be linearly dependent on sin2(t): d= K sin2(t)+ d0. Extrapolation towards zero tilt al-
Fig. 3. Left: Contour plots of the corrected X-ray intensities vs. the in-plane and the out-of-plane scattering vector components Qxy and Qz at different lateral pressures (indicated) at 15°C for compound 2. The inserts show the lattice and the tilt directions of the hydrocarbon chains. Right: Isotherm ( –) and molecular area ( ) calculated from the GIXD data for compound 2 at 15°C.
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Table 1 Lattice parameters calculated from the GIXD measurements Compound
T (°C)
y (mN m−1)
a (nm)
b (nm)
Axy (nm2)
t (°)
1
15
2
15
3
15
0 4 14.5 37 4 11 13 18 25 37 4 11 18 25 37
0.496 0.493 0.485 0.482 0.494 0.492 0.488 0.485 0.484 0.483 0.502 0.493 0.487 0.485 0.484
0.491 0.489 0.485 0.482 0.490 0.488 0.489 0.485 0.484 0.483 0.494 0.490 0.487 0.485 0.484
0.210 0.208 0.204 0.201 0.209 0.208 0.206 0.204 0.203 0.202 0.214 0.209 0.206 0.204 0.203
13.5 12.4 0 0 11.5 10.1 9.0 0 0 0 15.2 11.9 10.1 0 0
lows the separation of the two contributions. In Fig. 5, d is shown as a function of sin2(t). For all samples, the expected linear correlation has been approved. The introduction of methyl-groups on going from 1 to 2 and 3 induces a pronounced change in the slope and offset d0. The d0 values of compounds 2 and 3 deviate slightly from zero, whereas d0 of compound 1 is practically zero. This indicates that there is a small contribution of backbone ordering to the distortion of 2 and 3.
3.2. Lipid dispersions
sharp reflection in the SAXS and a symmetric gel peak in the WAXS are obtained corresponding to spacings of 6.12 and 0.427 nm, respectively (Fig. 6 bottom, left). The absence of higher order reflections for lamellar gel phases of PEs has also been observed previously in investigations of triplechain PEs with shorter acyl chains [27] and for the double-chain DMPE [28]. The sharp first order reflection and the absence of higher order peaks indicate a poor correlation between the layers but a good ordering within one layer. The gel peak is symmetric suggesting an orientation of the chains
The lipid/water dispersions have been characterized in the water saturated two-phase region (60 wt.% water). In the DSC measurements, compound 1 shows a main transition temperature of 48.2°C. With increasing degree of methylation, a shift towards higher Tm-values is observed (2: 50.5°C; 3: 50.9°C, for additional data see Table 2). Besides the main-transition, no further transitions have been obtained up to 95°C. For all compounds was shown that the main transition is reversible, however, a large hysteresis of about 13°C for the PE (1) and of about 10°C for compounds 2 and 3 was obtained. The X-ray diffraction measurements were performed between 10 and 95°C. Fig. 6 summarizes the results obtained for compound 1. Only one
Fig. 4. 1/cos(t) as function of the lateral pressure y for compounds 1 ( ), 2 () and 3 ( ) at 15°C. t is the tilt angle of the hydrocarbon chains.
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Fig. 5. The unit cell distortion d vs. sin2(t) for compounds 1 ( ), 2 () and 3 ( ) at 15°C. t is the tilt angle of the hydrocarbon chains.
normal to the bilayer plane. Therefore, it can be assumed that below Tm compound 1 exhibits a lamellar Lb phase. The cross-sectional area of the chains amounts to 0.209 nm2. Between 25 and 49°C, the spacings decrease with increasing temperature starting from 6.19 to 6.06 nm. Above the main transition, the diffraction profile is changed. The WAXS displays a broad scattering from molten chains with a maximum position at 2.27 nm − 1 that corresponds to a cross-sectional area of about 0.223 nm2. In the SAXS, a broad reflection with low intensity occurs. The peak width is six times larger than that observed below Tm. The temperature dependency of the d-values remains almost linear with a negative slope of about 0.0076 nm °C − 1. For compound 2 a comparable scattering profile was obtained. Below Tm, the maximum
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position of the first order reflection in the SAXS is shifted towards lower values (0.147 nm − 1 at 45°C). Thus, the introduction of the methyl-group in the hydrophilic area of the molecule results in remarkably increased d-spacings by approximately 0.8 nm. At 45°C, spacings of about 6.8 nm are obtained. The gel peak (WAXS) is again symmetric and has a high intensity as observed for compound 1, indicating non-tilted chains in an Lb gel phase. Above Tm, an La-phase with spacings between 5.38 nm (53°C) and 4.89 nm (93°C) occurs (Fig. 7). The dimethyl derivative 3 displays a changed phase sequence. Fig. 8 shows the d-spacings as function of temperature and typical diffraction patterns obtained below and above Tm. First exposures taken below Tm revealed two reflections in the SAXS corresponding to a lamellar gel phase with almost constant spacings of about 7 nm. At 15°C, a sharp reflection at 2.4 nm in the WAXS indicates a non-tilted b-conformation of the chains. On going to higher temperatures, this peak shifts towards lower values (2.34 nm at 49°C). Therefore, the cross-sectional area increases from 0.200 nm2 (15°C) to 0.211 nm2 (49°C). Above the phase transition, three reflections of the typical hexagonal diffraction pattern (a=2d/ 3 = 6.35 nm at 54°C) are obtained. The WAXS displays the diffuse scattering from molten chains. Since the head group is small compared to the large hydrophobic area an inverse hexagonal phase (HII) must be assumed. Increasing temperature leads to a shift of the peak positions towards lower values leading to a lattice constant of 6.04 nm at a high temperature of 93°C.
4. Discussion Table 2 Main phase transition temperatures (Tm), enthalpies (DH) and entropies (DS) for aqueous dispersions (60 wt.%) of compounds 1–3 Compound
Tm (°C)
DH (kJ mol−1)
DS (Jmol−1 K−1)
1 2 3
48.2 50.5 50.9
49.3 57.9 52.5
153 179 156
The Tm-values of double-chain phospholipids decrease with increasing degree of head group methylation [11,12]. In contrast, triple-chain lipids with a slightly shorter main-chain revealed an increase in the Tm values on going from the PE to the PC head group [13]. The same behavior was found in this work for the triple-chain lipids with a longer main-chain. For compounds 1 and 2, that exhibit the same phase sequence, the transi-
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Fig. 6. Top: Long spacings (d) of the lamellar gel (Lb) and lamellar liquid-crystalline (La) phases obtained for compound 1 as a function of temperature. Bottom: Typical small- and wide-angle (insert) X-ray diffraction patterns at 42.2°C (left) and 53.1°C (right).
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Fig. 7. Long spacings (d) of the lamellar gel (Lb) and lamellar liquid-crystalline (La) phases obtained for compound 2 as a function of temperature.
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tion enthalpy also increases with increasing methylation of the head group. Three chains connected to the glycerol backbone require more space compared to the small PE head group. Therefore, from the molecular shape concept one would expect the existence of an inverse hexagonal phase above the main transition. However, up to 95°C only a lamellar liquid-crystalline phase was obtained. Both compounds 1 and 2 show a broad reflection of weak intensity in the La phase, but sharp reflections in the gel-state. The comparison of the area requirements of the hydrophobic and hydrophilic parts leads to the assumption of interactions between the head groups from adja-
Fig. 8. Top: Long spacings (d) of the lamellar gel (Lb) and lattice constants (a) of the hexagonal phases obtained for compound 3 as a function of temperature. Bottom: Typical small- and wide-angle (insert) X-ray diffraction patterns at 45.3°C (left) and 55.1°C (right) are given.
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cent layers. A model of interdigitated head groups must be assumed at least for compound 1 which has a much smaller d-value in the gel phase. The head groups interdigitate and form a hydrogen-bonding network that remains upon heating above the main transition. This model has been proposed previously based on investigations of triple chain PEs with an equal but shorter acyl chain length of n = 16 and is confirmed by the results obtained on compound 1. Increasing flexibility and larger area requirements of the molten chains result in a breakdown of the lamellar structure into fragments. This argumentation is supported by the small coherence length observed in these lamellar systems. Assuming an exponential decay of the coherence length as known from liquid crystals, values of about 60 nm above Tm and at least 500 nm in the gel state can be estimated. This interdigitation should be responsible for the inverse behavior of Tm as a function of methylation compared to the double-chain lipids. In the case of compound 1, the system is not so flexible due to the stronger hydrogen bonds formed between the head groups what leads to the slightly lower transition temperature and enthalpy compared to 2. In the case of compound 2, it is also possible that the head group interdigitation occurs at the same time as the chain melting. This model is supported by the larger d-value in the gel phase, the comparison with the Tc value in monolayers (see below) and the occurrence of an La-phase. Two methyl groups are necessary for the appearance of the expected hexagonal phase. With increasing head-group methylation, the size of the hydrophilic part is increased. Additionally, the possibility to form a hydrogen-bonding network between adjacent head groups is restricted (compound 2) or impossible (compound 3). Therefore, in the case of compound 3 an interdigitation of the head groups is less favorable. The larger d-spacings in the gel phase also point to the formation of bilayers with non-interdigitated head groups. At this point, it should be noted that in case of the PC head group hexagonal phases have been obtained for triple chain analogs [26]. Therefore, it must be assumed that
in the case of PEs and monomethyl-PEs with large hydrophobic areas lamellar phases are stabilized by specific head-group interactions. The analysis of the isotherm measurements for compound 2 resulted in a critical temperature that is in the range of the main transition temperature of the lipid water dispersion. For compound 1 previous investigations revealed a Tc value of 64°C, which is much larger than Tm obtained in the DSC experiments. These findings suggest that the arrangement in condensed monolayers and gel phase bilayers is similar in the case of compound 2 and completely different in the case of compound 1 (interdigitated head groups). The transition pressure to the non-tilted phase increases continuously with increasing degree of methylation indicating that the increased head group size has also an influence on the chain packing in the condensed monolayers.
Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft (Emmy-Noether-Program grant: BR 1826/2-1 and BR 1378/6-1). We gratefully acknowledge the help with the settingup of the X-ray experiments by Kristian Kjaer and Gert Rapp and thank HASYLAB for providing very generous access to the experimental facilities.
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