Temperature-Dependent Phase Behavior and the Crystal-Forming

Institut für Biochemie der Universität Münster, Wilhelm-Klemm Strasse 2, and ... R. Mallouri , A.D. Keramidas , G. Brezesinski , E. Leontidis. Jour...
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Langmuir 2007, 23, 1880-1887

Temperature-Dependent Phase Behavior and the Crystal-Forming Nucleation Process of Ethyl 4-Fluoro-2,3-dihydroxystearate Monolayers Silke Steffens,† Udo Ho¨weler,‡,# Tim Jo¨dicke,# Jens Oldendorf,‡ Rainer Rudert,§ Gu¨nter Haufe,*,‡ and Hans-Joachim Galla*,† Institut fu¨r Biochemie der UniVersita¨t Mu¨nster, Wilhelm-Klemm Strasse 2, and Organisch-Chemisches Institut der UniVersita¨t Mu¨nster, Corrensstrasse 40, D-48149 Mu¨nster, Germany, CHEOPS, Zur Quelle 6, D-48341 Altenberge, Germany, and Federal Institute for Materials and Research and Testing, Unter den Eichen 44-46, D-12203 Berlin, Germany ReceiVed August 15, 2006. In Final Form: NoVember 13, 2006 The phase behavior of enantiomeric compounds as well as mixtures of enantiopure and racemic diastereomers of ethyl 4-fluoro-2,3-dihydroxystearates has been investigated using surface pressure-area isotherms and Brewster angle microscopy (BAM). All mixtures exhibit a small plateau region within the surface pressure-area isotherm at 20 °C, whereas the enantiopure compound shows an isotherm behavior similar to that of fatty acids. Corresponding to the film balance measurements, the BAM images demonstrate different shapes of the domains within the coexistence region of the liquid-condensed/liquid-expanded phase. The domain structures of the monolayers were visualized after Langmuir-Blodgett transfer on mica sheets by scanning force microscopy (SFM). From the SFM images it becomes obvious that small crystallites are formed for all investigated compounds; however, their molecular assembly is diverse for different enantiomers. Variations in the phase behavior can be correlated with interactions between the polar molecular moieties and the subphase and altered intermolecular interactions. Molecular modeling calculations were applied to elucidate the structural organization of these intermolecular interactions. Ab initio calculations of the minima conformers of (S,S,R)- and (S,S,S)-ethyl 4-fluoro-2,3-dihydroxystearates have been performed to predict with the HARDPACK program the two-dimensional lattice structure based on the P1 space group. These calculations showed that intermolecular hydrogen bridges are crucial for the interactions within and between the molecules.

Introduction Noncovalent interactions play an important role in biologically relevant molecules, such as in the recognition process of enzymes with substrates or in biological membranes. These include hydrogen bonds,1 steric repulsion, and ionic,2 dipolar,3 hydrophobic, and van der Waals forces.4 Much information can be obtained about noncovalent interactions through the investigation of the phase behavior and the morphology of monolayer films. Therefore combinations of different physical techniques have been used such as film balance measurements combined with Brewster angle microscopy (BAM),5 grazing incidence X-ray diffraction,6 infrared reflection absorption spectroscopy,7 or scanning force microscopy (SFM) after Langmuir-Blodgett (LB) transfer.8 * To whom correspondence should be addressed. (H.-J.G.) Phone: +49-251-83 33200; fax: +49-251-83 33206, e-mail: [email protected]. (G.H.) Phone: +49-251-83 33281; fax: +49-251-83 39772; e-mail: [email protected]. † Institut fu ¨ r Biochemie der Universita¨t Mu¨nster. ‡ Organisch-Chemisches Institut der Universita ¨ t Mu¨nster. # CHEOPS. § Federal Institute for Materials and Research and Testing. (1) Kollmann, P. A.; Allen, L. C. Chem. ReV. 1972, 72, 283. (2) Shiloach, A.; Blankschtein, D. Langmuir 1998, 14, 7166. (3) Thirumoorthy, K.; Nandi, N.; Vollhardt, D. J. Phys. Chem. B 2005, 109, 10820. (4) (a) Sharp, K. A.; Nichols, A.; Fine, R. F.; Honig, B. Science 1991, 252, 106. (b) Widom, B.; Bhimalapuram, P.; Koga, K. Phys. Chem. Chem. Phys. 2003, 5, 3085. (5) (a) Ho¨nig, D.; Mo¨bius, D. J. Phys. Chem. 1991, 95, 4590. (b) He´non, S.; Meunier, J. ReV. Sci. Instrum. 1991, 62, 936. (6) Brezesinski, G.; Dietrich, A.; Struth, B.; Bo¨hm, C.; Bouwman, W. G.; Kjaer, K.; Mo¨hwald, H. Chem. Phys. Lipids 1995, 76, 145. (7) (a) Dluhy, R. A.; Cornell, D. G. J. Phys. Chem. 1985, 89, 3195. (b) Mendelsohn, R.; Brauner, J. W.; Gericke, A. Annu. ReV. Phys. Chem. 1995, 46, 305.

In the past few decades, these methods have also been applied to study amphiphiles intensively as model systems of the cell membrane. Another focus of interest herein is the influence of chirality on the membrane organization.9 Especially for this task, amphiphiles at the air/water interface provide a good tool since they are organized as simpler two-dimensional (2D) systems. Long-chain fatty acids and their esters are ideal candidates for a bipolar model system. In earlier studies, chiral bipolar stearate esters with two vicinal hydroxyl groups as well as different fluorohydrins were investigated as model components.10 Fix et al. demonstrated that, depending on the chirality, dihydroxylated stearic acid esters (erythro or threo) form different distinct hydrogen-bond networks. The model component used here is a chiral ethyl stearate that bears two hydroxyl groups vicinal to each other and neighbored by a fluorine atom. The fluorine atom is the most electronegative element in the periodic system of elements. Inserting a fluorine atom into a molecule changes the electron-density distribution, because the fluorine atom is only slightly bigger than a hydrogen atom and is both isosteric and isopolar to the hydroxyl group. For this reason, it is possible for the C-F group to act as a weak (8) (a) Zasadzinski, J. A. N.; Helm, C. A.; Longo, M. L; Weisenhorn, A. L.; Gould, S. A. C; Hansma, P. K. Biophys. J. 1991, 59, 755. (b) Birdi, K. S.; Vu, D. T. Langmuir 1994, 10, 623. (9) Nandi, N.; Vollhardt.; D. Chem. ReV. 2003, 103, 4033. (10) (a) Fix, M.; Sieber, M.; Overs, M.; Scha¨fer, H.-J.; Galla, H.-J. Phys. Chem. Chem. Phys. 2000, 2, 4515. (b) Fix, M.; Lauter, R.; Lo¨bbe, C.; Brezesinski, G.; Galla, H.-J. Langmuir 2000, 16, 8937. (c) Overs, M.; Fix, M.; Jacobi, S.; Chi, L. F.; Sieber, M.; Scha¨fer, H. J.; Fuchs, H.; Galla, H.-J. Langmuir 2000, 16, 1141. (d) Jacobi, S.; Chi, L. F.; Plate, M.; Overs, M.; Scha¨fer, H.-J.; Fuchs, H. Thin Solid Films 1998, 327, 180. (e) Chen, X.; Wiehle, S.; Weygand, M.; Brezesinski, G.; Klenz, U.; Galla, H.-J.; Fuchs, H.; Haufe, G.; Chi, L. J. Phys. Chem. B 2005, 109, 19866. (f) Fix, M.; Wiehle, S.; Haufe, G.; Galla, H.-J. Colloids Surf., A 2002, 198-200, 151.

10.1021/la062406g CCC: $37.00 © 2007 American Chemical Society Published on Web 01/04/2007

Phase BehaVior and Nucleation of EFDS Monolayers

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Scheme 1. Configurations of Investigated EFDSs in Different Mixtures of Racemic or Highly Enantioenriched Diastereomers

hydrogen-bond acceptor,11,12 although this topic has been discussed controversially recently.13,14 Particularly in biological systems, this type of interaction is discussed as being important for understanding the reasons responsible for different activities of various types of biomolecules and their fluorinated analogues.15 In the present paper, one main objective is to elucidate the effect of a single fluorine atom in the 4-position of ethyl 2,3dihydroxystearates16,17 on the phase behavior at the air/water interface in comparison with unfluorinated compounds. Moreover, the influence of enantiopure or racemic mixtures of diastereomers on self-organization at the air/water interface is investigated. Ethyl 4-fluoro-2,3-dihydroxystearates (EFDSs) were investigated as a new class of amphiphatic molecules at the air/water interface using film balance measurements. In order to gain more detailed information on 2D phase transformations and monolayer morphology, special attention was given to the difference between the enantiopure compounds and mixtures of enantiopure or racemic pairs of diastereomers, and BAM and SFM were applied. The phase behavior has been also investigated by temperaturedependent film balance measurements to quantify the influence of fluorine. Additionally, energy differences of the conformers of the (S,S,S) and (S,S,R) EFDSs have been calculated by an ab (11) (a) Howard, J. A. K.; Hoy, V. J.; O’Hagan, D.; Smith, G. T. Tetrahedron 1996, 52, 12613. (b) Smart, B. E. J. Fluorine Chem. 2001, 109, 3. (c) HylaKryspin, I.; Haufe, G.; Grimme, S. Chem.sEur. J. 2004, 10, 3411. (d) Fro¨hlich, R.; Rosen, T. C.; Meyer, O. G. J.; Rissanen, K.; Haufe, G. J. Mol. Struct. 2006, 787, 50. (12) Yamazaki, T.; Kitazume, T. Coordination ability of fluorine to proton or metals based on experimental and theoretical evidence. In Enantiocontrolled Synthesis of Fluoro-organic Compounds; Soloshonok, V. A., Ed.; Wiley: Chichester, UK, 1999; p 575. (13) (a) Huang, J.; Hedberg, K. J. Am. Chem. Soc. 1989, 111, 6909. (b) Dunitz, J. D.; Taylor, R. Chem.sEur. J. 1997, 3, 89. (c) Michel D.; Witschard, M.; Schlosser, M. Liebigs Ann. Chem. 1997, 517. (d) Thalladi, V. R.; Weiss, H.-C.; Bla¨ser, D.; Bose, R.; Nangia, A.; Desiraju, G. R. J. Am. Chem. Soc. 1998, 120, 8702. (e) Dunitz, J. D. ChemBioChem 2004, 5, 614. (14) Schlosser, M. The chemical and physiological size of fluorine. In Enantiocontrolled Synthesis of Fluoro-organic Compounds; Soloshonok, V. A., Ed.; Wiley: Chichester, UK, 1999; p 613. (15) (a) Welch, J. T.; Evarakrishnan, S. Fluorine in Bioorganic Chemistry; Wiley: New York, 1991. (b) Filler, R.; Kobayashi, Y., Yagupolskii, L. M., Eds.; Organofluorine Compounds in Medicinal Chemistry and Biomedical Applications; Elsevier: Amsterdam, 1993. (c) Bo¨hm, H.-J; Banner, D.; Bendels, M.; Kansy, M.; Kuhn, B.; Mu¨ller, K.; Obst-Sander, U.; Stahl, M. ChemBioChem 2004, 5, 637. (16) Oldendorf, J. Ph.D. Thesis, University of Mu¨nster, Germany, 2002. (17) Oldendorf, J.; Haufe, G. Eur. J. Org. Chem. 2006, 4463.

initio method, leading to model calculations of the 2D lattice structure based on the P1 space group. Materials and Methods Materials. The stereomeric EFDSs were synthesized as described elsewhere.16,17 The enantiomeric, diastereomeric, or racemic/ diastereomeric EFDSs investigated in this paper are presented in Scheme 1. The chemical purity of the compounds was proven by high-resolution mass spectrometry and elemental analysis; its relative stereochemistry as well as the enantiomeric purity of the examined amphiphiles was determined by 19F NMR experiments.17 Ultrapure water (pH 5.5) was obtained from a Millipore system (Millipore, Bedford, MA) comprising reverse osmosis, ion exchange, and 0.22 µm ultra filtration cartridges. Lipid stock solutions were prepared in chloroform (HPLC grade, Sigma-Aldrich, Steinheim, Germany). Isotherm Measurements. Surface pressure-area isotherms (or simply “π-A isotherms”) were obtained with film balances equipped with a Wilhelmy plate system allowing an accuracy of 0.1 mN/m. The total surface area was 175 cm2 (Riegler & Kirstein, Wiesbaden, Germany) or 690 cm2 (NIMA, Coventry, UK). Both troughs were temperature controlled. The temperature dependence measurements were made with a self-made cover that could be temperature controlled as well. Both the trough and the cover were connected via the same water circulation for temperature control. Monolayers were prepared by spreading appropriate chloroform solutions onto ultrapure water using a 25 µL syringe (ILS, Stu¨tzerbach, Germany). After 10 min of solvent evaporation, the monolayer was compressed with a velocity of 5.8 cm2/min (Riegler & Kirstein) or 10.0 cm2/min (NIMA). Brewster Angle Microscopy. The monolayers were visualized by a Brewster angle microscope (BAM 2Plus, Nanofilm Technology GmbH, Go¨ttingen, Germany) mounted on the NIMA trough. The light source of the BAM was a Nd:YAG laser. The laser beam was reflected at the air/water interface at the Brewster angle. The reflected beam was recorded with a CCD camera, and the data were digitally saved. The 10× objective of the microscope provided a diffractionlimited lateral resolution of approximately 2 µm. The size of the BAM images in this paper is 430 × 537 µm2. Langmuir-Blodgett Transfer. LB films were prepared by spreading a compound mixture dissolved in chloroform onto an ultrapure water subphase of a Wilhelmy film balance (Riegler & Kirstein, Mainz, Germany) with an operational area of 39 cm2. Prior to spreading, a cleaved mica sheet (Electron Microscopy Science, Munich, Germany) was dipped into the subphase. After an equilibration period of 10 min, the film was compressed with a

1882 Langmuir, Vol. 23, No. 4, 2007 velocity of 1.8 cm2/min to a defined surface pressure above the phase transition and transferred onto the mica sheet (0.64 mm/min) under almost constant surface pressure (∆π max ( 0.15 mN/m). Scanning Force Microscopy. Surface images of LB films were obtained at ambient conditions using a Nanoscope IIIa Dimension 3000 microscope from Digital Instruments (Santa Barbara, CA) operating in tapping mode. Silicon tips (BS-Tap 300, Nanoscience Instruments, Inc., Phoenix, AZ) with a resonance frequency of 250300 kHz were used. The mean height of the surface was analyzed by WSxM software (Nanotec Electronica, Barcelona, Spain). Relative heights with respect to the lowest area on the surface are determined from piezo displacement. 2D Lattice Model Calculations. We used the HARDPACK program18 to predict the best packing of the EFDS within the space group P1. The molecular structures of the head groups (C5) of the (S,S,R) and the (S,S,S)-EFDS were built with MOBY.19 For these models, a conformational analysis within MAXIMOBY20 using the AMBER force field21 resulted in 11 and 10 conformations for (S,S,R) and (S,S,S)-EFDS, respectively. These conformers were optimized at the RHF/6-31G**22 level with Gaussian 98. The remaining C14 alkyl chain was connected to each optimized head group in the best conformation for the all-trans arrangements. These rigid molecules were packed according to the P1 space group symmetry with the HARDPACK program. Only the energetically lowest packings out of 25 simulated annealing runs for a 3 × 3 grid using the 6-exp atomic parameter set were analyzed further. In order to check the validity of the results, all packings were generated with the simple Mulliken23 distribution, and the partial charges were fitted to the quantum mechanical electrostatic potential24 (ESP). The results were analyzed with respect to the sum of the ab initio energy of the isolated molecule and their HARDPACK packingenergy (electrostatic and van der Waals).

Results and Discussion Scheme 1 presents an overview of the synthesized compounds.16,17 An asymmetric Sharpless dihydroxylation with catalytic amounts of AD-mix β or AD-mix R of ethyl (E)-4fluorooctadec-2-enoate was used as a key step, giving two diastereomeric pairs of enantiomeric EFDSs (Scheme 1, routes A and B). The racemic compounds were prepared analogously using potassium permanganate. In all compounds, the hydroxyl functions in the 2- and 3-positions are in syn-configuration to each other, like in the threo form of sugars (Scheme 1, 1-4). Different mixtures of diastereomers 1-4 with R or S configuration of the fluorine in the 4-position were investigated in this study. The (R,R,S)/(R,R,R) (5) mixture is named RDIA and possesses a diastereomeric ratio of 69:31. From this mixture of diastereomers, one enantiopure EFDS named ENAN was separated by HPLC. The absolute configuration of this isomer could not be (18) Rudert, R. Acta Crystallogr., Sect. A 1996, 52 (Suppl.), C-94. (19) Ho¨weler, U. MOBY, version 2.3; Universita¨t Mu¨nster and CHEOPS: Altenberge, Germany, 2005. (20) Ho¨weler, U. MAXIMOBY, version 7.60; Universita¨t Mu¨nster and CHEOPS: Altenberge, Germany, 2004. (21) (a) Weiner, S. J.; Kollmann, P. A.; Case, D. A.; Singh, U. C.; Ghio, C.; Alagona, G.; Profeta, S., Jr.; Weiner, P. J. Am. Chem. Soc. 1984, 106, 765. (b) Weiner, S. J.; Kollmann, P. A.; Nguyen, D. T.; Case, D. A. J. Comput. Chem. 1986, 7, 230. (22) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Zakrzewski, V. G.; Montgomery, J. A.; Stratmann, R. E., Jr.; Burant, J. C.; Dapprich, S.; Millam, J. M.; Daniels, A. D.; Kudin, K. N.; Strain, M. C.; Farkas, O.; Tomasi, J.; Barone, V.; Cossi, M.; Cammi, R.; Mennucci, B.; Pomelli, C.; Adamo, C.; Clifford, S.; Ochterski, J.; Petersson, G. A.; Ayala, P. Y.; Cui, Q.; Morokuma, K.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Cioslowski, J.; Ortiz, J. V.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Gomperts, R.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Gonzalez, C.; Challacombe, M.; Gill, P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Andres, J. L.; Gonzalez, C. Head-Gordon, M.; Replogle, E. S.; Pople, J. A. Gaussian 98, revision A.6, Gaussian, Inc.: Pittsburgh, PA, 1998. (23) (a) Singh, U. C.; Kollman, P. A. J. Comput. Chem. 1984, 5, 129. (b) Besler, B. H.; Merz, K. M., Jr.; Kollman, P. A. J. Comput. Chem. 1990, 11, 431. (24) Mulliken, R. S. J. Chem. Phys. 1955, 23, 1833.

Steffens et al.

Figure 1. Surface pressure-area isotherms of different mixtures of EFDSs in ultrapure water at 20 °C.

determined. This compound was used for comparison with the mixture of diastereomers. SDIA (6) is a 67:33 mixture of (S,S,R)/ (S,S,S)-diastereomers. All compounds within the diastereomeric mixtures were practically enantiopure (>98% ee). By dihydroxylation of the R,β-unsaturated ester with KMnO4 (Scheme 1, C), the racemic mixture (RAC, 7) was synthesized. This is composed of two enantiomeric pairs, (R,R,S)/(S,S,R) and (R,R,R)/(S,S,S), in a diastereomeric ratio of 60:40. Film Balance Measurements. In Figure 1 the π/A isotherms of RAC, RDIA, SDIA, and ENAN at 20 °C on ultrapure water are presented. All investigated compounds show surface activity. The lift-off areas per molecule are 1.0 nm2 for RAC, 1.1 nm2 for RDIA, 0.98 nm2 for SDIA, and 0.6 nm2 for ENAN. The π/A isotherms of RAC, RDIA, and SDIA are characterized by a small “plateau”. This is an indication of a coexistence area of the liquidexpanded (LE) and liquid-condensed (LC) phases. The plateaus of the mixtures differ in transition pressure and molecular area. Nevertheless, it indicates a first-order phase transition from an LE to an LC monolayer state.25,26 The highest transition pressure is observed with RAC (15 mN/m). After the plateau, the monolayer shows the same compressibility as before, reflected by the shape of the isotherm. The monolayer of RAC collapses at a pressure of 40 mN/m and an area per molecule of 0.23 nm2. For the two diastereomeric mixtures SDIA and RDIA, an identical π/A isotherm behavior would be expected because the mixtures are enantiomeric to each other. Unexpectedly, the shape of the isotherm is different yet similar. The two-phase coexistence regions are developed at pressures of 4.8 mN/m (SDIA) and 1.8 mN/m (RDIA). For RDIA, the collapse point of the monolayer occurs at 40 mN/m and a molecular area of 0.27 nm2, and it occurs at 38.6 mN/m/0.25 nm2 for SDIA. The enantiopure compound ENAN shows a behavior different from all mixtures. In the π/A isotherm, no plateau is performed. This means that a two-phase coexistence region already exists at a low pressure close to zero. Below a molecular area of 0.3 nm2, the surface pressure increases abruptly. Thus the monolayer collapses directly from the LC phase at a pressure of 41.2 mN/m and an area per molecule of 0.23 nm2. The behavior of the compound mixtures can be concluded as follows: RAC shows a transition behavior where the plateau is shifted to a smaller area per molecule and higher surface pressure than that of RDIA and SDIA. This indicates that the RAC mixture builds a more expanded and structurally disturbed monolayer film. Surprisingly, RDIA and SDIA exhibit different π/A isotherms, although the mixtures are enantiomeric to each other. On the other hand, they differ in their diastereomeric ratio within a 3% range. This is taken as evidence that the noncovalent (25) Albrecht, O.; Gruler, H.; Sackmann, E. J. Phys. 1978, 39, 301. (26) Huda, M. S.; Fujio, K.; Uzu, Y. Bull. Chem. Soc. Jpn. 1996, 69, 3387.

Phase BehaVior and Nucleation of EFDS Monolayers

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Figure 2. BAM images of RAC EFDS during compression (scale bar 100 µm): (A) 13.7 mN/m 0.48 nm2, (B) 15.4 mN/m 0.34 nm2, (C) 27.0 mN/m 0.30 nm2, and (D) 27.6 mN/m 0.31 nm2.

Figure 4. BAM images of RDIA EFDS during compression (scale bar 100 µm): (A) 13.0 mN/m 0.48 nm2, (B) 15.4 mN/m 0.34 nm2, (C) 26.1mN/m 0.30 nm2, and (D) 27.0mN/m 0.28 nm2.

Figure 3. BAM images of SDIA EFDS during compression (scale bar 100 µm): (A) 4.1 mN/m 0.66 nm2, (B) 5.0 mN/m 0.60 nm2, (C) 8.5 mN/m 0.47 nm2, and (D) 12.0 mN/m 0.41 nm2.

Figure 5. BAM-images of the ENAN EFDS during compression (scale bar 100 µm) A 0.1 mN/m 0.60 nm2 B 1.0 mN/m 0.38 nm2 C 2.0 mN/m 0.30 nm2 D 4.5 mN/m 0.23 nm2.

interactions between the molecules within the monolayer film react very sensitively to changes in their chiral surroundings. In comparison to ENAN, both diastereomeric mixtures RDIA and SDIA are less condensed, which is clearly visible by the small plateau at low pressures for the diastereomeric mixtures. This kind of π/A isotherm behavior is expected for homochiral monolayers.9 For the examined compounds, however, it is not possible to decide which kind of preference is exhibited because we investigated mixtures of enantiopure diastereomers and its racemates in comparison with one pure enantiomer. Since the isotherms clearly show different thermodynamic behaviors, we consider chiral discrimination. Brewster Angle Microscopy. To obtain more information about the morphology of the condensed phase domains formed in the two-phase coexistence region, BAM studies were performed. As expected, RAC, ENAN, and both diastereomeric mixtures show crucial differences in the shape of their domains. In Figure 2A-D, the BAM images of RAC are shown. Before the final plateau pressure is reached, the RAC monolayer forms small, fringed, rounded domains with a diameter of about 10 µm (Figure 2A). During compression, these domains increase only in size, finally reaching a diameter of about 50 µm (Figure 2BD). The BAM images of SDIA are presented in Figure 3. The first structure appears at a pressure of 4 mN/m (Figure 3A). The domains consist of branched, curved needles with a nucleation center. This nucleation center possesses thicker branches from which thinner lines protrude (Figure 3B). During compression,

these strings seem to connect to each other (Figure 3C). At higher pressures, these structures fill the complete surface (Figure 3D). The domains show an irregular inner texture because the needlelike branches are straight as well as curved. In Figure 4, the domains of the diastereomeric mixture RDIA are shown. The domains take the shape of long, curved needles with a length of over 100 µm. In comparison, the shape of the domain structures is similar to that of SDIA, which, on the other hand, seems to be more filigree. The domains of RDIA develop at a nucleation center and seem to be less curved. At higher pressures, the surface is completely covered with the domains. The images of ENAN are presented in Figure 5. The main difference with respect to the diastereomeric mixtures is that, at surface pressures near zero, domains are already detectable. This is clear evidence that the monolayer is already in a two-phase coexistence region. The shape of the domains at large areas is similar to small, elongated beads. During compression, short, straight branches grow from these nucleation centers. At very low pressures (Figure 5D), the surface is completely covered with the domain structure. Additionally, small highly reflecting buds are developed. This could be a hint for the formation of multilayers, which was also observed in the images of SDIA and RDIA. The different shapes of the domains within the coexistence region could be a hint for the increasing structural disturbance within the monolayer film. Scanning Force Microscopy. To gain more structural information on the domain structures formed by the EFDSs,

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Figure 6. (A,B) SFM images of RAC (transfer pressure: 19 mN/ m). (C,D) Zoom-in SFM images of RDIA (transfer pressure: 5 mN/m). (E) SFM image (derivative of height image) of ENAN (transfer pressure: 4 mN/m).

monolayer films were transferred after phase transition via the LB technique onto mica sheets. The results show that, in the overall appearance, all obtained images correspond to the BAM observations. This clearly shows that the transfer process did not disturb the domain formation at the aqueous surface of the film balance. For all profiles, the z-axis is defined as the height of the structures, whereas the x-axis is the length of the line placed in the SFM image. In Figure 6A,B, SFM images of a RAC monolayer film are shown. Obviously, rounded structures with fringed outer lines are formed. Additionally, material between the domains is clearly present, which was not detectable by the BAM investigations. Figure 6B is a zoomed-in image of the marked area in Figure 6A. Here, the focus lays on the material between the domains. The round structures are found to be assemblies of small crystallites. It can be shown that the crystallites have a linear appearance with a defined length. These crystallites have no preference in their direction. The profile shows that the crystallites have distances between 100 and 250 nm. The size of the crystallites in the RAC mixture is probably limited because of interactions between molecules that are enantiomeric, racemic, or diastereomeric to each other. During the formation of domains via the assembly of crystallites, the chance is high that a mismatching molecule could probably disturb the crystallization process. This could be the reason for the limited length of the crystallites and the missing order of their direction.

Steffens et al.

In Figure 6C,D, the SFM images of the RDIA mixture are presented. Image C shows filigree, line structuring of the surface with a preference in direction. To obtain a more detailed view, Figure 6D is a zoomed-in image of Figure 6C showing small, elongated crystallites, which, in the case of RDIA, are assembled to stripes. The distance between the stripes has been determined to be between 100 and 250 nm. Interactions between diastereomeric molecules probably enhance the process to assemble the crystallites to stripes. In Figure 6E, the derivative of the height SFM image of ENAN is shown. Due to the small height of the structures, the derivative of the height image has been digitally produced. The monolayer film shows a surface structure that includes crystallites with a length between 2 and 7 µm. These crystallites are not aligned. Nevertheless, the whole surface is covered with an irregular multilayer of these crystallites. This correlates with the observation we made in the BAM experiments. In comparison to the unfluorinated compounds, the results of the AFM-measurements are completely different. It has been shown that 2,3-dihydroxystearic acid esters show linear periodic structures with a distance of 29 nm.27 The fluorine possibly disturbs the formation of periodic structures, and only small crystallites are formed, which could be found for every mixture and the enantiopure compound. Temperature Dependence of the π/A Isotherm. The thermodynamic behavior of all EFDSs is investigated by temperature-dependent film-balance measurements. All π/A isotherms of the EFDSs at different subphase temperatures are shown in Figure 7A-D. RAC, RDIA, and SDIA monolayers exhibit expanded and condensed phases for all subphase temperatures investigated, except for ENAN at 20 °C, which also shows a coexistence area from LE to LC, indicated by a small plateau in the π/A isotherms, at 25 °C and upward. The π/A isotherms of the RAC were performed at 10-25 °C in 5 °C interval steps for every measurement (Figure 7A). The π/A isotherms show a very similar behavior in shape and curvature. The differences are only indicated by higher plateau pressures and shifted molecular areas of the phase transition with increasing temperature. The collapse points of all π/A isotherms are about 40 mN/m. For RDIA and SDIA, a similar behavior of the plateau height can be detected (Figure 7B,C). In contrast to RAC, both diastereomeric mixtures show a collapse point that is equivalent for 20 and 25 °C (RDIA 40 mN/m, SDIA 38.6 mN/m). At a higher temperature interval (30-35 °C), the collapse point is shifted to lower pressures and smaller molecular areas. Conversely, the length of the plateau is a crucial difference in the diastereomeric mixtures. Besides, for both mixtures of diastereomers, a spike can be detected in the π/A isotherm after the collapse point. This has been described for the formation of multilayers after the collapse point.28 The two-phase coexistence region in the π/A isotherm of ENAN shows a decreasing plateau length with increasing temperature. In comparison with the diastereomeric mixtures, the collapse point of the π/A isotherms shows a linear decrease in surface pressure and an increase in the molecular area. The collapse point of the enantiopure compound is very sensitive to temperature differences. (27) Chi, L.; Jacobi, S.; Anczykowski, B.; Overs, M.; Scha¨fer, H.-J.; Fuchs, H. AdV. Mater. 2000, 12, 25. (28) (a) McFate, C.; Ward, D.; Olmsted, J., III Langmuir 1993, 9, 1036. (b) Hui, S. W.; Yu, H.; Xu, Z. C.; Bittman, R. Langmuir 1992, 8, 2724. (c) Makino, M.; Kamiya, M.; Ishii, T.; Yoshikawa, K. Langmuir 1994, 10, 1287. (d) Mertesdorf, C.; Ringsdorf, H. Liq. Cryst. 1989, 5, 1757.

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Figure 7. Surface pressure-area isotherms of all EFDSs at different temperatures: (A) RAC, (B) RDIA, (C) SDIA, and (D) ENAN.

Figure 8. Temperature dependence of the main phase transition pressure (πt) of different mixtures of the EFDSs. Table 1. Temperature Coefficients dπ/dT [mN/m‚K] of the Different Racemic and Enantioenriched EFDSs Determined for the LE-LC Phase Transition from Temperature-Dependent Behavior (Figure 3) amphiphiles

dπ/dT [mN/m‚K]

T0 [°C]

RAC RDIA SDIA ENAN

0.92 ( 0.09 0.81 ( 0.08 0.89 ( 0.15 0.77 ( 0.07

4.0 18.5 15.0 21.0

As shown in Figure 8, all different EFDSs exhibit a linear temperature dependence of the main phase transition pressure πt. The temperature coefficient dπ/dT is similar for the different mixtures and the enantiomer (Table 1). Generally, amphiphiles with one alkyl chain have a dπ/dT value of about 1 mN/m‚K. Literature values of a racemic mixture of methyl dihydroxyoctadecanoates reach a value of 1.3 mN/m‚K.10a Another value is reported for a 2-hydroxystearic acid with a dπ/dT value of 1.72 mN/m‚K.29 Obviously, the incorporation of an additional fluorine has a fluidizing effect, because all EFDSs gain a dπ/dT value of about 0.85 mN/m‚K. This could be due to a distinct structure within the headgroup caused by the fluorine. Interestingly, the phase transition temperature differs strongly between the mixtures (RAC, RDIA, and SDIA) and the enantiopure compound. This value has been extrapolated from (29) Vollhardt, D.; Siegel, S.; Cadenhead, D. A. J. Phys. Chem. B 2004, 108, 17448.

Figure 9. Enthalpy difference of different mixtures of the EFDSs.

the diagrams given in Figure 8, and the values are shown in Table 1. There is a great difference between the 4-fold mixture RAC (4.0 °C), the 2-fold mixtures RDIA (18.5 °C) and SDIA (15.0 °C), and the enantiomeric compound (21.0 °C). The disturbance within the monolayer film is directly correlated to these values and even better to the ∆H values. Therefore, a calculation of the phase transition enthalpy ∆H is possible to determine the effect of the different EFDSs within the LE to a LC phase transition. A suitable form of the ClausiusClapeyron equation is applied to the isotherms to calculate the phase transition enthalpy:

dπ/dT ) ∆H/T(AE - AC) Here, T is the subphase temperature in Kelvin, AE is the molecular area of the plateau onset, AC is the molecular area at the transition of the LE/LC to the condensed phase, and π is the surface pressure within the two-phase coexistence region. In Figure 9, the ∆H dependence on the temperature for all EFDS is shown. All ∆H values decreased linearly with increasing temperature. This is considered to be due to an increased ordering of the condensed phase with decreasing temperature. The highest ∆H value for all temperatures is reached by RDIA. At a subphase temperature of 298 K, a value nearly twice that of SDIA was found. This indicates that the ordering of the monolayer decreases. This could be already detected by the dissimilarity of the plateau length.

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Table 2. Energy Differences in kcal/mol of All 11 Minima Conformations of the (S,S,R)-EDFS and the Tilt Angle of the HARDPACK-Calculated Modela ab initio energy HARDPACK energy difference ESP Mulliken A B C D E F G H I J K

0.00 (1) 0.80 (2) 2.42 (3) 3.07 (4) 5.66 (5) 6.76 (6) 7.99 (7) 8.17 (8) 8.87 (9) 9.20 (10) 20.85 (11)

1.17 (3) 2.72 (8) 0.42 (2) 0.00 (1) 5.32 (10) 3.55 (9) 1.32 (4) 2.41 (7) 1.88 (6) 1.54 (5) 6.33 (11)

ESP

Table 3. Energy Differences in kcal/mol of All 10 Minima Conformations of (S,S,S)-EDFS and the Tilt Angle of the HARDPACK-Calculated Modela

sum Mulliken tilt angle

2.50 (2) 0.00 (1) 4.76 (7) 2.35 (4) 3.09 (4) 1.67 (2) 3.96 (6) 1.90 (3) 6.00 (10) 9.81 (10) 5.39 (8) 9.14 (6) 0.00 (1) 8.13 (5) 3.60 (5) 9.41 (7) 5.93 (9) 9.58 (9) 2.91 (3) 9.57 (8) 7.67 (11) 26.01 (11)

0.00 (1) 3.06 (3) 3.01 (2) 4.53 (4) 9.16 (6) 9.65 (9) 5.49 (5) 9.27 (7) 12.30 (10) 9.60 (8) 26.02 (11)

72.18 80.36 74.45 80.02 69.94 69.74 66.14 67.62 95.80 99.85 73.83

a

Numbers in brackets indicate the order of the energy differences in every column.

Comparison of the shape of the isotherms and the temperature coefficients with non-fluorinated compounds shows that the incorporation of fluorine has a condensing effect on the monolayer film. This has also been demonstrated for other monofluorinated compounds in comparison to their non-fluorinated parents.10e Here we were able to show that this condensing effect is diminished by an increasing number of functional groups. The fluorine substituent in our compounds probably disturbs the formation of intra- and intermolecular hydrogen bridges between the hydroxyl groups of neighboring molecules because of electrostatic repulsion and/or the formation of close intramolecular O-H‚‚‚F contacts similar to those found in regioisomeric longchain methyl 2,3-fluorohydroxyalkanoates.30 Quantum chemical calculation of the lowest energy conformations of our monofluorinated compounds and their non-fluorinated counterparts support this suggestion. The results of quantum chemical calculations are presented in the following paragraph. Theoretical Calculations. To elucidate the effect of the conformational order of the compounds within the diastereomeric mixtures, ab initio calculations have been performed. In Table 2, all 11 obtained conformers of the (S,S,R)-EFDS are presented. In the first column, a letter is given for every conformer, whereas, in the second column, the ab initio energy difference of the conformers is ordered by the lowest difference, which is normalized to zero (conformer A). The HARDPACK differences of both partial charges are shown in the third (ESP) and fourth (Mulliken) columns. In the fifth and sixth columns the sum of the HARDPACK energy difference and the ab initio energy difference is calculated and normalized to zero. The tilt angle of the calculated HARDPACK models is given in the seventh column. We can conclude for (S,S,R)-EFDS that four conformers are energetically close to each other. For both partial charges (ESP or Mulliken), we got the same group of best conformations of the molecule in the sum, only the order differs. For (S,S,S)-EFDS, all these parameters are described in Table 3. Here conformer A is stabilized in comparison with the energetically subsequent conformers B and C by an energy difference higher than 6 kcal/mol. The molecular models of the best conformations of the (S,S,R)and (S,S,S)-enantiomers are given in the Supporting Information. In Figure 10, a molecular model based on the P1 space group of the lowest sum energy of the HARDPACK calculation and the ab initio calculation of the head groups of (S,S,R)-EFDS is shown. In addition to the two intramolecular hydrogen bridges, it might be possible that an intermolecular hydrogen bridge from (30) Wiehle, S.; Bergander, K.; Grimme, S.; Haufe, G. Institute of Organic Chemistry, University of Mu¨nster. Unpublished results, 2006.

ab initio energy HARDPACK energy difference ESP Mulliken A B C D E F G H I J

0.00 (1) 6.53 (2) 6.85 (3) 7.41 (4) 9.36 (5) 10.93 (6) 11.70 (7) 13.56 (8) 13.78 (9) 14.70 (10)

3.20 (7) 4.34 (10) 4.24 (9) 2.31 (4) 0.62 (2) 2.74 (5) 3.50 (8) 3.14 (6) 0.00 (1) 2.06 (3)

3.00 (7) 5.92 (10) 1.58 (3) 2.39 (6) 1.02 (2) 4.20 (8) 1.95 (4) 5.78 (9) 0.00 (1) 2.33 (5)

ESP

sum Mulliken tilt angle

0.00 (1) 7.67 (4) 7.89 (5) 6.52 (2) 6.78 (3) 10.47 (6) 12.00 (8) 13.50 (9) 10.58 (7) 13.57 (10)

0.00 (1) 99.64 9.45 (5) 67.16 5.42 (2) 103.59 6.79 (3) 58.99 7.38 (4) 70.10 12.13 (8) 69.88 11.70 (7) 64.74 16.33 (10) 102.58 10.77 (6) 68.58 14.02 (9) 68.73

a Numbers in brackets indicate the order of the energy differences in every column.

Figure 10. Predicted molecular model of the lowest energy P1 crystal structure of (S,S,R)-EFDS.

Figure 11. Predicted molecular model of the lowest energy P1 crystal structure of (S,S,S)-EFDS.

the hydroxyl group in the 3-position of one molecule is interacting with the hydroxyl function in the 2-position of the second molecule. The distances of the intermolecular hydrogen bridge here in this model are too long. It has to be kept in mind that the HARDPACK program only packs the conformers. An optimization of the shown model within HARDPACK would lead to the formation of the intermolecular hydrogen bridge. In Figure 11, a molecular model based on the P1 space group of the lowest sum energy of the HARDPACK calculation and the ab initio calculation of the head groups of (S,S,S)-EFDS is shown. In this model, the possible hydrogen bridge is between the hydroxyl function in the 3-position and the oxygen of the carbonyl group of the ester function. Also, in this model, the calculated intermolecular distances are too long for a hydrogen bridge, as we mentioned in the other model previously. By comparing the model of (S,S,R)-EFDS with the HARD-

Phase BehaVior and Nucleation of EFDS Monolayers

PACK model of (S,S,S)-EFDS, the different angle of the head groups in relation to the alkyl chain is noticeable. The calculation of these two models showed the exceptional possibility of the three functional groups to act within a network of hydrogen bonds within a 2D packing. Even more complex, a monolayer of such molecules should act as an enantiopure layer. The changes within the network of hydrogen bonds are obvious by the comparison of the two calculated diastereomers.

Conclusion The characteristic features of EFDS in mixtures with diastereomeric, racemic, or enantiomeric components essentially affect their surface behavior. We have shown that, in the monolayer film, the formation of intermolecular hydrogen bridges is crucial for the shape of the domain structures. The mechanism of the fascinating domain formation has yet to be investigated. Temperature-dependent measurements demonstrated the decrease of ordering due to the higher amount of chiral components. The fluorine substituent has been shown to have a condensing effect compared to ethyl 2,3-dihydroxyoctanoates. Calculation of the

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2D lattice structure displayed the possibilities of ordering within the monolayer. Furthermore, the possible disturbance by mismatching molecules due to their stereochemistry is considered. This led us to the final conclusion of a phase separation in different mixtures by matching or mismatching interactions within the monolayer. Acknowledgment. Financial support by the Deutsche Forschungsgemeinschaft (SFB 424) is gratefully acknowledged. The authors are grateful to Professors L. Chi and H. Fuchs and their groups for equipment and fruitful discussion. Special thanks are given to Dr. A. Kerth from the Institute of Physical Chemistry at the University of Halle for the fruitful discussions and his contagious enthusiasm. Supporting Information Available: Molecular models of the best conformations of the (S,S,R)- and (S,S,S)-enantiomers of EFDS. This material is available free of charge via the Internet at http:// pubs.acs.org. LA062406G