Assembly of New vic-Dihydroxyoctadecanoic Acid Methyl Esters at the

Marina Fix, Robert Lauter, Christian Löbbe, Gerald Brezesinski, and Hans-Joachim Galla ... Marina Fix , Susanne Wiehle , Günter Haufe , Hans-Joachim...
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Langmuir 2000, 16, 1141-1148

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Assembly of New vic-Dihydroxyoctadecanoic Acid Methyl Esters at the Air-Water Interface Michael Overs,† Marina Fix,‡ Sandra Jacobi,§ Li Feng Chi,§ Manfred Sieber,‡ Hans-Ju¨rgen Scha¨fer,† Harald Fuchs,§ and Hans-Joachim Galla*,‡ Organisch-Chemisches Institut der Universita¨ t Mu¨ nster, Corrensstrasse 40, D-48149 Mu¨ nster, Germany, Institut fu¨ r Biochemie der Universita¨ t Mu¨ nster, Wilhelm-Klemm-Strasse 2, D-48149 Mu¨ nster, Germany, and Physikalisches Institut der Universita¨ t Mu¨ nster, Wilhelm-Klemm-Strasse 10, D-48149 Mu¨ nster, Germany Received May 27, 1999. In Final Form: September 13, 1999 Members of a new class of amphiphiles with two vicinal hydroxyl groups as a second polar moiety have been synthesized by dihydroxylation of trans-octadecenoates made of natural fatty acids or synthetic fatty acid esters. The phase behavior is studied by measuring surface pressure-area isotherms (π-A isotherms) and equilibrium spreading pressures (ESP) as well as Brewster angle microscopy (BAM) and fluorescence microscopy. Both, π-A isotherms and the micrographs of all compounds show a two-phase coexistence region, indicating a first-order phase transition from an expanded to a condensed phase. Depending on the position of the vicinal hydroxyl groups along the hydrophobic alkyl chain, the film pressure in the plateau region as well as the onset of the plateau region differ. Additionally, the position of the hydroxyl groups influences the formation of a stable condensed phase: only the methyl dihydroxyoctadecanoates with no or the largest distance between the two polar groups show an increase in surface pressure at small molecular areas. A molecular model for the processes during film compression is developed, where the two-phase coexistence region is interpreted as the continuous removal of one polar group from the water surface during compression.

Introduction Fatty acids and their esters are known to form monolayers at the air-water interface. The different phases during film compression can be studied by film balance measurements,1 Brewster angle microscopy,2 and fluorescence microscopy.3 The stability of monolayers against collapse can be determined by equilibrium spreading pressure measurements.4 In the condensed phase at smaller molecular areas the polar carboxylic or ester group is located at the water subphase and the lipophilic alkyl chain projects into the air.1 The films of mono-polar molecules on the basis of octadecanoic acid are well investigated with regard to monolayer formation, monolayer stability, and monolayer properties.5 The insertion of a second polar moiety into an amphiphilic structure offers the possibility to examine the influence of changing molecular properties on the physical characteristics of the monolayer. Different bipolar amphiphiles were investigated in order to obtain information about molecules with different headgroups and polar substituents in the hydrocarbon chain and their intermolecular interaction in monolayers.5-14 The interest in these molecules is based * Corresponding author. Phone: +49-251-83-33201. Fax: +49251-83-33206. E-mail: [email protected]. † Organisch-Chemisches Institut. ‡ Institut fu ¨ r Biochemie. § Physikalisches Institut. (1) Gaines, G. L., Jr. Insoluble monolayers at liquid-gas interfaces; John Wiley & Sons: New York, London, Sidney, 1966; p 285. (2) (a) He´non, F.; Meunier, J. Rev. Sci. Instrum. 1991, 62 (4), 936. (b) Ho¨nig, D.; Mo¨bius, D. J. Phys. Chem. 1991, 95, 4590. (3) (a) Lo¨sche, M.; Sackmann, E.; Mo¨hwald, H. Ber. Bunsen-Ges. Phys. Chem. 1983, 87, 848. (b) Weis, R. M.; McConnell, H. M. Nature 1984, 310, 47. (4) Roberts, G. Langmuir-Blodgett Films; Plenum Press: New York, 1990; p 21. (5) Sackmann, H.; Do¨rfler, H.-D. Z. Phys. Chem. 1972, 251, 303. (6) Menger, F. M.; Richardson, S.; Wood, M. G., Jr.; Sherrod, M. J. Langmuir 1989, 5, 833.

on their significance as model components for biological membranes. A series of synthetic fatty acids and phospholipids bearing various substituents in their hydrocarbon chain was studied by Menger et al.6 Many amphiphiles with bulky unpolar groups have been described, but large substituents render the dense packing more difficult or lead to a change of the conformation of the alkyl chain. Huda et al. discussed the influence of different subphases on hydroxylated fatty acids.7 To learn more about the effect of noncovalent interactions such as hydrogen bonds or hydrophobic interactions on the film stabilization we have synthesized different methyl threo-dihydroxyoctadecanoates (DHO) with systematically displaced hydroxyl groups in racemic and enantioenriched form.15,16 On the basis of these substances a systematic examination of inter- and intramolecular interactions with regard to hydrogen bonds and hydrophobic interactions is possible by comparing the properties of the particular regioisomers. Variations in the phase behavior between the different dihydroxylated methyl octadecanoates are expected to correlate directly with distinct interactions between the polar and unpolar (7) Huda, M. S.; Fujio, K.; Uzo, Y. Bull. Chem. Soc. Jpn. 1996, 69, 3387. (8) Baret, J. F.; Hasmonay, H.; Firpo, J. L.; Dupin, J. J.; Dupeyrat, M. Chem. Phys. Lipids 1982, 30, 177. (9) Jacobi, S.; Chi, L. F.; Plate, M.; Overs, M.; Scha¨fer, H.-J.; Fuchs, H. Thin Solid Films 1998, 327-329, 180. (10) Rekshit, A. K.; Zografi, G.; Jalal, I. M.; Gunstone, F. D. J. Colloid Interface Sci. 1981, 80, 466. (11) Kellner, B. M. J.; Cadenhead, D. A. J. Colloid Interface Sci. 1978, 63, 452. (12) Kellner, B. M. J.; Cadenhead, D. A. Chem. Phys. Lipids 1979, 3, 41. (13) Tachibana, T.; Yoshizumi, T.; Hori, K. Bull. Chem. Soc. Jpn. 1979, 52, 34. (14) Matuo, H.; Rice, D. K.; Balthasar, D. M.; Cadenhead, D. A. Chem. Phys. Lipids 1982, 30, 367. (15) Plate, M. Thesis, Mu¨nster, 1998. (16) Plate, M.; Overs, M.; Scha¨fer, H.-J. Synthesis 1998, 9, 1255.

10.1021/la990655h CCC: $19.00 © 2000 American Chemical Society Published on Web 11/17/1999

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Figure 2. Surface pressure-area isotherms of different methyl rac-threo-dihydroxy-octadecanoates at the air-water interface at 293 K (compression velocity 5.8 cm2/min). Scheme 2. Synthesis of 12,13-DHO (4) Figure 1. Structures of investigated racemic methyl dihydroxyoctadecanoates. Scheme 1. Synthesis of 6,7-DHO (2)

molecular moieties. We have chosen the methyl esters instead of the fatty acids because of their lower water solubility. In this paper, we present the synthesis of the racemic mixtures of the dihydroxylated methyl octadecanoates 2 and 4 as well as investigations on the phase behavior of the racemic amphiphiles 1-5 (Figure 1), studied by film balance measurements (π-A isotherms), fluorescence and Brewster angle microscopy (BAM) of the monolayers, and determination of the equilibrium spreading pressure (ESP). The corresponding study using enantioenriched compounds will be published prospectively. Results and Discussion Synthesis. The synthetic routes leading to the methyl dihydroxyoctadecanoates 2 and 4 (6,7-DHO and 12,13DHO) are outlined in Schemes 1 and 2. The synthesis of the amphiphiles 1 (as ethyl ester), 3, and 5 has already been reported.15,16 The synthesis of 1 will be reported elsewhere. For the synthesis of 2, cis-6-octadecenoic acid (petroselinic acid) (6) is isomerized with sodium nitrite in nitric acid yielding a mixture of cis- and trans-acid. The transacid 7 can be precipitated from acetone, and the cis-form in the filtrate can be isomerized again. After esterification methyl trans-6-octadecenoate (8) can be dihydroxylated with osmium tetraoxide to give 2 in 97% overall yield. The synthesis of 12,13-DHO (4) starts with 12-hydroxydodecanoic acid (9) which is esterified and then oxidized to methyl 12-oxododecanoate (11). Wittig olefi-

nation with hexyltriphenylphosphonium bromide (12)/nbutyllithium afforded the methyl cis-/trans-12-octadecenoate (13) in moderate yield. Isomerization with sodium nitrite in nitric acid yielded after crystallization from acetone the trans-fatty acid 14 in high purity and 46% yield. Esterification and dihydroxylation with osmium tetraoxide afforded methyl rac-threo-12,13-dihydroxyoctadecanoate (4) in 97% overall yield from 14. Film Balance Measurements. Figure 2 shows π-A isotherms of different racemic methyl threo-dihydroxyoctadecanoates (DHO) on ultrapure water at 293 K. All compounds behaved similarly on compression: the film pressure was zero at large molecular areas and showed an increase on reduction of the molecular area. At a characteristic area AM the course of the isotherms changed into a plateau region with constant film pressure and a large compressibility. Depending on the position of the vicinal hydroxyl groups, both the film pressure and the onset point AM of the plateau region differed. The shorter the hydrophobic part of the amphiphiles between the two polar groups, the smaller is the molecular area at the beginning of the plateau. In contrast the plateau pressure showed the highest value for 6,7-DHO (2) (Figure 3) and

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Figure 3. Dependence of the film pressure in the plateau region Πexpanded/condensed on the distance between the two polar groups in methyl rac-threo-dihydroxyoctadecanoates. (n means the number of C atoms between the ester and the hydroxyl groups.)

decreased with increasing distance between the two polar moieties of the molecules except for 2,3-DHO (1). Only 2,3-DHO (1) and 17,18-DHO (5) exhibited a condensed phase at 293 K with a large increase of the surface pressure up to 50 mN/m below 0.25 nm2 (Figure 2 and 4A,E). Further compression led to film collapse. The films of 6,7-DHO (2), 9,10-DHO (3), and 12,13-DHO (4) showed no significant rise of film pressure at molecular areas of 0.2 nm2 or smaller. According to BAM, 6,7-DHO (2), 9,10-DHO (3), and 12,13-DHO (4) formed threedimensional aggregates instead of a condensed monomolecular phase with an increase of film pressure at molecular areas smaller than 0.2 nm2 (Figure 5). Depending on the position of the vicinal hydroxyl groups along the hydrophobic alkyl chain, the three amphiphiles showed different multilayered structures: the domains of 6,7DHO (2) were pushed together at the edges, and then the edges were squeezed out into the third dimension (Figure 5A). The domains of 9,10-DHO (3) were pushed together, and spots of three-dimensional structures emerged at the edges of the domains (Figure 5B). During compression of the 12,13-DHO film the small domains aggregated by emerging small three-dimensional spots (Figure 5C). During expansion the π-A isotherms of films of 6,7-DHO (2) and 9,10-DHO (3) first showed the disintegration of the monomolecular domains (dark areas in Figure 5A,B) and in a second step the disintegration of the threedimensional structures. This behavior caused the two plateaus during film expansion (Figure 4B,C). All methyl dihydroxyoctadecanoates except for 17,18-DHO (5) showed a pronounced hysteresis. The expansion isotherms of films of 17,18-DHO (5) were congruent to those obtained by compression (due to clearness only the first three expansions are shown). Repeated compression and expansion led to a neglectable small exclusion of material for 2,3DHO (1) and 17,18-DHO (5). In the case of 6,7-DHO (2), 9,10-DHO (3), and 12,13-DHO (4) repeated compressions and expansions showed different isotherms. The second hysteresis was shifted to smaller molecular areas. Additionally the second expansion course showed no second plateau. To elucidate the reason for the differences in the plateau length and height, we investigated monolayers of the dihydroxylated methyl octadecanoates 1-5 by Brewster angle and fluorescence microscopy. Figure 6 shows Brewster angle micrographs of the monolayers of the different amphiphiles taken in the plateau region of the π-A diagram. A typical fluorescence micrograph of the mono-

Figure 4. Repeated hysteresis of 2,3-DHO (A), 6,7-DHO (B), 9,10-DHO (C), 12,13-DHO (D), and 17,18-DHO (E, expansion not shown due to clearness) films on ultrapure water at 293 K (A, E, 3 cycles; B-D, 2 cycles).

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Figure 5. Brewster angle micrographs of films of 6,7-DHO (A), 9,10-DHO (B), and 12,13-DHO (C) at molecular areas smaller than 0.2 nm2. The brightest structures are threedimenional.

layer of 17,18-DHO (5) is also given to show the corresponding results from both techniques. Note that the bright domains in the images A-E obtained from the BAM measurements represent a condensed phase while the bright areas in the fluorescence micrograph (Figure 6F) represent the expanded phase due to a squeeze-out of the fluorescence dye from the condensed regions. The condensed domains appeared during film compression at the onset of the plateau region whereas their area fraction increased continuously during compression. Both BAM and fluorescence microscopy clearly showed the coexistence of two monolayer phases, and thus, that the phase transition is of first order from an expanded to a condensed state. The condensed domain shapes depended on the position of the two vicinal hydroxyl groups along the hydrophobic alkyl chain (Figure 6). The bolaamphiphile 17,18-DHO

Overs et al.

(5) formed many small condensed domains compared to all other substances (Figure 6E,F). This molecule showed the longest expanded/condensed phase coexistence region in comparison to the other bipolar amphiphiles 1-4. The reduced distance between the ester and the hydroxyl groups in 12,13-DHO (4) led to a shorter plateau and also to smaller domains (Figure 6D). In contrast, 6,7-DHO (2) and 9,10-DHO (3) showed larger domains (Figure 6B,C). 6,7-DHO (2) formed dendritic domains with a size of several hundred micrometers. The domains of 9,10-DHO (3) in the expanded/condensed phase showed a curved shape. At the onset of the expanded/condensed phase coexistence region only a small number of domains are formed in contrast to 12,13-DHO (4) and 17,18-DHO (5). Films of the amphiphiles 6,7-DHO (2) and 9,10-DHO (3) showed a very short plateau and no condensed phase on further compression. In contrast, 2,3-DHO (1) formed small domains but exhibited a condensed phase at small molecular areas. In summary the amphiphiles form small domains if they show a long expanded/condensed phase coexistence region in the π-A isotherms except 2,3-DHO (1). Large domains are formed if the plateau is shorter. This is due to a prevented nucleation for 6,7-DHO (2) and 9,10-DHO (3) in comparison to 12,13-DHO (4) and 17,18-DHO (5). The hydroxyl groups in 2,3-DHO (1) do not behave as an isolated functional group like the hydroxyl groups in the other molecules. Here, the hydroxyl groups and the methyl ester group form a combined and enlarged headgroup. Therefore the π-A isotherm shows a condensed phase at a larger molecular area in comparison to the bolaamphiphile 17,18-DHO (5) because of the larger area requirement of the extended headgroup. The π-A isotherms of 2,3-DHO (1), 6,7-DHO (2), and 9,10-DHO (3) (Figures 2 and 4) showed on compression an overshoot of the surface pressure at the beginning of the plateau. The magnitude of the bipolar conformational change in comparison to a monopolar amphiphile is such that overshoot of the equilibrium transition pressure can occur. Matuo et al.14 observed the same phenomenon with films of the bipolar molecules ethyl rac-12-hydroxyotadecanoate and racemic 12-hydroxyoctadecanoic acid. Film balance measurements and the BAM and fluorescence microscopy investigations are interpreted by the molecular model shown in Figure 7 explaining the phase transitions during film compression. According to our model in case of the bolaamphiphile 17,18-DHO (5), the two hydrophilic moieties of the molecule (shown as circles in Figure 7) are in contact with the water subphase at larger molecular areas. Due to intermolecular interactions the surface pressure increases on compression of the monolayer. At the characteristic molecular area AM some molecules turn into an orientation perpendicular to the air-water interface accompanied with the removal of one polar group from the water surface. At the corresponding surface pressure πM the parallel and the perpendicular orientations coexist. On further compression more and more molecules are transferred to the perpendicular orientation. No experimental evidence from π-A isotherms, BAM, and fluorescence microscopic investigations exists to support a looplike orientation of molecules as proposed by Vogel and Mo¨bius17 in the case of the bolaamphiphile monomethyl octadecanedioate. 2,3-DHO (1) has the constitution opposite to that of 17,18-DHO (5) (no hydrophobic part between the two polar groups), and both polar groups act as a single headgroup. In the plateau region we postulate a change between two headgroup conformations with different area require(17) Vogel, V.; Mo¨bius, D. Thin Solid Films 1985, 132, 205.

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Figure 6. Brewster angle micrographs (A-E) and fluorescence micrograph (F) of different monolayers in the expanded/condensed coexistence region.

ments in addition to the condensed domain formation (Figure 7). In the case of methyl 3-hydroxyhexadecanoate Kellner et al.12 postulated also a change of the ester group lying parallel to the plane of the interface at low pressures and being pushed into a vertical position upon compression. Film pressure rises again after passing the plateau region, indicating the formation of a condensed phase. The monolayer collapse occurs at a larger molecular area in comparison to 17,18-DHO (5) due to the additional hydroxyl groups in the headgroup. A possible scenario of what happens during the compression of the films of 6,7-DHO (2), 9,10-DHO (3), and 12,13-DHO (4) is deduced from the mechanistic model 17,18-DHO (5). At larger molecular areas, both polar groups are oriented to the water subphase (Figure 7). On compression, the hydrophobic residue of the molecule is squeezed out of the interface and directed to the air. At the onset of the expanded/condensed phase coexistence region the molecules start to erect due to the removal of the hydroxyl groups from the water surface. According to this model the condensed phase with erected amphiphiles

should be formed at similar molecular areas in comparison to 2,3-DHO (1) and 17,18-DHO (5) due to a similar area requirement of the molecules. Although a condensed monolayered phase was observed in the plateau region (Figure 6), the expected typical behavior with an increase in surface pressure passing the plateau to lower areas was not observed. The system turned into the third dimension instead of a stable condensed phase. A comparison of the equilibrium spreading pressures (πe) with the surface pressures in the plateau region (πM) for all methyl dihydroxyoctadecanoates is represented in Table 1. Large value differences for the DHO 1-4 and a difference of ∆π ) 0.5 mN/m for the bolaamphiphile 17,18-DHO (5) are found. All ESP values are smaller than the phase transition pressures πM but are almost identical for 17,18-DHO (5). According to the explanations of Mu¨llerLandau et al.18 the condensed phase is metastable with respect to the crystalline bulk solid for πe < πM. This is (18) Mu¨ller-Landau, F.; Cadenhead, D. A.; Kellner, B. M. J. J. Colloid Interface Sci. 1980, 73, 264.

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than πM, too. 16-HHA forms a plateau region without a condensed phase. The formation of three-dimensional structures at small molecular areas instead of a stable, condensed, and monolayered phase in the case of the methyl dihydroxyoctadecanoates 2-4 can be the reason for strong hysteresis and the deviation between first and second compression (Figure 4B-D). Obviously, the system changed to a stable multilayered structure after passing the plateau. This is not the case for 2,3-DHO (1) or 17,18-DHO (5). The difference between πe and πM is too low for 17,18-DHO (5). On the other hand 2,3-DHO (1) acts as a monopolar amphiphile. Although πe < πM is valid, 2,3-DHO (1) formed a stable condensed phase. Dipalmitoylphosphatidyl choline (DPPC) is another example for such a behavior.18 According to the bipolar structure of the investigated amphiphiles 2-4 another behavior during film compression in the plateau region is conceivable: the ester group instead of the vicinal hydroxyl groups is removed from the water surface. In this case a monolayer collapse should occur at a molecular area of no less than 0.4 nm2. The actual collapse occurred at about 0.2 nm2.

Figure 7. Mechanistic model to explain the phase transitions during film compression: 2,3-DHO (A); 6,7-DHO, 9,10-DHO, and 12,13-DHO (B); 17,18-DHO (C). Table 1. Results of the Equilibrium Spreading Pressure (ESP) Measurements in Comparison to the Surface Pressure Values πM in the Plateau Region compound

ESP πe (mN/m)

πM (mN/m)

2,3-DHO (1) 6,7-DHO (2) 9,10-DHO (3) 12,13-DHO (4) 17,18-DHO (5)

6.0 ( 0.4 13.0 ( 0.1 9.0 ( 0.3 8.6 ( 0.4 3.5 ( 0.1

13.0 ( 0.2 24.5 ( 0.2 23.0 ( 0.2 15.0 ( 0.2 4.0 ( 0.2

given for the amphiphiles 2-4. In the two-phase coexistence region the expanded phase stabilized the condensed phase. 16-Hydroxyhexadecanoic acid (16-HHA)11,12 is an additional example for such a behavior: here, πe is smaller

Conclusion The phase behavior of vicinally dihydroxylated methyl octadecanoates is strongly influenced by the distance between the two polar moieties along the hydrophobic alkyl chain. The longer the distance, the larger is the molecular area at the beginning of the plateau region. This suggests an attachment of both polar groups to the water subphase in the expanded phase. The dihydroxylated methyl octadecanoates 2-5 show different shaped condensed domains: the shorter the expanded/condensed phase coexistence region, the bigger are the domains. Within the plateau region we postulated the permanent removal of the two vicinal hydroxyl groups from the water subphase. Additionally the position of the hydroxyl groups along the alkyl chain influences the stability of the condensed phases. Due to large differences between the equilibrium spreading pressures πe and the transition pressures πM for 6,7-DHO (2), 9,10-DHO (3), and 12,13-DHO (4) they form three-dimensional structures at small molecular areas. 17,18-DHO (5) forms stable condensed films at small molecular areas and shows congruent hysteresis cycles: πe and πM possess nearly the same value. 2,3-DHO (1) acts differently. In contrast to the other bipolar amphiphiles 2,3-DHO (1) shows a short plateau region and small condensed domains. In this molecule the methyl ester and the two adjacent hydroxyl-substituted carbon atoms behave as one big headgroup. We assume that the behavior as a monopolar amphiphile can be the reason for the formation of a stable condensed phase although πe< πM. Experimental Section Methods. Preparation of Substances. Solvents and reagents were purified where necessary using literature methods. If not mentioned otherwise, the reagents were used as supplied by Aldrich, Fluka, Merck, Henkel, or Lancaster. Thin-layer chromatography (TLC) was performed on aluminum plates (5 × 7.5 cm) coated with Merck 1.05549 Kieselgel 60F254. Silica gel column chromatography was performed using Merck flash 60 Kieselgel. Microanalyses were performed by the department of Microanalytical Services of the Institute of Organic Chemistry, University of Mu¨nster. FT-IR spectra were recorded on a Bruker IFS 28 spectrometer. Mass spectra (GC/MS) were obtained from a Finnigan MAT 8230 instrument, linked with a Varian GC 3400 using data system SS 300. Mass spectra (MALDI-TOF) were obtained from LAZARUS III-DE (constructed by H. Luftmann, Institute of Organic Chemistry, University of Mu¨nster). 1H NMR

vic-Dihydroxyoctadecanoic Acid Methyl Esters spectra (300 MHz) and 13C NMR spectra (75.4 MHz) were recorded on a Bruker WM 300. In both cases the deuterated solvent was used as lock. 31P NMR spectra (121.5 MHz) were also recorded on a Bruker WM 300 with H3PO4 as an external standard. Film Balance Measurements. Surface pressure-area isotherms (π-A isotherms) were performed on a Teflon trough (Riegler-Kirstein film balance, MPI fu¨r Kolloid- und Grenzfla¨chenforschung, Berlin, Germany) with a total surface area of 144 cm2. The surface pressure was measured with a Wilhelmy plate system to better than 0.1 mN/m accuracy. The trough was temperature controlled in the range of (0.1 K. Ultrapure water (pH 5.5) for the experiments was obtained from a Millipore system comprising reverse osmosis, ion exchange, and 0.22 µm ultrafiltration cartridge. Monolayers were prepared by spreading appropriate chloroform solutions onto pure water using a 100µL syringe. After spreading, 10 min was allowed for solvent evaporation, and then the monolayer was compressed with a velocity of 5.8 cm2/min. Equilibrium Spreading Pressure Measurements. The equilibrium spreading pressure (ESP) was measured using the same thermostated film balance as for the film balance measurements. The surface area was adjusted to 30 cm2. Small crystals of the substance concerned were deposited on the water surface. When the surface pressure reached a constant value, more crystals were added to the surface. The equilibrium spreading pressure was taken to be the pressure at which a further addition of crystals to the surface resulted in no detectable change in the surface pressure within 45 min. Fluorescence Microscopy.3 The measurements were performed on the film balance described above fitted onto the stage of an Olympus microscope equipped with a mercury lamp and a CCD camera with camera controller (Hamamatsu Hamamatsu, Japan). The solutions for fluorescence microscopy contained 1 mol % of the fluorescence probe 12-(N-(7-nitrobenz-2-oxa-1,3diazol-4-yl(amino)dodecanoic acid (Molecular Probes, Eugene, OR). Brewster Angle Microscopy.2 The morphologies of the condensed phase structures of the monolayers were visualized by Brewster angle microscopy (BAM). The home-designed BAM (gift from Hoechst AG, Frankfurt, Germany) was mounted at a computer-interfaced film balance (NIMA, Coventry, Great Britain). The light source of the BAM was a He-Ne laser. The laser beam is reflected at a mirror and strikes the water surface under the Brewster angle. The reflected beam is recorded with a CCD camera. A video system was used for data storage. Materials. trans-6-Octadecenoate. (7) cis-6-Octadecenoate (6) (2.825 g, 10 mmol) and some crystals of sodium nitrite were dissolved in 30% nitric acid (30 mL). The flask was closed immediately and the mixture heated until the fatty acid was completely solved. Then the flask was shaken for nearly 10 min until the trans-fatty acid precipitated. The precipitate was filtered off, washed with water, and recrystallized from acetone to afford 7 as white crystals (1.300 g, 4.6 mmol, 46%). Mp: 342 K. Anal. Calcd (found): C, 76.30 (76.54); H, 12.49 (12.13). FT-IR (ν˜ , cm-1): 2917, 2850 (s, CH), 1743 (s, CdO), 1691 (s, CdC), 1470 (m, δ, CH), 964 (m), 718 (w, CHrocking). 1H NMR (CDCl3, 300 MHz) (δ, ppm): 0.88 (t, 3J ) 6.5 Hz, 3H, 18-H), 1.18-1.48 (m, 20 H, 4-H, 9-H-17-H), 1.63 (mc, 2H, 3-H), 1.63 (mc, 2H, 3-H), 1.98 (mc, 4H, 5-H, 8-H), 2.33 (t, 3J ) 7.4 Hz, 2H, 2-H), 5.38 (mc, 2H, 6-H, 7-H). 13C NMR (CDCl , 75.4 MHz) (δ, ppm): 14.1 (q, 18-C), 22.6 (t, 3 17-C), 25.7, 29.2, 29.3, 29.4, 31.3, 31.4 (6 t, 4-C-16-C), 52.2, 52.9 (2 d, 2-C, 3-C), 58.4 (q, OCH3), 169.7 (s, 1-C). MS (GC/MS (as trimethylsilyl ester)) [m/z (%)]: 355 (8), 339 (32), 264 (14), 129 (71), 117 (100), 75 (72), 73 (78), 41 (81). Methyl trans-6-Octadecenoate (8). trans-6-Octadecenoate (7) (1.130 g, 4 mmol) was dissolved in methanol (20 mL). 2,2Dimethoxypropane (10 mL) and 3 drops of concentrated hydrochloric acid pa were added. The mixture was stirred for 24 h at room temperature and the solvent removed in vacuo. The residue was then filtered over silica gel (1:1 petroleum ether/diethyl ether) to obtain 8 as a color less oil (1.186 g, 4 mmol, 100%). Anal. Calcd (found): C, 76.76 (76.97); H, 12.23 (12.24). FT-IR (ν˜ , cm-1): 2926, 2851 (s, CH), 1742 (s, CdO), 1459 (m, δ, CH), 1437 (m, δ, CH), 1362 (m, C-O), 966 (m, δ, dC-H), 721 (w, CHrocking). 1H NMR (CDCl3, 300 MHz) (δ, ppm): 0.88 (t, 3J ) 6.8 Hz, 3H, 18-H), 1.23-1.43 (m, 20 H, 4-H, 9-H-17-H), 1.63 (mc, 2H, 3-H), 1.98

Langmuir, Vol. 16, No. 3, 2000 1147 (mc, 4H, 5-H, 8-H), 2.30 (t, 3J ) 7.5 Hz, 2H, 2-H), 3.66 (s, 3H, OCH3), 5.38 (dt, 3J ) 5.2 Hz, 3J ) 4.5 Hz, 2H, 6-H, 7-H). 13C NMR (CDCl3, 75.4 MHz) (δ, ppm): 14.1, (q, 18-C), 22.6 (t, 17-C), 24.4, 29.1, 29.2, 29.3, 29.5, 31.9, 32.1, 32.6, 33.9 (9 t, 2-C-5-C, 8-C16-C), 51.3 (q, OCH3), 129.5, 131.0 (2 d, 6-C, 7-C), 174.1 (s, 1-C). MS (GC/MS) [m/z (%)]: 296 (10), 241 (6), 227 (15), 199 (10), 185 (12), 171 (13), 143 (23), 87 (70), 74 (100), 55 (37), 41 (60). Methyl rac-threo-6,7-Dihydroxyoctadecanoate (2). A mixture of potassium hexacyanoferrate(III) (1.000 g, 3 mmol), potassium carbonate (0.380 g, 2.7 mmol), and methylsulfonamide (0.095 g, 1 mmol) in water (5 mL) and tert-butyl alcohol (5 mL) was stirred at room temperature until both phases were clear. Then osmium tetraoxide (0.120 mL, 2.5 wt % solution in tertbutyl alcohol) and methyl trans-6-octadecenoate (8) (0.297 g, 1 mmol) were added. After being stirred for 24 h at room temperature, the reaction was quenched by addition of Na2SO3 (1.500 g, 12 mmol). The mixture was again stirred for 30 min and then extracted three times with ethyl acetate (15 mL). The organic layer was washed with 2 N NaOH (15 mL), dried (MgSO4), and evaporated in vacuo. After purification by flash chromatography (2:5 ethyl acetate/cyclohexane) the dihydroxy ester 2 is obtained as a white powder (0.321 g, 1 mmol, 97%). Mp: 338 K. Anal. Calcd (found): C, 69.00 (69.05); H, 11.83 (11.59). FT-IR (ν˜ , cm-1): 3291 (s, OH), 2914, 2846 (s, CH), 1734 (s, CdO), 1468 (s, δ, CH), 1437 (w, δ, CH), 1327 (w, δ, OH), 720 (w, CHrocking). 1H NMR (CDCl3, 300 MHz) (δ, ppm): 0.88 (t, 3J ) 6.7 Hz, 3H, 18-H), 1.30-1.72 (m, 26 H, 3-H-5-H, 8-H-17-H), 1.97 (s, 2H, OH), 2.33 (t, 3J ) 7.3 Hz, 2H, 2-H), 3.40 (m, 2H, 6-H, 7-H), 3.67 (s, 3H, OCH3). 13C NMR (CDCl3, 75.4 MHz) (δ, ppm): 14.1 (q, 18-C), 22.6 (t, 17-C), 24.8, 25.2, 25.6, 29.3, 29.6, 31.9, 33.2, 33.7, 33.9 (9 t, 2-C-5-C, 8-C-16-C), 51.4 (q, OCH3), 74.1, 74.5 (2 d, 6-C, 7-C), 174.2 (s, 1-C). MS (GC/MS (as trimethylsilyl esters)) [m/z (%)]: 443 (10), 369 (4), 290 (16), 257 (100), 219 (98), 185 (30), 147 (20), 103 (16), 73 (80). Methyl 12-Hydroxydodecanoate (10). 12-Hydroxydodecanoate (9) (4.326 g, 20 mmol) was dissolved in methanol (100 mL). 2,2-Dimethoxypropane (25 mL) and 5 drops of concentrated hydrochloric acid pa were added. The mixture was stirred for 24 h at room temperature and the solvent removed in vacuo. The residue was then filtered over silica gel (4:1 cyclohexane/ethyl acetate) to obtain 10 as white crystals (4.607 g, 20 mmol, 100%). Mp: 305 K. Anal. Calcd (found): C, 68.01 (67.79); H, 11.28 (11.38). FT-IR (ν˜ , cm-1): 3303 (s, OH), 2920, 2852 (s, CH), 1744 (s, CdO), 1471 (m, δ, CH), 734 (w, CHrocking). 1H NMR (CDCl3, 300 MHz) (δ, ppm): 1.25-1.39 (m, 14 H, 4-H-10-H), 1.51-1.67 (m, 5H, 3-H, 11-H, OH), 2.29 (t, 3J ) 7.5 Hz, 2H, 2-H), 3.62 (t, 3J ) 6.6 Hz, 2H, 12-H), 3.67 (s, 3H, OCH3). 13C NMR (CDCl3, 75.4 MHz) (δ, ppm): 24.9, 25.7, 29.1, 29.2, 29.4, 29.5, 29.5, 32.8, 34.1 (9t, 2-C-11-C), 51.4 (q, OCH3), 63.0 (t, 12-C), 174.3 (s, 1-C). MS (GC/ MS) [m/z (%)]: 200 (30), 171 (5), 143 (24), 87 (50), 74 (100), 69 (28), 55 (50), 43 (42). Methyl 12-Oxododecanoate (11). Oxalyl dichloride (1.750 mL, 20 mmol) was dissolved in dry CH2Cl2 (50 mL). The mixture was stirred and cooled to 213 K. A solution of DMSO (2.800 mL, 40 mmol) in dry CH2Cl2 (10 mL) and 10 min later a solution of methyl 12-hydroxydodecanoate (10) (4.200 g, 18.2 mmol) in dry CH2Cl2 (20 mL) were added. The mixture was stirred for 45 min. Triethylamine (12.70 mL, 91 mmol) was added slowly, and the suspension was allowed to warm to room temperature. After addition of water (50 mL) the phases were separated and the aqueous phase was washed three times with CH2Cl2 (100 mL). The combined organic layers were washed with brine and dried (MgSO4). The solvent was removed in vacuo and the residue purified by flash chromatography (4:1 petroleum ether/diethyl ether) to obtain 11 (3.128 g, 13.7 mmol, 75%). Mp: 333 K. Anal. Calcd (found): C, 68.32 (68.38); H, 10.49 (10.59). FT-IR (ν˜ , cm-1): 2919 (s, CH), 2851 (s, CH), 1741 (s, CdO), 1466 (m, δ, CH), 1437 (m, δ, CH), 721 (w, CHrocking). 1H NMR (CDCl3, 300 MHz) (δ, ppm): 1.25-1.40 (m, 12H, 4-H-9-H), 1.52-1.68 (m, 4H, 3-H, 10-H), 2.30 (t, 3J ) 7.6 Hz, 2H, 2-H), 2.41 (dt, 3J ) 1.9 Hz, 3J ) 7.4 Hz, 2H, 11-H), 3.66 (s, 3H, OCH3), 9.76 (t, 3J ) 1.9 Hz, 1H, 12-H). 13C NMR (CDCl3, 75.4 MHz) (δ, ppm): 22.0 (t, 10-C), 24.9 (t, 3-C), 29.4, 29.5, 29.6, 29.8 (4 t, 4-C-9-C), 34.1 (t, 2-C), 43.8 (t, 11-C), 51.3 (q, OCH3), 174.2 (s, 1-C), 202.7 (s, 12-C). MS (GC/ MS) [m/z (%)]: 200 (48), 185 (100), 153 (70), 135 (28), 87 (62), 74 (92), 69 (26), 55 (35), 43 (26).

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Hexyltriphenylphosphonium Bromide (12). A solution of triphenylphosphine (26.229 g, 100 mmol) and 1-bromohexane (15.680 g, 95 mmol) in toluene (150 mL) was heated for 24 h under reflux. An 80 mL volume of the solvent was removed in vacuo, and petroleum ether (200 mL) was added. The precipitate formed was filtered off, washed with petroleum ether, and recrystallized from toluene/petroleum ether to afford white crystals (30.000 g, 70 mmol, 70%). Mp: 471 K. Anal. Calcd (found): C, 67.79 (67.45); H, 7.08 (6.60). FT-IR (ν˜ , cm-1): 3051, 3007 (w, CHarom), 2930, 2854 (s, CH), 1587 (m, CdCarom), 1482 (m, δ, CH), 1434 (s, P-Ph), 1113 (s), 996 (m), 746, 723, 692 (s, δ, CHarom). 1H NMR (CDCl3, 300 MHz) (δ, ppm): 0.82 (t, 3J ) 7.0 Hz, 3H, 6-H), 1.24 (mc, 4H, 4-H, 5-H), 1.63 (mc, 4H, 2-H, 3-H), 3.72 (mc, 2H, 1-H), 7.64-7.90 (m, 15H, Harom). 13C NMR (CDCl3, 75.4 MHz) (δ, ppm): 13.7, (q, 6-C), 22.0, 22.5, 23.1 (3 t, 3-C-5-C), 29.8, 31.0 (2 t, 1-C, 2-C), 117.6, 118.8 (2 s, Carom), 130.3, 130.5, 133.4, 133.5, 134.9 (5 d, Carom). 31P (CDCl3, 121 MHz) (δ, ppm): 26.2. MS (MALDI-TOF) [m/z (%)]: 347 (M - Br-). Methyl 12-Octadecenoate (13). Hexyltriphenylphosphonium bromide (12) (6.170 g, 14.4 mmol) was dissolved in dry THF (50 mL) and cooled to 195 K before n-butyllithium (7.200 mL, 11.5 mmol) was slowly added. The mixture was stirred for 10 min, and the aldehyde 11 (2.322 g, 10.1 mmol), dissolved in dry THF (20 mL), was added via a dropping funnel. The mixture was stirred for 3 h and was then allowed to warm to room temperature. Some drops of ethanol were added to destroy residual n-butyllithium. Silica gel (15 mL) was added, and the solvent was removed in vacuo. Flash chromatographic purification (10:1 petroleum ether/diethyl ether) yielded 13 as a colorless oil (1.750 g, 5.9 mmol, 59%). Anal. Calcd (found): C, 76.75 (76.96); H, 12.24 (12.58). FT-IR (ν˜ , cm-1): 2926 (s, CH), 2856 (s, CH), 1745 (s, CdO), 1464 (m, δ, CH), 1437 (m δ, CH), 723 (w, CHrocking). 1H NMR (CDCl , 300 MHz) (δ, ppm): 0.89 (t, 3J ) 6.9 Hz, 3H, 3 18-H), 1.22-1.38 (m, 20H, 4-H-10-H, 15-H-17-H), 1.62 (mc, 2H, 3-H), 2.01 (mc, 4H, 11-H, 14-H), 2.30 (t, 3J ) 7.6 Hz, 2H, 2-H), 3.66 (s, 3H, OCH3), 5.34 (m, 2H, 12-H, 13-H). 13C NMR (CDCl3, 75.4 MHz) (δ, ppm): 14.0, (q, 18-C), 22.5 (t, 17-C), 24.9, 27.2, 29.1, 29.2, 29.5, 29.6, 29.7, 31.4, 31.5, 32.6, 34.1, 34.4 (12 t, 2-C11-C, 14-C-16-C), 51.3 (q, OCH3), 129.9, 130.3 (2 d, 12-C, 13-C), 174.2 (s, 1-C). MS (GC/MS) [m/z (%)]: 296 (14), 265 (24), 264 (44), 222 (18), 111 (22), 97 (38), 87 (38), 83 (46), 74 (54), 69 (63), 55 (100), 41 (60). trans-12-Octadecenoate (14). Methyl 12-octadecenoate (13) (1.482 g, 5 mmol) was dissolved in methanol (15 mL), 2 N NaOH (20 mL) was added, and the mixture was stirred for 24 h. The solution was neutralized with 2 N HCl before most of the alcohol was removed in vacuo. The aqueous layer was extracted three times with ethyl acetate (50 mL) and evaporated. The residue and some crystals of sodium nitrite were dissolved in 30% nitric acid (30 mL). The flask was closed immediately and the mixture heated until the fatty acid was completely solved. Then the flask was shaken for nearly 10 min until the trans-fatty acid precipitated. The precipitate was filtered off, washed with water, and recrystallized from acetone to afford white crystals of 14 (0.650 g, 2.3 mmol, 46%). Mp: 330 K. Anal. Calcd (found): C, 76.75 (76.54); H, 12.58 (12.13). FT-IR (ν˜ , cm-1): 2953 (w, CH), 2915, 2849 (s, CH), 1691 (s, CdO), 1471 (m, δ, CH), 1110 (m, C-OH), 965 (m, δ, dCH). 1H NMR (CDCl3, 300 MHz) (δ, ppm): 0.88 (t, 3J ) 6.9 Hz, 3H, 18-H), 1.20-1.40 (m, 20H, 4-H-10-H,

Overs et al. 15-H-17-H), 1.55-1.71 (m, 2H, 3-H), 1.88-2.07 (m, 4H, 11-H, 14-H), 2.34 (t, 3J ) 7.4 Hz, 2H, 2-H), 5.32-5.45 (m, 2H, 12-H, 13-H). 13C NMR (CDCl3, 75.4 MHz) (δ, ppm): 14.1 (q, 18-C), 22.5 (t, 17-C), 24.9, 29.1, 29.4, 29.5, 31.4, 32.6, 34.1, 34.1 (8 t, 2-C11-C, 14-C-16-C), 130.3 (d, 12-C, 13-C), 179.4 (s, 1-C). MS (GC/ MS (as trimethylsilyl ester)) [m/z (%)]: 355 (10), 339 (54), 264 (17), 129 (91), 117 (91), 75 (90), 73 (100), 55 (83), 41 (95). Methyl trans-12-octadecenoate (15). trans-12-Octadecenoate (14) (1.130 g, 4 mmol) was dissolved in methanol (20 mL). 2,2-Dimethoxypropane (10 mL) and 3 drops of hydrochloric acid pa were added. The mixture was stirred for 24 h at room temperature and the solvent removed in vacuo. The residue was then filtered over silica gel (1:1 petroleum ether/diethyl ether) to obtain 15 as a colorless oil (1.185 g, 4 mmol, 100%). Anal. Calcd (found): C, 76.30 (76.96); H, 12.49 (12.24). FT-IR (ν˜ , cm-1): 2925, 2854 (s, CH), 1744 (s, CdO), 1464 (m, δ, CH), 1436 (m, δ, CH), 1170 (m, COC), 967 (m, δ, dCH). 1H NMR (CDCl3, 300 MHz) (δ, ppm): 0.88 (t, 3J ) 6.9 Hz, 3H, 18-H), 1.22-1.39 (m, 20 H, 4-H-10-H, 15-H-17-H), 1.62 (mc, 2H, 3-H), 1.97 (mc, 4H, 11-H, 14-H), 2.30 (t, 3J ) 7.6 Hz, 2H, 2-H), 3.66 (s, 3H, OCH3), 5.39 (mc, 2H, 12-H, 13-H). 13C NMR (CDCl3, 75.4 MHz) (δ, ppm): 14.0 (q, 18-C), 22.5 (t, 17-C), 25.0, 29.1, 29.2, 29.3, 29.4, 29.5, 29.7, 31.4, 32.6, 34.1 (10 t, 2-C-11-C, 14-C-16-C), 51.4 (q, OCH3), 130.4 (d, 12-C, 13-C), 174.3 (s, 1-C). MS (GC/MS) [m/z (%)]: 296 (20), 265 (36), 264 (70), 222 (23), 180 (20), 111 (22), 97 (54), 87 (42), 74 (56), 69 (80), 55 (100), 41 (58). Methyl rac-threo-12,13-Dihydroxyoctadecanoate (4). A mixture of potassium hexacyanoferrate(III) (1.000 g, 3 mmol), potassium carbonate (0.380 g, 2.7 mmol), and methylsulfonamide (0.095 g, 1 mmol) in water (5 mL) and tert-butyl alcohol (5 mL) was stirred at room temperature until both phases were clear. Then osmium tetraoxide (0.120 mL, 2.5% in tert-butyl alcohol) and methyl trans-12-octadecenoate (15) (0.297 g, 1 mmol) were added. After being stirred for 24 h at room temperature the reaction was quenched by addition of Na2SO3 (1.500 g, 12 mmol). The mixture was again stirred for 30 min and then extracted three times with ethyl acetate (15 mL). The organic layer was washed with 2 N NaOH (15 mL), dried (MgSO4), and evaporated in vacuo. After purification by flash chromatography (2:5 ethyl acetate/cyclohexane) the dihydroxy ester 4 is obtained as a white powder (0.320 g, 1 mmol, 97%). Mp: 340 K. Anal. Calcd (found): C, 68.91 (69.05); H, 11.61 (11.59). FT-IR (ν˜ , cm-1): 3302 (s, br, OH), 2914, 2848 (s, CH), 736 (S, CdO), 1468 (s, δ, CH), 1437 (m, δ, CH), 1204 (m, C-O), 1179 (m, C-O), 1075 (m, C-O), 721 (CHrocking). 1H NMR (CDCl3, 300 MHz) (δ, ppm): 0.89(t, 3J ) 6.7 Hz, 3H, 18-H), 1.22-1.65 (m, 26 H, 3-H-11-H, 14-H-17-H), 2.00 (s, 2H, OH), 2.30 (t, 3J ) 7.5 Hz, 2H, 2-H), 3.40 (m, 2H, 12-H, 13-H), 3.66 (s, 3H, OCH3). 13C NMR (CDCl3, 75.4 MHz) (δ, ppm): 14.0 (q, 18-C), 22.6 (t, 17-C), 24.9, 25.3, 25.6, 29.1, 29.2, 29.4, 29.5, 29.6, 31.9, 33.7, 34.1 (11 t, 2-C-11-C, 14-C-16-C), 51.4 (q, OCH3), 74.5 (d, 12-C, 13-C), 174.3 (s, 1-C). MS (GC/MS CI) [m/z (%)]: 348 (100), 330 (78), 313 (60), 281 (20), 246 (14), 124 (6), 109 (3).

Acknowledgment. This work was supported by the Deutsche Forschungsgemeinschaft as a contribution from the Sonderforschungsbereich SFB 424. LA990655H