Monopolar Conformational Transitions of Selected

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Bipolar/Monopolar Conformational Transitions of Selected Hydroxyoctadecanoic Acids and Esters: A Fluorescence Microscopy Study B. Asgharian† and D. A. Cadenhead* Department of Chemistry, State University of New York at Buffalo, Natural Sciences Complex, Buffalo, New York 14260-3000 Received March 24, 1999. In Final Form: September 7, 1999 The bipolar/monopolar conformational transitions of the hydroxyoctadecanoic acids (HOAs) [6-hydroxy9-hydroxy- and 12-hydroxyoctadecanoic acid (6HOA, 9HOA, and 12HOA)] plus those of the methyl and ethyl esters of 12HOA have been studied using classical surface pressure (Π)/area per molecule (A) isotherms and fluorescence microscopy techniques. Most studies were carried out using identical compressional rates (5 Å2 mol-1 min-1) and identical amounts of a fluid-phase soluble fluorescent probe 1-palmitoyl-2-[6-(7nitro-2-1-3-benzoxadiazol-4-yl)amino] caproyl phosphatidylcholine (NBD-PC); however, the effect of both compressional rate and probe concentration was investigated. For 6HOA, condensed-phase domains are roughly diamond-shaped with both nucleation and apparent crystal growth being observed. Larger, essentially hexagonal, crystals were observed for 9HOA. For 12HOA the extent of nucleation was much greater, producing elongated needlelike crystals quite different from either 6- or 9HOA. This difference is ascribed to a greater stability of the condensed state, possibly due to better packing and enhanced hydrogen bonding. The highly brittle nature of the needlelike crystals support this interpretation. On the basis of the π/A isotherms the 12HOA methyl ester condensed state shows little indication of diminished stability vis-a`-vis 12HOA, but the ethyl ester does. However, both esters show substantially larger crystals, indicating that growth is favored over nucleation, and it is proposed that the reduced hydrogen bonding of the ester polar group is a primary cause.

Introduction In recent years the direct observation of the microstructure of monomolecular films has been made possible by fluorescence microscopy,1-4 phase contrast microscopy,5,6 and Brewster angle microscopy (BAM).7,8 These techniques have the advantage of permitting such studies to be carried out at the air/water interface where phase changes and surface pressure (π)/area per molecule (A) isotherms can be monitored simultaneously. Fluorescence microscopy utilizes trace amounts of a fluorescent probe, which is preferentially soluble in the fluid phase of a biphasic region. Fluorescence microscopy of phase transitions in monomolecular films of fatty acids and phospholipids with long hydrocarbon chains have been demonstrated to show liquid condensed/liquid expanded (LC/LE) transitions, resulting in condensed-phase domains that frequently have rounded boundaries.9 The shapes of such domains have been explained in terms of a competition between electrostatic forces between headgroups promoting crystalline growth and the effects of line tension.10-12 In turn, line tension may be affected by the presence of impurities,13 including that of the fluorescent probe itself. * To whom all correspondence should be addressed. † Present address: Alcon Laboratory, Ft. Worth, TX 76134. (1) Lo¨sche, M.; Sackmann, G.; Mo¨hwald, H. Ber. Bunsen-Ges. Phys. Chem. 1983, 87, 848. (2) Peters, R.; Beck, K. Proc. Natl. Acad. Sci. U.S.A. 1983, 80, 7183. (3) Weis, R. M.; McConnell, H. M. Nature (London) 1986, 310, 47. (4) Knobler, C. M. Science 1990, 249, 870. (5) Fisher, A.; Lo¨sche, M.; Mo¨hwald, H.; Sackmann, E. J. Phys. Lett. 1984, 45, 785. (6) Mo¨hwald, H. Thin Solid Films 1988, 159, 1. (7) Angelova, A.; Vollhardt, D.; Ionov, R. J. Phys. Chem. 1996, 100, 10710. (8) McConlogue, C. W.; Vanderlick, T. K. Langmuir 1997, 13, 7158. (9) Asgharian, B.; Cadenhead, D. A.; Tomoaia-Cotisel, M. Langmuir 1993, 9, 228.

In this publication we extend previous studies of selected bipolar lipids to better understand the formation and nature of condensed-phase domains and their relation to molecular packing. The selection of a series of racemic hydroxyoctadecanoic acids (HOAs) and esters was made for two reasons. First of all, as is indicated in Figure 1, they exhibit a large area/molecule change (much larger than typical LC/LE transitions). Second, they represent one of the simplest examples of bipolar/monopolar transitions. Thus these lipids show a fascinating conformational transition from an expanded fluid state, where both polar groups are in the air/water interface, to an erect condensed state, where the hydroxyl group is lifted out of the interface. A highly expanded bipolar state, however, is only obtained when the hydroxyl and carboxyl groups are sufficiently separated. Previous studies of racemic hydroxyhexadecanoic acids (HHAs) and their methyl esters14,15 show monopolar amphipathic film-like behavior when the hydroxyl group is positioned at the second or third carbon. Thus, only a normal LC/LE transition occurs because the hydroxyl and carboxyl polar groups act as one combined polar group. In contrast, when the hydroxyl group was positioned beyond the fifth or sixth carbon position the molecules showed bipolar behavior in the expanded state and underwent a conformational change. We were also interested in investigating the possibility that the presence of the hydroxyl group could increase (10) McConnell, H. M.; May, V. T. J. Phys. Chem. 1988, 92, 4520. (11) Keller, D. J.; McConnell, H. M.; Moy, V. T. J. Phys. Chem. 1986, 90, 2311. (12) Moy, V. T.; Keller, D. J.; McConnell, H. M. J. Phys. Chem. 1988, 92, 5233. (13) Heckl, W. M.; Cadenhead, D. A.; Mohwald, H. Langmuir 1088, 4, 1352. (14) Kellner, B. M. J.; Cadenhead, D. A. J. Colloid Interface Sci. 1978, 63, 452. (15) Kellner, B. M. J.; Cadenhead, D. A. Chem. Phys. Lipids 1979, 23, 41.

10.1021/la990348b CCC: $19.00 © 2000 American Chemical Society Published on Web 10/28/1999

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Experimental Section 6-Hydroxyoctadecanoic acid (6HOA) and 9-hydroxyoctadecanoic acid (9HOA) were purchased from NU-check Prep. 12HOA, 12-hydroxyoctadecanoic acid methyl ester (12HOAME), and 12hydroxyoctadecanoic acid ethyl ester (12HOAEE) were obtained from Sigma. These samples were used as supplied when they checked out as better than 99% pure. The fluorescence probe 1-palmitoyl-2-[6-(7-nitro-2-1-3-benzoxadiazol-4-yl)amino] caproyl phosphatidylcholine (NBD-PC) was purchased from Avanti Polar Lipids. All of these compounds were dissolved in either chloroform or 9:1 volume mixtures of chloroform/MeOH. The substrate in all measurements was quadruply distilled water (pH ) 5.6). A separate computer-interface system for detailed isotherm evaluation has already been described elsewhere.20 The fluorescence microscopy film balance consisted of a modified Nikon microscope with a fluorescence attachment powered by a 75 W xenon arc lamp, set on the stage of a Teflon trough of dimensions 12 × 32 × 1 cm. The resulting images were directed toward a SIT 66 series model camera (DAGE-MTI) and were subsequently stored on a Phillips VCR and printed by a Mitsibishi video copy processor. To ensure isotherm correspondence the surface pressure was monitored by a Wilhelmy plate suspended from a Cahn electrobalance. In most experiments, a Teflon collar was placed around the objective. The collar was extended below the air/water interface to reduce both subphase flow and air turbulance, respectively. It had a 2-mm slit to permit surface pressure equilibrium to be achieved. The films were compressed at rates 0.2-1.0 cm/min (5 Å2 mol-1 min-1) with the help of a motor-driven movable barrier.

Results and Discussion Figure 1. Surface pressure (π)/area per molecule (A) isotherms of 6-, 9-, and 12-hydroxyoctadecanoic acid (HOA) at 22.0 °C, showing the bipolar/monopolar phase transition.

molecular interactions in the condensed state through hydrogen bonding. In addition, the location of the hydroxyl group can affect the flexibility and tilt of the fatty acid chains, resulting in a more rigid condensed state. 12-Hydroxyoctadecanoic acid (12HOA) and its esters were previously examined16 as both pure and mixed monolayer films. The acid and its methyl ester exhibited near-ideal mixed film behavior, but the acid/ethyl ester showed significant deviations from ideality. Transmission electron microscopy studies of collapsed films of racemic 12-HOA showed platelike aggregates, whereas helical aggregates were observed with the corresponding optically active compounds.17 More recently, Sakai and Umemura18 have shown that in the biphasic region, the tilt of the acyl chains from the normal is reduced from 55° to 28° during compression. In addition, Jacobi et al. have studied the behavior of monolayers of methyl vic-dihydroxyoctadecanoates, obtaining compressional isotherms, studying relaxation behavior and BAM micrographs.19 These latter investigators found that the methyl erythro-9,10-dihydroxyoctadecanoate (E9,10DHOAME) should pack better than the methyl threo-9,10-dihydroxyoctadecanoate (T9,10DHOAME), and the enantiomers of the acid (T9,10DHOA) do not form stable monolayers. It was also found that the enantiomers of E9,10DHOAME had significantly lower transition pressures to a condensed state. Although both the enantiomers and the racemic form had similar limiting areas, the lower transition pressures of the former suggest that they pack more easily and showed similar feather-like BAM micrographs, but in an opposite sense. (16) Matuo, H.; Rice, D. K.; Balthasar, D. M.; Cadenhead, D. A. Chem. Phys. Lipids 1982, 30, 367. (17) Tachibana, T.; Hori, K. J. Colloid Interface Sci. 1977, 61, 398. (18) Sakai, H.; Umemura, J. Langmuir 1988, 14, 6249. (19) Jacobi, S.; Plate, M.; Overs, M.; Schafer, H.-J.; Fuchs, H. Thin Solid Films 1998, 327-329, 180.

The Π/A isotherms of 6-, 9-, and 12HOAs are shown in Figure 1. The lower area limit of the fluid expanded phase in all three HOAs correlates well with that of a molecular model in which both the hydroxyl and carboxylic acid groups are in contact with the water. Upon compression, the hydroxyl group is lifted away from the interface and a condensed state is formed. The limiting area of the condensed state at zero pressure is about 24 Å2/molecule for all three molecules, which is somewhat larger than that of a vertically oriented close-packed hydrocarbon chain. From this it is clear that the hydroxyl group does create a small chain-packing problem in the condensed state. Moreover, the decreasing transition pressure with higher hydroxyl chain substitution shows that the stability of the expanded state vis-a`-vis the condensed state decreases in the order 6HOA > 9HOA > 12HOA. This does not necessarily indicate an increasing stability of the condensed states of 6HOA < 9HOA < 12HOA, particularly with near-identical condensed areas/molecule. Nevertheless an equilibrium spreading pressure (ESP) study of microcrystals of HHAs spread at the air/water interface showed that the stability of the crystals was greater, that is, the ESP values decreased as the separation of the hydroxyl and carboxyl groups increased.14 Fluorescence microscopy should throw additional and significant light on this transition. The fluorescence probe used (NBD-PC) is soluble in the fluid phase and insoluble in the solid phase and has an overall concentration in each case of 1%. It is important to note that within the concentration range of 0.5 to 2.0% dye, there was no observable effect on the size or shape of the resultant condensed domains. Fluorescence micrographs of 6HOA are shown in Figure 2. Soon after the onset of the transition, the first solid nuclei are observed in a homogeneous fluid phase media (top left). The initial nonuniform crystal size distribution is a consequence of simultaneous nucleation and growth (20) Asgharian, B.; Cadenhead, D. A. J. Colloid Interface Sci. 1989, 134, 522.

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Figure 2. Fluorescence micrographs of 6HOA at 22.0 °C at various stages beyond the onset of phase transition. (See the text for discussion.)

and arose through a crystal growth rate that was slower than the compressional speed (5 Å2 mol-1 min-1). The faster the compression rate the greater the nucleation rate. The apparently crystalline diamond-like shape of the domains is clearly seen in Figure 2 (top center). Whereas at larger areas/molecule the domains gave no indication of long-range interactions, at lower areas/ molecule (top right and lower left) they show a much more uniform superstructural packing, indicating long-range electrostatic repulsions that ultimately determine the average domain size.21,22 At this point (top right) crystals appear to grow primarily along the short axis of domains; however, the growth that does occur along the long axis is not quite uniform, resulting in ridges at each end. Because such growth is also dependent on the rate of impurity (dye) diffusion away from the crystal faces, defects are observed at the boundaries (top right through lower right). Such diffusion also results in uneven growth along the short axis in some domains. Further compression will eventually overcome electrostatic repulsion and result in domain reorientation and binding (lower right). Here the domains, for the most part, align along the long axis because of the in-plane component of the molecular dipoles aligning along this axis. The presence of ridges, however, suggests that the dipoles make a slight angle to the long axis and this prevents the two faces from meeting. Although this seems to be a reasonable explanation, supporting evidence needs to be obtained from synchrotron X-ray diffraction studies. Figure 3 shows the fluorescence micrographs of 9HOA, which show generally similar patterns of nucleation, growth, and domain binding. However, here the crystals assume either dendritic or hexagonal shapes. Furthermore, the average domain size is larger than that for 6HOA even though the compression rate is the same (5 Å2 mol-1 min-1). It is seen that when the crystal reaches a critical size, elongation along the edges of the hexagon initiates (21) Lo¨sche, M.; Duwe, H.-P.; Mo¨hwald, H. J. Colloid Interface Sci. 1988, 126, 432. (22) Lo¨sche, M.; Mo¨hwald, H. J. Colloid Interface Sci. 1989, 131, 56.

Figure 3. Fluorescence micrographs of 9HOA at 22.0 °C at various stages beyond the onset of phase transition. (See the text for discussion.)

(top right through lower right). It is interesting to note that smaller domains do not lose symmetry, even at relatively low area/molecule where the domains begin to bind together (lower right). Higher compressional rates result in elongation along all six edges; however, a sixfold symmetry with 60° angles is still maintained. Such elongated crystals anneal in about 1 h to a somewhat more rounded shape similar to those found in Figure 3 (lower left), but with slightly sharper corners. These observations and the ESP studies14,15 and those of monoglycerides23 lead us to conclude that most condensed domains should be regarded as metastable. On the basis of the results for 6- and 9HOA, it is clear that factors other than the compressional speed and temperature can affect the domain size. Here, for example, (23) Balthasar, D. M.; Cadenhead, D. A. J. Colloid Interface Sci. 1985, 107, 567.

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Figure 4. Fluorescence micrographs of 12HOA at 22.0 °C at various stages beyond the onset of phase transition. (See the text for discussion.)

a hexagonal domain can grow to a larger size along all six sides as opposed to the four sides of a diamond-shaped domain. These differences in crystal habit must reflect differences at the molecular level. Figure 4 shows fluorescence micrographs of 12HOA. Here the domains are elongated primarily along one axis. Even at compressional rates of about 5 Å2 mol-1 min-1 there is a much larger number of nuclei, which in turn limits the domain size (top left), however, further compression does result in some growth in both length and the thickness of the domains. Figure 4 (lower left) shows that the solid-phase domains exhibit repulsion at low separations. Further compression beyond this point results in the breaking up of the condensed domains into smaller fragments. Compressional speeds even slower than that cited above failed to increase the domain size significantly. It is not clear why 12HOA shows such pronounced elongation while 6HOA and 9HOA do not. The isotherms suggest that 12HOA may have a more stable condensed state but not why. Fosbinder and Rideal24 measured the surface potential of 4HOA, and interpreted this as indicating that the hydroxyl group dipole is primarily oriented in the plane of the interface, and contributes very little to the vertical component of dipole moment. Studies of some isomeric HHAs by Kellner and Cadenhead14,15 indicated that the vertical component of the dipole moment (∆V) is greater at the 9- than the 16-position, suggesting that 6HOA and 9HOA may have smaller ∆V values than 12HOA. Although the surface potential measurements contain a presumably fixed contribution from the substrate water dipoles, when the hydroxyl group is removed from carboxylic acid group the overall change is directly related to the location and orientation of the hydroxyl group. In addition, the portion of the chain above the hydroxyl group may tilt at an altered angle to that below, with the degree of tilt decreasing as the perturbing group shifts away from the center of the chain. This in turn could promote elongation along the direction of the chain tilt. In the case of 12HOA, the condensed-phase domains are quite rigid and crystalline and resist deformation despite the relatively thin width of only a few micrometers. When a sufficient strain is imposed, fracturing occurs. This is consistent with the electron mi(24) Fosbinder, R. J.; Rideal, E. K. Proc. R. Soc. London 1933, A143, 61.

Figure 5. π/A isotherms of 12HOAME and 12HOAEE at 22.0 °C.

croscopy study of Tachibana and Hori17 of a collapsed film of racemic 12HOA. Better packing and possible hydrogen bonding could give rise to a more rigid 12HOA film. Certainly the HHA/ESP data14 are consistent with greater hydrogen bonding occurring with increasing hydroxyl/ carboxyl separation. The condensed-phase domain shape of phospholipids and fatty acids with long hydrocarbon chains frequently show circular, rounded boundaries. This arises through line tension effects due to the gellike nature of these domains. Synchrotron X-ray diffraction studies25 indicate low positional order in the condensed state of such films. Although crystalline solid domains with well-defined edges have been previously reported,26,27 symmetric crystals, similar to three-dimensional crystals, such as shown here, are very rare. In the case of the hydroxy fatty acids the improved molecular interactions and resultant crystalline structure and shape appear to be partially due to the presence and location of the hydroxyl group, and are not completely determined by some combination of electrostatic forces from neighboring domains and the fluid matrix surrounding the crystal domains. It is clear that the domains seen here have sufficient lattice energy to overcome the effects of such free energy-reducing forces as line tension and that the shape of the crystals is eventually determined by the lattice packing. The final habit is a compromise between electrostatic forces and molecular orientation and packing of headgroups and chains. The crystal symmetry shown here for all three HOAs arises from the symmetry of the molecules themselves. As the molecules’ asymmetry is increased, the tendency to form more extended domains is also expected to increase. (25) Kjaer, K.; Als-Nielson, J.; Helm, C. A.; Tippman-Krayer, P.; Mo¨hwald, H. Thin Solid Films 1988, 159, 17. (26) Gobel, H. D.; Mo¨hwald, H. Thin Solid Films 1988, 159, 63. (27) Mo¨hwald, H. Angew Chem. 1988, 100, 750.

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Figure 6. Fluorescence micrographs of 12HOAME at 22.0 °C showing elongated domains (top left), typical “y-type” linear elongation at slower speeds 5-10 Å2 mol-1 min-1 (top center and right), and fractured domains at higher compression speeds (bottom left and right).

The isotherms of 12HOAME and 12HOAEE are shown in Figure 5. As is expected and is evident from the isotherms, there is not much difference between the molecular areas in the fluid and in the condensed states of the two esters. The condensed areas of both esters are similar to that of 12HOA, but both fluid-state areas are significantly greater. There is, however, a distinct difference in the surface pressure at which the transition initiates for the two esters. That of the methyl ester is very similar to that of 12HOA, whereas that of the ethyl ester is significantly higher, indicating the decreased stability of the condensed state due to ethyl esterification. Fluorescence microscopy shows a resemblance in crystal shape of the two esters. Figure 6 shows fluorescence micrographs of 12HOAME with typically large elongated domains obtained at slow compressional speeds (top left) of >10 Å2 mol-1 min-1 . At faster compressional speeds, branching along the main axis is observed (top center). Generally, growth along both axes is observed at different rates. Along the axis, where the crystalline domains grow more rapidly, this results in ridges, as is illustrated in Figure 6 (top center and top right). These ridges may well be generated by differing growth rates parallel and perpendicular to each other, and may eventually result in the observed split ends (see also Figure 2). The latter shows typical domains of several millimeters long and a few hundred micrometers breadth. At similar compressional rates 12HOAEE produced similar but even larger domains (not illustrated). The decreased stability of the condensed state of ethyl ester of 12HOA could give rise to decreased nucleation and increased size, but the same kind of isotherm consideration does not explain the larger size of 12HOAME crystals. On the other hand, calculated vertical dipole moments of methyl esters of HHAs by Kellner and

Cadenhead15 show a more-than-two-fold increase over the comparable hydroxy fatty acids in the condensed state. Similar changes in the ESP values also occur on esterification of the HHAs,15 a clear indication that hydrogen bonding does play a role in the stabilities of the crystalline forms of these acids and is substantially reduced when esterification takes place. One thing that is clear about esterification is that the hydrogen bonding capability of the primary polar group is dramatically decreased. It would seem that such hydrogen bonding must play a major role in nucleation. Although crystal elongation is expected to be directly related to the degree of hydrogen bonding and the way the molecular dipoles align, the final shape of the elongated domains should presumably be looked at in terms of symmetry considerations. For 12HOAME and 12HOAEE any increase in asymmetry would hinder but not eliminate elongation. As molecular asymmetry is increased, the tendency to form extended domains with increasing out-of-plane growth should also increase. A more extended study of other members of various hydroxy fatty acid/ester series should throw additional light on the effect of their various microstructural order parameters. We intend to complement fluorescence microscopy with both Brewster angle microscopy28 and X-ray diffraction studies. Acknowledgment. We would like to acknowledge the financial support of the National Institutes of Health Division of Research Resources through the Biomedical Research Support Grant Program and grant BSRG 507 RR 07066 in the completion of this work. LA990348B (28) Henon, S.; Meunier, J. Thin Solid Films 1993, 234, 471.