Langmuir 1994,10, 3787-3793
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Fluorescence Microscopy Study of Chiral Discrimination in Langmuir Monolayers of N-Acylvaline and N-Acylalanine Amphiphiles Dennis P. Parazak, Jack Y.-J. Uang, Ben Turner, and Keith J. Stine* Department of Chemistry, University of Missouri-St. Louis, St. Louis, Missouri 63121 Received March 31, 1994. In Final Form: July 14, 1994@ Monolayers of L- and of racemicN-palmitoylvaline, N-stearoylvaline,andN-palmitoylalanine have been investigated using fluorescencemicroscopy and surface pressure isotherm measurements. The condensed phase domains formed from the racemic films of the valine derivativesexhibitedlarge arms curved clockwise or counterclockwisewith equal probability. Very rapid compressionresulted in the formation of globular domains in the racemic films of the valine derivatives. In contrast, the condensed phase domains formed from the racemic films of the alanine derivative were irregular and branched, indicating a sensitivity to the head-group size. The monolayers of the pure L enantiomers of all these compounds exhibited dendritic growth of the condensed phase.
Introduction The investigation of Langmuir monolayers provides a n opportunity to study the dependence of phase behavior and packing on molecular structure as the amphiphiles are oriented a t the interface and the surface density can be continuously varied over a wide range in the monolayer regime. Effects due to the molecular feature of chirality have been investigated in monolayers of a number of classes of compounds using surface pressure (n)versus area per molecule (A) isotherm measurements, generally by comparing racemic and enantiomeric fi1ms.l Differences as a function of enantiomeric content of the film are possible and constitute examples of “chiral discrimination”, a phenomenon for which there are many examples in the solid state.2 In monolayers, differences arise for the condensed phases where chirality-dependent interactions become significant. In the case of a molecule with a single chiral center, these differences can be either heterochiral, for which the D:L interaction is more favorable, or homochiral, for which the L:L (or D:D)interaction is more favorable. The “tripod amphiphile” model of Andelman is the sole theoretical treatment of these effects in monolayer^.^-^ The most biologically important class of compounds, the membrane phospholipids, have not been found to exhibit any differences in their thermodynamic behavior;6 however, a structural difference between enantiomeric and racemic monolayers of dipalmitoylphosphatidylethanolamine has recently been found in synchrotron X-ray diffraction experiments.’ Fluorescence microscopy studies of dipalmitoylphosphatidylcholine monolayers have shown that molecular chirality can be manifested as a macroscopic curvature in micrometer-sized condensed phase domains.8-10 The curvature is attributed to an
asymmetric line tension, with the curvature tending to shorten the domain edge of greater line tension. In these studies, the racemic film showed no chiral structures.9 The diacetylenic phospholipid 10,12-tricosadiynoic-snglycerophosphocholine (DCB,gPC), of considerable technological importance given its capability of forming tubular microstructures,ll exhibited chiral phase separation during the formation of tubules and bilayer helices from racemic solutions.12 Lefi- and right-handed helices were observed to form from a racemic solution in these experiments; in addition, DSC scans across the chain melting transition depended on the enantiomeric content. Monolayers of the L enantiomer of DC8,gPC were recently observed to exhibit counterclockwise curved needlelike crystalline domains.13 Mirror image oblique lattice structures were recently observed in atomic force microscopy studies of a racemic Langmuir-Blodgett monolayer film of a chiral tetracyclic alcoh01.l~Chiral symmetry breaking has also been observed in polarized fluorescence microscopy studies of Langmuir films of saturated fatty acids in the tilted hexatic L1’ phase, for which the structure is inherently chiral.15J6 Amino acid derived amphiphiles have been the most studied class of chiral compounds and the observed chiral discrimination effects can be quite large. Chiral discrimination in monolayers17 and in micelleP of N-acyl amino acid amphiphiles has been attributed to differing extents of amide-amide hydrogen bond network formation. A complication in the study of monolayers of these compounds is the occurrence of nonequilibrium effects in the thermodynamic behavior as evidenced by the persistently observed hysteresis in the ll-A isotherms. It should be noted that compression rate dependence or compression-expansion hysteresis has been observed for all monolayers thus far reported to exhibit chiral discrimina-
* Abstract published in Advance ACS Abstracts, September 1, 1994. (1)Stewart, M.; Amett, E. M. In Topics in Stereochemistry; Eleil, E. L., Allinger, N. L.,Eds.; Wiley: New York, 1982;Vol. 13,p 195. (2)Jacques, J.; Collet, A.;Wilen, S. A. Enantiomers, Racemates, and Resolutions; Wiley: New York,1981. (3)Andelman, D. J.Am. Chem. SOC.1989,111, 6536. (4)Andelman, D. Physica A 1990,168,172. (5)Andelman, D.; Orland, H. J.Am. Chem. SOC.1993,115,12322. (6)Amett, E. M.; Gold, J. M. J . Am. Chem. SOC.1982,104,636. (7)Bohm, C.; Mohwald, H.; Leiserowitz, L.; Als-Nielsen, J.; Kjaer, K. Bwphys. J . 1993,64,553. (8)Weis, R. M.; McConnell, H. M. Nature 1984,310,47. (9)Weis, R. M., McConnell, H. M. J.Phys. Chem. 1985,89, 4453. (10)McConnell, H.M. Annu. Rev. Phys. Chem. 1991,42,171.
(11)Schnur, J. M. Science 1993,262,1669. (12)Singh, A.;Burke, T. G.; Calvert, J. M.; Georger, J. H.; Herendeen, B.; Price, R. R.; Choen, P. E.; Yaeger, P. Chem. Phys. Lipids 1988,47, 135. (13)Bourdieu, L.;Chatenay, D.; Daillant, J.; Luzet, D. J.Phys. 11 1994, 4 , 37. (14)Eckhardt, C. J.; Peachey, N. M.; Swanson, D. R.; Takacs, J. M.; Khan, M. A.; Kim, J. H.; Wang, J.; Uphaus, R. A.Nature 1993,362,614. (15)Qiu, X.;Ruiz-Garcia, J.;Knobler, C. M. Mater. Res. Symp. Ser. 1992,237,263. (16)Selinger, J. V.;Wang, Z.; Bruinsma, R. F.; Knobler, C. M. Phys. Rev. Lett. 1993,70,1139. (17)Hanrey,N.G.;Amett,E.M.;Mirajovsky,D.;Rase,P.L.;Verbiar, R.; Amett, E. M. J.Am. Chem. SOC.1989,111,1115. (18)Shinitsky,M.;Haimovitz,R.J.Am.Chem.Soc. 1993,115,12545.
0 1994 American Chemical Society 0743-7463/94/2410-3787$04.50/0
3788 Langmuir, Vol. 10,No. 10, 1994
tion. We recently reported evidence from fluorescence microscopy for chiral phase separation in a racemic monolayer ofN-stearoylserine methyl ester,lga compound first studied by Arnett and co-workers.17 "he evidence reported was the observation of equal numbers of oppositely curved domain segments during compression of a racemic monolayer. In the studies of glycine crystal growth under monolayers reported by Landau and coworkers, the observation of both crystal orientations of glycine under a racemic film was interpreted as evidence for chiral phase separation in the monolayer.20 ll-A compression isotherm data for the compound N-myristoylalanine have been reportedz1 and there has been a study of dendritic growth of D-myristoylalanine monolayers.zz In this paper, we report a fluorescence microscopy study of monolayers of N-acylalanine and N-acylvaline derivatives. The formation of chiral domains in racemic monolayers of N-palmitoylvaline and N-stearoylvaline is observed but is not observed in the racemic monolayers of N-palmitoylalanine. This result indicates a structural difference between the condensed phases of the monolayers of the N-acylvaline and N-acylalanine compounds, possibly arising from the larger valine head-group favoring hydrocarbon chain tilt.
Experimental Section 1. Isotherm Measurements. Isotherms were measured in a Teflon trough enclosed in a Plexiglas housing. The surface pressure was measured by the Wilhelmy plate method using a Cahn Model 25 electrobalance and a small piece of filter paper as the Wilhelmy plate. The BCD output from the electrobalance was read by a PCL-720 32 bit digital I/O card (B&CMicrosystems) in a PC 386 computer. ATeflon barrier on a threaded rod attached to a universal joint was driven by avariable stepper motor. During an isotherm run, mass versus time data were recorded and subsequently converted t o surface pressure (lT)versus area per molecule (A). 2. Fluorescence Microscopy. A Teflon trough fitted onto the stage of an Olympus BH-2 microscope was utilized. A DageMTI SIT-68 camera mounted onto the microscope was used to observe the monolayers through 20 x or 40 x ultralong working distance objectives (Olympus CDPLANBO and CDPLAN40).The probe fluorescence was excited using a mercury lamp. Polarized fluorescence microscopy23 using the 488-nm line of a 150 mW argon ion laser (Omnichrome) was also carried out, but no fluorescence anisotropy was observed in the experiments on these monolayers. The temperature was controlledby circulating water through copper tubing sandwiched between two thin copper sheets beneath the Teflon trough. Between the Teflon trough and the upper copper sheet, a layer of six thermoelectric devices (Marlow Industries) was placed to allow convenient adjustment of the water temperature using a power supply. The temperature was measured using a fine thermocouple in a Teflon sheeth inserted through the side of the trough and positioned just beneath the water surface. The area of the monolayer was adjusted using two small Teflon barriers positioned beneath the coverglass. One of these could be pushed forward by moving two thin rods inserted into the edges of the trough. These rods could also be pushed forward at a controlled continuous rate using a linear actuator (Oriel Corporation). The trough coverglass was conductively coated and could be mildly heated using a Variac to burn off condensation. 3. Compound Preparation. D,L-Valine methyl ester hydrochloride and D,L-alanine methyl ester hydrochloride was (19)Stine, K. J.;Uang, J. Y.-H.; Dingman, S. D. Langmuir 1993,9, 2112. (20)Landau, E.M.; Levanon, M.; Leiserowitz, L. Lahav, M.; Sagiv, J.Nature 1986,318, 353. (21)Bouloussa, 0. Dupeyrat,M. Biochim. Biophys. Acta 1988,938,
Parazak et al. obtained from Sigma Chemical Co. (St. Louis, MO). L-Valine methyl ester HC1, Lalanine methyl ester HCl, palmitoyl chloride (98%), and stearoyl chloride (97%) were obtained from Aldrich Chemical Co. (Milwaukee, WI). All reagents were used as received. The procedures given by Zeelen24were followed. The structure of the resulting compounds were verified using 1% and proton NMR on a Varian XI,-300. IR spectra were recorded with a Perkin-Elmer 1600 series FTIR. D,L-Stearoyhaline. D,L-Valine methyl ester hydrochloride (0.876 g) was dissolved into a solution of 1.4 g of potassium carbonate in 10mLofwater, and 10 mL of chloroform was added. A solution of 1.33 g of stearoyl chloride in 10 mL of chloroform was added and the mixture stirred for 1h. The chloroform layer was separated and dried, and the solvent was removed by rotary evaporation. After this step, 1.67 g of D,L-stearoylvaline methyl ester in 10 mL of 1N NaOH was heated to boiling and dioxane was added until a clear solution resulted. After refluxing for 1 h, 95 mL of water acidified with concentrated HC1 was added. The stearoylvaline was extracted with ether. The ether layer was then separated and dried, and the solvent was removed by rotary evaporation. The remaining solid was recrystallized 3 times from ethedethyl acetate (W2)yielding1.32 gofthe product. The melting point for the racemic compound was 103.5-104.5 "C. The characteristic IR absorption bands (in cm-l) were 3332 (N-H), 1710 (C=O in carboxylic acid), 1653 (amide I), and 1543 (amide 11). L-Stearoylvaline. A similar procedure was followed as for the racemic compound. The melting point of the L-stearoylvaline was 88-89 "C. It is interesting to note that the melting point of L enantiomer of N-stearoylvaline is slightly lower than that ofthe L enantiomer ofN-palmitoylvaline, a trend consistent with the reported melting points of enantiomeric (L) N-acylleucine derivatives studied over the range N-decanoyl to N - ~ t e a r o y l . ~ ~ The characteristic IR absorption bands (in cm-l) were 3290 (N-HI, 1711 (C=O in carboxylic acid), 1652 (amide I), and 1541 (amide 11). D,L-Palmitoyhaline. A similar procedure was followed as was used for the preparation of D,L-stearoylvaline. The solvent used for recrystallization was pentane/chloroform (4/7). The melting point was determined to be 99.5-100.5 "C. The Characteristic IR absorption bands (in cm-l) are as follows: 3332 (N-H), 1710 (C=O in carboxylic acid), 1651 (amide I), 1550 (amide 11). L-Palmitoylvaline. A similar procedure was followed as was used for the preparation of D,L-stearoylvaline. The solvent used for recrystallization was pentane/chloroform (2/1). The melting point was determined to be 91-93 "C. The characteristic IR absorption bands (in cm-l) are as follows: 3290 (N-H), 1710 (C=O in carboxylic acid), 1652 (amide band I), 1542 (amide band 11). D,L-Palmitoylalanine. D,L-Alaninemethyl ester hydrochloride (0.72 g) was dissolved in a solution of 1.4g ofpotassium carbonate in 10 mL of water, and 10 mL of chloroform was added. After stirring for an hour, a similar procedure as described for the preparation of the stearoylvaline derivatives was followed. The recrystallization was performed 3 times from ethyl acetate. The melting point was 114.0-115.0 "C. It is interesting to note that the melting points of racemic N-acylvaline derivatives are reported to exhibit a large odd-even alternation, while the melting points of the racemic N-acylalanines increase with hydrocarbon chain length without exhibiting any odd-even alternation.26 The characteristic IR absorption bands were 3314 (N-HI, 1707 (C=O in carboxylic acid), 1637 (amide I), and 1537 (amide 11). L-Palmitoylalanine. By use of the same procedure as in the preparation ofthe racemic form, a melting point of 94.0-97.0 "C was obtained. The characteristic IR absorption bands were 3322 (N-HI, 1706 (C=O in carboxylic acid), 1645 (amide I), and 1537 (amide 11). 4. Monolayer Preparation. The compoundswere dissolved in9:l hexane/ethanol a t concentrations near 5.0 x 1014molecule~ pL-l, resulting in typical deposition volumes in the 20-30 pL range. The solutions for fluorescence microscopy contained 0.5 mol % of the fluorescent probe 4-(hexadecylamino)-7-nitroben-
395.
(22)Akamatsu, S.;Bouloussa, 0.; To, K.; Rondelez, F. Phys. Rev. A 1992. 46.4504.
(23)Moy,V. T.;Keller, D. J.;McConnell, H. M. J . Phys. Chem. 1988, 92,5233.
(24)Zeelen, F.J.;Havinga, E. Recl. Trau. Chim. Pays-Bas 1958,77, 267. (25)Iyer, V. N.;Sheth, G. N.; Subrahmanyam, V. V. R. J . Indian Chem. SOC.1982,59,856.
Study of Amphiphile Monolayers 25 r.
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Figure 1. (a, top) lT-A isotherms of L- and racemic Nstearoylvaline at 18"C on a pH 2 HCl subphase. The solid line represents the data for the monolayer of the L enantiomer, while the dashed line represents the data for the racemic monolayer. The isotherms were measured on compression followed by expansion at a rate of 16 k molecule-' min-l. (b, bottom) Transition pressure II, versus temperature; the trianglesrepresent the data for monolayers of the L enantiomer, while the squares represent the data for the racemic monolayers. zoxa-1,3-diazole(MolecularRobes). The monolayerswere spread using a 25;uL syringe.
Results 1. Isotherm Measurements. Figure l a shows II-A isotherms of L- and of racemic N-stearoylvaline at 18 "C on a pH 2 HC1 subphase measured on compressiod expansion cycles. The isotherm of the racemic monolayer is more expanded than that ofthe enantiomeric monolayer, as expected for a monolayer of a n amphiphile exhibiting homochiral behavior. There is a n overshoot in ll during the compression and hysteresis in the compressiod expansion cycle, as has been observed in several other isotherm studies of this class of compounds. The observation of a n overshoot in the lT-A variation on compression, attributed to slow rearrangement of the head-groups in the condensed phase following nucleation, has previously been observed and studied for substituted azocrown amphiphiles by Mertesdorf and Ringsdorf.26 In monolayers of tetrasubstituted N-myristoylcyclame, it was found that the overshoot vanished when the compression rate was decreased to 1Az molecule-' min-l. In mono(26)Mertesdorf, C.; Ringsdorf, H. Liq. Cryst. 1989,5, 1757.
0 0.0
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1
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0.2 0.3 0.4 MOLE FRACTION OF D ENANTIOMER 0.1
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Figure 2. Transition pressure vs mole fraction of the D enantiomer for mixtures of L and D N-stearoylvaline on a pH 2 HC1 subphase at 18 "C. layers of similar derivatives of hexacyclene, however, a slow enough compression rate to remove the overshoot in Il could not be reached within a time limit practical for isotherm measurements. Compressions of racemic monolayers of N-stearoylvaline a t 18 "C were performed a t several different compression rates in the range 2-15 Az molecule-' min-'. The surface pressure overshoot was observed to diminish as the compression rate was reduced below 10 A2 molecule-l min-' and was absent a t 5 A2 molecule-' min-'. The IT-A compression isotherms gave the same transition pressure observed during the faster compressions. In Figure l a , the expansion cycle does not rejoin the compression exactly at larger area per molecule, suggesting some small loss of material, most likely due to dissolution or partial irreversible collapse, during the compression. The surface pressure of the transition has been taken a s the minimum ll value after the overshoot on compression. The dependence of the transition pressure on temperature for N-stearoylvaline monolayers is shown in Figure lb. The slopes obtained for linear fits to the II versus T data are 1.61 f 0.08 dyn cm-' K-l for the racemic monolayers and 1.50 f 0.10 dyn cm-' K-' for the monolayers ofthe L enantiomer. The corresponding values of TO,the extrapolated temperature (triple point temperature) a t which the transition pressure becomes zero, are 278.8 K for the racemic monolayers and 289.1 K for the monolayers of the L enantiomer. Given the nonequilibrium nature of the isotherms, the calculation of AH values using the two-dimensional Clapeyron equation is not reported. The dependence of ITtr on the enantiomeric mole fraction between XD= 0 and XD= 0.5 at 18 "C is shown in Figure 2. The data shown in Figure 2 show a monotonic, but nonlinear, dependence on enantiomeric mole fraction similar to that reported for N-myristoylalanine21and for two heterochiral monolayer system^,^^*^^ where the transition pressure is a minimum at the racemic composition. Il-A isotherms were also measured for N-palmitoylvaline monolayers, where the transitions occur below room temperature. In Figure 3, l l - A isotherms are shown for N-palmitoylvaline at 10 "C. Figure 4a shows II-A isotherms of L- and of racemic N-palmitoylalanine a t 28 "C on a pH 2 HC1 subphase measured on compressiodexpansion cycles. The crossing of the racemic isotherm to areas lower than the enantiomeric isotherm may or may not be a real effect and is (27) Dvolaitzky,M.;Gudeau-Boudeville,M.Langmuir 1989,5,1200. (28)Stine, K. J.; Whitt, S. A.; Uang, J. Y.-J. Chem. Phys. Lipids 1994, 69,41.
Parazak et al.
3790 Langmuir, Vol. 10,No. 10,1994 35 ~
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Figure 3. ll-A isotherms ofN-palmitoylvaline on a pH 2 HC1 subphase at 10 "C. The solid line represents the data for the monolayer of the L enantiomer, while the dashed line represents the data for the racemic monolayer. The isotherms were measured on compression followed by expansion at a rate of 16 A 2 molecule-1 min-1. 25 n
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Figure 5. Fluorescence micrographs of condensed phase domains in N-palmitoylvaline monolayers on a pH 2 HC1 subphase: (a) single curved arm domain in a racemic monolayer at 5 "C, 36 A2 molecule-'; (b) domain with three arms in a racemic monolayer at 5 "C, 33 A2 molecule-'. The monolayers
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Figure 4. (a) ll-A isotherms of L- and racemic N-palmitoylalanine at 28 "C on a pH 2 HC1 subphase. The solid line represents the data for the monolayer of the L enantiomer, while the dashed line represents the data for the racemic monolayer. The isotherms were measured on compression followed by expansion at a rate of 16 A2 molecule-' min-l. (b) Transition pressure &,. versus temperature; the open circles represent the data for monolayers of the L enantiomer, while the filled circles represent the data for the racemic monolayers.
were observed in stepwise compressions. The monolayers contains 0.5 mol % of fluorescent probe. The bar in the lower left represents 250 pm.
within the reproducibility of the molecular areas for these isotherms. Figure 4b shows the dependence of the transition pressure on temperature. The slopes obtained from linear fits to the data are 1.69dyn cm-l K-l for the racemic monolayers and 2.31 dyn cm-l K-l for the monolayers of the L enantiomer. The correspondingvalues of To are 294.5 K for the racemic monolayers and 302.6 K for the monolayers of the L enantiomer. 2. Fluorescence Microscopy. Examples of condensed phase domains formed on compression of the racemic monolayers of N-palmitoylvaline are shown in Figure 5. The domains are seen to consist of arms, curved clockwise or counterclockwise. We have observed equal numbers of arms curved in either direction. Single arm domains, twins of two arms both curved in the same or opposite directions, and some domains with three arms have all been observed. In Figure 5a, the possible solidlike nature of the growing phase may be inferred from the apparent sharp break in the upper arm of the domain, which seems to have resulted from a collision with the growing lower arm. This was not a common finding in scanning around the trough, but its occurrence provides evidence for the highly ordered nature of the phase being formed. In Figure 5b, the brighter section of liquidexpanded phase between the two upper arms of this threearmed domain must be due to some trapping of the ejected NBD-HDA probe. The same kind of domain structures
Langmuir, Vol. 10, No. 10,1994 3791
Study of Amphiphile Monolayers
, -
Figure 6. Fluorescence micrographs of condensed phase domains in N-stearoylvaline monolayers on a pH 2 HCl subphase: (a) racemic monolayer compresseg at 22 "C to 37 A2 molecule-' at 11 k2molecule-' min-'; (b) racemic monolayer compressed at 22 "C to 33 A2 molecule-' at 24 A2 molecule-' min-'; ( c ) racemic monolayer manually compressed slowly untjl first observation of domain growth at 22 "C to 40 A2 molecule-'; (d) monolayer of L-enantiomer compressed at 22 "C to 35 A2 molecule-' at 2 k2 molecule-' min-'. The monolayer contains 0.5 mol % of a fluorescent probe. The bar in the lower left represents 250 pm.
are observed on cooling (0.3 "Cmin-l) into the coexistence region from the liquid-expanded phase; domains formed on cooling tend to be somewhat larger in size and fewer in number, as expected for nucleation on a calmer water surface. Given that the transition to the condensed phase occurs a t rather low temperatures (at T < 10 "C in the racemic monolayer) in N-palmitoylvaline, N-stearoylvaline was prepared and studied. The transition from the liquidexpanded phase to the coexistence with the condensed phase in the racemic monolayers was studied on compression at 22 "C a t continuous rates ranging from 3 to 25 Hi2 molecule-' min-l and also at 30 "C. The monolayers were also observed on stepwise compressions at the temperatures for which ll-A isotherms were measured. The domain growth exhibits a dependence on the compression rate. Domains formed at two different compression rates a t 22 "C are shown in parts a and b of Figure 6. At a compressionrate of 11A2molecule-' min-l, curved domains form (Figure 6a) from the racemic fluid phase. Increasing the compression rate to 24 Hi2 molecule-l min-l results in the formation of rounded domain structures (Figure 6b). If these monolayers are compressed rapidly, the formation of the curved domain arms can be completely suppressed. Subsequent slower compression results in formation of curved segments on the domain peripheries. An example of a twinned domain in the racemic monolayer a t 22 "C formed by manually moving and then stopping the barrier motion as soon as the nucleation was observed
is shown in Figure 6c. This type of "wishbone" twinned domain shape was very common. Observation of the domains in a racemic film at 22 "C over 3 h revealed surfactant dissolution as evidenced by a decrease of about one-third in the condensed phase area coverage;this effect caused fraying of the domains, but no net relaxation of the overall domain curvature could be seen. Monolayers of the L enantiomer ofN-stearoylvaline were studied on compression at 22 "C and continuous rates ranging from 1to 25 Hi2 molecule-' min-l and also at 27 "C. Again, the monolayers were also observed on stepwise compressions at the temperatures for which ll-A isotherms were measured. The condensed phase domains of the monolayers of the L-enantiomer exhibit dendritic growth forms, as shown in Figure 6d. Monolayers of the L enantiomer of N-palmitoylvaline similarly exhibit dendritic growth forms on compression or cooling, but a t appropriately lower temperatures. Observation of N-palmitoylalanine monolayers surprisingly revealed somewhat different results for the morphologies of the condensed phase domains. Whereas the domains formed from the racemic monolayers of the two valine derivatives exhibited curved arms, the domains formed on compression or cooling of racemic monolayers of N-palmitoylalanine are branched in an irregular manner, as shown in Figure 7a. These films were examined a t continuous compression rates of 2-15 A2 molecule-' min-l a t 30 "C, in stepwise compressions a t the temperatures where ll-A isotherms were measured,
3792 Langmuir, Vol. 10, No. 10,1994
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Figure 7. Fluorescence micrographs of condensed phase domains inN-palmitoylalanine monolayers on a pH 2 HC1 subphase: (a) racemic monolayer compressed at 30 "C to 40 Hir! molecule-' a t 16 k2molecule-' min-'. (b) monolayer of L enantiomer compressed at 35 "C to 41 molecule-' at 2 A2 molecule-' min-'. The monolayer contains 0.5 mol % of fluorescent probe. The bar in the lower left represents 250pm.
A2
and also on coolingfrom the liquid-expanded phase several degrees above the coexistence region. Monolayers of the L enantiomer exhibit regular dendritic growth for the condensed phase on compression or cooling, as shown in Figure 7b. The transition in the monolayers of the L enantiomer was studied a t 35 "C at a compression rate of 2 A2 molecule-' min-l. Dendritic growth has previously been reported and studied quantitatively as to the tip curvature and growth rate relationship for the D enantiomer of N-myristoylalanine.22
Discussion The results presented here for theN-stearoylvaline and N-palmitoylvalinemonolayersprovide additional evidence for the separation of the L and D enantiomers in racemic homochiral films. The domains observed to form from the racemicN-stearoylvaline films on slower compression exhibit arms with clockwise and counterclockwise curvature. This is similar to our previous finding for racemic N-stearoylserine methyl ester monolayers. l9 In these fluorescence microscopy studies, chiral phase separation is inferred via a n argument used to explain the chiral condensed phase domains observed in phospholipid monolayers, where condensed phase domains of R-DPPC (dipalmitoylphosphatidylcholine) curved counterclockwise while those ofS-DPPC curved c1ockwise.*-l0 It was argued that if the molecules of one enantiomer are regularly
oriented throughout the domain, then the two sides of the domain will differ in their line tension against the liquidexpanded phase leading to a tendency to curve the domain so as to reduce the interfacial length of the side of larger line tension. The observation of chiral domains in a racemic film can thus be interpreted as evidence for chiral phase separation in a two-dimensional monolayer; however, a lack of domain curvature cannot be interpreted as indicating that chiral separation has not occurred. If the molecular or crystalline orientation is not uniform over at least a few tens of micrometers, then a net domain curvature would not be observed by fluorescence microscopy even though a chiral separation may have occurred. The lack of uniformity could arise from defects, possibly resulting from incorporation of a fraction of the opposite enantiomer, or from a microcrystalline domain structure. The domains we observe are growth forms, with slower compression rates yielding the chiral forms in the racemic monolayers of the valine derivatives. The compression rate required to observe the chiral forms bears some correspondence with the compression rate below which the overshoot in ll in the isotherm is diminished. These chiral forms have not been observed to relax their curvature during the 2-3 h time periods during which the monolayers were observed. The observation of the globular condensed phase domains on rapid compression for the racemicN-steroylvaline films suggests that forcing the growth of the condensed phase to occur more rapidly can inhibit the separation of the enantiomers on the water surface. Given that the growth of a condensed phase domain or domain segment of one enantiomer from a racemic fluid phase involves the selective attachment of molecules of that enantiomer, the diffusion of the other enantiomer away from the advancing interface of the growing domain is a limiting factor. It is likely that even a small amount of occlusion of the opposite enantiomer into a growing chiral domain will disrupt the structure enough to influence the domain shape. In this regard, it has been reported in grazing incidence diffraction studies of (R)-palmitoyllysine films that addition of 3% of the opposite enantiomer reduced the positional coherence length s ~ b s t a n t i a l l y . ~ ~ The differencesbetween the domain structures observed in the racemic films of the alanine and valine derivatives are quite interesting and can be discussed in light of the solid-state behavior. The differences in the melting point behavior in the racemic solid state indicate a difference in head-group packing between the N-acylalanines and the N - a ~ y l v a l i n e salthough ,~~ crystal structure data are not available. The melting points of the racemic Nacylvalines exhibit a n odd-even alternation with increasing chain length, while the melting points of the N-acylalanines increase monotonically with chain length. In long-chain ethyl esters in the solid state, odd-even effects are observed for melting points and transition enthalpies for phases with tilted hydrocarbonchains, while a smooth monotonic dependence on chain length is observed for phases with vertical hydrocarbon chains.30 In Langmuir monolayers of long chain ethyl esters, oddeven effects in triple point temperatures involving tilted phases are observed.31 This indicates a structural difference between the two condensed phases arising from the larger size of the valine head-group. The larger size of the valine head-group will favor tilting of the hydro(29) Jacquemain, D.; Grayer-Wolf, S.; Leveiller, F.; Deutsch, M.; Kjaer, IC;Als-Nielsen, J.; Lahav, M.; Leiserowitz, L. Angew. Chen., Znt. Ed. Engl. 1992,31, 130. (30) Lundquist, M.Ark. Kemi 1970,32, 27. (31) Lundquist, M.Chem. Scr. 1971, 1, 197.
Study of Amphiphile Monolayers carbon chains,32as suggested by the solid-state observation of odd-even melting point alternation. Uniform tilt order promotes coherence in the crystalline orientation across the domain23 and allows for the observation of the macroscopic curvature arising from the asymmetric line tension. It seems unreasonable to argue that the Nacylvaline films exhibit chiral separation while the N-acylalanine films do not. It is possible to argue that the uniformity of the crystalline orientation does not extend over entire domain segments in the condensed phase of the N-palmitoylalanine films, as it appears to for the condensed phase of the N-acylvaline films. The range of orientation may be reduced or fragmented. The hypothesis that the hydrocarbon chains are tilted in the N-acylvaline condensed phase and vertical in the Nacylalanine condensed phase could be tested by performing Bragg rod scans in grazing incidence X-ray diffraction on these monolayers; Brewster angle microscopy may also prove highly useful on this point. We have not observed any evidence for molecular tilt using polarized fluorescence microscopy with NBD-HDA as the probe, but this may be due to a failure of the probe orientation to couple to the hydrocarbon chain orientation due to the very different nature of the head-groups involved. The monolayers of the pure enantiomers of all the compounds studied here have been found to exhibit dendritic growth of the condensed phase which does not anneal appreciably over observable times, indicating a highly ordered and possibly crystalline condensed phase. The individually curved domains seen in the enantiomeric N-stearoylserine methyl ester monolayers were not observed. In the N-stearoylserine methyl ester monolayers, these domains with curvature unique to the enantiomer were observed on slow or on secondary compressions after the initial explosive dendritic growth. It might be expected that such domains should be observable for the Nacylvaline monolayers. It is likely that we have not been able to access a slow enough compression rate to observe individually curved domains in the enantiomeric films. This difference in behavior must be attributable to some parameter in the nonequilibrium growth for these systems, which is not well understood. A predominant structural feature of the condensed phases of these molecules should be the amide-amide hydrogen bonding network.17J8 The differences in the H-A isotherms between the racemic and enantiomeric films are similar in magnitude for the alanine and valine derivatives, indicating that the larger size of the valine head-group has not reduced the apparent magnitude of the chiral discrimination. Given the nonequilibrium nature of the isotherms, comparisons of calculated thermodynamic quantities using the 2D Clapeyron equation do not seem justified. The observed hysteresis appears to arise primarily from the amide-amide hydrogen bonding resulting in a highly ordered surface phase on compression. Recently we have examined monolayers of N-stearoylglycine methyl ester, in which the head-group is based on the only achiral amino acid and for which amide-amide hydrogen bonding is still possible, and observed a similar magnitude of hysteresis in the com(32)Safran, S.A.;Robbing, M.0.; Garoff, S. Phys. Rev. A 1986,33, 2186.
Langmuir, Vol. 10, No. 10, 1994 3793 pressiodexpansion cycle.33 The observed hysteresis is not uniquely related to the chiral nature of the molecules or their packing but arises due the strong interaction of amide-amide hydrogen bonding. The driving force for the chiral discrimination in monolayers and micelles ofN-acyl amino acids is believed to be the capacity for establishing amide-amide hydrogen bonding networks. Circular dichroism studies of micelles of N-stearoylserine showed a strong band near 215 nm attributed to a supramolecular chiral organization of the amide-amide hydrogen bonds.18 It would be desirable to obtain CD spectra from these monolayer films using a n external reflection method; such an effort is currently being planned. Infrared spectra of these films on D2O subphases34 or polarization modulation IR,3Sin which the subphase contribution is eliminated, could also yield information on the extent of amide-amide hydrogen bonding. Additional experiments to confirm more directly the apparent chiral phase separation involvingBrewster angle microscopy will soon be initiated. We are attempting to see if the proper selection of a chiral probe molecule with a diastereomeric discrimination strong enough to result in contrast between separation domain segments of D and L enantiomers can be achieved. It is unlikely that a n NBD labeled phospholipid probe will yield a diastereomeric preference, given that no diastereomeric discrimination was observed for mixed monolayers ofN-myristoylalanine and DPPC.21 We have not observed any probe discrimination using NBD-DPPE. One possibility is to label one of the N-acyl amino acid enantiomers a t the end of the alkyl chain with a n NBD group and dope the monolayer with the usual small fraction of this probe with the hope of preserving some preferential incorporation of the probe into the domains of the labeled enantiomer on the basis of the stereospecific head-group interactions; such a synthetic effect is now underway. Another interesting possible experiment concerns attempting to observe the enantioselective action of a n enzyme on chiral and racemic films, as has been done for the case of phospholipase-A2 acting on phospholipid monolayer^.^^^^^ The fluorescence microscopy study by Ringsdorf and co-workers has shown that the stereospecific action of enzymes on phospholipid domains can be directly observed.36 It is intended to use the fluorescence microscopy experiments reported here as a basis for further characterization ofthese phenomena.
Acknowledgment. Acknowledgment is made to the donors of the Petroleum Research Fund, administered by the American Chemical Society, for support of this research. Financial support for a postdoctoral associate (J. Y.-J.Uang) from Monsanto Company is gratefully acknowledged. (33) Uang,J.Y.-J.;Parazak,D.P.;Chiu,H.Y.;Stine,K. J.Submitted for publication. (34) Flach, C. R.; Brauner, J. W.; Mendelsohn, R. Appl. Spectrosc. 1993,47, 982. (35)Blaudez, D.;Buffeteau, T.; Cornut, J. C.; Desnbat, B.; Escafre, N.; Pezolet, M.; Turlet, J. M. Appl. Spectrosc. 1993,47, 869. (36)Grainger, D.W.; Reichert, A.; Ringsdorf, H.; Salesse, C. Biochim. Biophys. Acta 1990,1023, 365. (37)De Haas, G. H.; Dijkman, R.; Lugtigheid, R. B.; Dekker, N.; Van den Berg, L.; Egmond, M. R.; Verheij, H. M. Biochim.Biophys. Acta 1993,1167,281.
