Langmuir 1998, 14, 4525-4534
4525
Temperature Dependence of Chiral Discrimination in Langmuir Monolayers of N-Acyl Amino Acids As Inferred from Π/A Measurements and Infrared Reflection-Absorption Spectroscopy Frank Hoffmann,† Heinrich Hu¨hnerfuss,*,† and Keith J. Stine‡ Institute of Organic Chemistry, University of Hamburg, Martin-Luther-King-Platz 6, D-20146 Hamburg, Germany, and Department of Chemistry, University of Missouri-St. Louis, 8001 Natural Bridge Road, St. Louis, Missouri 63121 Received March 5, 1998. In Final Form: May 26, 1998
N-Hexadecanoylalanine monolayers were investigated on pure aqueous subphases and in the presence of 1 mM solutions of CaCl2 and ZnCl2, respectively, in the temperature range between 293 and 308 K using Langmuir trough measurements and IR reflection-absorption spectroscopy (IRRAS). The overwhelming importance of the temperature for chiral recognition processes was particularly clearly shown in the presence of Zn2+ in the subphase, where a change from homo- to heterochiral preference was observed by IRRAS measurements within a temperature range of 5 deg only. This change was not reflected by corresponding changes in Langmuir curves. This result implies that chiral interactions on a molecular scale inferred from IRRAS and on a macroscopic scale (Langmuir curves) may be at variance because of a different importance of hydrogen bond and complex formation at these scales. Calcium ions exert strong expanding effects, thus weakening the homochiral effect on the macroscopic level and even inducing a heterochiral effect on the molecular level at 293 K. A comparison between the results obtained in the presence of N-hexadecanoylalanine and its methyl ester, respectively, supports the hypothesis that hydrogen bond formation via the carboxyl group also plays an important role for chiral recognition.
Introduction Investigations of chiral effects in monomolecular surface films are of considerable interest due to the influence of chiral molecules on many enzymatic processes at membrane surfaces. Thus far, emphasis has been placed upon investigations of monolayers consisting of N-acyl amino acid amphiphiles1-5 or of 1,2-dihexadecanoyl-sn-glycero3-phosphocholine6,7 (DPPC), that is, model substances that fulfill several important requirements for simulating biomembranes and interface processes at biomembrane surfaces. The enantioselective permeability of the bloodbrain barrier (BBB) for the environmental pollutant R-HCH,8,9 which is assumed to be closely related to the molecular structure of the chiral components of the endothelic membrane, stimulated the present investigations on chiral amphiphilic model substances. As amino acids are known to play an important role in membrane sections that allow transport into cells, for example, * Corresponding author. Phone: +49-40-4123- 4240. Fax: +4940-4123-2893. E-mail:
[email protected]. † University of Hamburg. ‡ University of Missouri-St. Louis. (1) Bouloussa, O.; Dupeyrat, M. Biochim. Acta 1988, 938, 395. (2) Harvey, N. G.; Mirajovsky, D.; Rose, P. L.; Verbiar, R.; Arnett, E. M. J. Am. Chem. Soc. 1989, 111, 1115. (3) Stine, K. J.; Uang, J. Y.-J.; Dingman, S. D. Langmuir 1993, 9, 2112. (4) Stine, K. J.; Whitt, S. A.; Uang, J. Y.-J.; Chem. Phys. Lipids 1994, 69, 41. (5) Gericke, A.; Hu¨hnerfuss, H. Langmuir 1994, 10, 3782. (6) Gutberlet, T.; Milde, K.; Bradaczek, H.; Haas, H.; Mo¨hwald, H. Chem. Phys. Lipids 1994, 69, 151. (7) McConlogue, C. W.; Vanderlick, T. K. Langmuir 1997, 13, 7158. (8) Mo¨ller, K.; Bretzke, C.; Hu¨hnerfuss, H.; Kallenborn, R.; Kinkel, J. N.; Kopf, J.; Rimkus, G. Angew. Chem. 1994, 106, 911; Angew. Chem., Int. Ed. Engl. 1994, 33, 882. (9) Mo¨ller, K.; Hu¨hnerfuss, H.; Rimkus, G. J. High Resolut. Chromatogr. 1993, 16, 672.
through protein channels, herein emphasis will be placed upon amino acid derivatives. For such surface-active compounds with a single chiral center, a preferential D/D or L/L interaction is denoted as “homochiral” behavior, while a preferential D/L interaction is called “heterochiral” behavior. Homochiral interactions are of particular interest, because they raise the possibility of phase separation into regions of the L- and D-enantiomers.4,10 This so-called “chiral symmetry breaking” was theoretically described by Selinger et al.11 The question whether or not hydrogen bond formation is crucial for chiral effects in monolayers is presently the subject of considerable scientific debate.12 Furthermore, Neumann et al.,13 Hu¨hnerfuss et al.,14,15 and Simon-Kutscher et al.16 showed that the presence of bivalent cations in the aqueous subphase may give rise to considerable compression or expansion of the film-forming molecules as well as to an increase or decrease in chiral discrimination in the respective monolayers depending on the kind of cation. However, the relative contributions of the different effects that may be of importance to chiral discrimination, that is, electrostatic interactions, hydrogen bond formation, and complex formation by counterions in the subphase, are not yet clear. This paper aims at supplying deepened insight into this particular question by systematically (10) Andelman, D. J. J. Am. Chem. Soc. 1993, 111, 6536. (11) Selinger, J. V.; Wang, Z. G.; Brunisma, R. F.; Knobler, C. M. Phys. Rev. Lett. 1993, 70, 1139. (12) Stine, K. J.; Leventhal, A. R.; Parazak, D. P.; Uang, J. Y.-J. Enantiomer 1996, 1, 41. (13) Neumann, V.; Gericke, A.; Hu¨hnerfuss, H. Langmuir 1995, 11, 2206. (14) Hu¨hnerfuss, H.; Gericke, A.; Neumann, V.; Stine, K. J. Thin Solid Films 1996, 284-285, 694. (15) Hu¨hnerfuss, H.; Neumann, V.; Stine, K. J. Langmuir 1996, 12, 2561. (16) Simon-Kutscher, J.; Gericke, A.; Hu¨hnerfuss, H. Langmuir 1996, 12, 1027.
S0743-7463(98)00267-4 CCC: $15.00 © 1998 American Chemical Society Published on Web 07/18/1998
4526 Langmuir, Vol. 14, No. 16, 1998
Hoffmann et al.
Figure 1. Π/A isotherms of N-hexadecanoylalanine (DL and L) on acidified aqueous subphases (pH ) 2) at (a) 293 K (20 °C), (b) 295 K (22 °C), (c) 298 K (25 °C), (d) 299 K (26 °C), (e) 301 K (28 °C), (f) 303 K (30 °C), (g) 305 K (32 °C), and 308 K (35 °C).
varying amino acid derivatives such that the contributions of these three interaction parameters can be studied in detail (see Figure 1 in ref 15). Special emphasis is placed upon the temperature dependence of chiral discrimination effects in two-dimensional systems. For example, Harvey et al.2 and Stine et al.3 showed that chiral discrimination effects appear in monolayers of N-octadecanoylserine methyl ester at 297 K, while this is not the case at 293 K. Herein, the temperature-dependent experiments were carried out both for pure aqueous subphases and for subphases containing Ca2+ and Zn2+ cations, respectively,
in order to study the influence of complex formation on chiral discrimination effects in amino acid monolayers. These two cations were chosen because of their importance for various processes at membrane surfaces; for example, Ca2+ ions are assumed to play a key role for the emission of acetylcholine at the praesynaptic membrane, and Zn2+ belongs to the so-called “essential heavy metals” both for animals and plants. Recent monolayer investigations performed in the presence of bivalent cations resulted in dramatic influences on both the compression and the chiral discrimination of the monolayer.14,16,17
Langmuir Monolayers of N-Acyl Amino Acids
With regard to possible methods that can be used for the investigation of chiral amphiphiles, fluorescence or Brewster angle microscopy is well-suited for the study of chiral discrimination in Langmuir monolayers, if chiral symmetry breaking is manifested in the shape of micrometer-sized domains of the ordered phase curving in either direction or showing dendritic morphologies.18 However, it should be noted that these observations are confined to mesoscopic scales and can determine neither molecular characteristics like the conformational order of the alkyl chains nor the structure and solvation of the headgroup of the film-forming compounds. A potential method well-suited for addressing issues related to the conformational order and organization of monolayers appears to be infrared reflection-absorption spectroscopy (IRRAS). The technique involves a single external reflection from the film-covered air/water interface under controlled conditions of surface tension. Thus, detailed information about the structure and interactions of headgroups becomes accessible. For a review of this spectroscopic technique, the reader is referred to a publication by Mendelsohn et al.19 Experimental Section The preparations of N-hexadecanoylalanine and N-hexadecanoylalanine methyl ester were described in a previous publication.3 The concentrations of the spreading solutions were 1.4 × 10-3 mol L-1. The water was deionized and purified (conductivity < 0.05 µS) by a Seralpur Pro 90C apparatus (Seral, Ransbach, Germany). The pH values of the subphase were adjusted by adding the appropriate volumes of highly diluted HCl solutions (analytical-reagent grade; Merck, Darmstadt, Germany) to the pure water. The pH value of the pure water was in the range 5.7-6.0 and is called “pH 6” throughout the present paper. With regard to the variability of the pH value the reader is referred to ref 20. About 15 min after spreading, the surface pressure/area (Π/ A) isotherms were recorded with the help of a Lauda FW2 Langmuir trough (Lauda, Germany) that was temperaturecontrolled in the limits of (0.1 K. The compression velocity was about 0.006 nm2 molecule-1 min-1. The external reflection-absorption spectroscopy was performed on a Bruker IFS 66 (Karlsruhe, Germany) spectrometer equipped with a MCT detector and using a modified external reflection attachment of SPECAC (Orpington, Great Britain) which includes a miniaturized Langmuir trough, permitting thermostatic measurements and an appropriate match for water vapor compensation by carefully controlling the humidity in the sample chamber of the spectrometer. The angle of incidence of the IR beam was set to 30°, and unpolarized radiation was used. Furthermore, the Blackman-Harris apodization function with a resolution of 8 cm-1 and a zero filling factor of 2 were used, and the spectra were taken by coadding 1024 scans (corresponding with a data collection time of about 2 min). The reflectanceabsorbance is defined as -log(R/R0), where R and R0 are the reflectivities of the film-covered and the pure water surfaces, respectively. The film-forming compounds were spread from chloroform solution at an area of about 0.7 nm2 per molecule, and then the monolayers were compressed discontinuously using about 20-30 steps of 0.01-0.03 nm2 molecule-1 each. After the respective compression step, the monolayer was allowed to relax for 1 min, before starting the scanning procedure. For an extensive description of the method, the reader should refer to refs 17 and 21. The method and the evaluation of the data are (17) Gericke, A.; Hu¨hnerfuss, H. Thin Solid Films 1994, 245, 74 (18) McConnell, H. M. Annu. Rev. Phys. Chem. 1991, 42, 171. (19) Mendelsohn, R.; Brauner, J. W.; Gericke, A. Annu. Rev. Phys. Chem. 1995, 46, 305. (20) Simon-Kutscher, J. Strukturelle und oberfla¨ chenrheologische Untersuchungen von Langmuir-Filmen auf metallionenhaltigen Subphasen; Shaker Verlag: Aachen, 1998; Chapter 6. (21) Gericke, A.; Hu¨hnerfuss, H.; Michailov, A. V. Vib. Spectrosc. 1993, 4, 335.
Langmuir, Vol. 14, No. 16, 1998 4527
Figure 2. IR reflection-absorption spectrum of N-hexadecanoyl-DL-alanine on an acidified aqueous subphase (pH ) 2) at 298 K (25 °C) and 0.331 nm2/molecule. based upon the theory of IRRAS at low-absorbing substrates.19 The peak positions of the methylene stretching vibrations were determined by the “center of gravity method”.22
Results and Discussion N-Hexadecanoylalanine/H2O. Surface pressure/ area (Π/A) isotherms of N-hexadecanoylalanine monolayers spread on an acidified water subphase (pH ) 2) were determined at eight distinct temperatures in the range between 293 and 308 K for both the racemic and enantiomeric films (Figure 1). At 293 K the isotherm of the L-enantiomer exhibits a characteristic of a more condensed monolayer than the isotherm of the racemate, which implies a slightly preferential homochiral interaction in the low compressible region (presumably LS) near the collapse point. At higher temperatures, the isotherm of the racemic monolayer shows a phase transition of firstorder and a two-phase coexistence plateau region, while the curve of the enantiomeric monolayer remains almost unaltered. Basically, four characteristic changes were observed for the racemic monolayer: With increasing temperature (i) the phase transition point is shifted to smaller area/molecule values and inversely the plateau region becomes smaller; (ii) the surface pressure of the LE/LC phase transition increases; (iii) the surface pressure at the collapse point decreases; and (iv) the homochiral discrimination increases. At 308 K a plateau region does not exist anymore and the isotherm shows a marked LE phase only (see Figure 1h). At this temperature a phase transition LE f LC/LE can be also inferred from the isotherm of the enantiomeric film, so that the isotherm of the L-film recorded at 308 K correlates with the racemic one recorded at 293 Ksa remarkable difference of 15 K. As an example, in Figure 2 a spectrum of a Nhexadecanoyl-DL-alanine monolayer at an acidified aqueous surface is shown. The strong bands at 2919 and 2851 cm-1 can be attributed to the antisymmetric νas(CH2) and symmetric νs(CH2) methylene stretching vibrations, respectively, and will be discussed below. A strong and sharp band of the amide I vibration appears at about 1650 cm-1, and the band at about 1539 cm-1 is attributed to the amide II vibration. Furthermore, the wide band of the methylene scissoring δ(CH2) vibration is located at 1461 cm-1. The position and shape of the band of the carbonyl stretching vibration is compression- as well as temperature-depend(22) Cameron, D. G.; Kaupinnen, J. K.; Moffat, D. J.; Douglas, J.; Mantsch, H. H. Appl. Spectrosc. 1982, 36, 245.
4528 Langmuir, Vol. 14, No. 16, 1998
Figure 3. IR reflection-absorption spectrum of N-hexadecanoyl-DL-alanine on an acidified aqueous subphase (pH ) 2) at 298 K (25 °C) and different area/molecule values in the range between 2000 and 1200 cm-1.
ent. For temperatures higher than 301 K (not shown), only a weak and asymmetric band close to 1738 cm-1 is found independent of the compression state. For temperatures below 301 K a similarly formed band is present for large areas, but with progressive compression this band splits into three components, namely 1739, 1725, and 1706 cm-1 (Figure 3), indicating that the carbonyl group is involved in three types of hydrogen bonds. It is tentatively assumed that this is the spectroscopic expression of a process in which stable two-dimensional networks of interamphiphilic hydrogen bonds are formed. This assumption is supported by the fact that the area/molecule value at which the splitting of the carbonyl band appears (ca. 0.3 nm2/molecule) matches with the value in the Π/A isotherms where the transition to less compressible phases occurs. Although we made these observations for both the racemic and enantiomeric monolayers, it should not be concluded that the mechanism as described could not be significant for the process of chiral discrimination. It is well-documented that the wavenumbers of the νas(CH2) vibration are conformation-sensitive and that they can be empirically correlated with the order (i.e., with the trans/gauche ratio) of the hydrocarbon chain as follows: lower wavenumbers are characteristic of highly ordered conformations with preferential all-trans characteristics, while the number of gauche conformers increases with increasing wavenumbers and width of the band.13,19 Basically, IRRAS (“molecular information”) and Langmuir trough (“macroscopic information”) measurements are expected to supply comparable results, if experimental periods allowing sufficiently long relaxation times for the monolayers are used. However, this condition is seldom fulfilled because it would often require periods of several days until thermodynamic equilibrium is attained. Accordingly, the maximum time scale of a few hours usually accepted for IRRAS and Π/A measurements almost never gives rise to thermodynamic equilibrium, which implies that different information may be obtained from IRRAS and Π/A measurements. For an extensive discussion on this problem the reader should refer to Hu¨hnerfuss et al.15 As a consequence, we will use the term “molecular order” not in an absolute sense, but we will distinguish between the “conformational order of the alkyl chains” as
Hoffmann et al.
determined by IRRAS and the “macroscopic order” inferred from Π/A isotherms. The plots of the νas(CH2) wavenumbers versus area/ molecule measured at an acidified water subphase at 293, 295, 298, 299, 301, 303, 305, and 308 K are shown in Figure 4. In accordance with the results obtained from Π/A measurements at 293 K, no preferential chiral interaction can be discovered. The compression of the monolayers does not give rise to significant increases in the conformational order of the alkyl chains in both the enantiomeric and racemic monolayers, and the wavenumbers exhibit a comparatively low level for this class of amphiphiles (∼2919 cm-1) even at large area/molecule values. In principle, in the plot for 295 K a first indication of a homochiral separation becomes apparent, as already inferred from the Π/A isotherms, but in contrast to the Langmuir curve results it is only present at large areas. At 298 K and higher temperatures the positions of the νas(CH2) band of the racemic film range between 2924 and 2925 cm-1 for large area per molecule values, indicating a conformational highly disordered state. At an area between 0.4 and 0.5 nm2/molecule the wavenumbers decrease and finally converge toward the low values for the enantiomeric film. With increasing temperature this coincidence point is shifted to lower area/molecule values, and the wavenumber of the enantiomeric film increases for large area/molecule values. It is worth noting that the Π/A isotherms for the enantiomeric film are still typical of a very condensed monolayer. A detailed comparison of the thermodynamic results with the spectroscopic ones reveals additional discrepancies: the characteristics that are apparent in the plots of the νas(CH2) wavenumber versus area/moleculesthe point from which the wavenumbers begin to drop for both the racemic and enantiomeric films as well as the convergence pointsonly find expression in the Π/A isotherms in small degree. It particularly applies to the enantiomeric film. Vice versa the macroscopically determined phase transitions, which suggest that there are structural changes on a microscopic scale too, are not reflected in the IRRA spectra. Concerning the chiral behavior, the same conclusions can be drawn from the spectroscopic investigations and from the Π/A measurements; that is, a preferential homochiral interaction exists above 293 K, but it appears in a different manner. The fact that the wavenumbers are identical for both compressed enantiomeric and racemic monolayers may be indicative of chiral phase separation processes that in the case of the racemic monolayer lead to pure enantiomeric domains of D- and L-stereoisomers, in which the molecules can achieve an equivalent packing density. N-Hexadecanoylalanine/Ca2+. The Π/A isotherms determined on an aqueous 1 mM CaCl2 solution are indicative of comparatively expanded monolayers. At 293 K (see Figure 5a) a clear homochiral discrimination was found in the region between 0.35 nm2/molecule and the collapse point. This range corresponds with the two-phase coexistence region of the enantiomeric film. With increasing temperature this preferential homochiral interaction decreases and finally vanishes at 303 K, where the Langmuir curves for both the racemic and enantiomeric films are identical in the course of the complete compression range (see Figure 5b and c). The observation that the collapse point is encountered at smaller values than the theoretical area/molecule to be expected for saturated alkyl chains can be explained by the assumption that a microscopic or so-called partial collapse has occurred before the thermodynamic one. This is plausible because crystalline particles were observed on the water surface at the end of the compression close to the collapse point.
Langmuir Monolayers of N-Acyl Amino Acids
Langmuir, Vol. 14, No. 16, 1998 4529
Figure 4. Wavenumbers of the antisymmetric methylene stretching vibration versus area/molecule for N-hexadecanoylalanine (DL, O; L, [) on acidified aqueous subphases (pH ) 2) at (a) 293 K (20 °C), (b) 295 K (22 °C), (c) 298 K (25 °C), (d) 299 K (26 °C), (e) 301 K (28 °C), (f) 303 K (30 °C), (g) 305 K (32 °C), and 308 K (35 °C).
In comparison with the Π/A isotherms obtained on a pure aqueous subphase, the calcium ions cause a pronounced expansion effect, which is typical of cations that form ionic carboxylate complexes with such amphiphiles. It is worth mentioning that the homochiral discrimination increases with increasing temperature in the case of the metal ion free subphase, while it appears to decrease with increasing temperature on the calcium ion-containing
subphase. At first glance, this result suggests an antagonistic effect of the Ca2+ cations; however, a detailed analysis of the complete data sets of the two experimental series allows an alternative explanation: in the Π/A isotherms the addition of calcium ions to the aqueous subphase may be reflected by a similar expansion effect, as observed for an increase in the temperature, although very different reasons have to be assumed; that is, the
4530 Langmuir, Vol. 14, No. 16, 1998
Hoffmann et al.
Figure 6. Wavenumbers of the antisymmetric methylene stretching vibration versus area/molecule for N-hexadecanoylalanine (DL, O; L, [) on 1 mM CaCl2 subphases (pH ) 6) at (a) 293 K (20 °C) and (b) 298 K (25 °C).
Figure 5. Π/A isotherms of N-hexadecanoylalanine (DL and L) on 1 mM CaCl2 subphases (pH ) 6) at (a) 293 K (20 °C), (b) 298 K (25 °C), and (c) 303 K (30 °C).
calcium ions cause a structural change at the molecular level while the temperature effect is a thermodynamic phenomenon. The plots of the νas(CH2) wavenumbers versus area/ molecule of a N-hexadecanoylalanine monolayer measured on an aqueous subphase containing 1 mM CaCl2 at 293 and 298 K are summarized in Figure 6. For 293 K the spectroscopic results are at variance with the thermodynamic ones: The Π/A isotherms suggest a slight homochiral preference above 0.35 nm2/molecule; upon further compression a pronounced homochiral interaction is encountered. In contrast, the IR spectra reflect a preferential heterochiral interaction. Furthermore, it is important to note that compression of the enantiomeric film does not give rise to significant increases in the conformational order of the alkyl chains; that is, no significant change in the wavenumbers of the νas(CH2) band is apparent, which exhibit a comparably high level for this class of amphiphiles. A completely different situation is encountered at 298 K. For both monolayers the νas(CH2) wavenumbers are comparable and slightly decreasing upon compression; that is, no preferential homo- or heterochiral interaction can be inferred from the IR reflection-absorption spectra. Similar results were obtained at 303 K, except that the νas(CH2) wavenumbers are slightly shifted to higher values (not shown). The Π/A isotherms still indicate a slightly preferential homochiral interaction at this temperature
which vanishes not before 303 K. In conclusion, a preferential homochiral behavior is observed on the macroscopic scale (Π/A isotherms; 293 and 298 K), while on the molecular scale heterochiral interactions appear to dominate (IRRAS; 293 K). N-Hexadecanoylalanine/Zn2+. On an aqueous 1 mM ZnCl2 solution at 293 K (see Figure 7a), the Π/A isotherms show an expanded characteristic which looks very similar to the results obtained on an aqueous 1 mM CaCl2 solution, with a preferential homochiral interaction between 0.41 nm2/molecule and the collapse point. However, in the presence of Zn2+ this effect remains until a temperature of 303 K (see Figure 7b). Admittedly, it is not yet clear whether the first discontinuity in the curves of the L-film is indicating a phase transition point of first-order (because of the relatively big pressure overshoot, the non-horizontal course after this point, and the linear temperature dependence23 (r ) 0.988)) or whether it is due to the relatively large relaxation times, which would imply that this effect disappears when applying sufficiently low compression velocities. The fact that the presence of Zn2+(aq) in the subphase leads to an expansion of Langmuir monolayers is unexpected insofar as previous investigations of alkanoic acids performed on zinc ion-containing subphases indicated very intensive ordering effects induced by the formation of zinc complexes with the carboxyl group.16 Concerning the chiral behavior, it can be concluded that at 293 K a weak preferential homochiral interaction exists within the LE phase and a clear one within the LE/LC coexistence region. At 303 K the Π/A isotherms suggest preferential homochiral interaction within the LE/LC coexistence region only. (23) Kellner, B. M. J.; Mu¨ller-Landau, F.; Cadenhead, D. A. J. Colloid Interface Sci. 1978, 66, 597.
Langmuir Monolayers of N-Acyl Amino Acids
Figure 7. Π/A isotherms of N-hexadecanoylalanine (DL and L) on 1 mM ZnCl2 subphases (pH ) 6) at (a) 293 K (20 °C) and (b) 303 K (30 °C).
Figure 8. IR reflection-absorption spectrum of N-hexadecanoyl-L-alanine on a 1 mM ZnCl2 subphase (pH ) 6) at 298 K (25 °C) and 0.323 nm2/molecule.
A typical spectrum of an N-hexadecanoyl-L-alanine monolayer on a subphase containing zinc ions at 293 K is shown in Figure 8, and the results of the analysis of the conformational order in the form of the νas(CH2) wavenumber versus area/molecule plots are summarized in Figure 9 for four different temperatures (293, 298, 300, and 303 K). At 293 K the values for both the racemic and enantiomeric monolayers are independent of the compression status and range between 2924 and 2925 cm-1. This result is remarkable in multiple respects. Firstly, in accordance with the Π/A isotherms, the films seem to be in a conformationally highly disordered state over the whole compression range, but in contrast to the case of the macroscopic level, no chiral discrimination can be inferred. This observation is at variance with the results which were obtained on the calcium ion-containing subphase at the same temperature. Furthermore, it is
Langmuir, Vol. 14, No. 16, 1998 4531
worth mentioning that the remarkable structural disorder remains until the collapse point is reached. An increase in temperature has drastic consequences for the conformational order as well as for the chiral behavior. At 298 K, all data points of the enantiomeric monolayer exhibit lower values than those of the racemic one, although the wavenumber differences are small at large area/molecule values, but they are significant for small areas so that a preferential homochiral interaction can be inferred. At 300 K the wavenumbers suggest a slight but not significant heterochiral preference at large areas, while upon compression below 0.27 nm2/molecule homochiral interactions appear to be more favorable. Thus, a change from a weak heterochiral to a weak homochiral preference occurs within an isothermal compression. And, finally, another notable change regarding the chiral behavior appears at 303 K, that is, only 3 deg higher than the previous temperature: While for the enantiomeric film no change in the wavenumbers of the νas(CH2) vibration and, thus, in the conformational order of the alkyl chains was observed, a very pronounced dependence on the compression state of the monolayer was determined for the racemic film (see Figure 9d). Obviously, in this case a sensitive equilibrium of forces is present: a variation of the temperature within an interval of 5 K only causes remarkable changes of the chiral behavior. As a diagnostic tool that may allow insight into the present complex type, the analysis of the separation ∆ between the antisymmetric and symmetric carboxylate vibrations,24,25 that is, ∆ ) νas(COO) - νs(COO), can be used. Although the significance of ∆ values as a diagnostic tool has been criticized,26 this experimental approach in many cases appears to represent a meaningful guide. The ∆ values usually assigned to the four main coordination types, ionic, monodentate, chelating bidentate, and bridging bidentate (see ref 16, Figure 6), were largely inferred from the infrared spectra of pure metal carboxylates and aqueous solutions of acetates (see Table 2 in ref 16). It is worth noting that the differences between these two sets of ∆ values are particularly large for monodentate coordination, because in aqueous solutions the uncoordinated carbonyl group may form hydrogen bonds to the adjacent water molecules, which are known to reduce the wavenumber of the νas(COO), thus giving rise to lower ∆ values. This type of coordination is often denoted as “Hbonded monodentate”. Due to the inconvenient orientation of the transition dipole momentum in general and the high residual water vapor level encountered in the present case, the νs(COO) band is weak, which implies that caution has to be applied with regard to the interpretations of the ∆ values, which are tentatively given in the subsequent section. In contrast to the spectra which were recorded on a subphase containing calcium ions, no carbonyl band of free acid groups was detected, and instead of a single and sharp νas(COO) band, a multiply split band appears with components at about 1561, 1545, and 1523 cm-1 (Figure 8). Therefore, it is reasonable to assume that different types of carboxylate complexes are present at the same time. The assignment of the peak at 1421 cm-1 to the νs(COO) vibration will result in ∆ values of 140, 124, and 102 cm-1, respectively. The first ∆ value may be attributed to a monodentate complex, while the last one may indicate (24) Nakamoto, K. Infrared and Raman Spectra of Inorganic and Coordination Compounds; Wiley: New York, 1986. (25) Tackett, J. E. Appl. Spectrosc. 1989, 43, 483. (26) Edwards, D. A.; Hayward, R. N. Can. J. Chem. 1968, 46, 3443.
4532 Langmuir, Vol. 14, No. 16, 1998
Hoffmann et al.
Figure 9. Wavenumbers of the antisymmetric methylene stretching vibration versus area/molecule for N-hexadecanoylalanine (DL, O; L, [) on 1 mM ZnCl2 subphases (pH ) 6) at (a) 293 K (20 °C), (b) 298 K (25 °C), (c) 300 K (27 °C), and (d) 303 K (30 °C).
Figure 10. Π/A isotherms of N-hexadecanoylalanine methyl ester monolayers (DL and L) on pure aqueous subphases (pH ) 6) at (a) 293 K (20 °C), (b) 298 K (25 °C), (c) 303 K (30 °C) and (d) 308 K (35 °C).
chelating-bidentate coordination. The second ∆ value of 124 cm-1 could be the spectroscopic reflection of an unsymmetrical chelating-bidentate complex. However, taking into account the limited transferability of the ∆ values and the proximity to the ∆ value of ionic complexes, it is also possible that this type of coordination is present. N-Hexadecanoylalanine Methyl Ester/H2O. With the esterification of the carboxyl group, one possibility to form intramolecular H-bonds is excluded. Thermodynamic measurements were performed within the same temperature region between 293 and 308 K, as in the case
of N-hexadecanoylalanine, but steps of 5 deg were chosen. The Π/A isotherms, recorded at 293, 298, 303, and 308 K, respectively, are shown in Figure 10. In comparison with the respective isotherms of the unesterified species, many identical features appear and a similar temperature dependence is present. Minor differences include kinks in the curves within the LS phase, which vanish at g298 (L) and g303 K (DL), respectively. As in the case of the unesterified derivative, a preferential homochiral interaction is encountered in the temperature range between 293 and 308 K, where this
Langmuir Monolayers of N-Acyl Amino Acids
Langmuir, Vol. 14, No. 16, 1998 4533
Figure 11. Comparison between IR reflection-absorption spectra of N-hexadecanoyl-DL-alanine methyl ester and the respective enantiomeric film on pure aqueous subphases (pH ) 6) at 303 K (30 °C) at 0.438 (DL) and 0.368 nm2/molecule (L), respectively, in the range between 2000 and 1200 cm-1.
effect increases with increasing temperature. At temperatures below 293 K no chiral effects were observed. Accordingly, it has to be concluded that H-bond networks formed by the carboxyl groups do not play a key role for the chiral discrimination processsat least for this special system and on the macroscopic level. However, we would like to emphasize that it is not possible to generalize this conclusion due to contrary results for other systems.12 Figure 11 shows two typical spectra of racemic and enantiomeric N-hexadecanoylalanine methyl ester monolayers, recorded on a pure aqueous subphase (pH ≈ 6) at 303 K. The analysis of the compression dependence of the νas(CH2) bands is summarized in Figure 12. At 293 K wavenumbers of 2921 cm-1 were found at large areas/ molecule for both the racemic and enantiomeric monolayers, they begin to drop at a value of about 0.48 nm2/ molecule, approaching 2919 cm-1 at e0.39 nm2/molecule. In accordance with the thermodynamic results, no sign of chiral discrimination was observed. A completely different situation is encountered at 298 K. The racemic film is in an extremely disordered state at large areas/molecule, as emerging from the band positions of 2925 cm-1. This value decreases within a compression interval of only 0.1 nm2/ molecule, and then it remains constant (∼2919 cm-1). The wavenumbers determined for the enantiomeric monolayer turned out to be independent of compression (note, for high area/molecule values, no analyzable band was present). Accordingly, the spectroscopic results confirm the beginning emergence of homochiral discrimination at this temperature, which is in line with the conclusions already drawn from the Π/A measurements. However, in the latter case homochiral preference was observed until the collapse point, while the νas(CH2) wavenumbers of the racemic and enantiomeric monolayers converge at medium area/molecule values. The decrease in the wavenumbers of the racemic filmsas in the case of the unesterified derivativescould be indicative of a so-called “chiral symmetry breaking process”, that is, the formation of domains consisting of pure D- and L-enantiomers, respectively. It is worth noting that the region in which the chiral phase separations seem to occur cannot be correlated with any characteristic features of the Π/A isotherms (for example phase transition points, width of the plateau, etc.).
Figure 12. Wavenumbers of the antisymmetric methylene stretching vibration versus area/molecule for N-hexadecanoylalanine methyl ester (DL, O; L, [) on pure aqueous subphases (pH ) 6) at (a) 293 K (20 °C), (b) 298 K (25 °C), and (c) 303 K (30 °C).
An additional increase in the temperature of 5 K changes the interaction parameters significantly. At areas above 0.5 nm2/molecule the positions of the νas(CH2) bands are independent of the compression status. However, those of the enantiomeric monolayer are slightly shifted to lower wavenumbers, which implies a weak homochiral interaction that increases upon further compression, as indicated by the decrease in the wavenumbers of the enantiomeric film, while those of the racemic one largely remain on the very high level at 2925 cm-1 and only decrease to 2922 cm-1 close to the collapse point. In conclusion, at 303 K the chiral discrimination effect can be observed over the complete compression range both at the molecular (IRRAS) and the macroscopic levels (Π/A isotherms). Another fundamental difference between the spectra of the racemic and enantiomeric monolayers, recorded at 303 K and at an area below 0.5 nm2/molecule (and above 0.3 nm2/molecule), is the position and intensity of the amide I band (Figure 11). While a sharp and strong band appears in the case of the enantiomeric film (∼1648 cm-1), no unambiguous assignment is possible for the racemic
4534 Langmuir, Vol. 14, No. 16, 1998
one due to the low signal-to-noise ratio (note the different compression status of the racemic and enantiomeric monolayers can be neglected in this specific case; see the Π/A isotherm of the L-enantiomer in Figure 10c). With high probability, it is shifted to lower wavenumbers. The shift of the amide I band in N-acyl amino acid methyl esters is well-known and is attributed to the different intra-/intermolecular H-bond formations in the racemic mixture.3 The latter hydrogen bond formation appears to be unfavorable to allow chiral phase separation in racemic monolayers under such experimental conditions. Conclusions The results presented herein are part of a systematic investigation on the contribution of the following parameters to chiral recognition processes in amino acid monolayers: intra- and intermolecular hydrogen bond formation in the headgroup as well as complex formation between the headgroup and cations in the subphase. The complete experimental approach of the ongoing investigation can be deduced from Figure 1 in ref 15. Thus far, the data set is limited, and it would be too early to draw a comprehensive conclusion from the available results. However, some aspects of general importance can already be stated: (i) The most notable result is the importance of the parameter “temperature” for chiral recognition processes. Within relatively small temperature ranges chiral discrimination may emerge or vanish. In the case of the monolayer system N-hexadecanoylalanine on a subphase containing 1 mM ZnCl2 even a change from homo- to heterochiral preference was observed by IRRAS measurements within a temperature range of 5 deg only. (ii) On a macroscopic scale (Π/A measurements) a result may or may not be obtained that is at variance with the
Hoffmann et al.
respective conclusion drawn from IRRAS data on a molecular scale. For example, the aforementioned change from preferential homo- to heterochiral interaction is not reflected by corresponding Π/A results. We tentatively assume that intermolecular interactions, be they homoor heterochiral, give rise to a specific order within the domains. The macroscopic parameters, for example, the Π/A curves, largely depend on the interaction between the domains. As a consequence, the respective shapes of the domains, for example, dendritic, plates, needles, etc., which are known to be enantioselectively different, are expected to lead to different macroscopic results. This aspect will be pursued by Brewster angle microscopy. (iii) A significant influence of cations in the subphase on chiral recognition processes was observed. Calcium ions exert strong expanding effects on the films of N-hexadecanoylalanine. The homochiral interaction is largely weakened at the macroscopic level. A weaker expanding effect is exerted by zinc ions on the monolayers of N-hexadecanoylalanine. On the molecular level a change from homo- to heterochiral preference occurs as the temperature rises. (iv) A comparison between the monolayers of Nhexadecanoylalanine and N-hexadecanoylalanine methyl ester supports the thesis that H-bond formation via the carboxyl group is crucial for the mutual recognition of the enantiomers among themselves. Acknowledgment. This work was supported by the Fonds der Chemischen Industrie, Germany. The authors thank J. Simon-Kutscher and A. Gericke for enlightening discussions. LA9802670
