Role of Hydrogen Bond and Metal Complex Formation for Chiral

The Journal of Physical Chemistry B 0 (proofing), ... Molecular Recognition of 1-(2-Octadecyloxycarbonylethyl)cytosine Monolayers to Guanosine at the ...
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Role of Hydrogen Bond and Metal Complex Formation for Chiral Discrimination in Amino Acid Monolayers Studied by Infrared Reflection-Absorption Spectroscopy Heinrich Hu¨hnerfuss,*,† Volker Neumann,† 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 MissourisSt. Louis, 8001 Natural Bridge Road, St. Louis, Missouri 63121 Received December 22, 1995. In Final Form: February 19, 1996X Comparison between the macroscopic order (inferred from surface pressure/area isotherms and fluorescence microscopy) and the molecular order [determined by infrared reflection-absorption spectroscopy (IRRAS)] for N-acylamino acid monolayers shows that hydrogen bond formation via the NH, COOH, and p-hydroxyphenyl groups, respectively, may lead to pronounced chiral discrimination. On a pure water surface, N-hexadecanoylalanine films exhibit preferential homochiral interactions, which may be strengthened by Pb2+ in the aqueous subphase, while Zn2+ disturbs this structure and suppresses the chiral discrimination. In N-octadecanoyltyrosine and N-octadecanoyltyrosine methyl ester monolayers, both homo- and heterochiral discrimination may be observed, depending on the available area per molecule, where the free fatty acid carboxyl group gives rise to additional film compression and, thus, to a higher conformational order for the alkyl chains. However, in the presence of both PbCl2 and ZnCl2 in the aqueous subphase, the pronounced chiral discrimination effect disappears. Analysis of the methylene scissoring δ(CH2) bands shows that the different effects of Pb2+ and Zn2+ cannot be explained on the basis of different subcell structures (in both cases an orthorhombic structure is prevailing), but on the basis of different complex formations between the bivalent cations and the carboxylic acid headgroup as inferred from the separation ∆ between the antisymmetric and the symmetric carboxylate vibrations, ∆ ) νa(COO) - νs(COO). The results summarized in the present paper suggest that theories like Andelman’s tripode theory should be expanded to account for potential metal complex formations.

Introduction Recent investigations of the enantioselective enrichment of (+)-R-1,2,3,4,5,6-hexachlorocyclohexane in the brain tissues of common eider ducks, sheep, and harbor seals1,2 revealed a preferential permeability of the blood-brain barrier (BBB) for the (+)-enantiomer of the chiral environmental pollutant R-HCH, while its (-)-enantiomer as well as other chiral pollutants including oxychlordane and heptachlorepoxide is largely held back by the BBB. This enantioselective effect, which is assumed to be closely related to the molecular structure of the chiral components of the endothelic membrane that separates the blood capillaries of the brain and the surrounding tissue, stimulated the present investigation on chiral amphiphilic model substances. As amino acids are known to play an important role in membrane sections that allow transport into the cells, e.g., through protein channels, emphasis will be placed upon amino acid derivatives. The molecular structure of the protein sections in turn is expected to be dependent on the electrostatic interactions and potential hydrogen bond formations between the respective amino acid molecules. This latter aspect, i.e., the question of whether or not hydrogen bond formation is crucial for the formation of chiral effects in monolayers, is presently the subject of considerable scientific debate.3-6 Furthermore, Neumann et al.,7 †

University of Hamburg. University of MissourisSt. Louis. X Abstract published in Advance ACS Abstracts, April 15, 1996. ‡

(1) 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. (2) Mo¨ller, K.; Hu¨hnerfuss, H.; Rimkus, G. J. High Resolut. Chromatogr. 1993, 16, 672. (3) Stine, K. J.; Whitt, S. A.; Uang, J. Y.-J. Chem. Phys. Lipids 1994, 69, 41. (4) Andelman, D. J. Am. Chem. Soc. 1989, 111, 6536. (5) Andelman, D.; Orland, H. J. Am. Chem. Soc. 1993, 115, 12322.

Hu¨hnerfuss et al.,8 and Simon-Kutscher et al.9 showed that the presence of bivalent cations in the aqueous subphase may give rise to considerable compression and increased chiral discrimination in monolayers. However, the relative contributions of the different effects that may be of importance to chiral discrimination, i.e., electrostatic interactions, hydrogen bond formation, and complex formation by counterions in the subphase, are not yet clear. This paper aims to supply deepened insight into this particular question by systematically varying amino acid derivatives such that the contributions of the three parameters summarized earlier can be studied in detail. Our experimental approach is depicted schematically in Figure 1: in the first part of this investigation, Nacylalanine (R1 ) C15H31; R2, R3 ) H) and N-acyltyrosine (R1 ) C17H35; R2 ) H; R3 ) PhOH) are studied, which may form the two intramolecular hydrogen bonds indicated in Figure 1, concurring with the respective intermolecular hydrogen bonds between the amide hydrogen and the carboxyl groups. In the case of N-acyltyrosine, additional intramolecular hydrogen bonds between the p-hydroxy group of the aromatic ring and the amide hydrogen and the carboxyl group have to be considered. The experiments were repeated by adding Pb2+ and Zn2+ cations to the aqueous subphases to study the influence of complex formation on chiral discrimination effects with amino acid monolayers. Recent monolayer investigations performed in the presence of these bivalent cations resulted in dramatic influences on both the compression and the chiral discrimination of the monolayer.7,9,10 (6) Harvey, N. G.; Mirajovsky, D.; Rose, P. L.; Verbiar, R.; Arnett, E. M. J. Am. Chem. Soc. 1989, 111, 1115. (7) Neumann, V.; Gericke, A.; Hu¨hnerfuss, H. Langmuir 1995, 11, 2206. (8) Hu¨hnerfuss, H.; Gericke, A.; Neumann, V.; Stine, K. J. Thin Solid Films 1996, in press. (9) Simon-Kutscher, J.; Gericke, A.; Hu¨hnerfuss, H. Langmuir 1996, in press.

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etc., became accessible. For a review of this spectroscopic technique, the reader is referred to the recent publication by Mendelsohn et al.12 Experimental Section

Figure 1. Schematic representation of intramolecular hydrogen bond formations conceivable for the alanine, serine, and tyrosine derivatives discussed in the present work: R1 ) C15H31, C17H35; R2 ) H; R3 ) H, OH, PhOH.

In the next part of the present investigation, the hydrogen bond formation is partly suppressed either by N-methyl substitution or by esterification of the carboxyl group, or it is completely excluded by both N-methyl substitution and esterification (see Figure 1). In the present paper, we will only include the N-acyltyrosine methyl ester to allow comparison between the tyrosine derivatives with free and with esterified carboxyl groups. The results of the other amino acid derivatives will be presented in a subsequent paper. With regard to possible methods that can be used for the investigation of chiral amphiphiles, fluorescence 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.3,11 However, it should be noted that fluorescence observations are confined to macroscopic scales and can determine neither molecular characteristics like the conformational order of the hydrophobic alkyl chains nor the structure and hydration of the headgroup of the film-forming compounds. These latter parameters are assumed to be important for the macroscopic chirality-dependent domain structures (e.g., direction of curvatures) as observed by fluorescence microscopy, and, therefore, a comprehensive study of chiral effects in Langmuir monolayers has to include these aspects. A potential method well-suited for addressing issues related to the conformational order and organization of monolayers and biological membranes 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, e.g., carboxylic acids, carboxylic acid esters, carboxylates, amides, (10) Gericke, A.; Hu¨hnerfuss, H. Thin Solid Films 1994, 245, 74.

The preparation and separation of N-hexadecanoylalanine, N-octadecanoyltyrosine, and N-octadecanoyltyrosine methyl ester were described in previous publications.3,13 The spreading solvent chloroform of Licrosolv grade (Merck, Darmstadt, Germany) was used as received. The water was deionized and purified (conductivity < 0.05 µS) by a Seralpur Pro 90C apparatus (Seral, Ransbach, Germany). The pH values of the subphases were adjusted by adding the appropriate volumes of 1 M HCl solutions (analytical reagent grade; Merck) to 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. In addition, aqueous subphases containing 1 mM PbCl2 (g99%; Aldrich, Steinheim, Germany) and 1 mM ZnCl2 (g99%; Merck) were used, ensuring the interaction of all film molecules with bivalent cations. The external infrared reflection-absorption spectroscopy was performed on a Bruker IFS 66 (Karlsruhe, Germany) spectrometer equipped with an MCT detector 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 (angle of incidence of the IR beam, 30°; unpolarized radiation; Blackman-Harris apodization function with a resolution of 8 cm-1; zero filling factor, 1; 1024 scans). The equilibrium period used at each molecular area comprised 60 s. The reflectance-absorbance is defined as -log(R/R0), where R and R0 are the reflectivities of the filmcovered and pure water surfaces, respectively. For an extensive description of the method, the reader should refer to ref 14. The method and evaluation of the data are based upon the theory of IRRAS at low-absorbing substrates.12,14

Results 1. N-Hexadecanoylalanine. The surface pressure/ area (Π/A) isotherms of N-hexadecanoylalanine monolayers spread on a pure water surface and on aqueous subphases containing 1 mM PbCl2 or 1 mM ZnCl2 are shown in Figure 2a-c, respectively, for both the racemic and the enantiomeric films. In the case of the pure aqueous subphase, the Π/A isotherms suggest a preferential homochiral interaction in the range between 0.8 and about 0.3 nm2/molecule. The isotherms approach the collapse point at comparable areas per molecule; however, the surface pressure of the collapse point is considerably higher for the enantiomeric monolayer. The Π/A isotherms determined on an aqueous 1 mM PbCl2 solution show comparatively compressed characteristics, even below the theoretical value of a saturated alkyl chain. As we observed crystallization effects on the water surface during the course of the compression procedure close to and below the collapse point, we assume that the extremely compressed characteristics of the Langmuir curves are not caused by losses to the subphase, but rather are due to the formation of three-dimensional structures of the compressed monolayers. Accordingly, the differences between the two curves of the racemic and enantiomeric monolayers observed below the collapse point do not necessarily reflect heterochiral discrimination; they may merely be the consequence of different speeds of crystallization in the three-dimensional space. (11) McConnell, H. M. Annu. Rev. Phys. Chem. 1991, 42, 171. (12) Mendelsohn, R.; Brauner, J. W.; Gericke, A. Annu. Rev. Phys. Chem. 1995, 46, 305. (13) Parazak, D. P.; Uang, J. Y.-J.; Turner, B.; Stine, K. J. Langmuir 1994, 10, 3787. (14) Gericke, A.; Hu¨hnerfuss, H.; Michailov, A. V. Vib. Spectrosc. 1993, 4, 335.

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Figure 2. Π/A isotherms of N-hexadecanoyl-DL-alanine and N-hexadecanoyl-L-alanine. Conditions: Compression rate ) 0.05 nm2 molecule-1 min-1; subphase temperature ) 301 K; spreading solvent was CHCl3. (a) On pure water, pH 2. (b) On an aqueous solution of 1 mM PbCl2, pH 6. (c) On an aqueous solution of 1 mM ZnCl2, pH 6.

On an aqueous 1 mM ZnCl2 solution, a very expanded characteristic is encountered with a preferential homochiral interaction between about 0.4 nm2/molecule and the collapse point. Upon further compression, the Π/A isotherm of the L-enantiomer shows a very steep portion, which we also attribute to the formation of threedimensional structures as already discussed for an aqueous PbCl2 subphase. As an example, in Figure 3 the spectra of the Nhexadecanoyl-L-alanine monolayer at a pure water surface and at aqueous subphases containing 1 mM PbCl2 and 1 mM ZnCl2 are shown. In the three spectra, the antisymmetric methylene stretching vibration νa(CH2) appears in the range 2918-2922 cm-1, while the symmetric methylene stretching vibration ranges between 2849 and 2853 cm-1. A detailed discussion of the relevance of the νa(CH2) vibration to the conformational order of the hydrophobic alkyl chains will be given later. In the case of the pure aqueous subphase, two bands in the spectral region of the carbonyl stretching vibrations at 1739 and

Figure 3. IR reflection-absorption spectra of N-hexadecanoylL-alanine at 301 K (spreading solvent was CHCl3): (a, top) on pure water, pH 2, 0.309 nm2/molecule; (b, middle) on an aqueous solution of 1 mM PbCl2, pH 6, 0.143 nm2/molecule; (c, bottom) on an aqueous solution of 1 mM ZnCl2, pH 6, 0.273 nm2/molecule.

1703 cm-1 are present, which Gericke and Hu¨hnerfuss15 tentatively assigned to the vibrations of an unprotonated and a diprotonated fatty acid carbonyl group. In accordance with this interpretation, these two bands disappear in the presence of bivalent Pb and Zn cations. Instead, strong bands at about 1540 and 1562 cm-1 are present, which are indicative of the antisymmetric carboxylate stretching vibrations. The strong band in the range 1616-1651 cm-1 is attributed to the amide I vibrations, the band in the region 1421-1423 cm-1 is attributed to the symmetric carboxylate stretching vibration, and the band at 1462-1467 cm-1 is attributed to the methylene scissoring δ(CH2) vibration. It is well-documented that the wavenumbers of the νa(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: 7,12 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. As a consequence, the results summarized in Figure 4a imply that the alkyl chains within the N-hexadecanoylL-alanine monolayer [νa(CH2) about 2919 cm-1] exhibit a significantly higher conformational order than those within the racemic surface film [νa(CH2) about 2924 cm-1]. The higher error bars of the data of the racemic film also indicate a less ordered film state. Furthermore, it is important to note that compression of the monolayer does not give rise to significant increases in the conformational order of the alkyl chains in both the enantiomeric and the racemic monolayer. Basically, the same conclusions can be drawn from the respective νa(CH2) data of N-hexadecanoylalanine monolayers at an aqueous subphase containing 1 mM PbCl2 (not shown). The characteristics of the curves are very comparable to those obtained at a pure water surface, (15) Gericke, A.; Hu¨hnerfuss, H. J. Phys. Chem. 1993, 97, 12899.

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Figure 4. Wavenumbers of the antisymmetric methylene stretching vibration vs area/molecule for N-hexadecanoyl-DLalanine (O) and N-hexadecanoyl-L-alanine (b) at 301 K: (a) pure water subphase, pH 2; (b) aqueous subphase of 1 mM ZnCl2, pH 6, spereading solvent CHCl3.

although the νa(CH2) wavenumbers were lower, i.e., for the racemic film about 2921 cm-1 was found and for the enantiomeric film about 2918 cm-1 was found, which are indicative of the compression effect induced by the Pb2+ cations and reflect a more ordered arrangement of the alkyl chains compared with the situation encountered at a pure aqueous subphase. By the way of contrast, a completely different result was obtained in the presence of an aqueous subphase containing 1 mM ZnCl2 (Figure 4b). Within the error of the method, no differences between the νa(CH2) vibrations of the racemic and the enantiomeric films were found [νa(CH2) about 2923 cm-1]. It is worth mentioning that this effect is at variance with previous observations by Simon-Kutscher et al.9 and by Gericke and Mendelsohn,16 who showed that bivalent Zn cations may induce a very high order for fatty acid monolayers. 2. N-Octadecanoyltyrosine. The Π/A isotherms of an N-octadecanoyltyrosine monolayer on a pure water subphase show an interesting feature (Figure 5a): between about 0.8 and 0.35 nm2/molecule a preferential heterochiral interaction is suggested, while upon further compression homochiral interactions appear to become more favorable. Quite different characteristics are observed on aqueous subphases containing 1 mM PbCl2 (Figure 5b) and 1 mM ZnCl2 (Figure 5c), respectively. In both cases, the respective curves of the racemic and enantiomeric films show the same characteristics, possibly with a slight tendency to a preferential homochiral interaction. The spectra of the N-octadecanoyl-L-tyrosine monolayer at a pure water surface and at aqueous subphases containing 1 mM PbCl2 and ZnCl2 are shown in Figure 6. The antisymmetric methylene stretching vibration νa(CH2) appears in the range 2919-2923 cm-1, while the symmetric methylene stretching vibration ranges between (16) Gericke, A.; Mendelsohn, R. Langmuir, submitted for publication.

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Figure 5. Π/A isotherms of N-octadecanoyl-DL-tyrosine and N-octadecanoyl-L-tyrosine. Conditions: Compression rate ) 0.05 nm2 molecule-1 min-1; subphase temperature ) 299 K; spreading solvent was CHCl3. (a) On pure water, pH 2. (b) On an aqueous solution of 1 mM PbCl2, pH 6. (c) On an aqueous solution of 1 mM ZnCl2, pH 6.

2850 and 2853 cm-1. In the case of the pure aqueous subphase, a sharp strong band of the carbonyl stretching vibration at 1738 cm-1 is present, which may be indicative of an unprotonated fatty acid carbonyl group, while the amide I band is located at 1598 cm-1. In the presence of PbCl2 and ZnCl2, the amid I vibration was encountered at about 1618-1619 cm-1, the antisymmetric carboxylate stretching vibrations in the range 1513-1561 cm-1 (three components), and the methylene scissoring δ(CH2) vibration at about 1461 cm-1. The plots of the νa(CH2) wavenumbers vs area/molecule measured at a pure water subphase (Figure 7a) in this case confirm the conclusions drawn from the Π/A isotherms: the results for the racemic monolayer suggest a preferential heterochiral interaction at large area/ molecule, while upon compression below about 0.35 nm2/ molecule, homochiral interactions appear to be more favorable. Another notable difference between the results for the racemic and the enantiomeric monolayers is their dependence on the compressional state: while for the

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Figure 6. IR reflection-absorption spectra of N-octadecanoylL-tyrosine at 299 K (spreading solvent was CHCl3): (a, top) on pure water, pH 2, 0.401 nm2/molecule; (b, middle) on an aqueous solution of 1 mM PbCl2, pH 6, 0.351 nm2/molecule; (c, bottom) on an aqueous solution of 1 mM ZnCl2, pH 6, 0.453 nm2/molecule.

racemic film no significant change in the wavenumber of the νa(CH2) vibration and, thus, in the conformational order of the alkyl chain was observed, a very pronounced dependence on the compressional status of the monolayers was determined for the enantiomeric film. However, in the presence of both PbCl2 (Figure 7b) and ZnCl2 (Figure 7c) in the aqueous subphase, these different characteristics disappear: in both cases, the νa(CH2) wavenumber of the racemic and enantiomeric films shows a dependence on the compressional state of the monolayer, and, furthermore, comparable results were obtained for the racemic and enantiomeric films, i.e., no preferential homo- or heterochiral interaction can be inferred from the IR reflection-absorption spectra. 3. N-Octadecanoyltyrosine Methyl Ester. Having in mind the conception depicted in Figure 1, we included N-octadecanoyltyrosine methyl ester in the present study to gain insight into the importance of hydrogen bond formation via the fatty acid carboxyl group and its suppression by esterification. The results of the measurements that were thus far only performed on a pure aqueous subphase are summarized in Figure 8a for the Π/A isotherms and in Figure 8b for the wavenumber/area per molecule plot of the νa(CH2) vibration. Basically, the results are similar to those obtained for the tyrosine derivative with a free carboxylic acid group. However, comparison of the wavenumber values of the compressed monolayers of N-octadecanoyltyrosine (racemic film, about 2918.5 cm-1; L-enantiomer, about 2917.5 cm-1) and N-octadecanoyltyrosine methyl ester (racemic film, about 2919.5 cm-1; L-enantiomer, about 2918 cm-1) seem to suggest that the free fatty acid carboxyl group gives rise to additional film compression and, thus, to a higher conformational order of the alkyl chains. Discussion 1. Molecular Order and Chiral Discrimination. The molecular order of the film-forming molecules is

Figure 7. Wavenumbers of the antisymmetric methylene stretching vibration vs area/molecule for N-octadecanoyl-DLtyrosine (O) and N-octadecanoyl-L-tyrosine (b) at 299 K: (a) pure water subphase, pH 2; (b) aqueous subphase of 1 mM PbCl2, pH 6; (c) aqueous subphase of 1 mM ZnCl2, pH 6, spreading solvent CHCl3.

closely related to the macroscopic characteristics of monoand bilayers, e.g., the domain sizes and shapes as well as the elasticity and permeability of the membranes. 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. For example, Kato et al.17 proved drastic changes of the shape of the Π/A isotherms of arachidic acid monolayers even at constant temperatures in the course of experimental periods of about 4.6 days. Accordingly, the maximum time scale of a few hours usually accepted for IRRAS and Langmuir trough measurements almost never gives rise to thermodynamic equilibrium. Furthermore, within the domains the film-forming molecules may have already attained a relatively high order at large areas per molecule (as inferred from IRRAS measurements), while the Π/A isotherms may reflect a relatively disordered state due to irregular distributions of domains. As a consequence, very different information may be inferred from IRRAS and (17) Kato, T.; Hirobe, Y.; Kato, M. Langmuir 1991, 7, 2208.

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Figure 8. (a) Π/A isotherms and (b) wavenumbers of the antisymmetric methylene stretching vibration vs area/molecule for N-octadecanoyl-DL-tyrosine methyl ester (O) and N-octadecanoyl-L-tyrosine methyl ester (b) at 299 K (pure water subphase, pH 6).

Langmuir trough measurements. In some cases, these two methods may even supply contradictory results, as recently shown by Hu¨hnerfuss et al.8 To overcome this awkward situation, Tredgold recently evoked a pragmatic approach (Tredgold, personal communication) and suggested that one should not plan experiments according to time scales that allow thermodynamic equilibrium, but rather one should use experimental periods that are typical of the respective method and, as a consequence, accept different results for the molecular order in dependence on the respective experimental approach. In the present paper, we will interpret our data according to Tredgold’s suggestion, i.e., the term “molecular order” will not be used in an absolute sense, but we will distinguish between the “conformational order of the alkyl chains” as determined by IRRAS and the “macroscopic order” inferred from Π/A isotherms and fluorescence microscopy studies, which were performed with the present N-acylamino acid derivatives as well.3,13 The latter method was specifically applied to the determination of chiral phase separation; however, it should be kept in mind that it supplies limited insight into the molecular order of monolayers. In fluorescence studies, chiral phase separation is inferred via an argument used to explain the chiral condensed phase domains observed in phospholipid monolayers, where condensed phase domains of R-DPPC (1,2-dihexadecanoyl-sn-glycero-3-phosphocholine, often referred to as dipalmitoylphosphatidylcholine) curved counterclockwise, while those of S-DPPC curved clockwise (see ref 13 and literature cited therein). It was argued that if the molecules of one enantiomer are oriented regularly throughout the domain, then the two sides of the domain will differ in their line tension against the liquid-expanded phase, leading to a tendency to curve the domain so as to reduce the interfacial length of the side

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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 will 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 the incorporation of a fraction of the opposite enantiomer, or from a microcrystalline domain structure. 2. N-Hexadecanoylalanine. With regard to the “macroscopic order” as inferred from the surface pressure/ area (Π/A) isotherms (Figure 2a), N-hexadecanoylalanine monolayers spread on a pure water surface appear to exhibit preferential homochiral interactions at large areas per molecule, greater than about 0.3 nm2/molecule. In spite of this clear preference, fluorescence microscopy studies that were published separately13 did not supply visual evidence for chiral phase separation in racemic monolayers of N-hexadecanoylalanine. The condensed phase domains formed from the racemic films of the alanine derivative were irregular and branched, indicating a sensitivity to the headgroup size. The monolayers of the pure L-enantiomer exhibited dendritic growth of the condensed phase. By the way of contrast, in fluorescence microscopy studies with very similar homochiral systems as used in the present investigation,18,19 N-octadecanoylserine methyl ester and N-octadecanoylalanine, visual evidence for chiral phase separation in racemic monolayers was observed. Clockwise and counterclockwise domains or domain segments were observed in monolayers compressed at sufficiently slow rates, while dendritic growth was observed in monolayers of a single enantiomer. These results imply that the macroscopic order, including chiral phase separation effects, may very sensitively respond to changes in the alkyl chain length (e.g., N-hexadecanoyl-/N-octadecanoylalanine) and the possible compression via an additional hydrogen bond formation (serine, R3 ) OH in Figure 1, instead of alanine, R3 ) H). The same conclusion may be drawn from the present results about the potential influence of metal complex formation on the macroscopic order: the addition of PbCl2 to the aqueous subphase (Figure 2b) gives rise to considerable compression of both the racemic and enantiomeric monolayers, while in the presence of ZnCl2 (Figure 2c) the expanded characteristics of the Π/A isotherms become more pronounced in comparison with a pure water subphase. However, in the latter case, stronger chiral discrimination with preferential homochiral interactions are suggested by the Langmuir curves. The respective information about the conformational order of the alkyl chains, i.e., on a molecular scale, can be inferred from the IR reflection-absorption spectra summarized in Figure 3 and from the νa(CH2) wavenumbers shown in Figure 4. On a pure aqueous subphase, N-hexadecanoylalanine monolayers (Figure 4a) exhibit a conformational order that reflects a clear homochiral preference. The same holds for the presence of PbCl2 in the aqueous subphase, which increases the conformational order with preferential homochiral interactions. However, in the case of aqueous subphases containing 1 mM ZnCl2, the molecular interactions change drastically; in particular, the conformational order within the enantiomeric film (18) Stine, K. J.; Uang, J. Y.-J.; Dingman, S. D. Langmuir 1993, 9, 2112. (19) Gericke, A.; Hu¨hnerfuss, H. Langmuir 1994, 10, 3782.

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is reduced such that no differences are discernible between racemic and enantiomeric monolayers. This effect induced by bivalent Zn cations is unexpected insofar as previous investigations of alkanoic acids performed in the presence of Zn2+ indicated very intensive ordering effects induced by the formation of Zn complexes with the carboxyl group. For example, Simon-Kutscher et al.9 reported unusually low νa(CH2) wavenumbers of 2914.3 cm-1 for the octadecanoic acid/Zn2+ system, and Gericke and Mendelsohn,16 who investigated hexadecanoic acid-d17 half-deuterated at the tail end, in the presence of Zn2+ found for the methylenes adjacent to the headgroup νa(CH2) wavenumbers as low as 2911.8 cm-1. The contradiction between previous results obtained with saturated alkanoic acid model substances and the present alanine derivatives implies that the conformational order in the former case is largely dependent on the formation of metal complexes between the carboxylic headgroup and Zn2+, while in the case of the alanine derivatives the additional formation of hydrogen bonds, be it between the amide and/or the carboxyl groups, seems to play an important role. The molecular structure thus induced appears to be strengthened in the presence of Pb2+ and weakened in the presence of Zn2+. Basically, detailed insight into the respective molecular structures can be inferred from the position of the scissoring band of the methylene group [δ(CH2)], which is known to be extremely sensitive to interchain interactions. Usually the following assignments are made:20 A sharp, narrow singlet at 1472 cm-1 is found in the spectra of systems in which the methylene chains are packed in an all parallel arrangement, described as a triclinic subcell. The band is split into a doublet (1472 and 1462 cm-1) when the chains adopt orthorhombic subcell packing, in which the planes of the carbon-carbon bonds of neighboring chains are oriented at about 90° to each other. The third subcell exhibited is the so-called hexagonal type, which is characteristic of the relatively more disordered “rotator” phase of alkanes. The interactions are considerably smaller in the hexagonal subcell due to higher concentrations of gauche defects that are incorporated in the chains, particularly near their ends. The scissoring band of chains packed in the hexagonal subcell is a somewhat broader singlet near 1468 cm-1. Fully distorted liquid-like chains exhibit a considerably broadened and relatively lower intensity scissoring band between 1468 and 1466 cm-1. In the present work, for N-hexadecanoyl-DL-alanine monolayers on a pure water surface, a band at 1462 cm-1 with a shoulder on the high-frequency side (not shown) and a weak doublet at about 1467 and 1462 cm-1 for the enantiomeric film (Figure 3a) may be inferred from our spectra for the δ(CH2) vibration. However, taking into account the resolution of 8 cm-1 used and the presence of water vapor rotation-vibration bands, no unambiguous assignment to an orthorhombic subcell structure is possible. In the presence of PbCl2, unusually strong δ(CH2) bands at about 1467 cm-1 with a shoulder on the low-frequency side (racemic film; not shown) and at 1464 cm-1 (enantiomeric film; Figure 3b) were observed. The values determined in the presence of ZnCl2 were 1464 (racemic film; not shown) and 1462 cm-1 (enantiomeric film; Figure 3c), respectively. These wavenumber values may be indicative of orthorhombic subcell structures; however, we refrain from giving an unambiguous assignment (20) Weers, J. G.; Scheuing, D. R. In Fourier Transform Infrared Spectroscopy in Colloid and Interface Science; Scheuing, D. R., Ed.; ACS Symposium Series 447; American Chemical Society: Washington, DC, 1991; p 87.

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because of the same limitations explained earlier for Figure 3a. It is important to note that the position of the δ(CH2) vibration summarized earlier for N-hexadecanoylalanine surface films responded relatively insensitively to compression of the monolayer. This observation confirms the conclusion drawn from the νa(CH2) wavenumber that a relatively high conformational order of the alkyl chains is attained within the domains already at large areas per molecule, whether it be on a pure water surface or on subphases containing PbCl2 or ZnCl2. The complete disappearance of the chiral discrimination as inferred from the νa(CH2) wavenumbers determined in the presence of a 1 mM ZnCl2 solution cannot be explained on the basis of different subcell structures. Therefore, the reason for this effect is assumed to be related to different metal complex formation between the bivalent cations and the headgroup of the alanine derivative. This assumption is supported by the effect that the carbonyl stretching vibrations observed on a pure water surface in the range 1703-1739 cm-1 disappear on aqueous subphases containing 1 mM PbCl2 or ZnCl2 and instead carboxylate vibrations become visible. Accordingly, as a diagnostic tool that may allow insight into the respective coordination type, the analysis of the separation ∆ between the antisymmetric and symmetric carboxylate vibrations,21,22 i.e., ∆ ) νa(COO) - νs(COO), can be used. Although the significance of ∆ values as a diagnostic tool has been criticized,23 this experimental approach in many cases appears to represent a meaningful guide.22,24 The ∆ values usually assigned to the four main coordination types, ionic, monodentate, chelating bidentate, and bridging bidentate (see ref 9, Figure 6), were largely inferred from the infrared spectra of pure metal carboxylates and aqueous solutions of acetates22 (see Table 2 in ref 9). 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 νa(COO) vibration,21,22 thus giving rise to lower ∆ values. This type of coordination is often denoted as “H-bonded monodentate”.22 In the present work, N-hexadecanoylalanine monolayers on an aqueous subphase containing 1 mM PbCl2 appear to form a strong complex with Pb2+ cations. In the case of the racemic monolayer (not shown), three components of the νa(COO) vibrations are visible in the range 15231557 cm-1, where the component at 1523 cm-1 is clearly dominant and relatively sharp. The νs(COO) vibration is located at 1418 cm-1, and thus for the two most prominent components, ∆ values of 105 and 139 cm-1 are obtained. The first wavenumber may be attributed to a chelating bidentate complex, while the second one may indicate H-bonded monodentate or unsymmetric chelating bidendate coordination.9,24 For the L-enantiomeric monolayer (Figure 3b), a pronounced peak for the νa(COO) vibration at 1540 cm-1 (with a shoulder at the higher frequency side) and a peak for the νs(COO) vibration at 1421 cm-1 give rise to a ∆ value of 119 cm-1, which indicates the preference of chelating bidentate complex formation.24 A completely different situation was encountered in the presence of ZnCl2 in the aqueous subphase. For the νa(COO) vibration sharp peaks at 1561 (racemic film; not shown) and 1562 cm-1 (enantiomeric film; Figure 3c) with (21) Nakamoto, K. Infrared and Raman Spectra of Inorganic and Coordination Compounds; Wiley: New York, 1986. (22) Tackett, J. E. Appl. Spectrosc. 1989, 43, 483. (23) Edwards, D. A.; Hayward, R. N. Can. J. Chem. 1968, 46, 3443. (24) Ramos Moita, M. F.; Duarte, M. L. T. S.; Fausto, R. J. Chem. Soc., Faraday Trans. 1994, 90, 2953.

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a small shoulder on the high-frequency side were determined; however, no νs(COO) vibration could be detected. These observations support the assumption that the interaction between the carboxylic acid headgroup and the Zn2+ cations is different. A clear assignment to a specific coordination type in this case is not possible because of the lack of a νs(COO) band. However, it should be noted that a comparable position of the νa(COO) band was also found for the Zn2+ complex formed with Noctadecanoyltyrosine, which may allow the tentative indirect assignment (see the following) to a H-bonded monodentate coordination. 3. N-Octadecanoyltyrosine. The Π/A isotherms of N-octadecanoyltyrosine monolayers (Figure 5) on a pure water subphase (Figure 5a) between about 0.8 and 0.35 nm2/molecule suggest a preferential heterochiral interaction, while upon further compression homochiral interactions appear to become more favorable. In accordance with these results, fluorescence microscopy showed that the racemic condensed phase domains differ from those of the enantiomeric compound in that they are more compact in shape.3 By the way of contrast, on aqueous subphases containing 1 mM PbCl2 (Figure 5b) and 1 mM ZnCl2 (Figure 5c), respectively, the curves of the racemic and the enantiomeric films show the same characteristics, possibly with a slight tendency to a preferential homochiral interaction. The respective information about the conformational order of the alkyl chains can be inferred from the νa(CH2) wavenumbers shown in Figure 7. The results for Noctadecanoyltyrosine monolayers on a pure water subphase (Figure 7a) in part confirm the conclusions drawn from the Π/A isotherms: the wavenumbers for the racemic monolayer suggest a preferential heterochiral interaction at large area/molecule, while upon compression below about 0.35 nm2/molecule homochiral interactions appear to be more favorable. However, a notable difference between the results for the racemic and enantiomeric monolayers has to be noted for the dependence on the compressional state: while for the racemic film no significant change in the wavenumber of the νa(CH2) vibration and, thus, in the conformational order of the alkyl chains was observed, a very pronounced dependence on the compressional state of the monolayers was determined for the enantiomeric film. However, in the presence of both PbCl2 (Figure 7b) and ZnCl2 (Figure 7c) in the aqueous subphase, the pronounced chiral discrimination effect disappears: in both cases, the νa(CH2) wavenumbers of the racemic and enantiomeric films show a dependence on the compressional state of the monolayer, and, furthermore, comparable results were obtained for the racemic and enantiomeric films, i.e., no preferential homoor heterochiral interaction can be inferred from the determination of νa(CH2) vibrations. The δ(CH2) vibrations of N-octadecanoyltyrosine monolayers (Figure 6) on a pure water surface as well as on subphases containing PbCl2 and ZnCl2 appear at about 1461 cm-1, which may be indicative of an orthorhombic subcell structure. However, for the reasons discussed earlier, we refrain from assigning this vibration to a specific subcell structure. With regard to the carboxylate vibrations, in the presence of the racemic monolayer on an aqueous PbCl2 subphase (not shown), three components of the νa(COO) vibrations are visible at 1518, about 1542, and 1565 cm-1, exhibiting comparable strengths. The νs(COO) vibration shows a relatively broad and weak band at about 1403 cm-1. Thus, for the three components, ∆ values of 115, 139, and 162 cm-1 are obtained. The first two ∆ values may be attributed to chelating bidentate and H-bonded

Hu¨ hnerfuss et al.

monodentate coordinations, respectively, as already found for the alanine derivative. The value of 162 cm-1 may possibly indicate additional complex formation of the Pb2+ cation with adjacent film-forming molecules, a so-called “bridging bidentate” complex. For the L-enantiomeric monolayer (Figure 6b), comparable peak positions were found for the three components of the νa(COO) vibration at 1513, 1542, and 1561 cm-1. The νs(COO) vibration band at about 1400 cm-1 was also very weak and broad. As a result, ∆ values of 113, 142, and 161 cm-1 are calculated, suggesting the same types of complex formation as in the racemic film. 4. N-Octadecanoyltyrosine Methyl Ester. In the present work, we included N-acyltyrosine methyl ester to allow a comparison between the tyrosine derivatives with free and with esterified carboxyl groups (see Figure 1). The characteristics of the Π/A isotherms of the enantiomeric and the racemic films (Figure 8a) look very different from those of the respective N-octadecanoyltyrosine monolayers exhibiting a free carboxylic acid group (Figure 5a). While the Π/A isotherms of the latter monolayers suggest a preferential heterochiral interaction between 0.8 and about 0.35 nm2/molecule, in the former case preferential homochiral interactions were observed between about 0.5 and 0.3 nm2/molecule. This result implies that a free or an esterified carboxylic acid group may give rise to very different chiral interactions. Qualitatively, the same results can be inferred from the respective νa(CH2) wavenumber curves (Figure 8b): compression of the L-enantiomer of N-octadecanoyltyrosine methyl ester gives rise to increasing conformational alkyl chain order, i.e., a decrease in gauche conformers. However, the conformational order of the corresponding racemic monolayers remains relatively constant upon compression until about 0.25 nm2/molecule. Furthermore, it is interesting to note that the νa(CH2) wavenumber data of the N-octadecanoyltyrosine methyl ester films are in line with the results concluded from the Π/A isotherms: in the presence of the L-enantiomeric and racemic films of these compounds, both methods suggest preferential homochiral interactions in the range between about 0.45 and 0.25 nm2/molecule. A detailed quantitative comparison between the νa(CH2) values of N-octadecanoyltyrosine (racemic films, about 2918.5 cm-1; L-enantiomer, about 2917.5 cm-1) and N-octadecanoyltyrosine methyl ester (racemic film, about 2919.5 cm-1; L-enantiomer, about 2918 cm-1) reveals that the free fatty acid carboxyl group gives rise to additional film compression and, thus, to a higher conformational order of the alkyl chains. This aspect and the potential modifications of this effect by the formation of metal complexes with bivalent cations will be pursued in a subsequent paper. Conclusions Comparison between the macroscopic order (inferred from surface pressure/area isotherms and fluorescence microscopy) and the molecular order [determined by infrared reflection-absorption spectroscopy (IRRAS)] for N-acylamino acid monolayers showed that hydrogen bond formation via the NH, COOH, and p-hydroxyphenyl groups may lead to pronounced chiral discrimination. Bivalent Pb2+ and Zn2+ cations may strengthen or weaken both the macroscopic and the molecular order and, thus, the chiral discrimination in N-acylamino acid monolayers: in N-hexadecanoylalanine monolayers, Pb2+ cations give rise to increased compression and increased conformational order, while the presence of Zn2+ cations in the aqueous subphase leads to a decrease in the conformational order and suppresses chiral discrimina-

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tion. Analysis of the methylene scissoring δ(CH2) bands shows that the different effects of Pb2+ and Zn2+ cannot be explained on the basis of different subcell structures (in both cases, an orthorhombic structure appears to prevail), but different complex formations between the bivalent cations and the carboxylic acid headgroup appear to be responsible. Insight into the respective coordination types was obtained by analysis of the separation (∆) between the antisymmetric and symmetric carboxylate vibrations, ∆ ) νa(COO) - νs(COO). For the racemic N-hexadecanoylalanine/Pb2+ complex chelating bidentate and H-bonded monodentate coordinations were determined, and for the L-enantiomeric monolayer the preference of chelating bidentate complex formation was found. The respective complex with Zn2+ cations appears to be much weaker, e.g., no νs(COO) vibration could be detected, and only an indirect assignment to H-bonded monodentate coordination can be suggested. In N-octadecanoyltyrosine and N-octadecanoyltyrosine methyl ester monolayers on a pure water surface, both homo- and heterochiral discrimination may be observed, depending on the available area per molecule. A detailed quantitative comparison between the νa(CH2) values of these two tyrosine derivatives reveals that the free fatty acid carboxyl group gives rise to additional film compression and, thus, to a higher conformational order of the alkyl chains.

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However, in the presence of both PbCl2 and ZnCl2 in the aqueous subphase, the pronounced chiral discrimination effect in the N-octadecanoyltyrosine monolayer disappears. In both cases, chelating bidentate and H-bonded monodentate coordinations, as already found for the alanine derivative, were proved. In the presence of PbCl2, additional complex formation of the Pb2+ cation with adjacent film-forming molecules, a so-called “bridging bidentate” complex, was detected, while the Zn2+ cations may form an additional complex that we presently cannot clearly assign. The results summarized in the present paper suggest that theories like Andelman’s tripode theory should be expanded to account for potential metal complex formations. Acknowledgment. This work was supported by the European Community as part of the “Human Capital and Mobility ProgrammesDynamic Network”, Contract No. ERBCHRXCT930322, as well as by the “Fonds der Chemischen Industrie”, Germany. The authors thank S. Kutz and J. Berndt, who performed some of the present measurements, as well as A. Gericke, R. Mendelsohn, J. Simon-Kutscher, and R. H. Tredgold for enlightening discussions. LA951567+