Lupane-Type Pentacyclic Triterpenes in Langmuir Monolayers: A

Feb 22, 2012 - Lupane-type pentacyclic triterpenes (lupeol, betulin, and betulinic acid) are natural products isolated from various plant sources. The...
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Lupane-Type Pentacyclic Triterpenes in Langmuir Monolayers: A Synchrotron Radiation Scattering Study Marcin Broniatowski,* Michał Flasiński, and Paweł Wydro Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Kraków, Poland S Supporting Information *

ABSTRACT: Lupane-type pentacyclic triterpenes (lupeol, betulin, and betulinic acid) are natural products isolated from various plant sources. The terpenes exhibit a vast spectrum of biological activity and are applied in therapies for different diseases, among which the anticancer, anti-HIV, antihypercholesteremic, and antiinflammatory are the most promising. These chemicals possess amphiphilic structure and were proved to interact strongly with biomembranes, which can be the key stage in their mechanism of action. In our studies, we applied Langmuir monolayers as versatile models of biomembranes. It turned out that the three investigated terpenes are capable of stable monolayer formation; however, these monolayers differ profoundly regarding their physicochemical characteristics. In our research, we applied the Langmuir technique (surface pressure−mean molecular area (π−A) isotherm registration) coupled with Brewster angle microscopy (BAM), but the main focus was on the synchrotron radiation scattering method, grazing incidence X-ray diffraction (GIXD), which provides information on the amphiphilic molecule ordering in the angström scale. It was proved that all the investigated terpenes form crystalline phases in their monolayers. In the case of lupeol, only the closely packed upright phase was observed, whereas for betulin and betulinic acid, the phase situation was more complex. Betulinic acid molecules can be organized in an upright phase, which is crystalline, and in a tilted phase, which is amorphous. The betulin film is a conglomerate of an upright crystalline monolayer phase, tilted amorphous monolayer phase, and a crystalline tilted bilayer. In our paper, we discuss the factors leading to the formation of the observed phases and the implications of our results to the therapeutic applications of the native lupanetype triterpenes.

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INTRODUCTION Isoprenoid cyclic lipids are abundant in the plant kingdom, and their biodiversity is enormous.1 They are important components of the waxy cuticle layer covering leaves and stems, are incorporated into cell walls and cellular membranes where they act as antioxidants, take part in different signaling paths, and last but not least exhibit antibiotic and fungicidal activity.2 Among different classes of isoprenoid lipids, lupane-type pentacyclic triterpenes are widely distributed among plant families.3 The structure of these chemicals is illustrated in Scheme 1. Four cyclohexane rings, all in the chair conformation, and one cyclopentane ring in envelope conformation are the building blocks of the molecule.4 All the compounds possess a hydroxylic group at carbon C3, which renders the molecule's amphiphilic properties. The three main representatives of the family, namely lupeol, betulin, and betulinic acid, differ in the oxidation state at the C28 carbon atom. Lupeol, whose name comes from lupine, is isolated from various plants and can be considered the ground structure.5 Betulin is the most abundant of these terpenes and can be easily isolated from white birch bark.6 Betulinic acid can be isolated from different plants, but it is usually prepared via the oxidation of betulin.7 These natural substances are biologically active and can be applied in therapies for various diseases. First of all, the anticancer activity © 2012 American Chemical Society

Scheme 1. General Structure of Lupane Pentacyclic Triterpenes (R = CH3, lupeol; R = CH2OH, betulin; R = COOH, betulinic acid)

should be underscored here, as a plethora of scientific articles is devoted to this subject.8−11 Depending on the tumor cell line, one of the native lupane terpenes can be more active than the Received: January 3, 2012 Revised: February 22, 2012 Published: February 22, 2012 5201

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(R&K) trough, a single barrier instrument with an area of 500 cm2, was installed. The investigated terpenes were dissolved in a chloroform/methanol 9/1 mixture. The concentrations of the solutions ranged from 2 to 2.5 mg/mL (ca. 5 × 10−6 M). The terpene solutions were spread on the water interface with a Hamilton microsyringe. At least 10 min was allowed for chloroform evaporation, and then the monolayers were compressed at a rate of 20 cm2/min (ca. 3.5 Å2·mol−1·min−1). The surface pressure was monitored during the experiments with a Wilhelmy-type tensiometer and a filter paper (Whatman ashless) stripe as the sensor. All the isotherms were measured at least three times. The temperature of the aqueous subphase was controlled with a circulating water bath at a precision of ±0.1 K. The compression modulus CS−1 was calculated following its definition:

others; however, many sources prove that betulinic acid exhibits the most potent and universal activity.12−14 Lupane-type triterpenes and their semisynthetic derivatives have also been tested and applied against different viruses,15,16 especially against HIV.17,18 The application of lupane terpenes in the therapies for hypercholesterolemia,19 urinary stones,20 or various inflammations6,19 is also finding wide coverage in scientific literature. There is agreement that these terpenes trigger apoptosis selectively in neoplasimic cells by interacting with mitochondrial membranes.21−23 Generally, these compounds have amphiphilic properties, are structurally similar to steroids, and therefore are easily incorporated into cellular and mitochondrial membranes.24,25 The interaction of these terpenes with membrane phospholipids can be the crucial step in the mechanism of their biological activity. Some preliminary research has already been performed in the model systems;26 however, the data are fragmentary and further studies are necessary to understand the interactions on the molecular level. Langmuir monolayers are often applied as simple but versatile models of cellular membranes.27 In contrast to other methods, in this technique the composition of the membrane and the lateral interactions of the hydrophobic moieties can be strictly controlled.28 The method has recently been applied to study the interactions of cardiolipin with betulinic acid in binary monolayers in studies focused on the elucidation of the interaction between this terpene and mitochondrial membrane.29 Unfortunately, one-component betulinic acid monolayers have not thoroughly been investigated, whereas lupeol and betulin have never been characterized in monolayers at the water/air interface. Therefore, we think that the systematic and comparative studies of one-component monolayers of these terpenes will shed new light on the biological activity of the compounds by broadening the physicochemical perspective and by the better understanding of the intermolecular interactions in such systems. In contrast to lupeol, betulin and betulinic acid have a second hydrophilic group at the opposite end of the molecule which endows them with bolaform surfactant character.30 Such compounds can acquire two orientations at the water/air interface, as only one of the hydrophilic groups can be immersed in the aqueous subphase. The elucidation of the orientation of betulin and betulinic acid at the water/air interface is thus one of the main aims of the studies, as it can be crucial for the understanding of the interaction of these terpenes with membrane lipids. In these studies, we would like to mainly stress the importance of synchrotron radiation scattering methods, as they enable the identification of the different molecular orderings in floating monolayers and can be conclusive for the problems we would like to explore.

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CS−1 = − A

dπ dA

(1)

Brewster Angle Microscopy. Brewster angle microscopy experiments were performed with an ultraBAM instrument (Accurion GmbH, Goettingen, Germany) equipped with a 50 mW laser emitting p-polarized light at a wavelength of 658 nm, a 10× magnification objective, polarizer, analyzer, and a CCD camera. The spatial resolution of the BAM was 2 μm. The instrument is coupled with the KSV 2000 Langmuir trough and installed on an antivibration table. Synchrotron Radiation Scattering. The experiments were performed at the BW1 beamline in HASYLAB (Hamburg, Germany) on a dedicated liquid surface diffractometer. The R&K Langmuir trough is placed in a gastight canister and mounted on the goniometer of the instrument. After the preparation of the monolayer, the canister is sealed and is flushed with helium to eliminate oxygen, which results in the reduction of the scattering background and the minimization of the beam damage during X-ray scans. The construction of the liquid surface diffractometer installed in BW1 is described in multiple scientific papers;31−34 therefore, to avoid repetitions, we only mention here some foundations of the grazing incidence X-ray scattering (GIXD) and X-ray reflectivity (XR) methods (for the XR technique, see also Supporting Information). Both methods are available on the same diffractometer and differ in the geometry of the experiments. GIXD provides the information about the lateral ordering of the molecules in the monolayer; if they are periodically ordered, a diffraction signal can be registered. XR provides information about the electron density profile along the monolayer normal; the reflectivity curve can be registered also for disordered monolayers. Both methods give insight into the angstrom scale ordering of the molecules within the film. GIXD. In GIXD, the scattered intensity is measured by scanning over a range of horizontal scattering vectors Qxy;

Q xy ≈

4π sin(2θxy/2) λ

(2)

where 2θxy is the angle between the incident and diffracted beam projected on the liquid surface. The GIXD intensity resulting from a powder of 2D crystallites can be represented as Bragg peaks, resolved in the Qxy direction, by integrating the scattered intensity over the Qz direction, which is measured by the position-sensitive detector placed perpendicular to the air−water interface. Conversely, the Bragg rod profiles can be resolved in the Qz direction:

EXPERIMENTAL SECTION

Materials. Lupeol, betulin, and betulinic acid (all of 98% purity) were purchased from Sigma-Aldrich and used as obtained. Chloroform (99.8%, spectroscopic purity, stabilized with ethanol) and methanol (99.5%) were provided by Sigma-Aldrich. The water used in the experiments was produced by the Milli-Q (Millipore) system, and its resistivity was 18.2 MΩ·cm. Langmuir Technique. Three different Langmuir troughs were applied during the experiments. The general characteristics of the monolayers were investigated with a NIMA 611 instrument, a doublebarrier trough with an area of 600 cm2. For BAM experiments, the monolayers were spread on a KSV 2000, a double barrier trough with a total area of 870 cm2, whereas in HASYLAB, a Riegler&Kirstein

Qz =

2π sin α f λ

(3)

where αf is the X-ray exit angle and obtained by integrating the scattered intensity over Qxy corresponding to the Bragg peak. The number of Qxy maxima and the relation between their intensities informs about the number of crystalline phases present at the water/air interface and about the symmetry of the crystal lattices describing these phases. The values at Qxy maxima provide the information about the unit cell parameters of the 2D lattice. The number of the maxima in the Bragg rod is of help in the discrimination over a monolayer and multilayer, whereas the location of the Qz maxima provides the 5202

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information about the molecular tilt. The calculation of the tilt depends on the symmetry of the 2D lattice. Additional important parameters are coherence length Lxy, being the diameter of a statistical crystalline domain, and Lz, which is the length of the coherently scattering moiety. Both Lxy and Lz are defined by the Scherrer formula: Lxy = 0.9 Lz = 0.9

2π fwhmQxy

2π fwhmQz

collapse of the film is reflected in the course of the isotherm not as a peak but only as a reduction in the surface pressure slope which can be explained by the assumption that the nucleation and growth of the 3D domains are relatively slow as compared with the film compression rate.36 The oxidation of the C28 carbon atom, from CH3 in the lupeol molecule to CH2OH in betulin or to COOH in betulinic acid, introduces profound changes to the π−A isotherm characteristics. This is reflected in the CS−1−π dependencies: for betulinic acid, it achieves the value of ca. 120 mN/m typical for the liquid condensed (LC) state of a monolayer; whereas for betulin CS−1, it does not achieve the limit of 100 mN/m, which according to the classical description is a feature of the liquid expanded state (LE).37 The isotherm of betulinic acid is shifted toward higher molecular areas as compared to the lupeol monolayer. The monolayer collapses at a molecular area of 41 Å2 and a surface pressure of 36 mN/m. In contrast to betulinic acid, the π−A isotherm of betulin is shifted toward lower mean molecular areas from the curve of lupeol. This monolayer collapses at a molecular area of ca. 26 Å2 and a surface pressure of 30 mN/m. This is a very interesting observation: the collapse areas of lupeol and betulinic acid are comparable (43 and 41 Å2/molecule, respectively), whereas for betulin, it is much smaller. In the Cambridge Structural Database (CSD),38 structural data of lupeol and some derivatives of betulin and betulinic acid can be found.4,39,40 On the basis of the crystalline structures, it can be estimated that if a molecule of the investigated terpenes was inscribed into a cylinder of uniform diameter (the rigid rod model), the basal area of the cylinder would range from 40 to ca. 45 Å2, depending on the terpene. An area of 26 Å2/molecule is much lower than the cross section of betulin molecule, which means that the investigated surface film, at least at higher surface pressures, is not a pure monolayer but also contains multilayer regions. It should be underscored that the π−A isotherm of betulin is smooth and no manifestations of phase transitions can be observed in its course. Therefore, it can be assumed that even at lower surface pressure values, different phases coexist at the air/water interface. Betulin and betulinic acid possess an additional hydrophilic group on the opposite side of the molecule with respect to the OH group located at the C3 carbon atom and thus can be treated as examples of bolaform surfactants (bolaforms have two hydrophilic head groups located at both termini of the hydrophobic moiety30). Here arises a problem typical for Langmuir monolayers formed from bolaamphiphiles: one of the hydrophilic groups is immersed in water while the second is in contact with the air.41 In the case of lupeol, there is only one OH group, which is in contact with water and enables the formation of Langmuir monolayers. If betulin and betulinic acid were oriented identically to lupeol, the π−A isotherms would be very similar to each other, but this is not the case. The high CS−1 values and very steep increase of the π value upon compression makes the upright orientation of lupeol molecules in its monolayer very probable, similar to the well-known monolayers of cholesterol.42 The expanded character of the betulinic acid monolayer suggests that either the film-forming molecules are significantly tilted from the monolayer normal or there is equilibrium between two different orientations: upright with the OH at C3 immersed in water and tilted with the COOH group having contact with water. It has to be underscored here that because of geometrical reasons, if the COOH group of betulinic acid or the CH2OH group of betulin is in contact with water, the molecules must be tilted to maximize the immersion of the

(4)

(5)

where fwhm are the full widths at half-maximum of the Bragg peak and Bragg rod, respectively. It is specific for GIXD that typically only the lowest order peaks are registered. The intensities of second and higher order peaks are very low and normally cannot be discerned from the background. Different reasons for such situations are specified here. The amount of matter intercepting the X-ray beam is limited, and there are various kinds of crystalline disorder.32 The large difference in intensities between the first- and second-order peaks cannot be explained by static chain tilt even when the Lorentz polarization factor is included but may be due to Debye−Waller-type effects, which are enhanced by the proximity of the liquid substrate.35 Apart from the low background to signal ratio, another factor extinguishing the higher-order signals is the roughness of the air/water interface. The roughness gives rise to diffuse scattering, and its impact on the measured Bragg rod can also be included in the Debye−Waller term (increasing with Qz).

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RESULTS AND DISCUSSION All three investigated terpenes from the lupane family turned out to be capable of Langmuir monolayer formation. The surface pressure−mean molecular area (π−A) isotherms together with compression modulus−surface pressure (CS−1−π) dependencies are gathered in Figure 1. Upon

Figure 1. π−A isotherms of the investigated terpenes. Inset: corresponding CS−1−π dependencies.

compression of the lupeol monolayer, the surface pressure starts to rise at about 55 Å2/molecule. The isotherm is very steep, which is correlated with the CS−1 values exceeding 250 mN/m, meaning that the film achieves the state described traditionally as solid. The monolayer collapses at the surface pressure of 24 mN/m and molecular area of 43 Å2. The 5203

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brightness are not large, but consecutive stripes at different gray levels can be seen in the magnified fragment of the BAM image. This can be interpreted as the manifestation of the presence of two kinds of condensed domains within the floating film, differing significantly in the tilt angle of the molecules. The thicker, brighter regions can be formed of upright molecules whereas the more obscure of the regions can be formed of tilted molecules. The presence of two kinds of domains in the monolayer of betulinic acid has recently been postulated by Chen and coauthors.29 They supposed that with the increasing surface pressure the tilted phase, in which COOH is in contact with water, transforms into the upright phase, in which OH is immersed in water. The situation is similar for the film of betulin; however, additional regions of very high brightness can often be observed at the edges of the domains as it is shown in the magnified fragment of image c in Figure 2. The presence of such a bright object in BAM images corroborates the hypothesis that in betulin films multilayer regions are present even at low surface pressures far from the film collapse. To have an insight into the organization of the terpene molecules in the angstrom scale and to verify the above formulated hypotheses, grazing incidence X-ray diffraction (GIXD) and X-ray reflectivity (XR) methods have been applied. It turned out that the GIXD results collected for betulin monlayers are more complex than for the other investigated terpenes; thus, we first present and discuss the GIXD data for lupeol and betulinic acid and later proceed to betulin. As visible in Figure 3a,d, there is only one intensive diffraction signal registered for the monolayers of lupeol and betulinic acid. Traditionally, the GIXD results are presented as Bragg peaks (I(Qxy) integrated over all Qz values and Bragg rods (I(Qz) integrated over all Qxy values. The Bragg peaks of both terpenes are symmetrical Lorenzian curves having the maximum virtually at the same value of Qxy. They differ only in the value of the peak width at half-maximum, as the peak of lupeol is much narrower than that of betulinic acid. The Bragg rods are nearly identical, with the maximum at 0 Å−1 and a width at half-maximum of ca. 0.4 Å−1. Only one symmetrical Bragg peak correlated with a Bragg rod having only a single maximum located at 0 Å−1 corroborates close packing of the film-forming molecules, which can be described by a hexagonal lattice. All the parameters calculated from the GIXD data are gathered in Table 1. It is interesting that the areas of the unit cell for both terpenes are very similar and correspond to the cross sections of the molecules acquired from CSD4,39,40 crystal structures. Moreover, the values are very close to the mean molecular areas observed in the proximity of the collapse pressure of these monolayers. This proves that at high surface pressures, practically all the terpene molecules present in the film are involved in the formation of the crystalline phase. The hexagonal lattice is typical for molecules oriented upright at the air/water interface, as molecular tilt lowers the symmetry of a two-dimensional lattice.31,43 Therefore, on the basis of the GIXD data, it can be stated that in the crystalline phase, betulinic acid molecules are oriented in the same manner as that of lupeol molecules, that is upright, parallel to the monolayer normal. The upright position of betulinic acid molecules means that the OH group at C3, and not the COOH group, is in contact with water. Moreover, the practically identical values of the mean molecular area at film collapse, the area of 2D unit cell, and the crystallographic cross section of the

head group and minimize the contact of the hydrophobic moiety with water. Both possible orientations of betulin and betulinic acid at the air/water interface are illustrated in Scheme 2. Scheme 2. Possible Orientations of Betulin and Betulinic Acid at the Air/Water Interface: (a) Upright with the OH at C3 Immersed in Water; (b) Tilted with the CH2OH or COOH at C17 Immersed in Watera

a

The hydrogen-subtracted structures in the wire-frame rendering used in the picture are taken from CSD.

In the case of betulin, the monolayer is also expanded but the π−A isotherm is shifted toward lower mean molecular areas as compared with lupeol; thus, as suggested above, apart from both possible betulin orientations in the monolayer, multilayer domains are also probably present here. All the monolayers were visualized upon compression with the application of Brewster angle microscopy, and the representative BAM images are gathered in Figure 2.

Figure 2. BAM images of the investigated terpenes collected at 10 mN/m: (a) lupeol; (b) betulinic acid; (c) betulin. The regions in red frames are magnified below. The size of the whole BAM picture is 400 × 700 μm.

In the case of lupeol, the monolayers are homogeneous until the film collapse. In contrast, in monolayers of betulin and betulinic acid, large condensed domains are observed. At zero surface pressure, they flow on the surface, separated from each other, while upon compression, they come into closer contact, but the monolayers remain cracked even at high surface pressures. In Figure 2, fragments of the images surrounded with red frames are magnified to emphasize the features of the domains. The most important observation is that they differ in brightness. The gray scale in BAM microscopy depends on the thickness of the investigated monolayer: the thicker the floating film, the brighter the image. For betulinic acid, the changes in 5204

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Figure 3. (a, d) Intensity maps I(Qxy,Qz), (b, e) Bragg peaks, and (c, f) Bragg rods for the monolayers of (a−c) lupeol and (d−f) betulinic acid registered at 10 mN/m. The Bragg peaks were fitted with the Lorenz, whereas the rods were fitted with Gauss curves (red continuous curves).

cannot be formed. The hydrogen bond between the COOH groups leads to the appearance of dimers in the monolayer. Such a monolayer is undulated and dynamic, and the molecules have translational and partial rotational freedom, so not every possible hydrogen bond is formed, which leads to defects in the periodic two-dimensional lattice and, in consequence, to the reduction of the Lxy value. As mentioned previously, the case of betulin is more difficult and is discussed separately. The intensity map registered for the betulin monolayer at 10 mN/m is presented in Figure 4. Four diffraction signals can be identified in the map: (1) a very strong signal at Qxy = 1.020 Å−1 with the intensity maximum at the horizon (Qz = 0 Å−1); (2) at Qxy = 0.895 Å−1 with the intensity smeared over a long-range of Qz values; (3) a higher order signal at Qxy = 1.346 Å−1 and intensity maximum at the horizon; (4) the signal smeared over the Scherrer arc,45 the intensity maximum of which lies at Qxy = 0.964 Å−1 and Qz = 0.371 Å−1. For a more detailed analysis of the GIXD data, the diffraction signals have been converted to Bragg peaks and Bragg rods, which are shown in Figure 5. By analogy to the above-discussed terpenes, the intense peak at 1.020 Å−1 is the manifestation of a condensed, closely packed phase of upright molecules (phase I). The parameters calculated for this phase are gathered in Table 1. The lattice parameters are identical with the crystalline phase of betulinic acid, differing only in the Lxy value (248 and 165 Å, respectively). The higher value of Lxy (but still lower than for lupeol) indicates that the statistical crystalline domains in the betulin film are larger than in the betulinic acid monolayer. In

molecule mean that apart from the upright, hexagonal phase no additional disordered phase is present in the monolayer at high surface pressure value, which is in agreement with the conclusions of Chen and co-authors.29 However, at lower surface pressures, the tilted domains directed with the COOH group toward water can be present in the film, which is manifested in the BAM images as slightly less bright areas (Figure 2b). The tilted molecules in such domains are not periodically ordered; thus, no signal connected with this phase can be observed in the GIXD intensity map. Lupeol and betulinic acid have very similar values of Lz. The Lz values are interpreted as the length of the fragment of a filmforming molecule which scatters X-rays coherently; it can be the whole hydrophobic tail or a shorter fragment if there is disorder in this moiety.44 The values of 13−14 Å observed for the investigated terpenes are in good agreement with the CSD crystallographic data.4 The length of 13.5 Å is exactly the length of the hydrophobic moiety, which is close to the length of the whole molecule. This correlation of the crystallographic data and our results is an additional proof of the upright orientation of the film-forming molecules in their monolayers. Lupeol and betulinic acid differ in the values of coherence length Lxy (420 and 165 Å, respectively). This result is interesting, as one could expect a reversed trend. The side COOH groups of two adjacent molecules can form a hydrogen bond. The formation of a hydrogen bond leads to stronger intermolecular attraction and closer contact and should cause the condensation of the monolayer. However, because of structural reasons a large network of hydrogen bonds overwhelming numerous molecules 5205

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0.1 0.1 0.1 0.2 02 0.1 ± ± ± ± ± ± 70 ± 9 105.2 ± 0.2 23.2 ± 0.1

13.9 14.3 13.8 23.6 22.6 22.4

Lxy (Å)

423 ± 9 165 ± 2 248 ± 9 :257 ± 20 ± ± ± ± 44.31 43,74 43.74 103.3 0 0 0 18.9 ± 0.1

Figure 4. Intensity map (I(Qxy,Qz) registered for the betulin monolayer at 10 mN/m.

both cases, the hydrogen bond formation between the terminal side polar groups at C28 is important. If the defect hypothesis proposed above is correct, it can be inferred from the Lxy values that the betuline monolayer is less prone to such defects. The difference can also be connected with the chemical properties of both side polar groups. The carboxylic group can ionize, but as it is a weak acid, this dissociation is only partial; therefore, in the film of betulinic acid some COOH groups can be ionized and some not. The ionization changes the preferences of a functional group toward hydrogen bond formation; thus, the partial dissociation of some random COOH groups increases the probability of defect formation and, in consequence, lowers the range of crystallinity in the floating film. The other factor which can also be important here is the binding of ions to COOH and salt formation. From the crystallographic point of view, every COOH group binding an ion is also a defect center. The hydroxyl group of betulin cannot ionize or form a salt, so the factors discussed here of additional disorder are eliminated and Lxy is visibly higher. The two other low-order peaks at 0.894 and 0.964 Å−1 are a manifestation of the presence of another crystalline phase in betulin monolayer. The fact that both signals have no intensity at the horizon indicates that this phase is tilted. The signal at Qxy = 0.894 Å−1 is narrow and smeared over a long range of Qz. It has two intensity maxima at Qz of ca. 0.3 and 0.6 Å−1 . The presence of two maxima in the course of a Bragg rod, obeying the relation Qz2 = 2Qz1 (Figure 5c), is often observed for crystalline bilayers,46 so phase II can be identified as a bilayer in which betulin molecules are tilted from the monolayer normal. Another feature very characteristic for this phase is the presence of a Scherrer arch in the diffractogram.45,47,48 We identified the location of the second peak of this phase as Qxy = 0.964 and Qz = 0.371 Å−1, but it is only approximation, as this peak is smeared, interfering partially with the long signal at Qxy = 0.895 Å−1. The presence of the Scherrer arch in the diffractogram proves that there is no identical azimuth of the tilt settled within phase II.48 Therefore, phase II, a crystalline tilted bilayer, can be treated as a set of domains differing in lattice parameters. The Lz value of ca. 22−23 Å corroborates the identification of the phase, as for an untilted bilayer, Lz should be ∼28 Å. Thus, the rough estimation of the tilt is 35°, assuming that the bilayer is not interdigitated. Generally, two limiting azimuths can be discussed in the case of the Scherrer arch: NN (nearest neighbor) and NNN (next nearest neighbor).47 In the NNN

a

1.014 1.021 1.021 0.964 0.894 1.021 0.894

w, full width of the peaks at half-maximum (FWHM); τ, tilt angle; A, area of the 2D unit cell.

90 12.320 ± 0.005 8.543 ± 0.006

120 120 120 90 ± ± ± ±

0.007 0.001 0.002 0.01 7.153 7.107 7.107 14.04 7.153 ± 0.007 7.107 ± 0.001 7.107 ± 0.002 7.36 ± 0.03

0.407 ± 0.001 0.396 ± 0.002 0.408 ± 0.002 0.253 ± 0.001 0.25 ± 0.01 0.408 ± 0.002 0.23 ± 0.02 0.0134 ± 0.0003 0.0382 ± 0.0002 0.023 ± 0.001 0.08 ± 0.01 0.022 ± 0.002 0.023 ± 0.001 0,022 ± 0.002 ± ± ± ± ± ± ± lupeol betulinic acid betulin phase I betulin phase II NNN NN

0.001 0.001 0.001 0.004 0.001 0.001 0.001

w (Å )

Qxy (Å ) compound

0 0 0 0.371 ± 0.002 0.605 ± 0.004 0 0.315 ± 0.004

Qz (Å )

0.04 0.1 0.01 0.2

A (Å2) γ (deg) b (Å) a (Å) w (Å )

lattice parameters

‑1

Bragg rod −1 ‑1

Bragg peak −1

Table 1. Structural Parameters Calculated for the Investigated Compounds from the GIXD Dataa

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τ (deg)

Lz (Å)

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Figure 5. Bragg peaks (a, b) and Bragg rods (c−f) extracted from the intensity map registered for the betulin monolayer at 10 mN/m: (a) Bragg peak integrated over Qz from 0 to 0.325 Å−1; (b) Bragg peak integrated over Qz from 0.325 to 0.650 Å−1; (c) Bragg rod at Qxy = 0.895 Å−1; (d) Bragg rod at Qxy = 0.964 Å−1; (e) Bragg rod at Qxy = 1.020 Å−1; (f) Bragg rod at Qxy = 1.346 Å−1. The solid lines in the plots are Lorentz (Bragg peaks) or Gauss (Bragg rods) best fits of the experimental, background-corrected data points.

Scheme 3. Postulated Orientation of Betulin Molecules in the Observed Crystalline Phasesa

a

The hydrogen subtracted structures in the wire-frame rendering used in the picture are taken from CSD.

case, there are two peaks in the diffractogram, both at Qz > 0, so the set of parameters, Qxy = 0.895, Qz = 0.605 Å−1 and Qxy = 0.964, Qz = 0.371 Å−1, is definitive for this limiting phase. In the NN case, two signals should be observed in the intensity map: one nondegenerated, centered at the horizon, and one degenerated, out of the horizon. One peak is present, Qxy = 0.895, Qz = 0.315 Å−1, but we also need the nondegenerated peak. Figure 4 shows that the signal at 1.02 Å−1 is broadened, and the lacking peak can be overlapped with the intense signal of the hexagonal monolayer phase. Thus, for the limiting NN case, we acquire 1.020 Å−1 as the Qxy value of the nondegenerated peak (Qz = 0), and Qxy = 0.895, Qz = 0.315 Å−1 as the coordinates of the maximum of the degenerated peak. The structural parameters calculated for the limiting cases are gathered in Table 1. The tilt angle is 18.9° for NNN and 23.2° for NN. Both values are lower than 35° estimated above, so some interdigitation of the lower and upper layer can be postulated. The bilayer is tilted, and it can be hypothesized that

it evolves from the tilted monolayer in which the OH at C28 is in contact with water. However, similarly to betulinic acid, there is no signal in the diffractogram which could be correlated with the tilted monolayer which is rather amorphous. The orientation of the molecules at the air/water interface in all the described phases is illustrated in Scheme 3. Regarding Scheme 3, a problem arises: does the OH group at C3 of the molecule from the bottom layer contact the OH group at C28 or C3 of the molecule from the upper layer? The structural consideration leads to the conclusion that if it was the former option, there would be a significant steric hindrance, as the methyl group located at C4 of the bottom molecule would collide with the cyclopentane ring of the upper molecule. Therefore, the latter possibility in which the hydroxyl groups located at the C3 carbon atoms of both molecules come in close contact is much more probable. It seems that the OH groups at C3 directed toward the air in the tilted domains can come in close contact and form intermolecular hydrogen 5207

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images for the former terpenes. On the other hand, the interactions between the side groups generate defects that lead to the lowering of periodicity, which was observed by GIXD. The results of our experiments prove that minor changes in the amphiphilic molecule structure can lead to profound differences in the monolayer characteristic. It could be predicted that the behavior of lupeol, which has only one polar group at one end of the molecule, can be different from betulin and betulinic acid, being bolaforms. However, the results collected for betulin and betulinic acid astonished us. Now it is obvious that we should not expect one common mechanism of action for all three investigated terpenes with biomembranes. Probably, the interactions will depend profoundly on the lipid composition of a particular membrane. Therefore, depending on the target cell or target organelle, one of these terpenes can be much more effective than the others, as frequently reported in pharmacological and medical studies. However, it seems possible to establish some trends regarding the interactions of the investigated triterpenes with different lipid classes. We have undertaken the studies of the interactions of these terpenes with model eukaryotic and bacterial membranes. The preliminary results are promising, but additional experiments are needed before publication, to obtain a more comprehensive picture of the investigated problem.

bonds. At the tilted phase, the cyclopentane ring is partially immersed in water, which from the thermodynamic point of view is not energetically favorable. Therefore, the f lip-flopping of some betulin molecules to the additional upper layer and the formation of a crystalline bilayer is convenient thermodynamically. In our experiments, we focused mainly on GIXD experiments; however, to have a deeper insight into the investigated systems we also applied the X-ray reflectivity (XR) technique, which is also available at the BW1 beamline in HASYLAB. The investigated terpenes have a very small (OH) head group, which results in quite uniform distribution of the electron density. This is reflected in the course of the reflectivity curves, which have only one weak fringe, and the contrast is rather low. However, this data can be informative for interested readers investigating similar compounds, and we attach the reflectivity data and its interpretation in a separate file as Supporting Information.

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CONCLUSIONS The physicochemical properties of the monolayers formed by the investigated lupane-type triterpenes at the water/air interface differ profoundly. Lupeol forms solid monolayers in which the molecules are oriented perpendicularly at the interface. This molecule has only one hydroxylic head group and behaves in the floating films similarly to cholesterol or other steroids investigated previously. The interactions of the hydrophobic moieties have purely van der Waals character. In contrast to lupeol, betulin and betulinic acid bear an additional polar function at the second pole of the hydrophobic moiety. This opens the possibility of two different orientations in Langmuir monolayers: perpendicular with the OH group at the carbon C3 atom in contact with water and tilted with the hydroxyl or carboxyl group at C28 immersed in water. There is not a distinct phase transition between the two phases; BAM observations proved that two kinds of domains are present even at zero surface pressure. Upon compression, the continuous reorientation of the molecules leads to the transformation of the tilted phase to the upright orientations in the case of betulinic acid, as the GIXD results correlated with π−A isotherms prove that at high surface pressures, only the upright phase is present. In the case of betulin, the interaction between the tilted and upright phases leads to the formation of a crystalline, tilted bilayer, which dominates at high surface pressures. It is interesting that betulin and betulinic acid monolayers behave so differently upon compression. The reason for the observations seems to be related to the mutual interaction of the side hydrophilic groups. In both cases, the tilted monolayer phase is thermodynamically unstable, as at this conformation, a part of the cyclopentane ring of the terpene skeleton is immersed in water. The potential energy of the system is lowered upon compression by the elimination of the tilted phase in the case of betulinic acid and by the formation of the tilted bilayer in the case of betulin. The experiments prove that the hydrogen bonds formed by the side COOH groups in the upright phase of betulinic acid are strong enough to stabilize the monolayer; whereas in the case of betulin it is more convenient for the system to form intermolecular hydrogen bonds stabilizing the bilayer. Because of the additional dipolar interactions and the possibility of hydrogen bond formation, betulin and betulinic acid molecules interact stronger with each other than lupeol molecules. This is manifested in the presence of large crystalline domains of sharp edges observed in BAM

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +48126632082. Fax: +48126340515. Funding

The research was carried out with equipment (UltraBAM) purchased from financial support by the European Regional Development Fund in the framework of the Polish Innovation Economy Operational Program (contract no. POIG.02.01.0012-023/08). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge HASYLAB, DESY (Hamburg), for granting us beamtime at BW1 beamline and express our gratitude to Dr. Bernd Struth for his help at BW1.

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REFERENCES

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