Phase Transition in Glycolipid Monolayers Induced by Attractions

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Phase Transition in Glycolipid Monolayers Induced by Attractions between Oligosaccharide Head Groups Kaoru Tamada, Hiroyuki Minamikawa, and Masakatsu Hato* Surface Engineering Laboratory, Department of Polymer Physics, National Institute of Materials and Chemical Research, 1-1 Higashi, Tsukuba, Ibaraki 305, Japan

Kenjiro Miyano Department of Applied Physics, Faculty of Engineering, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan Received April 24, 1995. In Final Form: December 11, 1995X The surface property of two kinds of synthetic glyceroglycolipids was investigated to confirm the stereoeffect of sugar residues on the phase behavior in monolayers: one is the maltooligosaccharide-containing lipids [MalN(C12)2] and the other is cellooligosaccharide-containing lipids [CelN(C12)2]. The two kinds of glycolipids exhibit the opposite dependence of the surface pressure-area (Π-A) isotherm on the number of glucose residues (N), in that MalN(C12)2 tends to be more expanded as N increases, while CelN(C12)2 tends to be more condensed. The liquid-expanded/liquid-condensed phase transition in Cel4(C12)2 was evaluated by the Brewster angle microscopy and the latent heat analysis with the Clausius-Clapeyron equation, as a model of film condensation induced by the attraction between oligosaccharide head groups. The film morphology of Cel4(C12)2 was unique in that it exhibits the optically isotropic and flexible fluid type of the LC phase, while Glc(C16)2 forms a two-dimensional tilted crystalline phase in the same way as fatty acids and phospholipids. The heat of transition (∆H) of Cel4(C12)2 does not converge toward zero even at high temperature. In other words, the main transition of Cel4(C12)2 does not become second order in contrast to the behavior usually encountered in monolayers. The refractive index of the oligosaccharide head groups in water as well as the alkyl chains in air must be properly taken into account to interpret the reflectivity.

Introduction It is quite recent that glycolipids constituting a biological membrane have been mentioned as molecules which play a key role in various physiological events like cell recognition and adhesion process.1,2 Various attempts have been made to ascertain the specific interaction between glycosphingolipids (GSLs) with biological experimental techniques.3,4 The adhesion behavior of liposomes containing Lewis x glycolipid (Lex, Galβ1f4[FucR1f3] GlcNAcβ1fR]) or other glycolipids onto the solid plates coated with Lex or GSLs was studied in the presence or absence of Ca2+ ion.5-10 The affinity binding technique was also applied to confirm the effect.11 Independently, the cluster structure of GSLs on the cell surface was determined by the cryogenic electron microscopy.12-14 Individual molecules assume a preferred con* Corresponding author. X Abstract published in Advance ACS Abstracts, February 15, 1996. (1) Hakomori, S. Annu. Rev. Biochem. 1981, 50, 733. (2) Fenderson, B. A.; Eddy, E. M.; Hakomori, S. BioEssays 1990, 12, 173. (3) Turley, E. A.; Roth, S. Nature 1980, 283, 268. (4) Brewer, G. T.; Thomas, P. D. Biochim. Biophys. Acta 1984, 776, 279. (5) Eggens, I.; Fenderson, B. A.; Toyokuni, T.; Dean, B.; Stroud, M. R.; Hakomori, S. J. Biol. Chem. 1989, 264, 9476. (6) Kojima, N.; Hakomori, S. J. Biol. Chem. 1989, 264, 20159. (7) Kojima, N.; Hakomori, S. J. Biol. Chem. 1991, 266, 17552. (8) Hakomori, S. Pure Appl. Chem. 1991, 63, 473. (9) Kojima, N.; Hakomori, S. J. Glycobiol. 1991, 1, 623. (10) Kojima, N.; Shiota, M.; Sadahira, Y.; Honda, K.; Hakomori, S. J. Biol. Chem. 1992, 267, 17264. (11) Kojima, N.; Fenderson, B. A.; Stroud, M. R.; Goldberg, R. I.; Habermann, R.; Toyokuni, T.; Hakomori, S. Glycoconjugate J. 1994, 11, 238. (12) Tillack, T. W.; Allietta, M.; Moran, R. E.; Young, W. W., Jr. Biochim. Biophys. Acta 1983, 733, 15. (13) Rock, P.; Allietta, M.; Young, W. W., Jr.; Thompson, T. E.; Tillack, T. W. Biochemistry 1990, 29, 8484.

formation by their multivalency, which is supposed to be an essential condition for their selective interactions.15 In spite of such a biological importance, the physicochemical properties of glycolipids have not been fully investigated, due to the difficulty in extracting sufficient amounts of chemically pure glycolipid from natural membranes. In general, natural membrane extracts exhibit a large distribution of chain length and unsaturation in the alkyl chain part, and a complicated isomeric mixture in the carbohydrate portion as well.16 This heterogeneity interferes with a clear understanding of their properties, since the influence of the detailed structure of lipids and the existence of impurities turn out to be unexpectedly high. Some pioneering works have been performed with these natural extracts; the surface property of GSLs17-20 and galactolipids21,22 have been investigated in monolayers17,19-22 and self-assembled films.18 However, no specific attraction between the oligosaccharide head groups could be determined in those monolayer systems. This is presumably because the functional group, which is located at the end of the sugar residue, cannot interact with others in the monolayer due to the steric effect of the (14) Rock, P.; Allietta, M.; Young, W. W., Jr.; Thompson, T. E.; Tillack, T. W. Biochemistry 1991, 30, 19. (15) Zhou, B.; Li, S.-C.; Laine, R. A.; Huans, R. T. C.; Li, Y.-T. J. Biol. Chem. 1989, 264, 12272. (16) Curatolo, W. Biochim. Biophys. Acta 1987, 906, 111 and 137. (17) Maggio, B.; Cumar, F. A.; Caputto, R. Biochem. J. 1978, 171, 559. (18) Maggio, B. Biochim. Biophys. Acta 1985, 815, 245. (19) Fidelio, G. D.; Maggio, B.; Cumar, F. A. Biochim. Biophys. Acta 1986, 854, 231. (20) Luckham, P.; Wood, J.; Froggatt, S.; Swart, R. J. Colloid Interface Sci. 1993, 156, 164. (21) Tomoaia, M.-C.; Zsako´, J.; Chifu, E.; Quinn, P. J. Chem. Phys. Lipids 1983, 34, 55. (22) Ali, S.; Brockman, H. L.; Brown, R. E. Biochemistry 1991, 30, 11198.

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whole head group, or possibly, the attraction is too small to be detected by the monolayer technique in their systems. The direct measurement of the interactions between glycolipid bilayers deposited on a pair of mica surfaces has been performed with a surface force apparatus (SFA).23-25 On the compressive approaches, forces were generally repulsive; the double layer repulsion at large surface separations and the steric and/or hydration forces at shorter separations. This was in part due to the presence of the negatively charged groups (N-acetylneuraminic acid) in the oligosaccharide head groups examined, so that the overall forces were overwhelmed by the strong double layer repulsion.23 In those systems, no adhesion was observed.23,24 Though Marra25 has discussed the attractive van der Waals interactions between the mono- and digaractosyl diglyceride lipid bilayers, these attractions appear not to be directly related to the “specific” interactions between oligosaccharide head groups. A synthetic route of glycolipids has been developed to prepare chemically well-defined glycolipids,26-29 and the physicochemical properties of these synthetic glycolipids have been reported.30-32 Recently, our research group developed an efficient route to synthesize glycolipids in a good yield and good stereoselectivity.33 This route was applied to prepare glycolipids bearing cello- or maltooligosaccharides with a definite number of glucose residues (N; N ) 1-6), in which the oligosaccharide head group is linked to the tail group through an O-glycosidic bond. The tail group is composed of saturated long double alkyl chains connected to glycerol at the 1 and 3 positions through ether bonds. In the previous communication,34 we have demonstrated that the stereochemistry of the head groups exhibits profound effects on the physical properties of the glycolipid/water systems: the cellooligosaccharide and the maltooligosaccharide head groups have opposite effects on the phase behavior of the aqueous glycolipids. The results strongly suggest that the interaction between the maltooligosaccharides is repulsive, while that between the cellooligosaccharides is attractive. In this study, we investigate the monolayer property of these well-defined glycolipids at the air/water interface. The objective of our studies is to determine the influence of the stereochemistry dependent interactions between oligosaccharide head groups on the phase behavior in monolayers. Through the systematic study of our glycolipids, we found the first direct evidence of attractive interactions between oligosaccharide head groups in the cellooligosaccharide-type glycolipid monolayers, where liquid-expanded (LE)/liquid-condensed (LC) phase transition induced by oligosaccharide head groups could be determined. The extremely simple structure of our gly(23) Luckham, P.; Wood, J.; Froggatt, S.; Swart, R. J. Colloid Interface Sci. 1993, 156, 173. (24) Parker, J. L. J. Colloid Interface Sci. 1990, 137, 571. (25) Marra, J. J. Colloid Interface Sci. 1986, 109, 11. (26) Six, L.; Russ, K.-P.; Liefla¨nder, M. Tetrahedron Lett. 1983, 24, 1229. (27) Mannock, D. A.; Lewis, R. N. H.; McElhaney, R. N. Chem. Phys. Lipids 1987, 43, 113. (28) Endo, T.; Inoue, K.; Nojima, S. J. Biochem. (Tokyo) 1982, 92, 953. (29) Jarrell, H. C.; Jovall, P. A° .; Giziewicz, J. B.; Turner, L. A.; Smith, I. C. P. Biochemistry 1987, 26, 1805. (30) Hintz, H.-J.; Kuttenreich, H.; Meyer, R.; Renner, M.; Fru¨nd, R.; Koynova, R.; Tenchov, B. G. Biochemistry 1991, 30, 5125. (31) Asgharian, B.; Cadenhead, D. A.; Mannock, D. A.; Lewis, R. N. A. H.; McElhaney, R. N. Biochemistry 1989, 28, 7102. (32) Mannock, D. A.; Lewis, R. N. A. H.; McElhaney, R. N. Biochemistry 1990, 28, 7102. (33) Minamikawa, H.; Murakami, T.; Hato, M. Chem. Phys. Lipids 1994, 72, 111. (34) Hato, M.; Minamikawa, H. Langmuir 1996, 12, xxx-xxx.

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colipids may have contributed to this successful observation. In the latter haf of this paper, we will focus our attention on the phase behavior induced by the oligosaccharide head groups in comparison with that induced by the long alkyl chains.35-38 Here, the classical latent heat analysis with Clausius-Clapeyron equation35,39 and the powerful new technique to visualize the film morphology, the Brewster angle microscopy (BAM),40-42 are applied to the characterization of these systems. This may shed some light on the molecular mechanism of the glycolipids in cell recognition process. Experimental Section (a) Materials and Monolayer Method. Two types of glycolipids, maltooligosaccharide-containing lipids [MalN(C12)2] (1) and cellooligosaccharide-containing lipids [CelN(C12)2] (2), were produced by the same synthetic route described previously,33 where N is the number of glucose residue in the oligosaccharide head group (N ) 1-5). In the maltooligosaccharide head groups [MalN], each glucose unit is linked through R-1,4-O-glycosidic bonds, while in the cellooligosaccharide head groups [CelN] they are linked through β-1,4-O-glycosidic bonds. The tail of 1,3-diO-dodecylglycerols is composed of saturated double alkyl chains containing 12 carbon atoms per single chain. In a series of glycolipids, a glycolipid comprising one glucose residue is named as [Glc(C12)2] (3) distinctively to avoid confusion, since both maltoand cello-type of glycolipids result in the same chemical structure (Glc(C12)2 ) Mal1(C12)2 ) Cel1(C12)2). A glycolipid, [Glc(C16)2] (4) containing one glucose residue and a long double alkyl chain (carbon number/single chain ) 16) was also prepared in the same way as the glycolipids having shorter alkyl chains.33 The purity of all synthetic glycolipids was estimated to be better than 99%.

These compounds were each dissolved in a CHCl3/MeOH mixture to make spreading solutions with a concentration of 0.2-0.3 mM. The monolayer mass density is varied by means of the successive addition method over a range of water temperature (T ) 8.5-42 °C),43 and surface pressure vs area per molecule (Π-A) measurements and Brewster angle microscopic observation are carried out for each film. The subphase water (35) Albrecht, O.; Gruler, H.; Sackmann, E. J. Phys. 1978, 39, 301. (36) Fischer, A.; Lo¨sche, M.; Mo¨hwald, H.; Sackmann, E. J. Phys. Lett. 1984, 45, L785 and references cited therein. (37) Bibo, A. M.; Knobler, C. M.; Peterson, I. R. J. Phys. Chem. 1991, 95, 5591. (38) Kenn, R. M.; Bo¨hm, C.; Peterson, I. R.; Mo¨hwald, H. J. Phys. Chem. 1991, 95, 2092. (39) Grainger, D. W.; Sunamoto, J.; Akiyoshi, K.; Goto, M.; Knutson, K. Langmuir 1992, 8, 2479. (40) Ho¨nig, D.; Mo¨bius, D. Thin Solid Films 1992, 210/211, 64. (41) He´non, S.; Meunier, J. Rev. Sci. Instrum. 1991, 52 (4), 936. (42) Ho¨nig, D.; Mo¨bius, D. J. Phys. Chem. 1991, 95, 4590. (43) Sauer, B. B.; Yu, H. Tien, C.; Hager, D. F. Macromolecules 1987, 20, 393.

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was obtained by the same method described in the previous paper.34,44 Surface pressure was determined by a Wilhelmy technique using a sandblasted platinum plate. The surface pressure was observed as a function of time until the time dependence (dΠ/dt) reached approximately 10-3 mN/m‚s, at which point we took the measured Π to be near equilibrium. The equilibrium spreading pressures were determined by the same method described in the previous paper.44 (b) Brewster Angle Microscopy. The instrument is the same as that reported previously.45 The Brewster angle microscope (BAM) was mounted on a Teflon trough, and the microscope images taken with a CCD camera were recorded on a video tape and digitized for the image analysis with a personal computer. The incident angle of the He-Ne laser beam was fixed at approximately 53°, which is the Brewster angle at the air/water interface (nair ) 1, nwater ) 1.33 at RT). The light reflectivity from the LE and LC phases on water was determined by the image analysis of the BAM pictures. The brightness of the BAM images was converted into the reflectivity using a calibration line determined by the incident angle dependence of the reflectivity on the pure water surface in the vicinity of the Brewster angle.45

Results and Discussion 1. Effect of the Stereochemistry of the Oligosaccharide Head Groups on the Phase Behavior in the Monolayers. A series of Π-A isotherms of the maltooligosaccharide-containing lipids [MalN(C12)2] and the cellooligosaccharide-containing lipids [CelN(C12)2] are summarized in Figure 1. In Figure 2, the Π-A isotherms of MalN(C12)2 and CelN(C12)2 measured at low temperature are superimposed for comparison, allowing the dependence of Π-A isotherms upon the number of glucose residues (N) to be exhibited more clearly. The Π-A isotherms of MelN(C12)2 tend to expand as N increases. The kink observed with Glc(C12)2 (N ) 1) corresponding to the phase transition disappears and the Π-A isotherms appear as the liquid-expanded type when N g 2 (Figure 2a). On the other hand, the Π-A isotherms of CelN(C12)2 exhibit the opposite trend: the film condensation proceeds as the number of glucose residue N increases, i.e. the LE/LC phase transition region becomes wider with the shape changing from a kink into a plateau and the transition pressure lowing as N increases (Figure 2b). In both cases of CelN(C12)2 and MalN(C12)2, the lift-off points of Π-A isotherms shift from A ) 1.0 nm2 to about 1.2 nm2 as N increases. These Π-A isotherms can be explained in terms of the different conformations of the oligosaccharide head groups shown in Figure 3.34 The molecular conformations were estimated by the intramolecular energy optimization performed by molecular mechanics (Allinger MM2), which are practically identical to the preferred conformations of the corresponding homopolysaccharides, amylose and cellulose, respectively.46 In MalN(C12)2, the oligosaccharide head groups take a “helical” conformation and forms bulky head groups (Figure 3a). This “helical” structure presumably prevents the attraction between head groups in monolayers, since the pyranose rings cannot approach each other due to the steric hindrance. Apart from the van der Waals forces, it has been often argued that the attraction between sugar head groups may be based on the hydrophobic interaction between the opposing faces of pyranose rings and/or the hydrogen bonding between pyranose rings hydroxyl groups.6 (44) Hato, M.; Minamikawa, H.; Okamoto, K.; Iwahashi, M. J. Colloid Interface Sci. 1993, 161, 155. (45) Hosoi, K.; Ishikawa, T.; Tomioka, A.; Miyano, K. Jpn. J. Appl. Phys. 1993, 32, L135. (46) Kennedy, J. F.; White, C. A. Carbohydrate Chemistry; Kennedy, J. F., Ed.; Clarendon Press: Oxford, 1988; p 3.

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According to the space-filling model, the maximum cross sectional areas of the maltooligosaccharide head groups normal to the long axis increase as the number of glucose residues N increases: A0 ≈ 0.4 nm2 (N ) 1), 0.4 nm2 (N ) 2), 0.6 nm2 (N ) 3), 0.95 nm2 (N ) 4), 1.2 nm2 (N ) 5). When N g 3, the cross sectional areas of the head groups become larger than that of the double alkyl chains (A0 > 0.40 nm2). The disappearance of the kink and the expansion of the lift-off point of the Π-A isotherms using MalN(C12)2 can be explained by the size effects of these bulky head groups. On the other hand, the oligosaccharide head groups of CelN(C12)2 take the “extended” conformation parallel to the alkyl chain tail groups. At least, in a compressed state, the attractive forces between glucose residues seem to operate more easily, since both the hydrophobic surfaces and the pyranose ring hydroxyl groups are free from the steric hindrance. In addition, the cross sectional areas of the cellooligosaccharide head groups are close to that of the double alkyl chains even when the number of sugar residues (N) increases: A0 ≈ 0.4 nm2 for all N values. Therefore, the packing of CelN(C12)2 alkyl chains is not interrupted by the size effect of the head group in contrast to MalN(C12)2. The expansion of the lift-off point of the Π-A isotherms in CelN(C12)2 may be explained by thermal fluctuation of the molecules, which is related to the total molecular length of the lipids that is a function of N. The film condensation (LE/LC phase transition) promoted as N increases is strong evidence of the attraction between the cellooligosaccharide head groups. Further important evidence is supported by the well-defined phase transition with clear distinction of LE, LE/LC coexistence, and LC phases. The different physical state can be observed as a function of surface molecular density, quite similar to the phase behavior of phospholipid and fatty acid monolayers.31,35,47,48 This proves that the molecules are spread in a monomer condition in dilute regime and the film starts to condense when the molecules are compacted into a defined concentration. This is because the attractive interactions between glucose residues are short-range forces whether they are van der Waals forces, hydrophobic attractions, or the hydrogen bonding. 2. Characterization of Film Condensations Induced by the Attraction between Oligosaccharide Head Groups. 2.1. Π-A Isotherms and BAM Images. To clarify the difference between the film condensation induced by the oligosaccharide head groups and that by the long alkyl chains, we performed detailed analysis of Cel4(C12)2 and Glc(C16)2 monolayers. Here, Cel4(C12)2 is used as a model of the film condensation induced by the oligosaccharide head groups, while Glc(C16)2 is used as a model of that induced by the long alkyl chains. The Π-A isotherms measured at the wide temperature range and the BAM images taken at the plateau (LE/LC phase transition regime) for Cel4(C12)2 and Glc(C16)2 monolayers are shown in Figures 4 and 5, respectively. Despite the superficial similarity, close inspection reveals that these isotherms have distinct characteristics. First, the transition pressure (Πt) of Cel4(C12)2 has smaller dependence on temperature than that of Glc(C16)2. Second, the width of the plateau with Cel4(C12)2 is much wider than that produced by Glc(C16)2 and less affected by temperature. The Cel4(C12)2 monolayer has one more distinguishing characteristic on the relaxation speed of Π at the LE/LC phase transition regime: Cel4(C12)2 required (47) Harkins, W. D.; Boyd, E. J. Phys. Chem. 1941, 45, 20. Boyd, G. E. J. Phys. Chem. 1958, 62, 536. (48) Mo¨hwald, H. Thin Solid Films 1988, 159, 1.

Phase Transition in Glycolipid Monolayers

Figure 1. A series of surface pressure-area (Π-A) isotherms of maltooligosaccharide-containing lipids [MalN(C12)2] and cellooligosaccharide-containing lipids [CelN(C12)2]: (0) HT (T ) 32 °C), (O) RT (T ) 22.5 °C), (4) LT (T ) 8.5 °C).

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Figure 2. Π-A isotherms taken at low temperature (LT, T ) 8.5 °C) for each glycolipids. (a) MalN(C12)2: (2) N ) 1, (O) N ) 2, (4) N ) 3, (0) N ) 5. (b) CelN(C12)2: (2) N ) 1, (O) N ) 2, (4) N ) 3, (0) N ) 4.

over 30 min to reach the equilibrium Πt at each measurement point, while Glc(C16)2 required less than 10 min under the same spreading condition. Since such a slow relaxation process is observed only in the LE/LC transition regime, it is regarded as a time for the nucleation and the nuclear growth of the LC phase rather than for evaporation of the spreading solvent (CHCl3/MeOH). This subject will be discussed in the appendix with the Π-A isotherms measured by the continuous compression method. The film morphologies are thoroughly related to the molecular state determined by the Π-A isotherms in both Cel4(C12)2 and Glc(C16)2 monolayers. The gas bubbles (LE/G coexistence films) can be observed in the dilute regime (Π ≈ 0 mN/m), and the films change to the homogeneous liquid expanded phase at Π g 0.1 mN/m in the same way as phospholipids and fatty acids.48-50 Subsequently, the LC domains start to appear at the beginning of the plateau. These domains grow as the surface molecular density increases until the end of the plateau, where the water surface is nearly homogeneously covered with the LC phase.36,48 In both Figures 4 and 5, the upper frames are the LE/ LC biphasic images taken at the middle of the plateau and the lower ones are those taken at the end of the plateau respectively. In all frames, the bright domains correspond to the LC phase and the dark matrix corresponds to the LE phase. These biphasic images give us clear distinction between Cel4(C12)2 and Glc(C16)2. The LC phase of Cel4(C12)2 exhibits the optically isotropic and highly viscoelastic fluid phase, which does not have a clear edge and changes shape according to the surface flow caused by the thermal convection of subphase water (Figure 4). On the other hand, the LC phase of Glc(C16)2 exhibits an optically anisotropic, two-dimensional crystalline phase similar to (49) Miyano, K.; Tamada, K. Langmuir 1992, 8, 160. (50) Tamada, K.; Kim, S.; Yu, H. Langmuir 1993, 9, 1545.

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phospholipids and fatty acids (Figure 5).40-42,51 Although we did not use an analyzer in front of the CCD camera, the different shade of brightness in LC domains is clearly visible with Glc(C16)2 through the p-polarized incidence beam. It corresponds to the different orientation of molecules in the film plane. Such a molecular tilt (film anisotropy) is typical of the LC phase of long alkyl chains. The isotropic LC phase of Cel4(C12)2 indicates that the molecular condensation in Cel4(C12)2 is not due to the attraction between the long alkyl chains but possibly due to the attraction between the cellooligosaccharide head groups. Such a direct transition from the isotropic LE phase to the isotropic LC phase observed with Cel4(C12)2 monolayers is novel and does not fit in the general phase diagram of monolayers.35 Some dye surfactants, such as cyanine dyes,49,52 multicyclic compounds,53 and azobenzene derivatives,54 are known to exhibit specific film characteristics due to the effect of functional groups that actively participate in the film condensation, similar to our glycolipids. However, the phase behavior of these compounds were not discussed in terms of the Π-A isotherms for lack of reproducibility. Compared with these studies, the Π-A isotherms of our system were very stable at all measurement temperatures. It is noted that the equilibrium spreading pressures ΠE of MalN(C12)2 at 22.5 °C, 35 mN/m (N ) 1), 45 mN/m (N ) 2), 38 mN/m (N ) 3), and 42 mN/m (N ) 5), are only marginally lower than the collapse pressure of each isotherm (see Figure 1). The ΠE of CelN(C12)2 at 22.5 °C, 28 mN/m (N ) 2), 32 mN/m (N ) 3), and 14 mN/m (N ) 4), are also close to the corresponding collapse pressure (for N ) 2, 3) or the pressure at the plateau (N ) 4) (see Figure 1). This indicates that the present measurements are close to the equilibrium conditions and that the Π-A isotherms of our system are of sufficient quality for the thermodynamic analysis. In addition, our systems are unique in that they show attraction in water of highly hydrophilic functional groups. In the next section, we perform the thermodynamic analysis of the Π-A isotherms with the ClausiusClapeyron equation to obtain the latent heat of transition. Furthermore, in the last section, we determine the light reflectivity from the boundary of air/monolayer/water using the BAM technique, to confirm the molecular condensation in water through the optical constant of the monolayer phase. 2.2. The Latent Heat of Transition. In Figure 6, the transition pressure (Πt) of Cel4(C12)2 and Glc(C16)2 is plotted against temperature (T), in which the Π value at the center of the plateau is used as Πt. The data of conventional phospholipids (DMPC, DPPC) are cited from references31,35 for comparison. The Πt-T plots of both phospholipids and Glc(C16)2 show quite a similar slope (dΠt/dT) over the respective transition temperature range. From this observation, we may infer that the LE/LC phase transition of these systems results from the same thermodynamic process. The shift in temperature scale is explained by the influence of the alkyl chain length and the nature of head groups. As expected from the Π-A isotherms, Cel4(C12)2 exhibits significantly different profile (see Figure 6), in which the slope of the Πt-T curve is much smaller than the former cases. The entropy change (∆S) and the latent heat (∆H) of the LE/LC phase transition is determined from the dΠt/ (51) Overbeck, G. A.; Mo¨bius, D. J. Phys. Chem. 1993, 97, 7999. (52) Kuroda, S.; Sugi, M.; Iizima, S. Thin Solid Films 1985, 133, 189 and references cited therein. (53) Miyano, K.; Tamada, K. Langmuir 1993, 9, 508. (54) Tabe, Y.; Yokoyama, H. J. Phys. Sci. Jpn. 1994, 63 (7), 2472.

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Figure 3. The space filling model of the minimum energy conformation of the cello- and the maltooligosaccharides: (a) maltooligosaccharide (maltotetraose, Mal4); (b) cellooligosaccharide (cellotetraose, Cel4).

Figure 4. Π-A isotherms of Cel4(C12)2 and BAM images taken at the plateau (LE/LC phase transition) in the corresponding Π-A isotherms at varied temperatures. In the BAM images, the bright regions correspond to the LC phase and the dark regions correspond to the LE phase: (9) T ) 42 °C, (2) T ) 38 °C, (]) T ) 32 °C, (×) T ) 27 °C, (0) T ) 22.5 °C, (4) T ) 14.5 °C, (O) T ) 8.5 °C.

dT through Clausius-Clapeyron equation in two dimensions:35,39

∆H ) ∆ST ) (dΠt/dT) ∆AT

(1)

where ∆A is the area change per molecule at the phase transition. In this study, we used the width of the plateau

Figure 5. Π-A isotherms of Glc(C16)2 and BAM images taken at the plateau (LE/LC phase transition) in the corresponding Π-A isotherms at varied temperatures. In the BAM images, the bright domains correspond to the LC phase and the dark matrix correspond to the LE phase: ([) T ) 32 °C, (×) T ) 27 °C, (0) T ) 22.5 °C, (4) T ) 14.5 °C, (O) T ) 8.5 °C.

in each Π-A isotherm as ∆A value. The calculated ∆H and ∆S values are plotted against temperature in Figure 7, where the data of DPPC cited from the reference35 is superimposed for comparison. The ∆H and ∆S values of Glc(C16)2 show a good agreement with those of DPPC over the whole temperature range, since the transition of both systems is driven by the van der Waals interaction between the long alkyl chains (∼C16). It is also important that the head groups in both systems have no net charges, since charges are known to strongly influence ∆H and ∆S.35

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Figure 6. The temperature dependence of the transition pressure (Πt) in the glycolipid and phospholipid monolayers. The data of phospholipids are cited from refs 31 and 35. The error for reading of each Πt is estimated to be within (10%: (b) Cel4(C12)2, (O) Glc(C16)2, (0) L-R-dimyristoylphosphatidylcholine (DMPC),31 (×) L-R-dipalmitoylphosphatidylcholine (DPPC).28

Figure 7. The entropy change (∆S) and the latent heat (∆H) at the LE/LC phase transition in the glycolipid and phospholipid monolayers calculated by the Clausius-Clapeyron equation. The data of phospholipids are cited from ref 35. The error in ∆S and ∆H values are estimated to be within (10%: (b) Cel4(C12)2, (O) Glc(C16)2, (4) L-R-dipalmitoylphosphatidylcholine (DPPC).

The ∆H-T profiles of Glc(C16)2 and DPPC show a feature typical of long alkyl chain monolayers, where ∆H appears to be constant at low temperature and then converges toward zero at higher temperature at the so-called tricritical point. Above this temperature, the LE/LC transition becomes second order, where the plateau changes to a kink in the Π-A isotherms. Albrecht et al.35 explained this phenomenon by the Landau theory55 in terms of the coupling between the lateral order (molecular density) and the chain orientation order (stretching vector).56 They cited the Rodbell-Bean effect57 in magnetism as a suitable analog, in which a decrease of coupling of those two factors leads to a transition from first to second order. This phenomenon, a change of transition from first to second order depending on temperature, cannot be observed in three-dimensional liquid-solid phase transition in general, because of the symmetry.55 (55) Landau, L. D.; Lifschitz, E. M. Statistical Physics; AddisonWesley: Reading, MA, 1960. (56) de Gennes, P. G. The Physics of Liquid Crystal; Clarendon Press: Oxford, 1974; p 316. (57) Bean, C.; Rodbell, D. Phys. Rev. 1962, 126, 104. (58) Toshev, B. V.; Platikanov, D.; Scheludko, A. Langmuir 1988, 4, 489. (59) Heavens, O. S. Optical Properties of Thin Films; Dover: New York, 1965. (60) Born, M.; Wolf, E. Principles of Optics; Pergamon: New York, 1970; p 100. (61) Swalen, J. D. J. Mol. Electron. 1986, 2, 155. (62) Pallas, N. R.; Pethica, B. A. Langmuir 1985, 1, 509.

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On the other hand, a specific feature of Cel4(C12)2 stands out clearly in this latent heat analysis. The ∆H and ∆S values of Cel4(C12)2 are much lower than those of Glc(C16)2 and DPPC below room temperature and exhibit an opposite dependence on temperature. With Cel4(C12)2, ∆H increases as the temperature increases, and it does not converge toward zero at higher temperature. This profile is rather similar to a three-dimensional system. The difference in phase behavior between Glc(C16)2 and Cel4(C12)2 seems to correlate with the molecular tilt suggested by the BAM observation (Figure 4). A molecular tilt in monolayers have been attributed to a spontaneous breaking of the chiral symmetry;63 however, it should be mentioned that the phase behavior in monolayers has not yet been accounted for thoroughly and it is far from being satisfactorily described by any theoretical explanations.64 2.3. The Light Reflectivity from the Boundary of Air/ Monolayer/Water Obtained by the BAM Technique. We performed not only the observation of film morphology but also the quantitative analysis of the BAM images. The refractive index (n) of the films was deduced from the light reflectivity (R) at each phase (LE, LC). First, a calibration line was determined by the incident angle dependence of the light reflectivity. The BAM images on pure water surface were taken at various incidence angles near the Brewster angle, and the brightness of those images was plotted against the theoretically calculated light reflectivity. A good linearity was confirmed between the experimental and theoretical values within the range of reflectivity required for our experiments. The light reflectivity from the monolayer films was determined from the BAM images by use of this calibration line. The reflectivities of Cel4(C12)2 and Glc(C16)2 monolayers along with the Π-A isotherms are shown in Figure 8a,b, respectively. Both figures show the same tendency; the reflectivity changes gradually as the surface molecular density increases within the homogeneous LE phase and then reaches two constant values at the plateau (LE/LC coexistence phase). The higher value corresponds to the reflectivity of the LC phase and the lower value corresponds to that of the LE phase. The difference of the light reflectivity between the LE/LC phase is slightly larger at low temperature than at high temperature. This result seems to be related to the domain size in Figure 5. The smaller domains at low temperature appear to compensate for the larger difference of surface pressure between the LE/LC phase.49,58 Note that the reflectivity of Cel4(C12)2 is distinctly higher than that of Glc(C16)2 with both the LE and LC phases. If the reflectivity is determined only by the tail groups in molecules, the reflectivity of Glc(C16)2 should be higher than that of Cel4(C12)2, since the alkyl chain length of Glc(C16)2 (d ) 1.9 nm) is much longer than that of Cel4(C12)2 (d ) 1.4 nm). This experimental result implies that the effect of the oligosaccharide head groups must be taken into account. This will be illustrated in the following calculation of the light reflection from the air/monolayer/ water boundary with the multilayer model.59,60 (a) Three-Phase Model. Here, we assume a single layer model, optically homogeneous and isotropic, at the air/ water interface. If we put the reasonable film thickness (d) into the equation, we can estimate the refractive index of the film (n) from the light reflectivity (R). Initially, we employed the alkyl chain length as a d value, ignoring the head group. With the alkyl chain length of C16 (d ) 1.9 nm), the refractive index of the LC phase with Glc(C16)2 (63) Selinger, J. V.; Wang, Z.-G.; Bruinsma, R. F.; Knobler, C. M. Phys. Rev. Lett. 1993, 70 (8), 1139. (64) Bell, G. M.; Combs, L. L.; Dunne, L. J. Chem. Rev. 1981, 81, 15.

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Figure 8. The light reflectivity (R) from the boundary of air/ monolayer/water: (a) Cel4(C12)2, (b) Glc(C16)2. The different symbols indicate the R values taken at different temperature: (0) HT (T ) 32 °C), (4) RT (T ) 22.5 °C), (O) LT (T ) 8.5 °C). The solid curves are the corresponding Π-A isotherms to each temperature.

is estimated to be n ) 1.50 using the light reflectivity from the BAM image (R ) (1.5-2.0) × 10-6). This is within a range of our expectation, which is slightly less than the refractive index of stearic acid (in solid), n| ) 1.55, n⊥ ) 1.52.61 However, when the reflectivity of Cel4(C12)2 (d ) 1.4 nm) is calculated using this n, we obtained R ) 1.2 × 10-6, which is much smaller than the experimental value determined by the BAM images (R ) (2.5-3.0) × 10-6). The necessity to consider the contribution of head groups is demonstrated by this calculation more quantitatively. (b) Four-Phase Model. In this model, we divide the glycolipid monolayers into two layers normal to the water surface. One is the layer formed by the hydrophobic tail groups and another is the layer formed by the oligosaccharide head groups, where the glycerol and ether linkages are not counted in either layer. First, we discuss Glc(C16)2 monolayer. To determine the refractive index of the oligosaccharide layer (n2) from the reflectivity (R), we should know all other parameters: the refractive index and the film thickness of tail groups (n1, d1) and the film thickness of the oligosaccharide head groups (d2). The n1 value is estimated by the ClausiusMossotti relation,

( - 1)/( + 2) ) NR/30

(2)

where  is the relative dielectric constant, N is the molecular density, R is the molecular polarizability, and 0 is a constant ()8.854 × 10-12 F m-1). Since ( - 1)/( + 2) is proportional to the molecular density N, the refractive index of monolayers can be deduced from that of solid by the ratio of molecular density in monolayers to solid. In this calculation, a reciprocal of “area per molecule” is used as a relative N, in which the cross

sectional area of one molecule in crystals (A0 ) 0.42 nm2) and the limiting area of the Π-A isotherms (A0 ) 0.52 nm2) is used as “area per molecule” in solid and monolayers, respectively. The dielectric constant of stearic acid (e ) 2.40) is used as  of solid.61 By this calculation, the refractive index of alkyl chains in the LC phase was determined to be n1 ) 1.43. By use of this n1 and the film thickness (d1 ) 1.9 nm, d2 ) 0.8 nm), the refractive index of oligosaccharide was estimated to be n2 ) 1.46-1.50 with the light reflectivity R ) (1.5-2.0) × 10-6 for Glc(C16)2 monolayers. Similarly, we obtained n2 ) 1.43-1.46 for Cel4(C12)2 monolayers (n1 ) 1.43, d1 ) 1.4 nm, d2 ) 2.2 nm, R ) (2.0-2.5) × 10-6). The refractive index of the oligosaccharide (in solid) is presumed to be around 1.55 from the refractive index of glucose (n ) 1.55) and cellulose (n| ) 1.53, n⊥ ) 1.60). Since the refractive indices of the oligosaccharides and the alkyl chains are similar in the solid phase, the upper layer (alkyl chain layer) and the lower layer (oligosaccharide layer) in monolayers are expected to have similar optical constants. However, the n2 values of Glc(C16)2 and Cel4(C12)2 are slightly higher than the n1 value in our calculation. One reason may be that we neglect the length of linkage part, omission of which leads to the overestimation of n2. This view can be supported by noting that the n2 value of Glc(C16)2 is somewhat higher than that of Cel4(C12)2, since the contribution of this linkage part should be larger against the smaller head groups. Another possible reason is the influence of the matrix phase. For actual monolayers, the free space in monolayers between the tail groups and the head groups must be filled with air and water, respectively. The overall refractive index of the head group layer (hydrophilic layer) should be higher than the tail group layer (hydrophobic layer) for a portion of the difference between the refractive index of water and air. In this paper, we performed only a rough estimation of the refractive index of monolayer in view of the uncertainty of each parameter. To perform more detailed analysis in 2D, we should at least know the physical property of the glycolipid crystals in 3D. Our glycolipids, in which both the alkyl chain length and the oligosaccharide chain length can be controlled, are expected to offer the direct information about the contribution of the depth (the vertical position) of molecules at the air/water interface, which has not been explored yet. Conclusion The direct evidence of the attraction between the oligosaccharide head groups was confirmed in the cellooligosaccharide-containing lipid (CelN(C12)2) monolayers. Such an attraction was not observed in the maltooligosaccharide-containing lipids (MalN(C12)2) because of the steric hindrance of the bulky head groups. The LE/LC phase transition in Cel4(C12)2 induced by the oligosaccharide head groups was distinguished from the conventional transition induced by the long alkyl chains, by use of the latent heat analysis and the BAM observation. The molecular tilting, which is a specific feature in the film condensation driven by the alkyl chain interaction, could not be observed in Cel4(C12)2. The light reflectivity measurement with the BAM made it clear that the oligosaccharide chains in water formed the dense phase in the same way as the alkyl chains in air for Cel4(C12)2 monolayers. We are planning to perform the direct measurement of the surface forces between Cel4(C12)2 monolayers deposited on the mica surface with the surface force apparatus (SFA).

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Figure 9. The Π-A isotherms for a compression-decompression cycle measured by the continuous compression method with a moving wall trough (268 mm (X) × 26 mm (Y)) at T ) 22.5 °C: (a) Cel4(C12)2, (b) Glc(C16)2 (solid lines). The compression speed is 10 mm/min (the upper line) and 1 mm/min (the lower line) in (a), and 10 mm/min in (b). The decompression speed is not controlled exactly but estimated to be 10 mm/min or above in both figures. The open spheres indicate the Π-A isotherms measured by the successive addition method, which are the extraction from Figure 4 and Figure 5.

Appendix Figure 9a,b shows the Π-A isotherms measured by the continuous compression method with the moving wall trough (268 mm (X) × 26 mm (Y)) for Cel4(C12)2 and Glc(C16)2 monolayers, respectively (T ) 22.5 °C). In Figure 9, the solid lines indicate the compression-decompression cycle: the compression speed to the X-direction is 10 mm/ min (the upper line) and 1 mm/min (the lower line) in Figure 9a, and 10 mm/min in Figure 9b, respectively. The decompression speed is not controlled exactly but estimated to be 10 mm/min or above in both figures. The open spheres indicate the Π-A isotherms measured by the successive addition method, which are extracted from Figure 4 and Figure 5 for a comparison. In Figure 9a, the peak in the compression process reveals that the relaxation speed of Π in the plateau is much

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slower than the compression speed, i.e., this peak indicates the supersaturation. The dependence of the peak height on the compression speed supports the above conclusion. The relaxation speed of the decompression process appears not so slow as the compression process that the Π-A isotherm in decompression process exhibits the clear plateau in the same way as the successive addition method. In contrast to Cel4(C12)2, both the compression and the decompression lines of Glc(C16)2 nearly agree with the result of the successive addition method in spite of the high compression speed (10 mm/min). The deviation between the compression and decompression lines is caused by the collapse of the film at the high Π region (>40 mN/m). The time dependence of the film morphology observed with the BAM is closely correlated with the relaxation phenomena of Π, which gives us the clear evidence that the nucleation and nuclear growth are the rate-determining step in these processes. No specific domains can be observed for Cel4(C12)2 monolayers at the moment of spreading, and then the LC phases appear gradually scores of minutes after spreading (the biphasic morphologies finally obtained are shown in Figure 4). On the other hand, for Glc(C16)2 monolayers, the stable domains can be observed immediately after the spreading solvent evaporates (see the images in Figure 5). There is no precedent for such a slow transition in previous studies of ordinary phospholipids and fatty acids31,35,45,62 except for a recent report concerning mixed monolayers consisting of docosandioic acid/eicosylamine (DDA/EA).63 In that case, the strong interaction between head groups (carboxylic acids and amines) induced an unusually slow transition and film instability. A nucleation-like nonequalibrium phenomenon and the squeezing out of EA molecules from monolayers were expected to occur at the plateau region. Although our system is distinct from that case by the stability of the Π-A isotherms, the reason of slow relaxation time may be partially in common. The unique feature of the film condensation proceeding in water, the lower mobility of molecules, and the desorption process of hydration water make the transition slow down. Acknowledgment. We thank Dr. A. Tomioka of the University of Tokyo and Dr. K. Kajikawa of the Institute of Physical and Chemical Research in Japan (RIKEN) for helping us in the image analysis of BAM pictures and the calculation of the light reflectivity respectively. We thank also our colleagues Dr. K. Yase, Mr. T. Baba, and Dr. K. Abe for valuable discussions. LA9503257