The Effects of Oligosaccharide Stereochemistry on the Physical

Akio Ohta , Kosuke Toda , Yu Morimoto , Tsuyoshi Asakawa , Shigeyoshi Miyagishi. Colloids and Surfaces A: Physicochemical and Engineering Aspects 2008...
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Langmuir 1996, 12, 1658-1665

The Effects of Oligosaccharide Stereochemistry on the Physical Properties of Aqueous Synthetic Glycolipids Masakatsu Hato* and Hiroyuki Minamikawa Surface Engineering Laboratory, National Institute of Materials and Chemical Research, 1-1, Higashi, Tsukuba, Ibaraki-305, Japan Received April 24, 1995. In Final Form: December 11, 1995X Effects of the stereochemistry of oligosaccharide head groups on the physical properties of aqueous synthetic glycolipids, 1,3-di-O-dodecyl-2-(β-glycosyl)glycerols bearing cellooligosaccharides (β-1,4-Oglycosidic bonds) and maltooligosaccharides (R-1,4-O-glycosidic bonds) as hydrophilic groups have been studied. The increase in the number of glucose residues, N, in the two different headgroups exhibited opposite effects on the physical properties of the aqueous glycolipids. For the maltooligosaccharide-containing lipid, MalN(C12)2, increasing N in the head groups decreases the hydrated solid/liquid crystalline phase transition temperature Tm and increases the “hydrophilicity” of the lipids. However, the Tm of the cellooligosaccharide-containing lipids CelN(C12)2 increases with increasing N of the cellooligosaccharide head groups. It is noteworthy that Tm jumps from 59 °C (for N ) 4) to above 160 °C (N ) 5), so that the Cel5(C12)2 cannot form a liquid crystalline phase and was totally insoluble in water. The results can be explained in terms of different conformations of the head groups, i.e., a “helical” conformation of the maltooligosaccharides and an “extended” conformation of the cellooligosaccharides.

Introduction Glycolipids are important amphiphiles that bear oligosaccharides as their hydrophilic head groups and are progressively gaining importance in scientific and technical fields as well.1-8 As glycopipids can be synthesized from renewable resources like oligosaccharides and fatty alcohol, they appear to be less environmentally damaging than many other synthetic surfactants.6,7 Moreover, as oligosaccharides are more hydrophilic than the conventional oligo(ethylene oxide) head groups,9,10 they are attractive as a new type of surfactant. Another important field is that of biological cell membranes where glycolipids are believed to be involved in a variety of physiological events1,2 such as molecular recognition at cell surfaces4,11,14 and stabilization of the membrane structure of say archaebacteria that grow under extreme environmental conditions.12 Though these functions are mainly attributable to the interactions between the oligosaccharide head groups, the molecular bases of their functions are as yet not well understood.1,2,11-14 In spite of the growing attention to glycolipids, surprisingly few studies have been performed so far to elucidate the general relation between their chemical structure and X Abstract published in Advance ACS Abstracts, February 15, 1996.

(1) Curatolo, W. Biochim. Biophys. Acta 1987, 906, 111. (2) Curatolo, W. Biochim. Biophys. Acta 1987, 906, 137 and references cited therein. (3) Hinz, H.-J.; Six, L.; Ruess, K.-P.; Liefla¨nder, M. Biochemistry 1985, 24, 806. (4) Hakomori, S. Pure Appl. Chem. 1991, 63, 473. (5) Koynova, R.; Caffrey, M. Chem. Phys. Lipids 1994, 69, 181 and references cited therein. (6) Nilsson, F. Thesis, University of Lund, 1993. (7) Fukuda, K.; So¨derman, O.; Lindman, B.; Shinoda, K. Langmuir 1993, 9, 2921. (8) Sugar Chains in Biological Cell Functions; Irimura, T., Ed.; Nikkei Science Pub.: Tokyo, 1994 (in Japanese). (9) Shinoda, K.; Fukuda, M.; Carlsson, A. Langmuir 1990, 6, 334. (10) Sakakibara, T. Yukagaku 1990, 39, 451 (in Japanese). (11) Eggens, I.; Fenderson, B.; Toyokuni, T.; Dean, B.; Hakomori, S. J. Biol. Chem. 1990, 264, 9476. (12) Kates, M. Handbook of Lipid Research 6, Glycolipids, Phosphoglycolipids, and Sulfoglycolipids; Kates, M., Ed.; Plenum Press: New York, 1990; pp 1-122. (13) Pascher, I. Biochim. Biophys. Acta 1976, 455, 433. (14) Kojima, N. Trends Glycosci. Glycotechnol. 1992, 4, 491.

the physical properties of glycolipid/water systems in a systematic manner.15 This is in part due to the difficulty of obtaining a sufficient amount of chemically pure compounds either from natural membrane extracts or by synthetic methods. Though the synthetic approach has proved promising in preparing well-defined compounds,16-19 the procedures to obtain stereochemically pure compounds are still demanding so that the kinds of oligosaccharide head groups so far synthesized and examined have been mainly mono- and disaccharides. We have recently proposed an efficient synthetic route for obtaining a new series of chemically pure glycolipids, 1,3-di-O-alkyl-2-O-(β-glycosyl)glycerols, bearing a series of oligosaccharides as their head groups.20 Provided an oligosaccharide possesses reducing terminus, the method allows us to couple a desired oligosaccharide β-glycosidically with a 1,3-di-O-alkylglycerol of a defined chain length. By employing this method, we are now able to synthesize the desired series of chemically pure glycolipids that allow us to investigate the physical properties of aqueous glycolipids in a systematic manner. In this paper, we report a first attempt to investigate general relationships between the stereochemistry of oligosaccharide head groups and the physical properties of the aqueous glycolipids. As oligosaccharides that form the head groups of the lipids can assume a variety of conformations, arising say from the multiple choices of isomeric linkages that join the monosaccharide groups,21 the stereochemistry and hence the conformations of the head groups are most relevant to the physical properties of glycolipid/water systems. We here choose two different series of oligosaccharides as the head groups, namely cellooligosaccharides and (15) Hinz, H.-J.; Kuttenreich, H.; Meyer, R.; Renner, M.; Fru¨nd, R.; Koynova, R.; Boyanov, A. I.; Tenchov, B. G. Biochemistry 1991, 30, 5125. (16) Mannock, D. A.; Lews, R. N. A. H.; McElhaney, R. N. Chem. Phys. Lipids 1987, 43, 113. (17) Six, L.; Russ, K.-P.; Liefla¨nder. Tetrahedron Lett. 1983, 24, 1229. (18) Endo, T.; Inoue, K.; Nojima, S. J. Biochem. (Tokyo) 1982, 92, 953. (19) Jarrell, H. C.; Jovall, P. A° .; Giziewicz, J. B.; Turner, L. A.; Smith, I. C. P. Biochemistry 1987, 26, 1805. (20) Minamikawa, H.; Murakami, T.; Hato, M. Chem. Phys. Lipids 1994, 72, 111. (21) Hendrickson, J. B.; Cram, D. J.; Hammond, G. S. Organic Chemistry, 3rd ed.; McGraw-Hill: New York, 1970; p 989.

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Figure 1. Chemical Structures of the 1,3-di-O-dodecyl-2-O(β-glycosyl)glycerols bearing a series of oligosaccharide as the head groups. Glc, MalN, and CelN represent glucose, maltooligosaccharids, and cellooligosaccharids head groups, respectively, where N is the number of glucose residues in the head groups. Note that Glc(C12)2 corresponds to Mal1(C12)2 and Cel1(C12)2.

maltooligosaccharides. It is noted that in homopolysaccharides their allowed conformations can be classified into four distinct types, i.e., the “extended ribbon” (e.g., 1,4β-D-glucan like cellulose), the “flexible helix” (e.g., 1,4R-D-glucan like amylose), the “crumpled ribbon”, (e.g., 1,2β-D-glucan) and the “flexible coil” (e.g., all 1,6-disubstituted homoglycans).22 According to this classification, the cellooligosaccharides (derived from cellulose), where all the glucose residues are linked through β-1,4-O-glycosidic bonds, have a preference for the “extended ribbon” conformation.22 The maltooligosaccharides (derived from amylose), where all the glucose residues are linked via R-1,4-O-glycosidic bonds, have a preference for the “flexible helix” conformation.22 The hydrophobic part employed was 1,3-di-O-dodecylglycerol. Since the only variable in the lipid structure is the number of glucose residues, N, in each oligosaccharide head group, the results obtained directly reflect the influence of the number of glucose residue in two different oligosaccharide head groups. The structures of aqueous amphiphiles being crucial to their properties and functions in many different ways,23-33 we also discuss how the structure of aqueous phases is controlled by the structural variations of the glycolipids at the molecular level. Experimental Section Materials. The chemical structures of the lipids used in this paper are given in Figure 1. All the glycolipids are the same materials reported in the previous paper where the details of the chemical properties of these lipids were described.20 We employed purest grade oligosaccharides and a dodecanol as starting materials. In short, we coupled a chromatographically purified oligosaccharide (Seikagaku Kogyo, Tokyo; >99%) with a 1,3-diO-dodecylglycerol which was prepared from dodecanol (Tokyo Kasei Kogyo; purest grade >99.5% by GC) and epichlorohy(22) Kennedy, J. F.; White, C. A. Carbohydrate Chemistry; Kennedy, J. F., Ed.; Clarendon Press: Oxford, 1988; p 3. (23) Prost, J.; Rondelez, F. Nature 1991, 350, 11. (24) Luzzati, V. Biological Membranes; Chapman, D., Ed.; Academic Press: New York, 1968; pp 71-123. (25) Andersson, S.; Hyde, S. T.; Larsson, K.; Lidin, S. Chem. Rev. 1988, 88, 221. (26) Lipowsky, R. Nature 1991, 349, 475. (27) Shinoda, K.; Lindman, B. Langmuir 1987, 3, 135. (28) Quinn, P. J.; Williams, W. P. Biochim. Biophys. Acta 1983, 737, 223. (29) Lindblom, G.; Rilfors, L. Biochim. Biophys. Acta 1989, 988, 221. (30) Larsson, K. J. Phys. Chem. 1989, 93, 7304. (31) Lindblom, G.; Rilfors, L. Adv. Colloid Interface Sci. 1992, 41, 101. (32) Verkleij, A. J. Biochim. Biophys. Acta 1984, 779, 43. (33) Siegle, D. P. Chem. Phys. Lipids 1986, 42, 279.

Langmuir, Vol. 12, No. 6, 1996 1659 drin.20,34 The number of glucose residues in the oligosaccharide head groups, N, was varied from 1 to 5, and they were linked β-glycosidically to the hydrophobic group. Thin-layer chromatography, elemental analysis, NMR, and IR spectroscopy confirmed that the purity of the glycolipids employed is more than 99%.20 Water employed in this work was purified as follows. Tap water was first treated by a water purification system designed by our institute (reverse osmosis, ion-exchange, adsorption treatment, filtration through a 0.22 µm filter), then distilled in an all-glass apparatus, and further treated with an Elga UHQ unit just before experiments. Purity was successively checked by surface tension measurement (72.4 mN/m at 22 °C).35 Microscopic Observation. The lipid/water systems were examined by an Olympus BHS-751-P polarizing microscope equipped with a Mettler FP82HT hot stage thermostated to an accuracy of (1 °C. Microscope slides were buffed with lint-free paper tissue immediately before use. A small amount of the 50 wt % aqueous lipid was transferred with a pipet from the vial onto the glass slide and was immediately covered with the slide cover. The sample was then sheared between the slide and the cover to a thickness of about 5-10 µm and was left for a few minutes on the constant temperature stage for relaxation and thermal equilibration.36 The different liquid crystalline phases were optically identified according to the birefringent textures observed between crossed polarizers.37,38 The temperatures at which hydrated solid/liquid crystalline phase transitions occurred (Tm) were determined by approaching the transition points both from higher and lower temperatures. The transition temperature Tm thus determined was in good agreement with the temperature at the main endothermic peak on each DSC thermogram. Differential Scanning Calorimetry (DSC). Calorimetric measurement of the aqueous glycolipids was performed with a Seiko SSC/560U differential scanning calorimeter over the temperature range from 5 to 105 °C. A 2-3 mg sample of the dry lipid dispersed in 50 µL of pure water was sealed in a silver DSC capsule. The capsule was then taken repeatedly through heating-cooling cycles between 5 and 105 °C to ensure complete hydration of the lipids.15 We confirmed that silica gel thin layer chromatography of the lipids exhibited no sign of decomposition after this pretreatment. Usually samples were incubated at 3 °C for 1-3 days before each measurement. By these pretreatments, the repeated heating runs gave reproducible results. As the thermodynamic parameters obtained at the heating rates of 0.5 and 1.0 agreed well with each other, we usually employed a rate of 1.0 °C/min. The peak temperature in the excess apparent heat capacity versus temperature curve (thermogram) was taken as the transition temperature Tm. Enthalpy changes ∆H associated with the transition were determined by using naphthalene as the standard.39 As the Tm of the Cel5(C12)2/water system was higher than 100 °C, we employed an 80 wt % aqueous lipid in a stainless steel capsule that enabled us to measure the thermogram up to 170 °C. The heating rate was 5.0 °C/min. X-ray Diffraction. Samples for X-ray diffraction of known concentrations were prepared by weighing the desired amount of lipid and pure water in a glass cell. Repeated cycles of freezing and thawing were then performed. The lipid dispersion was then filled into either a thin-walled quartz capillary (1.5 mm L, GLAS, Berlin) or cells with Kapton (25 µm thick) windows. The samples were then inserted into a Mettler HP82HT sample holder thermostated to an accuracy of (1 °C and exposed to the pinhole collimated X-ray beam (0.3 mm L) for 20 minutes. A Rigaku Rotaflex generator RU-200 served as the source for nickel-filtered Cu KR radiation (40 kV, 100 mA). X-ray diffraction patterns were recorded by a flat camera (the sample to film distance ) (34) Kuwamura, T. Kogyo Kagaku Zasshi 1961, 64, 1958 (in Japanese). (35) Hato, M.; Minamikawa, H.; Okamoto, K.; Iwahashi, M. J. Colloid Interface Sci. 1993, 161, 155. (36) Moucharafieh, N.; Friberg, S. E.; Larsen, D. W. Mol. Cryst. Liq. Cryst. 1979, 53, 189. (37) Rosevear, F. B. J. Am. Oil Chem. Soc. 1954, 31, 628. (38) Bellare, J. R.; Davis, H. T.; Miller, W. G.; Scriven, L. E. J. Colloid Interface Sci. 1990, 136, 305. (39) Ozawa, T.; Mita, T. Jikken Kagaku Kouza (Experimental Methods in Chemistry); The Chem. Soc. of Japan: Maruzen, Tokyo, 1992; pp 77-93 (in Japanese).

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300 mm) with an imaging plate (Fuji Film Ltd., HR-III N) and read by a Rigaku RINT2000 system.

Results Effect of Stereochemistry of the Oligosaccharide Head Groups on DSC Thermograms and Hydrated Solid/Liquid Crystalline Phase Transition Temperature Tm. In Figures 2 and 3, the DSC thermograms and the birefringent textures of the liquid crystalline phases of the two series of lipids MalN(C12)2 (Figure 2) and CelN(C12)2 (Figure 3) are summarized. It is first noted that most of the DSC thermograms exhibit only a single sharp endothermic peak at a characteristic temperature that depends upon the number of glucose residues N in each lipid. At the main endothermic peak temperature, the hydrated solid transforms into a liquid crystalline phase. As the Tm of Mal5(C12)2 is below 0 °C, the DSC measurement was carried out with a 70 wt % aqueous lipid. In this case, the thermogram exhibits at least three broad peaks at -10, -6, and -3 °C. A shoulder at 0 °C is due to the melting of ice. The numerical data and thermodynamic parameters obtained by these measurements are summarized in Table 1, and the Tm versus N curves are shown in Figure 4. The thermodynamic data listed in Table 1 are those associated with the hydrated solid/liquid crystalline phase transition (see later) except for the H(C12)2 (1,2-di-O-dodecylglycerol)/ water system. The H(C12)2/water system exhibits an isotropic liquid + water dispersion above its Tm that is practically identical to the melting point of the pure H(C12)2. Therefore, the parameters are those associated with the solid/isotropic phase transition. These results clearly indicate that the chemical structure of the head group has profound effects on the hydrated solid/liquid crystalline phase transition temperature Tm. The Tm of the MalN(C12)2 decreases with increasing number of glucose residues N. By increasing N from N ) 1 to N ) 5, the Tm decreases from 52 to -3 °C. While, with increasing N of CelN(C12)2 from N ) 1 to N ) 4, the Tm increases by ∆Tm = 14 °C. When N is further increased from N ) 4 to N ) 5, there is an abrupt jump in the Tm from 59 to above 160 °C. As Figure 3(5) indicates, the Cel5(C12)2/water system does not exhibit any peak (neither endothermic nor exothermic) on the DSC thermogram over the temperature range from 5 to 170 °C. Above 170 °C, the lipid decomposed into a brownish oily material. The microscopic photograph of Figure 3(5) indicates that the Cel5(C12)2 in excess water remains as crystals at least up to 80 °C (maximum operational temperature without severe water evaporation). For all the lipids studied, the Tm appears nearly constant up to Clipid ≈ 80 wt %. With further increase in the Clipid, the Tm starts to rise steeply,40 an indication that the hydration of the oligosaccharide head groups is crucial in depressing the melting points (Tm) of the glycolipid/water systems. Melting point depression caused by the hydration has generally been observed in other lipid/water systems such as the Ldipalmitoylphosphatidylcholine/water system.41 Finally, it is noted that the Tm of the CelN(C12)2 exhibits an “odd-even effect” of the hydrophilic groups, while the Tm of the MalN(C12)2 does not exhibit clear “odd-even” effect. Enthalpy Changes and Entropy Changes Associated with the Hydrated Solid/Liquid Crystalline Phase Transition. Figure 5 shows the effects of N on the enthalpy changes ∆H (Figure 5a) and entropy changes (40) Most of the dry glycolipids studied decomposed above 200 °C prior to the melting. (41) Kodama, M. Thermochim. Acta 1986, 109, 81.

Hato and Minamikawa

∆S (Figure 5b) associated with the hydrated solid/liquid crystalline phase transition of the two series of MalN(C12)2 and of CelN(C12)2. For CelN(C12)2, both the ∆H and the ∆S decrease appreciably with increasing N in the head group, an indication that the increase in the Tm of the CelN(C12)2/ water systems with increasing N is mainly ascribable to the decreasing entropy of transition (melting). The ∆S (∆H) values of the MalN(C12)2/water systems, on the other hand, are not significantly affected by increasing N values. The ∆S (∆H) values level off above N ) 2 at about 40 cal/K‚mol (11 kcal/mol), being twice as large as those of the corresponding CelN(C12)2. X-ray Diffraction and Birefringent Textures of the Liquid Crystalline Phase. Phase Behavior of the CelN(C12)2 and of the MalN(C12)2 in Water. Table 2 summarizes the X-ray diffraction lines and their ratios of the 50 wt % glycolipid/water mixtures. Types of the liquid crystalline phase as determined from the diffraction line ratio42 together with the typical birefringent textures in polarizing light observed are summarized. Owing to the insufficient amount of the glycolipids presently available, we could only investigate the crude outline of the phase behavior of the lipid/water systems. The phase behavior described here was derived mainly from optical microscopy observations of three to five samples, differing in concentration over the range from 1 wt % to 60 wt % of each lipid. Therefore, we were unable to determine the exact phase boundary. The temperature range examined was between 20 and 80 °C. The phase behavior of the glycolipids of the shorter oligosaccharide chain length, i.e., CelN(C12)2 with N ) 2, 3, 4 and MalN(C12)2 with N ) 2, 3, appears relatively simple. At a dilute lipid concentration regime, an LR phase + water dispersion occurs (a two-phase coexistence region of the LR and the dilute aqueous lipid solution). The lipid concentration in the excess water phase that was estimated from the surface tension measurement was low, possibly the order of 10-6 M or less. At higher lipid concentrations, the LR phase predominates to form a single phase. The birefringent textures of the LR phase observed consist of oily streaks37 or Malthesian crosses and are stable up to at least 80 °C (Figures 2, 3). However, the textures of the glycolipid/water systems of the longer oligosaccharide chain length, Cel4(C12)2/water and Mal5(C12)2/water systems, appear featureless. In the two-phase region, typical myelin figures spontaneously formed, and gentle sonication of the solution gave spherical closed vesicles. In this sense, the phase behavior of these synthetic glycolipids is similar to the ordinary phospholipid/water systems. The effects of different head groups become distinct when the N is larger than 4. Due to the abrupt jump in the Tm above 160 °C, the Cel5(C12)2/water system can no longer form a liquid crystalline phase. Therefore, only the solid + water dispersion is possible for the Cel5(C12)2. On the other hand, the Mal5(C12)2/water system can form a liquid crystalline phase at least above 50 wt % lipid. The liquid crystalline phase is most probably an LR phase as judged from the X-ray diffraction line ratio of 1:0.5. However, the LR phase of Mal5(C12)2 is distinctly different from that of MalN(C12)2 with shorter oligosaccharide chain length (N ) 1, 2, 3). First, the texture observed is rather featureless (Figures 2-5a): after strong shearing, a texture consisting of networks of extremely thin lines is observed (Figures 2-5b). Neither oily streaks nor Malthesian crosses are observed. Moreover, the LR phase of the Mal5(C12)2/water is unstable with respect to (42) Luzzati, P. V.; Mustacchi, H.; Skoulios, A.; Husson, F. Acta Crystallogr. 1960, 13, 660.

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Figure 2. The effects of the number of glucose residues N in the maltooligosaccharide head group on the DSC thermograms and the typical birefringent textures of the liquid crystalline phases formed in the 50 wt % lipid/water systems. The photographs were taken between crossed polarizers at temperatures about 5 deg above each Tm except for the Mal5(C12)2/water system where the photograph was taken at 22 °C. The number in the figure represents the N value.

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Figure 3. The effects of the number of glucose residues N in the cellooligosaccharide head groups on the DSC thermograms and the typical birefringent textures of the liquid crystalline phases formed in the 50 wt % lipid/water systems. The experimental conditions were identical to those of Figure 2. The number in the figure represents the N value.

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Table 1. Phase Transition Temperature Tm, Enthalpy ∆H, and Entropy ∆S Change Associated with the Hydrated Solid/Liquid Crystalline Phase Transition of the Aqueous CelN(C12)2 and MalN(C12)2 compound H(C12)2b Glc(C12)2c

Tm (°C)

∆H (kcal/mol)

42 16.4 ( 0.3 52 14.6 ( 0.4

∆S (cal/K‚mol) 52 ( 1 45 ( 1

CelN(C12)2 Cel2(C12)2 46 8.8 ( 0.3 28 ( 1 Cel3(C12)2 66 6.2 ( 0.3 18 ( 1 Cel4(C12)2 59 5.9 ( 0.3 18 ( 1 Cel5(C12)2d >160 Mal2(C12)2 Mal3(C12)2 Mal4(C12)2 Mal5(C12)2

45 15 15 -3

MalN(C12)2 11.4 ( 0.3 36 ( 1 11.9 ( 0.3 41 ( 1 10.9 ( 0.3 38 ( 1 11.0 ( 0.4 41 ( 1

birefringent texturea isotropicb

oily streaks oily streaks

malthesian crosses malthesian crosses thin lines

a

Nomenclature after ref 37. b 1,3-Di-O-dodecylglycerol, the hydrophobic part of the lipids, does not form a liquid crystalline phase in water. Above Tm, it transforms into an isotropic liquid + water dispersion. The Tm is identical to the melting point of the dry compound in air. c Glc(C12)2 ) Cel1(C12)2 ) Mal1(C12)2. d Tm is above 160 °C.

Figure 5. (a) Effect of the number of glucose residues N on the enthalpy changes ∆H associated with the hydrated solid/liquid crystalline phase transition: (4) MalN(C12)2 and (O) CelN(C12)2. (b) Effect of the number of glucose residues N on the entropy changes ∆S associated with the hydrated solid/liquid crystalline phase transition: (4) MalN(C12)2 and (O) CelN(C12)2. Table 2. Diffraction Lines Observed in the Small Angle X-ray Scattering Measurement of the CelN(C12)2/Water and the MalN(C12)2/Water Systemsa compound

diffraction lines (Å)

Figure 4. Tm as a function of the number of glucose residues N in the head groups: (4) MalN(C12)2 and (O) CelN(C12)2.

Glc(C12)2c

dilution. It does not exhibit an LR + water dispersion that is generally observed for the other glycolipid/water systems examined. Upon dilution, less and less of the birefringent textures become, and the LR phase appears to transform into, an isotropic (or a very weakly birefringent) phase + water dispersion. Below 20 wt % lipid, the solution appears almost isotropic. At 1.5 wt % aqueous lipid, the solution becomes clear and isotropic and at the same time rather viscous. However the solution is not a single phase but consists of regions of a highly swollen lipid phase and water that are visible only through the interference microscope. Though we could not identify the precise behavior or the structure of the lower lipid concentration regime, the features observed clearly indicate the enhanced hydrophilicity of the Mal5(C12)2. It is finally noted that though the mesophase structure so far unambiguously identified in the present study was an LR phase, the presence of other structures like an HII or a cubic phase is not completely excluded particularly in the Glc(C12)2/water and the Mal5(C12)2/water systems. The Glc(C12)2/water system exhibits a cubic phase (most probably a simple cubic phase59) below about 40 wt % lipid and at a temperature range below 70 °C. Above 70 °C, the cubic phase appears to transform into an HII phase. Above about 50 wt %, however, an X-ray diffraction of a liquid crystalline phase gives only a single diffraction at 30 Å.55 More detailed studies on the phase behavior of the MalN(C12)2/water and the CelN(C12)2/water systems are

Cel2(C12)2 53.0, 26.5 Cel3(C12)2 61.0, 30.5 Cel4(C12)2 72.2, 36.4 Cel5(C12)2d

30.0

type of liquid temp diffraction (°C)b line ratio crystalline phase 57 CelN(C12)2 53 1:0.50 67 1:0.50 80 1:0.504

LR (lamellar) LR (lamellar) LR (lamellar)

MalN(C12)2 Mal2(C12)2 50.5, 25.5, 16.0 50 1:0.50:0.32 LR (lamellar) Mal3(C12)2 53.0, 27.0, 18.0 37 1:0.51:0.34 LR (lamellar) Mal4(C12)2 Mal5(C12)2 57.0, 28.5, 45 1:0.50 LR (lamellar) a Data are for the 50 wt % aqueous lipids. b Experimental temperature. c Glc(C12)2 ) Cel1(C12)2 ) Mal1(C12)2. d As the Tm is above 160 °C, no liquid crystalline phase exists.

now going on and will be published in forthcoming communications. Discussion Possible Conformations of the Oligosaccharide Head Groups in the Aqueous Glycolipids. The most significant finding of the present work is that the celloand the maltooligosaccharide head groups exhibit opposite effects on the physical properties of the aqueous glycolipids. The increased size of the cellooligosaccharide head groups increases the Tm and finally leads to the insoluble lipid. On the other hand, the increased size of the maltooligosaccharide head groups decreases the Tm and appears to increase the “hydrophilicity” of the lipids. The present results can be explained in terms of the different conformations of the head groups. As a starting

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point for a discussion, let us first consider the minimum energy conformations of the malto- and cellooligosaccharides in vacuum ( ) 1.0) that are derived from Allinger’s MM2 calculation.43,44 As typical examples, the calculated minimum energy conformations of the maltopentaose and cellopentaose are illustrated in Figures 6 and 7, respectively. Each minimum energy conformation has much in common with of the corresponding homopolysaccharide.22 The “helical” structure of the maltooligosaccharides may correspond to the “flexible helix” conformation in amylose (the 1,4-RD-glucan),22 while the “extended” structure in the cellooligosaccharides to the “extended ribbon” conformation in cellulose (1,4-β-D-glucan).22 An important point predicted by the present MM2 calculation is the tendency of the cross section areas of the maltooligosaccharides (the areas of the projection of the oligosaccharides down z-axis) to increase with increasing N, while those of the cellooligosaccharides remain nearly constant irrespective of the N values. The cross section areas of the maltooligosaccharides are A ≈ 0.4, 0.4, 0.6, 0.95, and 1.2 (in nm2/molecule unit), for the N ) 1, 2, 3, 4, and 5, respectively, while those of the cellooligosaccharides are nearly constant, i.e., A ≈ 0.4 for all N values. By comparing the above-mentioned theoretical cross section areas with the molecular area information obtained either from pressure-area isotherms45 or the X-ray diffraction study, we can estimate the most plausible conformations of the oligosaccharide head groups in the liquid crystalline phases of the lipids. First, from the pressure-area isotherms at the airwater interface,45 we found that the fully compressed packing head group areas (A*) of MalN(C12)2 (at Π ≈ 40 mN/m) increased with increasing N, i.e., 0.4, 0.48, 0.55, 0.62 (nm2/molecule) for N ) 1, 2, 3, and 5, respectively (see Figures 1 and 2a in ref 45). While for all N values studied (N ) 1, 2, 3, 4; see Figures 2b and 4 in ref 45), those of CelN(C12)2 were approximately 0.4 nm2/molecule, which is close to the cross section areas of the extended double alkyl chains. The N dependence of the A* agrees semiquantitatively with the trend predicted from the MM2 calculation. The marked smaller A* value of Mal5(C12)2 may in part be due to staggering of the head groups. The above results strongly suggest that the cello- and maltooligosaccharide head groups in the fully compressed monolayers in fact adopt “extended” and “helical” conformations, respectively. Second, the interlayer spacing (first-order spacing d1) of the 50 wt % aqueous glycolipids increases nearly linearly with N. For CelN(C12)2, the increment of d1 per glucose residue is about 0.9 nm, about 1.7 times of the size (major axis) of a glucose residue, while that of MalN(C12)2 is 0.4 to 0.3 nm, smaller than the size of a glucose residue (Table 2).46 These diffraction data are compatible with the “extended” and “helical” conformations assumed. From the foregoing discussion, it is most plausible that the cello- and maltooligosaccharide head groups in the liquid crystalline phase also adopt “extended” and “helical” conformations, respectively, although we recognize the (43) Burkert, V.; Allinger, N. L. Molecular Mechanics; American Chemical Society: Washington, DC, 1982. (44) Computer Modeling of Carbohydrate Molecules; French, A. D., Brady, J. W., Eds.; American Chemical Society: Washington, DC, 1990. (45) Tamada, K.; Minamikawa, H.; Hato, M.; Miyano, K. Langmuir 1996, 12, 1666-1674. (46) As there is at present no data on the exact composition of the maximally hydrated LR phase of each lipid, we can only consider the first-order Bragg spacing ()bilayer thickness + water layer thickness). Therefore the increment described here includes the change in the water layer thickness as well.

Hato and Minamikawa

Figure 6. The space-filling model of the minimum energy conformation of the maltopentaose estimated by the MM2 calculation. (a) Longitudinal projection of the maltotpentaose (viewed from Y axis) showing a “helical” conformation similar to that of amylose. (b) Projection down long axis (Z axis), which corresponds to the cross section area of the maltopentaose. Owing to the “helical” conformation, the cross section areas of the maltooligosaccharides tend to increase with increasing N values.

Figure 7. The space-filling model of the minimum energy conformation of the cellopentaose estimated by the MM2 calculation. (a) Longitudinal projection of the cellopentaose (viewed through Y axis) showing an “extended” conformation similar to that of cellulose. (b) Projection down the long axis (Z axis), which corresponds to the cross section area of the cellopentaose. Owing to the “extended” conformation, the cross section areas of the cellooligosaccharides are nearly constant irrespective of the N values.

possibility that the conformations of the oligosaccharide head groups in the aqueous media may not be strictly identical to those illustrated in Figures 6 and 7, by virtue of thermal fluctuations and the hydration of the head groups. Effects of the Different Head Group Conformations on the Phase Behavior of the Glycolipid/Water Systems. The Spontaneous Curvature47-49 and the Packing Parameter50-54 of the Glycolipids. 1. MalN(47) Helfrich, W. Z. Naturforsch. Teil C 1973, 28, 693. (48) Olsson, U.; Wennerstro¨m, H. Adv. Colloid Interface Sci. 1994, 49, 113. (49) Gruner, S. M. J. Phys. Chem. 1989, 93, 7562. (50) Tartar, H. V. J. Phys. Chem. 1955, 59, 1195. (51) Tanford, C. Hydrophobic Effect: Formation of Micelles and Biological Membranes; John Wiley: New York, 1980. (52) Israelachvili, J. N.; Mitchell, J.; Ninham, B. J. Chem. Soc., Faraday Trans. 1976, 2, 72. (53) Mitchell, D. J.; Ninham, B. W. J. Chem. Soc., Faraday Trans. 2 1981, 77, 601. (54) Hyde, S. T. Pure Appl. Chem. 1992, 64, 1617.

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(C12)2. As the cross section areas of the maltooligosaccharide head groups increase with increasing N, and the cross section areas in the hydrocarbon chain regions are nearly constant for all the lipids, the spontaneous curvature47-49 of the MalN(C12)2 monolayer is expected to change toward the positive direction with increasing N, i.e., increasing tendency for the lipid monolayers to curve toward apolar regions (we count curvature toward oil as positive). In other words, a preference of a liquid crystalline phase may change from an HII to an HI via an LR with increasing N values. The packing parameter (v/ Alc)50-54 also predicts a similar trend. The volume, V, and the molecular length of the chains, 1c, are estimated from the empirical equations proposed by Tanford.51 Assuming the cross section areas of the head groups A are equal to A* of the corresponding lipids, the packing parameters of the MalN(C12)2 are ∼1.3, 1.1, 0.97, and 0.84 for N ) 1, 2, 3, and 5, respectively. As the estimated packing parameters depend on the choice of 1c and, in particular, A values, we are unable to discuss them quantitatively. However, with increasing N, the parameters in fact shift from the expected values for an HII (v/Alc > 1) via an LR phase or bilayers (0.5 < v/Alc < 1) toward those for nonspherical micelles (0.33 < v/Alc < 0.5).52 This is qualitatively in accord with the present experimental results. The foregoing discussion indicates that increasing N in the head groups results in a monotonous increase in the “hydrophilicity” of the lipids. In this sense, the maltooligosaccharide head groups behave like conventional oligo(ethylene oxide) head groups, though the oligosaccharide head groups appear to be far less sensitive to temperature and no clouding phenomenon was observed. In consequence, the increasing N in the maltooligosaccharide head groups may finally result in a soluble glycolipid (the formation of normal micelles). This is in fact observed for the homolog with shorter alkyl chain length, e.g., Mal5(C8)2 is soluble in water forming a micellar solution with good foaming stability.55 On the other hand, Mal2(C8)2 forms only an LR phase. The “helical” conformations may also affect their thermal behavior. Due to the steric hindrance, the increasing N of the head groups in the MalN(C12)2 enhances the hydration of the head groups, thus effectively reducing the transition temperature Tm. As seen in the DSC thermogram, the cooperativity of the melting transition considerably decreases at N ) 5. 2. CelN(C12)2. As the cross section areas at the head groups of the CelN(C12)2 are approximately equal to that of the hydrophobic regions, the spontaneous curvature of the CelN(C12)2 monolayer is expected to be close to zero, an indication that the CelN(C12)2/water systems prefer to form a planar monolayer (an LR phase). The packing parameters are close to 1.0 for all N in accord with the present experimental results. However, due to the abrupt jump in the Tm, the CelN(C12)2/water systems with N g 5 can no longer form a liquid crystalline phase. Therefore, the increasing N in the cellooligosaccharide head groups cannot contribute to enhance the “hydrophilicity” of the lipids. This is most probably due to an attractive interaction between the head groups. In this context, it is worthwhile to consider the recent SAXS measurement on the aqueous cellooligosaccharides (N ) 1-6; without hydrophobic part).56 Sano and coworkers have demonstrated that the shorter cellooligosaccharides (N ) 1-4) are soluble in a form of molecularly dispersed species, while the cellopentaose and (55) Seguer, J. B.; Minamikawa, H.; Hato, M. Unpublished results. (56) Sano, Y.; Sasaki, T.; Kajiwara, K.; Urakawa, H.; Inoue, H.; Hiragi, Y. Photon Factory Activity Rep. 1991, 9, 196.

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the cellohexaose are soluble only in an aggregated state. The aggregate size of the cellohexaose is larger than that of the cellopentaose, indicating a growing tendency toward molecular aggregation in aqueous media with increasing N. The apparent similarity between the enhanced spontaneous molecular association in the aqueous cellopentaose and the sudden jump of the Tm observed for the Cel5(C12)2/water system tempts us to assume a common molecular mechanism, i.e., inter head group attraction that is related to the cooperative interactions between cellulose chains to give the rigid structural features of plant cell walls. In the Cel5(C12)2/water systems, the effect appears much enhanced owing to the forced parallel alignment (and hence increased density) of the head groups that are chemically linked to the hydrophobic groups. The increase in N of the “extended” cellooligosaccharide head groups affords unique features in the physical properties of the CelN(C12)∞/water systems. The head group packing may restrin the hydration of the head groups so that the Tm rises above 160 °C at N ) 5. As seen from the decreasing ∆S associated with the hydrated solid/LR phase transition with increasing N values (Figure 5b), the difference between the molecular ordering in the LR phase and that in the hydrated solid state diminishes with increasing N. On the other hand, the SAXS measurement of the aqueous maltooligosaccharides (N ) 1-6) indicates that they are soluble in a form of molecularly dispersed state,56 just as expected from the present results. Concluding Remarks In this paper, we have demonstrated that the stereochemistry of the head groups, i.e., the maltooligosaccharides (“helical” conformation) and the cellooligosaccharides (“extended” conformation), have opposite effects on the phase behavior of the aqueous synthetic glycolipids. The increasing N of maltooligosaccharides decreases the Tm and tends to enhance the disorder of the lipid assembly, while the increasing N of cellooligosaccharides raises the Tm and enhances the order of the lipid assembly. Present results have some implications in the future investigation and application of the glycolipids. First, the maltooligosaccharides appear to be convenient and useful head groups for controlling the hydrophilic/ lipophilic balance of the molecules57 and hence the structure of aqueous glycolipids. In preliminary experiments, we confirmed that the MalN(Cn)2 could generate varying phases from an HII (reversed hexagonal phase) to an isotropic micellar solution by controlling the hydrophilic/hydrophobic balance of the molecule. For example, Glc(C16)2 formed an HII phase,58 and Mal5(C8)2 formed an isotropic micellar solution with good foaming stability.55 Second, the present results strongly suggest that the head group interactions depend on the stereochemistry of the head groups. The interaction between the maltooligosaccharide head groups appears repulsive, while that between the cellooligosaccharide head groups attractive. The subject of the stereochemistry dependent interactions will be further described in a following communication.45 Acknowledgment. We thank Dr. Y. Sasanuma and Mr. T. Baba for their discussion of the X-ray diffraction data. Thanks are also due to Dr. J. B. Seguer for his synthesis and the SAXS measurement of the Mal5(C12)2/ water system. LA950326Z (57) Shinoda, K. Prog. Colliod Polym. Sci. 1983, 68, 1. (58) Baba, T.; Minamikawa, H.; Hato, M. Unpublished results. (59) Klug, H. P.; Alexander, L. E. X-ray Diffraction Procedures; John Wiley & Sons, Inc.: New York, 1959; p 346.