Vesicle and Tubular Microstructure Formation from Synthetic Sugar

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Vesicle and Tubular Microstructure Formation from Synthetic Sugar-Linked Amphiphiles. Evidence of Vesicle Formation from Single-Chain Amphiphiles Bearing a Disaccharide Headgroup† Santanu Bhattacharya* and S. N. Ghanashyam Acharya Department of Organic Chemistry, Indian Institute of Science, Bangalore 560 012, India Received April 20, 1999. In Final Form: September 7, 1999 Altogether eight sugar-based amphiphiles were synthesized. Out of these, four amphiphiles contained only one hydrocarbon chain in their hydrophobic segment. Two of these single-chain amphiphiles contained fully cyclic, disaccharide headgroups, while the other two were made of one cyclic sugar unit connected to the hydrocarbon chain via open, reduced alditol units. The first two single-chain amphiphiles formed vesicles upon suspension in water as indicated by transmission electron microscopy and dye entrapment experiments. The diameters of these vesicles ranged from 500 to 800 Å. Upon aging, these vesicles transformed into tubular and lamellar microstructures. However, the other two single-chain amphiphiles produced micelles in water. Four double-chain systems including two glycolipid analogues, N-hexadecyl-(1hexadecylamido)-β-D-lactoside and N-hexadecyl-(1-hexadecylamido)-β-D-maltoside, were also synthesized. The other two double-chain (dihexadecyl ether) lipids were based on a pseudoglyceryl backbone where the headgroup residues were made of amino-β-D-maltoside and amino-β-D-lactoside, respectively. All the doublechain amphiphiles formed vesicles upon suspension in water as indicated by transmission electron microscopy and dye entrapment experiments. The diameters of these vesicles ranged from ∼1000 to 1100 Å. X-ray diffraction of the cast films of the vesicle-forming amphiphiles indicated a lamellar thickness of ∼58-60 Å for these membranous organizations. The vesicular aggregates showed sharp, solid-to-fluid thermal transitions that ranged from ∼52 to 62 °C. Formation of giant-sized vesicles from the single-chain amphiphiles could also be triggered by complex formation with Cu2+ ions under ambient pH conditions.

Introduction Glycolipids represent an important class of naturally occurring amphiphiles, which have been implicated in diverse intercellular recognition events.1 However, it is often difficult to isolate these lipids in pure form from natural sources. Therefore it is important to synthesize and examine sugar-based amphiphiles that generate vesicular membranes. This is particularly relevant because naturally occurring monoglycosylated lipids cannot form stable bilayer membranes by themselves. A vesicleforming supporting lipid is usually added to the glycolipid in order to produce stable bilayer vesicles.2 Several sugarderived amphiphiles are also attracting recent attention due to their significant immunomodulatory and antineoplastic activities.3 Recently a new family of sugar-linked amphiphiles such as N-alkylaldosylamines and related analogues derived from unprotected disaccharides have been introduced.4 These molecules are attractive since these are quite easy * To whom the correspondence should be addressed: fax, +9180-3443529; e-mail, [email protected]. Also at the Chemical Biology Unit of JNCASR, Bangalore 560 012, India. † Part of the Special Issue “Clifford A. Bunton: From Reaction Mechanisms to Association Colloids; Crucial Contributions to Physical Organic Chemistry”. (1) Curatolo, W. Biochim. Biophys. Acta 1987, 906, 137. (2) (a) Endo, T.; Inoue, K.; Nojima, S. Biochem. J. 1982, 92, 953. (b) Maggio, B.; Fridelso, G. D.; Guman, F. A.; Yu, R. K. Chem. Phys. Lipids 1986, 42, 49. (3) (a) Lockhoff, O. Angew. Chem., Int. Ed. Engl. 1991, 30, 1611. (b) Attard, G. S.; Blacksby, W. P.; Leach, A. R. Chem. Phys. Lipids 1994, 74, 83. (4) (a) Rico-Lattes, I.; Garrignes, J.-C.; Perez, E.; Barnes, C. A.; Dupuich, C. M.; Lattes, A.; Linas, M.-D.; Anbertin, A. M. New J. Chem. 1995, 19, 341. (b) Costes, F.; Ghaul, M. E.; Bon, M.; Lattes, I. R.; Lattes, A. Langmuir 1995, 11, 3644.

to synthesize and do not require long and expensive synthetic routes involving multiple protection and deprotection steps. Unfortunately however, some of these interesting amphiphiles have not been examined for vesicle formation and for other biochemical applications such as protein solubilization. This could be possibly due to their inherent hydrolytic instability in aqueous media.5 One can indeed utilize the hydrolytic lability of such molecules for the design of sustained release devices provided membrane formation is achieved from these systems. Thus one possible way to achieve successful exploitation of these molecules for specific biochemical applications is to explore the conditions in which the formation of vesicle or related aggregates is favored. In the present paper we demonstrate that it is possible to generate reasonably stable, lamellar, tubular, and “giant” vesicular microstructures from pure N-hexadecyllactosyl- and N-hexadecylmaltosylamines and related double-chain derivatives. Altogether we prepared eight sugar-linked amphiphiles. Four of these amphiphiles, 1-4 were based on single lipophilic chains (Chart 1). Two of the single chain amphiphiles, 1 and 2, contained disaccharide maltoside or lactoside headgroups while the other two were made of maltitol and lactitol headgroups. Another set of lipids was synthesized which were closer to that of typical glycolipid structure in the sense that they contained two hydrocarbon chains. Electron microscopy indicated that six of these (1, 2, 5-8) form vesicular membranes upon dispersion in aqueous media. Formation of the vesicles with closed aqueous compartments from these amphiphiles was confirmed by dye (methylene blue) entrapment studies. Formation of (5) Garelli-Calvet, R.; Latge, P.; Rico, I.; Lattes, A.; Puget, A. Biochim. Biophys. Acta 1992, 1109, 55.

10.1021/la990468j CCC: $19.00 © 2000 American Chemical Society Published on Web 12/10/1999

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Bhattacharya and Acharya Chart 1

giant-sized aggregates from some of these amphiphiles could also be triggered by complex formation with Cu2+ ions in ambient pH conditions. We also provide evidence of the presence of lamellar organizations from some of these amphiphiles by differential scanning calorimetry. Taken together the present findings provide ways to achieve vesiculation even from single-chain aldosylamines and also demonstrate the importance of headgroup structure in the elucidation of the microstructures they adopt upon suspension in water. Results and Discussion Background. Amphiphilic sugar derivatives are interesting due to their significance in areas of self-assembly and molecular recognition in biosystems.6 The sugarlinked amphiphiles are also often produced on a commercial scale, and its starting materials are obtained from renewable raw materials.7 The aldopyranose rings at the headgroup level of these molecules have multiple hydroxyl groups with defined orientation unlike their acyclic analogues. Consequently, the formation of strong cooperative hydrogen bonding “networks” between the amphiphiles is feasible. Spontaneous association of these amphiphiles is thus anticipated in water as a result of (6) Giulieri, F.; Guillod, F.; Greiner, J.; Krafft, M.-P.; Reiss, J. G. Chem. Eur. J. 1996, 1, 1335. (7) Aveyard, R.; Binko, B. P.; Chen, J.; Esquena, J.; Fletcher, P. D. J.; Buscall, R.; Davies, S. Langmuir 1998, 14, 4699.

cooperating noncovalent forces, i.e., hydrogen bonds among sugar moieties and hydrophobic association between the long hydrocarbon chains. The pronounced chiral character of the headgroup should also help generate novel nonspheroidal microstructures.8 An intriguing question remains why certain amphiphiles form vesicles and others form micellar or other type of nonvesicular aggregates. This point has been addressed in the literature by Israelachvilli,9a and empirical rationale correlating molecular architecture with possible aggregate morphology has been extended by Israelachvilli,9b Evans,9c and Ninham.9d In general, singlechain amphiphiles form micellar type aggregates and those with two hydrocarbon chains produce lamellar and vesicular aggregates. There are exceptions to such generalizations, and indeed some of these hypotheses have also been challenged.10 Covalent attachment of two hydrocarbon chains, instead of one, to a single headgroup generally enhances the order (8) O’Brien, D. F.; Armitage, B.; Benedicto, A.; Bennett, D. E.; Lamparski, H.; Lee, H. G.; Srisiri, W.; Sisson, T. M. Acc. Chem. Res. 1998, 37, 861. (9) (a) Israelachvilli, J. N. Intermolecular and Surface Forces, 2nd ed.; Academic Press: New York, 1992. (b) Israelachvilli, J. N.; Mitchell, D. J.; Ninham, B. W. J. Chem. Soc., Faraday Trans. 1976, 72, 1525. (c) Mitchell, D. J.; Ninham, B. W. J. Chem. Soc., Faraday Trans. 2 1981, 77, 601. (d) Evans, D. F.; Ninham, B. W. J. Phys. Chem. 1983, 87, 5025. (10) Fuhrhop, J.-H.; Koenig, J. Membranes and Molecular Assemblies: The Synkinetic Approach; The Royal Society of Chemistry: Cambridge, 1994, p 28.

Vesicle Formation from Sugar-Linked Amphiphiles

of the molecular orientation in aggregates restricting their conformational flexibilities. Despite this fact, instances are known where vesicle formation has been observed from single-chain amphiphiles. As a matter of fact, stable bilayer membranes have been generated from single-chain amphiphiles by restricting its conformational mobility via incorporation of “rigid” aromatic segments such as diphenylazomethine units in hydrocarbon chains or through the enhancement of intermolecular interactions via ionpairing between amphiphiles in aggregates. Thus bilayer membranes have been obtained either by the incorporation of a rigid segment11 or by the use of a hyperextended alkyl chain12 or via a perfluorinated chain.13a In this context the recent studies pertaining to the relation of amphiphilic properties to the morphology of supramolecular assemblies from glycolipid analogues with fluorocarbon chains also deserve special mention.13b Electrostatic attraction between oppositely charged headgroups,14 hydrogen bonding,15 protonation of the headgroups or coordination,16 and polymerization,16 etc., also bring about vesicle formation. These strategies generate special properties different from those of isolated, individual amphiphilic molecules, which offer important ways to structurally design fabricated, two-dimensional supermolecular systems with predictable functions. To compare the effect of introduction of double- versus single-hydrocarbon chains in these amphiphiles on their aggregation properties, we synthesized altogether eight sugar amphiphiles, 1-8. Four of these amphiphiles, 1-4, contain single lipophilic chains. Two of the single-chain amphiphiles, 1 and 2, have fully cyclic disaccharide maltoside or lactoside residues as headgroups while the other two were made of partially open-chain maltitol and lactitol headgroups. As is obvious, the double-chain molecules, 5 and 6, contain amide linkage at the backbone bearing headgroups. Presence of these amide connectors at the anomeric position of the sugar makes them hydrolytically stable even under variable pH conditions in water. The lipids 7 and 8 contain a pseudoglyceryl skeleton, making them closer to the structures of various naturally occurring glycerol based lipids. Synthesis. The synthesis began by converting the commercially available D-maltose and D-lactose to Nhexadecyl-D-maltosylamine (1) and N-hexadecyl-D-lactosylamine (2), respectively, on reacting with 1-aminohexadecane in ca. 65% and 85% yields. Compounds 1 and 2 were converted into N-hexadecyl-D-maltitol (3) and Nhexadecyl-D-lactitol (4), respectively in ca. 80% and 86% yields upon reduction with NaBH4 in MeOH. Upon reaction with palmitoyl chloride in THF/DMF, 1 and 2 were also converted to the double-chain compounds N-hexadecanoyl-N-hexadecyl-D-maltosylamide (5) and Nhexadecanoyl-N-hexadecyl-D-lactosylamide (6), respectively, in 47% and 55% yields (Scheme 1). To prepare 7 and 8, first benzylglycerol was converted into 1,2-dihexadecyloxy-3-propanol (9) by following a literature procedure.17 9 was reacted with p-toluenesulfonyl chloride to (11) Kunitake, T.; Okahata, Y.; Shimomura, M.; Yasunami S.; Takarabe, K. J. Am. Chem. Soc. 1981, 103, 5401. (12) Menger, F. M.; Yamasaki, Y. J. Am. Chem. Soc. 1993, 115, 3840. (13) (a) Krafft, M.-P.; Giulieri, F.; Reiss, J. G. Angew. Chem., Int. Ed. Engl. 1993, 32, 741. (b) Emmanouil, V.; Ghoul, M. E.; Andre-Barres, C.; Guidetti, B.; Rico-Lattes, I.; Lattes, A. Langmuir 1998, 14, 5389. (14) (a) Bhattacharya, S.; De, S. Chem. Commun. 1995, 651. (b) Bhattacharya, S.; De, S. Chem. Commun. 1996, 1283. (c) Bhattacharya, S.; De, S.; Subramanian, M. J. Org. Chem. 1998, 63, 7640. (15) (a) Lu, X.; Zhang, Z.; Liang, Y. Langmuir 1996, 12, 5501. (b) Lu, X.; Zhang, Z.; Liang, Y. Langmuir 1997, 13, 533. (16) Dodrer, N.; Regen, S. L. J. Am. Chem. Soc. 1990, 112, 2839. (17) Kates, M.; Chan, T. H.; Stanacev, N. Z. Biochemistry 1963, 2, 394.

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form 1,2-dihexadecyloxy-3-propyltosylate (10) in 80% yield. The monotosylate, 10, was reacted with NaN3 to afford 1,2-dihexadecyloxy-3-propanazide (11) in 72% yield. 10 was subjected to catalytic hydrogenation by using 5% Pd/charcoal to yield 1,2-dihexadecyloxy-3-aminopropane (12) in 72% yield. 12 was reacted with maltose and lactose in 2-propanol to afford 1,2-dihexadecyloxypropyl-3-N-Dmaltosylamine (7) and 1,2-dihexadecyloxypropyl-3-N-Dlactosylamine in 40 and 42% yield, respectively (Scheme 2). All new compounds were characterized extensively, and their analytical and spectroscopic data are consistent with their given structures. Aggregation Behavior in Water. To examine the nature of the aggregates formed from the aqueous suspensions of the presently described sugar amphiphiles, each of these compounds were dispersed in water either by probe sonication or by cosolubilization in MeOH-H2O (1:5) followed by heating with stirring as described in the Experimental Section. Generally sonication at pH 6.4 led to significant hydrolysis of the aldosylamine amphiphiles, 1, 2, 7, and 8, as indicated by TLC. However, sonication was the preferred method for preparing stable suspensions from the other double-chained compounds 5 and 6 where no hydrolysis was observed. For the single-chain compounds 1 and 2, a cosolubilization procedure was effective to furnish stable aqueous suspensions. To further understand the relationship between the headgroup architecture and the aggregate morphology, we reduced the disaccharide amphiphiles 1 and 2 with NaBH4 in MeOH to form their respective alditol amphiphiles, viz., maltitol and lactitol, 3 and 4. Compounds 3 and 4 dissolved in water quite readily and did not phase separate even without cosolubilization in MeOH or sonication. Aggregate morphologies and the particle sizes from the suspensions or solutions of each of the above amphiphile were examined by transmission electron microscopy (TEM) and dynamic light scattering (DLS). TEM experimentation revealed the existence of different morphologies obtained from aqueous suspensions of each of 1, 2, 5, 6, 7, and 8. No discernible microstructures could however, be seen from that of 3 and 4. Freshly generated aqueous suspensions of 1 under TEM examination on carbon Formvar coated copper grid (400 mesh) showed the existence of few vesicle-like aggregates, the diameters of which ranged from ∼750 to 800 Å (Figure 1a). Some larger structures were also found to be present. Another sample of the same suspension was kept for aging for ∼6 h at ambient temperature. Interestingly TEM examination of this aged suspension revealed the presence of tubular types of microstructures, the typical widths of which were ∼250 Å while the lengths were ∼6000 Å (Figure 1b). Freshly prepared suspension of 2 in water also showed the existence of several closely clustered aggregates under TEM, the diameters of which ranged from ∼400-500 Å (Figure 1c). We have also left the same suspension of 2 over >1 h for aging at ambient temperature. TEM examination indicated that upon aging there was a morphological change of the resulting aggregate. These aggregates maintained long lamellae-like microstructures, the widths of which were ∼54 Å and the lengths were ∼1000 Å (Figure 1d). Such a dimension of the width suggests the existence of an open bilayer-like arrangement in these microstructures (see below). Interconversion into nonspheroidal aggregates upon variation of conditions or aging is known. A welldocumented example is that of N-octyl-D-gluconamide, which forms spherical micelles at >80 °C in water but rearranges in part to extended helical micellar fibers and

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Bhattacharya and Acharya Scheme 1a

a Reaction, conditions, reagents, and yields: (i) n-C H NH , 2-propanol, stir, 24 h, 60 °C, 85%; (ii) NaBH , in MeOH, 30 min, 16 33 2 4 86%; (iii) n-C15H30COCl in THF/DMF, Na2CO3, stir, 24 h, 55%.

in part to vesicles of low curvature below 70 °C.18a Another example involves glutamate based amphiphiles which form helical microstructures upon fresh suspension in water. But these upon prolonged aging convert themselves into tubular microstructures.18b Data obtained from DLS studies were generally in accord with the particle dimensions obtained from TEM. The hydrodynamic diameters of freshly prepared aqueous suspensions of amphiphiles were ∼800-900 Å for 1 and 600-800 Å for 2. In contrast the aggregate sizes obtained (18) (a) Fuhrhop, J.-H.; Svenson, S.; Boettcher, C.; Rossler, E.; Vieth, H.-M. J. Am. Chem. Soc. 1990, 112, 4307. (b) Nakashima, N.; Kunitake, T. J. Am. Chem. Soc. 1985, 107, 509.

from the solutions of 3 and 4 were much smaller (∼70 ( 6 and 81 ( 4 Å). These dimensions are indicative of the formation of micellar aggregates from the suspensions of 3 and 4. We thought it worthwhile to compare the properties of the aggregates obtained from the presently described sugar-linked amphiphiles with that of the aggregates formed in water from nonionic, single-chain aldonamide amphiphiles that carry an open-chain aldohexose via amide linkages. Aldonamide amphiphiles were first reported by Pfannemiler and Welte, the aggregation properties of which were later extensively examined by Fuhrhop and co-workers.19 These aldonamides were found


Langmuir, Vol. 16, No. 1, 2000 91 Scheme 2a

a Reaction, conditions, reagents and yields: (i) p-TsCl, CHCl , pyridine, 0 °C; workup H O+, 80%; (ii) DMF, NaN , RT, 5 h, 72%; 3 3 3 (iii) EtOAc, 5% Pd/C, H2, 10 h, 72%; (iv) 4, 2-propanol, 60 °C, 24 h, 40%; (v) 4, 2-propanol, 60 °C, 24 h, 42%.

to be not soluble in water at ambient temperatures and could only be hydrated at elevated temperatures to generate micellar type organizations. O’Brien and coworkers also synthesized a number of amphiphilic aldonamides incorporating a diacetylene unit in the middle of their hydrophobic chains. Their aqueous suspensions could only be obtained by refluxing in aqueous media. UV irradiation of these suspensions afforded nonspheroidal aggregates from these types of aldonamide amphiphiles.20 Although these amphiphiles, 3 and 4, differ from the aldonamides in that they have one cyclic pyranose residue, the connection region between the headgroup and the (19) (a) Pfanemiler, B.; Welte, W. Chem. Phys. Lipids 1985, 37, 227. (b) Fuhrhop, J.-H.; Schneider, P.; Rosenberg, J.; Boekma, J. Am. Chem. Soc. 1987, 109, 3387. (c) Fuhrhop, J.-H.; Boettcher. C. J. Am. Chem. Soc. 1990, 112, 1768. (20) Frankel, D. A.; O’Brien, D. F. J. Am. Chem. Soc. 1990, 112, 7436. (b) Frankel, D. A.; O’Brien, D. F. J. Am. Chem. Soc. 1994, 116, 10057.

hydrocarbon chain bears a similar reduced open chain sugar in either case. While the presently described alditol amphiphiles, 3 and 4, also produced micellar type organizations, it should be noted that 3 or 4 could be readily solubilized in water under ambient conditions without heating, unlike the aldonamides. This is probably because these molecules are significantly protonated at pH ∼6.4. On the basis of geometrical and theoretical reasoning, vesicle formation was considered to require amphiphiles containing two hydrocarbon chains. The amphiphiles with one long alkyl chain were generally considered to associate only to form micellar type organizations.9 Therefore the formation of vesicles from the presently described singlechain disaccharide amphiphiles, 1 and 2, is not in accord with the above postulates although other examples of vesicle formation from aqueous suspensions of ionic, singlechain amphiphiles have also been reported.21 (21) Hargreaves, W. R.; Deamer, D. W. Biochemistry 1978, 17, 3759.

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Figure 1. Transmission electron microscopic images of the aggregates formed from various amphiphiles: (a) aggregates formed from a freshly prepared MeOH-H2O suspension of 1; (b) tubular structures formed from a MeOH-H2O suspension of 1 upon aging for ∼5 h; (c) vesicular structures obtained from a freshly prepared suspension of 2 in MeOH-H2O; (d) lamellar aggregates formed from an aged suspension of 2.

To further ascertain the onset of aggregation and to differentiate between the nature of aggregation between 1 and 2 with that of 3 and 4, we also carried out the following experiments. The solubilization process of the transfer of a suitable fluorescent probe from water to the aggregate interior is often accompanied by large changes in the fluorescence emission properties of the probe. Hence monitoring of the probe emission as a function of the surfactant concentration provides a simple method for the determination of the critical aggregate concentration.22 Solubilization of a lipophilic dye, 1,6-diphenylhexatriene, in different aggregates of 1-4 and examination of their emission spectra as a function of amphiphile concentration gave breaks due to the onset of aggregation (not shown). While such breaks were clearly seen at 1.3 ((0.5) × 10-4 M for 3 and 2.1 ((0.5) × 10-4 M for 4, no such inflection was seen with the aggregates of 1 and 2 at this concentration range. Instead at lower concentrations (∼1.2 × 10-5 M), breaks were observed with the suspensions of 1 and 2. The critical aggregate concentrations for 1 and 2 contrast significantly with that of 3 and 4 suggesting that suspensions of 1 and 2 are vesicular. This magnitude of critical aggregate concentrations is consistent with the reports of bilayer formation from other single-chain amphiphiles.11 The particle sizes of the respective suspensions as revealed by dynamic light scattering also support similar conclusion. Vesicle-like aggregates were also seen from the freshly prepared sonicated suspensions of 5, 6, 7, and 8 in water (Figure 2). However, vesicles of 7 and 8 were unstable (22) De. S.; Aswal, V. K.; Goyal, P. S.; Bhattacharya, S. J. Phys. Chem. 1996, 100, 11664.

and turned turbid and yellowish in ca. 30 min due to hydrolytic decomposition as indicated by TLC. Data obtained from DLS studies were also in reasonable accord with the particle dimensions obtained from TEM. The hydrodynamic diameters of freshly prepared aqueous suspensions of amphiphiles were 800-1200 Å for 5 and 6 and 1000-1100 Å for 8. Aggregate Complexation with Metal Ions. Many enzymatic reactions in biological systems have recently been elucidated to comprise of interactions involving sugar units with various metal ions. The investigations involving the chemistry of sugar-metal ion complexation have, however, remained little explored owing to their complicated stereochemistry and hygroscopic properties.23 Only the recent work of Yano24 revealed that N-glycosylamines could coordinate to transition metal ions such as Ni2+ ion through the oxygen atom of the hydroxyl group at the C-2 position of the sugar moiety and the nitrogen atom of the amine part. To our knowledge, so far no attempt has been made to examine the effects brought about by metal ion complexation on a glycolipid aggregate. Recently it was shown that at the molecular level, the competition and balance between the hydrophobic assembly of the long alkyl chains and the coordination structures of the Cu2+complexed headgroups lead to various types of bilayer organizations, which explains the reason for different type of membrane-level properties.15b It occurred to us that since the presently designed amphiphiles possess both amino and sugar units at the (23) Angyal, S. J. Adv. Carbohydr. Chem. Biochem. 1989, 47, 1. (24) Tanase, T.; Doi, M.; Nouchi, R.; Kato, M.; Sato, Y.; Ishida, K.; Kobayashi, K.; Sakurai, T.; Yamamoto, Y.; Yano, S. Inorg. Chem. 1996, 35, 4848 and references therein.


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Figure 2. Transmission electron microscopic images of the aggregates formed from sonicated, negatively stained aqueous supensions of (a) 6 and (b) 8.

Figure 3. (a, b) Giant vesicles obtained from two different samples of the aqueous suspensions of Cu2+ complexes of 2. (c) Microstructures with clumped textures obtained from a sample of an aqueous suspension of Cu2+ complex of 1.

level of their headgroups, it should be possible to explore the effects of inducing metal ion complexation with the aggregates. Toward this end, we prepared a mixture containing the aqueous suspensions of a given amphiphile (2 mmol) and Cu(NO3)2 (1 mmol) solution. Then sonication of the resulting mixtures afforded translucent, light blue colored aggregates the viscoelasticity of which increased significantly on aging. When we complexed these disaccharide amphiphiles (0.5 mM) with Cu(NO3)2 (molar ratio 2:1), huge vesicle-like organizations were produced with 2 (Figure 3a,b). On the other hand, the aggregates of 1 produced microstructures with clumped textures under comparable conditions in the presence of Cu2+ (Figure 3c) and the resulting complex was not particularly viscoelastic in character. The N-aldosylamines on the outer aggregate surfaces of 1 and 2 should normally coordinate to either Cu2+ or

Ni2+ ion in a bidentate fashion through the oxygen of the OH group at the C-2 position of the disacharide moiety and the N-atom of the amine part. We believe that in the present situation, the Cu2+ coordinates with headgroup residues more effectively in the aggregates that contain lactosyl units at the headgroup than the ones bearing the maltosylamine headroups. Interestingly, with NiCl2 at identical amphiphile/Ni2+ ratio, again 2 formed some sort of vesicle-like microstructures (not shown), while the maltose derivative 1, did not produce any such organizations. Because of the differences in the linkage orientation (axial vs equatorial) between the two sugar rings in maltosyl and lactosyl amphiphiles, their packing densities at or near the headgroup should be quite different. Under these circumstances, the influence of the Cu2+ or Ni2+ ions on the disaccharide residues at the aggregate surface

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would depend on the accessibility of the required functional groups at the headgroup for effective metal ion complexation. The architectural differences in the disaccharide residues should also affect the extent of hydration at the vesicular surfaces. This difference in hydration may also influence the efficiency with which a metal ion can ligate to a given vesicular surface. We are currently exploring these aspects in greater detail.

Figure 4. Gel-filtration profiles of the separation of the dye entrapped (small peaks) in vesicles of (a) 1, (b) 2, (c) 5, and (d) 6 from the corresponding free dye (methylene blue).

Possible modes of Cu2+ ion complexation to the maltosylamine (A) or lactosylamine (B) residues of the amphiphiles at the aggregate surfaces. Entrapment of Dye in Aggregates. To figure out whether these aggregates constituted closed aqueous shells as would be expected in a vesicle, we employed methylene blue,25 a water-soluble dye with high extinction coefficent (λmax ) 665 nm) for the examination of possible entrapment. This experiment is important as unstructured “clumps” or open lamellar fragments and dynamic micelles should not entrap an organic dye, and if there is a closed aqueous compartment inside these aggregates, then only one will observe dye retention. Various aqueous suspensions of different sugar amphiphiles at pH ) 6.4 were cosonicated with methylene blue. The resulting suspension was then loaded into a column packed with preequilibrated sephadex G-50 matrix and eluted with water. Translucent vesicular suspensions eluted right after the void volume. The filtration was continued until several eluent fractions later, free dye also got gel filtered. Thus the dye present in the bulk aqueous medium could be separated from the entrapped dye in the amphiphilic aggregates. The fractions containing a given type of vesicular fractions were pooled and were then lysed with aqueous solution of micellar TritonX-100 (1 mg/mL), and the amount of dye present was evaluated quantitatively by spectrophotometric methods. Gel-filtration profiles showing the separation of free dye, methylene blue, from the same loaded inside the aggregates of 1, 2, 5, and 6 are shown in Figure 4. The percent dye entrapments varied from 1 to 2%. While decomposition of vesicular 7 and 8 did not allow effective gel filtration, the samples from the aggregates of 3 and 4 failed to entrap any methylene blue dye. Thermotropic Behavior. To ascertain the presence of lamellar organizations in each of the above aggregates, the aqueous suspensions of the above sugar amphiphiles, 1-8 were examined by DSC. Since the presence of a small amount of impurity can affect the thermotropic properties (25) Dewall, S. L.; Wang, K.; Berger, D. R.; Watanabe, S.; Hernadez, J. C.; Gokel, G. J. Org. Chem. 1997, 62, 6784.

Figure 5. DSC trace for the thermal transition obtained from an aqueous suspension (1 mM) of 2.

and could affect the transition, all the amphiphiles were recrystallized prior to DSC scans. DSC heating scans were obtained at 1 mM concentration. Well-defined, sharp peaks due to thermotropic transition were obtained in most instances. A representative thermogram for aqueous suspension of 2 is given in Figure 5. A pronounced single endothermic peak indicates the solid gel-to-fluid phase transition, which is one of the basic physical chemical attributes of bilayer membranes. A similar peak was also observed from 1. Interestingly the main transition temperatures (Tm), transition enthalpies (∆H), and entropies (∆S) of aggregates of 1 and 2 are little different. Higher Tm values by a few degrees and larger magnitudes of other transition parameters for 1 probably indicate the differ-


Langmuir, Vol. 16, No. 1, 2000 95

Table 1. Thermotropic Phase-Transition Parameters and Lamellar Thicknesses of Aqueous Suspensions of Various Sugar-Linked Amphiphilesa

amphiphile 1 2 5 6 7 8

DSCb calcd Tm ∆H ∆S thicknessc lengthd (°C) (kcal/mol) (cal/K mol) (Å) (Å) 62.1 58.3 58.5 54.0 54.2 52.7

5.8 4.7 9.0 7.6 9.2 8.3

17.3 14.2 27.1 23.2 28.1 25.5

58.5 58.0 59.6 59.0

60.4 57.4 63.8 61.2

a See text for experimental details. b Concentration of amphiphile was 1.4 mM for all the samples. Tm represents the “main” transition peak; other minor peaks are also seen. The values ∆H are the averages of total enthalpy for successive runs. We estimate that transition enthalpies are accurate within (5%. ∆S values were calculated by dividing ∆H by Tm assuming the thermal phase transition as a first-order process. c As estimated from reflection X-ray diffraction of cast films. d Bimolecular thicknesses of amphiphiles on the basis of calculations of their energy-minimzed conformations.

ences in the hydration at the headgroup level due to the conformational variations between maltoside and lactoside units. Interestingly, a similar trend in the sense of slightly larger transition parameters was consistently seen with all the maltose-based lipids relative to their lactose-based analogues. The other notable feature is that introduction of two hydrocarbon chains in the molecular structures of the amphiphiles either through an amide connection or via anchoring through a pseudoglyceryl skeleton provide considerably higher ∆H and ∆S values for the thermal transitions relative to their single-chain counterparts. This is of course along the lines of expectation; a larger hydrophobic segment should provide greater transition enthalpies and entropies. The Tm values obtained from the double-chain lipids by DSC also were a few degrees lower than the corresponding single-chain analogues. Closer values of Tm and other transition parameters with either type of double-chain lipids indicate the existence of similar lateral packing in the molecular organizations of these amphiphiles. Significantly, under the conditions of study, neither of the aqueous suspensions of 3 and 4 gave any endothermic transition peak during the calorimetric scan. This is suggestive of the fact that aqueous suspensions of 3 and 4 did not produce lamellar type organizations, a fact already indicated from DLS, TEM, and dye entrapment studies. XRD Patterns of Cast Films from the Aggregates. It was described in several reports26 that the selfassembled organizations of amphiphiles in dilute aqueous suspensions are generally retained in their water cast films on support surfaces such as glass slides. To obtain the features of the aqueous aggregates, an X-ray diffraction method was applied to the cast films generated from the aqueous suspensions of the presently described amphiphiles. Periodic peaks indicative of ordered structures, which are similar to those reported, are observed in the XRD profiles of the cast films of the series of aggregates. Significantly, the observed lamellar thicknesses of 1 and 2 are quite comparable to that of 5 and 6. These values are given in Table 1. Notably the corresponding aggregates of 3 and 4 did not show any peaks in the scanned range, (26) (a) Kimizuka, N.; Kawasaki, T.; Kunitake, T. J. Am. Chem. Soc. 1993, 115, 4387. (b) Kunitake, T.; Shimomura, M.; Kajiyama, T.; Harada, A.; Okuyama, K.; Takayanagi, M. Thin Solid Films 1984, 121, L89.

which is in concurrence with the results obtained from TEM, DSC, DLS, and dye entrapment studies. On the other hand the aqueous aggregate samples of 7 and 8 on glass slide turned yellowish during drying within ca. 1 h, probably indicating the decomposition of the lipid molecules. Consequently, the corresponding sticky patches that were cast on the glass plates did not give reproducible results under XRD experiments. Molecular Modeling Studies. From molecular modeling calculations we found that the bimolecular layer thicknesses of these amphiphiles held linearly in an assembly of 1 to be 60.4 Å and for 2 to be 57.4 Å. In the case of 5 and 6, it was found to be 63.8 and 61.2 Å, respectively. These calculations are in general agreement with the experimental results obtained from the cast film XRD patterns (Table 1). Since each disaccharide headgroup consists of seven hydroxyl and one amino residues, a strong hydrogen bonding type of interaction is plausible at the headgroup level as the individual sugar residues remain within hydrogen bonding distances ranging from 2.1 to 3.4 Å in their preferred energy-minimized conformations of the amphiphiles. Conclusions In this report, we present evidence for vesicle-type aggregate formation from the aqueous suspensions of single-chain maltosyl- and lactosylhexadecylamines. The corresponding double-chain amphiphiles also form vesicles upon suspension in water as expected from their molecular structures. With the single-chain aldosylamine amphiphiles such as 1 or 2, the headgroup network should be organized by hydrogen bonding and water-associated bridging interactions. Such pronounced association at the level of the headgroup restricts the dynamics of chain flexing sufficiently so that even with the single-chain amphiphiles vesicle formation is feasible. In this situation, even with a single hydrocarbon chain per molecule of the amphiphile, the lamellar organization is supported, as the same has considerable strength in its packing. It also should be noted that the amines must be protonated in water at pH ∼6.4 although the extent of protonation of the headgroup nitrogen atom would be difficult to predict. Protonation extent will affect the headgroup’s effective size and charge and, in turn, its capacity to get solvated. These issues are obvious to recognize but difficult to quantify. Nevertheless these factors contribute to the overall stability of the resulting supramolecular entity. Present investigation leads us to suggest that in the present study the amphiphile headgroup is the important determinant of the aggregate nature, size, and organization. Single-tailed sugar amphiphiles form aggregates in their aqueous suspensions that adopt either a vesicular or micellar type of organization depending upon whether the headgroup structure is “fixed” or “fluxional”. In 1 and 2, the presence of two aldopyranose rings in either type of disaccharides allows the formation of strongly hydrogenbonded interamphiphile association via multiple and directional hydroxy residues of the cyclic sugar units. In alditol amphiphiles, 3 and 4, the second ring is reduced to acyclic open-chain form leaving only one aldopyranose ring intact. The open-chain part is amenable to wide conformational variations which might be responsible for greater flexibility and less tighter inter-headgroup association and overall enhancement in the aggregate dynamics. Taken together, these factors drive the formation of micellar type organizations in 3 and 4. Work is

96


currently underway employing these aggregates for lectin binding and membrane protein reconstitution studies. Experimental Section General Method. Melting points were recorded in open capillaries and are uncorrected. NMR spectra of CDCl3, DMSOd6 solutions were obtained at 300 or 400 MHz (1H). Descriptions of instruments used for various characterizations have been published.27 Synthesis. N-Hexadecyl-D-maltosylamine, 1. To a solution of (25 mmol) hexadecylamine in 50 mL of 2-propanol was added a solution of D-maltose monohydrate (15 mmol, 5.4 g) in 30 mL of water. The reaction mixture was stirred for 24 h with periodic heating to ∼60 °C at regular intervals as and when the solution turned turbid. At the end of this period, a precipitate was formed, which was separated from the solvent by filtration. The crude residue was dried first under vacuum, was then recrystallized from EtOH, and then was again freeze-dried to eliminate traces of water to avoid hydrolysis of 1 on prolonged storage (5.5 g, 65%). 1H NMR (CD3SOCD3, 400 MHz): 0.80 (t, 3H), 1.18 (m, 28H), 2.21 (br s, 1H), 2.50 (m, 1H), 2.8 (m, 2H), 3.12 (m, 2H), 3.23 (m, 1H), 3.37 (m, 1H), 3.65 (m, 2H), 4.1 to 4.8 (m, 4H). Anal. Calcd for C28H55O10N‚H2O: C, 57.61; H, 9.84; N, 2.4. Found: C, 57.81; H, 10.21; N, 2.45. N-Hexadecyl-D-lactosylamine, 2. A similar procedure as that described for 1 was followed for the synthesis of 2 except that D-lactose was used in place of D-maltose (7.2 g, 85%). 1H NMR (CD3SOCD3, 400 MHz): 0.79 (t, 3H), 1.17 (m, 28H), 2.2 (br s, 1H), 2.5 (m, 1H), 2.8 (m, 2H), 3.07 (m, 2H), 3.23 (m, 1H), 3.37 (m, 1H), 3.65 (m, 2H), 4.1 to 4.8 (m, 4H). Anal. Calcd for C28H55O10N‚H2O: C, 57.61; H, 9.84; N, 2.4. Found: C, 57.36; H, 10.03; N, 1.95. N-Hexadecylamino-1-deoxymaltitol, 3. A solution of N-hexadecyl-D-maltosylamine, 1 (1 mmol, 565 mg) in water (50 mL), was cooled in an ice bath. A solution of NaBH4 (1.2 mmol) in MeOH (15 mL) was then added dropwise. The mixture was stirred for 30 min and then treated with active carbon. After filtration through Celite, water from the filtrate was evaporated to leave a residue, which was taken up in methanol and evaporated five times in succession. A white flaky hygroscopic powder was obtained on freeze-drying (452 mg, 80%). 1H NMR (CD3SOCD3, 400 MHz): 0.82 (t, 3H), 1.15 (m, 28H), 2.2 (br s, 1H), 2.5 (m, 1H), 2.8 (m, 2H), 3.07 (m, 2H), 3.23 (m, 1H), 3.37 (m, 1H), 3.65 (d m, 2H), 4.1 to 4.8 (m, 4H). Anal. Calcd for C28H57NO10‚1.5H2O: C, 56.54; H, 10.17; N, 2.36. Found: C, 56.55; H, 10.08; N, 2.62. N-Hexadecylamino-1-deoxylactitol, 4. A similar procedure as that described for 3 was followed for the synthesis of 4 except that 2 was used instead of 1 (486 mg, 86%). 1H NMR (CD3SOCD3, 400 MHz): 0.79 (t, 3H), 1.17 (m, 28H), 2.2 (br s, 1H), 2.5 (m, 1H), 2.8 (m, 2H), 3.07 (m, 2H), 3.23 (m, 1H), 3.37 (m, 1H), 3.65 (m, 2H), 4.1 to 4.8 (m, 4H). Anal. Calcd for C28H57NO10‚H2O: C, 57.41; H, 10.15; N, 2.39. Found: C, 57.39; H, 10.20; N, 2.55. N-Hexadecanoyl-N-hexadecyl-D-maltosylamide, 5. To a solution of 1 (1 mmol, 565 mg) in 100 mL of dry THF containing sodium carbonate (117 mg, 1.1 mmol) was added dropwise a solution of hexadecanoyl chloride (1 mmol, 275 mg) in 10 mL of DMF at 0 °C, with stirring over a period of 45 min. The stirring was continued at 0 °C for 3 h and then for an additional 24 h at room temperature. The solvent was removed at pump to dryness and washed with water; solid was collected on filtration. The crude residue was dried first under vacuum and was purified by column chromatography on silica gel (378 mg, 47%). 1H NMR (CD3SOCD3, 400 MHz): 0.81 (t, 6H), 1.20 (m, 56H), 2.2 (br s, 1H), 2.5 (m, 1H), 2.8 (m, 2H), 3.07 (m, 2H), 3.23 (m, 1H), 3.37 (m, 1H), 3.70 (d + m, 2H), 4.1 to 4.8 (m, 4H). Anal. Calcd for C44H85NO11: C, 65.75; H, 10.67; N, 1.74; found C, 65.55, H. 10.61; N, 1.94. N-Hexadecanoyl-N-hexadecyl-D-lactosylamide, 6. A similar procedure as that described for 5 was followed for the synthesis (27) (a) Bhattacharya, S.; Snehalatha, K.; George, S. K. J. Org. Chem. 1998, 63, 27. (b) Bhattacharya, S.; Mandal, S. S. Biochim. Biophys. Acta (Biomembranes) 1997, 1323, 29. (c) Bhattacharya, S.; Haldar, S. Biochim. Biophys. Acta (Biomembranes) 1996, 1283, 21. (d) Bhattacharya, S.; Ghosh, S.; Easwaran, K. R. K J. Org. Chem. 1998, 63, 9140. (e) Bhattacharya, S.; Haldar, S. Langmuir 1995, 11, 4748.

Bhattacharya and Acharya of 6 except that 2 was used instead of 1 (442 mg, 55%). 1H NMR (CD3SOCD3, 400 MHz): 0.81 (t, 6H), 1.20 (m, 56H), 2.2 (br s, 1H), 2.5 (m, 1H), 2.8 (m, 2H), 3.07 (m, 2H), 3.23 (m, 1H), 3.37 (m, 1H), 3.70 (d + m, 2H), 4.1 to 4.8 (m, 4H). Anal. Calcd for C44H85NO11: C, 65.75; H, 10.67; N, 1.74. Found: C, 65.51; H, 10.9; N, 1.97. 1,2-Dihexadecyloxy-3-propanol, 9. This was prepared from 1-benzylglycerol by following a literature procedure.17 1,2-Dihexadecyloxy-propane-3-tosylate, 10. To a solution of 9 (1 mmol, 540 mg) in CHCl3 (10 mL) and pyridine (1 mL) at 0 °C was added p-TsCl (190 mg, 1 mmol), and the mixture was stirred for 2 h at 0 °C. After the completion of the reaction, the reaction mixture was poured into a solution of ice cold dilute HCl (5 mL) and extracted with CHCl3. This solution was washed and dried over anhydrous Na2SO4 and concentrated under vacuum (556 mg, 80%). IR (cm-1): 1430, 1380 (SdO). 1H NMR (CDCl3, 300 MHz): δ 6.5 (m, 4H), 3.6-3.4 (br m, 9H), 2.4 (s, 3H), 1.3 (br m, 56H), 0.81 (t, 6H). LRMS: m/e 695. 1,2-Dihexadecyloxypropane-3-azide, 11. To a solution of 10 (1 mmol, 694 mg) in DMF (5 mL) was added NaN3 (5 mmol, 325 mg), and the mixture was stirred at room temperature for 5 h followed by refluxing for 2 h. After the completion of the reaction, the reaction mixture was diluted with water and extracted with Et2O, dried over anhydrous Na2SO4, and concentrated under vacuum (414 mg, 72%). IR (cm-1): 2120 (azide str.). 1H NMR (CDCl3, 300 MHz): 0.81 (t, 6H), 1.3 (br m, 56H), δ 3.6 (br m, 9H). LRMS: m/e 565. 1,2-Dihexadecyloxy-3-aminopropane, 12. To a solution of 11 (1 mmol, 694 mg) in EtOAc (5 mL) was added 20 mg of Pd/ charcoal catalyst. This was connected to a hydrogenator under a pressure of 40 psi, and the hydrogenation was continued for 10 h. Once the reaction was over, the Pd/charcoal catalyst was filtered and the solvent from the filtrate was removed by rotary evaporation. This was acidified, filtered, and treated with Na2CO3, and the resulting solution was extracted with Et2O (414 mg, 72%). 1H NMR: δ 0.81 (t, 6H), 1.3 (br m, 56H), 3.6 (br m, 9H). LRMS m/e 539. 1,2-Dihexadecyloxypropyl-3-N-D-maltosylamine, 7. To a solution 3-amino-1,2-dihexadecyloxypropane, 12 (0.5 mmol, 270 mg), in 2-propanol (10 mL) was added a solution of D-maltose monohydrate (0.5 mmol, 180 mg) in 5 mL of water. The reaction mixture was stirred for 24 h with periodic heating to ∼60 °C at regular intervals as and when the solution turned turbid. At the end of this period, a precipitate was formed, which was separated from the solvent by filtration. The crude residue was dried first under vacuum, was then recrystallized from EtOH, and then was again freeze-dried to eliminate traces of water to avoid hydrolysis of 7, on prolonged storage (345 mg, 40%). 1H NMR (CD3SOCD3, 400 MHz): 0.80 (t, 6H), 1.18 (m, 56H), 2.21 (br s, 1H), 2.50 (m, 1H), 2.8 (m, 2H), 3.12 (m, 2H), 3.23 (m, 1H), 3.37 (m, 1H), 3.65 (m, 2H), 4.1 to 4.8 (m, 4H). MALDI-TOF mass spectrum: expected m/e 863; base peak found at m/e 863, an additional peak (886) due to (M + Na)+ was also observed. 1,2-Dihexadecyloxypropyl-3-N-D-lactosylamine, 8. A similar procedure as that described for 7 was followed for the synthesis of 8, except that D-lactose was used in place of D-maltose (362 mg, 42%). 1H NMR (CD3SOCD3, 300 MHz): 0.84 (t, 6H), 1.26 (m, 56H), 2.2 (br s, 1H), 2.5 (m, 1H), 2.8 (m, 2H), 3.07 (m, 2H), 3.23 (m, 1H), 3.37 (m, 1H), 3.65 (m, 2H), 4.1 to 4.8 (m, 4H). MALDITOF mass spectrum: expected m/e 863; base peak found at m/e 863, an additional peak 887.5 due to (M + Na)+ was also observed. Aggregate Preparation. The required amounts (1 mM) of the amphiphiles 1 and 2 were separately dispersed in water by cosolubilization in MeOH-H2O (1:5) followed by heating (70 °C) with stirring for 10 min which resulted in the formation of an opalescent solution. These vesicular solutions were used for DSC, DLS, and X-ray diffraction experiments. In the case of amphiphiles 5-8, a required amount of each of these compounds was dispersed in water by bath sonication for 15 min at ∼70 0 °C in order to afford 1 mM solutions. In all the cases, clear and optically translucent dispersions were obtained. Transmission Electron Microscopy Studies. For transmission electron microscopy (TEM) studies, individual aqueous suspensions of the amphiphiles were made in the following manner. Typically amphiphile suspensions (0.5 × 10-3 M) were generated in a pure, deionized water/MeOH (5:1) mixture by

Vesicle Formation from Sugar-Linked Amphiphiles cosolubilization of 1 and 2 and by a bath sonication method for 5-8, prepared in the presence of 0.5% uranyl acetate solution. Opalescent to optically clear aqueous dispersions were obtained in all the cases. Typically a given amphiphile was solubilized in MeOH which was rapidly injected into deionzed water at ∼70 °C such that the final concentration of the amphiphile was 0.5 mM. These produced stable opalescent aqueous suspensions from both 1 and 2, which also contained ∼16% MeOH. One drop of the above dispersion was placed into a carbon Formvar coated copper grid (400 mesh). Filter paper was employed to wick away excess water. It was then kept under a mechanical vacuum for approximately 0.5 h. A JEOL-TEM 200 CX electron microscope with an accelerating voltage of 120 keV was used for recording the micrograph images. Light Scattering Experiments. Mean hydrodynamic diameters of the aggregate particles were determined by dynamic laser light scattering using a Zetasizer 3000 (Malvern Instruments Ltd. Malvern, U.K.). Light scattering employed a He-Ne laser source at a wavelength of 633 nm keeping the detector angle at 90°. Each suspension was generated by reverse-phase evaporation as described previously. The data were analyzed using internal instrument software involving a Malvern 7132 digital (16-bit) autocorrelator. A 220 nm latex standard was used for calibration. Fluorescence Measurements. Steady-state fluorescence measurements were performed using a Hitachi F-4500 spectrofluorimeter with quartz cuvettes of 1 cm path length. Excitation wavelength was fixed at 360 nm and emission spectra of the region 390-480 nm were recorded with samples keeping the respective slit widths at 2.5 and 5 nm, respectively. One microliter of 10 mM DPH dissolved in THF was added to various amounts of sugar-linked amphiphiles dissolved or suspended in a total volume of 1 mL of water. Background samples lacking the probe were prepared in all cases, and corrections were done for scattering. The critical aggregate concentrations were determined for respective amphiphiles from the plot of the amphiphile concentration against fluorescence intensity at 430 nm as described.22 Differential Scanning Calorimetry (DSC). Individual sugar amphiphile suspensions (1.4 mM) in pure water (1 mL) were loaded into the sample cell, and an identical volume of

Langmuir, Vol. 16, No. 1, 2000 97 water was placed into the reference cell of an MC-2 ultrasensitive scanning calorimeter (Microcal Co., Amherst, MA). Samples were degassed under vacuum and then kept for scanning. The scan rate was maintained at 1 °C/min. The calorimeter was electronically calibrated, and the transition enthalpy, entropy, and van’t Hoff enthalpy were obtained using the data analysis software (Origin) coupled with the operating microcomputer. X-ray Diffraction Studies. Self-supported cast films for the X-ray diffraction studies were prepared by dispersing the amphiphile in water in order to prepare a 1 mM solution as described above. A few drops of this suspension were placed on a precleaned glass plate at ∼50 °C and air-dried at room temperature. Finally it was kept under vacuum for 15 min. X-ray diffraction was carried out by the reflection method with a X-ray diffractometer (model XDS-2000, Scintag, Inc., USA). The X-ray beam was generated with a Cu anode and the wavelength of the KR1 beam was 1.5406 Å. The X-ray beam was directed toward the film edge, and the scanning was continued up to the 2θ value of 20°. Molecular Modeling Studies. All the sugar-linked amphiphiles were drawn and their energy was minimized, and preferred conformations were calculated using the INSIGHT II 2.3.5 package (DISCOVER, Biosym Technologies). DISCOVER is a molecular simulation program that performs energy minimization to optimize initial geometries of different amphiphilic molecules constructed from appropriate fragments in INSIGHTII. The minimization path followed conjugate gradients optimization. Consistent valence force field was selected for computations. Calculated thicknesses of different amphiphilic monomers appear in Table 1.

Acknowledgment. This work was supported by a Swarnajayanti Fellowship Grant of the Department of Science and Technology, Government of India, awarded to S.B. S.N.G.A thanks CSIR for a research fellowship. We also thank Dr. G. N. Subbanna of MRC for recording TEM and the SERC at IISc for providing the Molecular Modeling Facility. LA990468J

Vesicle and Tubular Microstructure Formation from Synthetic Sugar

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