Vesicle Formation from Oligo(oxyethylene)-Bearing Cholesteryl

Cholesteryl Amphiphiles: Site-Selective Effects of. Oxyethylene .... yielded the amphiphiles 4a-d and 5b in moderate to good isolated .... peak which ...
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Langmuir 2001, 17, 2067-2075

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Vesicle Formation from Oligo(oxyethylene)-Bearing Cholesteryl Amphiphiles: Site-Selective Effects of Oxyethylene Units on the Membrane Order and Thickness Santanu Bhattacharya* and Yamuna Krishnan-Ghosh Department of Organic Chemistry, Indian Institute of Science, Bangalore 560 012, India Received April 3, 2000. In Final Form: June 29, 2000 Altogether 16 cholesterol-based amphiphiles were synthesized. In the first and second groups, nonionic oligo(ethylene glycol) appendages were covalently introduced either indirectly via a succinate spacer or directly respectively to the 3β-OH group of cholesterol. A third group of derivatives was prepared where the location of the cationic charge was conserved although the headgroup residue was progressively modified with increasing lengths of oligo(oxyethylene) units. In the fourth group, a cationic center was introduced onto the steroid backbone via increasing lengths of oligo(oxyethylene) spacers. In the fifth group, the poly(oxyethylene) segments of one amphiphile each of series 3 and 4 were replaced by the same length of polymethylene chain. Vesicle formation from the aqueous suspensions of these compounds was confirmed by TEM and dye entrapment. Fluorescence anisotropy and XRD studies revealed remarkable control of membrane characteristics by both the length and location of the oxyethylene segment.

Introduction Synthetic cationic cholesterol derivatives are currently the focus of attention of many workers. This is because some of this class of compounds are used as drugs and are now being employed for diverse purposes such as gene therapy,1-4 enzyme inhibition,5 membrane spanning conductors,6 or in medicinal applications.7 Moreover, aggregates composed of only cationic cholesterol derivatives have been shown to bring about DNA transfection in cells with greater efficiency than commercial transfection formulations comprising glycerol-based cationic amphiphiles.8,9 The interest in the utilization of cationic cholesterol derivatives as cytofectins over traditional cationic glycerol-based amphiphiles stems from the fact that they are normally employed as a mixture with a helper lipid such as DOPE (dioleoyl phosphatidyl ethanolamine) which limits shelf life as, over a period of time, these formulations tend to phase separate.10 It has also been shown that the efficiency of DNA transfection mediated by this class of molecules is highly dependent on the structure of the cholesterol monomer9,11 although not much * To whom correspondence should be addressed. Fax: +91-80360-0529. E-mail: [email protected]. Also affiliated with the Chemical Biology Unit of JNCASR, Bangalore 560 012, India. (1) Cooper, R. G.; Etheridge, C. J.; Stewart, L.; Marshall, J.; Rudginsky, S.; Cheng, S. H.; Miller, A. D. Chem. Eur. J. 1998, 4, 137. (2) Vigneron, J. P.; Oudrhiri, N.; Fauquet, M.; Vergely, L.; Bradley, J. C.; Basseville, M.; Lehn, P.; Lehn, J.-M. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 9682. (3) Gao, X.; Huang, L. Biochem. Biophys. Res. Commun. 1991, 179, 280. (4) Leventis, R.; Silvius, J. R. Biochim. Biophys. Acta 1990, 1023, 124. (5) Bottega, R.; Epand, E. M. Biochemistry 1992, 31, 9025. (6) Otto, S.; Osifchin, M.; Regen, S. L. J. Am. Chem. Soc. 1999, 121, 7276. (7) Kreimeyer, A.; Andre, F.; Guoyette, C.; Hyunh-Dinh, T. Angew. Chem., Int. Ed. Engl. 1998, 37, 2853. (8) Moradpour, D.; Schauer, J. I.; Zurawski, V. R., Jr.; Wands, J. R.; Boutin, R. H. Biochem. Biophys. Res. Commun. 1996, 221, 82. (9) Krishnan-Ghosh, Y.; Visweswariah, S. S.; Bhattacharya, S. FEBS Lett. 2000, 473, 341. (10) For more details see the following review: Miller, A. D. Angew. Chem., Int. Ed. Engl. 1998, 37, 1768. (11) Fichert, T.; Regelin, A.; Massing, U. Bioorg. Med. Chem. Lett. 2000, 10, 787.

is known how exactly these cationic cholesterol derivatives bring about DNA transfection. However, there is not a single report in the literature that addresses the relationship between the molecular structure of a given cholesteryl lipid with the properties manifested upon its aggregation in aqueous media. This paper present the results of the first such investigation involving aggregate properties of a series of cationic cholesterol derivatives whose molecular structures have been systematically varied. Cholesterol is weakly amphipathic since its hydroxyl group is polar and the steroid ring system along with its isopentyl tail at C-17 is nonpolar. However, the affinity of its OH group for water is much less than the affinity of an ionic headgroup of a typical lipid molecule. Hence cholesterol alone cannot aggregate in water to form membranes or related aggregates. Since cholesterol possesses a long, rigid hydrophobic segment, covalent attachment of a charged residue to the steroid backbone should result in molecules with amphiphilic properties, which on suspension in water should form thermally stable membranes. Indeed examples are known where polar derivatives of cholesterol have been shown to form vesicles or related aggregates.12-16 Herein we report the synthesis of five families of cationic and nonionic cholesterol-based lipids (cholamphiphiles) 1-5 (Chart 1), containing oxyethylene units. In view of the fact that there is an intimate relation between headgroup hydration and transfection efficiency of liposome-based transfection reagents,17-19 we chose oligo(oxyethylene) residues as elements of hydration modulation at the headgroup level. The newly synthesized (12) Davis, S. C.; Szoka, F. C., Jr. Bioconjugate Chem. 1998, 9, 783. (13) De Wall, S. L.; Wang, K.; Berger, D. R.; Watanabe, S.; Hernandez, J. C.; Gokel, G. W. J. Org. Chem. 1997, 62, 6784. (14) Echegoyen, L.; Hernandez, J. C.; Kaifer, A.; Gokel, G. W.; Echegoyen, L. E. J. Chem. Soc., Chem. Commun. 1988, 836. (15) Wu, P.-S.; Wu, H.-M.; Tin, G. W.; Schuh, J. R.; Croasmun, W. R.; Baldeschweiler, J. D.; Shen, T. Y.; Ponpipom, M. M. Proc. Natl. Acad. Sci. U.S.A. 1982, 79, 5490. (16) Lyte, M.; Shinitzky, M. Chem. Phys. Lipids 1979, 24, 45. (17) Webb, M. S.; Hui, S. W.; Steponkus, P. L. Biochim. Biophys. Acta 1993, 1145, 93. (18) Bloomfield, V. A. Curr. Opin. Struct. Biol. 1996, 6, 334. (19) Safinya, C. R.; Koltover, I.; Raedler, J. Curr. Opin. Coll. Interface Sci. 1998, 3, 69.

10.1021/la000498i CCC: $20.00 © 2001 American Chemical Society Published on Web 03/09/2001

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Langmuir, Vol. 17, No. 7, 2001 Chart 1

Bhattacharya and Krishnan-Ghosh Scheme 1a

a Reagents, reaction conditions, and yields: (i) (a) (COCl) , 2 2 h; (b) dry CHCl3, HO(CH2CH2O)nH, NEt3, 0 °C, 3 h (yield: 1a, 55%; 1b, 53%; 1c, 50%).

Scheme 2a

cholamphiphiles form closed vesicular membranes upon dispersion in water as evidenced by electron microscopy and dye entrapment studies. Unlike membranes from conventional fatty acid based lipids, the presently described systems do not show any thermal solid-to-fluid phase transitions. Membrane-level properties such as rigidity and bilayer thickness were found to depend on the length and position of oligo(oxyethylene) units in the lipid monomer. We explain such a significant influence of the headgroup on the membrane properties in these cholamphiphiles on the basis of temperature-dependent fluorescence anisotropy measurements and X-ray diffraction (XRD) methods. Results and Discussion Lipid Molecular Structures. The linker region between the cationic headgroup and the steroid backbone plays an important role in mediating gene transfer efficiency.9 Accordingly we have first synthesized two families of cationic cholamphiphiles, one where the linker region is an ester moiety and the other where there is an ether linkage. Correspondingly, in the first group, nonionic oligo(oxyethylene) appendages were introduced to the 3βOH group of cholesterol via covalent connection through a succinate spacer as in 1a-c. In contrast, oligo(oxyethylene) units are directly attached to 3β-site in cholesterol via an ether linkage to afford 2b-d. In the third group, i.e., in cholesteryl derivatives, 3a-d, the cationic headgroup is linked to the 3β-cholesterol via an ester connection. Here the positive charge is located at a fixed distance from the cholesteryl backbone although the headgroup hydration is continually modified with the progressive increase in the number of oxyethylene units on the NMe2+ center. To see the difference in properties if any depending on the sequence in which the two moieties were attached, a fourth series of cationic cholesteryl amphiphiles, 4a-d, was also synthesized. This series of cholesterol derivatives connects the cationic NMe3+ groups via an oligo(oxyethylene) spacer chain. The spacer connects itself via an ether link to the 3β-OH of cholesterol. The cationic NMe3+ group is therefore placed at incrementally greater distance from the cholesteryl backbone with the insertion of increasing number of oxyethylene units. In the fifth group, 5a,b, the corresponding tetraoxyethylene moieties in compounds 3d and 4d were replaced with a polymethylene chain of the same length. This was done to verify whether the observed membrane properties of 3a-d and 4a-d are largely due to the presence of the poly(oxyethylene) segment. Synthesis. Cholesterol possesses a long, hydrophobic region. Easy amenability of the 3β-OH group to chemical modification into polar moieties allows a convenient method of generating novel lipids that could act as cytofectins. We have synthesized the first series of

a Reagents, reaction conditions, and yields: (i) TsCl, dry pyridine, 0 °C, 6 h (93%); (ii) dry dioxane, HO(CH2CH2O)nH, reflux, N2, 4 h (yield: 2a, 86%; 2b, 80%; 2c, 90%; 2d, 93%); (iii) TsCl, dry pyridine/CHCl3, 0 °C, 3 h (yield: 7a, 92%; 7b, 87%; 7c, 96%; 7d, 85%); (iv) LiBr, dry DMF, N2, 65 °C, 4 h (yield: 8a, 95%; 8b, 96%; 8c, 97%; 8d, 90%); (v) NMe3, dry acetone/ EtOH, reflux, 24 h (yield: 4a, 90%; 4b, 91%; 4c, 80%; 4d, 75%, 5b, 59%); (vi) bromoacetyl chloride, dry C6H6, DMAP, Et3N, 0 °C, 24 h (77%); (vii) RNMe2, dry acetone/EtOH, reflux, 24 h (yield: 3a, 80%; 3b, 90%; 3c, 89%; 3d, 88%; 5a, 76%); (viii) dry dioxane, Br (CH2)11OH, reflux, N2, 4 h (78%).

cholesterol derivatives by converting cholesteryl hemisuccinate 6c into the corresponding acid chloride with oxalyl chloride and then treating with the appropriate oligo(ethylene glycol) in slight excess to give the corresponding nonionic amphiphiles, 1a-c (Scheme 1). Cholesteryl tosylate, 6a, upon refluxing in dry dioxane with the appropriate oligoethylene glycol yielded 2a-d in good yields12 (Scheme 2). Cholesteryl bromoacetate, 6b, was quaternized with the appropriate tertiary amine to yield 3a-d and 5a also in good yields (Scheme 2). 2a-d were then converted to the respective tosylates, 7a-d, which upon heating to ∼65 °C with 1 equiv of LiBr in dry DMF furnished the corresponding bromides, 8a-d, in excellent yields. Quaternization of the bromides with the appropriate tertiary amine in a 1:1 mixture of dry acetone/EtOH yielded the amphiphiles 4a-d and 5b in moderate to good isolated yields (Scheme 2). The intermediates and the final products were appropriately characterized by IR, NMR, mass spectrometry, and elemental analysis; cf. Experimental Section. Prediction of Aggregate Morphology. To predict the type of aggregation expected from an amphiphile, empirical models have been proposed on the basis of the relative sizes of headgroup and hydrophobic segments.20-22 Although the validity of such models has been questioned,23 it is often desirable to draw a relationship between the amphiphile molecular structure and the type of aggregates obtained upon suspension in water. According (20) Israelachvilli, J. N.; Mitchell, D. J.; Ninham, B. W. J. Chem. Soc., Faraday Trans. 2 1976, 72, 1525. (21) Mitchell, D. J.; Ninham, B. W. J. Chem. Soc., Faraday Trans. 2 1981, 77, 601. (22) Evans, D. F.; Ninham, B. W. J. Phys. Chem. 1983, 87, 5025. (23) Fuhrhop, J.-H.; Koenig, J. Membranes and Molecular Assemblies: The Synkinetic Approach; The Royal Society of Chemistry: Cambridge, U.K., 1994; p 28.

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Table 1. Packing Parameters for the Amphiphiles Using Theoretical Modelsa entry

compd

Vca (Å3)

L (Å)

Ae (Å2)

Pe (V/LAe)

Ac (Å2)

Pc (V/LAc)

1 2 3 4 5 6 7 8

1a 1b 1c 2b 2c 2d 3a 4a

735 735 735 735 735 735 735 735

23b 23b 23b 21 21 21 21 21

37 39 63 37 39 63 64 64

0.86 0.82 0.51 0.95 0.90 0.56 0.55 0.55

39 44 49 39 44 49 64 64

0.82 0.73 0.65 0.90 0.80 0.71 0.55 0.55

a V was taken as according to the rectangular box model where L was calculated as specified in text. b L was taken as the sum of the c c c lengths of steroid (21 Å) and the succinato spacer associated with the amphiphile.

to this model, if a dimensionless quantity called the packing parameter, p, for an amphiphile falls within the range 0.5-1, then vesicle formation is anticipated. The packing parameter, p, is given by the relation p ) V/LA, where V is the volume of the hydrophobic segments, L is the critical chain length which is roughly the length of the fully extended hydrocarbon chain, and A is the area of cross section of the headgroup. Data summarizing the theoretical and experimental parameters for a few representative amphiphiles in this study are presented in Table 1. For 1-4, the volume (V) of the hydrophobic portion (steroid) was considered to be 735 Å3 assuming the steroid to be a rectangular box of dimensions 5 × 7 × 21 Å.13 Surface pressure-area isotherms are known to provide experimentally determined information pertaining to the headgroup area of a given amphiphile with reasonable accuracy. The “measured” values (m) of headgroup areas were obtained from previously published literature on surface pressure area isotherms of compounds bearing similar headgroups. The experimental values of area of cross section (Ae) and theoretical areas of cross section (Ac) of the headgroups of the respective poly(ethylene glycol) segments in 1a-c and 2b-d could be obtained from surface tension measurements.24 For the -NMe3+bearing headgroups, 3a and 4a, Ae and Ac were taken from the literature where both Ae and Ac are in good agreement (64 Å2).22 In these instances, Pc and Pe (0.5 e P e 1.0) predict vesicle formation by these amphiphiles. Transmission Electron Microscopy. Bath sonication for ca. 10-15 min. at ∼ 60 °C yielded slightly translucent aqueous suspensions from these cholamphiphiles. To discern the nature of the aggregates obtained from various amphiphiles, the respective aqueous suspensions were examined with the aid of negative-stain transmission electron microscopy as described in the Experimental Section. TEM examination of the individual, air-dried vesicular suspensions of 1-5 layered on carbon-Formvarcoated copper grids revealed the existence of closed aggregate structures in all cases. Few representative micrographs are shown in Figure 1. All cholesteryl derivatives formed predominantly unilamellar and nearly spherical vesicles except 2b-d, 4a-d, and 5b that generated mostly multiwalled vesicles under identical conditions of aggregate preparation. Dye Entrapment in Vesicles. Having confirmed lamellar aggregate formation from these newly developed cholamphiphiles, dye entrapment was employed to examine whether these aggregates comprised closed inner aqueous compartments. Micellar or open lamellar aggregates do not have the ability to entrap hydrophilic or ionic molecules such as water-soluble dyes. Since the surfaces of the aggregates of 1-5 are either nonionic or (24) Sokolowski, A.; Burczyk, B. J. Colloid Interface Sci. 1983, 94, 369.

Figure 1. Negative stain transmission electron micrographs of sonicated aqueous suspensions of 0.4 mM cholamphiphiles at pH 6.8: (A) 4a; (B) 3d; (C) 3c; (D) 5a.

positively charged, the choice of dye molecule for the entrapment studies becomes important. We chose a cationic dye, methylene blue (MB), for this purpose in order to avoid the electrostatic association of the dye molecules with the vesicle surface. This dye (MB) has been previously used to show entrapment capacities of the aggregates made from steroidal azacrown derivatives by Gokel et al.13 To examine the entrapment abilities of the aggregates 1-5, we generated vesicles from 1-5 by sonication of a film of the amphiphile to give a final concentration of 5 mM in water containing 0.1 mM MB for 10 min at 60 °C. Upon elution with water, separate fractions (of 1 mL each) were collected. To reduce the background scatter from these opalescent fractions, TritonX-100 was introduced into each fraction as described in the Experimental Section. A gel filtration profile was obtained upon plotting the absorbance at 665 nm of the fractions obtained from the gel filtration column. A typical profile is shown in Figure 2. In all the cases it was observed that there was a small initial portion containing vesicles entrapping barely 0.5-5% of total dye followed by a large peak which was the free, untrapped dye. It is clear that the MB molecules associated with vesicles could be distinctly separated from the free dye. The percentages of dye entrapped inside vesicles ranged from ∼1.0 to 10 L/mol of total dye as given in the Table 2. Fluorescence Anisotropy Measurements. To probe the thermal response of these aggregates we then measured the fluorescence anisotropy as a function of temperature. This technique has been used extensively for probing the fluidity of the vesicle bilayer and for measuring

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Table 2. Transmission Electron Microscopy, Dye Entrapment, and X-ray Diffraction Data for Cholesteryl Amphiphiles 1-5 entry

compd

size of aggregatea (nm)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

1a 1b 1c 2b 2c 2d 3a 3b 3c 3d 4a 4b 4c 4d 5a 5b

170-330 50-270 440 85-240 100-300 120-500 50-400 130-300 130-300 160-200 40-135 40-120 25-132 75-300 30-80 180-390

entrapment capacityb (%) 0.8 0.9 1.4 1.4 1.5 2.0 1.2 0.5 1.6 4.5 2.7 1.6 3.8 1.6 1.1 2.6

aggregate layer widthsc [Å (%)] calcdd obsde 42.0 42.0 42.0 46.0 46.0 46.0 42.0 42.0 42.0 42.0 42.0 49.0 56.0 63.0 42.0 72.0

46.5 (100) 47.0 (100) 49.7 (100) 42.1 (100) 46.5 (100) 47.9 (100) 42.0 (100) 40 (36), 34 (64) 46 (58), 28 (14), 25 (28) 28 (82), 25 (18) 36.6 (100) 51.9 (100) 55.2 (100) 57.9 (100) 55.1 (100) 63.3 (40), 51.9 (60)

a [amphiphile] ) 4 mM; pH ) 6.8. b [amphiphile] ) 5 mM; methylene blue ) 0.1 mM; pH ) 6.8. c [1] - [5] ) 0.5 mg /mL. d Lengths of two molecular layers of lipids as obtained from models. e Bilayer width as obtained from the X-ray diffraction measurements of cast films. Values in parentheses indicate the percentage of a given morph.

Figure 2. Dye entrapment profile of aqueous suspension of 4b in methylene blue (MB). [4b] ) 5.0 mM; [MB] ) 0.1 mM. The absorbance at 665 nm was checked for all fractions.

the melting temperature (Tm) associated with the gel to liquid crystalline phase transition associated with membranes.25,26 DPH (1,6-diphenylhexa-1,3,5-E,E,E-triene) was used as the fluorescent probe which is known to intercalate between the alkyl chains in the hydrophobic interior of the bilayer. Hence it is reasonable to assume that DPH partitions into the hydrophobic interior in the aggregates formed by these amphiphiles. The relationship of steady-state anisotropy (r) due to doped DPH in a given membrane versus temperature is indicative of the Tm of a membrane as well as provides a reasonable measure of the membrane order. A higher value of r indicates lesser fluidity due to the restricted freedom of movement experienced by DPH in the bilayer especially in their solidlike gel states. However, in the cases of amphiphiles 1-5, irrespective of whether the headgroups were nonionic or cationic, a classical “melting” temperature profile was not obtained (Figure 3). Instead there was a gradual monotonic decrease of r value with the increase in temperature after which a plateau was reached. This is not surprising as a major contribution to the gel-to-liquid crystalline phase transition in membranes composed of conventional fatty acid based lipid molecules is due to a sudden, catastrophic increase in chain motion beyond Tm. In membranes generated from cholamphiphiles, the steroid backbone consists of fused rings, which render the entire backbone (25) Shinitzky, M.; Barenholz, Y. Biochim. Biophys. Acta 1978, 515, 367. (26) Andrich, M. P.; Vanderkooi, J. M. Biochemistry 1976, 15, 1257.

Figure 3. Fluorescence anisotropy (r) versus temperature (°C) plots due to DPH for different amphiphiles. [Amphiphile] ) 0.1 mM, [DPH] ) 1 µM, and pH ) 6.8.

immobile. Even on increase of the temperature, the steroid backbone being so conformationally rigid, there is no stark drop in the membrane rigidity at a particular temperature (phase transition) which is one of the most general characteristics of fatty acid based lipid membranes. It is evident from Table 3 that all the nonionic derivatives 1a-c and 2b-d possess consistently higher r values as compared to the corresponding charged derivatives 3-5. This is probably due to the repulsion faced by the charged cationic monomers at the headgroup level which is absent in the case of nonionic derivatives. Cationic derivatives 3a-d where the PEG segments are attached to the cholesterol backbone through a -NMe2+ center show a steady decrease in r value at 20 °C as the n value increases (Figure 4A). Since the PEG segment is located beyond the cationic center, it is reasonable to assume that, due to its propensity to get hydrated, it sweeps out a volume proportional to its length at the headgroup level. Consequently, the attachment of a PEG segment to a cationic center in these systems seems to result in the formation a large headgroup which limits the lateral distance of closest approach of the neighboring monomers.27 A headgroup with greater bulk leads to a

Oligo(oxyethylene)-Bearing Cholesteryl Amphiphiles Table 3. Fluorescence Anisotropy Values As Measured Using DPH as a Probe entry

compda

rb(20 °C)

η(20 °C)

r(62 °C)

η(62 °C)

1 2 3 2 3 4 5 6 7 8 9 10 11 12 14 15

1a 1b 1c 2b 2c 2d 3a 3b 3c 3d 4a 4b 4c 4d 5a 5b

0.264 0.268 0.260 0.281 0.279 0.274 0.268 0.255 0.247 0.232 0.221 0.244 0.252 0.264 0.254 0.214

42.9 44.9 41.1 52.2 51.0 48.1 44.9 38.9 35.7 30.6 27.3 34.6 37.7 42.9 39.3 25.5

0.203 0.215 0.213 0.260 0.253 0.256 0.203 0.213 0.213 0.179 0.184 0.194 0.183 0.181 0.215 0.147

22.8 26.2 25.2 41.1 38.1 39.3 22.8 25.2 25.2 17.9 18.8 20.8 18.7 18.3 26.2 12.9

a [amphiphile] ) 0.1 mM; [DPH] ) 1µM; pH ) 6.8. b Error in r values was (0.002 units.

Figure 4. (A) Plots of fluorescence anisotropy values (r) at 25 °C with n value of -(CH2CH2O)n- units in 3a-d and 4a-d. (B) Variation of membrane thickness with n value in the series 3a-d (open squares) and 4a-d (closed circles).

greater intermonomer separation which in turn leads to “looser” membrane packing. In the cationic derivatives 4a-d where the quaternary ammonium center is attached to the cholesteryl backbone through varying lengths of PEG segments, the anisotropy values at 20 °C increases as n value increases (Figure 4A). This reversal in trend may be due to the dual nature of the PEG segment as well as its specific location in the bilayer membranes of these systems. The PEG segment is known to be special for the reason that it can take on both hydrophilic and hydrophobic character.28,29 When located between the hydrophobic portion and cationic center, the PEG segment probably gets inserted into the bilayer while facilitating water-promoted hydrogen bond(27) Ringsdorf, H.; Laschewsky, A.; Elbert, R. J. Am. Chem. Soc. 1985, 107, 4134. (28) See: Gokel, G. W.; Murillo, O. In Comprehensive Supramolecular Chemistry; Pergamon: New York, 1996; Vol.1, Chapter 1, p 1. (29) Moyer, B. A. In Comprehensive Supramolecular Chemistry; Pergamon: New York, 1996; Vol.1, Chapter 10, p 377.

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ing at the interfacial region. The longer the PEG segment, the greater is its ability to remain embedded in the bilayer and allow the monomers to position their positive charges away from each other. This results in a closer approach of the adjacent monomers resulting in higher r value for greater n value. At 20 °C, 5b, which possesses a polymethylene segment of identical length, shows an r value of ∼0.28 at as compared to 4d which has a value of ∼0.26. This indicates that, at the location between the hydrophobic portion and the cationic center, the PEG segment takes on hydrophobic character and behaves akin to a polymethylene spacer. Differential Scanning Calorimetry. To examine the nature of the phase of the bilayers formed by these amphiphiles, DSC studies were carried out. Thermograms of 1 mM dispersions of all of the amphiphiles 1-5 showed flat traces in the temperature interval of 20-75 °C and did not show any evidence of a thermal transition (not shown). This is in accordance with the fluorescence anisotropy data and reconfirms the conclusions presented in the preceding section. X-ray Diffraction Studies. To figure out the orientation of the monomers in the bilayers and to know about their lamellar packing, X-ray diffraction studies were performed on supported cast films of 1-5. Reflections up to 10° were analyzed and interpreted in terms of higher order reflections of stacked bilayer structures (Table 2). A series of reflections was obtained for the aggregates of 1-5, the highest intensity peak being the long spacing (corresponds to the lowest 2θ value) as shown in Figure 5. Molecular modeling35 allowed computation of the theoretical length of amphiphile monomers. This calculation for two molecules of 3a oriented parallel to the bilayer normal showed a theoretical bilayer width ∼43 Å (Table 2). The diffraction patterns of 1a-c showed membrane thicknesses of 46.5, 47.1, and 49.7 Å, respectively, corresponding to 2θ values of ∼1.7-1.9 on the basis of higher order reflections. The above measured values suggest hydrated bilayer organization for both 1a-c and 2b-d. These cast films, however, showed no evidence of lipid polymorphism. In contrast the aggregates from 3b-d showed two different kinds of packing arrangements in their aggregates. Two series of reflections were observed. 3b showed a weaker reflection corresponding to the long spacing value of 40.1 Å and a stronger reflection corresponding to the long spacing value 33.9 Å. 3c,d showed reflections corresponding to ∼28 and 25 Å (Figure 5B). However, 3c showed stronger reflection at 25 Å while 3d showed a stronger reflection at 28 Å. It is noteworthy that among the amphiphiles 3a-d the lengthening of the PEG segment, apart from inducing lipid polymorphism, progressively shrinks the bilayer width (Figure 4B). In this series of amphiphiles 3a-d, the whole of the PEG segment most probably form part of the headgroup. The reason for this “shrinkage” may be due to the following. Although the charge remains constant, with the increase of the PEG segment, the volume swept out by the headgroup increases (Figure 6B,C). This limits the closer approach of the (30) Abrahamsson S.; Dahlen, B. Chem. Phys. Lipids 1977, 20, 43. (31) Gao, Q.; Craven, B. M. J. Lipid Res. 1986, 27, 1214. (32) Fahey, D. A.; Small, D. M.; Kodali, D. R.; Atkinson, D.; Redgrave, T. G. Biochemistry 1985, 24, 3757. (b) Small, D. M. The Physical Chemistry of Lipids from Alkanes to Phospholipids. Handbook of Lipid Research; Plenum Press: New York, 1986. (33) Dahlen, B. Chem. Phys. Lipids 1979, 23, 179. (34) Larsson, K. The Lipid Handbook, 2nd ed.; Gunstone, F. D., Harwood, J. L., Padley, F. B., Eds.; 1994; Chapter 8.11, p 461. (35) For details consult BIOSYM programs available from BIOSYM Technologies, 9685 Scranton Road, San Diego, CA 92121-3752.

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Figure 5. Representative grazing angle X-ray Diffraction spectra for vesicular suspensions of (A) 3c and (B) 4b.

Bhattacharya and Krishnan-Ghosh

between the steroid backbone and the hydrocarbon chain.30 The crystal structure of cholesteryl oleate31 shows packing similar to triglycerides.32 However, for the corresponding charged derivative 5a in an aqueous environment, the hydrophobic polymethylene spacer loops into the bilayer membrane (Figure 6D) in order to avoid unfavorable contact with water unlike its oxyethylene analogue 3d. This would result in a packing where hydrocarbon chains and the steroid skeleton are arranged in the same layer which has also been observed in derivatives such as cholesteryl laurate.33 This chain length fits well with the length of the cholesteryl moiety,34 and 5a incorporates an identical chain length at the quaternary ammonium center. Thus in 5a the headgroup now reduces to a -NMe2+ group. In 3a-d we see that the increase in length of PEG segment at the cationic center in this location increases the bulkiness of the headgroup which induces greater interdigitation due to looser packing leading to smaller bilayer widths. However, when the -NMe3+ center was attached at the end of the PEG segment as in 4a-d, the trend observed in their bilayer widths was reversed (Figure 4B). This may be due to the fact that, with increasing PEG lengths, a portion of the segment gets inserted into the bilayer as shown in Figure 6F,G. The addition of a single oxyethylene unit to 4a results in a 15 Å increase in bilayer width. This is reflective of the extent of flexibility conferred on the monomer by just one oxyethylene unit. This conformational flexibility allows the positioning of the charge far away so that with the charge repulsion minimized, monomers may pack closer to each other. Due to the lack of flexibility of the spacer between the steroid and the -NMe3+ in 4a, the monomers are unable to position their charged centers far enough from each other to minimize repulsion. To circumvent such an unfavorable scenario most probably an interdigitation takes place as indicated from the experimentally obtained bilayer width of 36 Å (Figure 6A). This reasoning is strengthened by the fact that 5b and 4d show longer bilayer widths indicating the ability of the intervening spacer to position the charges of adjacent monomers favorably. Thus it is evident that PEG segment in this location now behaves equivalent to a polymethylene spacer as the bilayer widths of 5b and 4d are quite similar. Conclusions

Figure 6. Schematic representation of possible aggregate layer packing plans of cast films of various cholamphiphiles. Key (plan, no.): A, 3a; B, 3b; C, 3c,d; D, 5a; E, 4a; F, 4b,c; G, 4d; H, 5b. The shaded areas in plans B and C represent the region swept by the oligooxyethylene segment. The oligo(oxyethylene) segment is represented by solid black lines in plans F, and G. The gray solid line represents the polymethylene segment. Dashed lines represent water.

monomers leading to greater intermonomer separation in the bilayer which induces interdigitation of the C-17 cholesteryl side chains of outer and inner leaflets of the bilayer to fill the “voids” and provide stabilization. In 5a, the replacement of the PEG segment by a polymethylene segment produces a bilayer of 55 Å width. Uncharged hydrocarbon chain appended derivatives of cholesterol arrange themselves in the solid phase such that the steroid backbone and hydrocarbon chains are not in the same layer. This is due to the length mismatch

In the present study we report the synthesis of 16 new cholesteryl amphiphiles. We also present their membrane forming properties. All the amphiphiles form stable, closed vesicles as evidenced by TEM and dye entrapment. These membranes show a departure from the behavior of conventional bilayer forming fatty acid based lipids in that the former do not exhibit a solid-fluid melting transition. The bilayer thickness in these membranes appears to be modulated by the nature or the size of the headgroup. Depending on its location in the cholamphiphile monomer, the oxyethylene group shows either hydrophilic or hydrophobic character.28,29 In the series 3a-d, the PEG segment is located outside the hydrophobic portion of the membrane. It takes on hydrophilic character and remains in a random orientation thereby increasing headgroup bulk. Increase in bulkiness of the headgroup increases intermonomer separation, reflected in the r values, which induces interdigitation as supported by XRD data. In the

Oligo(oxyethylene)-Bearing Cholesteryl Amphiphiles

series 4a-d, the -(CH2CH2O)n- unit is located between the cholesteryl backbone and the -N+Me3 headgroup. Here it acts like a hydrophobic spacer as an increase in the n value in 4 results in an increase in the length of the bilayer as evidenced from the XRD data. Thus the present study demonstrates that the introduction of an oligo(oxyethylene) unit into cholesterol-based amphiphiles can bring about ramifications at the membrane level far exceeding the seemingly trivial structural modification at the molecular level. The findings described herein illustrate a first step toward the generation of thermally stable organized assemblies of controllable order and thickness. Experimental Section General Methods. Melting points were recorded in open capillaries and are uncorrected. 1H NMR spectra were recorded in a JEOL-JANL-LA-300 NMR spectrometers. Chemical shifts are reported in ppm downfield from the internal standard, tetramethylsilane. IR spectra were recorded in a Perkin-Elmer model 781 spectrometer and are reported in wavenumbers (cm-1). Microanalyses were performed on a Carlo Erba elemental analyzer model 1106. Steam distilled water was used for all physical measurements, and pH measurements were made with a Schott Gerate Digital laboratory pH meter CG 825. UV-vis spectra were recorded on a Shimadzu model 2100 UV-vis recording spectrophotometer equipped with a TCC-60 temperature controller. Materials. All reagents, solvents, and starting materials were obtained from the best commercial sources and were distilled, recrystallized, or used without further purification, as appropriate.36 Thin layer chromatographic analyses were performed on silica gel-G (Merck) coated plates. Preparative chromatographic columns were packed with silica gel (60-120 mesh) obtained from Merck. Cholesteryl hemisuccinate (6c) was purchased from Sigma. Cholesteryl bromoacetate (6b),13 cholest-5en-3β-tosylate (6a),12 cholest-5-en-3β-oxyethan-2-ol (3a),12 cholest-5-en-3β-oxypent-3-oxa-an-5-ol (3b),12 cholest-5-en-3β-oxyoct-3,6-dioxaan-8ol (3c),12 and 11-bromocholest-5-en-3β-oxyundecane (8e)37 were prepared according to the procedures described in the literature. Synthesis. Cholest-5-en-3β-oxysuccinato-2-oligo(ethylene glycol) (1a-c). Solid 6c (0.5 g, 1.03 mmol) was dissolved in 5 mL of CH2Cl2 and cooled to 0 °C. Oxalyl chloride (1.00 mL, 11.6 mmol) was added and stirred for 10 min at 0 °C and then for 2 h at room temperature. The solvent was removed in vacuo, dry CHCl3 (10 mL) was added, and the solution was cooled to 0 °C. This precooled solution was then added dropwise to a solution of a given oligo(ethylene glycol) (3 equiv) containing triethylamine (Et3N) (0.11 g, 1.09 mmol) and stirred at room temperature for 3 h. The reaction mixture was diluted with CHCl3 (20 mL) shaken sequentially with 2 N HCl (15 mL), water (15 mL), and brine (15 mL) and finally dried over anhydrous Na2SO4. The solvent from the solution was evaporated under reduced pressure and the residue purified by column chromatography over silica gel (60120 mesh) using ethyl acetate/hexane as eluent to give the individual monoesters as transparent gums. The percentage yields isolated after purification and spectral and analytical details of each of 1a-c are given below. 1a: Isolated as a gum, 0.324 g, 55%. IR (CHCl3) (cm-1): 1730, 1160. 1H NMR (CDCl3, 90 MHz; δ): 0.62-2.45 (multiple peaks, 46 H), 2.65 (s, 4 H), 3.5-3.9 (m, 6H), 4.2-4.39 (m, 2H), 4.60 (m, 1H), 5.39 (d, 1H). MS (MALDI-TOF): m/e 614 (M + K), 598 (M + Na). Anal. Calcd for C35H58O6‚H2O: C, 70.91; H, 10.2. Found: C, 71.16; H, 10.21. 1b: Isolated as a gum, 0.336 g, 53%. IR (CHCl3) (cm-1): 1730, 1160. 1H NMR (CDCl3, 90 MHz; δ): 0.62-2.45 (multiple peaks, 46 H), 2.65 (s, 4 H), 3.5-3.9 (m, 10H), 4.2-4.39 (m, 2H), 4.60 (m, 1H), 5.39 (d, 1H). MS (MALDI-TOF): m/e 658 (M + K), 642 (M + Na). The elemental analysis could not be recorded due to the highly sticky nature of 1b. (36) Perrin, D. A.; Armarego, W. L.; Perrin, D. R. Purification of laboratory chemicals, 3rd ed.; Pergamon: New York, 1990. (37) Krishnan-Ghosh, Y.; Gopalan, R. S.; Kulkarni, G. U.; Bhattacharya, S. J. Mol. Struct. 2001, in press.

Langmuir, Vol. 17, No. 7, 2001 2073 1c: Isolated as a gum, 0.340 g, 50%. IR (CHCl3) (cm-1): 1730, 1160. 1H NMR (CDCl3, 90 MHz; δ): 0.62-2.48 (multiple peaks, 46 H), 2.65 (s, 4 H), 3.5-3.9 (m, 14H), 4.2-4.39 (m, 2H), 4.60 (m, 1H), 5.39 (d, 1H). MS (MALDI-TOF): m/e 702 (M + K), 686 (M + Na). Anal. Calcd for C39H66O8: C, 70.66; H, 10.04. Found: C, 71.05; H, 10.33. Cholest-5-en-3β-oxy-(2-N,N,N-trimethylammonium bromide) Acetate (3a). Cholesteryl bromoacetate (6b) (0.5 g, 0.98 mmol) was added to a saturated solution of gaseous Me3N in dry acetone (15 mL) in a screw top pressure tube which was subsequently sealed and heated at 90 °C for 24 h. The white precipitate obtained was filtered out, washed with cold dry acetone (50 mL), and recrystallized 3 times from dry ethyl acetate to give 3a as a white solid. Mp: 236.5 °C (dec) (0.884 g, 80%). IR (CHCl3) (cm-1): 1730. 1H NMR (CDCl , 90 MHz; δ): 0.62-2.45 (multiple peaks, 46 H), 3 3.65 (s, 9H), 4.65 (m, 1H), 4.9 (s, 2H) 5.40 (d, 1H). LRMS: m/e 486 (M - Br). Anal. Calcd for C32H56O2NBr‚H2O: C, 65.73; H, 10.0; N, 2.39. Found: C, 65.97; H, 10.11; N, 1.96. General Procedure for the Synthesis of Cationic Cholesterol Derivatives 3b-d and 5a. A solution of 6b (1 mmol) in dry acetone (20 mL) was refluxed for 12 h with the appropriate N-alkylN,N-dimethylamine (1.1 mmol). The white precipitate so formed in each case was filtered off, washed with dry acetone, and recrystallized several times from dry acetone to give white solid materials which were found to be pure by TLC; the yields ranged 88-95%. All compounds were found to be hygroscopic and existed as hydrates despite prolonged drying under vacuum. The spectroscopic and analytical data for these compounds are given below. Cholesteryl (2-Hydroxyethyl-N,N-dimethylammonium bromide) Acetate (3b). Mp: 194 °C, 0.537 g, 0.90 mmol, 90%. IR (CHCl3) (cm-1): 3650-3100, 1740. 1H NMR (CDCl3, 300 MHz; δ): 0.68-2.36 (multiple peaks, 46H), 3.54 (s, 6H), 3.90 (t, 2H), 4.04 (t, 2H), 4.67 (m, 3H), 5.40 (d, 1H). MALDI-TOF: m/e 518 (M+ - Br). Anal. Calcd for C33H58O3NBr‚H2O: C, 64.47; H, 9.84; N, 2.28. Found: C, 64.16; H, 9.58; N, 2.06. Cholesteryl ((1-Hydroxy-3-oxapentano)-5-N,N-dimethylammonium bromide) Acetate (3c). 3c was obtained as a white solid. Mp: 200 °C, 0.570 g, 0.89 mmol, 89%. IR (CHCl3) (cm-1): 36503100, 1750. 1H NMR (CDCl3, 300 MHz; δ): 0.68-2.36 (multiple peaks, 46H), 3.57-3.7 (multiple peaks, 10H), 4.01 (m, 4H), 4.7 (bs, 3H), 5.40 (d, 1H). MALDI-TOF: m/e 561 (M+ - Br). Anal. Calcd for C35H62O4NBr‚0.25 H2O: C, 65.14; H, 9.76; N, 2.17. Found: C, 65.08; H, 9.99; N, 1.81. Cholesteryl ((1-Hydroxy-3,6,9-oxaundecano)-11-N,N-dimethylammonium bromide) Acetate (3d). 3d was obtained as a white solid. Mp: 190 °C, 0.640 g, 0.88 mmol, 88%. IR (CHCl3) (cm-1): 3700-3100, 1745. 1H NMR (CDCl3, 300 MHz; δ): 0.68-2.36 (multiple peaks, 46H), 3.61-3.67 (m, 18H), 3.77 (t, 2H), 4.01 (t, 2H), 4.07 (t, 2H), 4.71-4.78 (m, 3H), 5.40 (d, 1H). MALDI-TOF: m/e 649 (M+ - Br). Anal. Calcd for C39H70O6NBr‚0.25 H2O: C, 63.87; H, 9.69; N, 1.91. Found: C, 63.83; H, 9.81; N, 1.65. Cholesteryl ((2-N,N-Dimethyl-N-(11-hydroxy-n-undecanyl)ammonium bromide) Acetate (5a). Mp: 185-7 °C; 76%. IR (CHCl3) (cm-1): 3650-3110, 1730. 1H NMR (CDCl3, 90 MHz; δ): 0.65 (s, 3H), 0.86-2.34 (multiple peaks, 58 H), 3.29 (s, 6H), 3.55 (t, 2H), 3.82 (t, 2H), 4.68 (m, 1H), 5.38 (d, 1H). MALDI-TOF: m/e 643 (M+ -Br). Anal. Calcd for C42H76O3NBr: C, 69.77; H, 10.60; N, 1.94. Found: C, 69.40; H, 10.66; N, 1.79. General Procedure for the Synthesis of Nonionic Cholesterol Derivatives 2a-d. To a suspension of cholest-5-ene-3β-tosylate (500 mg, 0.9 mmol) in anhydrous dioxane (9 mL) was added the respective alcohol (1 mL), and the mixture was stirred under reflux for 4 h in an inert atmosphere. The solution was cooled and the solvent removed in vacuo. The white residue was partitioned between CHCl3 (20 mL) and water (20 mL), washed sequentially with saturated NaHCO3 (2 × 10 mL), water (10 mL), and saturated brine (10 mL), and dried over anhydrous Na2SO4, and the solvent was removed in vacuo. The residue was purified by column chromatography on silica gel (60-120 mesh) using ethyl acetate/hexanes. Cholest-5-en-3β-oxyethan-2-ol (2a): white waxy solid, 346 mg, 0.81 mmol, 86%. Mp: 98-99 °C (lit mp12 97-98 °C). IR (CHCl3) (cm-1): 3369, 2930, 2866, 1466, 1380. 1H NMR (CDCl3, 300 MHz; δ): 0.69 (s, 3H), 0.86-1.57 (33H, m), 1.78-2.04 (6H, m), 2.192.22 (2H, m), 2.35-2.37 (1H, m), 3.17-3.21 (1H, m), 3.6(2H, t,

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Langmuir, Vol. 17, No. 7, 2001

J ) 4.5 Hz), 3.73 (2H, t, J ) 4.5 Hz), 5.36 (1H, d, J ) 4.5 Hz). MALDI-TOF: m/e 454 (M+ + Na+). Anal. Calcd for C29H50O2: C, 80.87; H, 11.7. Found: C, 80.48; H, 12.03. Cholest-5-en-3β-oxypent-3-oxa-an-5-ol (2b): white gummy solid, 382 mg, 0.80 mmol, 80%. IR (CHCl3) (cm-1): 3407, 2929, 2861, 1462. 1H NMR (CDCl3, 300 MHz; δ): 0.63 (s, 3H), 0.81-1.55 (33H, m), 1.76-1.99 (6H, m), 2.17-2.21 (2H, m), 2.35-2.37 (1H, m), 3.15-3.28 (2H, m), 3.53-3.69 (8H, m), 5.36 (1H, d, J ) 4.5 Hz). MALDI-TOF: m/e 498 (M+ + Na+). Anal. Calcd for C31H54O3‚ 0.5 H2O: C, 76.96; H, 11.46. Found: C, 77.00; H, 11.54. Cholest-5-en-3β-oxyoct-3,6-oxa-an-8-ol (2c): transparent gummy solid, 431 mg, 0.83 mmol, 90%. IR (CHCl3) (cm-1): 3425, 2932, 2859, 1460. 1H NMR (CDCl3, 300 MHz; δ): 0.63 (s, 3H), 0.811.55 (33H, m), 1.79-2.06 (6H, m), 2.18-2.29 (2H, m), 2.37-2.41 (1H, m), 3.16-3.23 (2H, m), 3.60-3.78 (12H, m), 5.37 (1H, d, J ) 4.5 Hz). LRMS: m/e 517 (M+ - H+). Anal. Calcd for C33H58O4: C, 76.40; H, 11.27. Found: C, 76.89; H, 10.82. Cholest-5-en-3β-oxyundeca-3,6,9-oxa-an-11-ol (2d): transparent gum, 486 mg, 0.86 mmol, 93%. IR (CHCl3) (cm-1): 3425, 2932, 2859, 1460, 1371. 1H NMR (CDCl3, 300 MHz; δ): 0.63 (s, 3H), 0.81-1.55 (33H, m), 1.79-2.06 (6H, m), 2.18-2.29 (2H, m), 2.37-2.41 (1H, m), 3.16-3.23 (2H, m), 3.60-3.78 (16H, m), 5.37 (1H, d, J ) 4.5 Hz). LRMS: m/e 560.9 (M+ - H+). Anal. Calcd for C35H62O5‚0.5 H2O: C, 73.51; H, 11.11. Found: C, 73.3; H, 11.00. General Procedure for the Tosylation of Alcohols (2a-d). The alcohol (200 mg) was taken in dry CHCl3 (10 mL), pyridine (1 mL) was added, and the mixture was cooled to 0 °C. To the cold solution, p-toluenesulfonyl chloride (1.1 equiv) was added and allowed to stir for 3 h at room temperature. The reaction mixture was poured into cold dilute HCl (25 mL of 6 N HCl) and extracted with CHCl3 (2 × 15 mL). The organic layers were dried over Na2SO4 (anhydrous), and solvent was removed in vacuo. Cholest-5-en-3β-oxyethanetosylate (7a): 248 mg, 0.42 mmol, 92%. IR (CHCl3) (cm-1): 3369, 2930, 2865, 1465, 1350, 1180, 1170. 1H NMR (CDCl3, 300 MHz; δ): 0.67 (s, 3H), 0.86-2.27 (m, 41H), 2.45 (s, 1H), 3.10 (m, 1H), 3.65 (t, J ) 4.5 Hz, 2H), 4.15 (t, J ) 4.5 Hz, 2H), 5.31 (d, J ) 4.5 Hz, 1H), 7.32 (d, J ) 8 Hz, 2H), 7.78 (d, J ) 8 Hz, 2H). Cholest-5-en-3β-oxypent-3-oxane-5-tosylate (7b): white waxy solid, 232 mg, 0.37 mmol, 87%. IR (CHCl3) (cm-1): 3370, 2930, 2865, 1465, 1350, 1180, and 1170. 1H NMR (CDCl3, 300 MHz; δ): 0.67 (s, 3H), 0.86-2.38 (m, 41H), 2.45 (s, 1H), 3.11 (m, 1H), 3.46 (s, 4H), 3.68 (t, J ) 4.5 Hz, 2H), 4.12 (t, J ) 4.5 Hz, 2H), 5.28 (d, J ) 4.5 Hz, 1H), 7.31 (d, J ) 8 Hz, 2H), 7.78 (d, J ) 8 Hz, 2H). Cholest-5-en-3β-oxyoct-3,6-oxane-8-tosylate (7c): transparent gummy solid, 250 mg, 0.37 mmol, 96%. IR (CHCl3) (cm-1): 3370, 2930, 2865, 1465, 1350, 1180, and 1170. 1H NMR (CDCl3, 300 MHz; δ): 0.67 (s, 3H), 0.86-2.36 (m, 41H), 2.46 (s, 1H), 3.11 (m, 1H), 3.47 (s, 8H), 3.68 (t, J ) 4.5 Hz, 2H), 4.13 (t, J ) 4.5 Hz, 2H), 5.30 (d, J ) 4.5 Hz, 1H), 7.32 (d, J ) 8 Hz, 2H), 7.78 (d, J ) 8 Hz, 2H). Cholest-5-en-3β-oxyundeca-3,6,9-oxane-11-tosylate (7d): transparent gummy solid, 217 mg, 0.30 mmol, 85%. IR (CHCl3) (cm-1): 3370, 2930, 2865, 1465, 1350, 1180, and 1170. 1H NMR (CDCl3, 300 MHz; δ): 0.67 (s, 3H), 0.86-2.35 (m, 41H), 2.45 (s, 1H), 3.14 (m, 1H), 3.57 (s, 12H), 3.68 (t, J ) 4.5 Hz, 2H), 4.12 (t, J ) 4.5 Hz, 2H), 5.30 (d, J ) 4.5 Hz, 1H), 7.32 (d, J ) 8 Hz, 2H), 7.78 (d, J ) 8 Hz, 2H). General Procedure for Halogenation of 7a-d. The tosylate (7a-d) (100 mg) was taken in dry DMF (5 mL) containing LiBr (1.1 equiv) and stirred under nitrogen atmosphere at 65 °C for 4 h. The reaction mixture was poured into water (25 mL) and extracted with CHCl3 (3 × 15 mL). The organic layers were dried over Na2SO4 (anhydrous) and removed in vacuo. The oily residue was purified by column chromatography over silica gel (60-120 mesh) with hexanes as eluent to give glassy solid melts that were found to be bromides. 2-Bromocholest-5-en-3β-oxyethane (8a): white solid, 80 mg, 0.16 mmol, 95%. IR (CHCl3) (cm-1): 3370, 2930, 2865, 1465, and 1350. 1H NMR (CDCl3, 300 MHz; δ): 0.67 (s, 3H), 0.86-2.38 (m, 41H), 3.22 (m, 1H), 3.45 (t, J ) 4.5 Hz, 2H), 3.78 (t, J ) 4.5 Hz, 2H), 5.35 (d, J ) 4.5 Hz, 1H). LRMS: m/e 494 (M+ + 2), 492 (M+). Anal. Calcd for C29H49OBr: C, 70.56; H, 10.0. Found: C, 70.6; H, 10.18.

Bhattacharya and Krishnan-Ghosh 5-Bromocholest-5-en-3β-oxypent-3-oxane (8b): glassy melt, 82 mg, 0.15 mmol, 96%. IR (CHCl3) (cm-1): 3370, 2930, 2865, 1465, and 1350. 1H NMR (CDCl3, 300 MHz; δ): 0.67 (s, 3H), 0.86-2.35 (m, 41H), 3.15 (m, 1H), 3.43 (t, J ) 4.5 Hz, 2H), 3.65 (t, J ) 4.5 Hz, 4H), 3.76 (t, J ) 4.5 Hz, 2H), 5.28 (d, J ) 4.5 Hz, 1H). LRMS: m/e 538 (M+ + 2), 536 (M+). Anal. Calcd for C31H53O2Br: C, 69.25; H, 9.94. Found: C, 69.92; H, 10.26. 8-Bromocholest-5-en-3β-oxyoct-3,6-oxane (8c): glassy melt, 84 mg, 0.14 mmol, 97%. IR (CHCl3) (cm-1): 3370, 2930, 2865, 1465, and 1350. 1H NMR (CDCl3, 300 MHz; δ): 0.67 (s, 3H), 0.86-2.40 (m, 41H), 3.20 (m, 1H), 3.48 (t, J ) 4.5 Hz, 2H), 3.67 (t, J ) 4.5 Hz, 8H), 3.82 (t, J ) 4.5 Hz, 2H), 5.34 (d, J ) 4.5 Hz, 1H). MALDITOF: m/e 606 (M + 2 + Na), 604 (M + Na). Anal. Calcd for C33H57O3Br: C, 68.13; H, 9.88. Found: C, 68.39; H, 10.02. 11-Bromocholest-5-en-3β-oxyundeca-3,6,9-oxane (8d): transparent gum, 79 mg, 0.13 mmol, 90%. IR (CHCl3) (cm-1): 3370, 2930, 2865, 1465, and 1350. 1H NMR (CDCl3, 300 MHz; δ): 0.67 (s, 3H), 0.86-2.36 (m, 41H), 3.14 (m, 1H), 3.43 (t, J ) 4.5 Hz, 2H), 3.63 (t, J ) 4.5 Hz, 12H), 3.78 (t, J ) 4.5 Hz, 2H), 5.28 (d, J ) 4.5 Hz, 1H). MALDI-TOF: m/e 650 (M + 2 + Na), 648 (M + Na). Anal. Calcd for C35H61O4Br: C, 67.18; H, 9.83. Found: C, 67.13; H, 10.08. 11-Bromocholest-5-en-3β-oxyundecane (8e). Mp: 68-70 °C, 82 mg, 0.13 mmol, 78%. IR (film) (cm-1): 2933, 2860, 1466, 1377, 1093. 1H NMR (CDCl3, 300 MHz; δ): 0.67 (s, 3H), 0.85-1.66 (m, 51H), 1.87-2.33 (8H, m), 3.03-3.11 (1H, m), 3.39 (2H, t, J ) 4.5 Hz), 3.60 (2H, t, J ) 4.5 Hz), 5.28 (1H, d, J ) 4.5 Hz). LRMS: m/e 620 (M+ + 2), 618 (M+). Anal. Calcd for C38H67OBr·0.25H2O: C, 73.1; H, 10.9. Found: C, 72.8; H, 10.98. General Procedure for the Quaternization of 8a-e. The respective bromide (8a-e) (100 mg) was taken in 1:10 dry EtOH/ dry acetone and heated with excess Me3N dissolved in acetone in a screw-top sealed tube for 24 h to yield the cationic cholesterol amphiphiles. These were purified by column chromatography on neutral alumina. Upon recrystallization from dry acetone, analytically pure amphiphiles were obtained as white solids in moderate to high yields. These amphiphiles were found to be extremely hygroscopic in their pure forms, precipitating as hydrates even from dry solvents upon exposure to ambient atmosphere, which could not be removed even upon prolonged drying under high vacuum. Melting points of these amphiphiles could not be recorded due to their hygroscopic nature. Cholest-5-en-3β-oxyethane-N,N,N-trimethylammonium Bromide (4a): white solid, 101 mg, 0.18 mmol, 90%. IR (CHCl3) (cm-1): 3370, 2930, 2865, 1465, and 1350. 1H NMR (CDCl3, 300 MHz; δ): 0.67 (s, 3H), 0.85-2.32 (m, 41H), 3.25 (s, 3H), 3.48 (s, 9H), 3.96 (s, 4H), 5.36 (d, J ) 4.5 Hz, 1H). MALDI-TOF: m/e 473.9 (M+ - Br). Anal. Calcd for C32H58ONBr‚H2O: C, 67.34; H, 10.6; N, 2.45. Found: C, 67.22; H, 10.51; N, 2.24. Cholest-5-en-3β-oxypent-3-oxane-5-N,N,N-trimethylammonium Bromide (4b): white waxy solid, 101 mg, 0.17 mmol, 91%. IR (CHCl3) (cm-1): 3370, 2930, 2865, 1465, and 1350. 1H NMR (CDCl3, 300 MHz; δ:) 0.67 (s, 3H), 0.86-2.32 (m, 41H), 3.15 (m, 1H), 3.49 (s, 9H), 3.63 (s, 4H), 3.99 (s, 4H), 5.35 (d, J ) 4.5 Hz, 1H). MALDI-TOF: m/e 517.3 (M+ - Br). Anal. Calcd for C34H62O2NBr‚2.5 H2O: C, 63.63; H, 10.52; N, 2.18. Found: C, 63.43; H, 10.69; N, 2.18. Cholest-5-en-3β-oxy-oct-3,6-oxane-8-N,N,N-trimethylammonium Bromide (4c): white gummy solid, 88 mg, 0.14 mmol, 80%. IR (CHCl3) (cm-1): 3370, 2930, 2865, 1465, and 1350. 1H NMR (CDCl3, 300 MHz; δ): 0.67 (s, 3H), 0.85-2.37 (m, 41H), 3.16 (m, 1H), 3.48 (s, 9H), 3.60 (s, 4H), 3.66 (m, 4H), 3.99 (s, 4H), 5.34 (d, J ) 4.5 Hz, 1H). MALDI-TOF: m/e 560.7 (M+ - Br). Anal. Calcd for C36H66O3NBr‚2H2O: C, 63.88; H, 10.43; N, 2.07. Found: C, 63.77; H, 10.39; N, 1.74. Cholest-5-en-3β-oxyundeca-3,6,9-oxane-11-N,N,N-trimethylammonium Bromide (4d): white gum, 82 mg, 0.12 mmol, 75%. IR (CHCl3) (cm-1): 3370, 2930, 2865, 1465, and 1350. 1H NMR (CDCl3, 300 MHz; δ): 0.67 (s, 3H), 0.85-2.38 (m, 41H), 3.18 (m, 1H), 3.47 (s, 9H), 3.61-3.69 (m, 8H), 3.98 (s, 4H), 5.34 (d, J ) 4.5 Hz, 1H). MALDI-TOF: m/e 605.1 (M+ - Br). Anal. Calcd for C38H70O4NBr‚2.5 H2O: C, 62.53; H, 10.36; N, 1.92. Found: C, 62.53; H, 10.33; N, 1.67. Cholest-5-en-3β-oxyundecane-11-N,N,N-trimethylammonium Bromide (5b): white gum, 94 mg, 0.12 mmol, 59%. IR (CHCl3) (cm-1): 3230, 2920, 2860, 1455, and 1350. 1H NMR (CDCl3, 300

Oligo(oxyethylene)-Bearing Cholesteryl Amphiphiles MHz; δ): 0.67 (s, 3H), 0.83-1.5 (m, 51H), 1.75-1.96 (m, 6H), 3.03 (m, 1H), 3.38 (t, 3H), 3.47 (s, 23H), 3.62 (t, 3H), 5.28 (d, 1H). MALDI-TOF: m/e 599 (M+ - Br). Anal. Calcd for C41H76ONBr· 2.5H2O: C, 68.01; H, 11.28; N, 1.94. Found: C, 67.73; H, 10.98; N, 1.78. Sample Preparation. A given amount of the amphiphile was dissolved in CHCl3 (0.5 mL) and then dried under a stream of N2 to yield a thin film of the amphiphile. The resulting film was further dried by keeping under high vacuum for another 1.5 h. After this, the requisite amount of water (Millipore) (pH ) 6.8) was added and left for hydration for 30 min. This was followed by vortex mixing for 10 min and bath sonication at >60 °C for 10 min. Dye Entrapment Studies. Films of a given amphiphile were prepared as described above, and 10 mL of 0.1 mM methylene blue (MB) (λmax ) 665 nm) was added such that amphiphile concentration was 5 mM. A 2-mL aliquot of the resulting suspension was loaded on to a column packed with preequilibrated Sephadex G-50. Gel filtration was performed using water as the eluent until elution of free dye was complete. Neat TritonX-100 was added to aliquots of fractions to give a 1 wt % concentration of Triton X 100, and the vesicles were lysed by bath sonication of the resulting solution for 2 min at room temperature. The absorbances at 665 nm for all the fractions containing lysed solutions was determined and plotted against the elution volume. Transmission Electron Microscopy. Vesicles were made by adding a known amphiphile from stock solutions in CHCl3, evaporating the organic solvent to form a film, and samples were prepared as mentioned above in water (Millipore) at pH ∼ 6.8. The concentration of the amphiphile was maintained at 4 mM for all samples. A 15 µL volume of the vesicular solution was loaded onto Formvar-coated, 400 mesh copper grids, allowing them to remain for 1 min. Excess fluid was wicked off the grids by touching their edges to filter paper, and 15 µL of 2% uranyl acetate was applied on the same grid for a after which the excess stain was similarly wicked off. The grid was air-dried for 10 min, and the specimens were observed under a bright-field TEM (JEOL 100 CX II) operating at an acceleration voltage of 80 kV. Fluorescence Depolarization Measurements. A solution of compound in CHCl3 and 1,6-diphenyl hexatriene (DPH) was made and evaporated to form a film. It was hydrated for 30 min with 1 mL of double distilled water (pH ) 6.8). The resulting concentration of the compound was 0.1 mM, and that of DPH was 1 µM. The solution was bath sonicated for 10 min at 60 °C to give a vesicular suspension which was excited at 360 nm, and the emission followed at 430 nm on a Hitachi model F-4500 spectrofluorometer. At each temperature the fluorescence emission spectra were recorded by adjusting the polarizers at 4 different positions. r values at different temperatures for the vesicular solutions were calculated using Perrin’s equation: r ) (I| - I⊥G)/(I| + 2I⊥G) where I| and I⊥ are the observed intensities measured with polarizers parallel and perpendicular to the vertically polarized exciting beam, respectively. G is the factor used to correct for inability of the instrument to transmit

Langmuir, Vol. 17, No. 7, 2001 2075 differently polarized light equally. Individual r vs T plots for each aggregate gave the information about its order-disorder transition as well as the rigidity as a function of temperature. Differential Scanning Calorimetry. Films of a given amphiphile were prepared as described in Sample Preparation. A 2 mL volume of Millipore water was added to yield a final amphiphile concentration of 1 mM. Vesicles were prepared by freezing to 0 °C for 15 min followed by thawing to 70 °C for 15 min followed by vortexing for 5 min. Samples were subjected to seven such freeze-thaw cycles. A 0.5 mL volume of these vesicular dispersions was loaded on a differential scanning calorimeter (Calorimetric Sciences Corp.) and scanned from 20 to 75 °C at a scan rate of 30 K/h. X-ray Diffraction Studies. Self-supported cast films for the XRD studies were prepared by dispersing the films of amphiphiles (0.5 mg /mL) of water as described previously.38 A 1 mL volume of this suspension was placed on a precleaned glass plate and air-dried at room temperature. Reflection XRD studies were carried out using an X-ray diffractometer (model XDS 2000, Scintag Inc.). The X-ray beam, generated with a Cu anode at the wavelength of KRl beam 1.540 598 Å, was directed toward the film edge, and scanning was done up to the 2θ value of 10°. Molecular Modeling Studies. The modeling studies were conducted with BIOSYM software running on a Silicon Graphics Indigo workstation. The atomic coordinates of the carbon and hydrogen atoms for the cholesteryl backbone have been extracted from the fractional atomic coordinates of cholesteryl laurate39 as has been done with previous theoretical treatments of cholesterol interaction in membrane bilayers.40 The molecules 1-5 were drawn in INSIGHT II using standard bond lengths, angles, and dihedral angles. The atoms within each molecule were assigned their proper hybridization, charge, and bond order by utilizing the Builder module of INSIGHT (version 2.3.5). The CVFF force field provided by the Discover module was chosen for minimization constraints. This force field was applied to the constructed derivative and evaluated with the conjugate gradient method. The interaction number for the conjugate gradient method was 200. The derivative (or convergence criterion) was chosen as 0.001 kcal mol-1. Each molecule was minimized first using the steepest gradient method (2000 iterations) followed by the conjugate gradient method (5000 iterations) at 300 K with a time interval of 1.0 fs.

Acknowledgment. This work was supported in the form of a Swarnajayanti Fellowship Grant of the Department of Science and Technology, Government of India, awarded to S.B. LA000498I (38) Kimizuka, N.; Kawasaki, T.; Kunitake, T. J. Am. Chem. Soc. 1993, 115, 4387. (39) Sawsik, P.; Craven, B. M. Acta Crystallog., Sect. B 1980, B36, 3027. (40) Dufourc, E. J.; Parish, E. J.; Chitrakorn, S.; Smith, I. C. P. Biochemistry 1984, 23, 6062.