Vesicle Formation from Oligo(oxyethylene)-BearingCholesteryl 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, nonionicoligo(ethylene glycol) appendages were covalently introduced either indirectly via a succinate spacer ordirectly respectively to the 3â-OH group of cholesterol. A third group of derivatives was prepared wherethe location of the cationic charge was conserved although the headgroup residue was progressively modifiedwith increasing lengths of oligo(oxyethylene) units. In the fourth group, a cationic center was introducedonto the steroid backbone via increasing lengths of oligo(oxyethylene) spacers. In the fifth group, thepoly(oxyethylene) segments of one amphiphile each of series 3 and 4 were replaced by the same lengthof polymethylene chain. Vesicle formation from the aqueous suspensions of these compounds was confirmedby TEM and dye entrapment. Fluorescence anisotropy and XRD studies revealed remarkable control ofmembrane characteristics by both the length and location of the oxyethylene segment.

Introduction

Synthetic cationic cholesterol derivatives are currentlythe focus of attention of many workers. This is becausesome of this class of compounds are used as drugs and arenow being employed for diverse purposes such as genetherapy,1-4 enzyme inhibition,5 membrane spanningconductors,6 or in medicinal applications.7 Moreover,aggregates composed of only cationic cholesterol deriva-tives have been shown to bring about DNA transfectionin cells with greater efficiency than commercial trans-fection formulations comprising glycerol-based cationicamphiphiles.8,9 The interest in the utilization of cationiccholesterol derivatives as cytofectins over traditionalcationic glycerol-based amphiphiles stems from the factthat they are normally employed as a mixture with a helperlipid such as DOPE (dioleoyl phosphatidyl ethanolamine)which limits shelf life as, over a period of time, theseformulations tend to phase separate.10 It has also beenshown that the efficiency of DNA transfection mediatedby this class of molecules is highly dependent on thestructure of the cholesterol monomer9,11 although not much

is known how exactly these cationic cholesterol derivativesbring about DNA transfection. However, there is not asingle report in the literature that addresses the relation-ship between the molecular structure of a given cholesteryllipid with the properties manifested upon its aggregationin aqueous media. This paper present the results of thefirst such investigation involving aggregate properties ofa series of cationic cholesterol derivatives whose molecularstructures have been systematically varied.

Cholesterol is weakly amphipathic since its hydroxylgroup is polar and the steroid ring system along with itsisopentyl tail at C-17 is nonpolar. However, the affinityof its OH group for water is much less than the affinityof an ionic headgroup of a typical lipid molecule. Hencecholesterol alone cannot aggregate in water to formmembranes or related aggregates. Since cholesterol pos-sesses a long, rigid hydrophobic segment, covalent at-tachment of a charged residue to the steroid backboneshould result in molecules with amphiphilic properties,which on suspension in water should form thermally stablemembranes. Indeed examples are known where polarderivatives of cholesterol have been shown to form vesiclesor related aggregates.12-16

Herein we report the synthesis of five families of cationicand nonionic cholesterol-based lipids (cholamphiphiles)1-5 (Chart 1), containing oxyethylene units. In view ofthe fact that there is an intimate relation betweenheadgroup hydration and transfection efficiency of lipo-some-based transfection reagents,17-19 we chose oligo-(oxyethylene) residues as elements of hydration modu-lation at the headgroup level. The newly synthesized

* To whom correspondence should be addressed. Fax: +91-80-360-0529. E-mail: sb@orgchem.iisc.ernet.in. Also affiliated withthe 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.

(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.

2067Langmuir 2001, 17, 2067-2075

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

cholamphiphiles form closed vesicular membranes upondispersion in water as evidenced by electron microscopyand dye entrapment studies. Unlike membranes fromconventional fatty acid based lipids, the presently de-scribed systems do not show any thermal solid-to-fluidphase transitions. Membrane-level properties such asrigidity and bilayer thickness were found to depend onthe length and position of oligo(oxyethylene) units in thelipid monomer. We explain such a significant influence ofthe headgroup on the membrane properties in thesecholamphiphiles on the basis of temperature-dependentfluorescence anisotropy measurements and X-ray diffrac-tion (XRD) methods.

Results and Discussion

Lipid Molecular Structures. The linker region be-tween the cationic headgroup and the steroid backboneplays an important role in mediating gene transferefficiency.9 Accordingly we have first synthesized twofamilies of cationic cholamphiphiles, one where the linkerregion is an ester moiety and the other where there is anether linkage. Correspondingly, in the first group, nonionicoligo(oxyethylene) appendages were introduced to the 3â-OH group of cholesterol via covalent connection througha succinate spacer as in 1a-c. In contrast, oligo(oxyeth-ylene) units are directly attached to 3â-site in cholesterolvia an ether linkage to afford 2b-d. In the third group,i.e., in cholesteryl derivatives, 3a-d, the cationic head-group is linked to the 3â-cholesterol via an ester connec-tion. Here the positive charge is located at a fixed distancefrom the cholesteryl backbone although the headgrouphydration is continually modified with the progressiveincrease in the number of oxyethylene units on the NMe2

+

center. To see the difference in properties if any dependingon 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 derivativesconnects the cationic NMe3

+ groups via an oligo(oxyeth-ylene) spacer chain. The spacer connects itself via an etherlink to the 3â-OH of cholesterol. The cationic NMe3

+ groupis therefore placed at incrementally greater distance fromthe cholesteryl backbone with the insertion of increasingnumber of oxyethylene units. In the fifth group, 5a,b, thecorresponding tetraoxyethylene moieties in compounds3d and 4d were replaced with a polymethylene chain ofthe same length. This was done to verify whether theobserved membrane properties of 3a-d and 4a-d arelargely due to the presence of the poly(oxyethylene)segment.

Synthesis. Cholesterol possesses a long, hydrophobicregion. Easy amenability of the 3â-OH group to chemicalmodification into polar moieties allows a convenientmethod of generating novel lipids that could act ascytofectins. We have synthesized the first series of

cholesterol derivatives by converting cholesteryl hemisuc-cinate 6c into the corresponding acid chloride with oxalylchloride and then treating with the appropriate oligo-(ethylene glycol) in slight excess to give the correspondingnonionic amphiphiles, 1a-c (Scheme 1). Cholesteryltosylate, 6a, upon refluxing in dry dioxane with theappropriate oligoethylene glycol yielded 2a-d in goodyields12 (Scheme 2). Cholesteryl bromoacetate, 6b, wasquaternized with the appropriate tertiary amine to yield3a-d and 5a also in good yields (Scheme 2). 2a-d werethen converted to the respective tosylates, 7a-d, whichupon heating to ∼65 °C with 1 equiv of LiBr in dry DMFfurnished the corresponding bromides, 8a-d, in excellentyields. Quaternization of the bromides with the appropri-ate tertiary amine in a 1:1 mixture of dry acetone/EtOHyielded the amphiphiles 4a-d and 5b in moderate to goodisolated yields (Scheme 2). The intermediates and the finalproducts were appropriately characterized by IR, NMR,mass spectrometry, and elemental analysis; cf. Experi-mental Section.

Prediction of Aggregate Morphology. To predictthe type of aggregation expected from an amphiphile,empirical models have been proposed on the basis of therelative sizes of headgroup and hydrophobic segments.20-22

Although the validity of such models has been ques-tioned,23 it is often desirable to draw a relationship betweenthe amphiphile molecular structure and the type ofaggregates 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 As-

semblies: The Synkinetic Approach; The Royal Society of Chemistry:Cambridge, U.K., 1994; p 28.

Chart 1 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

a Reagents, reaction conditions, and yields: (i) TsCl, drypyridine, 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) drydioxane, Br (CH2)11OH, reflux, N2, 4 h (78%).

2068 Langmuir, Vol. 17, No. 7, 2001 Bhattacharya and Krishnan-Ghosh

to this model, if a dimensionless quantity called thepacking parameter, p, for an amphiphile falls within therange 0.5-1, then vesicle formation is anticipated. Thepacking parameter, p, is given by the relation p ) V/LA,where V is the volume of the hydrophobic segments, L isthe critical chain length which is roughly the length of thefully extended hydrocarbon chain, and A is the area ofcross section of the headgroup. Data summarizing thetheoretical and experimental parameters for a few rep-resentative amphiphiles in this study are presented inTable 1. For 1-4, the volume (V) of the hydrophobic portion(steroid) was considered to be 735 Å3 assuming the steroidto be a rectangular box of dimensions 5 × 7 × 21 Å.13

Surface pressure-area isotherms are known to provideexperimentally determined information pertaining to theheadgroup area of a given amphiphile with reasonableaccuracy. The “measured” values (m) of headgroup areaswere obtained from previously published literature onsurface pressure area isotherms of compounds bearingsimilar headgroups. The experimental values of area ofcross section (Ae) and theoretical areas of cross section(Ac) of the headgroups of the respective poly(ethyleneglycol) segments in 1a-c and 2b-d could be obtainedfrom surface tension measurements.24 For the -NMe3

+-bearing headgroups, 3a and 4a, Ae and Ac were takenfrom the literature where both Ae and Ac are in goodagreement (64 Å2).22 In these instances, Pc and Pe (0.5 eP e 1.0) predict vesicle formation by these amphiphiles.

TransmissionElectronMicroscopy.Bath sonicationfor ca. 10-15 min. at ∼ 60 °C yielded slightly translucentaqueous suspensions from these cholamphiphiles. Todiscern the nature of the aggregates obtained from variousamphiphiles, the respective aqueous suspensions wereexamined with the aid of negative-stain transmissionelectron microscopy as described in the ExperimentalSection. TEM examination of the individual, air-driedvesicular suspensions of 1-5 layered on carbon-Formvar-coated copper grids revealed the existence of closedaggregate structures in all cases. Few representativemicrographs are shown in Figure 1. All cholesterylderivatives formed predominantly unilamellar and nearlyspherical vesicles except 2b-d, 4a-d, and 5b thatgenerated mostly multiwalled vesicles under identicalconditions of aggregate preparation.

Dye Entrapment in Vesicles. Having confirmedlamellar aggregate formation from these newly developedcholamphiphiles, dye entrapment was employed to ex-amine whether these aggregates comprised closed inneraqueous compartments. Micellar or open lamellar ag-gregates do not have the ability to entrap hydrophilic orionic molecules such as water-soluble dyes. Since thesurfaces of the aggregates of 1-5 are either nonionic or

positively charged, the choice of dye molecule for theentrapment studies becomes important. We chose acationic dye, methylene blue (MB), for this purpose inorder to avoid the electrostatic association of the dyemolecules with the vesicle surface. This dye (MB) has beenpreviously used to show entrapment capacities of theaggregates made from steroidal azacrown derivatives byGokel et al.13 To examine the entrapment abilities of theaggregates 1-5, we generated vesicles from 1-5 bysonication of a film of the amphiphile to give a finalconcentration of 5 mM in water containing 0.1 mM MBfor 10 min at 60 °C. Upon elution with water, separatefractions (of 1 mL each) were collected. To reduce thebackgroundscatter fromtheseopalescent fractions,Triton-X-100 was introduced into each fraction as described inthe Experimental Section. A gel filtration profile wasobtained upon plotting the absorbance at 665 nm of thefractions obtained from the gel filtration column. A typicalprofile is shown in Figure 2. In all the cases it was observedthat there was a small initial portion containing vesiclesentrapping barely 0.5-5% of total dye followed by a largepeak which was the free, untrapped dye. It is clear thatthe MB molecules associated with vesicles could bedistinctly separated from the free dye. The percentagesof dye entrapped inside vesicles ranged from ∼1.0 to 10L/mol of total dye as given in the Table 2.

Fluorescence Anisotropy Measurements. To probethe thermal response of these aggregates we then mea-sured the fluorescence anisotropy as a function of tem-perature. This technique has been used extensively forprobing the fluidity of the vesicle bilayer and for measuring

(24) Sokolowski, A.; Burczyk, B. J. Colloid Interface Sci. 1983, 94,369.

Table 1. Packing Parameters for the Amphiphiles Using Theoretical Modelsa

entry compdVc

a

(Å3)L

(Å)Ae

(Å2)Pe

(V/LAe)Ac

(Å2)Pc

(V/LAc)

1 1a 735 23b 37 0.86 39 0.822 1b 735 23b 39 0.82 44 0.733 1c 735 23b 63 0.51 49 0.654 2b 735 21 37 0.95 39 0.905 2c 735 21 39 0.90 44 0.806 2d 735 21 63 0.56 49 0.717 3a 735 21 64 0.55 64 0.558 4a 735 21 64 0.55 64 0.55

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

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

Oligo(oxyethylene)-Bearing Cholesteryl Amphiphiles Langmuir, Vol. 17, No. 7, 2001 2069

the melting temperature (Tm) associated with the gel toliquid crystalline phase transition associated with mem-branes.25,26 DPH (1,6-diphenylhexa-1,3,5-E,E,E-triene)was used as the fluorescent probe which is known tointercalate between the alkyl chains in the hydrophobicinterior of the bilayer. Hence it is reasonable to assumethat DPH partitions into the hydrophobic interior in theaggregates formed by these amphiphiles. The relationshipof steady-state anisotropy (r) due to doped DPH in a givenmembrane versus temperature is indicative of the Tm ofa membrane as well as provides a reasonable measure ofthe membrane order. A higher value of r indicates lesserfluidity due to the restricted freedom of movementexperienced by DPH in the bilayer especially in their solid-like gel states.

However, in the cases of amphiphiles 1-5, irrespectiveof whether the headgroups were nonionic or cationic, aclassical “melting” temperature profile was not obtained(Figure 3). Instead there was a gradual monotonic decreaseof r value with the increase in temperature after whicha plateau was reached. This is not surprising as a majorcontribution to the gel-to-liquid crystalline phase transi-tion in membranes composed of conventional fatty acidbased lipid molecules is due to a sudden, catastrophicincrease in chain motion beyond Tm. In membranesgenerated from cholamphiphiles, the steroid backboneconsists of fused rings, which render the entire backbone

immobile. Even on increase of the temperature, the steroidbackbone being so conformationally rigid, there is no starkdrop in the membrane rigidity at a particular temperature(phase transition) which is one of the most generalcharacteristics of fatty acid based lipid membranes.

It is evident from Table 3 that all the nonionicderivatives 1a-c and 2b-d possess consistently higherr values as compared to the corresponding chargedderivatives 3-5. This is probably due to the repulsionfaced by the charged cationic monomers at the headgrouplevel which is absent in the case of nonionic derivatives.Cationic derivatives 3a-d where the PEG segments areattached to the cholesterol backbone through a -NMe2

+

center show a steady decrease in r value at 20 °C as then value increases (Figure 4A). Since the PEG segment islocated beyond the cationic center, it is reasonable toassume that, due to its propensity to get hydrated, itsweeps out a volume proportional to its length at theheadgroup level. Consequently, the attachment of a PEGsegment to a cationic center in these systems seems toresult in the formation a large headgroup which limitsthe lateral distance of closest approach of the neighboringmonomers.27 A headgroup with greater bulk leads to a

(25) Shinitzky, M.; Barenholz, Y. Biochim. Biophys. Acta 1978, 515,367.

(26) Andrich, M. P.; Vanderkooi, J. M. Biochemistry 1976, 15, 1257.

Table 2. Transmission Electron Microscopy, Dye Entrapment, and X-ray Diffraction Data for Cholesteryl Amphiphiles1-5

aggregate layer widthsc [Å (%)]entry compd

size ofaggregatea (nm)

entrapmentcapacityb (%) calcdd obsde

1 1a 170-330 0.8 42.0 46.5 (100)2 1b 50-270 0.9 42.0 47.0 (100)3 1c 440 1.4 42.0 49.7 (100)4 2b 85-240 1.4 46.0 42.1 (100)5 2c 100-300 1.5 46.0 46.5 (100)6 2d 120-500 2.0 46.0 47.9 (100)7 3a 50-400 1.2 42.0 42.0 (100)8 3b 130-300 0.5 42.0 40 (36), 34 (64)9 3c 130-300 1.6 42.0 46 (58), 28 (14), 25 (28)

10 3d 160-200 4.5 42.0 28 (82), 25 (18)11 4a 40-135 2.7 42.0 36.6 (100)12 4b 40-120 1.6 49.0 51.9 (100)13 4c 25-132 3.8 56.0 55.2 (100)14 4d 75-300 1.6 63.0 57.9 (100)15 5a 30-80 1.1 42.0 55.1 (100)16 5b 180-390 2.6 72.0 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 oftwo 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 4bin methylene blue (MB). [4b] ) 5.0 mM; [MB] ) 0.1 mM. Theabsorbance at 665 nm was checked for all fractions.

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

2070 Langmuir, Vol. 17, No. 7, 2001 Bhattacharya and Krishnan-Ghosh

greater intermonomer separation which in turn leads to“looser” membrane packing.

In the cationic derivatives 4a-d where the quaternaryammonium center is attached to the cholesteryl backbonethrough varying lengths of PEG segments, the anisotropyvalues at 20 °C increases as n value increases (Figure4A). This reversal in trend may be due to the dual natureof the PEG segment as well as its specific location in thebilayer membranes of these systems. The PEG segmentis known to be special for the reason that it can take onboth hydrophilic and hydrophobic character.28,29 Whenlocated between the hydrophobic portion and cationiccenter, the PEG segment probably gets inserted into thebilayer while facilitating water-promoted hydrogen bond-

ing at the interfacial region. The longer the PEG segment,the greater is its ability to remain embedded in the bilayerand allow the monomers to position their positive chargesaway from each other. This results in a closer approachof the adjacent monomers resulting in higher r value forgreater n value. At 20 °C, 5b, which possesses a polym-ethylene 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 hydro-phobic portion and the cationic center, the PEG segmenttakes on hydrophobic character and behaves akin to apolymethylene spacer.

Differential Scanning Calorimetry. To examine thenature of the phase of the bilayers formed by theseamphiphiles, DSC studies were carried out. Thermogramsof 1 mM dispersions of all of the amphiphiles 1-5 showedflat traces in the temperature interval of 20-75 °C anddid not show any evidence of a thermal transition (notshown). This is in accordance with the fluorescenceanisotropy data and reconfirms the conclusions presentedin the preceding section.

X-ray Diffraction Studies. To figure out the orienta-tion of the monomers in the bilayers and to know abouttheir lamellar packing, X-ray diffraction studies wereperformed on supported cast films of 1-5. Reflections upto 10° were analyzed and interpreted in terms of higherorder reflections of stacked bilayer structures (Table 2).A series of reflections was obtained for the aggregates of1-5, the highest intensity peak being the long spacing(corresponds to the lowest 2θ value) as shown in Figure5. Molecular modeling35 allowed computation of thetheoretical length of amphiphile monomers. This calcula-tion for two molecules of 3a oriented parallel to the bilayernormal showed a theoretical bilayer width ∼43 Å (Table2). The diffraction patterns of 1a-c showed membranethicknesses of 46.5, 47.1, and 49.7 Å, respectively, cor-responding to 2θ values of ∼1.7-1.9 on the basis of higherorder reflections. The above measured values suggesthydrated bilayer organization for both 1a-c and 2b-d.These cast films, however, showed no evidence of lipidpolymorphism.

In contrast the aggregates from 3b-d showed twodifferent kinds of packing arrangements in their ag-gregates. Two series of reflections were observed. 3bshowed a weaker reflection corresponding to the longspacing value of 40.1 Å and a stronger reflection corre-sponding to the long spacing value 33.9 Å. 3c,d showedreflections corresponding to ∼28 and 25 Å (Figure 5B).However, 3c showed stronger reflection at 25 Å while 3dshowed a stronger reflection at 28 Å. It is noteworthy thatamong the amphiphiles 3a-d the lengthening of the PEGsegment, apart from inducing lipid polymorphism, pro-gressively shrinks the bilayer width (Figure 4B). In thisseries of amphiphiles 3a-d, the whole of the PEG segmentmost probably form part of the headgroup. The reason forthis “shrinkage” may be due to the following. Althoughthe charge remains constant, with the increase of the PEGsegment, the volume swept out by the headgroup increases(Figure 6B,C). This limits the closer approach of the

(27) Ringsdorf, H.; Laschewsky, A.; Elbert, R. J. Am. Chem. Soc.1985, 107, 4134.

(28) See: Gokel, G. W.; Murillo, O. In Comprehensive SupramolecularChemistry; 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.

(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 PhysicalChemistry of Lipids from Alkanes to Phospholipids. Handbook of LipidResearch; 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.

Table 3. Fluorescence Anisotropy Values As MeasuredUsing DPH as a Probe

entry compda rb(20 °C) η(20 °C) r(62 °C) η(62 °C)

1 1a 0.264 42.9 0.203 22.82 1b 0.268 44.9 0.215 26.23 1c 0.260 41.1 0.213 25.22 2b 0.281 52.2 0.260 41.13 2c 0.279 51.0 0.253 38.14 2d 0.274 48.1 0.256 39.35 3a 0.268 44.9 0.203 22.86 3b 0.255 38.9 0.213 25.27 3c 0.247 35.7 0.213 25.28 3d 0.232 30.6 0.179 17.99 4a 0.221 27.3 0.184 18.8

10 4b 0.244 34.6 0.194 20.811 4c 0.252 37.7 0.183 18.712 4d 0.264 42.9 0.181 18.314 5a 0.254 39.3 0.215 26.215 5b 0.214 25.5 0.147 12.9a [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 series3a-d (open squares) and 4a-d (closed circles).

Oligo(oxyethylene)-Bearing Cholesteryl Amphiphiles Langmuir, Vol. 17, No. 7, 2001 2071

monomers leading to greater intermonomer separationin the bilayer which induces interdigitation of the C-17cholesteryl side chains of outer and inner leaflets of thebilayer to fill the “voids” and provide stabilization.

In 5a, the replacement of the PEG segment by apolymethylene segment produces a bilayer of 55 Å width.Uncharged hydrocarbon chain appended derivatives ofcholesterol arrange themselves in the solid phase suchthat the steroid backbone and hydrocarbon chains are notin the same layer. This is due to the length mismatch

between the steroid backbone and the hydrocarbon chain.30

The crystal structure of cholesteryl oleate31 shows packingsimilar to triglycerides.32 However, for the correspondingcharged derivative 5a in an aqueous environment, thehydrophobic polymethylene spacer loops into the bilayermembrane (Figure 6D) in order to avoid unfavorablecontact with water unlike its oxyethylene analogue 3d.This would result in a packing where hydrocarbon chainsand the steroid skeleton are arranged in the same layerwhich has also been observed in derivatives such ascholesteryl laurate.33 This chain length fits well with thelength of the cholesteryl moiety,34 and 5a incorporates anidentical chain length at the quaternary ammoniumcenter. Thus in 5a the headgroup now reduces to a -NMe2

+

group. In 3a-d we see that the increase in length of PEGsegment at the cationic center in this location increasesthe bulkiness of the headgroup which induces greaterinterdigitation due to looser packing leading to smallerbilayer widths.

However, when the -NMe3+ center was attached at the

end of the PEG segment as in 4a-d, the trend observedin their bilayer widths was reversed (Figure 4B). Thismay be due to the fact that, with increasing PEG lengths,a portion of the segment gets inserted into the bilayer asshown in Figure 6F,G. The addition of a single oxyethyleneunit to 4a results in a 15 Å increase in bilayer width. Thisis reflective of the extent of flexibility conferred on themonomer by just one oxyethylene unit. This conforma-tional flexibility allows the positioning of the charge faraway so that with the charge repulsion minimized,monomers may pack closer to each other. Due to the lackof flexibility of the spacer between the steroid and the-NMe3

+ in 4a, the monomers are unable to position theircharged centers far enough from each other to minimizerepulsion. To circumvent such an unfavorable scenariomost probably an interdigitation takes place as indicatedfrom the experimentally obtained bilayer width of 36 Å(Figure 6A). This reasoning is strengthened by the factthat 5b and 4d show longer bilayer widths indicating theability of the intervening spacer to position the chargesof adjacent monomers favorably. Thus it is evident thatPEG segment in this location now behaves equivalent toa polymethylene spacer as the bilayer widths of 5b and4d are quite similar.

Conclusions

In the present study we report the synthesis of 16 newcholesteryl amphiphiles. We also present their membraneforming properties. All the amphiphiles form stable, closedvesicles as evidenced by TEM and dye entrapment. Thesemembranes show a departure from the behavior ofconventional bilayer forming fatty acid based lipids inthat the former do not exhibit a solid-fluid meltingtransition.

The bilayer thickness in these membranes appears tobe 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 hy-drophobic character.28,29 In the series 3a-d, the PEGsegment is located outside the hydrophobic portion of themembrane. It takes on hydrophilic character and remainsin a random orientation thereby increasing headgroupbulk. Increase in bulkiness of the headgroup increasesintermonomer separation, reflected in the r values, whichinduces interdigitation as supported by XRD data. In the

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

Figure 6. Schematic representation of possible aggregate layerpacking 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 regionswept 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.

2072 Langmuir, Vol. 17, No. 7, 2001 Bhattacharya and Krishnan-Ghosh

series 4a-d, the -(CH2CH2O)n- unit is located betweenthe cholesteryl backbone and the -N+Me3 headgroup.Here it acts like a hydrophobic spacer as an increase inthe n value in 4 results in an increase in the length of thebilayer as evidenced from the XRD data.

Thus the present study demonstrates that the intro-duction of an oligo(oxyethylene) unit into cholesterol-basedamphiphiles can bring about ramifications at the mem-brane level far exceeding the seemingly trivial structuralmodification at the molecular level. The findings describedherein illustrate a first step toward the generation ofthermallystableorganizedassemblies of controllableorderand thickness.

Experimental SectionGeneral Methods. Melting points were recorded in open

capillaries and are uncorrected. 1H NMR spectra were recordedin a JEOL-JANL-LA-300 NMR spectrometers. Chemical shiftsare reported in ppm downfield from the internal standard,tetramethylsilane. IR spectra were recorded in a Perkin-Elmermodel 781 spectrometer and are reported in wavenumbers (cm-1).Microanalyses were performed on a Carlo Erba elementalanalyzer model 1106. Steam distilled water was used for allphysical measurements, and pH measurements were made witha Schott Gerate Digital laboratory pH meter CG 825. UV-visspectra were recorded on a Shimadzu model 2100 UV-visrecording spectrophotometer equipped with a TCC-60 temper-ature controller.

Materials. All reagents, solvents, and starting materials wereobtained from the best commercial sources and were distilled,recrystallized, or used without further purification, as appropri-ate.36 Thin layer chromatographic analyses were performed onsilica gel-G (Merck) coated plates. Preparative chromatographiccolumns were packed with silica gel (60-120 mesh) obtainedfrom Merck. Cholesteryl hemisuccinate (6c) was purchased fromSigma. Cholesteryl bromoacetate (6b),13 cholest-5en-3â-tosylate(6a),12 cholest-5-en-3â-oxyethan-2-ol (3a),12 cholest-5-en-3â-oxy-pent-3-oxa-an-5-ol (3b),12 cholest-5-en-3â-oxyoct-3,6-dioxaan-8-ol (3c),12 and 11-bromocholest-5-en-3â-oxyundecane (8e)37 wereprepared according to the procedures described in the literature.

Synthesis. Cholest-5-en-3â-oxysuccinato-2-oligo(ethylene gly-col) (1a-c). Solid 6c (0.5 g, 1.03 mmol) was dissolved in 5 mLof CH2Cl2 and cooled to 0 °C. Oxalyl chloride (1.00 mL, 11.6mmol) was added and stirred for 10 min at 0 °C and then for 2h at room temperature. The solvent was removed in vacuo, dryCHCl3 (10 mL) was added, and the solution was cooled to 0 °C.This precooled solution was then added dropwise to a solutionof a given oligo(ethylene glycol) (3 equiv) containing triethylamine(Et3N) (0.11 g, 1.09 mmol) and stirred at room temperature for3 h. The reaction mixture was diluted with CHCl3 (20 mL) shakensequentially with 2 N HCl (15 mL), water (15 mL), and brine (15mL) and finally dried over anhydrous Na2SO4. The solvent fromthe solution was evaporated under reduced pressure and theresidue purified by column chromatography over silica gel (60-120 mesh) using ethyl acetate/hexane as eluent to give theindividual monoesters as transparent gums. The percentageyields isolated after purification and spectral and analyticaldetails 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 thehighly sticky nature of 1b.

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-3â-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 subsequentlysealed and heated at 90 °C for 24 h. The white precipitate obtainedwas filtered out, washed with cold dry acetone (50 mL), andrecrystallized 3 times from dry ethyl acetate to give 3a as a whitesolid. Mp: 236.5 °C (dec) (0.884 g, 80%). IR (CHCl3) (cm-1): 1730.1H NMR (CDCl3, 90 MHz; δ): 0.62-2.45 (multiple peaks, 46 H),3.65 (s, 9H), 4.65 (m, 1H), 4.9 (s, 2H) 5.40 (d, 1H). LRMS: m/e486 (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 CholesterolDerivatives 3b-d and 5a. A solution of 6b (1 mmol) in dry acetone(20 mL) was refluxed for 12 h with the appropriate N-alkyl-N,N-dimethylamine (1.1 mmol). The white precipitate so formedin each case was filtered off, washed with dry acetone, andrecrystallized several times from dry acetone to give white solidmaterials which were found to be pure by TLC; the yields ranged88-95%. All compounds were found to be hygroscopic and existedas hydrates despite prolonged drying under vacuum. Thespectroscopic and analytical data for these compounds are givenbelow.

Cholesteryl (2-Hydroxyethyl-N,N-dimethylammonium bro-mide) 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-dimethylammo-nium bromide) Acetate (3c). 3c was obtained as a white solid.Mp: 200 °C, 0.570 g, 0.89 mmol, 89%. IR (CHCl3) (cm-1): 3650-3100, 1750. 1H NMR (CDCl3, 300 MHz; δ): 0.68-2.36 (multiplepeaks, 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-dimeth-ylammonium bromide) Acetate (3d). 3d was obtained as a whitesolid. 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)am-monium 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 CholesterolDerivatives 2a-d. To a suspension of cholest-5-ene-3â-tosylate(500 mg, 0.9 mmol) in anhydrous dioxane (9 mL) was added therespective alcohol (1 mL), and the mixture was stirred underreflux for 4 h in an inert atmosphere. The solution was cooledand the solvent removed in vacuo. The white residue waspartitioned between CHCl3 (20 mL) and water (20 mL), washedsequentially with saturated NaHCO3 (2 × 10 mL), water (10mL), and saturated brine (10 mL), and dried over anhydrousNa2SO4, and the solvent was removed in vacuo. The residue waspurified by column chromatography on silica gel (60-120 mesh)using ethyl acetate/hexanes.

Cholest-5-en-3â-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), 2.19-2.22 (2H, m), 2.35-2.37 (1H, m), 3.17-3.21 (1H, m), 3.6(2H, t,

(36) Perrin, D. A.; Armarego, W. L.; Perrin, D. R. Purification oflaboratory chemicals, 3rd ed.; Pergamon: New York, 1990.

(37) Krishnan-Ghosh, Y.; Gopalan, R. S.; Kulkarni, G. U.; Bhatta-charya, S. J. Mol. Struct. 2001, in press.

Oligo(oxyethylene)-Bearing Cholesteryl Amphiphiles Langmuir, Vol. 17, No. 7, 2001 2073

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-3â-oxypent-3-oxa-an-5-ol (2b):whitegummysolid,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 ) 4.5Hz). 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-3â-oxyoct-3,6-oxa-an-8-ol (2c): transparent gummysolid, 431 mg, 0.83 mmol, 90%. IR (CHCl3) (cm-1): 3425, 2932,2859, 1460. 1H NMR (CDCl3, 300 MHz; δ): 0.63 (s, 3H), 0.81-1.55 (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 (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-3â-oxyundeca-3,6,9-oxa-an-11-ol (2d): transpar-ent 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 forC35H62O5‚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). Thealcohol (200 mg) was taken in dry CHCl3 (10 mL), pyridine (1mL) was added, and the mixture was cooled to 0 °C. To the coldsolution, p-toluenesulfonyl chloride (1.1 equiv) was added andallowed to stir for 3 h at room temperature. The reaction mixturewas poured into cold dilute HCl (25 mL of 6 N HCl) and extractedwith CHCl3 (2 × 15 mL). The organic layers were dried overNa2SO4 (anhydrous), and solvent was removed in vacuo.

Cholest-5-en-3â-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-3â-oxypent-3-oxane-5-tosylate (7b): white waxysolid, 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 ) 8Hz, 2H).

Cholest-5-en-3â-oxyoct-3,6-oxane-8-tosylate (7c): transparentgummy solid, 250 mg, 0.37 mmol, 96%. IR (CHCl3) (cm-1): 3370,2930, 2865, 1465, 1350, 1180, and 1170. 1H NMR (CDCl3, 300MHz; 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-3â-oxyundeca-3,6,9-oxane-11-tosylate (7d): trans-parent 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 ) 4.5Hz, 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 for4 h. The reaction mixture was poured into water (25 mL) andextracted with CHCl3 (3 × 15 mL). The organic layers were driedover Na2SO4 (anhydrous) and removed in vacuo. The oily residuewas purified by column chromatography over silica gel (60-120mesh) with hexanes as eluent to give glassy solid melts thatwere found to be bromides.

2-Bromocholest-5-en-3â-oxyethane (8a): white solid, 80 mg,0.16 mmol, 95%. IR (CHCl3) (cm-1): 3370, 2930, 2865, 1465, and1350. 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.

5-Bromocholest-5-en-3â-oxypent-3-oxane (8b): glassy melt, 82mg, 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 ) 4.5Hz, 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-3â-oxyoct-3,6-oxane (8c): glassy melt, 84mg, 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 ) 4.5Hz, 8H), 3.82 (t, J ) 4.5 Hz, 2H), 5.34 (d, J ) 4.5 Hz, 1H). MALDI-TOF: m/e 606 (M + 2 + Na), 604 (M + Na). Anal. Calcd forC33H57O3Br: C, 68.13; H, 9.88. Found: C, 68.39; H, 10.02.

11-Bromocholest-5-en-3â-oxyundeca-3,6,9-oxane (8d): trans-parent 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-3â-oxyundecane (8e). Mp: 68-70 °C, 82mg, 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 ) 4.5Hz), 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. Therespective bromide (8a-e) (100 mg) was taken in 1:10 dry EtOH/dry acetone and heated with excess Me3N dissolved in acetonein a screw-top sealed tube for 24 h to yield the cationic cholesterolamphiphiles. These were purified by column chromatographyon neutral alumina. Upon recrystallization from dry acetone,analytically pure amphiphiles were obtained as white solids inmoderate to high yields. These amphiphiles were found to beextremely hygroscopic in their pure forms, precipitating ashydrates even from dry solvents upon exposure to ambientatmosphere, which could not be removed even upon prolongeddrying under high vacuum. Melting points of these amphiphilescould not be recorded due to their hygroscopic nature.

Cholest-5-en-3â-oxyethane-N,N,N-trimethylammonium Bro-mide (4a): white solid, 101 mg, 0.18 mmol, 90%. IR (CHCl3)(cm-1): 3370, 2930, 2865, 1465, and 1350. 1H NMR (CDCl3, 300MHz; 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/e473.9 (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-3â-oxypent-3-oxane-5-N,N,N-trimethylammoni-um 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 C34H62O2-NBr‚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-3â-oxy-oct-3,6-oxane-8-N,N,N-trimethylammoni-um 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. Calcdfor C36H66O3NBr‚2H2O: C, 63.88; H, 10.43; N, 2.07. Found: C,63.77; H, 10.39; N, 1.74.

Cholest-5-en-3â-oxyundeca-3,6,9-oxane-11-N,N,N-trimethyl-ammonium 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 forC38H70O4NBr‚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-3â-oxyundecane-11-N,N,N-trimethylammoni-um Bromide (5b): white gum, 94 mg, 0.12 mmol, 59%. IR (CHCl3)(cm-1): 3230, 2920, 2860, 1455, and 1350. 1H NMR (CDCl3, 300

2074 Langmuir, Vol. 17, No. 7, 2001 Bhattacharya and Krishnan-Ghosh

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 wasdissolved in CHCl3 (0.5 mL) and then dried under a stream ofN2 to yield a thin film of the amphiphile. The resulting film wasfurther 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 followedby vortex mixing for 10 min and bath sonication at >60 °C for10 min.

Dye Entrapment Studies. Films of a given amphiphile wereprepared as described above, and 10 mL of 0.1 mM methyleneblue (MB) (λmax ) 665 nm) was added such that amphiphileconcentration was 5 mM. A 2-mL aliquot of the resultingsuspension was loaded on to a column packed with preequili-brated Sephadex G-50. Gel filtration was performed using wateras the eluent until elution of free dye was complete. Neat Triton-X-100 was added to aliquots of fractions to give a 1 wt %concentration of Triton X 100, and the vesicles were lysed bybath sonication of the resulting solution for 2 min at roomtemperature. The absorbances at 665 nm for all the fractionscontaining lysed solutions was determined and plotted againstthe elution volume.

Transmission Electron Microscopy. Vesicles were madeby adding a known amphiphile from stock solutions in CHCl3,evaporating the organic solvent to form a film, and samples wereprepared as mentioned above in water (Millipore) at pH ∼ 6.8.The concentration of the amphiphile was maintained at 4 mMfor all samples. A 15 µL volume of the vesicular solution wasloaded onto Formvar-coated, 400 mesh copper grids, allowingthem to remain for 1 min. Excess fluid was wicked off the gridsby touching their edges to filter paper, and 15 µL of 2% uranylacetate was applied on the same grid for a after which the excessstain was similarly wicked off. The grid was air-dried for 10 min,and the specimens were observed under a bright-field TEM (JEOL100 CX II) operating at an acceleration voltage of 80 kV.

Fluorescence Depolarization Measurements. A solutionof compound in CHCl3 and 1,6-diphenyl hexatriene (DPH) wasmade and evaporated to form a film. It was hydrated for 30 minwith 1 mL of double distilled water (pH ) 6.8). The resultingconcentration of the compound was 0.1 mM, and that of DPHwas 1 µM. The solution was bath sonicated for 10 min at 60 °Cto give a vesicular suspension which was excited at 360 nm, andthe emission followed at 430 nm on a Hitachi model F-4500spectrofluorometer. At each temperature the fluorescence emis-sion spectra were recorded by adjusting the polarizers at 4different positions. r values at different temperatures for thevesicular solutions were calculated using Perrin’s equation: r )(I| - I⊥G)/(I| + 2I⊥G) where I| and I⊥ are the observed intensitiesmeasured with polarizers parallel and perpendicular to thevertically polarized exciting beam, respectively. G is the factorused to correct for inability of the instrument to transmit

differently polarized light equally. Individual r vs T plots foreach aggregate gave the information about its order-disordertransition as well as the rigidity as a function of temperature.

Differential Scanning Calorimetry. Films of a givenamphiphile were prepared as described in Sample Preparation.A 2 mL volume of Millipore water was added to yield a finalamphiphile concentration of 1 mM. Vesicles were prepared byfreezing to 0 °C for 15 min followed by thawing to 70 °C for 15min followed by vortexing for 5 min. Samples were subjected toseven such freeze-thaw cycles. A 0.5 mL volume of these vesiculardispersions was loaded on a differential scanning calorimeter(Calorimetric Sciences Corp.) and scanned from 20 to 75 °C ata scan rate of 30 K/h.

X-ray Diffraction Studies. Self-supported cast films for theXRD studies were prepared by dispersing the films of amphiphiles(0.5 mg /mL) of water as described previously.38 A 1 mL volumeof this suspension was placed on a precleaned glass plate andair-dried at room temperature. Reflection XRD studies werecarried out using an X-ray diffractometer (model XDS 2000,Scintag Inc.). The X-ray beam, generated with a Cu anode at thewavelength of KRl beam 1.540 598 Å, was directed toward thefilm edge, and scanning was done up to the 2θ value of 10°.

Molecular Modeling Studies. The modeling studies wereconducted with BIOSYM software running on a Silicon GraphicsIndigo workstation. The atomic coordinates of the carbon andhydrogen atoms for the cholesteryl backbone have been extractedfrom the fractional atomic coordinates of cholesteryl laurate39 ashas been done with previous theoretical treatments of cholesterolinteraction in membrane bilayers.40 The molecules 1-5 weredrawn in INSIGHT II using standard bond lengths, angles, anddihedral angles. The atoms within each molecule were assignedtheir proper hybridization, charge, and bond order by utilizingthe Builder module of INSIGHT (version 2.3.5). The CVFF forcefield provided by the Discover module was chosen for minimiza-tion constraints. This force field was applied to the constructedderivative and evaluated with the conjugate gradient method.The interaction number for the conjugate gradient method was200. The derivative (or convergence criterion) was chosen as 0.001kcal mol-1. Each molecule was minimized first using the steepestgradient method (2000 iterations) followed by the conjugategradient method (5000 iterations) at 300 K with a time intervalof 1.0 fs.

Acknowledgment. This work was supported in theform of a Swarnajayanti Fellowship Grant of the Depart-ment 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.

Oligo(oxyethylene)-Bearing Cholesteryl Amphiphiles Langmuir, Vol. 17, No. 7, 2001 2075