Indian Journal of Chemistry Vol. 4OB, October 2001 , pp. 891 -894

Rapid Communication

Membrane formation from oxyethylene bearing cationic cholesterol derivatives t

Santanu Bhattacharya· & Yamuna Krishnan-Ghosh

Department of Organic Chemistry, Indian Institute of Science, Bangalore 560 0 1 2, India

·Ema'il: sb@orgchem.iisc.ernet.in Received 26 December 2000; accepted 23 May 2001

Eight cholesterol based amphiphiles, la-d and 2a-d, bearing differing lengths of oxyethylene units at different locations, have been synthesized. Membrane formation from the aqueous suspensions of these compounds has been confirmed by transmission electron microscopy and dye entrapment. X-Ray diffraction and fluorescence anisotropy measurements reveal modulation of the membrane characteristics by both the length and location of the oxyethylene segment.

Cholesterol is the third major component of animal membranes after phosphoglycerides and glycolipids. 1 Its polar OH group at 3-position gives it a weak amphiphilic character, while the fused rings of its hydrophobic portion provide it with greater rigidity than other lipids in membranes. Hence cholesterol is an important determinant of the membrane properties2

even though cholesterol on its own cannot aggregate in water to form membranes. One way to achieve vesicle formation from cholesterol derivatives alone would be through covalent attachment of a charged residue to the cholesteryl backbone. Indeed examples are known where polar derivatives of cholesterol have been shown to form vesicles or related aggregates3. However, so far there has been no attempt to exploit cholesterol based amphiphiles to develop membranes, the organization and thickness of which could be modulated by molecular design.

We have previously examined the behavior of electrostatically complementary amphiphiles4 and the effects of insertion of a polymethylene spacer chain between two lipid monomerss. An alternative concept in lipid molecular design is introduced herein by the incorporation of oligo-oxyethylene units either at the headgroup bearing cholesterol or between the cholesteryl backbone and the headgroup. Herein we report the synthesis of two families of cationic cholamphiphiles, la-d and 2a-d containing oxyethylene

tDedicated to Prof. U. R. Ghatak on his 70lh birthday.

units (Scheme I). In the first series of cholesteryl derivatives la-d,

the cationic headgroup is linked to the 3f}-cholesterol via an ester group. In all of these derivatives, 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. In order to see the difference in properties, if any, depending on the sequence in which the two moieties were attached another series of cationic cholesteryl amphiphiles 2a-d has also been synthesized. In the second series of cholesterol derivatives, the cationic NMe3 + groups are linked at the 3f}-OH of cholesterol via an ether link. The cationic NMe3

+

group is however, placed at incrementally greater distance from the cholesteryl backbone with the insertion of increasing number of oxyethylene units in 2a-d. Cholesteryl tosylate, 3 upon refluxing in dry dioxane with the appropriate oligoethylene glycol in slight excess yielded 4a-d in good yields. 4a-d were then converted to the respective tosylates, Sa-d which upon heating to -65 °C with 1 eq. LiBr in dry DMF furnished the corresponding bromides, 6a-d, in excellent yields. Quarternization of the bromides with the appropriate tertiary amine in a 1 : 1 mixture of dry acetonelEtOH yielded the amphiphiles 2a-d, in moderate to good isolated yields. Cholesteryl bromo­acetate was similarly quaternized with the appropriate tertiary amine to yield la-d also in good yields.*

Upon dispersal in water (0.4 mM) by sonication la-d and 2a-d gave opalescent suspensions§. TEM examination (JEOL 200CX) of the individual, air­dried suspensions of la-d or 2a-d layered on carbon­formvar coated copper grids revealed the existence of polydisperse, closed aggregate structures in all the cases. Interestingly the cholesteryl derivatives la-c formed predominantly unilamellar, nearly spherical

* All the amphiphiles were characterized by IR, IH NMR, MALDI-TOF and elemental analysis to establish their chemical purity and are consistent with their proposed structures. t-rhe desired weight of amphiphile was dissolved in CHCl3 (0.5 mL) and then dried first under a stream of N2 and then under high vacuum for another 1 .5 hr to yield a thin film of amphiphile. After this, requisite amount of water (Millipore, pH=6.8) was added and left for 10 min. Bath sonication (35 kHz) for 10 min at - 6O"C yielded translucent, stable aqueous suspensions.

892 INDIAN J CHEM, SEC B, OCTOBER 2001

Chol-OH --,(_i) ----..� CholOTs (ii) � Chol��H (iii) � ChOI�� Ts

(Vi)! 0

3 4a: n = 1 5a: n = 1 4b: n = 2 5b: n = 2 4c: n = 3 5c: n = 3

Chol� Sr 4<:1: n = 4 (iv) 5d: n = 4 , I 6b (Vii).

0 , CholON*"-, '*" _R

Sr

�� (v) Ch� , _ �

Sr ChoH-�Sr

1 a: R = CH3 1 b: R = CH2CH2OH 1 c: R = (CH2CH20)2-H 1 d: R = (CH2CH20)4-H

2a: n = 1 2b: n = 2 2c: n = 3 2d: n = 4

6a: n = 1 6b: n = 2 6c: n = 3 6d: n = 4

Scheme I - Reagents, reaction conditions and yields: (i) TsCl, dry pyridine, 0 °C, 6h (93 %) (ii) dry dioxane, HO(CH2CH20)nH, reflux, N2, 4h (yield for 4a, 86 %; 4b, 80 %; 4e, 90 %; 4d, 93 %) (iii) TsCl, dry pyridine/CHCl3, O °C, 3h (for 5a, 92 %; 5b, 87 %; 5e, 96 %; 5d, 85 %) (iv) LiBr, dry DMF, N2, 65 °C, 4h (for 6a, 95 %; 6b, 96 %; 6c, 97 %; 6d, 90 %) (v) NMe3, dry acetonelEtOH, reflux, 24h (for 2a, 90 %; 2b, 91 %; 2e, 80 %; 2d, 75 %) (vi) Bromoacetyl chloride, dry C6H6, DMAP, Et3N, 0 °C, 24h (77 %) (vii) RNMe2, dry acetonelEtOH, reflux, 24h (for la, 80 %; lb, 90 %; Ie, 89 %; ld, 88 %).

vesicles. In contrast, Id and 2a-d generated mostly multi lamellar vesicles (not shown). Closer scrutiny suggested that the thickness of each lamella was highly uniform in a given vesicular suspension for all the samples Id and 2a-d. Although there was no correlation in size of the vesicles so formed for a given series of amphiphile, the size distributions ranged from -40 nm to -400 nm.

Having confirmed vesicle formation from these newly developed cholesteryl derivatives, dye entrapment experiments tt were performed to examine whether these vesicles comprised closed, inner aqueous compartments. For this purpose, a water­soluble dye, methylene blue (MB), which has been encapsulated in inner aqueous compartments of vesicles of other amphiphiles,3b-c was used. Importantly in all the cases, MB entrapped vesicles could be distinctly separated from the 'free' MB molecules confirming the presence of closed structures containing an inner aqueous compartment. The percentages of entrapment ranged from 0.5-4.5% of total dye as given in Table 1. I nterestingly the percentage entrapment increased with the increase in

ttYesicles la-d and 2a-d were generated by sonication of a dry film of a given amphiphile (5 mM) in water (PH -6.8) containing MB (0. 1 mM) for 10 min at -60°C. Then, 2 mL of this translucent. blue suspension was loaded onto a pre-equilibrated Sephadex G-50 (Pharmacia) column and gel filtration was performed using water as eluent.

vesicle sizes as well as their multilamellar character. To examine the response of the temperature

variation on the presently described aggregates, fluorescence anisotropy values (r) due to each vesicle doped with 1 ,6-diphenylhexa- l ,3,5-triene (OPH) at various temperatures were then determined. Comparison of r vs. T plots (not shown) of the vesicles of la-d or 2a-d with that of naturally occurring dipalmitoyl phophatidylcholine (OPPC) clearly brings out the differences in the thermal responses between membranes produced from the cholamphiphiles and that from a hydrocarbon-chain based lipid, OPPC. No pronounced break in r vs. T plots from 1 5-65°C with la-d or 2a-d indicates lack of any detectable solid gel to fluid phase transitions. Conventional lipids such as OPPC, by contrast, undergo a thermally induced conversion from an ordered, solid-like, gel phase into a liquid-crystal like, fluid phase replete with s-gauche conformations in its hydrocarbon chains. Absence of any detectable thermal transition most likely stems from the conformationally immobile nature of the steroidal rings. Thus upon heating only temperature induced lateral separation of monomers coupled with local disordering due to trans-to-gauche isomerization of the side chain at the C- 1 7 position contribute to the low r-value.

The sonicated aqueous dispersions of 1-2 (5 mg/mL) were also converted to regular self-supporting films

RAPID COMMUNICATIONS 893

Table I -Entrapment capacity and membrane widths of aggregates formed by 1-2

Entry Compd Entrapment Unit Aggregate layer widthsb (A) capacity" (%) CalcdC Obsd (%)d

I la 1 .2 42.0 42.0 ( 100) 2 Ib 0.5 42.0 40 (36), 34 (64) 3 Ie 1 .6 42.0 46(58), 28( 14), 25 (28) 4 Id 4.5 42.0 28 (82), 25 ( 1 8) 5 2a 2.7 42.0 36.6 ( 100) 6 2b 1 .6 49.0 5 1 .9 ( 100) 7 2e 3.8 56.0 55.2 ( 100) 8 2d 1 .6 63.0 57.9 ( 100)

"Absorbance at 665 nm was measured for all fractions; [MB] = O. I mM; [amphiphile] = 5 mM; pH = 6.8. b [amphiphile] = 5 mglmL. c Lengths of two molecular layers of lipids as obtained from models. dAs obtained from the X-ray diffraction measurements of cast films. Values in parentheses indicate the percentage of a given morpho

by casting on glass slides as described6• Reflection X-ray diffraction (Scintag XDS-2(00) of these cast films gave the long spacings from individual lipid films as given in Table 1. The formation of regular bilayer-like arrangement is indicated with the aggregates of la as the layer length (42 A) obtained from XRD is in good agreement with the sum of calculated lengths (CPK model) of two molecular layers of la. Slight reduction in the layer length (40 A) of lb aggregates could be explained on the basis of the formation of a tilted bilayer arrangement. In contrast, however, the aggregates of Ie and ld in water showed evidences of complex polymorphism with significant reduction in the layer lengths of the predominant morphs. Such a dramatic reduction in aggregate layer width could be explained on the basis of pronounced interdigitation and tilting. Thus in the series la-d, there is steady decrease in the membrane thickness upon increasing in the number of oxyethylene units (n-value) in la before reaching a plateau at n = 4. Under comparable conditions, the XRD of self-supporting cast films from aqueous suspensions of 2a-d were also measured. Remarkably here the results indicated an exactly opposite trend to what was observed with la-d. 2a formed a rather thin aggregate (- 37 A) indicating a significant interdigi­tation and/or tilting in its bilayer plans. As the n-value is increased the aggregates gradually tend to adopt regular bilayer or hydrated bilayer arrangements.

Examination of the r-values at 25°C for the membranes formed from the two series of cholamphiphiles as a function of their molecular structures reveals strikingly opposing trends. In the series of vesicles of la-d, a steady decrease in r-value was observed as the n-value increased (Figure lA). Loss of r-value at a given temperature indicates

increasing disorder in the membrane packing. In contrast, in the series of 2a-d, a monotonous increase in r-value was observed as oxyethylene units were incrementally added to the spacer between the NMe3 + headgroup and the cholesteryl backbone (Figure lA.). Thus a gradual enhancement of lipid packing is manifested with increase in n-value in the series 2a-d. These findings demonstrate the remarkably opposing trends of the bilayer widths and membrane rigidity in the two series of cholamphiphiles la-d and 2a-d (Figues lA and lB), where the sequence of attachment of the -(CH2CH20)n- segment and the positive charge were interchanged. In la-d the attachment of a -(CH2CH20)n-H segment to the cationic -NMe2+ group progressively increases the size of the head group and enhances hydration. This also leads to greater charge dispersal. When the charge is concentrated to the small -NMe3 + group in la, the mutual repulsion at the head group level is so strong that only a bilayer arrangement is possible where the charges of the monomers in the inner and outer leaflets are farthest from each other. However, as the charge becomes more dispersed over increasingly bulky headgroup, in lb-d, repulsion between the charges of the inner and outer leaflets gets progressively reduced leading to the formation of strongly interdigitated and other non-bilayer organizations. Thus the membrane thickness in la-d decreases with the increase in n-value as seen in Figure lB. At the same ti�e, the increase in the size of the cationic headgrOlip increases the lateral separation between the adjacent monomers. This in tum leads to greater disorder (reflected in the loss.in r-value) with increase in n-value. As a consequence of the greater lateral separation with increase in n-value, the monomers from the inner and outer leaflets come

894 INDIAN J CHEM, SEC B, OCTOBER 2001

0.28 60 A .............. B

� C\ 55 -� � ,/

0.26 "- o.:t: r � h.. ,/ '-/ 50 -

0 , .� � / � w.</ � 45 - i '" 0.24 / " / . .... 0., �

, 11) / ' 0 § 40 -

\ /

� /

\( .. "6 35 -.2 0.22 11) � ::;g 30 - "-'0-----0

0.20 25 I I I I I

0 1 2 3 4 5 0 2 3 4 5 n - value n - value

Figure lA-Plots of fluorescence anisotropy values (r) at 25 DC with n-value of -(CH2CH20)n-units in 1a-d and 2a-d. B Variation of Membrane Thickness with n value in the series 1a-d (open squares) and 2a-d (closed circles)

closer to each other in order to allow the interdigi tation of the cholesteryl side chains to fill the 'voids' . The aggregate width thus decreases with greater interdigitation.

The oxyethylene group has unique ability to show both hydrophilic as well as hydrophobic character7• In the series 2a-d, the -(CH2C H20)n- unit is located in between the cholesteryl backbone and the N+Me3 headgroup. Here it appears to act like a hydrophobic spacer as an increase in the n-value in 2 results in an increase in the length of the hydrophobic portion of the lipid monomer. This in turn translates into an increase in the bilayer width with increase in n-value as evidenced from the XRD data. At the same time, since this moiety is located close to the interfacial region near the headgroup in 2a-d, it also has the propensity to get hydrated. This presumably facilitates water promoted hydrogen bonding7b between adjacent lipid monomers drawing them closer together leading to more efficient packing and enhancing r-value8. Thus a longer oxyethylene segment results in a longer "sticky strip" being made available for water which acts as a "glue" serving to increase membrane rigidity and order.

Acknowledgement This work was supported in the fonn of a

Swarnajayanti Fellowship Grant of the Department of

Science and Technology, Government of India, awarded to S.B.

References 1 Voet D & Voet J G, Biochemistry, 2nd Ed (John Wiley, New

York), 1995. 2 Cholesterol in membrane models, edited by L Finegold (CRC

Press, Boca Raton, FL), 1993. 3 (a) Davis S C & Szoka (Jr) F C, Bioconjugate Chem, 9, 1998,

783; (b) De Wall S L, Wang K, Berger D R, Watanabe S, Hernandez J C & Gokel G W, J Org Chem, 62, 1997, 6784; (c) Echegoyen L, Hernandez J C, Kaifer A, Gokel G W & Echegoyen L E, J Chem Soc Chem Commun, 1988, 836. (d) 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 USA, 79, 1982, 5490; (e) Lyte M & Shinitzky M, Chem Phys Upids, 24, 1979, 45.

4 (a) Bhattacharya S & De S; Chem Commun, 1995, 65 1 ; (b) Bhattacharya S, De S & Subramanian M, J Org Chem, 63, 1998, 7640; (c) Bhattacharya S & De S, Langmuir, 15, 1999, 3400.

5 (a) Bhattacharya S & De S, Chem Commun, 1997, 2287; (b) Bhattacharya S & De S, Chem Eur J, 5, 1999, 2335.

6 Bhattacharya S & De S, Chem Commun, 1996, 1283; (b) Kimizaka N, Kawasaki T & Kunitake T, J Am Chem Soc, 1 15, 1993, 4387; (c) Bhattacharya S & Krishnan-Ghosh Y, Langmuir, 17 , 2001, 206.

7 (a) See Gokel G W & Murillo 0, in Comprehensive supramolecular chemistry, Vol. 1 , Ch. 1 , (Pergamon: New York), 1996, p. l ; (b) ibid, Moyer, B A, Ch. 1 0, p. 377.

8 Bhattacharya S & Haldar S, Langmuir, 1 1, 1995, 4748.