Phase transition in a lipid bilayer. II. Influence of adamantane derivatives

C'nemist,'y and Physics o f Lipi~ 17 (1976) 71-78 © North-Holland Publishing Company

PHASE TRANSITION IN A LIPID BILAYEt~. II. INFLUENCE OF ADAMANTANE DEI~VATIVES Mahendra K. JA_IN, Nora YEN-MIN WU, Thomas K. MORGAN, Jr., Martha S. BRIGGS and Roger K. MURRAY, Jr. Division of Health Sciences and Department of Chemist~. , University of Delaware, Newark. Delaware 19 711. USA Received February 25, ,.976,

accepted March 30, 1976

The influen~ of thirty-four adama:~tane, proteadamalttane, and homoadamantane derivatives on the phase transition characteristics of the bilayer in dipalmitoyl lecithin liposomes has been determiaed by differential s c a ~ . g calorimetry. "Eachof these compounds induces a broadening of the phase tta~Jsition px'oxlleof the lipid bilayer that is dependent upon the concentration of the solute and its molecular structure. The concentration-response curves obtained for these solutes suggest that the cage compound den,atives modify the phase properties and under some conditions may induce a phase separation in the doped bilayer. The relati~'e activity sequences obtained for the compounds examined cannot be accounted for by simple considerations of lipid/water partition coefficients, substitution constants based on free energy relat,t, nships, or the relative pol&rities or sizes of suL~tituent groups. The observaqons are consistent with the hypothesis that the position and crientation of a solute within the bilayer are critical factors in de:ermining its relative potent. The position of a solute within the bilayer is significantly controlled by the pre~nce of polar substituet~ts and by the relative geometric relationships of these grouvs. For a given substituent group, the shape and size of the hydrocarbon cage becor,~es increasingly important. It is a p ~ e n t t.hat seemingly minor modifications in the structure of a solute can significantly aller its influence on the phase transition behavior of a bilayer.

I. Introduction Structure-activity correlations for solutes that interac ' with lipid bilayer regions of biomembranes h~_avegenerally met w~.th little success [ 1 ]. Recently we have shown that the pharmacological activities and membrane expanding ab~_ities of a series of drugs cart be correlated with their relative effectiveness in lowering the gel to liquid crystalline phase transition temperatuie o[ lecithin b~ayers [2]. Pievieusly it ha~ been reported that adamar, tane, a hydrophobic quasi-spherical molecule, is an effective lipophilic perturber which binders the cooperative axi'~ ordering of phosphol~pid alkyl chains [3]. It hab also been voted that adamantanone int~-oduces disorder in membranes by perturbing the lipid alkyi chains [4]. We now wish ~':oreport our obmrvations on the effect~s of thi'rty-four adamantane and adamantane-related de~wtives on the gel to liquid crystalline phase :ransition characteristicc of dipalmitoyl lecithin liposomes dGpe~ with ~hese solutes. 71

72

M. IC Jaln et al., Phase transition in a lipid bgayer

11. Experimental The DL.l,2.dipalmitoyl lecithin Used was obtained from the Sigma Chemical Company. It was chromatographed on neutral alumina and the lecithin thus obtained showed no impurity by thin layer chromatography. All of the l-halo and 2-ba!oadamantanes were prepared by the method of Olah and Welch:[5]. The other 1- and 2.substituted adamantanes employed in this study were commercial samples obtained from the Aldrich Chemical Company. 2.Protoadamantanone [6], 2-endo-Protoadamantanol [6], 2-exo-protoadamantanol: [7], 5,protoadamantanone [8], and the 5-endo- and 5-exo-protoadamantanols [9] were prepared according to known procedures. Synthetic routes leading to 2,5.protoadama_rttanedione and the diols related to it have recently been reported [10]. 4-Homoadamantanone and 4homoadamantanol were prepared by the method of Schleyer [11 ]. A recent report details the synthesis of 2-homoadamantanone and 2-exo-homoadamantanol [ 12]. The liposomes doped with adamantane derivatives were prepared for the differential scanning calorimetry studies as follows. A methanolic solution of an adamantane derivative was evaporated in a small test tube' Liposomes were trans. ferred onto the dry film of adamantane residue and the mixture was shaken with a 3 mm glass bead on a Vortex shaker under a nitrogen atmosphere at 45-50°C for several minutes. The sample was then allowed to equilibrate for 2-4 days at 4°C and finally shaken again for several minutes at 45-50°C. For comparison, some samples were prepared by premixing the lipid and adamantane solute in an organic solvent and then generating the liposomes from a mixed film. The transition profiles of the liposomes prepared by either method were identical. Control experiments showed that the adamantane derivatives were indeed incorporated into

the liposomes. All samples prepared for scanning were 75 mM in dipalmitoyl lecithin, 50 mM in potassium chloride, 5 viM in TRIS buffer, and were prepared at pH 7.3. The samples were analyzed using a Perkin.Elmer DSCI-B differential ~ g calorimeter operating at a sensiti~Aty of I mcal and a scanning rate of 1.25 °C/min towards increasing temperature. The interpretation of differential scanning calorimetry curves has been discussed previously [ 13,14]. II!. Results The phase transition profde for pure dipalmitoyl lecithin liposomes Shows a sharp transition commencing at 41.0°C(Tc) which is essentially (more than 99%) complete in 1.6°C. The phase transition profile of dipalmitoyl lecithin is significantly ~tered when an adamantane derivative is incorporated into the sample. As the concentration of the adamantane derivative is increased, T c decreases and the half height width (HHW) increases. Such behavior was found to be typical for the thirty-four substituted adamantanes, protoadamantanes, and homoadamantanes that we have examined.

M K .lain et al., Phase transition in a lipid bilayer 1

4 2

adamantine

73

3 5

protoadamantane

homoadama~ntane

The phase transition pro fries obtained for each of these compounds are characteristic and concentration dependent. The profiles do not change significantly with the scanning rate, the number of scans, or the age of the sample, i.e., the prof'des of a samp!e obtained eigtlt hours after initial mixing and several days later were indistinguishable. The inte~rated are~ of the phase transition profRes (linearly related to the enthalpy of transition) for each doped sample was found to be independent of the concentration of the additive. For the samples exam~ed, the HHW of the phase transition profile appears to be the parameter that is laost sensitive to the concentration of the solute in the brayer. The HHW can be determined accurately over a wide range of concentrations of the solute and, at least at low concentrations of the additives, appears to be linearly concentration dependent. In order to compare the relative potencies of adamaatane derivatives, we have defined an arbitrary constant, Hl-P~,¢100,which is the concentration of solute al which the HHW of the phase transition profile of pure dipalmitoyl lecithin liposomes is increased by 100%. Thus, the solutes which are most potent have fire sma;lest HHWI0o values. For most of the compounds examined, HHWI00 could be obtained directly from the measured HHW values. However, in a few cases it was necessary to extrapolate the plot of HHW versus concentration in order to obtain i~lWl00. In such cases the shape of the EHW versus concentration curve could be rationalized from the detailed phase diagram. The HHWlo0 values for the adamantane, protoadamantane, and homoadamantane derivatives studied are summarized in table 1.

IV. Discussion Previously it has been suggested that the incorporation of a solute in a lipid brayer creates a new phase with a lower T c [2]. Such is the characteristic behavior of the adamantane and rela:ed cage compound derivatives that we have investigated. The population of the new phase is dependent upon the conce~ltration of the solute in the bilayer. Examination of table 1 dearly shows that changes in the nature of the substituent group present in a given cage hydrocarbon or changes in the cage skeletal framework both markedly influence the effect of these solutes on the phase transition profile. In particular, it is apparent that (1) corresponding

Table 1 Relative changes induced by adamantane derivatives On the phase transition of dipalmitoyl lecithin. Substituent

HHWI00 (mM)a

l-Substituted Adamantanes H

CH3 F

>I00. b

~ 1408.b

CI Br CN COCI CH2CO2H I OH CO2H COCH3 CH2OH

15. 8.1 7.3 7.1 5.0 4.9 4.3 4.0 2.0 2.0

2-Substituted Adamantanes F O= CI Br I OH

18. b I0. 5.5 4.3 2.7 2.4

2-Substituted Protoadamantanes H 0== OH (exo) OH (endo)

>I00. b 7.0 2.8 2.6

S-Substituted Protoadamantanes 0== OH (exo) OH (endo)

9.6 3.0 2.4

2,5-Disubstituted Protoadamantanes dione diol ( 2-exo-5-endo ) diol (diendo)

>I00. b ~70. b 9.

2-Substituted Homoadamantanes H O= OH (exo)

~30. b 4.2 2.1

4-Substituted Homoadamantanes O:= OH

3.8 2.7

bRepxodacibility ±10% of the reported value, Value obtained via extrapolation due to saturation behavion

M.K. Jain et al., Phase transitio., in a lipid bilayer

75

homoadan antane derivatives are more potent than the cc rrespe.~-!ing protoadamantane derivatives which are more effective than eith~ ~. or 2-substituted adamantanes. ~2) Corresponding 2-adamantane derivatives are more potent than l-ada. mantane derivatives. (3) In both the l-halo and 2-haloadamantanes the order of effectiveness is I > Br > CI > F. (4) For a given cage skeleton, a hydroxyl substituent is more potent than a keto substituent. What general model of solute-lipid bilayer interaction might account for all of these observations? It is well known that many diverse membrane processes that are induced by small molecules are dependent on the lipophilicity of the solute [ 1, 15-18]. A common measure of lipophilicity is the partition coefficient for the distribution of the solute between aqueous and nonpolar phases. The maximum biological responses observed for a series of related solutes may thus be compared in terms of the molecular partitioning of the solutes between the aqueous and nonpolar regions of a biophase.~By means of such an approach, Hansch has generated a series of partition coefficients 0r-values) for substituent groups [ 19]. Accordingly, the greater the lipid-water partition coefficient of a substituent, the larger should be its membrane concentration and, consequently, the greater its activity or potency. Comparison of the relative activities of the thirteen l-substituted adamantanes that we have investigated with the Hansch ~r-values for these substituents shows no significant correlation. Moreover, it is apparent that the relative effectiveness of the 1-substituted adamantanes is not simply a function of the relative sizes of the substituent groups. Thus, polar substituents are much more potent than would be expected on the basis of size alone. It is generally recognized that the interaction energies between a solute and a bulk solvent may be considerably different than those between the solute and a lipid bilayer or biomembrane. Unlike a bulk solvent, a lipid bilayer is a highly organized structure in which the hydrocarbon chains are packed in a highly ordered hexagonal array and lie more or less perpendicular to the plane of the bilayer [20]. It is now clear that the fluidity of the lipid bilayer is a consequence of conformational changes in the hydrocarbon chains. Moreover, the mobility of the methylene growps composing the hydrocarbon chains changes dramatically along the length of the chain with the major effects being observed in methylene groups that are more than eight carbons removed from the carboxyl substituent [21-26]. Thus, both above and below the transition temperature, the portion of a hydrocarbon chain that is near the center of the bilayer is more "fluid" than the portion of the chain near the polar head group. This mobility gradient is a consequence of the close packing of the hydrocarbon chains and the presence of kinks along the chains = [27-31]. The number of kinks in the hydrocarbon chains increases abruptly just above the phase transition temperature [21-26]. At the phase transition temperature, the hydrocarbon chains of lecithin bilayers underl~o a change from a highly ordered tilted hexagonal array to a phase characterized by considerable molecular mobility. The degree to which this mobility is anisotropic varies significantly with the distance from the polar head group.

76

M.K. Jain et a£. Phase transition in a lipid bilayer

In view of these considerations, it is reasonable to expect that a solute induced perturbation in the phase transition of a lipid btlayer should be maximal when the solute is localized hi the most closely packed region of the bilayer, i.e., near the polar head groups. Of course, it follows that minimal perturbations should occur for solutes that are localized near the methyl end of the hydrocarbon chains as this is inherently asignificantly more disordered region of the lipid bilayer. The location of the solute in the lipid bilayer should be determined not only by the solubility of the solute in the hydrophobic environment but also by the orientation and accessibility of polar substituent groups in the solute molecule, Moreover, we would expect that the polar substituent of a solute should be oriented toward the polar head groups of the lipid bilayer provided that the hydrophobic interactions of the remainder of the solute molecule with the hydrocarbon portion of the lipid can accommodate the solute in this orientation. This suggests that subtle molecular structure parameters may significantly influence solute perturbations upon the phase transition of a lipid bflayer. In summary, we propose that solute induced changes in the phase transition characteristics o f a lipid bilayer are determined by the position and orientation o f the solute within the bilayer and that these param-

eters depend upon the amphipathic character of the solute and involve both the polar and hydrophobic interactions of the solute with the bilayer. The experimental results we h~ve obtained with variously substituted adamantanes, protoadamantanes, and homoadamantanes are consistent with these proposals. In particular: 1. The membrane/water partition coefficients of the adamantane derivatives cannot be correlated with their relative potencies. The relative concentrations of adamantane, 1.fluoroadamantane, 1-adamantanecarboxylic acid, 1-adamantanol, 1-adamantanemethano|, and 2-adamantanol ~ liposomes are virtually identical; the HHW100 values of these compounds differ by a factor of more than 50. Thus, the hydrophobicity of these solutes is not a sufficiently sensitive parameter to correlate their relative potencies. 2. The addition of a single polar substituent group to an otherwise nonpolar molecule increases its potency, e.g., compare any of the cage hydrocarbons with its alcohol derivatives. A polar substituent should locate the solute much closer to the polar head groups of the lipid bflayer and hence the potency of the solute should be enhanced. Consistent with this conchsion is the observation that for a given cage skeleton a hydroxyl substituent lea~ to a more potent solute than a keto substituent. 3. Comparison of the relative potencies of similarly substituted cage compounds follows the order: homoadamantane ~ protoadamantane > adamantane. Cage hydrocarbons with identical substitttents wouldbe expected to be located in approximately equivalent regions of the lipid bilayer and possess comparable orientations with respect to the bilayer. Under such circumstances, steric considerations should become important. Molecular m~elsdearly show that the effective volumes of the hydrocarbon cages follow the relative order of their potencies. These same

M.K. Jain et al., Phase transition in a lipid bilayer

77

factors account for the observations that 2-substituted adamantanes are more potent than comparably 1-substituted adamantanes and that in both the 1-halo and 2-haloadmnantanes the order of solute effectiveness is I > Br > C1 > F. 4. The relative potency of ~ solute containing.two polar substituents is determined by the geometric arrangement of the substituents. Thus, 2,5-diendo-protoadaman. tanediol is nearly eight times more potent than 2-exo-5-endo-protoadamantanediol. In the diendo.substituted compound both of the hydroxy substituents are much more closely oriented than is the case in the isomeric diol. By contrast, in 2,5-protoadamantanedione the two carbonyl groups cannot act in concert. This compound is significantly less potent than either 2- or 5.protoadamantanone. These results suggest that lipidic solvent/buffer partition coefficients are of only limited value in predicting either qualitatively or quantitatively relative solute effects on lipid brayers. Such coefficients do not take into consideration the aniso. tropy of brayer organization. It is apparent that the gradients of polarity and fluidity within the microenvironment of lipid brayers promote the subtle interplay of stereoelectronic factors in the interaction of solutes with lipid brayers which so strongly influence phase transition characteristics. Monitoring the influence of doping agents on the endothermic phase transition of a phospholipid brayer has been employed by several research groups as an effective model to assess the relatice pharmacological potencies, membrane expand. ing abilities, and partition coefficients of series of drugs [2, 32-34]. Of course, the biomembrane is the locus of action for several lipid-soluble drugs. Our results suggest that relatively minor modifications in the structure of a solute can significantly alter its impact on the phase transition behavior of a brayer. An understanding and elaboration of the molecular parameters involved in small molecule-bilayer interactions may help to define the molecular mechanisms that distinguish one type or class of lipid.soluble membrane-active drug from another, and may permit the design of more specific and selective membrane-active drugs, as well as non.perturbing membrane probes.

Acknowledgments This work was supported by grants from the donors of the Petroleum Research Fund, admtn~tered by the American ChemicaA Society, the University of Delaware Research Foundation, the Delaware Institute of Medical Education and Research, and the Research Corporation.

References [1] P. Seeman, Pharmacol. Rev. 24 (1972) 583 [21 M.IL lain, N.Y.-I~ Wu and LV. gray, Nature 255 (1975) 494

78

M.K. Jatn et al., Phase transition in a lipid bilayer

[3] S. Eletr, M.A. W ~ T. Watkins and A.D. Keith, Biochim. Biophys. Acta 339 (1974) 190 [41 J. Cupp, M. Klymkowski, J. Sands, A. Keith and W. Snipes, Biochim. Biophy3. Acta 389 (1975) 345 [5] G.A. Olah and J. Welch, Synthesis (1974) 653 [6] L.A. Spurlock and K.P. Clark, J. Am. Chem. Soc. 94 (1972) 5349 {7l R.K. Murray, Jr., B.L. Jelus and K.A. Babiak, Ckg. Mass Spectrom. 9 (1974) 710 [81 H.W. Whitlock, Jr. and bLW. Siefken, J. Am. Chem. Soc. 90 (1968) 4929 [9] J. Boyd and FLH. Overton, J. Chem. Soc., Perki_nTrans. 1 (1972) 2533 [IC] R.K. Munay, Jr. and T.FL Morgan, Jr., J. Or& Chem. 40 (1975) 2642 [II] P.v.R. Schleyer, E. Funke and S.H. Liggero, J. Am. Chem. Soc. 91 (1969) 3965 [12] R.K. Murray, Jr., K.A. Babiak and T.IL Moflgan,Jr., J. Oflg.Chem. 40 (1975) 2463 [13l B.D. Ladbrooke and D. Chapman, Chem. Phys. Lipids 3 (1969) 304 [14l M.C. Ph~i~, B.D. Ladbrooke and D. Chapman, Biochim. Biophys. Acta 196 (I970) 35 [15] J.M. Diamond and E.M. Wright, Annu. Rev. Physiol. 31 (1969) 581 [161 K.H. Sullivan, M.K. Jain and A.L. Koch, Biochim. Biophys. Acta 352 (1974) 287 [17l E.I. Eger, C. Lundgren and CL. Miller, Anesthesiology 30 (1969) 129 [181 B. Fourcans and M.K. Jain, Adv. Lipid Res. 12 (1974) 147 [19] A. Leo, C. Hansch and D. Elkins, Chem. Rev. 71 (1971) 525 [20] M.K. Jain, The Biomolecular Lipid Membrane: A System. Van Nostrand Reinhold (1972) [21] W.L Hubbell and H.M. McConnell, J. An~ C'nem. Soc. 93 (1971) 314 [22] B.G. McFarland and H.M. McConnell, l~oc. Natl. Acad. SoL U.S. 68 (1971) 1274 [23] P. Jost, L.J. Libertini, V.C. Hebert and O.H. Griffith, J. Mol. Biol. 59 (1971) 77 [241 J.C. Metcalfe, NJ.M. Birdsall, J. Feeney, A.G. Lee, Y.K. Levine and P. Partington, Nature 223 (1971.) 199 [251 A.F. Horwitz, W.J. Horsley and M.P. Klein, Proc. Natl. Acad. SoL U.S. 69 (1972) 590 {26] N.J.M. Birdsall, A.G. Lee, Y.K. Levine and J.C. Metcalfe, Biochim. Biophys. Acta 241 (1971) 693 [27] J.E. Rothman, J. Theoret. Biol. 38 (1973) 1 [28] H. Trauble, J. Membrane Biol. 4 (1971) 193 [29] D. Marsh, J. Membrane Biol. 18 (1974) 145 [30] S. Marcelja, Biochim. Biophys. Acta 367 (1974) 165 [31] J.F. Nagle, J. Chem. Phys. 58 (1973) 252 [32] M.W. Hill, Biochim. Biophys. Acta 356 (1974) 117 [33] B.R. Cater, D. Chapman, S.M. H_awesand J. Saville, Biochim. Biophys. Acta 363 (1974) 54 [34] D. Papahadjopoulos, :4. Jacobson, G. Poste and G. Shepherd, Biochim. BiophyL Acta 394 (1975) 504