Thermal Lipid OrderâDisorder Transitions in Complexes of Various

Langmuir 2000, 16, 9729-9737

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Thermal Lipid Order-Disorder Transitions in Complexes of Various Disulfide Tethered Macrocyclic Diacylglycerol Analogues and Dipalmitoyl Phosphatidyl Choline. Role of Diacylglycerol Chain Motions Sangita Ghosh,†,‡ Chittoor P. Swaminathan,‡ Avadhesha Surolia,‡ Kalpathy R. K. Easwaran,‡ and Santanu Bhattacharya*,† Department of Organic Chemistry and Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India Received April 19, 2000. In Final Form: August 30, 2000 The role of diacylglycerol (DAG) chain motions in binary mixtures of DAG with dipalmitoylphosphatidyl choline (DPPC) has been studied using differential scanning calorimetry (DSC). The DAGs used in this study were synthesized to have different extents of chain restriction. This was accomplished via tethering of two long hydrocarbon chains by a reducible disulfide bond at different depths in the chain. The disulfide tether provides the advantage of comparing the effect of incorporation of each of these chain restricted DAGs in DPPC vesicles with their reduced analogue, in which the S-S linkage could be opened up to -SH by in situ treatment with a reducing agent, dithiothreitol (DTT). DSC analysis with such mixtures reveals that restriction of chains at different depths in the DAGs lead to very different properties of the overall DPPC/DAG coaggregate. DAGs in which the disulfide tether is located at the middle of the chain have only partial chain restriction. Incorporation of this type of DAG in DPPC destabilizes the resulting vesicles while the end-tethered DAGs upon incorporation in DPPC vesicles lead to stabilization of the coaggregates. Similar studies with the reduced, open-chain analogues reveal that release in chain restriction leads to extra stabilization in the coaggregates.

Introduction The formation of 1,2-diacylglycerols (DAGs) from phospholipids are known to be stimulated by hormones and neurotransmitters, and its accumulation within the membrane acts to modulate the activity of phospholipase A2 (PLA2)1-4 and protein kinase C (PKC).5 Mammalian cells contain at least 50 structurally distinct molecular species of 1,2-diacylglycerol whose fatty acid composition can be varied.6 Kramer et al. reported7 that the global amount of DAG generated on the cytoplasmic membrane surface alone could be 2-6 mol %, which brings it within range of the concentrations where DAGs induce perturbations in the bilayer structure. Nature uses phospholipase C via a stimulus activation mechanism to generate higher concentrations of DAGs as required in cell membranes. A number of studies have shown that these DAGs induce structural changes in the membrane thus destabilizing the overall bilayer matrix.8,9 It is believed that * To whom correspondence should be addressed. Also at the Chemical Biology Unit, Jawaharlal Nehru Center for Advanced Scientific Research, Bangalore 560 012, India. E-mail: sb@ orgchem.iisc.ernet.in. Fax: +91-80-360-0529. Tel: +91-80-3092664. † Department of Organic Chemistry. ‡ Molecular Biophysics Unit. (1) Pelech, S.; Vance, D. E. Trends Biochem. Soc. 1989, 14, 28. (2) Cunningham, B. A.; Tsujita, T.; Brockman, H. L. Biochemistry 1989, 28, 32. (3) Dawson, R. M. C.; Hemington, N. L.; Irvine, R. F. Biochem. Biophys. Res. Commun. 1983, 117, 196. (4) Nishizuka, Y. Science 1986, 233, 305. (5) (a) Berridge, M. J. Annu. Rev. Biochem. 1987, 56, 159. (b) Das, S.; Rand, R. P. Biochemistry 1986, 25, 2882. (6) Hodgkin, M. N.; Pettitt, T. R.; Martin, A.; Michell, R. H.; Pemberton, A. J.; Wakelam, M. J. O. TIBS, pp 200-204, 23 June issue, 1998. (7) Kramer, R. M.; Checani, G. C.; Deykin, D. Biochemistry 1987, 26, 779. (8) Cheng, K.; Hui, S. W. Arch. Biochem. Biophys. 1986, 244, 382.

small regions of nonbilayer phases or regions of a higher curvature are formed within the bilayer.10-14 This induces membrane destabilization, rendering the bilayer more prone to attack by the membrane bound enzymes.2,3,15-18 The phase behavior of the parent phospholipid membranes on incorporation of DAGs having identical chain lengths as the phospholipid molecules has been characterized earlier using different biophysical techniques.19,20 Such perturbations have also been characterized in part by Ortiz et al.14 They incorporated DAGs of different chain lengths in dipalmitoylphosphatidylcholine (DPPC) vesicles in order to characterize the nature of distortion in each case. De Boeck and Zidovetzki et al.21,22 have extensively used 2H NMR to show that perturbation induced by the DAG into the bilayer structure depends strongly on the length of the fatty acid side chains. Marsh and Schorn23 have studied the effect of incorporation of DAG containing (9) (a) Siegel, D. P.; Banschbach, J.; Alford, D.; Ellens, H.; Lis, L. J.; Quinn, P. J.; Yeagle, P. L.; Bentz, J. Biochemistry 1989, 28, 3703. (b) Siegel, D. P.; Banschbach, J.; Yeagle, P. L. Biochemistry 1989, 28, 5010. (10) Das, S.; Rand, R. P. Biochem. Biophys. Res. Commun. 1984, 124, 491. (11) Dawson, R. M. C.; Irvine, R. F.; Bray, J.; Quinn, P. J. Biochem. Biophys. Res. Commun. 1984, 125, 836. (12) Ohki, K.; Sekiya, T.; Yamauchi, T.; Nozawa, Y. Biochim. Biophys. Acta 1982, 693, 341. (13) Epand, R. M. Biochemistry 1985, 24, 7092. (14) Ortiz, A.; Villalain, J.; Gomez-Fernandez, J. C. Biochemistry 1988, 27, 9030. (15) Burch, R. M. FEBS Lett. 1988, 234, 283. (16) Kramer, R. M.; Jakubowski, J. A.; Deykin, D. Biochim. Biophys. Acta 1988, 959, 269. (17) Kolesnick, R. N.; Paley, A. E. J. Biol. Chem. 1987, 262, 9204. (18) Zidovetzki, R.; Laptalo, L.; Crawford, J. Biochemistry 1992, 31, 7683. (19) Lopez-Garcia, F.; Villalain, J.; Gomez-Fernandez, J. C.; Quinn, P. J. Biophys. J. 1994, 66, 1991. (20) Heimburg, T.; Wu¨rz, U.; Marsh, D. Biophys. J. 1992, 63, 1369. (21) De Boeck, H.; Zidovetzki, R. Biochemistry 1989, 28, 7439. (22) De Boeck, H.; Zidovetzki, R. Biochemistry 1992, 31, 623. (23) Marsh, D.; Schorn, K. Biochemistry 1996, 35, 3831.

10.1021/la000587v CCC: $19.00 © 2000 American Chemical Society Published on Web 11/15/2000

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saturated chains in DPPC vesicles using ESR spectroscopy. They employed DAG or PC analogues that are spinlabeled at different depths within the hydrocarbon chain and have been able to comment on the way saturated diacylglycerols incorporate and destabilize the bilayer structure. However, several other questions remain unanswered. For example, nothing is currently known on how hydrocarbon chain motions in DAGs influence the thermal order-disorder transitions when incorporated in DPPC vesicles. It also remains to be seen whether it is possible to restrict chain mobility by macrocyclization at specific depths within the bilayers, and if so, whether one can utilize these findings for the design of PLA2 modulators. This is relevant as macrocyclic, interchain linked dialkylglycerols are also known to occur in archaebacteria. For instance, lipids of methanococcus jannaschii have been isolated that are based on the macrocyclic diether formed by the linkage between the terminal methyl groups of the phytanyl chains present in the 2,3-di-O-alkyl-sn-glycerol.24 Therefore, to conclusively correlate the nature of such membrane perturbations with DAG structure, there is a need for developing a variety of DAGs with specific modifications such that the location of perturbation within the membrane on incorporation of the DAG is defined. In our ongoing studies25 seeking to establish a possible relationship between lipid molecular structure and membrane properties, we have earlier reported syntheses of a series of novel diacylglycerols in which the two acyl chains are tethered at different depths via a reducible disulfide linkage so as to give macrocycles of various sizes.26,27 Due to this macrocycle formation, there would be restrictions in free motion of the acyl chains to different extents depending on the length of the hydrocarbon chain that is free. In addition to attributing chain perturbation at specific depths within the membrane, these DAGs have the extra advantage of being opened up from the macrocyclic, chain tethered disulfide DAG to the corresponding open chain thiol form, via a -S-S- to -SH reduction reaction. Hence one can conveniently compare the changes in thermotropic properties when the chain restriction is imposed as against the situation in which it is relaxed. In this paper we examine in detail how thermal properties of DPPC vesicles vary on incorporation of each of these DAGs at various concentrations. We have used six different tethered DAGs in this study (Chart 1). To place the effects of incorporation of macrocyclic DAGs into appropriate perspective, we have used in this study two saturated DAGs, dipalmitoylglycerol (DPG) and diundecanoylglycerol (DUG), respectively. Dioleoylglycerol (DOG) was also used to look for the effect of doping the DPPC membrane with an unsaturated DAG. Results In the present study, we have employed six different sized macrocyclic analogues of 1,2-diacylglycerols. DAG 1 has a total chain restriction due to the attachment of a disulfide linker at the end of the two acyl chains. DAG 5 also has a total chain restriction but has a carboncarbon linkage as against a disulfide in DAG 1. Thus while 1 could be opened up by treatment of dithiothreitol (DTT), (24) De Rosa, M.; Gambacorta, A.; Gliozzi, A. Microbiol. Rev. 1986, 50, 70. (25) (a) Bhattacharya, S.; Haldar, S. Langmuir 1995, 11, 4748. (b) Bhattacharya, S.; Haldar, S. Biochim. Biophys. Acta 1996, 1283, 21. (26) Ghosh, S.; Easwaran, K. R. K.; Bhattacharya, S. Tetrahedron Lett. 1996, 37, 5769. (27) Bhattacharya, S.; Ghosh, S.; Easwaran, K. R. K. J. Org. Chem. 1998, 63, 9232.

Ghosh et al. Chart 1

5 remains unaffected. Hence 5 is considered a control that cannot be reduced even in the presence of DTT. DAG 2 provides partial chain restriction due to a tethering approximately at the middle of the hydrocarbon chain, with part of it motionally unrestricted within the membrane. On the other hand, the two hydrocarbon chains in DAG 4 are joined near the chain-glycerol backbone linker region. Therefore in this molecule the hydrocarbon chains are free to dangle beyond the linker region. DAG 3 is a short-chain (C6) diacylglycerol, the chains in which are tethered at their termini and when incorporated within the membrane they are expected to show perturbation, very different from the other tethered DAGs with longer chain lengths. Effects of Incorporating Various Diacylglycerols. The consequences of the addition of a variety of diacylglycerols having different extents of restriction in chain mobility in the DPPC membrane were investigated. The values of the main transition and the heats of transition of pure DPPC suspensions obtained (main transition 7.9 kcal/mol) were found to be in good agreement with that reported.28-30 A variety of effects were observed depending on the molecular structure of the DAG employed and the amount of DAG included in DPPC coaggregates. However, an assumption is made following Chapman et al.29 that all of the added DAG is associated with the lipid matrix (28) Cater, B. R.; Chapman, D.; Hawes, S. M.; Saville, J. Biochim. Biophys. Acta 1974, 363, 54. (29) Chapman, D. Quart. Rev. Biophys. 1975, 8, 185. (30) Eliasz, A. W.; Chapman, D.; Ewing, D. F. Biochim. Biophys. Acta 1976, 448, 220.

DAG Chain Motion in DAG/DPPC by DSC

Langmuir, Vol. 16, No. 25, 2000 9731

Table 1. Gel to Fluid Thermal Phase Transition Properties of DAG/DPPCa Covesicles at Varying DAG 1 Concentrations [DAG 1] (mol %)

Tm1b (°C) pre

Tm2b (°C) main

∆Hcc (kcal/mol)

∆Sd (cal/K mol)

∆Hve (kcal/mol)

CUf (molecules)

0 1 3 10 15 30 3(red.)g 10(red.)g 30(red.)g

35.0 34.3 35.4 33.4 s s 35.1 s s

40.3 40.8 40.9 40.9 40.5 39.6 41.3 41.2 42.1

7.5 6.4 5.1 4.6 3.4 1.5 3.6 3.8 3.9

24.0 20.3 16.2 14.6 10.9 4.9 11.5 12.1 12.4

1493 599 421 282 320 146 330 257 137

199 94 83 62 94 96 92 68 35

a [DPPC] used in this study is 1 mM in water. b T refers to transition temperature (°C) and the values varied by (0.1 °C between m successive runs. c ∆Hcal values refer to the calorimetric enthalpies and are estimated to be accurate by (5%. d ∆S values refer to the entropy of transition and were calculated by dividing ∆Hc/Tm assuming phase transition to be a first-order process, and are accurate by (5%.f The CU values (cooperativity unit, determined from the ratio of ∆Hv/∆Hc) are accurate by (2%. g These samples contain DAG 1 in the specific mol % that was reduced to -SH form prior their incorporation in DPPC membranes.

Figure 1. Typical DSC endotherm showing the apparent excess heat capacity for the solid to fluid phase transition of covesicles comprising DPPC and DAG 4 (30 mol %). The concentration of DPPC was 1 mM in water and the scan rate was 90 °C/h. Deconvolution of progress baseline subtracted and concentration normalized DSC curve: (OsO) raw data (‚‚‚) first deconvoluted peak (- - -) second deconvoluted peak (s) fit curve.

of the membranes. Figure 1 shows a typical DSC endotherm along with the fit of the transition peak data for a coaggregate comprising DPPC and DAG 4. Deconvolution of the transition peak indicates that it consists of two closely distributed transition components differing by 2-3 °C. The results of the fits of the transition are given in Tables 1-6. End-Tethered Diacylglycerol, DAG 1. The structure of this molecule is shown in Chart 1. In this molecule (DAG 1), a 38-membered macrocyclic unit is present in which the two acyl chains, which are of similar length as that of DPPC, are joined at the chain termini by a disulfide bridge. Such tethering imposes practically complete chain restriction in the molecule. The disulfide tether is located ∼20.6 Å in the bilayer from the glycerol backbone of the DAG as revealed from the calculation of its energy

Figure 2. DSC thermograms for the coaggregates of DPPC/ DAG 1. Mole percent of DAG is indicated at the right end of the traces. Only heating experiments are shown. The Y-axis has been arbitrarily shifted.

minimized conformational studies using the INSIGHT II 2.3.5 package (DISCOVER, Biosym. Technologies) (not shown). Table 1 records the changes in thermotropic parameters upon incorporation of 1 in DPPC vesicles at different concentrations. The addition of increasing concentrations of 1 induces a gradual decrease in pretransition peak in DPPC with its complete disappearance at ca. 15 mol % 1 (Figure 2). The peak due to the main transition, however, shifts to higher temperatures up to 10 mol % of 1. At the same time, this peak also broadens, though the broadened peak maximum due to Tm is shifted only by about 0.5 °C. At >30 mol % of 1, there is a gradual shift of the main transition to a lower temperature (i.e., 39.6 °C), which is lower than that of pure DPPC. The

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Table 2. Gel to Fluid Thermal Phase Transition Properties of DAG/DPPCCovesicles at Varying DAG 2 Concentrationsa

a

[DAG 2] (mol %)

Tm1 (°C) pre

Tm2 (°C) main

∆Hc (kcal/mol)

∆S (cal/K mol)

∆Hv (kcal/mol)

0 5 10 30 10(red.)b 30(red.)b

35.0 s s s s s

40.3 39.8 39.8 39.9 39.3 39.0

7.5 5.8 5.7 5.3 6.2 7.4

24.0 18.6 18.1 17.0 19.6 23.8

1493 355 213 216 262 185

CU (molecules) 199 61 38 41 43 25

All conditions as in Table 1. b The corresponding reduced form of DAG 2.

Figure 3. Plot of main transition temperature (Tm) and cooperativity unit as a function of concentration of DAG 1. Empty circles represent Tm of the DPPC/DAG 1 coaggregates, Hollow squares represent the Tm of the coaggregates of DPPC and the reduced (dithiol) form of DAG 1. Filled circles represent the cooperativity unit of the DPPC/DAG 1 coaggregates. Filled squares represent the cooperativity unit of the coaggregates of DPPC and reduced (dithiol) form of DAG 1.

calorimetric enthalpy ∆Hc also decreases progressively with the increase in concentration of 1, being 1.5 kcal/mol at 30 mol % of 1. A look at the variation of cooperativity unit (CU) with increase in 1 also reveals an interesting trend. Starting with a highly cooperative DPPC bilayer (CU 199), incorporation of even 1 mol % of 1 in DPPC drastically reduces the CU to ∼94 in the resulting coaggregate. The same trend follows till the incorporated 1 reaches 10 mol %. Inclusion of 1 beyond 10 mol % however, shows a recovery of CU again. These dependencies as a function of incorporated DAG 1 concentration in DPPC bilayers are shown in Figure 3. The DAG 1, when reduced with DTT, produces a DAG derivative of 1 in which the disulfide tether is replaced by -SH groups at the terminus of either chain. This leads to withdrawal of the restriction of chain motion that is imposed due to disulfide tethering. Inclusion of DTT did not alter the existence of pretransition (35 °C) and main transition (40.5 °C) temperatures of DPPC although a very small posttransition peak was seen at 42 °C. Both the peaks due to the pretransition and posttransition, however, disappeared when DTT along with the reduced form of DAG 1 were included in DPPC (Figure 4). The main transition due to DPPC got broadened significantly with the inclusion of the reduced form of 1 and the small posttransition peak got merged with this transition abolishing the pretransition peak as well. A comparison of the CU at 10 mol % of macrocyclic disulfide linked 1 in DPPC bilayer and that of its reduced open-chain counterpart at the same concentration in DPPC bilayers

Figure 4. DSC thermograms for the coaggregates of DPPC and reduced (dithiol) form of DAG 1. The mole percent of DAG is indicated at the right end of the traces. Only heating experiments are shown. The Y-axis has been arbitrarily shifted.

show an enhanced cooperativity on relaxation of chain tethering due to reduction of the disulfide bond (Table 1). Middle Tethered Diacylglycerol, DAG 2. DAG 2 has a tethering approximately at the middle level of the hydrocarbon chain thus giving a 30-membered macrocycle which is smaller in ring size than that of DAG 1. Beyond the points of tether, the rest of the hydrocarbon chain remains free and motionally unrestricted. 2 therefore has only partial restriction in chain motion and its incorporation should induce perturbation around the middle of each leaflet in the bilayer. This tether should be located ∼14.7 Å deeper from either faces of the bilayer. Notably, the effects of incorporation of 2 in DPPC membranes appear to be quite different from that of 1. As seen from Table 2, as little as 5 mol % of 2 already makes the pretransition disappear, while the main transition gets broadened and shifted to lower transition temperatures (not shown). This indicates that incorporation of 2 in the DPPC bilayer leads to membrane destabilization and hence the resulting aggregate melts at a lower temperature. With a further increase in DAG 2 concentration, the ∆Hc of the composite also decreases progressively. However, the decrease is not as drastic as in the case of 1. The CU decreases with an increase in the concentration of 2 with a minimum observed at around 10 mol % of 2 and remains nearly the same up to 30 mol %. However, on incorporation of the reduced, open chain form of 2, there is an increase in the


Langmuir, Vol. 16, No. 25, 2000 9733

Table 3. Gel to Fluid Thermal Phase Transition Properties of DAG/DPPC Covesicles at Varying DAG 4 Concentrationsa

a

[DAG 4] (mol %)

Tm1 (°C) pre

Tm2 (°C) main

∆Hcal (kcal/mol)

∆S (cal/K mol)

∆Hv (kcal/mol)

CU (molecules)

0 5 15 30 5(red.)b 10(red.)b 30(red.)b

35.0 s s s s s s

40.3 39.5 39.5 39.3 41.5 44.6 47.1

7.5 7.5 7.2 5.4 7.5 8.3 7.4

24.0 24.0 23.0 17.4 23.8 26.1 23.8

1493 254 170 147 359 230 185

199 34 24 27 48 28 25



a

[DAG 3] (mol %)

Tm1 (°C) pre

Tm2 (°C) main

∆Hcal (kcal/mol)

∆S (cal/K mol)

∆Hv (kcal/mol)

CU (molecules)

0 5 10 20 30 5(red.)b 10(red.)b 30(red.)b

35.0 s s s s s s s

40.3 39.0 38.9 37.2 36.4 40.1 39.6 38.7

7.5 6.9 6.6 6.5 2.5 7.1 6.9 6.4

23.9 22.1 21.2 20.9 7.9 22.6 22.1 20.6

1493 617 423 190 203 809 546 423

199 90 64 29 83 115 79 66


Figure 5. Plot of main transition temperature (Tm) and CU as a function of concentration of DAG 4. Empty circles represent the Tm of the DPPC/DAG 4 coaggregates. Hollow squares represent the Tm of the coaggregates of DPPC and the reduced (dithiol) form of DAG 4. Filled circles represent the CU of the DPPC/DAG 4 coaggregates. Filled squares represent the CU of the coaggregates of DPPC and reduced (dithiol) form of DAG 4.

Figure 6. Plot of main transition temperature (Tm) and CU as a function of concentration of DAG 3. Empty circles represent Tm of the DPPC/DAG 3 coaggregates. Hollow squares represent the Tm of the coaggregates of DPPC and the reduced (dithiol) form of DAG 3. Filled circles represent the CU of the DPPC/ DAG 3 coaggregates. Filled squares represent the CU of the coaggregates of DPPC and reduced (dithiol) form of DAG 3.

cooperativity of the thermal transition at 10 mol % of the reduced 2, compared to its parent, disulfide tethered macrocyclic counterpart, 2. Linker-Tethered DAG 4. In this DAG, the disulfide linkage joins the two carbon atoms of the hydrocarbon chains that are located adjacent to the chain-linking ester carbonyl groups. Hence the motional chain restriction is expected to be a minimum with this molecule since it has most of the hydrocarbon chain remaining free and would thus offer perturbation around the linker region when incorporated in the bilayer. The effects of this DAG in DPPC membranes are quite similar to that of 2. Thus the Tm of the resulting DAG 4/DPPC coaggregates get progressively decreased with increasing concentration of 4 (Table 3). The pretransition of DPPC is abolished upon incorporation of ∼5 mol % of 4 and then decreases gradually with concentration but shows a slight increase at 30 mol % of 4 (Figure 5). Upon reduction, 4 is converted

to the corresponding dithiol where the chains are fully unrestricted. Interestingly, incorporation of the reduced form of 4 in DPPC membranes leads to an enhancement in the phase transition temperature of the resulting mixture. The increase in Tm becomes more pronounced with increasing concentration of the reduced 4 in DPPC. Short Chain End-Tethered DAG 3. Like DAG 1, the diacylglycerol derivative DAG 3 is also end-tethered through a disulfide connection. However, 3 differs from 1 in that it has shorter acyl (C6) chains comprising (CH2)5 units that are joined at the end with a disulfide tether, thereby giving a smaller macrocyclic ring. This DAG molecule would likely be located at a depth of ∼7 Å from the interfacial region of the membrane. It can be seen (Table 4) that the thermotropic behavior of the 3 incorporated DPPC membrane is quite similar to that of the mixture containing naturally occurring short chain DAG, 1,2-dicaproylglycerol (1,2-DCG), and DPPC.28 In this case

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a

[DAG 5] (mol %)

Tm1 (°C)

Tm2 (°C)

Tm3 (°C)

∆Hc (kcal/mol)

∆S (cal/K mol)

∆Hv (kcal/mol)

CU (molecules)

0 5 10 15 20 25 30

35.0 s s s s s s

40.3 40.3 40.5 40.8 s s s

s s s 42.2 43.0 43.2 44.3

7.5 7.0 6.7 s 6.3 6.9 5.9

23.9 22.3 21.4 s 19.9 22.1 18.6

1493 901 667 s 265 315 434

199 128 99 s 42 46 73

All conditions as in Table 1.


a

[DAG 6] (mol %)

Tm1 (°C) pre

Tm2 (°C) main

∆Hc (kcal/mol)

∆S (cal/K mol)

∆Hv (kcal/mol)

CU (molecules)

0 5 10 15 20 5(red.)b 10(red.)b 30(red.)b

35.0 s s s s s s s

40.3 41.0 41.9 42.3 42.4 41.2 42.5 49.2

7.5 4.4 3.8 2.8 1.1 4.7 4.0 5.7

23.9 14.0 12.1 8.9 3.5 15.1 12.7 17.5

1493 298 236 249 235 397 208 592

199 68 63 90 216 84 52 105

All conditions as in Table 1. b The corresponding reduced form of DAG 6. Table 7. Gel to Fluid Thermal Phase Transition Properties of Different Non-macrocyclic Occurring DAG/DPPC Coaggregates at Varying DAG Concentrationsa

lipid aggregate DUG

DPG

DOG

a

[DAG] (mol %)

Tm1 (°C) main

Tm2 (°C)

∆Hc (kcal/mol)

∆S (cal/K mol)

∆Hv (kcal/mol)

CU (molecules)

0 10 20 25 30 5 10 15 30 5 15 30

40.3 42.5 s s s 41.0 46.0 s s 41.0 40.4 39.3

s 44.6 45.2 45.4 45.5 47.0 53.4 53.7 57.0 s s s

7.5 3.2 8.0 8.1 9.0 5.1 6.2 11.7 14.7 9.1 7.7 8.8

23.9 10.1 25.3 25.4 28.3 16.2 19.4 35.8 44.5 29.0 24.6 28.2

1493 416 186 179 217 156 97.4 75.1 67.6 985 819 697

199 130 23 22 24 31 16 6 5 108 106 79

All conditions as in Table 1.

too, the pretransition is lost at ∼5 mol % of 3. The main transition temperatures and enthalpies decrease with an increase in the concentration of 3 up to 20 mol %. However, at >20 mol % concentrations extra peaks appear with Tm of 33.2 and 36.4 °C. Incorporation of the reduced form of 3 in DPPC also results in a gradual lowering in Tm values. However, by comparison of the macrocyclic versus the open-chain reduced analogue, incorporated in DPPC vesicles, there is an increase in the Tm with other reduced DAG/DPPC coaggregates. The cooperativities of the thermal transitions get reduced with the increase in 3 content irrespective of the fact that 3 is included in its disulfide state or in the corresponding reduced bis-thiol form. However, the loss in CU is more pronounced with 3 than that of its reduced analogue (Figure 6). Carbon-Carbon End-Tethered DAG 5. Unlike the previously described diacylglycerol derivatives with disulfide tether, the ends of the two acyl chains of DAG 5 are linked via a carbon-carbon bond rather than a disulfide bond. Hence this DAG could not be opened up by reduction with dithiothreitol as could be done for the other macrocyclic DAGs 1-4. However, the effect of incorporation of this macrocyclic 5 in DPPC can be compared with its corresponding open chain counterpart, 1,2-diundecanoyl glycerol (DUG), which was synthesized as described. As seen in Table 5, doping with an increasing amount of 5 raises the main transition temperature and at 15 mol % two peaks are observed. Beyond 15 mol %, the main transition is lost and only the phase that has a higher

melting temperature exists. The CU also follows this trend as seen before for the other DAGs. Starting with a considerably high CU of 128 at 5 mol %, the CU decreases to a value of 42 at 20 mol % after which it again increases to ∼73 at 30 mol % (Table 5). Diundecanoyl Glycerol, DUG. To compare the effects of macrocyclization, we recorded the thermotropic phase transition for diundecanoyl glycerol which has the same chain length as that of macrocyclic DAG 5. In this DUG, the terminal methyl groups of the undecanoyl chains remain untethered unlike in 5 where the two terminal carbon atoms are joined by an extra carbon-carbon bond to form the macrocyclic ring. The main transition temperature of its covesicles with DPPC shifts to higher values with increasing concentration of DUG. Beyond 10 mol %, the only phase that melts at higher temperature exists and shifts to still higher temperatures with further increase in concentration. Notably, the CU values of the coaggregates of DPPC and DUG remain almost the same despite increases in concentration of DUG, unlike what was observed with other macrocyclic DAGs including 5. End-Tethered Dialkylglycerol, DAG 6. This DAG, unlike the other diacylglycerol derivatives discussed above, has ether linkages instead of ester linkages between the hydrocarbon chain and the glycerol backbone. In addition, the hydrocarbon chains are tethered through a disulfide connection in this molecule. Despite differences in linkage function, the observed trend was similar to the other endtethered, diester DAGs 1 and 5 (Table 6). The Tm values


of the DPPC coaggregates shift to higher temperatures with an increase in concentration of 6. ∆Hc decreases progressively and the CU of the resulting aggregate shows the same trend as is observed with other DAGs. On reducing this DAG with DTT, the macrocycle opens up. Inclusion of increasing amounts of reduced derivative of 6 in DPPC shows a dramatic increase in Tm. With as low as 5 mol % of reduced 6 in DPPC, a higher melting phase other than the main transition is observed. The Tm of this phase also keeps increasing with 6 concentration. Upon incorporation of 30 mol % of reduced 6 in DPPC, the main transition temperature rises to ∼49 °C and an additional peak is also seen at ∼56 °C. The CU also shows an increased value upon reduction as compared to the corresponding macrocyclic analogue, 6 at the same concentration. Discussion To understand the process of lipid mediated signal transduction across biological membranes, it is necessary to know the physical details of mixed DAG/ phospholipid coaggregate systems. The array of lipid bilayers formed by the DPPC-water system could be affected due to incorporation of DAG in a variety of ways. Many techniques have been employed in the past to understand the phase behavior of the DAG incorporated phospholipid systems.19,20 The DAGs might change the van der Waals interactions between the phospholipid hydrocarbon chains.30 They can also spread apart polar headgroups of the phospholipids thereby changing the net charge separation or can even induce nonbilayer phases.10 When the DAGs are present in the bilayer, the extent of hydration at the level of the phospholipid headgroup is also modified. The present work was initiated to develop an understanding at the molecular level of how the imposition of specified restrictions of chain motions of diacylglycerols influence the thermally induced order-disorder transitions of DAG associated phospholipid membranes. In view of their presence in natural membranes24 and their proposed roles in the transduction of molecular signals across biological membranes, it is important to investigate the thermal properties of the complexes of chain motion restricted DAGs and phospholipids. We first discuss the effect of incorporation of DAG 1 in the DPPC bilayer. Incorporation of this molecule in the bilayer should be facile due to its rodlike shape. This should lead to a decrease in the ∆Hc upon inclusion of 1. Moreover, the Tm shifts to higher temperatures because in this DAG, the terminal methyl groups are rendered immobile due to end disulfide tethering. This in turn causes rigidification and consequent thermal stabilization of the membrane. Examination of the changes in the CU values indicates that on addition of 1 at concentrations up to 10 mol %, small DAG rich regions are probably formed within the bilayer framework of DPPC. This results in a loss of homogeneity of the bilayer composition and hence causes a decrease in CU. These patches also appear as isotropic phases in 31P NMR.31 On further increase in the concentration of 1 to 30 mol %, an apparently more homogeneous membrane is formed, where the small DAG-rich regions seem to disappear. Hence the resulting membrane melts more cooperatively. This resulting bilayer is more fluid compared to the parent DPPC membrane as evidenced from anisotropy measurements (as sensed from doped diphenylhexatriene in membranes).31 This can be further substantiated by the disappearance of the isotropic phases (31) Ghosh, S.; Easwaran, K. R. K.; Bhattacharya, S. Unpublished results.

Langmuir, Vol. 16, No. 25, 2000 9735

in 31P NMR. To further substantiate these points, we also measured leakage rates of entrapped dye 6-carboxyfluorescein (CF) from various DAG/DPPC complexes. This rate for 30 mol % 1/DPPC covesicles is less than that from 10 mol % 1/DPPC covesicles. This suggests that presumably due to the existence of microscopic patches in 1/DPPC (10 mol %) coaggregates, the membrane organization contains defects and hence the leakage rate of CF is faster. Microviscosity measurements due to doped 1,6-diphenylhexatriene also indicate that 1 doped membrane reaches maximum microviscosity at around 15 mol % after which it declines steadily till 30 mol %. In short, the incorporation of 1 up to ∼15 mol % into DPPC membranes gradually rigidifies the resulting bilayer though with a steady decrease of cooperativity. Beyond 15 mol % of 1, the membrane starts to accommodate 1 and becomes more homogeneous leading to fluidization of the overall membrane. Once reduced, the disulfide linkages in 1 are replaced with free thiol residues. Incorporation of the resulting reduced form of 1 in DPPC leads to the manifestation of phases that melt at higher temperatures. This behavior is similar to what was seen with naturally occurring saturated chain dipalmitoyl glycerol (DPG) as reported by Ortiz et al.14 Since calorimetric thermograms are sensitive to the experimental details of membrane preparation and also depend on the method employed, we repeated heating scans with DPG/DPPC bilayer systems under our experimental conditions. A new phase was observed from 5 mol % DPG and this phase kept increasing at the expense of the DPPC main transition and at 15 mol % of DPG, only the higher melting phase was observed (Table 7). On further increase in the DPG concentration, Tm phase shifts to higher values and at ∼30 mol % of DPG, the resulting DPG/DPPC coaggregate melts at 57 °C. Similarly when the reduced form of DAG 1 is incorporated in DPPC membranes, an increase in the main transition temperature along with the formation of two discrete phases is observed in DPG/DPPC covesicular systems. A close look at the CU of the 1/DPPC coaggregates and comparison with that of its reduced analogue clearly shows that on reductive opening of the disulfide macrocycle, there is a release in the chain restriction. This reinforces the destabilizing effect from the freedom of motion of terminal methyl groups in the resulting membranes. This makes it difficult to accommodate the open-chain DAG in DPPC and pack itself uniformly within the bilayer. This is also reflected in the formation of a different phase that melts at a higher temperature. However, the open-chain bis-thiol from of 1, due to its resemblance in terms of the hydrocarbon chain length and its mobility with that of DPPC molecules, gets distributed more evenly. Hence the change in bilayer properties due to 1 is mainly dictated by perturbations at the midplane of the bilayer where the acyl chains terminate and we observe an increased bilayer rigidity due to an imposition of restriction in chain motion. Moreover, this restriction also enables 1 to pack better in the DPPC bilayers as evidenced from the formation of one single phase. In comparison, the incorporation of its reduced analogue in DPPC bilayers leads to a phase separation suggesting an incompatibility of the reduced 1 in DPPC membranes. DAG 6 has a similar chain length to that of 1 and also has a disulfide linkage at the end of the hydrocarbon chains. But the hydrocarbon chains in 6 are connected to the glycerol backbone via an ether linkage rather than via an ester function. Thermotropic measurements indicate that major changes in perturbation as reflected by

9736


the melting behavior of the DAG/DPPC bilayer system comes from a restriction in chain motion and no major change is reflected by the phase properties on changing the linker region from a diester to a diether in the DAG. Here too, the Tm shifts to higher temperatures with increasing concentrations and CU changes as in the case of 1. DAG 5 in which the two acyl chains are joined at the end by a carbon-carbon tether also imposes practically total restriction in its chain mobility. This also yields a situation where its incorporation in a DPPC membrane leads to rigidification and stabilization of the resulting complex. Thus a higher melting temperature was observed with DAG 5/DPPC. Interestingly, the CU values of the binary aggregate 5/DPPC at any concentration of 5 are higher than that containing disulfide end-tethered 1 at the same concentrations. End tethering is an important factor in the thermal stabilization of bilayer membranes which also renders the resulting order-disorder a more cooperative one. This is observed with membranes organized from end-tethered phosphatidyl choline analogues.32 Notably however, doping of 1 in DPPC generated a membrane whose thermal transitions are less cooperative than that of the membrane in which 5 is doped. Both 1 and 5 are end-tethered diacylglycerols. However, 1 contains larger sized sulfur atoms at its terminal tether and hence might be less compatible in DPPC bilayers than 5.This explains why the thermal transitions of the 1/DPPC covesicles are less cooperative than that of 5/DPPC coaggregates. Interestingly, the thermal properties change totally in the case of 2 and 4 where the restrictions are at the middle of the acyl chains or close to the linker region, respectively. Calorimetric studies of their complexes with DPPC indicate that incorporation of these partially chain restricted DAGs make more destabilized bilayers as seen by the lowering of the Tm and also by the abolition of pretransition. In these cases also, the homogeneity of the resulting membranes remain similar up to DAG concentrations as high as 30 mol %, as found from the trends of CU variation as a function of DAG concentration. Taken together these findings point toward the fact that incorporation of a DAG with partial chain restriction, and perturbation around the middle of the hydrocarbon part of the bilayer as in 2 and 4, leads to fluidization of the membrane. This is in contrast to the situation involving the DAGs in which there is a total chain restriction. Also a DPPC membrane doped with 2 is less homogeneous compared to the membrane doped with 1. This might be due to the fact that 2 has a smaller sized macrocycle only up to twelve carbon atoms and after this the rest of the chains comprising the (CH2)6CH3 segment in 2 is free. This presumably leads to incompatibility in the structural features of hydrocarbon chain regions with that of DPPC in the bilayer. Moreover, in this case, the thermal stabilization due to restriction of chain motion of the chain termini is withdrawn. A brief molecular mechanics study for 4 indeed reveals that the glycerol backbone region of this molecule does not overlap with that of DPPC27 and this probably makes it difficult to pack itself, thereby leading to a loss in homogeneity of the resulting membrane. Here also, reduction and therefore opening of the macrocyclic ring has the same effect on the melting temperature and CU as that observed for 1. Opening of the macrocycle gives a saturated chain analogue of DAGs (32) Menger, F. M.; Chen, X. Y.; Brocchini, S.; Hopkins, H. P.; Hamilton, D. J. Am. Chem. Soc. 1993, 115, 6600.

Ghosh et al.

2 and 4 which now give a better organized membrane that melts at temperatures higher than that of the DPPC bilayer. This membrane now has a higher CU than its macrocyclic DAG doped counterpart. Another feature that we noted was the relatively higher values for ∆Hc for membranes doped with 4. We feel that this might be due to the presence of a hydrophobic disulfide tether close to the linker region that may also affect the headgroup hydration at the membrane interfaces. A previous report of fluidification of membrane due to DAG addition has been observed in 1,2-dioleoylglycerol (DOG). On repeating heating scans for DOG/DPPC systems, under our experimental conditions we observed a reduction in the main transition temperature to 39.3 °C at 30 mol % with loss of pretransition peak at 5 mol %, similar to what has been found by Ortiz et al.14 The short-chain DAG 3, though tethered through a disulfide linkage at the chain termini, is a completely different system from the other end-linked DAGs because of its small hydrocarbon length. In this case, upon incorporation of 3 in DPPC membranes, voids within the bilayer are created due to incompatibility in chain lengths. This might explain why a more fluid membrane is produced in this situation, which is indeed observed from the melting profiles of 3/DPPC mixed bilayers of varying concentration of 3. Upon reduction of 3 and incorporation of the same in DPPC, the Tm shifts to a higher temperature and the corresponding Tm values are higher than that of the DPPC coaggregates containing the tethered 3 as has been observed for all other DAGs. Conclusions Studies done in the past with simple saturated diacylglycerols,19,20 fatty acids, long-chain alcohols,30 and alkanes, show the induction of nonbilayer phases, thereby destabilizing the bilayer matrix. However, none of these reports addresses the issue of chain motions in diacylglycerols. Herein we report for the first time the variation in thermotropic phase properties of diacylglycerol analogue-DPPC complexes in which the extent of chain perturbation of the DAG has been modulated by using macrocyclic DAGs of different extents of chain restriction. Since diacylglycerols play a very important role in controlling the activities of various membrane bound enzymes such as protein kinase C (PKC) and phospholipase A2 (PLA2), it might be possible to find a putative basis for a role of diacylglycerols in terms of their molecular architectures. These findings indicate that those macrocyclic DAGs that destabilize the thermal properties of the DPPC bilayer and fluidize the resulting membranes might act as activators of these enzymes. However, both dicaproyl and its macrocyclic counterpart 3 impart comparable destabilization effects upon incorporation in to DPPC vesicles in terms of their thermal transition behavior. This indicates that the effects of restricting chain motions with shorter chain DAG are different from the DAGs in which the chain lengths are comparable to that of the matrix lipid (DPPC). In contrast, the end-tethered DAGs that upon incorporation in DPPC rigidify the resulting complex might show different effects on these enzymes. Introduction of tethered diacylglycerol analogues in these studies therefore represents a new approach with which it might be possible to investigate molecular events and specificity necessary for the control of PLA2 activities. Experimental Section General. DPPC (dipalmitoylphosphocholine) and DTT (dithiothreitol) were obtained from Sigma Chemical Co. Undecylenic acid, N,N-dicyclohexylcarbodiimide, and DMAP were obtained

DAG Chain Motion in DAG/DPPC by DSC from Aldrich and used without further purification. Water was distilled in an all-glass apparatus and deionized in a Milli-Q apparatus from Millipore Iberica (Spain). The disulfide containing macrocyclic diacylglycerols were synthesized in our laboratory as described elsewhere.26 Synthesis of 1,2-Diundecanoyl Glycerol (DUG) (Control DAG Derivative). (+)-Benzyl glycerol (1.5 mmol) was esterified with 10-undecenoic acid by adding a solution of the diol in dry CCl4 (0.1 g/mL) to 2.2 molar equivalents of the acid in dry CCl4 (0.1 g/mL) and a catalytic amount of 4-(dimethylamino)pyridine at 0 °C. To the resulting mixture was added N,N-dicyclohexylcarbodiimide (0.47 g, 2.2 mmol) and was stirred for 4 h. DCU formed during the reaction was filtered off and the precipitate washed with 2 mL CCl4. The filtrate and washings are taken together and the solvent was evaporated to give a crude product which was purified by column chromatography eluting with hexane/EtOAc (12:1). The product was characterized by IR, 1H NMR, and 13C NMR. The product, 1,2-bis-(10-undecenoyl)-3-O-benzyl glycerol, was then subjected to hydrogenation both to effect the removal of the O-benzyl protecting group and to reduce the olefinic double bond of the 10-undecenoyl chains. A solution of this compound (0.5 g), in THF-ethanol (20 mL, 4:1 v/v) was hydrogenated in the presence of 10% Pd/C (0.25 g, 50% w/w) at 40 psi for 3 h. The catalyst was filtered off, the solvent was evaporated, and the pure product was obtained as a solid in 85% yield by column chromatography eluting with hexane/EtOAc (6:1). The product was characterized by IR, 1H NMR, 13C NMR, and mass spectrometry. Differential Scanning Calorimetry (DSC). DPPC (1.47 mg, 0.002 mmol) and the DAGs (5-30 mol % relative to DPPC)

Langmuir, Vol. 16, No. 25, 2000 9737 were dissolved together in a solution of chloroform and methanol (2:1 v/v) in a wheaton glass vial. The solvent was evaporated under a stream of N2 so as to deposit the lipid as a film on the walls of the vial. The last traces of solvent were removed by keeping the film under vacuum for 3 h. In the case of the reduction experiments, DTT was also added (3 eq. per eq. of the DAG taken) as a methanol solution along with the solution of the phospholipid and DAG and dried as described above. Multilamellar vesicles were then prepared by dispersing the lipid film in 2 mL of water (Milli-Q). The vesicle suspension was subjected to three freezethaw cycles. DSC measurements were carried out with a Microcal MC-2 heat conduction differential scanning calorimeter which consists of a reference cell and a sample cell. The melting behavior was monitored using water (Milli-Q) as the reference. Heating scans were recorded between 30 and 60 °C at a scan rate of 1.5 deg/min. The calorimetric data were analyzed using Origin data analysis software for DSC supplied by the manufacturer. Tm is the temperature at half of the transition peak area. The ratio of the van’t Hoff enthalpy (∆Hv) to the calorimetric enthalpy (∆Hc) i.e., ∆Hv/∆Hc, provides the measure of the cooperativity of the transition. Analysis of DPPC, using this protocol, produces thermotropic data that are in agreement with those that were previously reported.28

Acknowledgment. This work was supported by the Department of Science and Technology Grant No. DST/INT/ISR/M-6/97. LA000587V

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