Phospholipids showing complex bilayer phase transitions. II. Four sulfur-containing phosphatidylcholines

Chemistry and Physics of Lipids, 55 (1990) 323--330

323

Elsevier Scientific Publishers Ireland Ltd.

Phospholipids showing complex bilayer phase transitions. II. Four sulfur-containing phosphatidylcholines* Joseph Hajdu" and Julian M. Sturtevant b •Department of Chemistry, California State University, Northridge, California 91330 (U.S.A.) and ~Department of Chemistry, Yale University, New Haven, Connecticut 06511 (U.S.A.) (Received March 28th, 1990; revision received June 15th, 1990; accepted June 15th, 1990)

The main phase transitions of aqueous dispersions of four synthetic phosphatidylcholines (PCs) containing sulfur atoms in thioester and thioether linkages have been studied by high sensitivity differential scanning calorimetry (DSC). The transition enthalpies ranged from 7.4 to 10.3 kcal mol "j, with values for t®, the temperature of maximal excess heat capacity, in the range 38.0 to 40.8°C. The corresponding values for dipalmitoyl PC (DPPC) are 8.5 kcal tool-' and 41.7°C. Curve fitting required the sum of from one to four two-state components to give an accurate representation of the observed DSC curves. Comparison of the results given here with those reported by B.Z. Chowdhry, (3. Lipka, J. Hajdu and J.M. Sturtevant, (1984) Biochemistry 23, 2044--2049, for PCs containing arnide, ether or carbamoyl linkages in place of the usual ester bonds shows that small changes in organic structure can result in large changes in thermotropic behavior. The complexity in the cases showing more than a single two-state component is presumably due to a series of sequential cooperative transitions the character of which is at present unknown.

Keywords: lipid phase transition; sulfur-containing phosphatidycholines; differential scanning calorimetry.

Introduction

Sulfur substituted phospholipid analogues represent a new class of synthetic compounds that recently began receiving increasing attention and growing significance in a number of areas of phospholipid biochemistry [1--7]. Specifically, replacement of the scissile carboxylic ester function by the corresponding thioester moiety has provided a series of chromogenic phospholipase substrates, allowing introduction of highly specific and sensitive spectrophometric assay methods for kinetic characterization and mechanisitic elucidation of lipolytic enzymes [4,8 --10]. Furthermore, the introduction of thioether substituents at the sn-I position of alkyl-ether phospholipids resulted in a number of new antitumor active analogues of plateletCorrespondence to: J.M. Sturtevant. *Paper i: B.Z. Chowdhry, G. Lipka, J. Hajdu and J.M. Sturtevant (1984) Biochemistry 23, 2044--2049.

activator factor (PAF) exhibiting substantially lower platelet aggregating and immunosuppressing chemotherapeutic side effects than the corresponding O-alkyl reference compounds [2,3,7]. A number of these compounds are currently under investigation as antileukemic phospholipids and one such thioether analogue (Iimofosine) has reached phase II clinical trials as a potential anticancer drug [6]. Recent structure-activity studies aimed at delineation of interactions between phospholipase enzymes and structurally modified nonhydrolyzable phospholipid analogues led to the development of sn-l-thioalkyl compounds showing a tenfold increase in binding to phospholipase A s enzymes compared to the corresponding sn-l-O-alkyi derivatives. Indeed, such thioether compounds with acylaminodeoxy substituents at the sn-2-position were found to be among the most potent phospholipase A s inhibitors developed to date [11--12].

0009-3084/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in lreland

324

Despite the rapidly broadening spectrum of enzymological, cell-biological and medicinal applications of sulfur containing phospholipid derivatives, very little has been accomplished in the area of characterization of the physicochemical properties o f these compounds. Elucidation of the thermotropic behavior of sulfur substituted phospholipids is of interest not only in comparison with their oxygen analogues but also with respect to their use as structural and mechanistic probes for the study o f membranes and membrane-dependent enzymic reactions. In the present paper we report the first series of studies of the phase transition properties of four synthetic sulfur-substituted phospholipids using high sensitivity differential scanning calorimetry (DSC) as a follow up to the characterization of the thermotropic behavior of the respective oxygen analogues of the compounds [13]. As in the previous work, three o f the lipids studied here were found to have transitions more complex than simple two-state systems. Materials and Methods

Phospholipids Stereospecific syntheses of compounds V - VIII, Table I were carried out as previously reported [14--16]. Elemental analyses, infrared and proton magnetic resonance data were consistent with the structures given in Table I. The purity of each compound was established according to conventional criteria (chromatography, including 'overloaded' TLC, elemental analysis and NMR). Thus, the phospholipids (0.05 mg/10 /al) dissolved in chloroform exhibited single spots on Whatman K6F silica gel plates developed in chloroform/methanol/water (65:25:4 v/v/v), and compounds VII and VIII without base-labile thioester functions were also checked on silica gel plates developed with chlor o f o r m / m e t h a n o l / a q u e o u s ammonia (1:9:1 v / v / v), the phospholipids being visualized by molybdic acid spray [17] a n d / o r iodine vapor. We have previously established [9] that the lipids containing thioester groups, which might be expected to be especially susceptible to hydrolysis or acyl chain migration, do not undergo

any degradation under conditions more severe than those encountered in the present work. All other compounds used were of reagent grade except organic solvents which were spectrograde.

Vesicle preparation Prior to vesicle preparation the lipids were kept in a vacuum oven at 60°C until no further weight-loss occurred. The lipids thus treated were in the form of hydrates [14--16] as indicated in Table I. Multilamellar liposomes (MLVs) were prepared by dispersing at 40-45°C at concentrations of 1--4 mg of lipid per ml of water or buffer in a vortex mixer for 30 s. This procedure was repeated twice and yielded cloudy suspensions identical in appearance with those obtained with dipalmitoyl PC.

Calorimetry The P~. -" L, lipid phase transitions were observed by DSC, some in the DASM-4 instrument (NPO Biopribor, Puschino, U.S.S.R.) [18] and some in the MC-2 instrument (Microcal, Inc. Northampton, MA 01060). In those cases where a lipid was run in both instruments, closely agreeing results were obtained. The multilamellar suspensions of the lipids were scanned at approximately 0.1 K/min. The same results were obtained with either doubly deionized water or 10 mM potassium phosphate pH 7.0 as suspending medium. Instrumental base lines obtained by scanning buffer or doubly deionized water in both sample and reference cells of the calorimeter were horizontal in the temperature range of interest. The noise level was 0.01--0.02 kcal K -~ mol -j. All samples were scanned under a nitrogen pressure of 1.5 atm to minimize bubble formation. In all cases rescan of the lipid suspension gave a DSC trace identical with the one obtained in the first scan, indicating that no significant decomposition took place during the scan.

Data analysis In order to estimate the level of impurity required to produce the asymmetry observed in the DSC curves for lipids V, VII and VIII, in each case we calculated a least squares fit of the

325 TABLE I Structures of lipids studied by DSC.

CH2Rs

I R2-CH

I

0

I

,

CH2OPOCH2CH2N(CHs) s

l O"

Ltpld

Anhydrous molecular w e i g h t

R.

No. moles of of water of crystalizatim

0

II I*

-O-(CH=)svCH =

CHg(CH=) za-C-NH-

0

0

! II*

-O-C- (CH:) s a C H s

U CHs(CH=) vO-C-NH-

0 -O-C- (CH=) aeCHs

u CHs(CH2) zv-NII-C-NB-

0 -O-C- (CH~) I=CH8

U CHs(CH=) ze-C-NH-

0

-O-C- (CH=) ,4CHs

789.2

0

U V

818.2

0

, IV*

679.2

0

a III*

747.1

II CH= (CH=) 14-C-S-

750.1

0

U VI

-O-(CH=)IsCH =

CHs(CH 2) z4-C-S-

736.2

2.5

O

U VII

-S-(CH2)IsCH s

CH= (CH=) =.-O-C-[~l-

737.2

0

U VIII

-S-(CH=)tsCH a

CHg (CH2) 14-C-HH-

735.2

*Results for these lipids are reported in Chowdhry et al. [13] and in Table IV.

observed data to a theoretical curve based on ideal solution theory with the assumption that the impurity has negligible solubility in the aqueous phase [19--20]. In this treatment the

value of the transition enthalpy, A/-/o~, obtained by planimeter integration, is used and a value for to, the transition temperature of the pure lipid, is assumed. The standard deviation of the

326

observed data from the theoretical curve is minimized by varying the total mole fraction of the impurity in the lipid, the ratio, K, of the mole fraction of the impurity in the gel phase of the lipid to that in the liquid crystal phase and AHvH, the van't H o f f enthalpy. The value of t o is then varied to see whether the minimization can be improved. The procedure followed here for analysis of the data in terms of multiple transitions was briefly outlined by Chowdhry et al. [131, with one difference. In the procedure as outlined by Chowdhry et al. one of the fitting parameters, the van't H o f f enthalpy, was allowed to vary freely. In the present work we have required that the ratio/J = AHvH/Ah,,, where Ahc~ is the calorimetric enthalpy in cal g-~, of a component transition, be the same for each component in the transition of a given lipid. The size in lipid molecules of the apparent cooperative unit for a transition is equal to ~ divided by the molecular weight of the lipid and it seems reasonable to take this quantity as a property of the overall

transition rather than of a particular component of the transition. This change in procedure does not significantly affect the values of the other two varied parameters, namely t~,z, the temperature of half completion of the component transition, and AH~, the enthalpy of that transition. Results and Discussion Tracings of the DSC curves for the main transitions of lipids V--VIII are shown in Fig. 1. At concentrations of 1--4 mg ml -] the DSC curves were practically noise free. The parameters characterizing the transitions of the lipids are given in Table II. All the lipids except lipid VI showed asymmetric DSC curves, and they all had values f o r tin, the temperature in °C at maximal excess specific heat, lower than that for DPPC and for Ate/z, the transition width at half maximal excess specific heat, much larger than that for DPPC. The enthalpies of transition for all the lipids except lipid V were lower than that of DPPC. There are two possible explanations of the

T

ot

r=1

m U

g I

3: U

U

Q

Q m

I

5

VII/

~

V~

m s U

x ILl 0

l 38

I M

I 40

42

Temperature / "C Fig. !. DSC curves for the main transitions of lipids V - - V I I i , giving the exc¢~ apparent specific heat as a function of temperature. The lipids (concentrations I - - 4 mg ml "L) were suspended in water or 10 mM potassium phosphate buffer, pH 7.0, by vortexing at 40---45 ~ .

327 T A B L E il Parameters for the main transitions of lipids. Lipid (no. of expts.)

Concentration mg ml -~

t,,, °C

V (3) VI (5) VII (2) VIII (3) DPPC b

I--4 I 2 2 I

40.83 39.41 37.96 39.70 41.69

AH,"

6t,:2,

°C

0.68 0.51 0.60 0.80 0.08

± ± ± ±

kcal mol-' ± 0.03 ± 0.01 ± 0.05 -I- 0.05

10.29 8.23 7.44 7.76 8.50

-t- 0.21 ± 0.12 ± 0.06 :t: 0.19

0.07 0.02 0.02 0.II

•Determined by planimeter integration. bDipalmitoylphosphatidylcholine. Mabrey and Sturtevant [22] reported 41.4°C and 8.74 kcal mol -~ for tm and hat/respectively.

breadth and asymmetry o f the transitions; either the lipids contain significant amounts o f impurities, or they are more complex than simple twostate processes, as was reported in Paper I [13] to be the case for lipids I - - I V (Table I) and is also the case for dipalmitoylphosphatidylethanolamine (DPPE) [21]. It was found that the DSC data for lipids V, VII and VIII could indeed be very well fitted, by the procedure outlined above under Data Analysis, to theoretical curves appropriate for the case of a water insoluble impurity forming ideal solutions in the lipid. The parameters obtained by curve fitting are listed in Table III. The DSC curve obtained with lipid VI was quite accurately symmetrical, indicating either a very low level of impurity in this lipid, or an impurity with approximately equal solubilities in the two lipid phases. It is seen that in all cases the impurity would partition almost exclusively into the liquid

crystal phase. The ratio of the enthalpy in the sixth column to that in the fifth column, divided by the lipid molecular weight, gives the values 190, 310 and 210 molecules, respectively, for the sizes o f the apparent cooperative units in the lipid transitions. The standard deviations listed in the last column of the table are given as percentages of the maximal values of the excess specific heat. The parameters in columns 3 and 4 of Table III are not firmly established, in that an increase in one can to some extent be offset by a decrease in the other. Although it is difficult to prove that the levels o f impurity required to account for the broadening and asymmetry o f the DSC curves actually exceed those occurring in our preparations, we nevertheless prefer to attribute these characteristics of the curves to the presence of multiple transitions. Application o f the least squares curve fitting procedure outlined

T A B L E II1 The parameters obtained in fitting the data for lipids V, VII and VIII to the theoretical curve for a water-insoluble impurity. Lipid

Transition temp. of pure lipid, °C

Total mole fraction of impurity in lipid

Distribution constant, K

Transition enthalpy, cat g-~

van't Hoff enthalpy kcal tool-~

Stand. dev., % C

V VII VIII

40.98 38.20 39.78

0.010 0.010 0.0075

0.050 0.035 0.060

13. I 9.5 10.2

1900 2150 1600

0.4 1.8 1.0

328

by Chowdhry et ai. [13] gave good fits of the experimental data with from one to four twostate components, with the parameters listed in Table IV. The table also gives the parameters obtained with lipids I--IV by Chowdhry et al. [131. The adequacy of the fits is illustrated in Fig. 2 for lipid V and in Fig. 3 for lipid VI1. In each figure the solid line represents the observed data, the dashed lines the calculated components and the dotted, line the sum of the components. The fact that good fits of the DSC data to theoretical curves based on models involving one or more two-state transitions could be obtained does not prove the unique validity of the models. The complex transitions are presumably made up of sequentially occurring alterations of the initial structure since the Gibbs phase rule, if it is indeed applicable to a bilayer system con:

I

I

6

~4

I

0 35

36

37

38

39

Tewerature/*C Fig. 3. The DSC curve observed for lipid Vll. Curve identification as for Fig. 2.

~B

~)

-

__

Tu~ature/~C Fig. 2. The DSC curve ( - - - - - ) observed for lipid V, with three components (. . . . ) obtained by curve fitting. The curve ( . . . . ) calculated as the sum of the component curves agrees well with the experimental curve.

taining cooperative units of only a few hundred molecules, would preclude the existence of more than two lipid phases in equilibrium with each other over a range of temperatures. Perhaps cooperative changes in the hydration states of the lipid are involved, or intermediate rearrangements o f either the gel phase or the liquid crystal phase or both. It should be noted that any such transitions involve significant enthalpy changes and are highly cooperative and thus cannot be assumed to be very minor in character. It is evident from a consideration of the phase transition properties reported here that what appear to be small changes in composition can have dramatic effects on the transition properties and by inference on the structures of the corresponding bilayers. Several illustations of this are discussed here. An interesting feature o f the data in Table IV is the extremely wide range of the total transi-

329

TABLE IV Parameters from curve fitting of the lipid DSC data. Lipid

Comt,/v ponent °C

1 2 3 4 5

~/.., kcal mol-'

39.8 44.4 46.5 47.3 47.75

M'/,. kcal mol "~ 4.92 7.30 4.40 5.53 5.57

Std. dev.

%c

140 220 700 1130 2040

Total 27.90 il

I 2

5.0 7.0

2.4 1.7 Total

11I

I 2 3

50.9 55.9 57.4

105 160

4.1 4.0 2.6 7.7

110 430 480

Total 14.30 IV

I 2

51.7 52.85

1.30 3.87 Total

V

i 2 3

39.44 40.38 40.80

295 1160

5.17 1.36 3.32 5.69

350 860 1480

1.7

1470 440 910 1580 250

2.4 2.9

Total 10.37 VI VII

1 I 2 3 4

39.40 36.93 37.40 37.92 38.67

8.32 1.00 2.07 3.59 0.97 Total

VIII

1 2

39.12 39.70

7.63 2.80 4.49

Total

700 1100

1.9

7.29

tion enthalpies listed in column 4, covering the range of 4.1 to 27.9 kcal mol -j. The low value for lipid II is presumably mainly due to the shortness of the alkyl chain in the R 2 group of

this lipid. It is difficult to explain the extraordinarily high value for lipid I, especially since the only other lipid in the table, having an ether linkage in the RI group, lipid VI, has a much smaller enthalpy. Replacement of the C~sH370-group in the sn-I position in lipid I by the CIsH33S-group and with no other change, yields a product, lipid VIII, for which only two components are indicated compared with five for lipid I and which has a total enthalpy of transition only about one-fourth that of lipid I. Changing the CH z group next to the O in the sn-I position of lipid VI, which appears to have a simple two-state transition, to a C=O group forms lipid V which has a threecomponent transition with a 25O7o increase in total enthalpy. Lipid IV is distearoylphosphatidylcholine (DSPC) with an amide linkage replacing the ester linkage at position sn-2; lipid V is DPPC with a thioester linkage replacing the normal ester bond. This changes a lipid with a two component transition into one with a three component transition and twice as large a total enthalpy of transition despite the decrease in the length of the alkyl chains. The sn-I groups in lipids I and Vl are very similar, differing only by two CH z groups, the sn-2 group in lipid I is a 16-carbon amide group while the corresponding group in lipid VI is a 16-carbon thioester group. These seemingly small differences change a five-component transition into a single component transition having only 0.3 as much enthalpy change. Replacement of an O atom in the group at sn-2 in lipid VII by a CH 2 group in VIII changes a four-component transition into a two-component transition, this time with only a 4% change in enthalpy. The large changes in transition characteristics produced by small changes in organic structures in the comparisons given here as well as in other comparisons amongst these lipids and those studied in Paper I [13] indicate not only rather large changes in bilayer structures but also that the observed effects will be difficult to explain. Certainly techniques other than DSC will have to be employed if answers to the questions raised in this report are to be found. High resolution NMR, for example, might be very useful

330

provided sufficiently fine temperature control can be achieved within the narrow temperature ranges covered by the transitions.

8 9

Acknowledgements 10

This research was supported in part by grants from the National Institutes of Health (DK36215 and CA46750 to J.H. and GMO4725 to J.M.S.) and the National Science Foundation (DMB8421173 to J.M.S.). References I

2

3

4 5 6

7

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