Langmuir 1996,11,101-107
101
Thermotropic Behavior of Structurally-Related Phospholipids and Phosphonolipid Analogs of Lung Surfactant Glycerophospholipids Huicai Liu,? J. G. Turcotte,S and R. H. Notter*pfF§ Departments of Chemical Engineering and Pediatrics, University of Rochester, Rochester, New York 14642, and Department of Medicinal Chemistry, University of Rhode Island, Kingston, Rhode Island 02881 Received August 2, 1994. In Final Form: October 14, 1994@ Gel to liquid crystal transition temperatures (T,) were measured by differential scanning calorimetry for a series of synthetic analogs of naturally occurring lung surfactant glycerophospholipidsat pH 2.6,5.6, and 11.5. Compounds all had equivalentc16 saturated chains, and included dipalmitoylphosphatidylcholine (DPPC),dipalmitoylphosphatidylethanolamine(DPPE),dihexadecylphosphatidylcholine(DHPC),dihexadecylphosphatidylethanolamine (DHPE), and eleven related phosphonolipids varying systematically in the N-head group [six ether-ether phosphonolipids(DEPN series) and five ether-amide phosphonolipids (EAPN series)]. Thermotropic behavior varied significantly with molecular structure and pH as follows: (1)DPPC, DHPC, and phosphonolipids with choline (DEPN-8,EAPN-9)or otherwise fully N-substituted head groups (DEPN-9)had transition temperatures that were highest at pH 2.6, decreased at pH 5.6, and relatively unchanged at pH 11.5; (2) DPPE, DHPE, and phosphonolipids with unsubstituted primary amine head groups (DEPN-13,EAPN-13) had much higher T,than fully N-substituted compounds at pH 2.6 and 5.6, with decreased and almost equivalent transition temperatures at pH 11.5;(3)phosphonolipids with one N-methyl substitution (DEPN-12, EAPN-12) had Tc values and pH-dependence similar to unsubstituted primary amine compounds;and (4) phosphonolipidswith twohr-methylsubstitutions (DEPN10, EAPN-10) or two N-hydroxyethyl substitutions (DEPN-11,EAPN-11) had Tcvalues that were lowest at pH 5.6, and increased at pH 2.6 and 11.5. These results show that phase transition temperatures increased as intermolecular hydrogen bonding between lipid head groups increased, and Tcalso increased as head group electrostaticrepulsion and hydration decreased. Differencesin molecular packing associated with chain-backbonejunctional group also affected T,, which was consistently highest in ether-linked (vs ester-linked or ether-amide linked) compounds of equivalent N-head group. These molecular factors have previously been found to affect strongly the surface pressure-area isotherms of synthetic phospholipids and phosphonolipids in surface films cycled at the air-water interface.
Introduction Glycerophospholipids are the major structural components of all biological membranes. They are also important functional components of pulmonary surfactant, a complex mixture of phospholipids and specific proteins necessary for normal lung The disaturated glycerophospholipid dipalmitoylphosphatidylcholine (DPPC) is the single most abundant constituent of mammalian pulmonary surfactant. In addition, a number of other phosphatidylcholines (PC),together with smaller amounts of phosphatidylethanolamines (PE) and other phospholipid classes, are also present in this complex biological As constituents of either cell membranes or materia1.l~~ lung surfactant, the physicochemical behaviors of glycerophospholipids in ordered aggregates in the aqueous phase are crucial in defining their ultimate biological activity. The particular polymorphic phase formation of phospholipids in water depends on the molecular structure both in the hydrophobic fatty chain region and in the polar + Department of Chemical Engineering, University o f b c h e s t e r .
* Department of Medicinal Chemistry, University of Rhode Island. * Department of Pediatrics, University of Rochester.
Abstract published in Advance ACS Abstracts, December 15, 1994. (1)King, R. J.; Clements, J. A. In Handbook of Physiology, The Respiratory System; Fishman, A. P., Ed.; Am. Physiol. SOC.Press: Bethesda, MD, 1985;Vol. I, p 309. (2)Notter, R. H.; Morrow, P. E. Ann. Biomed. Eng. 1975,3 , 119. (3)Notter, R. H.; Finkelstein, J. N. J.Appl. Physiology 1984,57, 1613. (4)Notter, R. H. In Surfactant Replacement Therapy;Shapiro, D. L., Notter, R. H., Eds.; A. R. Liss, Inc.: New York, 1989;p 19. @
0743-746319512411-0101$09.0010
head group region, as well as on head group hydration and ionization, pH, and t e m p e r a t ~ r e .A ~ variety of PC and PE molecules have been extensively studied for their thermotropic properties when dispersed in water. Differential scanning calorimetry (DSC)andX-ray diffraction, for instance, have been used in studies of phospholipid phase behavior at different hydration6-11 and pH.12-22 Investigations with specific structural analogs have also defined thermal effects resulting from different fatty ( 5 ) Lewis, R. N. A. H.; McElhaney,R. N. In The Structure ofBiological Membranes; Yeagle, P. Ed.; CRC Press, Inc.: Boca Raton, FL, 1992;p 73. (6)Janiak, M.J.; Small, D. M.; Shipley, G. G. J. Biol. Chem. 1979, 254,6068. (7)Bach, D.; Sela, B.; Miller, I. R. Chem. Phys. Lipids 1982,31,381. (8)Kodama, M.; Kuwabara, M.; Seki, S.Biochim.Biophys.Acta 1982, 689,567. (9)Kodama,M.; Hashigami, H.; Seki, S. Biochim.Biophys.Acta 1985, 814. 300. (10) Ruocco, M. J.; Siminovitch, D. J.; Griffin, R. G. Biochemistry 1985,24,2406. (11)Kim, J. T.; Mattai, J.; Shipley, G. G. Biochemistry 1987,26, R.543 ""I-. (12)Eibl, H.InPolyunsaturatedFatty Acids; Kunau, W. H., Holman, R. T., Eds.; American Oil Chemists Society: Champaign, IL, 1977;p 229. (13)Cevc, G.;Watts, A.; Marsh, D. Biochemistry 1981,20,4955. (14)Massari, S.;Folena, E.; Ambrosin, V.; Schiavo, G.; Colonna, R. Biochim. Biophys. Acta 1991,1067,131. (15)Stumpel, J.; Harlos, K.; Eibl, H. Biochim. Biophys. Acta 1980, 599,464. (16)Trtiuble, H.; Eibl, H. Proc.Natl.Acad. Sci. U S A . 1974,71,214. (17)Cevc, G. Biochemistry 1987,26,6305. (18)Eibl, H.; Woolley, P. Bwphys. Chem. 1979,10,261. (19)van Dijck, P.W. M.; de Kruijff, B.; Verkleij, A. J.; van Deenen, L. L. M.; de Gier, J. Biochim. Biophys. Acta 1978,512,84. (20)Harlos, K.; Eibl, H. Biochim. Biophys. Acta 1980,601,113. (21)Seddon, J. M.; Cevc, G.; Marsh, D. Biochemistry 1983,22,1280. (22)Boggs, J . M.Biochim. Biophys. Acta 1987,906,353.
0 1995 American Chemical Society
102 Langmuir, Vol. 11, No. 1, 1995
Liu et al.
chain-glycerol backbone linkage groups,10,21,23-35 from at the airlwater interface have also recently been reThe present study focuses on additional DSC and from differing head group N-methylati~n,~~,~~,~~-~~ other modification^.^^^^^^^^ These structural differences measurements defining changes in T,as a function of pH for diether and ether-amide phosphonolipids and for the in phospholipid molecules affect their interactions with related phospholipids DPPC, DPPE, DHPC, and DHPE. each other and with solvent, and alter aggregation states and phase transition behavior. In the present study, a Thermal results for these 15 compounds are discussed in terms of their specific molecular structural differences, series of glycerophospholipids and phosphonolipids with as well as pH-generated changes in intermolecular systematic molecular structural variations are investihydrogen bonding, and head group ionization state and gated for thermal behavior as a function of pH. hydration. Specific compounds studied here for gel to liquid crystalline (La) phase transition temperature (T,)are Materials and Methods DPPC, dipalmitoylphosphatidylethanolamine (DPPE); Synthetic Phospholipids and PhosphonolipidAnalogs. dihexadecylphosphatidylcholine and -phosphatidylethaSynthetic phospholipids(>99%pure) were obtained commercially nolamine (DHPC and DHPE), plus six diether phosphoas (R)-dipalmitoylphosphatidylcholine(DPPC) and (R)-dipalminolipid (DEPN) and five ether-amide phosphonolipid toylphosphatidylethanolamine(DPPE),Avanti Polar Lipids, Inc., (EAPN) compounds synthesized previously by Turcotte (DHPC), Pelham, AL,(RS)-di-0-hexadecylphosphatidylcholine and ~ o - w o r k e r s .Phosphonolipid ~~~~~ compounds differed Sigma Chemical Co., St. Louis, MO; and (R)-dihexadecylphosfrom naturally-occurring glycerophospholipids in (1)the phatidylethanolamine (DHPE),Fluka Chemical Corp., Ronkonkopresence of ether linkages at the sn-llsn-2 and sn-3lsn-2 ma, NY. Phosphonolipid analog compounds were synthesized individually as six racemic (RS) diether analogs (DEPN-8 to -13) positions of the racemic DEPN series and of ether and and 5 chiral (R) ether-amide analogs (EAPN-9 to -13),based on amide linkages at the sn-1 and sn-2 positions, respectively, the nomenclature and methods ofTurcotte et a l . 5 0 ~ 5The ~ N-head of the chiral EAPN series and (2) the presence of a groups of phosphonolipid analogs were6O~~~ DEPN-8, EAPN-9, phosphonate ester (isosteric methylene substitution) in N(CH&+; DEPN-9, N(CH~CH~OH)~CHS+; DEPN-10, EAPN-10, the polar head group. Phosphonolipids all had equivalent NH(CH3)2+ (protonated form); DEPN-11, EAPN-11, NH(CH216-carbon saturated fatty chains, with N-head groups CH20H)2+ (protonated form); DEPN-12, EAPN-12, NH2CHsf varying systematically from primary amine (PE-related) (protonated form); and DEPN-13, EAPN-13, N H 3 + (protonated through quarternary ammonium (PC-related). Phase form). Structure confirmation and purity of all phosphonolipid transition temperatures for these DEPN and EAPN compounds were determined at synthesis by elemental analysis (C, H, P, N), thin layer chromatography, and multinuclear (IH, compounds near pH 5.6 have been given by Lu et al.,52 and was generally >99%. The 13C,31P)NMR spectro~copy6~-~~ and their surface pressure-area isotherms in cycled films purity of all phospholipidsand phosphonolipidswas also routinely
verified periodically by a single spot on thin layer chromatography (23)Devlin, M. T.; Levin, I. W. Biochemistry 1989,28,8912. (24)Seddon, J . M.; Cevc, G.; Kaye, R. D.; Marsh, D. Biochemistry with solvent system C of Touchstone et al.55 1984,23, 2634. Differential Scanning Calorimetry (DSC). Thermal ex(25) Haas, N. S.; Sripada, P. K.; Shipley, G. G. Biophys. J. 1990,57, periments utilized a 910 DSC and Thermal Analyst 2000 system 117. (DuPont Instruments, Wilmington, DE). For DSC studies, 0.1(26) Hauser, H. Biochim. Biophys. Acta 1981, 646, 203. 0.5 & 0.01 mg of phospholipid or phosphonolipid was weighed (27)Boggs, J. M.; Stamp, D.; Hughes, D. W.; Deber, C. M. Biochemistry directly as a solid powder at room temperature in a sample pan 1981,20, 5728. with a Mettler Analytic Balance (Greifensee, Switzland). Buff(28) Vaughan, D. J.; Keough, K. M. FEBS Lett. 1974,47, 158. (29) Chen, S. C.; Sturtevant, J. M. Biochemistry 1981,20,713. ered or unbuffered aqueous subphase was then added and the (30) Dorset, D. L. Biochim.Biophys. Acta 1988, 938, 279. pan sealed and placed in the DSC cell along with a reference pan (31) Siminovitch, D. J.; Wong, P. T. T.; Mantsch, H. H. Biophys. J. containing the same amount of solvent without lipid. A minimum 1987,51, 465. of five preliminary heating and cooling scans was done over a (32) Schwarz, F. T.; Paltauf, F.; Laggner, P. Chem.Phys. Lipids 1976, wide temperature range spanning the expected main phase 17, 423. transition (from -20 to 60-95 "C) to ensure full equilibration (33)Hauser, H.; Guyer, W.; Paltauf, F. Chem.Phys.Lipids 1981,29, 103. and hydration (in excess water). Final thermal data were then (34) Ruocco, M. J.; Makriyannis, A.; Siminovitch, D. J.; Griffin, R. recorded at a heatingrate of5 "C/min. Transitions were described G. Biochemistry 1985,24,4844. in terms of onset temperature (To), peak temperature (T& and (35) Siminovitch, D. J.; Jeffrey, K. R.; Eibl, H. Biochim. Biophys. phase transition enthalpy (AH),with the onset temperature of Acta 1083. 727. 122. the main phase transition correspondingto the gel to liquid crystal ( 3 6 Brdwn, P. M.;Steers, J.; Hui, S. W.; Yeagle, P. L.; Silvius, J. R. transition temperature (T,) for the compounds studied. Biochemistry 1986,25, 4259. (37) Sisk, R. B.; Huang, C. H. Biophys. J. 1992, 61, 593. Subphases for DSC experiments used distilled, deionizedwater (38) Mason, J . T.; O'leary, T. J. Biophys. J 1990, 58, 277. (Milli-Q W Plus system, Millipore Corp., Bedford, MA). All (39) Gruner. S. M.: Tate, M. W.: Kirk. G. L.: So. P. T. C.: Turner. D. contained NaCl and a total ionic strength of -0.15: (1)0.018 M C.; Keane, D. T.; Tilcock, C. P. S.;CulliS, P. R.'Bidchemist& l986,27, NaH2P04 0.107 M NaCl 0.025M HCl, pH 2.6; (2) unbuffered 2853. 0.15 M NaC1, pH -5.6; and (3) 0.0025 M NazHP04 0.14 M (40) Mulukutla, S.; Shipley, G. G. Biochemistry 1984,23, 2514. NaCl 0.0075 M NaOH, pH 11.5. Additional DSC measure(41)Casal, H. L.; Mantsch, H. H. Biochim. Biophys. Acta 1983,735, 387. ments in buffer a t pH 5.6 (0.01 M NaH2P04 0.138 M NaCl (42)Pascher, I.; Sundell, S. Biochim. Biophys. Acta lB86,855, 68. 0.002 M NaOH) verified that thermal behavior was not changed (43) Brown, P. M.; Silvius, J. R. Biochim. Biophys. Acta 1980,980, significantly compared to unbuffered 0.15 M NaC1. Three 181. compounds (DPPC, DHPC, and DEPN-8) had identical T,values (44)Dorset, D. L.; Zhang, W. Biochim. Biophys. Acta 1990, 1028, in buffered and unbuffered subphases a t pH 5.6, while T,values 299. differed only minimally between the two subphases for DEPN(45) Gagne, J.; Stamatatos, L.; Diacovo, T.; Hui, S. W.; Yeagle, P. L.; Silvius, J. R. Biochemistry 1985,24,4400. 11 and DEPN-12 (0.2 and 0.6 "C, respectively). (46)Leventis, R.; Fuller, N.; Rand, R. P.; Yeagle, P. L.; Sen, A.; Zuckermann, M. J.; Silvius, J. R. Biochemistry 1991, 30, 7212. Results (47) Silvius,J. R.; Brown, P. M.; O'leary, T. J.Biochemistry 1986,25, To facilitate the presentation of data, phospholipids and 4249. (48) Dorfler, H. D.; Miethe, P.; Mops, A. Chem. Phys. Lipids 1990, phosphonolipidsare divided into groups aRer the notation 54. 171. ~(49)Bach, D.; Bursuker, I.; Eibl, H.; Miller, I. R. Biochim. Biophys. (52) Lu, R. Z.; Turcotte, J. G.; Lin, W. H.; Steim, J. M.; Notter, R. H. Acta 1978, 514, 310. J . Colloid Interface Sci. 1992, 154, 24. (50) Turcotte, J. G.; Lin, W. H.; Pivarnik, P. E.; Sacco, A. M.; Shirali, (53) Liu, H.; Lu, R. 2.; Turcotte, J. G.; Notter, R. H. J . Colloid Interface S. S.; Bermel, M. S.; Lu, R. Z.; Notter, R. H. Biochim. Bioph-ys. Acta Sci. 1994, 167, 378. _ _ 1991, 1084, 1. (54) Liu, H.: Turcotte, J. G.: Notter, R. H. J. Colloid Interface Sci. (51)Turcotte, J.G.;Lin, W.H.;Motola,N.C.;Pivarnik,P.E.;Bhongle, 1994,167,391. N. N.; Heyman, H. R.; Shirali,S. S.;Lu, R. Z.;Notter, R. H. Chem.Phys. (55) Touchstone, J. C.; Chen, J. C.; Beaver, K. M. Lipids 1980,15, 61. Lipids 1991, 58, 81.
+
+
--.
~
+
+
+
+
Thermotropic Behavior of Lipids
Langmuir, Vol. 11, No. 1, 1995 103
Table 1. Thermal Phase Transition Data for DPPC and Ita Analogs at DifPerent pH Value@ pretransition
main transition
AH
AH pH
To("C) T, ("C) (kcavmol) T,("C) T m ("C) (kcdmol) DPPC
2.6 5.6 11.5
35.1 35.4
36.8 37.1
2.6 5.6 11.5
33.5 33.0
35.3 34.9
2.6 5.6 11.5 2.6 5.6 11.5 2.6 5.6 11.5
48.2 1.2 41.3 1.2 41.7 DHPC 49.5 1.1 43.8 0.9 43.1 DEPN-8 48.5 45.3 45.0 DEPN-9 47.3 44.2 44.4 EAPN-9 44.0 41.8 41.9
50.4 41.7 42.0
8.9 7.8 8.1
51.5 44.4 43.8
7.7 9.2 7.4
50.6 45.8 45.3
8.3 12.2 9.2
48.6 44.6 44.8
7.6 7.3 9.3
46.0 42.3 42.4
8.1 7.9 9.7
To, onset temperature; T,, peak temperature; T,, onset temperature of the main transition. Data are averages of three to six experiments for each compound, with standard errors of 5 1% for the main transitiontemperature(except that for DEPN-9 at pH 2.6 was 52%),55%for the main transition enthalpy (except those for DHPC at pH 2.6 andDEPN-9 at pH 11.5 were 510%),52%for the pretransition temperature, and 5 15% for the pretransition enthalpy.
5
- l o t
15
30
(56) Papahadjopoulos, D. Biochem. Biophys. Acta 1988, 163, 240. (57) Lehniger,A. L.Biochemistry;Worth Publishers,Inc.:New York,
1977; p 198.
40
50
60
70
80
Temperature ("C)
-
o
d-
l
5-F 20
pH=11.5
730
40
50
60
t 70
Temperature (OC)
a
of Liu et a1.F group I (DPPC, DHPC, DEPN-8, DEPN-9, EAPN-9, quarternary ammonium), group I1 (DPPE, DHPE, DEPN-13, EAPN-13, primary amines), group IIIa (DEPN-12, EAPN-12, secondary amines), and group IIIb (DEPN-10, DEPN-11, EAPN-10, EAPN-11, tertiary amines). Compounds in all groups had a phosphate/ phosphonate moiety that was uncharged (protonated) at pH 2.6 and was negatively charged (deprotonated) at pH 5.6 and 11.5.17J8,56Primary, secondary, and tertiary compounds had N-head groups that were positively charged a t pH 2.6 and 5.6 and uncharged at pH 11.5.17J8@ Quaternary ammonium compounds had a fixed N-head group positive charge a t all pH value^.^^.^^ Thus, in terms of the predominant forms in solution, group I compounds had overall head groups that were net positively charged a t pH 2.6 and zwitterionic a t pH 5.6 and 11.5. Group 11, IIIa, and IIIb compounds had overall head groups that were net positively charged at pH 2.6, zwitterionic at pH 5.6, and net negatively charged at pH 11.5. Thermal data for group I compounds DPPC, DHPC, DEPN-8, DEPN-9, and EAPN-9 are summarized in Table 1as a function of pH, and representative heating curves for DEPN-8 and EAPN-9 are shown in Figure 1. All five group I compoundshad an increased main phase transition temperature at pH 2.6 compared to pH 5.6. The amount by which T , was increased at pH 2.6 compared to 5.6 was DPPC (-7 "C) > DHPC (-6 "C) > DEPN-8 (-3 "C) DEPN-9 (-3 "C) > EAPN-9 (-2 "C). The cooperativity of the main chain-melting phase transition of group I compounds was decreased at pH 2.6, as shown by the increased difference between the peak temperature and onset temperature (Table 1). The pretransition also disappeared at pH 2.6 for DPPC and DHPC (DEPN-8, DEPN-9, and EAPN-9 had no pretransition at any pH). Temperatures for the main transitions and pretransitions
f
DEPN-8
Figure 1. Representative calorimetric scans of group I compounds DEPN-8 and EAPN-9 at dispersionsolvent pH 2.6, 5.6, and 11.5. Each heating scan is an individual experiment from the averaged thermal data in Table 1.
Table 2. Thermal Phase Transition Data for DPPE and Its Analogs at Different pH Valuesa high temperature transition
main transition
AH
AH pH
To("C) T, ("C) (kcdmol) T,("C) T, ("C) (kcdmol) DPPE
2.6 5.6 11.5
68.5 63.0 41.6
69.5 63.9 43.7
7.0 8.9 9.4
71.0 68.0 43.5
73.0 69.2 44.3
6.7 8.5 8.6
65.9 64.4 44.3
68.3 64.9 45.2
6.3 7.0 9.6
56.6 47.7 43.1
59.9 49.9 44.5
9.0 4.3 10.4
DHPE 2.6 5.6 11.5
82.5
83.8
1.3 DEPN-13
2.6 5.6 11.5 EAPN-13 2.6 5.6 11.5
82.0
84.2
12.7
To, Onset temperature; T,, peak temperature; T,,onset temperatureof the main transition. Data are averages of three to six experiments for each compound, with standard errors of 5 1% for the main transition temperature, 55%for the main transition enthalpy, and 51% (temperature) and 515% (enthalpy) for the high-temperature transition.
of group I compounds did not change substantially when the pH was raised from 5.6 to 11.5 (Table 1). Thermal data for group I1 compounds DPPE, DHPE, DEPN-13, and EAPN-13 are summarized as a function of pH in Table 2, and representative thermograms for DEPN13and EAPN-13 are shown in Figure 2. The main phase transition temperatures of group I1compounds increased at pH 2.6 compared to 5.6, and the cooperativity of the transition decreased. The magnitude of T , increase at low pH was in the following order: EAPN-13 (-9 "C) > DPPE (5.5 "C) > DHPE (3 "C) > DEPN-13 (1.5 "C). At both pH 2.6 and 5.6, the phase transition temperatures of group I1 compounds were much higher than for group
Liu et al.
104 Langmuir, Vol. 11, No. 1, 1995
Table 3. Thermal Phase Transition Data for Other Analogs with One or Two N-substitutions at Different pH Value@ pre- or post-transition
main transition
AH
pH
AH To ("0 T m ("C) (kcaVmo1) Tc("0T, ("C) (kcal/mol) Monomethylated Compounds DEPN-12
50
40
30
60
70
80
2.6 5.6 11.5
i
2.6 5.6 11.5
,
58.2
60.3
3.5
52.7 52.0 42.6
55.5 53.1 43.2
8.3 8.4 10.7
53.1 45.5 48.8
54.8 46.3 49.5
6.7 6.5 15.7
48.7 43.6 47.3
50.7 44.1 47.8
9.2 8.6 10.9
47.4 42.4 43.2
50.1 43.2 43.7
9.5 6.8 12.4
44.5 39.9 42.4
45.5 40.3 43.1
5.4 5.6 10.8
DEPN-10 41.4
42.5
0.5 DEPN-11
EAPN-13
i.,
8.6 9.4 8.9
Disubstituted Compounds 2.6 5.6 11.5
20
60.2 59.4 47.7
EAPN-12
Temperature ('C)
-15
59.0 58.8 47.1
, ;.. I , ,, , 30 4 0 5 0
~ . , , ' j , , . ,!.,.
60 7 0
, , ,
80 90 100
Temperature ("C)
Figure 2. Representative calorimetric scans of group I1 compounds DEPN-13 and EAPN-13 at pH 2.6, 5.6, and 11.5. Heating scans are representative individual experimentsfrom the averaged thermal data in Table 2.
I (Table 2 vs Table 1). However, main phase transition temperatures for DPPE, DHPE, and DEPN-13 decreased tremendously at pH 11.5, givingvalues near those ofgroup I compounds at high pH. EAPN-13 also had a similarly lowered T, at pH 11.5, although compared to other group I1 molecules, this compound showed a more gradual pattern of decrease in T,as pH was increased (Table 2). None of the group I1 compounds studied had a pretransition before the main chain-melting phase transition. However, at pH 5.6, DHPE and EAPN-13 exhibited a second phase transition a t high temperatures ( ' 8 0 "C). This high temperature transition for EAPN-13 at pH 5.6 was reported previously as its gel to liquid crystal t r a n s i t i ~ n but , ~ ~ the more extensive data in Table 2 indicate that the lower temperature transition present at all pH values represents the appropriate gel to La transition for this compound. The high temperature EAPN-13 transition at pH 5.6 disappeared a t pH 2.6 and 11.5 (Table 2, Figure 2) and, in spite of its large enthalpy, showed more analogy with the high-temperature DHPE transition. The high-temperature transition for DHPE likely reflects a change from the Laphase to an inverted hexagonal phase ( H I I ) ,and ~ ~ this may also be true for the EAPN-13 high-temperature transition at pH 5.6. Table 3 gives averaged thermal data for compounds in group IIIa (DEPN-12, EAPN-12) and group IIIb (DEPN10, DEPN-11, EAPN-10, EAPN-111, and Figure 3 shows representative thermograms for selected molecules (DEPN12 and DEPN-10). The main phase transition temperatures of DEPN-12 and EAPN-12 showed little change between pH 2.6 and 5.6, but decreased significantly at pH 11.5 (Table 3). At pH 5.6, there was also a second hightemperature phase transition for EAPN-12 that was not found at pH 2.6 or 11.5. Main phase transition temperatures for all group IIIb compounds decreased as pH decreased by 7.6 "C for DEPNincreased from 2.6 to 5.6 (T, 10 and by -5 "C for DEPN-11, EAPN-10, and EAPN-11; Table 3). At high pH 11.5, T, values for group IIIb compounds increased compared to pH 5.6, but the magnitude of change was somewhat less than between
2.6 5.6 11.5
36.1
38.3
1.5 EAPN-10
2.6 5.6 11.5
37.5
39.1
1.4 EAPN-11
2.6 5.6 11.5
34.5
35.7
0.7
a To,onset temperature; Tm,peak temperature; Tc, onset temperature of main transition. Data are averages of three to six experiments for each compound, with standard errors of 5 1%for the main transition temperature, 55% for the main transition enthalpy, (1% for the pre- or post-transition temperature, and 520% for the pre- or post-transition enthalpy.
pH 2.6 and 5.6. All four group IIIb compounds had a pretransition from gel LF to ripple gel Pr at pH 5.6, which disappeared at pH 2.6 and 11.5 (Table 3). The thermal data in Tables 1-3 can be presented and compared in alternative ways to display differences in T, associated with fatty chain linkage group (ether, amide, ester), head group phosphonatelphosphate moiety, and N-head group structure (Figures 4-7). Comparisons of Corresponding Diether vs EtherAmide Phosphonolipid Pairs (Figure4). Differences between the T,values of corresponding DEPN and EAPN molecules isolated effects due to (racemic) sn-1, sn-2 and sn-2, sn-3 ether linkages compared to (chiral) sn-1 ether and sn-2 amide linkages. Although the magnitude of difference varied among corresponding pairs, each DEPN compound consistently had a higher T,than its EAPN counterpart at all pH values studied (Figure 4). Comparisons of CorrespondingDiether vs Diester Compounds (Figure 5). The effects of ether vs ester fatty chain linkages on T,were isolated by comparing calorimetric results for DPPC vs DHPC and for DPPE vs DHPE (Figure 5). At all pH, the diether phospholipids DHPC and DHPE had higher T,values than the corresponding diester phospholipids DPPC and DPPE (Figure 5). However, pretransition temperatures for DPPC were slightly higher than for DHPC (pH 5.6 and 11.5, Table 11, consistent with previous In addition, DPPE has an Lato HUtransition temperature above the upper scan limit of 95 "C studied here,21s58which is higher than the similar transition for DHPE (pH 5.6, Table 2).21 (58)McIntosh, T. J.; Simon,S.A. Biochemistry 1986,25, 4948.
Langmuir, Vol. 11, No. 1, 1995 105
Thermotropic Behavior of Lipids
+pH=5.6 -+- pH=11.5
~~"'i'"'~"""'""''i
-15 30
40
50 60 Temperature ('C)
70
g,
80
G e
4
75
c-
-
-
-15
30
40
50
70
60
50
80
45
Temperature ('C)
E
ri
pH=lI .5
I
8.9
10,lO
1
I
DEPN-(I
I
I
j
+pH=5.6 +pH=l1.5
40
spH=2.6 -+- pH=.5.6 -t-
;
0
DHPC
+pH=2.6
i
Figure 3. Representative calorimetric scans of group IIIa,b compounds DEPN-12 and DEPN-10 at pH 2.6, 5.6, and 11.5. Each heating scan is a representative individual experiment from the averaged thermal data in Table 3.
c"
0 DPPC
,
I
11.11
12,12
DPPE
DHPE
DEPN-13
Figure 5. The effects of the ether-ether and phosphonate linkages on T,at different pH. Data are graphed from Tables 1-3.
E
& pH=2.6
!
c"
I
13,13
Numbcr fw Each DEPN and EAPN Pair
Figure 4. Comparisons of the main phase transition temperature T,between corresponding DEPN-EAPN compound pairs with the same N-head group at pH 2.6,5.6, and 11.5.The ordinate AT, is the difference in T,between DEPN and EAPN compounds with the same N-head group. Comparisons of Corresponding Phosphate/phosphonate Compounds (Figure 6). The pairs of compounds DHPC vs DEPN-8 and DHPE vs DEPN-13 differed structurally only in the substitution of a head group phosphonate moiety in the phosphonolipids. "he effects of this structural difference on main phase transition temperature varied depending on pH and the choline vs ethanolamine N-head group. The T,of DEPN-8 was lower than DHPC at pH 2.6, but higher at pH 5.6 and 11.5 (Figure 5). In contrast, the T, of DEPN-13 was lower than DHPE at pH 2.6 and 5.6, and was slightly larger at pH 11.5. Comparisonsof Compounds with Different Head GroupN-Methylation (Figure 6). Phosphonolipids in the series DEPN-8, -10, -12, and -13, or EAPN-9, -10, -12, and -13, differed only in their degree of N-methyl substitution (three, two, one, and zero N-methyl groups, respectively). At pH 2.6, T, decreased almost linearly with N-methylation in each series, with DEPN-13 and EAPN13having the highest T, and DEPN-8 and EAPN-9 having the lowest (Figure 6). At pH 5.6, the pattern was similar, although EAPN-12 had a higher phase transition than EAPN-13. At pH 11.5, T , changed only slightly with
f
4 DEPN-8
0 DEI"-10 DEPN-12 DEPN-13
0
& pH=2.6 ---st --A-
pH=S.6 pH=11.5
I
I
EAPN-9
I
I
1
1
1
EAPN-10 ENN-12 E N N - 1 3
Figure 6. The effects of head group N-methylation on T,for DEPN and EAPN compounds at pH 2.6, 5.6, and 11.5. Data are graphed from Tables 1-3. N-methylation in both the DEPN and EAPN series, primarily because phase transition temperatures were decreased substantially at high pH for compounds with lower methylation number (Figure 6). Comparisons of Phosphonolipids with Methyl vs Hydroxyethyl N-Substitutions (Figure 7). Phosphonolipid pairs DEPN-8 and DEPN-9, DEPN-10 and DEPN-11, and EAPN-10 and EAPN-11 differed only in the N-substitution of two hydroxyethyl (CH2CH20H) groups for methyl groups in DEPN-9, DEPN-11, and
Liu et al.
106 Langmuir, Vol. 11, No. 1, 1995 10
a u^ e
5"
6
;
,
1 I
I
i
+p ~ = 5 . 6 +pH=11.5
DEPN-8,9
1
4
fi
-8- p H S . 6
DEPN-10.11 EAPN-I0,11
Analog Pairs for Companson
Figure 7. The effects of head group N-substitution of hydroxyethyl (CHzCHzOH)groups for methyl groups on the T,of phosphonolipids at different pH. The ordinate AT, is the difference in T,between each N-methylatedcompound (DEPN8, DEPN-10, or EAPN-10) and its N-hydroxyethylated counterpart (DEPN-9, DEPN-11, or EAPN-11). See the text for details. EAPN-11. N-Methylated DEPN-8, DEPN-10,and EAPN10 all had higher main phase transition temperatures than the corresponding hydroxyethyl compounds DEPN9, DEPN-11, or EAPN-11 at the pHvalues studied(Figure 7). The pretransition temperatures of DEPN-10 and EAPN-10 (Table 3, pH 5.6) were also higher than those of DEPN-11 and EAPN-11, respectively.
Discussion This study has demonstrated substantial variations in thermal behavior as a function of pH for 15 chainequivalent phospholipids and phosphonolipids. Changes in head group ionization state with pH gave rise to significant differences in the gel (or ripple gel) to liquid crystal T, values of individual compounds (Tables 1-3). In addition, significant differences in T,between individual compounds at fxed pH showed the importance of N-head group structure, chain-backbone linkage group (ether, amide, ester), and phosphatdphosphonate moiety on the Latransition (Tables 1-3, Figures 1-3). Additional phase transitions also depended on pH, including the pretransition from L p to Pp for several PC-related compounds (Table l),and the high-temperature phase transitions of DHPE and EAPN-13 (Table 2). Phase transition enthalpies varied less prominently with pH and molecular structure, and most AH values were in the range of -610 kcal/mol. Exceptions included the higher main transition enthalpies of DEPN-8 at pH 5.6 and group IIIb compounds at pH 11.5 (Tables 1and 3), and the enthalpy of the high temperature EAPN-13 transition at pH 5.6 (Table 2). A number of molecular characteristics and interactions, including intermolecular hydrogen bonding and head group charge (ionization), hydration, and conformation are affected by pH. Previous thermal studies of DEPN and EAPN compounds at pH -5.6 have suggested that intermolecular hydrogen bonding has a major influence on the value of T,.52 Transition temperature measurements for phosphonolipids at pH 5.6 in the present study agreed well with those published p r e v i o u ~ l yexcept , ~ ~ for reassignment of the T, for EAPN-13 at pH 5.6 to a lower melting peak (Table 2). In our experiments, group I1 compounds DPPE, DHPE, DEPN-13, and EMN-13 at pH 2.6 and 5.6 had the highest potential for intermolecular hydrogen bonding. Under these pH conditions, group I1 compounds had high T, values, which decreased substantially at pH 11.5 when intermolecular hydrogen bond formation was greatly reduced (Table 2). Charge repulsion between adjacent head groups in PE-related group I1 compounds also increased at pH 11.5 vs 5.6, since the
small N-head group can bend to reduce charge repulsion between adjacent zwitterionic molecules at pH 5.6.22 Reduced hydrogen bond formation, higher head group hydration,22and increased electrostaticrepulsion probably all contributed to the significant T, decreases for group I1 compounds at pH 11.5 (Table 2). Similar arguments also apply to the pH-dependent T, behavior of group IIIa compounds DEPN-12 and EAPN-12, which had the same changes in hydrogen bonding and ionization state with pH as group I1 molecules (compare Tables 2 and 3). Electrostatic interactions were particularly important in the pH-dependent thermal behavior of group I quarternary ammonium compounds (Table l), which could not hydrogen bond directly between head groups and had higher hydration than PE-like molecule^.^^-^^ PC-like group I molecules had a more stable gel phase and higher T, at pH 2.6 compared to pH 5.6 and 11.5 (Table 11, correlating with a relative decrease in charge repulsion and hydration at low pH. Although both PC and PE are zwitterionic at pH 5.6, charge repulsion in PC-like compounds is higher because of orientational constraints from the bulky N-head g r ~ u p . ~Charge ~ , ~ ~repulsion decreased rather than increased in group I compounds at pH 2.6 despite their net positive head g r o ~ p , and ~~,~~ polarity and hydration were also reduced.17 This also affected the pretransitions of DPPC and DHPC, which were present in the zwitterionic state a t pH 5.6 and 11.5, but disappeared at pH 2.6 (Table 1). Group IIIb compounds DEPN-10,ll and EAPN-10,ll also exhibited pretransitions when they were zwitterionic, but not at low or high pH when they were charged (Table 3). The PC pretransition is known to involve a change from a distorted hexagonal (two-dimensionalrectangular) tilted L ~ phase J to a true hexagonal, tilted, ripple bilayer Pp p h a ~ e . ~ The ~ , ~disappearance 3 of the pretransition could result from several factors, including conformational or interactional changes allowing direct transition from Lp to La,or a decrease in chain tilting in the Lp phase so that the pretransition is no longer obvious. Although the ionization states of group IIIb compounds DEPN-10,ll and EAPN-10,ll were the same as for group I1 and IIIa, their thermal behavior with pH was more complex (Table 3). T, values for Group IIIb molecules were lowest at pH 5.6, highest at pH 2.6, and intermediate at pH 11.5 (Table 3). The thermal behavior of compounds in groups I1 and IIIa was dominated by the effects of hydrogen bonding, but this factor is much less important in group IIIb molecules containing bulky, substituted N-head groups. Electrostatic repulsion in group IIIb compounds, which are more PC-related than PE-related, should decrease at pH 2.6 vs 5.6, tending to increase T, (Table 3). Electrostatic repulsion should also be reduced at pH 11.5vs 5.6 due to deprotonationofthe N-head group, again increasing T,. However, the relative increase in T, at high pH is not as large (Table 31, since repulsion between positively charged N-head groups in phospholipids is generally weaker than repulsion between negatively charged phosphate group^.^^^^^ Both hydrogen bonding and head group electrostatic interactions have been shown to affect strongly the surface pressure-area isotherm behaviors of phospholipid and phosphonolipid compounds in dynamically-compressed surface-excess films at the air-water i n t e r f a ~ e . Par~~?~~ ticularly affected as a function of pH is dynamic re(59) Damodaran, K. v.; Merz, Jr., K. M. Langmuir 1993, 9, 1179. (60) Janiak, M. J.; Small, D. M.; Shipley, G. G. Biochemistry 1976, 15,4575. (61) Janiak, M. J.; Small, D. M.; Shipley, G. G. J.LipidRes. 1979, 20, 183. (62) Scherer, J. R. Biophys. J . 1989, 55, 957. (63)Chowdhry, B. 2.;Lipka, G.; Hajdu, J.; Sturtevant, J. M. Biochemistry 1964,23, 2044.
Langmuir, Vol. 11, No. 1, 1995 107
Thermotropic Behavior of Lipids
where hydrogen bonding differencesare no longer maj0r.l' spreading during continuous cycling past c0llapse,5~ a film Both DEPN and EAPN compounds had T, values that behavior dependent on the character and properties of decreased monotonically with N-methylation at pH 2.6 collapse aggregates in the region of the interface. The (Figure 6). At pH 5.6, T,changes were also monotonic thermotropic properties of phospholipids and phosphonolipids in the present study also depend on interactions except that EAPN-13 had a lower transition than EAPNwithin bilayers or related aggregates, although these are 12 (Figure 6). At pH 11.5,where intermolecular hydrogen equilibrated in the aqueous phase and may differ in detail bonding effects were minimized, there was little variation from dynamically-generated film collapse structures in in T , with N-methylation (Figure 6), in agreement with the interfacial region. Despite potential differences, the study of Cevc17 with a series of diester and diether interfacial collapse aggregates and equilibrium bilayer phospholipids. At all pH values, compounds with two hydroxyethyl groups substituted for two methyl groups and aggregate forms should share some structural and interactional similarities, and it is reasonable that had lower La transition temperatures (Figure 7). This hydrogen bonding and electrostatic interactions that pattern of decreased T, over a range of ionization states strongly influence dynamic respreading in collapsed films (positive, zwitterionic, negative) was consistent with should also be important in thermal behavior. increased head group size and hydration from the introChain-backbonejunctional linkage (ether, amide, ester) duction of CHzCHzOH groups. Increased hydrogen bondand N-head group also affect the bilayer (aggregate)forms ing involving hydroxyethyl groups was unlikely, since it of phospholipid-like molecules and influence dynamic would tend to increase T, if present. respreading4 and thermal properties (Figures 4-7). All The presence of the phosphonate vs phosphate moiety five pairs of comparableDEPN-EAPN compoundsshowed in DEPN-8 vs DHPC and in DEPN-13 vs DHPE had a higher gel to La phase transition temperature for the relatively small effects on thermal behavior that varied ether linkage independent of pH (Figure 4). The etherwith pH and N-head group (Figure 5 ) . DEPN-8 had a linked phospholipids DHPC and DHPE also had higher slightly lower T, than DHPC at pH 2.6, but the transition T, values compared to ester-linked DPPC and DPPE temperature was increased in the phosphonolipid at pH (Figure 5). Differences in T , were largest between 5.6 and 11.5. DEPN-13 also had a lower T,than DHPE phosphonolipids DEPN-13 and EAPN-13 at pH 5.6 and at pH 2.6, but it remained lower at pH 5.6 and became 2.6, where intermolecular hydrogen bonding was signifiessentially equivalent at pH 11.5 (Figure 5). The phoscant (AT, 16.7 and 9.3 "C, respectively, Figure 4), and the phonate substitution increased head group hydrophobicity difference fell to only about 1"C at pH 11.5. Differences in otherwise equivalent phosphonolipidsvs phospholipids. in T,between other correspondingDEPN/EAPN molecules For DEPN-13, hydrogen bonding may have been reduced (Figure 41, or between ethervs ester phospholipids (Figure by the phosphonate substitution at both pH 2.6 and 5.6, 51, were also relatively small in magnitude but always leading to reduced T , compared to DHPE (Figure 5). The consistent with an increased T, for the ether linkage. phosphonate substitution had little effect on T , for DEPNThe chain-backbonejunctional group in phospholipids 13 vs DHPE at pH 11.5, where hydrogen bonding effects influences a number of properties including hydration in were small in both molecules. The bulky choline N-head the interfacial region and the depth of water penetration group and absence of intermolecular hydrogen bonding into the bilayer.62 The amide linkage is more polar than at any pH in DEPN-8 and DHPC probably also minimized the ether or ester linkage, leading to increased h y d r a t i ~ n . ~ ~ phosphonate-associated differences in T , between these The ether linkage is the most hydrophobic with the lowest compounds (Figure 5). hydratiod4and facilitates the formation of interdigitated In summary, these experiments have measured variabilayers with closer p a ~ k i n gand ~ ~ a, ~ more ~ flexible and tions in gel to liquid-crystal transition temperature with mobile charactereZ3Closer packed and less hydrated pH for a series of 15 structurally-related phospholipid bilayers are more stable in the rigid gel state, translating and phosphonolipid compounds. In spite of their fatty into a higher T , for DEPN vs EAPN compounds and for chain equivalence, substantial changes of T , with pH were DHPC vs DPPC and DHPE vs DPPE. In spite of higher observed for all compounds, indicating the importance of T,, diether phospholipids and phosphonolipids also have interactions involvingthe head group and backbone region. improved dynamic respreading compared to DPPC in films Intracompound differences in thermal behavior as a cycled in the collapse regime.50,53,54,65 There appears to be function of pH demonstrated that intermolecularhydrogen no simple correlation between film respreading and the bonding was a major factor in increasing T , in phosphomagnitude of T,. Unsaturated phospholipids with lower lipids and phosphonolipids that were primary or secondary phase transition temperatures than DPPC are also known amines. For PC-related quarternary ammonium comto have improved respreadmg in cycled interfacial pounds without intermolecular hydrogen bonding, T, was In addition, PC-related group I compounds have increased decreased at pH conditions where head group electrostatic T , at pH 2.6 vs 5.6 (Table l),but exhibit decreased repulsion was increased. Consideration of intercompound respreading at low pH in surface films.53 differences in T , at fixed pH showed that chain-backbone N-Head group methylation (or hydroxyethyl substitujunctional linkage group (ether, ester, amide) and phostion) strongly affected thermal behavior (Figures 6 and phonatdphosphate group also influenced thermal behav7). Previous studies have shown that the T , of phosphoior, in addition to N-head group structure. In particular, lipids decreases with increasing methylation nearly at all pH values, the ether linkage consistently resulted linearly at neutral or low pH,17,28,41,48 but not at pH 13, in increased T , compared to the ester or amide linkage for the phospholipids and phosphonolipids studied. (64) Boggs, J. M. Can. J.Biochem. 1980,58, 755. (65) Turcotte, J. G.; Sacco, A. M.; Steim, J. M.;Tabak, S. A.; Notter, R. H. Biochim. Bwphys. Acta 1977,488, 235. (66) Notter, R. H.; Tabak, S.A.; Mavis, R. D. J . Lipid Res. 1980,21, 10.
(67)Notter,R. H. InPulmonury Surfactant; Robertson, B.;van Golde, L. M. G.; Batenburg, J. J., Eds.; Elsevier: Amsterdam, 1984; p 17.
Acknowledgment. This work was supported in part by pulmonary SCOR grant HL-36543 from the National Institutes of Health. LA940613N
