Influence of Headgroup Chirality on the Mixing Behavior of

Phosphatidylglycerol (PG) molecules are a major component of cellular membranes. Because of their biological importance, they have been widely used in...
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Langmuir 2000, 16, 3491-3496

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Influence of Headgroup Chirality on the Mixing Behavior of Phosphatidylglycerol Mimics in Fluid Bilayers Maki Uragami, Yasuhito Miyake, and Steven L. Regen* Department of Chemistry and Zettlemoyer Center for Surface Studies, Lehigh University, Bethlehem, Pennsylvania 18015 Received September 28, 1999. In Final Form: December 10, 1999 The influence of headgroup chirality on the mixing behavior of a series of phosphatidylglycerol (PG) mimics has been investigated by use of the nearest-neighbor recognition method [Vigmond, S. J.; Dewa, T.; Regen, S. L. J. Am. Chem. Soc. 1995, 117, 7838]. For this purpose, a series of disulfide-based phospholipid dimers 1-10 were synthesized from 1,2-dimyristoyl-sn-glycero-3-phosphate and 1,2-stearoyl-sn-glycero3-phosphate, having the R-configuration at the sn-2 carbon, and either a R- or an S-configuration within the headgroup. Results of nearest-neighbor analyses indicate that chiral interactions between the headgroup and the glycerol backbone of neighboring PG mimics have a larger influence on lipid mixing than a chain length difference of four methylenes in analogous lipids, which are devoid of chirality and a hydroxymethylene moiety in the headgroup.

Introduction Phosphatidylglycerol (PG) molecules are a major component of cellular membranes. Because of their biological importance, they have been widely used in model systems for mechanistic studies.1 To date, suprisingly little attention has been paid to the chirality that exists within the headgroup region of such molecules. In particular, all synthetically derived PGs that have been prepared via exchange reactions, using phospholipase D as a catalyst and glycerol as a transphosphatidylation reagent, represent mixtures of diastereomers. When derived from natural phospholipids, these compounds have the Rconfiguration at the sn-2 carbon, and either the R- or the S-configuration within the pendant glycerol moiety. Recently, it has been found that only R,S-diastereomers (i.e., 1,2-diacyl-sn-glycero-3-phospho-1′-sn-glycerols) exist in cabbage and soybeans.2 In sharp contrast, significant proportions of R,R-diastereomers (i.e., 1,2-diacyl-sn-glycero-3-phospho-3′-sn-glycerols) have been identified in Escherichia coli (ca. 11%) and egg yolk (55%).2 These significant differences, by themselves, provide considerable impetus for examining the role that the headgroup chirality of phosphatidylglycerols plays in defining the structure and function of biomembranes as well as in model systems.2 We have been exploring the mixing behavior of phospholipids via the use of a chemical technique that we have termed nearest-neighbor recognition (NNR).3 A key advantage of this method over more classical techniques that have been used to probe the mixing of phospholipids (e.g., differential scanning calorimetry) is that it is directly applicable to the study of bilayers in the physiologically relevant fluid phase. Unlike other chemical approaches that have been used to investigate lipid mixing, the NNR method measures reversible chemical reactions. Thus, it yields thermodynamic information relating to nearestneighbor interactions in the bilayer state. It should also be noted that the NNR method, which is based on HPLC analyses, can detect energies of interactions that are as (1) Gennis, R. B. Biomembranes: Molecular Structure and Function; Springer-Verlag: New York, 1989. (2) Itabashi, Y.; Kuksis, A. Anal. Biochem. 1997, 254, 49. (3) Davidson, S. K. M.; Regen, S. L. Chem. Rev. 1997, 97, 1269.

low as ca. 100 cal/mol. In essence, therefore, the NNR method is a high-sensitivity technique that provides thermodynamic insight into lipid mixing, which cannot be obtained by any other means.3-5 In view of the recent stereochemical differences that have been found with natural PGs in various cellular membranes, we were intrigued with the possibility that chirality in the headgroup region of a PG could affect its mixing properties. We were also interested in this issue, because the more general question of whether diastereomeric interactions between a headgroup and a glycerol backbone of neighboring phospholipids can affect their mixing in the fluid bilayer state has not, to the best of our knowledge, previously been addressed.4 In this paper, we report experimental evidence that such diastereomeric interactions can, indeed, affect lipid mixing. Specifically, we report the results of a nearest-neighbor recognition (NNR) study that has been based on PG mimics having the R-configuration at the sn-2 carbon, and either the Ror the S-configuration within the headgroup. The PG and PG mimic can be seen in Chart 1. Experimental Section General Methods. Unless stated otherwise, all reagents were obtained from commercial sources and used without further purification. Boc-(S and R)-2-amino-3-phenylmethoxy-1-propanol were obtained from Advanced ChemTech Co. (Louisville, KY); 1,2-diacyl-sn-glycero-3-phosphatidic acids (monosodium salt) were obtained from Avanti Polar Lipids (Alabaster, AL). 2,4,6Triisopropylbenzenesulfonyl chloride (TPSCl) was recrystallized (4) In a previous report, it was shown that chirality in the glycerol backbone of a phospholipid can be recognized by nearest neighbors: Inagaki, M.; Shibakami, M.; Regen, S. L. J. Am. Chem. Soc. 1997, 119, 7161. (5) Nearest-neighbor recognition is defined as the thermodynamic preference for homodimer formation. The essence of the NNR method may be summarized as follows: when an equilibrium dimer distribution is found to be statistical (i.e., when the molar ratio of heterodimer to each homodimer is 2.0, starting from either pure heterodimer or a 1/1 mixture of homodimers), and when there is no driving force for transmembrane asymmetry (i.e., an uneven distribution of phospholipids between the inner and outer monolayer of the bilayer), this finding establishes that the monomeric components are randomly distributed throughout the membrane. When homodimers are favored (i.e., NNR exists), and when such recognition is eliminated by the presence of a diluent that does not alter the phase of the membrane, then the presence of lateral heterogeneity is indicated.

10.1021/la9912794 CCC: $19.00 © 2000 American Chemical Society Published on Web 02/15/2000

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Langmuir, Vol. 16, No. 7, 2000 Chart 1

from 1% thionyl chloride in hexane and dried under vacuum. All 1H NMR spectra were recorded on a Bruker 360 MHz instrument; chemical shifts are reported in ppm and are referenced to residual solvent. Activated Monomers (13). The following procedure that was used to prepare 13 (14S) was typical for the synthesis of all of the activated chiral monomers: 1,2-dimyristoyl-sn-glycero-3phosphate (200 mg, 0.32 mmol, monosodium salt) was dissolved in 2.0 mL of anhydrous pyridine with slight heating (40-50 °C). To this solution was added 2,4,6-triisopropylbenzenesulfonyl chloride (TPSCl, 297 mg, 0.98 mmol, 3 equiv), followed by addition of Boc-(R)-2-amino-3-phenylmethoxy-1-propanol (183 mg, 0.65 mmol, 2 equiv) as a powder. After stirring the mixture for 1 h at room temperature, 1.0 mL of water was added to quench the reaction. After additional stirring for 15 min, the solvent was removed under reduced pressure. The crude oil was then purified by column chromatography [SiO2, 25 g, CHCl3/CH3OH (30/1, v/v) and CHCl3/CH3OH/H2O (65/25/1)], followed by preparative TLC [Merck SiO2, CHCl3/CH3OH/H2O (13/6/1, v/v/v)] to give 247 mg (86%) of 11 (14S) as a colorless amorphous solid having Rf 0.66 [SiO2, CHCl3/CH3OH/H2O (13/6/1 (v/v/v)]; IR νmax (KBr) 2921.8, 2853.5, 1733.6, 1512.8, 1464.8, 1368.8, 1250.4, 1173.6, 1071.2, 1004.0 cm-1; 1H NMR (CDCl3) δ: 7.21 (m, 5 H), 5.48 (m, 1 H), 5.18 (m, 1 H), 4.45 (s, 2 H), 4.34 (d, J ) 11.8 Hz, 1 H), 4.14 (dd, J ) 6.35 and 12.0 Hz, 1 H), 3.89 (m, 4 H), 2.60 (m, 1 H), 2.50 (m, 1 H), 2.25 (m, 4 H), 1.52 (m, 4 H), 1.36 (s, 9H), 1.22 (m, 40 H), 0.85 (t, J ) 6.6 Hz, 6 H). Deprotection of the hydroxyl group was then carried out by dissolving 158 mg (0.184 mmol) of this compound in 5 mL of CH2Cl2, adding 120 mg of Pd-C (10 wt % Pd, dry basis) plus 0.1 mL of acetic acid. The mixture was then purged with H2 for 1 h at room temperature with constant stirring. Removal of Pd-C by filtration (along with careful washing with CHCl3), and concentration under reduced pressure, afforded a product having Rf 0.51 [SiO2, CHCl3/CH3OH/H2O, 13/6/1, (v/v/ v)]. The crude oil was then dissolved in 3.0 mL of CH2Cl2, and 1.0 mL of trifluoroacetic acid was then added to it. Subsequent stirring for 30 min at room temperature, followed by solvent evaporation, gave a residue that was diluted with CHCl3, washed, sequentially, with 5% Na2CO3 and water, and then concentrated (during the washing step, the organic layer formed an emulsion, which was broken up by centrifugation) to give a product (12,14S) having Rf 0.43 [SiO2, CHCl3/CH3OH/H2O, 13/6/1, (v/v/v)]. Finally, introduction of an activated disulfide group was accomplished by acylation of the amine moiety with N-[O-1,2,3-benzotriazin4(3H)one-yl]-3-(2-pyridyldithio) propionate [BPDP]). Thus 143 mg of 12 (14S) was dissolved in 3.0 mL of CHCl3, followed by

Uragami et al. addition of 76.2 mg (0.21 mmol) of BPDP. After the reaction mixture was stirred for 15 h at room temperature, it was diluted with CHCl3, washed with 5% Na2CO3, saturated with NaCl, and dried over anhydrous Na2SO4. Subsequent removal of solvent under reduced pressure afforded a crude yellow oil, which was purified by column chromatography [SiO2, 20 g, CHCl3/CH3OH, 10/1 (v/v) and CHCl3/CH3OH/H2O, 65/25/1 (v/v/v)] to give 114 mg of a pale yellow oil. Further purification via preparative TLC [Merck SiO2, CHCl3/CH3OH/H2O, 13/6/1 (v/v/v)] afforded 84.8 mg of 13 (14S) as a colorless oil (54%) having Rf 0.53 [SiO2, CHCl3/ CH3OH/H2O, 13/6/1 (v/v/v)]; IR νmax (KBr) 3430.3, 2926.5, 2850.3, 1735.9, 1649.9, 1555.0, 1460.1, 1418.2, 1239.4, 1102.6, 1067.3, 670.1 cm-1; 13C NMR (90 MHz, CDCl3) δ 173.7, 171.2, 159.8, 149.4, 137.2, 120.7, 119.7, 70.4, 63.6, 62.7, 60.5, 50.7, 35.3, 34.2, 31.9, 29.7, 24.8, 22.6, 14.0; 1H NMR (CDCl3) δ 8.42 (m, 1 H), 7.62 (m, 2 H), 7.01 (m, 1 H), 5.18 (m, 1 H), 4.35 (m, 1 H), 4.12 (m, 2 H), 3.90 (m, 4 H), 3.71 (m, 1 H), 3.62 (m, 1 H), 3.01 (m, 2 H), 2.62 (m, 2 H), 2.25 (m, 4 H), 1.52 (m, 4 H), 1.22 (m, 40 H), 0.85 (t, J ) 7.0 Hz, 6 H). HRMS for (C42H74N2O10Na2PS2) Calcd: 907.4318. Found: 907.4292. 13 (18S). Rf 0.54 [SiO2, CHCl3/CH3OH/H2O, 13/6/1 (v/v/v)]; IR νmax (KBr) 3414.0, 2917.8, 2850.3, 1737.0, 1650.1, 1557.7, 1462.6, 1415.2, 1242.6, 1169.2, 1106.8, 1066.0, 669.4 cm-1; 13C NMR (CDCl3) δ: 173.7, 171.2, 159.7, 149.1, 137.2, 120.7, 119.8, 70.2, 63.6, 60.5, 50.7, 44.2, 35.3, 34.2, 31.8, 29.7, 24.8, 22.6, 14.0; 1H NMR (CDCl3) δ 8.42 (m, 1 H), 7.63 (m, 2 H), 7.07 (m, 1 H), 5.19 (m, 1 H), 4.36 (m, 1 H), 4.13 (m, 2 H), 3.91 (m, 4 H), 3.71 (m, 1 H), 3.46 (m, 1 H), 3.01 (m, 2 H), 2.62 (m, 2 H), 2.25 (m, 4 H), 1.53 (m, 4 H), 1.22 (m, 56 H), 0.85 (t, J ) 6.7 Hz, 6 H). HRMS for (C50H90N2O10Na2PS2) Calcd: 1019.5569. Found: 1019.5569. The spectral properties and Rf values that were observed for the (14R) and (18R) analogues were identical to those of the above 14S and 18S forms of 13. Homodimer 1 (14S)(14S). Dithiothreitol (101.8 mg, 0.66 mmol) was dissolved in 0.5 mL of CHCl3, and the resulting solution cooled to 0 °C. A solution made from 30.0 mg (0.033 mmol) of the activated monomer 13 (14S) plus 1.0 mL of CHCl3 was then added dropwise to this solution, followed by addition of a 1 mL CHCl3 rinse. The reaction mixture was stirred at 0 °C for 30 min and at room temperature for 1h, and the solvent then removed under reduced pressure. The resulting crude yellow oil containing thiol monomer was purified by column chromatography [SiO2, CHCl3/CH3OH, 10/1 (v/v) and CHCl3/CH3OH/H2O (13/6/1)]. Further purification by prepartive TLC [Merck SiO2, CHCl3/CH3OH/H2O (13/6/1)] afforded the thiol monomer, which was then dissolved in 1.0 mL of CHCl3. To this solution was added a solution made from 32.6 mg (0.036 mmol) of the activated dimer 13 (14S) plus 0.5 mL of CHCl3 at room temperature. The mixture was then stirred at room temperature for 16 h, and the solvent removed under reduced pressure. Purification by preparative TLC [Merck SiO2, CHCl3/CH3OH/H2O (13/6/1)] afforded 24.4 mg (46%) of 7 having Rf 0.34 [SiO2, CHCl3/CH3OH/H2O (13/6/1)]; IR νmax (KBr) 3425.2, 2918.0, 2848.6, 1737.9, 1648.4, 1549.9, 1464.9, 1236.6, 1100.1, 1066.5, 844.2, 668.4 cm-1; 1H NMR (CDCl3) δ: 5.18 (m, 2 H), 4.35 (m, 2 H), 4.11 (m, 4 H), 3.90 (m, 8 H), 3.68 (m, 4 H), 3.14 (m, 4 H), 2.95 (m, 4 H), 2.27 (m, 8 H), 1.54 (m, 8 H), 1.22 (s, 80 H), 0.85 (t, J ) 7.0 Hz, 12 H). HRMS for (C72H140N2O20Na3P2S2) Calcd: 1571.8609. Found: 1571.8585. Homodimer 2 (18S)(18S). Experimental procedures that were used for the synthesis of 2 were exactly analogous to that used for 1. The desired homodimer had Rf 0.41 [SiO2, CHCl3/ CH3OH/H2O (13/6/1)]; IR νmax (KBr) 3425.2, 2918.0, 2848.6, 1737.9, 1648.4, 1549.9, 1464.9, 1236.6, 1100.1, 1066.5, 844.2, 668.4 cm-1; 1H (CDCl3) δ; 5.19 (m, 2 H), 4.38 (m, 2 H), 4.11 (m, 4 H), 3.92 (m, 6 H), 3.68 (m, 4 H), 2.98 (m, 4 H), 2.61 (m, 4 H), 2.28 (m, 8 H), 1.55 (m, 8 H), 1.23 (s, 112 H), 0.85 (t, J ) 7.6 Hz, 12 H); HRMS for (C90H172N2O20Na3P2S2) Calcd: 1796.1113. Found: 1796.1142. Heterodimer 3 (14S)(18S). Heterodimer 3 was prepared by reacting the thiol monomer derived from 13 (18S) with activated disulfide 13 (14S), using procedures similar to those described above. The desired product exhibited an Rf 0.38 [SiO2, CHCl3/ CH3OH/H2O (13/6/1)]; IR νmax (KBr) 3422.9, 2920.6, 2851.7, 1741.2, 1652.8, 1550.5, 1464.8, 1365.3, 1227.1, 1066.8, 842.9, 721.2 cm-1; 1H NMR (CDCl3) δ: 5.19 (m, 2 H), 4.37 (m, 2 H), 4.12 (m, 4 H), 3.92 (m, 7 H), 3.67 (m, 2 H), 3.46 (m, 2 H), 2.98 (m, 4

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H), 2.60 (m, 4 H), 2.27 (m, 8 H), 1.56 (m, 8 H), 1.23 (m, 96 H), 0.85 (t, J ) 6.7 Hz, 12 H). HRMS for (C82H156N2O20Na3P2S2) Calcd: 1683.9861. Found: 1683.9798.The spectral properties and Rf values that were observed for the (14R) and (18R) analogous dimers (i.e., 4, 5, 6, and 7) were identical to those of 1, 2, and 3. 2-Amino-1, 3-dihydroxy-2-pyridyldithiopropionamide. Serinol (134 mg, 1.46 mmol) was dissolved in 2 mL of anhydrous DMF. To this solution was added, dropwise, a solution made from N-[O-succinimidyl]-3-(2-pyridyldithio) propionate [SPDP] (550 mg, 1.76 mmol) in 2.0 mL of DMF at room temperature; an additional 1 mL of DMF, which was used to rinse the flask containing the SPDP solution, was also added to the reaction mixture. After stirring for 15 h at room temperature under a nitrogen atmosphere in the dark, the solvent was then removed under reduced pressure. The residue was then purified two times by column chromatography [SiO2, first chromatography: CHCl3/ CH3OH (30/1 to 20/1, v/v) and second chromatography: CHCl3/ CH3OH (50/1, v/v)], followed by purification by preparative TLC [Merck SiO2, CHCl3/CH3OH/H2O, 13/6/1 (v/v/v)] to give 229 mg (54%) of 2-amino-1, 3-dihydroxy-2-pyridyldithiopropionamide, having Rf 0.23 [Merck SiO2, CHCl3/CH3OH (10/1, v/v)]; IR νmax (KBr) 3289.2, 2938.3, 2874.3, 1644.8, 1557.9, 1448.6, 1415.7, 1261.5, 1116.3, 1043.7, 760 cm-1; 13C NMR (CD3OD) δ: 173.4, 161.2, 150.3, 139.1, 122.3, 121.1, 61.9, 54.5, 36.2, 35.5; 1H NMR (CD3OD) δ: 8.3 (m, 1 H), 7.72 (m, 2 H), 7.11 (m, 1 H), 3.83 (m, 1 H), 3.51 (m, 4 H), 2.96 (t, J ) 7.0 Hz, 2 H), 2.55 (t, J ) 7.0 Hz, 2 H). 13 (14 rac). 1,2-Dimyristoyl-sn-glycero-3-phosphate (monosodium salt) (96.7 mg, 0.157 mmol) was dissolved in 1.0 mL of anhydrous pyridine with heating. To this solution was added TPSCl (57.2 mg, 0.19 mmol) as a powder, and the mixture then heated for 15 min at 45 °C. To this solution was then added, dropwise, a solution made from 172 mg (0.260 mmol) of 2-amino1, 3-dihydroxy-2-pyridyldithiopropionamide plus 1.0 mL of dry pyridine, using an addition 0.5 mL of pyridine for rinsing. The mixture was then heated to 45 °C for 17 h with stirring. The product mixture was then cooled to room temperature, and 0.5 mL of water was added to decompose unreacted TPSCl. After stirring for 15 min, the solvent was removed under reduced pressure. The residue was then diluted with 15 mL of CHCl3, washed sequentially with 2 mL of 5% Na2CO3, 5 mL of saturated NaCl, and then concentrated under reduced pressure. Purification by preparative TLC (two times) [Merck SiO2, CHCl3/CH3OH/ H2O, 13/6/1 (v/v/v)] afforded 39.3 mg (28%) of 13 (14 rac) having an Rf and spectral properties that were identical to that of 13 (14S). Using similar procedures, 13 (18 rac) was obtained having identical Rf and spectral properties compared to that of 13 (18S). Phospholipid Dimers 8, 9, and 10. Procedures that were used to prepare 8, 9, and 10 were analogous to those used to prepare 1, 2, and 3, except that 13 (14 rac) and 13 (18 rac) were used as starting material. The Rf and spectral properties for 8, 9, and 10 were identical those of 1, 2, and 3, respectively. Mosher Esters. A Mosher ester of 13 (18 rac) was synthesized by reacting 20.6 mg (0.021 mmol) of 13 (18 rac) with 14.8 mg (0.064 mmol) of (S)-(-)-R-methoxy-R-(trifluoromethylphenylacetic) acid in 1.0 mL of CH2Cl2, in the presence of 6.6 mg (0.032 mmol) of DCC, and 0.3 mg (0.002 mmol) of DMAP for 6 h at room temperature. Removal of solvent under reduced pressure, followed by column chromatography [SiO2, CHCl3/CH3OH (100/1, v/v) and CHCl3/CH3OH/H2O (65/25/1)], and preparative TLC [Merck SiO2, CHCl3/CH3OH/H2O (13/6/1, v/v/v)] afforded 20 mg (80%) of the desired ester as a colorless oil having Rf 0.70 [SiO2, CHCl3/CH3OH/H2O (13/6/1, v/v/v)]; IR νmax (KBr) 2919.4, 2851.3, 1745.1, 1658.7, 1565.9, 1467.4, 1421.0, 1250.1, 1174.7, 1116.8, 1070.4, 850.2, 766.2, 722.7 cm-1; 1H NMR (CDCl3) δ: 8.38 (m, 1 H), 7.61 (m, 2 H), 7.43 (m, 2 H), 7.34 (m, 3 H), 7.04 (m, 1 H), 5.19 (m, 1 H), 4.41 (s, 2 H), 4.30 (m, 1 H), 4.11 (m, 1 H), 3.89 (m, 4 H), 3.44 (s, 3 H), 2.94 (m, 2 H), 2.57 (m, 2 H), 2.23 (m, 4 H), 1.51 (m, 4 H), 1.22 (m, 56 H), 0.85 (t, J ) 6.7 Hz, 6 H); 19F NMR (338 MHz, CDCl3) δ: 41.41 and 41.52. Mosher esters of 13 (18R) and 13 (18S) gave identical IR and 1H NMR spectra as that of the Mosher ester of 13 (18 rac). As expected, 13 (18R) gave only one 19F signal (41.37), as did 13

(18S) (41.50). For these measurements, fluorobenzene was used as an internal reference. Preparation of Liposomes and Initiation of ThiolateDisulfide Interchange. In a typical preparation, dichloromethane solutions of 1 and 2 (0.45 µmol of each dimer) were transferred to a test tube. The dichloromethane was then evaporated under a stream of argon over the solution, thereby leaving a thin film of lipid mixture. The resulting thin film was then dissolved with 150 µL of chloroform, and then diluted by 400 µL of diisopropyl ether. Subsequent addition of 50 µL of 3.3 mM borate buffer (47 mM NaCl and 0.7 mM NaN3, pH 7.4) result in an emulsion. After the emulsion was sonicated for 3 min by use of a mild (bath-type) sonicator, the organic phase was removed by gentle evaporation at 60 °C to afford a white gel in the bottom of the test tube. The gel was then collapsed by vigorous vortex mixing for 5 min, and 3.0 mL of additional buffer (10 mM borate, 140 mM NaCl, and 2.0 mM NaN3, pH 7.4) was added dropwise with vortex mixing. The vesicle dispersion was then degassed with an aspirator for 20 min, and the residual trace of organic solvent was removed by dialysis (Spectra/Por Membrane, MWCO 6000-8000) under an argon atmosphere using two 200 mL portions of degassed 10 mM borate buffer (pH 7.4) over the course of 18 h. The thiolate-disulfide interchange reaction was initiated, after the sample had thermally equilibrated at the desired temperature, by increasing the pH to 8.5 (addition of 20 µL of 0.15 M NaOH) followed by injection of 166 mL of an aqueous solution of 8.7 mM dithiothreitol (1.6 equiv relative to moles of lipid) and brief vortex mixing. All dispersions were maintained under an argon atmosphere throughout the course of the interchange reaction. Aliquots (0.45 mL) were removed at desired time intervals and quenched with 120 mL of 0.01 M HCl (final pH 5.0). After removal of water under reduced pressure, the resulting white salt was triturated with 2 mL of chloroform and centrifuged, and the chloroform was then removed under reduced pressure to yield a clear film. Samples were dissolved in 5 µL of chloroform plus 95 µL of mobile phase (HPLC) prior to injection. Analysis of Dimer Distributions by High-Performance Liquid Chromatography. Mixtures of lipid dimers were analyzed by HPLC using a Beckman Ultrasphere C18 reverse phase column (4.6 × 250 mm, 5 µm particle size). In general, the premixed mobile phase contained 80% 10 mM tetrabutylammonium acetate (TBA) in denatured ethanol, 12% water, and 8% hexane (v/v/v). The flow rate was 0.9 mL/min, and the column was maintained at 31.2 °C. Peaks were monitored at 205 nm using a Waters 996 photodiode array detector. Data were collected and processed using a Millennium workstation (Waters Corp.). In all cases, nearest-neighbor recognition values were determined from the integrated areas for the C18-homodimer relative to the corresponding C14/18 heterodimer. Differential Scanning Calorimetry. All calorimetry measurements were performed using a Microcal MC-2 calorimeter with DA-2 data acquisition and analysis software. Multilamellar vesicles were prepared by dispersing a thin lipid film (0.9 mmol) in 1.8 mL of 10 mM borate buffer (140 mM NaCl and 2.0 mM NaN3, pH 7.4), and their melting behavior was measured after four freeze-thaw (-196/+60 °C) cycles, using the same buffer solution as a reference. Heating scans were recorded between 10 and 60 °C at a scan rate of 30 deg/h. A borate buffer baseline was also collected and subtracted from each thermogram.

Results and Discussion Design and Synthesis of Exchangeable Phospholipid Dimers. Phosphatidylglycerol mimics that were designed for this study employed a 3-thiopropionamido moiety in place of the secondary hydroxyl group. Thus, disulfide-bridged dimers having controllable stereochemistry within the headgroup, and a capacity to undergo monomer exchange via thiolate-disulfide interchange reactions, were of immediate interest. Specific dimers that were chosen as synthetic targets were 1-10. Here, R, S, and rac designations refer only to the stereochemistry within the headgroup; in all cases, the

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Uragami et al. Scheme 1a

a Key: (a) Boc-(S or R)-2-amino-3-phenylmethoxy-1-propanol, triisopropylbenzene sulphonyl chloride, pyr; (b) Pd-C/H , CH Cl , 2 2 2 AcOH; CF3CO2H, CH2Cl2; (c) BPDP, CHCl3.

natural R configuration defines the glycerol backbone. In addition, the numbers that are indicated within the parentheses for 1-10 refer to the number of carbon atoms in each alkyl chain of the phospholipid monomer. For example, (14S) refers to a phospholipid that has been derived from 1,2-dimyristoyl-sn-glycero-3-phosphate, having an S-configuration in the headgroup. This backbone can be seen in Chart 2. Scheme 1 outlines the approach that was used to prepare 1-7. In brief, Boc-(S)-2-amino-3-phenylmethoxy-1-propanol or Boc-(R)-2-amino-3-phenylmethoxy-1-propanol was condensed with the appropriate phosphatidic acid, followed by deprotection of its hydroxyl and amine groups.6 Subsequent introduction of the 2-pyridyldithio-propionamido moiety (via condensation with N-[O-1,2,3-benzotriazin-4(3H)one-yl]-3-(2-pyridyldithio) propionate [BPDP]), followed by reductive cleavage with dithiothreitol, and coupling with either its precursor or a homologue afforded the requisite homodimers and heterodimers, respectively.7 Specific experimental procedures that were used for the dimer synthesis were similar to those previously described.8 The absence of epimerization throughout these transformations was confirmed by converting two dimer precursors (13) (used to prepare 2, 5, and 9) to Mosher esters [(S)-(-)-R-methoxy-R-(trifluoromethylphenyl acetic) acid] and measuring their 19F NMR spectra.9 Dimers 8-10 were synthesized by esterification of 1,2-dimyristoyl-snglycero-3-phosphate and 1,2-distearoyl-sn-glycero-3-phosphate with 2-amino-1, 3-dihydroxy-2-pyridyldithiopropionamide. The gel to liquid-crystalline phase transition temperatures for 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 were 21.2, 55.1, 33.9, 21.3, 55.6, 33.0, 33.0, 22.3, 56.4, and 33.9 °C, respectively. (6) Harbison, G. S.; Griffin, R. G. J. Lipid Res. 1984, 25, 1140. (7) Janout, V.; Lanier, M.; Regen, S. L. Tetrahedron Lett. 1999, 40, 1107. (8) Krisovitch, S. M.; Regen, S. L. J. Am. Chem. Soc. 1991, 113, 8175. (9) Dale, J. A.; Dull, D. L.; Mosher, H. S. J. Org. Chem. 1969, 34, 2543.

Chart 2

Nearest-Neighbor Recognition. Using experimental protocols similar to those previously described, we measured the equilibrium heterodimer/homodimer ratio for a series of these diastereomers in the fluid phase, as well as in the gel-fluid coexistence region.10-12 Thus, large unilamellar vesicles (1 mm average diameter, reverse

Headgroup Chirality Influence on PG Mimics in Bilayers

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Table 1. Nearest-Neighbor Recognition of Chiral Headgroupsa heterodimer/homodimerb equilibrating monomers

33 °C, gel-fluid phase

14S, 18S

0.94 ( 0.04

14R, 18R

0.92 ( 0.03

14R,18R + 82 mol % DPPG

14R, 18S

14 rac,18 rac

14 rac,18 rac + 75 mol % DPPG

0.96 ( 0.04

60 °C, fluid phase 1.86 ( 0.05 (1.839, 1.861, 1.903, 1.927, 1.870)c (1.880, 1.838, 1.800, 1.797, 1.920)d 1.66 ( 0.05 (1.616, 1.698, 1.605, 1.663)c (1.626, 1.633, 1.731, 1.737)d 1.65 ( 0.05 (1.701, 1.661, 1.688)c (1.582, 1.588, 1.695)d 1.96 ( 0.05 (1.888, 1.956, 1.914)c (2.010, 2.013, 1.993)d 1.81 ( 0.03 (1.778, 1.837, 1.774, 1.847, 1.827)c (1.794, 1.817, 1.758, 1.831, 1.825)d 1.80 ( 0.04 (1.832, 1.820, 1.735)c (1.823, 1.767, 1.802)d

Figure 1. Plot of the molar ratio of heterodimer/homodimer for 3/2 (b, O) and for 6/5 (9, 0) as a function of time. The open circles and squares are data from vesicles that originally contained pure heterodimer; the filled circles and squares were from an equimolar mixture of homodimers. In all cases, the temperature that was used for equilibration was 60 °C. The solid and dashed lines are simply intended to help guide the eyes of the reader. Chart 3

1.94 ( 0.04 (2.003, 1.953, 1.875)c (1.928, 1.908, 1.953)d

a Chemical equilibrium was generally reached in ca. 1 h at 60 °C and 15 h at 33 °C. b Molar ratio of heterodimer to each homodimer ( one standard deviations from the mean. c Raw equilibrium data obtained at increasing time intervals, starting from an initial 1/1 mixture of homodimers. d Raw equilibrium data obtained at increasing time intervals, starting from pure heterodimer.

evaporation methods, dynamic light scattering), which were initially composed of either a 1/1 molar ratio of homodimers, or the corresponding heterodimer, were treated with dithiothreitol to initiate monomer exchange. Equilibrium values were then determined from the convergence of both sets of data. A summary of our results are presented in Table 1. In the gel-fluid coexistence region, dimer distributions were independent of the stereochemistry within the headgroup. In contrast, a dependence on headgroup chirality could be detected in the fluid phase. Thus, greater nearestneighbor recognition (a lower heterodimer/homodimer ratio) was observed in the mixing of 14R with 18R, relative to 14S/18S, 14R/18S, and 14rac/18rac (Figure 1). It should be noted that the equilibrium values that are reported represent average values that have come from two vesicular membranes whose initial composition were entirely different; i.e., one originates from a mixture of homodimers, and the other from pure heterodimer. There is excellent agreement of the equilibrium values between both sets of data. Dilution studies that were carried out with 14R/18R, using 82 mol % of 1,2-dipalmitoyl-sn-glycero-3-[phosphorac-(1-glycerol)] (DPPG) (a phospholipid of intermediate chain length) resulted in the elimination of nearestneighbor recognition. Such a finding provides compelling evidence that, in the absence of this diluent, the mixing of 14R and 18R is nonrandom.11 Specifically, such a result indicates that the diluent produces a more homogeneously mixed bilayer by being able to mix more efficiently with (10) Krisovitch, S. M.; Regen, S. L. J. Am. Chem. Soc. 1992, 114, 9828. (11) Vigmond, S. J.; Dewa, T.; Regen, S. L. J. Am. Chem. Soc. 1995, 117, 7838. (12) Shibakami, M.; Inagaki, M.; Regen, S. L. J. Am. Chem. Soc. 1998, 120, 3758.

each of the exchangeable phospholipid monomers (i.e., 14R and 18R) in the fluid state. Similar dilution studies that were carried out with 14rac and 18rac also indicated the existence of nonrandom mixing in the absence of the diluent. The inability of 14S/18S, 14R/18R, and 14R/18S to recognize headgroup chirality in the gel-fluid coexistence region is a likely consequence of nearest-neighbor interactions that are dominated by strong van der Waals forces between the acyl chains. On going to the fluid phase, however, the magnitude of such forces is reduced and associative forces at the membrane surface (i.e., hydrogen bonding) can now make a greater contribution to the overall nearest-neighbor interaction. Since the headgroups in 14S/18S are enantiomerically related to those of 14R/ 18R, the observed difference in NNR must be due to the presence of the additional chiral center, which is located at the sn-2 position; i.e., the observed difference in nearestneighbor recognition must result from diastereomeric interactions.

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Although the magnitude of the effects reported herein is small (the difference in nearest-neighbor recognition between 14S/18S and 14R/18R corresponds to a ∆G° of ca. 145 ( 75 cal/mol at 60 °C), it is noteworthy that for analogous phospholipids such as 14, having large differences in alkyl chain length and melting behavior (i.e., the gel to liquid-crystalline phase transition temperatures for 14a and 14c differ by ca. 33 °C), random mixing has been observed; i.e., nearest-neighbor recognition could not be detected.8,13 Thus, these results indicate that chiral interactions between the headgroup and the glycerol backbone of neighboring PG mimics have a larger influence on lipid mixing than a chain length difference of four methylenes in analogous lipids, which are devoid of chirality and a hydroxymethylene moiety in the headgroup. The PG mimics 14a, 14b, and 14c can be seen in Chart 3. Finally, it should be noted that while the present findings show the existence of nonrandom mixing for these (13) Krisovitch, S. M.; Regen, S. L. J. Am. Chem. Soc. 1992, 114, 9828.

Uragami et al.

PG mimics, they do not yield insight into the nature of the domains or microdomains that may be formed. In particular, the size and size distributions of such domains, as well as their lifetimes are important (and also highly complex) structural issues that remain to be defined.14,15 Nonetheless, results reported herein lend strong support for the need to pay greater attention to headgroup chirality within phosphatidylglycerols. Acknowledgment. We are grateful to the National Institutes of Health (PHS Grant GM56149) for support of this research. LA9912794 (14) For a discussion of dynamic domain structure of lipid membranes, see: Jorgensen, K.; Mouritsen, O. G. Thermochim. Acta 1999, 328(12), 81. (15) For general reviews of lipid domains, which include a discussion of the use of related dimer distribution-based methods for probing lipid mixing, see: (a) Welti, R.; Glaser, M. Chem. Phys. Lipids 1994, 73, 121. (b) Tocanne, J.-F.; Cezanne, L.; Lopez, A.; Piknova, B.; Schram, V.; Tournier, J.-F.; Welby, M. Chem. Phys. Lipids 1994, 73, 139.