Synthesis and Study of Their

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PBN Derived Amphiphilic Spin-Traps. I/Synthesis and Study of Their Miscibility with Polyunsaturated Phospholipids Sandrine Morandat,‡ Gre´gory Durand,† Ange Polidori,† Le´a Desigaux,‡ Muriel Bortolato,‡ Bernard Roux,*,‡ and Bernard Pucci*,† Laboratoire de Chimie Bioorganique et des Syste` mes Mole´ culaires Vectoriels, Universite´ d’Avignon et des Pays du Vaucluse, Faculte´ des Sciences, 33 Rue Louis Pasteur, 84000 Avignon, France and Laboratoire de Physico-Chimie Biologique, UMR-CNRS 5013, Universite´ Claude Bernard Lyon I, 43 Boulevard du 11 Novembre 1918, Baˆ timent Chevreul 4e` me e´ tage, 69622 Villeurbanne Cedex, France Received March 18, 2003. In Final Form: July 15, 2003 The synthesis of new amphiphilic R-phenyl-N-tert-butylnitrone (PBN) derivatives bearing two hydrocarbon tails with 17 carbon atoms (compound A17) or 11 carbon atoms (compound B11) is reported. The amphiphilic and morphological properties of these potential antioxidant compounds were investigated at the airwater interface. The analyses were conducted using Langmuir film balance and Brewster angle microscopy. As it is possible to obtain pressure-area (π-A) isotherms from pure A17 and B11, this means that they can form stable monomolecular films at the air-water interface. Monolayers of pure PBN derivatives exhibited different interface behavior. Indeed, along with compression, the B11 monolayer showed only a liquid-expanded phase and the A17 monolayer showed a liquid-condensed phase. Brewster angle microscopy showed the presence of condensed domains within the A17 monolayer. The lateral interactions of these compounds with the polyunsaturated phospholipids, 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLoPC) and 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine (DLoPE), were evaluated by analysis of π-A isotherms and excess free energy of mixing. Each PBN derivative was miscible with DLoPC or DLoPE and formed nonideal mixed monolayers at the air-water interface. Only the A17 molecules exhibited favorable interactions at high surface pressures (over 20 mN/m) with both DLoPC and DLoPE, however. Under these conditions, A17 should be a better antioxidant than B11.

Introduction It is well established nowadays that free radicals act as pathological agents in a range of human diseases and various biological phenomena. Although the mechanism of cell membrane peroxidation has been well studied during recent decades, attention has recently been focused on the role of free radicals in senescence, atherosclerosis, and neurodegenerative conditions such as Alzheimer’s or Parkinson’s diseases. It is now well known that the polyunsaturated phospholipids, which are abundant within cytoplasmic membranes, are particularly sensitive targets for free-radical attack. The radical oxidation mechanism involves the abstraction of a hydrogen atom in bisallylic position yielding a new lipid-derived radical species, that is, polyunsaturated peroxyl radicals, which play a crucial role in the free-radical chain reaction of lipid peroxidation. Several studies1 have examined the effectiveness of various natural and artificial antioxidants in inhibiting freeradical degradation and in the process of photosensibilized oxidation. Spin-traps, such as nitrones, can react with short-lived lipid radicals, producing long-lived radical adducts and thus inhibit the peroxidation process. Nitrone spin-traps are widely used as free-radical scavengers in electron spin resonance (ESR) studies,2 but since the * Corresponding authors. B. Pucci: tel.: +33 4 90 14 44 42; fax: +33 4 90 14 44 49; e-mail: [email protected]. B. Roux: tel.: +33 4 72 44 80 78; fax: +33 4 72 43 15 43; e-mail: [email protected]. † Universite ´ d’Avignon et des Pays du Vaucluse. ‡ Universite ´ Claude Bernard Lyon I. (1) Frankel, E. N. Trends Food Sci. Technol. 1993, 4, 220.

reports of Novelli et al.,3 nitrones have also been used as potent antioxidants and anti-inflammatory agents in numerous animal models for neuroprotection,4 antiaging,5 and antidiabetic effects.6 In most cases, studies were performed with R-phenyl-N-tert-butylnitrone (PBN), but some authors have also reported the synthesis of related compounds to improve the efficiency of free-radical trapping.7 Our previous work focused on the synthesis of a PBN glycolipid analogue, for example.8 We showed that the amphiphilic character of these new compounds (2) For review see: (a) Janzen, E. G.; Haire, D. L. Two Decades of Spin Trapping. In Advances in Free Radicals Chemistry; Tanner, D., Ed.; JA1 Press: Greenwich, CT, 1990; Vol. 1, p 253. (b) Perkins, M. J. In Advances in Physical Organic Chemistry; Gold, V., Bethel, D., Eds.; Academic: New York, 1980; Vol. 17, p 1. (3) Novelli, G. P.; Angiolini, P.; Tani, R.; Consales, G.; Bordi, L. Free Radical Res. Commun. 1986, 1, 321. (4) (a) Asanuma, T.; Ishibashi, H.; Konno, A.; Kon, Y.; Inanami, O.; Kuwabara, M. Neurosci. Lett. 2002, 329, 281 and references cited. (b) Nakao, N.; Brundin, P. Neuroscience 1997, 76, 749. (c) Li, P. A.; He, Q. P.; Nakamura, L.; Csiszar, K. Free Radical Biol. Med. 2001, 31, 1191. (5) (a) Floyd, R. F.; Hensley, K.; Forster, M. J.; Kelleher-Anderson, J. A.; Wood, P. L. Mech. Dev. 2002, 123, 1021. (b) Sack, C. A.; Socci, D. J.; Crandall, B. M.; Arendash, G. W. Neurosci. Lett. 1996, 205, 181. (6) Ho, E.; Chen, G.; Bray, T. M. Free Radical Biol. Med. 2000, 28, 604. (7) (a) Bernotas, R. C.; Thomas, C. E.; Carr, A. A.; Nieduzak, T. R.; Adams, G.; Ohlweiler, D. F.; Hay, D. A. Biol. Med. Chem. Lett. 1996, 6, 1105. Thomas, C. E.; Ohlweiler, D. F.; Carr, A. A.; Nieduzak, T. R.; Hay, D. A.; Adams, G.; Vaz, R.; Bernotas, R. C. J. Biol. Chem. 1996, 271, 3097. Fevig, T. L.; Bowen, S. M.; Janowick, D. A.; Jones, B. K.; Munson, H. R.; Ohlweiler, D. F.; Thomas, C. E. J. Med. Chem. 1996, 39, 4988. (b) Sa´r, C. P.; Hideg, E Ä .; Vass, I.; Hideg, K. Biol. Med. Chem. Lett. 1998, 8, 379. (c) Dhainault, A.; Tizot, A.; Raimbaud, E.; Lockhart, B.; Lestage, P.; Goldstein, S. J. Med. Chem. 2000, 43, 2165. (d) Becker, D. A.; Ley, J. J.; Echegoyen, L.; Alvarado, R. J. Am. Chem. Soc. 2002, 124, 4678 and references cited. (8) Ouari, O.; Polidori, A.; Pucci, B.; Tordo, P.; Chalier, F. J. Org. Chem. 1999, 64, 3554.

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enhances their membrane-crossing ability.9 Furthermore, we recently reported a simple, efficient, and convergent synthetic route to amphiphilic single-chain PBN analogues.10 Despite the presence of bulky hydrophilic and hydrophobic groups, this new series of spin-traps kept their trapping activity and exhibited a potent antiapoptotic effect.11 However, because of their ability to cross cell membranes, these amphiphilic spin-traps are probably unsuitable for incorporation into the phospholipid membrane to inhibit its oxidation and thus protect the whole cell from exogenous free radicals. To provide an efficient protection of phospholipid membranes, the spin-traps should be able to remain in the cell membrane for long periods of time and should thus be highly miscible with natural phospholipids. With this in mind, we synthesized two PBN-derived amphiphilic spin-traps, which we chose to endow with two hydrocarbon tails (C17H35 for compound A17 and C11H23 for compound B11). The aromatic nitrone function acts as a bridge between the polar head, grafted onto the aromatic function, and the hydrophobic portion, bound to the PBN tert-butyl group. We predict that an insertion of this type, within the heart of the surfactant, will provide efficient protection of the lipid membrane. The present report deals with the synthesis and behavior of these compounds at the air-water interface. To determine their affinity for lipid membranes, the organization and homogeneity of the films they formed with polyunsaturated phospholipids were examined using a Langmuir film balance. The two-dimensional miscibility of these compounds with polyunsaturated phospholipids was evaluated by analysis of pressure-area isotherms and excess free-energy mixing.12-16 These experiments should provide us with a better understanding of the relationship between the interactions of these amphiphilic PBN derivatives with polyunsaturated phospholipids and their antioxidant efficiency. Experimental Section Materials. 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLoPC) was purchased from Sigma Chemicals (St Louis, MO), 1,2dilinoleoyl-sn-glycero-3-phosphoethanolamine (DLoPE) was from Avanti Polar Lipids Co (Alabaster, AL). Chloroform obtained from Sigma Chemicals was used as the spreading solvent for PBN derivatives and phospholipids were spread in hexane/ ethanol (9:1) from Merck (Darmstadt, Germany) and from Carlo Erba, respectively. All other chemicals and reagents were of the highest purity available from Merck (Darmstadt, Germany) and from Fluka Chemika (Buchs, Switzerland). The DLoPC, DloPE, and PBN derivatives solutions were protected against light and stored in a freezer until spreading. The distilled water was purified with a Millipore MilliQ filtering system, yielding a water resistance of 18.2 MΩ cm. Monolayer Technique. All experiments were performed at constant temperature (21 °C ( 0.1), in obscurity and under nitrogen-saturated atmosphere. The film balance was built by R&K (Riegler and Kirstein, Wiesbaden, Germany) and equipped with a Wilhelmy-type surface-pressure measuring system. The subphase was 150 mM KCl, 10 mM Tris-HCl, pH 7.2, buffer (TBS). (9) Partially published in Geromel, V.; Kadhom, N.; Cebalos-Picot, I.; Ouari, O.; Polidori, A.; Munnich, A.; Rotig, A.; Rustin, P. Hum. Mol. Genet. 2001, 10, 1221. (10) Durand, G.; Polidori, A.; Salles, J. P.; Pucci, B. Bioorg. Med. Chem. Lett. 2003, 13, 859 (11) Durand, G.; Polidori, A.; Ouari, O.; Tordo, P.; Geromel, V.; Rustin, P.; Pucci, B. J. Med. Chem. 2003, in press. (12) Angelova, A.; Ionov, R. Langmuir 1999, 15, 7199. (13) Chou, T.-H.; Chang, C.-H. Langmuir 2000, 16, 3385. (14) Chou, T.-H.; Chu, I.-M. Colloids Surf., A 2002, 211, 267. (15) Li, X. M.; Ramakrishnan, M.; Brockman, H. L.; Brown, R. E.; Swamy, M. J. Langmuir 2002, 18, 231. (16) Sanchez Macho, M. I.; Gonzalez, A. G.; Suarez Varela, A. J. Colloid Interface Sci. 2001, 235, 241.

Morandat et al. Lipids were spread, with a Hamilton syringe, at the air-water interface in hexane/ethanol 9:1 (v/v) for phospholipids and in chloroform for PBN derivatives to reach a final quantity of about 19 nmol of lipids. The solvent was allowed to evaporate for 20 min prior to compression. Then, the monolayer was compressed at 43 × 10-3 nm2 molecule-1 min-1 to obtain a pressure-area (π∠A) isotherm. The experiment was performed at least three times to ensure reproducibility of results. All lipid mixtures were used immediately after their preparation. Analysis of π-A Isotherms. The limiting molecular area of molecules was estimated by extrapolating the condensedlike curve to zero surface pressure. The surface elasticity moduli (Ks) were calculated from the pressure-area data obtained from the monolayer compressions using the following equation.17

Ks ) - A

dπ (dA )

where A is the molecular area at the indicated surface pressure π. The higher the Ks value of a monolayer, the more rigid it is. Analysis of PBN Derivative/Polyunsaturated Phospholipid Mixtures. At a given lipid mixing ratio, an ideal Ks-1 behavior can be determined by imparting a specific contribution of the Ks-1 value of each pure lipid on the reciprocal elasticity coefficient, depending on both molecular area fraction and mole fraction parameters. Thus, at a given constant surface pressure the ideal Ks-1 can be defined by the following equation:

Ks-112 )

( )

1 [(Ks-11 × A1) x1 + (Ks2-1 × A2) (1 - x1)] Aideal

where x1 is the mol fraction of component 1 and (A1) and (Ks-11) or (A2) and (Ks-12) are the molecular areas and surface elasticity moduli of pure components 1 and 2, respectively. The interaction between the two components and the thermodynamic stability of a mixed monolayer can also be investigated from an evaluation of excess free energy ∆Gex18:

∆Gex )

∫

π

0

∫

A12dπ - x1

π

0

∫

A1dπ - x2

π

0

A2dπ

Brewster Angle Microscopy (BAM). A commercial Brewster angle microscope (BAM) I-Elli2000, manufactured by NFT (Go¨ttingen, Germany), was equipped with a laser emitting polarized light at 532-nm wavelength. Light was reflected off the air-water interface at about 53.1° (Brewster angle). The lateral resolution of the microscope was 2 µm. The measurements were performed upon monolayer compression, at compression rates of about 13 × 10-3 nm2 molecule-1 min-1, with different shutter speeds (within the range of 1/50-1/1000 s). Synthesis. Detailed synthetic procedures are available as Supporting Information.

Results and Discussion Synthesis. Nitrones A17-B11 were prepared by condensing N-4-formyl benzyl lactobionamide 3 with the double-tail hydroxylamines 2a-2b, followed by hydrolysis of the lactobionamide acetyl groups. The synthesis of compound 3 was achieved with an overall yield of 26%, using the 4-cyanobenzaldehyde pathway, according to a previously described procedure.19 To synthesize the hydroxylamines 2a-2b, the alkyl isocyanates 1a-1b derived from either stearic (a) or lauric (b) acid were grafted onto the 2-methyl-2-nitro-propan-1,3-diol in toluene at 60 °C, in a first step. The carbamate derivatives 2a-2b were (17) Davies, J. T.; Rideal, E. K. Interfacial Phenomena, 2nd ed.; Academic Press: New York, 1963. (18) Goodrich, F. C. Proceedings of the Second International Congress on Surface Activity; Schulman, J. H., Ed.; Butterworth Press: London, 1957; Vol. 1, p 85. (19) Ouari, O.; Chalier, F.; Bonaly, R.; Pucci, B.; Tordo, P. J. Chem. Soc., Perkin Trans. 2 1998, 2299.

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Scheme 1. Synthesis of Nitrones A17 and B11

Figure 1. (A) π-A isotherms of pure A17 (a) and B11 (b). (B) represents surface elasticity modulus (Ks) plotted versus surface pressure for each pure monolayer of A17 (a) and B11 (b). Nineteen nmoles of each molecule were spread at the air-buffer interface. The subphase buffer was KCl 150 mM, Tris-HCl 10 mM, pH 7.2, thermostated at 21 °C.

obtained with a yield of 40 and 64%, respectively. The reduction of the nitro group under mild conditions with Kagan reagent21 led to the hydroxylamines 2a-2b with a good yield (60% for 2a and 78% for 2b), using Kende and Mendoza’s method.20 Next, nitronyl function was obtained by condensation of hydroxylamines 2a-2b with benzaldehyde derivative 3 in the dark. The reaction was carried out in an anhydrous mixture of THF/acetic acid (5:1, v:v) under argon for 6 days. Condensation did not occur after 72 h of reaction in pure THF. After purification by chromatography on a silica gel column, followed by LH20 size exclusion column chromatography, the nitrones 4a-4b were obtained with yields of 60 and 63%, respectively. Last, hydrolysis of the acetyl groups, using the Zemplen method, led to the amphiphilic double-chain nitrones A17-B11 with a yield of 75%. The structure of (20) Kende, A. S.; Mendoza, J. S. Tetrahedron Lett. 1991, 32, 1699. (21) Girard, P.; Namy, J. L.; Kagan, H. B. J. Am. Chem. Soc. 1980, 102, 2693.

4a-4b and A17-B11 were fully substantiated by NMR spectroscopy and FAB mass spectrometry. π-A Isotherms of Pure A17 and B11. The π-A isotherm of A17 and B11 indicated that these compounds formed stable surface monolayers because they could be compressed to surface pressures greater than 30 mN/m. The “lift-off” and collapse of the A17 π-A isotherm (Figure 1A) occurred near 116 Å2/molecule and 41 Å2/molecule, respectively. The A17 π-A isotherm showed a liquidexpanded (LE) to liquid-condensed (LC) phase transition between 4 and 6 mN/m surface pressure. Its collapse pressure was at the onset of 47 mN/m with a limiting molecular area of 50 Å2/molecule. The compression-decompression isotherm of B11 (Figure 1A) showed a limiting molecular area of 74 Å2/molecule and a collapse pressure of 40 mN/m. The π-A isotherm of B11 “lift-off” occurred near the same value as that of A17 and the collapse appeared at about 51 Å2/molecule. Thus, the molecular areas of collapse of A17 and B11 are significantly different. The cross-sectional areas of saturated hydrocarbon chains are about 20 Å2/molecule.15 The difference between A17 and B11 molecules is only the length of their hydrocarbon chains. The molecular areas of collapse of two phospholipids with the same polar headgroup and two different acyl chain lengths, DPPC and DMPC, were about 40 Å2/molecule16,22 and 50 Å2/molecule, respectively.23 DPPC molecules are able to reorganize at the air-water (22) Courrier, H. M.; Vandamme, T. F.; Krafft, M. P.; Nakamura, S.; Shibata, O. Colloids Surf., A 2003, 215, 33. (23) Smaby, J. M.; Momsen, M. M.; Brockman, H. L.; Brown, R. E. Biophys. J. 1997, 73, 1492.

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Figure 2. Brewster angle microscopy images of pure A17 (a and b) and pure B11 (c) monolayers on KCl 150 mM, Tris-HCl 10 mM, pH 7.2 at different surface pressures: 4.7 mN/m (a) (shutter speed 1/250 s) and 13.2 mN/m (d) (shutter speed 1/1000 s) for A17 and 20 mN/m (f) (shutter speed 1/50 s) for B11. Twentytwo nmoles of each molecule were spread at the air-water interface. The subphase buffer was thermostated at 21 °C.

Figure 3. (A) π-A isotherms of DLoPC/A17 mixtures. Molar percentage of A17 in the DLoPC monolayer 0 (a); 20 (b); 40 (c); 60 (d); 80 (e); and 100 (f). (B) Excess free energy of mixing (∆Gex) of binary DLoPC/A17 monolayers at different surface pressures versus the molar percentage of A17 in the mixtures. The surface pressures used were 10 (circles), 20 (squares), and 30 mN/m (triangles). Nineteen nmoles of lipids were spread on KCl 150 mM, Tris-HCl 10 mM, pH 7.2. The subphase buffer was thermostated at 21 °C.

interface during compression to induce a more condensed phase than DMPC. Thus, it seems that A17 has the same tendency to reorganize at the air-water interface as DPPC during compression. Moreover, unlike DMPC π-A isotherms, those of DPPC showed phase transition at a temperature of about 20 °C. This difference is also due to the behavior of DPPC, which is able to condense. A similar phenomenon could explain the phase transition obtained for the A17 π-A isotherm. Since the A17 collapse pressure was higher than that of B11, it seems that the increase in hydrocarbon chain length led to the stabilization of the monolayer. Furthermore, the Ks value (Figure 1B) of a B11 monolayer at 30 mN/m surface pressure was about 95 mN/m, whereas for an A17 monolayer, it was about 270 mN/m. This Ks increase as a function of chain length means that the A17 monolayer is more rigid than that of B11. For B11, the Ks increased quasi-linearly during compression, whereas for A17, the Ks values increased dramatically from 5 mN/m. This indicates that B11 was always in a LE phase and A17 in a LC phase. BAM of Pure PBN Derivatives. Figure 2 shows Brewster angle microscopy images of pure A17 and B11 at different surface pressures. BAM images of pure A17 (Figure 2a, b) showed domains which correspond to the coexistence of LE and LC regions. These domains disappeared with increasing surface pressure because all A17 molecules form a LC phase (Figure 2b). The BAM image of pure B11 (Figure 2c) did not show any domain formation as expected for a one-phase system (LE); the monolayer was homogeneous. Moreover, an ellipsometry measure-

ment on both molecules indicated an increase in membrane thickness during compression. The monomolecular film thickness of an A17 monolayer was about 2-fold greater than that of B11. π-A Isotherms of Mixtures of Phospholipids and PBN Derivatives. The measurement of compression isotherms was used to investigate lateral interactions between DLoPC or DLoPE and A17 or B11. We have chosen DLoPC and DloPE for our miscibility studies to determine the influence of the polar head size on the monolayer organization. The comparisons between the theoretical molecular areas and those determined at 10, 20, and 30 mN/m surface pressure are given as Supporting Information. DLoPC/A17 Mixtures. Figure 3A shows π-A isotherms of DLoPC mixed with various amounts of A17. DLoPC is a zwitterionic phospholipid forming fluid phase monolayers (Figure 3A, curve a). The addition of increasing amounts of A17 relative to DLoPC led to a decrease in molecular area. Moreover, a phase transition appeared with increasing A17 to phospholipid ratio. The limiting molecular area varied from 88.5 Å2/molecule for pure DLoPC to 50 Å2/molecule for pure A17. The collapse pressure of the DLoPC/A17 mixtures varies with monolayer composition (data not shown). This indicates that these two components are miscible. The excess intermolecular interactions were calculated by determining the ∆Gex at 10, 20, and 30 mN/m surface pressure (Figure 3B). At 20 and 30 mN/m surface pressure, the negative ∆Gex observed suggests that the interactions between DLoPC and A17 are of a predominantly attractive

PBN Derived Amphiphilic Spin-Traps

Figure 4. (A) π-A isotherms of DLoPE/A17 mixtures. Molar percentage of A17 in the DLoPE monolayer 0 (a); 20 (b); 40 (c); 60 (d); 80 (e); and 100 (f). (B) Excess free energy of mixing (∆Gex) of binary DLoPE/A17 monolayers at different surface pressures versus the molar percentage of A17 in the mixtures. The surface pressures used were 10 (circles), 20 (squares), and 30 mN/m (triangles). Nineteen nmoles of lipids were spread on KCl 150 mM, Tris-HCl 10 mM, pH 7.2. The subphase buffer was thermostated at 21 °C.

nature, whereas at 10 mN/m the interactions between these components are globally repulsive in character. Only the 80% mol A17 monolayer showed an attraction between the two components at 10 mN/m surface pressure. These results suggest that the 2D miscibility of these two components was nonideal. DLoPE/A17 Mixtures. π-A isotherms of DLoPE mixed with various amounts of A17 are represented in Figure 4A. Increasing amounts of A17 relative to DLoPE led to a molecular area decrease. The limiting molecular area varied from 78 Å2/molecule for pure DLoPE to 50 Å2/ molecule for pure A17. Moreover, increasing A17 to DLoPE ratios led to the appearance of a phase transition. These two components were miscible as shown by the collapse pressures of the DLoPE/A17 mixtures which vary with the monolayer composition. The excess intermolecular interactions were calculated by determining the ∆Gex at 10, 20, and 30 mN/m surface pressure (Figure 4B). At 20 and 30 mN/m surface pressure, the negative ∆Gex observed suggests that the interactions between DLoPE and A17 are predominantly attractive in character, whereas at 10 mN/m the interactions between these components are repulsive. Moreover, as for DLoPC/ A17 mixtures, only at 80% mol was A17 attracted to DLoPE at 10 mN/m surface pressure. It seems that whatever the nature of the polyunsaturated lipid, A17 exerts significant repulsive interactions with DLoPC or DLoPE at low surface pressure. This might be due to the coexistence of LE-LC phases in A17 monolayers at 10 mN/m surface pressure or below. At high surface pressure (in the LC phase of the monolayer), the interactions between components are attractive.

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Figure 5. (A) π-A isotherms of DLoPC/B11 mixtures. Molar percentage of B11 in the DLoPC monolayer 0 (a); 20 (b); 40 (c); 60 (d); 80 (e); and 100 (f). (B) Excess free energy of mixing (∆Gex) of binary DLoPC/B11 monolayers at different surface pressures versus the molar percentage of B11 in the mixtures. The surface pressures used were 10 (circles), 20 (squares), and 30 mN/m (triangles). Nineteen nmoles of lipids were spread on KCl 150 mM, Tris-HCl 10 mM, pH 7.2. The subphase buffer was thermostated at 21 °C.

DLoPC/ B11 Mixtures. The interface behavior of DLoPC/ B11 mixtures was also investigated and the relevant π-A isotherms are represented in Figure 5A. Because the molecular areas of these two components are not very different, the differences between π-A isotherms of the mixtures are relatively small. The limiting molecular area varied from 88.5 Å2/molecule for pure DLoPC to 74 Å2/ molecule for pure B11. Nevertheless, the increase in B11 content in the monolayer led to a decrease in molecular area. As for A17 containing mixtures, the collapse pressures of DLoPC/B11 mixtures were not significantly different from the theoretical collapse pressure, implying that B11 is miscible with DLoPC. The calculated ∆Gex (Figure 5B) exhibited only high positive values at all surface pressures, implying that interactions between DLoPC and B11 are repulsive in nature at each surface pressure and for each percentage of B11 in the monolayer. DLoPE/B11 Mixtures. Figure 6A shows π-A isotherms of DLoPE monolayers mixed with various amounts of B11. The limiting molecular area varied from 78 Å2/molecule for pure DLoPE to 74 Å2/molecule for pure B11. The ∆Gex values, represented in Figure 6B versus molar fraction of B11, indicate that at low surface pressure (10 mN/m), interactions between DLoPE and B11 are repulsive, but at 20 and 30 mN/m surface pressure interactions are attractive up to 20% mol B11 and repulsive after 40% mol B11. At 10 mN/m surface pressure, it seems that up to 20% mol B11 in a DLoPC or DLoPE monolayer the mixture

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Figure 7. Ks values determined for each DLoPC or DLoPE mixtures with A17 or B11 at 30 mN/m surface pressure minus theoretical Ks values at 30 mN/m (∆Ks30mN/m) plotted versus molar percentage of A17 or B11. ∆Ks30mN/m for DLoPC/A17 mixtures: black triangles; ∆Ks30mN/m for DLoPE/A17 mixtures: white triangles; ∆Ks30mN/m for DLoPC/B11 mixtures: black circles; and ∆Ks30mN/m for DLoPE/B11 mixtures: white circles. The subphase buffer was KCl 150 mM, Tris-HCl 10 mM, pH 7.2, and thermostated at 21 °C.

Figure 6. (A) π-A isotherms of DLoPE/B11 mixtures. Molar percentage of B11 in the DLoPE monolayer 0 (a); 20 (b); 40 (c); 60 (d); 80 (e); and 100 (f). (B) Excess free energy of mixing (∆Gex) of binary DLoPE/B11 monolayers at different surface pressures versus the molar percentage of B11 in the mixtures. The surface pressures used were 10 (circles), 20 (squares), and 30 mN/m (triangles). Nineteen nmoles of lipids were spread on KCl 150 mM, Tris-HCl 10 mM, pH 7.2. The subphase buffer was thermostated at 21 °C.

behaves ideally, but at 20 and 30 mN/m, the interactions between B11 and phospholipids are repulsive in nature. The B11 monolayer never reached an LC phase during compression, because of the length of the hydrocarbon chain. A17 is attracted to DLoPC or DLoPE at high surface pressures (20 and 30 mN/m), whereas B11 is repulsed at these surface pressures. Moreover, the range of ∆Gex variations were the same for A17, regardless of the nature of the headgroup of the phospholipid. B11 was significantly more repulsed by DLoPC than by DLoPE. This could be due to the polar headgroup of phosphatidylcholine, which occupies a larger surface than that of phosphatidylethanolamine, as shown by the molecular area of DLoPC (88.5 Å2/molecule) compared to that of DLoPE (78 Å2/molecule). Fluidity of Phospholipid/PBN Derivative Monolayers. In comparison to A17 (Figure 1B), B11 is unable to rigidify phospholipid membranes because its Ks value at 30 mN/m is about the same as that of a common phospholipid. Theoretical and calculated Ks values at 30 mN/m surface pressure were determined for all of the mixed monolayers. Figure 7 shows the difference between the calculated Ks30mN/m and the theoretical Ks30mN/m (∆Ks30mN/m) plotted versus the molar percentage of A17 or B11. It seems that the ∆Ks30mN/m for B11 mixtures is significantly smaller than that of A17 mixtures. Moreover, the DLoPC/B11 mixture had a ∆Ks30mN/m very close to 0, indicating that the calculated Ks value at 30 mN/m surface pressure was about the same as the theoretical value. For DLoPE/B11 mixtures the ∆Ks30mN/m was negative, indicating that the monolayer was more fluid than expected. The

∆Ks30mN/m for A17 containing mixtures were significantly higher than those obtained for B11. A DLoPC monolayer is made more fluid by A17 than a DLoPE monolayer, because the Ks30mN/m were lower for all molar percentages of A17 in the monolayer (data not shown). As one can see, only the B11 containing mixtures seem to behave as they ideally should. For all A17 containing mixtures, the ∆Ks30mN/m obtained were very low, that is, the determined Ks30mN/m were lower than the ideal ones. This indicated that the monolayer was less rigid than expected at all percentages. The A17 molecules, which had the greatest ability to change the physical properties of phospholipid monolayers, rigidified membranes, although not as well as expected, whereas B11, which is unable to change the membrane properties, showed significant repulsive interactions with phosphatidylcholine. This could be a problem for B11 because these repulsive interactions appeared at 20% mol B11 in the DLoPC monolayer, meaning that the interactions between these two components were not favorable. Conclusions In the condensed phase of the A17 π-A isotherm, the interactions with the two phospholipids (DLoPC and DLoPE) were attractive in character, whereas at low surface pressure (10 mN/m) the interactions were repulsive. For B11, the interactions with DLoPC were repulsive at all surface pressures. DLoPE/B11 mixtures showed very small attractive interactions at less than 20% mol B11 at 20 and 30 mN/m surface pressure and at 40% mol B11 or more the interactions with DLoPE were repulsive in nature. The polyunsaturated phospholipids/A17 monolayers were more fluid than expected. This implies that A17 was not able to rigidify the membrane as well as cholesterol.15 To study the potential antioxidant behavior of these PBN derivatives, we will use the method described by Morandat et al.24 for an another antioxidant, plasmalogen. The monolayer surface pressure must be fixed at 30 mN/m to mimic the internal pressure of biological membranes.25 Under these conditions, A17, which showed attractive interactions at this surface pressure, should be a better (24) Morandat, S.; Bortolato, M.; Anker, G.; Doutheau, A.; Lagarde, M.; Chauvet, J.-P.; Roux, B. Biochim. Biophys. Acta 2003, in revision. (25) Marsh, D. Biochim. Biophys. Acta 1996, 1286, 183.

PBN Derived Amphiphilic Spin-Traps

antioxidant than B11 because the interactions between A17 and DLoPC or DLoPE are favorable. Indeed, preliminary tests of the effect of A17 molecules on DLoPC UV-oxidation show that A17 is able to prevent DLoPC oxidation very efficiently. These studies are currently underway. Acknowledgment. We thank Karim El Kirat for helpful discussions. This work was supported by the “Centre National de la Recherche Scientifique” (CNRS), the “Ministe`re de l’Enseignement Supe´rieur et de la Recherche” (MESR), and in part by a grant of the “Re´gion

Langmuir, Vol. 19, No. 23, 2003 9705

Rhoˆne Alpes”. Brewster angle microscopy experiments were performed at the “Institut Multidisciplinaire de Biochimie des Lipides” (IMBL). Supporting Information Available: Synthetic procedures, analytical, and spectral characterization data. Variations of determined molecular area at 10, 20, and 30 mN/m as a function of the PBN derivatives molar fraction for mixed DLoPC/A17, DLoPE/A17, DLoPC/B11, and DLoPE/B11 monolayers. This material is available free of charge via the Internet at http://pubs.acs.org. LA034470T