A Surface Active Benzodiazepine Receptor Ligand for Potential

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Bioconjugate Chem. 2008, 19, 1888–1895

A Surface Active Benzodiazepine Receptor Ligand for Potential Probing Membrane Order of GABAA-Receptor Surroundings Anahı´ V. Turina,† Benjamı´n Caruso,† Gloria I. Yranzo,‡ Elizabeth L. Moyano,‡ and Marı´a A. Perillo*,† Biofı´sica-Quı´mica, Ca´tedra de Quı´mica Biolo´gica, Departamento de Quı´mica, Facultad de Ciencias Exactas, Fı´sicas y Naturales, and INFIQC-Departamento de Quı´mica Orga´nica, Facultad de Ciencias Quı´micas, Universidad Nacional de Co´rdoba. Av. Ve´lez Sarsfield 1611, 5016 Co´rdoba, Argentina. Received April 28, 2008; Revised Manuscript Received July 17, 2008

A conjugable analogue of the benzodiazepine 5-(2-hydroxiphenyl)-7-nitro-benzo[e][1,4]diazepin-2(3H)-one N1substituted with an aliphatic chain (CNZ acyl derivative, CAd) was synthesized. CAd inhibited FNZ binding to GABAA-R with an inhibition binding constant Ki ) 176 nM and expanded a model membrane packed up to 13 mN/m when penetrating from the aqueous phase. CAd exhibited surface activity with a collapse pressure π ) 18.8 mN/m and minimal molecular area Amin ) 49 Å2/molecule at the closest molecular packing, resulting in full and nonideal mixing with a phospholipid in a monolayer up to a molar fraction x = 0.1, decreasing its surface potential and contributing with a dipole that pointed its positive end toward the air and reoriented at the interface upon compression. These findings suggested that CAd could be stabilized at the membrane-water interface with its CNZ moiety stacked at the GABAA-R while its acyl chain can be inserted into the membrane depth.

1. INTRODUCTION 1

The 1,4-benzodiazepin-2-ones (BZDs) are drugs widely used as anxiolytics, hypnotics, and anticonvulsants. They act by enhancing the activity of the neurotransmitter γ-amino butyric acid (GABA) through an allosteric binding site at the integral membrane receptor GABAA (GABAA-R) (1). This is a pentameric protein that acts as a ligand-gated chloride channel and is the site of action of a variety of pharmacologically important drugs including benzodiazepines (BZDs) (2). Several nonspecific mechanisms are known to affect the conformation and activity of membrane-bound protein receptors, such as (i) the coupling between hydrophobic mismatch and curvature stress (3), (ii) changes in the lateral stresses profile (the depth-dependent distribution of lateral stresses within the membrane) which affect the conformation equilibrium and the activity of intrinsic proteins, the function of which involves a structural change accompanied by a depth-dependent variation in its cross-sectional area within the transmembrane domain (4), and (iii) the dipolar arrangement of the membrane which was shown to affect significantly the insertion, folding, conformation, and activity of membrane proteins (5, 6). With GABA-R as an example, studies on natural membranes support the hypothesis that the allosteric modulation of monoterpenes (6, 7) and detergents (8) on this receptor comprise effects caused by drug insertion or other sources of mechanical tension on the supramolecular organization of the receptor environment, through * Corresponding author. Biofı´sica-Quı´mica, Departamento de Quı´mica, Facultad de Ciencias Exactas, Fı´sicas y Naturales, Universidad Nacional de Co´rdoba, Av. Ve´lez Sarsfield 1611, 5016 Co´rdoba, Argentina. E-mail: [email protected]. Phone: +54-351-4344983 int 5. FAX: +54-351-4334139. † Departamento de Quı´mica. ‡ INFIQC-Departamento de Quı´mica Orga´nica. 1 Abbreviations: BZD, benzodiazepines; CAd, clonazepam acyl derivative; CNZ, clonazepam; dpPC, dipalmitoylphosphatidylcholine; DZ, diazepam; FNZ, flunitrazepam; GABA, gamma aminobutyric acid; GABAA-R, GABAA receptor; MGd, methyl glycinate derivative; NMR, nuclear magnetic resonance spectroscopy; sem, standard error of the mean; SM, synaptosomal membranes; TLC, thin layer chromatography; TMS, tetramethyl silane.

the mechanisms described above. Electrophysiological (9) and binding (10) experiments showed that the cholesterol content, a known buffering mechanism of membrane microsviscosity, affected the coupling between the binding sites for BZDs and the other drugs that interact with GABAA-R. Moreover, temperature-induced variations in membrane microviscosity also modulated the ligand binding to GABAA-R ((11) and refs therein). Membrane microviscosity is considered the main modulatory mechanism of membrane protein function. Changes in this general membrane property are accompanied by modifications in the hydrophobic thickness or in the lateral pressure profile of bilayers, which, as stated above, are indirect ways of connecting the protein structure with its function (12-17). It is known that the plasma membrane of cells is substantially ordered (e.g., RBL-2H3 cells (18)). It exhibits a limiting anisotropy (0.225 ( 0.005) significantly higher than that observed in the fluid phase of liposome bilayers. This indicates that, on average, a considerable amount of order exists in plasma membrane (19). However, far from homogeneous, many types of experiments have shown that, at the mesoscopic level, the plasma membranes of cells are patchy and locally differentiated into domains of different degrees of molecular order, some of which arise through lipid-lipid interactions (20). For this reason, typical evaluation of microviscosity through the analysis of general anisotropy is not enough to understand the GABAA-R sensitivity to membrane mechanical properties. The latter should include a scan of GABAA-R closest membrane surroundings and may be achieved by the use of molecular probes that can remain close to the protein and, at the same time, allow the scanning of the local molecular organization within the membrane depth. Hence, we propose to use a probe bearing, at one end, a GABAA-R ligand that will make the whole probe capable of stacking at the receptor and, at the other end, a hydrophobic tail with a fluorescent or spin label moiety covalently attached. The latter, once inserted in the membrane, would provide spectroscopic information about the molecular order within the membrane depth where it is located. This approach is based on similar studies on the nicotinic acetylcholine receptor (21). In our laboratory, we have tackled this challenge in two stages.

10.1021/bc800175z CCC: $40.75  2008 American Chemical Society Published on Web 08/13/2008

GABAA Receptor Surroundings’ Sensor Development Scheme 1. Synthesis of the CNZ-Acyl Derivative (CAd)

The first stage, consisting of the synthesis of a surface active GABAA-R ligand, will be followed by the addition of the sensing group in a second stage. In the present paper, we describe the synthesis, the GABAA-R binding kinetics, and the biophysical properties of a 5-(2hydroxiphenyl)-7-nitro-benzo[e][1,4]diazepin-2(3H)-one (clonazepam, CNZ) substituted at the N1 position with an aliphatic chain which was named CNZ acyl derivative (CAd). The ability of this compound to interact and stabilize at the membrane-water interface was studied in monomolecular layers at the air-water interface by the Wilhelmy plate method. Molecular parameters determined from surface pressure-area isotherms were used to predict not only the type of self-assembled structures that CAd would be able to form in aqueous dispersions, but also the molecular features that would contribute to enhancing the stability of this compound in the typical planar configuration that characterizes a membrane bilayer.

2. EXPERIMENTAL PROCEDURES 2.1. Materials. BZDs diazepam and clonazepam (DZ and CNZ) were kindly supplied by Products La Roche (Co´rdoba, Argentina). [3H]-FNZ was purchased from New England Nuclear Chemistry (E.I. DuPont de Nemours & Co. Inc., Boston, MA). Dipalmitoylphosphatidylcholine was obtained from Avanti Polar Lipids (Alabaster, AL). Other drugs and solvents were of analytical grade. Merck silica gel 60 was used for filtration and Aldrich silica gel 60 F254 was used for preparative TLC. 2.2. Procedure for Preparation of CNZ Derivative. A methanolic solution of sodium methoxide (1.85 mmol/mL methanol) was added to a solution of CNZ (1.52 mmol) in 25 mL of anhydrous methanol as described in ref 22. The mixture was refluxed for a period of 15 min. After this time, 2.26 mmol of 1-bromooctane was added and subsequently refluxed for 12 h. After cooling, methanol was evaporated and the crude was filtered using silica gel. This mixture was then purified by preparative TLC using hexane/ethyl acetate (80:20) and petroleum ether/ethyl acetate/ethanol (95:4:1) as eluents. The reaction yield was 50% compound 3 and 30% compound 4 as main products. These derivatives will be named CAd and MGd, respectively (see Scheme 1). 5-(2-Chlorophenyl)-6-nitro-1-octyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one (3, CAd). 1H NMR (200 MHz, CDl3): δ 0.85 (t, J ) 6.6 Hz, 3H), 1.24 (s, 10H), 1.60 (m, 2H), 4.31 (m, 2H), 4.37 (dd, J ) 11 and 228 Hz, 2H), 7.30-7.50 (m, 4H), 7.55 (d, J ) 9.1 Hz, 1H), 7.62-7.70 (m, 1H), 7.93 (d, J ) 2.6 Hz, 1H),

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8.35 (dd, J ) 2.6 and 9.1 Hz, 1H). 13C (50.33 MHz, CDCl3): δ ) 14.7, 23.2, 27.6, 29.1, 29.8, 29.8, 32.4, 48.2, 57.8, 123.2, 125.3, 126.6, 128.1, 131.0, 131.8, 132.4, 133.7, 137.8, 143.9, 147.8, 168.6, 169.2. Methyl-N-{(2-chlorophenyl)-[2-nitro-6-(octylamino)phenyl]methylene}glycinate (4, MGd). 1H NMR (200 MHz, CDCl3): δ 0.89 (t, J ) 6.6 Hz, 3H), 1.30 (s, 10H), 1.84 (q, J ) 7.3 Hz, 2H), 3.37 (m, 2H), 3.76 (s, 3H), 4.04 (q, J ) 19 and 47 Hz, 2H), 6.72 (d, J ) 9 Hz, 1H), 7.09-7.14 (m, 1H), 7.35-7.60 (m, 4H), 7.71 (d, J ) 2.6 Hz, 1H), 8.11 (dd, J ) 4 and 10 Hz), 11.08 (s, 1H). 13C (50.33 MHz, CDCl3): δ ) 14.1, 22.6, 27.3, 29.2, 29.3, 31.8, 43.5, 51.9, 54.2, 110.4, 115.9, 127.6, 128.6, 129.8, 130.3, 131.4, 133.8, 135.1, 154.4, 170.2, 170.6. 2.3. NMR Spectroscopy. All starting materials were commercially available and solvents were distilled and dried before use. 1H and 13C NMR spectra were recorded on a Bruker FT200 (1H at 200 MHz and 13C at 50.33 Hz) spectrometer using CDCl3. Chemical shifts are reported in parts per million (ppm) downfield from TMS. 2.4. Monolayer Studies. 2.4.1. Surface Pressure-Area and Surface Potential-Area Compression Isotherms. Monomolecular layers were prepared and monitored essentially according to Perillo et al. (23). Experiments were performed at room temperature. The surface pressure (π, Wilhelmy plate method via a platinized-Pt plate), surface potential (∆V, vibrating plate method), and the area enclosing the monolayer (A) were automatically measured with a Minitrough II (KSV, Helsinki, Finland). The Teflon trough used had 24 075 mm2 total area. Bidistilled water (230 mL total volume) was used as subphase. Lipid monolayers were formed by spreading, on the air-water interface, between 30 and 80 µL of 1 mg/mL chloroformmethanol 2:1 of (a) the newly synthesized CNZ acyl derivative (CAd), (b) a pure saturated phospholipid (dipalmitoylphosphatidylcholine, dpPC), or (c) a binary mixture dpPC-CAd at a molar fraction (x) varying between 0 and 1. The π-A and ∆V-A compression isotherms were recorded continuously, at a compression rate of 5 mm/min. Isotherms shown resulted from typical experiments repeated at least twice. 2.4.2. Compressional Modulus and Critical Packing Parameter Calculation. The compressional modulus (K) was calculated according to eq 1. δπ ( δA )

K ) - (Aπ)

π

(1)

The critical packing parameter (PC) was calculated according to the Israelachvili theory (24) by eq 2. Pc )

V a0 · lc

(2)

The average molecular area (a0) was experimentally determined from π-A isotherms, and optimal values for the hydrocarbon volume (V) and chain length (lc) were calculated from eqs 3 and 4, according to Perillo et al. ((23) and refs therein): V = (27.4 + 26.9n)nch

(3)

lc = 1.5 + 1.265n

(4)

where n and nch are the number of methylene groups in the hydrocarbon chain and the number of chains per molecule, respectively. The molecular area (a0) is a function of the interfacial free energy; hence, it was assigned the actual mean molecular areas (Mma) at specific values of surface pressure taken from the π-A isotherm of each monolayer at the different compositions studied. Pc values for the binary dpPC-CAd mixtures were calculated from the weighted mean values of (V) and (lc) of individual components.

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2.4.3. Penetration of CAd in Monomolecular Layers of dpPC at the Air-Water Interface. The aim of this experiment was to determine the maximum value of π that allowed drug penetration in the monolayer (πcutoff). These experiments were done in a circular Teflon trough (4.5 cm diameter and 0.5 cm depth). Between 5 and 30 µL of a chloroform-methanol 2:1 solution of phospholipid were spread on an aqueous surface (bidistilled water) and about 5 min were allowed for solvent evaporation. Monolayers were prepared at constant surface area but at different initial surface pressures (πi). The temporal variation of π induced by the CAd penetration into the monolayer after the injection of an ethanolic solution of the derivative in the subphase was measured until reaching a plateau (πmax). The values of ∆π ) πmax - πi were plotted against πi and a straight line was fitted to them. The πcutoff was determined from the intersection of the regression line with the abscissa axis. 2.5. Synaptosomal Membrane Preparations. Synaptosomal membranes (SM) were obtained from bovine brain cerebral cortex. Meninges were eliminated, the cortex dissected, and the SM were purified essentially according to the method of Enna and Snyder, modified by Perillo and Arce (25), lyophilized and stored at -20 °C. Immediately before use, membranes were resuspended in 50 mM pH 7 Hepes buffer containing 100 mM NaCl at a final protein concentration of 0.25 mg/mL. This SM suspension was used as membrane receptor preparation and GABAA-R source in the experiments that followed. 2.6. Competition Binding Experiments. The aim of this experiment was to assess the ability of the newly synthesized CAd to displace [3H]-FNZ from its binding site at the GABAA-R in SM. Binding was performed essentially as described previously (26). The whole procedure was carried out at 4 °C. The incubation system contained, in a final volume of 230 µL, the SM suspension at a final protein concentration of 0.25 mg/mL, 3 nM (minimum specific activity 74.1 Ci/mmol) [3H]-FNZ, 100 mM NaCl-50 mM Tris-HCl pH 7.4 buffer containing 9.4 µM DZ or 10 µM CAd (final concentrations). Samples were incubated at 4 °C in the dark for 1 h and then filtered through SS filters (Whatman GF/B type) with a Brandel automatic filtration apparatus (Brandel, Gaithersburg, MD). After filters were rinsed and dried in the air, they were placed in vials containing 2.5 mL of scintillation liquid (25% v/v Triton X-100, 0.3% w/v diphenyloxazole in toluene). The retained radioactivity was measured with a scintillation spectrometer Rackbeta 1214 (Pharmacia-LKB, Finland) at 60% efficiency for tritium. Specific binding (B) was calculated as the difference between total binding (TB) and nonspecific binding (NB) determined in the absence and in the presence of 9 nM DZ, respectively. Protein concentration was determined by the method of Lowry (27). 2.7. Statistical Analysis. Binding data were statistically analyzed using a two-tailed Student’s t-test for independent samples. p < 0.05 was considered to be statistically significant. Regression analysis was done by the least-squares method (28).

3. RESULTS AND DISCUSSION 3.1. Synthesis of the Probe Precursor. The probe precursor (3, CAd) was synthesized from the sodium salt (2) of 5-(2chlorophenyl)-7-nitro-1,3-dihydro-2H-1,4-benzodiazepin-2one (CNZ) (1) and 1-bromooctane (Scheme 1). The salt (2) was obtained by the treatment of CNZ with sodium methoxide according to the literature (22). The reaction of the BZD 1 and the bromoderivative was carried out in situ without isolation of the salt intermediate. The methyl N-methylene glycinate 4 (MGd) was also obtained in this reaction by the hydrolysis reaction of the benzodiazepinone ring in the presence of methanol. The formation of compound 4 could not be avoided

Turina et al.

Figure 1. Displacement of the [3H]-FNZ bound at synaptosomal membranes from bovine cortex induced by CAd and DZ at similar concentrations. The [3H]-FNZ and protein concentration used were 3 nM and 0.25 mg prot/mL, respectively. Other details were described in the Experimental Procedures section. Data shown are the mean ( sem of triplicates. *, significantly different with respect to E (p < 0.01, Student’s t test).

even when the reaction times and temperatures were modified. Therefore, compound 3 was purified as described in section 2.2 (purity was higher than 95%). Detailed spectra are provided as Supporting Information. 3.2. [3H]-FNZ Displacement Experiments. The ability of CAd to displace [3H]FNZ was evaluated by means of a radioreceptor binding assay. Results were analyzed in comparison with the well-known competitive inhibitor DZ. The results, depicted in Figure 1, show that DZ as well as CAd at similar concentrations (9 and 10 µM, respectively) were able to displace 80% and 59% of the total [3H]FNZ bound to SM, respectively. According to the denifition given in the Experimental Procedures section, the 20% residual binding in the presence of 9 nM DZ corresponded to nonspecific binding. Furthermore, CAd at the concentration assayed left a 21% undisplaced binding over the nonspecific indicating a lower affinity for the GABAA-R with respect to DZ. Assuming that CAd induces a competitive inhibition of [3H]FNZ binding, the inhibition binding constant (Ki) of this displacement agent can be calculated from eq 5 as follows: B)

BmaxL [I] Kd 1 + +L Ki

(

)

(5)

where L is the free radioligand concentration (3 nM), B and Bmax are the specific activities of bound radioligand (expressed in moles per protein mass units) at L or at saturating radioligand concentration, respectively, and Kd is the dissociation binding constant of the radioligand-receptor interaction. With Bmax ) 1404 fmol/mg protein and Kd ) 2.28 nM for [3H]FNZ binding to SM (26) and the experimental data shown in Figure 1, the Ki values for CAd were 176 nM. This value was similar to previously reported data (29) for other BZDs. This was a strong indication that the CNZ acyl derivative not only could interact with the BZD binding site at GABAA-R, but also that it was bound with an affinity comparable with that of a typical BZD. 3.3. Monomolecular Layers at the Air-Water Interface. 3.3.1. Surface Pressure-Molecular Area Isotherms. Surface pressure-area isotherms are shown in Figure 2. In Figure 2a, an isotherm of pure dpPC is shown as a reference. Dashed lines indicate the determination of the collapse pressure (πc ) 55.5 mN/m), the molecular area at the closest packing also known as minimal molecular area (Amin ) 40 Å2), and the transition surface pressure (πT ) 6 mN/m) corresponding to the bidimensional phase transition characteristic of dpPC.

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Figure 2. Behavior of dpPC/CAd binary mixtures at the air-water interface. (a) Collapse pressure (πc), transition pressure (πT), and minimal molecular area (Amin) determination exemplified over a dpPC π-A compression isotherm. (b) Surface pressure-Mma isotherms of dpPC-CAd binary mixtures. (c) Compressional modulus at 6 and 35 mN/m, as a function of monolayer composition. (d) Surface potential-Mma and (e) µ⊥-Mma compression isotherms of dpPC-CAd binary mixtures. Dipole moments (µ⊥) calculated according to eq 6 from ∆V values taken from panel (d) depicted as a function of Mma (f) Resultant dipolar modulus at 6 and 35 mN/m, as a function of monolayer composition. Numbers in panel (e) indicate the CAd molar fraction.

CAd was able to form monomolecular layers at the air-water interface both alone (Figure 2a) as well as in mixtures with dpPC at various molar fractions (x) (Figure 2b). From the analysis of the π-A compression isotherm of CAd (Figure 2a), a collapse pressure πc ) 18.8 mN/m and Amin ) 49 Å2 were determined and no bidimensional transitions were observed. In comparison with dpPC, CAd exhibited lower stability as reflected by its lower πc value, which may be due to the shorter length of its hydrocarbon chain. This provides a low London dispersion energy stabilizing the aggregate, which is not enough to counterbalance the repulsive interactions between the polar head groups. This leads to an inefficient molecular packing explaining the fact that, in spite of having only one hydrocarbon chain and not two as in the case of dpPC, CAd exhibits a minimal molecular area substantially higher than that of the phospholipid. The CAd/dpPC mixtures exhibited full miscibility up to xCAd = 0.1. Above xCAd ) 0.1, mixtures showed several bidimensional reorganizations indicating the occurrence of partial collapse processes of different bidimensional phases (enriched in dpPC, within the range 0.1 < xCAd < 0.4, or in CAd, within the range 0.4 < xCAd ) 1) coexisting in the monolayer. This indicated a partial miscibility of both components (Figure 2b). Further analysis was done through a phase diagram (see Figure 3 below). The effect of the proportion of CAd in the mixtures with dpPC on the monolayer elasticity was analyzed through the compressibility modulus K (Figure 2c) in the conditions of full miscibility at all compositions (6 mN/m) or compositionally

dependent miscibility (35 mN/m). At 6 mN/m, K increased continuously as a function of xCAd reflecting both the higher coherence of CAd films with respect to the liquid expanded phase of dpPC (corresponding to xCAd ) 0) as well as full miscibility of both compounds at this lateral pressure. At 35 mN/m, K decreased as a function of xCAd showing discontinuities that reinforced the different miscibility regimes exhibited by the phase diagram (miscibility of CAd in dpPC which is lost at xCAd e 0.1) and suggested a partial miscibility of dpPC in a continuous CAd phase within the range 0.4 < xdpPC < 0.6). 3.3.2. Surface Electrostatics. The surface potential ∆V-area isotherms (Figure 2d) showed that ∆V increased upon compression and decreased as a function of CAd in the mixtures. At the closest packing, ∆VdpPC ) 530 mV and ∆VCAd ) 296 mV, while the mixtures showed intermediate values. Surface potential is a measure of the electrostatic field gradient perpendicular to the membrane interface and thus varies considerably with the molecular surface density and with changes in orientation accompanying the monolayer compression. Taking the dielectric constant of the medium (water) as unity, the molecular dipole moment can be calculated according to eq 6 ∆V ) 12 ×

π µ + ψ0 A ⊥

(6)

where π ) 3.1416, A (molecular area), and V (surface potential) are expressed in Å2.molecule-1 and mV, respectively, µ⊥ (expressed in Debye units, mD) is the apparent (resultant) perpendicular dipole moment of the molecule, and ψ0 is the

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Figure 3. Surface pressure-composition phase diagram overlapped with critical packing parameter (Pc) for dpPC-CAd mixtures. Numbers on the graph refer to Pc values calculated for each mixture at the specified surface pressure. NT: no structure predicted in the theory (Pc > 1). The phase diagram (π-xCAd) was constructed using πc values (9), typical pure dpPC bidimensional phase transition pressure (b), and phase transitions pressures in the mixtures (1 and O), taken from Figure 2. The inset shows the Mma corresponding to those surface pressures at which bidimensional phase transitions and collapses occur, at each of the CAd molar fractions studied. (b) bidimensional phase transition characteristic of the pure dpPC, (O and 1) other bidimensional phase transitions observed in mixtures, and (9) Mma at the monolayer collapse point. Note that Pc values above the line joining the phase transition points, particularly in the range 0.2 < xCAd < 0.4, might be meaningless due to possible immiscibility.

electrostatic potential difference at the interface caused by the ionic double layer in ionized monolayers (for uncharged molecules ψ0 ) 0) (23, 30). The parameter µ⊥ contains different electrostatic contributions. In the case of uncharged molecules such as dpPC (a zwitterionic phospholipid) and CAd (uncharged at the pH assayed), main contributions arise from the resultant dipole moments of the monolayer components and those of the water hydration network (water molecules oriented at the polar headgroup). In turn, contributions from monolayer components include the polar headgroup and hydrocarbon chain contributions. An initial increase in the magnitude of µ⊥ was observed upon compression due to molecular reorientations at the surface (Figure 2e). Beyond a maximum, all µ⊥ vs Mma plots showed a decreasing trend which, in conjunction with the information taken from the π-Mma curves, can be interpreted as an indication of a monolayer compositional change due to instability occurring at high surface pressures. Figure 2f shows that, at π low enough to allow total mixing between dpPC and CAd, µ⊥ decreased upon an increase in xCAd. At a high surface pressure (35 mN/m), (a) within the xCAd range that allowed miscibility, µ⊥ was higher than at 6 mN/m, and (b) at xCAd > 0.1, µ⊥ values exhibited a decreasing tendency even more noticeable than at 6 mN/m which, in this case, would be reflecting the partial collapse of the monolayer (see phase diagram in Figure 3). 3.3.3. dpPC-CAd Binary Mixtures: Phase Diagram and Prediction of Their Self-Assembling Structures in Water. From data shown in Figure 2b, the π-x phase diagram was constructed (Figure 3). Lines in Figure 3 indicate the presence of π-x states at which phase transitions occur and delimit regions of single bidimensional phases that may coexist with an already collapsed phase. A single monolayer phase can be found at low pressures within the whole compositional range. This monolayer shows a πT that varies continuously with xCAd (triangles joint by a dotted line). This πT might be associated either with a bidimensional phase transition or with a partial collapse of the

Turina et al.

monolayer. The second hypothesis is supported by the shape inspection of π-A isotherms at the high xCAd (e.g., above 0.6) which suggests that after a collapse point monolayers go through overcompression states. At higher pressures, another partial collapse is observed at a πc that remains invariant up to xCAd ) 0.4 and then, at xCAd > 0.4, it decreases continuously with xCAd (hollow circles) up to the point where πc equaled the πc value of pure CAd. Hence, CAd seems totally miscible in dpPC up to xCAd ) 0.1, exhibiting a bidimensional phase transition between 6 and 8 mN/m. Within the xCAd range 0.1-0.2, the solubility of CAd in dpPC would be reached. This is based on the fact that, upon compression, the collapse pressure of mixtures with 0.1 < xCAd < 0.2 decreased from 39 to 37 mN/m, while at 0.1 < xCAd < 0.4, the πc value remained constant at πc ) 37 mN/m. Within this compositional range, there was a remaining monolayer that would be composed of a dpPC excess as indicated by a third collapse point which had the typical value of 55 mN/m. The interfacial stability of mixtures with xCAd < 0.4 decreased continuously as a function of the xCAd as indicated by the decreasing πc. Moreover, within this compositional range, after the monolayer collapsed no excess of dpPC remained stable at the air-water interface. Critical packing parameter values (Pc) allow the prediction of the type of self-assembling structures that would be formed when an amphipathic substance is dispersed in water. Pc values for the binary dpPC-CAd mixtures were calculated by eqs 2, 3, and 4 using values of mean molecular area which were taken from the isotherms shown in Figure 2b. Those Pc values were superimposed on the phase diagram (Figure 3). Pc < 0.5 predicts the self-assembling into micelles, 0.5 < Pc < 1 predicts the self-association into bilayer phases which would lead to the formation of vesicles, and Pc > 1 would correspond to phases with negative curvature. Although the latter are not predicted in Israelachvili’s theory, those phases are compatible with inverted vesicles, as well as with hexagonal II and cubic phases (23, 31). According to this interpretation in Figure 3, within the region located at the bottom-left corner of the π-xCAd phase space (at low xCAd and π), the resulting Pc values were compatible with the formation of bilayers. At the bottom-right corner (high xCAd and low π), Pc values predicted the formation of micelles. At the top of the π-xCAd phase space, in the region located above a line crossing the plane approximately along the diagonal from 45 mN/m at xCAd ) 0 through 28 mN/m at xCAd ) 0.4 to 18.8 at xCAd ) 1, Pc values were all above 1. In conditions of total miscibility, this may be interpreted as an indication of a tendency to form negative curvature phases when the mixture is dispersed in water. However, within regions of the phase diagram where components are partially miscible with one another, these calculated Pc values become meaningless. Hence, to obtain stable planar membranes at a π compatible with the equilibrium surface pressure of biomembranes (near 30-35 mN/m) the molar faction of CAd should not be higher than 0.2. The analysis of the variation of the Mma (mean molecular area) or the ∆V.A (surface potential per unit of molecular surface density) with the mole fraction (xCAd) at constant packing conditions (π ) 6, 18.5, or 35 mN/m) is shown in Figure 4. Straight dotted lines joined the values corresponding to the molecular parameters of pure dpPC and pure CAd. Experimental points lying on this line would represent either ideal mixing or immiscibility behavior of components in the monolayer. The compositional dependence or independence, respectively, of the collapse pressure might help to discriminate between both behaviors. At 6 mN/m, Mma (Figure 4a) as well as ∆V.A (Figure 4b) decreased as a function of xCAd. At this lateral pressure, these parameters showed positive deviations from ideality, suggesting repulsive interactions or other steric restrictions to

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Figure 4. Mean molecular area (a, c, and e) and surface potential per unit of molecular surface density (b, d, and f) vs composition plots at constant surface pressures. π ) 6 (a,b), 18.5 (c,d), or 35 mN/m (e,f). Straight lines represent the behavior expected for ideal mixing or complete segregation of the components. Deviations from this behavior, particularly at 6 mN/m, are more clearly reflected in Mma-x plots.

the ideal molecular packing between the monolayer components. While the expansion observed in Mma was very important, in the case of ∆V.A the expansion was less significant. These results indicate that the permanence of CAd at high proportions in the monolayer at low π disrupts the organization of the phospholipids (Mma is highly expanded) without acquiring a coherent organization (changes in ∆V.A were small). A similar behavior was in the Mma-xCAd plot at 18.5 mN/m (surface pressure higher but still within the miscibility region according to the phase diagram); however, no deviation from ideality was observed in ∆V.A (Figure 4c,d). At 35 mN/m, the monolayer phase exists up to xCAd ) 0.6 and at higher proportions of CAd a discontinuity was shown in Mma (there are no data at these π and xCAd, as shown in Figure 2b). Hence, at 35 mN/m, Mma and ∆V.A vs xCAd plots decreased up to the ordinate axis and reach zero. Negligible xCAd-dependent expansions were observed with respect to the ideality line not only in ∆V.A but also in Mma (Figure 4e,f). Either ideal mixing or total immiscibility may have exhibited this behavior. The inspection of the phase diagram suggested immiscibility within the range 0.2 < xCAd < 0.4 and partial miscibility within 0.4 < xCAd < 0.6 due to the compositional constancy and dependency of πc, respectively. Contrary to what happened within 0 < xCAd < 0.1, where we found CAd solubility in dpPC, the latter should be ascribed to dpPC mixed within a continuous CAd phase. Beyond xCAd ) 0.6, this miscibility continued with πc decreasing up to 18.8 mN/m at xCAd ) 1. 3.3.4. CAd Penetration in Phospholipid Monolayers. The ability of CAd to penetrate in the monomolecular layers of dpPC from the aqueous subphase was evidenced by the π increase at constant area at different initial surface pressures up to a πcutoff ) 13.08 ( 4.6 mN/m. This value was determined by extrapolating the plot of ∆π versus πi to ∆π ) 0 (Figure 5). On the other hand, the smooth variation of the monolayer compressibility (K) with the xCAd also showed that CAd was stable in the film at low molecular packings (low π). Above 13 mN/m, not only did CAd not penetrate in the film from the subphase (∆π ) 0), but also K at high π (Figure 2c) suffered a discontinuous variation with xCAd at xCAd > 0.2. Taken together, these results suggest that it was not possible to stabilize high amounts of CAd in highly packed monolayers independently of the direction from which the drug got access to the model membrane. However, small amounts of CAd can remain in the monolayer

Figure 5. Penetration of CAd in monomolecular layers of dpPC at different initial molecular packings. The line represents the fitness of a straight line to the experimental points by regression analysis by the least-squares method. The πcutoff value, indicated by the arrow, represents the maximum π allowing drug penetration and monolayer deformation. The regression line is defined by the equation ∆π ) a + b × πi, where a is the ordinate (17 ( 4 mN/m) and b the slope (-1.3 ( 0.6 mN/m). At ∆π ) 0, πi equals the πcutoff ) 13.08 ( 4.6 mN/m.

at lateral surface pressures compatible with the equilibrium pressure of bilayerssca. 30-35 mN/m (32)sas shown in the phase diagram (Figure 3) as well as by the resultant dipolar contributions at xCAd < 0.2 (Figure 2f). CAd was unstable at intermediate xCAd (0.2-0.4) leading to immiscibility behavior with dpPC, which at high xCAd (>0.4) became a phase inversion with dpPC partially miscible in a continuous CAd phase. This interpretation was strongly suggested by the linear A-xCAd and ∆V.A-xCAd plots (Figure 4e,f) in conjunction with πc independence (within 0.2 < xCAd < 0.4) or dependence (within 0.4 < xCAd e 1) on xCAd, respectively (Figure 3).

4. CONCLUSIONS The CAd synthesized in the present work was an amphipathic CNZ derivative capable of binding at the BZD site of the GABAA-R by means of its hydrophilic end and anchoring in a

1894 Bioconjugate Chem., Vol. 19, No. 9, 2008

Turina et al.

Table 1. Physicochemical Characteristics of CAd Compared with dpPC, Both at Their Respective Collapse Points property (units)

dpPC

CAd

πc (mN/m) Mma (Å2/molec) ∆Vπc (V) µ⊥ (mD) a Kc (mN/m) Pcc predicted self-organization xmax for 0.5 < Pc < 1 at π ) 30 mN/mb πcutoff (mN/m)

55.5 40 0.530 562 198 0.86 bilayer -

18.8 49 0.296 385 55 0.41 micelle 0.3 13

a Calculated as indicated in section 3.3.2. b Molar fraction to allow self-assembly into bilayers at a surface pressure of 30 mN/m. c Calculated at the πc of CAd.

biomembrane through the hydrophobic end represented by a hydrocarbon chain attached at the N1 position. The design of the BZD derivate was supported by theoretical studies about the BZD structure-activity relationship where high-affinity analogues interacted with three GABAA-R electrophilic groups located at C7, C2, and the iminic nitrogen N4. It is known that the N4 interaction is facilitated by the presence of halogens in C2 and when the phenyl ring is rotated in the greater coplanarity direction between the phenyl and the plane C′1 - C5 ) N4 (33). Consequently, some chemical alteration in the N1 position was not expected to affect (at least chemically) the BZD capacity to interact with the receptor binding site, and our results confirmed this hypothesis. Molecular parameters as well as conditions for self-assembly in water determined for CAd were summarized in Table 1 together with those of dpPC, which was taken as a reference. CAd as a Precursor to Obtain a Probe to Evaluate the Molecular Organization of Synaptosomal Membranes, In the Vicinity of GABAA-R. The present findings suggested that CAd could be stabilized at the membrane-water interface with its CNZ moiety stacked at the GABAA-R, while its methylene end inserted within the membrane depth may be useful for sensing the molecular order in the receptor surroundings provided a hydrophobic label is attached to it. Hence, such a chemical modification would make CAd a suitable molecular probe. The longer the hydrocarbon chain attached at N1 position of CNZ, the deeper the membrane regions that could be sensed would be. It is important to recall that a probe should provide information about the system properties without affecting them. That is why they are used at proportions sufficiently low (e.g., 0.5 mol % representing a xprobe ) 0.005). Our results indicate that this condition would be satisfied with CAd in bilayers which have an equilibrium surface pressure centered at 35 mN/m (32). CAd as a Precursor of an Affinity-Based Purification System for GABAA-R. Both the surface activity of CAd and its ability to interact with the BZD binding site at GABAA-R are equally important characteristics that would make CAd an appropriate molecule to develop an affinity-based precipitation method to purify GABAA-R based on CAd coated microparticles. The latter may be proposed as a useful substitute of traditional inmunoprecipitation procedures to avoid the requirement of monoclonal antibodies against specific epitopes of GABAA-R subunits (34).

ACKNOWLEDGMENT The present work was partially financed by grants from CONICET, SECyT-Universidad Nacional de Co´rdoba and ANPCyT from Argentina. B.C. is a Ph.D. student of the Doctorado en Ciencias Biolo´gicas of the Universidad Nacional de Co´rdoba. A.V.T. and B.C. are fellowship holders and E.L.M, G.I.Y., and M.A.P are Career Investigators from CONICET.

Supporting Information Available: All figures as well as H NMR and 13C NMR spectra. This material is available free of charge via the Internet at http://pubs.acs.org. 1

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