J. Phys. Chem. B 2008, 112, 10969–10975
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Luminescent Dipyridophenazine-Ruthenium Probes for Liposome Membranes Frida R. Svensson, Minna Li, Bengt Norde´n, and Per Lincoln* Department of Chemical and Biological Engineering/Physical Chemistry, Chalmers UniVersity of Technology, KemiVa¨gen 10, SE-41296 Gothenburg, Sweden ReceiVed: May 6, 2008; ReVised Manuscript ReceiVed: June 25, 2008
Ruthenium complexes with dipyridophenazine (dppz) type ligands have several characteristics that make them good candidates for use as luminescence probes for hydrophobic environments. Most studies have concerned DNA intercalation, but also lipid membrane fluidity and liposome orientation have been assessed. We report here dipyridophenazine derivatives ([Ru(phen)2dppz]2+) substituted with one or two alkyl ether chains of different lengths aimed at finding the optimum substitution for a high quantum yield when bound to a phospholipid membrane bilayer. The orientation of membrane bound molecules is studied using flow linear dichroism (LD) with phospholipid vesicles as membrane models. LD, excitation anisotropy, steady state luminescence and excited-state lifetime measurements are used to quantitatively investigate the insertion and orientation of the complexes in the vesicles. All complexes are inserted with their long axis of the dppz moiety mainly parallel to the lipid chains, and the degree of orientation is comparable to that of the orientation probe retinoic acid. The ruthenium “head group” with its positive charge functions as a buoy at the water-membrane interface while the hydrophobic chain part embeds the complex down into the bilayer. The complex with two hexyl ether substituents (named D6) has the optimal chain length regarding membrane insertion and orientation, and together with the highest quantum yield, is the best luminescence membrane probe in the two series. Introduction The lipid bilayer and membrane interactions are essential in biological processes in all living organisms which is why research concerning molecular interactions in the membrane is important.1-3 Studies of the membrane on a molecular level, including lipid chain orientation, and interaction and location of membrane bound molecules, are of vital importance for the understanding of more complex systems, for example, membrane protein function and transmembrane transport processes. The membrane bilayer also has applications in nanotechnology with the potential to function as a platform for DNA anchoring4-6 or to perform selective directed transport of electrons or protons from one side to the other.7 Unilamellar lipid vesicles composed of phospholipids are simple membrane models that can be used to mimic the membrane bilayer in spectroscopic measurements. The orientation of membrane bound molecules is conveniently studied using flow linear dichroism,8-14 and recently, retinoic acid was used as orientation probe to quantitatively determine transition moment orientations of membrane bound molecules.15,16 Ruthenium(II) complexes have the potential of being good orientation probes as well as luminescence probes since they have transition moments spanning three dimensions and a charge separated excited state with a long lifetime in the microsecond range.17 Ruthenium complexes with dipyridophenazine (dppz) ligands, for which the excited state is unusually sensitive to hydrogen bonding by solvent, have been frequently studied for their DNA binding properties.18,19 These complexes display no luminescence in aqueous buffer but show a strong luminescence increase in the presence of DNA, the “light switch effect”.20-22 This effect is also seen when ruthenium dppz complexes with no or unpolar substituents bind to a phospholipid membrane23 * Corresponding author. E-mail:
[email protected]. Phone: +46 (0) 31 772 30 55. Fax: +46 (0) 31 772 38 58.
making them useful as probes for lipid membrane fluidity24 or liposome orientation.8 Furthermore, photoexcitation of a ruthenium complex with a dppz ligand inserted into the phospholipid bilayer provides a simple means for directed electron transfer into the hydrocarbon core of the membrane, with potential applications for artificial light-harvesting systems. In this work, we investigate dipyridophenazine derivatives substituted with one or two alkyl ether chains of different lengths (Scheme 1) to find the optimum substitution for a high quantum yield when bound to a membrane bilayer. They are denoted Mn for monosubstituted and Dn for disubstituted derivatives, where n is the number of carbons in the alkyl chain. As membrane models unilamellar lipid vesicles (liposomes) with a net negative charge and a diameter of 100 nm are used. The insertion and orientation of the complex in the liposome is quantitatively investigated by linear dichroism and excitation anisotropy measurements. Moreover, steady state luminescence studies and excited-state lifetime measurements in liposomes and polyol solvents are used to compare the binding efficiency and insertion depth between different complexes. Experimental Methods Materials. Reagents and solvents for the synthesis of ligands and complexes were purchased from Sigma and used without further purification. 1,2-Dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phosphatidylglycerol (DOPG) were from Larodan (Malmo¨, Sweden). 1,2Propanediol (>99.5%) and ethylene glycol (>99.5%) were from Sigma. Methods. NMR spectra were obtained on a Varian spectrometer at 400 MHz for proton and 100 MHz for 13C. Chemical shifts are referenced to the chloroform peak (7.26 ppm) or acetonitrile peak (1.95 ppm). UV/vis absorption spectra were recorded on a Cary 4000 UV/vis spectrophotometer (Varian).
10.1021/jp803964x CCC: $40.75 2008 American Chemical Society Published on Web 08/13/2008
10970 J. Phys. Chem. B, Vol. 112, No. 35, 2008
Svensson et al.
SCHEME 1: Synthetic Routes and Structures for Ruthenium Dipyridophenazine Derivatives with One and Two Alkyl Ether Chains of Different Lengthsa
a
Phen ) 1,10-phenanthroline, pq ) 1,10-phenanthrolin-5,6-dione.
5-Bromomethyl-benzo[1,2,5]selenadiazole (MBr),25 5,6-bis-bromomethyl-benzo[1,2,5]selenadiazole (DBr),25 and [Ru(phen)2(1,10-phenanthrolin-5,6-dione)]Cl226 were prepared according to literature. General Procedure of Preparation of Alkoxymethyl Substituted Benzoselenadiazoles. A mixture of 5,6-bis-bromomethyl-benzo[1,2,5]selenadiazole (DBr; 0.3 mmol, 110 mg) and potassium hydroxide (1.2 mmol, 68 mg) in 2 mL of hexanol was heated at 100 °C for 10 min. A volume of 10 mL of water was added, and the resulting mixture was extracted with CH2Cl2 (3 × 10 mL). The combined extracts were dried (Na2SO4) and most of the solvents evaporated under reduced pressure. The oily residue was gently heated in a stream of nitrogen until the last traces of 1-hexanol had evaporated and subsequently purified by column chromatography on silica gel, (eluent hexane:ether, 5:1) to give DS6 as light yellow crystals after evaporation of solvent (82 mg, 65%). 1H NMR δH (400 MHz, CDCl3) 0.89 (6H, t, J ) 6.4 Hz), 1.25-1.35 (8H, m), 1.36-1.42 (4H, m), 1.62-1.68 (4H, m), 3.55 (4H, t, J ) 6.4 Hz), 4.64 (4H, s), 7.83 (2H, s); 13C NMR δc (100 MHz, CDCl3) 14.34, 22.89, 26.21, 30.01, 31.94, 70.59, 71.24, 121.83, 139.55, 160.41. The other compounds in the DSn and MSn series were similarly prepared from the appropriate alcohol and bromomethyl compound (see Supporting Information). General Procedure for Reduction of Benzoselenadiazoles.27 A mixture of zinc powder (250 mg, 3.4 mmol) and acetic acid (0.15 mL) was added in small portions in the solution of 5,6-bis-hexyloxymethyl-benzo[1,2,5]selenadiazole (DS6; 0.15 mmol, 62 mg) in 2.5 mL of EtOH under reflux. The color changed from yellow to red-brown and finally to colorless. After addition of the last portion of zinc powder, the heating was continued for 5 min. The excess zinc was filtered off and washed with 10 mL of hot ethanol 3 times. The combined filtrate was evaporated under reduced pressure, treated with 5 mL of aqueous sodium acetate (20%), and extracted with CH2Cl2 (3 × 5 mL). The extracts were combined, dried over anhydrous sodium sulfate, evaporated under reduced pressure, and purified by column chromatography on silica gel (eluent ether:methanol, 7:1) to give, after evaporation of solvent, DA6 as a light brown solid (28 mg, 55%) 1H NMR δH (400 MHz, CDCl3) 0.88 (6H, t, J ) 6.4 Hz), 1.25-1.38 (12H, m), 1.55-1.62 (4H, m), 3.43
(4H, t, J ) 6.4 Hz), 4.43 (4H, s), 6.86 (2H, s); 13C NMR δc (100 MHz, CDCl3) 14.36, 22.92, 26.23, 30.05, 31.99, 70.40, 70.55, 118.04, 128.97, 133.98. General Procedure of Preparation of Ruthenium Complexes.26 A solution of 4,5-bis-(hexyloxymethyl)-benzene-1,2diamine (DA6, 0.05 mmol, 17 mg) in CH3CN (0.5 mL) was added to [Ru(phen)2(1,10-phenanthrolin-5,6-dione)]Cl2 (0.025 mmol, 19 mg) in an amount of CH3CN containing a drop of acetic acid. The mixture color changed from original dark brown to dark red. After heating to 75 °C for 10 min, addition of NH4PF6 gave the crude product that was filtered off, washed with water, dried and purified by chromatography on aluminum oxide (activity grade 3) with CH3CN as eluent. After evaporation of solvent D6, hexafluorophosphate was obtained as an orangered solid (15 mg, 55%) 1H NMR δH (400 MHz, CD3CN) 0.98-1.04 (6H, m), 1.45-1.47 (8H, m), 1.54-1.58 (4H, m), 1.78-1.84 (4H, m), 3.77 (4H, t, J ) 6.0 Hz), 4.98 (4H, s), 7.72-7.78 (4H, m), 7.85 -7.89 (2H, m), 8.13 (2H, d, J ) 5.2 Hz), 8.21 (2H, d, J ) 5.2 Hz), 8.32 (2H, d, J ) 5.2 Hz), 8.38 (4H, s), 8.54 (2H, s), 8.71-8.74 (4H, m), 9.74 (2H, d, J ) 8.4 Hz). Preparation of Unilamellar Lipid Vesicles. DOPC and DOPG lipids, dissolved in chloroform, were mixed at a molar ratio of 4:1, and the solvent was evaporated under reduced pressure using a rotary evaporator. To remove any remaining traces of solvent, the phospholipid film was put under vacuum for at least 2 h. Vesicles were prepared by dispersion of the lipid film in buffer under vortexing. Thereafter, the vesicles were subjected to 5 freeze-thaw cycles (liquid nitrogen/37 °C) before extrusion 21 times through polycarbonate filters with a pore diameter of 100 nm using a LiposoFast-Pneumatic extruder (Avestin, Canada). Ruthenium complex hexafluorophosphates were dissolved in acetonitrile and added to the preformed vesicles (the final acetonitrile concentration was less than 1% in all experiments), prior to measurements. Unless otherwise stated, the buffer was 10 mM potassium phosphate (pH 7.4). Luminescence Measurements. Steady state luminescence measurements were performed on a SPEX τ-3 Fluorolog (Jobin Yvon Horiba, France) at 25 °C. A 20 µL sample of the ruthenium complex in acetonitrile was added to a 3 mL buffer solution with 100 µM of liposomes at a ruthenium-to-lipid molar
Ruthenium Based Luminescent Membranes Probes
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ratio of 1:100. The wavelength of excitation was 440 nm, and the emission was measured between 550 and 800 nm. Temperature dependent emission of ruthenium complex in polyol solvents were performed on a Cary Eclipse spectrofluorimeter (Varian) in the temperature range 10-90 °C with one emission spectrum measured every 10th degree. The samples were purged with argon for 10 min before measurements. Excited State Lifetime Measurements. The nanosecond emission decays were measured with a Nd:YAG laser (Continuum Surelite II-10, pulse width
