A Molecular Toolkit for Highly Insulating Tethered Bilayer Lipid

Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany. .... cushion has 77 repeat units) was used for detailed investigatio...
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Bioconjugate Chem. 2006, 17, 631−637

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A Molecular Toolkit for Highly Insulating Tethered Bilayer Lipid Membranes on Various Substrates Vladimir Atanasov, Petia P. Atanasova, Inga K. Vockenroth, Nikolaus Knorr, and Ingo Ko¨per* Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany. Received November 16, 2005; Revised Manuscript Received March 1, 2006

Tethered bilayer lipid membranes (tBLMs) are promising model architectures that mimic the structure and function of natural biomembranes. They provide a fluid, stable, and electrically sealing platform for the study of membrane related processes, specifically, the function of incorporated membrane proteins. This paper presents a generic approach toward the synthesis of functional tBLMs adapted for application to various surfaces. The central element of a tethered membrane consists of a lipid bilayer. Its proximal layer is covalently attached via a spacer unit to a solid support, either gold or silicon oxide. The membranes are characterized optically by using surface plasmon resonance spectroscopy (SPR) or ellipsometry and electrically by using electrochemical impedance spectroscopy (EIS). The bilayer membranes obtained show high electrical barrier properties and can be used to incorporate and study small membrane proteins in a functional form.

INTRODUCTION Model membrane systems mimic properties of a natural biomembrane. One type of model system, solid supported bilayer membranes, provides an architecture that is accessible to many (surface-) analytical methods. Thus, they can easily be used as biotic-abiotic interfaces in various biosensing applications (1-4). In the last years, tBLMs have proved to be a powerful tool for producing solid supported membranes. They consist of a lipid bilayer with the proximal layer at least to some extend covalently attached to a solid support. If this substrate is an electrode, electrical characterization of the system is possible. To be considered as a suitable model system, such architectures should possess specific key features of a natural membrane. One requirement is that the membrane must serve as a barrier between an inner- and an outer-membrane environment. This implies that the leakage of small molecules and ions across the lipid bilayer is prevented. Transport processes should only be governed by protein function and not be due to defects in the bilayer structure. Additionally, some lateral fluidity or mobility is necessary to allow for the incorporation and proper function of membrane proteins. As schematically depicted in Figure 1, the proximal layer of a tBLM can be divided into three distinct parts: (a) the lipid with a linking unit to (b) the spacer group and (c) the anchoring moiety. In previous tBLM architectures, either saturated (5, 6) or unsaturated (7-9) alkyl chains have been used as lipid tails. However, these lipids suffer from relatively high transition temperatures (e.g., the transition temperature of dipalmitoyl phosphatidylcholine is 41.5 °C (10)) and thus do not form fluid bilayer membranes at room temperature. To reduce the Krafft temperature (TK), it is common to use branched alkyl chains. TK is the critical temperature above which the solubility of the surfactant rapidly increases and micelles are formed. To modify this behavior, branched chains have been investigated by varying the branching types, including various short iso and antesio branches (11). Although the chain branching consistently lowers the TK values, this is often not sufficient for the formation of * Corresponding author. E-mail: [email protected]. Tel: +49-6131-379164. Fax: +49-6131-370503.

Figure 1. Schematic tBLM architecture. The proximal leaflet is formed by self-assembly of the anchor lipids, while the distal layer is completed by vesicle fusion. Defects and voids in the proximal leaflet might be filled during the fusion process.

fluid membranes, particularly for long alkyl chains. Highly branched alkyl chains show different properties. The most studied lipids are based on phytanyl with TK values below 0 °C (4, 12-14). The phytanyl functionality consists of a highly branched 3,7,11,15-tetramethylhexadecyl group. This moiety is well-known from extremophiles or archaea to form stable and fluid membranes in harsh environments (15). The hydrocarbon tail of the lipid is coupled to a polar headgroup. Since this linkage is in a dynamic interfacial region in an aqueous suspension, even small changes of this moiety can significantly modulate the aggregation properties. Among various linkage functionalities such as oxyethylenes (9, 16), sulfides (13), and 2-hydroxymethylglycerol (17), the glycerol linker is used most often (12). In natural lipids, this stable moiety occurs in a defined sn-2 stereochemistry configuration. The spacer unit separates the lipid layer from the solid substrate while serving as a polymer cushion that compensates for surface roughness effects, protects embedded biomaterial from touching the surface (and thus losing their functionality

10.1021/bc050328n CCC: $33.50 © 2006 American Chemical Society Published on Web 04/13/2006

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due to denaturation), and provides an ion reservoir underneath the membrane. Various materials such as oligosaccharides (18), polymers based on acrylamide and its derivatives (19), ethylene glycol esters/amides of succinic acid/succinamide (20), and poly(ethylene glycol) (21, 22) have been proposed. These moieties form a hydrophilic layer between the lipid membrane and the substrate surface. This reservoir shows a gellike structure that is permeable to ionic species (17). The characteristics of this reservoir have an important effect on the electrical properties of the tBLM (23). Finally, surface immobilization of the lipid-spacer molecule is achieved by an anchor group, which forms, by matching chemistry, a covalent bond with the surface. Several anchor groups have been tested on gold surfaces, most of them are based on gold-sulfur interactions (4, 14, 24). Gold substrates are interesting because they can be used as an electrode for electrical read-outs as well as for many other surface analytical techniques. However, most devices in the microelectronics and semiconductor field are silicon-based. These substrates, as well as the large number of silica-like substrates (glass, metallic oxides, etc.) are easily accessible, if not commercially available. They can also be integrated into standard microelectronics fabrications processes. Tamm et al. presented tBLMs on silicon oxide substrates using PEG-phospholipid conjugates with a triethoxisilane anchor group. The polymer-supported membrane (the PEG cushion has 77 repeat units) was used for detailed investigation on the lateral diffusion of proteins and lipids (25) and to study the vertical distances of the bilayer from the substrate (26). Investigations were carried out by using fluorescence recovery after photobleaching measurements and fluorescence interference-contrast microscopy. Similarly, Rossi et al. have presented a model membrane system with large polymeric spacer groups that can be anchored on a amino-silane treated glass substrate (27). Lateral mobility could be observed using fluorescence techniques. However, in both cases, no electrochemical measurements have been reported. We present here a generic approach, a molecular toolkit that allows for the systematic modification of certain key components of tethered membrane architectures. Starting from a universal precursor molecule, different anchor groups that are suitable either for noble metal surfaces (e.g., Au, Ag, Pt, Cu, Fe, Ni, or Hg) or for oxide surfaces (e.g., SiOx) can be added. The lipid headgroup consists of a diphytanyglycerol unit, while a short oligo(ethylene glycol) forms the spacer. The length of the spacer unit can easily be modified. These anchor lipids form stable bilayer membranes with high electrical sealing properties. The tBLMs can serve as a matrix for the functional incorporation of membrane proteins.

EXPERIMENTAL PROCEDURES Electrochemical Impedance Spectroscopy (EIS). Measurements were conducted using an IM6 spectrometer (Zahner Electrics) or an AUTOLAB PGSTAT 12 impedance spectrometer. Three electrode measurements were performed in Teflon cells with the substrates as the working electrode, a coiled platinum wire as the counter electrode, and DRIREF-2 reference electrodes (World Precision Instruments). Spectra were recorded for frequencies between 2 mHz and 1 MHz at a controlled potential with an AC modulation amplitude of 10 mV. Raw data were analyzed using the ZVIEW software package (version 2.70, Scribner Associates). The Teflon cells have a buffer volume of 0.5 mL for the silicon oxide substrates and 1 mL for the gold substrates and an electrochemically active area on the substrates of 0.385 and 0.2 cm2, respectively. The data obtained is fit using an equivalent circuit of capacitors and resistances. The final values are normalized to the electrode surface area.

Atanasov et al.

Contact Angle Measurements. Contact angles were determined using a DSA 10 contact angle measuring system (Kru¨ss, Germany). Software (Drop Shape Analysis, version 1.8) was employed to analyze the drops created by the sessile-drop method. Ellipsometry. Measurements were carried out using an EP3 ellipsometer (Nanofilm, Go¨ttingen, Germany) with a 532 nm laser source. The angle of incidence was 70° for measurements in air and 60° for measurements in a fluid cell. Thickness values were fit with the EP3View v2.01 software using a layer model with the following parameters: n ) 4.17 and k ) 0.049 for Si, n ) 1.4605 and k ) 0 for SiOx, n ) 1.50 and k ) 0 for the monolayers, and n ) 1.45 and k ) 0 for the bilayer. Surface Plasmon Resonance Spectroscopy (SPR). A setup in Kretschmann configuration, built in-house, with a 632 nm He/Ne laser was used. In the scan mode, reflectivity changes are monitored as a function of the angle of incidence of the incoming laser beam. In the kinetic mode, reflectivity changes occurring at a fixed angle are monitored as a function of time. SPR spectra were analyzed using a three layer model including the prism, gold, and the thiolipid monolayer. After vesicle fusion, a fourth layer corresponding to the outer leaflet of the bilayer was added. The refractive indices for the monoand bilayer were n ) 1.489 and n ) 1.423, respectively. Analytical Instrumentation. 1H and 13C NMR spectra were recorded on a Brucker DXP 250 MHz NMR spectrometer. If not otherwise stated, NMR spectra were recorded at ambient conditions at 250 and 62.5 MHz for 1H and 13C NMR, respectively. The spectra were calibrated to the solvent signal as follows: 7.24 (1H) and 77.0 (13C) ppm for CDCl3 and 5.23 (1H) and 54.0 (13C) ppm for CD2Cl2. Mass spectroscopy was performed using a VG ZAB2-SE-FPD Spectrofield spectrometer. IR spectra were measured on a Nicolet 730 FTIR spectrometer using an endurance diamant-ATR. Chemicals. (()-3-Benzyloxy-1,2-propanediol, tetra(ethylene glycol), and allylbromide (all purchased from Fluka) were dried over an A3 molecular sieve. Triethylamine (TEA, Acros) and tetrahydrofuran (THF, Fisher) were dried over CaH2 and potassium-benzophenone complex, respectively. Trichlorosilane (Acros), triethoxysilane (Lancaster), thioacetic acid (Acros), and chlorodimethylsilane (Lancaster) were distilled prior to use. p-Toluenesulfonyl chloride (TosCl, Acros), platinum on activated charcoal (Pt/C, Fluka), hexachloroplatinic acid (H2PtCl6, Fluka), platinum-divinyltetramethyldisiloxane complex as 2.12.4% solution in hexane (ABCR), 2,2’-azobis(2-methylpropionitrile) (AIBN, Acros), hexamethyldisilazane (Fluka), and 1,2di-O-phytanoyl-sn-glycero-3-phosphocholine (DphyPC) (Avanti Polar Lipids) were used as received. Synthesis. We will describe the synthetic steps depicted in Scheme 1, especially the final modifications of the anchor groups. Most reactions were carried out until complete consumption of the starting material as monitored by thin-layer chromatography (TLC) using a THF/petrolether (1:4) elution system. Precursor Synthesis, 9. The synthesis of the precursor molecule has been previously described (28). The synthetic pathway is depicted in Scheme 1. The product can be obtained at high purity. 1H NMR (CDCl3) δΗ, ppm: 5.9 (m, -CHd CH2), 5.3-5.1 (m, -CHdCH2), 4.0 (d, -OCH2CHdCH2), 3.6-3.4 (overlapping signals CH(2)O-), 1.5 (m, CH), 1.2 (overlapping signals -CH2-), 0.8 (t, CH3). 13C NMR (CD2Cl2) δC, ppm: 135.5, 116.8, 78.4, 72.4, 71.7, 71.2, 70.9, 70.2, 70.0, 69.0, 39.8, 37.8, 37.2, 33.2 30.3, 28.4, 25.2, 24.8, 22.9, 22.8, 19.9. IR (ATR): 2952, 2925, 2868, 1462, 1377, 1300, 1250, 1107, 1043, 995, 992, 879, 735 cm-1. Field desorption mass spectrum (FD-MS) (m/z) 869.8 (M+), calcd (C54H108O7)

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Scheme 1. Reaction Pathway for the Synthesis of Lipids with Different Anchor Groups via a Unique Precursor Molecule (9)

) 869.43; 1737.9 (M + M+), calcd (2×C54H108O7) ) 1738.86; 2607.7 (2M + M+), calcd (3×C54H108O7) ) 2608.29 (see Figure 2). Synthesis of the Thiol Anchored Lipid 10 (Step f). The conversion of a double bond to a mercapto group is a two step process: (a) radical addition of thioacetic acid to the double bond and (b) hydrolysis of the thioester to a thiol (29, 30). (a) A mixture of 9 (1.85 g), thioacetic acid (1.97 g), and azobisisobutyronitrile (AIBN) (7 mg) in toluene (6 mL) was degassed and heated to 70 °C for 24 h. The reaction was monitored by TLC (Rf of the product ) 0.35) of aliquots taken from the reaction mixture. After completion of the reaction, toluene and the remaining thioacetic acid were evaporated, and the residue was purified by flash chromatography (FC) (eluent THF/ petroleum ether ) 1:6). Yield: 1.53 g (76%). 1H NMR (CDCl3) δΗ, ppm: 3.6-3.4 (overlapping signals CH(2)O-), 2.9 (t, -CH2S-), 2.3 (s, -SC(O)CH3), 1.8 (p, -OCH2CH2CH2S-),

Figure 2. FD-MS spectrum of 9. The formation of doublets and triplets occur during analysis with the spectrometer.

1.5 (m, CH), 1.2 (overlapping signals -CH2-), 0.8 (t, -CH3). 13C NMR (CDCl ) δ , ppm: 77.9, 71.5, 70.9, 70.6, 70.2, 70.0, 3 C 69.6, 68.9, 39.4, 37.4, 36.7, 32.8, 30.6, 29.9, 29.8, 29.6, 28.0, 26.0, 24.8, 24.5, 24.4, 22.7, 22.6, 19.7. FD-MS (m/z): 945.4 (M+), calcd (C56H112O8S) ) 945.58. (b) The product of step a (1.53 g) was treated with NaOH (1 M in water, 6.3 mL) in THF (40 mL) for 24 h at 50 °C. After completion as seen by TLC (Rf(10) ) 0.53), the THF was evaporated, and the mixture was extracted with methylene chloride. The extracts collected were dried over CaCl2, the methylene chloride was evaporated, and the residue was purified by FC (THF/petroleum ether ) 1:4). Yield: 0.98 g (70%). 1H NMR (CDCl3) δΗ, ppm: 3.6-3.4 (overlapping signals -CH(2)O), 2.7 (t, -CH2S-), 1.9 (p, -OCH2CH2CH2S-), 1.5 (m, -CH), 1.2 (overlapping signals -CH2-), 0.8 (t, -CH3). 13C NMR (CD2Cl2) δC, ppm: 78.4, 71.7, 71.2, 70.9, 70.6, 70.2, 69.6, 69.0, 68.1, 39.7, 37.8, 37.1, 35.8, 33.2, 30.5, 30.3, 29.7, 28.4, 26.0, 25.1, 24.8, 24.7, 22.8, 22.7, 19.8. IR (ATR): br 2952, 2925, 2865, s 1462, s 1377, s 1292, s 1248, s 1111, s 1070, s 916, s 860, s 737 cm-1. FD-MS (m/z): 1804.5 (M + M+)s dimerization due to oxidation, calcd for 10 (C54H110O7S) ) 903.54; calcd for dimer (C108H218O14S2) ) 1805.06. Synthesis of 11 and 12 (Steps g and h) (31). A reaction mixture of freshly distilled chlorosilane (see table below), Ptcatalyst, and 9 was refluxed under inert atmosphere until complete consumption of 9 (Rf(9) ) 0.58, Rf(11, 12) ) 0.00). The mixture was filtered (0.2 µm PTFE) in a glovebox, and the residual chlorosilane was evaporated.

no.

chlorosilane (mL)

Pt catalyst (mg)

9 (g)

time (h)

yield (g, %)

11 12

chlorodimethylsilane (10) trichlorosilane (10)

H2PtCl6 (100) Pt/C (2%Pt) (100)

1.1 2.1

20 48

1.0, 82 2.3, 95

Characterization of 11. 1H NMR (CD2Cl2) δΗ, ppm: 3.63.4 (overlapping signals -CH(2)O-), 1.5 (m, -CH), 1.4-1.0

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(overlapping signals -CH2-), 0.8 (t, -CH3), 0.4 (s, Si-CH3). 13C NMR (CD Cl ) δ , ppm: 78.5, 73.8, 71.9, 71.4, 71.1, 70.7, 2 2 C 70.4, 69.2, 40.0, 38.0, 37.9, 37.4, 37.3, 33.4, 30.5, 28.6, 25.4, 25.0, 23.9, 23.0, 20.0, 15.8, 1.9. IR (ATR): br 2952, 2923, 2868, s 1462, s 1377, s 1299, s 1253, s 1108, s 941, s 845, s 845, s 735 cm-1. FD-MS (m/z): 945.9 (M+), calcd (C56H116O8Si) ) 945.63; 1874.2 (M + M+ - HCl)scondensation product due to hydrolysis of 11, calcd (C112H230O15Si2) ) 1873.24. Characterization of 12. 1H NMR (CD2Cl2) δΗ, ppm: 3.63.4 (overlapping signals -CH(2)O-), 1.8 (m, -SiCH2CH2) 1.5 (m, -CH), 1.2 (overlapping signals -CH2), 0.8 (t, -CH3). 13C NMR (CD2Cl2) δC, ppm: 78.5, 72.0, 71.9, 71.4, 71.1, 70.8, 70.6, 70.4, 69.2, 39.9, 38.1, 38.0, 37.9, 37.4, 37.3, 33.4, 30.5, 28.6, 25.4, 25.0, 23.3, 23.0, 21.5, 20.1, 20.0. IR (ATR): br 2952, 2924, 2867, s 1462, s 1377, s 1299, s 1250, s 1109, s 939, s 839, s 760, s 733, s 696, s588 cm-1. FD-MS: characterization was not possible due to condensation reaction during the analysis. Synthesis of 13 and 14 (Steps i and j) (32). Pt-complex (2.12.4% Pt), acetic acid, and 9 were added to freshly distilled trialkyloxysilane and refluxed under inert atmosphere (see table below). The reaction was monitored by TLC (Rf(13) ) 0.48, Rf(14) ) 0.53) of aliquots taken from the reaction mixture. After complete consumption of 9, the mixture was filtered (0.2 µm PTFE) in a glovebox, and the remaining alkyloxysilane was evaporated.

no.

alkyloxysilane (mL)

Pt complex acetic (µL) acid (µL)

13 14

trimethoxysilane (5) triethoxysilane (10)

1 1

3 3

9 (g)

time (h)

yield (g, %)

0.52 1.10

48 72

0.55, 92 0.94, 72

AlternatiVe Synthesis of 13 and 14 (Steps k and l) (33). Compound 12 diluted in toluene (2 mL, see table below) was added dropwise to an ice-cold mixture of alcohol, triethylamine, and toluene (3 mL) under inert atmosphere, and the reaction was left at RT overnight. The mixture was filtered (0.2 µm PTFE) in a glovebox, and the solvent was evaporated.

no.

12 (g)

alcohol (µL)

triethylamine (µL)

yield (g, %)

13 14

0.24 0.20

methanol (57) ethanol (66)

128 106

0.23, 98 0.18, 96

Both synthetic routes for 13 (i and k) and 14 (l and j) led to products with identical characteristics. Characterization of 13. 1H NMR δΗ, ppm: 3.6-3.4 (overlapping signals -CH(2)O), 3.5 (s, -SiOCH3), 1.5 (m, -CH), 1.2 (overlapping signals -CH2), 0.8 (t, -CH3), 0.6 (t, -SiCH2). 13C NMR δ , ppm: 77.9, 73.4, 71.5, 70.8, 70.6, 70.0, 68.9, C 50.5, 39.4, 37.4, 36.7, 32.8, 29.9, 28.0, 24.8, 24.5, 24.4, 22.7, 22.6, 19.7, 5.2. IR (ATR): br 2950, 2925, 2867, s 1461, s 1377, s 1301, s 1248, s 1194, s 1087, s 945, s 885, s 835, s 737 cm-1. FD-MS (m/z): 1011.8 (M + Na+), calcd (C57H118O10Si) ) 991.66. Characterization of 14. 1H NMR δΗ, ppm: 3.8 (q, -SiOCH2CH3), 3.6-3.4 (overlapping signals -CH(2)O), 1.5 (m, -CH), 1.2 (overlapping signals -CH2), 0.8 (t, -CH3), 0.6 (t, -SiCH2). 13C NMR δ , ppm: 77.9, 73.6, 71.4, 70.8, 70.6, 70.0, 68.9, C 58.3, 39.3, 37.4, 36.7, 32.8, 29.9, 28.0, 24.8, 24.5, 22.9, 22.7, 22.6, 19.7, 18.3, 6.4. IR (ATR): br 2952, 2925, 2868, s 1462, s 1377, s 1365, s 1296, s 1450, s 1105, s 1061, s 955, s 795, s 775 cm-1. FD-MS (m/z): 1056.2 (M + Na+), calcd (C60H124O10Si) ) 1033.74. tBLM Assembly. Monolayers of the different compounds were obtained via self-assembly. The thiol-based tBLMs were prepared on template stripped gold slides as described previously

(24). In short, 50 nm of gold is evaporated on a clean silicon wafer and glued to a clean glass slide using an epoxy glue. After curing, the silicon is stripped away, and the clean gold substrate is immersed for 24 h into an ethanolic self-assembly solution containing 10 (0.2 mg/mL). The substrates are thoroughly cleaned with ethanol and dried in a nitrogen stream. The chlorosilane-based (11 and 12) tBLMs were prepared in a self-assembly process on highly boron p-doped (0.01 Ω cm) silicon single crystal (100) wafers as described by Purrucker et al. (34). In short, 11 and 12 were assembled by immersion of the silicon wafer in a solution of the respective lipid in toluene (typically 2-40 mM) for 24 h. Et3N was used as a promoter and acid scavenger. For 11, dried substrates were used. The surface reaction of 12 requires a small amount of water at the substrate (35). The electrical backside contact of the wafer is made by a 250 nm thick aluminum electrode. First attempts using self-assembly of the alkoxysilanes (13 and 14) did not result in homogeneous mono- or bilayers. Further investigations will be made using Langmuir-BlodgettKuhn techniques.

RESULTS AND DISCUSSION Syntheses. From a chemical point of view, our approach consists of the synthesis of a “universal” lipid precursor. It possesses all advantages of diphytanyl glycerol, that is, it forms fluid membranes under ambient conditions. The tethering spacer consists of tetra(ethylene glycol), because this moiety prevents the nonspecific adsorption of proteins to surfaces (36-38), does not absorb to lipid bilayers (39), and interacts only minimally with quartz and glass surfaces (40). A terminal CdC double bound can be transformed in a few steps into thiol, chlorosilane, and alkyloxysilane anchors (see Scheme 1). We have previously published briefly the synthesis of the lipid precursor 9 as well as the synthesis of the lipids with chlorosilane anchors (28). The formation of tBLMs on silicon substrates and the functional incorporation of two different proteins have been reported. In the present study, we extend the chemical modification of the lipid anchor to thiols and alkoxysilanes for tBLM architectures on gold as well as on silicon substrates. Furthermore, the general synthetic strategy is discussed in more detail. Lipid Precursor, 9. The preparation of the lipid precursor 9 was achieved by the parallel synthesis of the hydrophobic lipid part (diphytanyl glycerol, 4) and the tetra(ethylene glycol) tethering unit with a double bond 8, followed by a Williamson reaction between both (Scheme 1, step e). The structure of the lipid precursor 9 could be shown by 1H NMR and 13C NMR; the purity is characterized by FD-MS. The related signal integrals at 5.9 and 5.3 ppm in 1H NMR and 135.5 and 116.8 ppm in 13C NMR show the double bond functional group. This will be important for the next steps, since the modification of the double bond functionality leads to the different anchor groups. A sequence of signals related to single, double, and triple m/z values of 870.2 (the calculated molecular weight of 9 is 869.43) are observed by FD-MS, as shown in Figure 2. This sequence may be due to the high affinity between the phytanyl chains. These physical interactions seem to be the main reason for the stability of membranes containing this residue. A similar sequence of signals was observed in the FD mass spectra of 1, 3, and 4 where phytanyl units were present. The formation of dimeric and trimeric macromolecules in terms of chemical interactions during the synthesis of the lipid precursor can be excluded because of the absence of any additional signals in 1H NMR and 13C NMR. However, the chemical interactions during the FD-MS measurements might be due to the high reactivity of the ionized lipid molecules that can undergo a number of chemical interactions (41). Thus, the purity of the lipid precursor 9 was determined to be higher than 98%.

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Lipids with -SH Anchor, 10. The transformation of a double bond into a thiol via thioacetals is a well-known reaction (30, 42). The reaction applied to our precursor lipid 9 leads to lipids with thiol anchors that can be used for tBLMs on gold surfaces. The complete conversion of the double bond to the thioacetal in the first step is shown indirectly by the disappearance of the 1H (5.9 and 5.2 ppm) and 13C NMR (135.5 and 116.8 ppm) signals corresponding to the double bond functionality. Additionally, signals at 2.3 in 1H NMR and 28.0 ppm in 13C NMR appeared. These signals are related to the methyl protons of the thioacetal groups. After FC isolation of 10, FD-MS was used to determine the structure. A single peak with m/z ) 1804.5 corresponds to a dimer of 10 (calculated molecular weight of 903.54), which is formed through oxidation of 10. The mechanism for the oxidation of thiols is a well-established process (42) resulting in -S-S- bridge formation and dimerization. The dimers are surface active and efficiently self-assemble on gold surfaces. Lipids with -Si(CH3)2Cl or -SiCl3 Anchors 11 and 12. Herein, we discuss the preparation of both lipids in more detail to understand their structure and chemical behavior and compare them with the other lipids (10, 13, and 14). Both lipids were prepared similarly using a hydrosilylation reaction in the presence of the corresponding hydrosilane (dimethylchlorosilane for 11 and trichlorosilane for 12) and a platinum-based catalyst (see Scheme 1, steps g and h). The hydrosilane is in large excess of the precursor lipid 9 and also serves as reaction medium. The use of a heterogenic catalyst, such as platinum over charcoal (Pt/C), requires longer reaction times (48 h) and larger amounts of the catalyst than using homogenic catalysts, such as hexachloroplatinic acid and platinum-divinyltetramethyldisiloxane complex. However, the heterogenic catalyst can easily be separated from the reaction mixture either by filtration or by centrifugation. It should be pointed out that due to extremely high reactivity of chlorosilanes with protic substances (water, acids, alcohols, amines, etc.), the presence of chlorosilane groups drastically reduces the number of suitable purification and analytical methods (e.g., flash chromatography). Thus, most of the purification steps, for example, filtration through 0.2 µm PTFE filter to remove the solid residues such as catalyst or threedimensional networks obtained by hydrolization of trichlorosilanes, were carried out in a glovebox. Excess of chlorosilanes, their dimers, and their trimers and of HCl (products of hydrolization of the chlorosilanes) could be removed in a vacuum (1 × 10-3 mbar). However, this does not purify the products (11 and 12) from unreacted 9. Therefore, the reactions are stopped only after complete consumption of the starting material. The reaction progress can be monitored either by TLC, FD-MS, or 1H NMR. The final products may still contain dimers, trimers, and oligomers of 11 and 12, as well as the main side products for both reactions, which is the lipid precursor with hydrogenated double bonds (isolated by FC). Because they will not interact with the substrate, the presence of saturated lipid precursors in the final product will not significantly influence the self-assembly process. Dimers, trimers, or 2Doligomers of 12 that possess available chlorosilane anchors can effectively self-assemble on a substrate. The relatively fast hydrolysis of 12 to 1D-, 2D-, and 3Doligomers causes the main difficulty during the analysis of the compound. It was impossible to detect any structure related fragments by FD-MS. Therefore, the disappearance of the proton signals corresponding to the double bond functionality of 9 by 1H NMR and the drop of the retention factor (R ) from 0.58 for f 9 to 0.00 for 12 indirectly prove the termination of the reaction. In the case of 11, FD-MS was more informative; dimers obtained by condensation reaction between hydrolyzed and nonhydrolyzed 11 were detected. The presence of signals at 0.39

Table 1. Surface Properties (Contact Angles (CA, deg) and Layer Thickness (nm)) of the Different Monolayersa static CA 10 104 ( 3 11 72 ( 4 12 87 ( 4

advancing receding modeled thickness thickness CA CA thickness (ellipsometry) (SPR) 105 ( 2 86 ( 4 91 ( 5

98 ( 3 59 ( 3 66 ( 4

4.5 4.6 4.6

b 1.3 ( 0.5 1.8 ( 0.6

3.3 ( 0.2 b b

a An approximation of fully stretched molecules is used to model the system. b Not applicable.

ppm corresponding to methylsilane protons in the 1H NMR were direct proof that the chlorodimethylsilane functionality was gained. Additionally, an indirect proof of the presence of chlorosilane anchors is accomplished by the formation of chemically adsorbed monolayers on silicon oxide surfaces, as well as by further modification of the trichlorosilane groups to alkoxysilanes. Lipid with -Si(OCH3)3 or -Si(OC2H5)3 Anchors, 13 and 14. The lipids 13 and 14 were obtained by applying either hydrosilylation of 9 with trimethoxysilane or triethoxysilane (Scheme 1, steps i and j (32)) or substitution of chlorine of the trichlorosilanated lipid 12 with methanol or ethanol (Scheme 1, steps k and l (33)). Since hydrosilylation occurs similarly in the cases of chlorosilanes and alkoxysilanes, the alternative method, that is, substitution of trichlorosilane anchors of 12 with alcohols, is much simpler and faster once 12 is obtained. Moreover, the method indirectly proves the quantitative content of trichlorosilyl anchors in 12. Similarly to 11 and 12, the hydrolization of 13 and 14 in ambient conditions renders the quantitative analysis of the structures difficult, especially in the case of 13, which hydrolyzes faster than 14 (43). However, FDMS, 1H NMR, and 13C NMR directly prove the presence of the methoxy- (13) and ethoxysilane (14) anchors. Indeed, both chlorosilanes (11 and 12) and alkoxysilanes (13 and 14) were prepared as lipids with appropriate anchors for silicon oxide surfaces. However, the different anchors differ in their hydrolyzation rates and, therefore, require individual immobilization techniques. Chlorosilanated lipids should be suitable for less homogeneous metal oxides (e.g., poorly oxidized metal) or less reactive (e.g., chemically modified) surfaces due to their high reactivity but poor control over the hydrolization process. On the other hand, alkoxysilanes hydrolyze more slowly, allowing a better control of the immobilization process. Additionally, the aggressive chlorosilanes can cause problems when adsorbed on surfaces obtained by layer deposition (e.g., plasma polymerized silicones or indium tin oxide (ITO) deposited on silicon wafers). Due to their high affinity for silicon oxide, these surface layers may get peeled off the substrate. We obtained self-assembled monolayers using the chlorosilanated lipids 11 and 12. Further experiments with 13 and 14 will be performed using Langmuir-Blodgett-Kuhn techniques and published separately. tBLM Formation and Characterization. The hydrophobic character of the different self-assembled monolayers was investigated using contact angle measurements. The results in Table 1 show that all samples possess relatively high contact angles. In our experiments, this is a prerequisite to allow for successful fusion of vesicles. Compound 10 forms a well-ordered monolayer, as can be seen by the small difference in advancing and receding contact angles. The further assembly and membrane formation of 10 is investigated by SPR. The formation of the outer leaflet of the lipid membrane by fusion with vesicles (50 nm by extrusion) results in an angular shift of the plasmon resonance. The kinetic scan recorded at 57.7° (Figure 3) shows the exponential character of the process, which reaches equilibrium after about 160 min. After 3 h, the vesicle fusion is completed at the final

636 Bioconjugate Chem., Vol. 17, No. 3, 2006

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CONCLUSION

Figure 3. SPR scans for vesicle fusion experiments on a monolayer of 10. The curve shows the evolution of the bilayer thickness with time. The inset shows the corresponding angular shift in the SPR curve before and after the addition of the vesicles. Table 2. Electrical Properties of the Different tBLMsa

10 11 12

R, MΩ cm2

C, µF cm-2

3.6 ( 1.1 1.5 ( 1.1 0.5 ( 0.5

1.1 ( 0.03 0.88 ( 0.04 0.78 ( 0.03

a The EIS data has been fit by an equivalent circuit. The values for the resistance and capacitance corresponding to the bilayer are listed.

thickness of the bilayer (6.7 nm). The inset in Figure 3 shows the angular scans before and after vesicle fusion. The fit parameters are summarized in Table 1. The optical properties of the bilayers composed of the lipids 11 and 12 were characterized using ellipsometry. As shown in Table 1, the experimental thickness values of the different monolayers (as measured from SPR or ellipsometry) and the theoretical thickness differ significantly. The latter are obtained for a model of a densely packed layer of fully stretched molecules. This lack of agreement between measured and calculated thickness might be due to an incomplete coverage of the surface. This assumption is supported by a hysteresis in advancing and receding contact angles, revealing a rather inhomogeneous surface in the case of the silane-based monolayers. Furthermore, the hydrophobic tails have a tilted conformation and the ethylene glycol units adopt a helical structure, as seen by IR spectroscopy (Lipkowski, unpublished results). By fusion of small unilamellar vesicles with monolayers of 10, 11, and 12, we were able to create bilayers and investigate their electrical properties using electrochemical impedance spectroscopy. The spectra were analyzed using a passive equivalent model circuit of resistors and capacitances. For membranes based on 11 and 12, a model with two RC elements in series with a feed resistance was used (R(RC)(RC)). Whereas for the thiol 10, one RC element in series with a capacitor and a feed resistance was sufficient (R(RC)C). The values we obtained were normalized to the electrode surface area and are listed in Table 2. The lower the capacitance is, the higher the charge separation. Here, the silane-based tBLMs show better electrical properties. However, the resistance values show an opposite trend, as the thiol-based tBLM shows the highest membrane resistance. The dense packing of the lipids is in agreement with the results from the contact angle measurements. Due to the high electrical resistances our membranes allow minimal leakage across the membrane. Therefore, we were able to show the functional incorporation of ion carriers and ion channels (37).

We developed a new synthetic strategy for the systematic study of tethered bilayer lipid membranes. A toolkit-like approach using a precursor strategy allows for the synthesis of anchor lipids with various end groups. We showed the synthesis of a thiol anchor and four different silane anchors and their ability to form monolayers on gold or silicon oxide surfaces. These monolayers can be used to form highly insulating bilayers offering a synthetic platform for the incorporation of membrane proteins. This approach can be used as a central element in biosensing applications when the coupling of biological elements to electronics is needed. Furthermore, the synthetic strategy allows adaptation of the lipids to various kinds of surface architectures by matching anchor groups. Similarly, spacer units of various types or of different lengths can be easily included in the synthesis to facilitate protein incorporation. This generic approach should allow for the design of tBLMs individually adapted to various substrates and biological compounds, for example, ion channels, receptors, and membrane proteins.

ACKNOWLEDGMENT Sven Ingebrandt is acknowledged for providing silicon wafers. Partial financial support came from the Defense Advanced Research Projects Agency (DARPA) through the MOLDICE program. I.K. acknowledges support from the Laboratoire Europe´en Associe´ (LEA) on “Polymers in Confined Geometries”.

LITERATURE CITED (1) Sackmann, E., and Tanaka, M. (2000) Supported membranes on soft polymer cushions: fabrication, characterization and applications. Trends Biotechnol. 18, 58-64. (2) Sackmann, E. (1996) Supported membranes: Scientific and practical applications. Science 271, 43-48. (3) Hillebrandt, H., Tanaka, M., and Sackmann, E. (2002) A novel membrane charge sensor: Sensitive detection of surface charge at polymer/liquid composite films on Indium Tin oxide electrodes. J. Phys. Chem. B 106, 477-486. (4) Cornell, B. A., Braach-Maksvytis, V. L. B., King, L. G., Osman, P. D. J., Raguse, B., Wieczorek, L., and Pace, R. J. (1997) A biosensor that uses ion-channel switches. Nature 387, 580-583. (5) Krueger, S., Meuse, C. W., Majkrzak, C. F., Dura, J. A., Berk, N. F., Tarek, M., and Plant, A. L. (2001) Investigation of Hybrid Bilayer Membranes with Neutron Reflectometry: Probing the Interactions of Melittin. Langmuir 17, 511-521. (6) Glazier, S. A., Vanderah, D. J., Plant, A. L., Bayley, H., Valincius, G., and Kasianowicz, J. J. (2000) Reconstitution of the Pore-Forming Toxin r-Hemolysin in Phospholipid/18-Octadecyl-1-thiahexa(ethylene oxide) and Phospholipid/n-Octadecanethiol Supported Bilayer Membranes. Langmuir 16, 10428-10435. (7) Tian, L., Yam, L., Wang, J., Tat, H., and Uhrich, K. E. (2004) Core cross-linkable polymeric micelles from PEG-lipid amphiphiles as drug carriers. J. Mater. Chem. 14, 2317-2324. (8) Morigaki, K., Baumgart, T., Jonas, U., Offenhaeusser, A., and Knoll, W. (2002) Photopolymerization of Diacetylene Lipid Bilayers and Its Application to the Construction of Micropatterned Biomimetic Membranes. Langmuir 18, 4082-4089. (9) Bhattacharya, S., and Dileep, P. V. (2004) Cationic Oxyethylene Lipids. Synthesis, Aggregation, and Transfection Properties. Bioconjugate Chem. 15, 508-519. (10) Pantusa, M., Bartucci, R., Marsh, D., and Sportelli, L. (2003) Shifts in chain-melting transition temperature of liposomal membranes by polymer-grafted lipids. Biochim. Biophys. Acta 1614, 165-170. (11) Mannock, D. A., Lewis, R. N. A. H., and McElhaney, R. N. (1990) The chemical synthesis and physical characterization of 1,2-di-Oacyl-3-O-(-D-glucopyranosyl -sn-glycerols), an important class of membrane glycolipids. Chem. Phys. Lipids 55 (3), 309-321.

Bioconjugate Chem., Vol. 17, No. 3, 2006 637

Highly Insulating Tethered Bilayer Lipid Membranes (12) Joo, C. N., Shier, T., and Kates, M. (1968) Characterization and synthesis of mono- and diphytanyl ethers of glycerol. J. Lipid Res. 9, 782-788. (13) Sita, L. R. (1993) Covalent Highly Stereoselective Syntheses of (3R,7R,11R)- and (3S,7R,11R)-3,7,11,15-Tetramethylhexadecanoic Acid (Phytanic Acid) and Corresponding 3,7,11,15-Tetramethylhexadecan-1-ols. J. Org. Chem. 58, 5285-5287. (14) Schiller, S. M., Naumann, R., Lovejoy, K., Kunz, H., and Knoll, W. (2003) Archaea Analogue Thiolipids for Tethered Bilayer Lipid Membranes on Ultrasmooth Gold Surfaces. Angew. Chem., Int. Ed. 42, 208-211. (15) Woese, C. R., and Fox, G. E. (1977) Phylogenetic Structure of the Prokaryotic Domain: The Primary Kingdoms. Proc. Natl. Acad. Sci. U.S.A. 74, 5088-5090. (16) Stewart, L. C., and Kates, M. (1989) Synthesis and characterization of deuterium-labeled dihexadecylglycerol and diphytanylglycerol phospholipids. Chem. Phys. Lipids 50, 23-42. (17) Cornell, B. A., Krishna, G., Osman, P. D., Pace, R. D., and Wieczorek, L. (2001) Tethered-bilayer lipid membranes as a support for membrane-active peptides. Biochem. Soc. Trans. 29, 613-617. (18) Hato, M. (2001) Synthetic glycolipid/water system. Curr. Opin. Colloid Interface Sci. 6, 268-276. (19) Theato, P., Zentel, R., and Schwarz, S. (2002) Synthesis of endfunctionalized lipopolymers and their characterization with regard to polymer-supported lipid memebrane. Macromol. Biosci. 2, 378394. (20) Burns, C. J., Field, L. D., Morgan, J., Petteys, B. J., Prashar, J., Ridley, D. D., Sandanayake, K. R. A. S., and Vignevich, V. (2001) Componenets for tethered bilayer membranes: synthesis of hydrophilically substituted phytanol derivatives. Aust. J. Chem. 54, 431-438. (21) Sharma, M. K., Jattani, H., and Lane M. Gilchrist, J. (2004) Bacteriorhodopsin Conjugates as Anchors for Suppported Membranes. Bioconjugate Chem. 15, 942-947. (22) Munro, J. C., and Frank, C. W. (2004) In situ formation and characterization of poly(ethylene glycol)-supported lipid bilayers on gold surface. Langmuir 20, 10567-10575. (23) Krishna, G., Schulte, J., Cornell, B. A., Pace, R. J., and Osman, P. D. (2003) Tethered Bilayer Membranes Containing Ionic Reservoirs: Selectivity and Conductance. Langmuir 19, 2294-2305. (24) Naumann, R., Schiller, S. M., Giess, F., Grohe, B., Hartmann, K. B., Ka¨rcher, I., Ko¨per, I., Lu¨bben, J., Vasilev, K., and Knoll, W. (2003) Tethered lipid bilayer on ultraflat gold surface. Langmuir 19, 5435-5443. (25) Wagner, M. L., and Tamm, L. K. (2000) Tethered PolymerSupported Planar Lipid Bilayers for Reconstitution of Integral Membrane Proteins: Silane-Poly(ethylene glycol)-Lipid as a Cushion and Covalent Linker. Biophys. J. 79, 1400-1414. (26) Kiessling, V., and Tamm, L. K. (2003) Measuring Distances in Supported Bilayers by Fluorescence Interference-Contrast Microscopy: Polymer Supports and SNARE Proteins. Biophys. J. 84, 408418. (27) Rossi, C., Homand, J., Bauche, C., Hamdi, H., Ladant, D., and Chopineau, J. (2003) Differential Mechanisms for Calcium-Dependent protein/membrane association as evidenced from SPR-binding studies on supported biomimetic membranes. Biochemistry 42, 15273-15283. (28) Atanasov, V., Knorr, N., Duran, R. S., Ingebrandt, S., Offenha¨usser, A., Knoll, W., and Ko¨per, I. (2005) Membrane on a Chip. A functional tethered lipid bilayer membrane on silicon oxide surfaces. Biophys. J. 89, 1780-1788.

(29) Mumbauer, P. D., Carey, D. H., and Ferguson, G. S. (1995) Organic Chemistry at Polymer Surfaces To Promote Adhesion to Gold and Copper: Surface-Modified Polybutadiene Having Functional Groups Containing Sulfur. Chem. Mater. 7, 1303-1314. (30) Romani, F., Passaglia, E., Aglietto, M., and Ruggeri, G. (1999) Functionalization of SBR copolymer by free radical addition of thiols. Macromol. Chem. Phys. 200, 524-530. (31) Prucker, O., Naumann, C. A., Ru¨he, J., Knoll, W., and Frank, C. W. (1999) Photochemical Attachment of Polymer Films to Solid Surfaces via Monolayers of Benzophenone Derivatives. J. Am. Chem. Soc. 121, 8766-8770. (32) Mamoru, T., and Kasumi, T. (1999) Method for making compounds containing hydrocarbonoxysilyi groups by hydrosilylation using hydrido (hydrocarbonoxy) silane compounds, Dow Corning Asia, Ltd., Tokyo, JP. (33) Hosomi, A., Kohra, S., Ogata, K., Yanagi, T., and Tominagat, Y. (1990) Pentacoordinate Allylsiliconates in Organic Synthesis: Synthesis of Triethylammonium Bis(catecho1ato)allylsiliconates and Selective Allylation of Aldehydes. J. Org. Chem. 55, 24152420. (34) Purrucker, O., Hillebrand, H., Adlkofer, K., and Tanaka, M. (2001) Deposition of highly resistive lipid bilayer on silicon-silicon dioxide electrode and incorporation of gramicidin studied by ac impedance spectroscopy. Electrochim. Acta 47, 791-798. (35) Silberzan, P., Le´ger, L., Ausserre, D., and Benattar, J. J. (1991) Silanation of Silica Surfaces. A New Method of Constructing Pure or Mixed Monolayers. Langmuir 7, 1647-1651. (36) Lee, J. H., Lee, H. B., and Andrade, J. D. (1995) Blood compatibility of poly(ethylene oxide) surfaces. Prog. Polym. Sci. 20, 1043-1079. (37) Du, H., Chandaroy, P., and Hui, S. W. (1997) Grafted poly(ethylene glycol) on lipid surfaces inhibits protein adsorption and cell adhesion. Biochim. Biophys. Acta 1326, 236-248. (38) Prime, K. L., and Whitesides, G. M. (1991) Self-assembled organic monolayers: model systems for studing adsorption of proteins at surfaces. Science 252, 1164-1167. (39) Arnold, K., Zschoernig, O., Barthel, D., and Herold, W. (1990) Exclusion of poly(ethylene glycol) from liposome surfaces. Biochim. Biophys. Acta 1022, 303-310. (40) Ariga, K., Shin, J. S., and Kunitake, T. (1995) Interaction af lipid monolayers with aqueous neutral polymers and the consequent monolayer stabilization and improved Langmuir-Blodgett transfer. J. Colloid Interface Sci. 170, 440-448. (41) Sabino, A. A., Eberlin, M. N., Moraes, L. A. B., and Laali, K. K. (2003) Gas-phase chemistry of the 2-tert-butyl-3-phenylphosphirenylium cation: novel onium ions by nucleophilic attack at phosphorus and de novo P-spiro bicyclic phosphonium ions via [4 + 2+] cycloaddition with dienes. Org. Biomol. Chem. 1, 395400. (42) Padeken, H.-G., and Klamann, D. (1985) In Methoden der Organischen Chemie, Organische Schwefel - Verbindungen., (Gundremann, K. D., and Hu¨mke, K., Eds.) Vol. E11, pp 38-62, Georg Thieme Verlag, Stuttgart, New York. (43) Kozerski, G. E., Gallava, R. H., and Ziemelis, M. J. (2003) Investigation of trialkoxysilane hydrolysis kinetics using liquid chromatography with inductively coupled plasma atomic emission spectrometric detection and nonlinear regression modeling. Anal. Chim. Acta 489, 103-114. BC050328N