Anticorrosive Lipid Monolayers with Rigid Walls around Porphyrin

Freie Universität Berlin, FB Biologie, Chemie, Pharmazie, Institut für ... Gold Nanoparticle Inclusion into Protein Nanotube as a Layered Wall Compo...
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Anticorrosive Lipid Monolayers with Rigid Walls around Porphyrin-Based 2 nm Gaps on 20 nm Gold Particles Guangtao Li and Ju¨rgen-Hinrich Fuhrhop* Freie Universita¨ t Berlin, FB Biologie, Chemie, Pharmazie, Institut fu¨ r Chemie/Organische Chemie, Takustrasse 3, D-14195 Berlin, Germany Received June 6, 2002 A meso-tetraphenylporphyrin derivative with two butanethiol and two carboxylate groups on opposite phenyl substituents was first covalently bound to citrate gold particles by a self-assembly procedure. In a second self-assembly step the remaining areas on the gold surface were covered with a monolayer of a bolaamphiphile (“bola”) containing a hydrosulfide end group for the attachment to gold and a triethylene glycol (TEG) end group for solubilization in water as well as in toluene. Bolas with octyl ends for dissolution only in toluene and with chiral gluconamide ends for water were also synthesized and applied. Two central secondary amide functions separated by 10 methylene units formed hydrogen bond chains within the monolayer and made it impermeable for cyanide ions. The porphyrin bottom of the water-filled gaps did not allow the passage of cyanide either. The holey membrane provided a complete protection against corrosion of the gold particles by 0.1 M cyanide in the bulk water. The porphyrin molecules on the bottom of the 2.2 nm wide gaps showed a weak fluorescence. It was quenched quantitatively by a tetracationic manganese porphyrinate, which fitted exactly into the gaps. A tetracationic porphyrin with a width of 3.4 nm caused no fluorescence quenching. The gaps thus have the uniform size of a monomeric porphyrin, and no domain formation was apparent. The same gaps with walls made of octadecanethiol did not discriminate between the two porphyrins of different size, both causing quantitative quenching. 1,2-trans-Cyclohexanediol or tyrosine were irreversibly immobilized within the gaps at pH 7 and stopped the entrance of the fitting porphyrin in both the rigid and the flexible gaps. The citrate gold particles thus proved to be smooth enough to allow the formation of well-defined gaps. It was also shown that 800 mg of gold colloid carries about 20 mg of membrane and gap material, which is enough for their characterization by solid-state NMR spectroscopy.

Introduction Chemisorbed alkanethiolates form rigid monolayers on flat gold surfaces, if their hydrophobic skeletons are connected at both ends by hydrogen bond chains between secondary amide groups. Such monolayers with a thickness of 2 nm become impermeable to methylamine1 and produce size-selective gaps around porphyrins lying flat on the gold surface.2 When the lipid material which formed the walls of the gaps contained a CdC double bond, it was possible to fixate a ring of ammonium groups within the gaps. A tetraanionic porphyrin was then fixated by charge interactions within such membrane pores at a distance of 0.8 or 2.0 nm from the bottom porphyrin.3 It was also shown that a porphyrin which was thus localized at the rim of the pore could be reversibly dislocated by changing the pH value of the bulk water. Such reversible formation of long-distance porphyrin heterodimers in aqueous media was considered as significant first step on the way to supramolecular systems for light-induced charge separation. The system must only be transferred from the small surface of the solid gold electrode to larger surfaces of colloidal particles in bulk water solutions to allow photochemical experiments. There was a second, even more important, motivation for us to copy the porphyrin heterodimer onto colloids. We have also found that electrons travel effectively from the gold electrode to ferricyanide ions in the bulk water * To whom correspondence should be addressed. (1) Bo¨hme, P.; Hicke, H.-G.; Bo¨ttcher, C.; Fuhrhop, J.-H. J. Am. Chem. Soc. 1995, 117, 5824. (2) Fudickar, W.; Zimmermann, J.; Ruhlmann, L.; Roeder, B.; Siggel, U.; Fuhrhop, J.-H. J. Am. Chem. Soc. 1999, 121, 9539. (3) Skupin, M.; Li, G.; Fudickar, W.; Zimmermann, J.; Roeder, B.; Fuhrhop, J.-H. J. Am. Chem. Soc. 2001, 123, 3454.

solution over the porphyrins and through the water-filled gaps above them. The passage is, however, totally blocked for weeks and months if the small water volume within the gaps contains 0.1 M cellobiose, trans-1,2-cyclohexanediol, tyrosine, or ascorbic acid. These blockade solutes do not equilibrate with bulk water that does not contain any organic solutes, the mechanism of entrapment of water-soluble compounds within the open pores that are in direct contact with the bulk water. The only means to elucidate the mobility and position of the solutes within the membrane gaps seems to be solid-state 1H and 2H NMR spectroscopy. At least 20 mg of membranes with water-filled gaps is needed for the characterization of the lipids and the entrapped water volumes, about 10 times more than for the fixated solutes. The main interest in the phenomenon of solute fixation comes first from the astonishing nonequilibrium behavior and second from the possibility that similar effects may also be responsible for glycoprotein recognition processes on cell surfaces. In a first step on the way to reactive membrane systems on the 10 mg scale, the rigid membranes were therefore transferred to colloidal gold particles. This selection allowed us to apply most of the chemistry developed for the solid gold electrodes. Only new, water-soluble headgroups had to be found, and the self-assembly procedure of the thiol-diamido bolaamphiphiles had to be established anew. Furthermore gold particles are most suitable for a direct analysis by transmission electron microscopy (TEM). The most problematic part seemed to be the first assembly step with the porphyrins, because an efficient method for avoiding domain formation was not known to us. Assembly of other than monomeric porphyrins will produce ill-defined gaps, which do allow neither the formation of defined long-distance heterodimers of por-

10.1021/la020526s CCC: $22.00 © 2002 American Chemical Society Published on Web 08/13/2002

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in comparison to the Brust gold and although we did not know whether long-lived colloidal solutions could be obtained with such massive gold particles. We report here on the first water- or toluene-soluble gold colloids of 20 nm diameter that are covered by closed hybrid membranes. “Hybrid” is meant here to indicate a fluid headgroup region, which is efficiently solvated in solution, and a rigid inside, which does not let anything pass (Figure 1b). Experimental Section

Figure 1. (a) Model of a Brust gold particle partially covered with a porphyrin, which is about the size of a surface plane, and small blocks of isolated hybrid monolayers. (b) Model of a citrate gold particle coated with a closed hybrid lipid monolayer and porphyrin-based gaps.

phyrins3 nor the immobilization of solutes. We quickly found out that the popular “Brust gold” particles of micellar size4,5 (typical diameter: 1-3 nm) are not appropriate for the establishment of complex membrane structures. Their plateaus are too small, and they produce too many edges (Figure 1a). Citrate gold particles with a typical diameter of 20-30 nm was considered to provide the minimal surface area of a few thousand square nanometers for the construction of a closed-surface monolayer comparable to that of vesicles. In the case of two parallel hydrogen bond chains separated by about 1.2 nm the hydrogen bond length would then rise only from 2.7 Å in the inner chain to 2.9 Å in the outer chain.6,7 About 1000 porphyrin molecules would then cover less than 0.5% of the surface and provide a convenient micromolar porphyrin concentration of 10-6 M in a nanomolar colloid solution. Citrate gold was therefore our first choice, although the mass ratio of gold to membrane rose by a factor of almost 100 (4) (a) Brust, M.; Walker, M.; Bethell, D.; Schiffrin, D. J.; Whyman, R. J. Chem. Soc., Chem. Comm. 1994, 801. (b) Brust, M.; Fink, J.; Bethell, D.; Schiffrin, D. J.; Kiely C. J. J. Chem. Soc., Chem. Commun. 1995, 1655. (5) (a) Hostetler, M. J.; Murray, R. W. Curr. Opin. Colloid Interface Sci. 1997, 2, 42. (b) Templeton, A. C.; Wuelfing, W. P.; Murray, R. W. Acc. Chem. Res. 2000, 33, 27. (6) (a) Turkevich, J.; Stevenson, P. C.; Hiller, J. Discuss. Faraday Soc. 1951, 11, 55. (b) Enustun, B.V.; Turkevich, J. J. Am. Chem. Soc. 1963, 85, 3317. (7) (a) Frens, G. Nat. Phys. Sci. 1973, 241, 20. (b) Grabar, K. C.; Freeman, R. G.; Hommer, M. B.; Natan, M. J. Anal. Chem. 1995, 67. (c) Garbar, K. C.; Brown, K. R.; Keating, C. D.; Stranick, S. J.; Tang, S. L.; Natan, M. J. Anal. Chem. 1997, 69, 471.

Chemicals. 1,10-Diaminodecane (Acros), bis(tert-butyl) dicarbonate (Aldrich), D-gluconic acid δ-lactone (Acros), triethylene glycol monomethyl ether (Aldrich), bromoacetic acid tert-butyl ester (Fluka), nonanoic acid (Fluka), N,N′-dicyclohexylcarbodiimide (DCC, Acros), 4-(dimethylamino)pyridine (DMAP, Merck), sodium borohydride (Merck), 3,3′-dithioldipropionic acid bis(Nhydroxysuccinimide ester) (Fluka), HAuCl4‚3H2O (Alfa), and sodium citrate dihydrate (Aldrich) were used as obtained. Syntheses of Functionalized Thiols (Scheme 1). The synthesis of porphyrin 11 will be described in a forthcoming publication;10 porphyrins 12 and 13 have been reported earlier.2,3 2,3,4,5,6-Pentahydroxyhexanoic Acid (10-Aminodecyl)amide (2). A 1 g (5.6 mmol) amount of D-gluconic acid δ-lactone was dissolved in 10 mL of water, and the solution was then diluted with 120 mL of methanol. This solution was dropped into 200 mL of a methanolic solution of 4.8 g (28 mmol) of 1,10diaminodecane at 65 °C within 2 h. After the addition the mixture was refluxed at 85 °C for 4 h. Then, the reaction mixture was cooled to 4 °C and left at this temperature for 12 h. The precipitate was filtered off and the methanol removed. A 100 mL volume of CHCl3 was added. The mixture was filtered and the solvent removed. Yield: 1.5 g (76.5%) of a white solid. 1H NMR (DMSO): δ 1.21-1.36 (m, 16H), 2.50 (m, 2H), 2.53 (m, 2H), 3.06 (m, 2H), 3.30-3.39 (m, 2H), 3.48 (m, 1H), 3.58 (m, 1H), 3.85 (m, 1H), 3.94 (m, 1H), 4.6-4.3 (m, broad, 5H), 7.54 (m, 1H). MS (FAB+): m/z ) 351 (M + 1). Anal. Calcd for C16H34N2O6 (Mr ) 350.24): C, 54.84; H, 9.78; N, 7.99. Found: C, 6.21; H, 10.21; N, 8.34. 2,3,4,5,6-Pentahydroxyhexanoic Acid [10-((3-Mercaptopropionyl)amino)decyl]amide (3). This compound was synthesized from 2 by the same method described for 6a as a white solid (44%). 1H NMR (DMSO): δ 1.19-1.35 (m, 16H), 1.59 (t, 1H), 2.44 (t, 2H), 2.84 (m, 2H), 3.30-3.39 (m, 6H), 3.35 (m, 1H), 3.58 (m, 1H), 3.85 (m, 1H), 3.94 (m, 1H), 4.3-4.6 (m, broad, 5H), 7.52 (m, 2H). MS (FAB+): m/z ) 439 (M + H). Anal. Calcd for C19H38N2O7S (Mr ) 438.24): C, 52.03; H, 8.73; N, 6.39. Found: C, 52.53; H, 9.01; N, 6.78. (10-Aminodecyl)carbamic Acid tert-Butyl Ester (4). A 3.8 g (22 mmol) amount of 1,10-diaminodecane was dissolved in 250 mL of CHCl3, and the solution was cooled in an ice-water bath. A 0.96 g (4.4 mmol) amount of bis(tert-butyl) dicarbonate was then dropped within 2 h. After the addition, the resulting mixture was stirred for further 24 h at room temperature. The white precipitate was filtered off, and the filtrate was concentrated to 10 mL on a rotatory evaporator. The residue was redissolved in 200 mL of ethyl acetate. The resultant solution was washed with 200 mL of brine and water and then dried over MgSO4. After filtration, solvent was removed under reduced vaccum, yielding 1.0 g of oil (83%). 1H NMR (CDCl3): δ 1.09 (s, 2H), 1.24 (s, 16H), 1.33 (s, 9H), 1.60 (t, 2H), 3.0 (q, 2H), 4.50 (s, broad, 1H). MS (FAB+): m/z ) 273 (M + H). Anal. Calcd for C15H32N2O2 (Mr ) 272.25): C, 66.13; H, 11.84; N, 10.28. Found: C, 66.54; H, 11.79; N, 10.35. N-(10-aminodecyl)acetamide (5a). A 1.0 g (3.7 mmol) amount of 4 and 0.24 g (4 mmol) of acetic acid were dissolved in 300 mL of CH2Cl2. After cooling at 0 °C for 15 min, 1.8 g (9 mmol) of DCC and 1.2 g (10 mmol) of DMAP were added. The reaction mixture was then stirred at room temperature for further 24 h. (8) Weisbecker, C. S.; Merritt, M. V.; Whitesides, G. M. Langmuir 1996, 12, 3763. (9) Templeton, A. C.; Hostetler, M. J.; Kraft, C. T.; Murray, R. W. J. Am. Chem. Soc. 1998, 120, 1906. (10) Li, G.; Fuhrhop, J. H. Angew. Chem., in press.

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Li and Fuhrhop Scheme 1

The white precipitate was filtered off and the filtrate washed successively with 0.1 M HCl, 8% NaHCO3, and water and dried over MgSO4. The solvent was removed in vacuo and the residue purified by silica column chromatography using CH2Cl2 as solvent, followed by crystallization from hexane/ethyl acetate. The resulting crystals were redissolved in 50 mL of CH2Cl2 containing 10 mL of TFA and stirred for 4 h to remove the Bocprotective group. Yield: 0.7 g (88%). 1H NMR (CDCl3): δ 1.29 (s, 14H), 1.42 (m, 4H), 1.91 (s, 3H), 2.63 (t, 2H), 3.22 (q, 2H), 5.58 (s, broad, 1H). MS (FAB+): m/z ) 215 (M + H). Anal. Calcd for C12H26N2O (Mr ) 214.20): C, 67.24; H, 12.23; N, 13.07. Found: C, 67.34; H, 12.53; N, 13.60. Nonanoic Acid (10-Aminodecyl)amide (5b). This compound was synthesized from nonaoic acid and 4 by the same method as described for 5a as a yellow solid (92%). 1H NMR (CDCl3): δ 0.9 (t, 3H), 1.10-1.60 (m, 30H), 2.21 (t, 2H), 2.51 (t, 2H), 3.18 (q, 2H), 6.30 (s, 1H). MS (FAB+): m/z ) 313 (M + H). Anal. Calcd for C19H40N2O (Mr ) 312.31): C, 73.02; H, 12.90; N, 8.96. Found: C, 73.12; H, 13.09; N, 9.12. N-(10-(Acetylamino)decyl)-3-mercaptopropionamide (6a). A 0.66 g (1.7 mmol) amount of 3,3′-dithioldipropionic acid bis(N-hydroxysuccinimide ester) was dissolved in 200 mL of CH2Cl2 containing 10 mmol of Et3N. A 100 mL volume of a CH2Cl2 solution of 0.7 g (3.3 mmol) of 5a was added dropwise at room temperature under an argon atmosphere. After the mixture was stirred at this temperature for 24 h, the white precipitate was filtered off, washed with CH2Cl2, and dried in air. Without further purification it was redissolved in 200 mL of 2-propanol containing 1 g of NaBH4, and the resulting suspension was refluxed under Ar gas for 4 h. After being cooled to room temperature, the reaction mixture was extracted three times with Ar-saturated CHCl3. The organic phase was washed with Ar-saturated water and dried over MgSO4. After removal of solvent under reduced pressure, the crude product was purified by crystallization from methanol, yielding 0.72 g (72%) of a white solid. 1H NMR (CDCl3): δ 1.27 (s, 12H), 1.51 (s, 4H), 2.0 (s, 3H), 2.49 (t, 1H), 2.82 (q, 2H), 3.27 (m, 4H), 5.70 (s, broad, 2H). MS (FAB+): m/z ) 303 (M + H). Anal. Calcd for C15H30N2O2S (Mr ) 302.20): C, 59.56; H, 10.00; N, 9.26. Found: C, 59.70; H, 10.32; N, 9.69. NonanoicAcid[10-((3-Mercaptopropionyl)amino)decyl]amide (6b). This compound was synthesized from 5b by the same method as described for 6a as a white solid (70%). 1H NMR (CDCl3): δ 0.91 (t, 3H), 1.30-1.50 (m, 28H), 1.60 (t, 1H), 2.12 (t, 2H), 2.46 (t, 2H), 2.82 (q, 2H), 3.30 (m, 4H), 5.69 (s. broad, 2H). MS (FAB+): m/z ) 401 (M + H). Anal. Calcd for C22H44N2O2S (Mr ) 400.31): C, 65.95; H, 11.07; N, 6.99. Found: C, 66.25; H, 11.45; N, 7.23.

{2-[2-(2-Methoxyethoxy)ethoxy]ethoxy}acetic Acid (8). To 100 mL of a stirred, dry THF solution of 2-[2-(2-methoxyethoxy)ethoxy]ethanol (1.64 g, 10 mmol) was added NaH (0.6 g, 15 mmol) at 0 °C, and stirring was continuated at this temperature for 15 min. Then the mixture was slowly brought to room temperature and stirred for another 1 h. After the addition of bromoacetic acid tert-butyl ester (2.9 g, 15 mmol), the reaction mixture was warmed to 80 °C and refluxed at this temperature for 10 h. The reaction solution was cooled to room temperature, and the formed precipitate was filtered off. The filtrate was concentrated to 10 mL and purified by silica column chromatography using hexane/ethyl acetate as eluent. The resulting protected form of target product was redissolved in 50 mL of CH2Cl2 containing 16 mL of TFA. After 4 h of stirring at room temperature, the solvent was evaporated and 2.8 g of oil was obtained (74.5%). 1H NMR (CDCl3): δ 3.32 (s, 3H), 3.52-3.76 (m, 12H), 4.16 (s, 2H), 9.92 (s, 1H). MS (FAB+): m/z ) 223 (M + H). Anal. Calcd for C9H18O6 (Mr ) 222.11): C, 48.64; H, 8.16. Found: C, 48.79; H, 8.43. N-(10-Aminodecyl)-2-{2-[2-(2-methoxyethoxy)ethoxy]ethoxy}acetamide (9). This compound was synthesized from 8 and 4 by the same method as described for 5a as an oil (43%). 1H NMR (CDCl ): δ 1.20-1.60 (m, 18H), 2.52 (m, 2H), 3.28 (m, 3 2H), 3.40 (s, 3H), 3.60 (m, 12H), 4.08 (s, 2H), 7.2 (s, 1H). MS (FAB+): m/z ) 377 (M + H). Anal. Calcd for C19H40N2O5 (Mr ) 376.29): C, 60.61; H, 10.71; N, 7.44. Found: C, 60.76; H, 10.95; N, 7.62. 3-Mercapto-N-[10-(2-{2-[2-(2-methoxyethoxy)ethoxy]ethoxy}acetylamino)decyl]propionamide (10). This compound was synthesized from 9 by the same procedure as described for 6a with an 80% yield of a white solid obtained. 1H NMR (CDCl3): δ 1.23-1.54 (m, 17H), 2.54 (t, 2H), 2.94 (q, 2H), 3.24 (m, 4H), 3.39 (s, 3H), 3.50-3.71 (m, 12H), 4.00 (s. 2H), 6.40 (s, 1H), 7.12 (s, 1H). MS (FAB+): m/z ) 465 (M + H). Anal. Calcd for C22H44N2O6S (Mr ) 464.29): C, 56.87; H, 9.54; N, 6.03. Found: C, 60.03; H, 9.76; N, 6.35. Gold Colloids. Colloidal Au nanoparticles with a mean diameter of 20 nm were prepared according to Turkevich6 and Natan7 with slight modifications. To 100 mL of boiling water containing 40 mg of HAuCl4 was added 120 mg of sodium citrate in 5 mL of water. The solution turned blue within 20 s and then to red-violet within 1 min. Refluxing was continued for 20 min and the solution cooled to room temperature and stored in a refrigerator at 5 °C. Preparation of Closed Membranes and Construction of Nanogaps on Colloidal Particles. Gold particles coated with closed membranes of 3, 6a,b, or 10 were prepared as follows: 5

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mL of the colloidal gold solutions was diluted with 10 mL of water and 40 mL of acetone. A 6 mg amount of the thiol amphiphile in 5 mL of acetone was added under vigorous stirring. The resulting suspension was stirred under N2 in the dark for 8 h. Membrane-coated nanoparticles precipitated and were centrifuged. The solid material was thoroughly washed with ethanol and acetone. The black amorphous solid was resuspended in DMSO, toluene, THF, or water. To generate nanogaps on colloidal gold, subsequent self-assembly of thiol porphyrin 11 and amphiphile 10 was applied. To a mixture of 5 mL of gold colloid + 15 mL of water + 60 mL of acetone was first added 80 µL of acetone solution of porphyrin 11 (10-5 M). After 15 min of stirring at room temperature 5 mL of acetone solution of thiol 10 (6 mg) was added. The reaction solution was stirred for 10 h. The precipitate formed was again thoroughly washed with ethanol and acetone. The black solid obtained was resuspended in water to yield a red solution. UV/Vis Spectroscopy of Gold Colloids. UV/vis absorption spectra of base citrate gold and membrane-coated gold particles were acquired using a Perkin-Elmer Lambda 16 spectrometer. Transmission Electron Microscopy (TEM). TEM samples were prepared by dropping 10 µL aliquots of colloidal solution onto a holey carbon coated copper grid. After deposition for 1 min, the remaining solution was blotted with a filter paper. The gold particles were imaged using a Philips M12 transmission electron microscope operated at 100 kV. Infrared Spectroscopy. About 3 mg of the solid gold particles was pulverized together with 1 g of KBr. A portion of this material was pressed into a transparent disk. The infrared spectra were collected in the transmission mode on a Nicolet 800 spectrometer. A total of 250 scans were taken, and a background spectrum was automatically subtracted. Fluorescence Quenching Experiments. The fluorescence measurements and quenching experiments were performed on a Perkin-Elmer spectrometer (LS50B). The gold particles coated with the perforated membranes were dispersed in water and placed in a quartz cuvette (3 mL). A 10 µL volume of aqueous solution (10-4 M) of quencher 12 was then added. For the quenching experiments with porphyrin 13 large quantities (30, 50, and 100 µL) of an aqueous solution (10-4 M) was used. In a control experiment 10 µL of aqueous solution (10-4 M) of 13 was added to a colloidal gold solution coated only with porphyrin 11. Chemical Etching of Colloidal Gold by Sodium Cyanide. A 2 mL sample of bare citrate gold or membrane-coated gold solution was quickly mixed with 0.5 mL of an aqueous NaCN solution (0.1 M). The decay in plasmon absorption band was monitored over 1 h on a UV/vis spectrometer. In the case of the Brust gold colloids, the loss of absorption at 500 nm was monitored.

Results Amphiphiles 3, 6a,b, and 10 were synthesized by standard procedures from 1,10-diaminodecane (Scheme 1). Amphiphile 6b contains a terminal SH group to be bound to the gold colloids and an octyl chain for the solvation of the monolayer by organic solvents, e.g. toluene. At the outer and inner parts of the hydrophobic core, two secondary amide groups form two parallel hydrogen bond chains in the monolayers.1-3 Bolaamphiphile 10 has a triethylene glycol ending for dissolution in both organic solvents and water. Glyconamide bolaamphiphile 3 was made for water solubility only and provides chiral centers for eventual recognition processes on the surface of the colloids. As a basis for nanometer membrane gaps, we first attempted to use meso-tetrakis[3,5-dicarboxyltetraphenyl]porphyrin, which had successfully been attached to planar gold electrodes earlier.2,3 It became, however, quickly apparent that this porphyrin did not form a stable monolayer on colloidal particles in bulk water. It separated from the gold surface during washing cycles until a colloidal solution of gold was obtained, which did not show a Soret band absorption. This was the case for both 2-3 nm (“Brust gold”) particles as well as for 20 nm citrate gold. We therefore synthesized porphyrin 11, which was covalently attached to gold by formation of two Au-S bonds via butanethiol ligands. It also contained two carboxylate groups for the binding of fluorescence quenchers, namely the manganese(III) meso-tetrapyridiniumylporphyrinates 12 and 13 (Chart 1). The best solvent mixture for self-assembly of the amphiphiles 3, 6a,b, or 10 on citrate gold particles was found to be a 4:1 mixture of acetone and water. The selfassembly of a stiff membrane made of the diamide 6a with an acetamide end group led to quantitative precipitation from acetone/water (Figure 2b). This surface monolayer did not swell and take up solvent molecules. The precipitate was, however, readily redissolved by DMSO, which broke the hydrogen bond chains. The maximum of the plasmon absorption band of the gold colloid then moved from 520 to 565 nm (Figure 2a). Amphiphile 6b with an octyl chain at the end also caused precipitation upon self-assembly in acetone/water mixtures. This precipitate was redissolved quantitatively in pure toluene, where a plasmon absorption at 550 nm was observed (Figure 2c). After the self-assembly of the triethylene glycol bolaamphiphile 10 the gold particles

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Figure 2. Models of citrate gold surface layers made of (a, b) bolaamphiphile 6a, (c) amphiphile 6b, (d) bolaamphiphile 10, (e) bolaamphiphile 3, and (f) bolaamphiphile 20 and porphyrin 11. Their solubility characteristics are indicated.

precipitated after about 2 h from acetone/water and were separated by short centrifugation at 3000 rpm. After centrifugation, the particles were redissolved and produced perfectly stable colloidal solutions in toluene (λmax ) 545 nm) as well as in water (λmax ) 536 nm; Figure 2d). The particles were then again precipitated quantitatively by the addition of cold acetone or ethanol, centrifuged, or filtered and could be quantitatively redissolved in water or toluene. No material was lost in these procedures. The gluconamide derivative 6 on gold behaved similarly to 10. But it was soluble only in water (Figure 2e) and not in organic solvents. Figure 2 reproduces model pictures of individual surface layers together with wavelengths of the plasmon absorption bands. The coated gold particles were then transferred to carbon-coated copper grids and characterized by transmission electron microscopy (TEM). The original citrate gold is thought to be covered mainly by citrate and/or acetonediacetic acid, which is formed in the reduction process of converting gold chloride to gold.6 The range of diameters of the particles is between 15 and 20 nm, the smallest distance between neighboring particles is very often below 1 nm, and many particles touch each other (Figure 3a). The surface layer consisting of citrate and/or acetonediacetic acid must be thin. In the case of the DMSOdissolved particles with a coating of diamide 6a, the interparticle separation seems to be even less. Some particles are separated by 4 nm, which would correspond to the thickness of two monolayers, but most particles seem to touch each other at the gold surface (Figure 3b). The DMSO-swollen surfaces keep the particles suspended but do not separate them. Partial interdigitation of the surface monolayers may occur here. The particle size remains the same with the octylamide cover 6b in toluene solution (Figure 3c). The closest distance between neigh-

Figure 3. TEMs of citrate gold deposited from different solutions (see text) onto carbon-coated grids and covered with different coatings: (a) citrate; (b) amphiphile 6a; (c) amphiphile 6b; (d, e) bolaamphiphile 10; (f) bolaamphiphile 3.

boring particles is now a constant 4-6 nm, which corresponds to twice the length of the rigid inner part of the membrane. The outer octyl chains presumably interdigitate and do not act as a distance-holder, but the diamide-linked rigid membrane parts do so. The same 4-6 nm distance between close neighbors is also found in the triethylene glycol 10 gold particles in toluene (Figure 3d) and water (Figure 3e) as well as in the gluconamide 3 coated particles in water (Figure 3f). In a few cases the TEM pictures of coated gold particles with and without porphyrin-based gaps were compared. No differences were observed, either for the monomeric particles or for the aggregates. The area of covered by the porphyrins was estimated to be roughly 0.5-2% of the total surface area

Anticorrosive Lipid Monolayers

of the gold particles, which is less than 5% of that of the solid gold electrodes investigated earlier. FTIR spectra of gold particles coated with 6a provided evidence for highly crystalline surface monolayers. The peak frequency for CH2 (as) and CH2 (sym) was located at 2918 and 2850 cm-1. These frequencies are the same as those found in crystalline ODT monolayers on planar gold.11 The narrow bandwidths of CH2 (as), 15 cm-1, and CH2 (sym), 9 cm-1, are characteristic of tightly packed chains with minimal gauche defects.11,12 The peak frequencies and bandwidths are indicative of a crystalline array of well-ordered methylene chains in all-trans conformations. The amide A band (N-H) appears at 3300 cm-1, amide I at 1650 cm-1, and amide II at 1560 cm-1. These frequencies are similar to those seen in solid polypeptides13 and are within the range of literature values for solid secondary amides in solid state.14 The high frequency of amide II implies a high degree of association between amide groups.15 It is evident that a network of lateral cross-links through hydrogen bonding was formed within these membranes. We assume that the hydrogen bonds detected here in the solid state are also present in an aqueous environment. One argument in favor of this assumption is the blockade of cyanide ion and probably also hydrocyanic acid transport, which is not matched by the more fluid octadecyl sulfide monolayer (see Figure 6). Direct FTIR measurements of the monolayer’s amide bands under a water film gave no reproducible results. The colloids were then further functionalized by the introduction of porphyrin-based gaps within the rigid inner parts of coatings. Attempts to attach meso-tetrakis[3,5dicarboxylphenyl]porphyrin to the gold particles failed. This octacarboxylato porphyrin was tightly bound by flat gold,2,3 but it dissociated from citrate gold particles upon washing with water, even at neutral pH. The porphyrin dihydrosulfide 11 was, however, fixated by self-assembly and remained bound to the particles after treatment with the bolaamphiphilic triethylene glycol hydrosulfide 10 (see Figure 2f). A small Soret band at 425 nm was observed (Figure 4f) next to the broad plasmon band, which had now shifted from 536 nm for the particles with closed monolayer (Figure 4d) to 568 nm. The plasmon band at 550 nm of the toluene dissolved gold covered with the octyl amphiphile 6b is also given as a comparison (Figure 4c). The interdigitation of the remote alkyl chains in 6bgold caused a slightly smaller red shift than that of the amide 6a chains, which were directly bound to the gold surface. The original, charged citrate gold particles may be most isolated from neighbors in aqueous solution (520 nm); next come triethylene glycol and gluconamide particles with closed membranes (all 536 nm) and the octyl particles (550 nm), and finally the DMSO-dissolved (565 nm) particles. The red shift to 568 nm in the case of the porphyrin-covered particles has probably a different origin. It is presumably caused by the polarizable π-system of the porphyrins, which takes part in the delocalization (11) (a) Nuzzo, R. G.; Dubois, L. H.; Allara, D. L. J. Am. Chem. Soc. 1990, 112, 558. (b) Laibinis, P. E.; Whitesides, G. M.; Allara, D. L.; Tao, Y.; Parikh, A. N.; Nuzzo, R. G. J. Am. Chem. Soc. 1991, 113, 7152. (c) Tao, Y. T. J. Am. Chem. Soc. 1993, 115, 4350. (12) (a) Clegg, R. S.; Hutchison, J. E. Langmuir 1996, 12, 5239. (b) Bent, S. F.; Schilling, M. L.; Wilson, W. L.; Katz, H. E.; Harris, A. L. Chem. Mater. 1994, 6, 122. (13) Bamford, C. H.; Elliott, A.; Hanby, W. C. Synthetic Polypeptides: Preparation, Structure, and Properties; Academic: New York, 1956. (14) (a) Clegg, R. S.; Hutchison, J. E. J. Am. Chem. Soc. 1999, 121, 5319. (b) Clegg, R. S.; Reed, S. M.; Smith, R. K.; Barron, B. L.; Rear, J. A.; Hutchison, J. E. Langmuir 1999, 15, 8876. (c) Valiokas, R.; Svedhem, S.; Svensson, S. C. T.; Liedberg, B. Langmuir 1999, 15, 3390. (15) Clegg, R. S.; Reed, S. M.; Hutchison, J. E. J. Am. Chem. Soc. 1998, 120, 2486.

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Figure 4. Absorption spectra of the original citrate gold particles as well as particles coated with different amphiphiles: (a) original citrate gold (in H2O); (b) methylamide 6a (in DMSO); (c) octylamide 6b (in toluene); (d) triethylene glycol 10 (in H2O); (e) gluconamide 3 (in H2O); (f) glycol 10 and porphyrin 11 (in H2O); (g) glycol 10 and porphyrin 11 and 12 heterodimer (in H2O).

of the gold electrons. Similar observations have been made with other dyes.16 Plasmon band shifts have, however, diverse origins.17 The interpretations considered here are only plausible to us and not necessarily correct. Fluorescence quenching experiments could be performed in aqueous solution using a routine spectrophotometer. The low-angle laser technology needed for the solid electrode experiments2,3 was not needed any more. Similar to the observations on planar gold electrodes, we (16) Makarova, O. V.; Ostafin, A. E.; Miyoshi, H.; Norris, J. R., Jr.; Meisel, D. J. Phys. Chem. B 1999, 103, 9080. (17) (a) Mulvaney, P. Langmuir 1996, 12, 788. (b) Heath, J. R.; Knobler, C. M.; Leff, D. V. J. Phys. Chem. B 1997, 101, 189. (c) Pileni, P. P. New J. Chem. 1998, 693.

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Figure 6. Time-dependent decrease of the plasmon absorption bands of citrate gold colloids in solution after addition of a 0.5 M NaCN-solution: (a-c) bolas 3, 10 (in H2O), and 6b (in THF/ H2O, 4:1); (d) bola 10 plus porphyrin 11 gaps (in H2O); (e) diamide 3 in DMSO; (f) octadecyl (THF/H2O, 4:1); (g) decrease of 500 nm absorption of Brust gold covered with bola 10 (THF/ H2O, 4:1); (h) bare citrate gold (in H2O).

Figure 5. Fluorescence spectra of porphyrin 11 on citrate gold particles: (a) on bare citrate gold before and after addition of manganese porphyrinate 13; (b) on membrane-coated gold before and after addition of 12; (c) same as (b) but with porphyrin 13; (d) same as (b) but after treating the gold particles with 0.1 M solutions of 1,2-trans-cyclohexanediol.

found that most of the fluorescence of porphyrin 11 was quenched by strong electronic coupling to the colloidal gold substrate. The remaining rest fluorescence of about 1% was, however, sufficient for our purposes (Figure 5a). It remained nearly constant after the self-assembly of bolaamphiphile 10 (Figure 5b). The particles covered by 10 and 11 were isolated as solid materials by acetone precipitation and produced the same UV/vis and fluorescence spectra after redissolution in water. The fluorescence was quenched to more than 90% on gold covered by porphyrin 11 and bola 10 by the addition of the fitting manganese-tetrapyridiniumylporphyrinate 12 (Figure 5b). Upon addition of the manganese porphyrin 12 and washing with water, the Soret bands of both porphyrins 11 and 12 became detectable (Figure 4f). Addition of the larger porphyrin 13 with an additional phenyl spacer caused quantitative fluorescence quenching on naked citrate gold (Figure 5a) but had no effect whatsoever on the porphyrin on the bottom of the rigid membrane gap (Figure 5c). This size-discrimination effect disappeared,

when 50% DMSO was added to the bulk water, which fluidized the monolayer. The gap was closed for the entering of the fitting porphyrin 12, when its water content was immobilized by addition of 0.1 M 1,2-trans-cyclohexanediol (Figure 5d)2,3,18 or tyrosine.3 The supposed tightness and impermeability of the hybrid monolayer was also tested with respect to cyanide ion attack of the gold surface.8,9 The decay of the plasmon absorptions was used to monitor the progress of citrate gold destruction; the loss of 500 nm absorption was monitored for Brust colloids. Bare citrate gold disappeared quickly after the addition of sodium cyanide (Figure 6h). Fluid octadecanethiol coatings in THF/water (4:1; Figure 6f) or amide 3 in DMSO/water (Figure 6e) had only a retarding effect for the same decomposition process. Rigid surface monolayers stabilized by two amide hydrogen bond chains on 20 nm particles were, however, not passed at all by cyanide ions. No gold dissolution was detectable after 24 h, when the particles were covered with 3, 6b, or 10 bolas (Figure 6a-c). After 1 month, less than 1% of the particles was destroyed (not shown). Even the particles with a large number of porphyrin-based holes were not attacked by cyanide ions (Figure 6d). The 4-5 Å high porphyrin layer obviously protected the Au-S bonds as effectively as the rigid C10 coating with two secondary amide groups. The diamide coatings thus provided a perfect corrosion protection shield for citrate gold in the time scale of several days to weeks. For 3 nm Brust-type gold particles, the same rigid membranes made of the triethylene glycol bola 10 had, however, no protective effect at all (Figure 6g). There were obviously too many unprotected edges. The models in Figure 6 summarize the experimental results with respect to the approach of cyanide ions to different gold particles. (18) Fuhrhop, J.-H.; Bedurke, T.; Gnade, M.; Schneider, J.; Doblhofer, K. Langmuir 1997, 13, 455.

Anticorrosive Lipid Monolayers

Solid-state spectra of 8oo mg of citrate gold particles coated with octadecanethiol gave a strong signal for the protons of the oligomethylene chain. Weak signals for the terminal methyl group and the methylene group neighboring the amide group were also detected.37 Discussion and Conclusion So far, very little attention has been given to functionalize the inside of monolayer coatings. Solubilization in organic solvents was achieved by alkanethiols,4,5 ureatype ligands on the surface were used to produce nanoparticle assemblies,19-22 metal complexes were applied as catalysts,23 and polymer multilayers24,25 were used as carrier for dyes. “Mixed SAMs” on flat gold electrodes, which are comparable to our hybrid SAMs, contained a fluid alkyl chain on the inside and an equally fluid oligoethylene glycol (OEG) chain on the outside, which rejected proteins.26,27 Nanoparticle assembly by interdigitation of the OEG moieties was not observed.8 Fluorescing dyes such as porphyrin,28 dansyl,29 and pyrene30 were located on top of a fluid alkane monolayer and then produced strong fluorescence bands. Rhodamine and fluorescing derivatives directly bound to the gold surface fluoresced as weakly as our porphyrin 11.31,32 What has been achieved with the hybrid membrane described here? From TEM pictures it is known that citrate gold surfaces contain no detectable edges but tend to form metal bridges to give assemblies with rough surfaces after some weeks.6 We have detected no such metal bridges between membrane-coated particles. Only the rest fluorescence of the porphyrins indicate some roughness, but it does not appear in the reaction with cyanide or in fluorescence quenching with nonfitting porphyrins. The TEM images of probes made by the same procedure were always close to identical. This reproducibility was not reached with the solid electrode surfaces. Many samples had to be dismissed there, because they were too rough. (19) Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J. Nature 1996, 382, 607. (20) Storhoff, J. J.; Mirkin, C. A. Chem. Rev. 1999, 99, 1849. (21) Teranishi, T.; Haga, M.; Shiozawa, Y.; Miyake, M. J. Am. Chem. Soc. 2000, 122, 4237. (22) Mann, S.; Shenton, W.; Li, M.; Connolly, S.; Fitzmaurice, D. Adv. Mater. 2000, 12, 147. (23) Li, H.; Luk, Y.-Y.; Mrksich, M. Langmuir 1999, 15, 4957. (24) Watson, K. J.; Zhu, J.; Nguyen, S. T.; Mirkin, C. A. J. Am. Chem. Soc. 1999, 121, 462. (25) Tedeschi, C.; Mo¨hwald, H.; Kirstein, S. J. Am. Chem. Soc. 2001, 123, 954. (26) Harder, P.; Grunze, M.; Dahint. R.; Whitesides, G.M.; Laibinis, P. E. J. Phys. Chem. B 1998, 102, 426. (27) Pertsin, A. J.; Grunze, M. Langmuir 2000, 16, 8829. (28) Imahori, H.; Arimura, M.; Hanada, T.; Nishimura, Y.; Yamazaki, I.; Sakata, Y.; Fukuzumi, S. J. Am. Chem. Soc. 2001, 123, 335. (29) Aguila, A.; Murray, R. W. Langmuir 2000, 16, 5949. (30) Fox, M. A.; Whitesell, J. K.; McKerrow, A. J. Langmuir 1998, 14, 816. (31) Templeton, A. C.; Cliffel, D. E.; Murray, R. W. J. Am. Chem. Soc. 1999, 121, 7081. (32) Chandrasekharan, N.; Kamat, P. V.; Hu, J.; Jones, G. J. Phys. Chem. B 2000, 104, 11103.

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Long-term water solubility of particles with PEG coatings has already been described.8 Both triethylene glycol and gluconamide headgroups avoid interdigitation and thereby prevent coagulation. A peculiar finding consists of the precipitation in acetone at temperatures below 20 °C, which is not understood. It is a property of the triethylene glycol unit itself. The total stability of colloidal solutions of the relatively heavy gold particles covered by gluconamide 3, which lasted for at least several months, is, to the best of our knowledge, unique. Entrapped water molecules in the gauche bent of the headgroup and internal hydrogen bond cycles may be responsible for strong hydration and permanent rejection of colliding particles. These structural details also stabilized quadruple helical fibers in water.35 The precipitation of triethylene glycol (TEG) particles by cold acetone and redissolution by heating to 32 °C has to the best of our knowledge not been described but occurs also in acetone solutions of TEG itself in the same manner as with the coated particles. Since the terminal oxygen atoms are methylated, we do not know the reason for this phenomenon. Perhaps there are trace amounts of alcohol groups present in commercial TEG, which form ketals with acetone. Most gratifying was the material character of the particles including stability against chemical attack in solution and their accessibility to 1H NMR measurements. Grams of colloids can be stored indefinitely, and each 1 g of gold carries about 20 mg of membrane material. These preparations allow for the first time comparative analyses of water-filled gaps with and without rigidifying solutes as well as differentiation of the flexibility of amphiphiles, which form the walls of the nanometer gaps. Transfer of very similar systems to amine-coated silica gel particles36 then also allows investigations of light-induced charge separation experiments between electron donors at the bottom of the gaps with acceptors attached to their walls. Work along these lines is in progress. Acknowledgment. Financial support by the Deutsche Forschungsgemeinschaft (SFB 448 “Mesoscopic Systems”), the Fonds der Deutschen Chemischen Industrie, and the FNK of the Free University is gratefully acknowledged. LA020526S (33) Hu, J.; Zhang, J.; Liu, F.; Kittredge, K.; Whitesell, J. K.; Fox, M. A. J. Am. Chem. Soc. 2001, 123, 1464. (34) Lahav, M.; Heleg-Shabtai, V.; Wasserman, J.; Katz, E.; Willner, I.; Du¨rr, H.; Hu, Y.; Bossmann, S. H. J. Am. Chem. Soc. 2000, 122, 11480. (35) Svenson, S., Kirste, B., Fuhrhop, J.-H. J. Am. Chem. Soc. 1994, 116, 11969. (36) (a) Beck, C.; Ha¨rtl, W.; Hempelmann, R. Angew. Chem. 1999, 9111. (b) Badley, R. D.; Ford, W. T.; McEnroe, F. J.; Assink, R. A. Langmuir 1990, 6, 792. (37) We thank Prof. H. H. Limbach for 1H NMR solid-state spectra of octadecanethiol-coated gold particles.