Self-Assembled Monolayers of Cavitand Receptors for the Binding of

Langmuir 1998, 14, 5457-5463

5457

Self-Assembled Monolayers of Cavitand Receptors for the Binding of Neutral Molecules in Water Arianna Friggeri, Frank C. J. M. van Veggel,* and David N. Reinhoudt* Supramolecular Chemistry and Technology and MESA Research Institute, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands

Rob P. H. Kooyman Biointerface Group and MESA Research Institute, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands Received April 10, 1998. In Final Form: June 22, 1998 Resorcin[4]arene cavitands 2-4 were synthesized starting from tetrol 5. With these cavitand tetrasulfides, self-assembled monolayers (SAMs) on gold were prepared and characterized by electrochemical capacitance and resistance measurements, contact angle (CA) measurements, grazing angle FT-IR spectroscopy, and X-ray photoelectron spectroscopy (XPS). In situ surface plasmon resonance (SPR) spectroscopy was employed to study the host-guest interactions at the monolayer-water interface, between receptor adsorbates 1-4 and p-toluic acid, benzoic acid, p-nitrobenzoic acid, p-hydroxybenzoic acid, p-cresol, p-nitrophenol, and p-methoxyphenol. SPR results show that the presence of functional groups on the upper rim of the adsorbate molecules induces selectivity in the guest recognition process. Furthermore, it is interesting to note that such interactions are not directly related to the degree of hydrophobicity of the receptor adsorbate or of the guest. Concentration-dependent experiments were performed with p-nitrophenol as guest for a monolayer of adsorbate 1. At a 3 mM concentration of p-nitrophenol, approximately five to eight guest molecules associate with each adsorbate molecule, implying the formation of 1-4 layers of guests on the receptor surface.

Introduction Self-assembled monolayers (SAMs) on gold can be used to create a variety of chemically well-defined, functionalized surfaces, interesting for the study of many different interface processes. They are particularly suitable for the study of antibody-antigene interactions,1 cell growth, and protein adsorption,2 as they can be considered synthetic models for organic membranes. Recently, SAMs of simple alkanethiol adsorbates have been shown to interact with surfactant molecules,3 whereas SAMs of macrocycles such as β-cyclodextrin can complex ferrocene4 as well as several azo derivatives and dye molecules.5,6 Other potential host molecules such as calixarenes have been employed in the detection of volatile organic compounds, with the aid of surface acoustic wave (SAW) devices.7 In our group, quartz crystal microbalance (QCM)8 and surface plasmon resonance (SPR)9 have been * To whom correspondence should be addressed. Fax: +31 53 4894645. Phone: +31 53 4892980. E-mail: [email protected]. (1) Morgan, H.; Taylor, D. M.; D’Silva, C. Thin Solid Films 1992, 209, 122. (2) Tidwell, C. D.; Ertel, S. I.; Ratner, B. D.; Tarasevich, B. J.; Atre, S.; Allara, D. L. Langmuir 1997, 13, 3404. (3) Sigal, G. B.; Mrksich, M.; Whitesides, G. M. Langmuir 1997, 13, 2749. (4) Rojas, M. T.; Ko¨niger, R.; Stoddart, J. F.; Kaifer, A. E. J. Am. Chem. Soc. 1995, 117, 336. (5) Maeda, Y.; Fukuda, T.; Yamamoto, H.; Kitano, H. Langmuir 1997, 13, 4187. (6) Weisser, M.; Nelles, G.; Wenz, G.; Mittler-Neher, S. Sens. Actuators B 1997, 38-39, 58. (7) Dermody, D. L.; Crooks, R. M.; Kim, T. J. Am. Chem. Soc. 1996, 118, 11912. (8) Schierbaum, K. D.; Weiss, T.; Thoden van Velzen, E. U.; Engbersen, J. F. J.; Reinhoudt, D. N.; Go¨pel, W. Science 1994, 265, 1413. (9) Huisman, B.-H.; Kooyman, R. P. H.; van Veggel, F. C. J. M.; Reinhoudt, D. N. Adv. Mater. 1996, 8, 561.

used to show how resorcin[4]arene derivatives selectively bind perchloroethylene in the vapor phase. SPR is a relatively simple, optical technique, long-term stable and very sensitive to changes in refractive index occurring at a metal interface.10 It is therefore suitable for the detection of molecular interactions with SAMs on gold. It is only in the last 10 years that SPR has become a well-established technique, especially in the field of biosensor technology.11,12 Recently, Whitesides et al. employed this technique to determine the affinity of detergents for a hydrophobic SAM, the number of detergent molecules on the SAM, the rate of desorption of these surfactants, and their efficiency in removing adsorbed protein.3 However, to the best of our knowledge, SPR has never been employed for the detection of host-guest interactions in water, between neutral molecules with MW < 200 Da and receptor molecules. In this paper, we describe the selective interactions occurring at the monolayer-water interface between resorcin[4]arene-based receptor adsorbates13 and low molecular weight, para-substituted phenols and benzoic acids, studied in situ by means of SPR. As receptor adsorbates, cavitands were chosen because the molecular structure of compound 1 (Figure 1) had previously been shown to give well-packed, ordered monolayers,14,15 capable of interacting with small guests such as tetrachlo(10) Welford, K. Opt. Quantum Electr. 1991, 23, 1. (11) Kooyman, R. P. H.; Lenferink, A. T. M.; Eenink, R. G.; Greve, J. Anal. Chem. 1991, 63, 83. (12) Malmqvist, M. Nature 1993, 361, 186. (13) Throughout this paper the trivial name cavitand will be used for the rigidified tetrameric resorcinarene framework of the adsorbates. The official IUPAC name is 7,11,15,28-tetrakis(XX)-1,21,23,25-tetra(YY)-2,20:3,19-dimetheno-1H,21H,23H,25H-bis[1,3]dioxacino[5,4i:5′,4′i′]benzo[1,2d:5,4d′]bis[1,3]benzodioxocin, in which XX refers to the aromatic rings substituents and YY to the methylene bridge substituents.

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Figure 1. Receptor adsorbates 1-4.

roethylene and toluene, in the vapor phase.9 In addition to this, the densely packed alkyl chains underneath the aromatic headgroups provide a good barrier toward possible interactions with the gold substrate or intercalation of guest molecules in the monolayer itself.16 Water was chosen as the medium to study host-guest interactions because monolayers in water can be considered model systems for biological interfaces. Moreover, with regards to sensor applications, the possibility of detecting organic species in water is also very interesting. So far, no previous studies have been carried out with cavitand receptors in aqueous media. The system described in this paper, used to detect cavitand-guest interactions in water, does not require water solubility of the hosts. This is advantageous with respect to measuring the same interactions, for example by NMR titration experiments. The choice of the category of guests was made on the basis of previous SPR studies9 as well as on complexation studies, using resorcinarenes and cavitands, carried out in organic solvents.17 This should allow us to determine whether particular functional groups show preferential interaction with certain receptors. Here we show that it is possible to detect weak, reversible interactions in water and that the association process is not controlled exclusively by the hydrophobic character of the adsorbates and/or of the guests. Experimental Procedures Chemicals. THF was freshly distilled from Na/benzophenone before use, and DMF was dried over molecular sieves (4 Å) for at least 3 days. For synthetic purposes dichloromethane, ethyl acetate, and hexanes (petroleum ether isomer mixture with boiling point between 60 and 80 °C) were distilled from calcium chloride. All other reagents were used as received; pa grade solvents were used for monolayer formation. Sodium hydride (60% in mineral oil) was washed with hexanes prior to use. Gold Substrates. Gold substrates were prepared on glass (25 mm in diameter) by evaporation of an adhesion layer of chromium (2 nm) followed by evaporation of gold (47.5 nm). Immediately before use, the substrate was cleaned with an oxygen plasma (10 min) and subsequently rinsed with copious amounts of ethanol. Monolayer Preparation. All glassware used to prepare monolayers was immersed in piran˜a solution. Warning: piran˜a solution should be handled with caution; it has been reported to (14) Thoden van Velzen, E. U.; Engbersen, J. F. J.; de Lange, P. J.; Mahy, J. W. G.; Reinhoudt, D. N. J. Am. Chem. Soc. 1995, 117, 6853. (15) Scho¨nherr, H.; Vancso, G. J.; Huisman, B.-H.; van Veggel, F. C. J. M.; Reinhoudt, D. N. Langmuir 1997, 13, 1567. (16) Thoden van Velzen, E. U.; Engbersen, J. F. J.; Reinhoudt, D. N. J. Am. Chem. Soc. 1994, 116, 3597. (17) Timmerman, P.; Verboom, W.; Reinhoudt, D. N Tetrahedron 1996, 52, 2663.

Figure 2. SPR setup used to measure simultaneously ∆RA and ∆Rref. detonate unexpectedly. Next, the glassware was rinsed with large amounts of high-purity water (Millipore). Monolayers were prepared by immersing the gold substrates in 1 mM adsorbate solutions (chloroform-ethanol, 1:1), which were then heated at 60 °C for 16 h. The samples were then allowed to cool to room temperature before being taken out of the adsorbate solutions and rinsed thoroughly with dichloromethane, ethanol, and water (Millipore), respectively. Monolayers of octadecanethiol were left in ethanolic solution for 16 h at room temperature and were then subjected to the same rinsing procedure as described above. Surface Plasmon Resonance. For all SPR experiments, a two-channel vibrating-mirror angle scan setup, based on the Kretschmann configuration,18 was used. This set up has previously been described by Kooyman et al.;19 therefore, a detailed description of the instrument will not be given in the present paper. It will suffice to say that light from a 2-mV HeNe laser (wavelength 632 nm) is directed onto the prism surface by means of a vibrating mirror and that the intensity of the reflected light is monitored by a large-area photodiode. Changes in plasmon angle (∆R) can be determined with an accuracy of 0.002°. Figure 2 shows how it is possible to measure simultaneously the changes in SPR angle occurring on the receptor and reference monolayers, upon addition of a certain solution of guest, with the aid of a two-compartment cell appropriately placed on both adsorbates. Before each measurement, the two cell compartments were filled with 800 µL of water each. After stabilization of the SPR signal (30 min), 700 µL of water were removed from each compartment and replaced with 700 µL of a certain guest solution. Once the signal was recorded, before measurement of a second signal, the solution of the first guest was removed and repeated washings with water (700 µL × 3 or 4) were carried out. SPR measurements were carried out four times for each receptor-guest system. For each experiment, the following guests were employed: p-toluic acid, benzoic acid, p-nitrobenzoic acid, p-hydroxybenzoic acid, p-cresol, p-nitrophenol, and p-methoxyphenol,20 all as 10 mM aqueous solutions, except for p-toluic acid and p-nitrobenzoic acid which are only very slightly soluble in water21 and, therefore, saturated solutions of these were used (≈2 mM). Instrumentation. Grazing-angle FT-IR was performed on a Biorad FTS60A spectrophotometer at an angle of incidence of 87°, in a nitrogen-purged chamber. For each spectrum 256 scans (18) Kretschmann, E.; Reather, H. Z. Naturforsch. 1968, 23, 2135. (19) Lenferink, A. T. M.; Kooyman, R. P. H.; Greve, J. Sens. Actuators B 1991, 3, 261. (20) Measurements with toluene were also carried out but did not give reliable signals for any of the adsorbate monolayers. The intensity of the signals was high, suggesting strong interaction with the adsorbates. However, they continuously changed and the layers showed memory effect for subsequent measurements. For these reasons the results obtained with toluene are not reported in this paper. (21) Morrison, R. T.; Boyd, R. N. Organic Chemistry, 2nd ed.; Allyn and Bacon, Inc.: Boston, MA, 1967; p 579.

Cavitand Receptors for Molecular Recognition were carried out, with a 2 cm-1 resolution. Contact angle (CA) measurements were carried out on a Kru¨ss contact angle measuring system G10. Measurements for a drop of water whose volume was gradually increased (advancing CA) and then decreased (receding CA) were repeated on 3 sites of the same sample. For each receptor adsorbate, 2-4, four samples were measured for a total of 12 drops, of which the average values are reported. X-ray photoelectron spectroscopy (XPS) was performed on a VG Escalab 220i-XL with monochromatic Al KR X-ray source. Electrochemical measurements were performed with an Autolab PGSTAT10, in a homemade electrochemical cell equipped with a platinum counter electrode, a mercury sulfate reference electrode (+0.61 VNHE) and a screw cap to position the gold electrode. For capacitance measurements the cell was filled with 50 mL of a 0.1 M K2SO4 electrolyte solution. Nitrogen was bubbled through the cell for at least 5 min before each measurement. Cyclic voltammograms were recorded between -0.3 and -0.1 mV at scan rates of 0.1, 0.2, and 0.5 V/s, and the capacitance was calculated from the voltammograms recorded at 0.2 V/s, at -0.2 VMSE. The values reported are the average of measurements on 3 individual samples. Heterogeneous electron transfer (HET) and impedance measurements were carried out in the presence of 50 mL of 0.1 M K2SO4, 1 mM K3[Fe(CN)6], and 1 mM K4[Fe(CN)6] electrolyte solution. HET cyclic voltammograms were recorded between 0 and -0.7 mVMSE with a scan rate of 0.1 V/s. Resistance values were obtained by analyzing the impedance spectra measured at -0.2 mV, between 0.1 Hz and 10 kHz, with the equivalent circuit method of Boukamp.22 The system can be described by a circuit consisting of a parallel resistance (Rml) and capacitance (Cdl), in series with a second resistance (Rel), where Rel is the resistance of the electrolyte, Rml is the resistance of the monolayer, and Cdl is the capacitance of the monolayer.23,24 Synthesis. All reactions were carried out under an argon atmosphere unless stated otherwise. For flash column chromatography, Merck silica gel 60 (0.040-0.063 mm, 230-400 mesh) was used. Melting points are uncorrected. Mass (FAB) spectra were recorded using m-nitrobenzyl alcohol as a matrix. The 1H NMR (250 MHz) and 13C NMR were recorded in CDCl3, and the chemical shifts are expressed relative to residual CHCl3. Elemental analyses are given where possible. Compounds 1 and 5 were prepared according to literature procedures.25,26 Methoxy Decylene Cavitand (6). To a suspension of 5 (0.20 g, 0.17 mmol) and sodium hydride (0.08 g, 1.74 mmol) in DMF was added methyl iodide (0.09 mL, 1.39 mmol). The mixture was stirred at 70 °C for 16 h, cooled to room temperature, and quenched by addition of methanol (5 mL). Subsequently, the solvent was evaporated in vacuo and the residue was triturated with MeOH. After filtration, 6 was obtained as a white powder in 91% yield: Mp 176-177 °C; 1H NMR δ 6.78 (s, 4H, ArH), 5.84 and 4.36 (AB-q, J ) 7.5 Hz, 4H, OCH2O), 5.90-5.70 (m, 4H, RCHdCH2), 4.98 (m, 8H, RCHdCH2), 4.70 (t, J ) 7.1 Hz, 4H, ArCHAr), 3.76 (s, 12H, OCH3), 2.25-2.10 (m, 8H, RCH2CdC), 2.10-1.98 (m, 8H, RCH2CHAr2), 1.50-1.24 (m, 48H, CH2); 13C NMR δ 148.1, 145.2, 138.9, 114.2 (Ar), 99.8 (OCH2O), 61.1 (OCH3), 36.9 (ArCHAr), 33.8-27.9 (CH2); FAB-MS m/z 1208.9 M+, 1209.9 [M + H]+. Anal. Calcd for C76H104O12: C, 75.5; H, 8.7. Found: C, 75.1; H, 8.2. Methoxy Didecyl Sulfide Cavitand (2). To a solution of 6 (0.18 g, 0.15 mmol) in THF (30 mL) at 0 °C was added decanethiol (0.31 mL, 1.50 mmol) and then 9-BBN (0.20 mL of a 0.5 M solution in THF, 0.10 mmol). The reaction was stirred for 1 h, during which it was allowed to warm to room temperature and subsequently for 15 h at room temperature. The solvent was evaporated in vacuo, and the crude product was purified by flash column chromatography (SiO2, CH2Cl2-EtOAc gradient 100:0 to 0:100). After recrystallization from CH2Cl2/MeOH, (22) (a) Equivalent Circuit version 4.55, 1996, Boukamp, B. A., University of Twente, Department Chemical Technology, Enschede, The Netherlands. (b) Boukamp, B. A. Solid State Ionics 1986, 18, 136. (c) Boukamp, B. A. Solid State Ionics 1986, 20, 31. (23) Lindholm-Sethson, B. Langmuir 1996, 12, 3305. (24) Nahir, T. M.; Bowden, E. F. Electrochim. Acta 1994, 39, 2347. (25) Thoden van Velzen, E. U.; Engbersen, J. F. J.; Reinhoudt, D. N. J. Am. Chem. Soc. 1994, 116, 3597. (26) Huisman, B.-H.; Rudkevich, D. M.; van Veggel, F. C. J. M.; Reinhoudt, D. N. J. Am. Chem. Soc. 1996, 118, 3523.

Langmuir, Vol. 14, No. 19, 1998 5459 product 2 was obtained as a clear oil, in 46% yield: 1H NMR δ 6.72 (s, 4H, ArH), 5.78 and 4.20 (AB-q, J ) 7.5 Hz, 4H, OCH2O), 4.64 (t, J ) 7.1 Hz, 4H, ArCHAr), 3.70 (s, 12H, OCH3), 2.44 (t, J ) 7.1 Hz, 16H, RCH2S), 2.19-2.04 (m, 8H, RCH2CHAr2), 1.611.45 (m, 16H, CH2CH2S), 1.45-1.12 (m, 112H, CH2), 0.82 (t, J ) 6.7 Hz, 12H, CH2CH3); 13C NMR δ 148.1, 145.2, 138.9, 114.1 (Ar), 99.6 (OCH2O), 61.1 (OCH3), 36.9 (ArCHAr), 32.2-27.9 (CH2), 22.7 (CH2CH3), 14.1 (CH2CH3); FAB-MS m/z 1906.6 [M + H]+, calcd for C116H192O12S4 1905.3. Acetyl Decylene Cavitand (7). To a suspension of 5 (0.20 g, 0.17 mmol) in acetic anhydride (2 mL, 17.35 mmol) was added pyridine (0.01 mL, catalytic amount). The mixture was heated for 1 h and then quenched by carefully adding MeOH (2 mL). The solvent was evaporated in vacuo, and the crude product was purified by recrystallization from MeOH. Compound 7 was obtained as a white powder in 65% yield: Mp 210-212 °C; 1H NMR δ 7.00 (s, 4H, ArH), 5.90-5.72 (m, 4H, RCHdCH2), 5.61 and 4.46 (AB-q, J ) 7.5 Hz, 4H, OCH2O), 5.06 (m, 8H, RCHd CH2), 4.72 (t, J ) 7.1 Hz, 4H, ArCHAr), 2.19 (s, 12H, C(O)CH3), 2.28-2.12 (m, 8H, RCH2CHAr2), 2.10-1.98 (m, 8H, RCH2CdC), 1.44-1.21 (m, 48H, CH2); 13C NMR δ 168.7 (CdO), 147.0, 138.5, 132.4, 114.4 (Ar), 99.6 (OCH2O), 36.8 (ArCHAr), 32.2-29.0 (CH2), 20.4 (OC(O)CH3); FAB-MS m/z 1321.5 [M + H]+, calcd for C80H104O16 1320.7, 1343.6 [M + Na]+. Acetyl Didecyl Sulfide Cavitand (3). To a solution of 7 (0.15 g, 0.11 mmol) in THF (30 mL) at 0 °C was added decanethiol (0.24 mL, 1.14 mmol) and then 9-BBN (0.06 mL of a 0.5 M solution in THF, 0.03 mmol). The reaction was stirred for 1 h, during which it was allowed to warm to room temperature and subsequently for 23 h at room temperature. The solvent was then evaporated in vacuo, and the crude product was purified by recrystallization from MeOH. Product 7 was obtained as a white powder in 66% yield: Mp 160-162 °C; 1H NMR δ 6.95 (s, 4H, ArH), 5.58 and 4.40 (AB-q, J ) 7.5 Hz, 4H, OCH2O), 4.69 (t, J ) 7.1 Hz, 4H, ArCHAr), 2.45 (t, J ) 7.1 Hz, 16H, RCH2S), 2.19 (s, 12H, C(O)CH3), 2.23-2.10 (m, 8H, RCH2CHAr2), 1.59-1.45 (m, 16H, CH2CH2S), 1.39-1.14 (m, 112H, CH2), 0.82 (t, J ) 6.7 Hz, 12H, CH3); 13C NMR δ 168.7 (CdO), 147.0, 138.5, 132.4, 114.4 (Ar), 99.6 (OCH2O), 36.8 (ArCHAr), 32.2-29.1 (CH2), 22.7 (CH2CH3), 20.4 (OC(O)CH3), 14.2 (CH2CH3); FAB-MS m/z 2018.0 [M + H]+. Anal. Calcd for C120H192O16S4: C, 71.4; H, 9.6; S, 6.4. Found: C, 71.4; H, 9.0; S, 6.3. N,N-Diethyl((aminocarbonyl)methoxy) Decylene Cavitand (8). To a solution of 2-chloro-N,N-diethylacetamide (0.19 mL, 1.36 mmol) in DMF (10 mL) was added sodium iodide (0.20 g, 1.36 mmol), and the mixture was stirred at 80 °C for 1 h. The reaction was then allowed to cool to room temperature, and 5 (0.20 g, 0.17 mmol) was added, followed by addition of sodium hydride (0.08 g, 1.70 mmol). The mixture was then stirred at room temperature for 24 h, after which 1 M HCl (2 mL) was added to quench the reaction. The solvent was evaporated in vacuo, and the residue was dissolved in CH2Cl2 (30 mL) and washed with water (3 × 50 mL). The organic layer was dried over MgSO4 and then concentrated under reduced pressure. The crude product was purified by flash column chromatography (SiO2, hexane-EtOAc gradient 10:100 to 0:100 followed by 9:1 EtOAc-Et3N) to afford 8 as a white powder in 35% yield: Mp 150-154 °C; 1H NMR δ 6.78 (s, 4H, ArH), 5.75 and 4.44 (AB-q, J ) 7.5 Hz, 4H, OCH2O), 5.89-5.68 (m, 4H, RCHdCH2), 4.98 (m, 8H, RCHdCH2), 4.69 (t, J ) 7.1 Hz, 4H, ArCHAr), 4.57 (s, 8H, OCH2C(O)N), 3.42-3.28 (m, 16H, NCH2CH3), 2.23-2.10 (m, 8H, RCH2CdC), 2.10-1.98 (m, 8H, RCH2CHAr2), 1.48-1.23 (m, 48H, CH2), 1.23 and 1.09 (m, 24H, NCH2CH3); 13C NMR δ 165.0 (Cd O), 147.7, 145.2, 139.1, 114.4 (Ar), 99.9 (OCH2O), 72.1 (OCH2C(O)N), 41.1 and 39.3 (NCH2CH3), 36.9 (ArCHAr), 34.1 (RCH2CHAr2), 29.9-24.7 (CH2), 14.4 and 12.8 (NCH2CH3); FAB-MS m/z 1606.1 [M + H]+, calcd for C96H140N4O16 1605.0, 1629.1 [M + Na]+. N,N-Diethyl((aminocarbonyl)methoxy) Didecyl Sulfide Cavitand (4). To a solution of 8 (0.06 g, 0.04 mmol) in THF (20 mL) at 0 °C was added decanethiol (0.08 mL, 0.46 mmol) and then 9-BBN (0.20 mL of a 0.5 M solution in THF, 0.10 mmol). The reaction was stirred for 1 h, during which it was allowed to warm to room temperature and subsequently for 3 h at room temperature after which no more starting material could be detected by TLC (ethyl acetate). The solvent was then evaporated

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Scheme 1. Synthesis of Adsorbates 2-4, Starting from Tetrol 5

Table 1. Advancing (θa) and Receding (θr) Contact Angle Data for Water Drops on Monolayers of Adsorbates 1-4 and Decanethiol decanethiol 1 2 3 4

θa (deg)

θr (deg)

∆θ (deg)

108 ( 1 107 ( 1 98 ( 2 109 ( 2 107 ( 3

91 ( 1 91 ( 1 68 ( 2 85 ( 1 88 ( 3

17 16 30 24 19

in vacuo, and the crude product was purified by preparative thinlayer chromatography (SiO2, EtOAc). After crystallization from CH2Cl2/MeOH, product 4 was obtained as a white powder in 90% yield: Mp 65-69 °C; 1H NMR δ 6.74 (s, 4H, ArH), 5.68 and 4.38 (AB-q, J ) 7.5 Hz, 4H, OCH2O), 4.63 (t, J ) 7.1 Hz, 4H, ArCHAr), 4.50 (s, 8H, OCH2C(O)N), 3.39-3.25 (m, 16H, NCH2CH3), 2.43 (t, J ) 7.1 Hz, 16H, RCH2S), 2.18-2.01 (m, 8H, RCH2CHAr2), 1.59-1.41 (m, 16H, CH2CH2S), 1.41-1.16 (m, 112H, CH2), 1.16-1.01 (m, 24H, NCH2CH3), 0.82 (t, J ) 6.7 Hz, 12H, CH2CH3); 13C NMR δ 165.0 (CdO), 147.7, 145.2, 138.8, 114.4 (Ar), 99.9 (OCH2O), 72.1 (OCH2C(O)N), 41.1 and 39.3 (NCH2CH3), 36.9 (ArCHAr), 34.1 (RCH2CHAr2), 32.2-29.1 (CH2), 22.7 (CH2CH3), 14.4 and 12.8 (NCH2CH3), 14.2 (CH2CH3); FAB-MS m/z 2326.1 [M + Na + H]+. Anal. Calcd for C136H228N4O16S4: C, 70.9; H, 10.0; N, 2.4; S, 5.6. Found: C, 70.7; H, 9.6; N, 2.2; S, 5.6.

Table 2. Capacitance and Resistance Values for Monolayers of Compounds 1-4 and Decanethiol 1 2 3 4 decanethiol

C (µF‚cm-2)a

R (106Ω)

1.14 1.27 2.23 1.63 1.24

0.38 2.14 0.34 1.08 0.80

a All capacitance values are the average of 3 measurements on 3 individual layers. The error on the values is within 10%.

Figure 3. Heterogeneous cyclic voltammograms of monolayers of adsorbates 1-4.

Synthesis. Receptor adsorbates 2-4 were synthesized starting from tetrol 5. The synthetic procedures are depicted in Scheme 1. Compound 6 was obtained by alkylation of 5 with methyl iodide in the presence of sodium hydride as a base. After trituration with methanol, 6 was obtained in very high yield. Compound 7 was synthesized by acylation of 5 with acetic anhydride, in the presence of a catalytic amount of pyridine and subsequently purified by recrystallization from methanol. Alkylation of 5, at room temperature, with 2-chloro-N,N-diethylacetamide in the presence of potassium iodide produced compound 7 in rather low yield.27 Finally, compounds 2-4 were obtained in medium to high yields by addition of decanethiol to compounds 6-8, using 9-BBN as radical initiator. Monolayer Characterization. Monolayers of receptor adsorbates 2-4 were prepared and characterized by wettability measurements, cyclic voltammetry and impedance spectroscopy, grazing angle FT-IR, and X-ray photoelectron spectroscopy (XPS). Contact angle results (Table 1) show that all monolayers have a relatively hydrophobic interface. An evaluation of the packing of the molecules in the monolayers can be made by consid-

ering the hysteresis values (∆θ).28 Decanethiol and adsorbate 1 monolayers, with ∆θ values of around 17, are relatively well ordered, while monolayers of compounds 2-4 have higher hysteresis values and are therefore somewhat less well ordered. Capacitance measurements performed on layers 2 and 4 are in the same range as the values reported for 1 and decanethiol layers, while 3 gives a slightly higher value indicating that a monolayer of the latter has relatively more defects than the others (Table 2). Figure 3 shows that all four layers are able to block the Fe(CN)64-/Fe(CN)63- redox couple, but 3 presents a slightly lower resistance. Therefore, these types of substituents on the upper rim of cavitands seem to interfere only slightly with the overall permeability and packing of the molecules in the monolayers. Grazing angle FT-IR spectra of monolayers of 3 and 4 are very similar whereas that of 2 shows slight differences (Table 3). In the spectra of 3 and 4 the C-H stretching vibrations can be easily observed, but the intensity of the ester and amide bands is too low to be detected. The CdO vibrations may not be observed in the spectrum due to a parallel orientation of the transition dipole moments of the carbonyls with respect to the gold substrate. Moreover,

(27) After purification by flash column chromatography, trace amounts of trialkylated product were also isolated.

(28) Pale-Grosdemange, C.; Simon, E. S.; Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1991, 113, 12.

Results and Discussion

Cavitand Receptors for Molecular Recognition

Langmuir, Vol. 14, No. 19, 1998 5461

Table 3. Grazing Angle FT-IR Data for Monolayers of Adsorbates 2-4 νas(CH3) νas(CH2) νs(CH3) νs(CH2) ν(C-Harom) 2

2959

2924

2874

2855

3 4

2964 2964

2921 2921

2879 2877

2850 2851

3005

δ(CH2) 1474, 1456, 1423

residual water adsorption in this region sometimes also causes difficulties in the identification of the peaks, due to overlap. In the spectrum of 2, apart from the C-H methyl and methylene stretching vibrations, a small peak due to C-H aromatic stretching vibrations can be observed at 3005 cm-1. In the region between 1420 and 1480 cm-1, C-H bending vibrations can also be clearly observed. XPS spectroscopy confirmed the presence of all the elements in the adsorbates, although the intensity of the nitrogen, for adsorbate 4, was low. Surface Plasmon Resonance. The interaction of several “small” molecules (MW < 200 Da) in aqueous solutions29 with the receptor adsorbates 1-4 was studied by surface plasmon resonance. SPR measurements performed with the setup described in the Experimental Procedures allow the detection of refractive index changes in the medium present at the monolayer interface, with a penetration depth of the evanescent field on the order of the wavelength employed.30 Therefore the signals obtained are due not only to the interaction of molecules with the monolayer but also to the presence of molecules dissolved in the medium (subsequently referred to as “bulk”). To account for the latter and simply for comparison purposes, a monolayer of octadecanethiol was used as a reference. Octadecanethiol was chosen because it forms a well-packed layer that contains no specific recognition sites and will interact with guests in water, mainly through hydrophobic forces.31 Each measurement was performed simultaneously on the reference layer and the receptor layer. Employing the same gold substrate for reference and receptor monolayers and measuring simultaneously ensures more reliable results because slight differences in the gold substrates can cause small differences in the intensities of the signals. Typical signals of an SPR experiment can be seen in Figure 4. The responses obtained upon addition of p-toluic acid, benzoic acid, p-nitrobenzoic acid, and p-hydroxybenzoic acid on receptor layer 1 (Figure 4a, top) and on the reference layer (Figure 4a, middle) are presented. Figure 4b shows the differences between the changes in SPR angle of the receptor layer (∆RA) and those of the reference layer (∆Rref). These differences (∆Rdiff) are also presented numerically in Table 4, along with the acidity constants (pKa) of the guests and their relative solubility in water.32 The positive values in the table indicate that the guests interact preferably with the receptor layer compared to the reference layer. A zero value represents (29) The solutions were not buffered because, after some initial measurements carried out with aqueous solutions of guests, no dependence of the acidity of the solutions on the interactions with receptors 1-4 could be detected. (30) It is beyond the scope of this paper to discuss the principles of SPR; for detailed studies, see: Mittler-Neher, S.; Spinke, J.; Liley, M.; Nelles, G.; Weisser, M.; Back, R.; Wenz, G.; Knoll, W. Biosens. Bioelectron. 1995, 10, 903. (31) For the comparison of the results obtained on the receptor layer, other reference layers could have been used. Different types of reference layers, for example hydroxyl-terminated and perfluorinated alkanethiol monolayers, are currently being studied to try to determine in which case the interactions with the guests are minimal. (32) From: Atlas of Spectral Data and Physical Constants for Organic Compounds; Grasselli, J. G., Ed.; CRC Press: Cleveland, OH, 1973. Key: 1, insoluble in water; 2, slightly soluble; 3, soluble.

Figure 4. (a) SPR responses obtained upon addition of p-toluic acid, benzoic acid, p-nitrobenzoic acid, and p-hydroxybenzoic acid on adsorbate layer 1 and on the octadecanethiol reference layer. (b) Difference (∆Rdiff) between the adsorbate (∆RA) and reference (∆Rref) signals. Typical responses to successive washings are indicated by first, second, and third, respectively. Asterisk indicates units of degrees.

the same degree of interaction with the two layers, and the negative values are due to stronger interactions of the guests with the reference layer than with the receptor layer. The errors shown for each measurement are mostly due to imprecisions arising from repeated additions of guest solutions in the measuring cell, followed by successive washings with water. Each guest showed fast response times (typically t90% < 1 s), and after an initial equilibration of the system, stable signals were obtained just after a few seconds. All signals were completely reversible, and the layers displayed no memory effect with any of the guests. Considering the values presented in Table 4, all of the guests except p-methoxyphenol show much greater interaction with adsorbate 1 than with the reference layer. Adsorbate 2 shows preferential interaction only with p-nitrobenzoic acid and p-nitrophenol; the other guests give ∆R’s similar to or lower than those measured on the reference layer. Adsorbate 3 shows moderately higher interactions with p-toluic acid and p-methoxyphenol, while for adsorbate 4 significant ∆R’s can be noted for p-nitrobenzoic acid and p-nitrophenol. Considering the responses obtained for each guest on the different adsorbates, it can be seen that, for example, p-toluic acid interacts more strongly with adsorbates 1 and 3, benzoic acid with adsorbate 1, and p-nitrobenzoic acid with all adsorbates except 3. A first observation that can be made regarding these results is that the introduction of relatively small functional groups on the upper rim of cavitand adsorbates induces selectivity for a particular guest. Most guests interact with the nonfunctionalized adsorbate 1, while only a few guests interact with the functionalized adsorbates. A second important observation is that the more

5462 Langmuir, Vol. 14, No. 19, 1998

Friggeri et al.

Table 4. SPR Angle Differences (∆rdiff) between SPR Angle Change on Adsorbates 1-4 (∆rA) and on Octadecanethiol Reference Layer (∆rref) upon Addition of Aqueous Solutions of Guests (p-Toluic Acid, Benzoic Acid, p-Nitrobenzoic Acid, p-Hydroxybenzoic Acid, p-Cresol, p-Nitrophenol, p-Methoxyphenol) ∆RA - ∆Rref ) ∆Rdiff (10-2 deg)b guest

solubilitya

1

4.36

1

6.3 ( 2.3

4.20

2

12.0 ( 2.0

3.41

1

8.8 ( 2.3

4.48

2

3.5 ( 0.5

10.17

2

4.3 ( 1.8

7.15

3

10.0 ( 2.0

pKa

>10

a

3

Key: 1, insoluble in water; 2, slightly soluble; 3, soluble.32

0 b

hydrophobic guests are not necessarily the ones that interact most significantly with the receptor adsorbates, nor does acidity determine the extent of receptor-guest interaction. Moreover, p-nitrobenzoic acid and p-nitrophenol give quite high ∆R’s with 1, 2, and 4, indicating that there is preferential interaction with guests containing nitro groups. However, there are no simple trends that can describe these results. It is important to note that the dominating factor determining the selectivity in the host-guest interactions measured is not hydrophobicity and that therefore other weak attractive forces such as dispersive interactions and hydrogen bonding also contribute to the association process.33,34 The changes in SPR angle measured for p-nitrophenol with adsorbates 1, 2, and 4 are quite large, considering the size of the guests. This suggests that there might be formation of multilayers of guest on the surface of these adsorbates. This prompted us to study the association process as a function of the concentration, and adsorbate 1 was chosen for the purpose.35 Other receptor-guest systems were not studied due to the poor solubility of the guests or to their low SPR responses. In Figure 5, it can be seen that the responses of both the receptor layer and the reference layer remain linear as the concentration of guest increases. However, the receptor curve has a steeper slope than the reference curve, indicating that p-nitrophenol has a higher affinity for adsorbate 1 than for the reference layer.36 Assuming that only one molecule of guest binds to one receptor molecule and that the refractive index of the monolayers is 1.45, a ∆R of ≈0.03-0.05° should be observed.37,38 However, the ∆R obtained for a 3 mM solution of guest, for example, is 0.24-0.25° (Figure 5). Therefore, ∆R ≈ 0.03° for a 1:1 binding implies that approximately eight guest molecules must be stacked or clustered on each receptor molecule. Assuming cofacial adsorption of the guests, only two guest molecules cover (33) Lemieux, R. U. Acc. Chem. Res. 1996, 29, 373. (34) Constantinos, P. M.; Tsiourvas, D. Adv. Mater. 1997, 9, 695. (35) Concentration dependence experiments were not carried out with adsorbates 2-4 because there was no reason to believe that these receptor layers would behave in a fundamentally different way from adsorbate 1. (36) At higher guest concentrations than those depicted in Figure 5, receptor curve and reference curve continue to diverge. (37) Kooyman, R. P. H.; Lechuga, L. M. Handbook of Biosensors and Electronic Noses; CRC Press: Boca Raton, FL, 1997; p 169. (38) Stenberg, E.; Persson, B.; Roos, H.; Urbaniczky, C. J. Colloid Interface Sci. 1991, 143, 513.

2

3

4

5.0 ( 1.0

0

-6.0 ( 2.0

0

0

10.0 ( 2.0

0

7.0 ( 2.0

0

0

-8.3 ( 2.3

0

0

0

13.0 ( 2.0

0

-4.0 ( 2.0

3.0 ( 2.0

0 10.0 ( 2.0 0

The values presented are the average of 4 independent measurements.

Figure 5. SPR angle changes on receptor layer 1 and on the reference layer, as a function of the concentration of pnitrophenol.

the area of one receptor molecule, thus accounting for the presence of four layers of guests on the receptor surface. However, if the guests adsorb perpendicularly to the receptor surface, four guest molecules can align on each adsorbate molecule; hence, two layers of guests are formed. Therefore, depending on the mode of adsorption, 2-4 layers of guests will be present on the receptor surface. Taking ∆R ≈ 0.05° implies that approximately five guest molecules interact with each adsorbate molecule. Therefore 1-2 layers of guests are formed, depending on the cofacial or perpendicular adsorption of the guests on the receptor surface. It seems highly probable that the formation of a first layer of p-nitrophenol on adsorbate 1 induces the growth of another 2-4 layers. As the concentration of guest increases, the number of layers also increases, and this gives rise to the linear dependence shown in Figure 5. Conclusions In summary, monolayers of compounds 2-4 have been prepared. Characterization of these layers by CA measurements, electrochemical techniques, and grazing angle

Cavitand Receptors for Molecular Recognition

FT-IR showed that they are hydrophobic and relatively well-ordered in comparison to a decanethiol monolayer. With XPS, presence of the receptor molecules on the gold surface was confirmed. It has been shown that it is possible to detect with surface plasmon resonance molecular interactions at the monolayer-water interface between a monolayer of cavitand receptor molecules and small aromatic guests. Moreover, upper-rim functionalized cavitand adsorbates 2-4 induce selectivity in the receptor-guest recognition process. It was found that the association between each adsorbate-guest system measured is specific and is not determined exclusively by

Langmuir, Vol. 14, No. 19, 1998 5463

the hydrophobic character of the receptor surfaces and/or the guests. Other attractive forces, such as dispersion forces and hydrogen bonding, must be contributing to the recognition process. Furthermore, the relatively large changes in SPR angles observed for some adsorbate-guest systems indicate that there is formation of clusters or multilayers of guests on such adsorbate layers. Acknowledgment. We thank Dr. P. J. de Lange (Akzo-Nobel Central Research) for XPS measurements. LA980409Q