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Accumulation of Functional Block Telomers on Metal Surfaces† Hiromi Kitano* and Kazuhiko Ohhori Department of Chemical and Biochemical Engineering, Toyama University, Toyama, 930-8555 Japan Received June 21, 2000. In Final Form: December 21, 2000 A disulfide-group-carrying novel iniferter (a compound which pursues initiation, chain transfer, and termination) (Cys-BDC) was prepared by coupling N,N-diethyldithiocarbamoylmethylbenzoic acid succinimidyl ester with cystamine dihydrochloride. Block copolymerization of methacrylic acid (MA) and 2-methacryloyloxyethyl D-glucopyranoside (MEGlc) was performed by successive UV-irradiation in the presence of the iniferter and N,N,N′,N′-tetraethylthiuram disulfide. The block telomer formed a selfassembled monolayer (SAM) on a gold electrode and vacuum-evaporated gold thin film, as confirmed by cyclic voltammetry and infrared reflection-absorption spectroscopy (IRRAS), respectively. The pHresponsiveness of the MA telomer block on the gold surface and silver colloid was confirmed by contact angle and ζ-potential measurements, respectively. Recognition of glucose residues in the MEGlc block by a lectin (Concanavalin A) was followed on the gold surface and silver colloid by IRRAS and turbidity measurements, respectively. Our results strongly support the usefulness of the iniferter for the preparation of various block-telomer-carrying SAMs with biorelated functions.
Introduction Block copolymers and telomers can often display multiple functions corresponding to each constituent block, and in some cases “synergism” of individual block units can be expected. Therefore, introduction of block copolymers is quite effective in expanding the usability of any materials. Polymeric compounds that can initiate the polymerization reaction (macroinitiators) have often been used as a convenient method to prepare block copolymers.1 Latex particles, for example, were prepared by emulsion polymerization of styrene using poly(ethylene oxide) azoinitiators.2 Recently, we examined the preparation of temperature-responsive polymer microspheres and linear block copolymers with a macroinitiator carrying poly(Nisopropylacrylamide) (PIPA) chains at its ends.3 The poly(2-methacryloyloxyethyl D-glucopyranoside) (PMEGlc)chain-carrying microspheres and linear block polymers were also prepared using the macroinitiator method.4 A controlled (or living) radical polymerization has been developed over the past decade to obtain well-defined polymers.5-7 Of various procedures for pursuing controlled * To whom correspondence should be addressed. E-mail: kitano@ eng.toyama-u.ac.jp. Tel: +81-76-445-6868. Fax: +81-76-445-6703. † Presented at the 49th Annual Meeting of the Society of Polymer Science, Nagoya, Japan, May 2000. (1) (a) Quirk, R. P.; Kim, J. In Ring Opening Polymerization; Brunelle, D. J., Ed.; Hanser: Munich, 1995; p 263. (b) Wang, Y.; Chen, S.; Huang, J. Macromolecules 1999, 32, 2480. (c) Weberskirch, R.; Hettich, R.; Void, B. Macromol. Chem. Phys. 1999, 200, 863. (d) Kops, J.; Ivan, B.; Batsberg, W. Macromol. Rapid Commun. 1998, 19, 15. (e) Zhang, S.; Hou, Z.; Gonsalves, K. E. J. Polym. Sci., Part A: Polym. Chem. 1996, 34, 2737. (2) Tauer, K. Polym. Adv. Technol. 1995, 6, 435. (3) (a) Kitano, H.; Kawabata, J. Macromol. Chem. Phys. 1996, 197, 1721. (b) Kitano, H.; Fukui, N.; Ohhori, K.; Maehara, Y.; Kokado, N.; Yoshizumi, A. J. Colloid Interface Sci. 1999, 212, 58. (4) (a) Kitano, H.; Kawabata, J.; Muramoto, T. Macromol. Chem. Phys. 1996, 197, 3657. (b) Kitano, H.; Maehara, Y.; Sugimura, M.; Matano, M.; Shigemori, K. Langmuir 1997, 13, 5041. (5) (a) Veregin, R. P. N.; Georges, M. K.; Kazmaier, P. M.; Hamer, G. K. Macromolecules 1993, 26, 5316. (b) Hawker, C. J.; Elce, E.; Dao, J.; Volksen, W.; Russel, T. P.; Barclay, G. G. Macromolecules 1996, 29, 2686. (c) Fukuda, T.; Terauchi, T.; Goto, A.; Tsujii, Y.; Miyamoto, T. Macromolecules 1996, 29, 3050. (d) Baethge, H.; Butz, S.; SchmidtNaake, G. Macromol. Rapid Commun. 1997, 18, 911.
radical polymerization, an iniferter (a compound which pursues initiation, chain transfer, and termination) has received much attention because of its ease of use.8-10 Of the iniferters used, N,N-diethyldithiocarbamoyl derivatives have been most extensively studied.8,10 Previously, for example, we synthesized a phospholipid carrying a benzyl-N,N-diethyldithiocarbamoyl (BDC) group as the iniferter and prepared novel block-telomer-carrying phospholipids by successive photoirradiation of various vinyl monomers in the presence of the iniferter.11 Organo-sulfur compounds such as alkyl or aromatic thiols and disulfides form a close-packed ordered monolayer, or self-assembled monolayer (SAM), on gold or silver surfaces via chemisorptive S-Au or S-Ag bonds.12 Recently, SAMs of alkanethiols and disulfides have been used as cell membrane mimetics because of their structural analogy to biomembranes,13 ease of preparation, and apparent stability.14 For example, a SAM of ω-mercaptoalkanoic acid15 and that of a ω-mercapto-polymer chain with many pendent glucose residues16 were constructed on silver colloids to analyze the interaction between proteins and biomembranes. In addition, a SAM of (6) (a) Wang, J.-S.; Matyjaszewski, K. J. Am. Chem. Soc. 1995, 117, 5614. (b) Matyjaszewski, K.; Wang, J.-S. Macromolecules 1995, 28, 7901. (c) Matyjaszewski, K.; Patten, T.; Xia, J.; Abernathy, T. Science 1996, 272, 866. (d) Matyjaszewski, K.; Nakagawa, Y.; Gaynor, S. G. Macromol. Rapid Commun. 1997, 18, 1057. (7) (a) Sawamoto, M.; Kamigaito, M. CHEMTECH 1999, 29, 30. (b) Sawamoto, M.; Kamigaito, M. In Synthesis of Polymers (Material Science and Technology Series); Schlu¨ter, A.-D., Ed.; Wiley-VCH: Weinheim, Germany, 1999; Chapter 6. (c) Senoo, M.; Kotani, Y.; Kamigaito, M.; Sawamoto, M. Macromolecules 1999, 32, 8005. (8) (a) Otsu, T.; Yoshida, M. Makromol. Chem., Rapid Commun. 1982, 3, 127, 133. (b) Otsu, T.; Yamashita, K.; Tsuda, K. Macromolecules 1986, 19, 287. (c) Otsu, T.; Matsunaga, T.; Kuriyama, A.; Yoshioka, M. Eur. Polym. J. 1989, 25, 643. (d) Doi, T.; Matsumoto, A.; Otsu, T. J. Polym. Sci., Polym. Chem. Ed. 1994, 32, 2911. (e) Hadziioannou, G.; Kroeze, E.; de Boer, B. Macromolecules 1996, 29, 8599. (9) Okawara, M.; Nakai, T.; Morishita, K.; Imoto, E. Kogyo Kagaku Zasshi 1964, 67, 2108. (10) (a) Nakayama, Y.; Matsuda, T. Macromolecules 1996, 29, 8622. (b) Nakayama, Y.; Takatsuka, M.; Matsuda, T. Langmuir 1999, 15, 1667. (c) Lee, J. H.; Nakayama, Y.; Matsuda, T. Macromolecules 1999, 32, 6989. (11) Kitano, H.; Chibashi, M.; Nakamata, S.; Ide, M. Langmuir 1999, 15, 2709.
10.1021/la000869v CCC: $20.00 © 2001 American Chemical Society Published on Web 02/14/2001
Functional Block Telomers on Metal Surfaces
cyclodextrin derivatives was used as a sensitive and specific sensing device because of the molecular recognition properties of cyclodextrin.17 In this study, we prepared a block telomer with many pendent carboxyl and sugar groups using the iniferter method and accumulated the block telomer on Au or Ag surfaces as a SAM. The responsiveness of the block telomer to an external stimulus (pH) and recognition processes of sugar residues in the telomer by a sugar-binding protein were examined on the metal surfaces.
Langmuir, Vol. 17, No. 6, 2001 1879 Scheme 1. Preparation of Block Telomers Using the Iniferter Method
Experimental Section Materials. Concanavalin A (Con A, from Canavalia ensiformis) was purchased from Sigma (St. Louis, MO). 2-Methacryloyloxyethyl D-glucopyranoside (MEGlc, mixture of R and β anomers (2.3:1)) was kindly donated by Nippon Fine Chemicals (Osaka, Japan).4,11,16,18 Poly(2-methacryloyloxyethyl D-glucopyranoside) with a disulfide group at the center of the molecule (DTPA-PMEGlc) was prepared using dithiodipropionic acid dip-nitrophenyl ester (DTPA-ONp) and poly(2-methacryloyloxyethyl D-glucopyranoside) with an amino group at its end (Mn ) 6.8 × 103, Mw/Mn ) 1.76) as described previously.16 Methacrylic acid from Wako Pure Chemicals (Osaka) was distilled in vacuo. Other reagents were obtained from commercial sources. Deionized water was distilled just prior to use for preparation of sample solutions. Preparation of Iniferter (Scheme 1).11 4-(N,N-Diethyldithiocarbamoylmethyl) benzoic acid (BDC) was prepared by the coupling of N,N-diethyldithiocarbamide sodium salt (2.01 g) and 4-chloromethylbenzoic acid (1.30 g) in dry acetone (140 mL) in the dark at 0 °C for 1 h and at room temperature for 23 h. The resultant white precipitates were filtrated and dissolved in chloroform. The chloroform solution was repeatedly washed with 3% (w/w) aqueous citric acid solution and with cold water. The chloroform phase was dehydrated with anhydrous sodium sulfate (12) (a) Nuzzo, R. G.; Allara, D. L. J. Am. Chem. Soc. 1983, 105, 4481. (b) Nuzzo, R. G.; Fusco, F. A.; Allara, D. L. J. Am. Chem. Soc. 1987, 109, 2358. (c) Porter, M. D.; Bright, T. B.; Allara, D. L.; Chidsey, C. E. D. J. Am. Chem. Soc. 1987, 109, 3559. (d) Bain, C. D.; Troughton, E. B.; Tao, Y.-T.; Evall, J.; Whitesides, G. M.; Nuzzo, R. G. J. Am. Chem. Soc. 1989, 111, 321. (e) Bain, C. D.; Whitesides, G. M. Adv. Mater. 1989, 1, 506. (f) Nuzzo, R. G.; Dubois, L. H.; Allara, D. L. J. Am. Chem. Soc. 1990, 112, 558. (g) Chidsey, C. E. D.; Loiacono, D. N. Langmuir 1990, 6, 682. (h) Laibinis, P. E.; Whitesides, G. M.; Allara, D. L.; Yu-Tai Tao.; Parkih, A. N.; Nuzzo, R. G. J. Am. Chem. Soc. 1991, 113, 7152. (i) Dubois, L. H.; Nuzzo, R. G. Annu. Rev. Phys. Chem. 1992, 43, 437. (j) Bain, C. D.; Whitesides, G. M. Adv. Mater. 1989, 1, 506. (k) Hill, W.; Wehling, B. J. Phys. Chem. 1993, 97, 9451. (13) (a) Garrett, R. H.; Grisham, C. M. Biochemistry; Saunders College Publishing: Fort Worth, 1995. (b) Gennis, R. B. Biomembranes: Molecular Structure and Function; Springer-Verlag: New York, 1989. (c) Voet, D.; Voet, J. G. Biochemistry, 2nd ed.; John Wiley & Sons: New York, 1995. (14) (a) Spinke, J.; Lileym, M.; Gunder, H.-J.; Angermaier, L.; Knoll, W. Langmuir 1993, 9, 1821. (b) Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 10714. (c) Lopez, G. P.; Albers, M. W.; Schreiber, S. L.; Carroll, R.; Peralta, E.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 5877. (d) Scho¨nher, H.; Vancso, G. J.; Huisman, B.-H.; van Veggel, F. C. J. M.; Reinhoudt, D. N. Langmuir 1997, 13, 1567. (e) Schierbaum, K.-D.; Weiss, T.; van Velzen, E. U. T.; Engbersen, J. F. J.; Reinhoudt, D. N.; Go¨pel, W. Science 1994, 265, 1413. (f) Huisman, B.-H.; Kooyman, R. P. H.; van Veggel, F. C. J. M.; Reinhoudt, D. N. Adv. Mater. 1996, 8, 561. (g) Jeon, N. L.; Finnie, K.; Branshaw, K.; Nuzzo, R. G. Langmuir 1997, 13, 3382. (h) Flink, S.; Boukamp, B. A.; van den Berg, A.; van Veggel, F. C. J. M.; Reinhoudt, D. N. J. Am. Chem. Soc. 1998, 120, 4652. (i) Beulen, M. J.; Kastenberg, M. I.; van Veggel, F. C. J. M.; Reinhoudt, D. N. Langmuir 1998, 14, 7463. (j) Gorman, C. B.; Miller, R. L.; Chen, K.-Y.; Bishop, A. R.; Haasch, R. T.; Nuzzo, R. G. Langmuir 1998, 14, 3312. (15) Maeda, Y.; Yamamoto, H.; Kitano, H. J. Phys. Chem. 1995, 99, 4837. (16) Yoshizumi, A.; Kanayama, N.; Maehara, Y.; Ide, M.; Kitano, H. Langmuir 1999, 15, 482. (17) (a) Maeda, Y.; Kitano, H. J. Phys. Chem. 1995, 99, 487. (b) Yamamoto, H.; Maeda, Y.; Kitano, H. J. Phys. Chem. B 1997, 101, 6855. (c) Maeda, Y.; Fukuda, T.; Yamamoto, H.; Kitano, H. Langmuir 1997, 13, 4187. (d) Fukuda, T.; Maeda, Y.; Kitano, H. Langmuir 1999, 15, 1887. (e) Kitano, H.; Taira, Y.; Yamamoto, H. Anal. Chem. 2000, 72, 2926. (18) Kitano, H.; Ohno, K. Langmuir 1994, 10, 4131.
and finally evaporated (BDC, 1.13 g, 51.5% yield). Anal. Calcd for C13H17O2NS2: C, 55.10; H, 6.05; N, 4.94. Found: C, 54.80; H, 5.86; N, 4.90. The obtained BDC (0.65 g) was coupled with N-hydroxysuccinimide (0.28 g) in the presence of N,N′-dicyclohexylcarbodiimide (DCC, 0.53 g) in dry CHCl3 (128 mL) in the dark at 0 °C for 1 h and at room temperature for 23 h and purified by silica gel chromatography with a chloroform-acetone mixture (9:1) as eluent (BDC-OSu, 0.53 g, 61.4% yield). Anal. Calcd for C17H20O4N2S2: C, 53.81; H, 5.30; N, 7.36. Found: C, 53.27; H, 5.23; N, 7.34. A mixture of cystamine dihydrochloride (0.061 g) and triethylamine (0.12 mL) dissolved in dry DMSO (2 mL) was added slowly to a BDC-OSu (0.290 g) solution dissolved in dry CHCl3 (6 mL), and the solution mixture was incubated at room temperature for 24 h. After evaporation, the product was purified by precipitation in CH3CN (Cys-BDC, 0.14 g, 73.4% yield). Anal. Calcd for C30H42O2N4S6: C, 52.75; H, 6.20; N, 8.20. Found: C, 52.88; H, 6.19; N, 8.23. 1H NMR (400 MHz, chloroform-d): δ 7.77 (d, 4H, -CH)CH-), 7.45 (d, 4H, -CH)CH-), 6.91 (t, 2H, -NH-), 4.60 (s, 4H, -C(dS)-S-CH2-), 4.04 (q, 4H, -C(dS)N-CH2-), 3.79 (q, 4H, -C(dO)NH-CH2-), 3.73 (q, 4H, -C(dS)-N-CH2-), 2.98 (t, 4H, -S-S-CH2-), 1.28 (t, 12H, -CH3). Telomerization of Methacrylic Acid (MA) and 2-Methacryloyloxyethyl D-Glucopyranoside (MEGlc) (Scheme 1 and Table 1). Cys-BDC (17.3 mg), MA (0.84 mL), and N,N,N′,N′tetraethylthiuram disulfide (TD, 15.3 mg, capping reagent)8 were dissolved in dry tetrahydrofuran (THF, 3.2 mL) and, after passing through N2 gas for several minutes, photoirradiated in a thermostated quartz cell (light path, 5 cm; volume, 5 mL) at 25 °C for 5 h with a high-pressure mercury lamp (UI-501C, 250 W, Ushio, Tokyo, Japan) at a distance of 25 cm (3.0 × 1014 quanta/s). After evaporation, the oily mixture was dried in vacuo and dissolved in water. After filtration (filter paper No. 2, Advantec Toyo, Tokyo) to remove unreacted Cys-BDC and TD, the filtrate was dialyzed against water (cellulose ester dialysis membrane; molecular weight cutoff, 103; Spectrum, Laguna Hills, CA) for several days in the dark and finally lyophilized in the dark (CysBDC-PMA, telomer 1, 0.17 g). 2-Methacryloyloxyethyl D-glucopyranoside (MEGlc, 50% (w/w) aqueous solution, 0.44 mL), telomer 1 (31.0 mg), and TD (4.6 mg) were dissolved in THF (3.5 mL) and poured into the thermostated quartz cell. After passing through N2 gas for several minutes, the solution mixture was photoirradiated at 25 °C for 10 min with the high-pressure mercury lamp. The reaction mixture was evaporated and dissolved in water. After filtration with filter paper to remove unreacted TD, the filtrate was ultrafiltrated for several days (membrane, Amicon YM10;
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Table 1. Characteristics of Prepared Telomers telomer telomer 1 telomer 2 telomer 3 b
Cys-BDC (mg) 17.3 31.0b 8.5
solvent (mL) 3.2 3.5 2.5
monomer (mL) 0.84 (MA) 0.44 (MEGlc)c 1.5 (MEGlc)c
irradiation time (min) 300 10 30
yield (g)
Mw (DP)a
Mw/Mn
0.17 0.075 0.42
6.3 × (7.5) 4.0 × 103 (13.7) 5.0 × 104 (171)
1.09 1.44 1.32
102
a Molecular weight and degree of polymerization of each telomer chain. The total M of the telomer prepared was twice this value. w Cys-BDC-PMA (telomer 1). c 50% (w/w) aqueous solution.
Scheme 2. Deposition of Block Telomer on Metal Surfaces
exclusion limit for globular protein, 104) and finally lyophilized (Cys-BDC-PMA-PMEGlc, telomer 2, 0.075 g). Similarly, CysBDC (8.5 mg) was photoirradiated with MEGlc (1.5 mL) and TD (7.5 mg) in THF (2.5 mL) at 25 °C for 30 min. The PMEGlctelomer-carrying disulfide was purified by ultrafiltration (membrane, Amicon YM1; exclusion limit, 103) and finally lyophilized (Cys-BDC-PMEGlc, telomer 3, 0.42 g). Molecular weight of the telomers was determined by GPC (telomers 1, 2, and 3; Waters HPLC System; column, Wako Beads G-30; mobile phase, 0.1 M NaBr; standard samples, pullulan from Showa Denko, Tokyo) and a matrix-assisted laser desorption-ionization time-of-flight mass spectrometer (telomer 1; MALDI-TOF, Voyager RP, PerSeptive Biosystems). Deposition of Telomer on the Gold Surface (Scheme 2a). The telomer was dissolved in water (3.7, 10.2, and 10.3 mg/mL for telomers 1, 2, and 3, respectively) and incubated with a gold electrode (AUE 6.0 × 1.6 mm; BAS, Tokyo) for 24 h at room temperature. Cyclic voltammetric (CV) measurements of the gold electrode were performed with a potentiostat (HA-301, HokutoDenko, Tokyo) and function generator (HA-104, Hokuto-Denko). Outputs of the potentiostat were digitized by using an analogto-digital converter and stored on a microcomputer (PC-486 SE, Epson, Suwa, Japan). Data analyses were performed with custommade application software. Gold and Pt electrodes and a KClsaturated calomel electrode (SCE) were used as working, counter, and reference electrodes, respectively. The cyclic voltammogram of the electrode was measured with a solution of potassium ferricyanide (K3[Fe(CN)6], 5 mM) as a probe. Preparation of Ag Colloids Covered with SAM of Telomer (Scheme 2b). A dispersion of silver colloid was prepared by the reduction of AgNO3 (2.3 mg) with NaBH4 (10.2 mg) in water (100 mL) at 0 °C as reported previously.17b The average hydrodynamic diameter (dhd) of the silver colloid was estimated as 29.1 ( 4.5 nm by the dynamic light scattering (DLS) technique (DLS-7000, Otsuka Electronics, Hirakata, Osaka; light source, He-Ne laser, 632.8 nm). The concentration of silver colloid
was evaluated as 326 µM by conductometric titration with a N/1000 HNO3 aqueous solution. The dispersion of silver colloid (15 mL) was incubated with telomer 1 (0.37 mg/mL water), telomer 2 (1.0 mg/mL water), or DTPA-PMEGlc (1.4 mg/mL water) for 24 h at room temperature and ultrafiltrated with an Amicon membrane (YM-10, telomer 1; YM-100 (molecular cutoff ) 105), telomer 2 and DTPA-PMEGlc). Modification with the telomer chain resulted in a red shift of the absorption peak of the silver colloid (absorption peak of bare and three types of SAM-carrying silver colloids (telomer 1-Ag, telomer 2-Ag, and DTPA-PMEGlc-Ag); 392, 411, 406, and 409 nm, respectively; Ubest-35 UV-visible spectrophotometer, Japan Spectroscopic Co., Tokyo). Radical Production on Photoirradiation of BDC Group. A mixture of 1,1′-diphenyl-2-picrylhydrazyl (DPPH, 100 µM) and Cys-BDC (100 µM) dissolved in THF (4 mL) was irradiated in the thermostated quartz cell at 25 °C using the high-pressure mercury lamp placed at a distance of 200 cm. The decrease in absorbance at 530 nm (molar absorption coefficient () of DPPH, log ) 4.16)19a,b was monitored (reference, 100 µM DPPH solution photoirradiated for 30 min beforehand). Radical production after photoirradiation of Cys-BDC-PMA was also examined. Turbidity Measurements. Aggregation of the SAM-carrying Ag colloids after mixing with Con A was examined by the change in turbidity (τ) (actually decadic absorbance) of the solution at 350 nm. The pH-responsiveness of the SAM-carrying silver colloid (telomer 1-Ag and telomer 2-Ag) was examined by stability of the colloid after addition of NaCl (which reduces the electrostatic repulsion between colloids) into the dispersion at various pH values. The logarithm of the initial turbidity change (dτ/dt)0 at 395 nm corresponding to the coagulation of colloids was plotted as a function of the logarithm of the concentration of NaCl added. The bending point in the plot of log(dτ/dt)0 versus log [NaCl] is called a critical flocculation concentration (cfc) above which there is no effective electrostatic repulsion between the particles, which results in a rapid coagulation where the process is diffusioncontrolled. The method of mixing using a polyethylene mixing rod was always constant to avoid any so-called ortho-kinetic effect.20 Measurements of Contact Angles. Static contact angles, θ, of water on the surface of block telomer SAMs were measured at 23 °C and 60% relative humidity by the sessile drop method (CA-D, Kyowa Interface Science, Tokyo).3,4 The θ values were determined five times to obtain a reliable average value. Measurements of ζ Potential. The ζ potentials of silver colloids at various pH values were measured using a PenKem Model 501 laser-zee meter (Bedford Hills, NY). The ζ potentials were determined 10 times to obtain a reliable average value. Ellipsometric Measurements.16 The thickness of SAMs accumulated on the evaporated gold thin film (thickness, 50 nm) formed above a glass plate (18 × 18 × 0.9 mm, Japan Laser Electronics, Nagoya, Japan) was measured using a DVA ellipsometer (Mizojiri Optical Co., Tokyo) employing a 632.8 nm HeNe laser at an incident angle of 70°. Data from five points were averaged for a given sample. The refractive indexes for the SAM of PMA and PMA-PMEGlc were assumed to be 1.489, in accordance with the value for poly(methyl methacrylate).21 Infrared Reflection-Absorption Spectroscopy (IRRAS) Measurements. The SAM of telomer 2 on the vacuumevaporated gold thin film (Japan Laser Electronics) was examined (19) (a) Kinoshita, M.; Imoto, M. Kobunshi Kagaku 1963, 20, 231. (b) Poirier, R. H.; Kahler, E. J.; Benington, F. J. Org. Chem. 1952, 17, 1437. (20) Everett, D. H. In Basic Principles of Colloid Science; Royal Society of Chemistry: London, 1998; p 143. (21) Bohn, L. In Polymer Handbook, 3rd ed.; Brandrup, J., Immegut, E. H., Eds.; John Wiley & Sons: New York, 1989; p VI/459.
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by infrared reflection-absorption spectroscopy (IRRAS) using an infrared spectrophotometer (Perkin-Elmer System 2000). Each spectrum was obtained by 5 × 103 scans at 16 cm-1 resolution, with a light incident on the metal substrate at 80° (VeeMax II, Pike Technologies, Madison, WI). Binding of Con A to the PMEGlc block in the SAM was detected as an increase in absorbance of the amide band around 1600 cm-1.
Results and Discussion A. Confirmation of Radical Production on Photoirradiation of BDC Group. When a mixture of 1,1′diphenyl-2-picrylhydrazyl (DPPH) and Cys-BDC dissolved in THF was irradiated in the quartz cell at 25 °C with the high-pressure mercury lamp, there was a rapid decrease in absorbance (-∆OD ) 0.69) at 530 nm19a in 30 min, which indicates the consumption of 47.8 µM of DPPH by coupling with radicals. This value indicates that 12% of the initial concentration of Cys-BDC (100 µM) was photocloven in 30 min (one Cyc-BDC molecule produces four radicals). Similarly, the radical production on photoirradiation of Cys-BDC-PMA (50 µM) was estimated to be 16.3 µM (8.2%) in 30 min. Because the radical production on photoirradiation of BDC derivatives could be confirmed, we performed photopolymerization of various vinyl monomers using Cys-BDC and Cys-BDC-PMA hereafter. B. Construction of A-B Block Telomer SAM on Metal Surfaces. Initially, our experiment was designed to simply construct a block telomer SAM directly on the metal surfaces by successive UV-irradiation of an iniferter SAM in the presence of different monomers. Therefore, we first deposited a Cys-BDC SAM on the gold surface and UV-irradiated the iniferter SAM in the presence of methacrylic acid. However, the formation of MA telomer on the gold surface following photoirradiation could not be confirmed by the increase in potential difference (∆Ep, difference between peaks of oxidation and reduction) of the cyclic voltammograms of ferricyanide. Furthermore, the ∆Ep value of the electrode photoirradiated with MA (64 mV) was smaller than that of the starting Cys-BDC SAM (81 mV) and close to that of the bare electrode (59 mV). This result showed that the iniferter SAM was mostly decomposed or forced to leave the gold surface during UVirradiation. Therefore, we adapted the strategy of our research to modification of the gold surface with the blocktelomer-carrying disulfide, which had previously been prepared in the solution phase. Three types of telomers prepared in the solution phase (telomers 1, 2, and 3) were separately dissolved in water and incubated with a gold electrode. Figure 1 shows cyclic voltammograms for the electrode incubated with the telomer solutions. Incubation with telomer 1 (Mw ) 6.3 × 102) markedly changed the voltammogram because of obstruction of diffusion of the probe (ferricyanide) to the electrode surface. In contrast, incubation with the block telomer, telomer 2 (Mw ) 4.0 × 103), induced a less significant change in the voltammogram than did telomer 1. These results suggest that the density of telomer 2 on the electrode surface was much less than that of telomer 1. Furthermore, incubation of telomer 3 with the gold electrode failed to modify the cyclic voltammogram compared to the bare electrode, indicating that telomer 3 did not attach to the gold surface via chemisorptive Au-S bonds. This is probably due to the steric hindrance of the neighboring gigantic PMEGlc chain (Mw ) 5.0 × 104)benzyl group conjugates for the disulfide group between the polymer conjugates to establish chemisorption to the gold surface. It was previously found that addition of 4 M
Figure 1. Cyclic voltammograms of gold electrode modified with various types of telomers: (solid line) bare gold electrode, (dashed-dotted line) modified with telomer 1, (dotted line) modified with telomer 2, and (dashed line) modified with telomer 3. Chart 1. Susceptible Structure of Prepared SAMs on Gold
urea (breaker of both hydrogen bonding and hydrophobic interaction)22 increased the intrinsic viscosity [η] of aqueous PMEGlc solution at 30 °C by 14%.23 Therefore, the PMEGlc block is not stretched by hydrophobic interaction and hydrogen bonding within the block. To exclude the likelihood of physical adsorption of the telomer, PMA (viscosity averaged molecular weight, Mv ) 6.2 × 104) and PMEGlc (Mn ) 2.9 × 104) prepared by conventional radical polymerization using 2,2′-azobisisobutyronitrile were separately incubated with the gold electrode. The voltammogram for ferricyanide was the same before and after the incubation. Therefore, we then proceeded to investigate in detail the surface properties of SAMs composed of telomer 1 or telomer 2. The voltammogram for reductive desorption measurements in 0.5 M KOH showed that the molecular occupation area (Γ value) for telomer 2 (5.5 nm2) was much larger than that of telomer 1 (2.3 nm2). Furthermore, ellipsometric measurements showed that the thicknesses of the telomer 1 SAM and the telomer 2 SAM were quite similar (2.7 and 2.8 nm, respectively, Chart 1). Because the PMEGlc block is much more bulky than the PMA block, (22) Creighton, T. E. In Proteins: Structure and Molecular Properties, 2nd ed.; Freeman: New York, 1993; p 293. (23) Ohno, K. Unpublished result.
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Figure 2. Effect of pH on the contact angle of gold thin film modified with telomer: (b) bare gold film, (2) modified with telomer 1, and (4) modified with telomer 2.
the telomer 2 molecule attached to the electrode might largely obstruct the approach of another block telomer to the electrode, resulting in the larger Γ value. As mentioned above, the sugar-carrying telomer block is not stretched by hydrophobic interaction and hydrogen bonding within the telomer block, which is in accordance with the CV and ellipsometric data for the telomer 2 SAM. Previously, by use of the ellipsometry and IRRAS techniques the tilt angles of n-alkyl thiol SAMs and terminally substituted alkanethiol SAMs from the surface normal of gold were estimated to be 20-30°12c and 2840°,12f respectively. Because of the noncrystalline and disordered structure, however, the tilt angle of the telomer chains prepared here could not be determined accurately. C. pH-Responsiveness of Block Telomer SAM. We next determined the contact angles, θ, of droplets of aqueous solutions of various pH values above the SAM on gold. Figure 2 shows that both telomers 1 and 2 exhibited a decrease in θ value at the alkaline region because of ionization of the PMA block in the telomer, which is in contrast to the almost constant θ value for the bare gold surface. The result for the telomer 1 SAM was expected, whereas the similar pH-dependence observed in the telomer 2 SAM system was unexpected, because the PMEGlc block in the telomer forms the upper layer, and the PMA block below the PMEGlc block does not appear to be significantly influenced by the outer solution. As discussed in section B, the surface density of the telomer 2 SAM on the gold electrode was very low. This means that telomer 2 anchored by the Au-S bond does not direct toward the bulk phase but largely bends over the gold surface, and consequently the PMA block in telomer 2 near the Au-S anchor can affect the θ value of the SAM. By use of the DLS technique, hydrodynamic diameters (dhd) of the silver colloids modified with the telomers (telomer 1-Ag and telomer 2-Ag) were estimated as 40.7 ( 6.7 and 41.2 ( 7.0 nm, respectively (mean ( SD). These values were larger than that for the bare silver colloid (29.1 ( 4.5 nm) by about 10 nm. Because the DP value of telomer 2 was larger than that of telomer 1, the similarity of the dhd values for the two types of telomercarrying colloids indicated that the telomer 2 chain anchors to the silver colloid at very low surface density and does not stretch out to the solution phase, which is consistent with the CV and ellipsometric data discussed above. The bare silver colloid exhibited a definite turbidity change after the addition of NaCl to have a cfc value of 0.035 M at pH 7.0, whereas no changes in turbidity could be detected, even after the addition of 0.5 M NaCl in the dispersion of telomer 1-Ag or telomer 2-Ag at any pH values. Therefore, surprisingly, no pH effect on the cfc
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Figure 3. ζ potential of silver colloids modified with telomer: (b) bare Ag colloid ([Ag] ) 3.26 µM), (2) telomer 1-Ag (0.80 µM), and (4) telomer 2-Ag (1.35 µM).
Figure 4. IRRAS spectra of gold thin film modified with telomer 2: (a, solid line) incubated with telomer 2 (10.1 mg/mL) for 24 h, (b, dashed line) (trace a) after incubation with Con A (2.9 mg/mL) for 24 h, and (c, dotted line) (trace b) after incubation with Me-Man (10 mM) for 1 h. The spectra were obtained in HEPES buffer (10 mM, pH 8.2) with [CaCl2] ) 0.2 mM and [MnCl2] ) 0.2 mM.
value in the telomer-carrying colloid system could be observed. This finding is due to the enormous steric stabilization effect of polymer chains attached to the silver colloids.4a,24 However, the ζ potential of the block-telomercarrying colloids (telomer 2-Ag) changed with pH in a manner similar to that of telomer 1-Ag (Figure 3), because of the bent structure of telomer 2 above the silver colloid surface. In contrast to this, the ζ potential of the bare silver colloid did not show significant pH-responsiveness. D. Recognition of Sugar Residues in Telomer 2 SAM by Con A. When a Con A solution was incubated with the telomer 2 SAM on a gold thin film, a novel amide band corresponding to the lectin molecule appeared in the IRRAS spectra (Figure 4, wave b). Because of the very small surface density of the telomer as mentioned above, the IRRAS spectrum was not clear. However, disappearance of the amide band was clearly observed after incubation of the film with a solution of small molecular weight sugar methyl R-D-mannopyranoside (Me-Man) (Figure 4, wave c). Because the R-mannose residue is preferentially bound by Con A rather than R- and β-glucose residues,25-31 the disappearance of the amide band after (24) Tamai, H.; Fujii, A.; Suzawa, T. J. Colloid Interface Sci. 1987, 118, 176. (25) So, L. L.; Goldstein, I. J. Biochim. Biophys. Acta 1968, 165, 398.
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Figure 6. Effect of pH on the initial turbidity change at 350 nm after mixing Con A solution with the sugar-carrying compounds. The measurements were pursued in HEPES buffer (8 mM, pH 5.38-8.01) or Tris buffer (8 mM, pH 8.38-10.67) with [Con A] ) 1 mg/mL, [CaCl2] ) 0.16 mM, and [MnCl2] ) 0.16 mM: (O) telomer 2-Ag ([Ag] ) 10.2 µM), (4) DTPA-PMEGlccarrying Ag ([Ag] ) 12.1 µM), and (b) telomer 3 (0.1 mg/mL).
Figure 5. (a) Effect of Con A concentration on the initial turbidity change of the telomer 2-carrying Ag colloid dispersion after mixing with Con A solution. The measurements were pursued in HEPES buffer (5 mM, pH 8.2) with [CaCl2] ) 0.1 mM and [MnCl2] ) 0.1 mM. (b) Turbidity of telomer 2-carrying Ag colloid (0.8 mL). Con A solution (2.1 mg/mL, 0.1 mL) was added at 0 s. Me-Man (100 mM, 0.1 mL) was added at the arrow.
incubation with Me-Man can be attributed to specific recognition of sugar residues in the telomer by Con A. Similarly, after mixing the Con A solution with the dispersion of telomer 2-Ag, there was a rapid increase in turbidity at 350 nm (Figure 5a). Furthermore, the addition of Me-Man to the dispersion of block-telomer-carrying colloid, which had been already aggregated by Con A, drastically reduced the turbidity (Figure 5b). These results indicate that the turbidity change is due to the recognition of sugar residues in the block telomer by a tetrameric protein, Con A. Figure 6 shows plots of the initial rate of turbidity change at 350 nm after mixing Con A solution (1 mg/mL) with the dispersion of telomer 2-Ag and DTPA-PMEGlc-carrying Ag at various pHs. The dispersion of DTPA-PMEGlc-Ag was used instead of telomer 3-Ag, because DTPA-PMEGlc (Mn ) 1.4 × 104) could be successfully fixed to the colloid surface whereas telomer 3 (Mw ) 105) could not. This is due to steric obstruction of the sugar-carrying polymerbenzyl group conjugates for the disulfide group at the (26) Lis, H.; Sharon, N. Annu. Rev. Biochem. 1986, 55, 35. (27) Barbet, J.; Machy, P.; Truneh, A.; Leserman, L. D. Biochim. Biophys. Acta 1984, 772, 347. (28) Ramkumar, R.; Surolia, A.; Podder, S. K. J. Biochem. 1995, 308, 237. (29) Chevenak, M. C.; Toone, E. J. Biochemistry 1995, 34, 5685. (30) (a) Mandal, D. K.; Kishore, N.; Brewer, C. F. Biochemistry 1994, 33, 1149. (b) Swaminathan, C. P.; Surolia, N.; Surolia, A. J. Am. Chem. Soc. 1998, 120, 5153. (31) Naismith, J. H.; Field, R. A. J. Biol. Chem. 1996, 271, 972.
center of the telomer 3 molecule to chemisorb to the metal surface, as mentioned in section B. For comparison, the rate of turbidity change of the aqueous telomer 3 solution (0.1 mg/mL) was also observed. The initial rates of turbidity change for telomer 3 solution and DTPAPMEGlc-carrying Ag were similarly pH-dependent, reflecting the intrinsic sugar-binding properties of the Con A molecule. In comparison, the optimum pH value for telomer 2-Ag was shifted to a more acidic region by 0.6 pH unit. We had expected a shift of the pH profile for the telomer 2-Ag system to a more alkaline region in comparison with the DTPA-PMEGlc-carrying Ag and telomer 3 systems, as often reported for the catalytic activity of enzymes immobilized to anionic polymer carriers.32 The results obtained were, therefore, opposite to our expectation. Several studies have demonstrated that a hydrophobic aglycon promotes recognition of sugars by lectin.33 Therefore, the results displayed in Figure 6 could be interpreted as follows: The PMA block in telomer 2 was comparatively hydrophobic in the acidic region, and the affinity for the sugar residue in the neighboring PMEGlc block by Con A was increased concomitantly. This phenomenon could be called a “cooperativity” between neighboring telomer blocks. In conclusion, the glucose-residue-carrying block telomer (telomer 2) accumulated on the metal surface was effectively recognized by the Con A molecule. The density of the telomer 2 SAM was not high because of the bulkiness of the PMEGlc block. The pH-responsiveness of the PMA block in the block telomer was retained because of the low density of the telomer SAM on the metal surfaces. Moreover, the cooperativity of neighboring telomer blocks was observed. The disulfide-carrying block telomer can be easily prepared from the disulfide-carrying iniferter (Cys-BDC) and any vinyl monomers. The iniferter is, therefore, very convenient to use for the preparation of block telomers of various compositions for the functionalization of metal surfaces. (32) (a) Goldstein, L.; Levin, Y.; Katchalski, E. Biochemistry 1964, 12, 1913. (b) Kitano, H.; Nakamura, K.; Ise, N. J. Appl. Biochem. 1982, 4, 487. (33) Ramkumar, R.; Surolia, A.; Podder, S. K. Biochem. J. 1995, 308, 237.
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Acknowledgment. This work was supported by Grants-in-Aid for Scientific Research (11167236 and 12450381) from the Ministry of Education, Science and Culture of Japan. The authors are grateful to Nippon Fine Chemicals for the kind donation of MEGlc and to the Rengo Company, Osaka, Japan, for financial support. The
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authors sincerely thank Professor T. Fukuda and Dr. Y. Tsujii, Institute for Chemical Research, Kyoto University, for ellipsometric measurements. LA000869V
