Langmuir 1998, 14, 4517-4524
4517
Self-Assembled Carbohydrate Monolayers: Formation and Surface Selective Molecular Recognition David J. Revell, Jonathan R. Knight, David J. Blyth, Alan H. Haines,* and David A. Russell* School of Chemical Sciences, University of East Anglia, Norwich, NR4 7TJ, United Kingdom Received February 27, 1998. In Final Form: May 18, 1998 The formation of self-assembled monolayers (SAMs) of representative thiocarbohydrate derivatives onto a gold surface has been investigated to build an artificial carbohydrate scaffold to mimic non-bonded molecular recognition phenomena. Three types of carbohydrate SAMs were formulated from (i) 1-β-Dthioglucose (1), (ii) 1-β-D-thioglucose tetraacetate (2), and (iii) 2-mercaptoethyl R-D-mannopyranoside (3). Subsequently, each SAM was spectroscopically characterized by reflection-absorption infrared spectroscopy (RAIRS). Deprotection of the acylated carbohydrate SAM was achieved in situ, indicating that chemical transformation may be performed without disruption of the sulfursgold bond. With the mannose derivative, in which the carbohydrate moiety was separated from the gold surface by the spacer unit sOCH2CH2SH, it was possible to demonstrate that such a carbohydrate SAM was able to interact selectively with a specific carbohydrate binding protein, i.e., concanavalin A (Con A). Exposure of the mannose derived SAM to a solution of Con A led to a specific binding interaction as measured by RAIRS and surface plasmon resonance (SPR). In contrast, on exposure of the mannose SAM to the L-fucose-specific lectin tetragonolobus purpureas, no such binding was observed. These results suggest that highly ordered SAMs of specifically designed carbohydrate derivatives can be formulated to mimic natural cell surface structures and used to study selective molecular recognition interactions.
Introduction Carbohydrates play a fundamental role in a large array of biological processes. In addition to their central position in metabolism, they are key components of glycoconjugates (glycoproteins and glycolipids), which are complex molecules that are present on the surfaces of cells and that are intimately involved in such important phenomena as the interactions of bacteria, viruses, and cancer cells with hosts, cell-cell recognition, the working of the immune system, and in tissue growth and repair.1 The carbohydrate components may, in general, be mono-, oligo-, or polysaccharides and act as recognition molecules for large biomolecules such as cell surface proteins (e.g., lectins), bacterial toxins, hormones, and antibodies. In the field of glycobiology, these carbohydrate-protein interactions have become the focus of intense interest over the last decade, especially with the development of methods for characterizing the complex carbohydrate structures on cell surfaces.2 It is now realized that these non-bonded interactions provide the key to an understanding of biological recognition phenomena.1 Thus, a detailed study of the interaction of proteins, such as lectins, with carbohydrates held in an ordered manner on a molecular scaffold or an artificial surface, to mimic natural cell surface carbohydrate structures, could provide vital information towards our understanding of this area of biology. Methods for engineering artificial surfaces that contain an ordered arrangement of selected carbohydrates is highly desirable, and various methods have been previously studied. Knowles et al.3 have prepared nonpolymeric structures based on m-substituted benzenes to generate inhibitors of the influenza virus hemagglutin. Polymer* Authors to whom correspondence should be addressed. (1) Varki, A. Glycobiology 1993, 3, 97-130. (2) Dwek, R. A. Chem. Rev. 1996, 96, 683-720. (3) Glik, G. D.; Toogood, P. L.; Wiley, D. C.; Skehel, J. J.; Knowles, J. R. J. Biol. Chem. 1991, 266, 23660-23669.
ization of monomers containing covalently attached carbohydrate structures offers another method to provide frameworks to support saccharide units. Spherical carbohydrate displays incorporating dendrimers and liposomes4 can be produced by the polymerization method. However, the most popular method for producing carbohydrate surfaces is through polymerization of modified acrylamides to produce linear displays of saccharidesubstituted acrylamide derivatives.5 Whitesides et al.6 used sialic acid-substituted acrylamide copolymers to investigate the mechanism of carbohydrate inhibition of hemagglutination. In addition to producing artificial cell surfaces, natural cell surfaces have been modified to present carbohydrates specifically designed for a number of applications. These modifications have been achieved through genetic approaches, external delivery methods, and via metabolic delivery.7 Self-assembled monolayers (SAMs) provide an alternative means by which molecules may be formulated as densely packed structures on a surface. The study of protein adsorption on SAMs is of considerable value because SAMs can provide highly ordered, multifunctional surfaces not unrelated to environments found on some cell surfaces. Fernandez et al.8 demonstrated the use of mixed SAMs to attach a fused β-galactosidase and choline receptor protein to a gold surface. Spinke et al.9 investigated the use of biotinylated alkanethiol SAMs to bind (4) Kiessling, L. L.; Pohl, N. L. Chem. Biol. 1996, 3, 71-77. (5) Kallin, E. Methods Enzymol. 1994, 242, 221-226. Kobayayashi, K.; Akaike, T.; Usui, T. Methods Enzymol. 1994, 242, 226-235. Nishimura, S. I.; Furuike, T.; Matswoka, K. Methods Enzymol. 1994, 242, 235-246. Tropper, F. D.; Romanowsk, A.; Roy, R. Methods Enzymol. 1994, 242, 257-271. (6) Mammen, M.; Dahmann, G.; Whitesides, G. M. J. Med. Chem 1995, 38, 4179-4190. Lees, W. J.; Spaltenstein, A.; Kingury-Wood, J. E.; Whitesides, G. M. J. Med. Chem. 1994, 37, 3419-3433. (7) Mahal, L. K.; Bertozzi, C. R. Chem. Biol. 1997, 4, 415-422. (8) Madoz, J.; Kuznetzov, B. A.; Medrano, F. J.; Garcia, J. L.; Fernandez, V. M. J. Am. Chem. Soc. 1997, 119, 1043-1051. (9) Spinke, J.; Liley, M.; Schmitt, F. J.; Guder, H. J.; Andermaier, L.; Knoll, W. J. Chem. Phys. 1993, 99, 7012-7019.
S0743-7463(98)00246-7 CCC: $15.00 © 1998 American Chemical Society Published on Web 07/17/1998
4518 Langmuir, Vol. 14, No. 16, 1998
Revell et al.
streptavidin. Whitesides et al.10 have shown that a mixed hydrophobic and hydrophilic SAM is a useful substrate for the adsorption of proteins, and Allara et al.11 have studied cell growth and protein adsorption on alkane thiolate SAMs. Gold derivatives of carbohydrates such as aurothioglucose, in which the saccharide-to-metal linkage is via a sulfur atom, have long been known to the pharmaceutical industry and are apparently stable in an aqueous environment. The existence of these derivatives suggested that an artificial cell surface might be produced with which to investigate carbohydrate-lectin interactions by the deposition of highly ordered, monomolecular thin films of thiocarbohydrates onto gold substrates using the technique of self-assembly.12,13 Indeed, the deposition of a thiol-terminated hexasaccharide unit onto a gold surface was recently reported by Fritz et al.14 SAMs are useful tools because the molecular orientation of the carbohydrate molecules on the gold surface may be determined. In addition, carbohydrates containing a thiol moiety either directly at the anomeric center or attached via a pendant “spacer” group are not difficult to prepare and a wide range of functionality can be built into the carbohydrate molecules, thereby enabling a surface to be tailored to produce molecular recognition sites for a range of different types of interaction. Thus, we envisaged that SAMs could be produced with the ability to bind specific lectins. The gold-thiol reaction may be regarded as an oxidative addition of the SsH bond to the surface followed by a reductive elimination of hydrogen.15 The sulfur-to-gold bond is strong; the homolytic bond strength has been reported as ∼40 kcal/mol.15 This bond strength is an important consideration for the success of our preferred experimental method involving interactions of the goldbound carbohydrates with aqueous solutions of lectins. Furthermore, elaboration of complex carbohydrate ligands inevitably involves protected intermediates that may require that reactions be performed while the sugars are attached to the metal surface to reveal the free carbohydrate. Therefore, as part of our study, we have also examined the feasibility of performing de-O-acylation on a monosaccharide ester, covalently linked to the gold surface via its anomeric center. Our results on this type of deprotection complement those in the recent related study.14 To investigate the feasibility of carbohydrate deposition on a gold-coated substrate, initial experiments were conducted with commercially available 1-β-D-thioglucose (Na salt) (1) and 1-β-D-thioglucose tetraacetate (2). For our lectin-binding studies, however, a mannose derivative (3) was prepared consisting of a mannopyranoside residue, carrying on the anomeric carbon atom the thiol-containing spacer sOCH2CH2SH (see structures). Our studies of protein interactions with the SAM from this thio-mannopyranoside were conducted with two lectins, concanavalin A (Con A) and tetragonolobus purpureas. Con
A is a multivalent R-D-mannopyranoside binding lectin. Both Mn2+ and Ca2+ ions are required for activity, and between pH 5.8 and 7.0 the lectin exists as a tetramer and is capable of binding four terminal R-D-mannopyranosyl residues. Con A will also bind terminal R-D-glucosyl residues, but with less affinity.16-18 Tetragonolobus purpureas has an affinity for R-L-fucosyl residues,18,19 and we have used this lectin as a control because it was expected to show little affinity for the self-assembled mannopyranosyl residues. We used two techniques to detect the carbohydrates deposited on gold-coated substrates and their interaction with the two lectins, reflection absorption infrared spectroscopy (RAIRS) and surface plasmon resonance (SPR). RAIRS enables ready detection of saccharide residues on the metal surface by measurement of CsO and HsO vibration absorption bands and complexed lectins through their amide I, II, and A bands. SPR is capable of measuring, with high sensitivity, surface interactions such as carbohydrate-protein binding on a gold-coated glass substrate. There have been some recent reports on the use of SPR to study protein binding interactions on SAMs but, in general, the interactions investigated were of a less specific nature than those reported in this paper. For example, Whitesides et al.20 used the SPR technique to measure the nonspecific adsorption interaction of RNAse, lysozyme, fibrinogen, and pyruvate kinase onto a mixed SAM [CH3(CH2)10SH and HO(CH2CH2O)6(CH2)11SH]. Imata et al.21 studied the interaction of Con A immobilized on an aminosilane-hydrogel surface (a nonSAM surface) of an optical biosensor, with oligosaccharidebranched cyclodextrins. Additionally, Shinohara et al.22 examined the association between various lectins and biotin-derivatized oligosaccharides, the latter being linked to a biosensor via binding to streptavidin which was itself immobilized on a gold-coated surface by linkage to a carboxymethyl dextran. In contrast to these studies, the work described here is concerned with carbohydrates orientated on a gold surface in a highly ordered manner via self-assembly, an arrangement that we believe may be more relevant in attempts to mimic the environment of cell surface carbohydrates.
(10) Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 10714-10721. (11) Tidwell, C. D.; Ertel, S. I.; Ratner, B. D.; Tarasevich, B. J.; Atre, S.; Allara, D. J. Langmuir 1997, 13, 3404-3413. (12) Ulman, A. An Introduction to Ultrathin Organic Films from Langmuir Blodgett to Self-Assembly; Academic: Boston, 1991. (13) Nuzzo, R. G.; Allara, D. L. J. Am. Chem. Soc. 1983, 105, 44814483. Porter, M. D.; Bright, T. B.; Allara, D. L.; Chidsey, C. E. D. J. Am. Chem. Soc. 1987, 109, 3359-3586. Bain, C. D.; Whitesides, G. M. J. Am. Chem. Soc. 1988, 110, 6560-6561; Bain, C. D.. Troughton, E. B.; Tao, Y. T.; Evall. J.; Whitesides, G. M.; Nuzzo, R. G. J. Am. Chem. Soc. 1989, 111, 321-335. (14) Fritz, M. C.; Ha¨hner, G.; Spencer, N. D.; Bu¨rli, R.; Vasella, A. Langmuir 1996, 12, 6074-6082. (15) Ulman, A. Chem. Rev. 1996, 96, 1533-1554.
(16) Edelman, G. M.; Cunningham, B. A.; Reek, G. N. Jr.; Becker, J. W.; Waxdel, M. J.; Wang, J. L. Proc. Nat. Acad. Sci., U.S.A. 1972, 69, 2580-2584. (17) Hardman, K. D.; Ainsworth, C. F. Biochemistry 1972, 11, 49104919. (18) Goldstein, I. J.; Hayes, C. E. Adv. Carbohydr. Chem. Biochem. 1978, 35, 127-340. (19) Pereira, M. E. A.; Kabat, E. A. Ann. N. Y. Acad. Sci. 1974, 234, 301-305. (20) Mrksich, M.; Sigal, G. B.; Whitesides, G. M. Langmuir 1995, 11, 4383-4385. (21) Imata, H.; Kubota, K.; Hatton, K.; Aoyagi, M.; Jindoh, C. BioMed. Chem. Lett. 1997, 7, 109-112. (22) Shinohara, Y.; Sota, H.; Kim, F.; Shimizu, M.; Gotoh, M.; Tosu, M.; Hasegawa, Y. J. Biochem. 1995, 117, 1076-1082.
Experimental Section Reagents. Solvents and chemicals, including 1-β-D-thioglucose (as Na salt‚2H2O; 1) and 1-β-D-thioglucose tetraacetate (2), were obtained from the Aldrich Chemical Company. Methanol was dried by addition of magnesium and a catalytic amount of iodine followed by reflux and then distillation from the so-formed
Self-Assembled Carbohydrate Monolayers magnesium methoxide. The lectins Con A isolated from jack bean (Canavalia ensiformis) and lotus agglutinin, tetragonolobus purpureas, from asparagus pea, were purchased from Sigma Chemical Company. Synthesis of 2-Mercaptoethyl r-D-Mannopyranoside (3). (2-Bromoethyl) -Tetra-O-acetyl-R-D-mannopyranoside. Acetyl chloride (0.5 mL) was added to D-mannose (5 g, 27.75 mmol) and 2-bromoethanol (20 mL) and the mixture was heated at 60 °C for 2 h and then cooled. Triethylamine (2 mL) was added and the mixture was concentrated under reduced pressure to an oil that was subjected to column chromatography. Elution with dichloromethane/methanol (9:1, v/v) gave crude (2-bromoethyl) R-D-mannopyranoside (7.2 g), which was dissolved in pyridine (50 mL) and treated with acetic anhydride (13 mL, 137.8 mmol). The mixture was poured into ice-water, the mixture was extracted with dichloromethane (3 × 50 mL), and the combined extracts were extracted with saturated aqueous sodium hydrogen carbonate (80 mL) and aqueous sodium chloride (80 mL), and then dried. The filtered solution was concentrated, toluene was repeatedly evaporated from the residue to remove pyridine, and the remaining solid was crystallized from ethyl acetate/light petroleum to give the tetraacetate (7.8 g, 62%); mp, 112-113 °C; [R]D +44 (c 0.51, CHCl3); 1H NMR (270 MHz; CDCl3) δ 2.00, 2.06, 2.11, 2.17 (4 × 3 H, 4 × s, 4 × MeCO), 3.52 (2 H, t, J1′,2′ 5.9 Hz, H-2′a, 2′b), 3.85-4.03 (2 H, m, H-1′a, 1′b), 4.11-4.15 (2 H, m, 5-H, H-6a), 4.28 (1 H, dd, J5,6b 5.9 Hz, J6,6b 12.9 Hz, H-6b), 4.88 (1 H, d, J1,2 1.6 Hz, H-1), 5.28 (1 H, dd, J2,3 3.3 Hz, H-2), 5.29 (1 H, t, J3,4 ) J4,5 ) 9.6 Hz, H-4), 5.36 (1 H, dd, H-3); 13C NMR (67.5 MHz; CDCl3) δ 20.72 (× 2), 20.75, 20.88 (4 × Me), 29.61 (CH2Br), 62.44 (6-C), 66.04 (CH), 68.52 (CH2CH2Br), 68.97, 69.05 (2 × CH), 69.45 (C-5), 97.77 (C-1), 169.77, 169.86, 170.04, 170.62 (4 × CO). Anal. Calcd for C16H23BrO10: C, 42.21; H, 5.09; Br, 17.55. Found: C, 42.42; H, 5.12; Br, 17.19. (2-Acetylthioethyl) Tetra-O-acetyl-R-D-mannopyranoside. A mixture of (2-bromoethyl) tetra-O-acetyl-R-D-mannopyranoside (1.5 g, 3.29 mmol) in butanone (30 mL) containing potassium thioacetate (2.63 g, 23.06 mmol) was heated under reflux for 2.5 h, then cooled and filtered through Celite. The crude product obtained on concentration of the filtrate was purified by column chromatography [ethyl acetate/light petroleum (3:7)] to give as a syrup the (2-acetylthioethyl) glycoside (1.32 g, 89%), [R]D +50 (c 0.39, CHCl3); 1H NMR (270 MHz; CDCl3) δ 2.00, 2.06, 2.10, 2.16 (4 × 3 H, 4 × s, 4 × MeCOO), 2.36 (3 H, s, MeCOS), 3.12 (2 H, br t, J1′,2′ ∼6.6, H-2′a, 2′b), 3.63 (1 H, dt, J1′a,1′b 10.2 Hz, J1′a,2′a ) J1′a,2′b ) 6.3 Hz, H-1′a), 3.78 (1 H, dt, J1′b,2′a ) J1′b,2′b ) 6.6 Hz, H-1′b), 4.01-4.07 (1-H, m, H-5), 4.11 (1 H, dd, J6a,6b 12.2 Hz, J5,6a 2.6 Hz, H-6a), 4.27 (1 H, dd, J5,6b 5.6 Hz, H-6b), 4.83 (1 H, d, J1,2 1.6 Hz, H-1), 5.24 (1 H, dd, J2,3 3 Hz, H-2), 5.25-5.35 (2 H, m, H-3,4); 13C NMR (67.5 MHz; CDCl3) δ 20.60 (× 2) 20.76, 20.92 (4 × MeCOO), 28.47 (CH2SAc), 30.44 (MeCOS), 62.32 (6C), 65.99 (CH), 66.81 (CH2CH2SAc), 68.64, 68.84 (2 × CH), 69.33 (C-5), 97.43 (C-1), 169.61, 169.72, 169.88, 170.49 (4 × CO), 194.91 (MeCOS). Anal. Calcd for C18H26O11S: C, 48.0; H, 5.82; S, 7.12. Found: C, 48.28; H, 5.90; S, 6.84. 2-Mercaptoethyl R-D-mannopyranoside. Sodium metal (∼50 mg) was added to a solution of (2-acetylthioethyl) tetra-O-acetyl R-D-mannopyranoside (1.28 g, 2.84 mmol) in methanol (15 mL) and the solution so formed containing sodium methoxide was stored at room temperature for 4 h. A pellet of solid carbon dioxide was added, and the mixture was concentrated to an oil which was purified by column chromatography [methanol/ dichloromethane (3:17)] to yield, as a colorless oil, title compound 3 (0.42 g, 61%), [R]D +50 (c 0.39, CHCl3); 1H NMR (270 MHz; CD3OD) δ 2.70 (2 H, t, J1′,2′ ∼6.3, H-2′a, 2′b), 3.54-3.86 (8 H, complex, H-2,-3,-4,-5,-6a, -6b, -1′a, -1′b), 4.80 (1 H, d, J1,2 1.6 Hz, H-1); 13C NMR (67.5 MHz; CD3OD) δ 24.72 (CH2SH), 62.82 (C6), 68.48 (C-4), 70.51 (OCH2CH2SH), 72.00 (C-2), 72.50 (C-3), 74.70 (C-5), 101.54 (C-1). Anal. Calcd for C8H16O6S: C, 39.99; H, 6.71; S, 13.34. Found: C, 39.83; H, 6.80; S, 13.08. Preparation of Gold-Coated Glass Substrates. To characterize the carbohydrate SAMs by RAIRS, glass microscope slides (BDH Ltd.) were used as the substrates. For the SPR studies, B270-superwite high-transparent glass (UQG Ltd.), cut to the same dimensions as a standard microscope slide, was used as the SAM substrate. The B270 glass substrates were used because it was essential that the optical properties of the substrate
Langmuir, Vol. 14, No. 16, 1998 4519 matched those of the prism used in our SPR instrument. In both cases, the glass subtrates were cleaned in a solution of KOH in aqueous methanol (100 g of KOH was dissolved in 100 mL of Millipore water then diluted to 250 mL with methanol). The slides were rinsed with Millipore water and then dried in a stream of refluxing propan-2-ol. The cleaned, dried slides were then coated with a layer of chromium (99.999% purity, Johnson Matthey Ltd.) followed by a layer of gold (99.999% purity, Johnson Matthey Ltd.) by thermal evaporation under reduced pressure (Edwards Auto 306 vacuum evaporator). The thickness of each metal layer was dependent on the spectroscopic technique employed (5 nm Cr, 45 nm Au for RAIRS; 1 nm Cr, 45 nm Au for SPR). Formation of Carbohydrate SAMs for Reflection Absorption IR Characterization. The chromium was deposited onto the cleaned optical slides (to ensure adhesion of the gold onto the glass surface) followed by the gold. The carbohydrate SAMs were formed by immersing for 20 h (to ensure the formation of well-organized SAM23) the freshly prepared gold-coated optical slides into a solution of ∼1.0 × 10-3 mol dm-3 of the respective carbohydrate dissolved in an appropriate solvent: specifically, ∼1.0 × 10-3 mol dm-3 solution of 1 in methanol; ∼9.0 × 10-4 mol dm-3 solution of 2 in dichloromethane; and ∼1.3 × 10-3 mol dm-3 solution of 3 in methanol. Reference substrates for SAMs were prepared by immersing freshly prepared gold-coated slides into methanol (for 1 and 3) or dichloromethane (for 2) for 20 h. Once formed, the carbohydrate SAMs and reference substrates were each washed in fresh methanol or dichloromethane, according to their previous treatment, and subsequently dried in a stream of argon for 30 min. RAIR spectra were obtained from the carbohydrate SAMs using p-polarized light at an incidence angle of 85° using a Bio-Rad FTS40 FT IR spectrometer coupled with a Spectra-Tech FT85 specular reflectance unit. Spectra were obtained from the coaddition of 1024 scans of 4096 data points, giving a resolution of 4 cm-1. De-O-acylation of a SAM of 1-β-D-Thioglucose Tetraacetate (2) on a Gold Surface. Following the formation of a SAM of 1-β-D-thioglucose tetraacetate (2), the SAM was immersed, under anhydrous conditions, for 5 h in a methanol solution containing a catalytic amount of sodium methoxide, prepared by adding sodium metal (0.05 g) to dried methanol (50 mL). The SAM was removed, and then immersed sequentially in three different portions of dried methanol, with each immersion lasting 10 min. Methanol was allowed to evaporate from the glass substrate in air for 1 min and the substrate was then stored in a vacuum desiccator over phosphorus pentoxide. Lectin Interaction with Carbohydrate SAMs. Carbohydrate SAMs of 3 were immersed in a buffered solution of Con A (0.5 mg/mL, 10 mM NaH2PO4/NaOH at pH 7.5) or a buffered solution of tetragonolobus purpureas (0.1 mg/mL, 10 mM NaH2PO4/NaOH at pH 7.5) for 20 h at 4 °C. Trace amounts of Ca2+ and Mn2+ ions (200 µM) were added to the Con A buffered solution as these ions are a requirement for the agglutination activity of Con A. Reference substrates were formed by immersing goldcoated optical slides into 10 mM NaH2PO4/NaOH buffer solution adjusted to pH 7.5 for 20 h. The gold substrates were then washed in fresh Millipore water and dried in a stream of argon for 30 min. These SAMs were again spectroscopically characterized by RAIRS. Surface Plasmon Resonance Analysis of Carbohydrate SAM (3): Formation and Subsequent Measurement of SAM-Lectin Interaction. Chromium followed by a layer of gold were deposited onto the cleaned glass substrate. A glass flow-through cell was attached to the substrate with a Viton O-ring to provide a liquid tight seal. The substrate/flow-through cell was mounted on a 3 cm 45° B270 prism (n ) 1.514 at λ ) 632.8 nm, UQG Ltd.) using index matching fluid of n ) 1.524 (Cargille Laboratories Inc.) in the “Kretschmann” configuration.24 The irradiation source of the SPR instrument was a p-polarized (23) (a) Simpson, T. R. E.; Revell, D. J.; Cook. M. J.; Russell, D. A. Langmuir 1997, 13, 460-464. (b) Horn, A. B.; Russell, D. A.; Shorthouse, L. J.; Simpson, T. R. E. J. Chem. Soc., Faraday Trans. 1996, 92, 47594762. (24) Kretschmann, E.; Raether, H. Z. Naturforschung 1968, 23a, 2135-2136.
4520 Langmuir, Vol. 14, No. 16, 1998
Revell et al.
Figure 1. RAIR spectrum of a SAM of 1 on a gold-coated glass substrate. 10 mW He/Ne laser (Uniphase) of λ ) 632.8 nm. The reflected p-polarized light was detected by a photodiode (Hamamatsu) with the output processed by a personal computer. The SPR instrument used in this work was based on the design of Knoll et al.25 The SPR analysis software and the goniometer interface were both supplied by Professor W. Knoll (Mainz, Germany). All surface plasmon resonance reflectivity curves measured as a function of internal angle were recorded, using the goldcoated glass (B270) substrates, from 30° to 80° (in increments of 0.005°, external angle, around the SPR reflectivity minima) using a θ/2θ goniometer (model 414, Huber Industries). Once the SPR reflectivity curve of the gold-coated substrate had been recorded, a ∼1.3 × 10-3 mol dm-3 solution of carbohydrate 3 in methanol was added to the glass flow-through cell and left for 20 h. The carbohydrate 3 solution was then removed and the gold substrate was washed with fresh methanol and dried for 45 min with a stream of argon in situ. The SPR reflectivity curve of the carbohydrate SAM (3) was recorded. A buffered solution of Con A (0.5 mg/mL, 10 mM NaH2PO4/NaOH at pH 7.5) containing trace amounts of Ca2+ and Mn2+ ions (200 µM) was then added to the glass flow-through cell and left for 20 h. The buffered Con A solution was removed and the SAM-gold substrate was washed with fresh Millipore water and dried for 45 min with argon in situ. The SPR reflectivity curve of the Con A bound to the SAM of 3 was recorded.
Results and Discussion Synthesis of 2-Mercaptoethyl r-D-mannopyranoside (3). Fischer glycosidation of D-mannose with 2-bromoethanol gave syrupy but homogeneous 2-bromoethyl D-mannopyranoside, determined, on the basis of 13C NMR spectrum of the derived 2-mercaptoethyl glycoside (3), to be the R-isomer. The glycoside was characterized as its crystalline tetraacetate. Displacement of the halide ion by treatment with potassium thioacetate in butanone afforded the corresponding 2-(acetylthio)ethyl glycoside tetraacetate, which on deesterification afforded glycoside 3. In mannopyranosides, anomeric configuration cannot be determined either from the coupling constant J1,2 or from the 13C chemical shift of C-1 but, compared with β-anomers, R-glycosides show a characteristic upfield shift (25) Nishida, N.; Hara, M.; Sasabe, H.; Knoll, W. Jpn. J. Appl. Phys. 1996, 35, L799-L802.
for C-3 and C-5 due to the well-recognized γ-effect. A comparison of the relevant 13C chemical shifts in 3 with literature values for methyl R- and β-D-mannopyranoside26 allows identification of 3 as the R-anomer. RAIR Characterization of the Carbohydrate SAMs. The characterization of the three carbohydrate SAMs was performed by RAIRS directly from the gold-coated glass substrates. The RAIR spectrum of 1 is shown in Figure 1. The broad band at 3391 cm-1 is assigned as an OsH stretching vibration. The broadness and the shift to lower frequency of this band indicates intermolecular, and possibly intramolecular,27a hydrogen bonding between the carbohydrate molecules, thereby suggesting a densely packed monolayer. Additionally, the following bands are also present: 1410 cm-1, OH bending; 1272 cm-1, ν(C6sOsH)/CH2OH related mode,27b,c and 1107 cm-1, ν(CsOsC) of the carbohydrate ring.28 The RAIR spectrum of 2 is shown in Figure 2. Bands for CH3 (νas) at 2966 cm-1 and CH3 (νs) at 2888 cm-1 are seen representing the acetate groups, although the signal: noise for these absorption bands is poor. The main band characterizing 2 is the ν (CdO) at 1763 cm-1. The following bands have also been assigned: 1370 cm-1, CH3 sym bending mode; 1259 cm-1, ν(CsOsC) of the acetoxy group;29 1102 cm-1 ν(CsOsC) of the carbohydrate ring28 and 1063 cm-1, ν(CsOsC) of the acetoxy group.29 The RAIR spectrum of 3 is shown in Figure 3a. The broad band centered at 3345 cm-1 is assigned to an OsH stretching vibration (with intermolecular, and possibly intramolecular, hydrogen bonding). Low-intensity absorption bands for CH2 (νas) at 2920 cm-1 and CH2 (νs) at 2852 cm-1 are seen representing the ethyl spacer chain, (26) Bock, K.; Pedersen, C. Adv. Carbohydr. Chem. Biochem. 1983, 41, 27-66. (27) (a) Forster, A. B. Annu. Rev. Biochem. 1961, 30, 45-70. (b) Vasko, P. D.; Blackwell, J.; Koenig, J. L. Carbohydr. Res. 1972, 23, 407-416, (c) Cael, J. J.; Koenig, J. L.; Blackwell, J. Carbohydr. Res. 1973, 29, 123-134. (28) Barker, S. A., Bourne, E. J.; Whiffen, D. H. Meth. Biochem. Anal. 1956, 3, 213-245. (29) Casu, A., Reggiani, M., Gallo, G. G.; Vigevani, A. Carbohydr. Res. 1970, 12, 157-170.
Self-Assembled Carbohydrate Monolayers
Langmuir, Vol. 14, No. 16, 1998 4521
Figure 2. RAIR spectrum of a SAM of 2 on a gold-coated glass substrate.
incorporated into the molecule to distance the carbohydrate from the gold surface. The following bands have also been assigned: 1448 cm-1, δ CH2; 1416 cm-1, OH bending; 1272 cm-1, ν(C6sOsH)/CH2OH related mode;27b,c 1134 cm-1, aliphatic νas(CsOsC)28 and 1095 cm-1, ν(Cs OsC) of the carbohydrate ring.28 The RAIR spectra (Figures 1-3) show that the carbohydrates 1, 2, and 3 all have been self-assembled onto the gold-coated glass substrates, forming densely packed monolayers as evidenced by the intermolecular hydrogen bonding. From these data it is clear that carbohydrates incorporating a variety of functionalities can be readily formulated as SAMs. The formation of a SAM of 1 also shows, possibily unsurprisingly, that a compound containing an sS-sNa+ moiety facilitates the formation of a thiolate species at a gold surface. Such a finding agrees with Feher et al.30 who concluded that a wide range of sulfur-containing molecules can be used to create goldsupported surfaces, without the need for the cleavage of an SsH or SsS bond. Although the exact nature of the orientation of the three carbohydrate monolayers in relation to the gold surface cannot be definitively obtained from the RAIR spectra, it is clear that SAM 3 is orientated in a different manner to SAMs 1 and 2. With reference to the metal surface selection rule, only vibrational modes with a non-zero transition dipole moment (TDM) component perpendicular to the surface will be observed.23b Therefore, the intense absorption band, seen in Figure 3a at 1095 cm-1, which is associated with the ν(CsOsC) of the carbohydrate ring, suggests that the carbohydrate ring of SAM 3 lies close to the surface normal. However, the same band in SAMs 1 and 2 (1107 and 1102 cm-1, Figures 1 and 2, respectively) is much less intense, suggesting that the carbohydrate ring in these SAMs lies in a more parallel orientation with respect to the gold surface. Further, the ν(C6-O-H)/CH2OH-related mode observed at 1272 cm-1 in RAIR spectra of both 1 and 3, but at significantly different intensities, suggests that this moiety lies in a different orientation for these two (30) Dishner, M. H.; Hemminger, J. C.; Feher, F. J. Langmuir 1996, 12, 6176-6178.
monolayers. The RAIR spectrum shown in Figure 3a also suggests that the anchor spacer moiety of 3 is orientated towards the normal with respect to the gold surface [as shown by the intense absorption band at 1134 cm-1 associated with the aliphatic νas(CsOsC)]. Such an orientation of the spacer moiety would be expected because straight-chain alkanethiols form monolayers on gold surfaces at ∼25° from the surface normal.12 It is probable that the difference in surface orientation between the SAMs of 3 and those of 1 and 2 stems from the alignment of the aliphatic spacer moiety. Although the formation of SAMs from the commercially available acetylated carbohydrate 2 is clearly feasible, hydroxylated surfaces are of primary importance for biological molecular recognition, especially in the carbohydrate-lectin interaction. Therefore, we investigated the possibility of performing in situ surface chemistry on the SAM of 2, in particular de-O-acetylation, resulting in the replacement of acetoxy with hydroxy moieties. Figure 4a shows a schematic of the reaction performed on the SAM of 2. Figure 4b shows the RAIR spectrum of a SAM of carbohydrate 2 before (A) and after (B) replacement of the acetoxy groups with an hydroxyl functionality. In figure 4b-A, the ν(Cd0) at 1763 cm-1, the CH3 asymmetric bending mode at 1455 cm-1, and the CH3 symmetric bending mode at 1370 cm-1 are seen in the RAIR spectrum giving a clear indication of the presence of the acetoxy moieties. However, in Figure 4b-B, these absorption bands are no longer present, indicating that the acetoxy groups have been removed. It should also be noted that the intense absorption bands at 1259 and 1272 cm-1 (Figure 4b, A and B, respectively) are associated with the conversion of the acetoxy group to the hydroxy moiety on the C6 atom of the carbohydrate ring. With the formation of the hydroxy carbohydrate derivative, the following absorption bands would be expected in the RAIR spectrum; a broad band centered at ∼3390 cm-1 associated with the OH stretching vibration and a band at 1410 cm-1 due to OH bending. These bands are not obviously apparent in Figure 4b-B. However, on comparison with the data shown in Figure 1, it is clear that the spectrum in Figure
4522 Langmuir, Vol. 14, No. 16, 1998
Revell et al.
Figure 3. (a) RAIR spectrum of a SAM of 3 on a gold-coated glass substrate and (b) the RAIR spectrum of SAM 3 following interaction of the lectin Con A.
4b-B has a poor signal:noise. It is possible, therefore, that the broad OH stretch absorption band is obscured by the noise associated with that region of the spectrum, whereas the OH bending band at 1410 cm-1 is hidden beneath the broad feature associated with the band at 1272 cm-1. RAIRS Characterization of the Interaction Between a SAM of 3 with the Lectins Concanavalin A and Tetragonolobus Purpureas. Although the successful in situ formation of a carbohydrate SAM containing hydroxy groups was shown to be feasible, carbohydrate 3 was designed and synthesized to establish whether a carbohydrate SAM could be used for selective molecular recognition of specific lectins. The mannose derivative 3 was chosen because the target lectin Con A is known to
bind strongly with this carbohydrate. To facilitate the binding of the lectin to the SAM of 3, a thiol containing spacer (sOCH2CH2SH) to distance the mannose residue from the gold surface, was incorporated at the anomeric carbon atom. Figure 3b shows the RAIR spectrum resulting from the interaction of a SAM of 3 with Con A. The characteristic bands for Con A are present.31 The ν(NH) ‘amide A’ band is seen at 3300 cm-1, the ν(CdO) ‘amide I’ band is present at 1664 cm-1, and the out-of-phase, in-plane NH bending vibration strongly coupled to ν(CN) ‘amide II’ band appears (31) Ockmann, N. Biochim. Biophys. Acta 1981, 643, 220-232, ErnstFonberg, M. L.; Worsham, L. M. S.; Williams, S. G. Biochim. Biophys. Acta 1993, 1164, 273-282. Bandekar, J. Biochim. Biophys. Acta 1992, 1120, 123-143.
Self-Assembled Carbohydrate Monolayers
Langmuir, Vol. 14, No. 16, 1998 4523
Figure 4. (a) Schematic reaction of in situ surface monolayer chemistry converting the terminal acetoxy moieties of (A) SAM 2 with (B) hydroxy moieties, and (b) the RAIR spectra of the SAM of 2 (A) before and (B) after conversion.
at 1540 cm-1. In addition to the absorption bands associated with the Con A, the characteristic absorption bands due to the carbohydrate 3 SAM (seen in Figure 3a) are also present in the RAIR spectrum. This presence suggests that the Con A has bound to the SAM rather than displacing the SAM from the gold surface. To show that the molecular recognition capability of the SAM of 3 was specific to mannose binding lectins, the lectin tetragonolobus purpureas, which is specific towards L-fucose, was similarly interacted with the SAM 3 surface. If selective binding was to be achieved using SAM 3, then the tetragonolobus purpureas lectin should not bind to the SAM and leave the surface intact. Following 20 h of exposure with a solution of the tetragonolobus purpureas, the RAIR spectrum of the carbohydrate 3 SAM was unchanged from that observed in Figure 3a. This result clearly suggests that the carbohydrate 3 SAM does indeed exhibit a degree of specificity towards different lectins. SPR of SAM (3) Formation and Interaction with Concanavalin A. SPR occurs at a dielectric - metal interface when p-polarized light resonantly induces a collective oscillation in the free electron plasma at the metal film boundary. This resonance is observed as a minimum of reflectivity of the p-polarized light, as a function of incidence angle when the momentum of the incident photon is equivalent to that of the excited plasmon. A change in the dielectric constant at the interface will cause a shift in the reflectivity minimum. SPR is extremely sensitive to changes that occur at the metal-dielectric interface and therefore represents an ideal probe technique for monitoring the formation of a SAM, such as carbohydrate 3, and its subsequent interac-
tion with Con A on a gold surface. In addition, SPR gives complementary information to RAIRS, thereby providing a further understanding of the molecular recognition interactions occuring between the carbohydrate 3 SAM and the lectin Con A. DeBono et al.32 have previously shown, using SPR and surface plasmon microscopy (SPM), that when adsorbed directly onto a polycrystalline gold surface, the size of Con A does not correspond to either a monomeric, dimeric, or tetrameric form. This result suggests that direct adsorption of Con A to a gold surface results in the denaturation of the lectin. Con A is known to have hydrophobic character33,34 and, as such, has the ability to adsorb nonspecifically to hydrophobic surfaces.32 Therefore, the adsorption of Con A to a gold surface, which is a moderately hydrophobic substrate,32 was to be expected. This adsorption was confirmed using RAIRS in that the ‘amide A’, ‘amide I’ and ‘amide II’ absorption bands used to characterize the Con A on the SAM 3 surface (Figure 3b) were observed when a native gold-coated substrate was exposed to a solution of the lectin. The adsorption of Con A onto hydrophobic surfaces was studied further, with RAIRS, using a hexadecyl alkanethiol SAM. The lectin was observed to bind to the hydrophobic surface offered by the C16 SAM. The characteristic absorption bands of Con A were observed, although the CH2/CH3 (νs (32) DeBono, R. F.; Krull, U. J.; Rounaghi, Gh. ACS Symp. Series 1992, 511, 121-136. (33) Ochoa, J. L.; Kristainsen, T.; Pa˚hlman, S. Biochim. Biophys. Acta 1979, 577, 102-109. (34) Isbister, B. D.; St. Hilaire, P. M.; Toone, E. J. J. Am. Chem. Soc. 1995, 117, 12877-12878.
4524 Langmuir, Vol. 14, No. 16, 1998
Revell et al.
corresponds to the actual molecular size of 3, giving further supporting evidence that a monolayer of 3 has been formed at the gold surface. The interaction of Con A with the SAM 3 resulted in a shift of the reflectivity minimum to 44.84° (Figure 5c, representing a shift of 0.61° from the reflectivity minimum of the 3 SAM). This shift was used to calculate the thickness of the lectin layer bound to the carbohydrate SAM. The Con A layer was determined to be 45 Å, which is approximately the size of a monomer of the lectin (estimated to be an elliptical prolate),32 suggesting that a single layer of Con A had bound to the 3 SAM. The Con A retains its monomer size and shape, implying that the lectin does not denature upon binding to the carbohydrate SAM surface.
Figure 5. SPR reflectivity curves for (A) the gold-coated substrate, (B) the gold-coated substrate with a SAM of 3, and (C) the SAM 3 following interaction with the Con A.
and νas) were also seen, suggesting that the lectin had bound to the SAM surface. The interaction between Con A and a hydrophilic surface was also investigated. The hydrophilic surface studied was a di-undecanol disulfide [HO(CH2)11SS(CH2)11OH] SAM. The adsorption of Con A onto this hydrophilic surface was significantly reduced compared with the C16 alkanethiol SAM, as evidenced by the intensity of the absorption bands measured using the RAIRS technique (data not shown). However, although it is clear that Con A binds more readily to a hydrophobic surface, it should be noted that the binding of Con A to the C16 alkanethiol SAM is not selective because other proteins have been shown, by Whitesides et al.,35 to bind to such artificial surfaces. The adsorption of Con A to a native gold surface was also studied by SPR. The difference between the reflectivity minima for the gold-coated glass substrate (44.03°) and that of the Con A-bound substrate (44.50°) was 0.47°. This shift in the reflectivity minimum can be used to determine the thickness, using the Fresnel equations, of the Con A on the gold surface. [For the calculations, the complex refractive index (n + ik) for gold and chromium were taken to be n ) 0.236, k ) 3.385, and n ) 3.558, k ) 2.839, respectively.36] The shift of 0.47° gives a calculated thickness of the adsorbed con A of ∼27 Å. Such a thickness does not correspond with the dimensions of a monomer, dimer, or tetramer of con A, in agreement with DeBono et al.,32 and therefore it can be assumed that the lectin has denatured at the gold surface. Figure 5 shows the surface plasmon resonance reflectivity curves as a function of internal angle for (A) the blank gold-coated glass substrate, (B) a SAM of 3 on the gold surface, and (C) the Con A bound to the SAM of 3. The reflectivity minimum for the gold-coated substrate was at 44.06°. This minimum shifted to 44.23° upon formation of the 3 SAM (representing a shift of 0.17°). The resonance minimum shift of 0.17° gave a calculated thickness, using the Fresnel equations, of 14 Å for the carbohydrate 3 monolayer. Such a calculated thickness (35) Seigel, R. R.; Harber, P.; Dahint, R.; Grunze, M.; Josse, F.; Mrksich, M.; Whitesides, G. M. Anal. Chem. 1997, 69, 3321-3328. (36) Printz, M.; Sambles, J. R. J. Modern Optics 1993, 40, 20952104.
Conclusions These results show that carbohydrates can be readily formulated as SAMs on gold-coated substrates and characterized using RAIRS and SPR techniques. To build carbohydrate structures to measure nonbonded molecular recognition, it is essential that the appropriate recognition groups are available to facilitate such interactions. For the lectin-carbohydrate interaction studied in this work, the binding specificity of Con A for mannose and glucose residues stems from these carbohydrates possessing similar hydroxy configurations at the 3, 4, and 6 carbons. The axial OH group at the C2 of the mannose derivative enhances the affinity of Con A for this carbohydrate.37,38 The SAM of 3 clearly exhibited selective interaction, which would suggest that the appropriate hydroxy moieties were available to facilitate the nonbonded recognition by Con A. The RAIR spectroscopic data obtained from the three carbohydrate derivatives suggest that the orientation of each carbohydrate SAM was determined by the thiol moiety attached to the anomeric carbon of the carbohydrate ring. Therefore, the OCH2CH2SH spacer group of the SAM 3 on the gold surface would appear to orientate the carbohydrate such that the hydroxy groups of the mannose derivative were readily accessible to the Con A lectin. Although we have shown that commercially available carbohydrates can be formulated as SAMs and that the carbohydrate functionality of these derivatives can be modified in situ, if the orientation of the carbohydrate on the gold surface and the subsequent availability of appropriate recognition groups is controlled by the thiol spacer, then such structural characteristics must be considered when designing artificial carbohydrate surfaces. It is clear however, that synthetically produced SAM surfaces such as SAM 3, with specifically designed functionalities, can provide a means to mimic natural cell structures to give an insight into nonbonded molecular recognition phenomena. Acknowledgment. The authors acknowledge the financial support of the EPSRC, in the form of a studentship to DJR, and a ROPA award that supports DJB and enabled the development of the SPR instrumentation used in this work, and the MRC for a grant that supports JRK. LA9802466 (37) Derewenda, Z.; Yariv, J.; Helliwell, J. R.; Kalb(Gilboa), A. J.; Dodson, E. J.; Papiz, M. Z.; Wan, T.; Campbell, J. EMBO J. 1989, 8, 2189-2193. (38) Goldstein, I. J.; Reichert, C. M.; Misaki, A. Anals. N.Y. Acad. Sci. 1974, 234, 283-296.
