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Synthesis and Lectin Recognition of Polystyrene Core-Glycopolymer Corona Nanospheres† Takeshi Serizawa, Satoshi Yasunaga, and Mitsuru Akashi* Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering, Kagoshima University, 1-21-40 Korimoto, Kagoshima 890-0065, Japan Received November 22, 2000; Revised Manuscript Received February 27, 2001
Polymeric nanospheres with a polystyrene core and a glucosyloxyethyl methacrylate (GEMA) oligomer corona were synthesized by the free radical coplymerization of styrene (M1) plus a GEMA macromonomer (M2) at various molar ratios (M1/M2 ) 50-150) in the presence of AIBN (1 mol % to the total monomer) in an ethanol/water (3/2, v/v) solvent. The size of the nanospheres was controlled from 300 to 620 nm by altering the monomer ratio. The size distributions were significantly narrow. The amount of glucose conjugated per unit surface area of the nanosphere, which was analyzed by the anthron-sulfuric acid method, was 1.01-2.28 µg cm-1, which increased with an increase in size. The transmittance of a solution of dispersed nanospheres (the corresponding glucose concentration was 73 µM) increased by the addition of the glucosebinding protein concanavalin A (Con A) (1-50 µM), indicating that the nanospheres were being precipitated by the cross-linking of ConA. An enzyme-linked lectin assay (ELLA) revealed that Con A bound to the glucose on the nanospheres 250-700-fold more than to monomeric glucose. The binding activity to the nanospheres was less than that to a GEMA oligomer, and decreased with an increase in the amount of GEMA oligomer grafted onto the nanosphere, possibly because of steric hindrance of the lectin binding to the glucose on the nanospheres. The polystyrene core-glycopolymer corona nanosphere is a useful material for studying sugar-biomolecule recognition. Introduction The conjugation of sugars, which have diverse functions in biological systems,1 onto certain structural supports has attracted much attention because of potential applications in the biomedical material field. In particular, many researchers have conjugated sugars onto water-soluble synthetic polymers (glycopolymers);2 the chemical structure and other characteristics such as molecular weight and stereo-regularities of these glycopolymers can be systematically controlled. One significant finding was that sugar-binding proteins (lectins) preferentially bound “clustered” sugars on the polymers as compared to monomeric sugars, due to multivalent binding with the sugars. Furthermore, the binding activity seemed to be dependent on the molar content of the polymers. The activity increased with an increase in the sugar content but was reduced if the sugar content exceeded a certain value, possibly because of steric hindrance. In our research group, polymeric nanospheres with hydrophobic polymer cores and hydrophilic polymer coronas have been simply synthesized by the free radical copolymerization of hydrophilic macromonomers and hydrophobic comonomers in a polar solvent such as alcohol or a water/ alcohol mixture.3 Other research groups have also synthesized * To whom correspondence should be addressed. Telephone: +81-99-285-8320. Fax: +81-99-255-1229. E-mail: akashi@ apc.kagoshima-u.ac.jp. † The present study is part 32 in the series: Graft Copolymers Having Hydrophobic Backbone and Hydrophilic Branches. Part 31: Hiwatari, K.; et al. Polym. J. 2001, 33, 424.
similar core-corona nanospheres using the same methodology.4 The nanospheres obtained were well dispersed for a long time in an aqueous phase due to steric stabilization by the corona polymer. Their size distributions were also quite narrow and uniform. The nanospheres had highly hydrated polymer coronas in the aqueous phase, allowing the covalent conjugation of biomolecules such as proteins,5 the noncovalent loading of peptide drugs,6 and the noncovalent immobilization of metal nanoparticles as catalysts.7 For example, nanospheres with a polystyrene core and a lectin concanavalin A (Con A)-conjugated poly(methacrylic acid) corona successfully captured HIV-1 gp120 and virions from an aqueous dispersion.5 Accordingly, the corona has great potential for the conjugation of biomolecules and for analyzing the subsequent recognition processes without any decomposition of the biomolecules. Research into the conjugation of sugars onto polymeric particles8 or dendric ones9 is much less common than research into linear polymer systems. In our previous study, we conjugated lactose onto nanospheres with a polystyrene core and a poly(vinylamine) corona through an amide linkage between a lactose-lactone and the primary amine group, and analyzed the Ricinus communis agglutinin (RCA120) lectin binding.8b,c The lectin bound the lactose well, showing a suitable amount of conjugation for the recognition. Although the sugars were subsequently conjugated after the nanosphere preparation in the previous study,8b,c there is a significant advantage in the nanosphere system in that we can design the macromonomer. Selecting a specific sugar-containing
10.1021/bm000131s CCC: $20.00 © 2001 American Chemical Society Published on Web 04/27/2001
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Figure 1. Schematic representation of the GEMA nanosphere and Con A.
monomer, we can easily introduce glycopolymers onto the nanospheres as a corona. Furthermore, if we utilize such a macromonomer, we can introduce a larger amount of sugar onto the nanosphere surface as compared to the subsequent conjugation of sugars onto the nanosphere in our previous study.8b,c This difference in the conjugation process or the amount of sugar induces a different binding of the lectins to the nanosphere. In particular, it is important to analyze the lectin binding to two-dimensionally assembled glycopolymers, which were designed on the polymeric particle in the present study. In this study, we synthesized a macromonomer from sugarcontaining monomers, and copolymerized it with a styrene comonomer in order to prepare polystyrene core-glycopolymer corona nanospheres, and then characterized them. The lectin binding to the conjugated sugars was also analyzed using an enzyme-linked lectin assay (ELLA). Glucosyloxyethyl methacrylate (GEMA) was utilized as the model sugarcontaining monomer because of its easy handling. We have previously analyzed the polymerization of GEMA and the biological activities of sulfated polyGEMA as an analogue for a naturally occurring polysaccharide.10 Con A was used as the glucose-binding lectin. A schematic representation of the present system is shown in Figure 1. Experimental Section Materials. GEMA was kindly donated by Nippon Fine Chemical Co. (Takasago, Japan). The molar ratio of R and β isomers (R/β) was 3/7. Ammonium peroxodisulfate (APS) (Nacalai Tesque), 2-mercaptoethylamine hydrochloride (Wako Pure Chemical), 4-vinylbenzoic acid (VBA) (Tokyo Kasei), 3-(3-dimethylaminopropyl)-1-ethylcarbodiimide hydrochloride (EDC) (Wako Pure Chemical), anthron (Wako Pure Chemical), and 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) (Organon Teknika) were used without further purification. 2,2′-Azobis(isobutyronitrile) (AIBN) was recrystallized from methanol and dried in a vacuum. Styrene (Wako Pure Chemical) was distilled under reduced pressure in a nitrogen atmosphere. All solvents were distilled before use. Horseradish peroxidase (HRP) labeled Con A (Sigma) and poly(N-p-vinylbenzyl-Dmaltonamide) (PVMA) (Seikagaku Kougyo) were used without further purification. GEMA Oligomer Preparation. A GEMA oligomer that terminated in a primary amine was prepared by the free radical polymerization of GEMA with APS as a radical
Serizawa et al.
initiator, in the presence of 2-mercaptoethylamine as a chain transfer reagent, as shown in Scheme 1. For example, 5.0 g of GEMA and 3.9 mg of APS (1 mol % of the total monomer) plus 3.8 g of 2-mercaptoethylamine hydrochloride (twice molar excess compared to the total monomer) in 50 mL of water were placed into a 200 mL three-necked, roundbottomed flask equipped with a mechanical stirrer, condenser, and a nitrogen inlet tube. After dry nitrogen gas was bubbled into the reaction medium at 0 °C for 30 min, the mixture was incubated at 60 °C for a further 6 h. The solution was then reprecipitated into 2-propanol, and the precipitate was filtered and dried in vacuo. The reprecipitaion was repeated twice. The polymer was then dissolved in distilled water and purified by being dialyzed in distilled water with a cellulose dialyzer for 3 days. The aqueous solution obtained was lyophilized and weighed. The number-average (Mn) and weight-average (Mw) molecular weights of the oligomer were determined by gel permeation chromatography (GPC) (Tosoh HLC-8120 system with a Tosoh Column TSK-GEL) with a pullulan standard in water in the presence of 0.1 M NaCl at 40 °C. GEMA Macromonomer Preparation. The GEMA macromonomer was prepared by an amide formation between the amine group of a GEMA oligomer and the carboxyl group of VBA in the presence of EDC as an activating reagent for the latter group, as shown in Scheme 1. For example, 2.5 g of the oligomer was stirred with 0.59 g of VBA (10-fold molar excess to the oligomer) in the presence of 1.5 g of EDC (20-fold excess molar) in 225 mL of DMF at 0 °C for 24 h. After the reaction, the macromonomer was purified by being dialyzed in DMF with a cellulose dialyzer for 3 days and subsequently in distilled water for 3 days. The percentage of VBA introduced into the total oligomer was analyzed by the absorption spectra of the phenyl group using a Jasco V-550 at 200 nm in DMF with a VBA reference. GEMA Nanosphere Preparation. Nanospheres with a polystyrene core and a GEMA oligomer corona (GEMA nanosphere) were prepared by the free radical copolymerization of the GEMA macromonomer plus styrene as a hydrophobic comonomer in the presence of AIBN as a radical initiator, in an ethanol/water mixed solvent, as shown in Scheme 1. For example, the appropriate amounts of GEMA macromonomer and styrene were weighed into a glass tube with a suitable amount of AIBN (1 mol % to total monomers) and 5 mL of ethanol/water (3/2). The mixture was degassed by freeze-thaw cycles on a vacuum apparatus, sealed off, and incubated at 60 °C for 24 h. The nanospheres were purified by being dialyzed in distilled water with a cellulose dialyzer for 3 days. For the ELLA (see below), the nanospheres were centrifuged at 10000 rpm and redispersed in distilled water. This purification for the ELLA was repeated three times. The sizes of the nanospheres were analyzed by transmission electron microscopy (TEM) after the solvent was evaporated off the nanosphere dispersion on a copper mesh, and with subsequent carbon sputtering at a 20-50 nm thickness. Glucose Quantification on the Nanosphere. The amount of glucose conjugated on the GEMA nanospheres was
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Polystyrene Core-Glycopolymer Corona Nanospheres Scheme 1
quantified by the anthron-sulfuric acid method, which we reported in our previous study,8b as follows. The appropriate amount of GEMA nanospheres was treated with 2 N HCl at 80 °C for 24 h in order to release the glucose residues from the nanosphere corona by acid hydrolysis. After filtration, an adequate amount of the anthron/sulfuric acid/water (20/ 1/19, v/v/v) solution was added into the filtrate and stirred at 100 °C for 10 min. The optical density of the solution at 620 nm was measured using a UV/vis spectrometer (Jasco, V550). The amount of glucose on the nanospheres was quantified using a commercial glucose reference that had been similarly treated by the anthron reagent. Lectin Recognition. The lectin recognition activity of the GEMA nanospheres was initially analyzed by changes in the transmittance with time at 500 nm and 37 °C following the addition of the various concentrations of Con A into the nanosphere dispersion (0.1 M Tris HCl, pH 7.5, 1 mM MnCl2, 1 mM CaCl2, 1 M NaCl). The binding activity of the glucose-specific lectin Con A to the glucose on the nanospheres was also analyzed by an enzyme-linked lectin assay (ELLA) according to the literature8b,c,9 as follows. The appropriate amounts of GEMA nanospheres and HRP-labeled Con A solution (1 mM PBS) were added into a PVMAcoated well and incubated at 37 °C for 2 h. After the well was rinsed, a suitable amount of ABTS as the substrate for HRP was added, and incubated at ambient temperature for 20 min. After the reaction was stopped by the addition of sulfuric acid, the optical density (OD) at 405 nm relative to that at 600 nm was measured. The same procedure was applied to monomeric glucose as a reference. The percentage inhibition of Con A binding to PVMA by the inhibitors was estimated by the following equation: % inhibition ) (ODno inhibitor - ODwith inhibitor)/ ODno inhibitor × 100 (1) Results and Discussion GEMA Nanosphere Preparation. We prepared the GEMA oligomer with a Mn of 6300 (Mw/Mn 1.2) by the free
Table 1. Copolymerization of Styrene (M1) with GEMA Macromonomer (M2) run no.
M2 (mmol)
M1 (mmol)
1 2 3 4 5
2.17 × 10-2 1.71 × 10-2 1.50 × 10-2 1.29 × 10-2 1.14 × 10-2
1.09 1.37 1.50 1.61 1.71
M1/M2
yield (%)
Dma (nm)
SDb (nm)
CVc (%)
50 80 100 125 150
80 85 70 83 79
300 440 620 520 420
22 56 75 29 24
7 13 12 6 6
a Dm: diameter. b SD: standard deviation. c CV: coefficient of variation; SD/Dm.
radical polymerization of GEMA, with APS as a radical initiator in the presence of 2-mercaptoethylamine hydrochloride as a chain transfer reagent (yield 8.1%). We then prepared the GEMA macromonomer from the oligomer by the subsequent combination of VBA with the terminal amine group, in the presence of EDC as an activating reagent for the carboxyl group (yield 58%). The percentage of VBA introduced was only 10% to the total hydroxyl group, which means 90% of the hydroxyl groups apparently remained at the terminus of the oligomer. In this case, we used the mixture without any further purification because of the following two reasons: (1) it was very difficult to purify the mixture, and (2) we could not prepare the polymeric particles via the dispersion polymerization of styrene in the presence of the NVA oligomer only, which means that a 10% ratio of the GEMA macromonomer was sufficient to prepare the core-corona nanospheres, as has been previously reported.3,8b,c However, the preparation of macromonomers should be sophisticated for clinical applications in the future. We prepared the GEMA nanospheres by the free radical polymerization of the macromonomer plus styrene at various molar ratios with AIBN in ethanol/water (3/2, v/v), as shown in Table 1. In all cases, the reaction medium was gradually suspended with an increase in the reaction time. The yields ranged between 70 and 85%, which were reasonable when compared to nanospheres prepared using other types of macromonomers under similar conditions.3 The TEM images of the nanospheres are shown in Figure 2, indicating that the nanospheres were spherical in shape in all cases. The
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Serizawa et al. Table 2. Glucose Amount in the GEMA Nanospherea run no. 1 2 3 4 5
M1/M2b
Dm (nm)
amt of glucose per 1 cm2 of nanosphere surface (µg cm-2)
50 80 100 150 125
300 440 620 520 420
1.01 1.63 2.28 1.51 1.08
a Estimated by the anthron-sulfuric acid method. b M : styrene mono1 mer. M2: GEMA macromonomer.
Figure 2. TEM images of the GEMA nanospheres prepared at various molar ratios (styrene/GEMA macromonomer): (a) 50; (b) 80; (c) 100; (d) 125; (e) 150.
Figure 3. Mean diameters of the GEMA nanospheres against styrene/macromonomer ratios.
range of the sphere sizes and their distributions were also reasonable when compared to our other systems.3 However, their sizes were slightly larger than those of polystyrene nanoparticles prepared by the free radical polymerization of styrene in the presence of glycopolymers as an emulsifier. However, their size distributions were significantly narrower than those of the nanoparticles.8a The dependence of the particle size on the molar ratio (M1/M2) between styrene (M1) and the macromonomer (M2) in the feed was also different from our other systems,3 as shown in Figure 3. The particle size increased with an increase in the M1/M2 ratio until a ratio of 100 was reached and then decreased above that value. When we analyzed the effect of the monomer ratio using a model system of a nanosphere preparation with poly(ethylene glycol) macromonomers and hydrophobic comonomers such as styrene3j or methyl methacrylate,3k the size of the nanosphere simply increased with an increase in the styrene content. We have obtained similar results with other macromonomer systems.3 Accordingly, the present system seems to be a specific case.
Figure 4. Glucose amounts per 1 cm2 of the nanosphere surface against styrene/macromonomer ratios.
It is difficult to discuss in detail on the reason we obtained this result. However, there seems to be a specific event that occurs above a molar ratio of 125, based on previous results.3 Some of the styrene in the feed above this specific ratio might be insoluble because of its solubility in ethanol/water (3/2, v/v), although both monomers seemed to be completely solubilized before the copolymerization by visual inspection. Further research into specific nanosphere preparations is needed. Consequently, we prepared GEMA nanospheres with various sizes by altering the molar ratio between the GEMA macromonomer and the styrene. Glucose Quantification on the Nanospheres. The amount of glucose conjugated onto the GEMA nanospheres was quantified by the anthron-sulfuric acid method. These analytical data for the nanospheres are shown in Table 2. We did not observe any relationship between the monomer ratio and the amount of glucose per unit area (1 cm2) of the nanosphere surface, which was estimated from the average size of the nanospheres, with the nanosphere density assumed to be 1 g cm-3 as a polystyrene solid.11 However, the dependence of the amount of glucose on the ratio was similar to the size dependence in Figure 3 (see Figure 4). In fact, the amount of glucose increased linearly with an increase in the nanosphere size, as shown in Figure 5, in which the coefficient of variation was 0.80. Therefore, we can prepare nanospheres with a larger density of glucose on their glycopolymer corona on their surface if we can prepare larger nanospheres using the present methodology. In summary, we found that the amount of glucose on the GEMA nanospheres was dependent on the size of the nanospheres. On the other hand, the amount of glucose was 1-2 times larger than that of a closely packed graft of GEMA macromonomers with full extension on the nanospheres (considering the unit number of the macromonomers from
Polystyrene Core-Glycopolymer Corona Nanospheres
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Figure 5. Glucose amounts per 1 cm2 of the nanosphere surface against mean diameters of the GEMA nanospheres.
Figure 6. Transmittance change at 500 nm of the dispersion of the GEMA nanosphere (glucose concentration: 73 µM) by the addition of various concentrations of Con A: (a) none; (b) 1 µM; (c) 10 µM; (d) 50 µM.
its Mn), assuming the cross-sectional area of the macromonomer was the same as the glucose one (8 × 8 Å), which was estimated from a CPK model. In our current study,3l TEM images of the cross section of core-corona nanospheres embedded in a suitable resin suggested the presence of a polymer monolayer at the nanosphere surface derived of macromonomer. The thickness of this layer was similar to or less than the length of the full extension of the corresponding macromonomer, depending on its molecular weight. This means that the nanospheres were covered with a monolayer of macromonomer-derived polymer in order to minimize the surface free energy in an aqueous phase. Although it is difficult to discuss the amount of GEMA on the nanosphere, this might be a reasonable mechanism if we consider the following two possibilities: (1) the surface area of the GEMA nanospheres was actually larger than their apparent one, which was calculated only from their macroscopic sizes on the TEM images, and (2) there were two layers of the GEMA oligomer on the nanospheres. Lectin Recognition. The binding activity of Con A against the GEMA on the nanosphere was initially analyzed by the transmittance change when we added various concentrations of Con A into the nanosphere dispersion, as shown in Figure 6. Before the addition of Con A, the transmittance had not changed even after 8 h, indicating excellent dispersion stability of the nanospheres. When we added Con A into the dispersion, the transmittance gradually increased and saturated after 8 h, indicating that Con A, which has four binding sites for glucose, cross-linked the GEMA nanospheres through a sugar-lectin recognition process. Finally,
Figure 7. Binding inhibition of Con A to PVMA: (a) by a monomeric glucose; (b) by the GEMA nanosphere, of which the surface densities of glucose are 1.01 (O), 1.08 (3), 1.51 (]), 1.63 (4), and 2.28 µg cm-2 (0). The glucose concentration for ELLA of the nanosphere was estimated by assuming the homogeneous solution of glucose on the nanospheres.
almost all of the nanospheres were precipitated on the cell bottom. These changes were dependent on the concentration of Con A added. The nanospheres precipitated more rapidly with an increase in the Con A concentration, possibly because of more cross-linking points at the higher concentration. On the other hand, the transmittance decreased transiently at the initial stage over 1 h, possibly because of smaller aggregate formation. To quantitatively analyze the Con A binding to the GEMA nanospheres, we subsequently utilized an ELLA. The ELLA analyzed the binding inhibition of Con A from an aqueous phase to PVMA on a dish in the presence of exogenous inhibitors such as the GEMA nanospheres and monomeric glucose. When the degree of binding inhibition to PVMA was larger, it meant that the added sugar was preferentially recognized by Con A. The inhibition data for glucose and the GEMA nanosphere are shown in Figure 7, in which the glucose concentration in the latter inhibition data represents the apparent concentration on the nanospheres, assuming a homogeneous glucose solution. It is clear that the nanospheres effectively inhibited the Con A binding to PVMA at a lower glucose concentration than monomeric glucose, indicating the preferential binding of Con A to the glucose residues on the nanosphere. This is the so-called “positive” cluster effect. The concentration for 50% inhibition (IC50) and the relative potency against monomeric glucose are summarized in Table 3. The IC50 values of the nanospheres were 250-700-fold larger than that of monomeric glucose. The relative potency, which was calculated by normalizing to the potencies of individual Glc molecules, was dependent
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Table 3. Binding Inhibition of Con A to PVMA by Inhibitor
run no.
inhibitor
1 2 3 4 5 6 7
glucose GEMA oligomer GEMA nanosphere GEMA nanosphere GEMA nanosphere GEMA nanosphere GEMA nanosphere
amt of glucose per 1 cm2 of nanosphere surface Dm (µg cm-2) (nm)
300 436 622 523 415
1.01 1.63 2.28 1.51 1.08
Conclusion
IC50a (mM)
relative potencyb
35 0.015 0.05 0.085 0.14 0.07 0.055
1 2300 700 412 250 500 636
a The inhibitor concentration at 50% inhibition. b IC 50 of glucose/IC50 of nanosphere.
Figure 8. Relative potency of the GEMA nanospheres against monomeric glucose.
on the amount of glucose per unit area, as shown in Figure 8. Significantly, the relative potency decreased linearly with an increase in the amount of glucose. In the present system, a greater density of the GEMA oligomer on the nanospheres resulted in lower binding of Con A, possibly due to steric hindrance. In other words, if a polystyrene core-glycopolymer corona nanosphere that is preferentially recognized by a lectin is desirable, then we should prepare nanospheres with a lower density of the GEMA oligomer on the nanosphere surface and/or a smaller nanosphere size (see Figure 5). However, when we utilized a monomer ratio smaller than M1/M2 ) 30 in order to obtain smaller nanospheres (see Table 1), the nanospheres became polydispersed (CV ) 55%), although their mean size was smaller. In fact, the relative potencies of the nanospheres were always smaller than that of the GEMA oligomer, also indicating steric hindrance at the nanosphere surface. On the other hand, the relative potencies at the present glucose concentrations on the nanospheres were similar to those of R. communis agglutinin (RCA120) binding to polystyrene core-poly(vinylamine) corona nanospheres, on which lactose was subsequently immobilized after the nanosphere preparation.8b As a consequence, we found that the GEMA oligomers on the nanospheres were recognized by Con A. However, depending on the two-dimensional density of the GEMA oligomer on the surface, there were different types of cluster effects for the sugar-lectin recognition process.
We have synthesized polymeric nanospheres with a polystyrene core and a GEMA oligomer corona by the free radical copolymerization of styrene plus GEMA macromonomer in a mixed ethanol/water solvent. The size of the nanospheres was controlled by the monomer ratio, and the size distribution was quite narrow. The amount of glucose conjugated onto the nanospheres was analyzed by the anthron-sulfuric acid method and increased with an increase in the nanosphere size. The transmittance of the nanosphere dispersion increased upon the addition of the glucose-binding protein (Con A), indicating that the nanospheres were precipitated by cross-linking with this protein. An ELLA revealed that Con A preferentially recognized the glucose residues derived from the GEMA oligomer on the nanosphere surface rather than monomeric glucose. Furthermore, this binding activity increased with a decrease in the amount of glucose conjugated. We found that the closely packed glycopolymer on certain substrates (the nanospheres in the present study) showed significant sugar-lectin recognition related to the steric hindrance for the lectin binding. An ELLA did not estimate the equilibrium binding constant and/ or binding capacity of Con A to the nanospheres. Those as well as detailed structural analysis of the nanospheres will appear by using other methods in the near future. The present type of core-corona nanosphere would be useful in column chromatography for separating sugar-binding biomolecules, and for capturing or analyzing of the sugar-binding biomolecules from an aqueous phase. Acknowledgment. This work was financially supported in part by a Grant-in-Aid for Scientific Research (No. 11480259) and in the Priority Areas of “Super-Biosystem Constructed by Cognitive Multidimensional Glyco-molecules” (No.285/09240105) and of “Molecular Synchronization for Design of New Materials System” (No. 404/ 11167270) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan. This work was also financially supported in part by the project entitled “High and Ecological Utilization of Regional Carbohydrates”, through special Coordination Funds for Promoting Science and Technology (Leading Research Utilizing Potential of Regional Science and Technology) of the Science and Technology Agency of the Japanese Government, 1999. The authors would like to thank Dr. Akio Kishida and Dr. Toshiro Uchida of Kagoshima University for experimental supports and helpful discussions. References and Notes (1) (a) Rademacher, T. W.; Parekh, R. B.; Dwek, R. A. Annu. Rew. Biochem. 1988, 57, 785. (b) Varki, A. Glycobiology 1993, 3, 97. (c) Yee, Y. C.; Yee, R. T. Acc. Chem. Res. 1995, 28, 321. (d) Yamazaki, N.; Kojima, S.; Bovin, N. V.; Andre´, S.; Gabius, S.; Gabius, H.-J. AdV. Drug. DeliV. ReV. 2000, 43, 225. (2) (a) Mortell, K. H.; Gingras, M.; Kiessling, L. L. J. Am. Chem. Soc. 1994, 116, 12053. (b) Bovin, N. V.; Gabius, H.-J. Chem. Soc. ReV. 1995, 24, 413. (c) Kitano, H.; Ohno, K. Langmuir 1994, 10, 4131. (d) Roy, R. Trends Glycosci. Glycotech. 1996, 8, 79. (e) Kiessling, L. L.; Pohl, N. L. Chem. Biol. 1996, 3, 71. (f) Cao, S.; Roy, R. Tetrahedron Lett. 1996, 37, 3421. (g) Sigal, G. B.; Mammen, M.; Dahmann, G.; Whitesides, G. M. J. Am. Chem. Soc. 1996, 118, 7389. (h) Mortell, K. H.; Weatherman, R. V.; Kiessling, L. L. J. Am. Chem.
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