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Biomacromolecules 2001, 2, 22-24
Tailored Glycopolymers: Controlling the Carbohydrate-Protein Interaction Based on Template Effect Noriko Nagahori and Shin-Ichiro Nishimura* Laboratory for Bio-Macromolecular Chemistry, Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan Received May 16, 2000; Revised Manuscript Received November 2, 2000
Carbohydrate-protein interaction is one of the most important events or mechanisms for biological information transfer between cells and cell substrata.1 A series of carbohydrate-binding proteins (CBPs) existing on cell surfaces as well as glycosyltransferases have been known to mediate the initial recognition processes in biological systems by interactions with oligosaccharide receptors. Despite the importance of the specific interactions of CBPs or glycosyltransferases with glycoconjugates, interaction of these proteins with simple glycosides as ligands or acceptor substrates is commonly of low affinity (Ka ) 103-104 M-1) and the binding specificity tends to be broad.2 Actually, some families of CBPs sometimes share the same or quite similar carbohydrate-binding specificty.3 What then determines binding specificity and strength of affinity in carbohydrateprotein interactions? Is there any specific mechanism for modulating or amplifying the generally low affinity by binding of simple carbohydrates? In addition to the “glycoside cluster effect”,4 it has become a popular notion in recent years that multiple sugar side chains displayed on polymertype glycoligands remarkably enhance the binding affinity of simple monovalent sugar chains with proteins.5-9 However, it is also suggested that random clustering of carbohydrates sometimes reduce the strength of affinity with guest proteins owing to the sterically hindered sugar branches.10 Moreover, these multivalent glycopolymers usually exhibited high affinity but broad specificity with a variety of CBPs.11,12 There might be specific mechanisms on the cell surfaces for controlling topology or density of glycoconjugates suited for successful binding with CBPs. Topologically or spatially designed glycoligands are expected to be useful tools for investigating biological functions of individual CBP and for modulating lectin-mediated cell adhesions. Our attention, therefore, is now directed toward the synthesis of tailor-made glycoligands having both high affinity and tight binding specificity against guest CBP by controlling the size and the shape of the lectin-binding caVities of synthetic glycopolymer networks. A simple concept employed for displaying and immobilizing functional glycotags with appropriate spacing and flexibility in the sugar-CBP adducts is illustrated in Figure 1a. A polymerizable glycomonomer and the template lectin as guest molecule were incubated in a buffer solution and copolymerized with acrylamide and cross-linker using TEMED and APS as promoters.10,13 Subsequently, the templates were denatured and removed from the polymer complex under
Figure 1. Concept of the template-based synthesis of glycopolymers. Reagents and conditions: (i) ammonium peroxodisulfate, N,N,N′,N′tetramethylethylenediamine, 5 h; (ii) 90 °C, 10 min, sonication for 1 h, purification by gel filtration. Parts b-d represent plausible models of glycopolymers prepared in this study.
the condition mentioned in the footnote.14 In the present communication, the mannose-mannose binding protein (MBP) system was selected because this type of interaction has been found widely in nature15 and MBP-mediated cellular recognition seems to be one of the most important processes for controlling immunological systems.3 Thus, concanavalin A (ConA)16 and lens culinaris agglutinin (LCA)17 were chosen as a suited set of MBPs that exhibit quite similar binding specificity against the mannopyranose residues of a variety of glycoconjugates. Synthetic routes to polymerizable mannose derivatives 9, 15, and 24 as glycotags and a novel cross-linker 17 having excellent solubility in water are indicated in Scheme 1.18 As illustrated in the plausible models of polymers (Figure 1), controls such as simply cross-linked glycopolymers obtained by copolymerization without template (Figure 1c) and linear glycopolymers (Figure 1d) were also prepared in order to demonstrate the significance of the template effect in the polymerization process. Here, ConA(+) polymer represents a glycopolymer prepared in the presence of ConA as a
10.1021/bm0000438 CCC: $20.00 © 2001 American Chemical Society Published on Web 12/28/2000
Biomacromolecules, Vol. 2, No. 1, 2001 23
Communications Scheme 1. Synthetic Routes to (a) 9, 15, and 17a and (b) 24b
a Reagents and conditions: (i) BnNH (1.5 equiv), THF, room temperature, 12 h, 96%; (ii) CCl CN (6 equiv), DBU (0.5 equiv), CH Cl , -10 °C f room 2 3 2 2, temperature, 2 h, 88 %; (iii) NaN3 (1.05 equiv), NaI (0.1 equiv), EtOH, reflux, 48 h; (iv) BF3OEt2 (47%) (0.2 equiv), CH2Cl2,, 4 Å molecular sieves, 0 °C f room temperature, 1.5 h, 60%; (v) p-TsOH‚H2O (1 equiv), Pd-C, H2 gas, MeOH, room temperature, 3 h; (vi) acryloyl chloride (1.2 equiv), triethylamine (2.2 equiv), THF:MeOH (1:1), 0 °C, 38% from 6; (vii) NaOMe (0.2 equiv), MeOH, room temperature, 2.5 h, q.y.; (viii) NaHCO3 (4 equiv), Z-Cl (1.1 equiv), H2O, 0 °C, 8 h, 95%; (ix) BF3OEt2 (47%) (0.2 equiv), CH2Cl2,, 4 Å molecular sieves, 0 °C f room temperature, 8 h, 60%; (x) Pd-C, H2 gas, MeOH, room temperature, 20 h; (xi) acryloyl chloride (1.2 equiv), triethylamine (1.8 equiv), THF:MeOH (1:1), 0 °C, 60% from 12; (xii) NaOMe (0.2 equiv), MeOH, room temperature, 4 h, q.y.; (xiii) acryloyl chloride (2.4 equiv), triethylamine (2.4 equiv), THF, 0 °C, 82%. b Reagents and conditions: (i) NaOMe (0.2 equiv), MeOH, room temperature, 5 h, q.y.; (ii) triethyl orthobenzoate (2.6 equiv), p-TsOH‚H2O (0.06 equiv), TFA (0.08 equiv), CH3CN, room temperature, 20 min, then evaporated and (iii) TFA (90% aq) (10 equiv), CH3CN, room temperature, 30 min, 37% for 19, 35% for 20; (iv) 3 (2.4 equiv), BF3‚OEt2 (47%) (0.2 equiv to 3), CH2Cl2,, 4 Å molecular sieves, 0 °C f room temperature, 10 h, 49%; (v) Pd-C, H2 gas, MeOH, room temperature, 48 h; (vi) acryloyl chloride (1.2 equiv), triethylamine (2.2 equiv), THF:MeOH (1:1), 0 °C, 40% from 21; (vii) NaOMe (0.4 equiv), MeOH, room temperature, 48 h, q.y.
Table 1. Ka Values (M-1) of Glycopolymers with ConA
Ka (M-1) glycotag
linear polymer
ConA(+) polymer
ConA(-) polymer
template effecta
9 24
1.2 × 104 1.9 × 107
1.8 × 104 3.3 × 108
1.8 × 102 1.1 × 106
100 300
a
Template effect ) Ka(ConA(+))/Ka(ConA(-)).
template molecule. On the other hand, ConA(-) polymer means a control compound prepared without ConA as shown in Figure 1c. The binding constants of glycopolymers with lectins were determined and evaluated by a fluorescence spectroscopic method.10,19 Table 1 shows Ka values of linear polymers, ConA(+) polymers, and ConA(-) polymers with ConA in
which the compounds 9 and 24 were employed as glycotags. Comparing with linear polymers, randomly cross-linked polymers [ConA(-) polymers] apparently reduced their affinity with ConA. However, ConA(+) polymers retained the original affinity with template lectin to almost the same level as those of linear polymers though they were also crosslinked. Moreover, ConA(+) polymer derived from mannotrisaccharide monomer 24 showed higher affinity with the guest than that of the linear glycopolymer. Here, Ka(ConA(+))/Ka(ConA(-)) was defined as the “template effect” in the case of the interaction with ConA, and the values were calculated to be 100 (glycotag 9) and 300 (glycotag 24), respectively. These results suggest that specific cavities of glycopolymers for the guest proteins were constructed by the “template effect” during the polymerization reactions.
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Biomacromolecules, Vol. 2, No. 1, 2001
Communications
Table 2. Ka Values (M-1) and Selectivity Coefficient of the Tailored Glycopolymers
Ka (M-1) for glycopolymers a guest
keff (selectivity coeff)
ConA LCA
ConA(+)
LCA(+)
9.6 × 7.5 × 106 12.8b
1.5 × 105 1.3 × 107 87c
107
Supporting Information Available. Text giving experimental details and characterization data, figures showing NMR spectra and fluorescence dependency, and tables of sugar ratios and molecular weights. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes
a
Glycomonomer 15 was used as a glycotag in both cases. ConA(+) polymer was prepared in the presence of ConA as a template molecule and LCA(+) polymer was prepared in the presence of LCA as a template molecule. b Keff ) Ka(ConA)/Ka(LCA). c Keff ) Ka(LCA)/Ka(ConA).
ConA(+) polymer derived from mannotrisaccharide monomer 24 showed much higher affinity with the guest protein than ConA(+) polymer prepared from monosaccharide monomer 9, suggesting that the “strength of the affinity” in the glycopolymer-protein interaction strongly depends on the strict oligosaccharide structures. Next, our interest was focused on the versatility of this procedure to create tailored glycopolymers that discriminate CBP used as template from other CBPs with a similar carbohydrate-binding specificity. Thus, LCA(+) polymer was prepared according to the procedure described above and employed for further binding assay. Ka values of glycopolymers [ConA(+) polymer and LCA(+) polymer] with ConA or LCA were measured and the results are listed in Table 2. While ConA(+) polymer exhibited preferential affinity with ConA to LCA, LCA(+) polymer had also a much higher affinity with LCA than with ConA. A selectivity coefficient expressed as Keff (Ka with template lectin/Ka with control lectin) helped us to understand this controlled interaction between glycopolymers and lectins. Keff values of the ConA(+) polymer and LCA(+) polymer were estimated to be 5.8 and 87, respectively. The results indicate that the “binding selectivity” of the glycopolymer is deeply dependent on the size and shape of the template as well as sugar-binding specificity. From these results, it is clearly demonstrated that novel type of glycoligands having both high selectivity and strong affinity with target CBP can be designed by means of template-based polymerization strategy. The present communication reports very preliminary results of the first example of the template-imprinted water-soluble polymers that can specifically recognize proteins.20,21 Further structural characterization of the specific cavity in an aqueous solution as well as the versatility of this method is under investigation, and the results will be reported as soon as possible. Acknowledgment. This work was partly supported by a grant from NEDO (New Energy and Industrial Technology Development Organization, Japan). We appreciate valuable comments and suggestions by Dr. Y. C. Lee of the Johns Hopkins University. We also thank Ms. A. Maeda, Ms. H. Matsumoto, and Ms. S. Oka of the Center of Instrumental Analysis, Hokkaido University, for measuring elemental analysis and mass spectroscopy data.
(1) Dwek, R. A. Chem. ReV. 1996, 96, 683-720. (2) Weis, W. I.; Drickamer, K.; Hendrickson, W. A. Nature 1992, 360, 127-134. (3) Lee, Y. C.; Lee, R. T. Acc. Chem. Res. 1995, 28, 321-327. (4) Lee, Y. C. FASEB J. 1992, 6, 3193-3200. (5) Nishimura, S.-I.; Matsuoka, K.; Kurita, K. Macromolecules 1990, 23, 4182-4184. (6) Nishimura, S.-I.; Lee, Y. C. Synthetic glycopolymers: New tools for glycobiology, In Polysaccharides: Structural DiVersity and Functional Versatility; Dumitriu, S., Ed.; Marcel Dekker Inc.: New York, 1998; p 523-537. (7) Roy, R.; Pon, R. A.; Tropper, F. D.; Andersson, F. O. J. Chem. Soc., Chem. Commun. 1993, 264-265. (8) Mammen, M.; Dahman, G.; Whitesides, G. M. J. Med. Chem. 1995, 38, 4179-4190. (9) Zeng, X.; Murata, T.; Kawagishi, H.; Usui, T.; Kobayashi, K. Carbohydr. Res. 1998, 312, 209-217. (10) Furuike, T.; Nishi, N.; Tokura, S.; Nishimura, S.-I. Macromolecules 1995, 28, 7241-7247. (11) Nishimura, S.-I.; Matsuoka, K.; Furuike, T.; Ishii, S.; Kurita, K.; Nishimura, K. Macromolecules 1991, 24, 4236-4241. (12) Matsuoka, K.; Nishimura, S.-I. Macromolecules 1995, 28, 29612968. (13) Copolymerization was carried out according to the condition reported previously10 [template:glycomonomer:acrylamide:cross-linker ) 1:100: 2000:10 (mol)]. The composition of polymers was calculated from the integration of the 1H NMR spectra. The average molecular weights of polymers were estimated by gel permeation chromatography (GPC) and the values were in the range from 33 to 44 kD. (14) The reaction mixture was boiled for 10 min to denature the template protein, and then 5 M NaCl (1 mL) was added. The solution was sonicated for 1 h and purified by chromatography on Sephadex G-25. It was demonstrated that this procedure gave template-free glycopolymers by using fluorescence-labeled lectin as guest molecule. Proton and 13C NMR spectra of glycopolymers also showed no significant signals due to the contamination by protein. (15) Kawasaki, N.; Kawasaki, T.; Yamashina, I. J. Biochem. 1983, 94, 937-947. (16) Peeks, G. N.; Becker, J. W.; Edelman, G. M. J. Biol. Chem. 1975, 250, 1525-1547. (17) Foriers, A.; Lebrun, E.; Rapenbusch, R. V.; Neve, R.; Strosberg, A. D. J. Biol. Chem. 1981, 256, 5550-5560. (18) All new compounds gave satisfactory elemental and spectroscopic analytical data. (19) Fluorescence emission of lectins at 340 nm by excitation at 280 nm was measured. Changes in the intensities of the emission spectra by varying the concentration of glycopolymers were carefully measured and the affinity constants were estimated by using the Steck-Wallach equation. (20) Polysaccharides have been used for the preparation of the templateimprinted nanostructured surfaces; Shi, H.; Tsai, W.; Garrison, M. D.; Ferrari, S.; Ratner, B. D. Nature 1999, 398, 593-597. (21) Polymer gels have also been discussed as a potent candidate for the protein-like polymers that can recognize and recover molecules; Tanaka, T.; Wang, C.; Pande, V.; Grosberg, A. Y.; English, A.; Masamune, S.; Gold, H.; Levy, R.; King, K. Faraday Discuss. 1996, 102, 201-206.
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