Recognition Specificity of Neoglycopolymers Prepared by Ring-Opening Metathesis Polymerization

Protein-saccharide interactions facilitate fundamental cellcell recognition events in processes as diverse as host-pathogen interactions, fertilization, development, and the mounting of an immune response.1 Despite their importance in cell recognition, individual protein-carbohydrate interactions are generally weak (i.e., with Ka ) 103-104 M-1).2 Additionally, carbohydratebinding proteins often exhibit broad recognition specificity because they generally bind a group of related saccharide structures.3 To enhance the strength of cell surface binding, Nature often assembles multiple protein-saccharide complexes to provide the necessary avidity.2,4 In biological systems, both affinity and specificity are critical components of cell-cell recognition. Proteins that mediate cell adhesion must select their biologically-relevant ligand in an environment containing other sugars for which they have some affinity (Figure 1). For example, influenza viral hemagglutinins bind preferentially to cells displaying either 2,3or 2,6-linked sialic acid residues, depending on the viral strain.5 If multivalent interactions contribute to the avidity of biological recognition processes, what is their effect on specificity? We are investigating the functional properties of a new class of polyvalent carbohydrate ligands, substances prepared by ringopening metathesis polymerization (ROMP).6,7 We demonstrated that such saccharide-substituted polymers act as polyvalent ligands for the mannose/glucose-binding protein concanavalin A.6 Moreover, these materials function as high-avidity ligands in cell agglutination assays. To examine the effect of multivalent binding on the specificity of protein-carbohydrate interactions, we synthesized several carbohydrate-bearing polymers and tested them for their ability to bind to concanavalin A. The inhibitory properties of the polyvalent ligands were compared with those of the corresponding monosaccharides to ascertain the effects of multivalent presentation on affinity and specificity. Concanavalin A can engage in monoand multivalent binding, rendering the lectin well suited for investigating structure/function relationships of both ligand classes.8 Concanavalin A recognizes R-glucoseand R-mannose-containing saccharides, with the manno configuration at C2 preferred.9 The protein causes agglutination of red blood cells through interaction with cell surface glycoproteins. Multiple concanavalin A-saccharide complexes are required for hemagglutination, but agglutination can be inhibited by either monovalent or polyvalent glucose and mannose derivatives.6,8,10 To probe the intrinsic affinity13 and specificity of concanavalin A, pairs of monovalent ligands of differing affinities for the protein were selected. For example, methyl R-D-mannopyranoside binds with 4-fold-higher affinity than does methyl R-Dglucopyranoside.8,11 The specificity of interaction of concanavalin A with R-C-allyl glycosides is diminished; the free energies of binding for R-C-allyl glucoside 1 (-4.7 kcal/mol) and R-Callyl mannoside 2 (-4.9 kcal/mol) differ by only 0.2 kcal/mol.12 Similarly, the difference between the binding free energies of R-O-ethyl mannopyranoside 6 and R-O-ethyl glucopyranoside 5 is 0.8 kcal/mol, but the binding free energies of the related R-C-propyl mannoside 4 and R-C-propyl glucoside 3 are indistinguishable.12 Thus, concanavalin A binds both Oand C-glycosides of the R-configuration, but higher diastereoselectivity is observed in the O-glycoside series. To elucidate the effect of multivalent presentation on the functional affinity13 of these ligands, we determined the inhibitory properties of mannoseand glucose-derived neoglycopolymers for concanavalin A-facilitated agglutination. Polyvalent Cand O-glycoside derivatives of the gluco or manno configuration were generated via an aqueous ROMP (Figures 2 and 3). The corresponding bicyclic oxanorbornene derivatives6 were polymerized to afford glucoseand mannose-substituted Cglycoside neoglycopolymers 9 and 10. To decrease the amount of residual metal in the resulting carbohydrate-substituted materials, the polymerization was effected using a catalyst solution derived from mixing the oxanorbornene substrate and RuCl3 in water.14,15 Application of this general strategy afforded the corresponding R-O-glucoside and R-O-mannoside polymers 11 and 12. For each polymer, the backbone was produced as a 1:1 ratio of cis and trans alkene isomers. The four resulting neoglycopolymers were subjected to gel filtration chromatography. By comparison of their migratory aptitudes with those of dextran standards, a relative molecular mass (Mr) of approximately 106 was obtained for each. Neoglycopolymers 9-12 were tested for their specificity as multivalent ligands for the glucose/mannose binding lectin con(1) For an overview of the biological roles of carbohydrates, see: Varki, A. Glycobiology 1993, 3, 97-130. (2) Lee, Y.; Lee, R. Acc. Chem. Res. 1995, 28, 321-7. (3) For example, E-, P-, and L-selectin all bind to the tetrasaccharide sialyl Lewis x; see: Berg, E. L.; Robinson, M. K.; Mansson, O.; Butcher, E. C.; Magnani, J. L. J. Biol. Chem. 1991, 266, 14869-72. (4) Drickamer, K. Nature: Struct. Biol. 1995, 2, 437. (5) (a) Rogers, G. N.; Paulson, J. C.; Daniels, R. S.; Skehel, J. J.; Wilson, I. A.; Wiley, D. C. Nature 1983, 304, 76. (b) Sauter, N. K.; Bednarski, M. D.; Wurzburg, B. A.; Hanson, J. E.; Whitesides, G. M.; Skehel, J. J.; Wiley, D. C. Biochemistry 1989, 28, 8388. (6) Mortell, K. H.; Gingras, M.; Kiessling, L. L. J. Am. Chem. Soc. 1994, 116, 12053-4. For a method of neoglycopolymer preparation by ROMP using defined ruthenium catalysts, see: Fraser, C.; Grubbs, R. H. Macromolecules 1995, 28, 7248-55. (7) For alternative synthetic strategies to polyvalent carbohydrate-substituted materials, see: (a) Schnarr, R. L.; Weigel, P. H.; Kuhlenschmidt, M. S.; Lee, Y. C.; Roseman, S. J. Biol. Chem. 1979, 253, 7940-51. (b) Kingery-Wood, J. E.; Williams, K. W.; Sigal, G. B.; Whitesides, G. M. J. Am. Chem. Soc. 1992, 114, 7303-5. (c) Roy, R.; Zanini, D.; Meunier, S. J.; Romanowska, A. J. Chem. Soc., Chem. Commun. 1993, 1869-72. (d) Spevak, W.; Nagy, J. O.; Charych, D. H.; Schaefer, M. E.; Gilbert, J. H.; Bednarski, M. D. J. Am. Chem. Soc. 1993, 115, 1146-7. (e) Kiessling, L. L.; Pohl, N. L. Chem. Biol. 1996, 3, 71-77 and references cited therein. (8) Bittiger, H. ConcanaValin A as a Tool; John Wiley & Sons, Ltd.: London, 1976. (9) Goldstein, I. J. Carbohydrate Binding Specificity of ConcanaValin A; Goldstein, I. J., Ed.; John Wiley & Sons, Ltd: London, 1976; pp 5565. (10) For a study of naturally-occurring polyvalent saccharides, see: Mandal, D. K.; Brewer, C. F. Biochemistry 1993, 32, 5116-20. (11) Chervenak, M. C.; Toone, E. J. Biochemistry 1995, 34, 5685-95. (12) Weatherman, R. V.; Kiessling, L. L. J. Org. Chem. 1996, 60, 5348. (13) For a discussion of intrinsic and functional affinities for multivalent systems, see: Greenspan, N. S.; Cooper, L. J. N. Springer Semin. Immunopathol. 1993, 15, 275-91. (14) Novak, B. M.; Grubbs, R. H. J. Am. Chem. Soc. 1988, 110, 75423. (15) For experimental details, see the supporting information. Figure 1. Multivalent protein-carbohydrate interactions may enhance the strength and specificity of cell-cell binding in an environment containing other ligands. 2297 J. Am. Chem. Soc. 1996, 118, 2297-2298