Synthesis and evaluation of affinity adsorbents for glycoproteins: an artificial lectin

A combination of rational design based on mimicking natural protein-carbohy drate interactions and solid-phase combinatorial chemistry has led to the i dentification of an affinity ligand which displays selectivity for the mann ose moiety of glycoproteins. The ligand, denoted 18/18 and comprising a tri azine scaffold bis-substituted with 5-aminoindan, has been synthesised in s olution, characterised by TLC, H-1-NMR and MS. When immobilised to amine-de rivatised agarose at concentrations >24 mu mol/g moist weight gel, ligand 1 8/18 selectively binds glucose oxidase. The adsorbed enzyme was quantitativ ely eluted with 0.5 M alpha-D-methyl-mannoside and to a lesser extent with the equivalent glucoside. An investigation of the comparative retention tim es of saccharidic solutes showed that significant retardation was observed for alpha-D-mannose, mannobiose and mannan, with little or no evidence for selective retention of other saccharides, with the exception of alpha-L-fuc ose. Interestingly, alpha-L-fucose and alpha-D-mannose share an identical c onfiguration of the hydroxyl groups on C-2, C-3 and C-4. Analysis of Scatch ard plots from partition equilibrium studies on the interaction of glucose oxidase and the p-nitrophenyl-glycosides of D-mannose, D-glucose, L-fucose and D-galactose with immobilised 18/18 establish that the affinity constant s (K-AX) for the enzyme, the glycosides of mannose, glucose and fucose, and the p-nitrophenyl-galactoside are 4.3x10(5) M-1, 1.9x10(4) M-1 and 1.2x10( 4) M-1 respectively. H-1-NMR studies on the interaction of alpha-D-methyl m annoside with ligand 18/18 in solution confirm the involvement of the hydro xyl group in the C-2 position. Molecular modelling suggests the formation o f four hydrogen bonds between the hydroxyl groups at positions C-2, C-3 and C-4 of alpha-D-methyl-mannoside and the bridging and ring nitrogen atoms o f the triazine scaffold, with aromatic stacking of a second ligand against the carbohydrate face. The greater specificity of ligand 18/18 for mannose and glucose than for galactose parallels that exhibited by concanavalin A. (C) 2000 Published by Elsevier Science B.V.