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

A combination of rational design based on mimicking natural protein–carbohydrate interactions and solid-phase combinatorial chemistry has led to the identification of an affinity ligand which displays selectivity for the mannose moiety of glycoproteins. The ligand, denoted 18/18 and comprising a triazine scaffold bis-substituted with 5-aminoindan, has been synthesised in solution, characterised by TLC, 1H-NMR and MS. When immobilised to amine-derivatised agarose at concentrations >24 μmol/g moist weight gel, ligand 18/18 selectively binds glucose oxidase. The adsorbed enzyme was quantitatively eluted with 0.5 M α-d-methyl-mannoside and to a lesser extent with the equivalent glucoside. An investigation of the comparative retention times of saccharidic solutes showed that significant retardation was observed for α-d-mannose, mannobiose and mannan, with little or no evidence for selective retention of other saccharides, with the exception of α-l-fucose. Interestingly, α-l-fucose and α-d-mannose share an identical configuration of the hydroxyl groups on C-2, C-3 and C-4. Analysis of Scatchard 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 constants (KAX) for the enzyme, the glycosides of mannose, glucose and fucose, and the p-nitrophenyl-galactoside are 4.3×105 M−1, 1.9×104 M−1 and 1.2×104 M−1 respectively. 1H-NMR studies on the interaction of α-d-methyl-mannoside with ligand 18/18 in solution confirm the involvement of the hydroxyl group in the C-2 position. Molecular modelling suggests the formation of four hydrogen bonds between the hydroxyl groups at positions C-2, C-3 and C-4 of α-d-methyl-mannoside and the bridging and ring nitrogen atoms of 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.