Optimization of the conditions for the immobilization of glycopolypeptides on hydrophobic silica particulates and simple purification of lectin using glycopolypeptide-immobilized particulates.

ABSTRACT

Glycopolypeptide-immobilized particulates exhibit high binding selectivities and affinities for several analytes. However, to date, the conditions for the synthesis of glycopolypeptide-immobilized particulates have not been optimized and the application of these particulates as carriers for affinity chromatography has not been reported. Accordingly, herein, as a model compound for determining the optimal conditions for the immobilization of an artificial glycopolymer on hexyl-containing hybrid silica particulates (HSPs), the glycopolypeptide poly [GlcNAcß1,4GlcNAc-ß-NHCO-(CH2)5NH-/CH3(CH2)9NH-/γ-PGA] (3) containing multivalent chitobiose moieties and multivalent decyl groups with a γ-polyglutamic acid backbone was synthesized. Immobilization of 3 on HSPs under each condition was evaluated by a lectin-binding assay using wheat germ (Triticum vulgaris) agglutinin (WGA), which is an N-acetylglucosamine-binding lectin. As a result, the optimal immobilization conditions for HSPs at 25 mg/mL were obtained at dimethyl sulfoxide (DMSO) concentration of reaction solvent in the range of 1(DMSO)9(water) to 4(DMSO)6(water) and a compound 3 concentration in the range of 125 nM-1250 nM. Furthermore, the influence of the alkyl group structure introduced into glycopolypeptide for imparting hydrophobicity to it on the immobilization of glycopolypeptide on HSPs was investigated. As a result of comparing three types, poly [GlcNAcß1,4GlcNAc-ß-NHCO-(CH2)5NH-/γ-PGA] (1) with no alkyl group, poly [GlcNAcß1,4GlcNAc-ß-NHCO-(CH2)5NH-/CH3(CH2)4NH-/γ-PGA] (2) with a pentyl group, and 3 with a decyl group, 3 showed the best immobilization efficiency on HSPs. Finally, 1 mg 3-immobilized HSPs prepared under the optimum conditions adsorbed approximately 7.5 µg WGA in a structure-specific manner. We also achieved a simple WGA purification from raw wheat germ extract as a practical example using 3-immobilized HSPs. We believe that in the future, these glycopolypeptide-immobilized particulates will be used not only for the purification of plant lectins, but also as specific adsorbents for various lectins-like substances such as in vivo lectins, pathogenic viruses, and toxin proteins.