High-density Glycosylation Of Microporous Polypropylene Membrane Surface Via Click Chemistry For Protein Separation

Affinity membrane is a new developed technology for the isolation and purification of biomolecules, which combines the advantages of convection mass transfer of membrane separation with the high sepcific recognition function of affinity chromatography. Microporous polypropylene membrane (MPPM) is a promising membrane, which has been widely used due to its high stability, well-controlled pores and low cost. On the other hand, carbohydrates (saccharides) can specifically recognize protein acceptors by carbohydrate-protein interactions. The selectivity and binding strength between saccharide and protein can be enhanced by the formation of glycoside cluster effect. Therefore, it is of great interesting to immobilize carbohydrates (saccharides) on the membrane surface at a high density for developing a new glycosylated affinity membrane, which can be used to the selective separation and purification of proteins.Considerable advances have been made with respect to the strategies of glycosylation on the polymer surfaces. However, a major disadvantage is the low reaction which yield limits the density of saccharide on the surface, and then suppresses the formation of glycoside cluster effect and decreases the selectivity and binding strength between saccharide and their protein acceptors. Therefore, the identification of an effective reaction is the critical determinant for fabrication of the glycosylated membrane surface with a high glycosyl density. Cu (I)-catalyzed 1,3-dipolar cycloaddition of azides and alkynes (click chemistry) has advantages of high yield and specificity, which has allowed a quantitative introduction of glycosyl groups onto polymeric surfaces. Therefore, click chemistry is introduced to fabricate glycosylated surfaces or carbohydrates-decorated surfaces with high density of saccharides which is used to mimick the "glycoside cluster effect" and enhance the binding strength and selectivity between carbohydrate ligands and their protein acceptors.In this thesis, versatile methods were developed to prepare glycosylated MPPM with high glycosyl density. The specific studies are mainly concentrated on the following aspects:1. Acrylic acid was grafted on MPPM by UV-induced graft polymerization. Subsequently, the glycosylated membrane was constructed by the coupling reaction between carboxyl group and amino-glucose derivative with the aid of EDC/NHS. The reaction conditions were optimized to improve the reaction efficiency. However, it is always lower than 40%, which is not beneficial to the formation of glycoside cluster effect.2. Cu (I)-catalyzed 1,3-dipolar cycloaddition of azides and alkynes was used to constructed glycosylated MPPM with a high glycosyl density. It is promising that the surface glycosyl density can be well controlled in a wide range and the maximum value is over 10μmol/cm2. The recognition capability of these glycosylated MPPMs to lectins indicates the occurrence of the "glycoside cluster effect" when the glycosyl density on the membrane surface exceeds 0.20μmol/cm2. However, the triazole moieties derived from this azide/alkyne click reaction seem to engage in hydrogen bonding and stacking interactions with the amino acid residues of proteins. These unexpected interactions unavoidably cause the non-specific adsorption of proteins, which decreases the recognition capability between the glycosylated membrane surface and its protein receptor.3. High-density glycosylated MPPM surface was successfully constructed by radical-mediated thiol-yne click chemistry. It shows that the glucosylated MPPM can specifically recognized Con A (Concanavalin A) rather than PNA (peanut agglutinin). In addition, the thiol-yne click chemistry shows the advantage for constructing glycosylated membrane surface with affinity adsorption towards corresponding lectin receptor and without non-specific interaction between the thioether groups and proteins.4.3-(Trimethylsilyl) propargyl methacrylate was synthesized and subsequently used to tether on the MPPM surface by the plasma pretreatment combined with UV-induced graft polymerization. The alkyne groups on the surface can be directly transfered into high-density glycosylated pendants by thiol-yne click chemistry. However, the efficiency of this click chemistry is lower than 55% due to the heterogeneous characteristic of reaction on the membrane surface. Moreover, the steric hindrance of glucose pendants increases the difficulty of bimolecular addition reaction with alkyne. The specific recognition properties of the glucosylatd surface were estimated and showed selective affinity adsorption capability to Con A.5. The high-density glycosylated MPPMs constructed by click chemisty were used as affinity membranes for Con A separation on the basis of the specific interactions between saccharide ligands and their lectin acceptors. The binding capacity of Con A on the glucosylated MPPM increases with glycosyl density. The Con A adsorbed onto glucosylated MPPM surface can be effectively desorbed by 1 M HAc.