Preparation Of Boronate Affinity Monoliths Based On Different Matrix And Its Application Forspecific Capture Of Glycoproteins

The thesis is involved in the preparation of boronate affinity monolith based on different matrix,and their applicationsfor specific capture of glycoproteins.It consists of five chapters.In chapter one, the general introduction include the development of boronate affinity technique and monolithic column, and the challenges during the researches. And the developing history and the preparation methods of boronate affinity monolith were introducted. Additionally, the aim, significance and innovation of this thesis were also presented.In chapter two, synthesis and application of a macroporous boronate affinity monolith using a metal-organic gel as a porogenic template for the specific capture of glycoproteins. Amacroporous boronate affinity monolithic column was prepared using3-acrylamidophenylboronic acid (AAPBA) as organic monomer,ethylene dimethacrylate (EDMA)as crosslinker, and metal-organic gels (MOGs) as porogenic template.The poly(AAPBA-co-EDMA) monoliths were synthesized in stainless steel columns by in-situ polymerization. In contrast to traditional porogenic methods,the new explored application of MOGs has proven to be a more convenient method for the formation of macropores. To fabricate the macroporous structure with uniformed open-channel network, the preparation conditions were systematically investigated including the ratio of monomers, reaction temperature and concentration of the MOGs. The prepared macroporous monoliths were characterizedby scanning electron micrographic (SEM), Fourier-transform infrared (FTIR) and mercury intrusion porosimeter, and applied to specific capture of cis-diol containing molecules.Horseradish peroxidase (HRP) and transferrin (TF) were chosen as test glycoproteins, and the chromatographic analysis demonstrated that the macroporous boronate affinity monoliths exhibited highspecificity and better dynamic binding capacity toward glycoproteins. The resulted poly(AAPBA-co-EDMA) affinity monolith was successfullyapplied to specifical capture of TF from a bovine serum sample.In chapter three, a boronate-silica hybrid affinity monolith was prepared for specific capture of glycoproteins at neutral pH condition. The monolith was synthesized via a facile "one-pot" procedure in a stainless steel column by concurrently mixing hydrolyzed alkoxysilanes tetramethoxysilane (TMOS) and vinyltrimethoxysilane (VTMS), organic monomer3-acrylamidophenylboronic acid (AAPBA) and initiator2,2’-azobisisobutyronitrile (AIBN) together. The polycondensation of alkoxysilanes and copolymerization of organic monomer and vinyl-silica monolith were carried out successively by reacting at different temperature. After optimizing the preparation conditions, the resulted hybrid affinity monolith was systematically characterized and exhibited excellent affinity to both cis-diol-containing small molecules and glycoproteins at neutral and physiological pH, including adenosine, horseradish peroxidase, transferrin and ovalbumin. The binding capacity of ovalbumin on monolith was measured to be2.5mg·g-1at pH7.0. Furthermore, the hybrid affinity monolith was applied to the separation of transferrin from bovine serum sample at a physiological condition. Good repeatability was obtained and the relative standard deviations (RSDs) of retention time was1.15%and4.77%(n=5) for run-to-run and column-to-column, respectively.In chapter four, anovel strategy for preparation of hybridboronate affinity monolith was developed by utilizing Cu(I)-catalyzed1,3-dipolar azide-alkyne cycloaddition reaction (CuAAC) of azide-functionalized monolithic intermediate andalkyne-boronate ligand.A "Single-Step" procedurewas first employed to synthesize azide-functionalized hybrid monolith to provide reactive sites for click chemistry,then the alkyne-boronate ligands were covalently immobilized on the azide-functionalized hybrid monolith via in-column CuAAC reaction under mild conditions to form "Clicked"hybrid boronate affinity monolith. The prepared boronate affinity monolith was characterized and evaluated by elemental analysis, Fourier-transform infrared spectroscopy, scanning electron microscopy. The resulted hybrid boronate affinity monolith exhibited excellent specificity toward nucleosides and glycoproteins under neutral conditions. The binding capacity of glycoprotein ovalbumin at pH7.0was measured to be2.36mg·g-1. The practicability of the boronate affinity hybrid monolithic material was demonstrated by specific capture of the glycoproteins ovalbumin and ovotransferrin from an egg sample.In chapter five, a novel strategy for preparation of organic-inorganic hybrid boronate affinity monolith was developed by thiol-eneclick chemistry. A mercaptopropyl-modified silica monolith was first synthesized via sol-gel processby in situ co-condensation using tetramethoxysilane (TMOS) and3-mercaptopropyltrimethoxysilane (MPTMS) as precusors, then the3-acrylamidophenylboronic acid (AAPBA)was covalently immobilized on the hybrid monolith via the thiol-eneclick reaction to form AAPBA-silica hybrid affinity monolith. The reaction conditions for the preparation of AAPBA-silica hybrid affinity monolith were carefully optimized, including the ratio of TMOS to MPTMS, the content of poly(ethylene glycol)(PEG) and methanol. The morphology and mechanical stability of the boronate affinity monolith was characterized and evaluated by scanning electron microscopy and fourier-transform infrared spectroscopy. The obtained boronate affinity hybrid monolith exhibited excellent specificity toward cis-diols containing nucleosides under neutral conditions, and was further applied to specific capture of the glycoproteins ovalbumin and horseradish peroxidase.