【作者】 杨帆；

【导师】 张玉奎；

【作者基本信息】 南开大学 ， 分析化学， 2013， 博士

【摘要】 本工作的主要内容是几种有机聚合物基质及有机-无机杂化基质硼酸亲和整体柱的制备,及其在糖蛋白的分离和富集中的应用。论文内容包括如下五部分：第一部分：绪论。综述了硼酸亲和技术以及整体柱技术的发展情况及其面临的挑战；对结合两者优势的硼酸亲和整体柱的发展现状和制备技术进行了综述。此外,还对本论文的研究意义、研究内容以及课题的创新性进行了简要的介绍。第二部分：以金属有机凝胶为致孔模板的大孔型硼酸亲和整体柱的制备及其应用。以金属有机凝胶(MOGs)作为新颖的致孔模板,3-丙烯酰胺基苯硼酸(AAPBA)作为硼酸配基,乙二醇二甲基丙烯酸酯(EDMA)作为交联剂,在不锈钢柱内原位聚合制备了大孔型有机基质的poly(AAPBA-co-EDMA)亲和整体柱。以MOGs致大孔的新颖方法比传统的致孔方法更加简易方便。通过系统地优化反应条件,包括单体比例、反应温度、MOGs用量,制备得到具有通透大孔结构的整体柱骨架,并结合扫描电镜、傅立叶红外光谱、压汞仪等表征手段对整体柱的形貌、孔径和性能进行评价。将poly(AAPBA-co-EDMA)亲和整体柱应用于对糖蛋白的分离和富集,考察糖蛋白辣根过氧化物酶(HRP)和转铁蛋白(TF)在整体柱上的色谱保留行为,结果表明该大孔型有机硼酸亲和整体柱对糖蛋白表现出良好的亲和性能和动态吸附容量。并将其成功地应用于加标牛血清样品中糖蛋白TF的分离和富集。第三部分：采用"One-Pot"法合成有机-无机杂化硼酸亲和整体柱并将其用于中性条件下分离富集糖蛋白。本工作四甲氧基硅烷(TMOS)和乙烯基三甲氧基硅烷(VTMS)作为硅烷前驱体,3-丙烯酰胺基苯硼酸(AAPBA)作为有机单体,采用“One-Pot"法制备一种新型的有机-无机杂化基质的硼酸亲和整体柱,制备过程简单。对AAPBA-硅胶杂化硼酸亲和整体柱的制备条件进行系统的优化,可以得到大孔均匀分布、通透性能良好的整体柱骨架结构。实验中对该硼酸亲和整体柱的骨架形貌、渗透性、动态吸附容量和亲和色谱保留行为等进行表征和评价。由于硅胶基质的引入,AAPBA-(?)胶杂化硼酸亲和整体柱表现出良好的亲水性能,无需在流动相中加入有机溶剂,这样的色谱分析条件有利于生物分子的分析。另外硼酸配基AAPBA中羰基氧原子与硼原子之间的配位作用使其在中性条件下即可与顺式二醇作用,因此该硼酸亲和整体柱在中性条件下即对含有顺式二醇的生物小分子和糖蛋白表现出良好的特异性亲和作用,并被成功的应用于实际样品的分析中。第四部分：基于Click Chemistry修饰的新型有机-无机杂化硼酸亲和整体柱的制备及其在糖蛋白分离中的应用。本工作发展了一种新颖的制备有机-无机杂化硼酸亲和整体柱的方法。首先通过"Single-Step"法合成带有叠氮活性基团的杂化整体柱骨架,随后利用叠氮杂化整体柱与炔基硼酸配基之间的铜(Ⅰ)催化叠氮-炔环加成反应(CuAAC)制备“Clicked "有机-无机杂化硼酸亲和整体杜。结合元素分析、红外光谱、扫描电镜等多种常规手段对制备的杂化硼酸亲和整体柱进行系统的表征和评价。“Clicked"有机-无机杂化硼酸亲和整体柱在中性条件下对核苷和糖蛋白表现出良好的亲和性能,pH7.0时糖蛋白卵清白蛋白的动态吸附容量为2.36mg·g-1。该整体柱被成功地应用于从实际样品蛋清中分离富集卵清白蛋白和卵转铁蛋白,具有实用性第五部分：基于thiol-ene点击化学修饰的有机-无机杂化硼酸亲和整体柱的制备及应用。本工作首先采用溶胶-凝胶法,以四甲]氧基硅烷硅烷(TMOS)和巯丙基三甲氧基硅烷(MPTMS)作为反应前驱体制备巯硅胶整体柱。然后利用巯基-烯之间的点击反应在整体柱上修饰硼配基3-丙烯酰胺基苯硼酸(AAPBA),制成AAPBA-硅胶杂化亲和整体柱。制备过程简单、安全。优化了硼酸亲和整体柱的制备条件,并以扫描电镜、红外光谱等多种手段对整体柱形貌和机械性能进行表征。AAPBA硅胶杂化亲和整体柱在中性条件下对含有顺式二醇的生物小分子表现出良好的特异性亲和能力,并被成功地应用于对糖蛋白的分离分析。点击化学的修饰方法新颖、可靠,可以用于制备多种不同类型的硼酸亲和整体柱,具有较大的应用前景。更多还原

【Abstract】 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.更多还原