桥连β-环糊精主体分子用于生物活性分子的研究与分析应用

【作者】 刘芳；

【导师】 唐波；

【作者基本信息】 山东师范大学 ， 分析化学， 2008， 硕士

【摘要】 环糊精具有疏水的空腔和亲水的表面,可以在水溶液中选择性结合各种具有适当极性和尺寸的有机、无机以及生物分子形成主-客体或超分子配合物。这种特性使其在分析分离、医药、模拟酶等许多领域得到广泛应用。天然环糊精与模型底物分子的配位作用一般相对较弱,即使在其边臂上引入某种功能基的简单修饰也很难显著提高其分子键合能力,这在很大程度上限制了环糊精在模拟酶和识别底物等方面的应用。但如果将两个环糊精单元通过适当的基团连接起来,可以得到具有双重疏水结合作用和多重识别功能的桥连双环糊精,由于结合位点的增加,以及可以通过调节连接基团以达到适宜的几何匹配,两个相邻的环糊精空腔可协同参与对形状和尺寸适合的客体分子进行包结,扩展分子键合能力,给出更高的配位稳定常数,这种特性使其有可能在酶-底物和抗原-抗体强键合相互作用模拟研究中得到重要应用。因此,基于桥连双环糊精的分子识别研究已成为当今环糊精化学的研究热点之一。基于桥连β-环糊精独特的结构和特性,本论文主要开展了两大方面的研究: (一)设计合成了一种新型的金属桥连β-环糊精,并通过1HNMR、IR、元素分析及氧化还原滴定等予以表征。金属桥连β-环糊精由于受二价铜离子的顺磁性及螯合作用的影响,与没有络合铜离子的主体桥连β-环糊精相比,荧光强度减弱。而谷胱甘肽也可以与铜离子产生络合作用,这种竞争络合作用会导致荧光恢复,且荧光强度随谷胱甘肽浓度变化而发生变化。基于此,建立了一种快速、简便的用于检测谷胱甘肽的荧光光度法。分析实验是在室温下,pH6.00的PBS缓冲溶液中进行的,线性范围为0.30 ~ 20.0μmol L-1,检出限为63.8 nmol L-1。血浆中主要成分对测定不产生干扰,应用本法测定人血浆样品中谷胱甘肽的含量,结果令人满意。(二)基于FRET原理,构建荧光探针:亲和素修饰纳米金-咪唑酮桥连β-环糊精-荧光素超分子主体化合物识别生物素。亲和素与生物素存在特异性结合,其结合部位是生物素上的咪唑酮环,因此本文设计合成了一种咪唑酮桥连的β-环糊精,通过桥连β-环糊精桥基上的咪唑酮环与亲和素特异性结合,可以将bis(β-CDs)连接到亲和素修饰纳米金上。而bis(β-CDs)通过两个相邻空腔的协同作用,大大增强了对荧光素的包结能力,使其靠近纳米金,FRET现象发生,荧光素的荧光猝灭;竞争性的客体分子生物素存在时,挤占桥连β-CDs桥基上的咪唑酮结合位点,桥连β-环糊精连同包结的荧光素远离纳米金,FRET难以发生,荧光素的荧光随之得到恢复。基于此,建立了一种高灵敏度、高选择性的用于生物素检测的荧光分析方法。分析实验是在室温下,pH 7.6的Tris-HCl缓冲溶液中进行的,线性范围为0.80 ~ 20.0μg/mL,检出限为57.0 ng/mL。该探针将应用于血浆样中生物素含量的测定,为发展一种新的血液中生物素含量的检测方法提供实验依据。与传统的生物素的检测方法相比,预计该方法具备荧光光谱法灵敏度高、选择性好、用样量少、方法简便等优点。更多还原

【Abstract】 Cyclodextrins (CDs) are a class of cyclic oligosaccharides with six to eight d-glucose units linked byα-1, 4-glucose bonds. They possess a hydrophilic exterior and a hydrophobic cavity capable of including a variety of inorganic/organic hydrophobic compounds which possess suitable polarity and dimension via host-guest complexation. CDs are extensively studied as not only excellent receptors for molecular recognition but also functional building blocks to construct molecular devices. Among various functional CDs, bridged bisCDs, which comprises two CDs cavities linked by a functional bridge, is a greatly promising candidate. In comparison with native CDs and mono-modified CDs, bridged bisCDs exhibit significant high binding ability and molecular recognition through the cooperative binding of two adjacent CDs units. Moreover, the linker can supply a well-organized pseudo-cavity that in turns provides additional binding interactions with accommodated guest molecules.Based on the intramolecular cooperative binding and multiple recognition of bridged bis (β-CDs) with functional linkers in solution, as well as their unique properties and wide applications, we have carried out two aspects of investigation: First, a novel metallobridged bis (β-CDs) was synthesized and characterized by means of 1H NMR, IR, element analysis and redox iodometric titration. The fluorescence of metallobridged bis (β-CDs) was weak compared with bis (β-CDs) which had not formed complex with copper (II) because of the paramagnetism of copper (II) ions. Glutathione could form complexes with copper (II) derived from the metallobridged bis (β-CDs). This competitive complexation with copper (II) can lead to a significant fluorescence recovery of the bis (β-CDs). Therefore, a rapid and simple spectrofluorimetric method was developed for the determination of glutathione. The analytical application for glutathione was investigated in PBS buffer solution of pH 6.00 at room temperature. The linear range of the method was 0.30 ~ 20.0μmol L-1 with the detection limit of 63.8 nmol L-1. There was no interference from the plasma constituents. The proposed method had been successfully applied to the determination of glutathione in human plasma.Second, avidin modified gold nanoparticles (AGNPs)– bis (β-CDs) - fluorescein has been designed as a selective fluorescent probe for detecting biotin content utilizing FRET. Inclusion of fluorescein into bis (β-CDs) makes FRET occur through donor and quencher nearby. FRET switches off by the addition of the competitive biotin which affords high binding ability with avidin. This competitive binding restores the fluorescence of the quenched fluorescein. This phenomenon suggests that the AGNPs- bis (β-CDs)-FL is effective as a fluorescent probe for biotin recognition. The analytical application for biotin was investigated in Tris-HCl buffer solution of pH 7.6 at room temperature. The linear range of the method was 0.80 ~ 20.0μg/mL with the detection limit of 57.0 ng/mL.更多还原