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辅酶Q类似物的合成及其电化学研究
Synthesis and Electrochemistry of Ubiquinone Analogue
【作者】 马巍;
【导师】 龙亿涛;
【作者基本信息】 华东理工大学 , 应用化学, 2012, 博士
【摘要】 辅酶Q是一类广泛存在于自然界真核生物细胞膜上的脂溶性醌类化合物,在细胞线粒体呼吸链上作为关键的电子传递中间体参与有关生物氧化的能量转换过程,是细胞呼吸和细胞代谢的激活剂,也是重要的抗氧化剂和非特异性免疫增强剂。研究表明,辅酶Q结构类似物的合成及其相关的生物活性是辅酶Q研究的一个重要方面。目前,基于电极和纳米材料仿生界面的构建在生物识别、生物传感、模拟生物过程以及理解生物分子在生物界面中的重要作用等方面都优于单独的有机或无机体系。从仿生学的观点来看,构建功能化的仿生界面为探索生物分子在生物过程中的功能起到了重要作用。基于此,本文合成了一系列辅酶Q类似物,对其电子传递机理和构效关系进行研究;同时以模仿线粒体呼吸链的初始阶段为契机,通过构建辅酶Q功能化的仿生界面研究了辅酶Q在呼吸链中重要的质子偶联电子传递作用,为帕金森病的早期诊断提供了新方法。具体内容如下:1.辅酶Q类似物的合成、电子传递性质及其生物活性研究通过应用简单、有效的方法合成了一系列6-位取代的辅酶Q类似物(UQAs),并检测了它们在质子惰性溶剂中的电子传递的性质与UQAs的生物活性。通过电化学和紫外-可见光谱电化学方法研究了UQAs在质子惰性溶剂中电子传递过程和半醌自由基性质。通过MTT实验研究了10种含不同取代基的UQAs对小鼠黑色素瘤B16F10细胞的毒活性得到结构-生物活性之间的关系。从UQAs抑制小鼠黑色素瘤B16F10细胞生长活性的IC50值可以看出,合成的UQAs能有效的诱导肿瘤细胞死亡。在细胞毒活性的测试中,6-乙烯基泛醌5和6-(4’氟代苯基)泛醌7的活性最高,其IC50值分别是6.1μM和6.2μM,而辅酶Q10的IC50值>100μM。通过现场电子自旋共振光谱技术来研究细胞毒活性,发现醌类化合物导致癌细胞死亡的能力与其半醌自由基的弛豫时间有关。值得注意的是,活性高的化合物5和6-苯基泛醌6的半醌自由基的弛豫时间更长。这为将来醌类化合物作为抗癌药物提供有价值的参考。2.亚甲基桥连的双辅酶Qo的电化学性质及其氢键作用研究合成了亚甲基桥连的双辅酶Q0化合物(Bis-CoQo),并采用电化学,现场紫外-可见和电子顺磁共振光谱电化学技术对Bis-CoQo及其所有还原产物:单自由基阴离子、反磁二价阴离子和四价阴离子性质进行表征。在无水乙腈溶液中,Bis-CoQo的循环伏安电化学性质为三步四电子的氧化还原过程。这一性质表明双醌内部有强的相互作用力。并通过变温循环伏安法,确认了Bis-CoQo的第三步电化学氧化还原过程中伴随了化学反应。在含有微量水的乙腈溶剂体系中,各电子转移的电位差随着乙腈溶剂中水含量的增加而减少。Bis-CoQo的还原产物和乙腈溶液中的水形成的氢键强度可通过峰电位的位移来计算。当加入适量水观察到一个还原峰时,双醌内强的分子内作用力消失。此外,也考察了Bis-CoQo在Hela细胞内的抗氧化能力。3.基于辅酶Q衍生物支撑仿生膜的构建及其在NADH/NAD+的氧化还原反应中的研究设计合成了三个不同烷基链长的泛醌二硫化物(QnS,n=1,5,10),通过QnS自组装修饰在金电极表面形成QnS-SAM后,将泛醌修饰的金电极放在含NADH/NAD+磷脂囊泡溶液中孵化,从而在SAM修饰的金电极表面形成悬浮的磷脂双层膜(QnS-HBM-NADH/NAD+)。在该仿生膜模式下,当表面修饰的泛醌作电子传递中间体和NADH/NAD+包埋在磷脂双层膜中时,不仅大大减小NADH的氧化过电势,而且也实现了NADH和NAD+的可逆转化。通过原位的表面增强拉曼光谱手段验证了NADH和NAD+的相互转化;紫外-可见光谱电化学实验表明在QnS-HBM-NADH/NAD+这种仿生膜模式下电化学氧化NADH得到的NAD+具有生物活性。该仿生膜界面的建立可作为一种应用平台研究生物相关的电活性分子嵌在生物膜中的性质为探究疏水环境下的氧化还原反应机制提供新思路。4.功能化量子点模拟呼吸链中辅酶Q电子传递反应及其在帕金森病检测中的应用通过设计和合成了一系列不同链长含1,2,3-三唑环的泛醌二硫化物(QnNS, n=2,5,10)。将其作为表面包裹配体功能化半导体量子点(QnNS-QDs),因为配体跟QDs之间的电子传递导致QDs的荧光变化。结果表明还原态HQnNS固定在QDs表面后增强了荧光,而氧化态QnNS修饰在QDs表面后猝灭了QDs的荧光。荧光和光谱电化学研究表明QnNS-QDs的荧光随氧化还原电位而变化,其变化程度取决于链长的不同,可以通过电化学方式可逆调控QnNS-QDs荧光的增强和猝灭效应。从生物学的角度研究泛醌系列化合物在呼吸链中重要的电子与质子传递作用,且对线粒体呼吸链的初始阶段进行仿生建立了检测复合体Ⅰ的光学生物传感器。实验结果表明在NADH存在的体系中,QnNS-QDs的荧光随复合体Ⅰ浓度的增大而增强。这表明在QDs表面的氧化包裹层QnNS在复合体Ⅰ和NADH作用下得到两电子、两质子而生物催化还原为HQDNS,从而增强QDs的荧光。流行病学研究表明线粒体中复合体Ⅰ功能失调是早期PD阶段的发病原因。其它证据也表明复合体Ⅰ缺失是PD诊断的一个关键指标。通过QnNS-QDs体系的荧光变化检测人成神经瘤SH-SY5Y细胞内复合体Ⅰ的缺失水平,这对于早期分子诊断和监测PD进展有重要意义和潜在的应用前景。
【Abstract】 Coenzyme Q, also known as ubiquinone. is a lipid-soluble compound, indispensable for optimal functioning of all living organisms. As the only nonprotein component of the mitochondrial electron-transport chain, CoQ is a central electron carrier, simultaneously transferring protons from the mitochondrial matrix to the intermembrane space. The concomitant proton gradient across the inner mitochondrial membrane is essential for ATP production. Apart from their main function. CoQ and several other CoQ family members have additional functions in the regulation of the cellular metabolism and antioxidant function as scavengers of free radicals. Some research indicates that systemic ubiquinone analogues (UQAs) studies for synthesis and biological application are generating interesting. Recently, biointerface fabrication and research on electrode and nano materials surface enjoys increasing interest in biosensing, bio-recognition, understanding molecule-biointerfaces interaction and biomimicking biological process, better than either solely organic or inorganic systems. From a biomimetic point of view, functionalized biointerfaces have played an important role in understanding of biomolecules behaviours in biological processes. On these account, the dissertation focuses on the Preparaiong of a series of UQAs. These unique compounds have been investigated to explore their electron-transfer processes and structure-activities relationships of UQAs were examined in this study. We construct the bio-interface using ubiquinone to represents a biomimetic electron-transfer system, modeling part of the mitochondrial respiratory chain the proton coupled electron transfer, and for Parkinson’s disease diagnosis and progression. The details are summarized as follows:1. In situ spectroeletrochemistry and biological activities of natural UQAsQuinones are a group of potent antineoplastic agents. Here we described effective and facile routes to synthesize a series of UQAs. These unique compounds have been investigated by electrochemistry and in situ UV-Vis spectroelectrochemistry to explore their electron-transfer processes and radical properties in aprotic media. The structure-activities relationships of inhibiting cancer cell proliferation of UQAs were examined in murine melanoma B16F10 cells. Our results revealed that UQAs had improved antiproliferative activity and displayed better inhibitory effects than natural ubiquinone 10. The cytotoxic activities of UQAs were correlated to the semiubiquinone radicals, which were confirmed by in situ electron spin resonance. In the cytotoxicity test,6-vinyl ubiquinone 5 and 6-(4’-fluorophenyl) ubiquinone 7 that possess half maximal inhibitory concentration value (IC50) of 6.1μ.M and 6.2μM. This would make them as valuable candidates for future pharmacological studies2. Electrochemical study and hydrogen bond interaction of bis-coenzyme QoA methylene-bridged bis-coenzyme Qo (Bis-CoQo) that shows intramolecular electronic communications has been for the first time synthesized. By employing electrochemical, in-situ UV-vis and electron paramagnetic resonance spectroelectrochemical techniques, the unstable reduced intermediate species:mono-radicals, diamagnetic dianions and tetra-anions of Bis-CoQo have been observed. The electron-transfer process can be defined as a three-step reduction process with a total of four-electron in CH3CN solution. The chemical reaction in the third redox step process was confirmed by variable temperature cyclic voltammetry. In an aprotic CH3CN solution, the peak potential separation between electron-transfer steps diminished sequentially with increasing concentration of water. The hydrogen bonding interactions between water and the electrochemical reduced intermediates of Bis-CoQo can be estimated by peak potential shifts. The electronic communications of Bis-CoQo may be blocked when one reduction peak observed with proper quantities of water in CH3CN solution. Bis-CoQo protected cellular antioxidant defense capacity is also assessed.3. Reversible redox of NADH and NAD+ at a hybrid lipid bilayer membrane using ubiquinoneWe synthesized three ubiquinone-terminated disulfides with different alkyl spacers (QnS, n =1,5,10). QnS is the modified on the gold electrode surfac using self-assembled techniques. Biomimetic membrane model in which ubiquinone is embedded in lipid bilayer membranes that contains the NADH/NAD+redox couple (QnS-HBM-NADH/NAD+) were then formed on the QnS-SAMs. Importantly, we have shown that reversible interconversion between NADH and NAD+could occur at a low overpotential when both ubiquinone, as a redox mediator, and NADH/NAD+ were embedded in a lipid bilayer. Further evidence for the reversible interconversion NADH/NAD+ was obtained by in situ surface enhanced Raman scattering, and spectroelectrochemical UV-vis experiments confirmed that the electrochemical NADH oxidation at the ubiquinone HBM allows for the regeneration of biologically active NAD+ Furthermore, this system can be used as a platform to examine biologically relevant electroactive molecules embedded in a natural membrane environment and provide new insights into the mechanism of biological redox cycling.4. Ubiquinone/ubiquinol coupled quantum Dots as switchable redox-fluorescent biosensor for Parkinson’s Disease diagnosisWe prepared surface-attached CdSe/ZnS QDs exploiting three ubiquinone-terminated disulphides (QnNS, n=2,5,10). The FL enhancement of reduced HQnNS and quenching of oxidized QnNS-modified QDs can be reversibly tuned with the redox potential of surface capping layer, following the transformation between QnNS and HQnNS state via electron transfer on the QDs surface. The FL and electrochemical properties are spacer dependent indicating the importance of the heterogeneous electron transfer kinetics from the surface capping layer to the QDs. Concerning synergy between ubiquinone and NADH in enzymatic reaction of electron transport chain, it enables us to follow the activities of complex I to mimic the initial electron-transfer process of respiratory chain and develop a unique optical sensor for the detection of complex I. We demonstrated the FL of QnNS-QDs light up with complex I in the presence of NADH arises from oxidized ubiquinone to reduced ubiquinol on the surface of QDs. Studies in human postmortem material indicate that impaired complex I activity of mitochondria are important in the pathogenesis of sporadic PD. Others have also demonstrated that complex I deficiency is a potential index for PD diagnosis. Importantly, the utility of the system is demonstrated by monitoring the FL change to trace complex I levels in human neuroblastoma SH-SY5Y cells. Our results demonstrated that our constructed QnNS-QDs sensor could be used for early detection of PD and monitoring disease progression. If our results are confirmed in other cohorts, there is no doubt that this biosensing approach is a significant step forward toward molecular diagnosis of PD.
【Key words】 Coenzyme Q/Ubiquinone; Eletrochemistry; Biomemitic membrane; Quantum dots; Respiratory chain;