海肾萤光素酶生物发光底物及探针的设计、合成和活性研究

发布时间：2018-03-20 22:07

本文选题：海肾萤光素酶　切入点：腔肠素　出处：《山东大学》2017年硕士论文　论文类型：学位论文

【摘要】：生物发光(Bioluminescence)是指生物体内的化学物质在酶的作用下产生可见光的现象,该过程不依赖于机体对光的吸收,是将生物能转化为光能的过程。而化学发光(Chemiluminescence)是不需要酶的参与,只依靠化学反应产生可见光的现象。生物发光现象广泛存在于自然界生物有机体中,包括细菌、昆虫和海洋生物等。生物发光成像(Bioluminescence imaging)是通过灵敏的光学检测仪器监控萤光素酶标记的细胞或基因在活体生物内的活动和行为过程的一种新兴的成像技术。生物发光成像技术具有操作简便快速、灵敏度高、能够实现实时动态观测以及非侵袭性等优点。生物发光成像技术在肿瘤生长监测和转移示踪、目标基因表达的检测、蛋白-蛋白相互作用、药物高通量筛选和细胞内ATP水平探测等领域有着不可替代的技术优势。海肾萤光素酶发光体系是常见的生物发光体系之一。该体系需要海肾萤光素酶(Renilla luciferase)、底物腔肠素(coelenterazine)以及分子氧的参与,最终产生可见光。海肾萤光素酶发光体系简单,不需要ATP、Mg2+等辅助因子,此外海肾萤光素酶能够表达于哺乳动物细胞中并且无细胞毒性。然而该体系在生命科学中若得到更广泛的应用,还需要克服以下缺点:第一,发射波长较短(450-475nm),容易被组织吸收,不利于动物成像;第二,稳定性较差,在中性或碱性介质中容易被氧化产生化学发光,从而升高了背景信号。目前,科学家们已经对腔肠素的C-2、C-5、C-6和C-8取代位置进行了修饰改造,然而具有较好性质的底物却寥寥无几,其主要原因是被改造的腔肠素类似物不能够很好地被海肾萤光素酶所识别。DeepBlueCTM是一种可以商业获得的腔肠素类似物,已经被用于生物发光共振能量转移研究,并且其化学结构简单,与天然的腔肠素相比少了两个羟基,所以在研究中科学家常以此为参照进行结构改造。本研究课题主要分为三个部分:首先以DeepBlueCTM为参照进行结构改造,得到了一个活性更好的腔肠素类似物;在此基础上,运用前药策略设计评价了一批酯类腔肠素衍生物;最后设计并合成了一个用于检测毒性物质苯硫酚的生物发光和化学发光探针。本研究对海肾生物发光成像技术的推广与应用具有有较重要的意义。第一部分(底物改造):以DeepBlueCTM为参照对其进行结构改造,以获得波长红移且稳定的海肾萤光素酶发光底物。分别合成了两个含氧和含硫的腔肠素类似物,因为属于同一主族的氧和硫具有相似的性质,都能够与杂环形成p-π共轭,这样可使发射波长红移。然后采用了经过纯化的海肾萤光素酶对这些化合物进行了体外生物发光性质研究,并在细胞水平上作了相关研究。结果表明,在体外含氧的腔肠素类似物显示了较大的红移(63 nm),但是量子产率有所下降。在细胞水平上,含氧的腔肠素类似物表现出较低的发光强度,而含硫的腔肠素类似物的生物发光强度是最强的。第二部分(酯类腔肠素衍生物的设计):为了提高腔肠素类似物的稳定性并且延长发光时间,在发光最好的含硫腔肠素类似物的3位羰基上引入了保护基团,形成羧酸酯和碳酸酯结构。此保护基团可以在细胞内被酯酶、脂肪酶、亲核性物质水解,进而释放出含硫的腔肠素类似物,该底物再与细胞内的海肾萤光素酶作用产生生物发光。被保护的腔肠素类似物不能直接被海肾萤光素酶识别作用,因此不能产生光信号。此部分总共合成了 10个酯类腔肠素类似物,由于保护基团的位阻不同,其在细胞内持续发光时间的时间不同。此外也进行了体内研究,构建了裸鼠腋下移植瘤模型,结果表明这些化合物能够延长生物发光时间,达到了最初的目的。第三部分(苯硫酚探针的设计):与荧光成像相比,生物发光成像具有一定的优势:不需要激发光照射、背景信号低、良好的生物相容性、灵敏度高等。因此,基于生物发光原理的探针已经成为了研究热点。目前大多数的生物发光探针都是基于对萤火虫萤光素结构的改造,而基于海肾萤光素酶体系的生物发光探针很少。我们设计并合成了一个用于检测毒性物质苯硫酚的生物发光和化学发光探针,在含硫的腔肠素类似物的3位羰基处引入了 2,4-二硝基苯醚,该保护基团能够被具有强亲核性的苯硫酚选择性地脱去,从而释放出含硫的腔肠素类似物。腔肠素类似物不仅能够产生生物发光,而且还能在体外通过DMSO等氧化物质产生化学发光。该探针具有较高的灵敏度和选择性,能够在水溶液、细胞以及血浆中检测毒性物质苯硫酚,是很有潜力的生物发光和化学发光探针。综上所述,为了找到量子产率高、发射波长红移的海肾萤光素酶发光底物,我们在DeepBlueCTM的C-8位引入了硫原子,使其与杂环形成p-π共轭,而且所改变的部分很小,尽量保证了底物与酶的识别作用,接着通过体外酶水平和细胞水平上的研究验证了这一设想。腔肠素3位羰基是其生物发光或化学发光的关键活性部位,如果运用前药原理先将羰基保护起来,并使其在特定的条件下被释放出来,就可以提高腔肠素及其类似物的稳定性。我们分别设计了 10个能够缓慢释放腔肠素类似物底物的酯类化合物和一个能够检测毒性物质苯硫酚的发光探针,接着对这些化合物进行了体内外活性评价。作为生物发光的重要成员,以腔肠素类似物为底物的生物发光还需要进一步地研究开发,而将前药原理运用到生物发光体系对海肾萤光素酶生物发光体系的应用和推广具有重要的意义。
[Abstract]:Bioluminescence (Bioluminescence) refers to the chemical substances in the body to produce visible light under the action of the enzyme, the process does not depend on the body's absorption of light, is the biological energy into light energy. The chemiluminescence (Chemiluminescence) is not required for enzyme participation, only rely on the production of visible light chemistry the reaction phenomenon. Bioluminescence phenomena widely exist in organisms in nature, including bacteria, insects and other marine organisms. Bioluminescence imaging (Bioluminescence imaging) is a new imaging technique through optical detection instrument monitoring fluorescence activity and behavior sensitive cells or genes in the in vivo biological enzyme marker in the process. Bioluminescence imaging technology has the advantages of simple operation, high sensitivity, can realize the advantages of real-time dynamic observation and non invasive. Bioluminescence imaging in tumor growth. Measurement and transfer of tracer, target detection of gene expression, protein - protein interactions, the field of drug high-throughput screening and intracellular ATP level detection technology has irreplaceable advantages. The Renilla luciferase luminescence system is one of the most common bioluminescence system. The system needs a Renilla luciferase (Renilla luciferase), substrate coelenterazine (coelenterazine) and molecular oxygen, resulting in visible light. The Renilla luciferase luminescence system is simple, do not need ATP, Mg2+ and other auxiliary factors, in addition to Renilla luciferase expression in mammalian cells and no cytotoxicity. However, the system has been applied in life science if more broadly, also need to overcome the following disadvantages: first, the emission wavelength is short (450-475nm), easily absorbed by tissues, is not conducive to animal imaging; second, poor stability, easy in neutral or alkaline medium by oxygen To produce chemiluminescence, thereby increasing the background signal. At present, scientists have to coelenterazine C-2, C-5, C-6 and C-8 substituted positions were modified, but the good properties of substrate is scanty, the main reason is the transformation of the coelenterazine analogs can not well be Renilla luciferase the enzyme.DeepBlueCTM recognition is a commercially available coelenterazine analogs have been used in bioluminescence resonance energy transfer research, and its chemical structure is simple, natural and coelenterazine less than two hydroxyl groups in the study, so scientists often used as a reference for structure transformation. This thesis is mainly divided into three a part: firstly, taking DeepBlueCTM as the reference for structural transformation, obtained a better activity of coelenterazine analogs; on this basis, the use of prodrug design strategy evaluation of a number of coelenterazine ester derivatives Finally, the design and synthesis of the organism; one for the detection of toxic substances thiophenol bioluminescence and chemiluminescence probe. This research is of important significance for the promotion and application of Renilla bioluminescence imaging technology is the first part. (the substrate transformation): using DeepBlueCTM as a reference on the structure transformation, in order to get the sea kidney the fluorescent wavelength and stable luciferase substrate respectively. Two compounds containing oxygen and sulfur coelenterazine analogs, because oxygen and sulfur are in the same family have similar properties, are capable of forming p- conjugated with heterocyclic compounds, which can make the emission wavelength red shift. Then adopted after Renilla fluorescence the purified cellulase of these compounds were studied in vitro biological properties of light, and the related research at the cellular level. The results showed that in vitro oxygen coelenterazine analogs showed a large red shift (63 nm), but The quantum yield decreased. At the cellular level, oxygen coelenterazine analogs exhibited lower luminescence intensity, and sulfur coelenterazine analogs of the bioluminescence intensity is the strongest. The second part (the design of coelenterazine esters derivatives): coelenterazine analogs in order to improve the stability and extend the time. The protection groups were introduced in 3 carbonyl sulfur coelenterate best luminescence hormone analogues on the formation of carboxylic acid esters and carbonates structure. This protection group can be in intracellular esterase, lipase, nucleophilic substances hydrolysis, releasing the sulfur content of the coelenterazine analogs, the substrate and cell in the sea renal luciferase bioluminescence was produced. The protection of coelenterazine analogs can not directly be Renilla luciferase recognition function, therefore cannot produce light signals. This part of the total synthesis of 10 esters of coelenterazine analogs, because The protection of steric groups, the emitting duration in cells at different time. In addition also were carried out in vivo, construct nude mouse armpit transplanted tumor model, the results showed that these compounds can prolong the bioluminescence time, achieved the purpose. The third part (design phenthiol probe): compared with fluorescence imaging. Bioluminescence imaging has certain advantages: no need for the excitation light, low background signal, good biological compatibility, high sensitivity. Therefore, based on the principle of bioluminescence probe has become the focus of research. Most of the bioluminescent probe is the firefly structure based on the transformation of the sea, and rarely kidney luciferase bioluminescence probe based on the system. We design and one for the detection of toxic thiophenol bioluminescence and chemiluminescence probe was synthesized, containing sulfur coelenterazine class Like the 3 carbonyl group introduced at the two 2,4- nitrobenzene ether, protecting groups can be strongly nucleophilic thiophenol selectively removed, thus releasing sulfur coelenterazine analogs. Coelenterazine analogs can produce bioluminescence, but also in vitro by DMSO oxidation material to produce chemiluminescence. The probe has high sensitivity and selectivity in aqueous solution, toxic substances in plasma cells and detection of thiophenol, is promising bioluminescence and chemiluminescence probe. To sum up, in order to find the high quantum yield, the fluorescence emission wavelength of Renilla luciferase substrate redshift, we introduce the sulfur atom in DeepBlueCTM C-8, the formation of p- conjugated with heterocyclic compounds, and the changed part is very small, the recognition of the substrate and enzyme as far as possible, followed by in vitro enzymatic and cellular level. To verify this assumption. Coelenterazine 3 carbonyl group is the key active site of bioluminescence or chemiluminescence, if using the prodrug principle to carbonyl protection, which is released under certain conditions, it can improve the stability of coelenterazine and its analogues. We design 10 to the slow release of coelenterazine ester compound substrate analogue and a luminescent probe can detect toxic thiophenol, followed by in vitro and in vivo activity evaluation of these compounds. As an important member of bioluminescence, substrate bioluminescence also needs further research and development with coelenterazine analogs, and the prodrug principle is of great significance to the popularization and application of bioluminescent system of Renilla luciferase bioluminescence system.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q6-33

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