本文選題：海腎螢光素酶　切入點(diǎn)：腔腸素　出處：《山東大學(xué)》2017年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：生物發(fā)光(Bioluminescence)是指生物體內(nèi)的化學(xué)物質(zhì)在酶的作用下產(chǎn)生可見(jiàn)光的現(xiàn)象,該過(guò)程不依賴(lài)于機(jī)體對(duì)光的吸收,是將生物能轉(zhuǎn)化為光能的過(guò)程。而化學(xué)發(fā)光(Chemiluminescence)是不需要酶的參與,只依靠化學(xué)反應(yīng)產(chǎn)生可見(jiàn)光的現(xiàn)象。生物發(fā)光現(xiàn)象廣泛存在于自然界生物有機(jī)體中,包括細(xì)菌、昆蟲(chóng)和海洋生物等。生物發(fā)光成像(Bioluminescence imaging)是通過(guò)靈敏的光學(xué)檢測(cè)儀器監(jiān)控螢光素酶標(biāo)記的細(xì)胞或基因在活體生物內(nèi)的活動(dòng)和行為過(guò)程的一種新興的成像技術(shù)。生物發(fā)光成像技術(shù)具有操作簡(jiǎn)便快速、靈敏度高、能夠?qū)崿F(xiàn)實(shí)時(shí)動(dòng)態(tài)觀測(cè)以及非侵襲性等優(yōu)點(diǎn)。生物發(fā)光成像技術(shù)在腫瘤生長(zhǎng)監(jiān)測(cè)和轉(zhuǎn)移示蹤、目標(biāo)基因表達(dá)的檢測(cè)、蛋白-蛋白相互作用、藥物高通量篩選和細(xì)胞內(nèi)ATP水平探測(cè)等領(lǐng)域有著不可替代的技術(shù)優(yōu)勢(shì)。海腎螢光素酶發(fā)光體系是常見(jiàn)的生物發(fā)光體系之一。該體系需要海腎螢光素酶(Renilla luciferase)、底物腔腸素(coelenterazine)以及分子氧的參與,最終產(chǎn)生可見(jiàn)光。海腎螢光素酶發(fā)光體系簡(jiǎn)單,不需要ATP、Mg2+等輔助因子,此外海腎螢光素酶能夠表達(dá)于哺乳動(dòng)物細(xì)胞中并且無(wú)細(xì)胞毒性。然而該體系在生命科學(xué)中若得到更廣泛的應(yīng)用,還需要克服以下缺點(diǎn):第一,發(fā)射波長(zhǎng)較短(450-475nm),容易被組織吸收,不利于動(dòng)物成像;第二,穩(wěn)定性較差,在中性或堿性介質(zhì)中容易被氧化產(chǎn)生化學(xué)發(fā)光,從而升高了背景信號(hào)。目前,科學(xué)家們已經(jīng)對(duì)腔腸素的C-2、C-5、C-6和C-8取代位置進(jìn)行了修飾改造,然而具有較好性質(zhì)的底物卻寥寥無(wú)幾,其主要原因是被改造的腔腸素類(lèi)似物不能夠很好地被海腎螢光素酶所識(shí)別。DeepBlueCTM是一種可以商業(yè)獲得的腔腸素類(lèi)似物,已經(jīng)被用于生物發(fā)光共振能量轉(zhuǎn)移研究,并且其化學(xué)結(jié)構(gòu)簡(jiǎn)單,與天然的腔腸素相比少了兩個(gè)羥基,所以在研究中科學(xué)家常以此為參照進(jìn)行結(jié)構(gòu)改造。本研究課題主要分為三個(gè)部分:首先以DeepBlueCTM為參照進(jìn)行結(jié)構(gòu)改造,得到了一個(gè)活性更好的腔腸素類(lèi)似物;在此基礎(chǔ)上,運(yùn)用前藥策略設(shè)計(jì)評(píng)價(jià)了一批酯類(lèi)腔腸素衍生物;最后設(shè)計(jì)并合成了一個(gè)用于檢測(cè)毒性物質(zhì)苯硫酚的生物發(fā)光和化學(xué)發(fā)光探針。本研究對(duì)海腎生物發(fā)光成像技術(shù)的推廣與應(yīng)用具有有較重要的意義。第一部分(底物改造):以DeepBlueCTM為參照對(duì)其進(jìn)行結(jié)構(gòu)改造,以獲得波長(zhǎng)紅移且穩(wěn)定的海腎螢光素酶發(fā)光底物。分別合成了兩個(gè)含氧和含硫的腔腸素類(lèi)似物,因?yàn)閷儆谕恢髯宓难鹾土蚓哂邢嗨频男再|(zhì),都能夠與雜環(huán)形成p-π共軛,這樣可使發(fā)射波長(zhǎng)紅移。然后采用了經(jīng)過(guò)純化的海腎螢光素酶對(duì)這些化合物進(jìn)行了體外生物發(fā)光性質(zhì)研究,并在細(xì)胞水平上作了相關(guān)研究。結(jié)果表明,在體外含氧的腔腸素類(lèi)似物顯示了較大的紅移(63 nm),但是量子產(chǎn)率有所下降。在細(xì)胞水平上,含氧的腔腸素類(lèi)似物表現(xiàn)出較低的發(fā)光強(qiáng)度,而含硫的腔腸素類(lèi)似物的生物發(fā)光強(qiáng)度是最強(qiáng)的。第二部分(酯類(lèi)腔腸素衍生物的設(shè)計(jì)):為了提高腔腸素類(lèi)似物的穩(wěn)定性并且延長(zhǎng)發(fā)光時(shí)間,在發(fā)光最好的含硫腔腸素類(lèi)似物的3位羰基上引入了保護(hù)基團(tuán),形成羧酸酯和碳酸酯結(jié)構(gòu)。此保護(hù)基團(tuán)可以在細(xì)胞內(nèi)被酯酶、脂肪酶、親核性物質(zhì)水解,進(jìn)而釋放出含硫的腔腸素類(lèi)似物,該底物再與細(xì)胞內(nèi)的海腎螢光素酶作用產(chǎn)生生物發(fā)光。被保護(hù)的腔腸素類(lèi)似物不能直接被海腎螢光素酶識(shí)別作用,因此不能產(chǎn)生光信號(hào)。此部分總共合成了 10個(gè)酯類(lèi)腔腸素類(lèi)似物,由于保護(hù)基團(tuán)的位阻不同,其在細(xì)胞內(nèi)持續(xù)發(fā)光時(shí)間的時(shí)間不同。此外也進(jìn)行了體內(nèi)研究,構(gòu)建了裸鼠腋下移植瘤模型,結(jié)果表明這些化合物能夠延長(zhǎng)生物發(fā)光時(shí)間,達(dá)到了最初的目的。第三部分(苯硫酚探針的設(shè)計(jì)):與熒光成像相比,生物發(fā)光成像具有一定的優(yōu)勢(shì):不需要激發(fā)光照射、背景信號(hào)低、良好的生物相容性、靈敏度高等。因此,基于生物發(fā)光原理的探針已經(jīng)成為了研究熱點(diǎn)。目前大多數(shù)的生物發(fā)光探針都是基于對(duì)螢火蟲(chóng)螢光素結(jié)構(gòu)的改造,而基于海腎螢光素酶體系的生物發(fā)光探針很少。我們?cè)O(shè)計(jì)并合成了一個(gè)用于檢測(cè)毒性物質(zhì)苯硫酚的生物發(fā)光和化學(xué)發(fā)光探針,在含硫的腔腸素類(lèi)似物的3位羰基處引入了 2,4-二硝基苯醚,該保護(hù)基團(tuán)能夠被具有強(qiáng)親核性的苯硫酚選擇性地脫去,從而釋放出含硫的腔腸素類(lèi)似物。腔腸素類(lèi)似物不僅能夠產(chǎn)生生物發(fā)光,而且還能在體外通過(guò)DMSO等氧化物質(zhì)產(chǎn)生化學(xué)發(fā)光。該探針具有較高的靈敏度和選擇性,能夠在水溶液、細(xì)胞以及血漿中檢測(cè)毒性物質(zhì)苯硫酚,是很有潛力的生物發(fā)光和化學(xué)發(fā)光探針。綜上所述,為了找到量子產(chǎn)率高、發(fā)射波長(zhǎng)紅移的海腎螢光素酶發(fā)光底物,我們?cè)贒eepBlueCTM的C-8位引入了硫原子,使其與雜環(huán)形成p-π共軛,而且所改變的部分很小,盡量保證了底物與酶的識(shí)別作用,接著通過(guò)體外酶水平和細(xì)胞水平上的研究驗(yàn)證了這一設(shè)想。腔腸素3位羰基是其生物發(fā)光或化學(xué)發(fā)光的關(guān)鍵活性部位,如果運(yùn)用前藥原理先將羰基保護(hù)起來(lái),并使其在特定的條件下被釋放出來(lái),就可以提高腔腸素及其類(lèi)似物的穩(wěn)定性。我們分別設(shè)計(jì)了 10個(gè)能夠緩慢釋放腔腸素類(lèi)似物底物的酯類(lèi)化合物和一個(gè)能夠檢測(cè)毒性物質(zhì)苯硫酚的發(fā)光探針,接著對(duì)這些化合物進(jìn)行了體內(nèi)外活性評(píng)價(jià)。作為生物發(fā)光的重要成員,以腔腸素類(lèi)似物為底物的生物發(fā)光還需要進(jìn)一步地研究開(kāi)發(fā),而將前藥原理運(yùn)用到生物發(fā)光體系對(duì)海腎螢光素酶生物發(fā)光體系的應(yīng)用和推廣具有重要的意義。
[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.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：Q6-33

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