氨基酸熒光探針的設(shè)計、合成及生物應(yīng)用研究
發(fā)布時間：2019-06-09 17:53
【摘要】：氨基酸(amino acid)作為生物體中的基本物質(zhì),與生物的生命活動有著十分重要的關(guān)系,調(diào)節(jié)著生命的一系列活動。不同種類的氨基酸生物功能各不相同,如巰基類氨基酸,半胱氨酸(Cysteine,Cys)和高半胱氨酸(Homocysteine,Hcy),分子結(jié)構(gòu)上只相差一個亞甲基,但功能方面相差很大:Cys是參與蛋白質(zhì)合成的人必須氨基酸,同時也是谷胱甘肽(GSH)的一份子。Hcy,甲硫氨酸代謝的產(chǎn)物,不參與蛋白質(zhì)的合成,卻與許多疾病相關(guān)聯(lián)。谷胱甘肽(GSH)也與許多疾病發(fā)生相聯(lián)系。因此,定量檢測氨基酸在生物體系中的濃度在化學(xué)、生物、醫(yī)學(xué)等領(lǐng)域中都具有十分重要的價值。本文成功設(shè)計并合成了一系列氨基酸熒光探針,并利用共聚焦熒光顯微鏡實現(xiàn)了其在活細胞和組織中的生物成像應(yīng)用,主要包括三方面工作。(一)描述了一種新型的檢測線粒體細胞器中硫醇的雙光子熒光探針CA-TPP。探針以新型雙光子染料咔唑作為熒光平臺,醛基作為識別硫醇類的反應(yīng)位點,修飾上線粒體的靶向基團三苯基膦而設(shè)計合成的,探針本身的熒光強度比較低,當(dāng)與巰基氨基酸發(fā)生作用后,熒光增強,從而實現(xiàn)對巰基氨基酸的識別作用,并對Cys有著較好的線性關(guān)系。該探針在雙光子穩(wěn)定性方面也表現(xiàn)了良好的檢測線粒體中RSH的特性,同時與線粒體商用探針MTR進行復(fù)染得到的共定位系數(shù)達0.90以上,并成功實現(xiàn)了對組織70μm深度的內(nèi)源性半胱氨酸的雙光子生物成像。(二)設(shè)計了一種在水溶液中對銅離子和Cys的雙檢測的熒光探針PI。基于傳統(tǒng)的菲醌-咪唑類熒光平臺,探針本身具有較高的熒光強度及量子產(chǎn)率,與銅離子形成螯合物后熒光消失,再與Cys結(jié)合后,銅離子得到釋放,熒光進而恢復(fù),從而達到選擇性識別Cys的目的,并對細胞內(nèi)的Cys進行了成像。成功實現(xiàn)了在水相中對Cu~(2+)的檢測及對Cys的高度選擇。(三)設(shè)計合成了一種特殊的巰基氨基酸類熒光探針,即檢測溶酶體H_2S的雙光子熒光探針TP-PMVC,該探針能對溶酶體和溶酶體中H_2S以雙通道形式同時檢測成像。我們選用典型的雙光子染料咔唑MVC作為反應(yīng)基體,在基體的3,6位置上引入H+和H_2S的反應(yīng)位點。探針本身在中性溶液中沒有熒光,在溶酶體的酸性環(huán)境中發(fā)出紅色熒光,而檢測溶酶體中H_2S時則發(fā)出綠色熒光,通過利用不同的激發(fā)波長從而實現(xiàn)對溶酶體和溶酶體硫化氫的雙通道檢測,并實現(xiàn)了較好的雙光子性質(zhì)。在細胞及組織中并以不同的顏色通過顯微鏡成像表現(xiàn)出來。
[Abstract]:Amino acid (amino acid), as the basic substance in organisms, has a very important relationship with the life activities of organisms and adjusts a series of activities of life. The biological functions of different kinds of amino acids are different, such as sulfhydryl amino acids, Cysteine,Cys and Homocysteine,Hcy, and the molecular structure is only one methylene. However, the function of Cys is very different: Hcy is a necessary amino acid involved in protein synthesis, and it is also a member of (GSH). Hcy, the metabolite of methionine, is not involved in protein synthesis, but it is associated with many diseases. Glutathione (GSH) is also associated with the occurrence of many diseases. Therefore, quantitative detection of amino acid concentration in biological system is of great value in chemistry, biology, medicine and other fields. In this paper, a series of amino acid fluorescence probes were successfully designed and synthesized, and their biological imaging applications in living cells and tissues were realized by confocal fluorescence microscope, including three aspects of work. (1) A novel two-photonic fluorescence probe CA-TPP. for the detection of mercaptan in mitochondrial organelle was described. The probe was designed and synthesized by using a new two-photonic dye carbazole as fluorescence platform and aldehyde group as the reaction site to recognize mercaptan. The fluorescence intensity of the probe itself was relatively low. After interaction with sulfhydryl amino acids, the fluorescence was enhanced, so as to realize the recognition of sulfhydryl amino acids, and had a good linear relationship with Cys. The probe also showed good characteristics in detecting RSH in mitochondria in terms of two-photonic stability. At the same time, the co-localization coefficient obtained by restaining with MTR, a commercial probe of mitochondria, was more than 0.90. at the same time, the co-localization coefficient was more than 0.90. The two-photonic bioimaging of endogenous cysteine at a depth of 70 渭 m was successfully realized. (2) A fluorescence probe PI. for the double detection of copper ion and Cys in aqueous solution was designed. Based on the traditional phenanthraquinolimidazolium fluorescence platform, the probe itself has high fluorescence intensity and quantum yield. After chelating with copper ion, the fluorescence disappears, and then binds to Cys, the copper ion is released and the fluorescence is restored. In order to achieve the purpose of selective recognition of Cys, the intracellular Cys was imaged. The detection of Cu~ (2) and the height selection of Cys in aqueous phase were successfully realized. (3) A special sulfhydryl amino acid fluorescence probe, TP-PMVC, was designed and synthesized, which can detect H 鈮，
本文編號：2495759
[Abstract]:Amino acid (amino acid), as the basic substance in organisms, has a very important relationship with the life activities of organisms and adjusts a series of activities of life. The biological functions of different kinds of amino acids are different, such as sulfhydryl amino acids, Cysteine,Cys and Homocysteine,Hcy, and the molecular structure is only one methylene. However, the function of Cys is very different: Hcy is a necessary amino acid involved in protein synthesis, and it is also a member of (GSH). Hcy, the metabolite of methionine, is not involved in protein synthesis, but it is associated with many diseases. Glutathione (GSH) is also associated with the occurrence of many diseases. Therefore, quantitative detection of amino acid concentration in biological system is of great value in chemistry, biology, medicine and other fields. In this paper, a series of amino acid fluorescence probes were successfully designed and synthesized, and their biological imaging applications in living cells and tissues were realized by confocal fluorescence microscope, including three aspects of work. (1) A novel two-photonic fluorescence probe CA-TPP. for the detection of mercaptan in mitochondrial organelle was described. The probe was designed and synthesized by using a new two-photonic dye carbazole as fluorescence platform and aldehyde group as the reaction site to recognize mercaptan. The fluorescence intensity of the probe itself was relatively low. After interaction with sulfhydryl amino acids, the fluorescence was enhanced, so as to realize the recognition of sulfhydryl amino acids, and had a good linear relationship with Cys. The probe also showed good characteristics in detecting RSH in mitochondria in terms of two-photonic stability. At the same time, the co-localization coefficient obtained by restaining with MTR, a commercial probe of mitochondria, was more than 0.90. at the same time, the co-localization coefficient was more than 0.90. The two-photonic bioimaging of endogenous cysteine at a depth of 70 渭 m was successfully realized. (2) A fluorescence probe PI. for the double detection of copper ion and Cys in aqueous solution was designed. Based on the traditional phenanthraquinolimidazolium fluorescence platform, the probe itself has high fluorescence intensity and quantum yield. After chelating with copper ion, the fluorescence disappears, and then binds to Cys, the copper ion is released and the fluorescence is restored. In order to achieve the purpose of selective recognition of Cys, the intracellular Cys was imaged. The detection of Cu~ (2) and the height selection of Cys in aqueous phase were successfully realized. (3) A special sulfhydryl amino acid fluorescence probe, TP-PMVC, was designed and synthesized, which can detect H 鈮，
本文編號：2495759
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