氨基酸荧光探针的设计、合成及生物应用研究
发布时间：2019-06-09 17:53
【摘要】：氨基酸(amino acid)作为生物体中的基本物质,与生物的生命活动有着十分重要的关系,调节着生命的一系列活动。不同种类的氨基酸生物功能各不相同,如巯基类氨基酸,半胱氨酸(Cysteine,Cys)和高半胱氨酸(Homocysteine,Hcy),分子结构上只相差一个亚甲基,但功能方面相差很大:Cys是参与蛋白质合成的人必须氨基酸,同时也是谷胱甘肽(GSH)的一份子。Hcy,甲硫氨酸代谢的产物,不参与蛋白质的合成,却与许多疾病相关联。谷胱甘肽(GSH)也与许多疾病发生相联系。因此,定量检测氨基酸在生物体系中的浓度在化学、生物、医学等领域中都具有十分重要的价值。本文成功设计并合成了一系列氨基酸荧光探针,并利用共聚焦荧光显微镜实现了其在活细胞和组织中的生物成像应用,主要包括三方面工作。(一)描述了一种新型的检测线粒体细胞器中硫醇的双光子荧光探针CA-TPP。探针以新型双光子染料咔唑作为荧光平台,醛基作为识别硫醇类的反应位点,修饰上线粒体的靶向基团三苯基膦而设计合成的,探针本身的荧光强度比较低,当与巯基氨基酸发生作用后,荧光增强,从而实现对巯基氨基酸的识别作用,并对Cys有着较好的线性关系。该探针在双光子稳定性方面也表现了良好的检测线粒体中RSH的特性,同时与线粒体商用探针MTR进行复染得到的共定位系数达0.90以上,并成功实现了对组织70μm深度的内源性半胱氨酸的双光子生物成像。(二)设计了一种在水溶液中对铜离子和Cys的双检测的荧光探针PI。基于传统的菲醌-咪唑类荧光平台,探针本身具有较高的荧光强度及量子产率,与铜离子形成螯合物后荧光消失,再与Cys结合后,铜离子得到释放,荧光进而恢复,从而达到选择性识别Cys的目的,并对细胞内的Cys进行了成像。成功实现了在水相中对Cu~(2+)的检测及对Cys的高度选择。(三)设计合成了一种特殊的巯基氨基酸类荧光探针,即检测溶酶体H_2S的双光子荧光探针TP-PMVC,该探针能对溶酶体和溶酶体中H_2S以双通道形式同时检测成像。我们选用典型的双光子染料咔唑MVC作为反应基体,在基体的3,6位置上引入H+和H_2S的反应位点。探针本身在中性溶液中没有荧光,在溶酶体的酸性环境中发出红色荧光,而检测溶酶体中H_2S时则发出绿色荧光,通过利用不同的激发波长从而实现对溶酶体和溶酶体硫化氢的双通道检测,并实现了较好的双光子性质。在细胞及组织中并以不同的颜色通过显微镜成像表现出来。
[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，
本文编号：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，
本文编号：2495759
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