基于FRET机理的pH荧光探针的设计合成及真菌细胞pH_i成像

发布时间：2018-05-07 14:24

本文选题：FRET型小分子荧光探针 + 设计与合成　；参考：《第二军医大学》2017年硕士论文

【摘要】：细胞内pH(intracellular pH,pH_i)和pH稳态对参与控制真核细胞的生长和代谢极为重要。真菌细胞的pH_i影响真菌细胞膜的跨膜转运、代谢酶的活性和生物大分子的合成,同时也是一些其它一些细胞反应的触发器,如诱使热敏蛋白的产生。一些具有抗真菌活性的药物如质子泵抑制剂奥美拉唑、抗心律失常药胺碘酮的作用机理也与降低真菌细胞pH_i改变有关。准确测定真菌细胞的pH_i有利于研究真菌细胞pH_i调控系统,和pH_i相关的药物作用机制。目前常用的测定真菌细胞内pH的方法主要有pH敏感的小分子参比荧光探针和pH敏感的参比荧光蛋白。参比荧光蛋白虽然具有非侵袭性的优点,但操作复杂耗时,在此不做深究。小分子荧光探针技术操作方便快捷,灵敏度高,但目前应用于真菌细胞的小分子荧光探针较少,且大都为商品化的染料。小分子自身也存在一些局限性:1.不容易透过真菌的细胞膜,需要做成非荧光形式的二乙酰基酯类分子进入细胞后再由酶水解得到荧光核,而且有些染料分子在细胞内水解不完全,影响在细胞内的成像;2.某些染料分子的pKa偏高,并不适合真菌细胞pH_i的成像和测定。本文的研究目的是设计合成新型的pH敏感的小分子荧光探针用于真菌细胞pH_i测定和成像。硼酯螺环可逆开关控制的荧光团R-B为本文课题组前期合成得到的pH敏感的小分子荧光探针,其结构和机理新颖,但其pKa为9.46,并不适合真菌细胞生理学范围内的pH_i测定与成像。为了改善pKa和光谱性质,我们考虑将荧光团R-B作衍生化处理,设计成一个基于FRET机理的参比型荧光探针。即将pH敏感的硼酯螺环母体结构作为能量受体分子,香豆素荧光核为能量供体分子,刚性的哌啶环为中间连接体,设计合成得到了基于FRET机理的第三代pH敏感的小分子荧光探针R-BC。通过光谱性质测定,我们得到R-BC的pKa为6.25,比较符合测定真菌细胞pH_i及酸化的pKa要求。同时该探针对H+的传感具有良好的可逆性和专一性。通过探针应用于真菌细胞,R-BC细胞膜通透性高、细胞毒性小,具有良好的真菌细胞生物相容性。经过真菌细胞原位校正,R-BC的两波段的荧光强度比Ired/Iblue在pH_i为5.0到8.0范围内与pH_i成良好的线性相关,能准确测定此范围内的pH_i。我们利用该探针测定了抗心率失常药胺碘酮对于受试白色念珠菌的pH_i的影响,发现胺碘酮同样能引起白色念珠菌的pH_i酸化,证明了胺碘酮发挥抗菌活性的部分作用机制与影响真菌细胞的pH_i有关。同时我们设计合成了哌啶端分别连有乙酰基和香豆素乙酰基的硼酯螺环探针分子R-BN-1和R-BC-1。通过光谱性质测定和比较其pKa,初步研究和探讨了结构修饰对硼酯螺环荧光核pKa的影响。结果表明接入的香豆素荧光核能促使化合物分子的pKa向酸性区域移动,中间增加一个亚甲基连接单位能使pKa继续向酸性区域移动,但由于两荧光团间隔距离增加及连接体刚性程度减弱,相应的FRET效能降低。基于FRET机理的小分子荧光探针目前已经广泛应用于哺乳动物细胞,但还未有报道涉及到真菌细胞尤其是临床上致病真菌的应用。本文设计合成了新颖的FRET型的小分子荧光探针R-BC并将其应用到了真菌细胞内pH_i的测定与成像。该探针相对于商品化的荧光探针细胞生物相容性好,易透过真菌细胞膜,准确测定的pH_i的范围广,为真菌细胞pH_i调控系统以及相关机理的研究提供了更有力的手段。
[Abstract]:The intracellular pH (intracellular pH, pH_i) and pH homeostasis are very important for controlling the growth and metabolism of eukaryotic cells. The pH_i of fungal cells affects the transmembrane transport of the fungal cell membrane, the activity of metabolic enzymes and the synthesis of biological macromolecules. It is also a trigger for some other cell counter responses, such as inducing the production of thermosensitive proteins. Antiarrhythmic drugs such as omeprazole, amiodarone, antiarrhythmic drug amiodarone, are also related to the reduction of pH_i changes in fungal cells. The accurate determination of pH_i of fungal cells is beneficial to the study of the pH_i regulatory system of fungal cells and the mechanism of drug action related to pH_i. Currently, it is commonly used for the determination of pH in fungal cells. The methods are mainly pH sensitive small molecular reference fluorescence probe and pH sensitive reference fluorescent protein. Although the reference fluorescence protein has the advantages of non invasive, it is complicated and time-consuming and does not study here. Small molecular fluorescence probe is convenient and fast and sensitive, but the small molecular fluorescent probes used in fungal cells are less, And most of them are commercialized dyes. Small molecules themselves also have some limitations: 1. it is not easy to penetrate the cell membrane of fungi and need to make a non fluorescent form of two acetyl ester molecules into the cell and then hydrolyze the fluorescent nucleus by enzymatic hydrolysis, and some dye molecules are not completely hydrolyzed within the cells, affecting the imaging in the cells; 2. some dyes are stained. The high pKa of the material molecules is not suitable for imaging and determination of pH_i in fungal cells. The purpose of this study is to design and synthesize a new pH sensitive small molecular fluorescent probe for the determination and imaging of pH_i in fungal cells. The fluorescent cluster R-B controlled by the reversible switch of borosate rings is a pH sensitive small molecule fluorophore synthesized in the earlier period of this group. The structure and mechanism of the needle are novel, but its pKa is 9.46, which is not suitable for pH_i determination and imaging in the scope of fungal cell physiology. In order to improve the pKa and spectral properties, we consider the fluorescence R-B as a derivative based fluorescence probe based on the FRET mechanism. The structure of the pH sensitive borate parent body is used as the energy. The receptor molecule, the coumarin fluorescent nucleus is an energy donor, and the rigid piperidine ring is the intermediate junction. The design and synthesis of the third generation pH sensitive small molecular fluorescent probe based on the FRET mechanism is determined by the spectral properties. We have obtained the pKa of R-BC of 6.25, which is in accordance with the pKa requirements for the determination of pH_i and acidification of fungal cells. The sensing of H+ has good reversibility and specificity. Through the application of the probe to fungal cells, the membrane permeability of R-BC cells is high, the cell toxicity is small, and the biocompatibility of the fungal cells is good. The fluorescence intensity in the two band of R-BC is better than that of the pH_i in the range of 5 to 8 pH_i in the range of pH_i in the range of 5 to 8. Sexual correlation, we can accurately determine the pH_i. in this range. We used the probe to determine the effect of amiodarone on the pH_i of Candida albicans. It is found that amiodarone can also cause pH_i acidification of Candida albicans. It is proved that the partial mechanism of amiodarone's antibacterial activity is related to the pH_i affecting the fungal cells. At the same time, we designed and synthesized the borate probe molecule R-BN-1 and R-BC-1. of piperidine and coumarin acetyl, respectively, to determine and compare their pKa through spectral properties. The effect of structural modification on the pKa of borate ring fluorescence nucleus was preliminarily studied and discussed. The pKa moves to the acidic region, and a methylene connection can be added to the pKa to move to the acid region, but the corresponding FRET efficiency is reduced due to the increase of the two fluorescent mass interval and the weakening of the rigidity of the connecting body. The small molecular fluorescent probe based on the FRET mechanism has been widely used in mammalian cells, but not yet. The report involves the application of fungal cells, especially the clinical pathogenic fungi. This paper has designed and synthesized a novel FRET type small molecular fluorescent probe R-BC and applied it to the determination and imaging of pH_i in the fungal cells. The probe is biocompatible with the commercialized fluorescent probe cells, and is easily permeable through the fungal cell membrane, and the accurate determination of P The wide range of H_i provides a powerful tool for the study of fungal cell pH_i regulation system and related mechanisms.

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R914

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