黄酮类乏氧荧光探针的设计、合成及其性质的研究

发布时间：2018-03-06 15:51

本文选题：肿瘤　切入点：乏氧　出处：《河北大学》2014年硕士论文　论文类型：学位论文

【摘要】：乏氧是实体肿瘤微环境的重要特征之一，导致实体肿瘤对放、化疗产生较强的抗性，是恶性肿瘤局部复发和恶性转移的危害因素。因此对乏氧区域的检测具有非常重要的意义。乏氧荧光成像法有较高的灵敏度，因此成为人们研究的热点。 3-羟基黄酮衍生物具有较大的斯托克斯位移值、良好的光稳定性、较高的量子产率等光学特性，是一类应用比较广泛的荧光物质。本文以3-羟基黄酮类衍生物为发色团，以硝基苯基和硝基咪唑基为乏氧响应基团，设计合成了系列乏氧荧光探针。因硝基的强淬灭作用，导致荧光分子荧光减弱。在乏氧环境下，，硝基化合物透过乏氧细胞脂膜，在硝基还原酶和烟酰胺腺嘌呤二核苷酸的作用下，硝基被还原为氨基，通过电子转移反应，响应基团与荧光分子发生断裂，释放出荧光分子，从而实现对乏氧的检测。但是，硝基乏氧响应基团的引入，导致探针的水溶性大大降低。为此，在3-羟基黄酮分子的6位和8位引入吗啉基侧链，来增大其水溶性。采用NMR，HRMS和IR等波谱技术对探针的结构进行了表征，利用循环伏安法测定了探针的氧化还原电位，利用HRMS研究了乏氧探针的检测机理，利用荧光光谱法探讨了缓冲溶液种类、pH值、溶剂性质和含量等因素对发色团荧光性质的影响，采用MTT法对探针的毒性进行了评价，并采用荧光成像法将其应用于乏氧细胞的检测。结果表明：乏氧响应基团和取代基的结构影响探针的半波还原电位，3-（1’-甲基-4’-硝基咪唑）甲氧基-6,8-二吗啉基甲基黄酮的半波还原电位（-0.802V）高于3-（4-硝基苯基）甲氧基-6,8-二吗啉基甲基黄酮其半波还原电位（-0.812V），硝基咪唑修饰的探针更易被还原。4-（4＇-二甲氨基）苯基-3-羟基黄酮在6位和8位引入吗啉基后，荧光发射波长由545nm红移至554nm。荧光分子的荧光强度随pH的增加而增大，4-（4＇-二甲氨基）苯基-6,8-二吗啉基甲基-3-羟基黄酮在pH为7.8溶液中的荧光强度是在pH为5.0溶液中荧光强度的5倍；荧光分子的荧光强度随助溶剂极性的增加而减小，4-（4＇-二甲氨基）苯基-6,8-二吗啉基甲基-3-羟基黄酮在THF中的荧光强度高于在DMSO中；荧光分子的荧光强度随助溶剂量的增加而增大。将浓度为10μmol/L的探针作用于Hela细胞24h后没有表现出毒性；在乏氧细胞中，探针被还原，释放出荧光分子，显示出乏氧选择性，可用于乏氧细胞的检测。
[Abstract]:Hypoxia is one of the important characteristics of solid tumor microenvironment, which leads to strong resistance of solid tumor to radiotherapy and chemotherapy. It is a harmful factor for local recurrence and malignant metastasis of malignant tumor. Therefore, it is of great significance to detect the hypoxic region. Hypoxia fluorescence imaging has a high sensitivity, so it has become a hot research topic. The 3-hydroxyflavone derivatives are a kind of widely used fluorescent substances with large Stokes shift value, good light stability, high quantum yield and other optical properties. In this paper, the chromogenic group of 3-hydroxyflavonoids derivatives is used as chromogenic group. A series of anoxic fluorescence probes were designed and synthesized using nitrophenyl and nitroimidazolyl as hypoxia responsive groups. The fluorescence of fluorescence molecules weakened due to the strong quenching of nitro. In hypoxic environment, nitro compounds passed through the lipid membrane of hypoxic cells. In the presence of nitro reductase and nicotinamide adenine dinucleotide, the nitro group is reduced to amino group. By electron transfer reaction, the responsive group breaks with the fluorescent molecule and the fluorescent molecule is released. The water solubility of the probe was greatly reduced with the introduction of nitro-hypoxia responsive groups. Therefore, the morpholine side chain was introduced at 6 and 8 positions of 3-hydroxyflavone molecule. The structure of the probe was characterized by NMR-HRMS and IR spectroscopy, the redox potential of the probe was measured by cyclic voltammetry, and the detection mechanism of the probe was studied by HRMS. The effects of pH value of buffer solution, solvent property and content on the fluorescence properties of chromophore were studied by fluorescence spectrometry. The toxicity of the probe was evaluated by MTT method. Fluorescence imaging was used to detect hypoxia cells. The results showed that the half-wave reduction potential of the probe was influenced by the structure of hypoxic responsive groups and substituents. The half-wave reduction potential of the probe was higher than that of 3-O4-nitrophenyl) methoxy-8-dimorpholinyl methyl flavonoids (-0.802V). The half-wave reduction potential of -6 and 8-dimorpholinyl methyl flavonoids was -0.812V, and the probe modified by nitroimidazole was easier to be reduced by introducing morpholine group into phenyl-3-hydroxy flavonoids at the 6th and 8th positions, and the probe modified by nitroimidazole was more likely to be reduced to phenyl-3-hydroxy flavonoids at the 6 and 8 sites. The fluorescence emission wavelength shifted from 545 nm to 554 nm. The fluorescence intensity of the fluorescence molecule increased with the increase of pH. The fluorescence intensity of the fluorescence molecule in the solution of pH 7.8 was 5 times higher than that in the solution of pH 5.0. The fluorescence intensity of the fluorescence molecule decreased with the increase of the polarity of the cosolvent. The fluorescence intensity in THF was higher than that in DMSO. The fluorescence intensity of the fluorescent molecule increased with the increase of the amount of cosolvent. The probe with the concentration of 10 渭 mol/L was not toxic to Hela cells after 24 hours, and the probe was reduced to release fluorescent molecules in hypoxic cells, which showed hypoxia selectivity. It can be used for the detection of hypoxia cells.
【学位授予单位】：河北大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R914;O657.3

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