基于迈克尔加成的硫醇荧光成像探针

发布时间：2018-12-24 07:38

【摘要】：生物活性硫醇小分子,包括半胱氨酸(Cys)、同型半胱氨酸(Hcy)和还原型谷胱甘肽(GSH),在生物体的许多生理活动和新陈代谢过程中,扮演着重要的角色。硫醇的代谢和运输与体内许多重要的酶和蛋白质功能表达密切相关,硫醇浓度异常预示着相应的酶和蛋白质发生异常,可能导致许多疾病的发生。因此,定量检测生物体中的硫醇浓度,可为疾病的预防和诊断起到提示作用,在生命科学和临床医学中具有非常重要的意义。在各种各样检测硫醇的方法中,荧光探针以其选择性好、灵敏度高、检出限低、仪器简单和操作简便等特点,被认为是最简便有效的手段,广泛应用于检测识别活体组织和细胞中的微量样品。鉴于硫醇的生理重要性,近年来利用荧光探针方法检测硫醇含量的研究非常活跃。本论文主要利用硫醇中巯基的强亲核性,设计合成了五种可与硫醇发生迈克尔加成反应的新型荧光成像探针,并通过研究探针分子与硫醇加成前后的紫外和荧光光谱变化,达到对硫醇识别检测的目的。具体研究工作如下:1.设计合成了一种基于方酸衍生物的近红外硫醇荧光成像探针2-1,硫醇分子中的巯基可以亲核加成缺电子的方酸四碳环,通过紫外吸收光谱和荧光发射光谱可以发现,该探针可以快速特异性识别Cys/Hcy,而GSH及其他氨基酸对识别几乎不产生干扰。共聚焦成像实验表明,探针2-1可用于活细胞内检测Cys/Hcy。2.以三苯胺为荧光团,马来酰亚胺为识别基团,设计合成了一种高选择性识别硫醇的荧光成像探针3-1,该探针基于迈克尔加成机理实现了对硫醇的快速和特异性识别,反应机理经核磁和质谱方法进一步确定,并成功应用于细胞成像实验。3.设计合成了一种以萘酰亚胺为荧光团,马来酰亚胺为识别基团的硫醇荧光成像探针4-1,可以对硫醇进行快速地高选择性识别,识别过程荧光表现为off-on。响应时间研究表明,探针对硫醇的识别可在120 s内达到平衡。共聚焦成像实验证明4-1可用于检测HepG2活细胞内的硫醇。4.设计合成了两个萘酰亚胺-马来酰亚胺同分异构体衍生物,通过研究它们与硫醇及其他氨基酸作用前后的荧光变化,筛选出能基于迈克尔加成机理,快速、特异性识别Cys的荧光成像探针5-1,检出限低至0.064μM,并成功应用于检测活细胞内的Cys。5.设计合成了一种新型双荧光团组合型硫醇荧光成像探针6-1,可用于在生理条件下特异性比率识别Cys/Hcy。探针本身表现为蓝色荧光,Cys/Hcy通过迈克尔加成催化分子内脱羧,进而诱导分子内开环重排,导致共轭体系被破坏,表现为荧光红移(125 nm),荧光强度比值(I560/I435)分别高达142倍(Cys)和133倍(Hcy)。响应时间研究表明,6-1与Cys的作用比与Hcy作用快,而与GSH几乎不发生反应,并且将其成功应用于细胞和活体成像实验。
[Abstract]:Bioactive mercaptan small molecules, including cysteine (Cys), homocysteine (Hcy) and reduced glutathione (GSH), play important roles in many physiological activities and metabolic processes. The metabolism and transport of mercaptan are closely related to the expression of many important enzymes and proteins in the body. The abnormal concentration of mercaptan indicates the abnormality of enzymes and proteins, which may lead to many diseases. Therefore, quantitative detection of mercaptan concentration in organisms can serve as a reminder for the prevention and diagnosis of diseases, and play a very important role in life sciences and clinical medicine. Among all kinds of methods for detecting mercaptan, fluorescent probe is considered to be the most convenient and effective method because of its good selectivity, high sensitivity, low detection limit, simple instrument and easy operation. It is widely used to detect and identify trace samples in living tissues and cells. Due to the physiological importance of mercaptan, the use of fluorescence probe method to detect mercaptan content is very active in recent years. Based on the strong nucleophilic property of mercapto in mercaptan, five novel fluorescent imaging probes were designed and synthesized to react with mercaptan. The changes of UV and fluorescence spectra before and after the addition of mercaptan were studied. To achieve the purpose of identification and detection of mercaptan. The specific research work is as follows: 1. A near infrared mercaptan imaging probe 2-1 was designed and synthesized based on the derivative of sulfonic acid. The thiol molecular mercapto can be nucleophilic and electron-deficient tetrachlorothioic acid, which can be found by UV absorption and fluorescence emission spectra. The probe can recognize Cys/Hcy, quickly and specifically, while GSH and other amino acids can hardly interfere with the recognition. Confocal imaging showed that probe 2-1 could be used to detect Cys/Hcy.2. in living cells. Using trianiline as fluorescence group and maleimide as recognition group, a highly selective mercaptan recognition fluorescence imaging probe 3-1 was designed and synthesized. The probe realized rapid and specific recognition of mercaptan based on Michael addition mechanism. The reaction mechanism was further determined by NMR and MS, and successfully applied to cell imaging experiment. 3. A mercaptan fluorescence imaging probe 4-1 with naphthalene imide as fluorescence group and maleimide as recognition group was designed and synthesized. It can be used to identify mercaptan quickly and highly selectively. The fluorescence of recognition process is off-on.. The response time study showed that the recognition of mercaptan by the probe could reach equilibrium within 120 s. Confocal imaging showed that 4-1 could be used to detect mercaptan in living cells of HepG2. Two naphthalenimide-maleimide isomers were designed and synthesized. By studying their fluorescence changes before and after their interaction with mercaptan and other amino acids, we have screened out that they can be quickly and rapidly based on Michael addition mechanism. Fluorescence imaging probe 5-1, which specifically recognizes Cys, has a detection limit of as low as 0.064 渭 m, and has been successfully used to detect Cys.5. in living cells. A novel fluorescent imaging probe 6-1 for mercaptan was designed and synthesized, which can be used for specific ratio recognition of Cys/Hcy. under physiological conditions. The probe itself showed blue fluorescence, and Cys/Hcy catalyzed intramolecular decarboxylation through Michael addition, which led to intramolecular ring opening rearrangement, resulting in the destruction of the conjugated system and the fluorescence red shift (125 nm),). Fluorescence intensity ratio (I560/I435) was as high as 142-fold (Cys) and 133-fold (Hcy)., respectively. Response time studies show that 6-1 acts more quickly with Cys than with Hcy, but does not react with GSH, and is successfully used in cell and in vivo imaging experiments.
【学位授予单位】：山西大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：O657.3

【相似文献】