【作者】 于法标；

【导师】 韩克利； 赵建章；

【作者基本信息】 大连理工大学 ， 物理化学， 2013， 博士

【摘要】 有氧生命体需要有机体保持严格可控的内部氧化还原状态。动态的氧化还原平衡与生理和病理过程有着密切的关系。若解决实时监测生物体内的氧化还原平衡的生物检测技术问题,将会对生态平衡、生理和病理研究都有着重要的科学意义。在多种生物检测技术手段之中,合成有机小分子探针已为高时空分辨率检测活体系内的生理活性物种提供了一种最强大的生物化学工具。针对.上述问题,本文首次报道了了一系列新型可逆有机小分子荧光探针,特异性地研究口标生理活性物种。本文可视化研究了水溶液和细胞内的氧化还原循环对：过氧化亚硝酰/谷胱甘肽、次溴酸/抗坏血酸、双氧水/谷胱甘肽和气体信号分子硫化氧的抗氧化应激功能。本文探索了分子探针选择性地结合在特定细胞器内的问题,实现了在分子、细胞和活体三个层次上对具有生理氧化还原活性的物种的原位、实时、动态荧光成像分析。过氧化亚硝酰(ONOO-)和谷胱甘肽(GSH)之间的氧化还原平衡与一些生理和病理过程有着密切的关系。我们报道了两种近红外荧光探针用于监测的ONOO-/GSH水平在细胞和体内的变化情况。该探针集成了硒/碲酶模拟物作为受体能可逆地响应ONOO-/GSH水平的变化。该探针被成功地应用到细胞和动物体内的ONOO爆发和抗氧化剂GSH修复间期的氧化还原循环变化的可视化检测。次溴酸(HBrO)被认为是中性粒细胞主机防御系统的重要组成部分。但是过度产生或者在错误的地方生成可导致宿主的组织损伤,进而引发许多的疾病,包括关节炎癌症、哮喘等疾病。本工作两种新型可逆次溴酸荧光探针的合成,光谱性质及其生物应用。我们将这两种探针用于模拟生理条件下水溶液中次溴酸的检测和活细胞内次溴酸的荧光成像。这种可逆荧光探针能在生理条件下监测次溴酸氧化和抗坏血酸还原事件。双氧水(H2O2)可作为细胞正常生长和增殖的第二信使。如果超过生理致毒水平的阈值,就会导致因H2O2和抗氧化防御系统之间的不平衡而引起的氧化应激。本工作描述了一个具有“开-关-开”特征的荧光探针的设计、合成、光谱性质及其生物应用。这个探针可以简单直接地监测活细胞和组织内H2O2的氧化应激和硫醇还原修复的过程。硫化氢(H2S),这个具有令人厌恶气味的气体,被确定为第三个具有生物活性的气体,参与调节血管张力,心肌收缩,神经传导,胰岛素分泌等生理过程。本工作报道了一种能选择性响应细胞内硫化氢的比色和比率荧光探针Cy-N3。此探针可以很容易地用于评估细胞内硫化氢水平,实现了对细胞内硫化氢的激光共聚焦比率成像。更多还原

【Abstract】 Aerobic organisms require their components to maintain the intracellular redox status strictly and controllably. The intracellular dynamical redox balances are closely related with many physiological and pathological processes. In order to monitor the in vivo redox cycles, the developments of reversible fluorescent probes are ideal inspection tools which are the right biological detection technology for the troublesome biomedical problems. We designed and synthesized a series of new organic small molecular fluorescent probes for researching physiologically active species specifically and exploring the probe molecules selectively positioned in specific organelles. We investigated the redox cycles within solution and cells visually:ONOO-/GSH, HBrO/Ascorbic Acid, H2O2/GSH, and gasotransmitters hydrogen sulfide against oxidative stress. Based on monitoring the physiological active species changes under the normal and disease states, we could achieve the in situ, real-time and dynamic fluorescence imaging analysis within the three levels of molecules, cells and tissues. The experimental results would reveal these functional reactive species’generation, metastasis and mechanisms, which had an important significance to provide a strong theoretical ideas and experimental basis for exploring the signal transduction of intracellular reactive species.The redox homeostasis between peroxynitrite and glutathione is closely associated with the physiological and pathological processes, e.g. vascular tissue prolonged relaxation and smooth muscle preparations, attenuation hepatic necrosis, and activation matrix metalloproteinase-2. We report a near-infrared fluorescent probe based on heptamethine cyanine which integrates with telluroenzyme mimics for monitoring the changes of ONOO-/GSH levels in cells and in vivo. The probe can reversibly respond to ONOO-and GSH, and exhibits high selectivity, sensitivity and mitochondrial target. It is successfully applied to visualize the changes of redox cycles during the outbreak of ONOO-and the antioxidant GSH repair in cells and animal. The probe would provide a significant advance on the redox events involved in the cellular redox regulation.We also develop another near-infrared reversible fluorescent probe, containing organoselenium functional group, for the highly sensitive and selective monitoring of peroxynitrite oxidation and reduction events under physiological conditions. The probe effectively avoids the influence of autofluorescence in biological systems and gives positive results when tested in both aqueous solution and living cells. The real-time images of cellular peroxynitrite were successfully acquired.Hypobromous acid (HOBr) has been regarded as a reactive bromine species (RBS) among endogenous reactive species. It is always thought to be a key component of the neutrophil host defence system. Although HOBr formation is critical for immune response, excessive or misplaced generation can cause host tissue damage, leading to a wide range of diseases, including arthritis, cardiovascular disease, cancers, asthma, neurodegenerative conditions, kidney disease, cystic fibrosis, and inflammatory bowel disease. We describe the synthesis, properties, and application of two reversible fluorescent probes,mCy-TemOH and Cy-TemOH, for HOBr sensing and imaging in live cells. The two probes contain hydroxylamine functional group for the monitoring of HOBr oxidation/ascorbic acid reduction events. Confocal fluorescence microscopy has established the HOBr detection in live-cells.H2O2serves as a second messenger for normal cellular growth and proliferation. Once the threshold of toxic level is exceeded, the oxidative stress is caused by imbalance between H2O2and antioxidant defense systems. Oxidativ stress always refers to aging, cancer, neurodegenerative and cardiovascular diseases. We present the design, synthesis, spectroscopy, and biological applications of DA-Cy, an on-off-on fluorescent probe to monitor H2O2oxidative stress and thiols repair in living cells and tissues simply and directly. The probe employs the near-infrared heptamethine cyanine dye as a fluorophore, equipped with a chemical redox-responsive dopamine unit. This fluorescent probe can selectively detect H2O2with fluorescence off. In addition, the oxidized state of the probe could deplete thiols via Michael addition to switch its fluorescence emission on. The Confocal microscopy experiments show that in the HL-7702and HepG2cell lines, DA-Cy is able to sense the different intracellular redox environments. The probe also offers the unique capability for H2O2oxidative stress and thiols repair in the fresh rat hippocampus tissues.Following nitric oxide (NO) and carbon monoxide (CO), H2S, with the repulsive odor, is identified as the third biologically active gas that is termed a gasotransmitter or a gasomediator. H2S is involved in a diverse array of physiological processes, including regulation of vascular tone, myocardial contractility, neurotransmission, insulin secretion and so on. We present a colorimetric and ratiometric fluorescent probe Cy-N3that exhibits a selective response to H2S. The probe employs a near-infrared cyanine as fluorophore, and is equipped with an operating azide unit. It is readily employed for assessing intracellular H2S levels, and confocal ratiometric imaging is achieved successfully.更多还原