【作者】 张丹；

【导师】 王俊儒；

【作者基本信息】 西北农林科技大学 ， 化学生物学， 2018， 博士

【摘要】 荧光分析方法具有操作简单、检测限低、实时检测、便于活体成像等优点,已广泛应用于生物小分子的微量检测、生物成像等领域。为了更好地了解生物小分子的生物功能和阐述其产生、运输及下游功能,有机荧光探针逐渐发展为检测和研究生物活性小分子生理、生化过程的重要工具。本论文围绕生物硫醇、抗坏血酸和金属离子的荧光微量检测展开研究,设计合成了一系列反应型及配位型荧光探针,在结构表征基础上,深入研究了探针的识别机理,并将其用于生物传感研究。1.以苯并恶唑为荧光团,叠氮基为识别基团,设计合成了一种简单的基于ESIPT机理的荧光传感器（AHBO）。在HEPES测试体系（50%CH₃CN,10 mM,pH 7.4）中,加入2 eq的Cys和GSH,探针AHBO在紫外可见光谱中344 nm处的吸光度明显减弱,在荧光光谱488 nm处荧光信号逐渐增强,在5 min后达到最大值,其检测限可达到90nM,用365 nm的手提式紫外灯可裸眼观察其探针溶液的荧光颜色从无色变为青色。通过叠氮基与硫醇的亲核取代和重排反应,该探针表现出对GSH和Cys较好的选择性以及高的灵敏度,被成功应用于人体血清中GSH的定量检测以及癌细胞中内源性GSH的荧光成像研究,展示出该探针的潜在应用价值。2.在此工作中,我们在β-咔啉生物碱的6位修饰了叠氮基,设计合成了一种新型的选择性地检测Cys及GSH的荧光传感器（KL-DN）。该探针在最大吸收波长310 nm处的吸光度值逐渐增强,荧光强度在2 min后达到最大值,其检测限可达到92 nM,远低于生理环境的GSH水平。检测机理通过核磁滴定、高效液相色谱、液质联用等分析手段验证了检测产物。通过氧化还原反应,GSH能够被叠氮基快速识别,具有低毒性、良好的膜渗透性以及水溶性更优异的KL-DN首次实现在癌细胞以及小麦胚芽组织中内源性GSH的高灵敏度地检测及荧光成像研究。3.抗坏血酸（AA）是许多水果和植物中的重要营养成分,而研究发现多种疾病与抗坏血酸的异常水平密切相关,开发一种能够在食品中荧光检测抗坏血酸的简便方法具有重要意义。本工作中,我们设计并合成了三种化学传感器,这三种传感器（1,2和3）是在不同位点修饰叠氮基的1,8-萘酰亚胺衍生物。通过氧化还原反应,加入2 eq的抗坏血酸后,探针2的荧光强度在1 min内达到最大值,其检测限达到74 nM具有很高的选择性以及灵敏度。尽管探针1的4位叠氮基团能够被抗坏血酸还原,但由于发生了PET过程,其荧光信号并没有增强。与3位叠氮修饰的探针3相比,4位叠氮修饰的探针2更容易被抗坏血酸还原,其还原产物已通过核磁氢谱、碳谱、质谱验证。通过一种叠氮基-抗坏血酸反应的新策略,探针2首次成功地用于大蒜切片组织中抗坏血酸的荧光成像研究。4.本工作根据分子杂交原理通过click反应将β-咔啉生物碱、咔唑和1,8-萘酐荧光团相结合,设计合成了两个系列的杂交分子并初步探讨了其光物理性质。结果表明,在水相比例逐渐增加时,杂交分子开始聚集,并伴随着荧光信号逐渐增强。光学性质和晶体结构分析以及扫描电镜观察,证明该类杂交分子通过π-π堆积发生聚集并具有聚集诱导荧光的性质。当在含有50%水的DMSO溶液中加入金属离子(Al³⁺)后,含有β-咔啉单元的杂交分子与金属离子发生配位整体带有的正电荷相互排斥从而使其在水相中进行解聚,同时能够作为新的比率型荧光探针,对Al³⁺实现了双通道荧光信号检测并初步研究了生活污水中Al³⁺的荧光检测定量分析。基于以上工作,本研究从不同叠氮基修饰的荧光团分子结构入手,开展了苯并恶唑、β-咔啉、萘酐等荧光探针的设计合成,并实现了与生命活动密切相关的生物硫醇、抗坏血酸及Al³⁺的高灵敏度、高选择性地检测。该研究为叠氮基修饰的小分子荧光探针在生物分析领域中用于巯基氨基酸的检测提供了新的研究思路以及实验基础,也为基于叠氮基修饰的荧光探针的设计及应用拓展新的研究方向。更多还原

【Abstract】 Possessing the advantages of simple operation,low detection limit,real-time detection and live imaging,fluorescent analysis method has been widely used in detection of small biological molecules and biological imaging.In order to get a better understanding of biological functions and elaborate the production,transportation and downstream functions of small biological molecules,the organic fluorescent probes have been gradually developed into important tools for testing bioactive molecules and researching on their physiological and biochemical process.Centreing on fluorescence detection study on biological thiols,ascorbic acid and metal ions,a series of fluorescent probes based on reaction types and coordination types were designed,synthesized and fully characterized.Moreover,we further studied the recognition mechanisms and biosensor applications of them.1.A simple fluorescent sensor（AHBO）based on ESIPT mechanism was designed and synthesized by using benzoxazole as the fluorophore and azide group as the recognition unit.In HEPES test system containing 50%CH₃CN（10 mM,pH 7.4）,the absorption intensity of AHBO at 344 nm decreased upon addition of 2 eq GSH and Cys,while the fluorescence intensity at 488 nm gradually increased,achieving the maximum after 5 min.The detection limit could reach to 90 nM.The solution color of fluorescent probe changed from colorless to cyan,which was observed with a 365-nm portable UV lamp.Through nucleophilic substitution-rearrangement reactions,the probe（AHBO）with good selectivity and high sensitivity had been successfully applied in quantitative detection of GSH in human serum and fluorescence imaging of endogenous GSH in cancer cells,showing the potential application value.2.In this work,modifying azide group at 6-C ofβ-carboline alkaloid,we reported herein the design and synthesis of the turn-on probe KL-DN for selective detection of Cys and GSH.Upon addition of 2 eq GSH,the absorption intensity of the probe at 310 nm increased,and the fluorescence intensity at 487 nm reached to the maximum in 2 min with the detection limit of92 nM which is far below the level of GSH physiological environment.The detection mechanism and product were verified by analysis of nuclear magnetic titration,high performance liquid chromatography and liquid mass.Via redox reaction,GSH could be quickly recognized by azide.The probe,which has low toxicity,good membrane permeability and excellent water solubility（comparing with AHBO）,was for the first time applied to the detection and fluorescence imaging of endogenous GSH in cancer cells and wheat germ tissues.3.Ascorbic acid（AA）is a vital nutritional factor in many fruits and plants,and abnormal levels of AA are closely associated with several diseases.Therefore,the development of convenient methods for monitoring AA levels in biological systems is of great importance.In this work,we designed and synthesized three chemosensors which were based on a1,8-naphthalimide moiety with the azide group at different sites（Probes 1,2,and 3）.The fluorescence intensity of Probe 2 reached to the maximum within 1 min upon addition of 2 eq AA through the redox reaction.Meanwhile,Probe 2 showed high selectivity toward AA,having an experimental limit of detection of 74 nM.Although Probe 1 could be reduced by AA,the fluorescence intensity of AA had no change due to PET process.The reduction product of Probe 2 with AA was verified by ¹H NMR,¹³C NMR and mass spectram.Its reduction was easier than that of Probe 3 with a 3-substituted azide group.Moreover,Probe 2was successfully used for imaging of AA in garlic slice tissues for the first time.4.According to molecular hybridization principle,we designed and synthesized two series of hybrid molecules and preliminary discussed on their physical properties.Through click reaction,carbazole,β-carboline and 1,8-naphthalene anhydride fluorophore were linked by triazole ring.The results showed that when the proportion of water phase gradually increased,hybrid molecules began to aggregate and the fluorescence signal of them gradually increased.The optical properties,crystal structure analysis and scanning electron microscopy indicated that hybrid molecules aggregated viaπ-πaccumulation resulting in AIE property.When added metal ions(Al³⁺)in DMSO solution containing 50%water,the hybrid molecules containingβ-carboline unit could coordinate with metal ions leading to repelling of positive charges and depolymerization in DMSO solution containing 50%water.Meanwhile,compound 5-9 could be used as a new ratio fluorescent probe for Al³⁺with dual-channel fluorescence signal and was successfully applied to fluorescence detection analysis of Al³⁺in sanitary sewage samples.Starting with different azide-modified fluorophore molecular structures,fluorescent probes based on benzoxazole,β-carboline alkaloid and 1,8-naphthalimide were designed and synthesized.They were further applied to high sensitive and selective detection of biological thiols,ascorbic acid and Al³⁺,which are closely related to biological life activities.This work provides new research ideas and experimental basis for azide-modified small molecular fluorescent probes with the detection of thiols in the field of biological analysis.Besides,it also develops new research directions for the design and application of azide-modified fluorescent probes.更多还原