【作者】 杨凌；

【导师】 葛健锋； 孙如；

【作者基本信息】 苏州大学 ， 化学， 2018， 硕士

【摘要】 荧光探针是由荧光团(主体),识别基团(客体)和连接基团所组成的。到目前为止,荧光分子探针的设计原理主要根据荧光淬灭的机理,有以下几种:光致电子转移(PET),分子内电荷转移(ICT),荧光共振能量转移(FRET),激发态分子内质子转移(ESIPT),聚集诱导发光(AIE)等。其中,PET机理是分子设计领域最常用的荧光淬灭机理,当主体荧光团和客体之间发生非辐射电子转移时,荧光淬灭。PET又分为两种:受体激发的PET(acceptor-excited PET;a-PET)和供体激发的PET(donor-excited PET;d-PET)。a-PET是指客体HOMO上的电子向激发态荧光团HOMO转移;相反地,d-PET是指激发态电子从受激发的荧光团LUMO转移到客体的LUMO上。目前验证和区分这两种机理的主要方法是依靠(含时)密度泛函理论计算,过程繁琐复杂,故开发一种PET荧光分子探针的快速判断方法具有重要的理论意义和价值。由于荧光团和客体基团上的相对电子云密度存在差异,不同的荧光团与相同的客体相连接,可以达到不同的检测效果;同样地,相同的荧光团与不同的客体基团相连接,往往也会得到不一致的结果。因此,可以建立一种基于主客体电子云密度差的方法来判断是否存在PET机理。实际上,利用PET机理设计各类探针用于检测生物小分子和大分子也是最近报道的热点。本文基于rosamine设计合成了三类分子,并对其光学性质及生物学应用进行了深入研究。主要分为三个部分:第一部分主要基于经典的有机价键理论建立PET荧光探针分子设计的简便方法,后两个部分主要从细胞器靶向功能的角度研究生物分子的检测,具体内容如下:1.将不同电性的杂环与派洛宁相连接得到5个rosamine衍生物(探针3a-3e),分别研究了它们的p H响应。派洛宁与供电子的吲哚基团相连接时,a-PET过程被开启;然而当吲哚被质子化时,PET被禁阻。当派洛宁与吸电子的喹啉或吡啶基团相连接时,d-PET禁阻,荧光开启,而在强酸性条件下杂环上的氮被质子化后生成强吸电子的鎓盐,d-PET允许,荧光淬灭。5种红外荧光p H探针都具有高选择性,快速响应和很好的可逆性,可用于溶液中的p H荧光滴定。其中,探针3d的相对荧光强度增强了263倍,针对探针3d进行生物相容性改进的探针3e具有合适的p Ka,因此在V79和He La细胞中具有溶酶体靶向能力,并且3e为ON-OFF型探针,在正常细胞和癌细胞中分别展现出强弱信号,可用于酸性条件下细胞内的p H监测。研究结果表明:主客体的电子云密度相差越大,发生PET的可能性就越大,从而提供一种基于经典的有机价键理论而设计ON-OFF型荧光探针的简便方法。2.前期工作中设计的探针3c,利用不同的癌细胞和正常细胞进行染色,结果表明在绝大多数细胞中该分子为线粒体靶向探针。于是,用于检测线粒体中的重要小分子的新型的开启型荧光探针5和9被设计和合成。吡啶盐与派洛宁之间存在电子转移,d-PET被允许,荧光处于关闭状态。当外源性加入H2O2或H2S时,反应位点离去,游离的派洛宁-吡啶被释放,荧光开启。探针具备荧光量子产率高,红光发射,稳定性好,水溶性好等优点,可用于外源性检测溶液和细胞中的H2O2和H2S。探针5和9的检测限分别为57 n M和110 n M,均处于已报道探针中的中等水平。这项工作利用PET机理为细胞器靶向探针的发展提供了依据,对生物医学中靶向功能和药物输送的研究具有重要意义。3.继续沿用前期工作中派洛宁-吡啶盐骨架的线粒体靶向功能,设计了新型的OFF-ON型荧光探针11用于检测线粒体中的生物酶。在溶液中,探针11对硝基还原酶具有2.2 ng/m L的低检测限。常氧条件下,主要存在双电子还原的I型NTR;低氧条件下,主要存在单个电子还原的II型NTR。通过细胞成像可以区分开I型和II型硝基还原酶,整个检测过程也都在线粒体中进行。更多还原

【Abstract】 Fluorescent probe is composed of a fluorophore(host),a recognition group(guest)and a linking group.So far,the design principles of fluorescent molecular probes based on the mechanism of fluorescence quenching mainly include the following: Photoinduced Electron Transfer(PET),Intramolecular Charge Transfer(ICT),Fluorescence Resonance Energy Transfer(FRET),Excited-State Intramolecular Proton Transfer(ESIPT)and Aggregation-Induced Emission(AIE),etc.Among them,PET mechanism is the most commonly used for fluorescence quenching mechanism in the field of molecular design.When non-radiative electron transfer occurs between fluorophore and the guest,fluorescence quenches.There are two types of PET,both a-PET(acceptor-excited PET)and d-PET(donor-excited PET)can cause fluorescence quenching.The a-PET refers that photoinduced electrons can transfer from the highest occupied molecular orbital(HOMO)of the guest to the excited host moiety.In contrast,d-PET refers that electrons transfer from the excited host moiety to the lowest unoccupied molecular orbital(LUMO)of the guest.At present,the main method for verifying and distinguishing these two mechanisms is(Time-Dependent)Density Functional Theory((TD)DFT)calculations,and the process is cumbersome and complicated.Therefore,it is of great theoretical significance and value to develop a rapid method for judging PET in fluorescent molecular probes.Due to the differences in the relative electron density on the fluorophore and guest groups,different fluorophores are linked to the same guest to achieve different detection purposes.Similarly,the same fluorophores are linked to different guest groups,often resulting in inconsistent results.So a simple method based on the relative electron density between the fluorophore and guest groups can be established to determine the PET effect.Actually,the use of the PET mechanism to design various types of probes for the detection of small molecules and enzyme is also a recent hot topic.In this paper,three types of molecules based on rosamine were synthesized and designed,and their optical properties and biological applications were studied in depth.There were mainly divided into three parts.The first part was based on the classical organic bond theory to establish a simple method for PET fluorescent probes designing.And the latter two parts mainly studied the detection of biomolecules from the perspective of organelle-targeting function.The detail contents are as follows.1.Five different rosamine derivatives(probes 3a-3e)were obtained by linking nitrogen-containing heterocycles with different electron donating abilities and pyronine,and their p H responses were studied.When pyronine was connected to an electron-donating indole ring,the a-PET process was turned on,but when the indole was protonated,PET was prohibited.When pyronine was linked to an electron-withdrawing quinoline or pyridine,d-PET was inhibited,fluorescence was turned on.While nitrogen was protonated to form a strong electron-withdrawing pyridinium under strong acidic conditions,and d-PET was allowed,fluorescence was quenched.Five kinds of infrared fluorescence p H probes,with high selectivity,fast response and good reversibility,could be used for p H fluorescence titration in solution.Among them,the relative fluorescence intensity of probe 3d was enhanced by 263-fold,and probe 3e with improved biocompatibility had a suitable p Ka,so that it had lysosome-targeting ability in V79 and He La cells,and probe 3e with ON-OFF-type exhibited strong and weak signals in normal cells and cancer cells,respectively.And it could be used for the monitoring of intracellular p H under acidic conditions.The results showed that the PET effects was more likely to happen if the bigger difference of the electronic density between the host and the guest,thereby we provided a convenient method for designing ON-OFF fluorescent probes according to classical organic bond theory.2.The probe 3c designed in the previous work was stained with different cancer cells and normal cells.The results showed that this molecule was the mitochondrial targeting probe for most cells.The novel open fluorescent probes 5 and 9 were designed and synthesized for detecting important small molecules in mitochondria.There was electron transfer between pyridinium and pyronine,d-PET was allowed,and fluorescence was closed.When H2O2 or H2 S was added exogenously,the reaction site leaved,free pyronine-pyridine was released,and fluorescence was turned on.The probes had the advantages of high fluorescence quantum yield,red light emission,good stability and good water solubility,and they could be used for exogenous detection of H2O2 and H2 S in solution and cells.The detection limits of probes 5 and 9 were 57 n M and 110 n M,were all in the medium level of the reported probes,respectively.This work provides a basis for the development of organelle-targeted probes using the PET mechanism and is of great significance for targeting functions and drug delivery in biomedicine.3.The mitochondrial targeting function of the pyronine-pyridinium skeleton in the previous work was continued,and a novel ON-OFF type fluorescent probe 11 was designed to detect biological enzymes in the mitochondria.In the solution,the probe 11 had a low detection limit of 2.2 ng/m L for nitroreductase.Under normoxic conditions,there is mainly a type I NTR with two-electron-reduced;under hypoxic conditions,there is a type II NTR with single electron-reduced.The type I and type II nitroreductases could be distinguished by cell imaging,and the whole detection process was also performed in mitochondria.更多还原