【作者】 许超；

【导师】 钱鹰；

【作者基本信息】 东南大学 ， 材料物理与化学， 2019， 博士

【摘要】 氟硼二吡咯（BODIPY）染料因其结构易于合成,具有良好的光和化学稳定性、较高的摩尔消光系数和荧光量子产率、狭窄的荧光发射峰等优异的光化学物理性质,已被广泛应用于生物成像、光动力治疗、激光染料、OLED发射材料和有机光伏等领域。然而,传统BODIPY染料也存在诸如发射单一、波长较短、Stocks位移较小、分子组合设计少等这样的不足,使其在细胞器和生物荧光成像,以及其它光学应用领域受到限制。因此,本论文对BODIPY发色团进行结构修饰,通过合理的分子组合、设计,构筑了多种基于BODIPY骨架的大π共轭体系和功能性分子;并且,基于分析物的特异性化学反应,开发了系列检测生物活性分子的BODIPY荧光探针,并对其荧光性质和生物成像应用进行了研究。相关内容如下:1.将硒代吗啉单元和BODIPY荧光团进行组合,设计、合成了一例新型的、检测溶酶体内过氧化氢（H₂O₂）的荧光探针BODIPY-Se。通过对硒代吗啉上氮原子的质子化,以及硒原子与过氧化氢的氧化反应,硒代吗啉上氮原子对BODIPY荧光团产生的光诱导电荷转移效应（PET）可以得到有效抑制,从而实现BODIPY-Se荧光信号的逐步增强。同时,BODIPY-Se能够在H₂O₂与谷胱甘肽（GSH）之间实现5次氧化还原循环。细胞和生物体荧光成像结果表明,BODIPY-Se能够很好的作用于细胞溶酶体（pearson系数为0.96）,并对细胞内源和外源性过氧化氢浓度的变化产生荧光响应。2.以BODIPY发色团为核心,设计、合成了基于氧化-成环反应的过氧化氢荧光探针BODIPY-BQ。BODIPY发色团双侧2,6-位引入的硼酸酯充当底物识别器,可实现与过氧化氢的特异性氧化还原化学反应。BODIPY发色团3,5-位以不饱和的丙烯酸酚羟基酯修饰,使BODIPY染料的吸收和发射波长明显红移。BODIPY-BQ能够选择和灵敏性的与过氧化氢发生作用,通过硼酸酯氧化-水解,继而发生分子内的酯交换-成环反应,促使探针分子形成新的共轭体系,并导致探针荧光明显降低。3.通过Knoevenagel缩合反应,将?（11）?-不饱和吡唑啉酮单元连接到BODIPY发色团2位,设计、合成了靶向线粒体和选择性检测次氯酸根的荧光探针BODIPY-Pyra。BODIPY荧光团与吡唑啉酮单元通过C=C双键进行桥接,形成了扭转的分子内电子转移（TICT）,具有较弱的红光。而环境黏度的增加将有效抑制TICT作用,促使探针红色荧光增强。此外,通过C=C双键与次氯酸根的氧化裂解反应,BODIPY-Pyra能够选择和灵敏性的与次氯酸根发生氧化还原反应,并促使探针的紫外和荧光发射蓝移。细胞和生物荧光成像实验表明,BODIPY-Pyra可作用于细胞线粒体（pearson系数为0.97）,也能够对细胞和生物体内次氯酸根浓度的变化进行荧光检测。4.通过Sonogashira偶联反应和Knoevenagel缩合反应,分别在BODIPY发色团两侧2,6-位引入了萘酰亚胺荧光团和不饱和吡唑啉酮结构,设计、合成了定位于溶酶体,且具有双识别位点和比率型变化的次氯酸/根荧光探针BODIPY-Np。在双识别位点作用下,BODIPY-Np能够与次氯酸/根迅速发生作用,使萘酰亚胺上的硫醚氧化为亚砜,萘酰亚胺单元荧光降低;同时,不饱和吡唑啉酮上的C=C双键断裂,BODIPY核荧光打开,荧光光谱呈现比率型变化。生物荧光成像实验表明,BODIPY-Np能够作用于细胞溶酶体（pearson系数为0.98）,并可对细胞和生物体中的次氯酸产生荧光响应。5.通过Sonogashira偶联反应,构筑了具有分子内电荷转移（ICT）效应和双波段、长波发射的BODIPY-三苯胺荧光平台,并设计、合成了具有不同光物理性质的荧光探针BTN和BTB。在BODIPY发色团双侧2,6-位对称性的引入三苯胺乙炔基,促使BODIPY染料发射波长明显红移至近红外区,并呈现D-A-D电子结构特征。BTN分子在三苯胺外围引入四个羟基肟作为识别器,实现了对次氯酸的多位点、快速（10s）和特异性反应,并在光谱上呈现比色和比率型变化特征。然而,在三苯胺外围引入不饱和丙烯酸叔丁醇酯替代羟基肟,得到具有固体荧光和聚集诱导发光性质的荧光分子BTB。BTB分子结构不仅呈现出明显的ICT特征和溶剂效应,而且具有较大的荧光发射波长和拟Stocks位移。在二甲基亚砜/水体系中,随含水量的增高,BTB分子呈现出明显的聚集诱导荧光增强性质。并且,BTB分子还表现出对半胱氨酸的选择性作用,可导致BTB分子的聚集诱导荧光淬灭。6.通过suzuki偶联反应,将短波BODIPY荧光团与长波BODIPY染料进行组合,构筑了具有跨键能量转移（TBET）性质的荧光分子BODIPY-By。BODIPY-By分子以短波BODIPY为能量供体,长波BODIPY为能量受体,刚性的联苯基为连接基团,呈现出跨键能量转移（ETE=86%）的性质。长波BODIPY染料上含有一对?（11）?-不饱和双键,具有一定亲核反应性;使BODIPY-By能够选择性的与半胱氨酸/同型半胱氨酸发生共轭加成反应,并呈现出比率型的荧光变化。相对于半胱氨酸,同型半胱氨酸与BODIPY-By分子表现出更快的反应速率;因此,BODIPY-By可实现选择性检测同型半胱氨酸的目的。生物体内的荧光成像实验表明,BODIPY-By分子具有较好的渗透性和生物相容性,并可用于生物体内Cys/Hcy的检测。更多还原

【Abstract】 BODIPY dyes have been widely used in bioimaging,photodynamic therapy,laser dyes,OLEDemission materials and organic photovoltaic and other fields because of excellent photochemical and physical properties,such as convenient synthesis,good photochemical and chemical stability,high molar extinction coefficient,fluorescence quantum yield,narrow fluorescence emission peak and so on.However,traditional BODIPY dyes also have disadvantages such as single emission,short wavelength,small Stocks shifyand,less molecular combination,which limit their application in organelle,bioimaging and other optical fields.Therefore,basing on BODIPY skeleton,we modified the structure of BODIPY chromophore,and constructed a variety of largeπ-conjugated systems and functional molecules through reasonable molecular combination and design.Moreover,according to the specific chemical reaction of the analytes,a series of BODIPY fluorescent probes were developed to detect reactive molecules,and study their fluorescence properties and bioimaging applications.As following:1.A novel fluorescent probe BODIPY-Se for lysosomal hydrogen peroxide（H₂O₂）was designed and synthesized by the combination of selenalmorpholine unit and BODIPY fluorophore.By protonation of nitrogen atoms on selenalmorpholine and oxidation reaction of selenium atoms with H₂O₂,photoinduced charge transfer effect（PET）from nitrogen atoms to BODIPY fluorophore can be effectively inhibited,thus realizing the gradual enhancement of fluorescence signal for BODIPY-Se.Meanwhile,BODIPY-Se can realize 5 redox cycles between H₂O₂ and glutathione（GSH）.The results of bioimaging showed that BODIPY-Se could target cell lysosomes（Pearson coefficient of 0.96）and detect endogenous and exogenous hydrogen peroxide in living cells.2.A hydrogen peroxide fluorescent probe BODIPY-BQ based on oxidation-cyclization reactionwas designed and synthesized with BODIPY chromophore as the core.The borate on the 2,6-sites of BODIPY chromophore acts as the receptor,to achieve specific redox chemical reactions with hydrogen peroxide.The absorption and emission wavelengths of BODIPY dyes were significantly red-shift when the 3,5-sites of BODIPY chromophore were modified with unsaturated acrylate phenolic hydroxy esters.BODIPY-BQ can selectively and sensitively interact with hydrogen peroxide,through the oxidation-hydrolysis of borate ester,and then the transesterification-cyclization reaction,which promotes the formation of a new conjugated system of probe molecules and significantly decrease s the fluorescence of the probe.3.By Knoevenagel condensation reaction,?（11）?-unsaturated pyrazolinone unit was connected to the 2-site of BODIPY chromophore,and a fluorescent probe BODIPY-Pyra targeting mitochondria and selective detection for hypochlorite was designed and synthesized.The BODIPY fluorophore bridged with the pyrazolinone unit through C=C double bond,forming a twisted intramolecular electron transfer（TICT）with a weak red light.However,the increase of ambient viscosity will effectively inhibit the TICT effect and enhance the red fluorescence of the probe.In addition,BODIPY-Pyra was able to selectively and sensitively undergo redox reaction with hypochlorite by oxidative cleavage of C=C double bond with hypochlorite,and produce blue shift of uv and fluorescence of probe.Bioimaging experiments have shown that BODIPY-Pyra can direct the mitochondria（Pearson coefficient 0.97）and can also detect hypochlorite in cells and organisms.4.By Sonogashira coupling and Knoevenagel condensation reactions,naphthalimide fluorophore and unsaturated pyrazolinone structures were introduced on both sides of the BODIPY chromophore,respectively,and a hypochlorous acid/hypochlorite fluorescent probe BODIPY-Np with double recognition sites and ratiometric changes was designed and synthesized to locate in lysosomes.Because of double recognition sites,BODIPY-Np can act quickly with hypochlorous acid/hypochlorite to oxidize the thioether on naphthalimide to sulfoxide and decrease the fluorescence of naphthalimide unit.At the same time,C=C bond on unsaturated pyrazolinone was broke,the fluorescence of BODIPY core was turned on with a ratiometric change.Bioimaging experiments have shown that BODIPY-Np can locate lysosomes（Pearson coefficient 0.98）and monitor hypochlorous acid in cells and organisms.5.Through Sonogashira coupling reaction,the fluorescence platform of BODIPY-triphenylamine with intramolecular charge transfer（ICT）effect and dual-band and long-wave emission was constructed,and fluorescent probes BTN and BTB with different photophysical properties were designed and synthesized.The introduction of triphenylamine acetylene group on both sides of BODIPY chromophore leads to the obvious red shift of the emission wavelength of BODIPY dye,and presents D-A-D structure.BTN introduced four hydroxyl oxime on the periphery of triphenylamine as the receptor,which realized the multi-site,fast（10s）and specific reaction to hypochlorous acid,and presented colorimetric and ratiometric on the spectrum.However,unsaturated tert-butanol acrylate was introduced to rep lace hydroxyl oxime in the periphery of triphenylamine to obtain BTB with solid fluorescence and aggregation induced luminescence.BTB not only presents obvious ICT characteristics and solvent effects,but also has a large fluorescence emission wavelength and pseudo-stocks displacement.In dimethyl sulfoxide/water,BTB showed obvious aggregation induced fluorescence enhancement with the increase of water fraction.In addition,BTB also showed a selective reaction on cysteine,which could lead to quench the fluorescence induced by aggregation of BTB.6.By suzuki coupling reaction,the short wavelength BODIPY fluorophores and long wavelength BODIPY dyes were combined to construct a fluorescent molecule BODIPY-By with through bond energy transfer（TBET）.BODIPY-By take short wavelength BODIPY as the energy donor,long wavelength BODIPY as the energy receptor and rigid Biphenyl structure as the connecting group,presenting the property of TBET（ETE=86%）.long wavelength BODIPY dye contains a pair of?（11）?-unsaturated double bonds,with a certain nucleophilic reactivity.The conjugated addition reaction of BODIPY-By with cysteine/homocysteine was performed selectively with ratiometric change.Compared with cysteine,homocysteine showed a faster reaction rate with BODIPY-By.Therefore,BODIPY-By can have a selective detection of homocysteine.更多还原