【作者】 黄兰；

【导师】 刘叔文；

【作者基本信息】 南方医科大学 ， 药理学， 2013， 博士

【摘要】 有机荧光化合物在医学、生物学、材料学、化学等多种领域有着广泛的用途,是学术界乃至工业界的研究热点。大多数有机荧光化合物在稀溶液中有很好的发光性能,但在聚集状态,尤其是在晶态时,荧光减弱甚至猝灭。这种现象被称为荧光“聚集诱导淬灭效应”(Aggregation-caused Quenching, ACQ)。ACQ限制了荧光物质在制备有机发光二极管(Organic Light-Emitting Diode, OLED)、化学与生物传感器等多种领域中的应用。因此,ACQ一直是有机荧光物质在实际应用过程中一个难以克服却又必须解决的难题。在人们致力发展各种性能更加优异的新型发光材料的过程中,唐本忠(Tang Ben Zhong)院士课题组于2001年发现了一种在溶液中不发光,而在固态时却呈现很强的发光特性的硅杂环戊二烯(Silole)。他们将这一现象命名为聚集诱导发光特性(aggregation-induced emission, AIE)。聚集诱导发光特性化合物的发现为消除ACQ提供了一种根本的解决方法。短短的十来年间,具有AIE性质的有机荧光物质已在OLED.生物检测以及化学检测等多种应用领域中显示了其独特性质的优势。但到目前为止,具有高发光效率的AIE化合物种类有限,主要有silole型和多苯基取代乙烯型等小分子化合物和少数高分子化合物。多组分反应(Multicomponent reactions, MCRs)具有原子经济性、高效性、总产率高以及其产物具有结构多样性和复杂性等特点,是一种符合绿色化学概念的理想反应模式,在多种领域,尤其是在新药发展过程中发挥了其独特的优势。本课题组近期发展了一种原料易得、操作简单、反应条件温和的五组分反应(Five-component Reaction,5CR)。该5CR产物五取代四氢嘧啶类化合物具有很强的AIE效应及很高的荧光量子产率,是首次由MCRs合成的环状AIE化合物,打破了环状AIE化合物难以合成的观点。本论文一方面利用该5CR在合成上的优势,合成了结构各异的五取代四氢嘧啶类化合物并研究了其光学特性,另一方面发现了该新型AIE化合物在pH和金属离子检测等方面的用途。本论文共分五章(文献综述、五取代四氢嘧啶类化合物的多组份反应合成、五取代四氢嘧啶类化合物光学特性、金属铜离子检测以及pH测定),包含三大部分实验内容(五取代四氢嘧啶类化合物的多组份反应合成、光学特性以及其应用研究)。第一部分五取代四氢嘧啶类化合物的多组份反应合成目的合成结构各异的五取代四氢嘧啶类化合物,以用于其光学性质和应用研究。方法利用本课题组近期发展的一种原料易得、操作简单、反应条件温和的5CR进行目标化合物的合成。结果(1)共合成了22种化合物。(2)对22种化合物进行了ESI-MS、1Hand1C3NMR等结构表征。(3)用紫外-可见分光光度法测定化合物在溶液和悬浊液中的吸收光谱(4)用稳态荧光法测定化合物在溶液和悬浊液中发射光谱结论(1)利用本课题组近期发展的5CR,以不同的伯胺、甲醛、不同的芳香醛和丁炔二酸二酯为原料,在温和的反应条件下,可方便的合成目标化合物,产物总产率在40%-60%之间。(2)我们发现6显示重要的AIEE性能,它在溶液中实际上是不发光的(ΦF,s→0),但是在聚集体中发出很强的荧光,荧光量子率达到93%(6bb)。第二部分五取代四氢嘧啶类荧光化合物的光学性质目的五取代四氢嘧啶类荧光化合物不仅具有特殊的聚集诱导发光性质,某些化合物还具有发射波长与形态无关的特性,以及在不同的条件下形成蓝绿荧光异构体的特性。因此,本部分实验拟通过测定五取代四氢嘧啶类荧光化合物在不同溶剂、温度、浓度等条件下的光学性质,培养荧光异构体单晶并解析晶体结构中分子堆积模式,以探测该类化合物的发光机理。方法(1)用紫外-可见分光光度法测定化合物在溶液和悬浊液中的吸收光谱(2)用稳态荧光法测定化合物在溶液和悬浊液中以及不同形态的(粉末、薄膜、晶体)激发和发射光谱(3)用X-射线单晶衍射法测定化合物的单晶结构(4)用diamond软件解析单晶结构中分子堆积模式及分子间作用力结果(1)溶剂、温度及浓度对蓝绿光晶体的形成有明显的影响。高温或者低浓度有利于蓝光晶体的形成。相反,低温或者高浓度有利于绿光晶体的形成。最开始形成的悬浊液粒子是以绿色荧光异构体,在高温条件下随着超声时间的延长,绿色荧光异构体就会全部转变为蓝色荧光异构体。这就意味着动力学有利于绿色荧光异构体的形成,热力学有利于蓝色荧光异构体的形成。(2)当水/乙醇比例增加到80%时,6aa的紫外吸收光谱红移且在长波区域尾部抬高,同时发光强度显著地增强。这表明形成了J-聚集。化合物6aa在乙醇溶液中不发光,即便扩大50倍,也只能得到噪声信号,然而,当水的体积比达到95%时荧光量子产率增加为0.52。6aa晶体和粉末的的最大发射波长与悬浮液最大发射波长相同。这些结果说明6aa在蓝光聚集体中发射波长具有形态无关的特性。(3)单晶-X射线衍射数据分析显示6aa的蓝绿光发射特性产生于不同的分子堆积模式：蓝光是R/S-S/R堆积模式,绿光是R/R-S/S。因此,在不同的条件下制备蓝光聚集体,绿光聚集体以及蓝绿光聚集体混合物实际上是R/S-S/R与R/R-S/S堆积模式比例的改变。(4)根据上述的实验研究结果,6的聚集诱导发光增强的性质可能是由它们特殊的结构和J-聚集造成的。因为6的三个苯环彼此不共轭,并通过C-C或者C-N单键链接非芳香中心环,它们处于单分子状态(在纯有机溶液中)时可以自由旋转,从而使相应激发态去活化,6在溶液中不发光。6在聚集态时发强的荧光是由于它非对称的立体分子结构有效的抑制了π-π堆积,同时J-聚集抑制了分子内的转动以及形成了分子间电荷转移态。(5)在蓝光及绿光晶体聚集体中不发光的表面分子可能由于6aa高度灵活性和非对称立体结构所决定。结论6聚集诱导发光增强特性及6aa与形态无关性质可能产生于：1)可高度自由旋转的连接苯基的单键(6中的三个相邻的芳基互不共轭；通过单键与非芳香四氢嘧啶环相连接)；2)不对称的立体结构(相邻的三个苯基与手性四氢嘧啶环相连,并且相互垂直)；3)J-聚集。第三部分五取代四氢嘧啶类荧光化合物的应用研究目的(1)金属铜离子是生物体内重要的微量元素,过多过少都会影响生物体内正常的生理功能。因此,检测铜离子浓度具有重要的临床意义。为发展高选择性和高灵敏度的铜离子荧光探针,我们测定了5CR产物的荧光强度对铜离子的浓度的相应,发现其中的一个5CR产物dimethyl1,2,3,6-tetrahydro-2-(4-hydroxy-3-methoxyphenyl)-1,3-diphenylpyrimidine-4,5-dicarboxylate,即6bh,可发展为一种新型的铜离子荧光探针。(2)细胞和细胞器的许多重要生理过程等都与pH值密切相关。正常的生理条件下细胞外液中H+的浓度40nmol/L(pH=7.40),其变动幅度一般在0.1-0.2个pH单位。酸性和碱性过强会导致心、肺病变或神经类疾病,严重时甚至会有生命危险,利用各种荧光参数(如荧光强度、荧光寿命等)的变化来测定pH值,不仅便于荧光显微镜学研究,而且可时实检测细胞内pH的动态分布和区域变化。我们发现其中的一个5CR产物,dimethyl1,2,3,6-tetrahydro-2-(4-hydroxy-3-methoxyphenyl)-1,3-diphenylpyrimidine-4,5-dicarboxylate,即6bh,及Dimethyl1,2,3,6-tetrahydro-2-(4-hydroxy)-1,3-diphenylpyrimidine-4,5-dicarboxylate,即6bj,其不仅具有AIE性能,因而可开发为pH荧光探针方法(1)在不同过渡金属离子存在下以及在不同铜离子浓度存在下,用紫外-可见分光光度法和稳态荧光法测定测定五取代四氢嘧啶6bh吸收、激发和发射光谱的变化,以研究6bh作为荧光探针对铜离子的选择性及灵敏性,并探究其作用机理。(2)在不同pH值的缓冲溶液中,用紫外-可见分光光度法和稳态荧光法测定测定五取代四氢嘧啶6bj吸收和发射光谱的变化,以确定6bj作为荧光探针用于检测pH值。结果(1)6bh(30μM)在没有金属离子存在(99%水/乙醇混合液)与有金属离子(300μM)(Ag+, Ca2+, Co2+, Cu2+, Mn2+, Ni2+, Zn2+, Eu3+, Gd3+, Pr3+, Mg2+, Ce3+and Fe3+)存在的条件下,其发射光谱显示只有铜离子才能引起荧光的显著增加,而其他的金属离子对其没有影响(Ag+和Ca2+)或者荧光淬灭(除了Cu2+, Ag+和Ca2+,其他金属离子)。Fe3+几乎是使6bh聚集体的荧光完全淬灭。当Cu2+浓度减少到30μM,6bh聚集体的荧光强度仍然显著的增加。该结果表明,6bh是一种具有很高灵敏性Cu2+荧光探针(2)在与其它金属离子共存的情况下,6bh对Cu2+的选择性没有受到影响,这些结果从而说明Cu2+与6bh的相互作用是有竞争性的,对Cu2+具有很高的选择性。(3)化合物6bj在pH>12的溶液中,几乎没有荧光,这是因为,在碱性条件下,化合物6bj的酚羟基被转化为醇钠盐的形式,使其溶解在混合溶液中,没有聚集体产生。随着混合溶液pH值的降低,化合物6bj的醇钠盐结构逐渐减少,部分失去醇钠盐结构的化合物6bj开始聚集,溶液的荧光强度也随之增强。当pH=4或者5时,溶液的荧光强度保持不变,是pH=11溶液荧光强度的20倍,在波长为365nm紫外光的照射下,肉眼可以看出两者的明显区别。pH=4荧光强度最大,此时化合物6bj形成了大量的聚集体。实验表明,化合物6bj对pH值的变化有着灵敏的响应。结论(1)6bh是一种新型的铜离子荧光增强荧光探针,其对铜离子具有高的选择性灵敏性。6bh荧光强度与铜离子浓度为0-60μM时具有好的线性关系。6bh可以用来定量分析环境水样中铜离子。一个有趣的现象是随着铜离子浓度的增加除了荧光强度增强外,粒子尺寸减小,这可能是配位诱导分子内氢键的离解。基于对铜离子的高选择性和灵敏性,铜离子诱导的伴随粒子大小减小荧光增强的现象,特殊的分子堆积模式及没有细胞毒性,6bh有望用于实际应用,新探针设计及理论研究。(2)我们研究了含有酚羟基的五取代四氢嘧啶类化合物对pH的响应。研究结果表明化合物6bj对pH值的变化有着灵敏的响应。6bj的细胞毒性相比6bh较大,因此,可以通过对6bj结构进行适当的修饰。得到细胞毒性小的pH生物荧光探针。更多还原

【Abstract】 BackgroundOrganic fluorophores have attracted great interest from both academia and industry due to their potential applications in many fields, such as chemistry, materials science, biology, and medicine.Most of organic dyes have good luminescent properties in dilute solution, but in aggregate state, especially when in crystalline state, the fluorescence becomes weakened or quenched. This phenomenon is called "aggregation induced quenching"(ACQ). The ACQ effect have greatly limited their scope of practical applications, such as organic light-emitting diodes, chemosensors/biosensors etc. the ACQ effect has been a thorny obstruct that must be overcome.The development of efficient luminescent materials is a topic of great current interest. In2001, Tang discovered such a system, in which luminogen aggregation played a constructive, instead of a destructive, role in the light-emitting process:a series of silole molecules were found to be non-luminescent in the solution state but emissive in the aggregated state (as nanoparticle suspensions in poor solvents or as thin films in the solid state). They coined the term "aggregation-induced emission"(AIE) for this novel phenomenon, because the non-luminescent silole molecules were induced to emit by aggregate formation. The novel AIE effect is exactly opposite to the notorious ACQ effect discussed above. The phenomenon is of academic value. In last decade, The AIE luminogens have already shown practical applications in OLEDs, chem-sensors, and bio-probes, due to their efficient preparation and unique properties. However, until now, the studies of utilizing AIE dyes as FL sensors mainly concentrate on TPE derivatives and silole derivatives, so that developing new AIE-active sensory materials would be very rewarding work. Multi-component reaction (MCRs) is an ideal reaction mode which is consistent with the concept of green chemistry, It has attracted in-creased attention in synthetic chemistry for their distinct advantages such as such as atom economy, simplified workup procedures, great efficiency, high overall yields, and structure diversity and complexity of products. We have recently reported an efficient new five-component reaction (5CR) with raw material acquire, simple operation, moderate reaction characteristics Therefore, our group utilizes Multicomponent reactions (MCRs) to synthesis of the nitrogen heterocycles compound, Importantly, the5CR products pentasubstituted tetrahydropyrimidines show unusual characteristics of aggregation-induced emission enhancement (AIEE) and high fluorescence quantum yield. On one hand, In this thesis a new series of structure diversity, AIE-active pentasubstituted tetrahydropyrimidines derivatives have been directly synthesized via five-component reaction (5CR) under mild conditions, and study their optical properties, On the other hand we find that the AIE-active pentasubstituted tetrahydropyrimidines derivatives can apply as pH sensor and metal ion probes. This thesis is divided into five chapters (literature review, Convenient synthesis of C-6unsubstituted tetrahydropyrimidine-4,5-dicarboxylates via five-component Reactions, the optical properties of C-6unsubstituted tetrahydropyrimidine-4,5-dicarboxylates, detection copper ion and pH detection. The content of this thesis is divided into three parts (synthesis of pentasubstituted tetrahydropyrimidines derivatives via MCRs, optical properties and their applications).Part1Convenient Synthesis of C-6UnsubstitutedTetrahydropyrimidine-4,5-dicarboxylates via Five-component Reactions and Its Optical PropertiesObjection:We synthetize structure diversity pentasubstituted tetrahydropyrimidines derivatives via five-component reaction, and study their optical properties and explore their applications.Methods(1) Our group utilize Multicomponent reactions (MCRs) with atom economy, high efficiency, structural diversity and complexity of compounds to synthesis of pentasubstituted tetrahydropyrimidines derivatives.Results:(1) We have synthetized22compounds(2) Structural characterization by ESI-MS、1H and13C NMR(3) Assay the absorption spectra of the compound in solution and suspension by UV-visible spectrophotometry(4) Measure the excitation and emission spectra of the compound in solution, and suspension by steady-state fluorescenceConclusion(1) We have developed efficient and simple five component reactions (5CRs) of but-2-ynedioates, primary amines, primary amines, formaldehyde and aromatic aldehydes using urea as a catalyst, AcOH as an additive and methanol as solvent for the synthesis of C-2substituted tetrahydropyrimidine-4,5-dicarboxylates. The5CRs proceeded smoothly at room temperature to afford target products in40%-60%yields.(2) we found that6exhibit important AIEE properties because they show practically no emission in solution (ΦF,→0) but strong emission in aggregates withΦF,a up to93%(6bb)。 a library of6with high fluorescence efficiency could be built fast and efficiently by taking the5CR advantages of mild reaction conditions, readily available reactants, operational simplicity and potential ability in building structure diversity products.Part2The Optical Properties of C-6Unsubstituted Tetrahydropyrimidine-4,5-dicarboxylates6aaObjection:Pentasubstituted tetrahydropyrimidines not only exhibit important AIEE properties but also unusual size-independent emission (SIE) characteristics, as well as two kinds of fluorescent aggregates, bf-and gf-aggregates in different conditions. Therefore, In this part of the experiment, we will measure the optical properties of pentasubstituted tetrahydropyrimidines derivatives under different solvents, temperature, concentration, culture fluorescence isomer crystal and parse the crystal structure to understand molecules stacking mode, and explore the luminescence mechanism of the compound.Methods(1) Assay the absorption spectra of the compound in solution and suspension by UV-visible spectrophotometry(2) Measure the excitation and emission spectra of the compound in solution, and suspension, as well as different forms (powder, thin film, crystal) by steady-state fluorescence(3) The single crystal structure of the compound was determined by X-ray diffraction method(4) Diamond software parsing the molecular stacking mode and intermolecular forces in the single crystal structureResults:(1) The solvent, temperature, and concentration have a significant effect on the formation of the blue-green crystals。 We found that a slower crystallization rate (at higher temperatures or lower concentrations) favored the formation of bf-crystals. In contrast contrast, a faster rate (at lower temperatures or higher concentrations) favored the formation of gf-crystals. At the same time, We found that the particles that formed first under the studied conditions were the gf-forms. However, they could transform to the bf-forms by increasing ultrasonic time at higher temperatures. This means that the gf-particles are the kinetically favored form and the bf-particles are the thermodynamically favored form.(2) When the volume fraction of water (fw) in ethanol increased to80%, at the same time, the emission intensity was enhanced dramatically. This indicates the formation of J-aggregate. Compound6aa in ethanol is practically no emissive because the50-fold enlarged emission spectrum of6aa in ethanol is a noisy signal line.However, the ΦF value of6aa increases to0.52, as the fw, value is up to95%. The emission spectra of6aa in crystals and in powder (gently ground crystals) possess the same λem.max value as that in suspensions, which indicates that6aa in bf-aggregates shows SIE characteristics.(3) the processes of preparing the pure bf-aggregates, pure gf-aggregates as well as bf-and gf-aggregate mixtures of6aa under different aggregation conditions are practically the processes that alter the ratio of R/S-S/R and R/R-S/S stacking modes. In other words, the MSMs of6aa in crystals and suspensions could be tuned by changing the aggregation conditions.(4) According to the experimental results mentioned above, the AIEE property of6may result from their special structure and/-aggregation. Since the three phenyls of6are not conjugated with each other and connected to a non-aromatic central ring via C-C or C-N single bonds, they could rotate freely in monomer (in solution), which deactivates the corresponding excited states. This makes6non-emissive in solution. The strong fluorescence emission of6in aggregates should be attributed to its asymmetric stereo molecule structure efficiently preventing π-π stacking as well as J-aggregation restricting intramolecular rotation and forming intermolecular charge transfer states.(5) The non-emission of the surface molecules in the bf-and gf-crystalline aggregates may result from the high flexible and asymmetric stereo structure of6aa.ConclusionThe AIEE of6and the SIE characteristics of6aa may be attributed to i) the high flexible structures (the three phenyls of6are not conjugated with each other and connected to the non-aromatic tetrahydropyrimidine ring via single bonds); ii) the asymmetric stereostructure (the three phenyls adjacently are connected to the chiral tetrahydropyrimidine ring and are perpendicular from each other); and iii) J-aggregation. Part3Applications of C-6Unsubstituted Tetrahydropyrimidine-4,5-dicarboxylatesObjection:(1) Copper ion (Cu2+) is an essential trace element for many biological processes and systems, the detection of copper ion concentration has important clinical significance. Therefore, it is of great significance to develop high selective fluorescent enhancement chemosensors for Cu2+. We measured the fluorescence intensity of the5CR product with response to the concentration of copper ions, we found that6bh, one of these racemates, dimethyl2-(4-hydroxy-3-methoxyphenyl)-1,3-diphenyl-1,2,3,6-tetrahydropyrimidine-4,5-dicarboxylate(6bh, TPD-OH), can be a sensitive and selective fluorescent probe for Cu2+.(2) many important physiological processes of cell and organelle are closely related with pH value. in normal physiological conditions The concentration of H+is40nmol/L in the extracellular fluid and the rate of change is0.1-0.2pH unit in general, Strong alkaline and acid leads to heart, lung disease or neurological diseases, seriously it even has life-threatening, so we can use a variety of fluorescence parameters (such as fluorescence intensity, fluorescence lifetime) changes to measure the pH value, it can not only be used for fluorescence microscopy study,but also can detect the changes of intracellular pH in real-time it has attracted widespread attention. we found that one of these racemates, Dimethyl1,2,3,6-tetrahydro-2-(4-hydroxy)-1,3-diphenylpyrimidine-4,5-dicarboxylate,6bj, can be a sensitive and selective fluorescent probe for pH.Methods(1) Determination the changes of the absorption spectra, excitation spectra and emission spectra of6bh in the presence of other metal ions and in the different concentration of Cu2+by UV-visible spectrophotometry and steady-state fluorescence, study the sensitivity and selectivity of6bh for Cu2+, and explore the possible mechanism.(2) Determination the changes of the excitation spectra and emission spectra of6bj in different pH value, study the fluorescence intensity via the pH value and developt a new kind of pH fluorescence probe.Results:(1) The emission spectra of6bh (30μM) in the absence or in the presence of other metal ions (300μM)(Ag+, Ca2+, Co2+, Cu2+, Mn2+, Ni2+, Zn2+, Eu3+, Gd3+, Pr3+, Mg2+, Ce3+and Fe3+) indicate that only Cu2+caused a significant fluorescence enhancement and others showed no influence (Ag+and Ca2+) or a fluorescence quenching (all determined metals except Cu+, Ag+and Ca2+) on the emission spectra of6bh. Fe3+almost turned off the fluorescence of6bh in aggregates. When the concentration of Cu2+was decreased to30μM, the fluorescence intensity of6bh in aggregate also increased significantly(2) The fluorescence intensity at maximum emission wavelength (471nm) of6bh(30μM) in ethanol-water mixture (fw,=99%) in the absence of metal ion and in the presence of Cu2+, other transition metal ions or Cu2+with another transition metal ion are shown in Figure3b. It can be seen:(i) the influences of other metal ions at100μM of concentration on the fluorescence intensity of6bh were almost the same as that at300μM;(ii) the sensitivity of6bh to Cu2+is not influenced significantly by these commonly coexistent ions, which shows the interaction of Cu2+with6bh is competitive in the presence of other transition metal ions with high concentration. These proved that6bh is a high sensitive and selective probe for Cu2+.(3) The phenolic hydroxyl of6bj and6bh exists an acidic dissociation equilibrium in aqueous solutions. The phenolic hydroxyl of6bj and6bh exists a acidic dissociation equilibrium in aqueous solutions. When the pH increased higher than12, it almost no fluorescence, the amount of the neutral form in the equilibrium will increase but that of the anion form will decrease with the decrease of pH Since neutral form is not soluble but anion form is well soluble in aqueous solution, when the pH is4or5, the fluorescence intensity remained unchanged, the fluorescence intensity is20times than pH at11, under the irradiation of ultraviolet light we can easily discriminate the two forms. When the pH value is4, the fluorescence intensity reaches the maximum, the compound6bh formed large aggregates. It shows that compound6bj has sensitive to pHConclusion(1) A novel fluorescence enhancement probe for Cu2+,6bh, has been developed. The probe possesses high sensitivity and selectivity for Cu2+. Moreover, a good linear relationship between the fluorescence intensity of6bb. and the concentration of Cu2+ranges from0-60μM. Therefore,6bh can be used for the quantitative analysis of Cu2+in environmental water samples. An interesting phenomenon is that in addition to the fluorescence enhancement, the particle sizes of6bh become smaller upon increasing the concentrations of Cu2+, which is expected to result from the coordination-induced intermolecular hydrogen-bond dissociation. Considering the high selectivity and sensitivity to Cu2+, the interesting phenomenon of Cu2+-induced fluorescence enhancement with size-decrease, the special molecular stacking mode and no cytotoxicity,6bh is expected to be useful for practical applications, new probe design and theoretical research.(2)6bj is a phenol-moiety-containing compound that shows aggregation at pH values smaller than12to result in a high FL intensity with a pKa of10.5. Its cell cytotoxicity is smaller higher than6bh, so we can modify the structure of6bj and developt it as the pH probe with low cytotoxicity.更多还原