【作者】 孙颖；

【导师】 任爱民；

【作者基本信息】 吉林大学 ， 物理化学， 2011， 博士

【摘要】 生物发光的发光机制包括细胞内发光和细胞外发光两大类。从生物体排放出来的某些腺体中含有能发光的物质,这种细胞外的发光引起了科学家们的广泛兴趣。海萤属于细胞外发光,由于高的生物发光效率和很高的灵敏度而成为研究热点,但是人们目前对海萤荧光素的化学发光反应机理尚不清楚,尤其是理论方面的研究显得更为缺乏。利用海萤荧光素及其类似物作荧光探针来标记细胞或DNA,直接监控活体细胞内的细胞活动和基因活动,这一方面的理论研究还是一个崭新的研究课题。因此,本论文的主旨是研究海萤荧光素衍生物发光机理,并在海萤发色团中心吡嗪环基础上设计并研究一系列不但具有良好荧光性质而且具有较高的双光子吸收截面值的新型探针材料分子。为了详细地从理论计算的角度阐述以上几点问题,我们采用量子化学密度泛函方法（DFT）对分子基态几何构型优化、吸收光谱、前线分子轨道等方面进行了计算,以及进行NBO分析和过渡态寻找,用含时密度泛函理论（TDDFT）对相关分子的激发态几何结构优化、发射光谱以及荧光寿命做了进一步研究。采用ZINDO方法和完全态求和公式（SOS）以及组内自编的FTRNLO程序计算相关分子的单光子与双光子吸收性质等相关信息。这些理论研究为探究海萤高效率发光机理问题,以及基于海萤荧光素,设计和合成具有良好的荧光性质和双光子性质的材料分子奠定了一定的基础。本论文的主要内容从三个方面进行探究:1.为了探究海萤发光反应机理问题,从理论上研究了一系列咪唑并吡嗪酮环衍生物（海萤荧光素的核心结构）在气相以及DMSO和二甘醇二甲醚溶剂中的化学发光反应机理问题。研究结果表明,给电子基团与中心结构的π共轭相互作用都强于吸电子基团,并且溶剂增强了给电子基团与中心结构片段之间的相互作用。通过计算基态分子的电子抽取能,结果表明在这种溶剂中其给电子能力增强、稳定性降低,从而更容易实现MIP–→MIP·这个过程。从微观角度说明了给电子基团（尤其是吲哚作为取代基时）可以增加海萤类似物发光反应过程的速率,对提高荧光效率起到了积极的作用。从动力学和热力学的角度对其化学发光反应过程中路径（Ⅰ） :DT^ˉ→DT→¹AAP^*→hν及路径（Ⅱ）:DT-→¹AAP^*-→hν进行了研究。对该反应的活化能和产物荧光寿命的分析结果表明:针对化学发光反应（吡嗪过氧化四圆环质子化和失去二氧化碳激发的过程）,DMSO溶剂可以使海萤化学发光反应快速进行,提高荧光效率,但是DG溶剂可以增加路径（?）的荧光效率。2.为了研究海萤荧光素是否可以作为双光子荧光探针材料,我们计算了一系列吡嗪衍生物的荧光及其双光子吸收性质,研究结果表明:向2, 3, 5-三取代吡嗪衍生物引入苯乙烯基团可以增加分子的共轭性,并且能增加双光子吸收截面值以提高双光子吸收性质。对比荧光性质的差别,2-苯乙烯吡嗪系列衍生物和5-苯乙烯吡嗪系列衍生物的荧光强度（激发态振子强度）均大于其他取代基（乙酰氨基和吲哚基）在C2位和在C5位取代的吡嗪系列物,而3-吲哚吡嗪系列衍生物的荧光强度大于3-苯乙烯吡嗪系列衍生物。因此我们可以得出这样的结论:苯乙烯基团对三取代吡嗪衍生物的2位和5位作用较强,尤其是荧光性质影响很大,但是当C3位由吲哚取代时,3-吲哚衍生物也表现出良好的荧光性质,总的来说,设计的这类2, 3, 5-三取代吡嗪衍生物中,2-苯乙烯基-3-吲哚基-5-苯乙烯基吡嗪、2-苯乙烯基-3, 5-吲哚基吡嗪和2, 3, 5-苯乙烯基吡嗪化合物既有良好的荧光性质,又有很好的双光子吸收性质。以上结果为研究生物体海萤荧光素作为双光子荧光探针材料提供了重要的理论线索。3.与DNA结合的高敏感的双光子荧光探针（TFP）材料近年来颇受研究者的关注,但双光子荧光探针与DNA结合形式还没有明确阐述。因此,从理论上研究了一系列有实验测试支持的3,6-双（4-乙烯基吡啶）-咔唑衍生物中性分子和阳离子以及3,6-双（4-乙烯基吡啶）-咔唑衍生物碘盐分子,对其几何结构、电子结构和单、双光子吸收性质进行了详尽的研究,研究结果表明,咔唑3, 6位的乙烯吡啶基团和9位的乙基吡啶上的N是活化位置,在活化位上连接不同的取代基对双光子吸收截面值的影响很大,尤其是碘离子的引入使整个分子的双光子吸收截面值发生明显的变化,而且在整个研究过程中还发现,在咔唑3, 6位的乙烯吡啶基团和9位的乙基吡啶上的N的活化位置引入了不同的取代基形成的三个系列的双光子吸收光谱均在近红外区域,所以这些系列的分子也可以作为近红外光区的双光子吸收材料。总之,当将研究的分子作为双光子荧光探针与DNA作用时,作用的活性部位应该有三个,即咔唑环的3,6,9位。并且3,6-双（4-乙烯基吡啶）-咔唑衍生物阳离子和6-双（4-乙烯基吡啶）-咔唑衍生物碘盐更可能是探针与DNA结合形式。更多还原

【Abstract】 Bioluminescene includes two classes of intracellular and extracellular luminescence. The extracellular bioluminescence usually is that the glands emitted from orgnisms contain some substances can luminesce. The extracellular bioluminescence has caused great interest of scientists. Cypridina can emit blue light in seawater, belongs to the extracellular bioluminescence. The cypridina bioluminescence has been a research focus due to its high bioluminescence efficiency and high sensitivity. But the chemiluminescence reaction mechanism of cypridina luciferin remains unclear, and especially, the theoretical research on it is scarce. Using Cypridina luciferin analogues to label cell nucleus or DNA as probe and monitor living cell and gene activity is a new research subject. Therefore, the thesis aims to study the chemiluminescent reaction mechanism of Cypridina luciferin, design a series of TPA probe materials which exhibit not only good fluorescence properties but also large two-photon cross section based on core structure of Cypridina luciferin. We calculated the ground-state geometries, the absorption spectra and frontier molecular orbitals, NBO analysis, and the transition state search by means of DFT method. Moreover, their optimized excited-state geometry, the emission spectra and fluorescence lifetime were obtained by TDDFT. The one- and two-photon absorption properties were calculated by using the Zerner’s intermediate neglect of differential overlap （ZINDO） method and the sum of states （SOS） formula and the FTRNLO program compiled by our group. These calculations will play an important role in the study how to enhance the luminescence efficiency of cypridina luciferin analogues. And on the basis of above results, one can further explore high efficiency bioluminescence of cypridina luciferin and the design and synthesis of related TPA materials with good fluorescence efficiency and large TPA cross sections. The main contents of the thesis are summarized as follows:1. In order to explore the chemiluminescent reaction mechanism of cypridina luciferin, a series of cypridina derivatives were theoretically investigated in detail in the gas phase, DMSO, and diethylene glycol dimethy. These derivatives are obtained by replacing the 6-aryl with different group based on 6-aryl-2-methyllimidazo [1,2-a] pyrazin-3-（7H）-ketone rings. We discussed the geometry, bond length alternation, HOMO-LUMO gaps, electron extraction potentials and natural charge population through the key steps of chemiluminescence reaction. The results indicates that introducing the electron donating group result in better conjugated effect than electron-withdrawing group, and the interactions between electron-donating groups and central structure can be enhanced in solvent, which is ascribed to that the solvent enhances the electron donating ability and stability, and in turn facilitate MIP–→MIP·process in diglyme （DG）. Form this point of view, introducing the electron-donating groups （especially indole as substituents） can increase the luminescent reaction rate of cypridina analogues and improve the efficiency of fluorescence.From the kinetic and thermodynamic perspective, we investigate the chemiluminescence reaction of pathes of （Ⅰ） DT^ˉ→DT→¹AAP^*→hνand （Ⅱ） DT^ˉ→¹AAP^*ˉ→hν. The analysis of the activation energy of the reaction and the flurescence lifetime suggests that the DMSO solvent can improve the chemiluminescence of cypridina luciferin analogues and enhance the fluorescence efficiency, but DG solvent can increase the fluorescence efficiency of path （?）.2. Whether cypridina luciferin analogues can be used as two-photon fluorescent probe? To resolve this problem, the fluorescence and two-photon absorption （TPA） properties of a series of pyrazine derivatives were investigated. The calculated results indicate that introducing styrene groups to 2, 3, 5-trisubstituted pyrazine derivatices can increase the conjugated effect and the TPA cross section values, which can enhance the TPA properties. Comparing their fluorescence properties, the fluorescent intensity (the excited state oscillator strength f_em) for the 2-styrene pyrazine derivatives and 5-styrene pyrazine derivatives are larger than the other substituents （acetylamino group and indole group） at the 2-site and 5-site of pyrazine core, and the f_em of 3-indole pyrazine derivatives is larger than that of 3-styrene pyrazine derivatives. Based on these results, we can conclude that the styrene group plays a positive important role in fluorescence of 2-, 5-sites of pyrazine derivatives. However, the series of 3-substituted pyrazine derivatives with indole group have also good fluorescence properties. Among the 2, 3, 5-trisubstituted pyrazine derivatives, 2-styryl-3-indolyl-5-styryl-pyrazine, 2-styryl-3,5-indolyl-pyrazine and 2, 3, 5- trisubstituted pyrazines have the good fluorescence properties and largly two-photon absorption（TPA） cross-section. The results above offer some important clues for the future investigation that cypridina luciferin analogues are to be as TPA fluorescence probe material.3. A highly sensitive two-photon fluorescent probes （TFP） for DNA detection attract great attention of many researchers. However, the binding form between two-photon fluorescence probe and DNA remains unclear. Thus, we theoretically studied a series of 3, 6-bis （4-vinylpydinium） carbazole derivatives （BMVC）, and their dication （BMVC-C） and iodization （BMVC-I）. The calculated results indicate that the N of 3, 6-vinylpyridinium carbazole and the 9-vinylpyridinium carbazole are all active sites, and the TPA spectra of the compounds with the different substituents at the active site are in the near infrared region （NIR）, which makes them promising NIR two-photon image materials. In summary, the positions of 3, 6 and 9 are all the active sites when the studied molecules interact with DNA as two-photon fluorescent probes. The BMVC-C and BMVC-I molecules are more likely to combine with DNA as two-photon fluorescent probes.更多还原