海萤化学发光机理及其相关pH荧光探针材料的理论研究

Recently, bioluminescence is widely applied in various fields such as gene expression, gene regulation and biosensors. Due to high sensitivity and high quantum yield of cypridina bioluminescence reactions, a wide range of in vitro and in vivo analytical techniques have been developed in biotechnology and biomedical research, such as reporter gene and bioimaging. However, the cypridina bioluminescence reaction is too complexity to obtain the electronic and geometric structures of the chemiluminescent oxyluciferin and the crystal structure of cypridina luciferase. Many details of the chemical luminescence mechanism of cypridina are ambiguous and challenge both theory and experiment. Their applications are seriously restricted in some important fields. .The quantum chemistry methods are adopt in this project, (1) to predict the distribution of all oxyluciferin species in the pH range of interest by determining the corresponding dissociation constants (pKa and pKa*) in the ground and excited states; (2) to optimize the excited geometries of complex of neutral-form oxyluciferin and basic amino acid, calculate their fluorescence spectra, discuss the possibility that transforms neutral-form oxyluciferin into anionic-form oxyluciferin on the effect of basic amino acid; (3) to design new cypridina oxyluciferin analogues, predict emission spectra and fluorescence lifetimes of those new derivatives in different polarity and basicity of the environment. Through these systematic investigations, ascertain the molecule structure of chemiluminescent and fluorescent emitters, assess the effects of solvent polarity and basic amino acid on the chemiluminescent and fluorescent spectra, obtain some alternative molecules suitable for fluorescent pH probe with multicolor, high quantum yield, good proton selectivity and reversibility. At last, the chemical mechanism for cypridina bioluminescence is clarified. The fluorescent pH probe materials are successfully used in a number of areas: testing the pH of environment, the pH change in cell and in vivo.

近年来的研究发现，由于海萤发光具有灵敏度高，量子产率高、发光物质稳定等特点，使其成为报告基因、生物成像等生物技术、生物医学研究中的一种重要分析工具。但是由于海萤发光反应特别复杂，目前尚难以获得氧化荧光素化学发光态分子结构及荧光素酶晶体结构，海萤的化学发光机理尚不清楚，使其在一些重要领域的应用受到了严重的限制。.本项目拟用量子化学方法(1)研究基态和激发态酸性解离常数，评估不同pH下氧化荧光素的存在形式；(2)阐明碱性氨基酸作用下中性与阴离子形式氧化荧光素间转化的可能性；(3)设计海萤氧化荧光素相似物分子，研究溶剂极性及酸碱性对发射光谱和荧光寿命的影响。通过本项目的研究确定海萤化学发光态分子结构，评估溶剂极性及碱性氨基酸对光谱性质的影响，得到发光颜色不同、荧光量子产率较高、对质子具有好的选择性和可逆性的pH荧光探针材料备选分子，力争使其成功用于检测环境pH、细胞和生物体内pH及其变化。

海萤生物发光由于具有高量子产率、不需要辅因子、高信噪比等优点使其成功应用于生物成像、生物分析及生物医药等领域。然而，海萤生物发光机理尚不清楚；环境作用始终被忽略；天然海萤氧化荧光素和大多数衍生物只能发出蓝光，上述因素阻碍了海萤生物发光在许多领域的进一步应用。.为了解决上述问题，我们用密度泛函理论和含时密度泛函理论方法研究了：(1)形成有效化学激发的二氧杂环丁酮及生物发光光发射体的存在形式；(2) 溶剂、碱性氨基酸和氢键对荧光光谱的影响; (3) 设计了三个系列的荧光素衍生物：2-乙酰氨基-5-芳基-3-（对甲氧基苯基）吡嗪、2-乙酰氨基-5-芳基-3-（对三氟甲基苯基）吡嗪及苯、萘、菲芳香基团取代相似物。.研究表明，(1) 在乙酰氨基吡嗪酮骨架氧化过程中，二氧杂环丁酮以中性氨基和胍基丙基阳离子形式存在。这一形式分解成相应的氧化荧光素的过程被认为是形成有效化学激发的直接因素。经过“黑暗”的化学发光态后，此种氧化荧光素转化荧光态。(2)碱性氨基酸和溶剂极性对结构和光谱性质的影响不大。荧光性质可以通过氢键的共价程度来调节。(3) 这些海萤氧化荧光素衍生物的荧光覆盖了整个可见光区域。特别是，2-乙酰氨基-5-（反式-2,3,6,7-四氢-1H，5H-苯并[i, j]喹啉嗪-乙烯基）-3-（对-三氟甲基苯基）吡嗪可以发出红光。通过比较发射光谱和振子强度，发现C3位置的给电子基团取代使波长蓝移并提高发光效率。通过引入共轭双键，增加共轭长度，发射光谱红移，发光强度变强；激发态解离常数的计算表明2-乙酰氨基-5-萘基-3-萘并吡嗪可能是pH荧光探针的备选材料。本项目为获得仿海萤pH荧光探针材料备选分子提供重要信息，最终使其成功用于检测环境pH、细胞和生物体内pH及其变化。.已发表学术论文13篇，其中SCI 收录12篇，JCR分区一区1篇及二区3篇；培养硕士研究生3名（1名已毕业，2名在读）。