水溶性的新型BODIPY近红外荧光染料的合成及细胞荧光成像研究

Near-infrared (NIR) fluorescent dyes which function in the NIR spectral region ranging from 700 nm to 2000 nm are of great interest due to their diverse applications. NIR fluorescent dyes have obvious advantages over traditional visible fluorescent probes, such as deeper light penetration, low light scattering, and low background interference, which make them widely used in bio-imaging and bio-labeling. However, most of reported NIR dyes (e.g., cyanine or polyene dyes) showed poor photostability and chemical stability. Therefore, it is highly desired to develop some new NIR dyes with high photostability..4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene, also known as boron dipyrromethene (BODIPY), is a class of highly fluorescent organic chromophore. Its extraordinary chemical and physical properties, such as high quantum yield, large extinction coefficients, good thermal and photochemical stability, make it attractive for wide applications including luminescent devices, biological imaging and labeling, sensors, and dye-sensitized solar cells. BODIPY core usually exhibit visible absorption and emission located between 470 nm to 550 nm. To push the absorption/emission wavelength to NIR region, a variety of strategies have been used: 1) extension of π-conjugation by fusing rigid ring to the pyrrole unit (e.g., replacing pyrrole with isoindole), 2) functionalization at the α-position to extend the π-conjugation, and 3) replacement of the 8-carbon atom with nitrogen atom to form aza-BODIPY dyes..In this proposal, some novel NIR dyes based on BODIPY will be achieved by functionalization at the 3，5-position and meso-position. We expect that the spectroscopic properties of these dyes, such as emission wavelength, quantum yield, Stokes shifts and photostability, can be tuned by attaching some substitutes at α- and/or meso-position. To get insight into the relationship between structure and photophysical properties of NIR dyes, we employ cyclic voltammetry and Time-dependent DFT (TD-DFT) calculation to investigate the HOMO energy level, LUMO energy level, band gap, the optimized molecular structure, dipole moment and frontier molecular orbital profiles. Some fluorophores with good spectroscopic properties will be functionalized to be water-soluble and used for imaging in cancer cells.

近红外荧光染料具有荧光发射波长长和光穿透力强等优点，可以最大限度地降低生物体内物质的背景干扰，提高检测的信噪比和可视化效果，因此日益受到研究者的青睐。为了得到光稳定性好和荧光量子效率高的近红外荧光染料，本项目通过在BODIPY的meso位共轭连接羧基苯乙炔基等缺电子基团来增加分子的共轭结构，调节分子的荧光发射波长和荧光量子效率；同时在其3，5位上连接芳香取代基使波长进一步红移，探讨取代基对染料分子的荧光发射波长、荧光量子效率、Stokes位移以及光稳定性的影响，并运用循环伏安法和理论化学计算研究染料分子的微观结构与光物理性质之间的关系，总结它们之间的变化规律，为有效地设计光稳定性好的近红外荧光染料提供理论指导。为了改善分子的水溶性，我们在BODIPY的3，5位芳香取代基上引入聚乙二醇，同时在它的meso位引入羧基，得到系列水溶性的近红外荧光染料，最后用于对肿瘤细胞的荧光成像。

尽管BODIPY荧光染料具有荧光量子效率高和光稳定性好等优点，但是此类荧光染料缺乏良好的水溶性并且荧光发射波长处于520 nm以下，容易受到生物体内物质的背景干扰。近红外荧光染料可以最大限度地降低生物体内物质的背景干扰，提高检测的信噪比和可视化效果，因此日益受到研究者的青睐。本项目通过在BODIPY的3，5位引入聚乙二醇功能化的芳香取代基来改善其水溶性，得到荧光发射波长为653 nm的新型BODIPY荧光染料。该荧光染料具有高荧光量子效率（Φf= 0.45）、良好的水溶性和光稳定性，并成功地应用于细胞成像。此外，我们通过在BODIPY的8位直接引入脂基、羧基便使普通BODIPY的荧光发射波长红移至596 nm；当在其3，5位再通过缩合反应连接聚乙二醇功能化的芳香取代基时，得到了荧光发射波长为736 nm的水溶性近红外BODIPY荧光染料。这类化合物普遍具有中等的荧光量子效率和较长的荧光发射波长。特别值得注意的是，8位脂基取代的BODIPY荧光染料具有高荧光量子效率和大的Stokes位移（~ 60 nm），可以有效地避免自吸收和生物样品的背景干扰。细胞成像实验结果表明这类BODIPY荧光染料具有很好的细胞膜渗透性和荧光性能，可以实现对细胞的荧光成像。我们在此基础上成功地设计合成了一种基于吲哚-BODIPY结构和4-氯-7-硝基苯并呋咱结合物的新型荧光探针BY-NBD。此探针通过亲核取代和重排反应对Cys显示出双荧光发射带，而对GSH 显示出荧光“turn-on”响应的单一的发射带，其可用于将Cys和GSH区分开。该探针具有一定的优势，如长发射波长（λem=635 nm），选择性好，并能同时检测Cys和GSH。这项研究不仅开发出可以同时检测Cys和GSH荧光探针，而且还提供了一种在生命系统分析中检测的方法。