Mader Oliver, Reiner Knut, Egelhaaf Hans-Joachim, Fischer Rainer, Brock Roland
Institute for Cell Biology, University of Tübingen, Auf der Morgenstelle 15, 72076 Tübingen, Germany.
Bioconjug Chem. 2004 Jan-Feb;15(1):70-8. doi: 10.1021/bc034191h.
A collection of nine pentamethine indocyanine dyes was synthesized, and the photophysical characteristics relevant to applications in cell biology and single molecule detection were analyzed in detail. Substituents at the aromatic system covering the auxochromic series and substitutions in the polymethine chain were investigated with respect to absorption and emission spectra, fluorescence lifetimes, fluorescence quantum yields, and fluorescence autocorrelations. Substitutions in the polymethine chain increased the nonradiative energy dissipation of the excited singlet state and decreased the fluorescence quantum yield, relative to the unsubstituted compound. For substituents at the aromatic rings the fluorescence quantum yield negatively correlates with the position of the substituents in the auxochromic series -SO(3)(-), -H, -F, -CH(3). Compounds with sulfonic acid groups or halogen atoms attached to the indolenine systems had the highest fluorescence quantum yields. The compound S0387 had nearly 70% of the quantum yield of Cy5 and comparable photostability. The free carboxylic acid of S0387 was attached to peptides in high yield and purity by established procedures of solid-phase synthesis. The dye-labeled peptides did not aggregate or bind to tissue culture cells and proteins unspecifically. The indocyanine dye S0387 is therefore an attractive new fluorophore for in vitro and cell-based detection of receptor ligand interaction at nanomolar concentrations by flow cytometry, fluorescence correlation spectroscopy, and laser scanning microscopy.
合成了一组包含九种五甲川吲哚菁染料的化合物，并详细分析了其与细胞生物学和单分子检测应用相关的光物理特性。研究了芳香体系中覆盖助色基团系列的取代基以及聚甲川链中的取代基对吸收光谱和发射光谱、荧光寿命、荧光量子产率和荧光自相关的影响。相对于未取代的化合物，聚甲川链中的取代增加了激发单重态的非辐射能量耗散并降低了荧光量子产率。对于芳香环上的取代基，荧光量子产率与助色基团系列 -SO(3)(-)、-H、-F、-CH(3) 中取代基的位置呈负相关。吲哚啉体系连接有磺酸基团或卤素原子的化合物具有最高的荧光量子产率。化合物 S0387 的量子产率接近 Cy5 的 70%，且光稳定性相当。通过成熟的固相合成方法，S0387 的游离羧酸以高产率和高纯度连接到肽上。染料标记的肽不会聚集或非特异性地结合到组织培养细胞和蛋白质上。因此，吲哚菁染料 S0387 是一种有吸引力的新型荧光团，可用于通过流式细胞术、荧光相关光谱和激光扫描显微镜在纳摩尔浓度下体外和基于细胞的受体配体相互作用检测。
