Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen (Germany), Fax: (+49) 551-2012505.
Chemistry. 2013 Dec 2;19(49):16556-65. doi: 10.1002/chem.201302037. Epub 2013 Nov 7.
Bright and photostable fluorescent dyes with large Stokes shifts are widely used as sensors, molecular probes, and light-emitting markers in chemistry, life sciences, and optical microscopy. In this study, new 7-dialkylamino-4-trifluoromethylcoumarins have been designed for use in bioconjugation reactions and optical microscopy. Their synthesis was based on the Stille reaction of 3-chloro-4-trifluoromethylcoumarins and available (hetero)aryl- or (hetero)arylethenyltin derivatives. Alternatively, the acylation of 2-trifluoroacetyl-5-dialkylaminophenols with available (hetero)aryl- or (hetero)arylethenylacetic acids followed by intramolecular condensation afforded coumarins with 3-(hetero)aryl or 3-[2-(hetero)aryl]ethenyl groups. Hydrophilic properties were provided by the introduction of a sulfonic acid residue or by phosphorylation of a primary hydroxy group attached at C-4 of the 2,2,4-trimethyl-1,2-dihydroquinoline fragment fused to the coumarin fluorophore. For use in immunolabeling procedures, the dyes were decorated with an (activated) carboxy group. The positions of the absorption and emission maxima vary in the ranges 413-480 and 527-668 nm, respectively. The phosphorylated dye, 9,CH=CH-2-py,H, with the 1-(3-carboxypropyl)-4-hydroxymethyl-2,2-dimethyl-1,2-dihydroquinoline fragment fused to the coumarin fluorophore bearing the 3-[2-(2-pyridyl)ethenyl] residue (absorption and emission maxima at 472 and 623 nm, respectively) was used in super-resolution light microscopy with stimulated emission depletion and provided an optical resolution better than 70 nm with a low background signal. As a result of their large Stokes shifts, good fluorescence quantum yields, and adequate photostabilities, phosphorylated coumarins enable two-color imaging (using several excitation sources and a single depletion laser) to be combined with subdiffractional optical resolution.
具有大斯托克斯位移的明亮且光稳定的荧光染料广泛应用于化学、生命科学和光学显微镜领域中的传感器、分子探针和发光标记物。在这项研究中，设计了新的 7-二烷基氨基-4-三氟甲基香豆素，用于生物偶联反应和光学显微镜。它们的合成基于 3-氯-4-三氟甲基香豆素与可用的（杂）芳基或（杂）芳基乙烯基锡衍生物的 Stille 反应。或者，用可用的（杂）芳基或（杂）芳基乙烯基乙酸与 2-三氟乙酰基-5-二烷基氨基苯酚进行酰化，然后进行分子内环化，得到具有 3-（杂）芳基或 3-[2-（杂）芳基]乙烯基的香豆素。通过引入磺酸基或通过磷酸化连接到与香豆素荧光团融合的 2,2,4-三甲基-1,2-二氢喹啉片段的 C-4 的伯羟基，提供了亲水性。为了用于免疫标记程序，染料用（活化）羧基修饰。吸收和发射最大值的位置分别在 413-480nm 和 527-668nm 范围内变化。用 1-（3-羧基丙基）-4-羟甲基-2,2-二甲基-1,2-二氢喹啉片段修饰的磷酸化染料 9,CH=CH-2-py,H，该片段与具有 3-[2-（2-吡啶基）乙烯基]残基的香豆素荧光团融合（吸收和发射最大值分别为 472nm 和 623nm），用于具有受激发射损耗的超分辨率光显微镜，提供了优于 70nm 的光学分辨率和低背景信号。由于其大的斯托克斯位移、良好的荧光量子产率和适当的光稳定性，磷酸化香豆素能够将双色成像（使用多个激发源和单个耗尽激光）与亚衍射光学分辨率相结合。
