Institute of Organic Chemistry, Karlsruhe Institute of Technology, Fritz-Haber-Weg 6 D-76131 Karlsruhe, Germany.
Biopolymers. 2011;96(5):694-701. doi: 10.1002/bip.21617.
The fluorophore rhodamine B is often used in biological assays. It is inexpensive, robust under a variety of reaction conditions, can be covalently linked to bioactive molecules, and has suitable spectral properties in terms of absorption and fluorescence wavelength. Nonetheless, there are some drawbacks: it can readily form a spirolactam compound, which is nonfluorescent, and therefore may not be the dye of choice for all fluorescence microscopy applications. Herein this spirolactam formation was observed by purifying such a labeled peptoid with high performance liquid chromatography (HPLC) and monitored in detail by making a series of analytical HPLC runs over time. Additionally, a small library of eight peptoids with rhodamine B as label was synthesized. Analysis of the absorption properties of these molecules demonstrated that the problem of fluorescence loss can be overcome by coupling secondary amines with rhodamine B.
荧光染料罗丹明 B 常用于生物分析。它价格低廉，在各种反应条件下都很稳定，可以与生物活性分子共价连接，并且在吸收和荧光波长方面具有合适的光谱性质。尽管如此，它还是存在一些缺点：罗丹明 B 容易形成无荧光的螺环酰胺化合物，因此可能不是所有荧光显微镜应用的理想染料。本文通过高效液相色谱（HPLC）对标记的缩肽进行纯化，观察到了这种螺环酰胺化合物的形成，并通过随时间进行一系列分析型 HPLC 运行对其进行了详细监测。此外，还合成了一个带有罗丹明 B 作为标记的 8 个缩肽的小文库。对这些分子吸收特性的分析表明，通过将二级胺与罗丹明 B 偶联可以克服荧光损失的问题。
