Development of natural product derivatives for the fluorimetric DNA detection in cells

有机染料对核酸的染色是细胞荧光分析的重要组成部分，对所用的材料和方法有很高的要求，因此对新型荧光团的需求很大。因此，合成荧光探针的低水溶性和有限的细胞渗透性往往限制了它们的适用性，必须加以克服。在这个项目中，将研究来自天然产物的荧光探针是否因其生物源性而表现出更好的生物相容性。根据我们之前的研究结果，应确定与喹诺嗪离子相关的天然产物衍生物，如小檗碱或永活碱，并具有与合成的喹诺嗪荧光团相似的发射特性。通过系统的结构变化，将得到一系列的衍生物，其光物理性质和适合作为dna敏感的荧光探针将用光谱方法和共聚焦和高分辨率荧光显微镜进行详细检查。具体来说，将评估那些控制底物与核酸的强烈和选择性结合的因素，以及结合配体的发射特性的显着变化，从而实现明确和敏感的检测。此外，一旦引入额外的介质敏感取代基，将检查靶向荧光探针的特殊光物理和DNA结合特性的组合是否也可用于DNA结合位点的直接化学环境的表征。为此目的，羟基取代的配体将被确定为在激发态中经历有效和荧光可检测的质子转移。这种强光酸被认为非常适合通过荧光法监测与周围介质的分子间质子转移反应来直接监测和表征dna结合配体的直接化学邻近。所得数据将首次用于空间和时间分辨荧光法测定DNA水合壳中游离或结合水分子的迁移率和浓度，无论是在凝胶型或水凝胶型模型系统中还是在细胞中。

The staining of nucleic acids with organic dyes is an essential element of the fluorimetric analysis of cells and requires high standards of the employed materials and methods, so that there is still a great need for novel fluorophores. Hence, the low water-solubility and the limited cell permeability of synthetic fluorescent probes often limit their applicability and have to be overcome. In this project, it will be investigated whether fluorescent probes, that are derived from natural products, exhibit an improved biocompatibility as provided by their biogenic origin. Based on the results of our previous studies, derivatives of natural products shall be identified that are related to the quinolizinium ion, such as berberine or sempervirine, and that have resembling emission properties as the annelated quinolizinium fluorophore. By means of systematic structure variations, a series of derivatives will be made available whose photophysical properties and suitability as DNA-sensitive fluorescent probes will be examined in detail with spectroscopic methods and with confocal and high-resolution fluorescence microscopy. Specifically, those factors will be assessed that govern a strong and selective association of the substrates with the nucleic acid, along with a significant change of the emission properties of the bound ligand, thus enabling the unambiguous and sensitive detection. Furthermore, it will be examined whether the combination of the special photophysical and DNA-binding properties of the targeted fluorescent probes may also be employed for the characterization of the direct chemical environment of the DNA binding site, once an additional medium-sensitive substituent is introduced. For that purpose, hydroxy-substituted ligands will be identified that undergo an efficient and fluorimetrically detectable proton transfer in the excited state. Such strong photoacids are proposed to be well suited for the direct monitoring and characterization of the immediate chemical vicinity of the DNA-bound ligand by means of fluorimetric monitoring of the intermolecular proton transfer reaction with the surrounding medium. The resulting data will be used for the first time for the spatial- and time-resolved fluorimetric determination of the mobility and concentration of free or bound water molecules in the hydration shell of the DNA, either in gel- or hydrogel-type model systems or in cells.