New fluorescent cholesterol analogs for cell biological research: synthesis, characterization, and application

用于细胞生物学研究的新型荧光胆固醇类似物：合成、表征和应用

• 批准号: 452842040
• 负责人: Dr. Peter Müller
• 金额: --
• 依托单位: Lehrstuhl für Bioorganische Chemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/452842040?language=en
• 关键词: New; fluorescent; cholesterol; analogs; cell

Sterols belong to the most relevant biological molecules due to their significant role in many physiological processes. Therefore, there is great demand for measuring these molecules on the different levels of biological organization, i.e. from the subcellular up to the whole organism. However, sterols are difficult to detect, among others owing to a lack of inherent structures which allow their detection. One strategy to follow sterols is the use of analogs, especially fluorescent analogs have been widely used. Some of the challenges when using analogs are a comparatively easy synthesis, a mimicking of their endogenous counterparts, and appropriate fluorescence properties for applying microscopical assays. To fulfill these demands, the present project will develop, synthesize and characterize fluorescent analogs for two particularly relevant groups of sterols, i.e. cholesterol and cholesteryl esters. The latter molecules will be equipped with two fluorescence moieties, i.e. an extended conjugated system in the sterol ring and a suitable fluorescence moiety in the esterified acyl chain. This allows us for the first time to follow both, sterol and cleaved acyl chain independently upon hydrolysis of the ester in living cells. Moreover, the new probe enables us to continuously measure hydrolysis reactions using relief of Förster resonance energy transfer (FRET) between both fluorescent groups. With regard to cholesterol, a couple of analogs has been used having additional double bonds by that making the molecules fluorescent. In the project the number and/or positions of double bonds will be changed. From these modifications, cholesterol analogs with improved fluorescence properties are expected allowing the application of various fluorescence microscopical approaches. The advantages of the new molecules will be proven by applying them for the investigation of a current biological question, i.e. the influence of Niemann Pick type C (NPC) proteins on the cellular cholesterol transport. We expect, that the analogs allow to decipher new molecular details and mechanisms of how sterols are involved in subcellular and cellular processes.

由于其在许多生理过程中的重要作用，固醇属于最相关的生物分子。因此，在生物组织的不同水平上测量这些分子，即从亚细胞到整个生物体，存在巨大的需求。然而，固醇难以检测，尤其是由于缺乏允许其检测的固有结构。跟踪甾醇的一种策略是使用类似物，特别是荧光类似物已被广泛使用。当使用类似物时，一些挑战是相对容易的合成，其内源性对应物的模拟，以及用于应用显微镜测定的适当的荧光性质。为了满足这些需求，本项目将开发，合成和表征两个特别相关的甾醇组，即胆固醇和胆固醇酯的荧光类似物。后一种分子将配备有两个荧光部分，即甾醇环中的扩展共轭系统和酯化酰基链中的合适荧光部分。这使我们能够第一次遵循这两个，甾醇和裂解的酰基链独立水解的酯在活细胞中。此外，新的探针使我们能够使用两个荧光基团之间的Förster共振能量转移（FRET）的救济连续测量水解反应。关于胆固醇，已经使用了几种具有额外双键的类似物，其使分子发荧光。在该项目中，双键的数量和/或位置将发生变化。从这些修改，具有改进的荧光性质的胆固醇类似物，预计允许各种荧光显微镜方法的应用。新分子的优点将通过将它们应用于当前生物学问题的研究来证明，即尼曼匹克C型（NPC）蛋白对细胞胆固醇转运的影响。我们期望，类似物可以破译新的分子细节和机制，甾醇是如何参与亚细胞和细胞过程。