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

由于甾醇在许多生理过程中起着重要的作用，因此它属于最相关的生物分子。因此，有很大的需求测量这些分子在不同水平的生物组织，即从亚细胞到整个生物体。然而，固醇很难检测，其中一个原因是缺乏允许检测的固有结构。替代甾醇的一种策略是使用类似物，尤其是荧光类似物已被广泛使用。使用类似物时的一些挑战是相对容易的合成，模仿其内源性对应物，以及适用于显微分析的适当荧光特性。为了满足这些需求，本项目将开发、合成和表征两种特别相关的固醇类，即胆固醇和胆固醇酯的荧光类似物。后一种分子将具有两个荧光片段，即甾醇环上的扩展共轭体系和酯化酰基链上的合适荧光片段。这使我们第一次能够在活细胞中独立地水解甾醇和裂解酰基链。此外，新的探针使我们能够连续测量水解反应，利用缓解Förster共振能量转移（FRET）在两个荧光基团之间。关于胆固醇，一些类似物已经被使用，它们有额外的双键，使分子荧光。在项目中，双键的数量和/或位置将被改变。从这些修饰，胆固醇类似物与改进的荧光性质是预期允许应用各种荧光显微方法。新分子的优势将通过将其应用于当前生物学问题的研究来证明，即Niemann Pick type C （NPC）蛋白对细胞胆固醇运输的影响。我们期望，类似物允许破译新的分子细节和机制的甾醇是如何参与亚细胞和细胞过程。

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.