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

• Applicants: Dr. Peter Müller; Professor Dr. Pablo Wessig
• Subject Area: Biophysics
• Term: since 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 452842040

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.

• DFG Programme: Research Grants