Labeling of Phosphatidylinositol Lipid Products in Cells via Metabolic Engineering using a Clickable myo-Inositol Probe

Phosphatidylinositol (PI) lipids control critical biological processes and thus aberrant biosynthesis often leads to disease. As a result, the ability to track the production and localization of these molecules in cells is vital for elucidating their complex roles. Herein, we report the design, synthesis and application of clickable myo- inositol probe 1a for bioorthogonal labeling of PI products. To validate this platform, we initially conducted PI synthase assays to show that 1a inhibits PI production in vitro. Fluorescence microscopy experiments next showed probe-dependent imaging in T-24 human bladder cancer and C. albicans cells. Growth studies in the latter showed that replacement of myo -inositol with probe 1a led to an enhancement in cell growth. Finally, fluorescence-based TLC analysis and mass spectrometry experiments support the labeling of PI lipids. This approach provides a promising means for tracking the complex biosynthesis and trafficking of these lipids in cells. A clickable myo -inositol probe has been developed for the labeling of phosphatidylinositol (PI) lipids in live cells. Validation of labeling was accomplished through PI synthase inhibition assays, cellular fluorescence microscopy, TLC imaging and mass spectrometry. Probe effects of cell growth and transport were also investigated. This platform provides an exciting avenue for tracking the biosynthesis and trafficking of PI lipids, which are commonly dysregulated in disease.