Labeling of Phosphatidylinositol Lipid Products in Cells through Metabolic Engineering by Using a Clickable myo‐Inositol Probe

Abstract

Phosphatidylinositol (PI) lipids control critical biological processes, so aberrant biosynthesis often leads to disease. As a result, the capability to track the production and localization of these compounds in cells is vital for elucidating their complex roles. Herein, we report the design, synthesis, and application of clickable myo-inositol probe 1 a for bioorthogonal labeling of PI products. To validate this platform, we initially conducted PI synthase assays to show that 1 a inhibits PI production in vitro. Fluorescence microscopy experiments next showed probe-dependent imaging in T-24 human bladder cancer and Candida albicans cells. Growth studies in the latter showed that replacement of myo-inositol with probe 1 a 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 metabolic labeling-based chemoproteomic platform unravels the physiological roles of choline metabolites

“…Several of our choline analogs that successfully label choline lipids are structurally considerably more elaborate than native choline (Figure 2), thereby demonstrating that the mammalian choline lipid biosynthetic machinery is remarkably promiscuous. These findings add to the rapidly expanding toolkit of lipidic metabolic labeling probes that include non-natural fatty acids for labeling lipidic hydrophobic tails, [15, 41] sphingosine derivatives for labeling sphingolipids, [42] an azido myo -inositol derivative for labeling inositol lipids, [43] and alkynols for labeling phosphatidic acid. [44] Another significant outcome of these studies is the discovery that choline lipid headgroups are susceptible to chemical remodeling via sequential dealkylation and methylation in mammalian cells (Figures 3).…”

Fluorescent Chemical Tools for Tracking Anionic Phospholipids

“…The concentration of PI ranges around 1 % of the total phospholipid level in mammalian cells [1b] . A clickable myo‐inositol probe has been developed for labeling PI in cells [106] . An alkyl linker with an azide moiety was tagged on the PI headgroup at the 2‐hydroxy position.…”

“…The 2‐hydroxy position was selected for azide tagging since the rest of the hydroxy groups are phosphorylated when PI is converted into phosphoinositides. The probe was applied to living cells followed by treatment with a clickable fluorophore to image PI [106] . In the absence of any fluorescent bio‐sensor for imaging PI, the lipid has been indirectly monitored via a bio‐sensor for diacylglycerol (DAG) [41b,107] .…”

Metabolic labeling of glycerophospholipids via clickable analogs derivatized at the lipid headgroup

“…Our group has been interested in labeling myo-inositol-containing phospholipids including phosphatidylinositol (PI) and its downstream phosphatidylinositol polyphosphate (PIP n ) products, which are critical regulators of several key biological processes (Ricks et al, 2019). In this case, we avoided modification of myo-inositol at the 3-, 4-, and 5-positions where these compounds are phosphorylated in nature to produce the seven core PIP n isomers, along with the 1-position as the site of phospholipid attachment.…”