A novel bioluminescent probe for detecting newly synthesized phosphatidylcholine

Phosphatidylcholine (PC), the most abundant phospholipid in eukaryotic membranes, plays critical roles in maintaining membrane integrity and mediating cellular signaling pathways. However, the absence of tools for spatiotemporal tracking and quantitative analysis of phospholipid dynamics poses a significant technical challenge. Herein, we present a bioorthogonal bioluminescence imaging system that combines metabolic labeling with caged chemistry for phospholipid detection. The methodology employs an aryl azide-derived choline analog (N-Cho) that incorporates into nascent PC through endogenous biosynthesis pathways. A caged luciferin derivative (TL-PDC), synthesized via diphenylphosphophenylacetic acid protection of D-luciferin's phenolic hydroxyl group, undergoes Staudinger ligation with membrane-embedded N-Cho, triggering site-specific release of active D-luciferin. This initiates luciferase-catalyzed bioluminescence reactions that produce intense luminescent signal. Systematic validation confirmed TL-PDC's exclusive molecular specificity and sensitivity towards N-Cho-labeled phospholipids, this methodology demonstrates significant potential for advancing biological studies requiring phospholipid detection and analysis.