Clickable Lipid Precursors as Chemical Tools for Imaging and Tracking Lipids

The regulation of lipid metabolism is crucial for maintaining the human body, as disruptions in lipid homeostasis have drastic implications. While lipids are known for their roles as energy stores as well as for cellular compartmentalization, certain lipid classes can serve as signaling agents that govern cellular behavior and physiology or as biomarkers whose concentration and spatial organization impacts cell fate. Thus, dysregulation in these processes coincide with a variety of diseases and cancers. However, the ability to track lipids has been a long-standing challenge in the area of chemical biology since lipids are chemically diverse and undergo continuous interconversion within their complex biosynthetic pathways. A popular method to address this challenge is the lipid metabolic labeling platform whereby click-tagged substrates are introduced to cells and metabolized to generate click-tagged lipids. These lipids can be modified through click chemistry to enable selective labeling, detection, and imaging. In Chapter 1, we review scientific endeavors that took advantage of this platform. In Chapters 2 and 3, we describe the development and application of new precursors to add to the chemical toolbox to label a particular phospholipid, phosphatidylserine (PS) by headgroup labeling, and to label several lipid classes by glycerol-tagging. We confirmed the successful infiltration of these precursors into the lipid pathways of S. cerevisiae via mass spectrometry (MS), fluorescence imaging after click reaction with fluorescent dye, and lipid class separation by fluorescence-based thin-layer chromatography (TLC). In Chapter 2, we have synthesized novel azido-serine precursors and confirmed its conversion to PS lipid products. In Chapter 3, the same techniques were applied to assess a panel of azide-tagged monoacylglycerol (MAG) precursors in their ability to label neutral lipids and phospholipids. Chapter 4 describes an extension to the goal of “bulk" glycerophospholipid labeling with the synthesis of an azide-tagged phosphatidic acid precursor. Finally, Chapter 5 describes cysteine and serine analogs equipped with electrophilic and clickable tags as substrate mimics to probe the PS synthase enzyme active site. All in all, this novel set of clickable reporters adds to the rapidly expanding toolbox of lipid probes for chemical biology applications.