Lipid biosynthesis is dysregulated in a variety of diseases including certain cancers. Moreover, certain lipid classes play important regulatory roles in cells through roles such as secondary messengers. Thus, developing tools for tracking lipid biosynthesis, metabolism, and transport is crucial. While many detection methods are known for studying DNA, RNA, and proteins, tracking lipids is a particular challenge due to the constant interconversion between lipid species that are not directly encoded in the genome. Our approach to overcome this challenge entails the development of a lipid metabolic labeling platform that takes advantage of click chemistry to enable selective labeling of target molecules within a biological environment. We have generated tagged versions of natural substrates corresponding to important lipids, which contain azide or alkyne labels. These click-tagged substrates are fed to live yeast or mammalian cells for cellular uptake and incorporation into lipid biosynthetic pathways. We hypothesize that a diverse set of probes will provide distinct labeling patterns that are reflective of the native lipids and respective lipid products. Subsequent lipid products can then be derivatized with reporters such as fluorescent dyes for visualization of subcellular localization using fluorescence microscopy, as well as validation of labeling via flow cytometry, fluorescence-based thin layer chromatography (TLC) and lipidomic mass spectrometry (MS). In this work, we will present the design, synthesis, and initial experiments using a series of probes designed to label different subsections of the lipidome.

Metabolic labeling via clickable substrates to study lipid biosynthesis in cells

ACS Fall 2021

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