Live‐Cell Imaging of Sterculic Acid—a Naturally Occurring 1,2‐Cyclopropene Fatty Acid—by Bioorthogonal Reaction with Turn‐On Tetrazine‐Fluorophore Conjugates**

Abstract

In the field of lipid research, bioorthogonal chemistry has made the study of lipid uptake and processing in living systems possible, whilst minimising biological properties arising from detectable pendant groups. To allow the study of unsaturated free fatty acids in live cells, we here report the use of sterculic acid, a 1,2‐cyclopropene‐containing oleic acid analogue, as a bioorthogonal probe. We show that this lipid can be readily taken up by dendritic cells without toxic side effects, and that it can subsequently be visualised using an inverse electron‐demand Diels–Alder reaction with quenched tetrazine‐fluorophore conjugates. In addition, the lipid can be used to identify changes in protein oleoylation after immune cell activation. Finally, this reaction can be integrated into a multiplexed bioorthogonal reaction workflow by combining it with two sequential copper‐catalysed Huisgen ligation reactions. This allows for the study of multiple biomolecules in the cell simultaneously by multimodal confocal imaging.

Photoreactive bioorthogonal lipid probes and their applications in mammalian biology

“…Over the past two decades, coupled with the advent in multiplexing mass spectrometry based chemoproteomics, ligandphoto-affinity based protein profiling techniques have provided an edge over the aforementioned conventional approaches in mapping protein-ligand interactions, in that these multifunctional lipid probes enable the screening and identification of hitherto unknown protein ligands of lipids at a proteome wide scale under various physiological conditions in mammalian cells and tissues. While lipid probes studied in the past have employed multiple strategies, [54][55][56][57] such as appending a photoreactive group to a radioactive 58,59 or fluorophore conjugated lipid tail, [60][61][62][63] in this section, we aim to specifically discuss lipid probes that possess both a photoreactive group and a bioorthogonal handle, and how they have been profiled in mammalian systems to discover and characterize protein-lipid interactions. Commonly in such chemoproteomics experiments involving a lipid probe that has a photoreactive group and a bioorthogonal handle, as the first step, after incubation with the probe, the cellular lysates or live cells are irradiated with UV light of an appropriate wavelength and for a suitable exposure time, to ensure efficient crosslinking of the lipid probe with the protein(s) of interest.…”

Bioconjugation Strategies for Revealing the Roles of Lipids in Living Cells

“…In the second step, the probes are reacted with a functionalized fluorescent dye via a bioorthogonal reaction. In that context, copper-free azide–alkyne cycloaddition as well as inverse electron-demand Diels–Alder (IEDDA) tetrazine ligations have seen frequent use in live-cell imaging. − Subsequently, there have been numerous bioconjugation applications as well as new synthetic procedures for accessing symmetrical and unsymmetrical tetrazines as well as novel reactive dienophiles such as cyclopropenes and trans -cyclooctene (TCO). − Despite the widespread adoption of tetrazine ligations in bioconjugation chemistry, a noted drawback is the challenge in gaining spatial and temporal control over the initiation of the IEDDA reaction. Furthermore, reactive tetrazines are often unstable in water or in the presence of strong nucleophiles, and degradation by hydrolysis constitutes a major issue for live-cell imaging. , One strategy to achieve stimuli responsive tetrazine ligations is to control the redox state of the tetrazine.…”

Chemical Probes to Control and Visualize Lipid Metabolism in the Brain

“…Chem., Int. Ed.202261 . This paper describes live cell imaging of an oleic acid analogue containing a 1,2-cyclopropene as a bio-orthogonal click handle using various quenched tetrazine-fluorophores, thereby showing its subcellular distribution.…”

“…56 Recently, we expanded the IEDDA substrate scope using sterculic acid, a natural oleic acid analogue containing a 1,2cyclopropene as a bio-orthogonal click handle, for live cell imaging employing various quenched tetrazine-fluorophores. 4 This allowed us to visualize its distribution in live cells and capture sterculic acid-modified proteins.…”

“… 2022 61 . 4 This paper describes live cell imaging of an oleic acid analogue containing a 1,2-cyclopropene as a bio-orthogonal click handle using various quenched tetrazine-fluorophores, thereby showing its subcellular distribution. The 1,2-cyclopropene holds great promise for incorporation in various fatty acids, allowing live-cell imaging of lipid distribution.…”