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.

A Lysosome-Targeted Tetrazine for Organelle-Specific Click-to-Release Chemistry in Antigen Presenting Cells

“…For this, we preincubated the BMDCs with the caged ligands followed by different tetrazines that we and others had previously shown to be capable of uncaging TCO on BMDCs (19−24). 5,7,10,66,80 It was found that addition of tetrazines 19, 20, 23, and 24 (Figure 4A) resulted in increased levels of IL-2 compared to the caged controls (Figure 4C). The improved recovery of the iNKT cell activation in living cells of 20 compared to 19 is in consensus with earlier work of Peng Chen et al 7 Tetrazine-functionalized dextran (21) displayed limited uncaging despite having been reported as an exclusively extracellular tetrazine.…”

“…While it is generally accepted that a lower pH results in faster and more efficient decaging, 22,66,83 this effect is perhaps more drastic for LysoTz 3 due to increased protonation of the morpholine moiety. Results for the other tetrazines investigated again emphasize the discrepancy between uncaging in a mere aqueous buffer and cellular systems 80 (Figures 4 and S8). Altogether, we show that ligands 1 and 2 do pass the lysosomes before being presented by DCs.…”