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.

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.

<div>The global pandemic caused by SARS-CoV-2 calls for a fast development of antiviral drugs </div><div>against this particular coronavirus. Chemical tools to facilitate inhibitor discovery as well as </div><div>detection of target engagement by hit or lead compounds from high throughput screens are </div><div>therefore in urgent need. We here report novel, selective activity-based probes that enable </div><div>detection of the SARS-CoV-2 main protease. The probes are based on acyloxymethyl ketone </div><div>reactive electrophiles combined with a peptide sequence including non-natural amino acids </div><div>that targets the non-primed site of the main protease substrate binding cleft. They are the first </div><div>activity-based probes for the main protease of coronaviruses and display target labeling within </div><div>in a human proteome without background. We expect that these reagents will be useful in the </div><div>drug development pipeline, not only for the current SARS-CoV-2, but also for other </div><div>coronaviruses. </div>

Bioorthogonal chemistry has allowed the study of biomolecules in living systems with minimal structural perturbations to the molecules under investigation. In the field of lipid research, this has allowed for the study of lipid uptake and processing, whilst minimising artefacts on their biology resulting from pendant detectable 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 here show that this lipid can be readily taken up by dendritic cells without toxic side-effects, and that it can subsequently be visualised in live cells using an inverse electron-demand Diels-Alder (IEDDA) reaction with quenched tetrazine fluorophores. Furthermore, 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.

<div>The global pandemic caused by SARS-CoV-2 calls for a fast development of antiviral drugs </div><div>against this particular coronavirus. Chemical tools to facilitate inhibitor discovery as well as </div><div>detection of target engagement by hit or lead compounds from high throughput screens are </div><div>therefore in urgent need. We here report novel, selective activity-based probes that enable </div><div>detection of the SARS-CoV-2 main protease. The probes are based on acyloxymethyl ketone </div><div>reactive electrophiles combined with a peptide sequence including non-natural amino acids </div><div>that targets the non-primed site of the main protease substrate binding cleft. They are the first </div><div>activity-based probes for the main protease of coronaviruses and display target labeling within </div><div>in a human proteome without background. We expect that these reagents will be useful in the </div><div>drug development pipeline, not only for the current SARS-CoV-2, but also for other </div><div>coronaviruses. </div>