Bioorthogonal deprotectionsare readily used to control biologicalfunction in a cell-specific manner. To further improve the spatialresolution of these reactions, we here present a lysosome... Show moreBioorthogonal deprotectionsare readily used to control biologicalfunction in a cell-specific manner. To further improve the spatialresolution of these reactions, we here present a lysosome-targetedtetrazine for an organelle-specific deprotection reaction. We showthat trans-cyclooctene deprotection with this reagentcan be used to control the biological activity of ligands for invariantnatural killer T cells in the lysosome to shed light on the processingpathway in antigen presenting cells. We then use the lysosome-targetedtetrazine to show that long peptide antigens used for CD8(+) T cell activation do not pass through this organelle, suggestinga role for the earlier endosomal compartments for their processing. Show less
Torres-Garcia, D.; Plassche, M.A.T. van de; Boven, E. van; Leeuwen, T. van; Groenewold, M.G.J.; Sarris, A.J.C.; ... ; Kasteren, S.I. van 2022
Bioorthogonal chemistry combines well with activity-based protein profiling, as it allows for the introduction of detection tags without significantly influencing the physiochemical and biological... Show moreBioorthogonal chemistry combines well with activity-based protein profiling, as it allows for the introduction of detection tags without significantly influencing the physiochemical and biological functions of the probe. In this work, we introduced methyltetrazinylalanine (MeTz-Ala), a close mimic of phenylalanine, into a dipeptide fluoromethylketone cysteine protease inhibitor. Following covalent and irreversible inhibition, the tetrazine allows vizualisation of the captured cathepsin activity by means of inverse electron demand Diels Alder ligation in cell lysates and live cells, demonstrating that tetrazines can be used as live cell compatible, minimal bioorthogonal tags in activity-based protein profiling. Show less
Bertheussen, K.; Plassche, M.A.T. van de; Bakkum, T.; Gagestein, B.; Ttofi, I.; Sarris, A.J.C.; ... ; Kasteren, S.I. van 2022
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... Show moreIn 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. Show less
Plassche, M.A.T. van de; Barniol-Xicota, M.; Verhelst, S.H.L. 2021
The global pandemic caused by SARS-CoV-2 calls for the fast development of antiviral drugs against this particular coronavirus. Chemical tools to facilitate inhibitor discovery as well as detection... Show moreThe global pandemic caused by SARS-CoV-2 calls for the fast development of antiviral drugs against this particular coronavirus. Chemical tools to facilitate inhibitor discovery as well as detection of target engagement by hit or lead compounds from high-throughput screens are therefore in urgent need. We here report novel, selective activity-based probes that enable detection of the SARS-CoV-2 main protease. The probes are based on acyloxymethyl ketone reactive electrophiles combined with a peptide sequence including unnatural amino acids that targets the nonprimed site of the main protease substrate binding cleft. They are the first activity-based probes for the main protease of coronaviruses and display target labeling within a human proteome without background. We expect that these reagents will be useful in the drug-development pipeline, not only for the current SARS-CoV-2, but also for other coronaviruses. Show less
Vanhoutte, R.; Plassche, M.A.T. van de; Verhelst, S.H.L. 2020
Serine hydrolases (SHs) are a large, diverse family of enzymes that play various biomedically important roles. Their study has been substantially advanced by activity-based protein profiling, which... Show moreSerine hydrolases (SHs) are a large, diverse family of enzymes that play various biomedically important roles. Their study has been substantially advanced by activity-based protein profiling, which makes use of covalent chemical probes for labeling the active site and detection by various methodologies. However, highly selective probes for individual SHs are scarce because probe synthesis usually takes place by time-consuming solution phase chemistry. We here report a general solid-phase synthesis toward SH chemical probes, which will speed up probe library synthesis. It involves the construction of a recognition element ending in a secondary amine followed by capping with different electrophiles. We illustrate the power of this approach by the discovery of selective chemical probes for the depalmitoylating enzymes APT-1/2. Overall, this study reports new methodologies to synthesize SH probes, while providing new reagents to study protein depalmitoylation. Show less
Serine proteases comprise about one-third of all proteases, and defective regulation of serine proteases is involved in numerous diseases. Therefore, serine protease inhibitors are promising drug... Show moreSerine proteases comprise about one-third of all proteases, and defective regulation of serine proteases is involved in numerous diseases. Therefore, serine protease inhibitors are promising drug candidates. Aminomethyl diphenyl phosphonates have been regularly used as scaffolds for covalent serine protease inhibition and the design of activity-based probes. However, they cannot make use of a protease's primed site. Therefore, we developed a facile two-step synthesis toward a set of phenyl phosphinates, which is a related scaffold but can interact with the primed site. We tested their inhibitory activity on five different serine proteases and found that a phenyl group directly attached to the phosphorus atom leads to superior activity compared with phosphonates. Show less
