Quantification of Bioorthogonal Stability in Immune Phagocytes Using Flow Cytometry Reveals Rapid Degradation of Strained Alkynes

Bio-organic Synthesi

Live-cell imaging of sterculic acid - a naturally occurring 1,2-cyclopropene fatty acid - by bioorthogonal reaction with turn-on tetrazine-fluorophore conjugates

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.NWOERC-CoG 865175Molecular PhysiologyBio-organic Synthesi

Methyltetrazine as a small live-cell compatible bioorthogonal handle for imaging enzyme activities in situ

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 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.Bio-organic Synthesi

Chemical Control over T-Cell Activation in Vivo Using Deprotection of trans-Cyclooctene-Modified Epitopes

Tumorimmunolog

Fluorogenic Bifunctional Trans-cyclooctenes as Efficient Tools for Investigating Click-to-Release Kinetics

The inverse electron demand Diels-Alder pyridazine elimination reaction between tetrazines and allylic substituted trans -cyclooctenes (TCOs) is a key player in bioorthogonal bond cleavage reactions. Determining the rate of elimination on alkylamine substrates has so far proven difficult. Here, we report a fluorogenic tool consisting of a TCO-linked EDANS fluorophore and a DABCYL quencher for accurate detection of both the click and release rates for any tetrazine at physiologically relevant concentrations.Metals in Catalysis, Biomimetics & Inorganic MaterialsBio-organic Synthesi

The development of a broad-spectrum retaining β-exo-galactosidase activity-based probe.

Acid β-galactosidase (GLB1) and galactocerebrosidase (GALC) are retaining exo-β-galactosidases involved in lysosomal glycoconjugate metabolism. Deficiency of GLB1 may result in the lysosomal storage disorders GM1 gangliosidosis, Morquio B syndrome, and galactosialidosis, and deficiency of GALC may result in Krabbe disease. Activity-based protein profiling (ABPP) is a powerful technique to assess the activity of retaining glycosidases in relation to health and disease. This work describes the use of fluorescent and biotin-carrying activity-based probes (ABPs) to assess the activity of both GLB1 and GALC in cell lysates, culture media, and tissue extracts. The reported ABPs, which complement the growing list of retaining glycosidase ABPs based on configurational isomers of cyclophellitol, should assist in fundamental and clinical research on various β-galactosidases, whose inherited deficiencies cause debilitating lysosomal storage disorders.Medical BiochemistryBio-organic Synthesi