Catalytic Activation of Bioorthogonal Chemistry with Light (CABL) Enables Rapid, Spatiotemporally-controlled Labeling and No-Wash, Subcellular 3D-Patterning in Live Cells using Long Wavelength Light

Described is the spatiotemporally controlled labeling and patterning of biomolecules in live cells through the catalytic activation of bioorthogonal chemistry with light, referred to as “CABL”. Here, an unreactive dihydrotetrazine (DHTz) is photocatalytically oxidized in the intracellular environment by ambient O2 to produce a tetrazine that immediately reacts with a trans-cyclooctene (TCO) dieno-phile. 6-(2-Pyridyl)-dihydrotetrazine-3-carboxamides were developed as stable, cell permeable DHTz reagents that upon oxidation pro-duce the most reactive tetrazines ever used in live cells with Diels-Alder kinetics exceeding k2 106 M-1s-1. CABL photocatalysts are based on fluorescein or silarhodamine dyes with activation at 470 or 660 nm. Strategies for limiting extracellular production of singlet oxygen are described that increase the cytocompatibility of photocatalysis. The HaloTag self-labeling platform was used to introduce DHTz tags to proteins localized in the nucleus, mitochondria, actin or cytoplasm, and high-yielding subcellular activation and labeling with a TCO-fluorophore was demonstrated. CABL is light-dose dependent, and 2-photon excitation promotes CABL at the sub-organelle level to selectively pattern live cells under no-wash conditions. CABL was also applied to spatially resolved live-cell labeling of an endogenous pro-tein target by using TIRF microscopy to selectively activate intracellular monoacylglycerol lipase tagged with DHTz-labeled small mole-cule covalent inhibitor. Beyond spatiotemporally controlled labeling, CABL also improves the efficiency of ‘ordinary’ tetrazine ligations by rescuing the reactivity of commonly used 3-aryl-6-methyltetrazine reporters that become partially reduced to DHTzs inside cells. The spatiotemporal control and fast rates of photoactivation and labeling of CABL should enable a range of biomolecular labeling applications in living systems.