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

Abstract

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.

Superfast Tetrazole–BCN Cycloaddition Reaction for Bioorthogonal Protein Labeling on Live Cells

“…The second-order rate constants for the tetrazole–BCN ligation reactions range from 11 400 M –1 s –1 to 39 200 M –1 s –1 , with tetrazole 1 giving the fastest reaction. Notably, while the tetrazole–BCN ligation is faster than other BCN-mediated bioorthogonal reactions, it is slower than some tetrazine–TCO ligation reactions. , …”

“…Notably, while the tetrazole−BCN ligation is faster than other BCN-mediated bioorthogonal reactions, it is slower than some tetrazine−TCO ligation reactions. 50,51 To probe the origin of the difference in reactivity between Sph and BCN, we used DFT to compute both the optimized geometries of the reactant complexes and transition states of the cycloaddition reactions. We considered the exo geometry of BCN in the reactant complex and the transition state because its free energy at 298 K is 5.0 kcal/mol lower than the endo.…”

Ligand-directed Photocatalysts and Far-red Light Enable Catalytic Bioorthogonal Uncaging inside Live Cells

“…82,83 Curtius rearrangement with DPPA produced carbamoyl azide 10, which could be reduced to the amine 11. 84,85 Me3Almediated coupling with ethyl 6-(2-pyridyl)-dihydrotetrazine-3carboxylate 77 provided DHTz-caged n-CA4 analog 1 as a single Estereoisomer after flash chromatography. An analogous sequence was used to produce the model compound 3 from allyl phenyl ether.…”

“…Our own group has developed catalytic activation of bioorthogonal chemistry with light (CABL) as a tool for inducing bioorthogonal chemistry in live cells and in vivo. [77][78][79] In these approaches, photocatalysis and long wavelength light was used to initiate dihydrotetrazine (DHTz) oxidation and thereby activate rapid bimolecular chemistry with a fluorescently-tagged trans-cyclooctene. Silarhodamine (SiR) or fluorescein dyes, commonly used as fluorophores, were repurposed as photocatalysts in combination with 660 nm or 470 nm light, respectively.…”

“…Silarhodamine (SiR) or fluorescein dyes, commonly used as fluorophores, were repurposed as photocatalysts in combination with 660 nm or 470 nm light, respectively. 77,78 In these studies, the DHTz was anchored to a biological target, and a freely soluble photocatalyst was utilized. Herein, we describe the first ligand-directed catalysts for the activation of bioorthogonal chemistry, , whereby catalytic groups are localized via a tethered ligand either to DNA or to a protein target in the subcellular environment and used to initiate selective, nonnatural reactions with spatiotemporal control (Fig 1 ).…”