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 O 2 to produce a tetrazine that immediately reacts with a trans-cyclooctene (TCO) dienophile. 6-(2-Pyridyl)dihydrotetrazine-3-carboxamides were developed as stable, cell permeable DHTz reagents that upon oxidation produce the most reactive tetrazines ever used in live cells with Diels−Alder kinetics exceeding k 2 of 10 6 M −1 s −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 were demonstrated. CABL is light-dose dependent, and two-photon excitation promotes CABL at the suborganelle level to selectively pattern live cells under no-wash conditions. CABL was also applied to spatially resolved live-cell labeling of an endogenous protein target by using TIRF microscopy to selectively activate intracellular monoacylglycerol lipase tagged with DHTz-labeled small molecule 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.

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The advent of total-body Positron EmissionTomography (PET) has vastly broadened the range of research and clinical applications of this powerful molecular imaging technology. 1 Such possibilities have accelerated progress in 18 F-radiochemistry with numerous methods available to 18 F-label (hetero)arenes and alkanes. 2 However, access to 18 F-difluoromethylated molecules in high molar activity (Am) is largely an unsolved problem, despite the indispensability of the difluoromethyl group for pharmaceutical drug discovery. 3 We report herein a general solution by introducing carbene chemistry to the field of nuclear imaging with a [ 18 F]difluorocarbene reagent capable of a myriad of 18 F-difluoromethylation processes. In contrast to the tens of known difluorocarbene reagents, this 18 F-reagent is carefully designed for facile accessibility, high molar activity and versatility. The issue of Am is solved using an assay examining the likelihood of isotopic dilution upon variation of the electronics of the difluorocarbene precursor. Versatility is demonstrated with multiple [ 18 F]difluorocarbene based reactions including O-H, S-H and N-H insertions, and cross-couplings that harness the reactivity of ubiquitous functional groups such as (thio)phenols, N-heteroarenes, and aryl boronic acids that are easy to install. Impact is illustrated with the labelling of highly complex and functionalised biologically relevant molecules and radiotracers.

Electrophilic small molecules that can reversibly modify proteins are of growing interest in drug discovery. However, the ability to study reversible covalent probes in live cells can be limited by their reversible reactivity after cell lysis and in proteomic workflows, leading to scrambling and signal loss. We describe how thiomethyltetrazines function as reversible covalent warheads for cysteine modification, and this dynamic labeling behavior can be "switched off" via bioorthogonal chemistry inside live cells. Simultaneously, the tetrazine serves as a bioorthogonal reporter enabling the introduction of tags for fluorescent imaging or affinity purification. Thiomethyltetrazines can label isolated proteins, proteins in cellular lysates, and proteins in live cells with second-order rate constants spanning 2 orders of magnitude (k 2 , 1−100 M −1 s −1 ). Reversible modification by thiomethyltetrazines can be switched off upon the addition of trans-cyclooctene in live cells, converting the dynamic thiomethyltetrazine tag into a Diels−Alder adduct which is stable to lysis and proteomic workflows. Time-course quenching experiments were used to demonstrate temporal control over electrophilic modification. Moreover, it is shown that "locking in" the tag through Diels−Alder chemistry enables the identification of protein targets that are otherwise lost during sample processing. Three probes were further evaluated to identify unique pathways in a live-cell proteomic study. We anticipate that discovery efforts will be enabled by the trifold function of thiomethyltetrazines as electrophilic warheads, bioorthogonal reporters, and switches for "locking in" stability.

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