Since tetrazines are important tools to the field of bioorthogonal chemistry, there is a need for new approaches to synthesize unsymmetrical and 3‐monosubstituted tetrazines. Described here is a general, one‐pot method for converting (3‐methyloxetan‐3‐yl)methyl carboxylic esters into 3‐thiomethyltetrazines. These versatile intermediates were applied to the synthesis of unsymmetrical tetrazines through Pd‐catalyzed cross‐coupling and in the first catalytic thioether reduction to access monosubstituted tetrazines. This method enables the development of new tetrazine compounds possessing a favorable combination of kinetics, small size, and hydrophilicity. It was applied to a broad range of aliphatic and aromatic ester precursors and to the synthesis of heterocycles including BODIPY fluorophores and biotin. In addition, a series of tetrazine probes for monoacylglycerol lipase (MAGL) were synthesized and the most reactive one was applied to the labeling of endogenous MAGL in live cells.

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.

Since tetrazines are important tools to the field of bioorthogonal chemistry, there is a need for new approaches to synthesize unsymmetrical and 3‐monosubstituted tetrazines. Described here is a general, one‐pot method for converting (3‐methyloxetan‐3‐yl)methyl carboxylic esters into 3‐thiomethyltetrazines. These versatile intermediates were applied to the synthesis of unsymmetrical tetrazines through Pd‐catalyzed cross‐coupling and in the first catalytic thioether reduction to access monosubstituted tetrazines. This method enables the development of new tetrazine compounds possessing a favorable combination of kinetics, small size, and hydrophilicity. It was applied to a broad range of aliphatic and aromatic ester precursors and to the synthesis of heterocycles including BODIPY fluorophores and biotin. In addition, a series of tetrazine probes for monoacylglycerol lipase (MAGL) were synthesized and the most reactive one was applied to the labeling of endogenous MAGL in live cells.

A deaminative reaction of Katritzky alkylpyridinium salts and sulfinimines has been developed to deliver enantiopure α-chiral amines. The success of this method relied on the discovery of a thermally promoted deamination via single-electron transfer of an anion−π complex of the alkylpyridinium cation with potassium carbonate. This method boasts excellent diastereoselectivity over the α-stereocenter as well as broad functional group and heterocycle tolerance.

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 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 two orders of magnitude (k2 1–100 M-1s-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.