Thiomethyltetrazines Are Reversible Covalent Cysteine Warheads Whose Dynamic Behavior can be “Switched Off” via Bioorthogonal Chemistry Inside Live Cells

Abstract

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.

Dynamic Phosphorus: Thiolate Exchange Cascades with Higher Phosphorothioates

“…Dynamic covalent exchange cascades [1] are emerging as a central process not only to access modern materials [2–15] but also for use in chemical biology, particularly to penetrate cells [16–36] . Exchange cascades operate with dynamic covalent exchangers, which, upon exchange, either produce a new covalently tethered exchanger or offer another exchanger, and thereby continue to exchange.…”

Minimalist Tetrazine N-Acetyl Muramic Acid Probes for Rapid and Efficient Labeling of Commensal and Pathogenic Peptidoglycans in Living Bacterial Culture and During Macrophage Invasion

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Use of pyridazinediones for tuneable and reversible covalent cysteine modification applied to peptides, proteins and hydrogels

“…For example, whilst activity-based protein profiling (ABPP) has proved to be a valuable research tool, 42–44 it has been mainly applied using acetamide probes; 45–49 this assay could gain from the development of novel reversible probes. 13,50,51…”