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

生物正交化学 化学 四嗪 半胱氨酸 电泳剂 共价键 组合化学 小分子 生物物理学 点击化学 生物化学 有机化学 生物 催化作用
作者
Amanda Tallon,Yingrong Xu,Graham M. West,Christopher W. am Ende,Joseph M. Fox
出处
期刊:Journal of the American Chemical Society [American Chemical Society]
卷期号:145 (29): 16069-16080 被引量:21
标识
DOI:10.1021/jacs.3c04444
摘要

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 (k2, 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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