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<p>Herein, we report a new class of electrophilic warheads pyridinyl-substituted alkynyl carbonyls (<b>PyACs</b>) for cysteine-targeting peptides and protein modification. Incorporating a pyridinyl group into acetylenic carbonyls achieves three effects: it blocks thiol exchange through steric hindrance, accelerates reaction kinetics by intermolecular hydrogen bonding, and stabilizes the adducts through intramolecular hydrogen bonding. This conjugation was conducted under biocompatible conditions with high kinetic rates and excellent selectivity. The resulting conjugates display good stability even in the presence of dithiothreitol (DTT). The <b>PyACs</b> were successfully utilized to label peptides and proteins specifically at cysteine sites and therefore applied to the construction of functional antibody-fluorophore conjugates (AFCs) for selective staining of antigen-presenting cells, the building rapid quantitative thiol reactivity profiling (QTRP) approaches for proteome-wide mapping of intrinsic cysteine activity and hydrogen peroxide sensitivity, as well as the live-cell visualization of protein thiols. We showcase the excellent cysteine selectivity of <b>PyACs</b> in mass spectrometry-based proteome-wide cysteine profiling. Given the facile preparation of the probe, ultrafast reaction kinetics, excellent selectivity, conjugate stability and mass compatibility of this conjugation, we anticipate that <b>PyACs</b> will find widespread application in constructing valuable protein conjugates, understanding the roles of protein cysteine residues, and advancing the drug discovery pipeline.</p>

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Fast and efficient cysteine-specific bioconjugation enables protein functionalization and rapid quantitative thiol reactivity profiling

  • 期刊论文

  • 发表日期：

    2026年4月1日

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  • Linfeng Chen,
  • Jixiang He,
  • Ziyi Wang,
  • Tianyang Wang,
  • Mengzhao Li,
  • Huiling Wang,
  • Changlin Liu,
  • Jing Yang,
  • Chunrong Liu

摘要

Herein, we report a new class of electrophilic warheads pyridinyl-substituted alkynyl carbonyls (PyACs) for cysteine-targeting peptides and protein modification. Incorporating a pyridinyl group into acetylenic carbonyls achieves three effects: it blocks thiol exchange through steric hindrance, accelerates reaction kinetics by intermolecular hydrogen bonding, and stabilizes the adducts through intramolecular hydrogen bonding. This conjugation was conducted under biocompatible conditions with high kinetic rates and excellent selectivity. The resulting conjugates display good stability even in the presence of dithiothreitol (DTT). The PyACs were successfully utilized to label peptides and proteins specifically at cysteine sites and therefore applied to the construction of functional antibody-fluorophore conjugates (AFCs) for selective staining of antigen-presenting cells, the building rapid quantitative thiol reactivity profiling (QTRP) approaches for proteome-wide mapping of intrinsic cysteine activity and hydrogen peroxide sensitivity, as well as the live-cell visualization of protein thiols. We showcase the excellent cysteine selectivity of PyACs in mass spectrometry-based proteome-wide cysteine profiling. Given the facile preparation of the probe, ultrafast reaction kinetics, excellent selectivity, conjugate stability and mass compatibility of this conjugation, we anticipate that PyACs will find widespread application in constructing valuable protein conjugates, understanding the roles of protein cysteine residues, and advancing the drug discovery pipeline.