Tunable Heteroaromatic Sulfones Enhance in-Cell Cysteine Profiling

Heteroaromatic sulfones
react with cysteine via nucleophilic aromatic
substitution, providing a mechanistically selective and irreversible
scaffold for cysteine conjugation. Here we evaluate a library of heteroaromatic
sulfides with different oxidation states, heteroatom substitutions,
and a series of electron-donating and electron-withdrawing substituents.
Select substitutions profoundly influence reactivity and stability
compared to conventional cysteine conjugation reagents, increasing
the reaction rate by >3 orders of magnitude. The findings establish
a series of synthetically accessible electrophilic scaffolds tunable
across multiple centers. New electrophiles and their corresponding
alkyne conjugates were profiled directly in cultured cells, achieving
thiol saturation in a few minutes at submillimolar concentrations.
Direct addition of des­thio­biotin-functionalized probes
to cultured cells simplified enrichment and elution to enable the
mass spectrometry discovery of >3000 reactive and/or accessible
thiols
labeled in their native cellular environments in a fraction of the
standard analysis time. Surprisingly, only half of the annotated cysteines
were identified by both iodo­acet­amide-des­thio­biotin
and methyl­sulfonyl­benzo­thiazole-des­thio­biotin
in replicate experiments, demonstrating complementary detection by
mass spectrometry analysis. These probes offer advantages over existing
cysteine alkylation reagents, including accelerated reaction rates,
improved stability, and robust ionization for mass spectrometry applications.
Overall, heteroaromatic sulfones provide modular tunability, shifted
chromatographic elution times, and superior in-cell cysteine profiling
for in-depth proteome-wide analysis and covalent ligand discovery.

杂芳基砜（Heteroaromatic sulfones）可通过亲核芳香取代反应与半胱氨酸作用，为半胱氨酸偶联提供兼具机制选择性与不可逆性的骨架结构。本研究针对一系列具有不同氧化态、杂原子取代模式，以及各类给电子与吸电子取代基的杂芳基硫醚（heteroaromatic sulfides）文库开展评估。与传统半胱氨酸偶联试剂相比，部分取代基可显著影响反应活性与稳定性，使反应速率提升超过3个数量级。本研究确立了一系列可通过多中心精准调控、且易于合成的亲电骨架。我们直接在培养细胞中对新型亲电试剂及其对应的炔基偶联物进行表征，结果显示在亚毫摩尔浓度下仅需数分钟即可实现硫醇位点的完全饱和。将脱硫生物素（desthiobiotin）功能化的探针直接添加至培养细胞中，可简化富集与洗脱流程，从而实现质谱（mass spectrometry）鉴定：在远短于标准分析流程的时间内，即可在天然细胞环境中标记并鉴定超过3000种具有反应活性或可及性的硫醇位点。令人意外的是，在重复实验中，仅有半数的注释半胱氨酸可同时被碘乙酰胺-脱硫生物素（iodoacetamide-desthiobiotin）与甲基磺酰基苯并噻唑-脱硫生物素（methylsulfonylbenzothiazole-desthiobiotin）所鉴定，这表明两种探针在质谱分析中具有互补的检测效果。相较于现有半胱氨酸烷基化试剂，此类探针具备诸多优势，包括更快的反应速率、更高的稳定性，以及适配质谱应用的优异电离性能。总体而言，杂芳基砜具备模块化可调性，可改变色谱洗脱时间，并在深层蛋白质组范围分析与共价配体发现中展现出更优异的胞内半胱氨酸表征性能，可支持全面的蛋白质组范围分析与共价配体发现。