Sortase‐mediated ligation (SML) is a widely used method for peptide and protein ligation due to ease of substrate preparation and fast enzymatic kinetics. But there are drawbacks that limit broader applications. Sorting motif in substrates may not be exposed to sortase efficiently due to folding or aggregation. In addition, the ligation is reversible under transpeptidation equilibrium that restricts ligation yield. Here we report a simple but robust method to overcome such limitations. By employment of sarkosyl, the detergent alters substrate conformation to raise sorting motif accessibility for sortase catalysis. Moreover, transpeptidation becomes irreversible presumably by formation of micelle to shield ligation products from sortase. In consequence, excellent yields were achieved from sortase variants with different substrate specificity. Notably, this method is compatible with peptides or proteins capable of forming liquid‐liquid phase separation (LLPS), presenting a powerful approach for the conjugation of aggregation‐prone substrates. Therefore, we believe the sarkosyl‐enhanced SML could be widely applied in peptide and protein chemistry and the unique irreversible transpeptidation mechanism offers an insight to detergent‐driven equilibrium.

中文翻译：

---

利用肌氨酰进行稳健且不可逆的分选酶介导的连接


分选酶介导的连接（SML）由于易于底物制备和快速的酶动力学而成为肽和蛋白质连接的广泛使用的方法。但也有一些缺点限制了更广泛的应用。由于折叠或聚集，底物中的分选基序可能无法有效地暴露于分选酶。此外，连接在转肽平衡下是可逆的，这限制了连接产量。在这里，我们报告了一种简单但强大的方法来克服这些限制。通过使用肌氨酸，去污剂改变底物构象，以提高分选基序对分选酶催化的可及性。此外，转肽作用可能通过胶束的形成而变得不可逆，以保护连接产物免受分选酶的影响。因此，具有不同底物特异性的分选酶变体获得了优异的产量。值得注意的是，该方法与能够形成液-液相分离（LLPS）的肽或蛋白质兼容，为易于聚集的底物的缀合提供了一种强大的方法。因此，我们相信肌氨酸增强的 SML 可以广泛应用于肽和蛋白质化学，并且独特的不可逆转肽机制为去污剂驱动的平衡提供了见解。