Robust and Irreversible Sortase-Mediated Ligation by Empolyment of Sarkosyl

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. Sarkosyl significantly enhances sortase-mediated ligation by promoting substrate solubility, sorting motif accessibility and rendering reaction irreversibility, thereby leading to excellent ligation yields. Notably, this method is compatible with peptides or proteins capable of forming liquid-liquid phase separation, offering a powerful approach for conjugating aggregation-prone substrates in peptide and protein chemistry. image