Azole reagents enabled ligation of peptide acyl pyrazoles for chemical protein synthesis

Native chemical ligation (NCL) has been playing an increasingly important role in chemical protein synthesis (CPS), more efficient ligation methods that circumvent the requirement of peptidyl thioester and thiol additive—which allow the following desulfurization or refolding in one pot—are urgently needed for the synthesis of more complex protein targets and in large quantities. Herein, we discover that the weak acyl donor peptidyl N-acylpyrazole can be activated by azole reagents like 3-methylpyrazole or imidazole to facilitate its ligation directly with an N-terminal cysteine peptide. As it requires no thioester and thiol additive, this ligation strategy can be conveniently combined with metal-free desulfurization (MFD) or oxidative protein folding to allow various one-pot protocols. The utility and generality of the strategy are showcased by the total synthesis of ubiquitin via an N-to-C sequential ligation-MFD strategy, the semi-synthesis of a copper protein azurin, and the efficient assembly of a sulfated hirudin variant and the cyclotide kalata B1, all in a one-pot fashion.