Protein Ligation and Labeling Enabled by a C‐Terminal Tetracysteine Tag

Abstract The hydrazinolysis of S‐cyanylated peptide provides an alternative way to afford protein α‐hydrazide, a key reagent used in native chemical ligation (NCL), without the aid of any inteins or enzymes. The currently used non‐selective S‐cyanylation, however, allows no other cysteine in the protein besides the one at the cleavage site. Herein, we report a regioselective S‐cyanylation and hydrazinolysis strategy achieved via the fusion of a tetracysteine tag to the C‐terminal of the protein of interest. We term it tetracysteine enabled protein ligation (TCEPL). While highly selective, the strategy is applicable for proteins expressed as inclusion bodies, and this was showcased by the efficient semi‐synthesis of an iron‐sulfur protein rubredoxin and the catalytic and hinge domains of matrix metalloprotease‐14 (MMP‐14) containing 207 amino acid residues. Furthermore, the TCEPL strategy was exploited for protein C‐terminal labeling with amino reagents bearing a variety of functional groups, demonstrating its versatility and generality.