Efficient N‐Terminal Labeling of Proteins by Use of Sortase

“Sorting out” N-terminal labeling: The reversibility of transpeptidase reactions makes protein N-terminal labeling challenging. Depsipeptide substrates for sortase A release alcohol by-products, which are poor nucleophiles for the reverse reaction, during ligation. Proteins with an unhindered N-terminal glycine residue can be labeled efficiently with only a minimal excess of the labeling reagent (see scheme).