Proteins with a
functionalized <i>C</i>-terminus such as a <i>C</i>-terminal thioester are key to the
synthesis of larger proteins via expressed protein ligation. They are usually
made by recombinant fusion to intein. Although powerful, the intein fusion
approach suffers from premature hydrolysis and low compatibility with denatured
conditions. To totally bypass the involvement of an enzyme for expressed
protein ligation, here we showed that a cysteine in a recombinant protein was
chemically activated by a small molecule cyanylating reagent at its <i>N</i>-side amide for undergoing nucleophilic
acyl substitution with amines including a number of L- and D-amino acids and
hydrazine. The afforded protein hydrazides could be used further for expressed
protein ligation. We demonstrated the versatility of this approach with the
successful synthesis of ubiquitin conjugates, ubiquitin-like protein
conjugates, histone H2A with a posttranslational modification, RNAse H that
actively hydrolyzed RNA, and exenatide that is a commercial therapeutic
peptide. The technique, which is exceedingly simple but highly useful, expands
to a great extent the synthetic capacity of protein chemistry and will
therefore make a large avenue of new research possible.
Analysis of Site‐Specific Phosphorylation of PTEN by Using Enzyme‐Catalyzed Expressed Protein Ligation