Reactivity of Intein Thioesters: Appending a Functional Group to a Protein

The success of genome sequencing has heightened the demand for new means to manipulate proteins. An especially desirable goal is the ability to modify a target protein at a specific site with a functional group of orthogonal reactivity. Here, we achieve that goal by exploiting the intrinsic electrophilicity of the thioester intermediate formed during intein‐mediated protein splicing. Detailed kinetic analyses of the reaction of nitrogen nucleophiles with a chromogenic small‐molecule thioester revealed that the α‐hydrazino acetyl group was the optimal nucleophile for attacking a thioester at neutral pH to form a stable linkage. A bifunctional reagent bearing an α‐hydrazino acetamido and azido group was synthesized in high overall yield. This reagent was used to attack the thioester linkage between a target protein and intein, and thereby append an azido group to the target protein in a single step. The azido protein retained full biological activity. Furthermore, its azido group was available for chemical modification by Huisgen 1,3‐dipolar azide–alkyne cycloaddition. Thus, the mechanism of intein‐mediated protein splicing provides the means to install a useful functional group at a specific site—the C terminus—of virtually any protein.