Reactivity of intein thioesters: appending a functional group to a protein

Chembiochem. 2006 Sep;7(9):1375-83. doi: 10.1002/cbic.200600150.


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 alpha-hydrazino acetyl group was the optimal nucleophile for attacking a thioester at neutral pH to form a stable linkage. A bifunctional reagent bearing an alpha-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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amines / chemistry
  • Azides / chemical synthesis
  • Azides / chemistry
  • Cross-Linking Reagents / chemical synthesis
  • Cross-Linking Reagents / chemistry
  • Esters
  • Fluoresceins / chemical synthesis
  • Fluoresceins / chemistry
  • Inteins*
  • Kinetics
  • Molecular Structure
  • Proteins / chemistry*
  • Ribonuclease, Pancreatic / chemistry
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Sulfhydryl Compounds / chemistry*


  • Amines
  • Azides
  • Cross-Linking Reagents
  • Esters
  • Fluoresceins
  • Proteins
  • Sulfhydryl Compounds
  • Ribonuclease, Pancreatic