Thioester-forming chemoselective reaction of unprotected peptide fragments containing cysteine residues has been investigated. This work shows that free sulfhydryl groups are compatible with the reactive components of thioester-forming ligation chemistry. This allows conjugation by chemical ligation of cysteine or other thiol-containing peptides, followed by postligation disulfide bond formation to form folded protein domains, or large multisubunit synthetic proteins. Under acidic conditions, peptides bearing bromoacetyl or a-thiocarboxylate groups did not undergo intermolecular reaction with the sulfhydryl group of cysteine. Intramolecular reaction also did not occur, provided a sufficient number of intervening residues separated the functionalities. The results of these studies have been used in the design and synthesis of a 22 kDa tethered dimer HIV-1 protease analogue, prepared by the convergent chemical ligation of four unprotected peptide segments. Two pairs of ~50 residue peptides were ligated via formation of thioester bonds to form the individual monomer polypeptide chains. The ligated monomers each possessed a two residue extension, either at the N-terminal or at the C-terminal, containing an unprotected sulfhydryl group. These were subsequently linked via directed formation of a disulfide bond. The resulting enzyme analogue retained full catalytic activity, showing that the placement of the backbone thioesters and the disulfide bond were in functionally unimportant parts of the molecule.
|Original language||English (US)|
|Number of pages||7|
|Journal||Journal of the American Chemical Society|
|State||Published - Jan 1 1995|
All Science Journal Classification (ASJC) codes
- Colloid and Surface Chemistry