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 similar to 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.