Chemical synthesis of circular proteins

Abstract

Circular proteins, once thought to be rare, are now commonly found in plants. Their chemical synthesis, once thought to be difficult, is now readily achievable. The enabling methodology is largely due to the advances in entropic chemical ligation to overcome the entropy barrier in coupling the N- and C-terminal ends of large peptide segments for either intermolecular ligation or intramolecular ligation in end-to-end cyclization. Key elements of an entropic chemical ligation consist of a chemoselective capture step merging the N and C termini as a covalently linked O/S-ester intermediate to permit the subsequent step of an intramolecular O/S-N acyl shift to form an amide. Many ligation methods exploit the supernucleophilicity of a thiol side chain at the N terminus for the capture reaction, which makes cysteine-rich peptides ideal candidates for the entropy-driven macrocyclization. Advances in desulfurization and modification of the thiol-containing amino acids at the ligation sites to other amino acids add extra dimensions to the entropy-driven ligation methods. This minireview describes recent advances of entropy-driven ligation to prepare circular proteins with or without a cysteinyl side chain.

FIGURE 1.

Chemical ligation and cyclization schemes. A, enthalpy-driven cyclization based on C-terminal activation. OA, active ester. B, entropy-driven ligation via an S-N acyl shift between the C-terminal thioester and N-terminal Cys. X and Y represent a nucleophile and electrophile pair. Z is the bond of the capture intermediate. R represents side chains. C, thia-zip cyclization mechanism for forming a circular protein through stepwise ring expansion of thiolactone intermediates to an end-to-end thiolactone to enable lactam formation via an S-N acyl shift. NT, N-terminal.