Synthesis and Protein Engineering Applications of Cyclotides

Abstract

Cyclotides are a group of plant-derived peptides with a head-to-tail cyclized backbone that is stabilized by three knotted disulfide bonds. Their exceptional stability and tolerance for residue substitutions have led to interest in their application as drug design scaffolds. To date, chemical synthesis has been the dominant methodology for producing cyclotides and their analogues. Native chemical ligation is the most common strategy to generate the cyclic backbone and has been highly successful at producing a wide range of cyclotides for studies of structure–activity relationships. Both this and other chemical approaches require a specific linker at the C-terminus and typically involve a non-directed folding (disulfide oxidation) regimen, which can sometimes be a limiting factor in final yields. Following the recent discovery of enzymes involved in peptide cyclization in planta, site-specific and highly efficient enzymatic ligations have been used for synthetic cyclotide backbone cyclization. In this review, chemical synthesis strategies and approaches involving cyclization via enzymes for the production of cyclotides are described.