Enzymatic tools for peptide ligation and cyclization Development and applications

The renaissance of peptides as prospective therapeutics has fostered the development of novel strategies for their synthesis and modification. Here we describe the development of novel approaches for the efficient chemo-enzymatic synthesis of linear and complex (multi-)cyclic peptides. The structure-guided design of novel peptiligase variants resulted in the discovery of novel enzymes, which were successfully used in the convergent, fragment-based synthesis of thymosin-α₁ and the GLP-1 analogue exenatide at larger scale with significantly increased yields as compared to using classical straight-through solid-phase peptide synthesis. In addition, the use of several peptiligase variants has been proven to be efficient for the scalable and modular synthesis of several disulfide-rich macrocyclic peptides. Several ligation sites per molecule underline the high flexibility of the enzymatic approach, which also shows very good compatibility with CLIPS and click chemistry, as well as oxime ligation. This was demonstrated by synthesizing a new class of tetracyclic peptides using a combination of these chemistries together with newly developed small molecule scaffolds.