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Strategies for the efficients ynthesis of peptide macrocyclesh ave been al ong-standing goal. In this paper, we demonstrate the use of the peptide ligase termedo mniligase-1 as av ersatile and broadly applicable enzymatic toolf or peptide cyclization. Severalh ead-to-tail (multi)cyclic peptides have been synthesized, including the cyclotide MCoTI-II. Cyclization ando xidative foldingo ft he cyclotide MCoTI-II were efficiently performed in ao ne-pot reactiono na1 -gram scale.T he native cyclotide was isolated andt he correct disulfide bonding pattern was confirmed by NMRs tructure determination. Furthermore,c ompatibility of chemo-enzymatic peptide synthesis( CEPS) using omniligase1w ith methods such as chemical ligation of peptides onto scaffolds( CLIPS)w as successfully demonstrated by synthesizing ak inase-inhibitor derived tricyclic peptide.O ur studies indicate that the minimal ring size for omniligase-1 mediated cyclization is 11 amino acids,w hereas the cyclization of peptides longer than 12 amino acids proceeds with remarkablee fficiency.I na ddition,s everal macrocycles containing non-peptidic backbones( e.g.,p olyethylene glycol), isopeptide bonds (aminoa cid sidechain attachment) as well as d-amino acids couldbe efficiently cyclized.Keywords: chemo-enzymatic peptide synthesis (CEPS);c yclic peptides; cyclization;c yclotide synthesis;c yclotides;e nzyme catalysis;h ead-to-tail cyclization; ligases;m acrocycles;o mniligase-1;p eptides Peptide macrocyclesr epresent an extremely diverse class of molecules that attracti ncreased attentiona s drug leads and prospective pharmaceuticals,w ith currently over 30 cyclic peptides registered or in clinical trials.[1-3] Togetherw ith linearp eptides, they fill the gap between small-molecule drugs (less than 500 Da) and biologics (over 5000 Da) andh avet he potential to address previously "undruggable" targets. Many cyclicp eptides are characterized by their unique structural and enhancedb iopharmaceutical properties,s uch as an improved metabolic stability due to ar educed sensitivity to proteolytic cleavage.T he increasing number of cyclic peptidesu sed as therapeutics is accompanied by the need for efficient andc osteffectiver outes that enable their synthesis on al arge scale.M oreover, efficients ynthesiso fl arge libraries of cyclic peptides (e.g.,f or screeningp urposes) or of cyclicp eptides containing non-canonical amino acids (e.g., d-o ru nnatural amino acids to increase stability and diversity) is of importance.H owever, efficient cyclization of peptidesu sing traditional synthetic methodsi ss till ac hallenge.[4] Classical coupling reagents are often used to cyclize side-chain protected peptides in anhydrous organic solvents.H owever, heavy dilution to prevent polymerization,r isk of epimerization and ap oors olubility of side-chain protected peptideslimitthis approach, especially for peptides longer than 20 amino acids.[5] Native chemical ligation (NCL) [6] is often used to cyclize unprotected peptides in aqueous solution. However, not all pep...

The renaissance of peptides as prospective therapeutics has fostered the development of novel strategies for their synthesis and modification. In this context, besides the development of new chemical peptide ligation approaches, especially the use of enzymes as a versatile tool has gained increased attention. Nowadays, due to their inherent properties such as excellent regio- and chemoselectivity, enzymes represent invaluable instruments in both academic and industrial laboratories. This mini-review focuses on natural- and engineered peptide ligases that can form a new peptide (amide) bond between the C-terminal carboxy and N-terminal amino group of a peptide and/or protein. The pro's and cons of several enzyme classes such as Sortases, Asparaginyl Endoproteases, Trypsin related enzymes and as a central focus subtilisin-derived variants are summarized. Most recent developments with regards to ligation and cyclization are highlighted.

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Exemplified by antidiabetic exenatide CEPS provides a highly efficient, easily scalable biocatalytic route to manufacture therapeutic peptides.