Poster with Rapid Fire Oral 12th Australian Peptide Conference 2017

Omniligase-1: A Powerful Tool for Peptide Head-to-Tail Cyclization (#187)

Marcel Schmidt 1 2 , Ana Toplak 2 , Jan H. van Maarseveen 1 , Timo Nuijens 2
  1. Van 't Hoff Institute of Molecular Sciences, University of Amsterdam, Amsterdam, The Netherlands
  2. EnzyPep B.V., Geleen, The Netherlands

Peptide macrocycles represent a diverse class of molecules that are attracting increased attention as drug leads and prospective pharmaceuticals.[1] The increasing number of macrocyclic peptides being investigated as therapeutics mandates efficient and cost-effective routes that enable their synthesis. However, both lab scale and efficient large scale manufacture of peptide macrocycles using traditional synthetic methods are still major challenges. Within this context, the use of enzymes for the head-to-tail cyclization of peptides represents a promising strategy and provides an elegant link between chemistry and biology.[2] We have recently developed several subtilisin variants that are capable of forming a peptide bond with high efficiency in aqueous solution between a C-terminal activated ester segment and the N-terminus of an acyl acceptor segment.[3]

In this study, the peptide ligase omniligase-1 has been used as a tool for the cyclization of several head-to-tail (multi)cyclic peptides, including the cyclotide MCoTI-II.[4] Cyclization and oxidative folding of the cyclotide MCoTI-II was efficiently performed in a one-pot reaction at gram scale. The native cyclotide was isolated and the correct disulfide bonding pattern was confirmed by NMR structure determination. Furthermore, compatibility of chemo-enzymatic peptide synthesis (CEPS) using omniligase-1 with methods such as chemical ligation of peptides onto scaffolds (CLIPS) was successfully demonstrated by synthesizing a kinase-inhibitor derived tricyclic peptide. Our studies indicate that the cyclization of peptides longer than 12 amino acids proceeds with remarkable efficiency. In addition, several macrocycles containing non-peptidic backbones (e.g. polyethylene glycol), isopeptide bonds (amino acid side-chain attachment) as well as D-amino acids could be efficiently cyclized.

In conclusion, the efficiency of omniligase-1 (>95% average cyclization yield) combined with its broad substrate scope allows traceless ligation and makes CEPS a flexible and generally applicable tool, which provides a viable and economically attractive route for the synthesis of cyclotides and other head-to-tail cyclic peptides.

 

 

  1. [1] A. Zorzi, K. Deyle, C. Heinis, Curr. Opin. Chem. Biol. 2017, 38, 24–29.
  2. [2] M. Schmidt, A. Toplak, P. J. J. L. M. Quaedflieg, T. Nuijens, Curr. Opin. Chem. Biol. 2017, 38, 1–7.
  3. [3] A. Toplak, T. Nuijens, P. J. Quaedflieg, B. Wu, D. B. Janssen, Adv. Synth. Catal. 2016, 358, 2140–2147.
  4. [4] M. Schmidt, A. Toplak, P.J.L.M. Quaedflieg, H. Ippel, G.J.J. Richelle T.M. Hackeng, J.H. van Maarseveen, T. Nuijens, Adv. Synth. Catal., 2017, in press.