Oral Presentation 6th Modern Solid Phase Peptide Synthesis & Its Applications Symposium 2017

Chemoenzymatic peptide synthesis (CEPS) using peptiligase variants: A more efficient and greener route for the synthesis of long peptides, proteins and conjugates (#7)

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

Economic commercial manufacture and laboratory scale synthesis of long peptides and proteins is still a major challenge. Solid phase peptide synthesis (SPPS) is constrained by the exponential loss of yield with increasing peptide length and recombinant technologies are typically limited to the use of proteogenic amino acids. As a result, the continuously increasing popularity of peptide therapeutics is accompanied by a growing demand for new and efficient processes for their production at industrial scale. A particularly promising approach to meet this demand and to significantly improve peptide yield and crude peptide purity is the use of chemo‑enzymatic peptide synthesis (CEPS), a combination of conventional synthetic strategies with an enzymatic, epimerization‑free coupling of unprotected peptide segments in water. We have recently developed several subtilisin variants (e.g. Omniligase-1), which are capable of forming a peptide bond between a C-terminal activated ester and the N-terminus of an acyl acceptor fragment with high efficiency.[1] They present a wide range of selectivity, spanning from acceptance of any amino acid (except Pro), to highly selective ligases that can discriminate between the N-terminal sequences of dimeric peptides such as insulin. These robust ligases catalyze the footprint-free coupling of peptide segments, tolerate a wide range of co-solvents (up to 50%) and denaturing agents, are thermostable and show a very high catalytic efficiency (<0.001 molar equivalents). These extremely fast and scalable reactions are kinetically driven and irreversible. Conversion to product typically exceeds 95%. Over 250 ligases have been characterized and used to assemble a number of long peptides (e.g. exenatide)[2], macrocyclic peptides (e.g. cyclotide MCoTI-II)[3] as well as to couple peptides or peptide-based tags (e.g. FITC labelled) to other polypeptides such as insulin, proteins like human serum albumin or monoclonal antibodies. These ligases clearly represent a valuable adjunct to traditional SPPS and ligation (e.g. NCL) approaches.

  1. [1] M. Schmidt, A. Toplak, P. J. J. L. M. Quaedflieg, T. Nuijens, Curr. Opin. Chem. Biol. 2017, 38, 1–7.
  2. [2] T. Nuijens, A. Toplak, P. J. L. M. Quaedflieg, J. Drenth, B. Wu, D. B. Janssen, Adv. Synth. Catal. 2016, 358, 4041–4048.
  3. [3] M. Schmidt, A. Toplak, P. J. L. M. Quaedflieg, H. Ippel, J. J. Gaston, T. M. Hackeng, J. H. Van Maarseveen, T. Nuijens, Adv. Synth. Catal. 2017, 359, 2050–2055.