Oral Presentation 7th Modern Solid Phase Peptide Synthesis & Its Applications Symposium 2019

Condensation of cyanopyridine and aminothiol derivatives enables biocompatible peptide cyclization (#43)

Christoph Nitsche 1 , Thomas Huber 1 , Dahai Luo 2 , Richard Morewood 1 , Hideki Onagi 1 , Gottfried Otting 1 , Jun-Ping Quek 2
  1. Australian National University, Canberra, ACT, Australia
  2. Nanyang Technological University, Singapore

Constrained peptides fill an underexplored area of chemical space between small molecule therapies and larger antibodies. Cyclization or stapling often enhances metabolic stability by greater resistance towards proteolysis and can promote biological uptake across cell membranes. Currently available chemical strategies for cyclization and stapling, however, are not trivial and often require heavy metal catalysis, organic solvents or other conditions that are bioincompatible. The condensation between 1,2-aminothiols and nitriles is a yet underexplored chemical ligation strategy to enable bioconjugation and macrocyclization.

We developed a method to spontaneously cyclise peptides under biocompatible conditions, using an N-terminal cysteine residue and the unnatural amino acid 3-(2-cyano-4-pyridyl)alanine (Cpa). Cpa is readily prepared from the commercial precursor 3-(4-pyridyl)alanine and easily introduced into peptides using standard Fmoc solid-phase peptide synthesis. While the linear peptide can be isolated at acidic pH, cyclisation proceeds under biocompatible conditions at neutral pH with nearly quantitative yield. Cyclization half-times were below 10 minutes for all peptides analyzed. The reaction is orthogonal to all proteinogenic amino acids, including cysteine residues that are not at the N-terminus. No polymeric side products were observed even for strained macrocycles comprised of only five amino acid residues. A substrate peptide of a viral protease cyclized in this way produced an active-site inhibitor of nanomolar affinity and remarkable proteolytic stability (ref 1).

We further explored this chemistry to allow for stapling and cyclization of any peptide sequence independently of N-terminal cysteine residues. For this purpose, new unnatural amino acids were designed and introduced into peptide chains by standard Fmoc solid-phase peptide synthesis (unpublished results).

In conclusion, the condensation reaction of aminothiol derivatives (as in N-terminal cysteine residues) with activated nitriles (as in 2-cyanopyridines) represents an extraordinarily straightforward and selective strategy for peptide cyclization and stapling.

  1. Nitsche, C.; Onagi, H.; Quek, J.-P.; Otting, G.; Luo, D.; Huber, T., Biocompatible macrocyclization between cysteine and 2-cyanopyridine generates stable peptide inhibitors. Org. Lett. 2019, in revision.