Poster Presentation International Peptide Symposium 2023

Biocompatible head-to-side chain peptide macrocyclisation using oxime ligation (#229)

Lani J Davies 1 , Laura Shuttleworth 1 , Sherry Peng 1 , Xiaobai Zheng 1 , Christoph Nitsche 1
  1. The Australian National University, Canberra, ACT, Australia

Constraining peptides into macrocycles can increase their metabolic stability and binding affinity compared to their linear analogues.1 This is advantageous as it can be used to prevent the breakdown of peptide drugs through proteolysis and prearranges peptides in their active conformation, thus increasing their affinity due to a reduced entropic barrier.2 Oxime ligation is a bioorthogonal technique that is frequently used for protein conjugation, but currently neglected for peptide macrocyclisation. Oximes are formed from the biocompatible reaction between ketones or aldehydes and alkoxyamines (Figure 1).3 The resulting ligation product consists of only three heavy atoms (C=N-O) and displays cis and trans isomers. It is this small size that allows the moiety to be incorporated into peptides without disturbing the natural properties.4

By adding an α-ketoamide to the N-terminus, and an unnatural amino acid containing the alkoxyamine moiety elsewhere in a peptide side chain, we have demonstrated a biocompatible one-pot macrocyclization technique via oxime formation.5 Formation of the cyclised peptide first requires cleavage from the solid support with a mild cleavage cocktail containing HFIP instead of TFA, allowing for the spontaneous cyclisation of the peptide while preventing hydrolysis.5 The peptide then requires a second, more acidic cleavage cocktail to ensure complete deprotection of the side chains. This technique produces only the cis and trans isomers of the cyclic product with little to no impurities and can be applied across a variety of amino acid sequences. We demonstrate the facile synthesis of protease inhibitors with varying conformational constraint. The most constrained peptide displayed an activity 2 orders of magnitude higher than its linear analog.5

  1. Streefkerk, D. E. et al. Synthesis of Constrained Tetracyclic Peptides by Consecutive CEPS, CLIPS, and Oxime Ligation. Org. Lett. 21, 2095–2100 (2019).
  2. Tsomaia, N. Peptide therapeutics: Targeting the undruggable space. Eur. J. Med. Chem. 94, 459–470 (2015).
  3. Duflocq, S., Zhou, J., Huguenot, F., Vidal, M. & Liu, W.-Q. One-pot oxime ligation from peptides bearing thiazolidine and aminooxyacetyl groups. RSC Adv. 10, 17681–17685 (2020).
  4. Kölmel, D. K. & Kool, E. T. Oximes and Hydrazones in Bioconjugation: Mechanism and Catalysis. Chem. Rev. 117, 10358–10376 (2017).
  5. Davies, L. J., Shuttleworth, L. M., Zhang, X., Peng, S. & Nitsche, C. Bioorthogonal Peptide Macrocyclization Using Oxime Ligation. Org. Lett. 25, 2806–2809 (2023).