Poster Presentation The 16th Australian Peptide Conference 2025

Non-Symmetric Cysteine Stapling in Native Peptides and Proteins (#123)

Bishvanwesha Panda 1 , Sven Ullrich 2 , Upamali Somathilake 1 , Doug Lawes 1 , Christoph Nitsche 1
  1. Australian National University, Canberra, ACT, Australia
  2. Chemistry, University of Tokyo, Tokyo

Peptide stapling is a powerful strategy for stabilising native confirmations to enhance their bioactivity, particularly in targeting protein-protein interactions (PPIs).1 Traditional stapling methods typically rely on symmetric reagents, whereas non-symmetric approaches are less common due to the need for precise chemo- and regioselectivity.2 Here, we introduce a novel protocol employing 2-chloromethyl-6-cyanopyridine for asymmetric stapling of peptides with N-terminal and internal cysteines. 3 This reagent enables selective nitrile conjugation with the N-terminal cysteine, while the chloromethyl substituent forms a thioether bond with an internal cysteine, generating a bis-heterocyclic bridge. This biocompatible one-step reaction, conducted at physiological pH, generates diverse peptide macrocycles with enhanced stability, binding affinity and inhibitory potency.

The robustness of the method is demonstrated through conversion of various native peptides into stapled analogs. Notably, this approach does not require unnatural amino acids and is likely compatible with genetically encoded systems, broadening its potential in macrocyclic peptide therapeutic development. For example, the minor coat protein pIII of M13 phage, widely used in phage display, was engineered with N-terminal and internal cysteines at i, i+7 positions for successful modification using 2-chloromethyl-6-cyanopyridine. The protocol also accommodates further modification via conventional click reactions, provided an alkyne is integrated into the macrocycle.3

Additionally, research on peptide libraries binding to streptavidin showed that stapled analogs display higher affinity (KD = 3.6 μM) than their linear counterparts (KD = 502 μM). In bioactivity tests, cyclic peptides targeting Zika virus protease showed improved inhibition (Ki = 291 nM) and 20-fold increased stability against proteolysis. 3

  1. Dayanara, N. L., Froelich, J., Roome, P. & Perrin, D. M. Chemoselective, Regioselective, and Positionally Selective Fluorogenic Stapling of Unprotected Peptides for Cellular Uptake and Direct Cell Imaging. Chem. Sci. (2024).
  2. Chen, F.-J., Lin, W. & Chen, F.-E. Non-symmetric Stapling of Native Peptides. Nat. Rev. Chem. 8, 304–318 (2024).
  3. Ullrich, S., ‡ Panda, B., ‡ Somathilake, U., Lawes, D. J. & Nitsche, C. Non-symmetric Cysteine Stapling in Native Peptides and Proteins. Chem. Commun. (2025).