Poster Presentation The 16th Australian Peptide Conference 2025

Unlocking the Potential of Hydrazide-Based Native Chemical Ligation with a Novel Dual-Linker Solubilising Platform (#132)

Keziah Poggenpoel 1 2 3 , Kevin Beijerling 3 4 , William Kelton 3 4 , Iman Kavianinia 1 2 3
  1. School of Chemical Sciences, The University of Auckland, Auckland, New Zealand
  2. School of Biological Sciences, The University of Auckland, Auckland, New Zealand
  3. Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, Auckland, New Zealand
  4. School of Pharmacy and Biomedical Sciences, The University of Waikato, Hamilton, New Zealand

Native Chemical Ligation (NCL), introduced by Dawson, Kent and colleagues in 19941, has since become a fundamental technique in synthetic protein chemistry. This powerful method chemoselectively joins two unprotected peptide fragments: one fragment containing an N-terminal cysteine residue and another bearing a C-terminal peptide α-thioester, in neutral aqueous solutions, offering a versatile approach for the synthesis of proteins and large peptides. The generation of a thioester can be perceived as the limiting step of a successful ligation. Recent advancements led to the development of Fmoc-compatible thioester surrogates—also known as crypto-thioesters—that can be activated to form reactive peptide thioester species in the presence of thiols. Hydrazide-based NCL2 was introduced as an approach where the hydrazide acts as a readily accessible precursor for a thioester. Upon cleavage from the resin, peptide hydrazides are selectively oxidised to reactive acyl azide intermediates then converted to peptide thioesters by thiolysis generating ligated proteins in near quantitative yields. Despite the widespread use of hydrazide-based NCL, the solubility and aggregation tendencies of certain peptide hydrazide intermediates can complicate both peptide purification and ligation.3

To address these challenges, we have developed a novel dual-linker strategy that integrates a hydrazinolysis-cleavable linker with a C-terminal hexalysine (K₆) solubilising tag. This modular platform enables efficient generation of highly aggregation-prone peptide hydrazides directly from the resin, significantly improving both peptide handling and ligation efficiency. The utility of this approach was demonstrated through the synthesis of an epidermal growth factor receptor (EGFR)-targeting affibody (~7 kDa) via hydrazide-based NCL. This affibody serves as a representative model system, underscoring the growing need for streamlined methods in the chemical synthesis of structured and functional peptide-based therapeutics and diagnostics. Overall, the dual-linker strategy offers a robust and scalable solution for accessing ligation-ready peptide hydrazides, improving the synthetic tractability and applicability of NCL for challenging targets.

  1. (1) Dawson, P. E., Muir, T. W., Clark-Lewis, I., & Kent, S. B. Synthesis of proteins by native chemical ligation. Science. 1994, 266, 776-779.
  2. (2) Fang, G. M., Li, Y. M., Shen, F., Huang, Y. C., Li, J. B., Lin, Y., Hong-Kui, C., & Liu, L. Protein Chemical Synthesis by Ligation of Peptide Hydrazides. Angew. Chem. Int. Ed. 2011, 50, 7645-7649.
  3. (3) Huang, Y. C., Fang, G. M., & Liu, L. Chemical synthesis of proteins using hydrazide intermediates. Ntl Sci Rev. 2016, 3, 107-116.