Azapeptide Synthesis Fully Integrated into Standard Solid-Phase Peptide Synthesis (SPPS): Case Study with Azapeptide-GLP-1

Therapeutic peptides could benefit significantly from the well-established azapeptide (substitution of backbone alpha atom(s) with N, producing an enzyme resistant semicarbazide bond), yet their broader application remains limited due to the challenges associated with their synthesis, particularly in generating diverse libraries for systematic functional evaluation. Traditional azapeptide synthesis methods rely on the activation of hydrazine derivatives using carbonyl-donating reagents such as phosgene, chloroformates, or in situ activation of thiocarbazates. Despite advances in peptide chemistry, no fully automated system for azapeptide synthesis has been available until now. Herein, we present a fully automated solid-phase azapeptide synthesis platform, employing Fmoc-protected benzotriazole esters as bench-stable, pre-activated aza-amino acid building blocks. This protocol enables the rapid and efficient synthesis of azapeptide libraries, considerably reducing reaction times while improving both crude purity and isolated yields. To rigorously test this platform, we synthesized azapeptide analogues of the 31-residue peptide hormone, glucagon-like peptide-1 (GLP-1), modifying the proteolytic enzyme-sensitive sites. This fully automated azapeptide synthesis platform marks a significant milestone in peptide chemistry, addressing key barriers to library generation and offering a scalable and efficient tool for the development of azapeptide-based therapeutics.