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

Azapeptide modification, achieved by substituting backbone -carbons with nitrogen atoms to form enzyme-resistant semicarbazide bonds, can markedly enhance peptide stability and therapeutic potential. However, broad application has been constrained by two major synthetic challenges: the lack of suitable building blocks and the reduced nucleophilicity of the semicarbazide amino group, which limits post-coupling efficiency in automated solid-phase peptide synthesis (SPPS). Here, we describe a fully automated azapeptide synthesis platform that employs Fmoc-protected benzotriazole esters as bench-stable, pre-activated aza-amino acid building blocks. Microwave-assisted synthesis was integrated to accelerate aza-residue incorporation and improve coupling efficiency. This plug-and-play approach enables rapid solid-phase assembly of azapeptides, substantially reducing reaction times and improving yields. To demonstrate its utility, we synthesized azapeptide analogues of glucagon-like peptide-1 (GLP-1) with targeted substitutions at protease-sensitive sites, achieving enhanced stability as demonstrated in our recent study (bioRxiv 2025). This automated platform overcomes long-standing barriers in azapeptide chemistry, providing a scalable route for the rapid generation of stabilized peptide therapeutics. Significance StatementAzapeptides are a promising class of peptidomimetics with enhanced enzymatic stability and therapeutic potential. Yet, their synthesis has been limited by two persistent barriers: the lack of suitable, stable building blocks and the reduced coupling efficiency caused by the semicarbazide backbone. We developed a fully automated, microwave-assisted solid-phase synthesis platform that overcomes both challenges by using bench-stable benzotriazole ester aza-amino acid building blocks. This plug-and-play system enables efficient incorporation of aza-residues under standard SPPS conditions, reducing reaction times while improving yields and reproducibility. The resulting platform allows rapid generation of azapeptide libraries for biological screening and drug development, representing a major step toward scalable production of protease-resistant peptide therapeutics.