Ullrich, SvenNitsche, Christoph2025-12-232025-12-23978-1-0716-4561-1978-1-0716-4562-81064-3745PubMed:40531445ORCID:/0000-0002-3704-2699/work/190440187ORCID:/0000-0003-4184-7024/work/190440826https://hdl.handle.net/1885/733796889Constrained peptides are emerging as promising structures in therapeutic development. Offering compatibility with genetically encoded libraries for drug discovery, biocompatible methods to constrain peptides are particularly attractive. While there are many such methods to construct cyclic and stapled peptides, the biocompatible generation of bicyclic peptides is less explored. Addressing this need for biocompatible and selective ways to generate peptide bicycles, we previously developed a strategy based on noncanonical amino acids leveraging the reactivity of cyanopyridine and 1,2-aminothiol. This protocol provides detailed step-by-step instructions for the synthesis of these peptide bicycles and is designed to be accessible even to laboratories with limited synthetic chemistry resources. It outlines the solid-phase peptide synthesis of linear peptide precursors that efficiently form bicyclic structures in aqueous buffer at physiological pH. Utilizing commercially available building blocks, we devised a method to synthesize the noncanonical amino acids that are essential for bicyclization directly on the solid support during peptide synthesis.We acknowledge Dr. Josemon George, Dr. Richard Morewood, and Alexandra Coram, who co-developed elements of the methods described in this chapter [13, 20, 31]. The authors are grateful for funding by the Australian Research Council for Assoc. Prof. Christoph Nitsche in the form of a Discovery Project (DP230100079) and a Future Fellowship (FT220100010). Dr. Sven Ullrich is supported through a Feodor Lynen Research Fellowship from the Alexander von Humboldt Foundation.13en© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2025.Biocompatible chemistryConstrained peptidesNoncanonical amino acidsPeptide macrocyclizationSolid-phase peptide synthesis2025-06-1910.1007/978-1-0716-4562-8_2105009235859