High Yield Branched Puromycin Linker Design Enables Efficient cDNA Display and Chemical Modification of Peptides

Peptide chemical modification is a valuable technique for improving peptide stability and bioactivity, particularly in drug discovery applications. Here, we report the development of a novel linker junction strategy using strain-promoted azide-alkyne cycloaddition (SPAAC) that enables the efficient formation of branched puromycin linkers with an average yield of 97%. This approach represents an improvement over traditional Michael-Addition methods, which typically yield ~15%-20%. The high yield of the SPAAC reaction and the near absence of by-products make the linkage by click reaction easy to purify. We demonstrate the utility of our linker design by successfully performing cDNA display and chemical modification strategies such as bicyclization of peptides. Our study demonstrates the functionality of the cDNA display system with the newly incorporated junction. In addition, the successful introduction of peptide bicyclization via tris-bromomethyl-benzene (TBMB) in cDNA display serves as a proof-of-concept for complex chemical modifications. Furthermore, the position of puromycin, which disrupts protein biosynthesis, has been determined. This approach offers novel insights into the discovery of chemically modified peptides and has the potential to accelerate the development of peptide-based therapeutics and diagnostics.