Formamidine as an Easy‐On and Easy‐Off Linker for Reversible Crosslinking of Two Alkyl Amines in Peptide Stapling and Conjugation

Abstract Amino groups are abundant in both natural and synthetic molecules, offering highly accessible sites for modifying native biorelevant molecules. Despite significant progress with more reactive thiol groups, methods for ligating two amino groups with reversible linkers for bioconjugation applications remain elusive. Herein, we report the use of oxidative decarboxylative condensation of glyoxylic acid to crosslink or ligate two alkyl amines via a compact formamidine linkage, applicable in both intra‐ and intermolecular contexts. This linking chemistry exhibits unique hetero‐coupling selectivity between primary and secondary alkyl amines. Although the formamidine linkage is stable under pH‐neutral buffers and acidic conditions, it can be readily cleaved with ethylenediamine or hydrazine under mild conditions in alcohol solvents or aqueous media, fully restoring the amino groups. This study introduces a rare ‘easy‐on and easy‐off’ strategy for connecting two native amines in peptide stapling and biomolecule conjugation.