N-terminal dual functionalization of proteins via copper-mediated [3 + 2] cycloaddition of triazolecarbaldehyde with maleimide

Abstract Site-specific dual-functionalization strategies represent a critical advancement in bioconjugation chemistry, enabling the installation of multiple functional moieties. We report the copper-mediated [3 + 2] cycloaddition of 1H-1,2,3-triazole-4-carbaldehyde (TA4C) derivatives with maleimides for efficient N-terminal dual modification under mild aqueous conditions. The strategy demonstrates exceptional specificity and wide compatibility with substrates, achieving >99% conversion for peptides and >80% for proteins. The versatility is exemplified through the formation of an assembly with streptavidin and the protein modified with biotin and a Cy5 dye at the N-terminus, providing a robust tool for applications in biomaterials and chemical biology.