Tuning Tetrazine Substituents to Enhance Vinyltetrazine Labeling Performance.

The site-specific labeling of peptides and proteins is a powerful tool for investigating biological processes. It is demonstrated that the powerful thiol-specific labeling reagent vinyltetrazine activates prodrugs and enables ¹⁸F and near-infrared dual-modality imaging. It is predicted that substitutions at the 3-position of tetrazine can affect the labeling efficiency, bioorthogonal kinetics, and stability. Six vinyltetrazines with different 3-position modifications exhibit rapid labeling and, subsequently, bioorthogonal kinetics and excellent stability. Vinyltetrazine labeling modifies the charge characteristics and lipophilicity of target peptides, thereby improving cellular uptake. This research on tuning tetrazine substituents will aid in the construction of a comprehensive vinyltetrazine library, leading to the development of new peptide conjugates and pretargeted immuno-positron emission tomography (immune-PET) imaging.