A Minimal Tetrazine Linchpin for Cysteine Bridging and Bioorthogonal Ligation

Site-selective bridging of cysteines and native disulfide bonds represents a powerful strategy for peptide and protein modification. However, most bis-electrophilic reagents rely on azide-alkyne cycloadditions for downstream bioconjugation, while existing tetrazine-based cysteine linkers can generate undesired byproducts, such as peptide cyclodimers, or incorporate additional components that are not directly involved in conjugation or ligation. Here, we report divinyl- s -tetrazine ( DvTz ) as a minimal linchpin in which the electron-deficient tetrazine core activates the vinyl groups as Michael acceptors for efficient cysteine conjugation, while simultaneously serving as a bioorthogonal handle for subsequent inverse-electron-demand Diels-Alder (IEDDA) ligation. DvTz enables high-yielding, cysteine-selective stapling of unprotected peptides, tolerates all twenty canonical amino acids, and accommodates a broad range of sequences and stapling distances. A one-pot stapling-IEDDA sequence enables facile installation of functional tags without intermediate purification. The chemistry was extended to antibody Fab fragments, achieving site-specific disulfide rebridging and subsequent IEDDA ligation across three distinct Fab proteins, with exclusive formation of singly conjugated species. A Fab-drug conjugate was constructed with retained antigen-binding activity and selective potency against HER2-positive cell lines, showcasing the application of DvTz in therapeutically relevant settings. Collectively, this work establishes DvTz as a simple and versatile linchpin for peptide and protein bioconjugation.