Thiol-Retaining N-Terminal Cysteine Chemistry for Dual Modification and Bicyclic Peptide Construction

N-terminal cysteine presents a uniquely reactive 1,2-aminothiol motif that enables site-specific modification of peptides and proteins composed solely of canonical amino acids. For both in vitro and in vivo applications, this operationally simple chemistry is an attractive alternative to bioorthogonal strategies that require noncanonical handles. However, most 1,2-aminothiol-selective reagents irreversibly consume both amine and thiol, yielding inert heterocycles and limiting downstream diversification. Here, we report a thiol-retaining N-terminal cysteine chemistry by repurposing 2-((alkylthio)(aryl/alkyl)methylene)malononitriles (TAMMs) to favor a thiol-containing conjugate over the canonical cyclized product. Through rational design and mechanistic analysis of ortho-substituted TAMMs (o-TAMMs), we established steric hindrance as a key determinant of thiol-retaining adduct stability. The retained thiol provides an immediate handle for sequential dual modification of peptides and proteins. Extending this concept to scaffold design, an electrophile-equipped o-TAMM cross-linker converts CX_mCX_nC peptides into compact bicyclic architectures comprising a thioether ring and a disulfide ring. Phage display using this chemistry affords high-affinity bicyclic binders of KEAP1, and the disulfide can be transformed into a redox-stable thioacetal without a loss of affinity. Collectively, this work establishes a mechanistically grounded platform for thiol-retaining N-terminal cysteine ligation, enabling dual functionalization and access to structurally distinctive bicyclic peptides.