Modular Assembly of Bioconjugates Enabled by a Pyridine‐Based Chemoselective Sequential Conjugation Platform

Site‐selective Cys bioconjugation is central to chemical biology and therapeutic development. Owing to the widespread availability of amine‐containing building blocks, the modular assembly of alkylamines and Cys holds significant promise for diverse applications. However, existing strategies often suffer from reversible linkages, stereochemical heterogeneity, or limited modularity. Here we report a programmable pyridine‐based chemoselective sequential conjugation platform that integrates a thianthrenium leaving group and a fluorine handle on a single pyridine scaffold, enabling sequential, orthogonal coupling of alkylamines and Cys to form non‐chiral, stable aryl‐Cys linkages. The reaction exhibits exceptional functional group compatibility and high chemoselectivity in both the alkylamine‐ and thiol‐conjugation steps. The platform supports late‐stage peptide diversification and stapling, protein functionalization, and streamlined construction of a HER2‐targeting ADC (MRG002) analog with a tunable drug‐to‐antibody ratio. The resulting ADC shows slightly enhanced cytotoxicity in HER2‐positive cells, comparable activity in antigen‐negative controls, and markedly improved payload retention in human serum.