Pyridine-based platform enables stable, non-chiral Cys bioconjugation for ADCs and peptide diversification

Site-selective cysteine (Cys) bioconjugation is crucial for developing advanced therapeutics like antibody-drug conjugates (ADCs) and functionalized peptides. Current strategies often face significant challenges, including reversible linkages, undesirable stereochemical heterogeneity, and limited modularity, which can compromise conjugate stability and therapeutic efficacy. Overcoming these limitations is essential for achieving precise control over bioconjugate structure and function, ensuring optimal drug delivery and reduced off-target effects in complex biological systems.

Researchers developed a programmable pyridine-based chemoselective sequential conjugation platform that integrates a thianthrenium leaving group and a fluorine handle on a single pyridine scaffold. This design enables sequential, orthogonal coupling of alkylamines and Cys residues to form non-chiral, stable aryl-Cys linkages. The platform's capabilities were demonstrated through late-stage peptide diversification and stapling, protein functionalization, and the streamlined construction of a HER2-targeting ADC (MRG002) analog with a tunable drug-to-antibody ratio. The ADC analog's cytotoxicity was assessed in HER2-positive cells and antigen-negative controls, and its payload retention was evaluated in human serum.

The pyridine-based platform exhibited exceptional functional group compatibility and high chemoselectivity in both the alkylamine- and thiol-conjugation steps. This robust reactivity allowed for efficient late-stage peptide diversification and stapling, as well as protein functionalization. The platform successfully facilitated the streamlined construction of a HER2-targeting ADC (MRG002) analog, demonstrating a tunable drug-to-antibody ratio. The resulting ADC analog showed slightly enhanced cytotoxicity in HER2-positive cells, with comparable activity observed in antigen-negative controls. Most notably, the ADC analog demonstrated markedly improved payload retention in human serum, indicating enhanced stability.

The platform enables the formation of non-chiral, stable aryl-Cys linkages, addressing key limitations of existing bioconjugation methods.