On-resin assembly of cysteine-reactive linkers for controlled site-selective antibody bioconjugation.

Antibody conjugates represent key advances in targeted biotherapeutics, combining the precision of antibodies with a range of functional payloads, including cytotoxic small molecules, enzymes and peptides, to achieve high selectivity, reduced off-target effects and an improved therapeutic window compared with conventional small-molecule drugs. Here we present a protocol for the solid-phase synthesis of tetra-divinylpyrimidine (tetraDVP) linkers, a modular and scalable strategy for generating cysteine-reactive linkers used in site-selective bioconjugation, enabling the rapid production of diverse antibody conjugates. This protocol describes a multistep synthetic workflow involving solution-phase intermediate synthesis, solid-phase assembly on resin, polyethylene glycol elongation, installation of divinylpyrimidine warheads, and mild cleavage conditions, enabling reproducible production of the final linker scaffold. TetraDVP linkers are designed to simultaneously rebridge all four interchain disulfide bonds of native IgG1 and IgG4 antibodies with a single molecule, enabling the controlled installation of functional payloads such as peptides, drugs or protein tags with controlled payload-to-antibody ratio. This approach provides the only reported strategy for conjugating a single payload to a native antibody without the need for chromatographic purification or genetic/glycan engineering. Compared with previous solution-phase routes, this solid-phase protocol improves yield, scalability and reproducibility while enabling rapid diversification of linker architecture. The complete procedure can be performed in ~2 weeks and provides a versatile platform for accessing tetraDVP linkers bearing a variety of functional handles for antibody conjugation.