Switchable pathways for precise carboxylic acid modification in proteins and antibodies

Summary Historically, organic chemistry has been biased toward irreversible transformations. However, they have struggled to meet the selectivity demands from recent biopharma growth. Here, we establish the potential of reversible reactions within a multi-equilibria system from a multicomponent reaction (MCR), offering three switchable pathways for selective carboxylate modification in proteins and antibodies. The classical Ugi reaction yields heterogeneous mixtures, lysine being a major competitor. To address this, we developed a four-component reaction to render a four-component product (4CR-4CP). The 4CR-4CP can be disrupted and switched to 4CR-2CP or 3CR-2CP (>36 examples). Pathway exclusivity and carboxylate selectivity remain intact even at >99% bioconjugation. The method enables single- or dual-site probe installation and E21-labeling of insulin without compromising receptor binding or downstream signaling. The strategy extends to monoclonal antibodies, generating carboxylate-selective antibody-fluorophore and drug conjugates (AFCs and ADCs) for antigen-specific labeling and antiproliferative activity, opening a unique platform for multi-equilibria driven protein modification.