Unsaturated Phosphine Oxides for Modular Antibody Rebridging and Single Reagent Peptide‐Cyclization‐Bioconjugation

Abstract Achieving modular, selective and homogeneous protein modifications is of utmost importance for the design of next generation biopharmaceuticals, especially in the context of antibody‐drug conjugates (ADCs). Here, we introduce unsaturated phosphine oxides as versatile triple‐reactive reagents, allowing orthogonal chemoselective bioconjugation schemes. Starting from triethynyl‐phosphine oxide, a variety of functionalized diethynyl‐triazolyl‐phosphine oxides (DTPOs) could be accessed by using Cu I ‐catalyzed azide‐alkyne cycloaddition (CuAAC). We showcase DTPO‐reagents in the fast and selective generation of various highly stable antibody‐conjugates via antibody disulfide rebridging. A highlight from this methodology is the synthesis of a DAR 4 ADC following a modular 2‐step strategy using bioorthogonal tetrazine‐labeling with bicyclo‐[6.1.0]non‐4‐yne (BCN) or trans ‐cyclooctene (TCO) containing payloads. Notably, the DTPO‐rebridged ADC exhibited potent cytotoxicity against Her2 + cancer cells. Moreover, we utilize triethynyl‐phosphine oxide to obtain ethynyl‐ditriazolyl‐phosphine oxides (EDPOs) which enable a unique, single‐reagent peptide‐cyclization‐bioconjugation protocol resulting in functional cyclic peptide‐protein conjugates. Overall, our work provides versatile and powerful chemoselective modalities for the controlled modification of antibodies, peptide‐cyclization and peptide‐protein conjugation, expanding the toolkit for chemical biology and therapeutic development.