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 CuI-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.
