Bis‐ethynylphosphonamidates as an Modular Conjugation Platform to Generate Multi‐Functional Protein‐ and Antibody‐Drug‐Conjugates

Abstract Bis‐ethynylphosphonamidates allow for a simple chemoselective addition of two thiol‐containing modules in row. We describe four such bis‐electrophiles that carry different functional O‐substituents with tunable hydrophilicity and enable further subsequent conjugations, thus facilitating protocol constructing protein‐protein conjugates. An increased spacer length between the ethynylphosphonamidates simplifies formation conjugate from bulky proteins. apply these reagents to obtain homogeneous Antibody‐Drug‐Conjugates (ADCs) DM1 trastuzumab excellent cytotoxicity selectivity targeted cell line. Moreover, bis‐ethynylphosphonamidate, carrying an additional alkyne triple conjugation, has been subjected fluorescent labeling ADC specifically at drug site give Antibody‐Drug‐Fluorophore‐Conjugate (ADFC), allowing observation intracellular trafficking after uptake into cell.