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