DFT-Guided Discovery of Ethynyl-Triazolyl-Phosphinates as Modular Electrophiles for Chemoselective Cysteine Bioconjugation and Profiling.

We report the density functional theory (DFT) guided discovery of ethynyl-triazolyl-phosphinates (ETPs) as a new class of electrophilic warheads for cysteine selective bioconjugation. By using Cu I -catalysed azide alkyne cycloaddition (CuAAC) in aqueous buffer, we were able to access a variety of functional electrophilic building blocks, including proteins, from diethynyl-phosphinate. ETP-reagents were used to obtain fluorescent peptide-conjugates for receptor labelling on live cells and a stable and a biologically active antibody-drug-conjugate. Moreover, we were able to incorporate ETP-electrophiles into an azide-containing ubiquitin under native conditions and demonstrate their potential in protein-protein conjugation. Finally, we showcase the excellent cysteine-selectivity of this new class of electrophile in mass spectrometry based, proteome-wide cysteine profiling, underscoring the applicability in homogeneous bioconjugation strategies to connect two complex biomolecules.