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 CuI‐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. By means of density functional theory calculations, ethynyl‐triazolyl‐phosphinates (ETPs) were discovered as modular and cysteine‐selective electrophiles for bioconjugation. Using CuI‐click chemistry in aqueous buffers, this functional group can be easily introduced into azide‐containing (bio‐)molecules. These reagents can be used for proteome‐wide cysteine profiling and to obtain functional peptide‐ and protein conjugates, as well as protein–protein conjugates .