Chemoselective placement of unsaturated phosphorus electrophiles into ubiquitin for proximity-induced protein targeting

Mildly reactive electrophiles have emerged as powerful functional groups for developing chemical probes to target pharmacologically important proteins and study key biological mechanisms. With the current manuscript, we introduce unsaturated N-alkyl-phosphonamidates as a new and powerful class of chemical warheads that enable proximity-induced labeling and the specific targeting of protein-protein interactions. In contrast to many other electrophilic warheads, these cysteine-selective electrophiles can be incorporated into specific amino acid side on the protein level by a chemoselective Staudinger-phosphonite reaction using unsaturated phosphonites as chemical reactants with azido-containing ubiquitins obtained by auxotrophic expression. Following this protocol, we prepared several ethynyl-N-alkyl-phosphonamidate-containing ubiquitins, which undergo proximity-induced labeling with deubiquitinases (DUBs) and other ubiquitin-interacting proteins, even in the presence of high thiol concentrations and cell lysates. Furthermore, we demonstrate that changing the position of the electrophile allows selective targeting, enrichment and distinct functional regulation of DUBs via covalent binding to individual distinct ubiquitin binding pockets as highlighted in the functional regulation of USP5, a DUB that contains multiple ubiquitin binding sites. Our study highlights the prospect of targeting proteinprotein interactions with electrophilic protein-based probes by simultaneously enlarging the repertoire of chemical probes beyond purely synthetically derived warheads.