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Abstract Target validation is key to the development of protein degrading molecules such as proteolysis‐targeting chimeras (PROTACs) to identify cellular proteins amenable for induced degradation by the ubiquitin‐proteasome system (UPS). Previously the HaloPROTAC system was developed to screen targets of PROTACs by linking the chlorohexyl group with the ligands of E3 ubiquitin ligases VHL and cIAP1 to recruit target proteins fused to the HaloTag for E3‐catalyzed ubiquitination. Reported here are HaloPROTACs that engage the cereblon (CRBN) E3 to ubiquitinate and degrade HaloTagged proteins. A focused library of CRBN‐pairing HaloPROTACs was synthesized and screened to identify efficient degraders of EGFP‐HaloTag fusion with higher activities than VHL‐engaging HaloPROTACs at sub‐micromolar concentrations of the compound. The CRBN‐engaging HaloPROTACs broadens the scope of the E3 ubiquitin ligases that can be utilized to screen suitable targets for induced protein degradation in the cell. 

Abstract Di‐ubiquitin (diUB) conjugates of defined linkages are useful tools for probing the functions of UB ligases, UB‐binding proteins and deubiquitinating enzymes (DUBs) in coding, decoding and editing the signals carried by the UB chains. Here we developed an efficient method for linkage‐specific synthesis of diUB probes based on the incorporation of the unnatural amino acid (UAA)N^ϵ‐L‐thiaprolyl‐L‐Lys (L‐ThzK) into UB for ligation with another UB at a defined Lys position. The diUB formed by the UAA‐mediated ligation reaction has a G76C mutation on the side of donor UB for conjugation with E2 and E3 enzymes or undergoing dethiolation to generate a covalent trap for DUBs. The development of UAA mutagenesis for diUB synthesis provides an easy route for preparing linkage‐specific UB‐based probes to decipher the biological signals mediated by protein ubiquitination.