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Efficient Semi-synthesis of Atypical Ubiquitin Chains and Ubiquitin-based probe Forged by thioether isopeptide bond.

The development of powerful and general methods to acquire Ub chains has prompted the deciphering of Ub-mediated processes. Herein, we extended and improved the cysteine-aminoethylation assisted chemical ubiquitination (CAACU) strategy to efficient semi-synthesis of atypical Ub chain analogs and Ub-based probe. Combining the Cys aminoethylation and the auxiliary-mediated protein ligation, several linkage- and length-defined atypical Ub chains including di-Ubs, K27C-linked tri-Ub, K11/K48C-branched tri-Ub and even the SUMOlated Ub are successfully prepared from recombinantly expressed starting materials at about 9-20 mg/L expression level. In addition, the utility of this strategy is demonstrated to the synthesis of a novel non-hydrolyzable di-Ub PA probe which may provide a new useful tool for the mechanistic studies of DUBs recognition.