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The development of powerful and general methods to acquire ubiquitin (Ub) chains has prompted the deciphering of Ub‐mediated processes. Herein, the cysteine‐aminoethylation assisted chemical ubiquitination (CAACU) strategy is extended and improved to enable the efficient semi‐synthesis of atypical Ub chain analogues and Ub‐based probes. 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 a 9–20 mg L−1 expression level. In addition, the utility of this strategy is demonstrated with the synthesis of a novel non‐hydrolyzable di‐Ub PA probe, which may provide a new useful tool for the mechanistic studies of deubiquitinase (DUB) recognition. [ABSTRACT FROM AUTHOR]

Native chemical ligation combined with desulfurization has become a powerful strategy for the chemical synthesis of proteins. Here we describe the use of a new thiol additive, methyl thioglycolate, to accomplish one-pot native chemical ligation and metal-free desulfurization for chemical protein synthesis. This one-pot strategy was used to prepare ubiquitin from two or three peptide segments. Circular dichroism spectroscopy and racemic protein X-ray crystallography confirmed the correct folding of ubiquitin. Our results demonstrate that proteins synthesized chemically by streamlined 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase peptide synthesis coupled with a one-pot ligation-desulfurization strategy can supply useful molecules with sufficient purity for crystallographic studies. [ABSTRACT FROM AUTHOR]