Semi-synthesis of disulfide-linked branched tri-ubiquitin mimics

Ubiquitin (Ub) chain isopeptide bond mimics are useful molecules for biochemical and biophysical studies. Herein, we report the semi-synthesis of the disulfide-linked K11/K48-branched tri-Ub (Ub311/48(S–S)), the first example of an isopeptide mimic for the branched Ub chains, which have recently emerged as an interesting category of Ub modifications. Our strategy comprised the E1-dependent synthesis of the Ub conjugate of aminoethanethiol, followed by disulfide formation with Ub(K11C, K48C). The structure of the synthetic isopeptide bond mimics was verified by the crystal structure of Ub311/48(S–S). Deubiquitination and pulldown assays indicated that the synthetic Ub311/48(S–S) could be hydrolyzed by linkage-specific deubiquitinases (K11-specific Cezanne and K48-specific OTUB1), and recognized by proteasomal ubiquitin receptor S5a.