A Stable Bioisostere of Ester‐Linked Ubiquitin Chains Enables Decoding of Protein Interactors

Protein ubiquitination is a pivotal posttranslational modification that regulates diverse biological processes depending on the type of ubiquitin chain linkage. Recently, ester-linked ubiquitin chains have been identified, yet their inherent hydrolytic instability has posed a significant challenge for biochemical investigations. In this study, a stable and isosteric amide analog of an ester-linked ubiquitin dimer, is chemically synthesized in which serine (Ser) at position 20 of the proximal ubiquitin is replaced with 2,3-diaminopropionic acid (Dap). The desired amide analog is synthesized using a convergent approach involving the sequential chemoselective ligation of three peptide fragments generated through Fmoc-based solid-phase peptide synthesis. Employing this chemically robust ubiquitin probe, a previously unrecognized interaction is uncovered between Ser20-linked ubiquitin chains and spliceosome-associated factors, notably ubiquitin-specific protease 39. These findings highlight the potential of the ester-to-amide bioisosteric strategy to unlock mechanistic insights into atypical ubiquitin modifications. The approach not only circumvents the intrinsic instability of ester-linked ubiquitin chains but also provides a broadly applicable framework for dissecting their biological roles, paving the way for future discoveries in ubiquitin signaling.