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

## TL;DR

Researchers created a stable version of an unstable ubiquitin chain to study its interactions with proteins, revealing new insights into ubiquitin signaling.

## Contribution

A stable amide analog of ester-linked ubiquitin chains was synthesized to overcome their hydrolytic instability for biochemical studies.

## Key findings

- A stable amide analog of ester-linked ubiquitin was chemically synthesized using a convergent approach.
- The probe revealed an interaction between Ser20-linked ubiquitin chains and spliceosome-associated factors like ubiquitin-specific protease 39.
- The ester-to-amide strategy provides a framework for studying atypical ubiquitin modifications.

## Abstract

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.

An ester‐to‐amide analog of an ester‐linked ubiquitin dimer is chemically synthesized through a convergent approach involving the sequential chemoselective ligation of three peptide fragments generated through Fmoc‐based solid‐phase peptide synthesis. The hydrolysis‐resistant ubiquitin dimer served as a chemical probe in the identification of potential reader of the ester‐linked ubiquitin chain.© 2026 WILEY‐VCH GmbH