Chemical Synthesis of Ubiquitinated Proteins and Ubiquitin Probes for Biochemical and Functional Analysis

Protein ubiquitination is a pivotal post-translational modification that plays vital roles in eukaryotic cellular regulation. Ubiquitination is orchestrated by a cascade of enzymes: E1 activating enzymes, E2 conjugating enzymes, and E3 ligases collectively facilitate ubiquitin conjugation, while ubiquitin-binding proteins interpret the ubiquitin signal. Deubiquitinases (DUBs) counterbalance this process by removing ubiquitin from substrate proteins, thereby reversing ubiquitination. In recent years, chemical protein synthesis-enabling the preparation of proteins with atomic-level precision-has emerged as a powerful approach to investigate protein ubiquitination. This review offers a comprehensive overview of recent progress in the development of strategies for the chemical synthesis of ubiquitinated proteins and the development of ubiquitin-based probes designed for covalent capturing of ubiquitin-modifying enzymes to facilitate biochemical and biophysical studies. We begin by outlining strategies for constructing native isopeptide-linked ubiquitinated proteins, covering both total chemical synthesis and semisynthetic routes. We also summarize existing methodologies for the semisynthesis of ubiquitinated proteins containing isopeptide bond mimics. Furthermore, we highlight recent advances in the design and synthesis of ubiquitin-based probes used to study ubiquitin-conjugating enzymes, ubiquitin-interacting proteins, and deubiquitinases. Finally, we offer perspectives on future directions, including the development of synthetic strategies for efficiently accessing more complex ubiquitinated proteins and studying noncanonical ubiquitination modification.