Synthetic Strategies for Activity‐Based Probes to Decode Ubiquitin‐Like Modifiers

Ubiquitin-like proteins (Ubls) such as SUMO, NEDD8, ISG15, URM1, UFM1, FAT10, ATG8/ATG12, and FUBI are essential regulators of cellular homeostasis, controlling processes from protein stability and trafficking to immune signaling and autophagy. Their conjugation-deconjugation cycles are mediated by cascades of E1, E2, and E3 enzymes and reversed by Ubl-specific proteases (ULPs), many of which are cysteine-dependent. Deciphering these dynamic and reversible pathways requires tools that directly capture the active forms of these enzymes. Activity-based probes (ABPs) have become indispensable for this task, providing covalent, mechanism-based snapshots of enzymatic activity in complex systems. This review highlights chemistry-centric strategies for the design and synthesis of Ubl-targeting ABPs. We summarize synthetic and semisynthetic approaches that install electrophilic warheads onto Ubl backbones, methods for C-terminal ligation (native chemical ligation, activated cysteine ligation, hydrazide chemistry), and strategies for incorporating reporter tags or bioorthogonal handles. Probe development is organized by target class, including Ubl isopeptidases, E1/E2 conjugating enzymes, and E3 ligases. Representative examples illustrate how chemical design choices are tailored for specific applications-ranging from live-cell activity profiling to proteomic mapping and inhibitor discovery. Together, these methodologies establish a versatile chemical toolkit for dissecting Ubl biology, enabling the discovery of novel enzymes, the mapping of substrate networks, and the development of potential therapeutic modulators.