Preparation of UFM1-Derived Probes through Highly Optimized Total Chemical Synthesis**

Graphical A total chemical synthesis strategy utilizing UFM1 C-terminal valine hydrazide as a versatile handle is reported for the facile preparation of UFM1-derived probes. This approach allows the convenient installation of electrophilic warheads, fluorescent reporters and unnatural purification tags onto the UFM1 sequence. Ufmylation is involved in various cellular processes and associated with many human diseases. The understanding of this modification relies on the use of customized UFM1-derived probes for activity-based profiling of its related enzymes. This study presents a highly optimized total chemical synthesis for the generation of diverse UFM1-derived probes including UFM1-PA, Biotin-UFM1-PA and UFM1-AMC, in which a UFM1 C-terminal valine hydrazide was readily prepared by hydrazide-based ligation and used as a versatile handle for the installation of enzyme-sensitive warheads and fluorescent reporters. The resulting probes display high reactivity and selectivity for UFM1-specific enzymes in cell lysates. This strategy facilitates the generation and diversity of the UFM1-derived toolkit that can be employed to profile UFM1-specific enzymes, thereby shining insights into the dynamics of ufmylation.