Efficient semi-synthesis of ubiquitin-fold modifier 1 (UFM1) derivatives

Facile Preparation of UFMylation Activity-Based Probes by Chemoselective Installation of Electrophiles at the C-Terminus of Recombinant UFM1

“…27,28 Alkynes have been shown to react with deconjugating enzymes, 29,30 although for UFM1, only the propargyl group (UFM1-PA) has previously been tested for UFSP1 and UFSP2 de-UFMylases. 16 UFM1-PA was shown to be inactive with human UFSP1 and required extended incubation to engage with human UFSP2. The acetylated Cterminal UFM1 6 serves as a negative control.…”

“…α-Chloroacetyl is a commonly used motif in small-molecule cysteine covalent modifiers and undergoes S N 2 reactions with thiols. , Vinyl methyl ester (VME) is a well-known moiety for ubiquitin and Ubl pathway profiling studies, , and the related fumarate 3 should be more electrophilic than the common VMEs. Epoxides are known as cysteine-reactive groups that undergo ring-opening upon reaction with nucleophilic thiols. , Alkynes have been shown to react with deconjugating enzymes, , although for UFM1, only the propargyl group (UFM1-PA) has previously been tested for UFSP1 and UFSP2 de-UFMylases . UFM1-PA was shown to be inactive with human UFSP1 and required extended incubation to engage with human UFSP2.…”

“…The two previously reported synthetic probes are limited to dehydroalanine and propargylamine and require solid-phase peptide synthesis (SPPS), native chemical ligation (NCL), and refolding for their preparation. 16 With the aim of identifying flexible routes to more accessible probes bearing a variety of electrophilic warheads, we sought to employ direct aminolysis of the corresponding thioesters. Unfortunately, the presence of a C-terminal Val−Gly, instead of the Gly−Gly motif found on Ub and almost all other Ubls, complicates this route to electrophilic UFM1 probes.…”

“…Inspired by the work of Ovaa, Ploegh, and others on the generation of C-terminally modified Ub and Ubls bearing electrophilic warheads as powerful tools to investigate and inhibit specific conjugating and deconjugating enzymes, we sought to identify synthetic or semisynthetic UFM1-derived probes that could undergo covalent coupling with proteins involved in UFMylation. The two previously reported synthetic probes are limited to dehydroalanine and propargylamine and require solid-phase peptide synthesis (SPPS), native chemical ligation (NCL), and refolding for their preparation . With the aim of identifying flexible routes to more accessible probes bearing a variety of electrophilic warheads, we sought to employ direct aminolysis of the corresponding thioesters.…”

“…Unfortunately, the presence of a C-terminal Val–Gly, instead of the Gly–Gly motif found on Ub and almost all other Ubls, complicates this route to electrophilic UFM1 probes. Recently, two UFM1 probes have been obtained via direct aminolysis; , however, in these instances, the electrophiles were attached to the C-terminal glycine, moving the probes further away, in terms of atomic register, from the desired modification site (Figure A). Our own efforts to prepare Val C-terminally modified UFM1 with this approach resulted largely in the formation of hydrolyzed thioesters as the major product.…”