Efficient semi-synthesis of ubiquitin-7-amino-4-methylcoumarin

Semisynthesis of Ubiquitin and SUMO-Rhodamine 110-Glycine through Aminolysis of Boc-Protected Thioester Counterparts

“…However, this method is limited by the N -hydroxysuccinimide activation, and the bisglycyl-rhodamine 110 aminolysis process takes 5 days, which results in the time-consuming and low yield for obtaining Ub-Rho110-G (estimated to be 0.75–1 mg Ub-Rho110-G from 1 L of LB culture) (Scheme b). Recently, we reported a newly efficient semisynthetic strategy to prepare Ub-AMC, which takes advantage of direct aminolysis of Ub-thioester in a mixed-solvent system . In a typical experiment starting with the expression of the Ub­(1–76C) mutant from 1 L of LB medium, we could generate 4–5 mg of Ub-AMC.…”

“…Recently, we reported a newly efficient semisynthetic strategy to prepare Ub-AMC, which takes advantage of direct aminolysis of Ub-thioester in a mixedsolvent system. 14 In a typical experiment starting with the expression of the Ub(1−76C) mutant from 1 L of LB medium, we could generate 4−5 mg of Ub-AMC. Considering the similar reactivity of AMC and Rho110, we aim to use the aminolysis-based semisynthetic strategy to obtain Ub-Rho110-G through an easy-to-implement manner.…”

“…17 With bisglycyl-rhodamine 110 and Ub(1−75)-Mesna in hand, we first tried to synthesize Ub-Rho110-G according to our direct aminolysis protocol (Figure 1a). 14 Thus, Ub(1−75)-Mesna (1 mM) and bisglycyl-rhodamine 110 (5 mM) were dissolved in a mixed solvent (6:1 v/v DMSO/6.0 M Gn-HCl, 1.0 M HEPES, pH 8.5, 2% v/v thiophenol). The reaction mixture was stirred at 37 °C for 6−8 h, followed by monitoring with HPLC.…”

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

“…Due to the poor solubility of Gly‐AMC in the aqueous solution, [16] the synthesis of UFM1‐AMC was conducted in two steps, in which UFM1 Val82 ‐thioester must be purified for subsequent aminolysis (Figure 5a). Using a similar protocol as the synthesis of Ub‐AMC, [18] we initially prepared thioester UFM1 Val82 ‐MesNa ( 6 ) from hydrazide 4 by MesNa thiolysis (Figure 5a). Then, we dissolved the purified 6 in a mixed solvent of DMSO and Gdn⋅HCl buffer (6.0 M Gdn⋅HCl, 1.0 M HEPES, pH 8.5) in a volume ratio of 6 : 1 and initiated the reaction by adding sodium thiophenolate to a concentration of 0.2 M and excess Gly‐AMC (30 equiv.).…”

Expedient Synthesis of Ubiquitin‐like Protein ISG15 Tools through Chemo‐Enzymatic Ligation Catalyzed by a Viral Protease Lbpro

“…Lb pro has cross activities with Ub, while a higher Lb pro concentration and a longer incubation time are required compared to ISG15 [9, 10] . Given its unique cleavage site, we wondered whether the Lb pro ligation strategy could also be used to synthesize Ub‐based protein tools (e.g., Ub‐PA, Ub‐AMC) (Figures 4e, S8c), which have previously been prepared via aminolysis of an Ub thioester made via either intein‐based technology or protein hydrozinolysis [7b, 25] —a slow process taking days and yielding less than 40 % for both tools. We were delighted that incubation of recombinant Ub (5 mM) and Gly‐PA (500 mM) with Lb pro (40 μM) in HEPES buffer (pH 8.0) at room temperature yielded Ub‐PA within 6 h in an HPLC yield of 90 % (Figures 4f, S8e), again emphasizing the vastly improved speed of Lb pro ‐promoted ligation compared with thioester aminolysis.…”