A novel prosthetic linking arm for the synthesis of diubiquitin and its synthesis method and application
A synthetic method and linker technology, applied in the field of protein synthesis, can solve problems such as limitations of synthetic strategies
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Embodiment 1
[0053] 1. Add 2-oxotetrahydro-1,3-oxazole (17.42g, 0.2mol) into a round bottom flask, add 200ml of water and stir, add bromine (31.96g, 0.2mol) after stirring for 25 minutes, and At the same time, 6M NaOH solution was added to adjust the pH value of the solution to 8.6. After reacting for 10 minutes, the precipitate was filtered out, washed with water, and dried under vacuum at room temperature. The aqueous layer was extracted with dichloromethane, the extract was dried, evaporated to dryness, and the product 3-bromo -2-Oxotetrahydro-1,3-oxazole (21.58 g, yield 65%).
[0054] 2. Add 3-bromo-2-oxotetrahydro-1,3-oxazole (16.6g, 0.1mol) to 2,2'-dicyano-2,2'-azirane (0.821g, 5mmol) 100mL of benzene suspension. A solution of freshly distilled styrene (10.4 g, 0.1 mol) in benzene (20 mL) was then added over 5 minutes at 60°C. After stirring for 5 minutes, the mixture was evaporated and the residue was stirred with petroleum ether (50 mL), filtered and then dried to give compound 1...
Embodiment 2
[0058] 110 mg of lyophilized UbK27C powder (final concentration: 10 mg / ml) was dissolved in 12.32 mL of degassed alkylation buffer (6M guanidine hydrochloride, 1M HEPES, 10 mM D / L-methionine, pH=8.5) and 27.8 mg of TCEP was added. Ubiquitin was reduced for 20 min at room temperature. Then 332 mg 2-((acetylaminomethyl)thio)-N-(2-bromoethyl)-2-phenylethyl-1-ammonium (dissolved in DMF; final concentration 20 mM) was added and the reaction pH was reset. Adjust to 8.5. After stirring at room temperature for 5 hours, an additional 18.5 mg of TCEP and 249 mg of 2-((acetamidomethyl)thio)-N-(2-bromoethyl)-2-phenylethan-1-ammonium were added to the mixture (20mM). And the reaction was allowed to proceed for another 5 hours at room temperature. Subsequently, the reaction was quenched with 2.5-3.75 mg excess DTT (final concentration 150-200 mM) and purified by semi-preparative RP-HPLC to give the alkylated product (UbK27C+Aux) (90.6 mg, 80% yield ).
Embodiment 3
[0060] First configure the reaction buffer (6M Gn-HCl, 0.2M Na 2 HPO 4 , pH=7.2 (ultrasonic degassing, through membrane)), 43mg freeze-dried protein sample in Example 4 was dissolved with 5mL reaction buffer, and 43mgPdCl was added 2 (5equiv)(PdCl 2 Ultrasonic dissolution with reaction buffer), adjust pH = 7.2, monitor the reaction, about 20minAcm group can be completely removed (the unreacted raw material in step 1 has a displacement with the Acm removal product, and can be separated).
[0061] After the reaction was completed, 45 mg of excess DTT (60 equiv) was added to quench the reaction to make it fully complexed with Pd. After the precipitation was complete, it was separated by high-speed centrifugation, and then half-prepared for separation after passing through the membrane. The purified solution was collected and lyophilized to obtain the target product 36.25g Ub(1-76)K27C+Aux-Acm removed. Yield 85%.
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