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A plecanatide and solid-phase synthesis technology, which is applied in the field of solid-phase synthesis of plecanatide, can solve problems such as inability to accurately position, achieve the effects of guaranteed yield, real-time controllable cyclization process, and improved efficiency
Inactive Publication Date: 2018-08-24
HANGZHOU GOTOP BIOTECH
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[0006] The purpose of the present invention is to solve the problems in the background technology, to provide a solid-phase synthesis method of plecanatide, which can overcome the p
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Embodiment 1
[0024] Embodiment 1: a kind of solid phase synthesis method of plecanatide, comprises the following steps:
[0025] (1) Use catalytic esterification conditions to serially load Fmoc-Leu-OH onto WANG resin;
[0026] (2) According to the plecanatide sequence, use a condensation reagent to sequentially couple and protect amino acids from the C-terminal to the N-terminal, wherein the plecanatide sequence sequence is: Asn(trt)16-Asp(tbu)15-Glu(otbu) 14-Cys(trt)13-Glu(otbu)12-Leu11-Cys(A cm)10-Val9-Asn(trt)8-VaL7-Ala6-Cys(trt)5-Thr(tbu)4-Gly3-Cys (Acm)2-Le u1-wang resin;
[0027] (3) Cleavage the polypeptide from the resin with a TFA cleavage reagent to obtain free polypeptides with Acm protection at the 2 and 10 positions, in which the sulfhydryl groups of the other pair (5, 13 positions) Cys have been exposed; under alkaline conditions, Use 30% hydrogen peroxide / hydrogen peroxide with a reaction solution volume of 0.5ml / g-1ml / g to oxidize the pair of sulfhydryl groups to obtain ...
Embodiment 2
[0038] Embodiment 2: Synthesis method of plecanatide linear chain crude peptide
[0039] 1. Preparation of resin: Fmoc-Leu-Wang Resin 6mmol with a substitution degree (loading) of 0.35
[0040] Accurately weigh 26.5g of Fmoc-Leu-OH, 10.1g of Hobt, and 0.92g of Dmap into the Erlenmeyer flask, and dissolve with a small amount of DMF; then weigh 85.7g of Wang resin with an original Loading of 0.96 into the Erlenmeyer flask, and adjust to an appropriate reaction with DMF Concentration, add 11.6ML of DIC, temperature control 28-30 ℃, shaker reaction 3h. Remove the solution with a sand core reaction column, wash with DMF, DCM, and DMF twice respectively, add 155.6ml of acetic anhydride and 132.4ml of pyridine, use DMF as the reaction solvent, and seal at a temperature of 28-30°C for 5 hours. After the sealing is completed, the solution is removed, washed three times with DMF, DCM, and methanol in turn, and dried for later use.
[0041] The resin substitution degree measured by ult...
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Abstract
The invention relates to a solid phase synthetic method of plecanatide. The method comprises the following steps: splitting off polypeptide from resin by using a TFA (trans fatty acid) splitting agentto obtain Acm-protected free peptides at sites 2 and 10, wherein another pair of thiols of (5, 13 sites) Cys is exposed; under an alkaline condition, oxidizing the pair of thiols by using 30 percentof hydrogen peroxide/hydrogen peroxide of which the volume of a reaction solution is 0.5ml/g to 1ml/g so as to obtain monocyclic plecanatide with a pair of 5-13 disulfide bonds. The formation of the rest pair of disulfide bonds comprises the following steps: slowly dripping an iodine solution with certain concentration into a solution under an acidic condition; carrying out reaction for 3 hours; forming the pair of 2-10 disulfide bonds while Acm removal is carried out at the same time; adding ascorbic acid after the reaction is ended, and then removing an excessive amount of iodine; filtering,purifying and freeze-drying the reaction solution to obtain the powdery plecanatide. According to the application, a cyclizing route and conditions are optimized, accurate positioning can be achievedwhile the yield is ensured at the same time, the steps are simple, a cyclization process is controllable in real time, and final products are not needed to wait for confirming right or wrong, so thatthe efficiency is effectively improved.
Description
technical field [0001] The invention relates to a preparation method of polypeptide drugs, in particular to a solid-phase synthesis method of plecanatide, which can accurately locate the cyclized plecanatide. Background technique [0002] Plecanatide is a cyclic polypeptide composed of 16 amino acids, which can regulate the acid-base ions in the gastrointestinal tract, increase the peristalsis of the gastrointestinal tract, and is suitable for the treatment of chronic idiopathic constipation in adults. The U.S. Food and Drug Administration (FDA) approved the listing on January 19, 2017, under the trade name Trulance. [0003] Plecanatide sequence: H-Asn-Asp-Glu-Cys(13)-Glu-Leu-Cys(10)-Val-Asn-Val-Ala-Cys(5)-Thr-Gly-Cys(2)- Leu-OH [0004] Structure diagram: [0005] At present, plecanatide is obtained through biological extraction technology. The biological extraction is affected by the existing limited biological resources. The extraction process is cumbersome, the eff...
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