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Preparation method of plecanatide

A technology of plecanatide and -OH, applied in the field of peptide synthesis, can solve the problems of severe resin shrinkage, low yield of final product, difficult coupling, etc., and achieve the effect of reducing coupling difficulty, simple and feasible operation, and improving purity.

Active Publication Date: 2020-02-25
HYBIO PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These schemes all have a significant disadvantage: the difficulty of synthesizing the linear peptide of plecanatide is neglected. According to the above scheme, the linear peptide of this peptide causes serious shrinkage of the resin due to the influence of its own secondary structure, and the coupling is difficult. The purity of the linear peptide is 40-55%, the yield of the final product obtained by bonding the disulfide bond after the linear peptide synthesis is relatively low, generally 20-25%

Method used

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  • Preparation method of plecanatide
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  • Preparation method of plecanatide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1: Preparation of Fmoc-Leu-Wang Resin

[0044] Weigh 150g of Wang resin with a degree of substitution of 1.03mmol / g in a solid-phase reaction column, wash it twice with DMF and swell the resin with DMF for 30 minutes, get 37.5g Fmoc-Leu-OH, 15.6g HOBt, and 1.29gDMAP in 400ml of a mixed solution of DCM and DMF with a volume ratio of 1:1 was stirred in an ice-salt water bath. When the temperature was controlled at 0-10°C, 17.4g of DIPCDI was added dropwise and activated for 2-3 minutes. The above solution was added to a solid-phase reaction column and reacted at room temperature for 1.5 h. After the reaction was finished, wash with DMF for 4 times, then add 140 mL of acetic anhydride, 120 mL of pyridine and an appropriate amount of DMF, and mix and seal the reaction for more than 8 hours. After the reaction, wash 4 times with DMF and 2 times with DCM. After shrinking by methanol twice, the resin was drained to obtain 160.5 g of Fmoc-Leu-Wang resin, and the de...

Embodiment 2

[0045] Embodiment 2: the preparation of peptide resin

[0046] The Fmoc-Leu-Wang resin 47.6g (10mmol) of 0.21mmol / g degree of substitution prepared by weighing Example 1 is placed in a solid-phase reaction column, washed with DMF for 2 times and then used to swell the resin with DMF for 30 minutes, and then use 150ml of DBLK was deprotected for 5min+7min, washed 6 times with 150mL DMF.

[0047] Weigh 30.8g (50mmol) Fmoc-Cys(Mmt)-OH and 8.1g (60mmol) HOBt dissolved in 100mL of DMF / DCM (1:1), add 10mL (65mmol) DIPCDI under ice-water bath for 2-3min activation, Add the mixed solution into the reaction column, react at room temperature for 2 hours, and detect the end of the reaction with ninhydrin (if the resin is colorless and transparent, stop the reaction; if the resin develops color, prolong the reaction for 1 hour). After the reaction is over, wash the resin with 150mL DMF for 3 times, add 150mL DBLK for deprotection for 6min + 8min, wash the resin with 150mLDMF for 6 times,...

Embodiment 3

[0053] Example 3: Cleavage of Peptide Resins

[0054] The 78.6g (10mmol) peptide resin (1) obtained in Example 2 is all placed in the cleavage reactor, and the cleavage reagent (TFA:TIS:H 2 O=92:4:4 (V / V)), stirred at room temperature for 2h. The reactant was filtered with a sand core funnel, after the filtration was completed, a small amount of TFA was added to wash the resin, the filtrate was collected and combined, and concentrated under reduced pressure to a certain volume. Add frozen anhydrous ether (100ml / g peptide resin) to precipitate the solution, centrifuge, remove the supernatant, wash the precipitate three times with anhydrous ether, and vacuum dry to obtain 17.2g of crude peptide as a white solid powder. The purity of the crude peptide is 81.3% (HPLC testing conditions: Waters C18 300A 1.7um 2.0*100mm; buffer: 50mM ammonium phosphate buffer (phosphoric acid adjusted pH=6.2); phase A: acetonitrile: buffer = 10:90 (V: V); Phase B: buffer solution = 50:50 (V:V), gr...

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Abstract

The invention relates to a preparation method of lecanatide, which comprises the following steps: 1) sequentially coupling 16th-7th amino acids to solid-phase synthetic resin according to an Fmoc solid-phase synthesis strategy; (2) after an Fmoc protecting group is removed, treating by using a reagent for removing a Mmt protecting group, and removing the side chain protecting groups Mmt of Cys atthe 7th position and the 15th position; 3) forming a disulfide bond between Cys at the 7th position and Cys at the 15th position through a bonding reaction; 4) according to the Fmoc solid-phase synthesis strategy, sequentially coupling the 6th-1st amino acids; 5) removing the Fmoc protecting groups, and completing disulfide bonds between Cys at the 4th position and Cys at the 12th position throughan oxidation reaction, and 6) removing all side chain protecting groups, cracking resin with a cracking reagent, and precipitating with diethyl ether to obtain plecanatide. The method is simple and high in efficiency.

Description

technical field [0001] The invention belongs to the field of polypeptide synthesis, and in particular relates to a preparation method of plecanatide. Background technique [0002] Plecanatide is a guanylate cyclase C (GC-C) receptor agonist developed by Synergy Pharmaceuticals, Inc., and its trade name is Trulance. The effect is similar to that of the natriuretic peptide uroguanylin, which can induce fluid secretion into the gastrointestinal tract, thereby increasing gastrointestinal motility, thereby treating constipation. It was approved for marketing by the US Food and Drug Administration (FDA) on January 19, 2017. [0003] The English name of plecanatide is Plecanatide, and its CAS number is 467426-54-6. It consists of 16 L-type amino acid residues, of which Cys 4 、Cys 12 and Cys 7 、Cys 15 linked by disulfide bonds. Its peptide sequence is as follows: [0004] [0005] Both CN103694320 A and CN104211777 A have been authorized to apply for patents by our company...

Claims

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Application Information

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IPC IPC(8): C07K7/08C07K1/20C07K1/06C07K1/04
CPCC07K7/08C07K1/04C07K1/06C07K1/20Y02P20/55
Inventor 邹卫星宓鹏程陶安进袁建成
Owner HYBIO PHARMA
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