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Continuous flow solid-phase reaction preparation of semaglutide

A technology of fluid-solid and condensation reaction, which is applied in the field of preparation of semaglutide combined with continuous flow solid-phase synthesis system, which can solve the problems of low total yield, low efficiency and good solubility of crude products

Active Publication Date: 2020-10-02
苏州金顶生物有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method also needs to prepare short peptides by solid-phase synthesis, which cannot fundamentally improve the production capacity of semaglutide. Secondly, it will be a very cumbersome task to separate small fragments, condensation reagents and impurities from the main fragment during liquid-phase synthesis, especially The polypeptide fragments have good solubility and cannot be mostly removed by adjusting the acid-base of the solution like protecting amino acids. The purification work is difficult and the actual efficiency is very low.
[0013] Usually, when the number of amino acids is greater than 10, the average incomplete rate of polypeptide coupling per step reaches 1%, and when the number of amino acids is greater than 20, the average rate of incomplete polypeptide coupling per step reaches 2% ("Solid Phase Organic Synthesis. Principles and Application Guide" Wang Dexin), the synthesis of semaglutide by step-by-step coupling method will inevitably result in the total yield of crude product being less than 50%, which is not advisable for industrial production
But too much pursuit of the fragment method, the material cost of the fragment is higher than that of a single amino acid coupling, and the efficiency is much lower
At the same time, fragment coupling exists for a long time, and the coupling is not easy to complete

Method used

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  • Continuous flow solid-phase reaction preparation of semaglutide
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  • Continuous flow solid-phase reaction preparation of semaglutide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0107] Step 1: Preparation of Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Psi(me.me)Pro)-OH Fragment 1

[0108]

[0109] 1) Fmoc-Val-Ser(Psi(me.me)Pro)-2-CTC-resin

[0110] 10g of 2-CTC-resin (substitution degree=0.72mmol / g) was added to the solid phase reactor, the resin was swollen with 120mL of dichloromethane for 5 minutes, and then Fmoc-Val-Ser(Psi(me.me)Pro) was added sequentially -OH 1g (2.16mmol), 10.2mL DIEA, stirred for 2 hours. Drain the solvent, add 100mL of 10:90 methanol / dichloromethane solution, stir for half an hour to block unreacted complete chloromethyl. Wash 3 times with 100 mL of dichloromethane each time. After vacuum drying, the substitution degree of Fmoc-Val-Ser(Psi(me.me)Pro)-2-CTC-resin was measured to be 0.61 mmol / g.

[0111] Fmoc-Val-Ser(Psi(me.me)Pro)-2-CTC-ChemMatrix resin was removed with 100mL 2%DBU / 3%1-octylthiol DMF solution for 12 minutes at room temperature, and H- Val-Ser(Psi(me.me)Pro)-2-CTC-resin.

[0112] 2) Fmoc-Thr(t...

Embodiment 2

[0146] Step 1: Fmoc-Gly-HMPA-ChemMatrix resin synthesis

[0147]

[0148] Weigh 15g of HMPA-ChemMatrix resin (degree of substitution = 0.53mmol / g) and add it to a solid-phase reactor, swell the resin with 100mL of dichloromethane for 5 minutes, then add 6.7g of Fmoc-Gly-OH, 8.2mL of DIEA, and stir for 2 hours . Drain the solvent, add 100mL of 10:90 methanol / dichloromethane solution, stir for half an hour to block unreacted complete chloromethyl. Wash 3 times with 100 mL of dichloromethane each time. After vacuum drying, the degree of substitution of the Fmoc-Gly-HMPA-ChemMatrix resin was measured to be 0.47 mmol / g.

[0149] Step 2: H-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(AEEA-AEEA-(γ-Glu-(OtBu))-monoButyl Octadecanate)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly-HMPA-ChemMatrix resin 3 synthesis

[0150] The Fmoc-Gly-HMPA-ChemMatrix resin synthesized in step 1 was used for semaglutide resin synthesis. Weigh 80 g of Fmoc-Gly-HMPA-...

Embodiment 3

[0166] Step 1: Fmoc-Gly-Trt-ThioPEG-AM-PS resin synthesis

[0167]

[0168] Weigh 15g of Cl-Trt-ThioPEG-AM-PS resin (degree of substitution = 0.39mmol / g) and add it to a solid-phase reactor, swell the resin with 100mL of dichloromethane for 5 minutes, then add 5.2g of Fmoc-Gly-OH in sequence, 7.4 mL DIEA, stirred for 3 hours. Drain the solvent, add 100mL of 10:90 methanol / dichloromethane solution, and stir for half an hour to block unreacted triphenylchloromethyl. Wash 3 times with 100 mL of dichloromethane each time. After vacuum drying, the degree of substitution of the Fmoc-Gly-Trt-ThioPEG-AM-PS resin was measured to be 0.32 mmol / g.

[0169] Step 2: H-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(AEEA-AEEA-(γ-Glu-(OtBu))-monoButyl Octadecanate)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Boc) 2 -Gly-Arg(Boc) 2 -Gly-Trt-ThioPEG-AM-PS resin 3 synthesis

[0170] The Fmoc-Gly-Trt-ThioPEG-AM-PS resin synthesized in step 1 was used for semaglutide resin synthes...

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Abstract

The invention discloses a method for preparing semaglutide by combining a continuous flow solid-phase synthesis system. The method comprises the following steps of firstly, introducing Octadecanedioic(OtBu) acid-gamma-Glu-(OtBu)-AEEA-AEEA into a side chain of Lys26, and then completing a strategy of a main chain, wherein Fmoc-Gly-resin is adopted as a main chain structure, and continuous flow solid-phase synthesis is adopted for polypeptide fragment and semaglutide peptide chain growth.

Description

technical field [0001] The invention belongs to the field of polypeptide drug synthesis, and in particular relates to a method for preparing semaglutide combined with a continuous-flow solid-phase synthesis system. Background technique [0002] Semaglutide is a long-acting GLP-1 receptor agonist that only needs to be injected once a week. It is the seventh GLP-1 receptor agonist to be marketed in the world after exenatide, liraglutide, albiglutide, dulaglutide, lixisenatide and beinaglutide. Semaglutide has dual effects on hypoglycemic and weight loss. The hypoglycemic and weight loss effects of weekly semaglutide injections in patients with type 2 diabetes were significantly better than those of placebo, sitagliptin, insulin glargine U100 or Extended-release exenatide. Semaglutide's performance in weight loss is even better than that of the same drug Saxenda (liraglutide 3mg). [0003] The polypeptide sequence of semaglutide is as follows: [0004] H-His 7 -Aib 8 -Glu...

Claims

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Application Information

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IPC IPC(8): C07K14/605C07K1/04C07K1/06
CPCC07K14/605Y02P20/55
Inventor 向双春高峰刘聪翀李云平
Owner 苏州金顶生物有限公司
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