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Preparation of Liraglutide by Continuous Flow Solid Phase Reaction

A liraglutide and fluid-solid technology, applied in the field of liraglutide combined with continuous flow solid-phase synthesis system, can solve the problems of long fragment coupling time, incomplete coupling and low efficiency

Active Publication Date: 2020-12-08
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 or liquid-phase synthesis, which cannot fundamentally improve the production capacity of liraglutide. Secondly, it will be a very cumbersome process to separate small fragments, condensation reagents and impurities from the main fragment during liquid-phase synthesis. The work, especially the good solubility of polypeptide fragments, 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.
[0011] 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 liraglutide by stepwise 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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  • Preparation of Liraglutide by Continuous Flow Solid Phase Reaction
  • Preparation of Liraglutide by Continuous Flow Solid Phase Reaction
  • Preparation of Liraglutide by Continuous Flow Solid Phase Reaction

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0109] Step 1: Preparation of Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Psi(me.me)Pro)-OH Fragment 1

[0110]

[0111] 1) Fmoc-Val-Ser(Psi(me.me)Pro)-2-CTC-resin

[0112] 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.

[0113] Fmoc-Val-Ser(Psi(me.me)Pro)-2-CTC-resin was removed with 100mL 2%DBU / 2%1-octylthiol and DMF solution for 12 minutes at room temperature, and H- Val-Ser(Psi(me.me)Pro)-2-CTC-resin.

[0114] 2) Fmoc-Thr(tBu)-Phe...

Embodiment 2

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

[0147]

[0148] Weigh 10 g of HMPA-PEGA resin (degree of substitution = 0.44 mmol / g) and add it to a solid-phase reactor, swell the resin with 100 mL of dichloromethane for 20 minutes, and filter with suction. Then, 5.0 g (17 mmol) of Fmoc-Gly-OH, 2.6 mL (34 mmol) of pyridine, and 2.3 mL (17 mmol) of 2,6-dichlorobenzoyl chloride were added in sequence, and stirred for 2 hours. Drain the solvent, add 100mL acetic anhydride:pyridine=1:1 to carry out resin blocking reaction solution, stir for half an hour to block unreacted complete methylol. Wash 3 times with 100 mL of dichloromethane each time. After vacuum drying, the degree of substitution of the Fmoc-Gly-HMPA-PEGA resin was measured to be 0.36 mmol / g.

[0149] Step 2: H-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(Palm-Glu-OtBu)-Glu(OtBu)-Phe-Ile -Ala-Trp(Boc)-Leu-Val-Arg(Boc) 2 -Gly-Arg(Boc) 2 -Gly-HMPA-PEGA resin fragment 3 synthesis

[0150] The Fmoc...

Embodiment 3

[0165] Step 1: Fmoc-Gly-HMPA-Thio PEG-UREA-PS resin synthesis

[0166]

[0167] Weigh 15g of HMPA-Thio PEG-UREA-PS resin (degree of substitution = 0.41mmol / g) and add it to a solid-phase reactor, swell the resin with 100mL of dichloromethane for 5 minutes, then add 5.4g of Fmoc-Gly-OH, 7.7 mLDIEA, 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-Thio PEG-AM-PS resin was measured to be 0.33 mmol / g.

[0168] Step 2: H-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(Palm-Glu-OtBu)-Glu(OtBu)-Phe-Ile -Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly-HMPA-Thio PEG-UREA-PS resin 3 synthesis

[0169] The Fmoc-Gly-HMPA-Thio PEG-UREA-PS resin synthesized in step 1 was used for liraglutide resin synthesis. Weigh 10 g of Fmoc-Gly-HMPA-Thio PE...

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Abstract

The invention discloses a method for preparing liraglutide by combination of a continuous flow solid phase synthesis system. A main chain adopts Boc-His(x)-Ala-Glu(OtBu)-Gly,Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Psi(me.me)Pro)-OH,H-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(Palm-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(y)-Gly-Arg(y)-Gly- resin fragments, and besides, a continuous flow solid phase is adopted for synthesis of a growth peptide chain.

Description

technical field [0001] The invention belongs to the field of polypeptide drug synthesis, and in particular relates to a method for preparing liraglutide combined with a continuous-flow solid-phase synthesis system. Background technique [0002] Liraglutide is a GLP-1 receptor agonist, suitable for the control of blood sugar in adult patients with type 2 diabetes; it is suitable for patients whose blood sugar is still poorly controlled after treatment with metformin or sulfonylureas alone. Combined use of metformin or sulfonylureas. Liraglutide is the drug with the best hypoglycemic effect among GLP-1 receptor agonists such as exenatide, albiglutide, lixisenatide, and benaglutide. Liraglutide was as effective as dulaglutide. Liraglutide is effective in reducing body weight. Compared with traditional hypoglycemic drugs, GLP-1 can also delay gastric emptying, and act on the central fullness center to reduce food intake, thereby producing weight loss effects by suppressing ap...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/605C07K1/04C07K1/06
CPCC07K14/605
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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