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Solid phase synthesis method of leuprorelin

A technology of leuprolide and leucyl, applied in the field of biochemistry, can solve the problems of peptide chain racemization, degradation, low yield of aminolysis, etc., and achieve the effect of improving yield

Inactive Publication Date: 2009-04-15
ADLAI NORTYE BIOPHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method uses the classic solid-phase peptide synthesis strategy. The modification of the carbon-terminal functional group is obtained by cleavage of the resin through aminolysis. Due to the use of a strong alkaline solution in this aminolysis process, it is easy to cause the elimination of the peptide chain in a high pH environment. Spin or cause degradation, etc., and the yield of aminolysis is not high

Method used

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  • Solid phase synthesis method of leuprorelin
  • Solid phase synthesis method of leuprorelin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Preparation of leuprorelin peptide resin

[0030] Operation: (1) 10g (5mmol) of 4-[(1-N-ethylamino)ethyl]phenoxy-butyramide resin (product of sigma-aldrich company) is placed in the peptide synthesis reactor, alternating with 50ml each of dichloromethane and methanol Wash twice, 2 minutes / time, and drain. Add 50 ml of 20% (v / v) piperidine / DMF solution, stir and react at room temperature for 20 minutes, and then drain. Wash the resin alternately with dichloromethane and methanol 50ml 3 times, 2 minutes per time, and drain.

[0031] (2) Dissolve 11.4g of Fmoc protected amino acid and HBTU (0-benzotriazole-N,N,N,N-tetramethylurea hexafluorophosphate, Shanghai Yanchang Biochemical Technology Development Co., Ltd.) in 50ml DMF solution , Then add 13ml of N,N-diisopropylethylamine, mix well and add to the resin, stir at room temperature for 3 hours.

[0032] (3) Drain dry. The resin was washed alternately with dichloromethane and methanol 60ml each for 3 times, 2 minut...

Embodiment 2

[0038] Example 2: Preparation of Leuprolide Crude Peptide

[0039] Operation: Add 18.6g of leuprorelin peptide resin to a 500ml round bottom flask, add 190ml of trifluoroacetic acid: water: 1,2-ethanedithiol (volume ratio 95:2.5:2.5), and stir at room temperature for 2 hours After filtering and washing the resin with 10ml of trifluoroacetic acid, the filtrates were combined, and the filtrate was poured into 2000ml of glacial ether. A large amount of white solid was precipitated. The white solid was collected by filtration and dried to obtain 5.44g of crude leuprolide peptide. The yield was 90%.

Embodiment 3

[0040] Example 3: Purification of crude leuprolide

[0041] Device: C 18 Preparation column (50×300mm)

[0042] Eluent A: 0.1% TFA / H 2 O

[0043] Eluent B: Acetonitrile

[0044] Flow rate: 200ml / min

[0045] UV detection wavelength: 230nm

[0046] Operation: (1) Leuprolide crude peptide is dissolved in 5% (v / v) acetic acid aqueous solution

[0047] (2) Filter with 0.45μm microporous membrane

[0048] (3) Filtrate loading

[0049] (4) 23~25%(v / v) acetonitrile-water mobile phase gradient elution

[0050] (5) Collect the target peptide fraction

[0051] (6) Concentration, freeze drying

[0052] (7) 4.84 g of leuprolide white solid.

[0053] The product was confirmed by MS mass spectrometry, with a total yield of 80.1%.

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Abstract

The invention relates to a solid phase synthesis method of leuprorelin, which uses 4-((1-N-Alkylamino)ethyl) phenoxy-butyramide resin as a solid phase carrier to carry out procedure reaction, and peptide whole protecting resin can be obtained by sequentially carrying out condensation reaction to connect protecting amino-acid; crude peptide can be obtained by cutting the resin by trifluoroacetic acid, and the crude peptide is dissolved in an acetic acid water solution of 5 percent, and the leuprorelin can be obtained by purifying, leaching, condensing and freeze drying the opposite phase high performance liquid chromatograph columns of C18 fillers. The invention uses a novel solid phase resin, and products decorated by peptide chain carbon end alkyl can be obtained directly after finishing assembling the peptide chain by the solid phase synthesis technology and using acid hydrolysis to cut the peptide from the resin after, and the solid phase synthesis method of the leuprorelin is convenient in operation, reduces synthesis steps for synthesizing the leuprorelin greatly and improves the yield.

Description

Technical field [0001] The invention belongs to the technical field of biochemistry, and specifically relates to a solid-phase synthesis method of leuprolide. Background technique [0002] Leuprolide chemical name: 5-oxo-L-prolyl-L-histidyl-L-tryptophan-L-seryl-L-tyrosyl-D-leucyl-L- Leucyl-L-arginyl-N-ethyl-prolineamide [0003] The chemical structural formula is: [0004] [0005] Molecular formula: C 59 H 84 N 16 O 12 [0006] Molecular weight: 1209.42 [0007] Leuprolide is a highly active derivative of luteinizing-releasing hormone (LH-RH). It can produce transient pituitary-gonadal system excitement (acute effect) immediately after the first administration, and then inhibit pituitary production and Release gonadotropins. It also further suppresses the response of the ovaries and testes to gonadotropins, thereby reducing the production of estradiol and testosterone (chronic effect). So it can effectively inhibit the function of the pituitary-gonadal system. Leuprolide is w...

Claims

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Application Information

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IPC IPC(8): C07K7/06C07K1/04
CPCY02P20/55
Inventor 赵学平杨东晖
Owner ADLAI NORTYE BIOPHARMA CO LTD
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