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Method for preparing Nesiritide

A technology of crude peptide and resin, which is applied in the field of solid phase-liquid phase preparation, can solve the problems of unfavorable nesiritide industrial application, low yield of nesiritide, large environmental pollution, etc., and achieve considerable economic and practical value, future Ease of handling and less input of raw materials

Inactive Publication Date: 2009-09-02
HYBIO PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Due to the reason of the selected resin and oxidation method in the prior art, the yield of nesiritide is low, the cost is high, and the environmental pollution is large, which is not conducive to the large-scale industrial application of nesiritide

Method used

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  • Method for preparing Nesiritide
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  • Method for preparing Nesiritide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1: Preparation of Fmoc-His(Trt)-HMPB-AM resin

[0026] Add 7.2 g of HMPB-AM resin, with a substitution degree of 1.1 mmol / g, into a solid-phase reaction column, wash once with DMF, and swell the resin with DCM for 30 minutes, add 4.9 g of Fmoc-His(Trt)-OH, Add it to the above-mentioned reaction column equipped with resin and react for 2 hours. In order to improve the reaction efficiency, you can choose to add DIEA5-15ml. After the reaction was completed, it was washed 3 times with DMF and 3 times with DCM, blocked with methanol for 30 minutes, and then shrunk with methanol to obtain Fmoc-His(Trt)-HMPB-AM resin. The degree of substitution was 0.19 mmol / g.

Embodiment 2

[0027] Example 2: Preparation of side chain fully protected nesiritide-HMPB-AM resin

[0028] Weigh 20 g of Fmoc-His(Trt)-HMPB-AM resin (0.19 mmol / g, 3.8 mmol) into the reactor, wash once with DMF, and swell with DCM for 0.5 hours. After swelling, 20% DBLK was used to remove Fmoc protection, and then washed 4 times with DMF and 2 times with DCM. Dissolve 6.16g Fmoc-Arg(pbf)-OH (9.5mmol), 1.5g HOBt (11.4mmol), 1.8g DIC (14.3mmol) in DCM (a small amount of DMF can be added to aid dissolution), and add it to the solid phase reactor , and react at room temperature for 2h (the end point of the reaction is determined by the ninhydrin method). Repeat the above steps to complete Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Gly-OH , Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc -Ile-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Met-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly -OH, F...

Embodiment 3

[0029] Example 3: Scale preparation of Nesiritide-HMPB-AM resin with fully protected side chains

[0030] Weigh 825g of Fmoc-His(Trt)-HMPB-AM resin (0.19mmol / g, 156.8mmol) into the reactor, wash once with DMF, and swell with DCM for 0.5 hours. After swelling, 20% DBLK was used to remove Fmoc protection, and then washed 4 times with DMF and 2 times with DCM. Dissolve 254.3g Fmoc-Arg(pbf)-OH (392mmol), 63.5g HOBt (470.4mmol), 74.3g DIC (588mmol) in DCM (a small amount of DMF can be added to aid dissolution), add to the solid phase reactor, room temperature Reaction for 2h (the end point of the reaction is determined by the ninhydrin method). Repeat the above steps to complete Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Gly-OH , Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc -Ile-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Met-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly -OH...

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Abstract

The invention relates to a method for preparing Nesiritide. In the method, HMPB-AM resin is taken as an initial raw material, amino acids with Fmoc blocking groups are sequentially connected in a solid phase synthesis way under the action of condensing agent and transpeptidase reagent to obtain side chain full-protection linear Nesiritide HMPB-AM resin; linear Nesiritide raw peptides are obtained by schizolysis, Nesiritide raw peptides are obtained by liquid phase oxidation, and Nesiritide fine peptides are obtained by purification, salt transfer and freeze drying. The invention has the advantages of simple operation, easy post treatment, few raw materials, low cost, high yield coefficient, and the like, thereby having considerable economy and utility value and also having extensive application prospect in the polypeptide drug design synthesis field.

Description

technical field [0001] The invention relates to a preparation method of a polypeptide, in particular to a preparation method of nesiritide in combination of solid phase and liquid phase. Background technique [0002] Nesiritide, also known as human brain natriuretic peptide, was isolated from pig brain tissue by Sudoh in 1988. It is one of the important members of the endogenous natriuretic family and is mainly secreted by the heart. Its structure is similar to that of atrial natriuretic peptide, it has the effects of natriuresis, diuresis, dilation of blood vessels and inhibition of renin secretion, etc. It is mainly used for the treatment of heart failure, etc. It has better effect and less side effects. It is a kind of polypeptide drug with great market prospect . [0003] Nesiritide (Nesiritide) sequence is as follows: [0004] [0005] In the prior art, due to the selected resin and oxidation means, the yield of nesiritide is low, the cost is high, and the environm...

Claims

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Application Information

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IPC IPC(8): C07K14/47A61P9/04C07K1/04C07K1/02
CPCY02P20/55
Inventor 欧阳陵李红玲马亚平袁建成
Owner HYBIO PHARMA
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