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A kind of solid-phase preparation method of nesiritide crude product

A technology for solid-phase preparation and crude product is applied in the field of solid-phase preparation of crude nesiritide, which can solve the problems of many impurities and difficult to obtain.

Active Publication Date: 2015-09-02
SHANGHAI AMBIOPHARM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods HMPB-AM resins provided in the prior art are not conventional resins, and are difficult to obtain on the market
HMPB-AM resin is easy to cause racemization when connecting Fmoc-His(Trt)-OH, and there are many impurities

Method used

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  • A kind of solid-phase preparation method of nesiritide crude product
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  • A kind of solid-phase preparation method of nesiritide crude product

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preparation example Construction

[0079] In one example of the present invention, the preparation method of solid-phase synthesis of nesiritide by the polypeptide of the present invention comprises the following steps:

[0080] The first step, the synthesis of 32#His residue

[0081] Use PL wang resin (substitution degree 0.5-1.0mmol / g) and 3.0-5.0 equivalents of PBr3 to stir and react; after reacting for 2-3 hours, remove the reaction solution and wash the resin with DMF;

[0082] Then add 1.0-3.5 equivalents of Fmoc-His(Trt)-OH, 1.0-3.5 times the amount of DIPEA and stir for 10-18 hours. There is no bromine group on the resin, capped with methanol, and finally the resin is obtained and dried. , the degree of substitution measured by UV is 0.20-0.40mmol / g;

[0083] According to the classic polypeptide solid-phase synthesis method, the amino acid residues are bonded in sequence, so that the peptide chain grows from the C-terminal to the N-terminal, but in the process of bonding amino acid residues, the invent...

Embodiment 1

[0117] Synthesis of crude nesiritide 1

[0118] Loading Fmoc-His(Trt)-OH

[0119] With 10 grams of PL Wang resin (substitution degree 0.7-0.9mmol / g) and 2.5 equivalents of PBr 3 Stir the reaction for 2 hours; then take out the reaction solution, wash the resin with DMF, then stir and react with 3.0 equivalents of Fmoc-His(Trt)-OH and 3.0 equivalents of DIPEA for 16 hours, and seal the unreacted bromine groups on the resin with methanol end, finally obtained 13 grams of resin, and the UV ultraviolet measurement substitution degree was 0.26mmol / g.

[0120] Deprotection

[0121] Use 10% piperidine / 5% DBU / 5% HOBt / DMF (v / v / w / v) for two consecutive deprotections, then wash the resin with DMF and methanol respectively, and monitor the Fmoc deprotection by Kaiser test after exhausting thoroughly. go to degree.

[0122] amino acid condensation

[0123] When condensing 31#-23#, 16-14#, 11#, 8# amino acids, add 2.0 times the amount of Fmoc protected amino acids, 2.0 times the a...

Embodiment 2

[0132] Synthesis of crude nesiritide 2

[0133] Loading Fmoc-His(Trt)-OH

[0134] With 10 grams of PL Wang resin (substitution degree 0.6-0.8mmol / g) and 4.0 equivalents of PBr 3Stir the reaction for 2 hours; then take out the reaction solution, wash the resin with DMF, and then stir and react with 3.0 equivalents of Fmoc-His(Trt)-OH and 3.0 equivalents of DIPEA for 15 hours, and the unreacted bromine on the resin is capped with methanol, Obtain 13 grams of resins at last, and UV ultraviolet measurement substitution degree is 0.26mmol / g.

[0135] Deprotection

[0136] Use 10% piperidine / 5% DBU / 5% HOBt / DMF (v / v / w / v) to deprotect twice consecutively, wash the resin with DMF and methanol respectively, and monitor the removal of Fmoc by Kaiser test after exhausting thoroughly degree.

[0137] amino acid condensation

[0138] When condensing 31#-23#, 16-14#, 11#, 8# amino acids, add 1.5 times the amount of Fmoc protected amino acid, 1.5 times the amount of HOBt, 1.5 times ...

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Abstract

The invention discloses a method for preparing a solid phase nesritide crude product. The method comprises the steps of: sequentially connecting amino acid with a Fmoc protecting group by taking solid phase synthesis resin as a starting material according to a solid phase synthesis method to obtain a protected 32-peptide resin; sequentially removing the Fmoc protecting group; carrying out a peptide-bounding reaction by using any of TBTU (tetramethyluronium tetrafluoroborate) / HOBt (hydroxbenzotriazole), HBTU (o-benzotriazole-n,n,n,n-tetramethyl-uronium-hexafluorophosphate) / HOBt, BOP (benzotriazol1yloxy)tris(dimethylamino)phosphonium hexafluophosphate) / HOBt, TBTU / HOAt (hydroxyazabenzotriazole), HBTU / HOAt, DIC (diisopropylcarbodiimide) / HOBt or BOP / HOAt as a condensing agent to obtain a protected 32-peptide resin; and synchronously removing a side chain protecting group and cutting peptides to obtain the nesritide crude product. The method has the characteristics that the solid phase synthesis resin is bromine epoxy resin; and a DMF (Dimethyl Formamide) / DCM (Dichloromethane) mixed solvent is used as an activated solvent, and amino acids with the Fmoc protecting group and the condensing agent are dissolved in advance, activated outside a reaction system and then added to the reaction system.

Description

technical field [0001] The invention relates to the chemical synthesis of medical polypeptide raw materials, in particular to a solid-phase preparation method of nesiritide crude product. Background technique [0002] Nesiritide ((nesiritide / Natrecor) is a new class of vasodilator, a cyclic peptide composed of 32 amino acids, and the 10-26 position is connected by S-S bond. A recombinant human brain natriuretic peptide in the treatment of acute heart failure Or B-type natriuretic peptide (BNP), which is exactly the same as the endogenous hormone, is mainly used for the treatment of dyspnea in acute decompensated congestive heart failure. Nesiritide has venous, arterial and coronary vasodilator effects to relieve the anterior and posterior Load and increase cardiac output in the absence of direct positive inotropic effects.Intravenous nesiritide in patients with chronic heart failure can promote sodium efflux and inhibit the renin-angiotensin-aldosterone system and sympatheti...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/575C07K1/06C07K1/04
CPCY02P20/55
Inventor 白俊才张国庆张若平方路
Owner SHANGHAI AMBIOPHARM