Method for preparing lepirudin

A technology of lepirudin and coupling method, applied in the field of peptide synthesis, can solve the problems of low yield, difficult purification, high energy consumption, etc.

Inactive Publication Date: 2012-10-17
HYBIO PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, the step-by-step coupling method is mainly used to prepare Lepirudin, but the preparation cycle is long, the energy consumption is high, the purity is only about 20-30%, and th

Method used

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  • Method for preparing lepirudin
  • Method for preparing lepirudin
  • Method for preparing lepirudin

Examples

Experimental program
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preparation example Construction

[0119] The raw materials and reagents used in the method for preparing lepirudin provided by the present invention can be purchased from the market.

[0120] The meanings of the abbreviations used in the method for preparing Lepirudin provided by the present invention are as follows.

[0121] Fmoc 9-fluorenylmethoxycarbonyl

[0122] Boc tert-butoxycarbonyl

[0123] tBu tert-butyl

[0124] Trt Trityl

[0125] NMP N-Methylpyrrolidone

[0126] DMSO dimethyl sulfoxide

[0127] DMF N,N-Dimethylformamide

[0128] DCM dichloromethane

[0129] DBLK 20% Hexahydropyridine / DMF Solution

[0130] DIC N,N-Diisopropylcarbodiimide

[0131] DIPEA N,N-Diisopropylethylamine

[0132] DMAP 4-dimethylaminopyridine

[0133] PYBOP Benzotriazol-1-yl-oxytripyrrolidinyl hexafluorophosphate

[0134] TBTU O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroboric acid

[0135] HOBT 1-Hydroxybenzotriazole

[0136] TFE trifluoroethanol

[0137] TFA trifluoroacetic acid

[0138] EDT 1,2-eth...

Embodiment 1

[0153] Fragment 1 (sequence shown in SEQ ID NO:1)

[0154] FMOC-Leu-Thr(tBu)-Tyr(tBu)-Thr(tBu)-Asp(OtBu)-Cys(Trt)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-Glycine-2-CTC Peptide Preparation of resin

[0155] Weigh 10 g of 2-CTC resin with a substitution degree of 0.5 mmol / g, add it to a solid-phase reaction column, wash it twice with DMF, and swell the resin with DMF for 30 minutes, then take 1.8 g of Fmoc-Gly-OH and dissolve it in DMF. Add 2.7mL DIPEA in ice-water bath to activate, add to the above-mentioned reaction column filled with resin, react for 60min, drain, wash three times, block with methanol three times, add 25mL each time, each time for 10min, and wash with DMF for 6 Second-rate. Fmoc protection was removed with DBLK, followed by 6 washes with DMF.

[0156] Dissolve 5.74g Fmoc-Ser(tBu)-OH, 2.43g HOBt, and 3.51ml DIC in a mixed solution of DCM and DMF with a volume ratio of 1:1, add it to a solid-phase reaction column, and react at room temperature for 2h (the end point of t...

Embodiment 2

[0208] Fragment 1 (sequence shown in SEQ ID NO:1)

[0209] FMOC-Leu-Thr(tBu)-Tyr(tBu)-Thr(tBu)-Asp(OtBu)-Cys(Trt)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-Gly-2-CTC peptide Preparation of resin

[0210] Weigh 10 g of 2-CTC resin with a substitution degree of 0.5 mmol / g, add it to a solid-phase reaction column, wash it twice with DMF, and swell the resin with DMF for 30 minutes, then take 1.8 g of Fmoc-Gly-OH and dissolve it in DMF. After adding 2.7mL DIPEA to activate in ice-water bath, add to the above-mentioned reaction column with resin, after 60min of reaction, drain, wash three times, block with methanol three times, add 25mL each time, each time for 10min, then wash with DMF for 6 Second-rate. Fmoc protection was removed with DBLK, followed by 6 washes with DMF.

[0211] Dissolve 5.74g Fmoc-Ser(tBu)-OH, 2.43g HOBt, and 3.51ml DIC in a mixed solution of DCM and DMF with a volume ratio of 1:1, add it to a solid-phase reaction column, and react at room temperature for 2h (the end poi...

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Abstract

The present invention relates to the field of polypeptide synthesis, and particularly to a method for preparing lepirudin. According to the method, combination of stepwise synthesis and fragment synthesis is adopted so as to greatly reduce synthesis difficulty, save synthesis time, reduce final impurities, improve purity and yield, provide considerable economic and practical values, and provide wide application prospects in the field of design and synthesis of multiple disulfide bond ring closing polypeptide drugs.

Description

technical field [0001] The invention relates to the field of polypeptide synthesis, in particular to a method for preparing lepirudin. Background technique [0002] In 1884, Haycraft first discovered that the extract of medical leeches contained anticoagulant substances. It was not until 1957 that Markwardt named the pure anticoagulant substance isolated from medical leeches as hirudin. Hirudin is the most active and most researched ingredient among the various active ingredients extracted from Leech and its salivary glands. It is a small molecular protein (polypeptide) composed of 65-66 amino acids . Hirudin has a strong inhibitory effect on thrombin, and is the strongest natural specific inhibitor of thrombin found so far. [0003] Animal experiments and clinical studies have shown that hirudin is highly effective in anticoagulation and antithrombosis, and prevents further blood stasis phenomena such as activation of coagulation factors catalyzed by thrombin and platelet...

Claims

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Application Information

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IPC IPC(8): C07K14/815C07K1/06C07K1/04
Inventor 欧阳陵黄志云马亚平袁建成
Owner HYBIO PHARMA
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