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Method for synthesizing nucleoside triphosphate and nucleoside diphosphate from all-protected nucleoside phosphoramidite intermediate through acid catalysis

A technology of nucleoside phosphoramide and nucleoside triphosphate, applied in chemical instruments and methods, organic chemistry, production of bulk chemicals, etc., can solve the problem of low synthesis yield, etc., and achieve the effect of improving reaction speed and yield

Inactive Publication Date: 2013-07-10
JIANGXI SCI & TECH NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the various known methods have their own suitable nucleoside substrate types, the general synthesis yield is not high, and so far there is no method that can be applied to the efficient synthesis of all nucleoside triphosphates, so as to truly completely solve the problem. Difficulties in the Chemical Synthesis of Nucleoside Triphosphates

Method used

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  • Method for synthesizing nucleoside triphosphate and nucleoside diphosphate from all-protected nucleoside phosphoramidite intermediate through acid catalysis
  • Method for synthesizing nucleoside triphosphate and nucleoside diphosphate from all-protected nucleoside phosphoramidite intermediate through acid catalysis

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Embodiment 1

[0018] N -( O -Benzyl- O -(2′,3′-dibenzyloxycarbonyl)uridine-5′-)phosphorylpiperidine (such as figure 1 Medium substance 11 ) synthesis: 2′,3′-dibenzyloxycarbonyluridine (such as figure 1 Medium substance 1 , 500 mg, 0.98 mmol) was dissolved in dry acetonitrile (20 ml), and dry diisopropylethylamine (0.43 ml, 2.5 mmol) was added with stirring. Benzyloxyphosphoramidite diisopropylamine chloride (such as figure 1 Medium substance 10 , 534 mg, 1.95 mmol) was dissolved in anhydrous dry acetonitrile (10 ml), and then slowly added dropwise to the reaction flask. After the dropwise addition was completed, the reaction was continued for 45 minutes. The reaction solution was concentrated under reduced pressure, and the residue was azeotroped twice with acetonitrile (10 ml). A small amount of ethyl acetate was added to the residue to precipitate the resulting diisopropylethylamine hydrochloride. After filtering to remove salt, the filtrate was concentrated to obtain a yellow o...

Embodiment 2

[0020] N -( O -Benzyl- O -(2′,3′-dibenzyloxycarbonyl)adenosine-5′-)phosphorylmorpholine (such as figure 1 Medium substance 31 ) synthesis: 2′,3′-dibenzyloxycarbonyladenosine (such as figure 1 Medium substance 3 , 1.0 g, 1.87 mmol) was dissolved in anhydrous dichloromethane (50 ml), and dry triethylamine (0.65 ml, 4.68 mmol) was added with stirring. Benzyloxyphosphoramidite diisopropylamine chloride (such as figure 1 Medium substance 10, 1.02 g, 3.74 mmol) was dissolved in anhydrous dichloromethane (20 ml), and then slowly added dropwise to the reaction flask. After the dropwise addition was completed, the reaction was continued for 30 minutes. The reaction solution was concentrated under reduced pressure, and the residue was azeotroped twice with 30 ml of acetonitrile. A small amount of ethyl acetate was added to the residue to precipitate the resulting triethylamine hydrochloride. After filtering to remove salt, the filtrate was concentrated to obtain a yellow oil ...

Embodiment 3

[0022] N -( O -Benzyl- O -(2′,3′-dibenzyloxycarbonyl)ribavirin-5′-)phosphoryl-(4-methylpiperidine) (such as figure 1 Medium substance 33 ) synthesis: 2′,3′-dibenzyloxycarbonyl ribavirin (such as figure 1 Medium substance 5 , 1.0 mg, 1.95 mmol) was dissolved in anhydrous dichloromethane (50 ml), and dry tri-n-butylamine (1.16 ml, 4.88 mmol) was added with stirring. Benzyloxyphosphoramidite diisopropylamine chloride (such as figure 1 Medium substance 10 , 1.07 g, 3.90 mmol) was dissolved in anhydrous dichloromethane (20 ml), and then slowly added dropwise to the reaction flask. After the dropwise addition was completed, the reaction was continued for 30 minutes. The reaction solution was concentrated under reduced pressure, and the residue was azeotroped twice with 30 ml of acetonitrile. A small amount of ethyl acetate was added to the residue to precipitate the resulting tri-n-butylamine hydrochloride. After filtering to remove salt, the filtrate was concentrated to o...

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Abstract

The invention discloses a method for synthesizing nucleoside triphosphate and nucleoside diphosphate from an all-protected nucleoside phosphoramidite intermediate through acid catalysis. The method comprises the following steps of: generating a phosphoramidite intermediate crude product under an alkaline condition by taking carbobenzoxy protected nucleoside and benzyloxydiisopropylamino phosphorochloridite as raw materials, subjecting the phosphoramidite intermediate crude product to weak acid catalytic hydrolysis to obtain a nucleoside- H-phosphonate intermediate crude product, subjecting the nucleoside-H-phosphonate intermediate crude product and alkylamine to oxidative coupling, carrying out column chromatography on the resulting product to obtain an all-protected nucleoside phosphoramidite precursor, subjecting the nucleoside phosphoramidite precursor to catalysis and hydrogenation in an N-N-dimethylfomamide solution to remove all protective groups, filtering the resulting product to remove palladium on an activated carbon, and reacting the resulting product with pyrophosphate or monophosphoric acid alkyl ammonium salt under a condition in the presence of a weak acid catalyst to obtain a nucleoside triphosphate product and a nucleoside diphosphate product. By adopting the method disclosed by the invention, reaction velocity and yield of the pyrophosphoric acid and monophosphoric acid reagents with the phosphoramidite intermediate are greatly improved, and an efficient, universal and novel method for the chemical synthesis of the nucleoside 5'- triphosphate and the nucleoside 5'-diphosphate is established.

Description

technical field [0001] The invention belongs to the technical field of chemical preparation of natural product biochemical reagents, and relates to a method for synthesizing nucleoside triphosphate and nucleoside diphosphate from a fully protected nucleoside phosphoramide intermediate through acid catalysis. Background technique [0002] Nucleoside triphosphates are the material basis for gene replication, transcription, and translation. In molecular biology and medical research, deoxynucleoside triphosphates are widely used. Polymerase chain reaction (Polymerase chain reaction, PCR) technology uses DNA polymerase to amplify specific DNA fragments in vitro, and is used for the establishment of genetic maps, paternity identification, gene cloning and disease detection. Four natural deoxynucleoside triphosphates are the indispensable reaction substrates for this technology. Currently, four natural deoxynucleoside triphosphates are mainly used in the production of PCR experime...

Claims

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Application Information

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IPC IPC(8): C07H19/10C07H19/20C07H19/056C07H1/00
CPCY02P20/55
Inventor 孙麒龚珊珊孙剑蒲守智刘国栋多树旺肖强
Owner JIANGXI SCI & TECH NORMAL UNIV
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