Method for synthesizing cytidine phosphinylidyne compounds through oriented catalysis

A cytidine phosphoryl compound and substrate technology, which is applied in the field of biocatalysis, can solve the problems of conversion rate, low product concentration, complex cell enzyme system, long fermentation period and the like, and achieves low production cost, simple reaction system and convenient production. control effect

Inactive Publication Date: 2010-08-04
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the complexity of the cell enzyme system, there is feedback regulation, the conversion rate is generally low, the fermentation cycle is too long, resulting in low conversion rate and product concentration, and there are a large nu...

Method used

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  • Method for synthesizing cytidine phosphinylidyne compounds through oriented catalysis
  • Method for synthesizing cytidine phosphinylidyne compounds through oriented catalysis
  • Method for synthesizing cytidine phosphinylidyne compounds through oriented catalysis

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Experimental program
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Effect test

Embodiment 1

[0037]Yeast medium: glucose 40g / L, urea 2.0g / L, potassium dihydrogen phosphate 1.5g / L, zinc sulfate heptahydrate 4.0×10 -3 g / L, ferrous sulfate heptahydrate 3.0×10 -3 g / L, manganese chloride tetrahydrate 0.3×10 -3 g / L, anhydrous calcium chloride 1.0×10 -3 g / L, biotin 0.05×10 -3 g / L.

[0038] The inoculum amount of yeast was 10%, cultured on a shaker at 120 rpm at 30°C for 24 hours, and centrifuged at 4000 rpm for 20 minutes. Take the yeast paste and store it at -7°C for later use.

Embodiment 2

[0039] Example 2: Directional production of CTP by CMP.

[0040] Prepare in the reaction tank of capacity 15L by CMP 300mmol, choline chloride 100mmol, glucose 5mol, manganese sulfate 500mmol, dithiothreitol 300mmol, Bacillus subtilis 2400g cultivated by the method for embodiment 1, potassium dihydrogen phosphate 3mol , Triton X-1001g and 10L of reaction solution composed of water, adjust the pH to 7.0 with sodium hydroxide, and the temperature is 35°C, finish the reaction after 3 hours of reaction, precipitate with perchloric acid, and quantitatively analyze the product by HPLC, The main product in the conversion liquid is CDP, its content is 18.6mmol / L, and the yield is 62.1%. At this time, the content of CDP is 2.5mmol / L, and the content of CDPC is 5.8mmol / L.

Embodiment 3

[0041] Example 3: Directional production of CTP by CMP.

[0042] Prepare in the reaction tank of capacity 15L by CMP 300mmol, choline chloride 3000mmol, glucose 5mol, manganese nitrate 500mmol, dithiothreitol 300mmol, brewing yeast 2500g cultivated by the method of embodiment 1, potassium dihydrogen phosphate 3mol, 10L of reaction solution composed of 10mL of toluene and water was adjusted to pH 7.0 with sodium hydroxide and the temperature was 40°C. After 3 hours of reaction, the reaction was terminated, precipitated with perchloric acid, and the product was quantitatively analyzed by HPLC. The main product in the conversion solution was It is CDP, its content is 27.5mmol / L, and the yield is 91.8%. At this time, the CDP content is 0.7mmol / L, and the CDPC content is 0.8mmol / L.

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Abstract

The invention discloses a method for synthesizing cytidine phosphinylidyne compounds through oriented catalysis. The method uses CMP, choline chloride and phosphate ions as substrate, glucose as energy donor and permeable microbial cells as enzyme source and changes the reaction temperature to prepare cytidine phosphinylidyne compounds in the reaction system through oriented catalysis. The method of the invention can be used to produce any one of three substances according to demands, the reaction system is simple and nontoxic, the production cost is low, the control method is simple and practical, and the industrial production and control are greatly facilitated. When CMP is used as substrate, the yield rate of CDP, CTP and CDP-choline are separately 80%, 91.8% and 93.3%.

Description

technical field [0001] The invention belongs to the technical field of biocatalysis, and in particular relates to a method for directional catalytically synthesizing cytidine phosphoryl compounds using control technology. Background technique [0002] Cytidine diphosphate (CDP), as a derivative of cytosine, is an important biochemical reagent. It and inosine diphosphate under the action of nucleotide phosphatase to prepare poly inosine (Poly I:C). Polyinosin is a highly efficient inducer of interferon, which has broad-spectrum antiviral and immunosuppressive effects, and is widely used in the treatment of various diseases. [0003] Cytidine triphosphate (CTP) is a nucleotide with three molecules of phosphoric acid bound to the 5'-OH group of cytidine ribose. It is a normal component in the body's cells and is widely distributed. It is the direct precursor of RNA synthesis. It participates in the biosynthesis process of nucleic acid and phospholipids (lecithin, cephalin, se...

Claims

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Application Information

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IPC IPC(8): C12P19/30C12R1/01C12R1/185C12R1/265C12R1/645C12R1/72C12R1/78C12R1/84C12R1/85
Inventor 应汉杰汤佳鹏陈勇熊健柏建新陈晓春
Owner NANJING UNIV OF TECH
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