CN101792786B - Method for synthesizing cytidine phosphinylidyne compounds through oriented catalysis - Google Patents
Method for synthesizing cytidine phosphinylidyne compounds through oriented catalysis Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 44
- UHDGCWIWMRVCDJ-UHFFFAOYSA-N 1-beta-D-Xylofuranosyl-NH-Cytosine Natural products O=C1N=C(N)C=CN1C1C(O)C(O)C(CO)O1 UHDGCWIWMRVCDJ-UHFFFAOYSA-N 0.000 title claims abstract description 29
- UHDGCWIWMRVCDJ-PSQAKQOGSA-N Cytidine Natural products O=C1N=C(N)C=CN1[C@@H]1[C@@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-PSQAKQOGSA-N 0.000 title claims abstract description 29
- UHDGCWIWMRVCDJ-ZAKLUEHWSA-N cytidine Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-ZAKLUEHWSA-N 0.000 title claims abstract description 29
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- 238000006243 chemical reaction Methods 0.000 claims abstract description 49
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- 239000008103 glucose Substances 0.000 claims abstract description 14
- 239000001763 2-hydroxyethyl(trimethyl)azanium Substances 0.000 claims abstract description 12
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- SGMZJAMFUVOLNK-UHFFFAOYSA-M choline chloride Chemical compound [Cl-].C[N+](C)(C)CCO SGMZJAMFUVOLNK-UHFFFAOYSA-M 0.000 claims abstract description 12
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- 239000000758 substrate Substances 0.000 claims abstract description 9
- RZZPDXZPRHQOCG-OJAKKHQRSA-M CDP-choline(1-) Chemical compound O[C@@H]1[C@H](O)[C@@H](COP([O-])(=O)OP([O-])(=O)OCC[N+](C)(C)C)O[C@H]1N1C(=O)N=C(N)C=C1 RZZPDXZPRHQOCG-OJAKKHQRSA-M 0.000 claims abstract 2
- ZWIADYZPOWUWEW-XVFCMESISA-N CDP Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(O)=O)O1 ZWIADYZPOWUWEW-XVFCMESISA-N 0.000 claims description 32
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- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
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- 229950004354 phosphorylcholine Drugs 0.000 description 2
- PYJNAPOPMIJKJZ-UHFFFAOYSA-N phosphorylcholine chloride Chemical compound [Cl-].C[N+](C)(C)CCOP(O)(O)=O PYJNAPOPMIJKJZ-UHFFFAOYSA-N 0.000 description 2
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- IERHLVCPSMICTF-UHFFFAOYSA-N cytidine monophosphate Natural products O=C1N=C(N)C=CN1C1C(O)C(O)C(COP(O)(O)=O)O1 IERHLVCPSMICTF-UHFFFAOYSA-N 0.000 description 1
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
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
The invention belongs to the biocatalysis technology field, be specifically related to utilize the control technique method of synthesizing cytidine phosphinylidyne compounds through oriented catalysis respectively.
Background technology
Cytidine diphosphate(CDP) (CDP) is a kind of important biochemical reagents as the verivate of cytosine(Cyt).It prepares Polyinosinic-polycytidylic acid (Poly I:C) with the di-phosphate inosine under the effect of nucleotide phosphodiesterase enzyme.Polyinosinic-polycytidylic acid is a kind of interferon inducers efficiently, has broad-spectrum antiviral and immunosuppressive action, is widely used in multiple treatment of diseases.
Cytidine triphosphate(CTP) (CTP) is that termolecular phosphoric acid is combined in the Nucleotide on cytidine ribose 5 '-OH base, is a kind of normal composition in the body cell, distribute extensively, and be the direct precursor of RNA synthetic.It participates in the biosynthetic process of nucleic acid and phospholipids (Yelkin TTS, kephalin, serine phosphatide, sphingophospholipid etc.) in vivo; Phosphatide is the important component of nervous tissue; Also be a kind of natural fat emulsifier, have the brain of perfecting and nervous tissue, increase intelligence with memory and the transportation that promotes body inner cholesterol, fat is arranged; Improve metabolism of fat, thereby prevent the effect of piling up.CTP is widely used in the multiple nerve of treatment, vascular disease as biochemical drug.
Cytidine diphosphate (CDPC) is claimed CITICOLINE SODIUM again, is the agent of brain metabolic activation, can promote the synthetic of neuron membrane Yelkin TTS, has the reparation brain injury, and anoxia improves memory, the effect that increases intelligence, and clinical application is wider.So the synthetic technology of cytidine diphosphate is the research topic that people relatively are concerned about.
CDP, CTP and CDPC all belong to the category of cytidine phosphinylidyne compounds.
At present, the above CDP preparation method of document has two kinds of chemical synthesis and biological synthesis process.Chemical synthesis is to be reactant with CMP, with hexichol phosphoryl chloride phosphorylation, re-refine CDP.This method is used multiple volatile toxic reagent, severe operational environment, and exist yield low, the problem that cost is high (Biochim.Biophys.Acta., 1964,91 (1): 1-13).Biological synthesis process was divided into for two steps, with the synthetic CTP of CMP, again the CTP degraded was generated CDP earlier, can obtain highly purified CDP, and yield is in (chemistry and biotechnology, 2005,22 (7): 52-54) more than 70%.
CTP can utilize mikrobe (is main with cereuisiae fermentum) cellular enzymes system synthetic.The source of this technology required yeasts is wide, and cost is low, receives season limit few; And productive rate is higher, has to excavate to improve potentiality.It is one of main mode of production of present cytidine.According to reports, Kilajima etc. utilize yeast, and from the synthetic CTP of CMP, the reaction yield is 80% (HakkoKogaku Zasshi.1970,48 (12): 753-762).
CDPC compound method at present commonly used is a microbe transformation method, utilizes microbial cell enzyme system synthetic.In yeast cell, CMP can synthesize CDP under the catalysis of nucleoside monophosphate kinase; CDP can generate CTP under the effect of nucleoside diphosphokinase, choline can be under the catalysis of choline kinase synthesizing choline phosphate.CTP and phosphorylcholine can generate CDPC and tetra-sodium through choline phosphate cytidylyltransferase catalysis then.But because cellular enzymes system is complicated, have the feedback regulation effect, transformation efficiency is generally on the low side, and fermentation period is oversize, causes transformation efficiency, product concentration lower, has a large amount of by products in the reaction solution, comprises CMP, CDP and CTP.The at present domestic relevant report of also not utilizing same reaction system through control reaction temperature and the synthetic cytidine phosphinylidyne compounds of time orientation catalysis.
Summary of the invention
Technical problem to be solved by this invention provides the method for the synthetic above-mentioned three kinds of cytidine phosphinylidyne compounds of a kind of microorganism cells directional catalyzing.
For solving the problems of the technologies described above, thinking of the present invention is:
In view of in the preparation process of CDPC, need to consume lot of energy (ATP), therefore in the preparation process of cytidine phosphinylidyne compounds, needing two enzyme systems is regeneration system and the cytidine phosphinylidyne compounds synthetase series of ATP.The regeneration system of ATP is a substrate with the glucose of cheapness, and (EMP) realizes through glycolytic pathway, and this approach is one of most economical approach of energy regeneration; The cytidine phosphinylidyne compounds synthetase series is made up of nucleoside monophosphate kinase, nucleoside diphosphokinase, choline kinase and choline phosphate cytidylyltransferase.
In the whole process of CDPC synthetic, the cytidine phosphinylidyne compounds synthetase series plays keying action by nucleoside monophosphate kinase, nucleoside diphosphokinase, choline kinase and choline phosphate cytidylyltransferase.These enzymes in the cereuisiae fermentum have different optimum temperutures respectively.Four kinds of enzyme optimum temperutures are respectively cytidylate kinase, 25 ℃; Nucleoside diphosphokinase, 37 ℃; Choline kinase, 30 ℃; Choline phosphate cytidylyltransferase, 30 ℃.We can produce any one product among CDP, CTP and the CDPC through control reaction temperature and time.
Key of the present invention is:
1) to have adopted whole-cell catalytic technology, the own enzyme that has directly utilized cell be the synthetic three kinds of cytidine phosphinylidyne compounds of catalysis CMP in the present invention, can produce any one of three kinds of materials according to demand, and reaction system is simple, and is nontoxic, low production cost.
2) the present invention as means, influences the activity of relevant enzyme in the cytidine phosphinylidyne compounds building-up process through control reaction temperature.Under differing temps, the enzymic activity that can help synthetic a kind of material is strengthened, and the enzymic activity that helps generating other materials simultaneously then weakens, and the active selectivity of this kind of enzyme has determined the final product that reacts different.Control device is simple, has greatly made things convenient for the production control in the industriallization.
Concrete technical scheme of the present invention is following:
A kind of method of synthesizing cytidine phosphinylidyne compounds through oriented catalysis; With CMP, choline chloride 60 and phosphate anion is substrate; With glucose as energy donor; The microorganism cells of having property of utilization is the enzyme source, is means through changing temperature of reaction, impels the reaction system biocatalysis to synthesize cytidine phosphinylidyne compounds.
Wherein, said cytidine phosphinylidyne compounds is cytidine diphosphate(CDP) (CDP), cytidine triphosphate(CTP) (CTP) or CDP-choline (CDPC), and its structural formula is following:
Wherein, cytidine monophosphate, i.e. CMP, its structural formula is following:
The method of above-mentioned synthesizing cytidine phosphinylidyne compounds through oriented catalysis is: in the aqueous solution of pH5~10, carry out one of following three kinds of reactions, directed synthetic cytidine phosphinylidyne compounds:
(a) under 28~40 ℃, reacted 3 hours, primary product is CTP;
(b) under 28~40 ℃, reacted 3 hours; Adjust temperature to 28~34 ℃ again, reacted 9~15 hours, primary product is CDPC;
(c) under 28~40 ℃, reacted 3 hours; Adjust temperature to 28~34 ℃ again, reacted 9~15 hours; Adjust temperature to 20~27 ℃ again, reacted 6~10 hours, primary product is CDP.
Wherein, the initial action concentration of substrate CMP is 10~100mmol/L, preferred 20~50mmol/L; The initial action concentration of choline chloride 60 is 10~300mmol/L, preferred 20~150mmol/L; The initial action concentration of phosphate anion is 0.1~2mol/L, preferred 0.2~0.5mol/L; The initial action concentration of glucose is 0.1~1mol/L, preferred 0.2~0.5mol/L; The add-on of microorganism cells is by wet thallus 100~800g/L, preferred 200~600g/L.Phosphate ion can be selected from Tripyrophosphoric acid such as ortho-phosphoric acid, tetra-sodium, tripolyphosphate, inorganic phosphates such as potassium primary phosphate, SODIUM PHOSPHATE, MONOBASIC, Sodium phosphate, dibasic.
Wherein, reaction system also adds Mn
2+The compsn of ion and WR 34678; Mn
2+Initial action concentration is 1~200mmol/L, preferred 10~100mmol/L; WR 34678 initial action concentration is 1~200mmol/L, preferred 10~100mmol/L.
Wherein, described microorganism cells is meant the mikrobe that can utilize the synthetic CDPC of CMP, comprises the bacterium of aerobacter, Escherichia, Serratia, micrococcus sp; The yeast that yeast belong, mycocandida, Pichia, torulopsis, Debaryomyces, zygosaccharomyces genus, genus kluyveromyces, Hansenula and Brettanomyces belong to.Preferred intestinal bacteria, subtilis, yeast saccharomyces cerevisiae or Torulopsis candida.
Wherein, The microorganism cells of described having property is meant the microorganism cells that the permeability changes of the cytolemma of handling through chemistry, physics or biological method is crossed, and concrete grammar comprises surfactant method, organic solvent method, freeze-thaw method, ultrasonication method, aeration drying, freeze-drying or bacteriolyze enzyme process.
The tensio-active agent that uses in the surfactant method is non-ionics polyethylene oxide amines or triton x-100, cationic surfactant hexadecyl trimethylamine bromide, perhaps AS Sarkosyl L salt, and usage quantity is 0.1~50g/L; Preferred 1~20g/L; When being surfactant method processing yeast cell, tensio-active agent directly being added reaction solution, is the reaction solution of 1L for TV; Add 0.1~50g, preferably add 1~20g.
The organic solvent that uses in the organic solvent method is YLENE, toluene, Fatty Alcohol(C12-C14 and C12-C18), acetone or ETHYLE ACETATE; Usage quantity is 0.1~50mL/L, and preferred 1~20mL/L is promptly during organic solvent method process for producing bacterial strain; Organic solvent is directly added reaction solution; For TV is the reaction solution of 1L, adds 0.1~50mL, preferably adds 1~20mL.
It can be the dry thing of yeast cell, the centrifugal cell that obtains of culture of isolated, the lyophilized products of cell, commercially available yeast powder, air-dry yeast or waste yeast mud by fermentation that above-mentioned zymic utilizes form.
Beneficial effect of the present invention is:
The invention provides a kind of method of synthesizing cytidine phosphinylidyne compounds through oriented catalysis; With CMP, choline chloride 60 and phosphate anion is substrate; With glucose as energy donor; The microorganism cells of having property of utilization is the enzyme source, is means through changing temperature of reaction, impels the synthetic cytidine phosphinylidyne compounds of the directed biocatalysis of reaction system.The present invention can produce any a kind of of three kinds of materials according to demand, and reaction system is simple, and is nontoxic, low production cost, and control device is simple, has greatly made things convenient for the production control in the industriallization.The yield of CDP, CTP and CDP-choline can reach 80%, 91.8%, 93.3% respectively when doing substrate with CMP.
Embodiment
According to following embodiment, can understand the present invention better.Yet, those skilled in the art will readily understand that the described concrete material proportion of embodiment, processing condition and result thereof only are used to explain the present invention, and the present invention that should also can not limit in claims to be described in detail.
Embodiment 1:
Yeast culture base: glucose 40g/L, urea 2.0g/L, potassium primary phosphate 1.5g/L, Zinc vitriol 4.0 * 10
-3G/L, Presfersul 3.0 * 10
-3G/L, four hydration Manganous chloride tetrahydrates 0.3 * 10
-3G/L, Calcium Chloride Powder Anhydrous 1.0 * 10
-3G/L, vitamin H 0.05 * 10
-3G/L.
Yeast-inoculated amount 10% was cultivated centrifugal 4000rpm, 20 minutes 24 hours in 30 ℃ of following 120rpm shaking tables.Get yeast slurry ,-7 ℃ of preservations are subsequent use.
Embodiment 2: utilize the directed CTP of production of CMP.
In the reactive tank of capacity 15L, modulate by CMP 300mmol, choline chloride 60 100mmol, glucose 5mol, manganous sulfate 500mmol, WR 34678 300mmol, press the subtilis 2400g of the method cultivation of embodiment 1, potassium primary phosphate 3mol, the reaction solution 10L that triton x-100 1g and water are formed; Using sodium hydroxide to transfer pH is 7.0, and temperature is 35 ℃, reacts and finishes reaction after 3 hours; Use the perchloric acid precipitation, with HPLC product is carried out quantitative analysis, primary product is CDP in the conversion fluid; Its content is 18.6mmol/L; Yield is 62.1%, and this moment, CDP content was 2.5mmol/L, and CDPC content is 5.8mmol/L.
Embodiment 3: utilize the directed CTP of production of CMP.
The reaction solution 10L that yeast saccharomyces cerevisiae 2500g, potassium primary phosphate 3mol, toluene 10mL and the water that modulation is cultivated by CMP 300mmol, choline chloride 60 3000mmol, glucose 5mol, manganous nitrate 500mmol, WR 34678 300mmol, by the method for embodiment 1 in the reactive tank of capacity 15L is formed, use sodium hydroxide accent pH is 7.0, temperature is 40 ℃; React and finish reaction after 3 hours; Use the perchloric acid precipitation, with HPLC product is carried out quantitative analysis, primary product is CDP in the conversion fluid; Its content is 27.5mmol/L; Yield is 91.8%, and this moment, CDP content was 0.7mmol/L, and CDPC content is 0.8mmol/L.
Embodiment 4: utilize the directed CDPC of production of CMP.
In the reactive tank of capacity 15L, modulate by CMP 300mmol, choline chloride 60 3mol, glucose 5mol, manganous sulfate 500mmol, WR 34678 300mmol, press the intestinal bacteria 2500g of the method cultivation of embodiment 1, potassium primary phosphate 3mol, the reaction solution 10L that acetone 10mL and water are formed; Using sodium hydroxide to transfer pH is 7.0, and temperature is 35 ℃, reacts after 3 hours; Temperature is adjusted into 28 ℃, continues reaction and finishes reaction in 9 hours, uses the perchloric acid precipitation; With HPLC product is carried out quantitative analysis, primary product is CDPC in the conversion fluid, and its content is 28mmol/L; Yield is 93.3%, and this moment, CDP content was 0.8mmol/L, and CTP content is 0.3mmol/L.
Embodiment 5: utilize the directed CDPC of production of CMP.
In the reactive tank of capacity 15L, modulate by CMP 300mmol, choline chloride 60 3mol, glucose 5mol, manganous sulfate 500mmol, WR 34678 300mmol, press the pichia spp 2500g of the method cultivation of embodiment 1, the reaction solution 10L that potassium primary phosphate 3mol, ETHYLE ACETATE 10mL and water are formed, using sodium hydroxide to transfer pH is 7.0; Temperature is 40 ℃, reacts after 3 hours, and temperature is adjusted into 34 ℃; Continue reaction and finished reaction in 15 hours, use the perchloric acid precipitation, product is carried out quantitative analysis with HPLC; Primary product is CDPC in the conversion fluid, and its content is 21mmol/L, and yield is 70.0%; This moment, CDP content was 1.2mmol/L, and CTP content is 0.7mmol/L.
Embodiment 6: utilize the directed CDP of production of CMP.
The reaction solution 10L that intestinal bacteria 1000g, SODIUM PHOSPHATE, MONOBASIC 2mol, triton x-100 50g, toluene 50mL and the water that modulation is cultivated by CMP 100mmol, choline chloride 60 100mmol, glucose 1mol, manganous sulfate 20mmol, WR 34678 10mmol, by embodiment 1 method in the reactive tank of capacity 15L is formed, use sodium hydroxide accent pH is 6.0, temperature is 35 ℃; React after 3 hours, temperature is adjusted into 28 ℃, continues reaction 9 hours; Temperature is adjusted into 20 ℃ again, continues reaction and finishes reaction in 6 hours, uses the perchloric acid precipitation; With HPLC product is carried out quantitative analysis; Primary product is CDP in the conversion fluid, and its content is 8mmol/L, and yield is 80.0%; This moment, CTP content was 1mmol/L, and CDPC content is 0.8mmol/L.
Embodiment 7: utilize the directed CDP of production of CMP.
Modulation is pressed the reaction solution 10L of the Torulopsis candida of embodiment 1 method cultivation through 3 8000g of multigelation, SODIUM PHOSPHATE, MONOBASIC 5mol and water composition by CMP 1000mmol, choline chloride 60 3000mmol, glucose 10mol, Manganous chloride tetrahydrate 500mmol, WR 34678 2mol in the reactive tank of capacity 15L, and using sodium hydroxide to transfer pH is 9.0; Temperature is 40 ℃, reacts after 3 hours, and temperature is adjusted into 34 ℃; Continue reaction 15 hours, temperature is adjusted into 27 ℃ again, continues reaction and finishes reaction in 10 hours; Use the perchloric acid precipitation, with HPLC product is carried out quantitative analysis, primary product is CDP in the conversion fluid; Its content is 42mmol/L; Yield is 42.0%, and this moment, CTP content was 0.5mmol/L, and CDPC content is 11mmol/L.
Claims (3)
1. the method for a synthesizing cytidine phosphinylidyne compounds through oriented catalysis; It is characterized in that with CMP, choline chloride 60 and phosphate anion be substrate; With glucose as energy donor; The microorganism cells of having property of utilization is the enzyme source, is means through changing temperature of reaction, impels the reaction system biocatalysis to synthesize cytidine phosphinylidyne compounds;
Wherein, said cytidine phosphinylidyne compounds is cytidine diphosphate(CDP), cytidine triphosphate(CTP) or CDP-choline, and its structural formula is following:
Wherein, described microorganism cells is meant the mikrobe that can utilize the synthetic CDPC of CMP, comprises the bacterium of aerobacter, Escherichia, Serratia, micrococcus sp; The yeast that yeast belong, mycocandida, Pichia, torulopsis, Debaryomyces, zygosaccharomyces genus, genus kluyveromyces, Hansenula and Brettanomyces belong to;
Wherein, The microorganism cells of described having property is meant the microorganism cells that the permeability changes of the cytolemma of handling through chemistry, physics or biological method is crossed, and concrete grammar comprises surfactant method, organic solvent method, freeze-thaw method, ultrasonication method, aeration drying, freeze-drying or bacteriolyze enzyme process;
Wherein, the tensio-active agent that uses in the surfactant method is non-ionics, cationic surfactant or AS, and usage quantity is 0.1~50g/L.
Wherein, the organic solvent that uses in the organic solvent method is YLENE, toluene, Fatty Alcohol(C12-C14 and C12-C18), acetone or ETHYLE ACETATE, and usage quantity is 0.1~50mL/L;
Wherein, in the aqueous solution of pH5~10, carry out one of following three kinds of reactions, directed synthetic cytidine phosphinylidyne compounds:
(a) under 28~40 ℃, reacted 3 hours, primary product is CTP;
(b) under 28~40 ℃, reacted 3 hours; Adjust temperature to 28~34 ℃ again, reacted 9~15 hours, primary product is CDPC;
(c) under 28~40 ℃, reacted 3 hours; Adjust temperature to 28~34 ℃ again, reacted 9~15 hours; Adjust temperature to 20~27 ℃ again, reacted 6~10 hours, primary product is CDP.
2. according to the method for any described synthesizing cytidine phosphinylidyne compounds through oriented catalysis in the claim 1; The initial action concentration that it is characterized in that substrate CMP is 10~100mmol/L; The initial action concentration of choline chloride 60 is 10~300mmol/L; The initial action concentration of phosphate anion is 0.1~2mol/L, and the initial action concentration of glucose is 0.1~1mol/L, and the add-on of microorganism cells is by wet thallus 100~800g/L.
3. according to the method for any described synthesizing cytidine phosphinylidyne compounds through oriented catalysis in the claim 1, it is characterized in that described reaction system also adds Mn
2+The compsn of ion and WR 34678; Mn
2+Initial action concentration is 1~200mmol/L, and WR 34678 initial action concentration is 1~200mmol/L.
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EP0553821A1 (en) * | 1992-01-30 | 1993-08-04 | Kyowa Hakko Kogyo Co., Ltd. | Process for producing cytidine diphosphate choline |
CN101555509A (en) * | 2009-04-17 | 2009-10-14 | 南京工业大学 | Method for catalyzing and synthesizing uridine phosphinylidyne compound in an oriented way |
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EP0553821A1 (en) * | 1992-01-30 | 1993-08-04 | Kyowa Hakko Kogyo Co., Ltd. | Process for producing cytidine diphosphate choline |
CN101555509A (en) * | 2009-04-17 | 2009-10-14 | 南京工业大学 | Method for catalyzing and synthesizing uridine phosphinylidyne compound in an oriented way |
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