CN114262726A - Method for synthesizing citicoline sodium by using cytidine enzymatic method - Google Patents
Method for synthesizing citicoline sodium by using cytidine enzymatic method Download PDFInfo
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- YWAFNFGRBBBSPD-OCMLZEEQSA-M sodium;[[(2r,3s,4r,5r)-5-(4-amino-2-oxopyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-oxidophosphoryl] 2-(trimethylazaniumyl)ethyl phosphate Chemical compound [Na+].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 YWAFNFGRBBBSPD-OCMLZEEQSA-M 0.000 title claims abstract description 81
- 229960004774 citicoline sodium Drugs 0.000 title claims abstract description 75
- 238000000034 method Methods 0.000 title claims abstract description 54
- 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 30
- 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 30
- 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 30
- 230000002194 synthesizing effect Effects 0.000 title claims abstract description 15
- 238000006911 enzymatic reaction Methods 0.000 title claims abstract description 12
- 238000006243 chemical reaction Methods 0.000 claims abstract description 38
- 239000000047 product Substances 0.000 claims abstract description 28
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 22
- 238000001728 nano-filtration Methods 0.000 claims abstract description 17
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 13
- 238000001914 filtration Methods 0.000 claims abstract description 9
- 238000010438 heat treatment Methods 0.000 claims abstract description 8
- 238000007670 refining Methods 0.000 claims abstract description 6
- 239000003480 eluent Substances 0.000 claims abstract description 3
- 102100020797 UMP-CMP kinase Human genes 0.000 claims description 27
- 239000007788 liquid Substances 0.000 claims description 27
- 102000004190 Enzymes Human genes 0.000 claims description 25
- 108090000790 Enzymes Proteins 0.000 claims description 24
- 238000003756 stirring Methods 0.000 claims description 23
- 238000000926 separation method Methods 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 14
- 239000007787 solid Substances 0.000 claims description 13
- 239000001763 2-hydroxyethyl(trimethyl)azanium Substances 0.000 claims description 12
- 235000019743 Choline chloride Nutrition 0.000 claims description 12
- SGMZJAMFUVOLNK-UHFFFAOYSA-M choline chloride Chemical compound [Cl-].C[N+](C)(C)CCO SGMZJAMFUVOLNK-UHFFFAOYSA-M 0.000 claims description 12
- 229960003178 choline chloride Drugs 0.000 claims description 12
- 239000000758 substrate Substances 0.000 claims description 10
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- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 8
- 238000001816 cooling Methods 0.000 claims description 8
- 230000035484 reaction time Effects 0.000 claims description 7
- 229920000388 Polyphosphate Polymers 0.000 claims description 6
- 238000012544 monitoring process Methods 0.000 claims description 6
- 239000001205 polyphosphate Substances 0.000 claims description 6
- 235000011176 polyphosphates Nutrition 0.000 claims description 6
- 239000011347 resin Substances 0.000 claims description 6
- 229920005989 resin Polymers 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 6
- 239000012535 impurity Substances 0.000 claims description 5
- 239000012528 membrane Substances 0.000 claims description 5
- 238000001035 drying Methods 0.000 claims description 4
- 238000005406 washing Methods 0.000 claims description 4
- FGUUSXIOTUKUDN-IBGZPJMESA-N C1(=CC=CC=C1)N1C2=C(NC([C@H](C1)NC=1OC(=NN=1)C1=CC=CC=C1)=O)C=CC=C2 Chemical compound C1(=CC=CC=C1)N1C2=C(NC([C@H](C1)NC=1OC(=NN=1)C1=CC=CC=C1)=O)C=CC=C2 FGUUSXIOTUKUDN-IBGZPJMESA-N 0.000 claims description 3
- 235000019982 sodium hexametaphosphate Nutrition 0.000 claims description 3
- GCLGEJMYGQKIIW-UHFFFAOYSA-H sodium hexametaphosphate Chemical compound [Na]OP1(=O)OP(=O)(O[Na])OP(=O)(O[Na])OP(=O)(O[Na])OP(=O)(O[Na])OP(=O)(O[Na])O1 GCLGEJMYGQKIIW-UHFFFAOYSA-H 0.000 claims description 3
- 235000019832 sodium triphosphate Nutrition 0.000 claims description 3
- 239000001577 tetrasodium phosphonato phosphate Substances 0.000 claims description 3
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 2
- 239000003957 anion exchange resin Substances 0.000 claims description 2
- 238000004821 distillation Methods 0.000 claims description 2
- 239000011734 sodium Substances 0.000 claims description 2
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- 239000012141 concentrate Substances 0.000 claims 2
- 239000012043 crude product Substances 0.000 abstract description 4
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- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 12
- 101710100179 UMP-CMP kinase Proteins 0.000 description 9
- 101710119674 UMP-CMP kinase 2, mitochondrial Proteins 0.000 description 9
- IERHLVCPSMICTF-XVFCMESISA-N cytidine 5'-monophosphate Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(O)=O)O1 IERHLVCPSMICTF-XVFCMESISA-N 0.000 description 9
- 229950004354 phosphorylcholine Drugs 0.000 description 9
- PYJNAPOPMIJKJZ-UHFFFAOYSA-N phosphorylcholine chloride Chemical compound [Cl-].C[N+](C)(C)CCOP(O)(O)=O PYJNAPOPMIJKJZ-UHFFFAOYSA-N 0.000 description 9
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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 6
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- PCDQPRRSZKQHHS-CCXZUQQUSA-N Cytarabine Triphosphate 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)OP(O)(O)=O)O1 PCDQPRRSZKQHHS-CCXZUQQUSA-N 0.000 description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
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- 238000011069 regeneration method Methods 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 238000001308 synthesis method Methods 0.000 description 3
- BFUNVIKGWITYHK-UHFFFAOYSA-N 4-dichlorophosphorylmorpholine Chemical compound ClP(Cl)(=O)N1CCOCC1 BFUNVIKGWITYHK-UHFFFAOYSA-N 0.000 description 2
- YYROPELSRYBVMQ-UHFFFAOYSA-N 4-toluenesulfonyl chloride Chemical compound CC1=CC=C(S(Cl)(=O)=O)C=C1 YYROPELSRYBVMQ-UHFFFAOYSA-N 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- 108010078853 Choline-Phosphate Cytidylyltransferase Proteins 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- YNAVUWVOSKDBBP-UHFFFAOYSA-N Morpholine Chemical compound C1COCCN1 YNAVUWVOSKDBBP-UHFFFAOYSA-N 0.000 description 2
- 206010039966 Senile dementia Diseases 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- 150000001450 anions Chemical class 0.000 description 2
- 210000004556 brain Anatomy 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 229910052791 calcium Inorganic materials 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000002386 leaching Methods 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
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- 150000004712 monophosphates Chemical class 0.000 description 2
- 229960005010 orotic acid Drugs 0.000 description 2
- 150000003904 phospholipids Chemical class 0.000 description 2
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- 239000008399 tap water Substances 0.000 description 2
- 235000020679 tap water Nutrition 0.000 description 2
- ZKHQWZAMYRWXGA-KQYNXXCUSA-N Adenosine triphosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-N 0.000 description 1
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- 108090000364 Ligases Proteins 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 1
- 208000018737 Parkinson disease Diseases 0.000 description 1
- 101710135451 Probable cytidylate kinase Proteins 0.000 description 1
- 241000075117 Thrips orientalis Species 0.000 description 1
- 108020000553 UMP kinase Proteins 0.000 description 1
- WETWJCDKMRHUPV-UHFFFAOYSA-N acetyl chloride Chemical compound CC(Cl)=O WETWJCDKMRHUPV-UHFFFAOYSA-N 0.000 description 1
- 239000012346 acetyl chloride Substances 0.000 description 1
- LIPOUNRJVLNBCD-UHFFFAOYSA-N acetyl dihydrogen phosphate Chemical compound CC(=O)OP(O)(O)=O LIPOUNRJVLNBCD-UHFFFAOYSA-N 0.000 description 1
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- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
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- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
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- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 description 1
- LFGREXWGYUGZLY-UHFFFAOYSA-N phosphoryl Chemical group [P]=O LFGREXWGYUGZLY-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 citicoline sodium by a cytidine enzymatic method. And when the reaction conversion rate is more than 99%, heating and filtering the reaction solution, carrying out nanofiltration, concentrating, purifying by using a column, concentrating the eluent, adding 95% ethanol, crystallizing to obtain a crude product of citicoline sodium, and further refining the crude product to obtain a finished product of citicoline sodium. The HPLC purity of the finished product is more than 99 percent, and the total yield is more than 90 percent.
Description
Technical Field
The invention belongs to the technical field of biological pharmacy and biochemical engineering, and particularly relates to a method for synthesizing citicoline sodium by a cytidine enzymatic method.
Background
Citicoline sodium (Citicoline or CDP-choline) is the main component in cell membrane phospholipids, and can promote synthesis of nerve cell membrane phospholipids as an intermediate for endogenously synthesizing phosphatidylcholine. It can be used as brain metabolism activator, and has effects in repairing brain injury, resisting anoxia, improving memory, and improving intelligence. The traditional Chinese medicine composition is mainly used for treating acute craniocerebral trauma and post-operation conscious disturbance of brain clinically; treating Parkinson's disease; nerve deafness; has certain curative effects on senile dementia, depression, senility delaying, learning effect and memory improvement, etc.
At present, three methods for producing citicoline sodium mainly exist, including a chemical synthesis method, a microbial conversion method and an enzymatic synthesis method. The chemical synthesis method has the problems of low reaction conversion rate, more byproducts, high production cost, serious environmental pollution, multiple potential safety hazards and the like.
For example, chinese patent document CN201410250694.4A discloses a method for producing citicoline sodium by condensing citicoline and phosphorylcholine as substrates and p-toluenesulfonyl chloride as a condensing agent in the presence of N-dimethylformamide. The process route is as follows:
the method has the defects that the citicoline sodium is difficult to separate from the condensing agent, and the product is not suitable for medical use.
The preparation method disclosed in chinese patent document CN102016000492243A is to use choline chloride calcium salt as raw material, remove crystal water in the choline chloride calcium salt by binary azeotropic distillation with benzene, react with sodium carbonate to generate calcium carbonate precipitate, react the filtered solid sticky substance with acetyl chloride, and react the product with cytidylic acid to obtain citicoline sodium.
Although the method solves the problem of difficult separation of citicoline sodium and the condensing agent to a certain extent, the method has the defects of complicated steps and great environmental pollution in the synthesis process.
In the preparation method disclosed in chinese patent document CN201610158305.4A, dichlorophosphoryl morpholine is used as a raw material, and reacts with ethylene glycol to obtain ethylene glycol ester phosphoryl morpholine; then directly condensing with cytidine monophosphate to obtain ethylene glycol ester phosphorylcytidine monophosphate, and finally, carrying out ring opening on the ethylene glycol ester of the ethylene glycol ester phosphorylcytidine monophosphate by tributylamine to obtain citicoline sodium. The process route is as follows:
the method has the defects that the raw material of dichlorophosphoryl morpholine is small in market quantity, difficult to purchase and high in price, and is not beneficial to industrial production.
The principle of the microbial transformation method is that a multi-step enzyme catalytic reaction is carried out on raw material cytidylic acid and phosphorylcholine in microbial cells by utilizing an enzyme system in the microbial cells, and then a target product citicoline sodium is obtained. The microbial conversion method has the problems of low product concentration, more impurities, low yield and the like.
The preparation method disclosed in chinese patent document CN201110232740.4B is to prepare citicoline sodium by using phosphorylcholine and 5' -cytidylic acid as substrates, glucose as an energy donor, increasing ATP regeneration rate by inorganic salts, and using isasatchenia orientalis (i.orientalis) permeable cells. The process route is as follows:
although the method can utilize an enzyme system in the yeast and ATP in the yeast to generate citicoline sodium, the conversion rate is not high, and the product concentration is low; the reaction solution contains a large amount of glucose and various inorganic salts, which brings great difficulty to subsequent separation and purification. In chinese patent document CN201310358526.2B, a saccharomyces cerevisiae strain with high activity CMP kinase and high activity phosphorylcholine cytidylyltransferase is bred by chemical mutagenesis to improve the conversion rate of citicoline sodium, but the above problems still exist although the conversion rate is improved.
The method disclosed in chinese patent document CN201110052603.2A is a method of producing citicoline sodium by incorporating yeast-derived phosphorylcholine transferase (CTT), and orotate pyrophosphorylase (pyrE), orotate decarboxylase (pyrF), uridylate kinase (pyrH), Nucleoside Diphosphate Kinase (NDK), and cytidine triphosphate ligase (pyrG) into e.coli engineering bacteria to obtain recombinant bacteria K1-E/pggg-CCT, and performing biotransformation with the recombinant bacteria in a liquid containing glucose, orotate, phosphorylcholine, various inorganic salts, and an organic solvent.
According to the method, the conventional 5' -cytidylic acid is replaced by the low-cost orotic acid serving as a substrate, so that the cost is reduced to a certain extent, but the reaction is difficult to control due to the fact that a biological route from the orotic acid to the citicoline is too long, the required enzyme systems are too numerous, and the expression conditions of various enzymes in recombinant bacteria are different, so that the conversion rate and the yield are influenced. This method is well-thought, but is a long distance away from production scale-up.
The earliest enzyme-catalyzed synthesis method is to synthesize citicoline sodium by catalyzing Cytidine Triphosphate (CTP) and phosphorylcholine with phosphorylcholine transferase (CTT), and the method is too high in cost.
In recent years, a mild, efficient, economic, convenient and environment-friendly preparation process method of citicoline sodium is always explored by vast biochemistry workers. Aiming at the disadvantage of higher cost of the enzyme catalysis synthesis method, a method for synthesizing citicoline sodium by a multi-enzyme system catalysis with cytidylic acid and choline chloride as raw materials is developed by biological workers (Appl Microbiol Biotechnol 101:1409-1417), and the reaction formula is shown as follows:
the method has the advantages that 5' -cytidylic acid and choline chloride with lower cost replace expensive CTP and phosphorylcholine, ATP is recycled by an ATP regeneration system to reduce the addition amount of ATP, and the production cost is reduced to a certain extent. However, this method has a disadvantage that the addition of acetyl phosphate to the ATP regeneration system inhibits the catalytic activity of the enzyme, resulting in a low conversion rate or a low substrate concentration. The method has no industrialization condition.
Disclosure of Invention
The first technical problem to be solved by the invention is that the preparation method of citicoline sodium in the prior art has the problems of complicated reaction steps, low yield, high cost, serious environmental pollution and more potential safety hazards, and further provides the method for preparing citicoline sodium, which has the advantages of simple reaction steps, high yield, low cost, environmental protection and safety.
The second problem to be solved by the invention is to provide the enzyme required for preparing citicoline sodium by an enzyme method.
The third problem to be solved by the invention is to provide a production process for preparing citicoline sodium, which can be applied industrially.
The invention provides a method for synthesizing citicoline sodium by a cytidine enzymatic method, wherein the citicoline sodium has the following structure:
the method for preparing citicoline sodium by using the cytidine enzymatic method comprises the following steps:
a. dissolving cytidine in water, adding ATP, polyphosphate, UCK enzyme solution and PPK enzyme solution, and reacting for a period of time under the conditions of preset temperature and PH value to obtain reaction solution A;
b. continuously adding choline chloride, CMK enzyme liquid, NDK enzyme liquid, CCT enzyme liquid and CKI enzyme liquid into the reaction liquid A, continuously reacting for a period of time under the conditions of preset temperature and pH value to obtain reaction liquid B, and monitoring the reaction process by HPLC;
c. when the conversion rate of the reaction liquid B reaches a preset value, heating and stirring the reaction liquid B, filtering, and carrying out nanofiltration on filtrate to obtain nanofiltration concentrated liquid;
d. passing the nanofiltration concentrated solution through column resin, washing with water to remove impurities, eluting with HCl, and concentrating the eluate to obtain concentrated solution;
e. adjusting the pH value of the concentrated solution to a preset value, adding a solvent, stirring, cooling and crystallizing, and performing solid-liquid separation to obtain a solid, namely a crude citicoline sodium product;
f. and further refining the crude citicoline sodium product to obtain a finished citicoline sodium product.
Specifically, in the step a, the preset temperature is 35-37 ℃, the pH is 6.0-8.0, and the adding amount of ATP and polyphosphate is 0.01-0.03 eq and 1-3 eq of the molar equivalent of substrate cytidine respectively; the addition amount of the UCK enzyme solution and the PPK enzyme solution is 10-30% and 10-30% of the weight of cytidine respectively; the reaction time is 8-12 h.
Specifically, the polyphosphate in the step a is one of sodium tripolyphosphate, sodium tetrapolyphosphate or sodium hexametaphosphate.
Specifically, the addition amount of choline chloride in the step b is 1-1.3 eq of the molar equivalent of substrate cytidine, and the addition amounts of the CMK enzyme liquid, the NDK enzyme liquid, the CCT enzyme liquid and the CKI enzyme liquid are 10-30%, 10-30% and 10-30% of the weight of cytidine respectively; the reaction time is 12-16 h.
Specifically, in the step c, the heating temperature is 70-90 ℃, and the stirring time is 10-30 min; nanofiltration membrane of 200 daltons is selected for nanofiltration.
Specifically, the resin in the step d is strong-base anion exchange resin, and the concentration of HCl eluent is 0.05-0.1M; and concentrating by reduced pressure distillation until the concentration of citicoline sodium is 200-400 g/L.
In the step e, the pH value of the concentrated solution is adjusted to 6.0-8.0, the solvent is 95% ethanol, and the addition amount of the solvent is 3-5 times of the volume of the concentrated solution; the stirring speed is 50-150 rpm; the temperature is 0-10 ℃; the separation time is 10-20 h.
Specifically, the step of further refining in the step f is as follows: and adding pure water into the crude citicoline sodium product for redissolving, adding activated carbon, stirring and filtering, adding 95% ethanol with the volume being 3-4 times that of the filtrate, cooling to 0-10 ℃ under stirring, separating solid from liquid after 10-20 hours, and drying the solid in vacuum to obtain the finished citicoline sodium product.
The reaction formula for preparing citicoline sodium by the enzyme method is as follows:
compared with the prior art, the technical scheme of the invention has the following advantages:
(1) compared with the prior production process which utilizes cytidylic acid and choline phosphate as starting raw materials, the invention selects cytidine and choline chloride as the starting raw materials, and the latter has low price, thereby greatly reducing the cost;
(2) the substrate conversion rate is more than 99%, so that high yield is ensured, and subsequent separation and purification are simplified;
(3) the proportion of each enzyme can be controlled in the reaction process, so that the reaction balance is optimal, and no by-product is generated;
(4) the reaction conditions are normal temperature and normal pressure, the water phase has no organic solvent, and the method is safe and environment-friendly and is suitable for industrial production.
Detailed Description
In order that the invention may be more fully understood, reference will now be made to the following description. The present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Example 1
a. Enzymatic reaction of citicoline sodium:
940ml of tap water is taken and put into a 2000ml three-necked bottle, 50g of cytidine, 100g of sodium hexametaphosphate and 1.1g of ATP.2Na are added, the temperature is raised to 37 ℃, the pH value is adjusted to 7.0, 10g of UCK enzyme solution and 10g of PPK enzyme solution are added, the mixture is stirred and reacted for 8 hours at 37 ℃, and the reaction progress is monitored by HPLC.
b. Enzymatic sustained catalytic reaction of citicoline sodium:
after reacting for 8h, adding 34g of choline chloride, 10g of CMK enzyme solution, 10g of NDK enzyme solution, 10g of CCT enzyme solution and 10g of CKI enzyme solution, stirring and reacting for 16h at 37 ℃, sampling and detecting in the process, and monitoring the reaction process by HPLC.
c. The enzyme-catalyzed reaction of citicoline sodium is terminated:
after 16h, monitoring the cytidine conversion rate by HPLC to be more than 99%, heating the reaction solution to 80 ℃, keeping the temperature and stirring for 15min, and filtering to remove solid insoluble substances; and (4) carrying out nanofiltration on the filtrate, and selecting a 200-Dalton nanofiltration membrane.
d. And (3) carrying out aftertreatment crude on citicoline sodium reaction liquid:
and (3) loading the nanofiltration concentrated solution on strong-base anion resin, washing with water to remove impurities, eluting citicoline by 0.05M HCl, and concentrating under reduced pressure until the concentration of citicoline sodium is 300 g/L.
e. And (3) solid-liquid separation of the concentrated solution of the crude citicoline sodium product:
and d, adjusting the pH value of the concentrated solution obtained in the step d to 7.0, adding 95% ethanol with 4 times of volume, cooling to 0 ℃, keeping the temperature, stirring, crystallizing for 16 hours, and then carrying out solid-liquid separation to obtain a solid, namely a crude product of citicoline sodium. The HPLC purity of the crude citicoline sodium product was > 99%.
f. Refining citicoline sodium:
dissolving the crude citicoline sodium product with pure water until the concentration is 300g/L, adding activated carbon accounting for 1% of the weight of the crude citicoline sodium product, stirring and decoloring for 30min, filtering, adding 95% ethanol with the volume being 5 times of that of the filtrate, cooling to 0 ℃, keeping the temperature, stirring and crystallizing for 16h, then carrying out solid-liquid separation, leaching the solid with 80% ethanol, and drying in vacuum to obtain the finished citicoline sodium product with 95g in total. The HPLC purity of the finished citicoline sodium product is more than 99.5 percent, and the total yield is more than 90 percent.
Example 2
The UCK, PPK, CMK, NDK, CCT and CKI enzyme solutions used in steps a and b in example 1 were prepared as follows:
inoculating single colony of recombinant E.coli strain respectively expressing UCK, PPK, CMK, NDK, CCT and CKI enzymes into 5ml of liquid LB culture medium, carrying out shake culture at 37 ℃ for 12h, taking culture solution after shake culture, transferring the culture solution into 100ml of liquid LB culture medium by 1% of inoculum size, carrying out shake culture at 37 ℃ until OD600 value reaches 0.6, adding IPTG with final concentration of 1mM, and then carrying out shake culture at 37 ℃ for 16 h. And after the culture is finished, centrifuging the culture solution at 8000rpm for 10min to collect cells, adding 10ml of water to resuspend the cells, placing the resuspended cells in an ice bath to ultrasonically break the cells, centrifuging the broken solution at 8000rpm for 10min, and collecting supernatant to obtain the enzyme solution of UCK, PPK, CMK, NDK, CCT and CKI required by the synthesis of citicoline sodium.
Example 3
Based on example 1, the enzyme-catalyzed reaction of citicoline sodium in step a can replace the following method:
940ml of tap water is taken and put into a 2000ml three-necked bottle, 50g of cytidine, 50g of sodium tripolyphosphate and 1.1g of ATP.2Na are added, the temperature is raised to 35 ℃, the pH value is adjusted to 6.0, 10g of UCK enzyme solution and 10g of PPK enzyme solution are added, the mixture is stirred and reacted for 12 hours at 35 ℃, and the reaction process is monitored by HPLC.
Example 4
Based on example 1, the enzymatic sustained catalytic reaction of citicoline sodium in step b can replace the following method:
after reacting for 12h, adding 34g of choline chloride, 10g of CMK enzyme solution, 10g of NDK enzyme solution, 10g of CCT enzyme solution and 10g of CKI enzyme solution, stirring and reacting for 15h at 37 ℃, sampling and detecting in the process, and monitoring the reaction process by HPLC.
Example 5
Based on example 1, the enzyme-catalyzed reaction termination of citicoline sodium in step c can replace the following method:
after 15h, monitoring the cytidine conversion rate by HPLC to be more than 99%, heating the reaction solution to 90 ℃, keeping the temperature and stirring for 20min, and filtering to remove solid insoluble substances; and (4) carrying out nanofiltration on the filtrate, and selecting a 200-Dalton nanofiltration membrane.
Example 6
Based on example 1, the post-treatment of the crude citicoline sodium reaction solution in step d can replace the following method:
and (3) loading the nanofiltration concentrated solution on strong-base anion resin, washing with water to remove impurities, eluting citicoline by 0.1M HCl, and concentrating under reduced pressure until the concentration of citicoline sodium is 400 g/L.
Example 7
Based on example 1, the solid-liquid separation of the crude concentrated solution in step e can replace the following method:
adjusting the pH value to 7.0, adding 95% ethanol with the volume of 5 times of that of the filtrate, cooling to 10 ℃, keeping the temperature, stirring, crystallizing for 20 hours, and then carrying out solid-liquid separation to obtain a solid, namely a crude product of citicoline sodium. The HPLC purity of the crude citicoline sodium product was > 99%.
Example 8
Based on example 1, where the f step refinement of citicoline sodium can replace the following method:
dissolving the crude citicoline sodium product with pure water until the concentration is 400g/L, adding activated carbon accounting for 1% of the weight of the crude citicoline sodium product, stirring and decoloring for 30min, filtering, adding 95% ethanol with the volume being 3 times of that of the filtrate, cooling to 5 ℃, keeping the temperature, stirring and crystallizing for 12h, then carrying out solid-liquid separation, leaching the solid with 80% ethanol, and drying in vacuum to obtain the finished citicoline sodium product with 95g in total. The HPLC purity of the finished citicoline sodium product is more than 99.5 percent, and the total yield is more than 90 percent.
It should be noted that, in the step a of the above step embodiment 1, the temperature is raised to 37 ℃ and may be replaced by any value between 35 ℃ and 37 ℃, the pH is adjusted to 7.0 and may be replaced by any value between 6.0 and 8.0, and 8 hours and may be replaced by any value between 8 and 12 hours, according to the sequence.
In the step b of the step example 1, the reaction time of 8h may be replaced by any value between 8 and 12h, the reaction time of 37 ℃ may be replaced by any value between 35 and 37 ℃, and the reaction time of 16h may be replaced by any value between 12 and 16h according to the sequence.
In the step c of the step 1, 16h can be replaced by any value between 12 and 16h, the heating to 80 ℃ can be replaced by any value between 70 and 90 ℃, and the stirring for 15min can be replaced by any value between 10 and 30min according to the sequence.
In the step d of the above step embodiment 1, 0.05M HCl may be replaced by any value between 0.05M and 0.1M HCl, and 300g/L may be replaced by any value between 200 g/L and 400g/L, in order.
It should be noted that, in the step e of the above step example 1, the ph is adjusted to 7.0 and any value between 6.0 and 8.0 can be replaced, 4 times of the volume can be replaced by any value between 3 and 5, the temperature is reduced to 0 ℃ and any value between 0 and 10 ℃ can be replaced, and any value between 10 and 20 hours can be replaced in 16 hours.
It should be noted that, in the step f of the above step embodiment 1, in order, 300g/L may be replaced by any value between 200 and 400g/L, 30min may be replaced by any value between 10 and 30min, 5 times of volume may be replaced by any value between 3 and 5, when the temperature is reduced to 0 ℃, any value between 0 and 10 ℃ may be replaced, and 16h may be replaced by any value between 10 and 20 h.
In conclusion, compared with the prior production process which utilizes cytidylic acid and phosphorylcholine as starting raw materials, the invention selects cytidine and choline chloride as the starting raw materials, and the latter has low price, thereby greatly reducing the cost; the substrate conversion rate is more than 99%, so that high yield is ensured, and subsequent separation and purification are simplified; the proportion of each enzyme can be controlled in the reaction process, so that the reaction balance is optimal, and no by-product is generated; the reaction conditions are normal temperature and normal pressure, the water phase has no organic solvent, and the method is safe and environment-friendly and is suitable for industrial production.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications in light of the above teachings may occur to those skilled in the art. And are neither required nor exhaustive of all embodiments. And obvious modifications or variations such as would be obvious to one skilled in the art are intended to be included within the scope of the invention.
Claims (8)
1. A method for synthesizing citicoline sodium by a cytidine enzymatic method is characterized by comprising the following steps:
a. dissolving cytidine in water, adding ATP, polyphosphate, UCK enzyme solution and PPK enzyme solution, and reacting for a period of time under the conditions of preset temperature and PH value to obtain reaction solution A;
b. continuously adding choline chloride, CMK enzyme liquid, NDK enzyme liquid, CCT enzyme liquid and CKI enzyme liquid into the reaction liquid A, continuously reacting for a period of time under the conditions of preset temperature and pH value to obtain reaction liquid B, and monitoring the reaction process by HPLC;
c. when the conversion rate of the reaction liquid B reaches a preset value, heating and stirring the reaction liquid B, filtering, and carrying out nanofiltration on filtrate to obtain nanofiltration concentrated liquid;
d. passing the nanofiltration concentrated solution through column resin, washing with water to remove impurities, eluting with HCl, and concentrating the eluate to obtain concentrated solution;
e. adjusting the pH value of the concentrated solution to a preset value, adding a solvent, stirring, cooling and crystallizing, and performing solid-liquid separation to obtain a solid, namely a crude citicoline sodium product;
f. and further refining the crude citicoline sodium product to obtain a finished citicoline sodium product.
2. The method for synthesizing citicoline sodium by a cytidine enzymatic method according to claim 1, wherein the predetermined temperature in step a is 35 to 37 ℃, the PH is 6.0 to 8.0, and the amounts of ATP and polyphosphate added are 0.01 to 0.03eq and 1 to 2eq, respectively, based on the molar equivalent of the substrate cytidine; the addition amount of the UCK enzyme solution and the PPK enzyme solution is 10-30% and 10-30% of the weight of cytidine respectively; the reaction time is 8-12 h.
3. The method for synthesizing citicoline sodium by the cytidine enzymatic method according to claim 1, wherein the polyphosphate in the step a is one of sodium tripolyphosphate, sodium tetrapolyphosphate and sodium hexametaphosphate.
4. The method for synthesizing citicoline sodium by using the cytidine enzymatic method according to claim 1, wherein the predetermined temperature in step b is 35 to 37 ℃, the pH is 6.0 to 8.0, the addition amount of choline chloride is 1 to 1.3eq of the molar equivalent of the substrate cytidine, and the addition amounts of the CMK enzyme solution, the NDK enzyme solution, the CCT enzyme solution and the CKI enzyme solution are 10 to 30%, 10 to 30% and 10 to 30% of the weight of cytidine, respectively; the reaction time is 12-16 h.
5. The method for synthesizing citicoline sodium by using the cytidine enzymic method according to claim 1, wherein in the step c, when the conversion rate is more than 99%, the heating temperature is 70-90 ℃, and the stirring time is 10-30 min; nanofiltration membrane of 200 daltons is selected for nanofiltration.
6. The method for synthesizing citicoline sodium by the cytidine enzymic method according to claim 1, wherein the column resin in the step d is a strongly basic anion exchange resin; the concentration of the HCl eluent is 0.05-0.1M; and concentrating by reduced pressure distillation until the concentration of citicoline sodium is 200-400 g/L.
7. The method for synthesizing citicoline sodium by a cytidine enzymatic method according to claim 1, wherein in the step e, the pH value of the concentrate is adjusted to 6.0 to 8.0, the solvent is 95% ethanol, and the addition amount of the solvent is 3 to 5 times of the volume of the concentrate; the stirring speed is 50-150 rpm; the temperature is 0-10 ℃; the separation time is 10-20 h.
8. The method for synthesizing citicoline sodium by the cytidine enzymic method according to claim 1, wherein the step of further refining in the step f is: and adding pure water into the crude citicoline sodium product for redissolving, adding activated carbon, stirring and filtering, adding 95% ethanol with the volume being 3-5 times that of the filtrate, cooling to 0-10 ℃ under stirring, separating solid from liquid after 10-20 h, and drying the solid in vacuum to obtain the finished citicoline sodium product.
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| CN116144559A (en) * | 2022-09-26 | 2023-05-23 | 天津科技大学 | Genetically engineered bacterium for producing citicoline and construction method and application thereof |
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| CN101906126A (en) * | 2010-02-09 | 2010-12-08 | 南京工业大学 | Method for separating purified cytidine diphosphate choline by hydrophobic chromatography |
| CN103849666A (en) * | 2013-05-08 | 2014-06-11 | 开平牵牛生化制药有限公司 | Method for catalytically producing citicoline sodium with immobilized enzyme |
| CN112481233A (en) * | 2020-10-23 | 2021-03-12 | 天津科技大学 | Enzyme preparation for preparing citicoline and method for preparing citicoline through enzyme catalysis |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101906126A (en) * | 2010-02-09 | 2010-12-08 | 南京工业大学 | Method for separating purified cytidine diphosphate choline by hydrophobic chromatography |
| CN103849666A (en) * | 2013-05-08 | 2014-06-11 | 开平牵牛生化制药有限公司 | Method for catalytically producing citicoline sodium with immobilized enzyme |
| CN112481233A (en) * | 2020-10-23 | 2021-03-12 | 天津科技大学 | Enzyme preparation for preparing citicoline and method for preparing citicoline through enzyme catalysis |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116144559A (en) * | 2022-09-26 | 2023-05-23 | 天津科技大学 | Genetically engineered bacterium for producing citicoline and construction method and application thereof |
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