CN108892699B - Refining method of high-purity nucleotide - Google Patents
Refining method of high-purity nucleotide Download PDFInfo
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Abstract
The invention provides a refining method of high-purity nucleotide, which at least comprises the following steps: (1) primary refining: adding a crude nucleotide product and a solvent into a reactor, adjusting the pH value to 7.0-9.0, heating to 50-70 ℃, adding a decolorizing agent, reacting for 0.5-1h at a constant temperature, filtering, and cooling the filtrate to room temperature to obtain a primary refined nucleotide solution; (2) secondary refining: treating the primary refined solution of nucleotide obtained in the step (1) by a strong-base anion exchange resin column; then eluting with a top washing liquid, and collecting a secondary refined solution; (3) and (3) crystallization: adding a salting-out agent and an organic solvent into the secondary refined solution obtained in the step (2), crystallizing, filtering, leaching and drying to obtain high-purity nucleotide; the obtained nucleotide has high purity, high yield and less impurities.
Description
Technical Field
The invention relates to the technical field of product refining, in particular to a refining method of high-purity nucleotide.
Background
Guanylic acid or adenylic acid, cytidylic acid, uridylic acid and inosinic acid can be prepared by fermentation, RNA enzymolysis, chemical synthesis and the like. They can be applied in the fields of medicine, nutrition enhancer and food additive, etc., in particular, disodium guanylate and disodium inosinate are widely applied in food flavor enhancer with larger dosage. The production technology of nucleotide such as guanylic acid, adenylic acid, cytidylic acid, uridylic acid, inosinic acid and the like is mature, but the defects of color, luster, purity and the like exist in the aspect of product quality. In particular, products obtained by fermentation or RNA enzymolysis often contain impurities such as pigments which are difficult to remove, and the quality of the products is affected.
Regarding the separation and purification method of nucleotide produced by fermentation or enzyme method, patent No. CN101993466 discloses a preparation method of disodium 5' -guanylate, which is a method of separation and purification by using strong base anion exchange resin, and the purpose is to separate disodium guanylate from guanosine or a guanosine derivative by using resin, perform ion exchange adsorption, and then analyze. Patent No. CN1177859 discloses a method for separating nucleotide from ribonuclease hydrolysate by using anion exchange resin, which uses resin to separate and purify 4 kinds of nucleotide, and obtains single nucleotide by exchange adsorption and analysis. Patent No. CN105348347 discloses a refining method of disodium 5' -guanylate, which is relatively complicated, and adopts 30-35% hydrochloric acid and 0.1-1.0mol/L sodium chloride mixed solution for elution, and GMP is unstable under acidic conditions; at such a high concentration of hydrochloric acid, disodium guanylate is decomposed into guanosine, guanine, and the like, and the dye is eluted simultaneously with elution of 0.1 to 1.0mol/L sodium chloride, and thus the purpose of removing the dye cannot be achieved.
The pigment can be removed by activated carbon decolorization, macroporous resin, decolorizing resin, etc. However, for the disodium 5 '-guanylate obtained by fermentation or RNA enzymolysis, other methods have not been able to obtain the desired effect, for example, common activated carbon must be used for decolorization under acidic conditions, while disodium 5' -guanylate forms a gel-like substance at about pH3-4, and thus cannot be handled.
Accordingly, the present invention has been made keeping in mind the above problems occurring in the prior art, and an object of the present invention is to provide a method for purifying a high-purity nucleotide, which can produce a nucleotide having a high purity and a high yield with less impurities.
Disclosure of Invention
The present invention proposes the following solutions to the problems existing in the current production processes. The nucleotide obtained by the fermentation method mainly contains impurities such as pigment, nucleoside and the like. The nucleotide obtained by RNA degradation mainly contains impurities such as pigment and other nucleotides.
In view of the above problems, the present invention provides a method for purifying a high-purity nucleotide, comprising at least the steps of:
(1) primary refining: adding a crude nucleotide product and a solvent into a reactor, adjusting the pH value to 7.0-9.0, heating to 50-70 ℃, adding a decolorizing agent, reacting for 0.5-1h at a constant temperature, filtering, and cooling the filtrate to room temperature to obtain a primary refined nucleotide solution;
(2) secondary refining: treating the primary refined solution of nucleotide obtained in the step (1) by a strong-base anion exchange resin column; then eluting with a top washing liquid, and collecting a secondary refined solution;
(3) and (3) crystallization: and (3) adding a salting-out agent and an organic solvent into the secondary refined solution obtained in the step (2), crystallizing, filtering, leaching and drying to obtain the high-purity nucleotide.
In one embodiment, the decolorizing agent in step (1) comprises one of medical activated carbon, activated carbon 301 and activated carbon 303.
In one embodiment, the strong basic anion exchange resin column packed in step (2) is a 201 strong basic anion exchange resin, an Amberlite IRA-400 anion exchange resin, a DIAION HP20 anion exchange resin, a PA312 anion exchange resin, a guanidinium strong base anion exchange resin.
In one embodiment, the ratio of the primary nucleotide refined solution to the strongly basic anion exchange resin in the step (2) is (0.3-2): 1; the nucleotide mass is in Kg and the volume of cation exchange resin is in L.
In one embodiment, the top wash in step (2) is water or a sodium chloride solution.
In one embodiment, the weight ratio of the secondary refined solution to the organic solvent and the salting-out agent in the step (3) is 1: (1-2): (0.01-0.05).
In one embodiment, the organic solvent in step (3) comprises one or more of acetone, ethanol and methanol.
In one embodiment, the salting-out agent in step (3) comprises one or more of sodium sulfate, sodium phosphate, sodium chloride, potassium formate, and sodium formate.
In one embodiment, the strongly basic anion exchange resin in step (2) is a guanidinium strongly basic anion exchange resin; the preparation raw materials of the guanidino strong base anion exchange resin comprise 4-hydroxy phenformin, 2-methyl-1- (3-methyl-2-butylene-1-yl) -1H-benzimidazole, styrene, divinylbenzene and N, N-dimethyl hexadecylamine.
In one embodiment, the method for refining high-purity nucleotide is applied to the technical field of refining 5' -disodium guanylate, adenylate, cytidylate, uridylate and inosinic acid.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a graph of example 15-disodium guanylate determined by HPLC;
FIG. 2 is a graph of adenosine measured by HPLC in example 2;
FIG. 3 is a graph of cytidylic acid of example 3 measured by HPLC;
FIG. 4 is a chromatogram obtained by HPLC of uridylic acid in example 4;
FIG. 5 is a graph of inosinic acid according to example 5 measured by HPLC;
FIG. 6 is a graph showing the relationship between the amount of strongly basic anion resin used and the decolorization rate and recovery rate.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides a refining method of high-purity nucleotide, which at least comprises the following steps:
(1) primary refining: adding a crude nucleotide product and a solvent into a reactor, adjusting the pH value to 7.0-9.0, heating to 50-70 ℃, adding a decolorizing agent, reacting for 0.5-1h at a constant temperature, filtering, and cooling the filtrate to room temperature to obtain a primary refined nucleotide solution;
(2) secondary refining: passing the primary refined solution of nucleotide obtained in step (1) through a strongly basic anion exchange resin column; then eluting with a top washing liquid, and collecting a secondary refined solution;
(3) and (3) crystallization: and (3) adding a salting-out agent and an organic solvent into the secondary refined solution obtained in the step (2), crystallizing, filtering, leaching and drying to obtain the high-purity nucleotide.
In one embodiment, the solvent in step (1) is purified water or deionized water.
In one embodiment, the reagent for adjusting the pH in the step (1) is selected from one of sodium hydroxide solution and ammonia water; preferably, the agent for adjusting the pH in step (1) is a sodium hydroxide solution.
In one embodiment, the sodium hydroxide solution is a 1mol/L aqueous sodium hydroxide solution.
In one embodiment, the decolorizing agent in step (1) comprises one of medical activated carbon, activated carbon 301, and activated carbon 303; preferably, the decolorizing agent in step (1) is activated carbon 303.
In one embodiment, the crude nucleotide in step (1) is a nucleotide obtained by fermentation or RNA enzymolysis.
In one embodiment, the weight ratio of the crude nucleotide to the solvent and the decolorizing agent in step (1) is 1: (10-30): (0.005-0.015); preferably, the weight ratio of the crude nucleotide to the solvent and the decolorizing agent in the step (1) is 1: 24: 0.01.
in one embodiment, the strong basic anion exchange resin column packed in step (2) is a 201 strong basic anion exchange resin, an Amberlite IRA-400 anion exchange resin, a DIAION HP20 anion exchange resin, a PA312 anion exchange resin, a guanidinium strong base anion exchange resin.
In one embodiment, the ratio of the primary nucleotide refined solution to the strongly basic anion exchange resin in the step (2) is (0.3-2): 1; the mass of nucleotide is measured by Kg, and the volume of cation exchange resin is measured by L; preferably, the ratio of the primary nucleotide refined solution to the strong-base anion exchange resin in the step (2) is 1: 1, the mass of nucleotides is in Kg and the volume of cation exchange resin is in L.
In one embodiment, the top wash in step (2) is a sodium chloride solution.
In one embodiment, the sodium chloride solution is a sodium chloride aqueous solution with a mass fraction of 2-5%; preferably, the sodium chloride solution is a 3% sodium chloride aqueous solution by mass fraction.
In one embodiment, the top washing liquid in the step (2) has a washing speed of 40-80L/h; preferably, the rinsing speed of the top rinsing liquid in the step (2) is 60L/h.
In one embodiment, the weight ratio of the secondary refined solution to the organic solvent and the salting-out agent in the step (3) is 1: (1-2): (0.01-0.05); preferably, the weight ratio of the secondary refined solution to the organic solvent and the salting-out agent in the step (3) is 1: 1: 0.02.
in one embodiment, the organic solvent in step (3) comprises one or more of acetone, ethanol, methanol; preferably, the organic solvent in step (3) is ethanol.
In one embodiment, the organic solvent ethanol in the step (3) is 95% ethanol or absolute ethanol by volume fraction.
In one embodiment, the salting-out agent in step (3) comprises one or more of sodium sulfate, sodium phosphate, sodium chloride, potassium formate, sodium formate; preferably, the salting-out agent in the step (3) is sodium phosphate and sodium chloride.
In one embodiment, the weight ratio of the sodium phosphate to the sodium chloride is 1: (0.5-1); preferably, the weight ratio of the sodium phosphate to the sodium chloride is 1: 0.75.
the crystallization steps in the invention are as follows:
adding a salting-out agent into the secondary refined solution obtained in the step (2), heating to 50-70 ℃, preserving heat for 1-3h, then slowly adding an organic solvent, preserving heat for 0.5-1h, slowly cooling to 40-45 ℃, preserving heat for 0.5-1h, cooling to 30-35 ℃, preserving heat for 0.5-1h, cooling to 20-25 ℃, preserving heat for 0.5-1h, filtering, leaching with 75% by volume of ethanol, 85% by volume of ethanol and 95% by volume of ethanol in sequence, and drying under reduced pressure to obtain the high-purity nucleotide.
In one embodiment, the strongly basic anion exchange resin in step (2) is a guanidinium strongly basic anion exchange resin; the preparation raw materials of the guanidine base strong base anion exchange resin comprise 4-hydroxy phenformin (CAS number: 17426-94-7), 2-methyl-1- (3-methyl-2-butene-1-yl) -1H-benzimidazole (CAS number: 95481-04-2), styrene, divinylbenzene and N, N-dimethyl hexadecylamine.
The preparation method of the guanidino strong base anion exchange resin comprises the following steps:
(1) adding purified water, gelatin and sodium phosphate into a reactor, and heating to 50 ℃ to obtain a water phase; uniformly mixing styrene, divinylbenzene, benzoyl peroxide and a pore-foaming agent to obtain an oil phase; adding the uniformly mixed oil phase into a water phase, heating to 80 ℃, carrying out heat preservation reaction for 0.5h, heating to 90 ℃, carrying out heat preservation reaction for 0.5h, heating to 95 ℃, carrying out heat preservation reaction for 0.5h, cooling to 85 ℃, carrying out heat preservation reaction for 0.5h, cooling, washing with purified water, and drying to obtain a first prefabricated material, wherein a pore-forming agent is methylcyclopropane, methylisobutylketone, fatty alcohol and paraffin, and the weight ratio of the methylcyclopropane to the methylisobutylketone to the fatty alcohol to the paraffin is 3: 2: 1: 1; the weight ratio of the purified water to the gelatin to the sodium phosphate is 100: 1: 2; the weight ratio of the styrene to the divinylbenzene, the benzoyl peroxide and the pore-foaming agent is 20: 10: 0.2: 23; the weight ratio of the purified water to the styrene is 5: 1;
(2) adding the first prefabricated material obtained in the step (1), N-dimethylformamide, benzoyl peroxide and 2-methyl-1- (3-methyl-2-butene-1-yl) -1H-benzimidazole into a reactor, heating to 90 ℃, carrying out heat preservation reaction for 1H, heating to 95 ℃, carrying out heat preservation reaction for 0.5H, cooling to 85 ℃, carrying out heat preservation reaction for 1H, cooling, washing with absolute ethyl alcohol, and drying to obtain a second prefabricated material; the weight ratio of the first preform to the N, N-dimethylformamide, the benzoyl peroxide, the 2-methyl-1- (3-methyl-2-buten-1-yl) -1H-benzimidazole is 1: 5: 0.01: 0.3;
(3) adding the second prefabricated material obtained in the step (2), dichloroethane and N, N-dimethylhexadecylamine into a reactor, heating to 95 ℃, carrying out heat preservation reaction for 10 hours, filtering, washing and drying, adding 4-hydroxy-phenformin, N-dimethylformamide and sodium ethoxide with the concentration of 2.5mol/L, heating to 80 ℃, carrying out heat preservation reaction for 5 hours, cooling to room temperature, filtering and washing to obtain a third prefabricated material; the weight ratio of the second preform to the dichloroethane, the N, N-dimethylhexadecylamine, the N, N-dimethylformamide, the 4-hydroxyphenylbiguanide, the sodium ethoxide with a concentration of 2.5mol/L is 1: 3: 0.1: 3: 2.4: 0.3;
(4) adding n-heptanol, paraformaldehyde and dichloromethane into a reactor, slowly dropwise adding thionyl chloride, controlling the temperature to be 0-5 ℃, heating to 5-10 ℃ after dropwise adding, and carrying out heat preservation reaction for 3 hours; then swelling the third prefabricated material in the step (3) for 10 hours by using chlorine dioxide, dropwise adding stannic chloride, carrying out heat preservation reaction at 15-20 ℃ for 24 hours, filtering, washing by using 20% hydrochloric acid aqueous solution, dioxane and purified water respectively, and drying under reduced pressure at 60 ℃ to obtain a fourth prefabricated material; the weight ratio of the n-heptanol to the paraformaldehyde, the dichloromethane, the thionyl chloride, the third preform, the tin tetrachloride is 1: 0.3: 1.2: 1.6: 0.4;
(5) adding the fourth prefabricated material obtained in the step (4) and dioxane into a reactor, stirring for 0.5h, slowly dropwise adding a mixed solution of 10% by mass of a sodium chloride aqueous solution and triethylamine, heating to 30 ℃ after dropwise adding, carrying out heat preservation reaction for 14h, filtering, washing with a 2% by mass sodium chloride aqueous solution, 1% by mass of a hydrochloric acid aqueous solution and purified water in sequence, and drying at 60 ℃ under reduced pressure to obtain a guanidyl strong base anion exchange resin; the weight ratio of the fourth preform to the dioxane, the 10% sodium chloride aqueous solution and the triethylamine is 1: 5: 0.3: 2.
in one embodiment, the method for refining high-purity nucleotide is applied to the technical field of refining 5-disodium guanylate, adenylate, cytidylate, uridylate and inosinic acid.
The disodium 5' -guanylate is a strong freshener and can be widely applied to seasonings such as specially fresh monosodium glutamate, chicken essence, specially fresh soy sauce and the like; in addition, the disodium 5' -guanylate can also be used as an additive of infant milk powder.
In the invention, about 70% of pigment with strong negative charges exists in the 5 '-guanylate disodium enzymolysis solution or fermentation liquor, the pigment can be exchanged and adsorbed only by strong-base anion exchange resin, and when the pigment is top-washed by water or 3% sodium chloride, the 5' -guanylate disodium and other nucleotides can be washed off, and the pigment is difficult to elute. This can remove almost all of these pigments which are difficult to remove, and a high yield can be obtained.
The invention can remove about 30% of pigment by adopting the active carbon for decolorization under neutral conditions of 767, 303 and the like. About 70% of pigment impurities are found to be negatively charged through research, the primary purified solution of the nucleotide obtained in the step (1) is passed through a strongly basic anion exchange resin column to make the pigment almost completely adsorbed on the column, and the nucleotide is hardly exchange-adsorbed due to excess; stopping when the column is saturated by pigment exchange adsorption, top-washing with water or NaCl solution with mass fraction of 3%, and collecting the nucleotide solution passing through the column as secondary refined solution.
In addition, the thermal stability of the strongly basic anion exchange resin is poor, and the electropositivity of carbon atoms is increased mainly because benzyl carbon atoms of the resin are directly connected with quaternary amine nitrogen atoms, so that the thermal stability is reduced, and the application range of the strongly basic anion exchange resin is limited.
Therefore, aiming at the problem, the invention prepares the guanidino strong base anion exchange resin by 4-hydroxy phenformin, 2-methyl-1- (3-methyl-2-butylene-1-yl) -1H-benzimidazole, styrene, divinylbenzene and N, N-dimethyl hexadecylamine, and the resin not only ensures higher strong base exchange capacity, but also improves the thermal stability and mechanical strength of the resin and prolongs the service life of the resin.
In addition, since guanidine cations have three nitrogen atoms symmetrically distributed around carbon, have three C — N bonds of the same length and are shorter than common C — N bonds or C ═ N bonds, and since guanidine cations have a conjugation effect and have positive charges distributed evenly over three nitrogen atoms, the bond lengths are averaged, and compounds having such a structure are stable, guanidine has a strong tendency to form such a stable structure by receiving hydrogen ions, and exhibits strong basicity. According to the invention, the 2-methyl-1- (3-methyl-2-butene-1-yl) -1H-benzimidazole and 4-hydroxy-phenformin resin spheres are added into the raw materials for preparing the guanidine-based strong-base anion exchange resin, the surfaces and the interiors of the spheres have pore structures, the specific surface area of the whole sphere is improved, the purpose of improving the reaction rate is achieved, the deformation resistance of the spheres is increased, the thermal stability and the mechanical strength of the guanidine-based strong-base anion exchange resin can be improved, the adsorption capacity of impurities and the analysis capacity of nucleotides are promoted, and the purity and the yield of the nucleotides are improved.
The method for calculating the yield and the decolorization rate comprises the following steps:
and measuring the A430 and A260 values of the solution before and after strong-base anion exchange by using a spectrophotometer. And calculating the recovery rate and the decolorization rate of the nucleotide.
Af260Represents the nucleotide content of the solution after anion exchange; a. thes260Represents the nucleotide content of the initial solution;
Vfrepresents the volume of the solution after exchange; vsRepresenting an initial volume;
Af430representing the pigment content of the solution after the exchange; a. thes430Indicates the initial solution pigment content.
The resin dosage by the static method is explored:
100mL of GMP solution with the concentration of 100g/L decolorized by activated carbon is respectively added with 0, 5, 10, 15, 20 and 25mL of wet chlorine type strong anion resin, and the mixture is put into a shaking table for oscillation. After 2 hours, the mixture is filtered through kieselguhr, the volume is measured, and the filtrate A is determined430、A260A value; the results are shown in FIG. 6.
The liquid phase detection conditions of the nucleotide in the invention are as follows: the chromatographic column used was Hypersil ODS25 μm (4.6X 250mm), the mobile phase was 0.025mol/L ammonium acetate solution (pH adjusted to 5.5 with acetic acid), the flow rate was 0.8ml/min, at 254nm wavelength, and the detection was carried out with UV detector.
In the present invention, all the raw materials are commercially available and purchased from national chemical reagents unless otherwise specified.
To further illustrate the present invention, the following examples are provided for illustration.
Example 1
The method for refining high-purity nucleotide at least comprises the following steps:
(1) primary refining: adding a crude nucleotide product and a solvent into a reactor, adjusting the pH value to 8.5-9.0, heating to 60 ℃, adding a decolorizing agent, carrying out heat preservation reaction for 0.5h, filtering, and then cooling the filtrate to room temperature to obtain a primary refined nucleotide solution; the solvent in the step (1) is purified water; the reagent for adjusting the pH value in the step (1) is sodium hydroxide solution; the sodium hydroxide solution is 1mol/L sodium hydroxide solution; the decolorizing agent in the step (1) is activated carbon 303; the crude nucleotide in the step (1) is nucleotide obtained by fermentation liquor or RNA enzymolysis liquid; the weight ratio of the crude nucleotide product to the solvent to the decolorizing agent in the step (1) is 1: 24: 0.01;
(2) secondary refining: treating the primary refined solution of nucleotide obtained in the step (1) by a strong-base anion exchange resin column; then top washing with a top washing liquid, and collecting a secondary refined solution; the ratio of the primary refined solution of nucleotide to the strongly basic anion exchange resin in the step (2) is 1: 1, the mass of nucleotide is measured by Kg, and the volume of cation exchange resin is measured by L; the resin in the strong-base anion exchange resin column is guanidino strong-base anion exchange resin; the top washing liquid in the step (2) is a sodium chloride solution; the sodium chloride solution is a sodium chloride aqueous solution with the mass fraction of 3%; the leaching speed of the top washing liquid in the step (2) is 60L/h;
(3) and (3) crystallization: adding a salting-out agent into the secondary refined solution obtained in the step (2), heating to 60 ℃, preserving heat for 2 hours, then slowly adding an organic solvent, preserving heat for 0.5 hour, slowly cooling to 40-45 ℃, preserving heat for 0.5 hour, cooling to 30 ℃, preserving heat for 0.5 hour, cooling to 20-25 ℃, preserving heat for 1 hour, filtering, leaching with 75% by volume of ethanol, 85% by volume of ethanol and 95% by volume of ethanol in sequence, and drying under reduced pressure to obtain high-purity nucleotide; the weight ratio of the secondary refined solution to the organic solvent to the salting-out agent in the step (3) is 1: 1: 0.04; the organic solvent in the step (3) is ethanol; the organic solvent ethanol in the step (3) is ethanol with the volume fraction of 95 percent; the salting-out agent in the step (3) is sodium phosphate and sodium chloride; the weight ratio of the sodium phosphate to the sodium chloride is 1: 0.75.
the preparation method of the guanidino strong base anion exchange resin comprises the following steps:
(1) adding purified water, gelatin and sodium phosphate into a reactor, and heating to 50 ℃ to obtain a water phase; uniformly mixing styrene, divinylbenzene, benzoyl peroxide and a pore-foaming agent to obtain an oil phase; adding the uniformly mixed oil phase into a water phase, heating to 80 ℃, carrying out heat preservation reaction for 0.5h, heating to 90 ℃, carrying out heat preservation reaction for 0.5h, heating to 95 ℃, carrying out heat preservation reaction for 0.5h, cooling to 85 ℃, carrying out heat preservation reaction for 0.5h, cooling, washing with purified water, and drying to obtain a first prefabricated material, wherein a pore-forming agent is methylcyclopropane, methylisobutylketone, fatty alcohol and paraffin, and the weight ratio of the methylcyclopropane to the methylisobutylketone to the fatty alcohol to the paraffin is 3: 2: 1: 1; the weight ratio of the purified water to the gelatin to the sodium phosphate is 100: 1: 2; the weight ratio of the styrene to the divinylbenzene, the benzoyl peroxide and the pore-foaming agent is 20: 10: 0.2: 23; the weight ratio of the purified water to the styrene is 5: 1;
(2) adding the first prefabricated material obtained in the step (1), N-dimethylformamide, benzoyl peroxide and 2-methyl-1- (3-methyl-2-butene-1-yl) -1H-benzimidazole into a reactor, heating to 90 ℃, carrying out heat preservation reaction for 1H, heating to 95 ℃, carrying out heat preservation reaction for 0.5H, cooling to 85 ℃, carrying out heat preservation reaction for 1H, cooling, washing with absolute ethyl alcohol, and drying to obtain a second prefabricated material; the weight ratio of the first preform to the N, N-dimethylformamide, the benzoyl peroxide, the 2-methyl-1- (3-methyl-2-buten-1-yl) -1H-benzimidazole is 1: 5: 0.01: 0.3;
(3) adding the second prefabricated material obtained in the step (2), dichloroethane and N, N-dimethylhexadecylamine into a reactor, heating to 95 ℃, carrying out heat preservation reaction for 10 hours, filtering, washing and drying, adding 4-hydroxy-phenformin, N-dimethylformamide and sodium ethoxide with the concentration of 2.5mol/L, heating to 80 ℃, carrying out heat preservation reaction for 5 hours, cooling to room temperature, filtering and washing to obtain a third prefabricated material; the weight ratio of the second preform to the dichloroethane, the N, N-dimethylhexadecylamine, the N, N-dimethylformamide, the 4-hydroxyphenylbiguanide, the sodium ethoxide with a concentration of 2.5mol/L is 1: 3: 0.1: 3: 2.4: 0.3;
(4) adding n-heptanol, paraformaldehyde and dichloromethane into a reactor, slowly dropwise adding thionyl chloride, controlling the temperature to be 0-5 ℃, heating to 5-10 ℃ after dropwise adding, and carrying out heat preservation reaction for 3 hours; then swelling the third prefabricated material in the step (3) for 10 hours by using chlorine dioxide, dropwise adding stannic chloride, carrying out heat preservation reaction at 15-20 ℃ for 24 hours, filtering, washing by using 20% hydrochloric acid aqueous solution, dioxane and purified water respectively, and drying under reduced pressure at 60 ℃ to obtain a fourth prefabricated material; the weight ratio of the n-heptanol to the paraformaldehyde, the dichloromethane, the thionyl chloride, the third preform, the tin tetrachloride is 1: 0.3: 1.2: 1.6: 0.4;
(5) adding the fourth prefabricated material obtained in the step (4) and dioxane into a reactor, stirring for 0.5h, slowly dropwise adding a mixed solution of 10% by mass of a sodium chloride aqueous solution and triethylamine, heating to 30 ℃ after dropwise adding, carrying out heat preservation reaction for 14h, filtering, washing with a 2% by mass sodium chloride aqueous solution, 1% by mass of a hydrochloric acid aqueous solution and purified water in sequence, and drying at 60 ℃ under reduced pressure to obtain a guanidyl strong base anion exchange resin; the weight ratio of the fourth preform to the dioxane, the 10% sodium chloride aqueous solution and the triethylamine is 1: 5: 0.3: 2.
the nucleotide is 5-disodium guanylate, the content of the finally obtained finished product is 99.87% by HPLC (high performance liquid chromatography), and the infrared spectrum is consistent with that of a standard product.
Example 2
The method for refining high-purity nucleotide at least comprises the following steps:
(1) primary refining: adding a crude nucleotide product and a solvent into a reactor, adjusting the pH value to 8.0-8.5, heating to 60 ℃, adding a decolorizing agent, carrying out heat preservation reaction for 0.5h, filtering, and then cooling the filtrate to room temperature to obtain a primary refined nucleotide solution; the solvent in the step (1) is purified water; the reagent for adjusting the pH value in the step (1) is sodium hydroxide solution; the sodium hydroxide solution is 1mol/L sodium hydroxide solution; the decolorizing agent in the step (1) is activated carbon 303; the crude nucleotide in the step (1) is nucleotide obtained by fermentation liquor or RNA enzymolysis liquid; the weight ratio of the crude nucleotide product to the solvent to the decolorizing agent in the step (1) is 1: 24: 0.01;
(2) secondary refining: treating the primary refined solution of nucleotide obtained in the step (1) by a strong-base anion exchange resin column; then top washing with a top washing liquid, and collecting a secondary refined solution; the ratio of the primary refined solution of nucleotide to the strongly basic anion exchange resin in the step (2) is 1: 1, the mass of nucleotide is measured by Kg, and the volume of cation exchange resin is measured by L; the resin in the strong-base anion exchange resin column is guanidino strong-base anion exchange resin; the top washing liquid in the step (2) is a sodium chloride solution; the sodium chloride solution is a sodium chloride aqueous solution with the mass fraction of 3%; the leaching speed of the top washing liquid in the step (2) is 60L/h;
(3) and (3) crystallization: adding a salting-out agent into the secondary refined solution obtained in the step (2), heating to 60 ℃, preserving heat for 2 hours, then slowly adding an organic solvent, preserving heat for 0.5 hour, slowly cooling to 40-45 ℃, preserving heat for 0.5 hour, cooling to 30 ℃, preserving heat for 0.5 hour, cooling to 20-25 ℃, preserving heat for 1 hour, filtering, leaching with 75% by volume of ethanol, 85% by volume of ethanol and 95% by volume of ethanol in sequence, and drying under reduced pressure to obtain high-purity nucleotide; the weight ratio of the secondary refined solution to the organic solvent to the salting-out agent in the step (3) is 1: 1: 0.04; the organic solvent in the step (3) is ethanol; the organic solvent ethanol in the step (3) is ethanol with the volume fraction of 95 percent; the salting-out agent in the step (3) is sodium phosphate and sodium chloride; the weight ratio of the sodium phosphate to the sodium chloride is 1: 0.75.
the preparation method of the guanidino strong base anion exchange resin is the same as that of example 1.
The nucleotide is adenylic acid, the content of the finally obtained finished product is 100% by HPLC (high performance liquid chromatography), and the infrared spectrum is consistent with that of a standard product.
Example 3
The method for refining high-purity nucleotide at least comprises the following steps:
(1) primary refining: adding a crude nucleotide product and a solvent into a reactor, adjusting the pH value to 7.0-7.5, heating to 60 ℃, adding a decolorizing agent, carrying out heat preservation reaction for 0.5h, filtering, and then cooling the filtrate to room temperature to obtain a primary refined nucleotide solution; the solvent in the step (1) is purified water; the reagent for adjusting the pH value in the step (1) is sodium hydroxide solution; the sodium hydroxide solution is 1mol/L sodium hydroxide solution; the decolorizing agent in the step (1) is activated carbon 303; the crude nucleotide in the step (1) is nucleotide obtained by fermentation liquor or RNA enzymolysis liquid; the weight ratio of the crude nucleotide product to the solvent to the decolorizing agent in the step (1) is 1: 24: 0.01;
(2) secondary refining: treating the primary refined solution of nucleotide obtained in the step (1) by a strong-base anion exchange resin column; then top washing with a top washing liquid, and collecting a secondary refined solution; the ratio of the primary refined solution of nucleotide to the strongly basic anion exchange resin in the step (2) is 1: 1, the mass of nucleotide is measured by Kg, and the volume of cation exchange resin is measured by L; the resin in the strong-base anion exchange resin column is guanidino strong-base anion exchange resin; the top washing liquid in the step (2) is a sodium chloride solution; the sodium chloride solution is a sodium chloride aqueous solution with the mass fraction of 3%; the leaching speed of the top washing liquid in the step (2) is 60L/h;
(3) and (3) crystallization: adding a salting-out agent into the secondary refined solution obtained in the step (2), heating to 60 ℃, preserving heat for 2 hours, then slowly adding an organic solvent, preserving heat for 0.5 hour, slowly cooling to 40-45 ℃, preserving heat for 0.5 hour, cooling to 30 ℃, preserving heat for 0.5 hour, cooling to 20-25 ℃, preserving heat for 1 hour, filtering, leaching with 75% by volume of ethanol, 85% by volume of ethanol and 95% by volume of ethanol in sequence, and drying under reduced pressure to obtain high-purity nucleotide; the weight ratio of the secondary refined solution to the organic solvent to the salting-out agent in the step (3) is 1: 1: 0.04; the organic solvent in the step (3) is ethanol; the organic solvent ethanol in the step (3) is ethanol with the volume fraction of 95 percent; the salting-out agent in the step (3) is sodium phosphate and sodium chloride; the weight ratio of the sodium phosphate to the sodium chloride is 1: 0.75.
the preparation method of the guanidino strong base anion exchange resin is the same as that of example 1.
The nucleotide is cytidylic acid, the content of the finally obtained finished product is 100 percent by HPLC (high performance liquid chromatography), and the infrared spectrum is consistent with that of a standard product.
Example 4
The method for refining high-purity nucleotide at least comprises the following steps:
(1) primary refining: adding a crude nucleotide product and a solvent into a reactor, adjusting the pH value to 7.5-8.0, heating to 60 ℃, adding a decolorizing agent, carrying out heat preservation reaction for 0.5h, filtering, and then cooling the filtrate to room temperature to obtain a primary refined nucleotide solution; the solvent in the step (1) is purified water; the reagent for adjusting the pH value in the step (1) is sodium hydroxide solution; the sodium hydroxide solution is 1mol/L sodium hydroxide solution; the decolorizing agent in the step (1) is activated carbon 303; the crude nucleotide in the step (1) is nucleotide obtained by fermentation liquor or RNA enzymolysis liquid; the weight ratio of the crude nucleotide product to the solvent to the decolorizing agent in the step (1) is 1: 24: 0.01;
(2) secondary refining: treating the primary refined solution of nucleotide obtained in the step (1) by a strong-base anion exchange resin column; then top washing with a top washing liquid, and collecting a secondary refined solution; the ratio of the primary refined solution of nucleotide to the strongly basic anion exchange resin in the step (2) is 1: 1, the mass of nucleotide is measured by Kg, and the volume of cation exchange resin is measured by L; the resin in the strong-base anion exchange resin column is guanidino strong-base anion exchange resin; the top washing liquid in the step (2) is a sodium chloride solution; the sodium chloride solution is a sodium chloride aqueous solution with the mass fraction of 3%; the leaching speed of the top washing liquid in the step (2) is 60L/h;
(3) and (3) crystallization: adding a salting-out agent into the secondary refined solution obtained in the step (2), heating to 60 ℃, preserving heat for 2 hours, then slowly adding an organic solvent, preserving heat for 0.5 hour, slowly cooling to 40-45 ℃, preserving heat for 0.5 hour, cooling to 30 ℃, preserving heat for 0.5 hour, cooling to 20-25 ℃, preserving heat for 1 hour, filtering, leaching with 75% by volume of ethanol, 85% by volume of ethanol and 95% by volume of ethanol in sequence, and drying under reduced pressure to obtain high-purity nucleotide; the weight ratio of the secondary refined solution to the organic solvent to the salting-out agent in the step (3) is 1: 1: 0.04; the organic solvent in the step (3) is ethanol; the organic solvent ethanol in the step (3) is ethanol with the volume fraction of 95 percent; the salting-out agent in the step (3) is sodium phosphate and sodium chloride; the weight ratio of the sodium phosphate to the sodium chloride is 1: 0.75.
the preparation method of the guanidino strong base anion exchange resin is the same as that of example 1.
The nucleotide is uridylic acid, the content of the finally obtained finished product is 99.88 percent by HPLC (high performance liquid chromatography), and the infrared spectrum is consistent with that of a standard product.
Example 5
The method for refining high-purity nucleotide at least comprises the following steps:
(1) primary refining: adding a crude nucleotide product and a solvent into a reactor, adjusting the pH value to 7.5-8.0, heating to 60 ℃, adding a decolorizing agent, carrying out heat preservation reaction for 0.5h, filtering, and then cooling the filtrate to room temperature to obtain a primary refined nucleotide solution; the solvent in the step (1) is purified water; the reagent for adjusting the pH value in the step (1) is sodium hydroxide solution; the sodium hydroxide solution is 1mol/L sodium hydroxide solution; the decolorizing agent in the step (1) is activated carbon 303; the crude nucleotide in the step (1) is nucleotide obtained by fermentation liquor or RNA enzymolysis liquid; the weight ratio of the crude nucleotide product to the solvent to the decolorizing agent in the step (1) is 1: 24: 0.01;
(2) secondary refining: treating the primary refined solution of nucleotide obtained in the step (1) by a strong-base anion exchange resin column; then top washing with a top washing liquid, and collecting a secondary refined solution; the ratio of the primary refined solution of nucleotide to the strongly basic anion exchange resin in the step (2) is 1: 1, the mass of nucleotide is measured by Kg, and the volume of cation exchange resin is measured by L; the resin in the strong-base anion exchange resin column is guanidino strong-base anion exchange resin; the top washing liquid in the step (2) is a sodium chloride solution; the sodium chloride solution is a sodium chloride aqueous solution with the mass fraction of 3%; the leaching speed of the top washing liquid in the step (2) is 60L/h;
(3) and (3) crystallization: adding a salting-out agent into the secondary refined solution obtained in the step (2), heating to 60 ℃, preserving heat for 2 hours, then slowly adding an organic solvent, preserving heat for 0.5 hour, slowly cooling to 40-45 ℃, preserving heat for 0.5 hour, cooling to 30 ℃, preserving heat for 0.5 hour, cooling to 20-25 ℃, preserving heat for 1 hour, filtering, leaching with 75% by volume of ethanol, 85% by volume of ethanol and 95% by volume of ethanol in sequence, and drying under reduced pressure to obtain high-purity nucleotide; the weight ratio of the secondary refined solution to the organic solvent to the salting-out agent in the step (3) is 1: 1: 0.04; the organic solvent in the step (3) is ethanol; the organic solvent ethanol in the step (3) is ethanol with the volume fraction of 95 percent; the salting-out agent in the step (3) is sodium phosphate and sodium chloride; the weight ratio of the sodium phosphate to the sodium chloride is 1: 0.75.
the preparation method of the guanidino strong base anion exchange resin is the same as that of example 1.
The nucleotide is inosinic acid, the content of the finally obtained finished product is 99.93 percent by HPLC (high performance liquid chromatography), and the infrared spectrum is consistent with that of a standard product.
Example 6
The method for refining high-purity nucleotide at least comprises the following steps:
(1) primary refining: adding a crude nucleotide product and a solvent into a reactor, adjusting the pH value to 8.5-9.0, heating to 60 ℃, adding a decolorizing agent, carrying out heat preservation reaction for 0.5h, filtering, and then cooling the filtrate to room temperature to obtain a primary refined nucleotide solution; the solvent in the step (1) is purified water; the reagent for adjusting the pH value in the step (1) is sodium hydroxide solution; the sodium hydroxide solution is 1mol/L sodium hydroxide solution; the decolorizing agent in the step (1) is activated carbon 303; the crude nucleotide in the step (1) is nucleotide obtained by fermentation liquor or RNA enzymolysis liquid; the weight ratio of the crude nucleotide product to the solvent to the decolorizing agent in the step (1) is 1: 24: 0.01;
(2) secondary refining: treating the primary refined solution of nucleotide obtained in the step (1) by a strong-base anion exchange resin column; then top washing with a top washing liquid, and collecting a secondary refined solution; the ratio of the primary refined solution of nucleotide to the strongly basic anion exchange resin in the step (2) is 0.3: 1, the mass of nucleotide is measured by Kg, and the volume of cation exchange resin is measured by L; the resin in the strong-base anion exchange resin column is guanidino strong-base anion exchange resin; the top washing liquid in the step (2) is a sodium chloride solution; the sodium chloride solution is a sodium chloride aqueous solution with the mass fraction of 3%; the leaching speed of the top washing liquid in the step (2) is 60L/h;
(3) and (3) crystallization: adding a salting-out agent into the secondary refined solution obtained in the step (2), heating to 60 ℃, preserving heat for 2 hours, then slowly adding an organic solvent, preserving heat for 0.5 hour, slowly cooling to 40-45 ℃, preserving heat for 0.5 hour, cooling to 30 ℃, preserving heat for 0.5 hour, cooling to 20-25 ℃, preserving heat for 1 hour, filtering, leaching with 75% by volume of ethanol, 85% by volume of ethanol and 95% by volume of ethanol in sequence, and drying under reduced pressure to obtain high-purity nucleotide; the weight ratio of the secondary refined solution to the organic solvent to the salting-out agent in the step (3) is 1: 1: 0.04; the organic solvent in the step (3) is ethanol; the organic solvent ethanol in the step (3) is ethanol with the volume fraction of 95 percent; the salting-out agent in the step (3) is sodium phosphate and sodium chloride; the weight ratio of the sodium phosphate to the sodium chloride is 1: 0.75.
the preparation method of the guanidino strong base anion exchange resin is the same as that of example 1.
Example 7
The method for refining high-purity nucleotide at least comprises the following steps:
(1) primary refining: adding a crude nucleotide product and a solvent into a reactor, adjusting the pH value to 8.5-9.0, heating to 60 ℃, adding a decolorizing agent, carrying out heat preservation reaction for 0.5h, filtering, and then cooling the filtrate to room temperature to obtain a primary refined nucleotide solution; the solvent in the step (1) is purified water; the reagent for adjusting the pH value in the step (1) is sodium hydroxide solution; the sodium hydroxide solution is 1mol/L sodium hydroxide solution; the decolorizing agent in the step (1) is activated carbon 303; the crude nucleotide in the step (1) is nucleotide obtained by fermentation liquor or RNA enzymolysis liquid; the weight ratio of the crude nucleotide product to the solvent to the decolorizing agent in the step (1) is 1: 24: 0.01;
(2) secondary refining: treating the primary refined solution of nucleotide obtained in the step (1) by a strong-base anion exchange resin column; then top washing with a top washing liquid, and collecting a secondary refined solution; the ratio of the primary refined solution of nucleotide to the strongly basic anion exchange resin in the step (2) is 2: 1, the mass of nucleotide is measured by Kg, and the volume of cation exchange resin is measured by L; the resin in the strong-base anion exchange resin column is guanidino strong-base anion exchange resin; the top washing liquid in the step (2) is a sodium chloride solution; the sodium chloride solution is a sodium chloride aqueous solution with the mass fraction of 3%; the leaching speed of the top washing liquid in the step (2) is 60L/h;
(3) and (3) crystallization: adding a salting-out agent into the secondary refined solution obtained in the step (2), heating to 60 ℃, preserving heat for 2 hours, then slowly adding an organic solvent, preserving heat for 0.5 hour, slowly cooling to 40-45 ℃, preserving heat for 0.5 hour, cooling to 30 ℃, preserving heat for 0.5 hour, cooling to 20-25 ℃, preserving heat for 1 hour, filtering, leaching with 75% by volume of ethanol, 85% by volume of ethanol and 95% by volume of ethanol in sequence, and drying under reduced pressure to obtain high-purity nucleotide; the weight ratio of the secondary refined solution to the organic solvent to the salting-out agent in the step (3) is 1: 1: 0.04; the organic solvent in the step (3) is ethanol; the organic solvent ethanol in the step (3) is ethanol with the volume fraction of 95 percent; the salting-out agent in the step (3) is sodium phosphate and sodium chloride; the weight ratio of the sodium phosphate to the sodium chloride is 1: 0.75.
the preparation method of the guanidino strong base anion exchange resin is the same as that of example 1.
Comparative example 1
The method for refining high-purity nucleotide at least comprises the following steps:
(1) primary refining: adding a crude nucleotide product and a solvent into a reactor, adjusting the pH value to 8.5-9.0, heating to 60 ℃, adding a decolorizing agent, carrying out heat preservation reaction for 0.5h, filtering, and then cooling the filtrate to room temperature to obtain a primary refined nucleotide solution; the solvent in the step (1) is purified water; the reagent for adjusting the pH value in the step (1) is sodium hydroxide solution; the sodium hydroxide solution is 1mol/L sodium hydroxide solution; the decolorizing agent in the step (1) is activated carbon 303; the crude nucleotide in the step (1) is nucleotide obtained by fermentation liquor or RNA enzymolysis liquid; the weight ratio of the crude nucleotide product to the solvent to the decolorizing agent in the step (1) is 1: 24: 0.01;
(2) secondary refining: treating the primary refined solution of nucleotide obtained in the step (1) by a strong-base anion exchange resin column; then top washing with a top washing liquid, and collecting a secondary refined solution; the ratio of the primary refined solution of nucleotide to the strongly basic anion exchange resin in the step (2) is 1: 1, the mass of nucleotide is measured by Kg, and the volume of cation exchange resin is measured by L; the resin in the strong basic anion exchange resin column is 201 strong basic anion exchange resin; the top washing liquid in the step (2) is a sodium chloride solution; the sodium chloride solution is a sodium chloride aqueous solution with the mass fraction of 3%; the leaching speed of the top washing liquid in the step (2) is 60L/h;
(3) and (3) crystallization: adding a salting-out agent into the secondary refined solution obtained in the step (2), heating to 60 ℃, preserving heat for 2 hours, then slowly adding an organic solvent, preserving heat for 0.5 hour, slowly cooling to 40-45 ℃, preserving heat for 0.5 hour, cooling to 30 ℃, preserving heat for 0.5 hour, cooling to 20-25 ℃, preserving heat for 1 hour, filtering, leaching with 75% by volume of ethanol, 85% by volume of ethanol and 95% by volume of ethanol in sequence, and drying under reduced pressure to obtain high-purity nucleotide; the weight ratio of the secondary refined solution to the organic solvent to the salting-out agent in the step (3) is 1: 1: 0.04; the organic solvent in the step (3) is ethanol; the organic solvent ethanol in the step (3) is ethanol with the volume fraction of 95 percent; the salting-out agent in the step (3) is sodium phosphate and sodium chloride; the weight ratio of the sodium phosphate to the sodium chloride is 1: 0.75.
the nucleotide is 5-disodium guanylate.
Comparative example 2
The method for refining high-purity nucleotide at least comprises the following steps:
(1) primary refining: adding a crude nucleotide product and a solvent into a reactor, adjusting the pH value to 8.5-9.0, heating to 60 ℃, adding a decolorizing agent, carrying out heat preservation reaction for 0.5h, filtering, and then cooling the filtrate to room temperature to obtain a primary refined nucleotide solution; the solvent in the step (1) is purified water; the reagent for adjusting the pH value in the step (1) is sodium hydroxide solution; the sodium hydroxide solution is 1mol/L sodium hydroxide solution; the decolorizing agent in the step (1) is activated carbon 303; the crude nucleotide in the step (1) is nucleotide obtained by fermentation liquor or RNA enzymolysis liquid; the weight ratio of the crude nucleotide product to the solvent to the decolorizing agent in the step (1) is 1: 24: 0.01;
(2) secondary refining: treating the primary refined solution of nucleotide obtained in the step (1) by a strong-base anion exchange resin column; then top washing with a top washing liquid, and collecting a secondary refined solution; the ratio of the primary refined solution of nucleotide to the strongly basic anion exchange resin in the step (2) is 1: 1, the mass of nucleotide is measured by Kg, and the volume of cation exchange resin is measured by L; the resin in the strong basic anion exchange resin column is PA312 anion exchange resin; the top washing liquid in the step (2) is a sodium chloride solution; the sodium chloride solution is a sodium chloride aqueous solution with the mass fraction of 3%; the leaching speed of the top washing liquid in the step (2) is 60L/h;
(3) and (3) crystallization: adding a salting-out agent into the secondary refined solution obtained in the step (2), heating to 60 ℃, preserving heat for 2 hours, then slowly adding an organic solvent, preserving heat for 0.5 hour, slowly cooling to 40-45 ℃, preserving heat for 0.5 hour, cooling to 30 ℃, preserving heat for 0.5 hour, cooling to 20-25 ℃, preserving heat for 1 hour, filtering, leaching with 75% by volume of ethanol, 85% by volume of ethanol and 95% by volume of ethanol in sequence, and drying under reduced pressure to obtain high-purity nucleotide; the weight ratio of the secondary refined solution to the organic solvent to the salting-out agent in the step (3) is 1: 1: 0.04; the organic solvent in the step (3) is ethanol; the organic solvent ethanol in the step (3) is ethanol with the volume fraction of 95 percent; the salting-out agent in the step (3) is sodium phosphate and sodium chloride; the weight ratio of the sodium phosphate to the sodium chloride is 1: 0.75.
the preparation method of the guanidino strong base anion exchange resin is the same as that of example 1.
The nucleotide is 5-disodium guanylate.
Comparative example 3
The method for refining high-purity nucleotide at least comprises the following steps:
(1) primary refining: adding a crude nucleotide product and a solvent into a reactor, adjusting the pH value to 8.5-9.0, heating to 60 ℃, adding a decolorizing agent, carrying out heat preservation reaction for 0.5h, filtering, and then cooling the filtrate to room temperature to obtain a primary refined nucleotide solution; the solvent in the step (1) is purified water; the reagent for adjusting the pH value in the step (1) is sodium hydroxide solution; the sodium hydroxide solution is 1mol/L sodium hydroxide solution; the decolorizing agent in the step (1) is activated carbon 303; the crude nucleotide in the step (1) is nucleotide obtained by fermentation liquor or RNA enzymolysis liquid; the weight ratio of the crude nucleotide product to the solvent to the decolorizing agent in the step (1) is 1: 24: 0.01;
(2) secondary refining: treating the primary refined solution of nucleotide obtained in the step (1) by a strong-base anion exchange resin column; then top washing with a top washing liquid, and collecting a secondary refined solution; the ratio of the primary refined solution of nucleotide to the strongly basic anion exchange resin in the step (2) is 1: 1, the mass of nucleotide is measured by Kg, and the volume of cation exchange resin is measured by L; the resin in the strong-base anion exchange resin column is guanidino strong-base anion exchange resin; the top washing liquid in the step (2) is water or a sodium chloride solution; the sodium chloride solution is a sodium chloride aqueous solution with the mass fraction of 3%; the leaching speed of the top washing liquid in the step (2) is 60L/h;
(3) and (3) crystallization: adding a salting-out agent into the secondary refined solution obtained in the step (2), heating to 60 ℃, preserving heat for 2 hours, then slowly adding an organic solvent, preserving heat for 0.5 hour, slowly cooling to 20-25 ℃, preserving heat for 2 hours, filtering, leaching with 75% by volume of ethanol, 85% by volume of ethanol and 95% by volume of ethanol in sequence, and drying under reduced pressure to obtain high-purity nucleotide; the weight ratio of the secondary refined solution to the organic solvent to the salting-out agent in the step (3) is 1: 1: 0.04; the organic solvent in the step (3) is ethanol; the organic solvent ethanol in the step (3) is ethanol with the volume fraction of 95 percent; the salting-out agent in the step (3) is sodium phosphate and sodium chloride; the weight ratio of the sodium phosphate to the sodium chloride is 1: 0.75.
the preparation method of the guanidino strong base anion exchange resin is the same as that of example 1.
The nucleotide is 5-disodium guanylate.
Comparative example 4
The method for refining high-purity nucleotide at least comprises the following steps:
(1) primary refining: adding a crude nucleotide product and a solvent into a reactor, adjusting the pH value to 8.5-9.0, heating to 60 ℃, adding a decolorizing agent, reacting for 0.5-1h under heat preservation, filtering, and cooling the filtrate to room temperature to obtain a primary refined nucleotide solution; the solvent in the step (1) is purified water; the reagent for adjusting the pH value in the step (1) is sodium hydroxide solution; the sodium hydroxide solution is 1mol/L sodium hydroxide solution; the decolorizing agent in the step (1) is activated carbon 303; the crude nucleotide in the step (1) is nucleotide obtained by fermentation liquor or RNA enzymolysis liquid; the weight ratio of the crude nucleotide product to the solvent to the decolorizing agent in the step (1) is 1: 24: 0.01;
(2) and (3) crystallization: adding a salting-out agent into the primary refined solution of the nucleotide obtained in the step (1), heating to 60 ℃, preserving heat for 2 hours, then slowly adding an organic solvent, preserving heat for 0.5 hour, slowly cooling to 40-45 ℃, preserving heat for 0.5 hour, cooling to 30 ℃, preserving heat for 0.5 hour, cooling to 20-25 ℃, preserving heat for 1 hour, filtering, leaching with 75% by volume of ethanol, 85% by volume of ethanol and 95% by volume of ethanol in sequence, and drying under reduced pressure to obtain high-purity nucleotide; the weight ratio of the primary refined solution to the organic solvent to the salting-out agent in the step (2) is 1: 1: 0.04; the organic solvent in the step (2) is ethanol; the organic solvent ethanol in the step (3) is ethanol with the volume fraction of 95 percent; the salting-out agent in the step (3) is sodium phosphate and sodium chloride; the weight ratio of the sodium phosphate to the sodium chloride is 1: 0.75.
the nucleotide is 5-disodium guanylate.
Comparative example 5
The method for refining high-purity nucleotide at least comprises the following steps:
(1) dissolving: adding a crude nucleotide product and a solvent into a reactor, adjusting the pH value to 8.5-9.0, heating to 60 ℃, carrying out heat preservation reaction for 0.5h, filtering, and then cooling the filtrate to room temperature to obtain a crude nucleotide product solution, wherein the solvent in the step (1) is purified water; the reagent for adjusting the pH value in the step (1) is sodium hydroxide solution; the sodium hydroxide solution is 1mol/L sodium hydroxide solution; the crude nucleotide in the step (1) is nucleotide obtained by fermentation liquor or RNA enzymolysis liquid; the weight ratio of the crude nucleotide product to the solvent to the decolorizing agent in the step (1) is 1: 24;
(2) secondary refining: treating the crude nucleotide solution obtained in the step (1) by a strong-base anion exchange resin column; then top washing with a top washing liquid, and collecting a secondary refined solution; the ratio of the primary refined solution of nucleotide to the strongly basic anion exchange resin in the step (2) is 1: 1, the mass of nucleotide is measured by Kg, and the volume of cation exchange resin is measured by L; the resin in the strong-base anion exchange resin column is guanidino strong-base anion exchange resin; the top washing liquid in the step (2) is a sodium chloride solution; the sodium chloride solution is a sodium chloride aqueous solution with the mass fraction of 3%; the leaching speed of the top washing liquid in the step (2) is 60L/h;
(3) and (3) crystallization: adding a salting-out agent into the secondary refined solution obtained in the step (2), heating to 60 ℃, preserving heat for 2 hours, then slowly adding an organic solvent, preserving heat for 0.5 hour, slowly cooling to 40-45 ℃, preserving heat for 0.5 hour, cooling to 30 ℃, preserving heat for 0.5 hour, cooling to 20-25 ℃, preserving heat for 1 hour, filtering, leaching with 75% by volume of ethanol, 85% by volume of ethanol and 95% by volume of ethanol in sequence, and drying under reduced pressure to obtain high-purity nucleotide; the weight ratio of the secondary refined solution to the organic solvent to the salting-out agent in the step (3) is 1: 1: 0.04; the organic solvent in the step (3) is ethanol; the organic solvent ethanol in the step (3) is ethanol with the volume fraction of 95 percent; the salting-out agent in the step (3) is sodium phosphate and sodium chloride; the weight ratio of the sodium phosphate to the sodium chloride is 1: 0.75.
the preparation method of the guanidino strong base anion exchange resin is the same as that of example 1.
The nucleotide is 5-disodium guanylate.
And (3) performance testing:
the method for calculating the yield and the decolorization rate comprises the following steps:
and measuring the A430 and A260 values of the solution before and after strong-base anion exchange by using a spectrophotometer. And calculating the recovery rate and the decolorization rate of the nucleotide.
Af260Represents the nucleotide content of the solution after anion exchange; a. thes260Represents the nucleotide content of the initial solution;
Vfrepresents the volume of the solution after exchange; vsRepresenting an initial volume;
Af430representing the pigment content of the solution after the exchange; a. thes430Indicates the initial solution pigment content.
Table 1 results of performance testing
As is clear from the above data, the method for purifying a high-purity nucleotide according to the present invention can provide a nucleotide having a high purity, a high yield and a small amount of impurities.
The foregoing examples are merely illustrative and serve to explain some of the features of the method of the present invention. The appended claims are intended to claim as broad a scope as is contemplated, and the examples presented herein are merely illustrative of selected implementations in accordance with all possible combinations of examples. Accordingly, it is applicants' intention that the appended claims are not to be limited by the choice of examples illustrating features of the invention. Also, where numerical ranges are used in the claims, subranges therein are included, and variations in these ranges are also to be construed as possible being covered by the appended claims.
Claims (6)
1. A method for purifying a high-purity nucleotide, comprising at least the steps of:
(1) primary refining: adding a crude nucleotide product and a solvent into a reactor, adjusting the pH value to 7.0-9.0, heating to 50-70 ℃, adding a decolorizing agent, reacting for 0.5-1h at a constant temperature, filtering, and cooling the filtrate to room temperature to obtain a primary refined nucleotide solution;
(2) secondary refining: treating the primary refined solution of nucleotide obtained in the step (1) by a strong-base anion exchange resin column; then eluting with a top washing liquid, and collecting a secondary refined solution;
(3) and (3) crystallization: adding a salting-out agent and an organic solvent into the secondary refined solution obtained in the step (2), crystallizing, filtering, leaching and drying to obtain high-purity nucleotide;
the decolorizing agent in the step (1) is one of medical active carbon, active carbon 301 and active carbon 303;
the strongly basic anion exchange resin filled in the strongly basic anion exchange resin column in the step (2) is guanidino strongly basic anion exchange resin;
the preparation raw materials of the guanidino strong base anion exchange resin are 4-hydroxy phenformin, 2-methyl-1- (3-methyl-2-butylene-1-yl) -1H-benzimidazole, styrene, divinylbenzene and N, N-dimethyl hexadecylamine;
and (3) the top washing liquid in the step (2) is a sodium chloride aqueous solution with the mass fraction of 3%.
2. The method for purifying a high-purity nucleotide as claimed in claim 1, wherein the ratio of the primary purified solution of nucleotide to the strongly basic anion exchange resin in the step (2) is 0.3 to 2: 1; the nucleotide mass is in Kg and the volume of anion exchange resin is in L.
3. The method of purifying a high-purity nucleotide according to claim 1, wherein the weight ratio of the secondary purification solution to the organic solvent to the salting-out agent in the step (3) is 1: 1-2: 0.01-0.05.
4. The method of claim 1, wherein the organic solvent used in step (3) comprises one or more of acetone, ethanol, and methanol.
5. The method of claim 1, wherein the salting-out agent in step (3) comprises one or more of sodium sulfate, sodium phosphate, sodium chloride, potassium formate, and sodium formate.
6. The method of purifying high-purity nucleotide according to claim 1, wherein the method of purifying high-purity nucleotide is applied to 5，Disodium guanylate, adenylate, cytidineAcid, uridylic acid and inosinic acid refining technology.
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| US20220340611A1 (en) * | 2019-10-17 | 2022-10-27 | Cj Cheiljedang Corporation | Method for separating disodium 5'-inosinate |
| CN111704637A (en) * | 2020-06-28 | 2020-09-25 | 南京工业大学 | Method for refining nucleotide by adopting membrane distillation crystallization |
| CN111961102B (en) * | 2020-09-22 | 2022-10-04 | 南京工业大学 | 5' -uridine monophosphate crystal and preparation method thereof |
| CN112341503B (en) * | 2020-11-07 | 2022-05-20 | 潍坊华诺医药科技有限公司 | Method for separating and purifying deoxyribonucleoside triphosphate |
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| CN113831379B (en) * | 2021-09-24 | 2023-06-30 | 上海蔚之星生物科技有限公司 | RNA enzymolysis liquid chromatographic separation method and system based on intelligent control |
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