CN101665525B - Synthetic method of nucleotide - Google Patents
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- CN101665525B CN101665525B CN 200910192314 CN200910192314A CN101665525B CN 101665525 B CN101665525 B CN 101665525B CN 200910192314 CN200910192314 CN 200910192314 CN 200910192314 A CN200910192314 A CN 200910192314A CN 101665525 B CN101665525 B CN 101665525B
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Abstract
The invention discloses a synthetic method of nucleotide, which has the following steps: A. adding nucleoside metal salt into a reactant for reaction in a reaction bottle, reducing temperature to -20 DEG C to -5 DEG C and adding phosphorylation agent for reaction, and carrying out reaction for 3 to 8 hours at the temperature of 10 DEG C to 30 DEG C so as to phosphorylate nucleotide metal salt; B. slowly adding reaction solution from step A into cracked ice and mixing and hydrolyzing to obtain hydrolysate, with the hydrolyzing temperature of 4 DEG C to 9 DEG C and hydrolyzing time of 1 hour plus or minus 0.5 hour; and C. adding solvent water into the hydrolysate from step B, adopting the method of microwave extraction to extract nucleotide extract phase, adding substances of strong basicity to regulate pH value of the solution from step B to 8 to 10, and regulating the temperature of the reaction solution to 50 DEG C plus or minus 5 DEG C, reducing the temperature of the extract phase from step C to -8 DEG C to -4 DEG C, collecting filtrate by centrifugation, using acidic solution to regulate the PH value to 7.8 to 8.0, freezing to crystallize, and obtaining nucleotide by centrifugation. The entire synthetic process is simple, easy in operation, low in cost and good in environmental protection.
Description
Technical field
The present invention relates to a kind of synthetic method of Nucleotide, relate in particular to the synthetic method of the Nucleotide processed that a kind of dietetic food uses.
Background technology
Nucleotide has widely purposes in agricultural, food and medicine industry.Especially in the application of infant food and field of medicaments, irreplaceable function is arranged.In infant food, it can obviously improve baby's immunological competence as the additive of infant food, promotes the maturation of enteron aisle, promotes the synthetic of lipoprotein and polyunsaturated fatty acid, reduce the generation of the diseases such as baby's flu and diarrhoea, be conducive to baby's normal growth and growth.At field of medicaments, clinical experiment shows that Nucleotide participates in body metabolism, promotes internal organs to improve and recovery, improves hemopoietic function of bone marrow, can be used as the ancillary drug for the treatment of cancer virus, is a kind of very important medical material.Nucleotide can make the excessive hyperplasia of white corpuscle, for symptoms such as various radioactive substances or drug-induced leukopenia, non-specific thrombopenia good curative effect is arranged, and also can be used for the treatment of acute hepatitis, chronic hepatitis.Except pharmaceutical industries is used, also play an important role in fine chemistry industry and food service industry center acid.Nucleic acid material is as one of composition of makeup, and skin care, the effect such as wrinkle resistant are arranged.The health that it is nontoxic, nonirritant and biological activity are conducive to human body.Nucleic acid material is used as the plant nutrition additive, can obviously improve the output of the farm crop such as paddy rice, soybean after the use, and the highest volume increase can reach 40%.The raising that is used for animal can improve rate of body weight gain and the feed rate of utilization of animal, and the material consumption obviously reduces.5 '-Nucleotide is of many uses, and is closely bound up with people's life, studies its different synthesis path to medicine, food, and agricultural etc. has very important meaning.Nucleotide prepares the generation method at present has: chemical synthesis, microbe fermentation method, enzymolysis process and enzyme catalysis method are respectively to have studied the synthetic of Nucleotide from different angles, but also cut both ways.Chemical method mainly is to carry out phosphating reaction take nucleosides as raw material, and general desired raw material or catalyzer are more difficult to get, and productive rate is lower, and aftertreatment also bothers, but this is little to substrate nucleosides structural limitations, has widely sphere of action.Microbe fermentation method mainly is to utilize the biosynthetic pathway of microorganism strains to produce Nucleotide.By product is few, and cost is low, but the application of this method is subject to the considerable restraint of microbiological property, and it is the production method that history is the longest, technology is the most ripe that enzymolysis process is produced Nucleotide.Become the preparation classical way of various natural nucleotides but this method because the film of reactor easily stops up and the membrane reactor maintenance cost is high, so be not suitable for large-scale commercial production.The product that enzymatic process has microbial method simultaneously is single, the advantage that cost is low, the sphere of action that chemical method arranged again is characteristics widely, just are being subject to increasing attention, the weak point of this method is the screening for enzymes workload very large, and the substrate-function scope of enzyme also has certain restriction simultaneously.
Summary of the invention
For above-mentioned shortcoming, the technical problem to be solved in the present invention provides a kind of new Nucleotide synthetic method, and its production cost is low, transformation efficiency is high.
For solving the problems of the technologies described above, the technical solution adopted in the present invention is: a kind of synthetic method of Nucleotide, step is: A, will the nucleosides metal-salt add in the reagent and react in reaction flask, add again the phosphorylation agent reaction after cooling to-20～-5 ℃, 3～8 hours reaction times, temperature of reaction is 10～30 ℃, makes nucleosides metal-salt phosphorylation; B, the reaction solution of steps A slowly added to stir in the trash ice be hydrolyzed to get hydrolyzate, hydrolysis temperature is 4～9 ℃, hydrolysis time 1 ± 0.5 hour; C, in the hydrolyzate of step B, add aqueous solvent, adopt the method for microwave extracting to extract the Nucleotide extraction phase, add the pH to 8-10 of strong alkaline substance regulating step B solution, the conditioned reaction liquid temp is 50 ± 5 ℃, described microwave irradiation power 525 ± 50W, radiated time 7 ± 2 minutes; D, the extraction phase of step C is cooled to-8～-4 ℃, centrifugal collection filtrate is regulated pH value to 7.8～8.0 with acid solution again, freezing and crystallizing, centrifugal must Nucleotide.
Further: in the synthetic method of above-mentioned Nucleotide, described nucleosides metal-salt is one or more in inosine sodium salt, inosine sylvite, guanosine sodium salt, the guanosine sylvite.Described reagent is alkyl phosphate.Described alkyl phosphate is one or both in triethyl phosphate, the trimethyl phosphite 99.The weight ratio of described nucleosides metal-salt and reagent is 1: 10～30.Described phosphorylation agent is tripoly phosphate sodium STPP STP, Vanadium Pentoxide in FLAKES P
2O
5, phosphoric acid H
3PO
4, phosphorus oxychloride POCl
3In one or more.The weight ratio of described nucleosides metal-salt and phosphorylation agent is 1: 1～5.The strong alkaline substance of described step C is one or both among NaOH, the KOH, and the weight ratio of described hydrolyzate and aqueous solvent is 1: 16～19; The acid solution of described step D is HCl, H
2SO
4, HNO
3In one or more.
Compare with the synthetic method of existing Nucleotide, the present invention selects the nucleosides metal-salt to add in the reagent to react in reaction flask, adds the phosphorylation agent reaction, 3～8 hours reaction times after cooling to-20～-5 ℃ again, temperature of reaction is 10～30 ℃, makes nucleosides metal-salt phosphorylation.Get Nucleotide by hydrolysis, extraction and filtration at last.Reaction time of the present invention is short, only needs 3～5 hours, and general reaction needed 5～7 hours.General reaction conversion ratio 85%～90%, reaction conversion ratio of the present invention is up to 90%～97%.And adopt up-to-date microwave-assisted extraction technique, and greatly shortened the production time of Nucleotide, obviously improved the productive rate of Nucleotide, water has been saved cost as extraction agent during owing to extraction, reduces the discharging of process waste.Therefore, this technique is that environmental protection is economical again, is suitable for extensive explained hereafter.Whole building-up process is simple, easy to operate, cost is low, the feature of environmental protection is good.
Figure of description
Fig. 1 is the liquid chromatographic detection figure of embodiment 1;
Fig. 2 is the liquid chromatographic detection figure of embodiment 2;
Fig. 3 is the liquid chromatographic detection figure of embodiment 3.
Embodiment
Purport of the present invention is to select the nucleosides metal-salt to add in the reagent to react in reaction flask, adds the phosphorylation agent reaction after cooling to-20～-5 ℃ again, and in 3～8 hours reaction times, temperature of reaction is 10～30 ℃, makes nucleosides metal-salt phosphorylation.Get Nucleotide by hydrolysis, extraction and filtration at last.The present invention also adopts microwave-assisted extraction technique, has greatly shortened the production time of Nucleotide, has obviously improved the productive rate of Nucleotide, and water has been saved cost as extraction agent during owing to extraction, reduces the discharging of process waste.Below in conjunction with embodiment content of the present invention is described in further detail, mentioned content is not limitation of the invention among the embodiment, and each raw-material selection can be suited measures to local conditions and the result be there is no substantial effect in the preparation process.
At first, summary preparation method's of the present invention general planning: a kind of synthetic method of Nucleotide, step is: A, will the nucleosides metal-salt add in the reagent and react in reaction flask, add again the phosphorylation agent reaction after cooling to-20～-5 ℃, 3～8 hours reaction times, temperature of reaction is 10～30 ℃, makes nucleosides metal-salt phosphorylation; B, the reaction solution of steps A slowly added to stir in the trash ice be hydrolyzed to get hydrolyzate, hydrolysis temperature is 4～9 ℃, hydrolysis time 1 ± 0.5 hour; C, in the hydrolyzate of step B, add aqueous solvent, adopt the method for microwave extracting to extract the Nucleotide extraction phase, the pH to 8 of adding strong alkaline substance regulating step B solution～10, the conditioned reaction liquid temp is 50 ± 5 ℃, described microwave irradiation power 525 ± 50W, radiated time 7 ± 2 minutes; D, the extraction phase of step C is cooled to-8～-4 ℃, centrifugal collection filtrate is regulated pH value to 7.8～8.0 with acid solution again, freezing and crystallizing, centrifugal must Nucleotide.
In the 500ml there-necked flask, drop into 10g inosine sodium salt, add the 150g triethyl phosphate, stir and cool to-10 ℃, slowly add the 15g phosphorus oxychloride, after 4 hours, sampling detects, calculate transformation efficiency through the HPLC liquid chromatographic detection and reach 95%, end is reacted, and reaction solution is slowly added in the trash ice be hydrolyzed, hydrolysis temperature is 7 ℃, stirs hydrolysis time 1 hour, then transfer pH to 9 with NaOH, reacting liquid temperature is 50 ℃, microwave irradiation power 525W, radiated time 7min.The ratio of reaction solution and aqueous solvent 1: 18, the extraction yield can have more than 98%.The extraction phase cooling is refrigerated to-5 ℃, centrifugal collection filtrate, with HCl filtrate pH value is transferred to 7.8～8.0 and get reaction solution, this reaction solution is detected (1200 flow velocity 1.2ml/ seconds of Agilent Technologies model with high performance liquid chromatograph, 29 degrees centigrade of peak pressure 200bar of 5min column temperature moving phase: 0.5% potassium dihydrogen phosphate), purity is very high, shown in the liquid chromatographic detection figure of Fig. 1.With the reaction solution freezing and crystallizing, centrifugal, obtain t-inosinic acid.
Drop into 10g guanosine sodium salt in the 500ml there-necked flask, add the 150g triethyl phosphate, stir and cool to-10 ℃, slowly add the 15g phosphorus oxychloride, after 4 hours, sampling detects, and calculates transformation efficiency through the HPLC liquid chromatographic detection and reaches 94%, finishes reaction; Reaction solution slowly added in the trash ice be hydrolyzed, hydrolysis temperature is 7 ℃, stirs, and then hydrolysis time 1 hour transfers pH to 9 with NaOH, and reacting liquid temperature is 50 ℃, microwave irradiation power 525W, radiated time 7min.The ratio of reaction solution and aqueous solvent is 1: 20, and the extraction yield can have 99%; The extraction phase cooling is refrigerated to-5 ℃, centrifugal collection filtrate, with HCl filtrate pH value is transferred to 7.8～8.0 and get reaction solution, this reaction solution is detected (1200 flow velocity 1.2ml/ seconds of Agilent Technologies model with high performance liquid chromatograph, 29 degrees centigrade of peak pressure 200bar of 5min column temperature moving phase: 0.5% potassium dihydrogen phosphate), purity is very high, shown in the liquid chromatographic detection figure of Fig. 1.With the reaction solution freezing and crystallizing, centrifugal, obtain guanylic acid.
Embodiment 3
In the 500ml there-necked flask, drop into 5g inosine sodium salt and 5g guanosine sodium salt, add the 150g trimethyl phosphite 99, stir and cool to-10 ℃, slowly add the 15g phosphorus oxychloride, after 4 hours, sampling detects, HPLC liquid chromatography t-inosinic acid and guanylic acid chromatographic peak and reach 96% finish reaction; Reaction solution slowly added in the trash ice be hydrolyzed, hydrolysis temperature is 7 ℃, stirs, and then hydrolysis time 1 hour transfers pH to 9 with NaOH, and reacting liquid temperature is 50 ℃, microwave irradiation power 525W, radiated time 7min.The ratio of reaction solution and aqueous solvent is 1: 18, and the extraction yield can have 98%; The extraction phase cooling is refrigerated to-5 ℃, centrifugal collection filtrate, with HCl filtrate pH value is transferred to 7.8～8.0 and get reaction solution, this reaction solution is detected (1200 flow velocity 1.2ml/ seconds of Agilent Technologies model with high performance liquid chromatograph, 29 degrees centigrade of peak pressure 200bar of 5min column temperature moving phase: 0.5% potassium dihydrogen phosphate), purity is very high, shown in the liquid chromatographic detection figure of Fig. 1.With the reaction solution freezing and crystallizing, centrifugal, obtain guanylic acid, t-inosinic acid mixture.
Claims (6)
1. the synthetic method of a Nucleotide, step is:
A, will the nucleosides metal-salt add in the reagent and react in reaction flask, add the phosphorylation agent reaction after cooling to-20～-5 ℃ again, in 3～8 hours reaction times, temperature of reaction is 10～30 ℃, makes nucleosides metal-salt phosphorylation;
B, the reaction solution of steps A slowly added to stir in the trash ice be hydrolyzed to get hydrolyzate, hydrolysis temperature is 4～9 ℃, hydrolysis time 1 ± 0.5 hour;
C, in the hydrolyzate of step B, add aqueous solvent, adopt the method for microwave extracting to extract the Nucleotide extraction phase, the pH to 8 of adding strong alkaline substance regulating step B solution～10, the conditioned reaction liquid temp is 50 ± 5 ℃, described microwave irradiation power 525 ± 50W, radiated time 7 ± 2 minutes;
D, the extraction phase of step C is cooled to-8～-4 ℃, centrifugal collection filtrate is regulated pH value to 7.8～8.0 with acid solution again, freezing and crystallizing, centrifugal must Nucleotide;
Described nucleosides metal-salt is one or more in inosine sodium salt, inosine sylvite, guanosine sodium salt, the guanosine sylvite;
Described reagent is alkyl phosphate.
2. the synthetic method of Nucleotide according to claim 1, it is characterized in that: described alkyl phosphate is one or both in triethyl phosphate, the trimethyl phosphite 99.
3. the synthetic method of Nucleotide according to claim 1, it is characterized in that: the weight ratio of described nucleosides metal-salt and reagent is 1: 10～30.
4. the synthetic method of Nucleotide according to claim 1, it is characterized in that: described phosphorylation agent is tripoly phosphate sodium STPP STP, Vanadium Pentoxide in FLAKES P
2O
5, phosphoric acid H
3PO
4, phosphorus oxychloride POCl
3In one or more.
5. the synthetic method of Nucleotide according to claim 1, it is characterized in that: the weight ratio of described nucleosides metal-salt and phosphorylation agent is 1: 1～5.
6. the synthetic method of Nucleotide according to claim 1, it is characterized in that: the strong alkaline substance of described step C is one or both among NaOH, the KOH, the weight ratio of described hydrolyzate and aqueous solvent is 1: 16～19; The acid solution of described step D is HCl, H
2SO
4, HNO
3In one or more.
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| CN101993466B (en) * | 2010-10-14 | 2012-08-15 | 广东肇庆星湖生物科技股份有限公司 | Method for preparing 5'-disodium guanylate |
| CN102199182A (en) * | 2011-04-10 | 2011-09-28 | 浙江钱江生物化学股份有限公司 | One-step extraction method for disodium 5'-inosinate |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0453597A1 (en) * | 1988-10-25 | 1991-10-30 | Ajinomoto Co., Inc. | Process for producing a mixture of inosinic acid and guanosinic acid by direct phosphorylation |
| CN1086219A (en) * | 1992-07-08 | 1994-05-04 | 武田药品工业株式会社 | 5 '-production method of Nucleotide |
| CN1539846A (en) * | 2003-10-29 | 2004-10-27 | 徐昌洪 | Technique for preparing 5'nucleotide bi-sodium |
-
2009
- 2009-09-10 CN CN 200910192314 patent/CN101665525B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0453597A1 (en) * | 1988-10-25 | 1991-10-30 | Ajinomoto Co., Inc. | Process for producing a mixture of inosinic acid and guanosinic acid by direct phosphorylation |
| CN1086219A (en) * | 1992-07-08 | 1994-05-04 | 武田药品工业株式会社 | 5 '-production method of Nucleotide |
| CN1539846A (en) * | 2003-10-29 | 2004-10-27 | 徐昌洪 | Technique for preparing 5'nucleotide bi-sodium |
Non-Patent Citations (1)
| Title |
|---|
| 孙夏容等.微波萃取技术.《中外医疗》.2009,(第19期), * |
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