CN103421865A - Preparation method for modified cytidine antibiotic - Google Patents
Preparation method for modified cytidine antibiotic Download PDFInfo
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- CN103421865A CN103421865A CN2012101619062A CN201210161906A CN103421865A CN 103421865 A CN103421865 A CN 103421865A CN 2012101619062 A CN2012101619062 A CN 2012101619062A CN 201210161906 A CN201210161906 A CN 201210161906A CN 103421865 A CN103421865 A CN 103421865A
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- cytidine
- antibiotic
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Abstract
Provided is a modified cytidine antibiotic. The modified cytidine antibiotic is a nitrogen substituting cytidine antibiotic with a side chain -C4H8ClO9. The preparation method comprises steps: streptomyces ahygroscopicus is taken as a starting strain and subjected to fermentation cultivation in medium; after separation, purification and chemical modification, the modified cytidine antibiotic is obtained. The modified cytidine antibiotic has high biological resistance and structural stability. The preparation method has good chemoselectivity and mild technology conditions, and production processes are environmentally friendly.
Description
Technical field: the invention belongs to the microbial pesticide technical field, relate to a kind of preparation method who modifies the cytidine element.
Background technology:
Agricultural antibiotic (agricultural antibiotics), be called for short agriculture anti-, refer to by the microorganism fermentation produce, have the agricultural chemicals function, for the secondary metabolites of the harmful organisms such as insect pests grass mouse on agricultural.With general chemical synthetic pesticide, compare, agricultural antibiotic has following characteristics: 1. complex structure; 2. active high, consumption is little, selectivity good; 3. easily by biology or natural cause, decomposed not accumulation or residual in environment; 4. raw materials for production are the agricultural-food such as starch, carbohydrate, belong to the reproducibility energy; 5. adopt fermentation engineering production, same set of equipment can be produced different microbiotic as long as change bacterial classification, and producing bacterium is the actinomycetes in soil mostly, and fungus and bacterium is also arranged.
Nucleoside antibiotics is the important microbiotic of a class, they are large class compounds to nucleosides and nucleotide structure modification, take nucleosides as its common parent nucleus, have antibacterium, antimycotic, anti-trypanosome, antitumor, hoe up weeds, the biological activity of the wider range such as desinsection, strengthening immunity.The difference of different nucleoside antibioticss is the side-chain radical beyond parent nucleus.On agricultural, the nucleoside antibiotics of application mainly contains blasticidin S (Blasticidin S) and Polyoxin (Polyoxins).Modifying the cytidine element for side chain is-C
4H
8ClO
9Nitrogen replace the cytidine element.
Up to now, from various biologies (comprising bacterium, fungi, all kinds of plant), isolate more than 3800 kind of halogenide, approximately 95% halogenide is chlorination and bromide, fluoridize with iodide seldom, a few compounds is both chloride also containing iodine.Much halogenide, as paraxin (chloramphenicol), 7-Uromycin (7-chlotetracycline), vancomycin (vancomycin) etc. are all important microbiotic.Although the type of halogen atom, quantity and position are on compound, bioactive impact does not have specific rule, and some halogenide is compared with corresponding non-halide, and halogenation can strengthen its biological activity.As, the anti-mycotic activity of 2-chlorine pyrrolnitrin only has 10% of pyrrolnitrin (containing two chlorine atoms); With it, containing chlorine derivative, compare, not chloride butterfly mycin does not have anti-microbial activity.
Summary of the invention:
The purpose of this invention is to provide a kind of preparation method who modifies the cytidine element.
The present invention realizes by following method:
Bacterial strain: the S510 bacterial strain that streptomyces hygroscopicus Hainan mutation S101 bacterial strain of take obtains after natural separation is starting strain.
Substratum: Semen Maydis powder 15～25g, soybean cake powder 20～35g, glucose 15～25g, starch 8～12g, ammonium chloride 1～5g, calcium carbonate 1～5g, sal epsom 0.1～0.5, ferrous sulfate 0.1～0.5, sodium-chlor 0.1～0.5g, Sodium phosphate dibasic 0.1～0.5g, add tap water to 1000mL.Through 121 ℃ of high pressure moist heat sterilization 20min.
Fermentation culture: the shaking table concussion is cultivated, and rotating speed is 150～250 rev/mins, leavening temperature: 25～35 ℃, and tank pressure 0.01～0.15MPa, air flow 1:0.5～1, fermentation time 50～100h.
Tunning separates and purifying: the fermented liquid extraction, with Sephadex G-25 column chromatography, water elution, elutriant is (COSMOSIL C18 Econopak post after HPLC detects, moving phase 0.03% trifluoroacetic acid aqueous solution, flow velocity 0.4mL/min, ultraviolet detection wavelength 220nm), the main ingredient content that retention time wherein is about to 5.5min merges respectively in 75～85% and 60～75% part, then can obtain first isolate after vacuum lyophilization.Content again through a normal pressure column chromatography, can bring up to 80～85% at 60～75% primary extract by purity, merges the content of twice gained at the sample more than 75%, with the HPLC semipreparative column, is prepared, and finally can obtain the elaboration that purity is 90.12%.By elaboration solution cold dry after, the gained micro-yellow powder is side chain-C
4H
9O
9Nitrogen replace the cytidine element.Titration: cup-plate method.
Chemically modified: after the tunning separation and purification, with the KCl reaction, take propyl carbinol as solvent, FeCl
3For catalyzer, by the coordination catalysis halogenating reaction, prepare band side chain-C
4H
8ClO
9Nitrogen replace the cytidine element.Titration: cup-plate method.
Modification cytidine element of the present invention has higher biotic resistance and structural stability, and this preparation technology's chemo-selective is good, processing condition gentleness, production process environmental friendliness.Gained side chain-C
4H
9O
9Nitrogen to replace that the cytidine element tires be 2800 μ g/L, side chain-C
4H
8ClO
9Nitrogen to replace that the cytidine element tires be 6418 μ g/L.Based on above-mentioned advantage, band side chain-C of the present invention
4H
8ClO
9Nitrogen replace the cytidine element and will play an important role in field of biological pesticide, have a extensive future.
Embodiment
Below in conjunction with specific embodiment, the invention will be further elaborated, but be not limited to these specific embodiments, and all embodiment are all by above-mentioned operation steps operation.
Embodiment 1
Bacterial strain: the S510 bacterial strain that streptomyces hygroscopicus Hainan mutation S101 bacterial strain of take obtains after natural separation is starting strain.
Substratum: Semen Maydis powder 20g, soybean cake powder 25g, glucose 18g, starch 10g, ammonium chloride 3g, calcium carbonate 3g, sal epsom 0.3, ferrous sulfate 0.3, sodium-chlor 0.3g, Sodium phosphate dibasic 0.3g, add tap water to 1000mL.Through 121 ℃ of high pressure moist heat sterilization 20min.
Fermentation culture: the shaking table concussion is cultivated, and rotating speed is 220 rev/mins, leavening temperature: 28 ℃, and tank pressure 0.12MPa, air flow 1:0.5, fermentation time 72h.
Tunning separates and purifying: the fermented liquid extraction, with Sephadex G-25 column chromatography, water elution, elutriant is (COSMOSIL C18 Econopak post after HPLC detects, moving phase 0.03% trifluoroacetic acid aqueous solution, flow velocity 0.4mL/min, ultraviolet detection wavelength 220nm), the main ingredient content that retention time wherein is about to 5.5min merges respectively in 75～85% and 60～75% part, then can obtain first isolate after vacuum lyophilization.Content again through a normal pressure column chromatography, can be brought up to 80-85% at 60～75% primary extract by purity, merges the content of twice gained at the sample more than 75%, with the HPLC semipreparative column, is prepared, and finally can obtain the elaboration that purity is 90.12%.By elaboration solution cold dry after, the gained micro-yellow powder is side chain-C
4H
9O
9Nitrogen replace the cytidine element.Titration: cup-plate method.
Chemically modified: after the tunning separation and purification, with the KCl reaction, take propyl carbinol as solvent, FeCl
3For catalyzer, by the coordination catalysis halogenating reaction, prepare band side chain-C
4H
8ClO
9Nitrogen replace the cytidine element.Titration: cup-plate method.Reaction conditions is: side chain-C
4H
9O
9Nitrogen replace cytidine element: KCl:FeCl
3(mol ratio)=20:40:3, the consumption of solvent, n-butanol is every mole of side chain-C
4H
9O
9Nitrogen replace cytidine element 40ml, temperature of reaction is 120 ℃, yield is 91%.
Embodiment 2
Bacterial strain: the S510 bacterial strain that streptomyces hygroscopicus Hainan mutation S101 bacterial strain of take obtains after natural separation is starting strain.
Fermention medium: Semen Maydis powder 25g, soybean cake powder 35g, glucose 15g, starch 12g, ammonium chloride 5g, calcium carbonate 5g, sal epsom 0.1, ferrous sulfate 0.1, sodium-chlor 0.1g, Sodium phosphate dibasic 0.5g, add tap water to 1000mL.Through 121 ℃ of high pressure moist heat sterilization 20min.
Fermentation culture: the shaking table concussion is cultivated, and rotating speed is 250 rev/mins, leavening temperature: 25 ℃, and tank pressure 0.15MPa, air flow 1:0.5, fermentation time 64h.
Tunning separates and purifying: the fermented liquid extraction, with Sephadex G-25 column chromatography, water elution, elutriant is (COSMOSIL C18 Econopak post after HPLC detects, moving phase 0.03% trifluoroacetic acid aqueous solution, flow velocity 0.4mL/min, ultraviolet detection wavelength 220nm), the main ingredient content that retention time wherein is about to 5.5min merges respectively in 75～85% and 60～75% part, then can obtain first isolate after vacuum lyophilization.Content again through a normal pressure column chromatography, can be brought up to 80-85% at 60～75% primary extract by purity, merges the content of twice gained at the sample more than 75%, with the HPLC semipreparative column, is prepared, and finally can obtain the elaboration that purity is 90.12%.By elaboration solution cold dry after, the gained micro-yellow powder is side chain-C
4H
9O
9Nitrogen replace the cytidine element.Titration: cup-plate method.Side chain-C
4H
9O
9Nitrogen replace the cytidine element 2567 μ g/L that tire.
Chemically modified: after the tunning separation and purification, with the KCl reaction, take propyl carbinol as solvent, FeCl
3For catalyzer, by the coordination catalysis halogenating reaction, prepare band side chain-C
4H
8ClO
9Nitrogen replace the cytidine element.Titration: cup-plate method.Reaction conditions is: side chain-C
4H
9O
9Nitrogen replace cytidine element: KCl:FeCl
3(mol ratio)=20:40:3, the consumption of solvent, n-butanol is every mole of side chain-C
4H
9O
9Nitrogen replace cytidine element 40ml, temperature of reaction is 105 ℃, yield is 85.7%.
Embodiment 3
Bacterial strain: the S510 bacterial strain that streptomyces hygroscopicus Hainan mutation S101 bacterial strain of take obtains after natural separation is starting strain.
Fermention medium: Semen Maydis powder 15g, soybean cake powder 35g, glucose 15g, starch 8g, ammonium chloride 1g, calcium carbonate 1g, sal epsom 0.5, ferrous sulfate 0.5, sodium-chlor 0.5g, Sodium phosphate dibasic 0.1g, add tap water to 1000mL.Through 121 ℃ of high pressure moist heat sterilization 20min.
Fermentation culture: the shaking table concussion is cultivated, and rotating speed is 250 rev/mins, leavening temperature: 35 ℃, and tank pressure 0.01MPa, air flow 1:0.5, fermentation time 84h.
Tunning separates and purifying: the fermented liquid extraction, with Sephadex G-25 column chromatography, water elution, elutriant is (COSMOSIL C18 Econopak post after HPLC detects, moving phase 0.03% trifluoroacetic acid aqueous solution, flow velocity 0.4mL/min, ultraviolet detection wavelength 220nm), the main ingredient content that retention time wherein is about to 5.5min merges respectively in 75～85% and 60～75% part, then can obtain first isolate after vacuum lyophilization.Content again through a normal pressure column chromatography, can be brought up to 80-85% at 60～75% primary extract by purity, merges the content of twice gained at the sample more than 75%, with the HPLC semipreparative column, is prepared, and finally can obtain the elaboration that purity is 90.12%.By elaboration solution cold dry after, the gained micro-yellow powder is side chain-C
4H
9O
9Nitrogen replace the cytidine element.Titration: cup-plate method.
Chemically modified: after the tunning separation and purification, with the KCl reaction, take propyl carbinol as solvent, FeCl
3For catalyzer, by the coordination catalysis halogenating reaction, prepare band side chain-C
4H
8ClO
9Nitrogen replace the cytidine element.Titration: cup-plate method.Reaction conditions is: side chain-C
4H
9O
9Nitrogen replace cytidine element: KCl:FeCl
3(mol ratio)=20:40:3, the consumption of solvent, n-butanol is every mole of side chain-C
4H
9O
9Nitrogen replace cytidine element 40ml, temperature of reaction is 105 ℃, yield is 82.2%.
Claims (4)
1. a side chain is-C
4H
8ClO
9Nitrogen replace the cytidine element, its preparation method comprises:
With streptomyces hygroscopicus not for the bacterium that sets out, fermentation culture in substratum,
Separation, purified fermentation broth,
Tunning passes through FeCl after separation and purification
3The coordination catalysis halogenating reaction carries out chemically modified and obtains side chain for-C
4H
8ClO
9Nitrogen replace the cytidine element.
2. method according to claim 1, it is characterized in that substratum is: Semen Maydis powder 15～25g, soybean cake powder 20～35g, glucose 15～25g, starch 8～12g, ammonium chloride 1～5g, calcium carbonate 1～5g, sal epsom 0.1～0.5g, ferrous sulfate 0.1～0.5g, sodium-chlor 0.1～0.5g, Sodium phosphate dibasic 0.1～0.5g, add tap water to 1000mL.
3. method according to claim 1 is characterized in that the condition of fermentation culture is: shaking table concussion cultivation and fermentation, and leavening temperature: 25～35 ℃, tank pressure 0.01～0.15MPa, air flow 1:0.5～1, fermentation time 50～100h.
4. method according to claim 1, it is characterized in that: chemically modified is to take KCl as raw material, take propyl carbinol as solvent.
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| CN2012101619062A CN103421865A (en) | 2012-05-23 | 2012-05-23 | Preparation method for modified cytidine antibiotic |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1436787A (en) * | 2002-02-04 | 2003-08-20 | 中国农业科学院植物保护研究所 | Antibiotic and its prepn and application |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1436787A (en) * | 2002-02-04 | 2003-08-20 | 中国农业科学院植物保护研究所 | Antibiotic and its prepn and application |
Non-Patent Citations (3)
| Title |
|---|
| 何鑫等: "抗真菌类抗生素的研究进展", 《安徽农业科学》 * |
| 崔增杰等: "抗真菌农用抗生素有效成分研究进展", 《中国农学通报》 * |
| 王心等: "微生物生物活性卤代化合物的研究进展", 《中国海洋药物》 * |
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Application publication date: 20131204 |