CN102321678A - The method of 1 alpha-hydroxy vitamin D is made in a kind of microbial transformation - Google Patents
The method of 1 alpha-hydroxy vitamin D is made in a kind of microbial transformation Download PDFInfo
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- CN102321678A CN102321678A CN201110279293A CN201110279293A CN102321678A CN 102321678 A CN102321678 A CN 102321678A CN 201110279293 A CN201110279293 A CN 201110279293A CN 201110279293 A CN201110279293 A CN 201110279293A CN 102321678 A CN102321678 A CN 102321678A
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Abstract
The invention discloses the present invention is the medicine 1-alpha-hydroxy vitamin D of relevant treatment osteoporosis
2Or D
3, promptly alfacalcidol (paricalcitol) and the degree vitamine d3 (Doxercalciferol) a kind of new industrial production process.Adopt false Nocardia CGMCC No.5098 that 1 of vitamins D is carried out microbial transformation, a step directly obtains the 1-alpha-hydroxy vitamin D.The inventive method is gentleer than chemical method, need not protect and use deleterious tin anhydride to carry out oxidation, and have regioselectivity; Need not carry out radical protection, the bio-transformation yield is up to 40-50%, environmental friendliness; Through a step conversion reaction, shorten operational path greatly, improve yield; Reduce manufacturing cost, the chemical reaction about 6 steps of known synthetic alfacalcidol and degree vitamine d3 is shortened to a step, total recovery is brought up to 40-50%.
Description
Technical field
The present invention relates to chemistry and bio-pharmaceuticals, especially treat the medicine 1-alpha-hydroxy vitamin D of osteoporosis
2Or D
3, i.e. alfacalcidol (paricalcitol) and degree vitamine d3 (Doxercalciferol) preparing technical field.
Background technology
Alfacalcidol (paricalcitol) and degree vitamine d3 (Doxercalciferol) active vitamin medicine are used for rickets, osteomalacia that osteoporosis and a variety of causes cause clinically.
1-alpha-hydroxy vitamin D class drug main will be used to treat other diseases such as osteoporosis and chronic renal failure.Main at present employing chemical synthesis process is manufactured.Adopting the 1-alpha-position of vitamins D to carry out directly hydroxylation of chemistry, is very difficult, and productive rate is very low.The known synthetic chemical synthesis that adopts mostly of 1-alpha-hydroxy vitamin D mainly adopts vitamins D as raw material, through over-churning protection, ring and, polystep reactions such as oxidation, open loop, purifying generate.Chemosynthesis need be carried out the radical protection and the deprotection of multistep, the utilization illumination reaction, and open loop and isomerizing, step is various, and separation and purification is complicated.Complicated operation on producing, yield is on the low side, about 10%.
Summary of the invention
Above deficiency in view of prior art the purpose of this invention is to provide a kind of preparation 1-alpha-hydroxy vitamin D
2Or D
3, promptly alfacalcidol (paricalcitol) and degree vitamine d3 (Doxercalciferol) make it to have production process simplification, yield height, environmental friendliness and advantage of low manufacturing cost.
The objective of the invention is to realize through following means.
The method of 1 alpha-hydroxy vitamin D is made in a kind of microbial transformation, adopts false Nocardia CGMCC No.5098 that 1 of vitamins D is carried out microbial transformation, and a step directly obtains the 1-alpha-hydroxy vitamin D:
Substratum is in fermentation culture in the time of 3 days, vitamins D joined in the nutrient solution after as substrate and Schardinger dextrins and double solvents mixed dissolution, and concentration of substrate 1-15g/ liter, the ratio of Schardinger dextrins and vitamins D is 5: 1 to 2: 1; Continue to produce in the above-mentioned culturing process 1 hydroxylate, the time 1-10 of conversion days; Last through filtering, concentrated, silicagel column obtains target 1 α position hydroxylation product after separating.
The present invention adopts false Nocardia CGMCC No.5098 that 1 of vitamins D is carried out microbial transformation, and a step directly obtains the 1-alpha-hydroxy vitamin D
2Or D
3Gentleer than chemical method, need not protect and use deleterious tin anhydride to carry out oxidation, and have regioselectivity; Need not carry out radical protection, the bio-transformation yield is up to 40-50%, environmental friendliness; Through a step conversion reaction, shorten operational path greatly, improve yield; Reduce manufacturing cost, the chemical reaction about 6 steps of known synthetic alfacalcidol and degree vitamine d3 was shortened to for 1 step, total recovery is brought up to 40-50%.Biotransformation method can carry out that some chemical process is difficult to carry out or can not the synthetic reaction, and conversion condition is gentle, and is simple to operate.
The present invention directly introduces hydroxyl at 1 alpha-position of vitamins D, has solved the problem that organic synthesis is difficult to realize, directly introduces hydroxyl at short notice and generates active result.The bacterial classification that the present invention utilized is actinomycetes; False Nocardia CGMMC No.5098, through the control conversion condition, the cytochrome P 450 enzymes of microorganisms can be directly to the 1-α position hydroxylation of substrate vitamins D; It is high to have the product stereoselectivity, characteristics such as yield height.The present invention also provides a new operational path for the industrial preparation that solves alfacalcidol and degree vitamine d3, has shortened operational path, has improved yield, reduces manufacturing cost.
Description of drawings
Fig. 1 is the process route chart of the inventive method.
Embodiment
Below in conjunction with embodiment enforcement of the present invention is done further to describe.
Technical scheme route of the present invention is seen Fig. 1, though only provide the reactant vitamins D
2The preparation title product is the 1-alpha-hydroxy vitamin D
2Reaction formula, but reactant vitamins D
3The preparation title product is the 1-alpha-hydroxy vitamin D
3Reaction formula be complete equity.
Bacterial strain uses therefor is different from the used amycolata Amycolata of Japanese Patent JP2005323618 bacterial strain; Be that the contriver separates from the soil sample that Sichuan two E Shan take and obtains a strain bacterium SWJTUQL-3721021; Through identifying that bacterial strain belongs to false Nocardia bacteria actinomyces actinoides; Name for false Nocardia bacteria SWJTUQL-3721021 (Pseudonocardia autotrophica SWJTUQL-3721021), deposit number is CGMCC No.5098.
Technology of the present invention relates to bacterial strain CGMCC No.5098 carries out bio-transformation to vitamins D in the substratum of ventilation, stirring, carbon source, nitrogenous source, mineral salt, obtain the 1-hydroxy-vitamine D.
Transform and cultivate and to carry out shaking in bottle or the fermentor tank of ventilation; Nutritive substance comprises that inorganic salt, nitrogenous source, sugar or other soluble substance are as carbon source; Inorganic salt comprise muriate, nitrate salt, carbonate, vitriol or the phosphoric acid salt of basic metal, earth alkali metal such as magnesium, iron, zinc, manganese; MgSO4 preferably, KH
2PO4.Nitrogenous source can be an ammonium salt, comprises Citrate trianion, tartrate, malate, SUMATRIPTAN SUCCINATE, oxalate, acetate etc.; Amino acid and mixture, peptide or protein and hydrolyzate thereof, the water soluble extract of meat extract, cereal such as corn, wheat; Malted maize extract, corn impregnation liquid, bean cake powder, peanut powder, hawk bean powder, cottonseed meal.Carbon source can be glucose, sucrose, starch, dextrin, sorbose, seminose, lactose or the like.No matter solid culture still is that deep layer is cultivated and all under aeration condition, carried out, and leavening temperature 22-30 ℃, preferably 27 ℃, pH scope 6.5-7.5, preferably 7.0.
Process of the present invention is easy to carry out, with vitamins D
2Or D
3After mixing as substrate and γ or beta-cyclodextrin, join in the nutrient solution, concentration of substrate does not have particular requirement, preferably the 1-15g/ liter; The ratio of Schardinger dextrins and vitamins D is 5: 1 to 2: 1; Be preferably 3: 1, continue to produce 1 hydroxylate in the process of above-mentioned cultivation, the time of conversion is depended on the composition and the bacterial strain of substratum; Usually continue to cultivate preferably 4 days 1-10 days.
The time of substrate adding substratum is extremely important, normally in the time of 3 days, adds substrate the best in fermentation culture, also can control through the concentration of measuring sugar in the carbon source, normally drops to 0.05-0.002%.
The adding form of substrate is that former medicine solid is dissolved in the double solvents of organic solvent and tensio-active agent composition; Organic solvent is the alcohol organic solvent like low carbon number such as ethanol; Tensio-active agent is a polyoxyethylene sorbitan lipid acid non-ionic surfactant ester; Such as Triton X-100, Triton X-114 etc.Surface-active consumption is the 0.1-1 grams per liter with respect to culture volume than (g/L).
The present invention has used Schardinger dextrins, can comprise beta-cyclodextrin, γ-Huan Hujing.Cyclodextrin derivative in addition, hydroxypropyl-beta-cyclodextrin, glycosyl-beta-cyclodextrin with or methylated cyclodextrin.Wherein, best methylated cyclodextrin.Here, methyl flamprop is exhaustive methylation beta-cyclodextrin or part methyl beta-cyclodextrin.In the present invention, it is any selection between one or more these methylated cyclodextrin, preferably 2, and the 6-DM-.The interpolation of solubility promoter such as Schardinger dextrins etc. more helps the dissolving of substrate in nutrient solution, helps to improve transformation efficiency, and with respect to fermentation culture, consumption is 0.5～5 grams per liter.For keeping the aerobic condition of mikrobe, rotating speed was set to 200rpm when shaking table was cultivated.
Converted product adopt to filter, to mycelium with organic solvent such as organic chloroparaffin, ester class, fragrant alkane like extractions such as methylene dichloride, ETHYLE ACETATE, toluene, concentrated; Filtrate with non-polar macroporous resin like absorption such as 101 resins; With aforesaid organic solvent such as ethanol, acetone desorption, concentrate bullion, again through silica gel column chromatography; Organic solvent-normal hexane, sherwood oil etc. and ethyl ester gradient or 2: 1 are as eluent, and separation and purification obtains 1 pure α OH) VD.
Embodiment one
500ml shakes bottled 100 milliliters of nutrient solutions, 1.5% glucose, and 1.5% soybean cake powder, 0.5% steeping water, 0.5%NaCl, 0.2%CaCO3, pH 7.0.With 121 ℃ of substratum, sterilization 30min.2 milliliters of the spore suspensions of access bacterial strain CGMCC No.5098,27 ℃ of rotational oscillations are cultivated, and rotating speed 240rpm cultivated the 50mg vitamins D 4 days
3Be dissolved in 2mL ethanol with 10 milligrams beta-cyclodextrin, 10mgTriton X-100, this mixture is added in the nutrient solution, continue to cultivate 3 days; Filter, filtrating is with ETHYLE ACETATE 2 * 50mL extraction, and extracting solution concentrates; With silicagel column [column chromatography silica gel; ETHYLE ACETATE: sherwood oil (1: 2)] separate, obtain 1 α position hydroxylation product alfacalcidol 25mg, yield 50%.
UV：λmax＝265nm，λmin＝211nm，
1H?NMR(400MHz，DMSO)6.38(1H，d)，6.02(1H，d)，5.33(1H，s)，5.01(1H，s)，4.444(1H，m)，4.23(1H，m)，2.82(1H，m)，2.60(1H，m)，2.32(1H，m)，0.92(3H，d)，0.87(6H，m)。m/z：400[M]
+。
Embodiment two
Liquid culture based formulas: 1.5% glucose, 1.5% soybean cake powder, 0.5% steeping water, 0.7% yeast extract paste, 0.5%NaCl, 0.2%CaCO3, pH nature.With 121 ℃ of substratum, sterilization 30min.In the aforesaid liquid substratum of 100mL, at 28 ℃, 200rpm, shaking table cultivate 60h, with substrate VD bacterial classification inoculation
2340mg uses the 5ml anhydrous alcohol solution, adds the beta-cyclodextrin that methylates, 100mgTriton X-100 and the 5mlw sterilized water of 1 gram, and the dissolving mixing adds in the fermented liquid.Continue to cultivate 72h.The extraction of fermented liquid: add 30ml acetone in the 100mL fermented liquid, add 50mL ETHYLE ACETATE again, fully shake up, leave standstill; Get supernatant liquid, concentrate, carry out chromatography; Ethyl acetate/petroleum ether=1/2 obtains 1 α position hydroxylation product degree vitamine d3 140mg, yield about 40%.
UV：λmax＝265nm，λmin＝211nm，MS(EI)(m/z)：412(M
+)，
1H?NMR(400MHz，CDCl
3)δ0.57(s，3H)，0.76-0.91(m，9H)，4.25(m，1H)，4.45(m，1H)，5.02(s，1H)，5.21(m，2H，)，5134(s，1H)，6.03(d，1H)，6.39(d，1H)。
Embodiment three
250ml shakes bottled 50 milliliters of nutrient solutions, 1.5% glucose, and 1.5% soybean cake powder, 0.5% steeping water, 0.5%NaCl, 0.2%CaCO3, pH 7.0.With 121 ℃ of substratum, sterilization 30min.Insert bacterial strain CGMCC No.5098 bacterium colony, 27 ℃ of rotational oscillations are cultivated, and rotating speed 200rpm cultivated the 30mg vitamins D 3 days
2Be dissolved in the mixed solvent of 2mL ethanol and 1m sterilized water with beta-cyclodextrin and 5 milligrams of γ-Huan Hujings, 5mgTriton X-100 of 10 milligrams, this mixture is added in the nutrient solution, continue to cultivate 3 days; Filter, filtrating is with ETHYLE ACETATE 2 * 50mL extraction, and extracting solution concentrates; With silicagel column [column chromatography silica gel; ETHYLE ACETATE: sherwood oil (1: 2)] separate, obtain 1 α position hydroxylation product degree vitamine d3 10mg, yield 33%.
Embodiment four
500ml shakes bottled 100 milliliters of nutrient solutions, 1.5% glucose, 1.5% peptone, 1.5% soybean cake powder, 0.5% steeping water, 0.5%NaCl, 0.2%CaCO
3, pH 7.0.With 121 ℃ of substratum, sterilization 30min.Insert bacterial strain CGMCC No.5098 bacterium colony, 27 ℃ of rotational oscillations are cultivated, and rotating speed 240rpm cultivated the 100mg vitamins D 4 days
3Be dissolved in the mixed solution of 5mL ethanol and 2 ml sterile waters composition with 300 milligrams part methyl beta-cyclodextrin, 100mgTriton X-100, this mixture is added in nutrient solution, continue to cultivate 3 days; Filter, filtrating is with ETHYLE ACETATE 2 * 50mL extraction, and extracting solution concentrates; With silicagel column [column chromatography silica gel; ETHYLE ACETATE: sherwood oil (1: 2)] separate, obtain 1 α position hydroxylation product alfacalcidol 57mg, yield 57%.
The present invention provides a new operational path for the industrial preparation of alfacalcidol and degree vitamine d3; A kind of direct hydroxylated bioconversion method in 1-α position of vitamins D is provided; Chemical reaction about 6 steps of just known synthetic alfacalcidol and degree vitamine d3 shortens to a step, and total recovery is brought up to 40-50%.Technological operation is easy, reduces manufacturing cost, has got rid of poisonous reagent, environmental friendliness.
Claims (7)
1. the method for 1 alpha-hydroxy vitamin D is made in a microbial transformation, adopts false Nocardia CGMCC No.5098 that 1 of vitamins D is carried out microbial transformation, and a step directly obtains the 1-alpha-hydroxy vitamin D:
Substratum is in fermentation culture in the time of 3 days, vitamins D joined in the nutrient solution after as substrate and Schardinger dextrins and double solvents mixed dissolution, and concentration of substrate 1-15g/ liter, the ratio of Schardinger dextrins and vitamins D is 5: 1 to 2: 1; Continue to produce in the above-mentioned culturing process 1 hydroxylate, the time 1-10 of conversion days; Last through filtering, concentrated, silicagel column obtains target 1 α position hydroxylation product after separating.
2. the method for 1 alpha-hydroxy vitamin D is made in a kind of microbial transformation according to claim 1, it is characterized in that, said reactant vitamins D can be vitamins D
2Or vitamins D
3The respective objects product is the 1-alpha-hydroxy vitamin D
2Or 1-alpha-hydroxy vitamin D
3
3. the method for 1 alpha-hydroxy vitamin D is made in a kind of microbial transformation according to claim 1, it is characterized in that, the double solvents of said dissolving vitamins D substrate is made up of polyoxyethylene sorbitan lipid acid non-ionic surfactant ester and ethanol.
4. the method for 1 alpha-hydroxy vitamin D is made in a kind of microbial transformation according to claim 1, it is characterized in that, said Schardinger dextrins adopts beta-cyclodextrin or/and γ-Huan Hujing.
5. the method for 1 alpha-hydroxy vitamin D is made in a kind of microbial transformation according to claim 1, it is characterized in that, the ratio of Schardinger dextrins and vitamins D is for being preferably 3: 1.
6. the method for 1 alpha-hydroxy vitamin D is made in a kind of microbial transformation according to claim 1, it is characterized in that, produces preferably 4 days time of conversion of 1 hydroxylate in the culturing process.
7. the method for 1 alpha-hydroxy vitamin D is made in a kind of microbial transformation according to claim 4, it is characterized in that, said Schardinger dextrins adopts 2, the 6-DM-.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102660618A (en) * | 2012-04-17 | 2012-09-12 | 四川汪氏动物保健有限责任公司 | Method for preparing 25-hydroxyvitamin D by microbial transformation |
| CN103451242A (en) * | 2013-07-23 | 2013-12-18 | 西南交通大学 | Method for preparing 1alpha-hydroxy vitamin D through microbial conversion |
| CN103898004A (en) * | 2013-11-21 | 2014-07-02 | 中国医药集团总公司四川抗菌素工业研究所 | Pseudonocardia and method thereof for producing calcifediol by fermentation |
| CN105348164A (en) * | 2015-10-29 | 2016-02-24 | 无锡福祈制药有限公司 | Preparation method of 1alpha-hydroxyvitamin D2 |
| CN106748942A (en) * | 2016-11-28 | 2017-05-31 | 无锡福祈制药有限公司 | Doxercalciferol analog WXFQ 65 and its synthetic method |
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| CN114686450A (en) * | 2020-12-28 | 2022-07-01 | 苏州引航生物科技有限公司 | Modified vitamin D hydroxylase mutant and application thereof |
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2011
- 2011-09-20 CN CN201110279293A patent/CN102321678A/en active Pending
Cited By (13)
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| CN102660618B (en) * | 2012-04-17 | 2013-12-11 | 四川汪氏动物保健有限责任公司 | Method for preparing 25-hydroxyvitamin D by microbial transformation |
| CN102660618A (en) * | 2012-04-17 | 2012-09-12 | 四川汪氏动物保健有限责任公司 | Method for preparing 25-hydroxyvitamin D by microbial transformation |
| CN103451242A (en) * | 2013-07-23 | 2013-12-18 | 西南交通大学 | Method for preparing 1alpha-hydroxy vitamin D through microbial conversion |
| CN103898004B (en) * | 2013-11-21 | 2016-04-13 | 中国医药集团总公司四川抗菌素工业研究所 | The method of Selective medium and fermentative production U-32070E thereof |
| CN103898004A (en) * | 2013-11-21 | 2014-07-02 | 中国医药集团总公司四川抗菌素工业研究所 | Pseudonocardia and method thereof for producing calcifediol by fermentation |
| CN105348164B (en) * | 2015-10-29 | 2017-03-29 | 无锡福祈制药有限公司 | A kind of 1 α hydroxy-vitamine Ds2Preparation method |
| CN105348164A (en) * | 2015-10-29 | 2016-02-24 | 无锡福祈制药有限公司 | Preparation method of 1alpha-hydroxyvitamin D2 |
| CN106748942A (en) * | 2016-11-28 | 2017-05-31 | 无锡福祈制药有限公司 | Doxercalciferol analog WXFQ 65 and its synthetic method |
| CN106748942B (en) * | 2016-11-28 | 2018-06-29 | 无锡福祈制药有限公司 | Doxercalciferol analog WXFQ-65 and its synthetic method |
| CN108048493A (en) * | 2017-12-11 | 2018-05-18 | 四川菲派生物科技有限公司 | A kind of microorganism conversion manufactures 1,25- dihydroxyvitamin Ds3Method |
| CN108048493B (en) * | 2017-12-11 | 2021-04-27 | 四川菲派生物科技有限公司 | Preparation of 1, 25-dihydroxy vitamin D by microbial conversion3Method (2) |
| CN114686450A (en) * | 2020-12-28 | 2022-07-01 | 苏州引航生物科技有限公司 | Modified vitamin D hydroxylase mutant and application thereof |
| CN114686450B (en) * | 2020-12-28 | 2024-04-16 | 苏州引航生物科技有限公司 | Modified vitamin D hydroxylase mutants and uses thereof |
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Application publication date: 20120118 |