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Method for preparing 1alpha-hydroxyl vitamin D through microbial transformation

A technology for the transformation of hydroxyvitamins and microorganisms, which is applied in the fields of drugs for the treatment of osteoporosis, chemistry and biopharmaceuticals. It can solve the problems of complicated separation and purification, complicated operations, and numerous steps, and achieve high yields, simple operations, and reduced manufacturing costs. cost effect

Inactive Publication Date: 2012-01-18
SOUTHWEST JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chemical synthesis requires multi-step group protection and deprotection, using light reaction, ring opening and isomerization, many steps, complicated separation and purification
The production operation is complicated, and the yield is low, around 10%.

Method used

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  • Method for preparing 1alpha-hydroxyl vitamin D through microbial transformation
  • Method for preparing 1alpha-hydroxyl vitamin D through microbial transformation
  • Method for preparing 1alpha-hydroxyl vitamin D through microbial transformation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] 100 milliliters of culture fluid in 500 ml shake flasks, 1.5% glucose, 1.5% soybean cake powder, 0.5% corn steep liquor, 0.5% NaCl, 0.2% CaCO3, pH 7.0. The culture medium was sterilized at 121°C for 30 minutes. Insert 2 ml of the spore suspension of the strain CGMCC No.5098, culture with rotary shaking at 27°C at 240 rpm, culture for 4 days, 50 mg of vitamin D 3 Dissolve 10 mg of β-cyclodextrin and 10 mg Triton X-100 in 2 mL of ethanol, add this mixture into the culture medium, continue to cultivate for 3 days, filter, extract the filtrate with ethyl acetate 2×50 mL, concentrate the extract, and use silica gel Column [column chromatography silica gel, ethyl acetate:petroleum ether (1:2)] was separated to obtain 25 mg of alfacalcidol, a hydroxylated product at the 1α position, with a yield of 50%.

[0024] UV: λmax=265nm, λmin=211nm, 1 H 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), 0.9...

Embodiment 2

[0026] Liquid medium formula: 1.5% glucose, 1.5% soybean cake powder, 0.5% corn steep liquor, 0.7% yeast extract, 0.5% NaCl, 0.2% CaCO3, natural pH. The culture medium was sterilized at 121°C for 30 minutes. Inoculate the bacteria in 100mL of the above-mentioned liquid medium, culture at 28°C, 200rpm, on a shaker for 60h, and the substrate VD 2 Dissolve 340 mg in 5 ml of absolute ethanol, add 1 gram of methylated β-cyclodextrin, 100 mg Triton X-100 and 5 mlw sterile water, dissolve and mix well, and add to the fermentation broth. Continue to cultivate for 72h. Extraction of fermentation broth: add 30ml of acetone to 100mL of fermentation broth, add 50mL of ethyl acetate, shake well, let stand, take the supernatant, concentrate, perform chromatography, ethyl acetate / petroleum ether=1 / 2, get 1α-hydroxylation product calciferol 140mg, the yield is about 40%.

[0027] UV: λmax=265nm, λmin=211nm, MS(EI)(m / z): 412(M + ), 1 H NMR (400MHz, CDCl 3)δ0.57(s, 3H), 0.76-0.91(m, 9H), ...

Embodiment 3

[0029] 50 milliliters of culture solution in 250 ml shaker flasks, 1.5% glucose, 1.5% soybean cake powder, 0.5% corn steep liquor, 0.5% NaCl, 0.2% CaCO3, pH 7.0. The culture medium was sterilized at 121°C for 30 minutes. Insert the colony of strain CGMCC No.5098, culture with rotary shaking at 27°C, rotation speed 200rpm, culture for 3 days, 30mg vitamin D 2 Dissolve 10 mg of β-cyclodextrin, 5 mg of γ-cyclodextrin, and 5 mg of Triton X-100 in a mixed solvent of 2 mL of ethanol and 1 m of sterile water, add this mixture to the culture medium, continue to cultivate for 3 days, and filter , the filtrate was extracted with ethyl acetate 2 × 50mL, the extract was concentrated, and separated with a silica gel column [column chromatography silica gel, ethyl acetate: petroleum ether (1: 2)] to obtain 10 mg of hydroxylated product calciferol at the 1α position. Yield 33%.

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Abstract

The invention discloses a novel industrial preparation method for a medicament 1-alpha-hydroxyl vitamin D2 or D3, namely paricalcitol and doxercalciferol, for treating osteoporosis. Site 1 of the vitamin D is subjected to microbial transformation by using pseudonocardia CGMCC No. 5098 to directly obtain 1-alpha-hydroxyl vitamin D in one step. The method is milder than a chemical method; protection is not required and toxic selenium dioxide is not required to be used for oxidation; the method has regional selectivity; radical protection is not required; the biotransformation yield reaches 40-50 percent; the method is environmentally-friendly; the process route is greatly shortened through one-step transformation reaction, the yield is improved, and the preparation cost is reduced; the known chemical reaction of about six steps for synthesizing the paricalcitol and the doxercalciferol is shortened into one-step reaction; and the total yield is increased to be 40-50 percent.

Description

technical field [0001] The invention relates to chemical and biopharmaceuticals, especially 1-alpha-hydroxyvitamin D for the treatment of osteoporosis 2 or D 3 , that is, the technical field of preparation of alfacalcitol (paricalcitol) and degree calciferol (Doxercalciferol). Background technique [0002] Alfacalcitol (paricalcitol) and doxercalciferol (Doxercalciferol) active vitamin drugs are clinically used for osteoporosis and rickets and osteomalacia caused by various reasons. [0003] 1-alpha-hydroxyvitamin D drugs are mainly used to treat other diseases such as osteoporosis and chronic renal failure. At present, chemical synthesis methods are mainly used for production and manufacture. It is very difficult to carry out chemical direct hydroxylation using the 1-α-position of vitamin D, and the yield is very low. The known synthesis of 1-α-hydroxyvitamin D mostly adopts chemical synthesis method, mainly using vitamin D as raw material, which is produced through mul...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/02C12R1/01
Inventor 陆群冯海婷周傲群李正龙
Owner SOUTHWEST JIAOTONG UNIV
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