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A kind of spironolactone derivative and its microbial conversion preparation method and application

A microbial transformation, spironolactone technology, applied in biochemical equipment and methods, microorganism-based methods, microorganisms, etc., to achieve the effects of high conversion yield, easy large-scale cultivation, and mild reaction conditions

Active Publication Date: 2015-10-28
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional organic chemical synthesis can hardly carry out such a direct hydroxylation reaction

Method used

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  • A kind of spironolactone derivative and its microbial conversion preparation method and application
  • A kind of spironolactone derivative and its microbial conversion preparation method and application
  • A kind of spironolactone derivative and its microbial conversion preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Preparation of Biocatalyst

[0033](1) Bacterial activation culture: Cunninghamella elegans ATCC9245 (purchased from American Type Culture Collection) was inoculated into potato agar medium, and cultured at 28°C for 3 days to obtain the spores of the fungus. The potato agar (PDA) medium (purchased from EMD Millipore Chemicals in the United States) was dissolved in distilled water according to the instructions, and sterilized by autoclaving at 121°C for 15min;

[0034] (2) Seed expansion culture: Dip the spores from the activated plate in step (1) with a cotton swab and transfer it to a conical flask containing 50 mL of seed medium, and culture at 28 °C and 250 r / min constant temperature shaking for 3 day to obtain seed liquid. The seed medium was potato liquid (PDB) medium (purchased from EMD Millipore Chemicals, USA), dissolved in distilled water according to the instructions, 50mL was divided into a 250mL conical flask, and sterilized by high pressure stea...

Embodiment 2

[0036] The tracking analysis of the biotransformation product of embodiment 2 spironolactone

[0037] Take 50 mL of bacterial cell-containing fermentation broth prepared by the method of Example 1, and 1.64 g of wet bacterial cells; 1 mg of spironolactone was dissolved in 0.1 mL of anhydrous ethanol, and then added to the fermentation broth to form a reaction system (the total volume was 50mL), incubate at 28°C and 250r / min for 4 days of constant-temperature shaking transformation. After the transformation is completed, filter the cells with gauze to separate the cells from the culture solution, and use 50mL of anhydrous methanol at 25°C and 100KHz for the cells. Ultrasonic leaching was performed for 30 min, the bacteria were removed by filtration, and the filtrate was evaporated to dryness under reduced pressure with methanol to obtain a concentrate. The concentrate was dissolved with 0.5 mL of anhydrous methanol and filtered to obtain extract a, which was to be analyzed; the ...

Embodiment 3

[0042] Example 3: Preparation of Biocatalysts

[0043] (1) Bacterial activation culture: Cunninghamella elegans ATCC9245 was inoculated into potato agar (PDA) medium, and cultured at 28°C for 3 days to obtain the spores of the fungus. The PDA medium was dissolved in distilled water according to the instructions, and sterilized by high-pressure steam at 121 °C for 15 min;

[0044] (2) Seed expansion culture: Dip the spores from the activated plate in step (1) with a cotton swab and transfer them to 4 Erlenmeyer flasks containing 50 mL of seed medium, and culture at 28°C and 250r / min constant temperature shaking for 3 day to obtain seed liquid. Described seed culture medium is PDB medium, add distilled water to dissolve according to the instruction, 50mL is divided into 250mL Erlenmeyer flask, 121 ℃ of high pressure steam sterilization 15min;

[0045] (3) Bacterial fermentation culture: the seed liquid prepared in step (2) was sequentially inoculated into 20 bottles containing...

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Abstract

The invention discloses a method for preparing hydroxy spironolactone by microbial transformation, which comprises: forming a transformation system in the original fermentation liquid or a phosphate buffer with a pH of 6.8 by using wet bacteria obtained by fermentation culture of cunninghamella elegans ATCC 9245 as catalysts, using spironolactone as a substrate, and using anhydrous alcohol as a cosolvent, performing a transformation reaction under a constant temperature vibration condition of 25-32 DEG C and 150-300 r / min, after the reaction, separating and purifying the reaction liquid to obtain 12[beta]-hydroxy-spironolactone and 2[alpha]-hydroxy-spironolactone. The microbial transformation method of the invention synthesizes two hydroxy spironolactones which have a certain degree of improvement in diuretic effect when compared with original spironolactone; the microbial strain used as the catalyst is safe and nontoxic, simple in culture condition, strong in mixed bacterium contamination resistance, and suitable for large-scale culture; the transformation operation is simple; no other reagent is necessary in the reaction process; the method is high in transformation yield, low in cost, and easy to realize industrial application.

Description

(1) Technical field [0001] The invention relates to a method for synthesizing hydroxyspironolactone, in particular to a new method for preparing 12β-hydroxy-spironolactone and 2α-hydroxy-spironolactone by using Cunninghamella elegans ATCC9245 as a biological catalyst and spironolactone as a substrate . (2) Background technology [0002] Spironolactone, chemical name is 17β-hydroxy-3-oxo-7α-(acetylthio)-17α-pregna-4-ene-21-carboxylic acid-γ-lactone, clinical drug is also called spironolactone Antisterone, the molecular structure is shown in formula (III): [0003] [0004] Spironolactone competes with aldosterone for binding to aldosterone-specific receptors at the cellular level, thus attenuating the biological effects of aldosterone; it competes with aldosterone for receptor binding at the distal renal tubule, thereby inhibiting the exchange of sodium and potassium , has a diuretic effect. In the more than 40 years since its introduction in 1957, because of its weak d...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07J31/00C12P33/20C12P33/06A61P7/10C12R1/645
Inventor 梅建凤占纪勋王罗医王思渊张书伟
Owner ZHEJIANG UNIV OF TECH
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