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Method for preparing 7beta-hydroxyl-3beta cholesterol acetate from hydroxylase 3beta-cholesterol acetate

A technology of cholesterol acetate and β-cholesterol, which is applied in the field of enzymatic hydroxylation of 3β-cholesterol acetate to prepare 7β-hydroxy-3β-cholesterol acetate, which can solve problems such as industrial production difficulties, environmental pollution, and complex synthesis operations , to achieve good technical application and industrialization prospects, reduce production costs, and simple operation

Active Publication Date: 2015-05-13
杭州馨海生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0020] The present invention aims at the disadvantages in the prior art that the starting materials are not easy to obtain, the yield is low, the reagents used in the synthesis process are many and expensive, the synthesis operation is complicated, the synthesis process is poisonous or pollutes the environment, and the industrial production is relatively difficult. A mild and easy-to-control reaction condition, relatively simple synthetic operation, low cost, high yield, elimination of highly toxic chemical reagents, environmentally friendly enzyme hydroxylation of 3β-cholesterol acetate to prepare 7β-hydroxyl-3β-cholesterol acetate Methods

Method used

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  • Method for preparing 7beta-hydroxyl-3beta cholesterol acetate from hydroxylase 3beta-cholesterol acetate
  • Method for preparing 7beta-hydroxyl-3beta cholesterol acetate from hydroxylase 3beta-cholesterol acetate
  • Method for preparing 7beta-hydroxyl-3beta cholesterol acetate from hydroxylase 3beta-cholesterol acetate

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Effect test

Embodiment 1

[0043] A method for enzyme hydroxylation of 3β-cholesterol acetate to prepare 7β-hydroxyl-3β-cholesterol acetate, the specific steps of the method are as follows:

[0044]10 mg of crude enzyme of cytochrome P450 hydroxylase (cytochrome P450, Cyp27b1, locus tag: NP_034139 from gene bank) was dissolved in 10 mL (2 mM, pH=8.9) of PBS buffer solution.

[0045] Take out 10mL and add in the Erlenmeyer flask of 100mL, add 20mL PBS phosphate buffer solution (2mM, pH=8.9), continue to add 5mL concentration and be the 3β-cholesterol acetate solution (pH=8.9, 30% ethanol) of 10mM, 8 mL of hydrogen peroxide solution (pH=8.9) with a concentration of 10 mM was fully stirred in a shaker, the stirring speed was 200 r / min, the temperature was fixed at 37 ° C, and the reaction was performed for 2 hours. The mixed reagent of ether is used as a developing agent, and the end point of the reaction can be judged by monitoring the concentration of newly generated spots and the concentrations of remai...

Embodiment 2

[0049] A method for enzyme hydroxylation of 3β-cholesterol acetate to prepare 7β-hydroxyl-3β-cholesterol acetate, the specific steps of the method are as follows:

[0050] 20 mg of crude enzyme of cytochrome P450 monooxygenase (CYP98A3, locus tag: AT2G40890 from gene bank) was dissolved in 10 mL (5 mM, pH=8.0) of PBS buffer solution.

[0051] Take out 10mL and add it to a 100mL Erlenmeyer flask, add 20mL of PBS phosphate buffer solution (5mM, pH=8.0), and continue to add 5mL of 10mM 3β-cholesterol acetate solution (pH=8.0, 20% ethanol ), 8 mL of hydrogen peroxide solution (pH=8.0) with a concentration of 10 mM, fully stirred and reacted in a dynamic stirring device, the stirring speed was 200 r / min, and the temperature was fixed at 35 ° C for 0.5 hours. The formation of compound 7β-hydroxy-3β-cholesterol acetate and the disappearance of compound 3β-cholesterol acetate in phase chromatography were used to determine the end point of the reaction.

[0052] After the reaction, th...

Embodiment 3

[0055] A method for enzyme hydroxylation of 3β-cholesterol acetate to prepare 7β-hydroxyl-3β-cholesterol acetate, the specific steps of the method are as follows:

[0056] 15 mg of crude enzyme of Caenorhabditis elegans monooxygenase (Caenorhabditis elegans monooxygenase, locus tag: T19B4.1 from gene bank) was dissolved in 10 mL (5 mM, pH=7.0) of PBS buffer solution.

[0057] Take out 10mL and add in the Erlenmeyer flask of 100mL, add 20mL PBS phosphate buffered solution (5mM, pH=7.0), continue to add 3β-cholesterol acetate solution (pH=7.0, 25% acetone) that 5mL concentration is 10mM, 8 mL of hydrogen peroxide solution (pH=7.0) with a concentration of 10 mM was fully stirred in a shaker to react at a stirring speed of 130 r / min, and the temperature was fixed at 35°C for 1.5 hours. During the reaction, TLC silica gel plate was used to monitor, using ethyl acetate and petroleum ether The mixed reagent is used as a developing agent, and the end point of the reaction is judged by...

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Abstract

The invention relates to a method for preparing 7beta-hydroxyl-3beta cholesterol acetate, in particular to the method for preparing the 7beta-hydroxyl-3beta cholesterol acetate from hydroxylase 3beta-cholesterol acetate. The method comprises the following steps of: dissolving the hydroxylase 3beta-cholesterol acetate into a cosolvent; feeding the dissolved 3beta-cholesterol acetate solution in a buffer solution; adding an enzyme preparation and a hydrogen peroxide solution; adjusting the pH value to be 6-10; fully stirring and reacting at the temperature of 15-50DEG C for 0.5-9h; tracking and monitoring the reaction process by adopting a TLC (Thin-Layer Chromatography) silica gel plate or high performance liquid chromatography until the reaction is finished; extracting the reaction solution with an organic solvent; and removing the organic solvent by decompressing to obtain a crude product of the compound of the 7beta-hydroxyl-3beta cholesterol acetate. The invention has the advantages of mild and controllable reaction conditions, simple synthetic operation, low cost and high yield, and is friendly to the environment because a high-toxicity chemical reagent is removed.

Description

technical field [0001] The invention relates to a method for preparing 7β-hydroxy-3β-cholesterol acetate, in particular to a method for preparing 7β-hydroxy-3β-cholesterol acetate by enzymatically hydroxylating 3β-cholesterol acetate. Background technique [0002] Vitamin D is a sterol derivative. Its main function in the human body is to promote bone calcification, regulate calcium and phosphorus metabolism, and cell growth and differentiation. It is also a good selective immunomodulator. Among them, the physiological effect of active vitamin D3 is the strongest, and the one shown in figure (III) is a kind of vitamin D3. [0003] [0004] Vitamin D3, also known as niacinamide, cholecalciferol. Vitamin D mainly has the following physiological functions: 1. Improve the body's absorption of calcium and phosphorus, and make the levels of plasma calcium and phosphorus reach saturation. 2. Promote growth and bone calcification, and promote healthy teeth; 3. Increase phosphor...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P33/06
Inventor 于洪巍王博徐红娜
Owner 杭州馨海生物科技有限公司
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