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Method for synthesizing calcitriol through oxidase hydroxylation

A technology of oxidase hydroxylation and calcitriol, which is applied in fermentation and other directions, can solve the problems of not being able to meet the concept of green synthesis, complicated separation and purification, and low yield, and achieve high atom economy, step economy, and treatment process The effect of simplicity and convenient preparation process

Active Publication Date: 2022-07-08
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the total synthesis method needs to synthesize the A-ring and C / D-ring synthons separately, the synthetic route is tedious, the yield is low, it is not conducive to mass production, the separation and purification are complicated, and it cannot meet the concept of green synthesis

Method used

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  • Method for synthesizing calcitriol through oxidase hydroxylation
  • Method for synthesizing calcitriol through oxidase hydroxylation
  • Method for synthesizing calcitriol through oxidase hydroxylation

Examples

Experimental program
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Effect test

Embodiment 1

[0038] In a 1 mL reaction flask, add 550 microliters of phosphate buffer solution (pH=7), add 350 microliters of acetone, add 50 microliters of acetone solution of alfacalcidol (2.5mmol / L), add 50 microliters of acetone solution Oxidase (AaeUPO enzyme, 500 nmol / L). In the system, the concentration of hydrogen peroxide was 1.0 mmol / L.

[0039] The above reaction system was reacted in a shaker at 30°C for 24 hours. After the reaction, take 100 microliters of the reaction solution, add 200 microliters of ethyl acetate, extract, dry with anhydrous sodium sulfate, and analyze by liquid chromatography. figure 1It can be seen that with the prolongation of the reaction time, the concentration of calcitriol increased, and the supernatant was collected for liquid chromatography (HPLC) to detect the content of calcitriol. HPLC detection conditions are: chromatographic column: shim-pack GIST-C18 shim-pack GIST-C18 4.6×250 mm×5 μM; mobile phase: water: acetonitrile=45:55; flow rate: 1 mL...

Embodiment 2

[0049] In a 1 mL reaction flask, add 550 microliters of phosphate buffer solution (pH=7), add 350 microliters of acetone, add 50 microliters of acetone solution of alfacalcidol (5 mmol / L), and add 75 microliters of peroxide Enzyme (MroUPO enzyme, 500 nmol / L). In the system, the concentration of hydrogen peroxide was 1.0 mmol / L at 40°C.

[0050] After the above reaction system was reacted in a shaking table at 40°C for 36 hours, the reaction was terminated, 100 μl of the reaction solution was taken, 200 μl of ethyl acetate was added, extracted, dried over anhydrous sodium sulfate, and analyzed by liquid chromatography. The yield was 55%. .

[0051] Preparation of MroUPO enzyme

[0052] Recombinant protein expression: The MroUPO nucleic acid sequence (SEQ ID NO. 9) was subcloned into the pPICZαA vector with a His-tag at the C-terminus. The pPICZαA-MroUPO recombinant plasmid was transformed into P. pastoris strain X-33, and 100 μL of competent cells were added to the linearize...

Embodiment 3

[0058] In a 1mL reaction flask, add 550 microliters of phosphate buffer solution (pH=8), add 350 microliters of acetone, add 50 microliters of acetone solution of alfacalcidol (7.5mmol / L), add 20 microliters of acetone solution Oxidase (MfeUPO enzyme, 500 nmol / L). In the system, the concentration of hydrogen peroxide was 1.0 mmol / L at 30°C.

[0059] After the above reaction system was reacted in a shaking table at 30°C for 16 hours, the reaction was terminated, 100 μl of the reaction solution was taken, 200 μl of ethyl acetate was added, extracted, dried over anhydrous sodium sulfate, and analyzed by liquid chromatography. The yield was 75%. .

[0060] Preparation of MfeUPO enzyme:

[0061] Recombinant protein expression: The MfeUPO nucleic acid sequence (SEQ ID NO. 5) was subcloned into pPICZαA vector with a His-tag at the C-terminus. The pPICZαA-MfeUPO recombinant plasmid was transformed into P. pastoris strain X-33, and 100 μL of competent cells were added to the lineari...

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Abstract

The invention discloses a non-coenzyme-dependent enzymatic synthesis method for synthesizing calcitriol through oxidase hydroxylation, and 1, 25-dihydroxy vitamin D3 (calcitriol) which can be used as an intermediate or a raw material medicine of various medicines is directly prepared from alpha calcitriol through a one-pot one-step method. The preparation process is simple and convenient, the yield is high, the downstream treatment process is simple, and no other solvent or cofactor is used except water and acetone (or other organic cosolvents) in the preparation process. The method is a green biosynthesis method and has relatively high atom economy and step economy.

Description

technical field [0001] The invention belongs to the technical field of biocatalysis, and in particular relates to an enzyme-catalyzed synthesis method of an active vitamin D3 (calcitriol) compound. Background technique [0002] Vitamin D3 is a kind of vitamin D. It is a fat-soluble vitamin. It is also regarded as a hormone precursor that acts on calcium and phosphorus metabolism. It is mainly synthesized by the human body itself. In addition, vitamin D3 can also be obtained from foods of animal origin. Cholecalciferol (English: Cholecalciferol, also known as vitamin D3 or cholecalciferol) is a kind of vitamin D, and 7-dehydrocholesterol generated by dehydrogenation of cholesterol can form cholecalciferol by ultraviolet irradiation, so it is also That is, the source of vitamin D for cholecalciferol is 7-dehydrocholesterol. At present, vitamin drugs based on active vitamin D3 mainly promote the absorption and deposition of calcium and phosphorus in the intestine; maintain th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/22
CPCC12P7/22
Inventor 张武元李元滢张鹏鹏李欢欢袁波孙周通马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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