阅读说明：本技术 一种阿托伐他汀酸的合成方法 (Synthetic method of atorvastatin acid ) 是由 陈本顺 李大伟 徐春涛 石利平 尹斌 汪浩 钱若灿 张维冰 张凌怡 许�鹏 于 2021-09-03 设计创作，主要内容包括：本发明涉及药物合成领域,特别是涉及阿托伐他汀酸的合成方法。针对阿托伐他汀酸的化学合成中存在对环境不友好,以及容易产生杂质的问题,本发明公开了一种阿托伐他汀酸的合成方法,通过在含有式II化合物的溶剂中添加酶LP001实现由式II化合物生物合成式I化合物,该方法与现有技术中的单纯化学合成法相比,具有对环境友好、专一性强、不易生成脱酰胺杂质的优势,通过本发明公开的技术方案合成式I化合物,可以显著提高产物纯度,无需复杂的纯化工艺,具有广阔的工业化应用前景。(The invention relates to the field of drug synthesis, in particular to a synthetic method of atorvastatin acid. Aiming at the problems of environmental unfriendliness and easy generation of impurities in the chemical synthesis of atorvastatin acid, the invention discloses a synthetic method of atorvastatin acid, wherein the biosynthesis of a compound in a formula I from a compound in a formula II is realized by adding an enzyme LP001 in a solvent containing the compound in the formula II.)

1. A method for synthesizing atorvastatin acid is characterized by comprising the following steps of carrying out an enzyme catalyzed reaction on a compound of a formula II under the action of an enzyme LP001 to generate a compound of a formula I, wherein the reaction formula is as follows:

2. the method for synthesizing atorvastatin acid according to claim 1, wherein the amino acid sequence of the enzyme LP001 is as set forth in SEQ ID NO: 1 is shown.

3. The method for synthesizing atorvastatin acid according to claim 2, wherein the nucleotide sequence of the enzyme LP001 is as set forth in SEQ ID NO: 2, respectively.

4. The method for synthesizing atorvastatin acid according to claim 1, wherein the mass ratio of the compound II to the enzyme LP001 is 1 (1-30).

5. The method for synthesizing atorvastatin acid according to claim 1, wherein the enzymatic reaction of atorvastatin acid is performed in a mixed solution of alcohol and buffer, wherein the alcohol is one or more of isopropanol, methanol or ethanol, and the volume ratio of the alcohol to the buffer is 1: 10-20.

6. The method for synthesizing atorvastatin acid according to claim 5, wherein the mass-to-volume ratio of the compound II to the mixed solvent is 1:10-100 g/mL.

7. The method of synthesizing atorvastatin acid of claim 5, wherein the buffer is Tris-HCl buffer.

8. The method for synthesizing atorvastatin acid according to claim 1, wherein the enzyme LP001 is obtained by constructing a LP001 genetic engineering strain, culturing the strain by fermentation, and collecting the strain.

9. The method for synthesizing atorvastatin acid according to claim 1, wherein Ca is further added in the enzyme-catalyzed reaction²⁺Further preferred is the Ca²⁺The calcium chloride is added into the reaction system.

10. The method of synthesizing atorvastatin acid of claim 9 wherein the Ca is²⁺The amount of Ca added to the enzyme LP001 was 8g of Ca added to the enzyme LP001²⁺5-15mM。

Technical Field

The invention relates to the field of drug synthesis, in particular to a synthetic method of atorvastatin acid.

Background

Atorvastatin calcium (structural formula shown below) is hydroxymethyl glutaryl coenzyme A (HMG-CoA) reductase inhibitor. Mainly inhibits the synthesis of HMGCoA reductase, thereby inhibiting the synthesis of cholesterol in vivo and reducing the contents of serum low-density lipoprotein cholesterol and triglyceride. Because the atorvastatin calcium inhibits the cell synthesis of cholesterol, interferes with the generation of lipoprotein, lowers the serum total cholesterol level, can effectively lower the serum triglyceride level and can also raise the serum high-density lipoprotein cholesterol level. Can reduce plasma LDL cholesterol levels in certain homozygous familial hypercholesterolemia patients, and this type of population is less responsive to treatment with other lipid lowering agents. The composition is clinically used for treating primary hypercholesterolemia, mixed hyperlipidemia, hypertriglyceridemia, homozygous familial hypercholesterolemia and atherosclerosis. Statins have become a research hotspot in the 70 th century, the first statin lovastatin was approved by the FDA in the world in 1987, and new drugs such as simvastatin, pravastatin and atorvastatin are then appeared in succession like bamboo shoots in the spring after rain.

Atorvastatin acid is a salifying precursor of atorvastatin calcium, and the method adopted in the prior art is a chemical synthesis method and is mainly prepared through strong alkali. The chemical synthesis needs to use a large amount of chemical reagents, and the reagents have the problems of environmental unfriendliness in industrial production and do not meet the requirement of modern green production. Meanwhile, in the existing chemical preparation process, impurities with the following structural formula are easily generated, and the cost of subsequent separation and purification is increased.

Compared with a chemical synthesis method, the biological synthesis method has the advantages of milder reaction conditions and higher specificity of reaction products. Therefore, the method is an industrial synthesis method which accords with the modern green synthesis concept. The enzyme is an important participant enzyme in the biosynthesis process, and has specificity and high biological efficiency. However, specifically in one synthetic route, finding specific enzymes that can be adapted to this synthetic route is central to the research in the field of biosynthesis. This is also a technical difficulty that those skilled in the art need to overcome and develop the biosynthesis method for different synthetic routes.

Disclosure of Invention

The invention aims to solve the technical problems of environmental unfriendliness and easy generation of impurities in the chemical synthesis of atorvastatin acid.

In order to solve the technical problem, the invention discloses a method for synthesizing atorvastatin acid, which comprises the following steps that a compound in a formula II is subjected to an enzyme catalysis reaction under the action of an enzyme LP001 to generate a compound in a formula I, wherein the reaction formula is as follows:

further preferably, the amino acid sequence of the enzyme LP001 is as set forth in SEQ ID NO: 1 is shown.

More preferably, the nucleotide sequence of the enzyme LP001 is as set forth in SEQ ID NO: 2, respectively.

Further preferably, the mass ratio of the compound II to the enzyme LP001 is 1 (1-30).

Further preferably, the enzymatic reaction of the atorvastatin acid is carried out in a mixed solution composed of alcohol and buffer solution, wherein the alcohol is one or more of isopropanol, methanol or ethanol, and the volume ratio of the alcohol to the buffer solution is 1: 10-20; the mass-volume ratio of the compound II to the mixed solvent is 1:10-100 g/mL.

Further preferably, the buffer is Tris-HCl buffer.

In a preferred technical scheme, the enzyme LP001 is obtained by constructing an LP001 genetic engineering strain, performing fermentation culture and collecting thalli.

Specifically, the enzyme LP001 was prepared as follows: after PCR amplification is carried out on a fully-synthesized LP001 enzyme sequence, NdeI and XhoI enzyme cutting sites of a plasmid pET30a (+) are introduced to obtain a recombinant expression vector, the recombinant expression vector is electrically transferred into an LP001 expression cell to obtain LP001 expression engineering bacteria, the LP001 expression engineering bacteria are coated and screened by an antibiotic resistance flat plate to obtain a clone strain LP001-11, the clone strain LP001-11 is checked and sequenced to confirm that the clone strain is correct, fermentation culture is carried out after activation, bacteria are collected centrifugally, washed and resuspended, ultrasonically crushed and freeze-dried to obtain LP001 freeze-dried powder.

The method realizes the biosynthesis of the compound of the formula I from the compound of the formula II by adding the enzyme LP001 in the solvent containing the compound of the formula II, has the advantages of environmental friendliness, strong specificity and difficult generation of deamidation impurities compared with a single-purification chemical synthesis method in the prior art, can obviously improve the product purity by synthesizing the compound of the formula I by the technical scheme disclosed by the invention, does not need a complex purification process, and has wide industrial application prospect.

Drawings

FIG. 1 is a schematic representation of a recombinant expression vector.

Detailed Description

In order that the invention may be better understood, we now provide further explanation of the invention with reference to specific examples.

EXAMPLE 1 preparation of enzyme LP001

Step 1: preparation of LP001 gene engineering bacteria

According to the artificial sequence SEQ ID NO: 2, and (3) a nucleotide sequence shown in the specification, wherein the complete sequence of the synthetase LP 001. The synthesized sequence was PCR-amplified and cloned into NdeI and XhoI double restriction sites of expression plasmid pET-30a (+). The primers are respectively shown as SEQ ID NO: 3-4 is shown as follows:

F：cgccatatgactggtggacagcaaatg

R：ccgctcgagttacaggcaggtgccaatcaga

and (3) transferring the recombinant expression plasmid into an E.coli BL21(DE3) susceptible strain, selecting a positive transformant, sequencing and identifying to obtain the LP001 expression engineering bacterium.

Step 2: preparation of LP001

The obtained LP001 engineering strain is inoculated into LB liquid culture medium containing antibiotic kana resistance and cultured at 37 ℃ overnight to obtain seed culture solution. 1ml of seed liquid is inoculated into 100ml of TB fermentation liquid culture medium. Then placing the mixture at 37 ℃ for culture until the OD600 value is 0.6, adding IPTG with the final concentration of 0.05mol, placing the mixture at 16 ℃ for further culture for 16h, and then carrying out centrifugation at 12000rmp at 4 ℃ to collect thalli, namely the enzyme LP001 used in the example 2 and the example 3, wherein the amino acid sequence of the enzyme LP001 is shown in SEQ ID NO: 1 is shown.

And (3) adding the obtained enzyme LP001 into a liquid nitrogen tank for quick freezing for 5min, and freeze-drying to obtain freeze-dried powder for later use.

EXAMPLE 2 preparation of Compound I

In a 250mL Erlenmeyer flask, 30mL of Tris-HCl buffer, pH8.0, are added, followed in succession by 8g of enzyme LP001, 1g of Compound II, 2mL of isopropanol, 10 mL of MCaCl₂Stirring at 220rpm and reacting at 35 ℃ for 24h to obtain the compound I. The reaction result was checked by HPLC, the conversion was 61% and the recrystallization purity was 94%.

EXAMPLE 3 preparation of Compound I

30mL of Tris-HCl buffer (pH8.0) was added to a 250mL Erlenmeyer flask, followed by addition of 8g of the enzyme LP001, 1g of the compound II, and 2mL of isopropanol, followed by stirring at 220rpm and reaction at 35 ℃ for 24 hours to obtain the compound I. The reaction result was checked by HPLC, the conversion was 53% and the recrystallization purity was 90%.

By the fact thatAs can be seen from the experimental results of examples 2 and 3, Ca is added in the present invention²⁺And the LP001 has better catalytic effect.

The enzyme prepared by the invention has excellent stereoselectivity, can effectively improve the yield and the optical purity of the product, reduces the generation amount of impurities, and has good application prospect.

What has been described above is a specific embodiment of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and such improvements and modifications are also considered to be within the scope of the present invention.

Sequence listing

<110> Jiangsu Furuikangtai pharmaceutical Co., Ltd

<120> synthetic method of atorvastatin acid

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Gln Tyr Ser Ala Ala Ala Tyr Cys Gly Lys Asn Asn Asp Ala Pro Ala

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Gly Thr Asn Ile Thr Cys Thr Gly Asn Ala Cys Pro Glu Val Glu Lys

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Ala Asp Ala Thr Phe Leu Tyr Ser Phe Glu Asp Ser Gly Val Gly Asp

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Val Thr Gly Phe Leu Ala Leu Asp Asn Thr Asn Lys Leu Ile Val Leu

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Ser Phe Arg Gly Ser Arg Ser Ile Glu Asn Trp Ile Gly Asn Leu Asn

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Phe Asp Leu Lys Glu Ile Asn Asp Ile Cys Ser Gly Cys Arg Gly His

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Asp Gly Phe Thr Ser Ser Trp Arg Ser Val Ala Asp Thr Leu Arg Gln

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Lys Val Glu Asp Ala Val Arg Glu His Pro Asp Tyr Arg Val Val Phe

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Thr Gly His Ser Leu Gly Gly Ala Leu Ala Thr Val Ala Gly Ala Asp

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Leu Arg Gly Asn Gly Tyr Asp Ile Asp Val Phe Ser Tyr Gly Ala Pro

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Arg Val Gly Asn Arg Ala Phe Ala Glu Phe Leu Thr Val Gln Thr Gly

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Gly Thr Leu Tyr Arg Ile Thr His Thr Asn Asp Ile Val Pro Arg Leu

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Glu Gly Ile Asp Ala Thr Gly Gly Asn Asn Gln Pro Asn Ile Pro Asp

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