CN113881727A - Synthetic method of atorvastatin acid - Google Patents
Synthetic method of atorvastatin acid Download PDFInfo
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- CN113881727A CN113881727A CN202111030461.XA CN202111030461A CN113881727A CN 113881727 A CN113881727 A CN 113881727A CN 202111030461 A CN202111030461 A CN 202111030461A CN 113881727 A CN113881727 A CN 113881727A
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- enzyme
- atorvastatin acid
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/10—Nitrogen as only ring hetero atom
Abstract
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.
Description
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
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Met Thr Gly Gly Gln Gln Met Gly Arg Gly Ser Glu Phe Ser Pro Ile
1 5 10 15
Arg Arg Glu Val Ser Gln Asp Leu Phe Asn Gln Phe Asn Leu Phe Ala
20 25 30
Gln Tyr Ser Ala Ala Ala Tyr Cys Gly Lys Asn Asn Asp Ala Pro Ala
35 40 45
Gly Thr Asn Ile Thr Cys Thr Gly Asn Ala Cys Pro Glu Val Glu Lys
50 55 60
Ala Asp Ala Thr Phe Leu Tyr Ser Phe Glu Asp Ser Gly Val Gly Asp
65 70 75 80
Val Thr Gly Phe Leu Ala Leu Asp Asn Thr Asn Lys Leu Ile Val Leu
85 90 95
Ser Phe Arg Gly Ser Arg Ser Ile Glu Asn Trp Ile Gly Asn Leu Asn
100 105 110
Phe Asp Leu Lys Glu Ile Asn Asp Ile Cys Ser Gly Cys Arg Gly His
115 120 125
Asp Gly Phe Thr Ser Ser Trp Arg Ser Val Ala Asp Thr Leu Arg Gln
130 135 140
Lys Val Glu Asp Ala Val Arg Glu His Pro Asp Tyr Arg Val Val Phe
145 150 155 160
Thr Gly His Ser Leu Gly Gly Ala Leu Ala Thr Val Ala Gly Ala Asp
165 170 175
Leu Arg Gly Asn Gly Tyr Asp Ile Asp Val Phe Ser Tyr Gly Ala Pro
180 185 190
Arg Val Gly Asn Arg Ala Phe Ala Glu Phe Leu Thr Val Gln Thr Gly
195 200 205
Gly Thr Leu Tyr Arg Ile Thr His Thr Asn Asp Ile Val Pro Arg Leu
210 215 220
Pro Pro Arg Glu Phe Gly Tyr Ser His Ser Ser Pro Glu Tyr Trp Ile
225 230 235 240
Lys Ser Gly Thr Leu Val Pro Val Thr Arg Asn Asp Ile Val Lys Ile
245 250 255
Glu Gly Ile Asp Ala Thr Gly Gly Asn Asn Gln Pro Asn Ile Pro Asp
260 265 270
Ile Pro Ala His Leu Trp Tyr Phe Gly Leu Ile Gly Thr Cys Leu
275 280 285
<210> 2
<211> 864
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
atgactggtg gacagcaaat gggtcgcgga tccgaattca gtccgattcg tcgcgaagtg 60
agtcaggatc tgtttaatca gtttaatctg tttgcccagt atagtgccgc agcctattgc 120
ggtaaaaata atgatgcccc ggcaggtacc aatattacct gtaccggtaa tgcctgcccg 180
gaagttgaaa aagcagatgc cacctttctg tatagttttg aagatagcgg cgtgggtgac 240
gttaccggct ttctggcact ggataatacc aataagctga ttgttctgag ttttcgtggc 300
agccgcagca ttgaaaattg gattggtaat ctgaatttcg atctgaaaga aattaacgat 360
atctgtagtg gctgtcgtgg ccatgatggt tttaccagta gttggcgtag cgttgcagat 420
accctgcgtc agaaagttga agatgccgtt cgcgaacatc cggattatcg tgtggtgttt 480
accggccata gtctgggcgg cgcactggcc accgtggctg gtgcagatct gcgtggtaat 540
ggttatgata ttgatgtttt tagctacggc gccccgcgtg tgggtaatcg cgcctttgcc 600
gaatttctga ccgttcagac cggtggtacc ctgtatcgta ttacccatac caatgatatt 660
gttccgcgcc tgccgccgcg tgaatttggt tatagccata gcagcccgga atattggatt 720
aagagtggca ccctggttcc ggttacccgt aatgatattg tgaaaattga aggtatcgac 780
gccaccggcg gtaataatca gccgaatatt ccggatattc cggcacatct gtggtatttt 840
ggtctgattg gcacctgcct gtaa 864
<210> 3
<211> 27
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
cgccatatga ctggtggaca gcaaatg 27
<210> 4
<211> 31
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ccgctcgagt tacaggcagg tgccaatcag a 31
Claims (10)
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。
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| CN202111030461.XA CN113881727A (en) | 2021-09-03 | 2021-09-03 | Synthetic method of atorvastatin acid |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108315365A (en) * | 2018-02-08 | 2018-07-24 | 浙江宏元药业股份有限公司 | The biological synthesis method of Atorvastatin intermediate |
| CN109536468A (en) * | 2019-01-04 | 2019-03-29 | 浙江宏元药业股份有限公司 | Dicarbapentaborane reductase and its application in statins drug midbody synthesis |
| CN109776686A (en) * | 2019-03-27 | 2019-05-21 | 云南师范大学 | A kind of pattern of fusion lipase and its preparation method and application that thermostability improves |
| CN111647591A (en) * | 2020-06-24 | 2020-09-11 | 湖州颐盛生物科技有限公司 | Method for preparing statin intermediate by using immobilized enzyme |
-
2021
- 2021-09-03 CN CN202111030461.XA patent/CN113881727A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108315365A (en) * | 2018-02-08 | 2018-07-24 | 浙江宏元药业股份有限公司 | The biological synthesis method of Atorvastatin intermediate |
| CN109536468A (en) * | 2019-01-04 | 2019-03-29 | 浙江宏元药业股份有限公司 | Dicarbapentaborane reductase and its application in statins drug midbody synthesis |
| CN109776686A (en) * | 2019-03-27 | 2019-05-21 | 云南师范大学 | A kind of pattern of fusion lipase and its preparation method and application that thermostability improves |
| CN111647591A (en) * | 2020-06-24 | 2020-09-11 | 湖州颐盛生物科技有限公司 | Method for preparing statin intermediate by using immobilized enzyme |
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