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A kind of preparation method of (2s,3s)-2-hydroxyl-4-phenylbutane derivative

A technology of phenylbutane and its derivatives, which is applied in the field of bio-enzyme catalysis, can solve the problems of increasing the contact area between the substrate and the enzyme, the product purity is only 99%, and the difficulty of product separation and purification.

Active Publication Date: 2021-05-18
CHANGXING PHARMA
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  • Abstract
  • Description
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Problems solved by technology

[0008] Chinese patent CN102482648B discloses that selection comes from Novosphingobium aromaticivorans Carbonyl reductase gene construction engineering bacteria catalyze (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-2-butanone, the substrate concentration is 100g / L, the yield is greater than 95%, and the product The diastereomeric excess de is greater than 99.9%, but the patent only provides the technology to prepare the RS configuration, not the SS configuration
For the biological preparation of the SS configuration, Chinese patent CN104745649A discloses the use of (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-2-butanone as the substrate, and the substrate dosage concentration It is 50g / L, and the fluidity of the conversion system is promoted through the aid of co-solvents and surfactants, and the contact area between the substrate and the enzyme is increased. If no co-solvent is added, the fluidity of the conversion system will become poor, thereby affecting the conversion rate
However, in order to aid dissolution in this patent, additional substances such as toluene and Tween 60 added to the system are not conducive to the final separation and purification of the product, so the product purity is only 99%
Chinese patent CN104745649A also discloses a method for preparing biological enzymes in SS configuration, but the conversion system of this method uses glucose as the hydrogen supply system, the substrate concentration is 100g / L, and the reaction system is almost entirely in the water phase. Due to the substrate and SS configuration The products are hardly soluble in water. With the increase of the substrate concentration and no co-solvent, the fluidity of the system will gradually deteriorate, and the conversion rate will gradually decrease, which will limit the dosage of the substrate concentration, and the reaction will end in the system. Contains by-product gluconate, which brings difficulties to product separation and purification, and is difficult to be used in industrial production
[0009] To sum up, (2S,3S)-2 -Hydroxy-4-phenylbutane derivatives, the substrate concentration generally cannot exceed 100g / L due to solubility problems, or additional co-solvents are required to bring other impurities into the system, which will affect the separation and purification of SS configuration products and purity, the complex production process also increases the production cost

Method used

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  • A kind of preparation method of (2s,3s)-2-hydroxyl-4-phenylbutane derivative
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  • A kind of preparation method of (2s,3s)-2-hydroxyl-4-phenylbutane derivative

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Embodiment 1

[0043] Carbonyl reductase CXP I, whose amino acid sequence is shown in SEQ ID No.1, contains a polypeptide chain consisting of 253 amino acids, and is obtained by performing site-directed mutation on the gene of CX-LAC II strain.

[0044] The CX-LAC II strain was preserved in the China Type Culture Collection Center (CGMCC) on February 24, 2020, with the preservation number NO.19432, and was identified as Lactobacillus glutinum ( Lactobacillus farraginis ), facultative anaerobic, in the MRS solid medium, the colony is raised, slightly white, moist, with neat edges, and the colony is round, with a diameter of 3.0 mm±1.0 mm.   Microscopically, Gram-positive, generally short rods arranged in chains, usually immobile. CX-LAC II contains gene sequence CX II as shown in SEQID No.3.

[0045] Conduct site-directed mutation design on the gene sequence of CX II, mutate the 24th base A to T, the 519th base T to A, the 568-570th base TAT to CCG, and the 678th base The base T at position...

Embodiment 2

[0051] With (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-2-butanone as a substrate, the crude enzyme solution of carbonyl reductase obtained in Example 1 was used for enzyme activity experiments, and the preparation The reaction system is as follows: 50 mL system: add 1g (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-2-butanone, add different concentrations of isopropanol, stir at 40°C, add 2.4 g magnesium sulfate, 130 mg NAD + , use potassium phosphate or sodium buffer solution (100 mmol / L, pH8.0) to make up to 50 mL, and add 1 g of crude enzyme solution. After reacting for 24 hours, the conversion rate and de value were detected by high performance liquid chromatography (HPLC). The results are shown in Table 1. It can be seen that CXP I can be normally converted to almost exhausted substrate under 60% (m / V) isopropanol concentration, and the product de value is higher. Although general carbonyl reductases also exhibit excellent de values, their activity decreas...

Embodiment 3

[0055] The present invention increases the input amount of isopropanol in the system by providing a carbonyl reductase that is tolerant to high-concentration alcohol solutions. As an excellent substrate solvent and a hydrogen donor for the regeneration cycle of cofactors, it can further improve the substrate yield. Concentration, promote the conversion rate to break through the limit of the existing technology, can be further improved.

[0056] In a 1L catalytic system, add 80g (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-2-butanone, 2.4g magnesium sulfate, 60g glucose, 130 mg NAD +, and then adjust the pH to 7.0 with sodium carbonate solution, stir at 40° C., add 20 g of glucose dehydrogenase, and 4 g of the carbonyl reductase CXP I crude enzyme solution obtained in Example 1 above to start the reaction. Sodium carbonate solution was used for the reaction to maintain a pH of about 7.5. After 40 hours of reaction, 70% of the residual acid substrate was detected in the r...

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Abstract

The invention discloses a preparation method of (2S,3S)-2-hydroxyl-4-phenylbutane derivatives. (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-benzene Base-2-butanone, carbonyl reductase and excess isopropanol carry out biological enzyme-catalyzed reaction to obtain conversion liquid, and then obtain target product crystallization through continuous concentration and crystallization; the carbonyl reductase has high concentration of isopropanol tolerance through directed mutation The characteristics, so as to realize the solubilization of the substrate by excess isopropanol, and then increase the dosage of the substrate, improve the conversion rate, the product yield is not less than 95%, and the chiral purity is not less than 99.95%. In the present invention, through continuous concentration and crystallization of the conversion liquid, the crystallinity of the obtained product is more than 95%, the crystal is uniform, the impurity purity is not less than 99.95%, and the content is not less than 99%.

Description

technical field [0001] The invention relates to the field of biological enzyme catalysis technology, in particular to a preparation method of (2S,3S)-2-hydroxyl-4-phenylbutane derivatives. Background technique [0002] Non-peptide HIV protease inhibitors (PI) are the first choice for the treatment of HIV, such as saquinavir, nelfinavir, fosamprenavir, darunavir, etc. The process of releasing new and mature virus particles from the surface of infected host cells inhibits the protease of the virus. In June 2006, the FDA approved Darunavir in combination with other antiretroviral drugs for the treatment of HIV infection. For adult patients, darunavir was launched in 27 EU member states in March 2007. [0003] The core structures of the above compounds are all (2S,3S)-2-hydroxyl-4-phenylbutane derivative structures. For example, darunavir is mainly prepared through the compound of formula III (chemical name: (2S,3S)-N-tert-butoxycarbonyl-3-amino-1-chloro-2-hydroxy-4-phenylbuta...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P13/02C07C269/08C07C271/16
CPCC07C269/08C12P13/02C07C271/16Y02P20/55
Inventor 杨卫华谈聪张利坤钱敏帆金力倪建洲葛文强
Owner CHANGXING PHARMA
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