Method for catalyzing synthesis of atazanavir intermediate by carbonyl reductase CLEAs

A carbonyl reductase and atazanavir technology, which is applied in the field of medicine, can solve the problems of unreusable carbonyl reductase, high requirements on reaction conditions, low reaction efficiency, etc., and achieves low cost, good temperature stability, and simple reaction system. Effect

Inactive Publication Date: 2019-07-09
SHANGHAI UNIV OF MEDICINE & HEALTH SCI
View PDF5 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, in the existing methods for the biosynthesis of atazanavir intermediates, there are problems...

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for catalyzing synthesis of atazanavir intermediate by carbonyl reductase CLEAs
  • Method for catalyzing synthesis of atazanavir intermediate by carbonyl reductase CLEAs
  • Method for catalyzing synthesis of atazanavir intermediate by carbonyl reductase CLEAs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] This example discloses a method for catalytically synthesizing an atazanavir intermediate, using (S)-tert-butyl (4-chloro-3-carbonyl-1-phenylbutyl-2-yl) carbamate as Substrate, using carbonyl reductase NaSDR cross-linked aggregates as a catalyst, catalyzes the synthesis of tert-butyl ((2S,3R)-4-chloro-3-hydroxyl-1 -phenylbutyl-2-yl)carbamate.

[0051] Wherein, the method for generating the cross-linked aggregate of the carbonyl reductase NaSDR comprises the following steps:

[0052] S1: obtaining the crude enzyme solution of carbonyl reductase NaSDR;

[0053] S2: using a precipitating agent to precipitate the crude enzyme solution of carbonyl reductase NaSDR;

[0054] S3: adding a cross-linking agent and an additive at the same time to carry out a cross-linking reaction to obtain a cross-linked aggregate of carbonyl reductase NaSDR.

[0055] The precipitating agent is tert-butanol, and the crosslinking agent is pentylene glycol.

[0056] In S3, the crosslinking time...

Embodiment 2

[0062] This embodiment discloses a method for preparing carbonyl reductase NaSDR crude enzyme liquid, comprising the following steps:

[0063] 1. Activation of E.coli-NaSDR bacteria

[0064] On the LB solid plate containing kanamycin (50μg / ml), the bacterial solution was coated by the method of partitioning or dilution. After culturing at 37°C for 20 hours, a single colony of activated E.coli-NaSDR was obtained.

[0065] 2. Fermentation and cultivation of E.coli-NaSDR bacteria

[0066] (1) Inoculate a single colony of E.coli-NaSDR bacteria into 5ml primary seed medium, and cultivate it at 37°C and 220rpm for 11 hours, and the OD600 reaches 0.9-1.2, which is the primary seed solution.

[0067] (2) Inoculate 5 ml of the primary seed solution into the fermentation medium (2.5% inoculum size), and cultivate at 37° C. and 220 rpm for 2-3 hours. When the OD600 of the fermentation medium reaches 0.9-1.2, add 400 μL of IPTG (isopropylthiogalactopyranoside, concentration 100 mmol / L)...

Embodiment 3

[0073] This embodiment discloses the method for preparing CLEAs:

[0074] 1. Screen the most suitable precipitation conditions

[0075] The inventor screened a variety of precipitants, including acetone, ammonium sulfate saturated solution, tert-butanol and PEG600. 4ml precipitation system: including 400 μL of crude enzyme solution, the final volume fractions were 30%, 40%, 50%, 60% , 70%, 80% and 90% of the precipitant, and supplement the TEA-HCL buffer solution to 4ml. After stirring slowly for 1 h in an ice-water bath, the mixed solution was centrifuged at 3000 rpm for 2 min, and the supernatant was discarded, then resuspended with 2 mL of buffer, and the enzyme activity of the precipitate was determined. The results showed that the recovery rate of enzyme activity was the highest when the volume fraction of tert-butanol was 60%.

[0076] 2. Screen the optimal concentration of cross-linking agent

[0077] This step studies the optimum concentration of glutaraldehyde, whi...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention relates to the field of medicine, in particular to a method for catalyzing synthesis of an atazanavir intermediate by a carbonyl reductase CLEAs. The method uses (S)-tert-butyl (4-chloro-3-carbonyl-1-phenylbutyl-2-yl) carbamate as a substrate and a carbonyl reductase NaSDR crosslinked aggregate as a catalyst, and the catalyzing synthesis of tert-butyl ((2S, 3R)-4-chloro-3-hydroxy-1-phenylbutyl-2-yl)) carbamate is carried out in a system in which a co-substrate, a co-solvent and a buffer solution are present. By the adoption of the method for the catalyzing synthesis of the atazanavir intermediate by the carbonyl reductase CLEAs, a reaction system is simpler, additional expensive coenzymes are not necessary, and glucose is not required to participate in a coenzyme cycle; in addition, the reaction temperature is closer to room temperature; the carbonyl reductase NaSDR crosslinked aggregate as a biocatalyst has better temperature stability and pH stability and can be reusedin 6 batches, so that the method is more energy-saving and environmentally friendly, and cost is further reduced.

Description

technical field [0001] The invention relates to the field of medicine, in particular to a method for synthesizing an atazanavir intermediate catalyzed by carbonyl reductase CLEAs. Background technique [0002] Atazanavir sulfate (CAS: 198904-31-3) is currently the main anti-AIDS drug in the world. It was developed by Bristol-Myers Squibb in the United States. It was first listed in the United States in June 2003. It is mainly combined with other antiretroviral A combination of drugs to treat HIV infection. Because it is only taken once a day, it has the advantages of rapid absorption and fewer adverse reactions than other anti-HIV virus infection drugs, and compared with other protease inhibitors, atazanavir does not interfere with normal diet during medication. After the FDA approved the listing in 2003, the global sales of the drug in 2009 were 5.2 billion US dollars, ranking 70th. With the frequent outbreaks of virus epidemics worldwide and the deepening of AIDS prevent...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12P13/02C12N11/02C07D213/42
CPCC12P13/02C12N11/02C12N9/0006C07D213/42
Inventor 邵雷吴锴杨志钧
Owner SHANGHAI UNIV OF MEDICINE & HEALTH SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap