Preparation method of fosamprenavir intermediate

A system and hydrogen donor technology, applied in fermentation and other directions, can solve the problems of non-biological method reporting, and achieve the effects of short reaction time, low pollution and simple operation

Active Publication Date: 2015-07-01
ENZYMEWORKS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But the reaction of synthetic fosamprenavir intermediate (configuration is S, S) has no biological method report yet

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  • Preparation method of fosamprenavir intermediate
  • Preparation method of fosamprenavir intermediate
  • Preparation method of fosamprenavir intermediate

Examples

Experimental program
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Effect test

Embodiment 1

[0024] The gene fragment (synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.) containing the ketoreductase gene (SEQ ID No.1-20) was ligated with the digested product of pET28a plasmid (purchased from Invitrogen), and transformed into competent E. The coli BL21 (DE3) strain was screened to obtain positive clones, which were inoculated into 4 mL of liquid LB medium containing ampicillin resistance and activated overnight (37° C., 200 rpm). From the overnight culture, transfer 100 mL of liquid LB medium containing ampicillin resistance at 1 / 100 inoculum, culture at 37°C and 200 rpm with shaking until the OD600 value reaches 0.6-0.8, add IPTG and continue culturing overnight at 30°C. The cells were collected by centrifugation, and suspended in 10 mL of phosphate buffer (2 mM, pH 7.0). The cell suspension was ultrasonically disrupted in an ice bath for 10 minutes, centrifuged, the supernatant was pre-frozen overnight, and freeze-dried for 24h-48h to obtain the recombinant ket...

Embodiment 2

[0026] In a 10ml reactor, add 1.65mL 0.1M pH 7.0 phosphate buffer, dissolve 20mg of ketoreductase enzyme powder (SEQ ID No.1), 2mg of NADP or NAD in turn; substrate (N-protected (S)- 3-amino-1-chloro-4-phenyl-2-butanone) 0.1g dissolved in 0.25mL isopropanol, added to the reactor and stirred until completely dissolved, stirred at 1000rpm, reacted at 25°C for 24 hours, HPLC The conversion rate was detected, and the conversion rate of the NAD sample was 13.6%, and the conversion rate of the NADP sample was 61.3%.

Embodiment 3

[0028] In a 10ml reactor, add 1.65mL 0.1M pH 6.0-9.0 phosphate or triethanolamine buffer solution, dissolve 20mg of ketoreductase enzyme powder (SEQ ID No.1) and 2mg of NADP in sequence; the substrate (N-protected (S)-3-Amino-1-chloro-4-phenyl-2-butanone) 0.1g was dissolved in 0.25mL isopropanol, added to the reactor and stirred until completely dissolved, stirred at 1000rpm, and reacted at 25°C 20 hours, HPLC detects conversion rate, 6.0 (phosphate) sample conversion rate is 44.2%, 6.5 (phosphate) sample conversion rate is 52.8%, 7.0 (phosphate) sample conversion rate is 47.9%, 7.5 (phosphate) sample conversion rate The conversion rate was 56.3%, the conversion rate of the 8.0 (triethanolamine) sample was 57.3%, the conversion rate of the 8.5 (triethanolamine) sample was 62.1%, and the conversion rate of the 9.0 (triethanolamine) sample was 48.2%.

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Abstract

The invention relates to a preparation method of N-protected (2S, 3S)-3-amino-1-chloro-4-phenyl-2-butanol. The preparation method comprises the following steps: by taking N-protected (S)-3-amino-1-chloro-4-phenyl-2-butanone as a substrate, performing a reducing reaction on the substrate to generate the N-protected (2S, 3S)-3-amino-1-chloro-4-phenyl-2-butanol, wherein the reducing reaction is performed in the presence of a ketoreductase, a cofactor and a hydrogen donor, in an aqueous phase having the pH of 8-10 and at a temperature ranging from 30 to 50 DEG C; and the DNA sequence of the ketoreductase is as shown in any one of SEQ ID No. 1-20. According to the preparation method, as the specific ketoreductase is used for catalyzing the reduction reaction, the preparation method has the advantages of small enzyme dosage, mild reaction condition, simplicity in operation, short reaction time, high yield, and environmental protection. Compared with the chemical methods in the prior art, the preparation method is high in economic efficiency and low in pollution, and has an important application value.

Description

technical field [0001] The invention relates to a preparation method of a fosamprenavir intermediate, in particular to a method for enzymatically preparing N-protected (2S, 3S)-3-amino-1-chloro-4-phenyl-2-butanol. Background technique [0002] AIDS is currently the most serious infectious disease in the world, and more than 40 million people in the world are living with AIDS virus (Human Immunodeficiency Virus, HIV). Existing antiviral drugs can be divided into nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors and protease inhibitors according to their mechanism of action. Clinically, one or two reverse transcriptase inhibitors plus a protease inhibitor treatment plan (also known as cocktail therapy) is generally used. The research and development of protease inhibitors is later than that of reverse transcriptase inhibitors, but the growth rate is fast. In 2011, the sales of protease inhibitors were 2.3 billion US dollars, with a ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P13/00
Inventor 陶军华梁晓亮
Owner ENZYMEWORKS
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