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Method for preparing (S)-N-t-butyloxycarboryl-3-hydroxypiperidine

A carbonyl and oxidized coenzyme technology, which is applied in fermentation and other fields, can solve the problems of difficult catalyst acquisition, poor substrate tolerance, and low space-time yield, and achieve important industrial application value, mild reaction conditions, and high reaction efficiency.

Inactive Publication Date: 2016-03-23
CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, most of these catalysts are difficult to obtain, the stereoselectivity is not high, the substrate tolerance is poor, and the conversion rate is low, resulting in low space-time yield and no good industrial economic benefits (Org.Lett.2009, 11, 1245-1248 ; OPRD.2014,18,827-830; CN104603278A, CN103276027A, CN103923957A, CN103571908A, CN103131734A, WO2013190341A1)

Method used

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  • Method for preparing (S)-N-t-butyloxycarboryl-3-hydroxypiperidine

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Embodiment 1

[0032] Embodiment 1 Preparation of recombinant carbonyl reductase crude enzyme solution

[0033] The biocatalyst of the present invention can be prepared by conventional methods in the art.

[0034] Construction of recombinant bacteria with carbonyl reductase: The fragment containing the carbonyl reductase gene and the pET28a plasmid were double-digested with the same restriction endonuclease, ligated and the ligated product was transferred into a competent E.coliBL21(DE3) strain and screened to obtain Positive clones were inoculated into LB liquid medium containing kanamycin resistance (50 μg / mL) and activated overnight (37° C., 220 rpm) to prepare seed liquid. Transfer the seed solution to 100 mL of LB liquid medium containing kanamycin resistance (50 μg / mL) with 1% inoculum, and culture it with shaking at 37 ° C and 220 rpm until OD 600 When the temperature is between 0.6 and 0.8, add IPTG (0.5 mM) and continue culturing overnight at 20°C. The cells were collected by cent...

Embodiment 2

[0035] Embodiment 2 carbonyl reductase biocatalysis

[0036] A certain amount of substrate and 50 μL of methanol were added to a 10 mL reaction bottle, and then 0.1 mL of potassium phosphate buffer (1M, pH 7.0), 0.1 mL of recombinant carbonyl reductase crude enzyme solution (30 mg / mL, 6.27 U / mg ), 0.025mLNADP + Solution (stock solution concentration 40mM), 0.1mL glucose dehydrogenase crude enzyme solution (30mg / mL, 10U / mg), 0.3mL glucose syrup (stock solution concentration 100%, w / v), add water to 1mL, mix well Afterwards, the reaction was magnetically stirred at 30°C, and the pH of the reaction system was adjusted to 6.5-7.0 with 1M sodium hydroxide solution every 15 minutes. Carry out HPLC analysis after reaction finishes, and detection result is as follows:

[0037] serial number

Embodiment 3 100

[0038] Embodiment 3 100 milligram level preparation technology

[0039] Add 150mg substrate and 50μL methanol to a 10mL reaction bottle, then add 0.1mL potassium phosphate buffer (1M, pH7.0), 0.1mL recombinant carbonyl reductase ChKRED03 crude enzyme solution (30mg / mL, 4.5U / mg ), 0.025mLNADP + Solution (stock solution concentration 40mM), 0.1mL glucose dehydrogenase crude enzyme solution (30mg / mL, 10U / mg), 0.18mL glucose syrup (stock solution concentration 100%, w / v), make up to 1mL with water, mix well Afterwards, the reaction was magnetically stirred at 30°C, and the pH of the reaction system was adjusted to 6.5-7.0 with 1M sodium hydroxide solution every 15 minutes. After reacting for 1h and 2.5h, samples were taken for HPLC analysis, and the spectra were shown in the attached instructions. figure 2 with image 3 . Instructions attached figure 2The retention time of 14.040min is (S)-N-tert-butoxycarbonyl-3-hydroxypiperidine, the retention time of 15.443 is the R conf...

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Abstract

The invention relates to a method for preparing (S)-N-t-butyloxycarboryl-3-hydroxypiperidine through carbonyl reductase biocatalysis. According to the method, reaction conditions are moderate, conversion efficiency is high, stereoselectivity is high, and the product ee value is 99.5% or above. The concentration of a substrate can be increased to 250 g / L, the space time yield can be as high as 1600 g / L / d, production efficiency is greatly improved, and the method has important industrial application value.

Description

technical field [0001] The invention relates to a method for preparing (S)-N-tert-butoxycarbonyl-3-hydroxypiperidine by biocatalysis technology, which belongs to the technical field of biopharmaceutical industry. Background technique [0002] (S)-N-tert-butoxycarbonyl-3-hydroxypiperidine is an important pharmaceutical intermediate, which is widely used in the synthesis of analgesic, antipsychotic, antitumor and other drugs, such as ibrutinib. The drug was launched in the United States at the end of 2013, and it is conservatively estimated that the global sales in 2018 will reach 2.32 billion US dollars. [0003] Currently, optically active (S)-N-tert-butoxycarbonyl-3-hydroxypiperidine compounds can be obtained by chemical or enzymatic resolution of racemates. However, the limitation of the resolution method is that the maximum theoretical yield is 50%, and the separation and extraction are relatively cumbersome (USpatent2011092698A1, WO2011036280A1). [0004] Biocatalytic ...

Claims

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Application Information

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IPC IPC(8): C12P17/12
Inventor 吴中柳许光鹏刘艳裴小琼
Owner CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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