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Biological preparing method of key chiral intermediate of levonorgestrel

A technology for a chiral intermediate, levonorgestrel, is applied in the field of preparing a key chiral intermediate of levonorgestrel, can solve the problems of catalyst pollution, unsatisfactory stereoselectivity and the like, and achieves high substrate concentration, high Good stereoselectivity

Active Publication Date: 2018-04-06
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing chemical synthesis method is to use sodium borohydride as a reducing agent, and the ethyl condensate is reduced into an ethyl hydroxylate. During the reaction, 4 kinds of isomers will be produced, the stereoselectivity is not ideal, and the catalyst pollutes the environment; Martina et al. used oxazoborane as a catalyst, added 6.5mM ethyl condensation substrate, the yield was 25%-30%, and the e.e. value was 75%.

Method used

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  • Biological preparing method of key chiral intermediate of levonorgestrel
  • Biological preparing method of key chiral intermediate of levonorgestrel
  • Biological preparing method of key chiral intermediate of levonorgestrel

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Screening of microbial strains for catalytic reduction of 2,6-dichloro-3-fluorophenethyl alcohol

[0038] 1. Strain screening

[0039] Enrichment culture: Add 1g of fresh soil samples (collected from the campus of Zhejiang University of Technology (Hangzhou, Zhejiang)) into a 250mL shake flask containing 50mL enrichment medium, culture at 30°C and 200rpm for 5 days, and take 1mL for culture The culture medium was transferred to fresh enrichment medium, and the culture was continued for 5 days, and the enrichment was repeated twice. The enrichment medium consists of: (NH 4 ) 2 SO 4 2g / L, KH 2 PO 4 2g / L, NaCl 1g / L, MgSO 4 ·7H 2 O 0.5g / L, the compound of formula (II) (2g / L) is the only carbon source, the solvent is distilled water, pH6.5.

[0040] Plate primary screening: the enriched culture solution was diluted 10 with normal saline 4 -10 6 After doubling, it was spread on the plate to select medium, and cultured at 30°C for 2 days. Pick a single ...

Embodiment 2

[0058] Embodiment 2: Catalyst preparation for bioreduction of ethyl condensates

[0059]1) Slant culture: Inoculate Geotrichum candidum ZJPH1704 into the slant medium, culture at 30°C for 1-2 days, and store in a refrigerator at 4°C. Slant medium formula: glucose 15g / L, peptone 7.5g / L, yeast extract 6g / L, (NH 4 ) 2 SO 4 3g / L, KH 2 PO 4 1.5g / L, NaCl 0.75g / L, MgSO 4 ·7H 2 O 0.75g / L, agar 20g / L, solvent is distilled water, pH 6.5.

[0060] 2) Seed culture: pick a ring of bacteria from the mature slant and insert it into a 250 ml shake flask containing 100 ml of seed medium, culture at 30° C. and 200 rpm for 12 hours to obtain seed liquid. Seed medium formula: glucose 15g / L, peptone 20g / L, yeast extract 10g / L, (NH 4 ) 2 SO 4 2g / L, KH 2 PO 4 2g / L, NaCl 1g / L, MgSO 4 ·7H 2 O0.5g / L, solvent is distilled water, pH 6.5.

[0061] 3) Fermentation culture: transfer the seed liquid to a 250ml shake flask equipped with 70ml fermentation medium with an inoculum size of 7% i...

Embodiment 3

[0062] Example 3 Bioreduction of Geotrichum candidum ZJPH1704 to prepare ethyl hydroxylate

[0063] 3g of Geotrichum candidum ZJPH1704 wet thallus obtained by the method of Example 2 was suspended in 10ml of 0.1M, pH 6.5 phosphate buffer, the final concentration of the wet thallus was 300g / L by wet weight, and a final concentration of 7.5g was added / L The ethyl condensate represented by formula (Ⅲ) is used as the substrate, cultured with shaking at 30°C and 200rpm for 72 hours, tracked and detected by thin-layer chromatography, centrifuged the reaction solution after the reaction, took the supernatant, and added an equal volume of ethyl acetate The ester was extracted twice, and the extracts were combined, and the solvent was evaporated with a rotary evaporator. Gained concentrate is dissolved in anhydrous methanol and then detected by HPLC, see Figure 10 . Simultaneous detection of the substrate ethyl condensate III ( Figure 8 ) and product ethyl hydroxylate IV standard...

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Abstract

The invention discloses a biological preparing method of a key chiral intermediate of levonorgestrel. The method includes the steps that wet cells obtained by fermenting cultivation of cryytococcus neoformans ZJPH1704 is used as an enzyme source, an ethyl condensation compound is used as a substrate, phosphate buffer with the pH of 5.8-8.0 is used as a reaction medium, a reaction system is formed,reaction is conducted at the temperature of 30-40 DEG C and the rotating speed of 150-250 rpm, and after the reaction is completed, reaction liquid is separated and purified to obtain the key chiralintermediate of levonorgestrel. The cryytococcus neoformans ZJPH1704 strain is used for catalytic reduction to prepare the intermediate, the key chiral intermediate has the advantages that the catalytic reduction substrate is high in concentration, and the stereoselectivity is high. When the substrate concentration is 7.0 g / L (22.4 mM), the yield reaches 73%, and the ee value is 100%.

Description

(1) Technical field [0001] The invention relates to a method for preparing a key chiral intermediate of levonorgestrel by biological reduction of Geotrichum candidum ZJPH1704 strain. (2) Background technology [0002] Ethyl condensate (chemical name: 2-[2-(3,4-dihydro-6-methoxy-1(2H)-naphthylidene)ethyl]-2-ethylcyclopentane-1, 3-Diketone, CAS No.: 850-92-0) structural formula: [0003] [0004] Ethyl Hydroxide (chemical name: 2-ethyl-3-hydroxy-2-[2-(6-methoxy-3,4-dihydro-1(2H)-naphthalene) ethyl]cyclopenta Ketone, CAS number: 51773-49-0) structural formula: [0005] [0006] Ethyl hydroxylate is used in the synthesis of progesterone drug levonorgestrel (chemical name: D(-)-17α-ethynyl-17β-hydroxy-18-methylestr-4-en-3-one) The key chiral intermediate of the drug, the drug mainly acts on the hypothalamus and pituitary gland, has good progesterone activity, and a strong ability to bind to androgen receptors. Used in combination with estrogen, it can be used as short-a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/26C12R1/645
CPCC12P7/26
Inventor 王普肖美娟陈和洁繆彦斐
Owner ZHEJIANG UNIV OF TECH
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