WO2014190956A1 - Procédé de production de cathine - Google Patents

Procédé de production de cathine Download PDF

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Publication number
WO2014190956A1
WO2014190956A1 PCT/DE2014/000145 DE2014000145W WO2014190956A1 WO 2014190956 A1 WO2014190956 A1 WO 2014190956A1 DE 2014000145 W DE2014000145 W DE 2014000145W WO 2014190956 A1 WO2014190956 A1 WO 2014190956A1
Authority
WO
WIPO (PCT)
Prior art keywords
transaminase
formula
alcohol dehydrogenase
carried out
reaction
Prior art date
Application number
PCT/DE2014/000145
Other languages
German (de)
English (en)
Inventor
Dörte ROTHER
Martina Pohl
Thorsten SEHL
Original Assignee
Forschungszentrum Jülich GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Forschungszentrum Jülich GmbH filed Critical Forschungszentrum Jülich GmbH
Priority to EP14717091.4A priority Critical patent/EP3004364A1/fr
Publication of WO2014190956A1 publication Critical patent/WO2014190956A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/001—Amines; Imines
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01001—Alcohol dehydrogenase (1.1.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y206/00—Transferases transferring nitrogenous groups (2.6)
    • C12Y206/01—Transaminases (2.6.1)

Definitions

  • the invention relates to a process for the preparation of cathine.
  • Cathin ((1 p.2S) - norpseudoephedrine) belongs to the class of ephedra alkaloids and can be obtained from the leaves of the cat shrub. It is used as a cardiovascular agent because of the sympathomimetic, stimulating the autonomic nervous system, and is often used as an appetite suppressant.
  • cathin having an enantiomeric purity and diastereomeric purity of 99% or more can be produced. It can be achieved a turnover of up to 61, 4%.
  • the preparation proceeds in a two-stage process, is simple, inexpensive and requires no elaborate workup. Commercially available starting materials can be used and the two reaction steps can be carried out in a reaction vessel without isolation of the intermediate product.
  • the synthesis steps can be carried out either with preferably purified and lyophilized enzymes or with recombinant E. coli
  • Cells are carried out containing the enzymes used in the invention.
  • the process can be scaled up well and is therefore suitable for large-scale processes.
  • An S-selective enzyme in the sense of the invention means an enzyme which converts a substrate into an S-configured product.
  • Methylbenzylamine is an example of an amine donor.
  • amine donors may be used.
  • the transaminase may be from a chromobacterium, for example Chromobacterium violacaeum, preferably CV2025. Furthermore, transaminases from Alealigens denitrificans, Arthrobacter citreus, Bacillus megaterium, Pseudomonas fluorescens, Vibrio flurialis or Caulobacter crescentus.
  • purified and lyophilized enzymes are used. This has the advantage that products with significantly higher optical purities, ie higher enantiomeric and diastereomeric excesses, are produced with purified enzymes.
  • the enzymes are stable and can convert high substrate concentrations.
  • the reaction may be carried out in an aqueous medium in a pH range of 6 to 11, preferably pH 7.5 to 8.5.
  • buffers such as HEPES, potassium phosphate, MOPS, TEA or TRIS-HCl can be used.
  • the preferred temperature range is room temperature, the reaction can be performed well in a temperature range of 20 ° C to 30 ° C.
  • the reaction is preferably carried out at atmospheric pressure.
  • the pyridoxal-5 'phosphate concentration is between 100-200 ⁇ .
  • the process can be performed in vitro.
  • the enzymatic reaction can be done in vivo.
  • E. coli bacteria can be used as production organisms.
  • Genes coding for a transaminase can be ligated into a vector.
  • the E. coli strains are recombinant and contain plasmids carrying genes for a (S) -selective transaminase.
  • the plasmids may contain the genes for the o.
  • G. Transaminases includes.
  • plasmids it is possible to use, for example, pET29a or pKK233 basic bodies which contain the corresponding transaminase genes.
  • the production organisms secrete the desired product according to formula (2) into the aqueous solution.
  • the transaminase is deactivated after the first reaction step.
  • an acid preferably a non-oxidizing acid, for example HCl or H 2 S0 4 , can be added.
  • the inactivation can also be carried out by ultrafiltration of the enzyme.
  • a membrane with a molecular exclusion of, for example, 10 kDa (kilo daltons) can be used.
  • this reaction a reductive hydrogenation
  • alcohol dehydrogenases which convert the substrate and which are (S) -selective.
  • the alcohol dehydrogenase may be from Lactobacillus, preferably from Lactobacillus brevis (LbADH).
  • Lactobacillus brevis LbADH
  • alcohol dehydrogenases from Lactobacillus kefir, Candida magnoliae, Leifsonia species, Sporobolomyces salmonicolor or Trichosporon cutaneum can be used.
  • purified and lyophilized enzymes are used. This has the advantage that products with significantly higher optical purities, ie higher enantiomeric and diastereomeric excesses, are produced with purified enzymes.
  • the enzymes are stable and can convert high substrate concentrations.
  • the reaction can be carried out in an aqueous medium in a pH range from 6.5 to 8.5, preferably pH 7.5 to 8.5.
  • Suitable buffers such as HEPES (2- (4- (2-hydroxyethyl) -1-piperazinyl) ethanesulfonic acid), potassium phosphate, TEA (triethanolamine), TRIS-HCl (tris (hydroxymethyl) aminomethane) or MOPS (3- (N-morpholino) -propansulphonic acid).
  • HEPES (4- (2-hydroxyethyl) -1-piperazinyl) ethanesulfonic acid
  • potassium phosphate potassium phosphate
  • TEA triethanolamine
  • TRIS-HCl tris (hydroxymethyl) aminomethane
  • MOPS 3- (N-morpholino) -propansulphonic acid
  • the reaction is preferably carried out at atmospheric pressure.
  • NADPH + H + or NADP + can be used with magnesium ions (Mg 2+ ), eg as magnesium sulphate, if the enzyme LbADH is used.
  • NADH + H + , NAD + and other ions such as Ca 2+ , Mn 2+ , K + , Na + , Co 2+ , Li + , Zn 2+ or Rb + may be used.
  • the addition of metal ions is optional and has a positive effect on cofactor regeneration.
  • a formate dehydrogenase (FDH) with formate as cosubstrate can be used for cofactor regeneration, with TbADH from Thermoanaerobium brockii with ethanol or isopropanol as cosubstrate or with GDH (glucose dehydrogenase) with glucose as cosubstrate ,
  • the process can be performed in vitro.
  • the enzymatic reaction can be done in vivo.
  • E. coli bacteria can be used as production organisms.
  • genes which code for an alcohol dehydrogenase can be ligated into a vector.
  • the E. coli strains are recombinant and contain plasmids carrying genes for alcohol dehydrogenase. If vectors, for example plasmids are used, then these genes must be for a
  • the Alokholdehydrogenasen can be for example from Lactobacillus, preferably from Lactobacillus brevis (LbADH).
  • the vectors or plasmids may include alcohol dehydrogenases from Lactobacillus kefir, Candida magnolia, Leifsonia species, Sporobolomyces salmonicolor or Trichosporon cutaneum.
  • plasmids for example, pET21a or pKK233 basic bodies can be used which carry the corresponding alcohol dehydrogenase genes.
  • the production organisms secrete the desired product according to formula (2) into the aqueous solution.
  • the production method according to the invention can be operated both in vitro and in vivo.
  • the partial steps may either be either in vivo or in vitro, or the partial steps may be mixed in vivo or in vitro.
  • the process can be carried out in a one-pot reaction, so that it is easy to carry out and requires only a few work-up steps for the product according to formula 3.
  • Fig.1 A formula scheme for a method according to the invention.
  • Fig. 2 The time course of the Eduktabddling (1, 2 - PPDO) and a byproduct increase (acetophenone) for reaction step 1.
  • Figure 3 The time course of the decrease of APPO (intermediate) and the
  • the 2-step synthesis is carried out with the combination of the S-selective transaminase CV2025 from Chromobacterium violaceum in the first step and the S-selective LbADH from Lactobacillus brevis in the second step.
  • the conversion over both steps with a reaction time of 24 hours is 57.3%.
  • the resulting cathine has a very high optical purity: ee and de> 99%.
  • In Ganzzellsatz a turnover of> 95% in the first step ( Figure 2) and ⁇ 65% in the second step ( Figure 3) was achieved. Over both steps, sales were 61, 4%.
  • the resulting cathin has an ee of 97.4% and a de of 89.8%.
  • Step 1 10 mM 1-phenylpropane-1,2-dione, 15 mM (S) -alpha-methylbenzylamine, 10 mg / ml CV2025 (or 1 mg / ml purified enzyme), 100 mM HEPES with 0.1 mM pyridoxal 5'-phosphate, pH 7.5; Reaction time 24 h; Room temperature.
  • reaction solution after pH-shift 100 mM sodium formate, 10 mg cells / ml LbADH (or 1 mg / ml purified enzyme), 10 ⁇ / ml FDH, 0.2 mM NADP + , reaction time 24 h.

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  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biotechnology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Microbiology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

L'invention concerne un procédé de production de cathine, dans lequel la l-phénylpropan-1-2-dione de formule (1) est convertie dans une première étape, avec une transaminase S-sélective, en (S)-2-amino-l-phénylpropan-l-one de formule (2) et la (S)-2-amino-l-phénylpropan-l-one de formule (2) est en outre réduite, dans une deuxième étape, avec une alcool déshydrogénase S-sélective, en (1 S,2S)-norpseudoéphédrine (cathine) de formule (3).
PCT/DE2014/000145 2013-05-31 2014-03-19 Procédé de production de cathine WO2014190956A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP14717091.4A EP3004364A1 (fr) 2013-05-31 2014-03-19 Procédé de production de cathine

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102013009145.4 2013-05-31
DE201310009145 DE102013009145A1 (de) 2013-05-31 2013-05-31 Verfahren zur Herstellung von Cathin

Publications (1)

Publication Number Publication Date
WO2014190956A1 true WO2014190956A1 (fr) 2014-12-04

Family

ID=50483342

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/DE2014/000145 WO2014190956A1 (fr) 2013-05-31 2014-03-19 Procédé de production de cathine

Country Status (3)

Country Link
EP (1) EP3004364A1 (fr)
DE (1) DE102013009145A1 (fr)
WO (1) WO2014190956A1 (fr)

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
GWON-II HWANG ET AL.: "Efficient Synthesis of Ephedra Alkaloid Analogous Using an Enantiomerically Pure N-[(R)-(+)-a-Methylbenzyl]aziridine-2-carboxaldehyde", J. ORG. CHEM., vol. 61, 1996, pages 6183 - 6188
GWON-IL HWANG ET AL: "Efficient synthesis of Ephedra alkaloid analogues using an enantiomerically pure N-[(R)-(+)-a-methylbenzyl]aziridine-2-carboxaldehyde", THE JOURNAL OF ORGANIC CHEMISTRY, vol. 61, 6 September 1996 (1996-09-06), pages 6183 - 6188, XP055124911, ISSN: 0022-3263 *
HYEON-KYU LEE ET AL.: "Stereoselective Synthesis of Norphenedrine and Norpseudoephedrine by Using Asymmetrie Transfer Hydrogenation Accompanied by Dynamic Kinetic Resolution", J. ORG. CHEM., 2012, pages 5454 - 5460
HYEON-KYU LEE ET AL: "Stereoselective Synthesis of Norephedrine and Norpseudoephedrine by Using Asymmetric Transfer Hydrogenation Accompanied by Dynamic Kinetic Resolution", THE JOURNAL OF ORGANIC CHEMISTRY, vol. 77, no. 12, 15 June 2012 (2012-06-15), pages 5454 - 5460, XP055124650, ISSN: 0022-3263, DOI: 10.1021/jo300867y *
JUSTYNA KULIG ET AL: "Stereoselective synthesis of bulky 1,2-diols with alcohol dehydrogenases", CATALYSIS SCIENCE & TECHNOLOGY, vol. 2, no. 8, 2012, pages 1580, XP055124851, ISSN: 2044-4753, DOI: 10.1039/c2cy20120h *
MASAAKI NISHIMURA ET AL.: "Asymetric N1 Unit Transfer to Olefins with a Chiral Nitridomanganese Complex: Novel Stereoselective Pathways to Aziridines or Oxazolines", J. ORG. CHEM., vol. 67, 2002, pages 2101 - 2110, XP055124651, DOI: doi:10.1021/jo016146d
TORSTEN SEHL ET AL: "Efficient 2-step biocatalytic strategies for the synthesis of all nor(pseudo)ephedrine isomers", GREEN CHEMISTRY, vol. 16, no. 6, 2014, pages 3341, XP055124877, ISSN: 1463-9262, DOI: 10.1039/c4gc00100a *

Also Published As

Publication number Publication date
EP3004364A1 (fr) 2016-04-13
DE102013009145A1 (de) 2014-12-04

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