EP0826032A1 - BIOTECHNICAL PRODUCTION PROCESS OF PIPERAZINE R-$g(a)-CARBOXYLIC ACIDS AND PIPERAZINE S-$g(a)-CARBOXYLIC ACID AMIDE - Google Patents

BIOTECHNICAL PRODUCTION PROCESS OF PIPERAZINE R-$g(a)-CARBOXYLIC ACIDS AND PIPERAZINE S-$g(a)-CARBOXYLIC ACID AMIDE

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Publication number
EP0826032A1
EP0826032A1 EP96915025A EP96915025A EP0826032A1 EP 0826032 A1 EP0826032 A1 EP 0826032A1 EP 96915025 A EP96915025 A EP 96915025A EP 96915025 A EP96915025 A EP 96915025A EP 0826032 A1 EP0826032 A1 EP 0826032A1
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Prior art keywords
microorganisms
piperazine
piperazinecarboxamide
formula
optionally substituted
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Legal status (The legal status 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 status listed.)
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EP96915025A
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German (de)
French (fr)
Inventor
Andreas Kiener
Jean-Paul Roduit
Klaus Heinzmann
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Lonza AG
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Lonza AG
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Publication of EP0826032A1 publication Critical patent/EP0826032A1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • 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
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom
    • C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring
    • 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
    • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
    • C12P41/006—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by reactions involving C-N bonds, e.g. nitriles, amides, hydantoins, carbamates, lactames, transamination reactions, or keto group formation from racemic mixtures
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/38—Pseudomonas

Definitions

  • the invention relates to new microorganisms which are capable, optionally substituted, of (RS) - ⁇ -piperazinecarboxamides of the formula
  • microorganisms are capable of utilizing optionally substituted ⁇ -piperazinecarboxamides of the formula I above, in the form of their racemate or their optically active isomers, in particular R- ⁇ -piperazinecarboxamides, as the only nitrogen source.
  • These microorganisms or their cell-free enzymes are optionally substituted for a new process for the preparation of R- ⁇ -piperazinecarboxylic acids (formula II) and / or for the preparation of S- ⁇ -piperazinecarboxamides, optionally substituted, of the formula
  • R- ⁇ -piperazinecarboxylic acid of the formula II is an important intermediate for the preparation of (R) -2-carboxy-4- (3-phosphonopropyl) piperazine, a selective antagonist of N-
  • the object of the present invention is to provide a simple and technically viable biotechnological process for the production of optically pure R- ⁇ -piperazinecarboxylic acids, the S- ⁇ -piperazinecarboxamide also being able to be isolated in good purity at the same time.
  • microorganisms according to the invention can be isolated from soil samples, sludge or waste water with the aid of customary microbiological techniques. According to the invention, these microorganisms are isolated in such a way that they are the only ones in a medium with an optionally substituted ⁇ -piperazinecarboxamide (formula I) in the form of its racemate or its optically active isomers
  • Nitrogen source preferably with an R- ⁇ -piperazinecarboxamide as the sole nitrogen source, and grown with a suitable carbon source in the usual way; b) then those selected from the culture obtained by cultivation are those which are stable and are capable, in (RS) - ⁇ -piperazinecarboxamides (formula I), of the R- ⁇ -piperazinecarboxamide into the corresponding R- ⁇ -piperazinecarboxylic acid (formula II ) to transfer.
  • the microorganisms can use, for example, sugar, sugar alcohols or carboxylic acids as a growth substrate as a carbon source.
  • Hexoses such as glucose or pentoses can be used as sugar.
  • Di- or tricarboxylic acids or salts thereof such as citric acid or succinate can be used as carboxylic acids.
  • glycerin can be used as the sugar alcohol.
  • a sugar alcohol such as glycerol is preferably used as the carbon source.
  • the selection and growth medium which can be used are those customary in the art, such as, for example, the mineral salt medium according to Kulla et al. (Arch. Microbiol., 135, 1-7, 1983) or that described in Table 1. Preferably, the one described in Table 1 is used.
  • Piperazine carboxamide can be used as an enzyme inducer.
  • the cultivation and selection is expediently carried out at a temperature of from 15 to 55 ° C., preferably from 20 to 45 ° C. and at a pH between pH 5 and pH 11, preferably between pH 6 and pH 10.
  • microorganisms with specific R-piperazinecarboxylic acid amidase activity are microorganisms of the genus Burkholde ⁇ a and their functionally equivalent variants and mutants.
  • Microorganisms of the genus Burkholderia as deposited on April 20, 1995 with the German Collection of Microorganisms and Cell Cultures GmbH, Mascheroder Weg lb, D-38124 Braunschweig, in accordance with the Budapest Treaty, and their functionally equivalent variants and mutants are particularly preferred.
  • Taxonomic properties of the microorganisms of the genus Burkholderia (DSM 9925)
  • “Functionally equivalent variants and mutants” are understood to mean microorganisms which have essentially the same properties and functions as the original microorganisms. Such variants and mutants can happen to z. B. are formed by UV radiation.
  • the corresponding R- ⁇ -piperazinecarboxamide is converted and isolated from the microorganisms into the corresponding R- ⁇ -piperazinecarboxylic acid by means of the specific microorganisms already described or by means of cell-free enzymes
  • Piperazinecarboxamides of the formula I can be used as the piperazinecarboxamides of the formula I.
  • Representatives of substituted piperazine carboxamides can be -C-C4-alkyl-substituted such.
  • Piperazinecarboxamide or 4-methylpiperazinecarboxamide is preferably used.
  • the enzymes for the cell-free system can be obtained by disrupting the microorganisms in the usual manner. For example, the ultrasonic, French press or lysozyme method can be used for this. These cell-free enzymes can also be immobilized on a suitable carrier material.
  • DSM 9925 The previously described specific microorganisms of the genus Burkholderia (DSM 9925) and their functionally equivalent variants and mutants are particularly suitable for the method.
  • the biotransformation can also be dormant
  • the medium used for the method with resting cells can be the facl-usual ones, such as, for example, the mineral salt medium described above according to KuUa et al., 1983 (ibid), low-molecular phosphate buffer, HEPES buffer, or the medium described in Table 1.
  • a medium containing a carbon and nitrogen source such as, for example, commercially available media or the medium according to Table 1, is usually used for the process with growing cells. The method is preferably carried out in the medium according to Table 1.
  • the biotransformation is expediently carried out with a single or continuous addition of corresponding (RS) - ⁇ -piperazinecarboxamide in such a way that the concentration of (RS) - ⁇ -piperazinecarboxamide does not exceed 20% by weight, preferably 10% by weight.
  • the pH of the medium can be in a range from pH 5 to pH 11, preferably from pH 6 to pH 10.
  • the biotransformation is expediently carried out at a temperature of from 15 to 55 ° C., preferably from 20 to 50 ° C.
  • the R- ⁇ -piperazinecarboxamide of the formula I has been converted completely to the R- ⁇ -piperazinecarboxylic acid, with S- ⁇ -piperazinecarboxamide also being obtained.
  • the R- ⁇ -piperazinecarboxylic acid obtained in this way and / or the S- ⁇ -piperazinecarboxylic acid amide can be prepared by conventional workup methods such as, for. B. isolated by acidification, electrodialysis, chromatography or extraction. The isolated S- ⁇ -piperazinecarboxamide can then be hydrolyzed chemically to the S- ⁇ -piperazinecarboxylic acid.
  • the A-N medium was used for the isolation of microorganisms which are capable of utilizing racemic piperazinamide as the sole nitrogen source. 100 ml of this medium was placed in a 300 ml Erlenmeyer flask and mixed with various soil samples (2 g) from the LONZA AG plant in Visp, Switzerland. The flasks were incubated at 30 ° C for 5 days at rest. Then 1 ml of the A-N medium was used to make a fresh one
  • the cells were grown in 11 A-N medium at 30 ° C. and then harvested and washed. 5 g cells (wet weight) were in
  • the cells were grown in A-N medium (Table 1) and then washed once with physiological saline. After resuspending in 69 mM phosphate buffer, pH 8.0 and setting a cell density of 10 at OD650, 20 gH (RS) -piperazinecarboxamide were added at a temperature of 47 ° C. Then it was incubated for 18 h (total volume:
  • Detector Hewlett-Packard diode array detector Buffer: 10 mM disodium hydrogen phosphate, 10 mM boric acid,
  • Electrolyte 900 ml buffer plus 100 ml methanol
  • Capillary HP Gl 600-61211 Electrical field: 20 kV current: approx. 24 - 30 ⁇ A
  • Oven temperature 20 ° C detector setting: 210 nm (bandwidth 5 nm) migration time: approx.17.1 min (S-acid) approx. 17.7 min (R-acid)

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  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

New micro-organisms are capable of using as single nitrogen source piperazine alpha -carboxylic acid amides in the form of their racemate or optically active isomers having general formula (I) and of transforming piperazine (RS)- alpha -carboxylic acid amide having the above-mentioned formula (I) into a piperazine R- alpha -carboxylic acid having the formula (II).

Description

Biotechnologisches Verfahren zur Herstellung von R-α-Piperazincarbonsäure und S-α- PiperazincarbonsäureamidBiotechnological process for the production of R-α-piperazinecarboxylic acid and S-α-piperazinecarboxamide
Die Erfindung betrifft neue Mikroorganismen, die befähigt sind, in (RS)-α-Piperazincarbonsäu- reamiden, gegebenenfalls substituiert, der FormelThe invention relates to new microorganisms which are capable, optionally substituted, of (RS) -α-piperazinecarboxamides of the formula
das R-α-Piperazincarbonsäureamid, gegebenenfalls substituiert, in die entsprechende R-α- Piperazincarbonsäure, gegebenenfalls substituiert, der Formelthe R-α-piperazinecarboxamide, optionally substituted, in the corresponding R-α-piperazinecarboxylic acid, optionally substituted, of the formula
zu überführen. Die Mikroorganismen sind befähigt, gegebenenfalls substituierte α-Piperazincar- bonsäureamide der obigen Formel I, in Form ihres Racemats oder ihrer optisch aktiven Isomere, insbesondere R-α-Piperazincarbonsäureamide, als einzige Stickstoff quelle zu verwerten. Diese Mikroorganismen bzw. deren zellfreie Enzyme werden für ein neues Verfahren zur Herstellung von R-α-Piperazincarbonsäuren (Formel II), gegebenenfalls substituiert, und/oder zur Herstellung von S-α-Piperazincarbonsäureamiden, gegebenenfalls substituiert, der Formelto convict. The microorganisms are capable of utilizing optionally substituted α-piperazinecarboxamides of the formula I above, in the form of their racemate or their optically active isomers, in particular R-α-piperazinecarboxamides, as the only nitrogen source. These microorganisms or their cell-free enzymes are optionally substituted for a new process for the preparation of R-α-piperazinecarboxylic acids (formula II) and / or for the preparation of S-α-piperazinecarboxamides, optionally substituted, of the formula
eingesetzt. R-α-Piperazincarbonsäure der Formel II ist ein wichtiges Zwischenprodukt zur Herstellung von (R)-2-Carboxy-4-(3-phosphonopropyI)piperazin, einem selektiven Antagonisten von N-used. R-α-piperazinecarboxylic acid of the formula II is an important intermediate for the preparation of (R) -2-carboxy-4- (3-phosphonopropyl) piperazine, a selective antagonist of N-
Methyl-D-aspartat (Synlett, 1996, 143 - 144).Methyl D-aspartate (Synlett, 1996, 143-144).
Ein mikrobiologisches Verfahren zur Herstellung von R-α-Piperazincarbonsäure ist bisher in der Literatur nicht beschrieben.A microbiological process for the production of R-α-piperazinecarboxylic acid has not hitherto been described in the literature.
Aufgabe der vorliegenden Erfindung ist, ein einfaches und technisch gangbares biotechno¬ logisches Verfahren zur Herstellung von optisch reinen R-α-Piperazincarbonsäuren zur Verfügung zu stellen, wobei gleichzeitig das S-α-Piperazincarbonsäureamid ebenfalls in guter Reinheit isoliert werden kann.The object of the present invention is to provide a simple and technically viable biotechnological process for the production of optically pure R-α-piperazinecarboxylic acids, the S-α-piperazinecarboxamide also being able to be isolated in good purity at the same time.
Diese Aufgabe wird mit den Mikroorganismen gemäss Patentanspruch 1 und mit dem Verfahren gemäss Patentanspruch 3 gelöst.This object is achieved with the microorganisms according to claim 1 and with the method according to claim 3.
Die erfindungsgemässen Mikroorganismen können aus Bodenproben, Schlamm oder Abwasser unter Zuhilfenahme üblicher mikrobiologischer Techniken isoliert werden. Erfindungsgemäss erfolgt die Isolation dieser Mikroorganismen derart, dass man diese a) in einem Medium mit einem gegebenenfalls substituierten α-Piperazincarbonsäureamid (Formel I) in Form seines Racemats oder seiner optisch aktiven Isomere als einzigerThe microorganisms according to the invention can be isolated from soil samples, sludge or waste water with the aid of customary microbiological techniques. According to the invention, these microorganisms are isolated in such a way that they are the only ones in a medium with an optionally substituted α-piperazinecarboxamide (formula I) in the form of its racemate or its optically active isomers
Stickstoffquelle, vorzugsweise mit einem R-α-Piperazincarbonsäureamid als einziger Stickstoff quelle, und mit einer geeigneten Kohlenstoffquelle auf übliche Weise züchtet; b) aus der durch Züchtung erhaltenen Kultur dann jene selektioniert, die stabil sind und befähigt sind, in (RS)-α-Piperazincarbonsäureamiden (Formel I) das R-α-Piperazincar- bonsäureamid in die entsprechende R-α-Piperazincarbonsäure (Formel II) zu überführen.Nitrogen source, preferably with an R-α-piperazinecarboxamide as the sole nitrogen source, and grown with a suitable carbon source in the usual way; b) then those selected from the culture obtained by cultivation are those which are stable and are capable, in (RS) -α-piperazinecarboxamides (formula I), of the R-α-piperazinecarboxamide into the corresponding R-α-piperazinecarboxylic acid (formula II ) to transfer.
Demzufolge können alle Mikroorganismen angewendet werden, die spezifisch die R- Piperazincarbonsäure-Amidasen enthalten.Accordingly, all microorganisms which specifically contain the R-piperazinecarboxylic acid amidases can be used.
Als Kohlenstoffquelle können die Mikroorganismen beispielsweise Zucker, Zuckeralkohole oder Carbonsäuren als Wachstumssubstrat nützen. Als Zucker können Hexosen wie beispielsweise Glucose oder Pentosen angewendet werden. Als Carbonsäuren können Di- oder Tricarbonsäuren bzw. deren Salze verwendet werden wie beispielsweise Citronensäure oder Succinat. Als Zuckeralkohol kann beispielsweise Glycerin verwendet werden. Vorzugsweise wird als Kohlenstoffquelle ein Zuckeralkohol wie Glycerin eingesetzt. Als Selektions- und Anzuchtmedium können die in der Fachwelt üblichen verwendet werden, wie beispielsweise das Mineralsalzmedium gemäss Kulla et al. (Arch. Microbiol., 135, 1 - 7, 1983) oder das in Tabelle 1 beschriebene. Vorzugsweise wird das in Tabelle 1 beschriebene angewendet.The microorganisms can use, for example, sugar, sugar alcohols or carboxylic acids as a growth substrate as a carbon source. Hexoses such as glucose or pentoses can be used as sugar. Di- or tricarboxylic acids or salts thereof such as citric acid or succinate can be used as carboxylic acids. For example, glycerin can be used as the sugar alcohol. A sugar alcohol such as glycerol is preferably used as the carbon source. The selection and growth medium which can be used are those customary in the art, such as, for example, the mineral salt medium according to Kulla et al. (Arch. Microbiol., 135, 1-7, 1983) or that described in Table 1. Preferably, the one described in Table 1 is used.
Während der Anzucht und Selektion werden zweckmässig die wirksamen Enzyme der Mikroorganismen induziert. Als Enzym-Induktor kann Piperazincarboxamid angewendet werden.The effective enzymes of the microorganisms are expediently induced during the cultivation and selection. Piperazine carboxamide can be used as an enzyme inducer.
Zweckmässig erfolgt die Anzucht und Selektion bei einer Temperatur von 15 bis 55 °C, vorzugsweise von 20 bis 45 °C und bei einem pH- Wert zwischen pH 5 und pH 11 , vorzugsweise zwischen pH 6 und pH 10.The cultivation and selection is expediently carried out at a temperature of from 15 to 55 ° C., preferably from 20 to 45 ° C. and at a pH between pH 5 and pH 11, preferably between pH 6 and pH 10.
Bevorzugte Mikroorganismen mit spezifischer R-Piperazincarbonsäure-Amidase- Aktivität sind Mikroorganismen der Gattung Burkholdeήa, sowie deren funktioneil äquivalente Varianten und Mutanten. Besonders bevorzugt sind Mikroorganismen der Gattung Burkholderia, wie sie am 20.04.1995 bei der Deutschen Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg lb, D-38124 Braunschweig, gemäß Budapester Vertrag hinterlegt wurden, und deren funktionell äquivalente Varianten und Mutanten.Preferred microorganisms with specific R-piperazinecarboxylic acid amidase activity are microorganisms of the genus Burkholdeήa and their functionally equivalent variants and mutants. Microorganisms of the genus Burkholderia, as deposited on April 20, 1995 with the German Collection of Microorganisms and Cell Cultures GmbH, Mascheroder Weg lb, D-38124 Braunschweig, in accordance with the Budapest Treaty, and their functionally equivalent variants and mutants are particularly preferred.
Taxonomische Eigenschaften der Mikroorganismen der Gattung Burkholderia (DSM 9925)Taxonomic properties of the microorganisms of the genus Burkholderia (DSM 9925)
Zellform StäbchenCell shape chopsticks
Breite μm 0,7 - 0,8Width μm 0.7 - 0.8
Länge μm 1,5 - 3,5Length μm 1.5 - 3.5
Gram-Reaktion -Grief reaction -
Lyse durch 3% KOH +Lysis by 3% KOH +
Aminopeptidase (Cerny) +Aminopeptidase (Cerny) +
Sporen -Spores -
Oxidase +Oxidase +
Catalase +Catalase +
Pigmente gelbPigments yellow
Wachstum anaerob PNPG +Growth anaerobic PNPG +
ADHADH
UreaseUrease
Hydrolyse von GelatineHydrolysis of gelatin
SubstratverwertungSubstrate recycling
Adipat +Adipat +
Citrat wCitrate w
Malat +Malat +
Glucose -Glucose -
Adonitol -Adonitol -
Mannitol -Mannitol -
Suberat +Suberat +
Acetamid +Acetamide +
2,3-Butylenglycol + Abkürzungen: m-Hydroxybenzoat + α-Amylamin + PNPG: p-Nifc2,3-butylene glycol + abbreviations: m-hydroxybenzoate + α-amylamine + PNPG: p-Nifc
Tryptamin - ADH: AlkohTryptamine - ADH: alcohol
Unter "funktioneil aequivalente Varianten und Mutanten" werden Mikroorganismen verstanden, die im wesentlichen dieselben Eigenschaften und Funktionen wie die Ursprungs¬ mikroorganismen besitzen. Derartige Varianten und Mutanten können zufällig z. B. durch UV- Bestrahlung gebildet werden."Functionally equivalent variants and mutants" are understood to mean microorganisms which have essentially the same properties and functions as the original microorganisms. Such variants and mutants can happen to z. B. are formed by UV radiation.
Das erfindungsgemässe Verfahren zur Herstellung von R-α-Piperazincarbonsäure, gegebenenfalls substituiert, der FormelThe inventive method for the production of R-α-piperazinecarboxylic acid, optionally substituted, of the formula
und / oder von S-α-Piperazincarbonsäureamiden, gegebenenfalls substituiert, der Formel and / or of S-α-piperazinecarboxamides, optionally substituted, of the formula
erfolgt derart, dass man im (RS)-α-Piperazincarbonsäureamid, gegebenenfalls substituiert, der Formelis carried out in such a way that (RS) -α-piperazinecarboxamide, optionally substituted, of the formula
das entsprechende R-α-Piperazincarbonsäureamid mittels der spezifischen bereits beschriebenen Mikroorganismen oder mittels zellfreier Enzyme aus diesen Mikroorganismen zur entsprechenden R-α-Piperazincarbonsäure umsetzt und isoliert, wobei bei derthe corresponding R-α-piperazinecarboxamide is converted and isolated from the microorganisms into the corresponding R-α-piperazinecarboxylic acid by means of the specific microorganisms already described or by means of cell-free enzymes
Biotransformation neben der R-α-Piperazincarbonsäure das S-α-Piperazincarbonsäureamid anfällt, welches gegebenenfalls isoliert wird.Biotransformation in addition to the R-α-piperazinecarboxylic acid, the S-α-piperazinecarboxamide is obtained, which is optionally isolated.
Die Edukte, die (RS)-α-Piperazincarbonsäureamide der FormelThe starting materials, the (RS) -α-piperazinecarboxamides of the formula
die gegebenenfalls substituiert sind, können aus den entsprechenden aromatischen Amiden durch fachmännisch übliche Hydrierung gewonnen werden.which are optionally substituted, can be obtained from the corresponding aromatic amides by conventional hydrogenation.
Als Piperazincarbonsäureamide der Formel I können substituierte und unsubstituierte angewendet werden. Vertreter von substituierten Piperazincarbonsäureamiden können sein Cι-C4-Alkyl-substituierte wie z. B. 4-Methylpiperazincarbonsäureamid, H2N-CH2-substi- tuierte wie beispielsweise 4-Aminomethylpiperazincarbonsäureamid oder Acyl-substituierte wie 4-Acetylpiperazincarbonsäureamid. Vorzugsweise wird Piperazincarboxamid oder 4- Methylpiperazincarbonsäureamid verwendet. Die Enzyme für das zellfreie System können durch fachmännisch übliches Aufschliessen der Mikroorganismen gewonnen werden. Hierzu kann beispielsweise die Ultraschall-, French-Press oder Lysozym-Methode verwendet werden. Diese zellfreien Enzyme können auch auf einem geeigneten Trägermaterial immobilisiert werden.Substituted and unsubstituted can be used as the piperazinecarboxamides of the formula I. Representatives of substituted piperazine carboxamides can be -C-C4-alkyl-substituted such. B. 4-methylpiperazinecarboxamide, H2N-CH2-substituted such as 4-aminomethylpiperazinecarboxamide or acyl-substituted such as 4-acetylpiperazinecarboxamide. Piperazinecarboxamide or 4-methylpiperazinecarboxamide is preferably used. The enzymes for the cell-free system can be obtained by disrupting the microorganisms in the usual manner. For example, the ultrasonic, French press or lysozyme method can be used for this. These cell-free enzymes can also be immobilized on a suitable carrier material.
Für das Verfahren besonders geeignet, sind die zuvor beschriebenen spezifischen Mikro¬ organismen der Gattung Burkholderia (DSM 9925), sowie deren funktionell aequivalente Varianten und Mutanten.The previously described specific microorganisms of the genus Burkholderia (DSM 9925) and their functionally equivalent variants and mutants are particularly suitable for the method.
Die Biotransformation kann nach üblichem Anzüchten der Mikroorganismen mit ruhendenAfter normal cultivation of the microorganisms, the biotransformation can also be dormant
Zellen (nicht wachsende Zellen, die keine Kohlenstoff- und Energiequelle mehr benötigen) oder mit wachsenden Zellen durchgeführt werden.Cells (non-growing cells that no longer need a carbon and energy source) or with growing cells.
Als Medium für das Verfahren mit ruhenden Zellen können die facl männisch üblichen dienen, wie beispielsweise das zuvor beschriebene Mineralsalzmedium gemäss KuUa et al., 1983 (ibid), niedermolare Phosphatpuffer, HEPES-Puffer, oder das in Tabelle 1 beschriebene Medium. Für das Verfahren mit wachsenden Zellen wird üblicherweise ein Medium enthaltend eine Kohlenstoff- und Stickstoffquelle wie beispielsweise handelsübliche Medien oder das Medium gemäss Tabelle 1 angewendet. Vorzugsweise wird das Verfahren in dem Medium gemäss Tabelle 1 durchgeführt.The medium used for the method with resting cells can be the facl-usual ones, such as, for example, the mineral salt medium described above according to KuUa et al., 1983 (ibid), low-molecular phosphate buffer, HEPES buffer, or the medium described in Table 1. A medium containing a carbon and nitrogen source, such as, for example, commercially available media or the medium according to Table 1, is usually used for the process with growing cells. The method is preferably carried out in the medium according to Table 1.
Zweckmässig wird die Biotransformation unter einmaliger oder kontinuierlicher Zugabe von entsprechendem (RS)-α-Piperazincarbonsäureamid so durchgeführt, dass die Konzentration an (RS)-α-Piperazincarbonsäureamid 20 Gew.%, vorzugsweise 10 Gew.%, nicht übersteigt.The biotransformation is expediently carried out with a single or continuous addition of corresponding (RS) -α-piperazinecarboxamide in such a way that the concentration of (RS) -α-piperazinecarboxamide does not exceed 20% by weight, preferably 10% by weight.
Der pH- Wert des Mediums kann in einem Bereich von pH 5 bis pH 11, vorzugsweise von pH 6 bis pH 10, liegen.The pH of the medium can be in a range from pH 5 to pH 11, preferably from pH 6 to pH 10.
Zweckmässig wird die Biotransformation bei einer Temperatur von 15 bis 55 °C, vorzugsweise von 20 bis 50 °C durchgeführt.The biotransformation is expediently carried out at a temperature of from 15 to 55 ° C., preferably from 20 to 50 ° C.
Nach einer üblichen Umsetzungszeit von 1 bis 100 h ist das R-α-Piperazincarbonsäureamid der Formel I vollständig zur R-α-Piperazincarbonsäure umgewandelt, wobei auch S-α- Piperazincarbonsäureamid anfallt. Die auf diese Weise gewonnene R-α-Piperazincarbonsäure und / oder das S-α-Piperazincarbon¬ säureamid können durch übliche Aufarbeitungsmethoden wie z. B. durch Ansäuern, Elektrodialyse, Chromatographie oder Extraktion isoliert werden. Das isolierte S-α- Piperazincarbonsäureamid kann dann chemisch zur S-α-Piperazincarbonsäure hydrolysiert werden.After a customary reaction time of 1 to 100 h, the R-α-piperazinecarboxamide of the formula I has been converted completely to the R-α-piperazinecarboxylic acid, with S-α-piperazinecarboxamide also being obtained. The R-α-piperazinecarboxylic acid obtained in this way and / or the S-α-piperazinecarboxylic acid amide can be prepared by conventional workup methods such as, for. B. isolated by acidification, electrodialysis, chromatography or extraction. The isolated S-α-piperazinecarboxamide can then be hydrolyzed chemically to the S-α-piperazinecarboxylic acid.
Beispiele:Examples:
Beispiel 1example 1
a) Isolation von Mikroorganismen, die befähigt sind racemisches Piperazincarboxamid als einzige Stickstoffquelle zu verwerten:a) Isolation of microorganisms that are able to use racemic piperazine carboxamide as the only nitrogen source:
Für die Isolation von Mikroorganismen, die befähigt sind, racemisches Piperazinamid als einzige Stickstoffquelle zu verwerten, wurde das A-N-Medium, dessen Zusammensetzung in Tabelle 1 gegeben ist, verwendet. 100 ml dieses Mediums wurden in einen 300 ml Erlenmeyerkolben gegeben und mit verschiedenen Erdproben (2 g) aus dem Werksareal der LONZA AG in Visp, Schweiz versetzt. Die Kolben wurden ruhend bei 30 °C während 5 Tagen inkubiert. Dann wurde 1 ml des A-N-Mediums verwendet, um einen frischenThe A-N medium, the composition of which is given in Table 1, was used for the isolation of microorganisms which are capable of utilizing racemic piperazinamide as the sole nitrogen source. 100 ml of this medium was placed in a 300 ml Erlenmeyer flask and mixed with various soil samples (2 g) from the LONZA AG plant in Visp, Switzerland. The flasks were incubated at 30 ° C for 5 days at rest. Then 1 ml of the A-N medium was used to make a fresh one
Kolben mit dem gleichen Medium zu beimpfen. Dieser Kolben wurde wiederum unter den gleichen Bedingungen inkubiert. Insgesamt wurde dieser Anreicherungszyklus 5mal wiederholt. Danach wurden die Anreicherungen auf Agar-Medium (A-N-Medium mit zusätzlich 16 gH Agar) zu Einzelkolonien ausgestrichen. Die isolierten Mikroorganismen wurden im folgenden qualitativen Testsystem auf stereoselektive Amidasen hin untersucht. Einzelkolonien wurden verwendet, um 100 ml A- N-Medium in 300 ml Erlenmeyerkolben zu beimpfen. Diese Kolben wurden drei Tage auf einer Schüttelmaschine bei 30 °C inkubiert, wobei die Kulturen bereits nach einem Tag ausgewachsen waren. Danach wurden die zellfreien Kulturüberstände mittels Dünn- Schichtchromatographie (Kieselgel, Laufmittel: 11 Teile Ethanol, 6 Teile CHCI3, 6 TeileInoculate flask with the same medium. This flask was again incubated under the same conditions. In total, this enrichment cycle was repeated 5 times. The enrichments were then spread onto agar medium (A-N medium with an additional 16 gH agar) to form individual colonies. The isolated microorganisms were examined for stereoselective amidases in the following qualitative test system. Single colonies were used to inoculate 100 ml A-N medium in 300 ml Erlenmeyer flasks. These flasks were incubated for three days on a shaking machine at 30 ° C., the cultures having grown out after only one day. The cell-free culture supernatants were then removed by thin layer chromatography (silica gel, mobile phase: 11 parts of ethanol, 6 parts of CHCl 3, 6 parts
NH4OH (25%), Nachweis mittels Ninhydrin) auf den Gehalt an Piperazincarbonsäure und Piperazincarboxamid hin untersucht. Mikroorganismen, die etwa die Hälfte der einge¬ setzten Menge (RS)-Piperazincarboxamid umgesetzt hatten, wurden für biochemische Untersuchungen verwendet, um festzustellen, welche Stämme R-spezifische Amidasen enthielten. b) Biochemische Untersuchungen zur Identifizierung von Mikroorganismen mit R- spezifischen Amidasen:NH4OH (25%), detection using ninhydrin) for the content of piperazine carboxylic acid and piperazine carboxamide. Microorganisms which had reacted about half of the amount used (RS) -piperazinecarboxamide were used for biochemical tests to determine which strains contained R-specific amidases. b) Biochemical investigations to identify microorganisms with R-specific amidases:
Zur Herstellung von Protein-Rohextrakt wurden die Zellen in 11 A-N-Medium bei 30 °C angezogen und anschliessend geerntet und gewaschen. 5 g Zellen (Nassgewicht) wurden inTo produce crude protein extract, the cells were grown in 11 A-N medium at 30 ° C. and then harvested and washed. 5 g cells (wet weight) were in
10 ml 69 mmol Phosphatpuffer, pH 7,0, resuspendiert und mittels einer FRENCH®-Presse aufgeschlossen. Der Rohextrakt wurde nach einer Zentrifugation von 2 h bei 40O00 x g in Portionen bei -20 °C eingefroren. Zur Bestimmung der Stereoselektivität wurden die Hydrolyseraten von R-Prolinamid und S-Prolinamid verglichen. Dazu wurde folgender Enzymtest verwendet: Assayvolumen 1 ml, enthaltend 69 mmol Phosphatpuffer, pH 7,0,10 ml 69 mmol phosphate buffer, pH 7.0, resuspended and digested with a FRENCH® press. The crude extract was frozen in portions at -20 ° C. after centrifugation for 2 h at 40,000 × g. To determine stereoselectivity, the hydrolysis rates of R-prolinamide and S-prolinamide were compared. The following enzyme test was used for this: Assay volume 1 ml, containing 69 mmol phosphate buffer, pH 7.0,
100 - 800 μg Protein-Rohextrakt, 2 mg S- resp. R-Prolinamid-HCl, Inkubationszeit 1 -24 h, Inkubationstemperatur 30 °C, Detektion mit Ninhydrin nach Dünnschichtchromatographie (siehe oben). Die Amidase des Stammes mit der Bezeichnung DSM 9925 zeigte eine schnelle Hydrolyse von R-Prolinamid. Dieser Stamm wurde für die Herstellung optisch aktiver Piperazincarbonsäurederivate verwendet.100 - 800 μg protein crude extract, 2 mg S or R-prolinamide HCl, incubation time 1 -24 h, incubation temperature 30 ° C, detection with ninhydrin after thin layer chromatography (see above). The strain amidase called DSM 9925 showed rapid hydrolysis of R-prolinamide. This strain was used for the production of optically active piperazine carboxylic acid derivatives.
Durch Veränderung der Inkubationstemperatur und des pH- Wertes der Assay-Lösung wurde festgestellt, dass die spezifische Aktivität der Amidasen zwischen einer Temperatur von 30 - 60 °C und einem pH-Wert von 7 - 10 am grössten war.By changing the incubation temperature and the pH of the assay solution, it was found that the specific activity of the amidases was greatest between a temperature of 30-60 ° C. and a pH of 7-10.
Tabelle 1Table 1
A-N-MediumA-N medium
Zusammensetzung KonzentπComposition Konzentπ
(RS)-Piperazinamid 2000(RS) -piperazinamide 2000
Glycerin 10000Glycerin 10000
Hefeextrakt 500Yeast extract 500
Na2S04 100Na 2 S04 100
Na2HPθ4 2000Na 2 HP04 2000
KH2PO4 1000KH 2 PO 4 1000
NaCl 3000NaCl 3000
MgCl2x6H2O 400MgCl2x6H 2 O 400
CaCl2x2H2O 14,5CaCl 2 x2H 2 O 14.5
FeCl3x6H2O 0,8 ZnSθ4χ7H2θ lOOxlO"3 FeCl 3 x6H 2 O 0.8 ZnSθ4χ7H2θ lOOxlO " 3
H3BO3 300x10-3H3BO3 300x10-3
CoCl2x6H2O 200x10-3CoCl 2 x6H 2 O 200x10-3
CuCl2x2H2O 10x10-3CuCl 2 x2H 2 O 10x10-3
NiCl2x6H2O 20x10-3NiCl 2 x6H 2 O 20x10-3
NaMoO x2H O 30x10-3NaMoO x2H O 30x10-3
Beispiel 2Example 2
Herstellung von R-Piperazincarbonsäure:Production of R-piperazine carboxylic acid:
Für die Herstellung von R-Piperazincarbonsäure mit den Stämmen mit der Bezeichnung DSM 9925 wurden folgende Bedingungen gewählt.The following conditions were chosen for the production of R-piperazinecarboxylic acid with the strains with the designation DSM 9925.
Die Zellen wurden im A-N-Medium (Tabelle 1) angezogen und dann lmal mit physiologischer Kochsalzlösung gewaschen. Nach Resuspendieren in 69 mM Phosphatpuffer, pH 8,0 und Einstellen einer Zelldichte von 10 bei OD650 wurde bei einer Temperatur von 47 °C 20 gH (RS)-Piperazincarboxamid hinzugegeben. Dann wurde 18 h lang inkubiert (Totalvolumen:The cells were grown in A-N medium (Table 1) and then washed once with physiological saline. After resuspending in 69 mM phosphate buffer, pH 8.0 and setting a cell density of 10 at OD650, 20 gH (RS) -piperazinecarboxamide were added at a temperature of 47 ° C. Then it was incubated for 18 h (total volume:
200 ml, 31 mmol (RS)-Piperazincarboxamid (4 g)). Nach dem Entfernen der Zellen wurde die zellfreie Lösung unter reduziertem Druck auf 20 ml konzentriert und dann mit HC1 auf ca. pH 0 angesäuert, wobei das Produkt ausfiel. Die isolierte Säure wurde in 0,1 M HC1 umkristallisiert und getrocknet. Es wurden 1,38 g (6,8 mmol) R-Piperazincarbonsäure als Dihydrochlorid isoliert, entsprechend einer Ausbeute von 44% bezogen auf die eingesetzte Menge R-Amid im (RS)-Piperazincarboxamid. Der ee-Wert, d.h. die Enantiomerenreinheit, war größer als 99% nach Derivatisierung mit 2,3,4,6-Tetra-O-acetyl-ß-D-glucopyranosylisothiocyanat, gemessen mittels Kapillar-Elektrophorese.200 ml, 31 mmol (RS) -piperazinecarboxamide (4 g)). After removing the cells, the cell-free solution was concentrated to 20 ml under reduced pressure and then acidified to about pH 0 with HCl, the product precipitating. The isolated acid was recrystallized from 0.1 M HC1 and dried. 1.38 g (6.8 mmol) of R-piperazinecarboxylic acid were isolated as dihydrochloride, corresponding to a yield of 44% based on the amount of R-amide used in (RS) -piperazinecarboxamide. The ee value, i.e. the enantiomeric purity was greater than 99% after derivatization with 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl isothiocyanate, measured by capillary electrophoresis.
Tabelle 2Table 2
Kapillar-Elekrophorese BedingungenCapillary electrophoresis conditions
CE Apparatur: Hewlett-Packard HP DCECE equipment: Hewlett-Packard HP DCE
Detektor: Hewlett-Packard Diode-Array-Detektor Puffer: 10 mM Di-Natriumhydrogenphosphat, 10 mM Borsäure,Detector: Hewlett-Packard diode array detector Buffer: 10 mM disodium hydrogen phosphate, 10 mM boric acid,
150 mM Natriumdodecylsulfat, pH 9,0 Elektrolyt: 900 ml Puffer plus 100 ml Methanol Kapillare: HP Gl 600-61211 Elektrisches Feld: 20 kV Strom: ca. 24 - 30 μA150 mM sodium dodecyl sulfate, pH 9.0 Electrolyte: 900 ml buffer plus 100 ml methanol Capillary: HP Gl 600-61211 Electrical field: 20 kV current: approx. 24 - 30 μA
Ofentemperatur: 20 °C Detektoreinstellung : 210 nm (Bandbreite 5 nm) Migrationszeit: ca. 17,1 min (S-Säure) ca. 17,7 min (R-Säure) Oven temperature: 20 ° C detector setting: 210 nm (bandwidth 5 nm) migration time: approx.17.1 min (S-acid) approx. 17.7 min (R-acid)

Claims

Patentansprüche:Claims:
Mikroorganismen, dadurch gekennzeichnet, daß sie befähigt sind, in (RS)-α-Piperazincar- bonsäureamiden, gegebenenfalls substituiert, der FormelMicroorganisms, characterized in that they are capable, optionally substituted, of (RS) -α-piperazinecarboxamides of the formula
das R-α-Piperazincarbonsäureamid, gegebenenfalls substituiert, in die entsprechende R- α-Piperazincarbonsäure, gegebenenfalls substituiert, der Formel the R-α-piperazinecarboxamide, optionally substituted, in the corresponding R-α-piperazinecarboxylic acid, optionally substituted, of the formula
zu überführen.to convict.
2. Mikroorganismen nach Anspruch 1 der Gattung Burkholderia, insbesondere wie hinterlegt unter der Nr. DSM 9925, sowie deren funktionell äquivalente Varianten und Mutanten.2. Microorganisms according to claim 1 of the genus Burkholderia, in particular as deposited under No. DSM 9925, and their functionally equivalent variants and mutants.
3. Verfahren zur Herstellung von R-α-Piperazincarbonsäuren, gegebenenfalls substituiert, der Formel3. Process for the preparation of R-α-piperazinecarboxylic acids, optionally substituted, of the formula
und/oder von S-α-Piperazincarbonsäureamiden, gegebenenfalls substituiert, der Formeland / or of S-α-piperazinecarboxamides, optionally substituted, of the formula
dadurch gekennzeichnet, daß man in einem (RS)-α-Piperazincarbonsäureamid, gegebenenfalls substituiert, der Formel characterized in that in a (RS) -α-piperazinecarboxamide, optionally substituted, of the formula
das R-α-Piperazincarbonsäureamid mittels der Mikroorganismen gemäß Anspruch 1 oder mittels zellfreier Enzyme aus diesen Mikroorganismen zur entsprechenden R-α-Piperazin¬ carbonsäure umsetzt und dieses und/oder das bei dieser Biotransformation neben der R- α-Piperazincarbonsäure anfallende S-α-Piperazincarbonsäureamid isoliert. the R-α-piperazinecarboxamide is converted by means of the microorganisms according to claim 1 or by means of cell-free enzymes from these microorganisms to the corresponding R-α-piperazinecarboxylic acid and this and / or the S-α- obtained in this biotransformation in addition to the R-α-piperazinecarboxylic acid Piperazine carboxylic acid amide isolated.
4. Verfahren nach Patentanspruch 3 , dadurch gekennzeichnet, daß man die Biotransformation mittels Mikroorganismen der Gattung Burkholderia, insbesondere wie hinterlegt unter der Nr. DSM 9925, oder mit deren funktionell äquivalenten Varianten und Mutanten oder mit zellfreien Enzymen aus diesen Mikroorganismen durchführt.4. The method according to claim 3, characterized in that one carries out the biotransformation by means of microorganisms of the genus Burkholderia, in particular as deposited under the number DSM 9925, or with their functionally equivalent variants and mutants or with cell-free enzymes from these microorganisms.
5. Verfahren nach mindestens einem der Patentansprüche 3 und 4, dadurch gekennzeichnet, daß man die Biotransformation unter einmaliger oder kontinuierlicher Zugabe des (RS)- α-Piperazincarbonsäureamids durchführt, so daß die Konzentration an (RS)-α-Piperazincar- bonsäureamid im Kulturmedium 20 Gew.-% nicht übersteigt.5. The method according to at least one of claims 3 and 4, characterized in that one carries out the biotransformation with a single or continuous addition of the (RS) - α-piperazinecarboxamide, so that the concentration of (RS) -α-piperazinecarboxamide in the culture medium Does not exceed 20% by weight.
6. Verfahren nach mindestens einem der Patentansprüche 3 bis 5, dadurch gekennzeichnet, daß man die Biotransformation bei einem pH- Wert von 6 bis 10 und bei einer Temperatur von 15 bis 55° C durchführt. 6. The method according to at least one of claims 3 to 5, characterized in that one carries out the biotransformation at a pH of 6 to 10 and at a temperature of 15 to 55 ° C.
EP96915025A 1995-05-08 1996-05-07 BIOTECHNICAL PRODUCTION PROCESS OF PIPERAZINE R-$g(a)-CARBOXYLIC ACIDS AND PIPERAZINE S-$g(a)-CARBOXYLIC ACID AMIDE Withdrawn EP0826032A1 (en)

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