JP2002523107A - Method for producing (2R) -piperidine derivative - Google Patents

Abstract

  (57) [Summary] Formulas (I) and (VII) wherein R¹Is a hydroxy group, an amino group or C_1-6-Alkoxy group, R^FourIs a hydroxy group or an amino group]. The (R) -piperidine derivative has the general formula (II):^TwoIs a hydroxy group, an amino group or C _1-6-Alkoxy groups] can be obtained using microorganisms in the presence of trace elements or using cell-free enzymes obtained from these microorganisms. Embedded image

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention has the general formula:

Embedded image

And the general formula:

Embedded image

[Wherein R ¹ is a hydroxy group, an amino group or a C _1-6 -alkoxy group; R ⁴ is a hydroxy group or an amino group], a novel production of a (2R) -piperidine derivative represented by the following formula: About the method.

A (2R) -piperidic acid derivative represented by the general formula I, for example, (R) -pipecolic acid is, for example, a growth hormone secretion stimulant (WO-A-9513069) or an anxiolytic (DE)
-A-37 02 943) is an important starting material for synthesizing biologically active compounds.

EP-A-686 698 discloses an S-α-aminocarboxylic acid such as S-α-pipecolic acid by reacting racemic RS-α-aminocarboxylic acid amide with microorganisms of the genus Klebsiella and Pseudomonas. Is biotechnologically manufactured and the corresponding R
A method is described in which -α-aminocarboxylic acid amide is also by-produced. However, R-
In order to purify the α-aminocarboxylic acid amide, the S-α-aminocarboxylic acid must be separated in a relatively time-consuming and expensive manner.

[0006] Culture of a microorganism of the genus Alcaligenes in a medium containing (RS) -pipecolic acid
A method for producing R) -pipecolic acid by biotechnology is also known (Mochizuki. K).
; Yamazaki, Y .; Maeda, H .: Agric. Biol. Chem., 1988, 52 (5), 1113). The disadvantage of this method is that (R) -pipecolic acid is obtained in a mixture with (S) -2-aminoadipic acid and must be separated from the formed aminoadipic acid after production.

The method for producing (R) -pipecolic acid ester is disclosed in RJ Kazlauskas et al. (J. Org. Ch.
em., 1994, 59, 2075-2081). According to that, the corresponding racemic ester is converted to the desired product by a stereoselective lipase isolated from black Aspergillus niger. The disadvantage of this method is the low enantiomeric purity and yield of the desired product. In addition, very large amounts of enzymes are required.

JP-A-63 248 393 describes a process for producing (R) -pipecolic acid from racemic pipecolic acid using a microorganism of the genus Pseudomonas, Kurthia or Alcaligenes. The disadvantage of this method is that the reaction time is long.

It is an object of the present invention to eliminate this drawback and to provide a simple method for producing (2R) piperidine derivatives, which gives a high yield and is practical for many technologies. .

The object of the present invention is solved by a novel biotechnological method according to claim 1. According to the invention, the general formula III:

Embedded image

Wherein A forms a 5- or 6-membered saturated heterocyclic ring together with —NH and —CH, a racemate or its optical activity In one form of the various isomers, a microorganism is used that has the ability to serve as the sole source of nitrogen. Such microorganisms are known in the state of the art and have already been described in detail in EP-A-686 698.

[0012] Preferred microorganisms having the above characteristics are microorganisms of the genus Klebsiella and Pseudomonas. Among them, Pseudomonas putida (putida) is particularly preferred, and DSM
A Pseudomonas putida species designated 9923 and its functionally equivalent variants and mutants are used.

A microorganism of the species Pseudomonas putida, designated DSM 9923, was produced according to the Budapest Treaty on April 20, 1995, D-38124 Braunschweig, No. 1b, Macerode Street, German Microbial and Cell Culture Collection GmbH (GmbH).
DSM).

As used herein, “functionally equivalent varieties and variants” are microorganisms that have essentially the same properties and functions as the original microorganism. Such variants and variants may be formed accidentally, for example, by UV irradiation.

Surprisingly, these microorganisms, or cell-free enzymes obtained therefrom, have the general formula II:

Embedded image

In the formula, R ² is a hydroxy group, an amino group or a C _1-6 -alkoxy group, and an S isomer of the (RS) -piperidine derivative represented by the formula: It has been found that it can be opened and cleaved under aerobic conditions and used as the sole carbon and energy source in the case of microorganisms. Sufficient reaction time, usually 1 to 48
As time passes, cyclic S-α-aminocarboxylic acid or S-α-aminocarboxylic acid is contained in the reaction medium.
Other troublesome open-chain by-products in the reaction medium, such as 2-aminoadipic acid, are no longer present or present at all. General formula I accumulated by the reaction:

Embedded image

The (2R) -piperidine derivatives of the formula wherein R ¹ is a hydroxy group, an amino group or a C _1-6 -alkoxy group can be obtained without difficulty and in particular with time-consuming and costly separation processes. Can be separated without the need.

A C _1-6 -alkoxy group here and hereafter means a straight-chain or branched alkoxy group containing 1 to 6 carbon atoms. Specific group names mean methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy, pentoxy and its isomers, and hexoxy and its isomers.

A cell-free enzyme can be obtained by disrupting a microorganism according to a method commonly used by those skilled in the art. For this purpose, for example, the ultrasonic method, the French press method or the lysozyme method can be used. These cell-free enzymes can be immobilized on a suitable carrier.

The term “trace element” as used herein means, for example, zinc, manganese, cobalt, copper, nickel, molybdenum, calcium, magnesium, boron, iron or sulfur, the optimum composition of which depends on the microorganism used. I do. Zinc, manganese, cobalt,
Preferably, copper, nickel, molybdenum, calcium, magnesium, boron, iron and sulfur are used cooperatively.

The reaction between the (RS) -piperidine derivative of the general formula II and the growing microorganism proceeds as appropriate for the purpose. Preferred substrates of the general formula II are (RS) -pipecolic acid, (RS) -pipecolic acid ethyl ester, (RS) -isopropyl pipecolate and (RS
) -Pipecolinamide.

The term “aerobic conditions” as used herein means culture conditions for supplying oxygen to microorganisms. This can be performed by shaking culture with air oxygen or submerged culture in which molecular oxygen or air oxygen is aerated.

The reaction can be carried out by directly adding the (RS) -piperidine derivative represented by the general formula II without culturing the microorganism. Alternatively, the microorganism can be cultured normally and then reacted with a suitable carbon and energy source. As a carbon source and an energy source therefor, for example, sugars, carboxylic acids, sugar alcohols or amino acids can be used. As the sugar, for example, hexose such as glucose or pentose can be used. As the carboxylic acid, dicarboxylic acid or tricarboxylic acid and salts thereof can be used, and citric acid or succinate can be mentioned as an example. As the sugar alcohol, a trihydric alcohol can be used, and glycerin can be mentioned as an example.
Glutamic acid is used as an amino acid, for example.

The reaction is preferably performed by directly adding the microorganism to the (RS) -piperidine derivative represented by the general formula II without culturing the microorganism according to a conventional method. Microorganisms can utilize the S isomer of the (RS) -piperidine derivative of the general formula II as the sole carbon and energy source and the only nitrogen source during the reaction.

The medium for carrying out the method of the present invention includes a medium such as an inorganic salt medium well-known to those skilled in the art to which a necessary trace element has been added, for example, an inorganic salt medium of Kulla et al. (Arch. Mic.
robiol. 135, 1-7, 1983), the medium described in Table 1 or a low concentration buffer, for example a 10-100 mM phosphate buffer. The method in the medium according to Table 1 is preferred.

The reaction conditions suitable for the purpose are selected such that the microorganism utilizes the S isomer of the (RS) -piperidine derivative represented by the general formula II, preferably as a carbon source and an energy source. . Therefore, it is preferable that the culture medium does not contain any compounds other than the compound II that can be easily used as a carbon source and an energy source.

The reaction can be carried out while adding the (RS) -piperidine derivative represented by the general formula II only once or in several portions, or continuously. The addition of the (RS) -piperidine derivative represented by the general formula II is performed so that the concentration does not exceed 30% by mass, preferably 10% by mass, particularly preferably 5% by mass.

The pH value of the medium is preferably from 4 to 9, and particularly preferably from 6 to 8. Reaction temperature is 1
5 ° C to 80 ° C is suitable for the purpose, and particularly preferably 25 ° C to 40 ° C. What final product of the general formula I is obtained in the reaction depends on the starting materials and on the reaction conditions, in particular on the reaction time and on the supply of oxygen.

From (RS) -pipecolic acid [(RS) -piperidine derivative II, wherein R ² is a hydroxy group], only (2R) -pipecolic acid is obtained. Similarly (RS
) -Pipecolinamide [(RS) -piperidine derivative II, wherein R ² is an amino group], to obtain (2R) -pipecolinamide and (2R) -pipecolic acid. (2R) -Pipecolic acid ester and (2R) -Pipecolic acid can be obtained from (RS) -Pipecolic acid ester [(RS) -Piperidine derivative II, wherein R ² is a C _1-6 -alkoxy group]. Can be.

Whether (2R) -pipecolinamide or (2R) -pipecolic acid can be obtained from (RS) -pipecolinamide can be controlled, for example, by the reaction time. In the reaction of (RS) -pipecolinamide with the microorganism according to the present invention, (2R) -pipecolinamide is first accumulated and, if necessary, it can be isolated. When the reaction time is further extended, (2R) -pipecolic acid is produced from (2R) -pipecolic acid amide. The progress of the reaction can be followed by analysis, for example by chromatography. The same thing (
It is also adapted to the reaction of (RS) -pipecolic acid ester, where (2R) -pipecolic acid ester is accumulated first, and then (2R) -pipecolic acid is accumulated.

After a usual reaction time, preferably 1 to 48 hours, a (2R) -piperidine derivative represented by the general formula I, preferably (R) -pipecolic acid, (R) -pipecolic amide And (R) -pipecolic acid ethyl ester and (R) -pipecolic acid isopropyl ester can be obtained in a medium in a very high yield or quantitatively.

The (2R) -piperidine derivative represented by the general formula I thus obtained is
It can be isolated by conventional work-up methods, such as separation, acidification, chromatography, electrodialysis or crystallization of biomass.

The progress of the reaction can also be controlled through the supply of oxygen. For example,
From (RS) -pipecolinamide or (RS) -pipecolic acid ester,
When R) -pipecolic acid is produced, as described above, the reaction is carried out under aerobic conditions until (R) -pipecolinamide or (R) -pipecolic acid ester is accumulated, and then the supply of oxygen is stopped. , (R) -Pipecolic acid may be formed.

Equation V:

Embedded image

Wherein R ³ is an amino group or a C _1-6 -alkoxy group, wherein the C _1-6 -alkoxy group is as defined above, from a (RS) -pipecolic acid derivative according to formula IV:

Embedded image

The reaction to convert to (R) -pipecolic acid according to formula VI is:

Embedded image

The reaction is carried out through a (R) -pipecolic acid derivative according to the formula wherein R ³ is as defined above. At this time, the pipecolic acid derivative is accumulated in the medium while increasing the enantiomeric excess, and can be isolated if necessary. Here, if an (R) -pipecolic acid derivative according to formula VI is to be produced from an (RS) -pipecolic acid derivative according to formula V, this is isolated after accumulation. And if it is desired to produce (R) -pipecolic acid according to formula IV from a (RS) -pipecolic acid derivative according to formula V, then follow (R) according to formula VI
Performing the reaction under aerobic conditions until the pipecolic acid derivative has accumulated, and then stopping the supply of oxygen.

To determine whether the (R) -pipecolic acid derivative according to formula VI has accumulated in the medium, the progress of the reaction can be monitored by analysis, for example by chromatography. The reaction is preferably terminated when the accumulation of the (R) -pipecolic acid derivative according to formula VI is at a maximum.

Furthermore, the present invention provides, as another component, a compound of the general formula VII from a (2R) -pipecolic acid derivative according to the general formula VI:

Embedded image

[Wherein R ⁴ is a hydroxy group or an amino group], which includes a reduction reaction to a (2R) -piperidine derivative represented by the following formula:

As the reducing agent, usually, an alkali metal hydride or an alkaline earth metal hydride such as lithium aluminum hydride, sodium borohydride, potassium aluminum hydride, sodium aluminum hydride, magnesium borohydride or Calcium borohydride is used. In particular, lithium aluminum hydride is used.

The reduction is carried out at 0 to 100 ° C., preferably at the reflux temperature of the corresponding solvent. As is well known to those skilled in the art, the reduction can be performed in a polar organic solvent, such as ether. Examples of suitable ethers include diethyl ether, diisopropyl ether, tetrahydrofuran or 1,4-dioxane.

Usually, after a reaction time of 1 to 16 hours, the desired product of general formula VII, ie, (R) -2- (aminomethyl) piperidine or (R) -2- (Hydroxymethyl) piperidine can be isolated.

Table 1 Medium 1 (NH ₄ ) ₂ SO ₄ 2.0 g / l Na ₂ HPO ₄ 2.0 g / l KH ₂ PO ₄ 1.0 g / l NaCl 2.0 g / l MgCl ₂ .6H ₂ O 0 _{.4g / l CaCl 2 · 2H 2} O 14.5mg / l FeCl 3 · 6H 2 O 0.8mg / l ZnSO 4 · 7H 2 O 0.1mg / l MnCl 2 · 4H 2 O 0.09mg / l H 3 BO ₃ 0.3 mg / l CoCl ₂ .6H ₂ O 0.2 mg / l CuCl ₂ .2H ₂ O 0.01 mg / l NiCl ₂ .6H ₂ O 0.02 mg / l Na ₂ MoO ₄ .2H ₂ O _{03mg / l FeSO 4 · 7H 2} O 2.0mg / l EDTA-Na 2 · 2H 2 O 5.0mg / l.

Medium 2 KH ₂ PO ₄ 1.3 g / l NH ₄ Cl 5 g / l (NH ₄ ) ₂ SO ₄ 2 g / l Na ₂ SO ₄ 0.25 g / l MgCl ₂ .6H ₂ O 0.8 g / l _{CaCl 2 · 2H 2 O 0.16g /} l ZnSO 4 · 7H 2 O 9mg / l MnCl 2 · 4H 2 O 4mg / l H 3 BO 3 2.7mg / l CoCl 2 · 6H 2 O 1.8mg / l CuCl _{2 · 2H 2 O 1.5mg / l} NiCl 2 · 6H 2 O 0.18mg / l Na 2 MoO 4 · 2H 2 O 0.2mg / l FeSO 4 · 7H 2 O 30mg / l EDTA-Na 2 · 2H 2 O 175 mg / l.

Medium 3 Na ₂ SO ₄ 0.1 g / l Na ₂ HPO ₄ 2.0 g / l KH ₂ PO ₄ 1.0 g / l NaCl 2.0 g / l MgCl ₂ .6H ₂ O 0.4 g / l CaCl _{2 · 2H 2 O 14.5mg / l} FeCl 3 · 6H 2 O 0.8mg / l ZnSO 4 · 7H 2 O 0.1mg / l MnCl 2 · 4H 2 O 0.09mg / l H 3 BO 3 0.3mg / 1 CoCl ₂ .6H ₂ O 0.2 mg / l CuCl ₂ .2H ₂ O 0.01 mg / l NiCl ₂ .6H ₂ O 0.02 mg / l Na ₂ MoO ₄ .2H ₂ O 0.03 mg / l FeSO _{4 · 7H 2 O 2.0mg / l EDTA} -Na 2 · 2H 2 O 5.0mg / l.

(Example) (Example 1) Production of (R) -pipecolic acid from (RS) -pipecolic acid 1.1 Production by shaking culture method (RS) -pipecolic acid containing 1.5% concentration 400 ml of Medium 1 was placed in a 1 L Erlenmeyer flask, and Pseudomonas putida DSM 9923 was added. The contents were placed on a shaker and incubated at 30 ° C. at 140 revolutions per minute. After 24 hours, the cell-free supernatant was subjected to HPLC at an OD650 value of 3.7 to determine the content of (R) -pipecolic acid. Based on the (RS) -pipecolic acid used, the yield of (R) -pipecolic acid was> 45% and the ee value was> 99%.

1.2 Production in a Fermenter 100 ml of medium 3 containing (RS) -pipecolic acid at a concentration of 1% is placed in a 500 ml Erlenmeyer flask with baffles and Pseudomonas putida DSM
9923 was added. The contents were placed on a shaker and incubated at 30 ° C. and 140 revolutions per minute for 16 hours. 1.5 g of (RS) -pipecolic acid (45 g)
3 L of the medium 2 containing 5% in a 5% concentration was placed in a 5 L culture tank, and Pseudomonas putida DSM 9923 which had been cultured in advance was added. After 7 hours, an additional 45 g of (RS)
-Pipecolic acid was added continuously as a 10% solution over 5 hours. After a further 4 hours, the bioconversion reaction was stopped, the cell-free supernatant was subjected to HPLC and (R
) -Pipecolic acid content was quantified. The yield of (R) -pipecolic acid was quantitative, with an ee value> 97%.

Example 2 Production of (R) -pipecolic acid from (RS) -pipecolic acid ethyl ester 100 ml of 40 ml of medium 3 containing 1.5% of (RS) -pipecolic acid ethyl ester with baffles Place in Erlenmeyer flask and place Pseudomonas
Putida DSM 9923 was added. The contents were placed on a shaker and incubated at 30 ° C. at 140 revolutions per minute. After 26 hours, the cell-free supernatant was subjected to HPLC at an OD650 value of 2.0 to determine the content of (R) -pipecolic acid.
Based on the (RS) -pipecolic acid ethyl ester used, the yield of (R) -pipecolic acid was> 45% and the ee value was> 80%. After 44 hours, an enantiomerically pure (R) -pipecolic acid with an OD650 value of 3.2 was obtained.

Example 3 Production of (R) -Pipecolinamide from (RS) -Pipecolinamide by Shaking Culture Method Forty ml of medium 3 containing (RS) -pipecolinamide at a concentration of 1.5% was prepared. In a 100 ml Erlenmeyer flask with baffles, Pseudomonas putida DSM 9923 was added. Place the contents on a shaker at 30 ° C for 14 minutes per minute.
Incubation was performed at 0 rotation. After 20 hours, the cell-free supernatant was subjected to HPLC at an OD650 value of 3.0, and the content of (R) -pipecolinamide was quantified. Based on the (RS) -pipecolinamide used, the yield of (R) -pipecolinamide was> 45% and the ee value was> 99%.

Example 4 Production of (R) -Pipecolinamide and (R) -Pipecolic Acid from (RS) -Pipecolinamide in a Cultivation Vessel
mat) in a culture tank overnight. 1% (RS) at OD650 value of 14
-Pipecolinamide was added. GC analysis after 2 hours indicated the presence of enantiomerically substantially pure (R) -pipecolinamide. Pipecolic acid was not detected. The solution sample was placed on a shaker with the air cut off, at 37 ° C. and 140 minutes per minute.
Incubate with rotation. GC analysis for 48 hours indicated the presence of enantiomerically pure (R) -pipecolic acid. Pipecolinamide was not detected.

(Example 5) Production of (R) -pipecolic acid from (RS) -pipecolinamide by shaking culture method A 40 ml culture medium 3 containing (RS) -pipecolinamide at a concentration of 1% was provided with a baffle plate. In a 100 ml Erlenmeyer flask of Pseudomonas putida D
SM 9923 was added. Place the contents on a shaker at 30 ° C, 140
Incubate with rotation. After 26 hours, the cell-free supernatant was analyzed by GC at an OD650 value of 3.9. The yield of (R) -pipecolic acid is about 10% and the ee value is>
98%.

Example 6 (R) from (RS) -Pipecolic acid isopropyl ester by the shake culture method
Preparation of) -pipecolic acid isopropyl ester 100 m containing (RS) -pipecolic acid isopropyl ester at a concentration of 4 g / l
l of the minimal medium 3 was placed in a 500 ml baffled Erlenmeyer flask, and Pseudomonas putida DSM 9923 was added. Put the contents on a shaker, 3
Incubation was performed at 0 ° C. and 140 revolutions per minute. OD6 after 4.5 hours
The cell-free supernatant was subjected to GC at a value of 1.4 to determine the content of pipecolic acid and pipecolic acid isopropyl ester and the enantiomeric excess. The produced (R) -pipecolic acid isopropyl ester was enantiomerically pure, and the yield of (R) -pipecolic acid isopropyl ester relative to the (R) -pipecolic acid isopropyl ester used was 88%. Several percent of enantiomerically pure (R) -pipecolic acid was detected as a by-product.

After removing the biomass by centrifugation (8000 * g for 20 minutes), the aqueous solution was concentrated under reduced pressure to 1/3 volume, then adjusted to pH 10 with 30% sodium hydroxide and then with 20 ml each of diethyl ether. Extracted three times. The combined organic layers were dried over sodium sulfate and concentrated. Hydrogen chloride was introduced to obtain 0.195 g of crystals of (R) -pipecolic acid isopropyl ester hydrochloride (yield by GC: 8
0%; ee> 98%).

Example 7 Production of (R) -pipecolic acid from (RS) -pipecolic acid isopropyl ester 100 m containing (RS) -pipecolic acid isopropyl ester at a concentration of 4 g / l
1 of Minimal Medium 3 was placed in a 500 ml baffled Erlenmeyer flask and Pseudomona putida DSM 9923 was added. Put the contents on a shaker,
Incubation was performed at 140 ° C./min. OD65 after 7.5 hours
At a value of 0, 1.4, a sample was withdrawn and cultured with the air shut off. After 22 hours, the cell-free supernatant was subjected to GC to determine the content of pipecolic acid and pipecolic acid isopropyl ester and the enantiomeric excess. The (R) -pipecolic acid produced was enantiomerically pure, and the yield of (R) -pipecolic acid relative to the (R) -pipecolic acid isopropyl ester used was 80%. Several percent of enantiomerically pure (R) -pipecolic acid isopropyl ester was detected as a by-product.

Example 8 Reduction of (R) -Pipecolinamide to (R) -2- (aminomethyl) piperidine In a 2 L flask, under a nitrogen atmosphere, 11.8 g of lithium aluminum hydride was added to 500 ml of diethyl ether Was suspended. 20 g of (R) -pipecolinamide (see Example 3) were added slowly at 5 ° C. The mixture was stirred at room temperature for 3 hours and then at reflux for 5 hours. Cool to room temperature, 12 ml water, 12 ml 15
% Sodium hydroxide and 36 ml of water were successively slowly added dropwise. At room temperature, the mixture was stirred until it became snow-white. 120 g of sodium sulfate was added and stirred overnight at room temperature. The solid was filtered off and washed four times with 250 ml each time of diethyl ether. The organic layers were combined and concentrated under reduced pressure. 12.5 g of (R) as a yellow oil
2- (Aminomethyl) piperidine (content by GC: 66%) was obtained.
Distillation at 52-53 ° C./18 mbar afforded 3.6 g (20%) of a GC-pure (R) -2- (aminomethyl) piperidine colorless liquid.

Example 9 Reduction of (R) -Pipecolic Acid Isopropyl Ester to (R) -2- (Hydroxymethyl) piperidine In a 250 ml flask, under nitrogen atmosphere, 0.2 g of lithium aluminum hydride and 20 ml of diethyl Suspended in ether. 90 mg of (R) -pipecolic acid isopropyl ester hydrochloride were slowly added at 5 ° C. The mixture was stirred at room temperature for 1 hour and then at reflux for 4 hours. Cool to room temperature, add 0.2 ml water, 0 ml
. 2 ml of 15% sodium hydroxide and 0.4 ml of water were successively slowly added dropwise. At room temperature, the mixture was stirred until it became snow-white. 2 g of sodium sulfate was added and stirred at room temperature overnight. The solid was filtered off and washed four times with 20 ml each time of diethyl ether. The organic layers were combined and concentrated under reduced pressure. 47m as pale yellow oil
g of (R) -2- (hydroxymethyl) piperidine (content by GC:
95%; ee> 97%).

[Procedural Amendment] Submission of translation of Article 34 Amendment of the Patent Cooperation Treaty

[Submission date] October 18, 2000 (2000.10.18)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

Embedded image [Wherein R ¹ is a hydroxy group, an amino group or a C _1-6 -alkoxy group], a method for producing a (2R) -piperidine derivative represented by the general formula:

Embedded imageWhere R^TwoIs a hydroxy group, an amino group or C_1-6-Alkoxy group, R¹
And R^TwoMay be the same or different substituents,¹Is Ami
In the case of a no group, R^TwoIs a hydroxy group or C_1-6-Not an alkoxy group, but R¹Is C _1-6 In the case of -alkoxy, R^TwoIs not a hydroxy group or an amino group], and a (RS) -piperidine derivative represented by the general formula:

Embedded image [Wherein A together with -NH or -CH forms an optionally substituted 5- or 6-membered saturated heterocyclic ring]. (RS)-characterized by reacting with a growing microorganism that can be used as a sole nitrogen source in the form of an optically active isomer in the presence of trace elements and under aerobic conditions.
A method wherein the S isomer of piperidine derivative II undergoes ring opening and is utilized or decomposed as a carbon source and energy source.

Embedded image A method for producing (R) -pipecolic acid represented by the general formula:

Embedded image [Wherein R ³ is an amino group or a C _1-6 -alkoxy group], using a (RS) -pipecolic acid derivative represented by the following formula as a substrate:

Embedded image [Wherein A together with -NH and -CH forms an optionally substituted 5- or 6-membered saturated heterocycle], a racemate or an optical form thereof. A growing microorganism that can be used as the sole nitrogen source in one form of the active isomer;
Characterized by reacting under aerobic conditions in the presence of a trace element, wherein the reaction comprises a formula which can be isolated if necessary:

Embedded image Wherein the R ³ is as defined above, under aerobic conditions until the corresponding (R) -pipecolic acid derivative accumulates, the S of the (RS) -piperidine derivative V A method in which the isomer undergoes ring opening and is used or decomposed as a carbon source and energy source, followed by stopping the supply of oxygen.

Embedded image [Wherein R ⁴ is a hydroxy group or an amino group], a method for producing a (2R) -piperidine derivative represented by the following formula:
General formula:

Embedded image [Wherein R ³ is an amino group or a C _1-6 -alkoxy group], using a (RS) -pipecolic acid derivative represented by the following formula as a substrate:

Embedded image [Wherein A together with -NH and -CH forms an optionally substituted 5- or 6-membered saturated heterocyclic ring], an α-aminocarboxylic acid amide represented by the following formula: A growing microorganism that can be used as the sole nitrogen source in one form of its optically active isomer;
Under aerobic conditions in the presence of trace elements, the S isomer of the (RS) -piperidine derivative V undergoes ring opening and is utilized as a carbon and energy source or decomposed to give the general formula:

Embedded image Wherein R ³ is an amino group or a C _1-6 -alkoxy group, which is converted into a (R) -pipecolic acid derivative represented by the following formula:
A method characterized in that the compound is reduced to a desired compound represented by the general formula VII.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme court ゛ (Reference) C12R 1:40) C12R 1:40) (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV , MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Heinzmann, Klaus Switzerland, CH-3332 Bispertaminen, Unterstalden (no address) F-term (reference) 4B064 AE49 CA02 CB01 CD12 CD27 DA01

Claims (9)

[Claims] 1. A compound of the general formula:Where R¹Is a hydroxy group, an amino group or C_1-6-Alkoxy group], a method for producing a (2R) -piperidine derivative represented by the following general formula:Where R^TwoIs a hydroxy group, an amino group or C_1-6-Alkoxy group, R¹
And R^TwoMay be the same or different substituents,¹Is Ami
In the case of a no group, R^TwoIs a hydroxy group or C_1-6-Not an alkoxy group, but R¹Is C _1-6 In the case of -alkoxy, R^TwoIs not a hydroxy group or an amino group.] A (RS) -piperidine derivative represented by the following general formula:Wherein A is together with —NH or —CH, an optionally substituted 5-membered ring or 6-membered
Α-aminocarboxylic acid amide represented by the formula:
A microorganism that can be used as the sole source of nitrogen in one form, or
Cell-free enzymes isolated from these microorganisms, in the presence of trace elements, and under aerobic conditions
Wherein the S isomer of the (RS) -piperidine derivative II is ring-opened.
Used or decomposed as carbon and energy sources
Law. 2. A method according to claim 1, wherein (RS) -pipecolic acid, (RS
2. The process according to claim 1, wherein ())-pipecolic acid ethyl ester, (RS) -pipecolic acid isopropyl ester or (RS) -pipecolic acid amide are used. 3. The method according to claim 1, wherein the microorganism is reacted with a microorganism of the genus Pseudomonas, particularly a microorganism of the species Pseudomonas putida. 4. The method according to claim 1, wherein the reaction is carried out with a microorganism of Pseudomonas putida (DSM 9923) or a functionally equivalent variant and variant. 5. The method according to claim 1, wherein the method is reacted with a growing microorganism. 6. The method according to claim 1, wherein (RS) -pipecolic acid represented by the general formula II is added so that its concentration does not exceed 30% by mass. Method. 7. The method according to claim 1, wherein the reaction is carried out at a pH of 4 to 9 and a temperature of 15 ° C. to 80 ° C. 8. The formula: embedded image A method for producing (R) -pipecolic acid represented by the general formula: [Wherein, R ³ is an amino group or a C _1-6 -alkoxy group], using a (RS) -pipecolic acid derivative represented by the following formula as a substrate: [Wherein A together with -NH and -CH forms an optionally substituted 5- or 6-membered saturated heterocycle], a racemate or an optical form thereof. Characterized by reacting with microorganisms that can be used as a sole nitrogen source in the form of active isomers, or cell-free enzymes isolated from these microorganisms, under aerobic conditions in the presence of trace elements Wherein the reaction is of the formula: Wherein R ³ is as defined above, under aerobic conditions until the accumulation of the corresponding (R) -pipecolic acid derivative, the S of the (RS) -piperidine derivative II A method in which the isomer undergoes ring opening and is used or decomposed as a carbon source and energy source, followed by stopping the supply of oxygen. 9. A compound of the general formula: [Wherein R ⁴ is a hydroxy group or an amino group], a method for producing a (2R) -piperidine derivative represented by the following formula:
General formula: [Wherein R ³ is an amino group or a C _1-6 -alkoxy group], using a (RS) -pipecolic acid derivative represented by the following formula as a substrate: [Wherein A together with -NH and -CH forms an optionally substituted 5- or 6-membered saturated heterocyclic ring], an α-aminocarboxylic acid amide represented by the following formula: In a form of its optically active isomers, microorganisms which can be used as sole nitrogen source, or cell-free enzymes isolated from these microorganisms, under aerobic conditions in the presence of trace elements (R
The S isomer of the S) -piperidine derivative II undergoes ring opening and is utilized as a carbon source and energy source or decomposed to give a compound of the general formula: Wherein R ³ is an amino group or a C _1-6 -alkoxy group, which is converted into a (R) -pipecolic acid derivative represented by the general formula VII: Wherein the compound is reduced to a compound of the formula: