CA2341554A1 - Process for the preparation of (2r)-piperidine derivatives - Google Patents
Process for the preparation of (2r)-piperidine derivatives Download PDFInfo
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- CA2341554A1 CA2341554A1 CA002341554A CA2341554A CA2341554A1 CA 2341554 A1 CA2341554 A1 CA 2341554A1 CA 002341554 A CA002341554 A CA 002341554A CA 2341554 A CA2341554 A CA 2341554A CA 2341554 A1 CA2341554 A1 CA 2341554A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
- C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
- C07D211/08—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms
- C07D211/18—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with substituted hydrocarbon radicals attached to ring carbon atoms
- C07D211/20—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with substituted hydrocarbon radicals attached to ring carbon atoms with hydrocarbon radicals, substituted by singly bound oxygen or sulphur atoms
- C07D211/22—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with substituted hydrocarbon radicals attached to ring carbon atoms with hydrocarbon radicals, substituted by singly bound oxygen or sulphur atoms by oxygen atoms
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
- C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
- C07D211/08—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms
- C07D211/18—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with substituted hydrocarbon radicals attached to ring carbon atoms
- C07D211/26—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with substituted hydrocarbon radicals attached to ring carbon atoms with hydrocarbon radicals, substituted by nitrogen atoms
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
- C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
- C07D211/36—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D211/60—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
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- 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
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- 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
- C12P41/001—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by metabolizing one of the enantiomers
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- General Engineering & Computer Science (AREA)
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Hydrogenated Pyridines (AREA)
Abstract
The invention relates to a method for preparing (R)-piperidine derivatives of the general formulas (I) and (VII), where R1 is hydroxy, amino or C1-6-alkoxy and R4 is hydroxy or amino. Said (R)-piperidine derivatives are obtained from (RS)-piperidine derivatives of the general formula (II), in which R2 is hydroxy, amino or C1-6-alkoxy, in the presence of trace elements and using micro-organisms or cell-free enzymes from said micro-organisms.
Description
Process for the preparation of (2R)-piperidine derivatives Description The present invention relates to a novel process for the preparation of (2R)-piperidine derivatives of the general formulae ..,,. O I
R~
and VII
in which R1 is hydroxyl, amino or C1_6-alkoxy and R9 is hydroxyl or amino.
(2R)-Piperidine derivatives of the general formula I, such as, for example, (R)-pipecolic acid, are important synthesis units for biologically active compounds, such as, for example, stimulants of growth hormone secretion (WO-A-9513069) or anti-anxiety agents (DE-A-37 02 943).
EP-A 686 698 describes a biotechnological process for the preparation of S-a-aminocarboxylic acids, for example S-a-pipecolic acid, by reaction of the racemic RS-a-aminocarboxamides with microorganisms of the genera Klebsiella and Pseudomonas, the corresponding R-a-aminocarboxamides also being obtained. For the preparation of the R-a-aminocarboxamides in pure form, the S-a-aminocarboxylic acids, however, have to be removed by relatively complicated processes.
R~
and VII
in which R1 is hydroxyl, amino or C1_6-alkoxy and R9 is hydroxyl or amino.
(2R)-Piperidine derivatives of the general formula I, such as, for example, (R)-pipecolic acid, are important synthesis units for biologically active compounds, such as, for example, stimulants of growth hormone secretion (WO-A-9513069) or anti-anxiety agents (DE-A-37 02 943).
EP-A 686 698 describes a biotechnological process for the preparation of S-a-aminocarboxylic acids, for example S-a-pipecolic acid, by reaction of the racemic RS-a-aminocarboxamides with microorganisms of the genera Klebsiella and Pseudomonas, the corresponding R-a-aminocarboxamides also being obtained. For the preparation of the R-a-aminocarboxamides in pure form, the S-a-aminocarboxylic acids, however, have to be removed by relatively complicated processes.
A biotechnological process for the preparation of (R)-pipecolic acid by culturing microorganisms of the genus Alcaligenes in (RS)-pipecolic acid-containing medium is also known (Mochizuki, K.; Yamazak:i, Y.; Maeda, H.;
Agric. Biol. Chem. 1988, 52(5), 1113). This process has the disadvantage that the (R)-pipecolic acid is obtained as a mixture with (S)-2-aminoadipic acid and has to be removed from the aminoadipic acid formed after the preparation.
A process for the preparation of (R)-pipecolic acid esters is described by R.J. Kazlaus~;as et al (J. Org.
Chem., 1994, 59, 2075-2081). In this process, the corresponding racemic esters are reacted by means of isolated stereoselective lipases from Aspergillus niger to give the desired product. The disadvantage in this process is that the desired products are in poor enantiomeric purity and are obtained in low yield. In addition, very large amounts of enzyme are also necessary.
JP-A-63 248 393 describes a process for the preparation of (R)-pipecolic acid starting from racemic pipecolic acid by means of microorganisms of the genera Pseudomonas, Kurthia or Alcaligenes. A disadvantage in this process is the long reaction times.
It is the object of the present invention to eliminate these disadvantages and to make available a simple process for the preparation of (2R)-piperidine derivatives, with which high yields can be achieved and which is feasible on a large scale.
This object is achieved by the novel biotechnological process according to Claim 1.
According to the invention, microorganisms are employed which have the property of being able to utilize a-aminocarboxamides in the form of the racemate or its optically active isomers of the general formula III
A\
~CH~NHl NH U
in which A, together with -NH and -CH, is an optionally substituted 5- or 6-membered saturated heterocyclic ring, as the only nitrogen source. Microorganisms of this type are known in the prior ar_t and are already described in detail in EP-A-686 698.
Preferred microorganisms having the properties described above are microorganisms of the genera Klebsiella and Pseudomonas. Microorganisms which are particularly preferably employed are those of the species Pseudomonas putida, in particular those of the species Pseudomonas putida having the designation DSM 9923, and their functionally equivalent variants and mutants.
The microorganisms of the species Pseudomonas putida having the designation DSM 9923 were deposited on 20.04.1995 at the Deutsche Sammlung fur Mikroorganismen and Zellkulturen GmbH (DSM), Mascherodeweg lb, D-38124 Brunswick, according to the Budapest Convention.
"Functionally equivalent variants and mutants" are understood as meaning microorganisms which essentially have the same properties and functions as the original microorganisms. Variants and mutants of this type can be formed by chance, for example, by t1V irradiation.
Surprisingly, it has been found that these microorganisms, or cell-free enzymes thereof, can degrade the S-isomer in (RS)-piperidine derivatives of the general formula II
Agric. Biol. Chem. 1988, 52(5), 1113). This process has the disadvantage that the (R)-pipecolic acid is obtained as a mixture with (S)-2-aminoadipic acid and has to be removed from the aminoadipic acid formed after the preparation.
A process for the preparation of (R)-pipecolic acid esters is described by R.J. Kazlaus~;as et al (J. Org.
Chem., 1994, 59, 2075-2081). In this process, the corresponding racemic esters are reacted by means of isolated stereoselective lipases from Aspergillus niger to give the desired product. The disadvantage in this process is that the desired products are in poor enantiomeric purity and are obtained in low yield. In addition, very large amounts of enzyme are also necessary.
JP-A-63 248 393 describes a process for the preparation of (R)-pipecolic acid starting from racemic pipecolic acid by means of microorganisms of the genera Pseudomonas, Kurthia or Alcaligenes. A disadvantage in this process is the long reaction times.
It is the object of the present invention to eliminate these disadvantages and to make available a simple process for the preparation of (2R)-piperidine derivatives, with which high yields can be achieved and which is feasible on a large scale.
This object is achieved by the novel biotechnological process according to Claim 1.
According to the invention, microorganisms are employed which have the property of being able to utilize a-aminocarboxamides in the form of the racemate or its optically active isomers of the general formula III
A\
~CH~NHl NH U
in which A, together with -NH and -CH, is an optionally substituted 5- or 6-membered saturated heterocyclic ring, as the only nitrogen source. Microorganisms of this type are known in the prior ar_t and are already described in detail in EP-A-686 698.
Preferred microorganisms having the properties described above are microorganisms of the genera Klebsiella and Pseudomonas. Microorganisms which are particularly preferably employed are those of the species Pseudomonas putida, in particular those of the species Pseudomonas putida having the designation DSM 9923, and their functionally equivalent variants and mutants.
The microorganisms of the species Pseudomonas putida having the designation DSM 9923 were deposited on 20.04.1995 at the Deutsche Sammlung fur Mikroorganismen and Zellkulturen GmbH (DSM), Mascherodeweg lb, D-38124 Brunswick, according to the Budapest Convention.
"Functionally equivalent variants and mutants" are understood as meaning microorganisms which essentially have the same properties and functions as the original microorganisms. Variants and mutants of this type can be formed by chance, for example, by t1V irradiation.
Surprisingly, it has been found that these microorganisms, or cell-free enzymes thereof, can degrade the S-isomer in (RS)-piperidine derivatives of the general formula II
0 i( R
in which R2 is hydroxyl, amino or Cl-s-alkoxy, in the presence of trace elements and under aerobic conditions with ring-cleavage and, in the case of the microorganisms, can utilize it as the only carbon and energy source. After an adequate reaction period, customarily after 1 to 48 hours, only a few cyclic S-a-aminocarboxylic acids or other interfering open-chain by-products such as S-2-aminoadipic acid or none at all are then present in the reaction medium. The (2R)-piperidine derivatives accumulated in the reaction, of the general formula I
..,,. ~ f R' in which R1 is hydroxyl, amino or C1_6-alkoxy, can then be isolated without difficulties, in particular without complicated separation processes.
Here and in the following, C1_6-alkoxy has the meaning of a straight-chain or branched alkoxy group having from 1 to 6 C atoms. Methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy, pentaxy and its isomers and also hexoxy and its isomers may be mentioned by name.
The enzymes for the cell-free system can be obtained by technically customary breakdown of the microorganisms.
For this, for example, it is possible to use the ultrasonic, French press or lysozyme method. These cell-free enzymes can also be immobilized on a suitable carrier material.
Trace elements are to be understood, for example, as meaning zinc, manganese, cobalt, copper, nickel, molybedenum, calcium, magnesium, boron, iron or sulphur, the optimal composition of the trace elements being dependent on the microorganism used. Preferably, zinc, manganese, cobalt, copper, nickel, molybdenum, calcium, magnesium, boron, iron and sulphur are jointly employed.
Expediently, the reaction of (RS)-piperidine derivatives of the general formula II is carried out with growing microorganisms.
The most preferred substrates of the general formula II
are (RS)-pipecolic acid, ethyl (RS)-pipecolate, isopropyl (RS)-pipecolate and (RS)-pipecolamide.
Aerobic conditions are understood as meaning culturing conditions in which the microorganisms are supplied with oxygen. This can be carried out, for example, in shaker culture by means of atmospheric oxygen or in submerse culture by blowing in molecular oxygen or atmospheric oxygen.
The reaction can be carried out without customary culturing directly by addition of the microorganisms to (RS)-piperidine derivatives of the general formula II.
Alternatively, the reaction can be carried out after customary culturing of the microorganisms using a suitable carbon and energy source. The carbon and energy source employed here can be, for example, sugars, carboxylic acids, sugar alcohols or amino acids. Sugars which can be used are hexoses such as, for example, glucose or pentoses. Carboxylic acids which can be used are di- or tricarboxylic acids and their salts such as, for example, citric acid or succinate. A sugar alcohol which can be used is a trihydric alcohol, such as, for example, glycerol. An amino acid which can be employed is, for example, glutamate.
Preferably, the reaction is carried out without customary culturing directly by addition of the microorganisms to (RS)-piperidine derivatives of the general formula II.
In the reaction, the microorganisms can utilize the (S) isomer of the (RS)-piperidine derivative of the general formula II as the only carbon and energy source and as the only nitrogen source.
Media which can be used for the process according to the invention are the technically customary media such as mineral salt media, for example the mineral salt medium according to Kulla et al., (Arch. Microbiol.
135, 1-7, 1983) , the media described in Table 1 or low molar buffers, such as, for example, 10 to 100 mM
phosphate buffer, to which the necessary trace elements have been added. The process is preferably carried out in the media as in Table 1.
Expediently, the conditions are chosen such that the S
isomer of the (RS)-piperidine derivatives of the formula II is preferably utilized as a carbon and energy source by the microorganisms. Preferably, besides the compound II, the medium therefore does not contain any other compounds readily utilizable as a carbon and energy source.
Expediently, the reaction is carried out with single, repeated or continuous addition of (RS)-piperidine derivatives of the general formula II. The addition of (RS)-piperidine derivatives of the general formula II
is carried out in such a way that the concentration does not exceed 30% by weight, preferably 10% by weight, particularly preferably 5% by weight.
The pH of the medium is expediently in a range from 4 to 9, preferably from 6 to 8. Expediently, the reaction is carried out at a temperature from 15 to 80°C, preferably from 25 to 40°C.
The final products of the general formula I which is obtained in the reaction depends on the choice of the starting material and the reaction conditions, in particular the reaction time and the oxygen supply.
Starting from (RS)-pipecolic acid [(RS)-piperidine derivative II in which R2 is hydroxyl], only (2R)-pipecolic acid can be obtained. Starting from (RS)-pipecolamide [(RS)-piperidine derivative II in which RZ
is amino], both (2R)-pipecolamide and (2R)-pipecolic acid can be obtained. Starting from an (RS)-pipecolic acid ester [(RS)-piperidine derivative II in which Rz is C1_6-alkoxy] , both the (2R) -pipecolic acid ester and (2R)-pipecolic acid can be obtained.
Whether (2R)-pipecolamide or (2R)-pipecolic acid is obtained from (RS)-pipecolamide can be controlled, for example, by means of the reaction time. Thus in the reaction of (RS)-pipecolamide with the microorganisms according to the invention, (2R)-pipecolamide first accumulates, which can then optionally be isolated.
After a longer reaction time, the acid is then formed from the (2R)-pipecolamide. The reaction can be monitored analytically, for example chromatographically. The same applies to the reaction of (RS) -pipecolic acid esters in which first the (2R) -pipecolic acid ester and then the acid accumulates.
After a customary reaction time of preferably 1 to 48 hours, the (2R)-piperidine derivatives of the general formula I, preferably (R)-pipecolic acid, (R)-pipecolamide and ethyl and isopropyl (R)-pipecolates are preferably obtained in the medium in very high to quantitative yield.
The (2R)-piperidine derivatives of the general formula I obtained in this manner can be isolated by customary working-up methods such as, for example, by removal of the biomass, acidification, chromatography, electrodialysis or crystallization.
Alternatively, the course of the conversion reaction can be regulated by means of the oxygen supply. If, for example, (R)-pipecolic acid is to be prepared from (RS)-pipecolamide or (RS)-pipecolic acid ester, the reaction is expediently carried out under aerobic conditions until the accumulation of the (R)-pipecolamide or of the (R)-pipecolic acid ester, respectively, as described above and the oxygen supply is then stopped, the acid being formed thereafter.
The reaction of the (RS)-pipecolic: acid derivative according to formula V
V
~3 in which R3 is amino or C1_6-alkoxy, C1_6-alkoxy having the abovementioned meaning, to give (R)-pipecolic acid according to formula IV
H
_ g _ thus takes place via the (R)-pipecolic acid derivative according to formula VI
,l.,,, p VI
in which R3 has the abovementioned meaning, which temporarily accumulates in the medium with increasing enantiomeric excess and can optionally be isolated. If the (R)-pipecolic acid derivative according to formula VI is to be prepared from the (RS)-pipecolic acid derivative according to formula V, it is isolated after accumulation. If (R)-pipecolic acid according to formula IV is to be prepared from the (RS)-pipecolic acid derivative according to formula V, the reaction is expediently carried out under aerobic conditions until the accumulation of the (R)-pipecolic acid derivative according to formula VI and the oxygen supply is then stopped.
In order to determine whether the (R)-pipecolic acid derivative according to formula VI has accumulated in the medium, the reaction can be monitored analytically, for example chromatographically. The reaction should be terminated as soon as the maximum accumulation of the (R)-pipecolic acid derivative according to formula VI
is achieved.
A further component of the invention is the further reaction, the reduction, of the (2R)-pipecolir_ acid derivatives of the general formula VI to give the (2R)-piperidine derivatives of the general formula VII
VII
,,, .~R.
in which R' is hydroxyl or amino.
Customarily, the reducing agent used is an alkali metal hydride or an alkaline earth metal hydride such as, for example, lithium aluminium hydride, sodium borohydride, potassium or sodium aluminium hydride, or magnesium or calcium borohydride. Lithium aluminium hydride is used in particular.
The reduction is expediently carried out at a temperature from 0 to 100°C, preferably at the reflux temperature of the corresponding solvent.
As known technically, the reduction can be carried out in a polar organic solvent such as, for example, in an ether. Suitable ethers are, for example, diethyl ether, dipropyl ether, tetrahydrofuran or 1,4-dioxane.
After a customary reaction time of 1 to 16 h, the desired products of the general formula VII, such as (R)-2-aminomethylpiperidine or (R)-2-hydroxymethyl-piperidine, can be isolated by known working-up methods.
Table 1 Medium 1 (NH4) 2504 2. 0 g/1 Na2HP09 2.0 g/1 KH2P09 1.0 g/1 NaCl 2.0 g/1 MgCl2 6H20 0. 4 g/1 CaCl2 2H20 14 . 5 mg/1 FeCl3 6H20 0 . 8 mg/1 ZnS04 7H20 0. 1 mg/1 MnCl24H20 0.09 mg/1 H3BO3 0.3 mg/1 CoCl26H20 0.2 mg/1 CuCl22H20 0.01 mg/1 NiCl26Hz0 0.02 mg/1 Na2Mo09 2H20 0. 03 mg/1 FeS04 7H20 2 . 0 mg/1 EDTA-Na22H20 5.0 mg/1 Medium 2 KH2P09 1.3 g/1 NH4C1 5 g/1 (NH4 ) 2SOn 2 g/1 Na2S04 0.25 g/1 MgCl26H20 0.8 g/1 CaCl22H20 0.16 g/1 ZnS04 7Hz0 9 mg/1 MnCl2 4H20 4 mg/1 H3B03 2.7 mg/1 CoCl26H20 1.8 mg/1 CuCl22H20 1.5 mg/1 NiCl26H20 0.18 mg/1 Na2Mo0~ 2H20 0 . 2 mg/1 FeS09 7H20 30 mg/1 EDTA-Na22H20 175 mg/1 Medium 3 Na2S04 0.1 g/1 Na2HP04 2.0 g/1 KH2P09 1.0 g/1 NaCl 2.0 g/1 MgCl2 6H20 0 . 4 g/1 CaCl22H20 14.5 mg/1 FeCl3 6H20 0. 8 mg/1 ZnS04 7H20 0 . 1 mg/1 MnCl24H20 0.09 mg/1 H3BO3 0.3 mg/1 CoCl26H20 0.2 mg/1 CuClz2H20 0.01 mg/1 NiCl26H20 0.02 mg/1 Na2Mo09 2H20 0. 03 mg/1 FeS04 7H20 2 . 0 mg/1 EDTA-Na22H20 5.0 mg/1 Examples Example 1 Preparation of (R)-pipecolic acid from (RS)-pipecolic acid 1.1 in shaker culture 400 ml of Medium 1 having a concentration of 1.5% of (RS)-pipecolic acid were introduced into a 1 1 Erlenmeyer flask and treated with Pseudomonas putida DSM 9923. The batch was incubated at 30°C and 140 revolutions/min on a shaker. After 24 h, the cell-free supernatant was investigated for the content of (R)-pipecolic acid at an OD650 of 3.7 using HPLC. (R)-Pipecolic acid was obtained in a yield of >45% based on the (RS)-pipecolic acid employed and with an ee value of >99$ .
1.2 in a fermenter 100 ml of Medium 3 having a concentration of 1% of (RS)-pipecolic acid were introduced into a 500 ml Erlenmeyer flask with baffles and treated with Pseudomonas putida DSM 9923. The batch was incubated for 16 h at 30°C and 140 revolutions/min on a shaker.
3 1 of Medium 2 having a concentration of 1.5% of (RS)-pipecolic acid (45 g) were introduced into a 5 1 fermenter and treated with the preculture of Pseudomonas putida DSM 9923. After 7 h, a further 45 g of (RS)-pipecolic acid were continuously added as a 10%
solution in the caurse of 5 h. After a further 4 h, the biotransformation was stopped and the cell-free supernatant was investigated for the content of (R)-pipecolic acid using HPLC. (R)-pipecolic acid was obtained in quantitative yield and with an ee value of >97$.
Example 2 Preparation of (R)-pipecolic acid from ethyl (RS)-pipecolate 40 ml of Medium 3 having a concentration of 1.5$ of ethyl (RS)-pipecolate were introduced into a 100 ml Erlenmeyer flask having baffles and treated with Pseudomonas putida DSM 9923. The batch was incubated at 30°C and 140 revolutions/min on a shaker. After 26 h, the cell-free supernatant was investigated for the content of (R) -pipecolic acid at an OD650 of 2 . 0 using HPLC. (R)-Pipecolic acid was obtained in a yield of >45$ based on the ethyl (RS)-pipecolate employed and with an ee value of >80$. After 44 h, enantiomerically pure (R)-pipecolic acid was obtained at an OD650 of 3.2.
Example 3 Preparation of (R)-pipecolamide from (RS)-pipecolamide in shaker culture 40 ml of Medium 3 having a concentration of 1.5$ of (RS)-pipecolamide were introduced into a 100 ml Erlenmeyer flask having baffles and treated with Pseudomonas putida DSM 9923. The batch was incubated at 30°C and 140 revolutions/min on a shaker. After 20 h, the cell-free supernatant was investigated for the content of (R)-pipecolamide at an OD650 of 3.0 using HPLC. (R) -Pipecolamide was obtained i.n a yield of >45$
based on the (RS)-pipecolamide employed and having an ee value of >99$.
Example 4 Preparation of (R)-pipecolamide and (R)-pipecolic acid from (RS)-pipecolamide in a fermenter Pseudomonas putida DSM 9923 was cultured in a fermenter overnight on Medium 3 with 20 g/1 of glutamate. (RS)-Pipecolamide was added at an OD650 of 14. After 2 h, according to GC analysis virtually enantiomerically pure (R)-pipecolamide was present. Pipecolic acid could not be detected.
A sample of this solution was incubated at 37°C and 140 revolutions/min. in a shaker with exclusion of air.
After 48 h, according to GC analysis enantiomerically pure (R)-pipecolic acid was present. Pipecolamide could no longer be detected.
Example 5 Preparation of (R)-pipecolic acid from (RS)-pipecolamide in shaker culture 40 ml of Medium 3 having a concentration of 1% of (RS)-pipecolamide were introduced into a 100 ml Erlenmeyer flask having baffles and treated with Pseudomonas putida DSM 9923. The batch was incubated on a shaker at 30°C and 140 revolutions/min. After 26 h, the cell-free supernatant was investigated by GC at an OD650 of 3.9.
(R) -Pipecolic acid with ee >98% was found in about 10%
yield.
Example 6 Preparation of propyl (R)-pipecolate from propyl (RS)-pipecolate in shaker culture 100 ml of minimum Medium 3 having a concentration of 4 g/1 of propyl (RS)-pipecolate were introduced into a 500 ml Erlenmeyer flask having baffles and treated with Pseudomonas putida DSM 9923. The batch was incubated on a shaker at 30°C and 140 rpm. After 4.5 h, the cell-free supernatant was investigated for the content and enantiomer excess of pipecolic acid and isopropyl pipecolate at an OD650 of 1.4 using GC. Isopropyl (R)-pipecolate was obtained enantiomerically pure and in a yield of 88$ based on isopropyl (R)-pipecolate employed. A few per cent of enantiomerically pure (R)-pipecolic acid were found as a by-product.
After separation of the biomass by centrifugation (20 min at 8000xg), the aqueous solution was evaporated to one third in vacuo, adjusted to pH 10 using 30$
strength sodium hydroxide solution and immediately extracted three times with 20 ml of diethyl ether each time. The combined organic phases were dried using sodium sulphate and concentrated. By introduction of hydrochloric acid, 0.195 g of isopropyl (R)-pipecolate were crystallized as the hydrochloride (80~ yield; ee >98~ acc. to GC).
Example 7 Preparation of (R)-pipecolic acid from isopropyl (RS)-pipecolate 100 ml of minimal Medium 3 having a concentration of 4 g/1 of isopropyl (RS)-pipecolate were introduced into a 500 ml Erlenmeyer flask having baffles and treated with Pseudomonas putida DSM 9923. The batch was incubated on a shaker at 30°C and 140 rpm. After 7.5 h, a sample was taken at an OD650 of 1.4 and incubated with exclusion of air. After 22 h, the cell-free supernatant was investigated for the content and enantiomeric excess of pipecolic acid and isopropyl pipecolate using GC. (R)-Pipecolic acid was obtained enantiomerically pure and in a yield of 80o based on isopropyl (R)-pipecolate employed. A few per cent of enantiomerically pure isopropyl (R)-pipecolate were found as a by-product.
Example 8 Reduction of (R)-pipecolamide to (R)-2-aminomethylpiperidine 11.8 g of lithium aluminium hydride were suspended in 500 ml of diethyl ether under a nitrogen atmosphere in a 2 1 flask. 20 g of (R)-pipecolamide (cf. Ex. 3) were slowly added at 5°C. The mixture was stirred at room temperature for 3 h and then under reflux for 5 h.
After cooling to room temperature, 12 ml of water, 12 ml of 15~ strength sodium hydroxide solution and 36 ml of water were slowly added dropwise in succession. The mixture was stirred at room temperature until it was snow-white. 120 g of sodium sulphate were added and the mixture was stirred overnight at room temperature. The solid was filtered off and washed four times with 250 ml of diethyl ether each time. The combined organic phases were evaporated in vacuo.
12.5 g of (R)-2-aminomethylpiperidine were obtained as a yellowish oil (content: 66% according to GC).
Distillation at 52-53°C/18 mbar yielded 3.6 g (200) of GC-pure (R)-2-aminomethylpiperidine as a colourless liquid.
Example 9 Reduction of isopropyl (R)-pipecolate to (R)-2-hydroxymethylpiperidine 0.2 g of lithium aluminium hydride were suspended in 20 ml of diethyl ether under a nitrogen atmosphere in a 250 ml flask. 90 mg of isopropyl (R)-pipecolate hydrochloride were slowly added at 5°C. The mixture was stirred at room temperature for 1 h and then under reflux for 4 h. After cooling to room temperature, 0.2 ml of water, 0.2 ml of 15% strength sodium hydroxide solution and 0.4 ml of water were slowly added dropwise in succession. The mixture was stirred at room temperature until it was snow-white. 2 g of sodium sulphate were added and the mixture was stirred at room temperature overnight. The solid was filtered off and washed four times with 20 ml of diethyl ether each time. The combined organic phases were evaporated in vacuo. 47 mg of (R)-2-hydroxymethylpiperidine were obtained as a slightly yellowish oil (content: 95%; ee >97% according to GC).
in which R2 is hydroxyl, amino or Cl-s-alkoxy, in the presence of trace elements and under aerobic conditions with ring-cleavage and, in the case of the microorganisms, can utilize it as the only carbon and energy source. After an adequate reaction period, customarily after 1 to 48 hours, only a few cyclic S-a-aminocarboxylic acids or other interfering open-chain by-products such as S-2-aminoadipic acid or none at all are then present in the reaction medium. The (2R)-piperidine derivatives accumulated in the reaction, of the general formula I
..,,. ~ f R' in which R1 is hydroxyl, amino or C1_6-alkoxy, can then be isolated without difficulties, in particular without complicated separation processes.
Here and in the following, C1_6-alkoxy has the meaning of a straight-chain or branched alkoxy group having from 1 to 6 C atoms. Methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy, pentaxy and its isomers and also hexoxy and its isomers may be mentioned by name.
The enzymes for the cell-free system can be obtained by technically customary breakdown of the microorganisms.
For this, for example, it is possible to use the ultrasonic, French press or lysozyme method. These cell-free enzymes can also be immobilized on a suitable carrier material.
Trace elements are to be understood, for example, as meaning zinc, manganese, cobalt, copper, nickel, molybedenum, calcium, magnesium, boron, iron or sulphur, the optimal composition of the trace elements being dependent on the microorganism used. Preferably, zinc, manganese, cobalt, copper, nickel, molybdenum, calcium, magnesium, boron, iron and sulphur are jointly employed.
Expediently, the reaction of (RS)-piperidine derivatives of the general formula II is carried out with growing microorganisms.
The most preferred substrates of the general formula II
are (RS)-pipecolic acid, ethyl (RS)-pipecolate, isopropyl (RS)-pipecolate and (RS)-pipecolamide.
Aerobic conditions are understood as meaning culturing conditions in which the microorganisms are supplied with oxygen. This can be carried out, for example, in shaker culture by means of atmospheric oxygen or in submerse culture by blowing in molecular oxygen or atmospheric oxygen.
The reaction can be carried out without customary culturing directly by addition of the microorganisms to (RS)-piperidine derivatives of the general formula II.
Alternatively, the reaction can be carried out after customary culturing of the microorganisms using a suitable carbon and energy source. The carbon and energy source employed here can be, for example, sugars, carboxylic acids, sugar alcohols or amino acids. Sugars which can be used are hexoses such as, for example, glucose or pentoses. Carboxylic acids which can be used are di- or tricarboxylic acids and their salts such as, for example, citric acid or succinate. A sugar alcohol which can be used is a trihydric alcohol, such as, for example, glycerol. An amino acid which can be employed is, for example, glutamate.
Preferably, the reaction is carried out without customary culturing directly by addition of the microorganisms to (RS)-piperidine derivatives of the general formula II.
In the reaction, the microorganisms can utilize the (S) isomer of the (RS)-piperidine derivative of the general formula II as the only carbon and energy source and as the only nitrogen source.
Media which can be used for the process according to the invention are the technically customary media such as mineral salt media, for example the mineral salt medium according to Kulla et al., (Arch. Microbiol.
135, 1-7, 1983) , the media described in Table 1 or low molar buffers, such as, for example, 10 to 100 mM
phosphate buffer, to which the necessary trace elements have been added. The process is preferably carried out in the media as in Table 1.
Expediently, the conditions are chosen such that the S
isomer of the (RS)-piperidine derivatives of the formula II is preferably utilized as a carbon and energy source by the microorganisms. Preferably, besides the compound II, the medium therefore does not contain any other compounds readily utilizable as a carbon and energy source.
Expediently, the reaction is carried out with single, repeated or continuous addition of (RS)-piperidine derivatives of the general formula II. The addition of (RS)-piperidine derivatives of the general formula II
is carried out in such a way that the concentration does not exceed 30% by weight, preferably 10% by weight, particularly preferably 5% by weight.
The pH of the medium is expediently in a range from 4 to 9, preferably from 6 to 8. Expediently, the reaction is carried out at a temperature from 15 to 80°C, preferably from 25 to 40°C.
The final products of the general formula I which is obtained in the reaction depends on the choice of the starting material and the reaction conditions, in particular the reaction time and the oxygen supply.
Starting from (RS)-pipecolic acid [(RS)-piperidine derivative II in which R2 is hydroxyl], only (2R)-pipecolic acid can be obtained. Starting from (RS)-pipecolamide [(RS)-piperidine derivative II in which RZ
is amino], both (2R)-pipecolamide and (2R)-pipecolic acid can be obtained. Starting from an (RS)-pipecolic acid ester [(RS)-piperidine derivative II in which Rz is C1_6-alkoxy] , both the (2R) -pipecolic acid ester and (2R)-pipecolic acid can be obtained.
Whether (2R)-pipecolamide or (2R)-pipecolic acid is obtained from (RS)-pipecolamide can be controlled, for example, by means of the reaction time. Thus in the reaction of (RS)-pipecolamide with the microorganisms according to the invention, (2R)-pipecolamide first accumulates, which can then optionally be isolated.
After a longer reaction time, the acid is then formed from the (2R)-pipecolamide. The reaction can be monitored analytically, for example chromatographically. The same applies to the reaction of (RS) -pipecolic acid esters in which first the (2R) -pipecolic acid ester and then the acid accumulates.
After a customary reaction time of preferably 1 to 48 hours, the (2R)-piperidine derivatives of the general formula I, preferably (R)-pipecolic acid, (R)-pipecolamide and ethyl and isopropyl (R)-pipecolates are preferably obtained in the medium in very high to quantitative yield.
The (2R)-piperidine derivatives of the general formula I obtained in this manner can be isolated by customary working-up methods such as, for example, by removal of the biomass, acidification, chromatography, electrodialysis or crystallization.
Alternatively, the course of the conversion reaction can be regulated by means of the oxygen supply. If, for example, (R)-pipecolic acid is to be prepared from (RS)-pipecolamide or (RS)-pipecolic acid ester, the reaction is expediently carried out under aerobic conditions until the accumulation of the (R)-pipecolamide or of the (R)-pipecolic acid ester, respectively, as described above and the oxygen supply is then stopped, the acid being formed thereafter.
The reaction of the (RS)-pipecolic: acid derivative according to formula V
V
~3 in which R3 is amino or C1_6-alkoxy, C1_6-alkoxy having the abovementioned meaning, to give (R)-pipecolic acid according to formula IV
H
_ g _ thus takes place via the (R)-pipecolic acid derivative according to formula VI
,l.,,, p VI
in which R3 has the abovementioned meaning, which temporarily accumulates in the medium with increasing enantiomeric excess and can optionally be isolated. If the (R)-pipecolic acid derivative according to formula VI is to be prepared from the (RS)-pipecolic acid derivative according to formula V, it is isolated after accumulation. If (R)-pipecolic acid according to formula IV is to be prepared from the (RS)-pipecolic acid derivative according to formula V, the reaction is expediently carried out under aerobic conditions until the accumulation of the (R)-pipecolic acid derivative according to formula VI and the oxygen supply is then stopped.
In order to determine whether the (R)-pipecolic acid derivative according to formula VI has accumulated in the medium, the reaction can be monitored analytically, for example chromatographically. The reaction should be terminated as soon as the maximum accumulation of the (R)-pipecolic acid derivative according to formula VI
is achieved.
A further component of the invention is the further reaction, the reduction, of the (2R)-pipecolir_ acid derivatives of the general formula VI to give the (2R)-piperidine derivatives of the general formula VII
VII
,,, .~R.
in which R' is hydroxyl or amino.
Customarily, the reducing agent used is an alkali metal hydride or an alkaline earth metal hydride such as, for example, lithium aluminium hydride, sodium borohydride, potassium or sodium aluminium hydride, or magnesium or calcium borohydride. Lithium aluminium hydride is used in particular.
The reduction is expediently carried out at a temperature from 0 to 100°C, preferably at the reflux temperature of the corresponding solvent.
As known technically, the reduction can be carried out in a polar organic solvent such as, for example, in an ether. Suitable ethers are, for example, diethyl ether, dipropyl ether, tetrahydrofuran or 1,4-dioxane.
After a customary reaction time of 1 to 16 h, the desired products of the general formula VII, such as (R)-2-aminomethylpiperidine or (R)-2-hydroxymethyl-piperidine, can be isolated by known working-up methods.
Table 1 Medium 1 (NH4) 2504 2. 0 g/1 Na2HP09 2.0 g/1 KH2P09 1.0 g/1 NaCl 2.0 g/1 MgCl2 6H20 0. 4 g/1 CaCl2 2H20 14 . 5 mg/1 FeCl3 6H20 0 . 8 mg/1 ZnS04 7H20 0. 1 mg/1 MnCl24H20 0.09 mg/1 H3BO3 0.3 mg/1 CoCl26H20 0.2 mg/1 CuCl22H20 0.01 mg/1 NiCl26Hz0 0.02 mg/1 Na2Mo09 2H20 0. 03 mg/1 FeS04 7H20 2 . 0 mg/1 EDTA-Na22H20 5.0 mg/1 Medium 2 KH2P09 1.3 g/1 NH4C1 5 g/1 (NH4 ) 2SOn 2 g/1 Na2S04 0.25 g/1 MgCl26H20 0.8 g/1 CaCl22H20 0.16 g/1 ZnS04 7Hz0 9 mg/1 MnCl2 4H20 4 mg/1 H3B03 2.7 mg/1 CoCl26H20 1.8 mg/1 CuCl22H20 1.5 mg/1 NiCl26H20 0.18 mg/1 Na2Mo0~ 2H20 0 . 2 mg/1 FeS09 7H20 30 mg/1 EDTA-Na22H20 175 mg/1 Medium 3 Na2S04 0.1 g/1 Na2HP04 2.0 g/1 KH2P09 1.0 g/1 NaCl 2.0 g/1 MgCl2 6H20 0 . 4 g/1 CaCl22H20 14.5 mg/1 FeCl3 6H20 0. 8 mg/1 ZnS04 7H20 0 . 1 mg/1 MnCl24H20 0.09 mg/1 H3BO3 0.3 mg/1 CoCl26H20 0.2 mg/1 CuClz2H20 0.01 mg/1 NiCl26H20 0.02 mg/1 Na2Mo09 2H20 0. 03 mg/1 FeS04 7H20 2 . 0 mg/1 EDTA-Na22H20 5.0 mg/1 Examples Example 1 Preparation of (R)-pipecolic acid from (RS)-pipecolic acid 1.1 in shaker culture 400 ml of Medium 1 having a concentration of 1.5% of (RS)-pipecolic acid were introduced into a 1 1 Erlenmeyer flask and treated with Pseudomonas putida DSM 9923. The batch was incubated at 30°C and 140 revolutions/min on a shaker. After 24 h, the cell-free supernatant was investigated for the content of (R)-pipecolic acid at an OD650 of 3.7 using HPLC. (R)-Pipecolic acid was obtained in a yield of >45% based on the (RS)-pipecolic acid employed and with an ee value of >99$ .
1.2 in a fermenter 100 ml of Medium 3 having a concentration of 1% of (RS)-pipecolic acid were introduced into a 500 ml Erlenmeyer flask with baffles and treated with Pseudomonas putida DSM 9923. The batch was incubated for 16 h at 30°C and 140 revolutions/min on a shaker.
3 1 of Medium 2 having a concentration of 1.5% of (RS)-pipecolic acid (45 g) were introduced into a 5 1 fermenter and treated with the preculture of Pseudomonas putida DSM 9923. After 7 h, a further 45 g of (RS)-pipecolic acid were continuously added as a 10%
solution in the caurse of 5 h. After a further 4 h, the biotransformation was stopped and the cell-free supernatant was investigated for the content of (R)-pipecolic acid using HPLC. (R)-pipecolic acid was obtained in quantitative yield and with an ee value of >97$.
Example 2 Preparation of (R)-pipecolic acid from ethyl (RS)-pipecolate 40 ml of Medium 3 having a concentration of 1.5$ of ethyl (RS)-pipecolate were introduced into a 100 ml Erlenmeyer flask having baffles and treated with Pseudomonas putida DSM 9923. The batch was incubated at 30°C and 140 revolutions/min on a shaker. After 26 h, the cell-free supernatant was investigated for the content of (R) -pipecolic acid at an OD650 of 2 . 0 using HPLC. (R)-Pipecolic acid was obtained in a yield of >45$ based on the ethyl (RS)-pipecolate employed and with an ee value of >80$. After 44 h, enantiomerically pure (R)-pipecolic acid was obtained at an OD650 of 3.2.
Example 3 Preparation of (R)-pipecolamide from (RS)-pipecolamide in shaker culture 40 ml of Medium 3 having a concentration of 1.5$ of (RS)-pipecolamide were introduced into a 100 ml Erlenmeyer flask having baffles and treated with Pseudomonas putida DSM 9923. The batch was incubated at 30°C and 140 revolutions/min on a shaker. After 20 h, the cell-free supernatant was investigated for the content of (R)-pipecolamide at an OD650 of 3.0 using HPLC. (R) -Pipecolamide was obtained i.n a yield of >45$
based on the (RS)-pipecolamide employed and having an ee value of >99$.
Example 4 Preparation of (R)-pipecolamide and (R)-pipecolic acid from (RS)-pipecolamide in a fermenter Pseudomonas putida DSM 9923 was cultured in a fermenter overnight on Medium 3 with 20 g/1 of glutamate. (RS)-Pipecolamide was added at an OD650 of 14. After 2 h, according to GC analysis virtually enantiomerically pure (R)-pipecolamide was present. Pipecolic acid could not be detected.
A sample of this solution was incubated at 37°C and 140 revolutions/min. in a shaker with exclusion of air.
After 48 h, according to GC analysis enantiomerically pure (R)-pipecolic acid was present. Pipecolamide could no longer be detected.
Example 5 Preparation of (R)-pipecolic acid from (RS)-pipecolamide in shaker culture 40 ml of Medium 3 having a concentration of 1% of (RS)-pipecolamide were introduced into a 100 ml Erlenmeyer flask having baffles and treated with Pseudomonas putida DSM 9923. The batch was incubated on a shaker at 30°C and 140 revolutions/min. After 26 h, the cell-free supernatant was investigated by GC at an OD650 of 3.9.
(R) -Pipecolic acid with ee >98% was found in about 10%
yield.
Example 6 Preparation of propyl (R)-pipecolate from propyl (RS)-pipecolate in shaker culture 100 ml of minimum Medium 3 having a concentration of 4 g/1 of propyl (RS)-pipecolate were introduced into a 500 ml Erlenmeyer flask having baffles and treated with Pseudomonas putida DSM 9923. The batch was incubated on a shaker at 30°C and 140 rpm. After 4.5 h, the cell-free supernatant was investigated for the content and enantiomer excess of pipecolic acid and isopropyl pipecolate at an OD650 of 1.4 using GC. Isopropyl (R)-pipecolate was obtained enantiomerically pure and in a yield of 88$ based on isopropyl (R)-pipecolate employed. A few per cent of enantiomerically pure (R)-pipecolic acid were found as a by-product.
After separation of the biomass by centrifugation (20 min at 8000xg), the aqueous solution was evaporated to one third in vacuo, adjusted to pH 10 using 30$
strength sodium hydroxide solution and immediately extracted three times with 20 ml of diethyl ether each time. The combined organic phases were dried using sodium sulphate and concentrated. By introduction of hydrochloric acid, 0.195 g of isopropyl (R)-pipecolate were crystallized as the hydrochloride (80~ yield; ee >98~ acc. to GC).
Example 7 Preparation of (R)-pipecolic acid from isopropyl (RS)-pipecolate 100 ml of minimal Medium 3 having a concentration of 4 g/1 of isopropyl (RS)-pipecolate were introduced into a 500 ml Erlenmeyer flask having baffles and treated with Pseudomonas putida DSM 9923. The batch was incubated on a shaker at 30°C and 140 rpm. After 7.5 h, a sample was taken at an OD650 of 1.4 and incubated with exclusion of air. After 22 h, the cell-free supernatant was investigated for the content and enantiomeric excess of pipecolic acid and isopropyl pipecolate using GC. (R)-Pipecolic acid was obtained enantiomerically pure and in a yield of 80o based on isopropyl (R)-pipecolate employed. A few per cent of enantiomerically pure isopropyl (R)-pipecolate were found as a by-product.
Example 8 Reduction of (R)-pipecolamide to (R)-2-aminomethylpiperidine 11.8 g of lithium aluminium hydride were suspended in 500 ml of diethyl ether under a nitrogen atmosphere in a 2 1 flask. 20 g of (R)-pipecolamide (cf. Ex. 3) were slowly added at 5°C. The mixture was stirred at room temperature for 3 h and then under reflux for 5 h.
After cooling to room temperature, 12 ml of water, 12 ml of 15~ strength sodium hydroxide solution and 36 ml of water were slowly added dropwise in succession. The mixture was stirred at room temperature until it was snow-white. 120 g of sodium sulphate were added and the mixture was stirred overnight at room temperature. The solid was filtered off and washed four times with 250 ml of diethyl ether each time. The combined organic phases were evaporated in vacuo.
12.5 g of (R)-2-aminomethylpiperidine were obtained as a yellowish oil (content: 66% according to GC).
Distillation at 52-53°C/18 mbar yielded 3.6 g (200) of GC-pure (R)-2-aminomethylpiperidine as a colourless liquid.
Example 9 Reduction of isopropyl (R)-pipecolate to (R)-2-hydroxymethylpiperidine 0.2 g of lithium aluminium hydride were suspended in 20 ml of diethyl ether under a nitrogen atmosphere in a 250 ml flask. 90 mg of isopropyl (R)-pipecolate hydrochloride were slowly added at 5°C. The mixture was stirred at room temperature for 1 h and then under reflux for 4 h. After cooling to room temperature, 0.2 ml of water, 0.2 ml of 15% strength sodium hydroxide solution and 0.4 ml of water were slowly added dropwise in succession. The mixture was stirred at room temperature until it was snow-white. 2 g of sodium sulphate were added and the mixture was stirred at room temperature overnight. The solid was filtered off and washed four times with 20 ml of diethyl ether each time. The combined organic phases were evaporated in vacuo. 47 mg of (R)-2-hydroxymethylpiperidine were obtained as a slightly yellowish oil (content: 95%; ee >97% according to GC).
Claims (8)
1. Process for the preparation of (2R)-piperidine derivatives of the general formula in which R1 is hydroxyl, amino or C1-6-alkoxy, characterized in that an (RS)-piperidine derivative of the general formula in which R2 is hydroxyl, amino or C1-6-alkoxy and R1 and R2 can be the same or different, with the proviso that if R1 is amino, R2 is not hydroxyl or C1-6-alkoxy and if R1 is C1-6-alkoxy, R2 is not hydroxyl or amino, is reacted with growing microorganisms which are capable of utilizing .alpha.-aminocarboxamides, in the form of the racemate or of one of its optically active isomers, of the general formula in which A together with -NH and -CH is an optionally substituted 5- or 6-membered saturated heterocyclic ring, as the only nitrogen source, in the presence of trace elements and under aerobic conditions, the S isomer of the (RS)-piperidine derivative II being utilized or broken down with ring cleavage as a carbon and energy source.
2. Process according to Claim 1, characterized in that, as a substrate of the formula II, (RS)-pipecolic acid, ethyl (RS)-pipecolate, isopropyl (RS)-pipecolate or (RS)-pipecolamide is used.
3. Process according to any one of Claims 1 and 2, characterized in that the reaction is carried out using microorganisms of the genus Pseudomonas, in particular of the species Pseudomonas putida.
4. Process according to any one of Claims 1 to 3, characterized in that the reaction is carried out using microorganisms of the species Pseudomonas putida (DSM 9923) or its functionally equivalent variants and mutants.
5. Process according to any one of Claims 1 to 4, characterized in that the addition of (RS)-pipecolic acid derivatives of the general formula II is carried out in such a way that the concentration does not exceed 30% by weight.
6. Process according to any one of Claims 1 to 5, characterized in that the reaction is carried out at a pH of 4 to 9 and a temperature of 15 to 80°C.
7. Process for the preparation of (R) -pipecolic acid of the formula characterized in that (RS)-pipecolic acid derivatives of the general formula in which R3 is amino or C1-6-alkoxy, are reacted as substrate with growing microorganisms which are capable of utilizing .alpha.-aminocarboxamides, in the form of the racemate or one of its optically active isomers, of the general formula in which A together with -NH and -CH is an optionally substituted 5- or 6-membered saturated heterocyclic ring, as the only nitrogen source, in the presence of trace elements and under aerobic conditions, the reaction being carried out until the accumulation of the corresponding (R)-pipecolic acid derivative of the formula in which R3 has the meaning mentioned, which can optionally be isolated, under aerobic conditions, the S isomer of the (RS)-piperidine derivative V
being utilized or broken down with ring cleavage as a carbon and energy source, and the supply of oxygen is then stopped.
being utilized or broken down with ring cleavage as a carbon and energy source, and the supply of oxygen is then stopped.
8. Process for the preparation of (2R)-piperidine derivatives of the general formula in which R4 is hydroxyl or amino, characterized in that in the first stage (RS)-pipecolic acid derivatives of the general formula in which R3 is amino or C1-6-alkoxy, are converted as substrates using growing microorganisms which are capable of utilizing .alpha.-aminocarboxamides, in the form of the racemate or one of its optically active isomers, of the general formula in which A together with -NH and -CH is an optionally substituted 5- or 6-membered saturated heterocyclic ring, as the only nitrogen source, in the presence of trace elements and under aerobic conditions, under which the S isomer of the (RS)-piperidine derivative V is utilized or broken down with ring cleavage as a carbon and energy source, into an (R)-pipecolic acid derivative of the general formula in which R3 is amino or C1-6-alkoxy, which is reduced in the second stage to the desired compound of the general formula VII.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP98116061.7 | 1998-08-26 | ||
| EP98116061 | 1998-08-26 | ||
| PCT/EP1999/006277 WO2000012745A1 (en) | 1998-08-26 | 1999-08-26 | Method for preparing (2r)-piperidine derivatives |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2341554A1 true CA2341554A1 (en) | 2000-03-09 |
Family
ID=8232514
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002341554A Abandoned CA2341554A1 (en) | 1998-08-26 | 1999-08-26 | Process for the preparation of (2r)-piperidine derivatives |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP1108053A1 (en) |
| JP (1) | JP2002523107A (en) |
| AU (1) | AU5741599A (en) |
| CA (1) | CA2341554A1 (en) |
| WO (1) | WO2000012745A1 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| MXPA05008857A (en) | 2003-02-18 | 2006-03-09 | Metabolic Explorer Sa | Method for the production of evolved microorganisms which permit the generation or modification of metabolic pathways. |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS63248393A (en) * | 1987-04-02 | 1988-10-14 | Agency Of Ind Science & Technol | Production of d-pipecolic acid by microorganism |
| CA2150526C (en) * | 1994-06-09 | 2005-11-15 | Andreas Kiener | Biotechnological process for the preparation of cyclic s-.alpha.-amino carboxylic acids and r-.alpha.-amino carboxamides |
-
1999
- 1999-08-26 CA CA002341554A patent/CA2341554A1/en not_active Abandoned
- 1999-08-26 JP JP2000567728A patent/JP2002523107A/en active Pending
- 1999-08-26 AU AU57415/99A patent/AU5741599A/en not_active Abandoned
- 1999-08-26 WO PCT/EP1999/006277 patent/WO2000012745A1/en not_active Application Discontinuation
- 1999-08-26 EP EP99944524A patent/EP1108053A1/en not_active Ceased
Also Published As
| Publication number | Publication date |
|---|---|
| JP2002523107A (en) | 2002-07-30 |
| WO2000012745A1 (en) | 2000-03-09 |
| EP1108053A1 (en) | 2001-06-20 |
| AU5741599A (en) | 2000-03-21 |
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| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Discontinued |