WO2006117057A1 - Process for preparing enantiomerically enriched e-(2s)- and (2r)-alkyl-5-halopent-4-enecarboxylic acids or their esters - Google Patents

Process for preparing enantiomerically enriched e-(2s)- and (2r)-alkyl-5-halopent-4-enecarboxylic acids or their esters Download PDF

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WO2006117057A1
WO2006117057A1 PCT/EP2006/003267 EP2006003267W WO2006117057A1 WO 2006117057 A1 WO2006117057 A1 WO 2006117057A1 EP 2006003267 W EP2006003267 W EP 2006003267W WO 2006117057 A1 WO2006117057 A1 WO 2006117057A1
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alkyl
halopent
formula
enecarboxylic
enantiomerically enriched
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PCT/EP2006/003267
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French (fr)
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Gerhard Steinbauer
Wolfgang Skranc
Peter Pojarliev
Michael Stanek
Marcel Gerhardus Wubbolts
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Dsm Fine Chemicals Austria Nfg Gmbh & Co Kg
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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
    • C12P41/003—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions
    • C12P41/005—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions by esterification of carboxylic acid groups in the enantiomers or the inverse reaction
    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters

Definitions

  • the present invention relates to a process for preparing enantiomerically enriched E- (2S) - and
  • (2R) -alkyl-5-halopent-4-enecarboxylic acids or their esters at an optical purity of up to e . e . > 99% and in a yield of up to 98% of theory.
  • E- (2S) - and (2R) -alkyl-5-halopent-4-enecarboxylic acids and their esters constitute valuable intermediates for preparing pharmaceuticals as, for example, delta-amino- gamma-hydroxy-omega-arylalkanecarboxamides, which exhibit renin-inhibiting properties and can be used as antihypertensives in pharmaceutical preparations.
  • WO 01/09079 describes a variant method for preparing alkyl-5-halopent-4-enecarboxylic esters, according to which the desired esters are obtained as racemates in a yield of 84% by reacting isovaleric esters with 1, 3-dihalo-l-propene in the presence of a strong base, such as alkali metal amides (LDA) for example.
  • LDA alkali metal amides
  • the desired enantiomer is obtained from the racemate in yields of from about 32 to 46% by treating with esterases, for example with pig liver esterase (PLE) .
  • J " . Agric. Food Chem. , 32 (1), pp. 85 - 92 describes, for example, the preparation of different haloalkenecarboxylic acids, such as racemic 2-isopropyl-5-chloropent-4-enecarboxylic acid, from the corresponding isopropyldialkyl malonate.
  • the malonate is first of all alkylated with 1, 3-dichloro-l-propene, after which a decarboxylation takes place, with the ester being converted into racemic 2-isopropyl-5-chloropent-4-enecarboxylic acid. No racemate resolution is described.
  • WO 2004/052828 slightly modifies the method described in J. Agric. Food Chem. , 32 (1), 1, pp. 85 - 92 with regard to some process parameters.
  • a disadvantage of this method is, once again, the racemate resolution, as described in the WO specification, by using the enzyme pig liver esterase (PLE) .
  • the object of the present invention was to find a process for preparing enantiomerically enriched E- (2S) - and (2R) -alkyl-5-halopent-4-enecarboxylic acids or their esters which enables the desired compounds to be prepared, in a simple manner and while avoiding the esterase of animal origin, at higher optical purities, of up to e.e. > 99%, and in higher yields, of up to 98% of theory, as compared with the prior art .
  • the present invention accordingly relates to a process for preparing enantiomerically enriched E- (2S) - and (2R) -alkyl-5-halopent-4-enecarboxylic acids or their esters of the formula (Ia) or (Ib)
  • R is a d-C 6 -alkyl radical
  • A is H
  • R 1 where R 1 can be C 1 -C 4 -alkyl, or R 2 , where R 2 is an alkyl group but is not identical to R 1
  • X is chlorine, bromine or iodine, wherein an enantiomeric mixture of a 2-alkyl-5-halopent- 4-enecarboxylic ester of the formula (II)
  • R, R x and X are defined as above, is reacted with a stereoselective hydrolase, which is obtained from microorganisms or plants, in the presence of water or an alcohol of the formula R 2 OH, where R 2 is an alkyl group which is not identical to R 1 , as nucleophile, and a) either the remaining enantiomerically enriched E- (2S) - or (2R) -alkyl-5-halopent-4-enecarboxylic ester of the formula (Ia) or (Ib) , where A is Ri, is isolated, or b) if an alcohol is used as nucleophile, the resulting enantiomerically enriched E- (2S) - or
  • the process according to the invention is used to prepare enantiomerically enriched E- (2S) - or (2R) -alkyl-5-halopent-4-enecarboxylic acids or their esters of the formula (Ia) or (Ib) .
  • R is a Ci-C 6 -alkyl radical such as methyl, ethyl, n- and i-propyl, n- , i- and t-butyl, pentyl and hexyl .
  • Preference is given to C 1 -C 4 -alkyl radicals, with particular preference being given to the i -propyl radical .
  • A is H, Ri, where R x is a Ci-C 4 -alkyl radical, preferably a Ci-C 2 -alkyl radical and particularly preferably a methyl radical, or R 2 , where R 2 is an alkyl group which is not identical to Ri.
  • R 2 is preferably a
  • Ci-Cg-alkyl radical Ci-Cg-alkyl radical.
  • X is chlorine, bromine or iodine, preferably chlorine.
  • the enantiomerically enriched (S) - or (R) -carboxylic acids or their esters of the formula (Ia) or (Ib) are prepared, in accordance with the invention, by reacting an enantiomeric mixture of a 2-alkyl-5-halopent-4-enecarboxylic ester of the formula (II) with a stereoselective hydrolase which is obtained from microorganisms or plants.
  • enantiomerically enriched compounds are to be understood as compounds which exhibit a (S) - or (R) -enantiomeric excess (ee) of > 80%, preferably of 90% and particularly preferably of > 97%.
  • One enantiomer is preferably hydrolyzed, depending on the choice of the enzyme possessing a specific stereoselectivity.
  • the stereoselectivity of the enzyme can be described by the E ratio, as known, for example, from Chen et al . , J. Am. Chem. Soc . (1982), 104, 7294 - 7299. Preference is given, according to the invention, to using hydrolases having an E ratio of > 5, preferably > 10, particularly preferably > 50 and in particular preferably > 100.
  • the hydrolases which are used are of nonanimal origin.
  • stereoselective hydrolases examples include esterases, lipases, proteases, peptidases or acylases, etc . These enzymes are obtained from microorganisms
  • bacteria bacteria or fungi
  • suitable sources are wheat germs, molds, such as Absidia; Aspergillus; Fusarium; Gibberella; Mucor; Neurospora; Trichoderma; Rhizopus; Rhizomucor, for example Rhizomucor miehei; Thermomyces, for example Thermomyces lanuginosus; bacteria, such as Achromobacter; Alcaligenes; Bacillus, for example Bacillus licheniformis; Brevibacterium; Corynebacterium; Providencia; Pseudomonas, for example Pseudomonas fluoresceins, Pseudomonas cepase or Pseudomonas alcaligenes; Serratia, Rhodococcus, or yeasts, such as Candida, for example Candida rugose or Candida antarctica; and yeasts of the Actinomycete genus Nocardia.
  • enzymes which are classified as carboxylic ester hydrolases (EC 3.1.1) or as peptidases, for example EC 3.4.1., EC 3.4.11, EC 3.4.21, preferably EC 3.4.21.62, EC 3.4.22 or EC 3.4.23.
  • Enzymes which are suitable for being used in accordance with the invention can, for example, be obtained commercially.
  • Examples of commercially obtainable enzymes are : enzymes obtainable from Fluka: Candida cylindracea lipase, Pseudomonas fluorescens lipase, Aspergillus oryzae lipase, Rhizopus niveus lipase, Rhizomucor miehei lipase, Candida antarctica lipase, Mucor javanicus lipase, Rhizopus arrhizus lipase, Penicillium rogueforti lipase, Candida lipolytica lipase, Pseudomonas sp.
  • the enzymes can be used in any form, for example as a dispersion, as a solution, immobilized, as a crude enzyme, as a commercially obtainable enzyme, as an enzyme which has been further purified from a commercially obtainable preparation, as an enzyme which has been obtained from its source using a combination of known purification methods, as whole cells (where appropriate immobilized and/or permeabilized) which possess the requisite enzymic activity (naturally or by means of genetic modifications) , or in a lysate of such cells. It is furthermore also possible to use mutants of the naturally occurring (wild-type) enzymes which possess the corresponding activity.
  • Mutants of wild-type enzymes can be obtained, for example, by altering the DNA which encodes the wild-type enzyme, or by means of known mutagenesis techniques (random mutagenesis, site- directed mutagenesis, direct evolution, gene shuffling, etc.) such that the DNA encodes an enzyme which at least differs from the wild-type enzyme in one amino acid, and subsequently expressing the modified DNA in a suitable host cell.
  • Such mutants may exhibit improved properties with regard to (stereo) selectivity and/or activity and/or stability and/or resistance to solvents and/or resistance to pH and/or resistance to temperature.
  • enzymes which exhibit an amino acid sequence as described, for example, in WO 2005/032496, WO 2004/035729, WO 02/057411 or US 5,942,430.
  • the enzymes from WO 2005/032496, WO 2004/035729, WO 02/057411 and US 5,942,430 are hereby included by reference .
  • An enzyme which is suitable for the reaction according to the invention can be selected, for example, by screening several enzymes. Performing screenings is prior art. Usually, the conditions under which the substrate (racemic ester of the formula (II) ) and the enzyme are brought together for the purpose of selecting a suitable enzyme are chosen such that a good compromise is found between the stability of the enzyme, of the substrate and of the end product, on the one hand, and the reaction rate (which normally increases with increasing temperature) , on the other hand. Screenings can be carried out on any scale. For practical reasons, preference is given to using a reaction volume of between 0.15 ml and 10 ml if a large number of enzymes are being screened.
  • the ratio of enzyme to substrate is usually not critical for the screening and can be chosen to be between 1/20 and 2/1.
  • the quantity of substrate employed is likewise not critical and can, for example, be between 5 mM and 1.5 M.
  • the pH is not critical, either, and can, for example, be adjusted to a value of between 4 and 11 and can be kept constant by using an aqueous buffer solution.
  • the temperature during screening is once again not critical and can, for example, be between 10 and 60 0 C.
  • reaction conditions for the process according to the invention depends on the choice of the enzyme. Normally, the temperature of the reaction according to the invention is between 0 and 9O 0 C, preferably between 10 and 60 0 C.
  • the pH of the reaction solution is between 4 and 11, preferably between 6 and 9.
  • the choice of the solvent depends on the nucleophile which is used.
  • solvents which can then be used are water, a mixture of water and a solvent which is miscible with water, for example an alcohol, such as methanol, ethanol, isopropanol, tert-butanol, etc., dioxane, tetrahydrofuran, acetone or diemthyl sulfoxide, or a two-phase system composed of water and a solvent which is not miscible with water, for example an aromatic compound, such as toluene, xylene, etc., an alkane, such as hexane, heptane, cyclohexane, etc., or an ether, such as diisopropyl ether, methyl tert-butyl ether, etc.
  • an alcohol such as methanol, ethanol, isopropanol, tert-butanol, etc.
  • dioxane tetrahydrofuran
  • the solvent which is then preferably used is the alcohol R 2 OH, where R 2 is an alkyl group which is not, however, identical to R 1 .
  • R 2 is an alkyl group which is not, however, identical to R 1 .
  • an organic solvent such as tetrahydrofuran, heptane, toluene, hexane, CH 3 CN, methyl tert-butyl ether, etc.
  • This end product can either the remaining enantiomerically enriched E- (2S) - or (2R) -alkyl- 5-halopent-4-enecarboxylic ester of the formula (Ia) or (Ib) , where A is R 1 , or, if water is the nucleophile, the resulting enantiomerically enriched E- (2S) - or (2R) -alkyl-5-halopent-4-enecarboxylic acid of the formula (Ia) or (Ib) , where A is H, or if the alcohol is the nucleophile, the enantiomerically enriched E- (2S) - or (2R) -alkyl-5-halopent-4-enecarboxylic ester of the formula (Ia) or (Ib) where A is R 2 .
  • the end product can be isolated, for example, using customary methods such as extraction, crystallization, column chromatography, distillation, etc.
  • Example 1 Determining the activity and enantioselectivity of 3 hydrolytic enzymes:
  • reaction mixtures were diluted by adding in each case 750 ⁇ l of acetonitrile containing 50 mM phosphoric acid.
  • the resulting mixtures were centrifuged at 2500 rpm for 5 minutes and analyzed by reversed phase LC on a Varian Inertsil ODS-3 column (50 x 4.6 mm I. D. , 3 ⁇ m) .
  • the compounds were eluted using a gradient of 50 mM phosphoric acid in water (pH 2.3) and acetonitrile (1.0 ml/min, at 4O 0 C) .
  • the initial conditions for the gradients were 60/40% v/v buffer/acetonitrile at 0 minutes, with the percentage of acetonitrile increasing to 50% in 5 minutes and being kept at 50% for a further 2 minutes.
  • the injection volume was 5 ⁇ l.
  • Detection was effected using a spectrophotometer at 220 nm UV. The retention times were 3.2 min and 7.6 min, respectively, for 5-chloro-2-isopropylpent-4-enecarboxylic acid and methyl 5-chloro-2-isopropylpent-4-enecarboxylate .
  • the three enzymes were used for determining, by HPLC analysis, the preferred enantioselectivity of the enzyme and the enantiomeric excess of the 5-chloro- 2-isopropylpent-4-enecarboxylic acid which was formed.
  • the enantiomeric excess was determined on an EKA.
  • Chemicals Kromasil KRlOO-5CHI -TBB 250 mm x 4.6 mm I. D., 5 ⁇ m
  • 98/2/0.1% v/v n-heptane/allyl alcohol/trifluoroacetic acid as the eluent (0.8 ml/min at room temperature) .
  • the injection volume was 5 ⁇ l and detection was performed using a spectrophotometer at 210 nm UV.
  • Samples of the aqueous enzyme reaction were freeze- dried and then first of all dissolved in 100 ⁇ l of allyl alcohol and after that admixed with 500 ⁇ l of eluent for the analysis.
  • HPLC data were used to calculate the intrinsic enantioselectivity of the enzymes, expressed as an E ratio.

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Abstract

A process for preparing enantiomerically enriched E-(2S) - and (2R)-alkyl-5-halopent-4-enecarboxylic acids or their esters of the formula (Ia) or (Ib) in which R is a C1-C6-alkyl radical, A is H, R1, where R1 can be C1-C4-alkyl, or R2, where R2 is an alkyl group but is not identical to R1, and X is chlorine, bromine or iodine, in which an enantiomeric mixture of a 2-alkyl-5-halopent-4-enecarboxylic ester of the formula (II) in which R, R1 and X are defined as above, is reacted with a stereoselective hydrolase, which is obtained from microorganisms or plants, in the presence of water or an alcohol of the formula R2OH, where R2 is an alkyl group which is not identical to R1, as nucleophile.

Description

Process for preparing enantiomerically enriched E- (2S) - and (2R) -alkyl-5-halopent-4-enecarboxylic acids or their esters
The present invention relates to a process for preparing enantiomerically enriched E- (2S) - and
(2R) -alkyl-5-halopent-4-enecarboxylic acids or their esters at an optical purity of up to e . e . > 99% and in a yield of up to 98% of theory. E- (2S) - and (2R) -alkyl-5-halopent-4-enecarboxylic acids and their esters constitute valuable intermediates for preparing pharmaceuticals as, for example, delta-amino- gamma-hydroxy-omega-arylalkanecarboxamides, which exhibit renin-inhibiting properties and can be used as antihypertensives in pharmaceutical preparations.
WO 01/09079, for example, describes a variant method for preparing alkyl-5-halopent-4-enecarboxylic esters, according to which the desired esters are obtained as racemates in a yield of 84% by reacting isovaleric esters with 1, 3-dihalo-l-propene in the presence of a strong base, such as alkali metal amides (LDA) for example. The desired enantiomer is obtained from the racemate in yields of from about 32 to 46% by treating with esterases, for example with pig liver esterase (PLE) .
An important disadvantage of this method is the use of the enzyme pig liver esterase (PLE) , which is of animal origin.
J". Agric. Food Chem. , 32 (1), pp. 85 - 92 describes, for example, the preparation of different haloalkenecarboxylic acids, such as racemic 2-isopropyl-5-chloropent-4-enecarboxylic acid, from the corresponding isopropyldialkyl malonate. In this connection, the malonate is first of all alkylated with 1, 3-dichloro-l-propene, after which a decarboxylation takes place, with the ester being converted into racemic 2-isopropyl-5-chloropent-4-enecarboxylic acid. No racemate resolution is described.
WO 2004/052828 slightly modifies the method described in J. Agric. Food Chem. , 32 (1), 1, pp. 85 - 92 with regard to some process parameters. A disadvantage of this method is, once again, the racemate resolution, as described in the WO specification, by using the enzyme pig liver esterase (PLE) .
The object of the present invention was to find a process for preparing enantiomerically enriched E- (2S) - and (2R) -alkyl-5-halopent-4-enecarboxylic acids or their esters which enables the desired compounds to be prepared, in a simple manner and while avoiding the esterase of animal origin, at higher optical purities, of up to e.e. > 99%, and in higher yields, of up to 98% of theory, as compared with the prior art .
The present invention accordingly relates to a process for preparing enantiomerically enriched E- (2S) - and (2R) -alkyl-5-halopent-4-enecarboxylic acids or their esters of the formula (Ia) or (Ib)
Figure imgf000003_0001
in which R is a d-C6-alkyl radical, A is H, R1, where R1 can be C1-C4-alkyl, or R2, where R2 is an alkyl group but is not identical to R1, and X is chlorine, bromine or iodine, wherein an enantiomeric mixture of a 2-alkyl-5-halopent- 4-enecarboxylic ester of the formula (II)
Figure imgf000003_0002
in which R, Rx and X are defined as above, is reacted with a stereoselective hydrolase, which is obtained from microorganisms or plants, in the presence of water or an alcohol of the formula R2OH, where R2 is an alkyl group which is not identical to R1, as nucleophile, and a) either the remaining enantiomerically enriched E- (2S) - or (2R) -alkyl-5-halopent-4-enecarboxylic ester of the formula (Ia) or (Ib) , where A is Ri, is isolated, or b) if an alcohol is used as nucleophile, the resulting enantiomerically enriched E- (2S) - or
(2R) -alkyl-5-halopent-4-enecarboxylic ester of the formula (Ia) or (Ib) , where A is R2, is isolated, or c) if water is used as nucleophile, the resulting E- (2S) - or (2R) -alkyl-5-halopent-4-enecarboxylic acid of the formula (Ia) or (Ib) , where A is H, is isolated.
The process according to the invention is used to prepare enantiomerically enriched E- (2S) - or (2R) -alkyl-5-halopent-4-enecarboxylic acids or their esters of the formula (Ia) or (Ib) .
In the formulae (Ia) and (Ib) , R is a Ci-C6-alkyl radical such as methyl, ethyl, n- and i-propyl, n- , i- and t-butyl, pentyl and hexyl . Preference is given to C1-C4-alkyl radicals, with particular preference being given to the i -propyl radical .
A is H, Ri, where Rx is a Ci-C4-alkyl radical, preferably a Ci-C2-alkyl radical and particularly preferably a methyl radical, or R2, where R2 is an alkyl group which is not identical to Ri. R2 is preferably a
Ci-Cg-alkyl radical.
X is chlorine, bromine or iodine, preferably chlorine. According to the invention, the enantiomerically enriched (S) - or (R) -carboxylic acids or their esters of the formula (Ia) or (Ib) are prepared, in accordance with the invention, by reacting an enantiomeric mixture of a 2-alkyl-5-halopent-4-enecarboxylic ester of the formula (II) with a stereoselective hydrolase which is obtained from microorganisms or plants.
In this connection, enantiomerically enriched compounds are to be understood as compounds which exhibit a (S) - or (R) -enantiomeric excess (ee) of > 80%, preferably of 90% and particularly preferably of > 97%.
One enantiomer is preferably hydrolyzed, depending on the choice of the enzyme possessing a specific stereoselectivity.
The stereoselectivity of the enzyme can be described by the E ratio, as known, for example, from Chen et al . , J. Am. Chem. Soc . (1982), 104, 7294 - 7299. Preference is given, according to the invention, to using hydrolases having an E ratio of > 5, preferably > 10, particularly preferably > 50 and in particular preferably > 100.
According to the invention, the hydrolases which are used are of nonanimal origin.
Examples of suitable stereoselective hydrolases are esterases, lipases, proteases, peptidases or acylases, etc . These enzymes are obtained from microorganisms
(bacteria or fungi) or from plants. Examples of suitable sources are wheat germs, molds, such as Absidia; Aspergillus; Fusarium; Gibberella; Mucor; Neurospora; Trichoderma; Rhizopus; Rhizomucor, for example Rhizomucor miehei; Thermomyces, for example Thermomyces lanuginosus; bacteria, such as Achromobacter; Alcaligenes; Bacillus, for example Bacillus licheniformis; Brevibacterium; Corynebacterium; Providencia; Pseudomonas, for example Pseudomonas fluoresceins, Pseudomonas cepase or Pseudomonas alcaligenes; Serratia, Rhodococcus, or yeasts, such as Candida, for example Candida rugose or Candida antarctica; and yeasts of the Actinomycete genus Nocardia.
Preference is given to enzymes which are classified as carboxylic ester hydrolases (EC 3.1.1) or as peptidases, for example EC 3.4.1., EC 3.4.11, EC 3.4.21, preferably EC 3.4.21.62, EC 3.4.22 or EC 3.4.23.
Enzymes which are suitable for being used in accordance with the invention can, for example, be obtained commercially. Examples of commercially obtainable enzymes are : enzymes obtainable from Fluka: Candida cylindracea lipase, Pseudomonas fluorescens lipase, Aspergillus oryzae lipase, Rhizopus niveus lipase, Rhizomucor miehei lipase, Candida antarctica lipase, Mucor javanicus lipase, Rhizopus arrhizus lipase, Penicillium rogueforti lipase, Candida lipolytica lipase, Pseudomonas sp. type B lipoprotein lipase, Pseudomonas cepacia lipoprotein lipase, Chromobacterium viscosum lipoprotein lipase, Bacillus stearothermophilus esterase, Bacillus thermoglucosidasius esterase and Mucor miehei esterase, enzymes obtainable from Altus : Candida rugosa lipase, Mucor miehei lipase, Candida antarctica B lipase, Candida antarctica A lipase, Chiro-CLEC-CR, Chiro-
CLEC-CR (slurry) , penicillin acylase, Carlsberg subtilisin, Chiro~CLEC-BL (slurry) , Chiro-CLEC-PC
(slurry) Chiro-CLEC-EC (slurry) , Aspergillus oryzae protease, PeptiCLEC-TR (slurry) ; enzymes obtainable from Recombinant Biocatalysis : ESL-001-07, ESLOOl-01, ESL-001-01 with stabilizer, ESL-001-02, ESL-001-03 and ESL-001-05; enzymes obtainable from Boehringer-Mannheim: Chirazyme L4 (Pseudomonas sp.) , Chirazyme L5 (Candida antarctica fraction A) , Chirazyme Ll (Burkholderia) , Chirazyme L7 and Chirazyme L8; enzymes obtainable from DSM (formerly Gist-Brocades) : Naproxen esterase, Lipomax, Genzyme and lipoprotein lipase; enzymes obtainable from Novo: Novozyme 868, Novozyme 435, immobilized Candida antarctica lipase, nagase enzyme, lipase A-IOFG (Rhizopus javanicus) ; enzymes obtainable from Amano: Amano AYS, Amano PS, Amano PSD, Amano AKDIl, Amano AKDlIl and Aspergillus melleus protease.
The enzymes can be used in any form, for example as a dispersion, as a solution, immobilized, as a crude enzyme, as a commercially obtainable enzyme, as an enzyme which has been further purified from a commercially obtainable preparation, as an enzyme which has been obtained from its source using a combination of known purification methods, as whole cells (where appropriate immobilized and/or permeabilized) which possess the requisite enzymic activity (naturally or by means of genetic modifications) , or in a lysate of such cells. It is furthermore also possible to use mutants of the naturally occurring (wild-type) enzymes which possess the corresponding activity. Mutants of wild-type enzymes can be obtained, for example, by altering the DNA which encodes the wild-type enzyme, or by means of known mutagenesis techniques (random mutagenesis, site- directed mutagenesis, direct evolution, gene shuffling, etc.) such that the DNA encodes an enzyme which at least differs from the wild-type enzyme in one amino acid, and subsequently expressing the modified DNA in a suitable host cell. Such mutants may exhibit improved properties with regard to (stereo) selectivity and/or activity and/or stability and/or resistance to solvents and/or resistance to pH and/or resistance to temperature. It is also possible to use enzymes which exhibit an amino acid sequence as described, for example, in WO 2005/032496, WO 2004/035729, WO 02/057411 or US 5,942,430. The enzymes from WO 2005/032496, WO 2004/035729, WO 02/057411 and US 5,942,430 are hereby included by reference .
An enzyme which is suitable for the reaction according to the invention can be selected, for example, by screening several enzymes. Performing screenings is prior art. Usually, the conditions under which the substrate (racemic ester of the formula (II) ) and the enzyme are brought together for the purpose of selecting a suitable enzyme are chosen such that a good compromise is found between the stability of the enzyme, of the substrate and of the end product, on the one hand, and the reaction rate (which normally increases with increasing temperature) , on the other hand. Screenings can be carried out on any scale. For practical reasons, preference is given to using a reaction volume of between 0.15 ml and 10 ml if a large number of enzymes are being screened.
The ratio of enzyme to substrate is usually not critical for the screening and can be chosen to be between 1/20 and 2/1. The quantity of substrate employed is likewise not critical and can, for example, be between 5 mM and 1.5 M. The pH is not critical, either, and can, for example, be adjusted to a value of between 4 and 11 and can be kept constant by using an aqueous buffer solution.
The temperature during screening is once again not critical and can, for example, be between 10 and 600C.
The choice of the reaction conditions for the process according to the invention depends on the choice of the enzyme. Normally, the temperature of the reaction according to the invention is between 0 and 9O0C, preferably between 10 and 600C. The pH of the reaction solution is between 4 and 11, preferably between 6 and 9.
The choice of the solvent depends on the nucleophile which is used.
If, for example, water is the nucleophile, solvents which can then be used are water, a mixture of water and a solvent which is miscible with water, for example an alcohol, such as methanol, ethanol, isopropanol, tert-butanol, etc., dioxane, tetrahydrofuran, acetone or diemthyl sulfoxide, or a two-phase system composed of water and a solvent which is not miscible with water, for example an aromatic compound, such as toluene, xylene, etc., an alkane, such as hexane, heptane, cyclohexane, etc., or an ether, such as diisopropyl ether, methyl tert-butyl ether, etc.
If the nucleophile is an alcohol, the solvent which is then preferably used is the alcohol R2OH, where R2 is an alkyl group which is not, however, identical to R1. However, it is also possible to use mixtures of the alcohol and an organic solvent, such as tetrahydrofuran, heptane, toluene, hexane, CH3CN, methyl tert-butyl ether, etc.
After hydrolysis has taken place, the desired end product is then isolated.
This end product can either the remaining enantiomerically enriched E- (2S) - or (2R) -alkyl- 5-halopent-4-enecarboxylic ester of the formula (Ia) or (Ib) , where A is R1, or, if water is the nucleophile, the resulting enantiomerically enriched E- (2S) - or (2R) -alkyl-5-halopent-4-enecarboxylic acid of the formula (Ia) or (Ib) , where A is H, or if the alcohol is the nucleophile, the enantiomerically enriched E- (2S) - or (2R) -alkyl-5-halopent-4-enecarboxylic ester of the formula (Ia) or (Ib) where A is R2. The end product can be isolated, for example, using customary methods such as extraction, crystallization, column chromatography, distillation, etc.
Using the process according to the invention results in the corresponding acids or esters of the formula (I) being obtained in theoretical yields of up to 98% yield and having a e.e. of up to > 99%, with enzymes of animal origin being avoided.
Example 1: Determining the activity and enantioselectivity of 3 hydrolytic enzymes:
In order to test the enzymes for their activity, and to determine their intrinsic enantioselectivity (E ratio) , in each case 2 mg of enzyme were suspended in 225 μl of 50 mM Kpi buffer (pH 7.5). The enzymic reaction was started by adding in each case 25 μl of a 10% v/v mixture of methyl (R, S) -5-chloro-2-isopropylpent- 4-eneoate in tert-butanol . The reaction mixtures were kept in an incubator for in each case 24 h at room temperature while being shaken at 700 rpm on a Labinco LD-45 microtiter plate vibrator. After the incubation had been completed, the reactions were stopped and the reaction mixtures were diluted by adding in each case 750 μl of acetonitrile containing 50 mM phosphoric acid. The resulting mixtures were centrifuged at 2500 rpm for 5 minutes and analyzed by reversed phase LC on a Varian Inertsil ODS-3 column (50 x 4.6 mm I. D. , 3 μm) .
The compounds were eluted using a gradient of 50 mM phosphoric acid in water (pH 2.3) and acetonitrile (1.0 ml/min, at 4O0C) . The initial conditions for the gradients were 60/40% v/v buffer/acetonitrile at 0 minutes, with the percentage of acetonitrile increasing to 50% in 5 minutes and being kept at 50% for a further 2 minutes. The injection volume was 5 μl. Detection was effected using a spectrophotometer at 220 nm UV. The retention times were 3.2 min and 7.6 min, respectively, for 5-chloro-2-isopropylpent-4-enecarboxylic acid and methyl 5-chloro-2-isopropylpent-4-enecarboxylate .
The three enzymes were used for determining, by HPLC analysis, the preferred enantioselectivity of the enzyme and the enantiomeric excess of the 5-chloro- 2-isopropylpent-4-enecarboxylic acid which was formed. The enantiomeric excess was determined on an EKA. Chemicals Kromasil KRlOO-5CHI -TBB (250 mm x 4.6 mm I. D., 5 μm) using 98/2/0.1% v/v n-heptane/allyl alcohol/trifluoroacetic acid as the eluent (0.8 ml/min at room temperature) .
The injection volume was 5 μl and detection was performed using a spectrophotometer at 210 nm UV.
The retention times for the (R) - and (S) -5-chloro-
2-isopropylpent-4-enecarboxylic acids were 9.3 and
9.9 min, respectively.
Samples of the aqueous enzyme reaction were freeze- dried and then first of all dissolved in 100 μl of allyl alcohol and after that admixed with 500 μl of eluent for the analysis.
The HPLC data were used to calculate the intrinsic enantioselectivity of the enzymes, expressed as an E ratio.
Results :
Figure imgf000012_0001

Claims

Patent claims
1. A process for preparing enantiomerically enriched E- (2S) - and (2R) -alkyl-5-halopent-4-enecarboxylic acids or their esters of the formula (Ia) or (Ib)
Figure imgf000013_0001
in which R is a Ci-C6-alkyl radical, A is H, Ri, where R1 can be Ci-C4-alkyl, or R2, where R2 is an alkyl group but is not identical to Ri, and X is chlorine, bromine or iodine, wherein an enantiomeric mixture of a 2-alkyl-5-halopent-
4-enecarboxylic ester of the formula (II)
Figure imgf000013_0002
in which R, Ri and X are defined as above, is reacted with a stereoselective hydrolase, which is obtained from microorganisms or plants, in the presence of water or an alcohol of the formula R2OH, where R2 is an alkyl group which is not identical to R1, as nucleophile, and a) either the remaining enantiomerically enriched E- (2S) - or (2R) -alkyl-5-halopent-4-enecarboxylic ester of the formula (Ia) or (Ib) , where A is Rx, is isolated, or b) if an alcohol is used as nucleophile, the resulting enantiomerically enriched E- (2S) - or (2R) -alkyl-5-halopent-4-enecarboxylic ester of the formula (Ia) or (Ib) , where A is R2, is isolated, or c) if water is used as nucleophile, the resulting E- (2S) - or (2R) -alkyl-5-halopent-4-enecarboxylic acid of the formula (Ia) or (Ib) , where A is H, is isolated.
2. The process as claimed in claim 1, wherein stereoselective hydrolases obtained from wheat germs, from molds, from the group Absidia; Aspergillus; Fusarium; Gibberella; Mucor; Neurospora; Trichoderma; Rhizopus; Rhizomucor and Thermomyces, from bacteria, from the group Achromobacter; Alcaligenes; Bacillus; Brevibacterium; Corynebacterium; Providencia; Pseudomonas; Serratia and Rhodococcus, or from yeasts, from the group Candida, and those of the Actinomycete genus Nocardia, or mutants thereof, are employed.
3. The process as claimed in claim 1 or 2 , wherein stereoselective hydrolases having an E ratio of > 100 are employed.
PCT/EP2006/003267 2005-05-02 2006-04-10 Process for preparing enantiomerically enriched e-(2s)- and (2r)-alkyl-5-halopent-4-enecarboxylic acids or their esters WO2006117057A1 (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000039324A2 (en) * 1998-12-23 2000-07-06 Lonza Ag Method for producing optically active 1-amino-4-(hydroxymethyl)-cyclopent-2-ene derivatives
WO2004052828A1 (en) * 2002-12-09 2004-06-24 Asahi Glass Company, Limited Processes for producing (4e)-5-chloro-2-isopropyl-4-pentenoic ester and optically active isomer thereof
US6777574B1 (en) * 1999-07-29 2004-08-17 Speedel Pharma Ag 2-alkyl-5-halogen-pent-4-ene carboxylic acids and their production
EP1626093A1 (en) * 2004-08-11 2006-02-15 Dow Global Technologies Inc. Process for the production of (S)-5-chloro-2-isopropylpent-4-enoic acid esters

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000039324A2 (en) * 1998-12-23 2000-07-06 Lonza Ag Method for producing optically active 1-amino-4-(hydroxymethyl)-cyclopent-2-ene derivatives
US6777574B1 (en) * 1999-07-29 2004-08-17 Speedel Pharma Ag 2-alkyl-5-halogen-pent-4-ene carboxylic acids and their production
WO2004052828A1 (en) * 2002-12-09 2004-06-24 Asahi Glass Company, Limited Processes for producing (4e)-5-chloro-2-isopropyl-4-pentenoic ester and optically active isomer thereof
EP1571138A1 (en) * 2002-12-09 2005-09-07 Asahi Glass Company Ltd. Processes for producing (4e)-5-chloro-2-isopropyl-4-pentenoic ester and optically active isomer thereof
EP1626093A1 (en) * 2004-08-11 2006-02-15 Dow Global Technologies Inc. Process for the production of (S)-5-chloro-2-isopropylpent-4-enoic acid esters

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CHEN C-S ET AL: "QUANTITATIVE ANALYSES OF BIOCHEMICAL KINETIC RESOLUTIONS OF ENANTIOMERS", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 104, no. 25, 1982, pages 7294 - 7299, XP002393204, ISSN: 0002-7863 *

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