HK1135434A - Specific hydrolysis of the n-unprotected (r) -ester of (3) -amin0-3-arylpr0pi0nic acid esters - Google Patents

Description

Specific hydrolysis of (R) esters of N-unprotected (3) -amino-3-arylpropionic acid esters

Technical Field

The present invention relates to a process for the production of amino acid esters enriched in the (S) enantiomer by biological decomposition in the presence of ester hydrolases.

Background

Beta-amino acids and esters, such as 3-amino-3-arylpropionic acid esters, have a wide range of applications as synthetic intermediates, particularly for the production of pharmaceutical agents. Such molecules are chiral and preferably are available in the form of a single enantiomer or at least in enantiomerically enriched form.

The enantiomerically enriched forms of such molecules and the corresponding carboxylic acids can be obtained by chemical methods. For example, U.S. patent No. 6,673,926B 2 describes a process for preparing 3S-amino-3-arylpropionic acids (e.g., aryl ═ 3-pyridyl) and derivatives thereof using the classical resolution (resolution) method, which requires multiple steps including protection of the 3-amino group.

Alternative biocatalytic processes for the preparation of enantiomerically enriched beta-amino acids and esters have been described which are more economically and ecologically advantageous. The use of the lipase PS enzyme for the production of (S) -acids is described, for example, in U.S. Pat. No. 2004/0029236A 1, and in preparion of organic enzymatic acids via enzymatic hydrolysis, Tetrahedron Letters 41(2000) 2679-.

Other references describe methods for preparing enantiomerically pure (enantiopure) β -amino acids and/or β -amino esters, including, for example, WO 01/16090a1, which includes a description of the molecule, but does not include a description of the method of synthesizing the enantiomerically enriched compounds. The following references describe the use of Candida antarctica (Candida antarctica) lipase to catalyze the formation of a enantioselective amide between the amino group of a racemic substrate and 2, 2, 2-trifluoroethyl ester in diisopropyl ether: lui et al, Recent Advances in the stereoselective synthesis of beta-amino acids, Tetrahedron 58(2002) 7991-8035; gedey et al, Preparation of highlyenantiopure β -amino esters by Candidaantarctica lipaseA, tetrahedron: asymmetry 10(1999) 2573-2581. Thus, in the methods described in these references, the enrichment of (S) -unreacted substrate and the formation of (R) -enriched amide is only caused by acylation of the amino group, preferably in the (R) -center, by using an activated ester. Sanchez et al, in Candida antarctica lipase catalyzed resolution of ethyl (. + -.) -3-aminobutyrate, Tetrahedron: a similar pathway to the Gedey reference, but producing enantiomerically pure 3-aminobutyric acid, is described in Asymmetry, Vol.8, No.1, pp.37-40, 1997.

European patent application EP 1057894 a2, A3 describe the use of the aspergillus flavus (aspergillus flavus) esterase ATCC 11492 to produce N-benzyl-L-azetidinecarboxylate for the production of (S) -acid, but such enzymes have not been described for the production of β -amino acids or β -amino esters.

The biocatalytic pathways described in the above prior art, which involve biocatalytic hydrolysis of beta-amino acid esters, all produce (S) -acid as a hydrolysate. If the corresponding (S) -ester is desired as the desired product, re-esterification of the (S) -acid is required, usually by chemical means. Therefore, these biocatalytic pathways require multiple steps and are therefore relatively long and expensive.

Disclosure of Invention

The present invention provides an improved enzymatic process for the preparation of amino acids.

In one aspect, the invention is a process for preparing a 3-amino-3-aryl propionate enriched in the (S) -enantiomer of at least 80% ee, comprising the steps of: enantioselective hydrolysis (enantioselective hydrolysis) of an enantiomeric mixture of a 3-amino-3-arylpropionic acid ester, in the presence of an ester hydrolase, said 3-amino-3-arylpropionic acid ester being represented by the formulae (S) - (1) and R- (1):

wherein Ar is C_4-30Unsubstituted or substituted aromatic radical, R being C_1-10A linear or branched alkyl group; and recovering the optically active ester (S) - (1) which has not been hydrolyzed.

In a second aspect, the present invention is the use of an ester hydrolase obtained from Aspergillus (Aspergillus) for the preparation of a 3-amino-3-arylpropionate ester enriched in the (S) -enantiomer in at least 80% ee.

In a third aspect, the present invention is a process for preparing a 3-amino-3-arylpropionic acid enriched in the (R) -enantiomer in an enantiomeric excess (ee) of at least 80%, comprising the steps of: in the presence of an ester hydrolase, a mixture of enantiomers of a 3-amino-3-arylpropionic acid ester, said 3-amino-3-arylpropionic acid ester being represented by the formulae (S) - (1) and R- (1):

wherein Ar is C_4-30Unsubstituted or substituted aromatic radical, R being C_1-10A linear or branched alkyl group; and recovering the optically active acid (R) - (2) as a hydrolysate.

In a fourth aspect, the present invention is the use of an ester hydrolase obtained from Aspergillus in the preparation of a 3-amino-3-arylpropionate ester enriched in the (R) -enantiomer in at least 80% ee.

The present invention relates to the decomposition of esters to leave unreacted (S) -ester or to directly produce (R) -acid, thus reducing the number of steps compared to the processes described in the prior art.

Detailed Description

The compounds prepared according to the present invention are (S) -enantiomer-enriched amino acid esters represented by the formulae (S) - (1):

an amino acid is prepared from an enantiomeric mixture of a 3-amino-3-arylpropionate, said 3-amino-3-arylpropionate being represented by the formulae (S) - (1) and R- (1):

wherein Ar is C_4-30An aromatic group, R is C_1-10An alkyl group.

Suitable Ar groups include aryl, heteroaryl, substituted aryl or substituted heteroaryl. As used herein, "aryl" means an aromatic group, such as phenyl, naphthyl, and the like, unless otherwise specified. An aryl group may be unsubstituted or substituted with one or more substituents. Suitable substituents on the aryl group include, but are not limited to, groups independently selected from: halogen, alkyl, alkoxy, alkaryl, amino, amide, nitro, thio groups, carboxyl, hydroxyl. As used herein, unless otherwise indicated, "heteroaryl" represents any five or six membered monocyclic ring structure containing at least one heteroatom selected from O, N or S, or a bicyclic ring system wherein the heteroaryl ring is fused to another aryl ring. Examples of suitable heteroaryl groups include, but are not limited to, pyrrolyl, pyridyl, pyrazinyl, pyrimidinyl, pyrazolyl, pyridazinyl, furanyl, pyranyl, imidazolyl, thiophenyl, oxazolyl, isothiazolyl, isoxazolyl, furguryl, benzothienyl, benzofuranyl, indolyl, isoindolyl, indolinyl, indazolyl, purinyl, isoquinolyl, quinolinyl, isothiazolyl, and the like. Preferably, Ar is phenyl or substituted phenyl, most preferably phenyl.

Suitable R groups include C_1-10Linear or branched alkyl groups. As used herein, "alkyl" whether used alone or as part of a substituent group, shall include straight and branched chains containing from 1 to 10 carbon atoms. For example, alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, and the like. Preferably, R is methyl, ethyl, propyl, isopropyl, butyl, isobutyl or sec-butyl, most preferably methyl or ethyl.

In a particularly preferred embodiment, (S) -ethyl 3-amino-3 phenylpropionate is prepared from a mixture of (S) -ethyl 3-amino-3 phenylpropionate and (R) -ethyl 3-amino-3 phenylpropionate, with (S) -ethyl 3-amino-3 phenylpropionate and (R) -ethyl 3-amino-3 phenylpropionate being represented by the following formulae:

(S) -Ethyl 3-amino-3 phenylpropionate (R) -Ethyl 3-amino-3 phenylpropionate

The 3-amino-3-aryl propionates of the present invention have an enantiomeric excess or "ee" of the (S) -enantiomer. "enantiomeric excess" or "ee" is a measure of how much of one enantiomer is present relative to the other, and is represented by the following formula:

the% enantiomeric excess (moles of the main enantiomer-moles of the other enantiomer/total moles of both enantiomers) 100.

Preferably, the 3-amino-3-arylpropionic acid esters of the present invention are enriched in the (S) -enantiomer in at least 80% ee, more preferably in at least 90% ee, and even more preferably in at least 95% ee.

The enzyme used in the present invention is an ester hydrolase. Preferably, the ester hydrolyzing enzyme is obtained from Aspergillus, more preferably Aspergillus flavus species. Even more preferably, the ester hydrolase is an ester hydrolase derived from Aspergillus flavus var. columnaris, with Aspergillus flavus var. strain UKNCC 095253 and UKNCC 015958 being most preferred. The enzyme may be in aqueous solution, whole cells, frozen stem cells, immobilized on a solid support, held in a membrane bioreactor, or a combination thereof.

An exemplary reaction sequence for preparing (S) -ethyl 3-amino-3 phenylpropionate is as follows:

the choice of reaction parameters is highly discretionary, and suitable conditions may be found using methods known and conventional in the art. As a guide, the pH of the reaction should range from 4 to 10, preferably from 5 to 9, more preferably from 6 to 8. If the pH is too high, there will be a tendency for non-selective chemical hydrolysis of the ester to occur. If the pH is too low, the enzyme will degrade.

Preferably, the reaction is carried out at a temperature above about 10 degrees celsius, more preferably at least about 15 degrees celsius. The reaction temperature is preferably below 100 degrees celsius, preferably below about 40 degrees celsius.

The reaction of the present invention may be carried out in any reaction vehicle provided for this purpose, provided that the reaction vehicle has suitable pH and temperature control. For example, the reaction of the present invention may be in a conventional batch reactor, a loop reactor or an enzyme membrane reactor.

The reaction is typically carried out under aqueous conditions, for example in water and/or an aqueous buffer. If solubility of the reactants or reaction products is a consideration, the reaction may also be carried out in an organic cosolvent, for example, a hydrophobic or hydrophilic organic solvent is added in addition to water or an aqueous buffer. Such hydrophobic organic solvents may be, for example, ethers such as t-butyl methyl ether, isopropyl ether or hydrocarbons such as toluene, hexane, cyclohexane, heptane and isooctane; and the hydrophilic organic solution may be, for example, alcohols such as t-butanol, methanol, ethanol, isopropanol and n-butanol, ethers such as tetrahydrofuran, sulfoxides such as dimethyl sulfoxide, ketones such as acetone, or nitriles such as acetonitrile. Each of these hydrophobic or hydrophilic organic solvents may be used as a single co-solvent or in combination with each other, although a combination of a hydrophobic and a hydrophilic organic solvent is also contemplated.

The desired end product is retained after hydrolysis has occurred and must be recovered. Preferably, the desired end product is recovered by extraction, filtration, or a combination thereof.

Examples

The following examples illustrate some specific embodiments of the present invention and are intended to be illustrative only and not limiting.

Example 1 substrate solution (10.0 g/L3-Ammonia in sodium citrate phosphate buffer) Ethyl 3-phenyl-propionate) preparation:

the substrate solution was prepared by adding 0.2g of an ester (prepared according to Tetrahedron, 2002, 58, 7449) to 20ml of citric acid/sodium phosphate buffer (prepared according to McIlvaine, j.bio.chem., 1921, 49, 183); the pH is adjusted to the desired value with phosphoric acid.

Example 2 preparation of Aspergillus cells

Aspergillus flavus stigma variant strain 015958 (originally from the Collection of fungal Plant Pathological Bacteria (CABI)) was obtained from the United Kingdom National Culture Collection (UKNCC) and stored in glycerol at-70 ℃. This storage (250. mu.l) was used to inoculate 50ml of sterile potato dextrose broth in a 250ml Erlenmeyer flask with a barrier. Cultures were grown at 25 ℃ and 250rpm for 72 hours. This culture was then used to inoculate 2.0L of sterile potato dextrose broth in a bench top fermenter, and the culture was grown at 25 ℃, 500rpm, and 1.0L of air/min for 72 hours without pH control. The cells were harvested by filtration to yield 35.7g of wet cells, which were then frozen at-20 ℃. The cells were then lyophilized to yield 7.25g of stem cells.

Example 3: preparation of the biotransformation reaction mixture:

200mg of harvested lyophilized A.flavus cells were incubated with a substrate stock (20ml) at 27 deg.C (as a blank, in one reaction, the cells were replaced with buffer).

Example 4: achiral analysis of biotransformations

The reaction was stopped by transferring 100. mu.l of the biotransformation mixture to 900. mu.l of HPLC solvent (20% acetonitrile +10mM potassium phosphate, pH 3.0) and analyzed achirally.

And (3) analysis:

achiral HPLC analysis was performed using a C18(BDS) column (150 × 4.6mM, or equivalent) and an HPLC solvent (20% acetonitrile +10mM potassium phosphate, pH 3.0) to monitor absorbance at 210nm at 2 ml/min.

Example 5 chiral analysis of biotransformation

For chiral analysis, 100. mu.l of the biotransformation reaction were transferred to 900. mu.l of heptane, mixed, dried over anhydrous sodium sulfate and filtered, and the organic layer was analyzed by HPLC.

And (3) analysis:

chiral analysis can be performed to distinguish between the two isomers of the ester substrate. A Diacel Chiralcel OD-H column (250X 4.6mm, or equivalent) was used with 90% heptane/10% isopropanol as the mobile phase. Detection was performed at 210nm using a flow rate of 1 ml/min.

As a result:

for the biotransformation carried out at pH 7.0, a reaction time of 17 hours resulted in a 50% conversion (7% conversion blank) and an isolated purity of the (S) -ester of > 98.5% ee.

Claims (29)

1. A process for the preparation of a 3-amino-3-arylpropionic acid ester enriched in the (S) -enantiomer in an enantiomeric excess (ee) of at least 80%, comprising:

in the presence of an ester hydrolase, a mixture of enantiomers of a 3-amino-3-arylpropionic acid ester, said 3-amino-3-arylpropionic acid ester being represented by the formulae (S) - (1) and R- (1):

wherein Ar is C_4-30Unsubstituted or substituted aromatic radical, R being C_1-10A linear or branched alkyl group; and

the unhydrolyzed optically active ester (S) - (1) is recovered.

2. The method of claim 1, wherein Ar is phenyl or substituted phenyl.

3. The method of claim 1, wherein R is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, or sec-butyl.

4. The process of claim 1, wherein the enriched 3-amino-3-arylpropanoate ester is ethyl (S) -3-amino-3-arylpropanoate.

5. A process according to any one of claims 1 to 4, wherein the enantiomeric excess of the (S) ester produced is 90% or more.

6. A process according to any one of claims 1 to 5, wherein the ester hydrolase is obtained from Aspergillus oryzae.

7. The method according to claim 6, wherein the ester hydrolase is from Aspergillus flavus var.

8. The method of any one of claims 1-7, wherein the enzyme is in an aqueous solution, whole cells, frozen stem cells, immobilized on a solid support, held in a membrane bioreactor, or a combination thereof.

9. The process according to claim 8, wherein the enzyme of Aspergillus flavus stigma var.

10. The process according to any one of claims 1-9, wherein the hydrolysis is carried out at a pH of 4 to 10.

11. The method of claim 10, wherein the hydrolysis is performed at a pH of 6 to 8.

12. The process according to any one of claims 1-11, wherein the hydrolysis is carried out at a temperature of from +5 ℃ to +100 ℃.

13. The process of any one of claims 1-12, wherein the hydrolysis is carried out at a temperature of from +15 ℃ to +40 ℃.

14. Use of an ester hydrolase obtained from Aspergillus in the preparation of a 3-amino-3-arylpropionate enriched in the (S) -enantiomer in at least 80% ee.

15. Use of an ester hydrolase obtained from Aspergillus flavus var.

16. A process for preparing a 3-amino-3-arylpropionic acid enriched in an (R) -enantiomer having an enantiomeric excess (ee) of at least 80%, comprising:

in the presence of an ester hydrolase, a mixture of enantiomers of a 3-amino-3-arylpropionic acid ester, said 3-amino-3-arylpropionic acid ester being represented by the formulae (S) - (1) and R- (1):

wherein Ar is C_4-30Unsubstituted or substituted aromatic radical, R being C_1-10A linear or branched alkyl group; and

recovering the optically active acid (R) - (2) as the hydrolysate.

17. The method of claim 16, wherein Ar is phenyl or substituted phenyl.

18. The method of claim 16, wherein the enriched 3-amino-3-arylpropionic acid is (R) -3-amino-3-phenylpropionic acid.

19. A process according to any one of claims 16 to 18, wherein the enantiomeric excess of the (R) acid produced is 90% or more.

20. A process according to any one of claims 16 to 19, wherein the ester hydrolase is obtained from Aspergillus.

21. The method according to claim 20, wherein the ester hydrolyzing enzyme is from Aspergillus flavus var.

22. The method of any one of claims 16-21, wherein the enzyme is in an aqueous solution, whole cells, frozen stem cells, immobilized on a solid support, held in a membrane bioreactor, or a combination thereof.

23. The process according to claim 22, wherein the enzyme of Aspergillus flavus var.

24. The method of any one of claims 16-23, wherein the hydrolysis occurs at a pH of 4 to 10.

25. The method of claim 24, wherein the hydrolysis is performed at a pH of 6 to 8.

26. The process of any one of claims 16-25, wherein the hydrolysis is carried out at a temperature of +5 ℃ to +100 ℃.

27. The process of claim 26, wherein the hydrolysis is carried out at a temperature of +15 ℃ to +40 ℃.

28. Use of an ester hydrolase obtained from Aspergillus in the preparation of 3-amino-3-arylpropionic acids enriched in the (R) -enantiomer in at least 80% ee.

29. Use of an ester hydrolase obtained from Aspergillus flavus var.