WO2007039079A1

WO2007039079A1 - Process for the synthesis of beta amino acids

Info

Publication number: WO2007039079A1
Application number: PCT/EP2006/009050
Authority: WO
Inventors: Oreste Ghisalba; Kurt Laumen
Original assignee: Novartis Ag
Priority date: 2005-09-20
Filing date: 2006-09-18
Publication date: 2007-04-12
Also published as: GB0519200D0

Abstract

A process for the synthesis of beta amino acids comprising the hydrolysis of N-acetyl methyl ester derivatives using a biocatalyst.

Description

Process for the synthesis of beta amino acids

The present invention relates to a process for the synthesis of beta-amino acids using a microorganism as a biocatalyst and to the biocatalyst catalyzing this process.

Beta-amino acids are used in medicinal chemistry and show biological activity. Beta-amino acids may be found in e.g. beta-lactam antibiotics, HIV-protease inhibitors and further enzyme inhibitors. Classical organic synthesis or enzyme-catalyzed resolution may be used to synthesise beta-amino acids. Enzymes commonly used to resolve derivatives of alpha- amino acids have narrow substrate tolerance towards beta-amino acids. Due to the narrow substrate tolerance the biocatalytic approach is less used. Some examples using lipases or penicillin G acylase as catalysts are described.

There is a need to provide improved processes to synthesize beta-amino acids using a biocatalyst.

The present invention therefore provides a process for the synthesis of beta amino acids comprising the hydrolysis of N-acetyl methyl ester derivatives, characterized in that a biocatalyst is used.

In another aspect the present invention provides a process for the synthesis of beta amino acids comprising the hydrolysis of N-acetyl methyl ester derivatives using a biocatalyst characterized in that the biocatalyst is stereoselective^ hydrolyzing the N-acetyl methyl ester derivatives.

The advantages of the process of the invention are that whole cells as biocatalysts as well as isolated enzymes may be used to catalyze the process and the reaction medium may be distilled water. The product may easily be separated by simple extraction with organic solvents under acidic conditions without pH control or pH adjustment during the hydrolysis.

The stereoselective hydrolysis of N-acetyl methyl ester derivatives provides easy separation of the enantiomers. With an entiomerically pure pharmaceutical or agrochemical drug side effects e.g. triggered by the therapeutically ineffective enantiomer can be avoided. As used herein, the term "biocatalyst" means a purified enzyme, from original or recombinant source, a partially purified enzyme, a crude cell extract, enzymes immobilized or caught in partially broken cells or cell debris, a protein with enzymatic activity, a polypeptide with enzymatic activity, a living microorganism or dead microorganism containing a protein with enzymatic activity or a cell extract of such a microorganism.

The biocatalyst may be used in solution, suspension or in immobilized form according to routine processes, e.g. batch or continuous, known in the art.

A preferred embodiment is a biocatalyst, the substrate of which may be hydrolysed to beta- amino acids. The biocatalyst according to the invention is preferably selected from the group of (a) a protein with said enzymatic activity and (b) a living microorganism or dead microorganism containing a protein with said enzymatic activity or a cell extract of such a microorganism. A dead microorganism in context with the present invention is e.g. a microorganism in a disintegrated form in which the cell wall and/or cell membrane is mechanically or chemically disrupted or removed.

The biocatalyst of the invention may be an amido hydrolase or enzyme. If not stated otherwise, all these terms include not only the naturally occurring, authentic sequence of the protein of the enzyme, which are preferred embodiments of the invention, but also all mutants, variants and fragments thereof which exhibit amido hydrolase activity, preferably the same stereoselective activity as the natural enzyme.

The biocatalyst may be obtainable from a microorganism selected from the group of Rhodococcus globerulus and Rhodococcus equi. Preferably the biocatalyst is selected from the group consisting of Rhodococcus globerulus K1/1, DSM 10337, and Rhodococcus equi Ac6, DSM 10278 as disclosed in the International Application WO 97/41214 which is incorporated by reference.

Isolation of a microorganism naturally expressing amido-hydrolase activity of the invention may be achieved by a selection process comprising inoculating a selection medium with natural samples such as soil, water, or plant silage, said selection medium comprising N- acetyl methyl ester derivatives as sole carbon source. In the preferred embodiments of the selection process, the racemic substrates described above in the context with the hydrolysis reaction are used for the selection.

Apart from the carbon source, the selection medium also contains all essential ingredients necessary for allowing growth of microorganisms, such as mineral salts, N-sources and trace elements.

A suitable selection medium used for performing the invention comprises 3 g racemic N- acetyl-1-phenylethylamine, 3 ml Trace element solution SL-6, and 1 I mineral salt solution ML1 (pH 7.0), whereby the composition of the mineral salt solution ML1 is 5 g K2HPO4, 0.2 g MgSθ4^*7H2θ, 20 mg CaCtø, 20 mg FeSθ4^*7H2θ, 1.5 g (NH4)2SO4_i and 1 I Aqua deion (pH 7.0); and the composition of the trace element solution SL-6 is 20 mg NiCl2^*6H2θ, 200 mg CoCl2^*6H2θ, 30 mg MnCl2*4H2θ, 10 mg CuCl2^*2H2θ, 300 mg H3BO3, 30 mg Na2Moθ4^*2H2θ, 100 mg ZnSθ4^*7H2θ, and 1 I Aqua deion. This recipe may be varied as long as sufficient trace elements, mineral salts, and N-source are provided.

The enzyme of the invention which may be a polypeptide may be used in enriched or, preferably, purified form. In one embodiment of the invention an enzyme of the invention is particularly capable of catalyzing the following stereoselective reaction (A)

S- (or R) amine acid R- (or S) amide

¹ 2 3 4 or

cis (or trans) wherein the racemic acylamide of the formula (1) is stereoselective^ hydrolyzed with an amido hydrolase of the invention, which is a polypeptide with amido hydrolase enzymatic activity but without lipase- or esterase-activity, and which is capable of stereoselective^ hydrolysing the racemic acylamide of formula (1 ), and wherein said hydrolysis results in the R- (or S-) amine of the formula (2) and the acid of formula (3) and the S- (or R-) amide of formula (4), wherein in formula (1) to (4) .

R¹ is NH₂ or O-alkyl

R² is aryl or d-C₄aryl; unsubstituted or substituted by C₁-C₄ alkyl, C₁-C₄BIkOXy, C₁-C₄ hydroxyalkyl, C₁-C₄ aminoalkyl, C₁-C₄ haloalkyl, hydroxy, amino, halogeno, nitro, sulfo or cyano; or wherein the rings may contain one or two heteroatoms selected from nitrogen, sulfur and oxygen; preferably indane, tetraline or chromane, unsubstituted or substituted with methoxy, ethoxy, halogeno, methyl, ethyl, nitro, cyano, amino, hydroxy, trifluoromethyl; and

R³ is H or an aliphatic acyl residue; preferably C₁-C₄ alkyl; and more preferably methyl.

Aryl is, for example a homo- or heterocyclyl. A suitable ring system is, for example, a single or double ring system having from 3 to 10 ring atoms, is bonded via a carbon atom or via a nitrogen atom and contains up to 4 hetero atoms selected from oxygen, nitrogen, sulfur, and sulfur linked to 1 or 2 oxygen atoms; which in addition may also be fused with 1 or 2 phenyl radicals or with 1 or 2 cycloalkyl radicals, cycloalkyl preferably having from 5 to 7 ring atoms; and which may be unsaturated or partially or fully saturated. Examples for aryl are phenyl, napthyl, biphenylyl, anthryl, fluorenyl, thienyl. furyl, pyrrolyl, imidazolyl, pyrazolyl, oxazolyl, thiazolyl, tetrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolyl, benzimidazolyl, quinolyl, isoquinolyl, 3,1-benzofuranyl, chromanyl, cyclohexa- [b]pyrrolyl, cyclohexa[b]pyridyl, [b]pyrimidinyl, pyrrolidinyl, pyrrolinyl, cyclohexa[b]pyrazinyl, cyclohexa[b]pyrimidinyl, imidazolidyl, piperidyl, piperazinyl, morpholinyl, thiomorpholinyl, S,S-dioxo-thiomorpholinyl, indolinyl, isoindolinyl, 4,5,6,7-tetrahydro indolyl, 1 ,2,3,4- tetrahydroquinolyl or 1 ,2,3,4-tetrahydroisoquinolyl, for example one of the last-mentioned radicals, being unsubstituted or substituted by one or more substituents selected from lower alkyl, e.g. methyl, phenyl, 1- or 2-naphthyf, phenyl-lower alkyl, e.g. benzyl, hydroxy-lower alkyl, e.g. hydroxymethyl or 2-hydroxyethyl, hydroxy, lower alkoxy, e.g. methoxy or ethoxy, amino, lower alkylamino, e.g. methyl-, ethyl- or tertbutyl- amino, di-iower alkylamino, e.g. dimethyl- or diethyl-amino, carboxy, lower alkoxycarbonyl, for example methoxy-, isopropoxy-, sec-butoxy- or tert-butoxy-carbonyl, phenyl- or naphthyl-lower alkoxycarbonyl, e.g. benzyloxycarbonyl, halogen, for e fluorine, chlorine, bromine or iodine, especially chlorine or bromine, lower alkanoyl, e.g. acetyl or pivaloyl, nitro, oxo and/or by cyano.

In a preferred embodiment aryl is unsubstituted or substituted by one or more substituents selected from methyl, hydroxymethyl, 2-hydroxyethyl, hydroxy, methoxy; ethoxy, amino, methyl-amino, ethyl-amino, tert-butyl-amino, dimethylamino, diethyl-amino, carboxy, methoxy-carbonyl, isopropoxy-carbonyl, sec-butoxy-carbonyl, tert-butoxy-carbonyl, fluorine, chlorine, bromine, acetyl or pivaloyl, nitro, oxo and/or by cyano.

In a preferred embodiment of the above reaction A, a racemic acylamide is hydrolyzed wherein R³ is H or Ci-C₃ alkyl, more preferably Cialkyl. In particular, if an hydrolase obtainable from Rhodococcus globerulus or Rhodococcus equi is used, R³ is most preferably d-Caalkyl.

The term 'substituted' means that the moiety in question can be substituted by one to three identical or different substituents, preferably one or two identical or different, most preferably by one substituent selected from the group consisting of Ci-C₈ alkyl (preferably methyl), haloalkyl (preferably trifluoromethyl), halogen (preferably fluorine or chlorine), amino, nitro, and Ci-C₈ alkoxy (preferably methoxy).

In accordance with the present invention aryl standing alone or being member or an aralkyl moiety is a carbocyclic radical in which at least one ring is in the form of a 6-membered aromatic ring (i.e. a benzene ring). Preferred are phenyl, naphthyl, such as 1- or 2- naphthyl, biphenylyl, such as, especially, 4-biphenylyl, anthryl, and fluorenyl, and also such ring systems having one or more fused saturated rings.

In a preferred embodiment aralkyl stands for an aliphatic radical substituted by an aryl moiety, wherein the aliphatic radical is an unbranched or branched Ci-C₈ alkylene (preferably Ci-C₃ alkylene, most preferably methylene or ethylene) and the aryl moiety is a carbocyclic radical as defined above. In a more preferred embodiment aralkyl stands for a radical of the formula

wherein

R4 and R5 are each independent of each other hydrogen, C₁-C₄ alkyl, C₁-C₄ haloalkyl, halogeno, amino, cyano, nitro or C₁-C₄ alkoxy; preferably hydrogen, methyl, ethyl, trifluoromethyl, fluorine, chlorine, bromine, iodine, amino, nitro, cyano or methoxy; more preferably hydrogen, methyl, fluorine, chlorine, cyano, nitro or methoxy.

Further preferred compounds are

wherein n is an integral number selected from 0, 1 and 2; R4 and R5 are each independent of each other hydrogen or cyano.

Further preferred compounds are of formulae

wherein X is oxygen, carbon or nitrogen; preferably oxygen and carbon; and R4 and R5 are each independent of each other hydrogen, C₁-C₄ alkyl, C₁-C₄ haloalkyl, . halogeno, amino, cyano, nitro or C.-C4 alkoxy; preferably hydrogen, methyl, ethyl, iodo, bromo, trifluoromethyl, chloro, fluoro, amino, cyano, nitro, methoxy or ethoxy.

In a preferred embodiment the present invention provides the hydrolysis of trans- and cis-N- acetyl-2-aminocyclohexane carboxylic acid methyl ester:

(±)-trans-fa 1 S,2S-1 1R,2R-1a

(±)-cis-2a 1S.2S-2 1R,2R-2a

In another preferred embodiment of the invention an enzyme of the invention is capable of catalyzing the following stereoselective reaction:

(±)-3a-"/5a (R)-3a-15a (S)-3-15

(S)-3a-^5a

wherein a N-acetyl methyl ester derivative of the formula 3a-15a is stereoselective^ hydrolised with an amido hydrolase of the invention, which is a polypeptide with amine hydrolase enzymatic activity and which is capable of stereoselective^ hydrolysing the racemic substrate of formula 3a to 15a, wherein 3a to 10a is

and R1 is

3a: R1 is hydrogen

4a: R1 is methyl formate

5a: R1 is tert-butyl

6a R 1 is methoxy

7a: R1 is fluoro

8a: R1 is chloro

9a: R1 is phenyl

10a: R1 is nitrogen dioxide

and wherein 11a to 14a is

wherein

11a: R1 is hydrogen, R2 is methoxy and R3 is hydrogen 12a: R1 is methoxy, R2 is methoxy and R3 is hydrogen 13a: R1 is R2 is ethylene oxide, R3 is hydrogen 14a: R1 is hydrogen, R2 is methoxy, R3 is methoxy

and wherein 15a is

The biocatalyst may be obtainable from a microorganism selected from the group of Rhodococcus globerulus K1/1 , DSM 10337, and Rhodococcus equi Ac6, DSM 10278.

Example

400mg of each racemic substrate (±)-3a-15a was dissolved in 5 ml of methanol and the solutions were added to 50 ml of distilled water. Finally 50ml cell suspension of Rhodococcus equi DSM 10278 or Rhodococcus globerulus DSM 10337, obtained by addition of 8.5g wet cells in 900 ml phosphate buffer pH7.0, was added and the mixtures were shaken at 200 rpm and 30°C. After 64 hours the pH was adjusted to 2 by adding cone. HCI and extracted 3 times with CH₂CI₂. The organic phases were dried (MgSO₄) and the solvent removed under vacuum. The obtained residues were analyzed by chiral HPLC. The pH of the aqueous phases containing the amines 3-15 was adjusted to 8 and acetic acid anhydride was added. Usual workup gave the enantiomeric N-acetyl derivatives which were also submitted for HPLC-analysis.

The selectivities (E) for each substrate are summarized in table 1 :

Table 1

Deposition of Microorganisms

The following microorganisms are deposited according to the Budapest Treaty with the DSM - Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig. The deposition has been done as disclosed in the filing of the published International Application WO 97/41214.

Rhodococcus globerulus K1/1 , DSM 10337 (deposited September 23, 1995) Rhodococcus equiAcβ, DSM 10278 (deposited September 23, 1995)

Claims

1. A process for the synthesis of beta amino acids comprising the hydrolysis of N-acetyl methyl ester derivatives, characterized in that a biocatalyst is used.

2. The process of claim 1 wherein the biocatalyst is obtainable from a microorganism selected from the group of Rhodococcus globerulus and Rhodococcus equi.

3. The process of claim 1 or 2 wherein the biocatalyst is obtainable from a microorganism selected from the group of Rhodococcus globerulus, DSM 10337, and Rhodococcus equi Ac6, DSM 10278.

4. The process of any preceding claim wherein the biocatalyst is stereoselective^ hydrolyzing the N-acetyl methyl ester derivatives.

5. The process according to any preceding claim, which has the following reaction scheme,

R- (or S) amide

4 or

cis (or trans) wherein the racemic acylamide of the formula (1) is stereoselective^ hydrolyzed with an amido hydrolase of the invention, which is a polypeptide with amido hydrolase enzymatic activity but without lipase- or esterase-activity, and which is capable of stereoselective^ hydrolysing the racemic acylamide of formula (1 ), and wherein said hydrolysis results in the R- (or

S-) amine of the formula (2) and the acid of formula (3) and the S- (or R-) amide of formula (4), wherein in formula (1 ) to (4) .

R¹ is NH2 or lower alkyl

R² is aryl or Ci-C₄aryl; unsubstituted or substituted by Ci-C₄alkyl, Ci-C₄alkoxy, C₁-

C₄hydroxyalkyl, C₁-C₄aminoalkyl, Ci-C₄haloalkyl, hydroxy, amino, halogeno, nitro, sulfo or cyano; or wherein the rings contain one or two heteroatoms selected from nitrogen, sulfur and oxygen; preferably indane, tetraline or chromane, unsubstituted or substituted with methoxy, ethoxy, halogeno, methyl, ethyl, nitro, cyano, amino, hydroxy, trifluoromethyl; and

R³ is H or an aliphatic acyl residue; preferably d-C₄alkyl; and more preferably methyl.

6. The process of claim 1 to 5 wherein the reaction medium is distilled water.

7. A biocatalyst catalyzing the process of claims 1 to 6.

8. The biocatalyst according to claim 7 being an amido hydrolase.

9. The biocatalyst of claim 7 or 8 obtainable from a microorganism selected from the group consisting of Rhodococcus globerulυs K1/1 , DSM 10337, and Rhodococcus equi Ac6, DSM 10278.

10. The beta amino acids obtained by the process of claim 5.