WO1989012688A1

WO1989012688A1 - Process for synthesising l-alpha-amine or l-alpha-amino-n-hydroxyl acids by means of enzymes

Info

Publication number: WO1989012688A1
Application number: PCT/FR1989/000309
Authority: WO
Inventors: Jean-Claude Guilleux; Gilbert Renard; Jean Grimaud
Original assignee: Ecole Nationale Superieure De Chimie
Priority date: 1988-06-16
Filing date: 1989-06-16
Publication date: 1989-12-28
Also published as: FR2632969A1; FR2632969B1

Abstract

The process of the invention is remarkable in that a derivative of the transacrylic acid of formula (I) is reacted in an aqueous solution of ammonia or hydroxylamine in the presence of microbian ammonia-lyase produced by a culture of micro-organisms with such an activity, and the product formed of formula (II) is separated. In these formulae, R1 is a hydrogen atom or an alcoyl, R2 is a hydrogen atom or a hydroxyl and R3 is an aromatic or pseudo-aromatic radical. The products obtained are useful especially in peptide synthesis.

Description

The invention relates to a process for the synthesis by bioconversion of L-alpha-amino or L-alpha-amino acids. -N-hydroxylated.

The synthesis of alpha-amino acid analogues of very high optical purity or of L-alpha-amino acids not belonging to the 20 natural amino acids is of great importance, in particular in peptide synthesis for incorporation of these derivatives to oligopeptides. In isolated form, they can be used as enzyme inhibitors, as intermediates for the preparation of antibacterial agents, pharmaceutical specialties and chemical reagents.

Obtaining these amino acids is usually carried out by organic synthesis. In the case of chiral compounds, it is then necessary to have, beforehand, starting compounds which themselves have a chirality center, namely a carbon atom having four different substituents, the synthesis of which is not always easy. Biotechnological methods have also been proposed, for example the splitting of raciques by the action of an enzyme such as pancreatic carboxypeptidase. However, these techniques are difficult to implement on an industrial scale.

Acid bioconversion techniques catalyzed by enzymes produced by microorganisms have been reported, but only to obtain natural amino acids. Thus, in patent FR 2262 110 in the name of Pfizer Inc., the production of L- is described. phenylalanine enzymatically from trans-cinnamic acid using microorganisms capable of producing phenylalanine ammonia-lyase, in particular of the genus Rhodotorula rubra, Rhodotorula gracilis, Rhodotorula texensis, Rhodotorula marina, Rhodotorula minuta and Fusarium oxysporum.

Patent EP 0140713-A2 in the name of Genex Corporation describes the use of ammonium carbonate or carba ate for obtaining phenylalanine from trans-cinnamic acid.

Patent JP 8643993 in the name of Kyowa Hakko Kogyo Co. Ltd. also describes the enzymatic bioconversion of cinnaic acid, p-coumaric acid or fumaric acid to produce L- phenylalanine, L-tyrosine and L-aspartic acid using Sporobolo yces roseus cells, IFO 1040. Bioconversion is carried out in a column, the cells of microorganisms being immobilized on carageenan-KCl.

The inventors have found that it is possible to use an ammonia lyase for the synthesis of amino acids other than the twenty natural amino acids, in particular amino acids with quaternary chiral carbon, ie active on polarized light, using certain acrylic acid derivatives as starting materials.

The object of the invention is therefore to provide a method of synthesis, by enzymatic route, of easy implementation, of non-natural chiral alpha-amino acids, that is to say different from the twenty natural amino acids, of structure S corresponding to the optical isomer L. It also aims to provide a process for obtaining L-alpha-amino-N-hydroxylated acids. She has further intended to provide L-alpha amino acids, alpha methylated.

The synthesis process of the invention is characterized in that a derivative of the trans-acrylic acid of formula (I) is reacted:

in an aqueous ammonia or hydroxylamine solution, in the presence of microbial amia lyase, and the L-alpha-amino acid formed of formula (II) is separated from the reaction mixture:

COOH

c H ₂

R ₃

The substituents Ri and R3 have the following meanings in formulas (I) and (II):

Ri: hydrogen atom, alkyl radical in particular from C 1 to C, in particular methyl radical,

- Rz: hydrogen atom or hydroxyl group, - R3: aromatic or pseudo-aromatic radical, in particular phenyl radical, optionally substituted in ortho and / or meta and / or para position in particular by a halogen atom or a group nitro, a 2- thienyl, -2-furyl or 3-pyridyl, 1-naphthyl, 2-naphthyl radical, it being understood that, when R3 represents a unsubstituted phenyl radical and R 2 a hydrogen atom, R 1 represents an alkyl radical.

According to a preferred embodiment of the invention, the trans-acrylic acid of formula (I) is bioconverted with the cells of a strain of microorganisms having L-pseudo-aromatic or L-aromatic activity. microbial ammonia lyase. Unexpectedly, the microbial ammonia lyase which catalyzes in the microorganism strain the synthesis of natural products, proves capable of ensuring the bioconversion of products which are not naturally synthesized by the cell.

Advantageously, it turns out that the use of an acrylic acid derivative comprising as group R3, that is to say in the trans position relative to the carboxyl group, a group of aromatic or pseudo-aromatic character , makes it possible to have an acid convertible by the enzymatic route and to obtain an alpha-amino or alpha-amino-N-hydroxylated acid of high optical purity.

It will also be noted that the starting acrylic acid derivative may comprise a group R 1 having an alkyl meaning.

The process of the invention thus makes it possible to obtain amino acids which are not part of the twenty natural amino acids, with quaternary chiral carbon which are inaccessible by the methods of transamination or by the methods of α ino-oxidase in reverse mode.

To carry out bioconversion, we use strains from cultures on media rich in varied amino acids, but poor in mineral salts, particularly in chlorides, and poor in carbohydrates.

The microorganism strain used which must be compatible with the acrylic derivative to be transformed which penetrates into its cells and must ensure the excretion of the transformed product, advantageously exhibits an ammonia lyase activity of at least 3 U / mg of dry cells (approximately 0 .5 U / mg cells

^ ° wet) including 3 to 18 U / mg of dry cells.

As a strain of microorganisms, use is preferably made of a strain of the genus Rhodotorula, in particular Rhodotorula gracilis or glutinis. A strain particularly suitable for implementing the invention consists of the strain of Rhodotorula gracilis or glutinis NRRL Y1091.

The cells of the strain used are washed and suspended in an isotonic solution. It is particularly suitable

²⁰ to use a sodium hydrogen carbonate solution.

The cells can be free. As a variant, they can be immobilized for example according to the karageenan method as described in patent JP 8643993 which makes it possible to conserve the cellular properties.

According to the invention, the bioconver¬ sion is carried out in the presence of ammonia or hydroxylamine. These reagents are advantageously added to the reaction medium before introducing the

30 derivative of acrylic acid (I).

The use of hydroxylamine in place of ammonia allows the desired amino acid to be obtained with greater flexibility, at basic pHs. Indeed, hydroxylamine is not volatile at room temperature, its recovery is easier than that of ammonia during evaporation and recovered medium can be reused immediately for a new bioconversion with possibly a pH adjustment. In addition, the rate of formation of the desired amino acid turns out to be faster.

Advantageously, it is possible to synthesize chiral substituted hydroxylamines when the hydroxylamine is used at an acidic pH, in particular less than 6. To prepare derivatives in which

Rz is a hydrogen atom, that is to say alpha-amino acids, ammonia or hydroxylamine is used, by bringing the pH of the bioconversion medium, before the addition of the derivative of acrylic acid (I), respectively between 10.2 and 10.8, or between 9.0 and 10.5. To this end, carbon dioxide, acetic acid or sulfuric acid are advantageously used.

When it is desired to prepare derivatives of alpha-amino-N-hydroxylated acids, hydroxylamine is used and the pH is reduced to acid values, of the order of 5 to 6.

The adduct which forms in the presence of ammonia or hydroxylamine, after adding the acrylic acid derivative (I) to the reaction medium, corresponds to formula (III):

The counterion A- comes from the reaction medium and is advantageously formed by a carbamate, an acetate or a sulfate.

The bioconversion is advantageously carried out under anaerobic conditions at a temperature above ambient, in particular of the order of 30 to 40 ° C. and more especially with stirring.

A temperature of 30 ° C gives a slower bioconversion, but preserves the solidity of the cell. A temperature of 40 ° C allows a faster reaction, but increases cellular waste. A satisfactory separation of the amino acid of formula (II) is obtained, after lowering the pH of the medium to 3 and centrifuging to remove the insolubles, by ion exchange on ionic resin.

According to an aspect of great interest, the extremely narrow stereospecificity of the site of action leads to the strict L isomer, of structure (S) without trace of the D isomer.

Because of their high optical purity, the L-alpha amino and L-alpha-amino-hydroxy acids obtained according to the invention are particularly useful in particular in peptide synthesis.

The incorporation of these unnatural amino acids into a chain of amino acids thus proves to be particularly advantageous for the manufacture in particular of peptide hormones, especially for veterinary use and of peptide lures. One can operate for example as described by Nestor J. Jo et al in "Peptides synthesis, structure, function", DH Rich and E. Gross, Illinois, eds, p.109-112, 1981 or in Int. J. Peptide, Protein, 2 £, 1987, 118-125.

The quaternary chiral carbon amino acids useful in themselves, can also constitute synthesis intermediates of great interest. Indeed, decarboxylation provides the corresponding amine, the synthesis of which by other routes proves difficult.

Other characteristics and advantages of the invention will appear in the examples of synthesis which follow. EXAMPLE 1

Obtaining L-3- (2-thienyl) -alanine or (S) -2-amino-3- (2-thienyl) -propionic acid of formula

Bioconversion 1

At 6 ml of a R. glutinis cell suspension having pseudo-aromatic ammonia-lyase activity, thawed, washed and equilibrated with 1.3% NaHCO 3, containing 3 g of wet cells and 3 ml of 1.3% NaHCO 3, add 24 ml of an 8 M ammonia solution, brought to pH 10.2 to 10.8 approximately, by bubbling carbon dioxide, and 100 mg of trans-3- (2-thienyl) -acrylic acid. The bioconversion is carried out in a 50 ml bottle tightly sealed and shaken in an incubator between 30 and 37 ° C, at 200 rpm. After 15 h of bioconversion, the mixture is centrifuged to remove the cells, and the supernatant removed in vacuo at 35 ° C under 0.5 mm Hg until dry. After taking up in 15 ml of water, the pH is lowered from 8 to 5 by addition of 12 M HCl with vigorous stirring. The precipitate, filtered through millipore, contains practically only unreacted 3- (2-thienyl) -acrylic acid and small quantities of peptides from the cell medium. After lowering the pH of the filtrate to 3, and elimination of the slight precipitate which appears, the solution is deposited on a cation exchange column of 2 g of Sephadex SP-C 25 ^R , washed with hydrochloric acid at pH 3. Elution with water saturated with cold carbon dioxide (pH 3.96) removes the last fractions of unreacted acrylic acid. By adding NH <CO3H to pH 6 of the eluting solvent, pH 4.9, 47.9 mg of L-3 (2-thienyl) -alanine or (S) -2-amino- acid are recovered. 3-propionic as described by Ferger et al in J. Biol., 174, 214-242 (1948), Beil. E III / IV 18 H631 p.8191, identified by its FAB mass spectrum, its nuclear magnetic resonance spectrum of the proton in D2O medium, the uniqueness of the dark green-blue spot by coloring with ninhydrin in layer chromatography thin on MACHEREY-NAGE chiral plate, Allenmark, J. Biochem. Biophys. Methods, £, 1-25 (1984), with an Rf of 0.66 for the methanol-water-acetonitrile eluting solvent (50/50/200), (D-3- (2-thienyl) -alanine having under the same conditions an Rf of 0.51), and finally a specific rotary power in water at 25 ° C for the sodium D line (c = 2) of -41.3 °. Bioconversion 2

The conditions for bioconversion are taken over with the exception of the replaced carbon dioxide with acetic acid to bring the pH of the 6M ammonia solution between 10.2 and 10.8. 44 mg of L-3- (2-thienyl) -alanine are obtained. Bioconversion 3

In another variant, the conditions of bioconversion 2 are repeated with the exception of the ammonia solution replaced by a solution in 8 M hydroxylamine water brought to pH between 9.0 and 10.4 by acetic acid to lead to 40 mg of L-3- (2-thienylyl) -alanine. EXAMPLE 2

Obtaining (S) -2-amino-N-hydroxyl-3- (2-thienyl) propionic acid of formula

The conditions of bioconversion 3 are repeated, but the pH is lowered between 5 and 6 by acetic acid to lead to 25 mg of a product identified in C.C.M. such as (S) -2-amino-N-hydroxyl-3- (2-thienyl) propionic acid, without L-3- (2-thienyl) -alanine. Various syntheses are indicated below, operating under the conditions of bioconversion 1, but replacing 3- (2-thienyl) acrylic acid with the appropriate acrylic acids. For each of these acids, the product formed is indicated respectively:

- trans-3- (2-furyl) acrylic acid:

L-3- (2-furyl) -alanine or (S) -2-amino-3- (2-furyl) propionic acid

- trans-3- (3-pyridyl) acrylic acid:

L-3- (3-pyridyl) -alanine or (S) -2-amino-3- (3-pyridyl) -propionic acid - alpha-methyl trans-cinnamic acid:

L-alpha-methyl beta-phenyl alanine, or (S) -2- a ino, 2-methyl, 3-phenyl propionic acid. - trans-3- (1-naphthyl) acrylic acid:

L-3- (1-naphthyl) -alanine or (S) -2 amnio-3- (l-naphthyl) propionic acid

-Trans-2- (1-naphthyl) acrylic acid:

L-3- (2-naphthyl) -alanine or (S) -2 amino-3- (2- ° naphthyl) propionic acid

By replacing, under the conditions of bioconversion 1, carbon dioxide with sulfuric acid, and 3- (2-thienyl) acrylic acid with the appropriate acrylic acids, we obtain: 5 -with acids 2-, 3 -, 4-fluoro trans-cinnamiques: respectively L-β— (o-), (m-,) - (p-fluorophenyl) - with respectively 87%, 79% and 59% of yield

- with 2-, 3-, 4-chloro-cinnamic acids: respectively L-β— (o-), (m-,) - (p-chlorophen ₁ ) - with respectively 84%, 65% and 24 % yield

- with 2-, 3-, 4-nitro trans-cinnamic acids: respectively L-β— (o-), (m -) - (p-nitrophenyl) - with respectively 49%, 45% and 17% of yield. EXAMPLE 3 Obtaining of 2-amino N-hydroxy-3-phenyl propionic acid (S) a) Preparation of immobilized cells 11.2% (w / v) of cells, 5% (v / v) of glycerol, ⁰ 0.2% (w / v) sodium glutamate,

1.5% (w / v) of sorbitol, 30% (w / v) of polyethylene glycol then bubbling with nitrogen of the solution called solution A

- preparation of a solution of sodium alginate at 4% ^ (w / v) bubbled with nitrogen called solution B,

REPLACEMENT SHEET - mixing solutions A and B at 25 ° C with vigorous stirring for 15 minutes,

- percolation of the mixture obtained in a solution (solution C) of 0.2M calcium chloride bubbled with nitrogen via a peristaltic pump and a tapered Pasteur pipette.

Beads are thus obtained, the average diameter of which is 2 mm. (A ball has an average mass of 20 mg and contains on average 3.56 mg of dry matter, i.e. 17.8 mg of wet matter).

The beads are allowed to stabilize in solution C for 1 hour. They are then collected by filtration and washed with water. ^ The beads can then be stored in a 0.1M calcium chloride solution at 4 ° C. b) Testing of immobilized cells

The reaction medium contains:

- 2.1 g by wet weight of immobilized cells

- 20 ml of hydroxylamine hydrochloride 6M pH = 5.3

- 100 mg of trans-cinnamic acid

After 2 h, a compound identified in TLC and HPLC appears as being (S) 2-amino N-hydroxy-3-phenyl propionic acid. 5

0

5

Claims

CLAIMS 1. Process for the synthesis of an L-alpha-amino acid different from the twenty essential amino acids, or of an L-alpha-amino-N-hydroxylated acid, using ammonia lyase, characterized in that that a trans-acrylic acid derivative of formula (I) is reacted:

R ₃ - CH = C - COOH (I)

in an aqueous solution of ammonia or of ydroxylamine, in the presence of microbial ammonia-lyase, and the L-amino acid formed, of formula (II), is separated from the bioconversion mixture:

the substituents Ri to R3 having in the formulas (I) and (II) the following meanings:

Ri: hydrogen atom, alkyl radical, in particular methyl, - R2: hydrogen atom or hydroxyl group,

R3: aromatic or pseudo-aromatic radical, in particular phenyl radical, optionally substituted in the ortho and / or meta and / or para position with a halogen atom or a nitro group, 2-thienyl, -2-furyl or 3 radical -pyridyle, 1-naphthyle, 2-naphthyle, it being understood that, when R3 represents a phenyl group and R2 a hydrogen atom, Ri is an alkyl radical. 2. Process for the synthesis of an L-alpha-amino acid different from the twenty natural amino acids, or of an L-alpha-amino-N-hydroxylated acid, characterized in that a derivative of the acid is reacted trans-acrylic of formula (I):

R ₃ - CH = C - COOH (I)

in an aqueous solution of ammonia or of hydroxylamine, in the presence of cells of a strain of microorganisms exhibiting in particular an L-pseudo-aromatic or L-aromatic activity of microbial ammonia lyase, and the bioconversion mixture is separated from the L-amino acid formed, of formula (II):

the substituents Ri to R3 having in the fc. mules

(I) and (II) the following meanings:

- Ri: hydrogen atom, alkyl radical, in particular methyl,

- R2: hydrogen atom or hydroxyl group,

- R3: aromatic or pseudo-aromatic radical, in particular phenyl radical, optionally substituted in the ortho and / or meta and / or para position by a halogen atom or a nitro group, 2-thienyl, -2-furyl radical or 3-pyridyle, 1-naphthyle,

2-naphthyl, it being understood that when R3 represents a phenyl group and

R2 is a hydrogen atom, Ri is an alkyl radical.

3. Method according to claim 2, characterized in that a strain of microorganisms having an ammonia lyase activity is used, advantageously at least 3U / mg of dry cells, in particular from 3 to 18 U / mg of dry cells, this strain being chosen in particular from the genus Rhodotorula, in particular Rhodotorula gracilis or glutinis, preferably the strain NRRL Y1091.

4. Method according to any one of claims 1 to 3, characterized in that to prepare derivatives of formula II in which R ₂ is a hydrogen atom, an aqueous solution of ammonia or hydroxylamine is used, in reducing the pH of the bi.oconversi.on medium respecti.vement between 10.2 and

10.8 or between 9.0 and 10.5.

5. Method according to any one of claims 1 to 3, characterized in that to prepare the derivatives of formula II in which Ri is a hydroxyl group, an aqueous solution of ydroxylamine is used by reducing the pH of the bioconversion medium between 5 to 6 approximately.

6. Method according to any one of claims 1 to 5, characterized in that the derivative of formula (II) recovered from the bioconversion medium is subjected to at least one purification step in particular using ion exchange columns .

7. Intermediate product corresponding to formula (III): NHs * A "or HONH ₂ ⁺ A"

R ₃ - CH ₂ - COOH (III)

in which R1 and R2 are as defined above and A "is an acetate, a carbamate or a sulfate.