DE2627069A1 - Sulphur contg. amino acids prodn. - by incubating substd. amino acids with methioninase and sulphur cpds. - Google Patents

Abstract

In prodn. of substd. S-contg. L-amino acids in which the sulphur is bound to the beta or gamma carbon atom of the amino acid, (a) a beta- or gamma-substd. L-amino acid is contacted with an S-contg. cpd. in a methioninase-contg. aq.reaction medium in order to accumulate the substd. S-contg. L-amino acid in the reaction medium; and (b) the substd. S-contg. L-amino acid is recovered from the medium. The beta- or gamma-substd. L-amino acid must be one which is converted into pyruvic acid or alpha-ketobutryric acid when it is held in an aqueous medium with methioninase. The S-contg. cpd. is one which contains an active H-atom and in which at least one free electron pair is assigned to the S-atom. The substd. S-contg. L-amino acid obtd. as prod. is a cpd. in which the original beta- or gamma-substitution is replaced by the entire nucleophilic moiety of the S-contg. cpd. remaining after removal of the active H-atom. Certain of the prods. are physiologically active, while others are used as additives for foods on account on their nutrient effect or their aroma properties. Further prods. are useful as intermediates for therapeutic agents or as experimental or analytical cpds. In an example, various amino acids are incubated with n-propyl mercaptan in a culture of a methioninase- produsing microorganisms. Incubation of L-homocystein with Pseudomonas taetrolens 1FO 3460 gives S-propyl-L-homocystein in a molar yield of 20%.

Description

Process for the preparation of substituted on sulfur-containing L-amino acids The invention relates to processes for the production of L-amino acids such as L-cysteine, L-homocysteine and their S-substituted derivatives.

Sulphurous. Amino acids in which the sulfur atom is attached to the ß- or the γ-position of the aliphatic chain is a known class of compounds that are useful for many purposes. Certain of these connections are known e.g. for their physiological activities. Some of them are as additives to standard mammalian foods either because of them inherent nutritional value cder as a flavor generator or -amplifier useful. They are also used as intermediates in synthetic manufacture of therapeutic agents usable You can also study and analyze, especially by comparing their physiological activities with the activities used by other materials0 There is therefore a need for methods for the preparation of such compounds, both experimentally and in technical scale.

It has now been found that substituted sulfur-containing L-amino only n, in which the sulfur is healthy on a ß- or γ-carbon atom, thereby Can be made by having a precursor amino acid in a methioninase containing aqueous medium and a precursor of the substituent in contact brings. In particular, methods have been found according to which γ-substituted Sulfur derivatives of γ-substituted -α-aminobutyric acid can be obtained can.

The term "substituted sulfur-containing L-amino acid" as it is in this context when describing the process according to the invention L-amino acids generated is used, means that in the generated amino acid a sulfur is bonded to the ß- or γ-carbon atom, and that this Sulfur itself with another atom like hydrogen or a group like phenyl, is substituted.

The method according to the invention is carried out by ß- or g-substituted L-amino acid in an aqueous medium, the methioninase contains, in the present of the precursor of the substituent in contact brings.

There can be a great variety of chemical sulphurous Compounds as a precursor of the substituent in the practice of Invention come to use. In general, they can be called sulfur-containing Compounds are defined that have an active hydrogen with at least one an undivided pair of electrons assigned to the sulfur atom.

In the course of the reaction, the entire molecule with the exception of the active hydrogen to the original ß- or g-substituted amino acid bound by union of the sulfur atom with the ß- or g-carbon atom. The active hydrogen combines with the original ß- or g-substituent to form a molecular by-product. In a sense, the response is a nucleophilic displacement reaction in which the substituent precursor minus the active hydrogen atom acts as the nucleophilic part.

Those sulfur-containing ones useful in practicing the invention Compounds can be represented by the general formula Y-H, in which Y is a sulfur-containing, nucleophilic fraction in which at least one undivided Electron pair (one unshared electron pair) is assigned to the sulfur atom.

Typical of the compounds that act as substituent precursors The practice of the invention can be used, are hydrogen sulfide and hydrogen persulfide, sulfurous acid, thiols such as alkyl, alkenyl, aryl and Aralkyl mercaptans, sulfenic acids including alkyl, alkenyl, aryl and aralkylsulfenic acids, Sulfinic acids including alkyl, alkenyl, aryl and aralkyl sulfinic acids. In these Compounds can preferably contain alkyl and alkenyl radicals up to 6 carbon atoms and aryl and aralkyl radicals can contain up to 12 carbon atoms and can be substituted by substituents which are inert towards the reaction.

Special compounds that may be used include Oysteine thiol, cysteine, homocysteine, cysteine sulfenic acid, homocysteine sulfenic acid, Homocysteine sulfinic acid, propyl mercaptan, phenyl mercaptan, o-methylphenyl mercaptan, Benzyl mercaptan, α-naphthyl mercaptan, hexyl mercaptan, hexenyl mercaptan, phenylsulfenic acid, p-ethylphenylsulfinic acid and α-thio-β-methylbaphthalene. Other typical compounds are in the examples illustrated.

The amino acid starting compounds that are used in the method according to the Invention can be applied by the general formula X (CH2) nCH (NH2) COOH in n, where n is 1 or 2, can be represented.

They are therefore ß-substituted alanines or γ-substituted α-amino acids. X can have a wide variety of substituents mean, many of which are illustrated in the examples.

The characteristic feature, however, by which experts recognize may include the alanine or α-aminobutyric acid derivative for the purposes of In accordance with the invention, if it is useful, it is ammonium ion to XH and either pyruvic acid or α-ketobutyric acid can be converted, if it is kept in an aqueous medium with methioninase0 It can therefore be so the usefulness of a compound as a starting compound can be determined by that the compound at a concentration of about 0.5 g / dl for about 16 Hours at about 25 to 30 ° C in an aqueous medium containing 1 g / dl KH2PO4, 2 g / dl K2PO4, 0.001 g / dl pyridoxal phosphate and 0.5 g / dl freeze-dried cells of Pseudononas ovalis IFO 3738 contains.

Other known sources of methioninase or methioninase preparations of different degrees of purity can be used, such as this for the Experts can be seen.

The presence of an α-ketobutyric acid in the aqueous solution can be determined by the known MTBH method, in which 3-methyl-2-benzothiazolone hydrazine is used as a test reagent (Agr. Biol. Chem. (Japan) 31, 1054 (1967)) pyruvic acid can be made according to the Friedeman T.E. and Haugen, G.E.J. Biol. Chem. 147, 415-442, (1943).

Typically useful substituents represented by X can include those represented by halogen unc by R1O-, R2S-, R2SO-, R2S02- and HS03, where R1 is hydrogen, acyl, alkyl, aryl and aralkyl and R2 is hydrogen, alkyl, aryl, aralkyl, HOOC CH (NH2) CH2S- or HOOC CH (NH2) CH2CH2S-, In these definitions, alkyl, aryl and aralkyl have the same meanings as above and acyl refers to substituents including formyl up to 5 Contain carbon atoms.

Methioninase (methionine-α-desamino- g-mercapt methane lyase) is a well-known enzyme that converts L-methionine to methyl mercaptan, ammonium ion and α-ketobutyric acid decomposed. The enzyme can in a known manner by various microorganisms such as Clostridium sporogenes (Cance, Research 33, 1862-1865), Eschericia coli (Medical Journal of Osaka University 2, 111-117), Pseudomonas ovalis, Pseudomonas taetrolens, Pseudomonas striata and Pseudomonas desmolytica (Summary of Report for the Annual Meeting of Agricultural Chemical Society of Japan 96, 1974). That Enzyme can be isolated from these sources by known methods.

For practical experience of the invention, methioninase can be used in various forms, including purified or crude methioninase preparations, intact cells of the microorganism that contain the methioninase activity, freeze-dried cells of the microorganism, cells of the microorganism that dehydrated with acetone, homogenates of cells of the microorganism and sonicates of the cells of the microorganism are used.

The aqueous reaction medium contains the enzyme or the enzyme source, the amino acid of the formula I and Y-H and typically pyridoxal phosphate and / or inorganic Ions.

The preferred concentration of the amino acid of the formula in the reaction mixture is 0.1 to 20%, and the concentration of Y-H is usually less than 10%. The substituent precursor can be in the form of a salt, preferably an alkali salt such as potassium or sodium salt.

The reaction temperature is preferably kept at 1 to 45 ° C. During the reaction, the pH of the reaction mixture is kept between 5 and 12. If the reaction is carried out for 10 to 40 hours, the yields are high of the Schefel-containing L-amino acid according to the invention collected in the reaction mixture.

The sulfur-containing L-amino acid in the reaction medium can by any known method such as precipitating at the pH of the isoelectric point be won.

Example 1 An aqueous culture medium was prepared which per dl 0.25 g L-methionine, 0.1 g peptone, 0.2 g glycerine, ol g KH2PO4, 0.1 g K2HPO4, o, ol g of MgS04X7H20 and o, o25 g of yeast extract, adjusted to a pH of 8.0 with NaOH, of which looo ml was placed in a 2000 ml shake flask and heated by steam.

Each of the microorganisms shown in Table I was poured into a pouring bottle inoculated and grown at 27 ° C. for 24 hours, cells in the resulting Culture broths were collected by centrifugation and freeze-dried.

Aqueous reaction media were prepared containing 32 inM of the amino acid, which is shown in Table I, 50 mM n-propyl mercaptan, o, ol mM pyridoxal phosphate and 0.5 g / dl of the freeze-dried cells contained, and there were lo ml aliquots of each mixture placed in 500 ml shake flasks and shaken at 370C for 16 hours, There were the molar yields of propylthio-substituted L-amino acid in the resulting reaction media was accumulated, determined and in the table I recorded.

Table 1 Amino Acid Microorganism Molar Yield (%) L-homocysteine Pseudomonas taetrolens IFO 3460 20 Pseudomonas ovalis IFO 3738 75 Pseudomonas striata IFO 12996 60 Pseudomonas desmolitica IFO 12570 51 L-γ-chloro-α-aminobutyric acid IFO 3460 28 IFO 3738 85 IFO 12996 60 IFO 12570 50 L-methionine IFO 3460 25 IFO 3738 80 IFO 12996 63 IFO 12570 57 Example 2 The aqueous growth medium of Example 1 (40 l) was placed in a 50 L fermentation tank and sterilized.

1 1 of a culture broth of Pseudomonas ovalis, prepared in a similar way Manner as in the procedure shown in example 1, was transferred to the fermentation tank, and breeding was carried out at 270C for 24 hours.

The cells in the resulting culture broth were centrifuged collected. All cells obtained were in 5 l of an o, ol M-phosphate buffer solution suspended, the 10-5 5 M-pyridoxal phosphate and o, ol% mercaptoethanol in one pH 7.2., suspended and the suspension of the cells was homogenized.

Purified methioninase preparations were homogenized from the Mixture by salting out with ammonium sulfate DEAE cellulose chromatography and hydroxyapatite chromatography obtained according to the usual working practices.

An aqueous reaction medium was prepared containing 20 mM of the amino acids shown in Table II, 50 wM ethyl mercaptan and o, ol mPI pyridoxal phosphate and adjusted to a pH of 8.0 with KOH. lo ml of the aqueous reaction media were mixed with 0.4 mg of the purified methioninase preparation and at 37 ° C. shaken for 16 hours.

The sulfur-containing L-amino acid generated in the reaction media was isolated and made using an ion exchange resin ("Amberlite 1R-120" (H #)), and identified by comparison with authentic samples.

Molar yields in Table II are the yield from ethionine the L-isomer of the amino acid shown in Table II Tabel II Amino Acid Molar Yield (%) (X- (CH2) n-CH (NH2) -COOH) DL-γ-fluoro-α-aminobutyric acid 70 L-γ-chloro-α-aminobutyric acid 95 DL- g-bromo-α-aminobutyric acid 83 DL-t-iodo-i-aminobutyric acid 13 DL-ß-fluoroalanine 30 L-ß-chloralanine 55 DL-B-bromoalanine 21 DL-ß-iodalanine 8 L-O-formylhomoserine lo L-O-acetylhomoserine 20 L-homoserine 81 L-O-methylhomoserine 55 L-O-phenylhomoserine 45 L-O-benzylhomoserine 85 L-O-formylserine 12 L-O-acetylserine lo L-azaserine 8 L-serine 67 L-O-methylserine 63 L-O-ethylserine 62 L-O-phenylserine 25 L-O-benzylserine 31 DL-homocysteine 75 DL-homocysteine sulfenic acid 13 DL-homocysteine sulfinic acid 24 DL-homocysteine sulfonic acid 30 L-methionine 92 Tabel II (continued) Amino Acid Molar Yield (%) (X- (CH2) n-CH (NH2) -COOH) DL-methionine sulfoxide 42 DL-methionine sulfone 74 DL-nethionine ul foximin 30 L-cystine 12 L-cystine sulfonic acid 10 L-cystinic acid 8 L-S-methylcysteine 67 Example 3 The purified methioninase preparation (0.1 mg) was added to 1 ml of an aqueous reaction medium containing 20 mM of the amino acid shown in Table III, 0.2 g dl Na2SO3, 50 mM of the sulfur-containing Compound shown in Table III, o, ol mM pyoxal phosphate with a pH-IJert of 8.0 (KOH) contained. The aqueous reaction mixture was at 37 ° C during Shaken for 16 hours.

Ten µl of the resulting reaction medium was poured onto a silica gel thin-layer plate applied in spots and developed with a solvent containing 40 parts of isobutyl alcohol, 20 parts of methyl ethyl ketone, 20 parts of ethanol, 1 part of water, 14 parts of concentrated Ammonia water and 5 parts acetone.

The Rf value of the ninhydrin positive spot on the plate was identical with that of the authentic sulfur-containing L-amino acid listed in Table III is shown.

The presence of radioisotopes in S35-methionine and 5 35-L-cysteine was determined by a liquid scintillation counter, Ten ml @ 20mM, 15 Mm, 10mM, 5mM and 1mM L-methionine solutions were also placed on the same silica gel plate sprayed as a stain and the color development of the sulfur-containing L-amino acids were compared to the spots of L-methionine. The symbols in the last column show that the degree of color development is close to the sulfur-containing L-amino acid that of 20 mM to 15 mM L-methionine, 15 ir to lo mM L-merthionine, lo mM to 5 with L-methionine, hzw. 5 to 1 mM L-irethionine pads.

In a manner analogous to that described above, L-methionine, 3-Ch lor-i, - aminohutters acid and γ-methoxy-α-aminobutyric acid each with Thiocysteine, γ-cresol, thioacetic acid, thioglycolic acid and thiolactic acid in Brought in touch. The Rf values of the ninhydrin positive spot of the compounds produced was from L-methionine, γ-chloro-α-aminobutyric acid and γ-methoxy-α-aminobutyric acid different and so new amino acids were created. Table III Used Sulfur used- Amount of sulfur-containing product in amino acid produced Compound containing L-amino acid reaction solution L-γ-chloro-α-methyl mercaptan L-methionine ++++ aminobutyric acid S35-methyl mercaptan S35-L-methionine ++++ methylsulfinic acid L-methionine sulfone +++ ethyl mercaptan L-ethionine ++++ propyl mercaptan L-propionine +++ isopropyl mercaptan S-isopropyl-L-homocysteine ++ n-butyl mercaptan S-n-butyl-L-homocysteine +++ tert-butyl mercaptan S-tert-butyl-L-homocysteine ++ sec-butyl mercaptan S-sec-butyl-L-homocysteine ++ thiophenol S-phenyl-L-homocysteine ++ benzyl mercaptan S-benzyl-L-homocysteine +++ ß-mercaptoethanol S- (ß-hydroethyl) -L-homocysteine + cysteamine S- (ß-aminoethyl) -L-homocysteine + ß-mercaptopropion S- (ß-carboxyethyl) -L-homoacid cysteine + dithiothreitol γ-dithiothreitoyl-L-α-aminobutyric acid + Methylsulfenic acid L-methionine sulfoxide ++ methanesulfinamide L-methionine sulfoximine + Table III (continued) Used Used sulfur-generated Amount of sulfur-containing product in amino acid-containing compound L-amino acid reaction solution Sodium sulfide L-homocysteine + hydrogen persulfide L-homocysteine thiol + L-ß-chloralanine Sodium sulfide L-cysteine ++ S35-sodium sulfide S35-L-cysteine ++ methyl mercaptan S-methyl-L-cysteine +++ benzyl mercaptan S-benzyl-L-cysteine ++ cysteamine S- (ß-aminoethyl) -L-cysteine ++ Propenylsulfenic acid S-methyl-L-cysteine sulfoxide + ß-carboxyisopropyl- S- (ß-carboxyisopropyl) mercaptan cysteine + allyl mercaptan S-allyl-L-cysteine ++ H2N-C (O) SH S-carbamyl-L-cysteine + Hydrogen persulfide L-cysteine thiol + sulphurous acid L-cysteic acid + L-homoserine S35-methyl mercaptan S35-L-methionine ++++ methylsulfenic acid L-methionine sulfone ++ Ethyl mercaptan L-ethionine ++++ propyl mercaptan L-propionine ++ isopropyl mercaptan S-isopropyl-L-homocysteine + Table III (continued) Used Used sulfur- Generated amount of sulfur-containing product in amino acid containing Compound L-amino acid reaction solution n-butyl mercaptan S-butyl-L-homocysteine +++ tert-butyl mercaptan S-tert-butyl-L-homocysteine + sec-butyl mercaptan S-sec-butyl-L-homocysteine + Thiophenol S-phenyl-L-homocysteine ++ benzyl mercaptan S-benzyl-L-homocysteine +++ ß-mercaptoethanol S- (ß-hydroethyl) -L-homocysteine + cysteamine S- (ß-aminoethyl) -L-homocysteine + ß-mercaptopropion S- (ß-carboxyethyl) -L-homoacid cysteine + dithiothreitol γ-dithiothreitoyl-L-α-aminobutyric acid + Methylsulfenic acid L-methionine sulfoxide + methanesulfinamide L-methionine sulfoximine + L-O-methylserine S35-hydrogen sulfide S35-L-cysteine ++ methyl mercaptan S-methyl-L-cysteine ++ Benzyl mercaptan S-Benzyl-L-cysteine ++ Cysteamine S- (ß-aminoethyl) -L-cysteine + Propenylsulfenic acid S-propenyl-L-cysteine sulfoxide + Table III Used Sulfur used- Amount of sulfur-containing product in amino acid produced containing compound L-amino acid reaction solution allyl mercaptan S-allyl-L-cysteine ++ H2N-C (O) SH S-carbamyl-L-cysteine + L-methionine S35-methyl mercaptan S35-L-methionine ++++ methylsulfinic acid L-methionine sulfone +++ ethyl mercaptan L-ethionine ++++ propyl mercaptan L-propionine +++ isopropyl mercaptan S-isopropyl-L-homocysteine ++ n-butyl mercaptan S-n-butyl-L-homocysteine +++ tert-butyl mercaptan S-tert-butyl-L-homocysteine ++ sec-butyl mercaptan S-sec-butyl-L-homocysteine ++ thiophenol S-phenyl-L-homocysteine ++ Benzyl mercaptan S-Benzyl-L-homocysteine +++ ß-Mercaptoethanol S- (ß-Hydroethyl) -L-homocysteine + Cysteamine S- (ß-aminoethyl) -L-homocysteine + ß-mercaptopropionic acid S- (ß-carboxyethyl) -L-homocysteine + Dithiothreitol γ-Dithiothreitoyl-L-α-aminobutyric acid + Tabel III (continued) Used Sulfur used- Amount of sulfur-containing product produced in amino acid containing compound L-amino acid reaction solution methylsulfenic acid L-methionine sulfoxide ++ methanesulfinamide L-methionine sulfoximine + L-cysteine S35-sulfur water- S35-L-cysteine +++ substance S-methyl mercaptan S-methyl-L-cysteine +++ benzyl mercaptan S-benzyl-L-cysteine ++ cysteamine S- (ß-aminoethyl) -L-cysteine ++ propenylsulfenic acid S-propenyl-L-cysteine sulfoxide + allylsulfenic acid S-allyl-L-cysteine sulfoxide ++ methylsulfenic acid S-methyl-L-cysteine sulfoxide + ß-carboxyisopropyl-S- (ß-carboxyisopropyl) -cysteine + mercaptan allyl mercaptan S-allyl-L-cysteine ++ H2N-C (O) SH S-carbamyl-L-cysteine +

Claims (1)

Claims 1 Process for the production of substituted sulfur-containing L-amino acids in which the sulfur is attached to the ß- or r -carbon atom of the amino acid is bound, characterized in that (a) a ß- or g-substituted L-amino acid in an aqueous reaction medium containing methioninase together with a sulfur-containing compound brings into contact to the substituted, sulfur-containing L-amino acid accumulate in the medium, and (b) the substituted sulfur-containing L-amino acid is recovered from the medium, with the ß- or γ-substituted L-amino acid is one that is converted to pyruvic acid or oC-ketobutyric acid, when kept in an aqueous medium with methioninase, the sulfur-containing Compound an active hydrogen atom and at least one undivided pair of electrons assigned to the sulfur contains, and the substituted sulfur-containing L-amino acid is one in which the original B or γ substitution by the entire nucleophilic part of the sulfur-containing compound is replaced, which after Removal of hydrogen from her remains, 2, procedure according to Claim 1, characterized in that the ß- or g-substituted L-amino acid is represented by the formula X (CH2) n CH (NH2) COOH, where n is 1 or 2 and X consists of halogen, R1O-, R2S- R2SO-, R2S02- and HS03, where R1 is hydrogen, acyl, Alkyl, aryl and aralkyl, and R2 is hydrogen, alkyl, aryl, aralkyl, HOOC CH (NH2) CH2S- and HOOC is CH (NH2) CH2CH2S-. 3. The method according to claim 1 or 2, characterized in that the sulphurous compound of hydrogen sulphide and sulfur persulphide, sulphurous Acid, alkyl, alkenyl, aryl and aralkyl mercaptans, alkyl, alkenyl, aryl and Aralkylsulfenic acids and alkyl, alkenyl, aryl and aralkylsulfinic acids. 4, method according to claim 3, characterized in that the alkyl and the alkenyl radicals contain up to 6 carbon atoms, and that the aryl and aralkyl radicals Contain up to 12 carbon atoms. 5. The method according to claim 2, characterized in that X is halogen is. 6. The method according to any one of claims 1 to 5, characterized in that that methioninase is shown by a microorganism belonging to the genus Pseudomonas is.