JPH10215890A - Production of optically active alpha-(n-butoxycarbonyl)-diaminopropionic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain in an industrially advantageous way, the subject compound useful as a raw material for synthesizing medicines, agrochemicals or their intermediates by treating a, α-β-(n-butoxycarbonyl)-diaminopropionic acid with specific microbial cells and/or a product therefrom without changing its steric configuration. SOLUTION: This optically active α-(n-butoxycarbonyl)-diaminopropionic acid of formula II is obtained by treating α,β-di-(n-butoxycarbonyl)- diaminopropionic acid of formula I with a strain of microorganisms that selectively deprotect only its β-site, e.g. Brevibacterium linens IFO012142 or Pseudomonas sp. MC13361 (FERM P-15968) and/or a product therefrom.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an optically active α- (n
More specifically, the present invention relates to an efficient method for producing optically active α- (n-butoxycarbonyl) -diaminopropionic acid using a specific microorganism.

[0002]

2. Description of the Related Art Optically active α- (n-butoxycarbonyl) -diaminopropionic acid is an optically active compound used for various pharmaceuticals, agricultural chemicals and the like or a synthetic intermediate thereof. It is useful as a raw material for synthesis. In producing this optically active α- (n-butoxycarbonyl) -diaminopropionic acid, L-
Chemical synthesis method of methyl ester from α-benzyloxycarbonyldiaminopropionic acid as a starting material (Bioorg
anic & Medicinal Chemistry Letters 1996,6, p339)
There is. However, this method has a long process and the starting materials are not commercially available at the industrial production level, and cannot be said to be an inexpensive mass synthesis method.

[0003] Under such circumstances, optically active α- (n-
It has been desired to establish an economically excellent method for producing (butoxycarbonyl) -diaminopropionic acid. The present invention has been made in view of the above, and has an optically active α- (n-
An object of the present invention is to provide a novel method for efficiently producing (butoxycarbonyl) -diaminopropionic acid.

There have been several reports on methods for deprotecting N-protected amino acids using microorganism enzymes (J. Bio.
technology 1988, 7, p49, Biotechnology & Applied Bio
chemistry 1987, 9, p251, Bioorganic & Medicinal Chem
istry Letters 1992,2, p697, J. Biotechnology 1988,
9, p29, Can J. Microbiology 1984, 30, p1301, Agric. Bi
ol. Chem. 1986, 50, 1563), in these reports,
Examples of the N-protecting group include a benzoyl (Bz) group, a benzyloxycarbonyl (Cbz) group, a methoxycarbonyl group, an ethoxycarbonyl group, and a t-butoxycarbonyl (tBoc) group. The (nBoc) group has not been implemented.
In addition, there is an example in which N-α, ε- (protected) lysine is deprotected regioselectively, but there is no example in which N-α, β- (protected) diaminopropionic acid is used.

[0005]

According to the preliminary results of the present inventors, it has been found that α, β-di- (n-butoxycarbonyl) -diaminopropionic acid can be deprotected only from the α-position from a number of microorganisms. It was found that those which deprotect both α-position and β-position were easily found. about that,
Those which selectively deprotect α, β-di- (n-butoxycarbonyl) -diaminopropionic acid only at the β-position are not easily found.

In order to solve the above-mentioned problems, the present inventors conducted further intensive studies to develop an efficient method for producing α- (n-butoxycarbonyl) -diaminopropionic acid. Of β-di- (n-butoxycarbonyl) -diaminopropionic acid to selectively deprotect only the β-position to obtain α- (n-butoxy Carbonyl)
-Diaminopropionic acid was found to be obtained, and the present invention was completed. That is, the gist of the present invention is that the formula (1):

[0007]

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The α, β-di- (n-butoxycarbonyl) -diaminopropionic acid represented by the formula (1) is replaced by the α, β-di- (n-butoxycarbonyl) -diaminopropionic acid represented by the above formula (1) Characterized by the following formula (2) characterized by reacting cells of a microorganism having the ability to selectively deprotect β at the β-position and / or a preparation thereof to selectively deprotect only the β-position thereof:

[0009]

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The present invention relates to a method for producing an optically active α- (n-butoxycarbonyl) -diaminopropionic acid represented by the following formula (3):

[0011]

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L-α, β-di- (n-butoxycarbonyl) -diaminopropionic acid represented by the above formula is added to L-α, β-di- (n-butoxycarbonyl) represented by the above formula (3). The following formula, wherein a cell of a microorganism having the ability to selectively deprotect diaminopropionic acid at the β-position and / or a preparation thereof are allowed to act to selectively deprotect only the β-position thereof; (4):

[0013]

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A method for producing L-α- (n-butoxycarbonyl) -diaminopropionic acid represented by the following formula, and the following formula (5):

[0015]

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The DL-α, β-di- (n-butoxycarbonyl) -diaminopropionic acid represented by the above formula is added to the DL-α, β-di- (n-butoxycarbonyl) represented by the above formula (5). The following formula, wherein a cell of a microorganism having the ability to selectively deprotect diaminopropionic acid at the β-position and / or a preparation thereof are allowed to act to selectively deprotect only the β-position thereof; (6):

[0017]

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The present invention relates to a method for producing D-α- (n-butoxycarbonyl) -diaminopropionic acid represented by the formula: As a preferred embodiment of the present invention, the above formula (4)
In producing a compound represented by in Brevibacterium (Brevibacterium) genus and Pseudomonas (Pseudom
ona ) A method for treating cells of a microorganism selected from the group consisting of the genus and / or a preparation thereof; and, when producing the compound represented by the above formula (6), the genus Alcaligenes and Comamonas ( Comamonas ), and a method of acting on cells of a microorganism selected from the group consisting of the genus Comamonas and / or a preparation thereof.

[0019]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
The microorganisms used in the production method of the present invention include α, β
-Di- (n-butoxycarbonyl) -diaminopropionic acid is not particularly limited as long as it has the ability to selectively deprotect at the β-position. For example, genus Brevibacterium , Pseudomonas
(Pseudomonas) genus Alcaligenes (Alcaligenes) genus,
Examples of such microorganisms include microorganisms conforming to the genus Comamonas, and more specifically, Brevibacterium linens.
ibacterium linens ), Pseudomonas sp. ( Pseu
domonas sp.), Alcaligenes xylosoxydans (Alcaligenes xylosoxydans), and Comamonas testosteroni (Comamonas testosteroni), and the like.

Note that L-α, β represented by the above equation (3)
-Di- (n-butoxycarbonyl) -diaminopropionic acid is reacted with microbial cells of a microorganism and / or a preparation thereof to selectively deprotect at the β-position, thereby obtaining L represented by the above formula (4). When producing α- (n-butoxycarbonyl) -diaminopropionic acid, microbial cells and / or preparations thereof include Brevib
acterium ) and microorganisms selected from the group consisting of the genus Pseudomonas and / or preparations thereof, and DL-α, β represented by the above formula (5).
By reacting -di- (n-butoxycarbonyl) -diaminopropionic acid with microbial cells and / or a preparation thereof to selectively deprotect at the β-position, the optical represented by the above formula (6) is obtained. Active D-α- (n-butoxycarbonyl)
-When producing diaminopropionic acid, the cells of organisms and / or their preparations include alkaligenes ( Al
Caligenes) genus and Comamonas (Comamonas) cells of an organism selected from the group consisting of genus and / or their preparation thereof.

[0021] As a specific strain of the above microorganisms, Brevibacterium Rinensu (Brevibacterium line
ns) IFO12142 Pseudomonas sp. (Pseudomonas sp.) MCI3361 Alcaligenes xylosoxydans (Alcaligenes x
ylosoxydans ) IAM12600 Alcaligenes x
ylosoxydans) IAM12684 Comamonas testosteroni (Comamonas testosteron
i ) A strain such as ATCC11996 can be mentioned. The microorganism is a wild strain, UV irradiation, N-methyl-N'-nitrosoguanidine (NTG) treatment, ethyl methanesulfonate (EMS)
Any strain such as a mutant strain obtained by treatment, nitrite treatment, acridine treatment, or the like, or a recombinant strain derived by a genetic technique such as cell fusion or gene recombination may be used.

Further, among the above strains, Pseudomonas sp.
SP ( Pseudomonas sp.) All strains other than MCI3361 are known strains, respectively, (Fund) Fermentation Research Institute (IFO),
It can be easily obtained from the Institute for Molecular and Cellular Biology (IAM) of the University of Tokyo and the American Type Culture Collection (ATCC). The Pseudomonas sp. (Pseudomon
as sp.) For MCI3361 is a strain by the present inventors have found from the newly nature, it has been deposited as FERM P-15968 to the Ministry of International Trade and Industry Agency of life Institute of Advanced Industrial Science and Technology. The bacteriological properties of this strain are shown below.

<Mycological properties of MCI3361 strain> 1. Morphological properties (1) Cell shape Rod (2) Cell polymorphism None (3) Motility, monopolar flagella (4) Presence or absence of spores Cultural properties (1) Broth agar plate culture Circular, lenticular, smooth, light brown, translucent colony formation after culturing at 30 ° C for 2 days (2) Broth liquid culture Good growth, no surface growth (3) Broth gelatin puncture culture Liquefaction None (4) Litmus milk No change

3. Physiological properties (1) Gram stain negative (2) Nitrate reduction positive (3) Devaginal reaction positive (4) MR test negative (5) VP test negative (6) Indole formation negative (7) Hydrogen sulfide formation negative (8) Starch hydrolysis Negative (9) Utilization of citric acid Positive (10) Generation of dye No fluorescent dye was generated on King A and B media. (11) Urease-negative (12) Oxidase-positive (13) Catalase-positive (14) Range of growth: temperature 15 to 41 ° C: pH pH 5 to 9 (15) Attitude to oxygen No growth under anaerobic conditions (16) O- F test oxidation (17) Generation of acid from saccharide L-arabinose ± D-xylose ± D-glucose + D-mannose ± D-fructose ± D-galactose + maltose ± sucrose + lactose-trehalose-D-sorbitol-D -Mannitol-inositol-glycerin ± starch-(+: produces acid,-: does not produce, ±: cannot be said either)

4. 4. Other tests (1) Arginine hydrolysis positive (2) Esculin degradation negative (3) Galactosidase negative Chemical taxonomic properties (1) G + C content in DNA 64.7 mol% (2) Major isoprenoid quinone ubiquinone Q9 (3) Cell fatty acids C _{16: 0} , C _{16: 1} , C _{18: 0} 2-OH -C12: 0 3-OH-C _{10: 0} 3-OH-C _{12: 0}

6. Taxonomic considerations (1) Higher order identification MCI3361 strain is 1) Gram-negative bacillus, 2) aerobic, 3) does not form spore, 4) has motility by monopolar flagella,
5) The major isoprenoid quinone has ubiquinone Q9; 6) The G + C content in DNA is 64.7 mol%. Based on these characteristics, this strain is known as Bergey's Manual of Systematic Bacteriol.
ogy], vol. 1, p. 140-219 (1984), suggesting that it belongs to the family Pseudomonadaceae.

(2) Identification of the genus At present, the genus Pseudomonadaceae ( Pseudomonadas ) and the genus Xanthomonas ( Xant
homonas), Zooglea genus (Zoogloea) and phratry genus (F
rateuria ). Among them, this strain is characterized by having ubiquinone Q9,
( Pseudomonas ). Also even in a physiological nature, the presence of nitrate-reducing ability and denitrification ability and the genus Xanthomonas (Xanthomonas), Zooglea genus (Zoogl
oea) urease with the production of, further phratry genus (Frate
uria ) in terms of oxidase production and acid resistance. Based on the above, the strain MCI3361 was identified as Pseudomonas sp.

In the production method of the present invention, one or two of microorganisms having the ability to selectively deprotect at the β-position by acting on the α, β-di- (n-butoxycarbonyl) -diaminopropionic acid described above. More than one species is used in the form of cells and / or their preparation. Specifically, the cells obtained by culturing the microorganisms as they are, or the cells obtained by culturing are treated with acetone, freeze-dried, or physically or enzymatically crushed. Preparations such as those prepared can be used. Further, from these cells or preparations, they act on the α, β-di- (n-butoxycarbonyl) -diaminopropionic acid represented by the above formula (1) to selectively deprotect at the β-position, The enzyme fraction having the ability to convert to optically active α- (n-butoxycarbonyl) -diaminopropionic acid represented by the above formula (2) can be extracted and used as a crude product or a purified product. Furthermore, it is also possible to use those obtained by immobilizing the cells, preparations, enzyme fractions and the like thus obtained on a carrier such as polyacrylamide gel and carrageenan gel. Therefore, in this specification, the term "microbial cells and / or their preparation" is used as a concept including all the above-mentioned microbial cells, preparations, enzyme fractions, and immobilized products thereof.

Hereinafter, the method for producing the optically active α- (n-butoxycarbonyl) -diaminopropionic acid of the present invention will be specifically described. In the production method of the present invention, “α, β-di- (n-butoxycarbonyl) -diaminopropionic acid” represented by the above formula (1) is used as a raw material,
The cells of the specific microorganism and / or their preparation are allowed to act on this, and the “optically active α” represented by the above formula (2) is obtained.
-(N-butoxycarbonyl) -diaminopropionic acid "is produced.

In the production method of the present invention, microorganisms are usually used after being cultured, and this culture can be used as usual. Examples of the medium used for culture include a carbon source such as glucose, fructose, sucrose, glycerin and citric acid, an inorganic nitrogen source such as ammonium sulfate and sodium nitrate, yeast extract, peptone, urea,
What contains an appropriate combination of an organic nitrogen source such as meat extract and corn steep liquor, inorganic salts such as magnesium and potassium, and phosphoric acid may be used. In addition to these components, it is also possible to add inorganic salts, trace metals, amino acids, or vitamins as substances for promoting the reaction activity. Further, α, β-di- (n-butoxycarbonyl) -diaminopropionic acid or n-butoxycarbonyl β-alanine can be used as an enzyme inducer. The culture is performed by adjusting the pH of the medium to a range of 3 to 10 and at a temperature of 10 to 45 ° C.
It is preferred to carry out until the activity is maximized in the range of 1 to 10 days.

In the present invention, the cells of the microorganism obtained by culturing as described above and / or a preparation thereof are combined with the above formula (1)
Α, β-di- (n-butoxycarbonyl)-represented by
Reaction is carried out by bringing diaminopropionic acid into contact with an aqueous medium to obtain an optically active α- (n-butoxycarbonyl) -diaminopropionic acid represented by the above formula (2) as a reaction product. As the aqueous medium used here, water,
A buffer solution or a culture solution may, for example, be mentioned, and the aqueous medium may appropriately contain a water-soluble organic solvent or a fat-soluble organic solvent.

The amount of α, β-di- (n-butoxycarbonyl) -diaminopropionic acid to be added to the reaction solution is such that the concentration in the reaction solution becomes 0.01 to 50% by weight. The α, β-di- (n-butoxycarbonyl) -diaminopropionic acid thus obtained does not have to be completely dissolved in the aqueous medium in the reaction solution. When substrate inhibition occurs in the reaction, α, β-di- (n-butoxycarbonyl) -diaminopropionic acid, which is consumed as the reaction proceeds, is added continuously or intermittently in the amount consumed. By proceeding, it is possible to further improve the accumulation amount of the product. The amount of the microbial cells and / or a preparation thereof added to the reaction solution is such that when the cells are added, the microbial cells are added to the reaction solution so that the cell concentration is about 0.01 to 20% by weight, or When a preparation such as an enzyme is used, the specific activity of the enzyme is determined, and an amount is added so that the enzyme concentration when added is equivalent to the above-mentioned cell concentration. Preferred reaction conditions for this reaction include a reaction temperature of freezing point to 70 ° C, preferably 10 to 40 ° C,
1, preferably 5 to 9, and the reaction time is about 1 to 100 hours.

As a reaction product of the above reaction, α- (n
-Butoxycarbonyl) -diaminopropionic acid is obtained, and the optically active α- (n
As a method for isolating (-butoxycarbonyl) -diaminopropionic acid, after removing cells and / or a preparation thereof by centrifugation or the like, an acid such as hydrochloric acid is added to the reaction solution to adjust the pH to 1-2. The salt of α- (n-butoxycarbonyl) -diaminopropionic acid which precipitates and is separated, or α- (n-butoxycarbonyl) -diaminopropionic acid is extracted with an organic solvent such as chloroform or ethyl acetate, and crystallized. And other known methods.

[0034]

Embodiments of the present invention will be described below. Production Example 1 α, β-di- (n-butoxycarbonyl)-
Synthesis of diaminopropionic acid 10 g of DL-diaminopropionic acid hydrochloride (Tokyo Kasei) was dissolved in 85 mL of an aqueous solution of NaOH (17 g), and 25 g of n-butoxycarbonyl chloride was added while stirring under ice-cooling. Adjusted to pH 2, extracted with ethyl acetate, dehydrated with sodium sulfate, concentrated and dried, and
22 g of a white solid of α, β-di- (n-butoxycarbonyl) -diaminopropionic acid was obtained. : ¹ H-NMR
(400 MHz, CD ₃ OD) δ 0.93 (m, 6
H), 1.39 (m, 4H), 1.60 (m, 4H),
3.39 to 3.70 (m, 4H), 4.02 (m, 2
H), 4.04 (m, 2H), 4.27 (m, 1H)

Production Example 2 Synthesis of L-α, β-di- (n-butoxycarbonyl) -diaminopropionic acid Except that the starting material was changed to L-diaminopropionic acid,
In the same manner as in Example 1, L-α, β-di- (n-butoxycarbonyl) -diaminopropionic acid was obtained.

Production Example 3 Synthesis of a sample of α- (n-butoxycarbonyl) -diaminopropionic acid According to the method described in J. Med. Chem. 1981, 24, p554, L-β-Cbz was prepared from L-diaminopropionic acid. -Diaminopropionic acid, which is reacted with n-butoxycarbonyl chloride to give L-α- (n-butoxycarbonyl) -β-
It was converted to Cbz-diaminopropionic acid and subjected to Pd-C catalytic reduction to obtain L-α- (n-butoxycarbonyl) -diaminopropionic acid. : ¹ H-NMR (400 MHz, D ₂
O) δ 0.78 (t, J = 7.2 Hz, 3H), 1.2
4 (m, 2H), 1.49 (m, 2H), 3.08 (d
d, J = 12.8, 8.4 Hz, 1H), 3.29
(Dd, J = 12.8, 5.2 Hz, 1H), 3.9
7 (m, 2H) and 4.10 (m, 1H) Similarly, DL-α
-(N-Butoxycarbonyl) -diaminopropionic acid was obtained.

[0037] Example 1 Brevibacterium Rinensu (Brevibacterium line
ns ) IFO12142 cells were harvested with 2.0% fructose, 1.0% yeast extract, 0.5% peptone, 0.5% meat extract and 0.02% n-butoxycarbonyl-β-alanine.
And inoculated into 100 ml of a medium having a pH of 7.0,
Aerobic shaking culture was performed at 30 ° C. for 48 hours. After cultivation,
The cells were collected by centrifugation, and a phosphate buffer (0.1 M,
After washing at pH 8.0), the cells were washed with L-α, β-di-
(N-butoxycarbonyl) -diaminopropionic acid 2
Suspended in 20 mL of the same buffer containing
For 24 hours.

After completion of the reaction, the reaction supernatant from which the cells were removed by centrifugation was concentrated to 5 mL under reduced pressure. The pH was adjusted to pH 2 by dropwise addition of concentrated hydrochloric acid to obtain 35 mg of a precipitated hydrochloride. ¹ H
As a result of -NMR spectrum measurement, it was in agreement with a sample of α- (n-butoxycarbonyl) -diaminopropionic acid.
In addition, reverse phase HPLC analysis of the reaction supernatant after completion of the reaction [Inertosyl ODS2, eluent: phosphate buffer 10 mM (pH
8.0) + 25 mM aqueous solution of sodium lauryl sulfate, flow rate; 0.5 ml / min, UV; 208 nm], and the resulting α- (n-butoxycarbonyl) -diaminopropionic acid (retention time: 10.5 min) It was 1.9 mg / mL when the amount of production in a liquid was measured.

Further, optical resolution HPLC [MCIGEL
CRS10W, eluent; 2 mM CuSO4-acetonitrile 7% aqueous solution, flow rate: 0.5 ml / min, UV; 25
4 nm] (retention time: D-isomer 14.2 minutes, L-isomer 16.6 minutes), it was 100% eee of the L-isomer.

Example 2 Pseudomonas sp. MCI3361
The same operation as in Example 1 was performed using the cells. The amount of the generated α- (n-butoxycarbonyl) -diaminopropionic acid was 2.0 mg / mL. The optical purity was L-form 100% ee.

Example 3 Using the cells of the microorganisms shown in Table 1, the same culture operation as in Example 1 was performed, and DL-α, β-di- (n-butoxycarbonyl) -diaminopropionic acid was used as a substrate. , And the same reaction operation as in Example 1 was performed. The amount of generated α- (n-butoxycarbonyl) -diaminopropionic acid and the optical purity were measured in the same manner as in Example 1. Table 1 shows the obtained results.

[0042]

[Table 1] Table 1 量 Production amount Optical purity Absolute configuration Microorganism [ mg / mL] [% ee] Alkaligenes xylosoxidans IAM12600 2.0 100 D Alcaligenes xylosoxidans IAM12684 1.8 100 D Comamonas testosteroni ATCC 11996 1.5 100 D ───────────────────────────────結果 From the above results, it can be seen that according to the production method of the present invention, optically active α- (n-butoxycarbonyl) -diaminopropionic acid can be produced efficiently.

[0043]

According to the production method of the present invention, an optically active α
It is possible to efficiently produce-(n-butoxycarbonyl) -diaminopropionic acid, which is industrially advantageous.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI (C12P 13/04 C12R 1:38) (C12P 13/04 C12R 1:05) (C12P 13/04 C12R 1:01) (C12P 41/00 C12R 1:13) (C12P 41/00 C12R 1:38) (C12P 41/00 C12R 1:05) (C12P 41/00 C12R 1:01) (C12N 1/20 C12R 1:13) (C12N 1/20 C12R 1:38) (C12N 1/20 C12R 1:05) (C12N 1/20 C12R 1:01) (72) Inventor Makoto Ueda 1000 Kamoshida-cho, Aoba-ku, Aoba-ku, Yokohama-shi, Kanagawa-ken Mitsubishi Chemical Corporation Yokohama Within the Research Institute

Claims (5)

[Claims] (1) Formula (1): Α, β-di- (n-butoxycarbonyl)-represented by
The diaminopropionic acid has α, represented by the above formula (1),
Microbial cells having the ability to selectively deprotect β-di- (n-butoxycarbonyl) -diaminopropionic acid at the β-position and / or a preparation thereof are allowed to act, and only the β-position is regioselectively selected. The following formula (2) characterized by deprotection: Optically active α- (n-butoxycarbonyl) represented by
-A process for producing diaminopropionic acid. (2) Formula (3): Is added to L-α, β-di- (n-butoxycarbonyl) -diaminopropionic acid represented by the following formula (3): L-α, β-di- (n-butoxycarbonyl) -diaminopropione The following formula (4) characterized in that cells of a microorganism having the ability to selectively deprotect an acid at the β-position and / or a preparation thereof are allowed to act to selectively deprotect only the β-position. : A method for producing L-α- (n-butoxycarbonyl) -diaminopropionic acid represented by the formula: 3. The method according to claim 1, wherein the microorganism is selected from the group consisting of a genus Brevibacterium and a genus Pseudomonas. (4) Formula (5): The DL-α, β-di- (n-butoxycarbonyl) -diaminopropionate represented by the above formula (5) is added to the DL-α, β-di- (n-butoxycarbonyl) -diaminopropionic acid represented by The following formula (6) characterized in that cells of a microorganism having an ability to selectively deprotect an acid at the β-position and / or a preparation thereof are allowed to act to selectively deprotect only the β-position. : A method for producing D-α- (n-butoxycarbonyl) -diaminopropionic acid represented by the formula: 5. The method according to claim 1, wherein the microorganism is selected from the group consisting of a genus Alcaligenes and a genus Comamonas.
Or the production method according to 4.