JP3081649B2 - Process for producing S-(+)-mandelamide and its derivatives - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing corresponding S-(+)-mandelamide and S-(+)-mandelamide derivative from mandelonitrile and a mandelonitrile derivative using a microorganism. S-(+)-
Mandelamide and its derivatives can be converted to S-(+)-mandelic acid and its derivatives by hydrolysis. S-(+)-mandelamide and S
-(+)-Mandelic acid and derivatives thereof are important substances as raw materials for synthesizing medical and agricultural chemicals.

[0002]

2. Description of the Related Art There are many inventions relating to a technique for producing a corresponding amide by hydrating a nitrile compound with a microorganism, and are disclosed in JP-B-56-17918, JP-B-59-37951, JP-A-62-91189 and JP-A-62-91189. 61-16219
No. 4 gazette. However, these reports are directed to nitrile compounds having no asymmetric carbon in the molecule.

With respect to the production of amides from nitriles having an asymmetric carbon in the molecule, microorganisms belonging to the genera Bacillus, Bacterium, Micrococcus and Brevibacterium act on D, L-α-aminonitrile. L-
A method for obtaining a mixture of an α-amino acid and a D-α-aminoamide (see Japanese Patent Publication No. 56-50031), a racemic α-amino-γ-methylthiobutiate using an A4 mutant strain of Brevibacterium sp. Lonitrile, α-aminopropionitrile, α-aminobutyronitrile, α-
Amino-β-phenylpropionitrile, α-amino-
A method of obtaining corresponding L-form amino acids and D-form aminoamides from γ-methylpentylnitrile and α-aminoisovaleronitrile at a mixing ratio of 50% [Adv. Bio
chem. Engineer. 14 see 1 (1980)), Pseudomonas, Rhodococcus, the genus Nocardia by the D, L-aminonitrile from optically active α- amino acid and / or α- aminoamide method for producing (JP-2- No. 31694) and about 40% ee from D, L-α-aminonitrile by a stereospecific hydrolase.
To obtain the L-α-aminoamide of JP-A-63-50
0004) is known.

[0004]

In order to obtain an optically active amide using these methods, the separation operation from coexisting amino acids is complicated, and the yield of the amide is, in principle, limited by the substrate. There was a problem that it could only be reduced to half.

[0005]

In view of the above-mentioned circumstances, the present inventors have conducted various studies to develop an efficient method for producing optically active mandelamide and its derivatives. As a result, using a microorganism, mandelonitrile and its derivatives, or It has been found that almost all of benzaldehyde and its derivative and a mixture of hydrocyanic acid can be converted to S-(+)-mandelamide and its derivative, thereby completing the present invention.

That is, the present invention relates to a method for preparing a racemic mandelonitrile and its derivative represented by the following general formula (1) or a mixture of benzaldehyde and its derivative represented by the following general formula (2) and hydrocyanic acid: Alternatively, in a basic polar medium, a microorganism having a capability of stereospecifically hydrating a nitrile group of racemic mandelonitrile and a derivative thereof represented by the general formula (1) or a treated product thereof is acted on. From the starting material, a racemic mandelonitrile and its derivative represented by the general formula (1) or a mixture of benzaldehyde and its derivative represented by the general formula (2) and a hydrocyanic acid, which is directly superior to the following general formula (3) Producing an optically active S-(+)-mandelamide or a derivative thereof represented by the formula:
A process for producing-(+)-mandelamide and its derivatives,
It is.

Embedded image [X represents hydrogen, a methyl group, a methoxy group, a hydroxyl group and a halogen. ]

According to the present invention having the above-described features, the mandelonitrile represented by the general formula (1) and its derivative easily racemize by dissociation equilibrium in a neutral or basic polar medium. By utilizing this, by conjugating the racemization reaction system with a microorganism capable of stereospecifically hydrating the nitrile group of racemic mandelonitrile and its derivative represented by the general formula (1), It is based on the finding found by the inventors that in principle all substrates can be converted stoichiometrically to S-(+)-mandelamide and its derivatives.

The microorganism used in the present invention belongs to, for example, the genus Rhodococcus, and specifically includes Rhodococcus s.
p. HN6-1 (Microtechnical Laboratory No. 11773), Rhodococcus sp. HT40-6 ( FERM BP-523)
1 ) and Rhodococcus sp. PN42-2 (Microtechnical Laboratory No. 11775) can be mentioned. All of these microorganisms have been newly discovered by the present inventors and have been deposited with the above-mentioned number at the National Institute of Bioscience and Biotechnology, National Institute of Advanced Industrial Science and Technology. It is as follows.

[Table 1]

[0009] The above mycological properties were determined by using the Bergez Manual of Systematic Bacteriology (Be
rgey's Manual of Systematic Bacteriology, 198
When classified according to 6), all three strains were identified as bacteria belonging to the genus Rhodococcus.

Next, an embodiment of the present invention will be described.
Culture of the microorganism used in the present invention can be assimilated glucose, glycerol, a carbon source such as saccharose, urea,
The reaction is performed using a normal medium containing a nitrogen source such as ammonium sulfate and ammonium nitrate, and inorganic nutrients such as magnesium chloride, calcium chloride and iron chloride essential for growth. Yeast extract, meat extract,
A medium to which a natural medium such as molasses is added can also be used. High enzyme activity by adding nitriles such as benzonitrile, benzyl cyanide, isobutyronitrile, or amides such as isobutylamide and phenylacetamide as enzyme inducers at concentrations that do not significantly inhibit growth from the beginning to the middle of culture Is obtained.

The pH of the medium to be used may be selected within the range of 4 to 10 and the culture temperature may be selected within the range of 5 to 50 ° C. The culture may be performed aerobically for about 1 to 14 days and continued until the activity becomes maximum.

The hydration reaction of the mandelonitrile or the like represented by the general formula (1) can be carried out by the method of the above-mentioned method for culturing the cells of microorganisms or the treated cells (crushed cells, crude / purified enzymes, immobilized enzymes, And a mixture of mandelonitrile or a derivative thereof or benzaldehyde or a derivative thereof and hydrocyanic acid in a polar medium, for example, an aqueous medium such as water or a buffer solution.

In the present invention, as described above, in order to racemize the substrate, mandelonitrile or its derivative, it is essential to keep the reaction system near neutral or basic, and the pH is 4 to 11, preferably 6 to 11. Adjust to 10. Although the concentration of the substrate cannot be specified unconditionally due to the sensitivity of the enzyme to benzaldehyde or hydrocyanic acid, usually, the amount of mandelonitrile and its derivative in the reaction solution is 0.1 to 10% by weight, preferably 0.2 to 5.0%. %, Benzaldehyde or a derivative thereof is 0.1 to 10% by weight, preferably 0.2 to 5.0% by weight, and hydrocyanic acid is 0.1 to 1.0% by weight, preferably 0.1 to 0.5% by weight. %. The amount of microorganisms and the like to be used as a raw material substrate is 0.
The reaction temperature is 0 to 50 ° C, preferably 10 to 30 ° C, and the reaction time is about 0.1 to 24 hours.

Thus, optically active S-(+)-is obtained in high yield from a mixture of racemic mandelonitrile and its derivative, or a mixture of benzaldehyde and its derivative and hydrocyanic acid.
Mandelamide is produced and accumulated. The product can be isolated using a known method such as concentration, extraction, or crystallization.

[0015]

According to the present invention, a predominant amount (50 to 1) is obtained from racemic mandelonitrile and its derivative, or a mixture of benzaldehyde and its derivative and hydrocyanic acid.
00%) of S-(+)-mandelamide, and all of the raw materials can be stoichiometrically converted to S-(+)-mandelamide, which is extremely efficient S-(+)
-It can provide a method for producing mandelamide and its derivatives.

[0016]

The present invention will be described below in more detail with reference to examples, but the examples do not limit the present invention.

Example 1 (1) Culture Rhodococcus sp. PN42-2, HN6-1 and H
Three strains of T40-6 were each inoculated and cultured aerobically at 30 ° C. for 72 hours.

Medium (pH 7.5) Glycerol
5g yeast extract 0.2g cyanide _{0.5g K 2 HPO 4 5gMgCl 2 0.2g} CaCl 2 40mg MnSO 4 · 4H 2 O 4mg FeCl 3 · 7H 2 O 0.7mg ZnSO 4 · 7H 2 O 0.1mg distilled 1000 ml of water

(2) Cells were collected from the hydration reaction medium, and each of the cells was centrifuged to separate the cells from a 20 mM phosphate buffer (pH
Washed 3 times in 7.5). The precipitated cells were suspended in 1.5 ml of the above buffer containing 14 mM mandelonitrile,
The reaction was carried out with shaking at 150C for 15 hours. The cell concentration used was OD ₆₁₀ = 15-30. After completion of the reaction, each reaction solution was centrifuged to remove cells, and the content of mandelamide in the supernatant was determined by liquid chromatography (column;
SHODEX ODSF511A, carrier; 0.2M
H ₃ PO ₄ : acetonitrile = 4: 1, monitor; 20
8 nm). The optical purity of the produced mandelamide was determined using an optical resolution column (CHIRALCEL CA).
-1, Daicel Chemical Industries, carrier; 100% ethanol). The results are shown in Table 1.

[Table 2]

Example 2 Rhodococcus sp. HT40-
Six strains were cultured and collected in the same manner as in Example 1, and washed by centrifugation. The precipitated cells were treated with 14 mM 2-chloromandelonitrile, 3-chloromandelonitrile, 4-chloromandelonitrile, 4-bromomandelonitrile,
-Including hydroxymandelonitrile, 4-methylmandelonitrile and 4-methoxymandelonitrile.
The suspension was suspended in 5 ml of a 20 mM phosphate buffer (pH 7.5), and the reaction was carried out at 30 ° C. with shaking for 15 hours. The bacterial concentration used was OD ₆₁₀ = 22. After the completion of the reaction, the quantification of the mandelamide derivative contained in the supernatant from which the cells were removed by centrifugation and the measurement of the optical purity were carried out by the methods described in Example 1. The results are shown in Table 2.

[Table 3]

Example 3 Rhodococcus sp. HN6-1
The strain and the PN42-2 strain were cultured and collected in the same manner as in Example 1, and washed by centrifugation. 14 precipitated cells
The suspension was suspended in 1.5 ml of 20 mM phosphate buffer (pH 7.5) containing mM 2-chloromandelonitrile, 3-chloromandelonitrile and 4-chloromandelonitrile, and the mixture was shaken at 30 ° C. for 15 hours. The reaction was performed. The bacterial concentration used was HN6-1 strain OD ₆₁₀ = 21, PN4
The 2-2 strain had an OD ₆₁₀ = 17. The quantification of the produced mandelamide derivative and the measurement of the optical purity were performed by the methods described in Example 1. The results are shown in Table 3.

[Table 4]

Example 4 Rhodococcus sp. HT40-
Six strains were cultured and collected in the same manner as in Example 1 and centrifuged to obtain 1 strain.
Washing was performed using a 00 mM phosphate buffer (pH 7.5). The precipitated cells were treated with 14 mM benzaldehyde,
-Chlorobenzaldehyde, 3-chlorobenzaldehyde, 4-chlorobenzaldehyde, 4-methylbenzaldehyde and 4-hydroxybenzaldehyde and 14
The suspension was suspended in 1.5 ml of 100 mM phosphate buffer (pH 7.5) containing mM potassium cyanide and reacted at 30 ° C. with shaking for 24 hours. The bacterial concentration used was OD ₆₁₀
= 14. The quantification of the produced mandelamide and its derivatives and the measurement of the optical purity were carried out by the methods described in Example 1. The results are shown in Table 4.

[Table 5]

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Claims (1)

(57) [Claims] 1. A neutral or basic polar solvent for a racemic mandeloniloryl and its derivative represented by the following general formula (1) or a mixture of benzaldehyde and its derivative represented by the following general formula (2) and hydrocyanic acid: In the above , Rhodococcus (Rhod) is added to the nitrile group of racemic mandelonitrile and its derivative represented by the general formula (1).
ococcus) or a treated product thereof, whereby a racemic mandelonitrile represented by the general formula (1) and a derivative thereof, or a benzaldehyde represented by the general formula (2) and a derivative thereof represented by the general formula (1) are reacted with the microorganism. The following general formula (3) in which a dominant amount is directly obtained from a mixture of hydrocyanic acid
Producing an optically active S-(+)-mandelamide or a derivative thereof represented by the formula:
A method for producing (+)-mandelamide and its derivatives.