JP3313000B2 - Process for producing S-(+)-mandelamide and its derivatives - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a process for producing optically active S-(+)-mandelamide and its derivatives from racemic cyanohydrin or an aldehyde corresponding to the cyanohydrin and hydrocyanic acid 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 their derivatives are important substances as raw materials for synthesis of medical and agricultural chemicals.

[0002]

2. Description of the Related Art Hydration of nitrile compounds by microorganisms
Many inventions related to the technology to produce the corresponding amide
Production of amides from carbon-containing nitriles
Bacillus, Bacterium, Microco
Microorganisms belonging to the genus Kas and Brevibacterium are D, L
L-α-amino acid and D
-Method of obtaining a mixture of α-aminoamides (Table 1)
No. 50031), Brevibacterium R31
Using two A4 mutants, racemic α-amino-γ-
Methylthiobutyronitrile, α-aminopropionitrile
, Α-aminobutyronitrile, α-amino-β-fe
Nylpropionitrile, α-amino-γ-methyl pent
From nitrile and α-aminoisovaleronitrile
The corresponding L-form amino acids and D-form aminoamides are
A method of obtaining the mixture at a mixing ratio of% [Adv. Biochem. Engineer.
14 1 (1980)), Pseudomonas, Rhodo
D, L-Aminonito by the genus Coccus and Nocardia
Optically active α-amino acid and / or α-amino acid
Method for producing minoamide (JP-A-2-31694)
) And D, L-α-aminonitrile
About 40% ee of L-α-amino by specific hydrolase
Method for obtaining amide (see Japanese Patent Publication No. 63-500004)
Are known.

Further, when producing L-amino acids from α-aminonitriles, a method of selectively producing L-amino acids by suppressing the by-production of amides by the presence of the corresponding aldehyde (Japanese Patent Laid-Open No. However, the addition of aldehyde in this method is intended to prevent by-products of amides,
It is not intended to improve the reaction rate and productivity by adding the corresponding aldehyde.

[0004] On the other hand, some of the present inventors have argued that since these methods give a mixture of an optically active amide and an amino acid, a complicated separation operation is required to obtain one pure compound. In terms of production, there are various problems that need to be solved technically, such as the fact that the yield of amide can only reach half in principle, so all of the raw materials are converted to one optically active substance Development of a method to produce superior dominant amounts of optically active S-(+)-mandelamide and its derivatives directly from racemic mandelonitrile and its derivatives, or a mixture of benzaldehyde and its derivatives and hydrocyanic acid, using microorganisms (Japanese Patent Laid-Open No. 4-222)
No. 591).

[0005]

The problem to be solved by the present invention is disclosed in Japanese Patent Laid-Open No. 4-222.
The method described in No. 591 utilizes the property that racemic cyanohydrin as a raw material easily racemizes by dissociation equilibrium into benzaldehyde and hydrocyanic acid in an aqueous solution, and has a racemization and optically specific nitrile hydration activity. By combining with microorganisms, all of the cyanohydrin can be converted to S
-Conversion to mandelamide and its derivatives. However, the enzyme activity gradually decreased during the reaction, and the productivity of mandelamide per cell was not sufficiently high. Therefore, a problem was how to suppress the inactivation of the enzyme and increase the productivity of S-(+)-mandelamide and its derivatives.

[0006]

Means for Solving the Problems The present inventors have conducted studies to solve the above problems, and as a result, it has been found that by adding a specific amount of aldehyde corresponding to the starting material cyanohydrin during the reaction, the inactivation of the enzyme is suppressed. We have found that we have completed the present invention.

That is, the present invention provides a neutral or basic aqueous solution of a racemic cyanohydrin represented by the following general formula (1) or an aldehyde and a hydrocyanic acid represented by the following general formula (2) corresponding to the cyanohydrin. In a medium, a microorganism having the ability to stereospecifically hydrate the cyano group of the cyanohydrin or a treated product thereof is allowed to act,
The optically active S- represented by the following general formula (3), which is directly superior to the racemic cyanohydrin represented by the general formula (1) or the aldehyde represented by the general formula (2) and hydrocyanic acid,
In a process for producing (+)-mandelamide or a derivative thereof, a compound represented by the general formula (2) corresponding to the cyanohydrin:
Is within the range in which the aldehyde can be dissolved in the reaction system, and is 1 to 10 moles relative to the racemic cyanohydrin represented by the general formula (1), and 2 moles relative to the hydrocyanic acid.
A method for producing S-(+)-mandelamide and its derivatives, characterized in that the compound is added in an amount of about 11-fold mol.

[0008]

Embedded image [X is hydrogen, methyl, methoxy, isopropyl,
Represents a hydroxyl group, a nitro group, a sulfone group or a halogen]

In the present invention, as a substrate for a microbial reaction, racemic cyanohydrin represented by the general formula (1) or the following general formula (2) corresponding to the cyanohydrin, that is, dissociating equilibrium with the cyanohydrin in an aqueous solution: The aldehyde and hydrocyanic acid represented by are used.

As the cyanohydrin of the general formula (1),
For example, mandelonitrile, 2-methylmandelonitrile, 2-methoxymandelonitrile, 4-isopropylmandelonitrile, 4-hydroxymandelonitrile,
3- and 4-nitromandelonitrile, 3-sulfomandelonitrile, 2-, 3- and 4-chloromandelonitrile, 2-, 3- and 4-bromomandelonitrile, and the general formula (2) The aldehyde represented by
These are aldehydes corresponding to cyanohydrin of the general formula (1), such as benzaldehyde.

When using cyanhydrin as a substrate,
The concentration of the cyanohydrin is usually 0.1 to 10% by weight, preferably 0.2 to 5.0% by weight, and is within a range in which the aldehyde corresponding to the cyanohydrin can be dissolved in the reaction system. 1 to 10 times the molar amount,
Preferably, it is added in a molar amount of 1 to 5 times. When aldehyde and hydrocyanic acid are used as substrates, the hydrocyanic acid concentration is usually 0.1 to 1.0% by weight, preferably 0.1 to 0.5% by weight, and the aldehyde is dissolved in the reaction system. Within the range to be obtained, and 2 to 11 times mol, preferably 2 to 1 mol of hydrocyanic acid.
Add 6 times mol.

The amount of the aldehyde to be added varies depending on the type and concentration of the substrate used, but can be appropriately determined within the above range. If the amount added exceeds the range that can be dissolved in the reaction system, oil droplets of aldehyde will be generated, and the reaction rate will decrease, probably because the substrate is transferred to these oil droplets. The effect is small when the value is less than the above lower limit, while the effect level out even when the value exceeds the upper limit.

As a reaction medium, an aqueous medium such as water or a buffer is used to promote the racemization of cyanhydrin as a substrate.
That is, the pH is adjusted to 4 to 11, preferably 6 to 10.

The microorganism used in the present invention is, for example, ATCC 3327 as a microorganism belonging to the genus Rhodococcus.
8, Rhodococcus sp. HN6-1
1773), Rhodococcus sp. HT40-6 ( F
ERM BP-5231 ), Rhodococcus sp. PN
42-2 (Piercian Laboratories No. 11775) and Pseudomonas microorganisms include Pseudomonas chlororafis B23 (Pierce Laboratories No. 187).

Next, a general embodiment of the present invention will be described. Culture of microorganisms used in the present invention can be assimilated glucose, glycerol, a carbon source such as saccharose,
Nitrogen sources such as urea, ammonium sulfate and ammonium nitrate, magnesium chloride and calcium chloride essential for growth,
The reaction is performed using a normal medium containing an inorganic nutrient such as iron chloride. Further, those obtained by adding a natural medium such as yeast extract, meat extract, molasses, etc. to these mediums can also be used. Furthermore, enzymatic induction of nitriles such as benzonitrile, benzyl cyanide, isobutyronitrile, lactams such as ε-caprolactam, and amides such as isobutylamide and phenylacetamide at concentrations that do not significantly inhibit growth from the initial to the middle of culture. As a substance,
In addition, high enzyme activity can be obtained by adding cobalt ions, iron ions and the like as prosthetic groups of the enzyme.

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

The hydration reaction of the cyanohydrin or the like represented by the general formula (1) is carried out according to the method of the present invention. (Body, enzyme, etc.) in an aqueous medium such as water or a buffer solution adjusted to a predetermined pH, with a predetermined amount of a substrate and an aldehyde.

The amount of microorganisms and the like to be used is 0.01 to 5.0% by weight as dry cells based on the substrate, and the reaction temperature is 0 to 5.0%.
50 ° C., preferably 10-30 ° C., reaction time 1-72
About an hour.

Thus, optically active S-(+)-mandelamide and its derivatives are produced in high yield from racemic cyanohydrin of the general formula (1) or aldehyde of the general formula (2) and hydrocyanic acid. Stored. The product can be isolated using a known method such as concentration, extraction, or crystallization.

[0020]

The present invention will be described below in detail with reference to examples.

Example 1 (1) Culture In the following medium, Rhodococcus sp. PN42-2, HN6-1
And HT40-6 strain were inoculated, respectively, and aerobically cultured at 30 ° C. for 96 hours. Medium (pH 7.4) Glucose 27g polypeptone 4g Yeast extract 2g ammonium sulfate 0.2g ammonium nitrate _{2g MgCl 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 0.1 mg ε-caprolactam 4 g CoCl ₂ .6H ₂ O 30 mg 30 mM phosphate buffer (pH 7.4) 1000 ml

(2) Activity measurement Cells were collected from the culture medium, and each cell was centrifuged to remove 2 cells.
The plate was washed three times with 0 mM phosphate buffer (pH 7.5). The precipitated cells were suspended in the above buffer, and the suspension was added to a final concentration of 10%.
mM mandelonitrile and 0-40 mM benzaldehyde were added to a phosphate buffer (pH 7.5).
The reaction was performed while stirring at 0 ° C. for 30 minutes. The reaction was stopped by centrifuging the reaction solution to remove the cells. The content of mandelamide in the supernatant was analyzed by liquid chromatography (HPLC) (column; SHODEXODS F511A carrier; 0.1MH ₃ PO ₄ : acetonitrile = 4: 1, monitor; 254 nm). The activity was determined by defining the ability of 1 mg of dried cells at 30 ° C. to give 1 μmol of a product per 1 ml of the reaction solution per minute as 1 unit (1 U).
The activity of the benzaldehyde-added group is shown in Table 1 as a relative activity with respect to the non-added group as 100.

(3) Productivity To a phosphate buffer (pH 7.5) containing 10 mM mandelonitrile and 0 to 40 mM benzaldehyde, a very small amount (OD630 = 0.005 at the time of reaction) was added, and the mixture was stirred at 30 ° C. for 72 hours. While reacting. After the reaction, the content of mandelamide in the supernatant was measured, and the amount of amide produced per cell weight used was calculated. Table 1 shows the relative productivity when the productivity in the benzaldehyde-free area was set to 100.

Example 2 (1) Culture The following medium was inoculated with Pseudomonas chlororafis B23 strain and cultured aerobically at 25 ° C. for 96 hours. Medium (pH 7.2) sucrose 10g taste solution 10g methacrylamide _{2g FeSO 4 · 7H 2 O 10mg} MgSO 4 · 7H 2 O 20mg Distilled water 1000ml

(2) Measurement of activity and productivity The measurement was carried out in the same manner as in Example 1. The results are shown in Table 1.

Example 3 (1) Culture Rhodococcus HT40-6 strain was cultured in the same manner as in Example 1.

(2) Activity measurement The HT40-6 cells washed and suspended in the same manner as in Example 1 were
The mixture was added to 20 mM phosphate buffer (pH 7.5) containing 2 or 3 mM of cyanohydrin and 2 to 9 mM of aldehyde corresponding to each cyanohydrin, and the reaction was carried out with stirring at 30 ° C. for 30 minutes. Activity was determined. The results are shown in Table-2.

(3) Measurement of Productivity The HT40-6 strain washed and suspended in the same manner as in Example 1 was
It was added to 20 mM phosphate buffer (pH 7.5) containing 2 or 3 mM of cyanohydrin and 2 to 9 mM of aldehyde corresponding to each cyanohydrin, and the reaction was carried out with stirring at 30 ° C. for 72 hours. Relative productivity was determined. The results are shown in Table-2.

Example 4 (1) Culture Rhodococcus HT40-6 strain was cultured in the same manner as in Example 1.

(2) Accumulation reaction The HT40-6 strain (0.6 g as dry cells) washed and suspended in the same manner as in Example 1 was prepared by adding 300 ml of 20 ml of 10 mM mandelonitrile and 30 mM benzaldehyde.
M was added to a phosphate buffer (pH 7.5), the reaction was carried out with stirring at 15 ° C., and while the progress of the reaction was confirmed by HPLC as described in Example 1, mandelonitrile was supplied manually for 48 hours. An accumulation reaction was performed. For comparison, a similar experiment was performed without adding benzaldehyde. The results are shown in Table-3.

In Examples 1 to 4, the optical purity of the produced mandelamide and its derivative was measured using an optical resolution column (CHIRALCEL CA-1, Daicel Chemical Industries, carrier; 100% ethanol). As a result, in each of the examples, the optical purity was 90% ee or more as the S-(+) form.

[0032]

[Table 1]

[0033]

[Table 2]

[0034]

[Table 3]

[0035]

According to the present invention, inactivation of the enzyme can be suppressed, and racemic cyanohydrin, or S-(+)-mandelamide or the like, which is directly superior to aldehyde and hydrocyanic acid (50 to 100%), can be obtained. Can be produced and accumulated in yield,
It is also possible to convert all of the raw materials stoichiometrically to the desired product.

Continued on the front page (58) Fields investigated (Int. Cl. ⁷ , DB name) C12P 13/02 CA (STN) REGISTRY (STN) BIOSIS / WPI (DIALOG)

Claims (2)

(57) [Claims] 1. A racemic cyanohydrin represented by the following general formula (1), or an aldehyde represented by the following general formula (2) and a hydrocyanic acid corresponding to the cyanohydrin, in a neutral or basic aqueous medium: By reacting the cyanohydrin cyanide group with a Rhodococcus genus bacterium or a processed product thereof, a racemic cyanohydrin represented by the general formula (1) or an aldehyde represented by the general formula (2) and an aldehyde and a hydrocyanic acid, which are superior in amount, In a method for producing an optically active S-(+)-mandelamide represented by the formula (3) or a derivative thereof, an aldehyde represented by the general formula (2) corresponding to the cyanohydrin may be dissolved in a reaction system. Within the range, 1 to 10 times the molar amount of racemic cyanohydrin represented by the general formula (1), and 2 to 11 times the molar amount of hydrocyanic acid. S- characterized by adding
A method for producing (+)-mandelamide and its derivatives. Embedded image [X is hydrogen, methyl, methoxy, isopropyl,
Represents a hydroxyl group, a nitro group, a sulfone group or a halogen] 2. A racemic cyanohydrin represented by the following general formula (1) or an aldehyde represented by the following general formula (2) and a hydrocyanic acid corresponding to the cyanohydrin in a neutral or basic aqueous medium: By reacting the cyano group of the cyanohydrin with Pseudomonas chlororaphis B23 or a processed product thereof, the cyanide group has a direct superiority to the racemic cyanohydrin represented by the general formula (1) or the aldehyde and hydrocyanic acid represented by the general formula (2). In a method for producing an optically active S-(+)-mandelamide represented by the general formula (3) or a derivative thereof, an aldehyde represented by the general formula (2) corresponding to the cyanohydrin is dissolved in a reaction system. Within the range obtained, and in general formula (1)
1 to 1 for the racemic cyanohydrin represented by
A process for producing S-(+)-mandelamide and its derivatives, characterized in that it is added in a molar amount of 0-fold and 2 to 11 times the molar amount of cyanuric acid. Embedded image [X is hydrogen, methyl, methoxy, isopropyl,
Represents a hydroxyl group, a nitro group, a sulfone group or a halogen]