JPH05211880A - Production of optically active compound by enzymatic technique - Google Patents

Abstract

PURPOSE:To obtain an optically active compound useful as an intermediate for synthesizing medicines, pesticides, etc., by asymmetrically reducing the carbonyl group of an isonicotinic acid anilide derivative using a specific enzyme. CONSTITUTION:An enzyme obtained from microorganisms belonging to Pseudoplea having ability to synthesize a compound of formula II (A and A' are each H or OH; X is halogen or lower alkyl; R is phenyl or lower alkyl; (m) is 0-3) by asymmetrically reducing the carbonyl group of an isonicotinic acid anilide derivative of formula I is brought into contact with the compound of the formula I to produce the compound of the formula II, which is then collected.

Description

Detailed Description of the Invention

The present invention has the general formula (I)

[Chemical 6] (In the formula, X is the same or different and is a halogen atom or a lower alkyl group; R is a phenyl group or a lower alkyl group;
Represents an integer of 0 to 3. ) Asymmetrically reduces the carbonyl group of the isonicotinic acid anilide derivative to give the general formula (II)

[Chemical 7] (In the formula, A and A ′ are different from each other to represent a hydrogen atom or a hydroxyl group, and X, R and m have the same meanings as described above) From a microorganism belonging to the genus Pseudoprea having the ability to synthesize The prepared enzyme is contacted with the compound represented by the general formula (I) to convert it into the compound represented by the general formula (II), and then the compound represented by the general formula (II) is collected. The present invention relates to a method for producing an optically active compound represented by formula (II).

The present invention also has the general formula (III)

[Chemical 8] (In the formula, X ′ is the same or different and is a halogen atom, a hydroxyl group, an amino group, a lower alkyl group or a nitro group, R ′ is a phenyl group, a halogen-substituted phenyl group, a lower alkyl group or a cyclopropane group, and n is Indicates an integer from 0 to 3,

[Chemical 9] And the group R ′ are limited to those in which the carbon atom of O═ <is asymmetric carbon when the compound of the general formula (III) is reduced. ) Is asymmetrically reduced to the carbonyl group of the benzophenone derivative represented by the general formula (IV)

[Chemical 10] (In the formula, A and A'are different from each other to represent a hydrogen atom or a hydroxyl group, and X ', R'and n have the same meanings as described above.) Pseudo having the ability to synthesize a benzohydrol derivative An enzyme prepared from a microorganism belonging to the genus Pleia is contacted with a compound represented by the general formula (III) to convert it into a compound represented by the general formula (IV), and then the compound represented by the general formula (IV) is collected. And a method for producing an optically active compound represented by the general formula (IV).

Furthermore, the present invention provides 1-acetonaphthone with an enzyme prepared from a microorganism belonging to the genus Pseudoprea having the ability to asymmetrically reduce the carbonyl group of 1-acetonaphthone to synthesize optically active α-hydroxyethylnaphthalene. The present invention relates to a method for producing optically active α-hydroxyethylnaphthalene, which comprises contacting to convert to optically active α-hydroxyethylnaphthalene, and then collecting this.

General formula (V)

[Chemical 11] (In the formula, X is the same or different and is a halogen atom or a lower alkyl group; R is a phenyl group or a lower alkyl group;
Represents an integer of 0 to 3. The compound represented by the formula () shows an excellent plant growth regulating action, and in particular, the optically active substance represented by the general formula (II) is a useful substance which can be used as an active substance when used as a plant growth regulator. ..

Further, the optically active benzohydrol derivative represented by the general formula (IV) and the optically active α-hydroxyethylnaphthalene are synthetic intermediates for pharmaceuticals and agricultural chemicals (eg plant growth regulators) which require optical activity. It is an extremely useful substance that can be used as

[0006]

2. Description of the Related Art Hitherto, as a method for producing an optically active compound represented by the general formula (II) or (IV) or an optically active α-hydroxyethylnaphthalene, L-tartaric acid or the like was used from a corresponding racemic compound. The method of optical division was performed.

Among the optically active compounds represented by the general formula (II) and their racemates, for example, 4'-chloro-
2 ′-(α-hydrochitobenzyl) isonicotinic acid anilide (hereinafter abbreviated as inabenfide) exhibits an excellent plant growth regulating action, and in particular, its optically active S-form is active when used as a plant growth regulator. It is a useful substance that can be used as the main body. (See JP-A-2-28156)
Among the optically active compounds represented by the general formula (IV) and their racemates, for example, 2-amino-5-chlorobenzohydrol is a useful substance that can be used as a synthetic intermediate for inabenfide.

Heretofore, as a method for producing optically active inabenfide, optically active 2-amino-5-chlorobenzohydrol was obtained from racemic 2-amino-5-chlorobenzohydrol using L-tartaric acid. After that, a method of reacting this with isonicotinic acid was performed, but the method for producing optically active 2-amino-5-chlorobenzohydrol by an asymmetric synthesis method using an enzyme is more The method for enzymatically producing active inabenfide is not generally known. Of course, other than the above compounds, as a method for producing an optically active compound represented by the general formulas (II) and (IV) or an optically active α-hydroxyethylnaphthalene, these compounds are prepared by an asymmetric synthesis method using an enzyme. The production method for obtaining the compound is not known.

[0009]

[Problems to be Solved by the Invention] General formulas (II) and (IV)
When a conventional optical resolution method using tartaric acid or the like is used to obtain the compound represented by, or optically active α-hydroxyethylnaphthalene, both the optical purity and the yield are low, and the number of steps is large, and further recrystallization is performed. Since a complicated operation such as having to perform the operation twice is required, it is industrially disadvantageous in terms of equipment and cost.

[0010]

SUMMARY OF THE INVENTION In view of these circumstances, the present inventors have considered a compound represented by the general formula (II) containing an optically active inabenfide which is particularly excellent as a plant growth regulator,
A compound represented by the general formula (IV) containing an optically active 2-amino-5-chlorobenzohydrol that can be used as a synthetic intermediate for an optically active inabenfide, or an optically active α
As a result of earnest studies to obtain -hydroxyethylnaphthalene by an industrially excellent method, the carbonyl group of the compound represented by the general formula (I) is asymmetrically reduced to give the compound represented by the general formula (II). By contacting an enzyme prepared from a microorganism belonging to the genus Pseudoplea capable of synthesizing a compound with a compound represented by the general formula (I), the compound represented by the general formula (I
A method for obtaining an optically active compound represented by I) was found.
In addition, the present inventors have obtained a pseudopread (Pseudopl having the ability to asymmetrically reduce a compound represented by the general formula (III) to synthesize a compound represented by the general formula (IV).
ea) A method for obtaining an optically active compound represented by the general formula (IV) with high optical purity and a short process by contacting an enzyme prepared from a microorganism belonging to the genus ea) with a compound represented by the general formula (III) It was Furthermore, the present inventors
Asymmetrically reducing the carbonyl group of acetonaphthone, an enzyme prepared from a microorganism belonging to the genus Pseudoplea having the ability to synthesize optically active α-hydroxyethylnaphthalene, by contacting with 1-acetonaphthone, A method for obtaining optically active α-hydroxyethylnaphthalene with high optical purity and a short process has been found. The present invention has been completed based on these findings.

Next, the content of the present invention will be described.

The compound represented by the general formula (II) is a known plant growth regulator, for example, JP-A-58-4767,
JP-A-58-41869, JP-A-59-122402
Or the method described in JP-A-59-122469 or the like.

The enzyme used in the production of the compound represented by the general formula (II) according to the present invention belongs to the genus Pseudoplea having the ability to asymmetrically reduce the compound represented by the general formula (I). It is an enzyme derived from microorganisms. Of these microorganisms, Pseud is used for enzyme preparation.
olea trifolii, about 5 × 10 ⁴ KDa prepared from IFO 6691, isoelectric point 5.5 to 6.
0 enzymes are particularly preferred.

For culturing the microorganisms for preparing these enzymes, any nutrient source that can normally be utilized by these microorganisms can be used. For example glucose, sucrose,
Carbohydrates such as fructose, alcohols such as ethanol and glycerol, hydrocarbons such as paraffin, organic acids such as acetic acid and propionic acid, carbon sources such as soybean oil, or mixtures thereof, yeast extract, peptone, meat extract, corn steep liquor It is possible to use a normal medium in which nitrogen-containing inorganic or organic nutrient sources such as ammonium sulfate and ammonia, inorganic nutrient sources such as phosphate, magnesium, iron, manganese, potassium and vitamins such as biotin and thiamine are appropriately mixed. it can. As a culture method, a liquid medium having a pH adjusted to around 6 is aerobically aerated at 20 to 30 ° C., preferably 2
Culture with shaking at around 7 ° C for about 3 days. It is preferable to use a microorganism that has been passaged every 3 days.

The enzyme is prepared by filtering the culture solution, disrupting the cells with ultrasonic waves, and then purifying the supernatant obtained by ultracentrifugation by salting out with ammonium sulfate or by ion exchange chromatography. To do.

In the asymmetric synthesis reaction of the optically active compound represented by the general formula (II), the enzyme solution is diluted with a buffer solution having a pH of about 6 to 8 to prepare the substrate represented by the general formula (I) and NADP.
After H is added as a coenzyme, the process proceeds by gently shaking overnight at 20 ° C to 60 ° C (about 30 ° C in the case of inabenfide).

The optically active isonicotinic acid anilide derivative represented by the general formula (II) produced by the reaction is collected as follows.
The reaction solution is extracted with a solvent such as ethyl acetate and dichloromethane and subjected to silica gel chromatography to obtain a chemically and optically pure target compound.

The optical purity of the obtained compound represented by the general formula (II) can be determined by high performance liquid chromatography equipped with an optical isomer resolution column.

On the other hand, the enzyme used in the production of the optically active compound represented by the general formula (IV) according to the present invention has a pseudo ability to asymmetrically reduce the carbonyl group of the compound represented by the general formula (III). Pleasure (Pseudopl
It is an enzyme purified from a microorganism belonging to genus ea), and an enzyme prepared from Pseudoplea trifolii is particularly preferable. This microorganism, as described above,
It has been deposited under the number O 6691.

For culturing the microorganism of the genus Pseudoplea, the same culture method as described above can be used.

The enzyme can be prepared in the same manner as the above-mentioned method, and the asymmetric synthesis reaction of the compound represented by the general formula (IV) can also be carried out by the asymmetric synthesis reaction of the compound represented by the general formula (II). It can be carried out in exactly the same manner as in the case of the synthetic reaction.

The optically active benzohydrol derivative represented by the general formula (IV) produced by the reaction is collected by extracting the reaction solution with a solvent such as ethyl acetate or dichloromethane and performing silica gel chromatography to obtain the chemical and optical properties. A highly pure target compound is obtained. The optical purity of the obtained benzohydrol derivative represented by the general formula (IV) can be determined by high performance liquid chromatography equipped with an optical isomer resolution column.

The enzyme used for the production of the optically active α-hydroxyethylnaphthalene according to the present invention is purified from a microorganism belonging to the genus Pseudoplea having the ability to asymmetrically reduce the carbonyl group of 1-acetonaphthone. Enzymes, especially Pseu
Enzymes prepared from doplea trifolii are preferred. This microorganism is, as described above, IFO 6691.
Have been deposited.

For culturing the microorganism of the genus Pseudoplea, the same culture method as described above can be used.

The enzyme can be prepared in the same manner as described above, and the asymmetric synthesis reaction of optically active α-hydroxyethylnaphthalene can be performed by the asymmetric synthesis reaction of the compound represented by the general formula (II). It can be performed exactly as in the case.

To collect the optically active α-hydroxyethylnaphthalene produced by the reaction, the reaction solution was diluted with ethyl acetate,
By extracting with a solvent such as dichloromethane and performing silica gel chromatography, a chemically and optically pure target compound can be obtained. The optical purity of the obtained optically active α-hydroxyethylnaphthalene can be determined by high performance liquid chromatography equipped with an optical isomer resolution column. Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

Example 1 Glucose 40 g and polypeptone 10 in 1 L of distilled water
g, yeast extract _{5g, KH 2 PO 4 5g,} MgSO 4 · 7
The liquid medium containing 2 g of H ₂ O was put in a 100 ml Erlenmeyer flask in an amount of 20 ml and sterilized, and then Pseudop
Lea trifolii was inoculated and subcultured at 27 ° C. every 3 days. Next, this was inoculated into 25 of 200 ml of the above medium placed in a 500 ml Erlenmeyer flask and cultured with shaking for 3 days. Culture at 27 ° C, shaking speed 200
It was performed under aerobic conditions of rpm. After culturing, the cells were collected using a filter paper, and about 1 L of 100 mM phosphate buffer (pH = 7.
0), ultrasonically disrupted, and ultracentrifuged to obtain a supernatant. This was salted out with 70% saturated ammonium sulfate and the salted out enzyme was purified by DEAE ion exchange chromatography to prepare about 100 ml of enzyme solution. This enzyme solution was added to 900 mL of 100 mM Tris buffer (pH = 8.
0), where 2 g of NADPH and 1 g of 4'-
Chloro-2′-benzoylisonicotinic acid anilide was added, and the reaction was carried out by gently shaking at 30 ° C. overnight.
Next, this reaction solution was extracted with ethyl acetate, and then the solvent was distilled off. High performance liquid chromatography equipped with a Daicel CHIRALCEL-OG column (elution solvent = hexane: ethanol {4: 1}, flow rate = 1.0 ml / mi)
When the inabenfide was analyzed according to (n), the R-form was 10.
After 4 minutes, the S form was eluted at a retention time of 15.3 minutes, and the substrate 4'-chloro-2'-benzoylisonicotinic acid anilide was eluted at a retention time of 17.6 minutes. When the above reaction product was analyzed by high performance liquid chromatography under these conditions, 0.618 g of inabenfide was obtained,
The optical purity of the S form is 99% e. e. That was all.

Example 2 Glucose 40 g and polypeptone 10 in 1 L of distilled water
g, yeast extract 5g, KH ₂ PO ₄ 5g, MgSO ₄ ·
Liquid medium containing 2 g of 7H ₂ O was sterilized by putting 20 ml in a 100 ml Erlenmeyer flask and sterilizing it.
Pleia trifolii was inoculated and subcultured at 27 ° C. every 3 days. Then, this was inoculated into 25 of 200 ml of the above medium placed in a 500 ml Erlenmeyer flask, and 3
The cells were shake-cultured for one day. Culture at 27 ° C, shaking speed 20
It was performed under aerobic conditions of 0 rpm. After culturing, the bacterial cells were collected using a filter paper, and about 1 L of 100 mM phosphate buffer solution (pH =
It was suspended in 7.0), sonicated, and ultracentrifuged to obtain a supernatant. This was salted out with 70% saturated ammonium sulfate and the salted out enzyme was purified by DEAE ion exchange chromatography to prepare about 100 ml of enzyme solution.

10 ml of this enzyme solution was added to 990 ml of 100
Dilute in mM Tris buffer (pH = 8.0) and add 2
00 mg of NADPH and No. in the chemical formula list below. 1
100 mg of the compound shown in 1 was added, and the reaction was carried out by gently shaking at 30 ° C. overnight. Next, this reaction solution was extracted with ethyl acetate, and then the solvent was distilled off. The obtained reaction mixture was separated by silica gel chromatography to obtain No. 61.2 mg of the compound shown in 2
Got High performance liquid chromatography equipped with a Daicel CHIRALCEL-OG column (elution solvent = hexane: ethanol {8: 1}, flow rate = 1.0 ml / m).
No. in the attached sheet. When the compound shown in 2 was analyzed, the optical isomers were eluted at retention times of 6.3 minutes and 7.3 minutes. When the above reaction product was analyzed by high performance liquid chromatography under these conditions, No. 1 in the chemical formula list was found at 7.3 minutes. The optically active substance of the compound shown in 2 was detected, and the optical purity was 99% e. e. That was all.

Example 3 900 mL of the enzyme solution prepared in the same manner as in Example 1 was used.
It was diluted with 00 mM Tris buffer (pH = 8.0), 2 g of NADPH and 1 g of 2-amino-5-chlorobenzophenone were added thereto, and the reaction was carried out by gently shaking at 30 ° C. overnight. .. Next, this reaction solution was extracted with ethyl acetate, and then the solvent was distilled off. Daicel CHIRALPA
When 2-amino-5-chlorobenzohydrol was analyzed by high performance liquid chromatography equipped with a C-OP column (elution solvent = hexane: ethanol {10: 1}, flow rate = 1.0 ml / min), the R-form was found to be 15.1 minutes, S
The body elutes at a retention time of 13.7 minutes and is the substrate 2
-Amino-5-chlorobenzophenone eluted at a retention time of 9.7 minutes. When the above reaction product was analyzed by high performance liquid chromatography under these conditions, 0.324 g of 2-amino-5-chlorobenzohydrol was obtained, and the optical purity was 99% e. e. That was all.

Example 4 1 ml of the enzyme solution prepared in the same manner as in Example 2 was 999 m
L in 100 mM Tris buffer (pH = 8.0) and diluted with 200 mg NADPH and N in the chemical formula table.
o. 100 mg of the compound shown in 3 was added, and the reaction was carried out by gently shaking at 30 ° C. overnight. After the reaction solution was extracted in the same manner as in Example 2, it was separated by silica gel chromatography to obtain the chemical formula no. 25.2 mg of the compound shown in 4 was obtained. Daicel CHIRALCE
Chemical formula list No. by high performance liquid chromatography equipped with an L-OG column (eluting solvent = hexane: ethanol {40: 1}, flow rate = 1.0 ml / min). When the compound shown in 4 was analyzed, the optical isomers were eluted at the retention times of 20.9 minutes and 22.2 minutes. When the above reaction product was analyzed by high performance liquid chromatography under these conditions, the chemical formula no. The optically active substance of the compound shown in 4 was detected, and the optical purity was 99% e. e. That was all.

Example 5 5 ml of the enzyme solution prepared in the same manner as in Example 2 was added to 995 m
L in 100 mM Tris buffer (pH = 8.0) and diluted with 200 mg NADPH and N in the chemical formula table.
o. 100 mg of the compound shown in 5 was added, and the reaction was carried out by gently shaking at 30 ° C. overnight. After the reaction solution was extracted in the same manner as in Example 2, it was separated by silica gel chromatography to obtain No. 1 in the chemical formula list. 29.6 mg of the compound shown in 6 was obtained. Daicel CHIRA
High performance liquid chromatography equipped with an LCEL-OD column (elution solvent = hexane: ethanol {4: 1},
Flow rate = 1.0 ml / min) N in the chemical formula list
o. When the compound shown in 6 was analyzed, the optical isomer was found to be 7.
Elution occurred at retention times of 8 minutes and 8.1 minutes. When the compound obtained above was analyzed by high performance liquid chromatography under these conditions, No. 1 in the chemical formula list was found at 7.8 minutes. The optically active substance of the compound shown in 6 was detected, and the optical purity was 99%.
e. e. That was all.

Example 6 10 ml of the enzyme solution prepared in the same manner as in Example 2 was used for 990
Dilute to 100 mL of Tris buffer (pH = 8.0) with 200 mg of NADPH and N in the chemical formula table.
o. 100 mg of the compound shown in 7 was added, and the reaction was carried out by gently shaking at 30 ° C. overnight. After the reaction solution was extracted in the same manner as in Example 2, it was separated by silica gel chromatography to obtain No. 1 in the chemical formula list. 29.4 mg of the compound shown in 8 was obtained. Daicel CHIRA
High performance liquid chromatography equipped with an LCEL-OG column (elution solvent = hexane: ethanol {10:
1}, flow rate = 1.0 ml / min). When the compound shown in 6 was analyzed, the optical isomers were eluted at the retention times of 8.7 minutes and 9.5 minutes. When the compound obtained above was analyzed by high performance liquid chromatography under these conditions, No. 1 in the chemical formula list was found at 8.7 minutes. The optically active substance of the compound shown in 6 was detected, and the optical purity was 99.
% E. e. That was all.

Example 7 5 ml of the enzyme solution prepared in the same manner as in Example 2 was 995 m
L in 100 mM Tris buffer (pH = 8.0) and diluted with 200 mg NADPH and N in the chemical formula table.
o. 100 mg of 1-acetonaphthone shown in 9 was added, and the reaction was carried out by gently shaking at 30 ° C. overnight. The reaction solution was extracted in the same manner as in Example 2 and then separated by silica gel chromatography to obtain N in the chemical formula list.
o. Α-hydroxyethylnaphthalene 4 shown in 10
5.3 mg was obtained. Daicel CHIRALCE
By high performance liquid chromatography equipped with an L-OD column (elution solvent = hexane: ethanol {10: 1}, flow rate = 1.0 ml / min), No. 1
When α-hydroxyethylnaphthalene shown in 0 was analyzed, optical isomers were eluted at retention times of 5.4 minutes and 7.5 minutes. When the compound obtained above was analyzed by high performance liquid chromatography under these conditions, No. 1 in the chemical formula list was found at 5.4 minutes. The optically active substance of α-hydroxyethylnaphthalene shown in 10 was detected, and the optical purity was 99% e.
e. That was all.

[0035]

INDUSTRIAL APPLICABILITY According to the present invention, an optically active isonicotinic acid anilide derivative represented by the general formula (II), which is useful as a plant growth regulator, and synthetic intermediates such as pharmaceuticals and agricultural chemicals (eg, plant growth regulator) The general formula (I
Since V) and α-hydroxyethylnaphthalene can be produced with extremely high optical purity and the number of purification steps is small, it has become possible to produce an industrially excellent optically active substance.

[0036]

[Chemical 12]

Claims (6)

[Claims] 1. A compound represented by the general formula (I): (In the formula, X is the same or different and is a halogen atom or a lower alkyl group; R is a phenyl group or a lower alkyl group;
Represents an integer of 0 to 3. ) Is asymmetrically reduced by the carbonyl group of the isonicotinic acid anilide derivative represented by the general formula (II): (In the formula, A and A'are different from each other to represent a hydrogen atom or a hydroxyl group, and X, R and m have the same meanings as described above.) In the genus Pseudoplea having the ability to synthesize a compound represented by An enzyme prepared from a microorganism belonging to the microorganism is contacted with a compound represented by the general formula (I) to convert it into a compound represented by the general formula (II), and then the compound represented by the general formula (II) is collected. And a method for producing an optically active compound represented by the general formula (II). 2. An optically active 4′-chloro-4′-benzoylisonicotinic acid anilide is used as a substrate.
An enzyme prepared from a microorganism belonging to the genus Pseudoprea having the ability to asymmetrically synthesize chloro-2 '-(α-hydroxybenzyl) isonicotinic acid anilide was prepared as 4'-chloro-2'-benzoylisonicotinic acid anilide. 2. The production method according to claim 1, wherein the optically active 4′-chloro-2 ′-(α-hydroxybenzyl) isonicotinic acid anilide is converted into an optically active 4′-chloro-2′-anilide, and then this is collected. 3. A compound represented by the general formula (III): (In the formula, X ′ is the same or different and is a halogen atom, a hydroxyl group, an amino group, a lower alkyl group or a nitro group, R ′ is a phenyl group, a halogen-substituted phenyl group, a lower alkyl group or a cyclopropane group, and n is Indicates an integer from 0 to 3, And the group R ′ are limited to those in which the carbon atom of O═ <is asymmetric carbon when the compound of the general formula (III) is reduced. ), The carbonyl group of the benzophenone derivative is asymmetrically reduced to give a compound represented by the general formula (IV): (In the formula, A and A'are different from each other to represent a hydrogen atom or a hydroxyl group, and X ', R'and n have the same meanings as described above.) Pseudo having the ability to synthesize a benzohydrol derivative An enzyme prepared from a microorganism belonging to the genus Pleia is contacted with a compound represented by the general formula (III) to convert it into a compound represented by the general formula (IV), and then the compound represented by the general formula (IV) is collected. A process for producing an optically active compound represented by the general formula (IV), which comprises: 4. Prepared from a microorganism belonging to the genus Pseudoprea having the ability to asymmetrically reduce 2-amino-5-chlorobenzophenone as a substrate to optically active 2-amino-5-chlorobenzohydrol. Enzyme, 2-
4. Contact with amino-5-chlorobenzophenone to convert to optically active 2-amino-5-chlorobenzohydrol, which is then collected.
The manufacturing method described. 5. An enzyme prepared from a microorganism belonging to the genus Pseudoprea having the ability to asymmetrically reduce the carbonyl group of 1-acetonaphthone to synthesize optically active α-hydroxyethylnaphthalene is contacted with 1-acetonaphthone. , Optically active α-hydroxyethylnaphthalene, and then collected.
-A method for producing hydroxyethylnaphthalene. 6. The enzyme according to claim 1, wherein the enzyme is about 5 × 10 ⁴ KDa prepared from Pseudoplea trifolii and has an isoelectric point of 5.5 to 6.0. The manufacturing method described.