JPH04207197A - Production of optically active cyanohydrin and optically active lactone - Google Patents

Abstract

PURPOSE:To obtain the subject cyanohydrin useful for medical drugs, reagents of clinical analysis, etc., with industrial advantage by reacting an aldehyde with hydrocyanic acid in the presence of a lipase. CONSTITUTION:An aldehyde of formula I [R is (substituted)alkyl, (substituted) aryl, etc.] is reacted with hydrocyanic acid in a reaction solvent such as water or an acetate buffer in the presence of a microorganism-derived lipase (e.g. lipase derived from Achromobacter genus or Aspergillus genus), thus giving the objective cyanohydrin of formula II.

Description

Detailed Description of the Invention [Field of Industrial Application] The present invention relates to a method for producing cyanohydrin and a method for producing lactone, and in particular, a method for producing optically active cyanohydrin and an optically active lactone based on biochemical techniques. Relating to a manufacturing method.

[Prior Art] Cyanohydrin, which is widely used as a raw material for synthesizing lactones, α-oxyacids, unsaturated carboxylic acids, etc., has conventionally been produced by a method in which hydrocyanic acid (hydrogen cyanide) is applied to an aldehyde or ketone, or by a method in which aldehyde Alternatively, it has been synthesized by chemical methods such as a method in which an alkali cyanide is applied to an adduct of a ketone and sodium hydrogen sulfite.

Cyanohydrin synthesized by such chemical methods is generally racemic, and the target substance synthesized by chemical methods using racemic cyanohydrin as a raw material is also racemic. For this reason, when using cyanohydrin synthesized by chemical methods as a raw material to obtain pharmaceuticals, test drugs, food additives, veterinary drugs, feed additives, etc. that require the use of one optical enantiomer. It is necessary to optically resolve racemic cyanohydrin to obtain one optical antipode and then chemically synthesize the target compound, or to obtain the target compound as a racemate and then optically resolve this racemate. Ta.

By the way, in recent years, methods have been developed to biochemically synthesize optically active organic substances (organic substances rich in one optical enantiomer or one optical enantiomer) using microorganisms and enzymes, and various methods have been developed. Attempts are being made to apply this to organic synthesis.

For example, to synthesize optically active cyanohydrin, aldehyde and hydrocyanic acid are synthesized using oxynitrilase (E,
A method of reacting in the presence of C, 4, 1, 2, 10) is disclosed in JP-A-63-219388.

[Problem to be solved by the invention] However, oxynitrilase is easily deactivated when using an organic solvent that is suitable as a reaction solvent in terms of increasing the reaction rate and recovering reaction products, and the selection of the reaction system is extremely difficult. The problem is that it is limited. In addition, since this oxynitrilase is extracted and purified from plants, it must be prepared and isolated from sources (plants, seeds) before it can be used industrially.
There are concerns about the amount of manpower involved, and there is also the drawback that it is expensive.

Therefore, an object of the present invention is to provide a method for producing optically active cyanophintrines and a method for producing optically active lactone, which can be carried out industrially more stably.

[Means for Solving the Problem] As a result of intensive research to achieve the above object, the present inventor has found that
The present invention has been achieved by discovering that lipase solves the above-mentioned difficulties and concerns and can be suitably applied to the production of optically active cyanophintrines and optically active lactones.

That is, the method for producing an optically active cyanohydrin of the present invention is based on the following general formula (I) R-CHO...(I) (wherein R is an alkyl group, a substituted alkyl group, an arylalkyl group, a substituted arylalkyl group, A substituent selected from the group consisting of an aryl group and a substituted aryl group.) is characterized by reacting an aldehyde represented by the following with hydrocyanic acid in the presence of lipase. The optically active cyanohydrin obtained has the following general formula (II) R-C-CN
...(n)■H (In the formula, R is the same as in the above general formula (I).).

Further, the method for producing an optically active lactone of the present invention can be carried out using the following general formula (i) R+ R3 HO-C-C-CHO... (i) 2 R4 (wherein, R+, R2, R3 and R4 are each hydrogen represents an atom or an alkyl group having 1 to 4 carbon atoms, R+,
R1, R3 and R4 may be the same or different. ) and hydrocyanic acid are reacted in the presence of lipase to form the following general formula (it) R' R3H R2R40H (wherein, R1, R2, R3 and R4 are represented by the general formula (i
). ) After obtaining optically active cyanohydrin, the method is characterized in that this cyanohydrin is subjected to acid hydrolysis treatment. The obtained optically active lactone has the general formula (iii) (wherein R1, R2, R3 and R4 are represented by the general formula (i)
). ).

The present invention will be explained in detail below.

First, the method for producing optically active cyanohydrin of the present invention will be explained. In this method, as described above, the following general formula (I) R-CHO...(I) (wherein R is an alkyl group or a substituted alkyl group) , an arylalkyl group, a substituted arylalkyl group, an aryl group, and a substituted aryl group.) An aldehyde represented by the following is used as a raw material.

The alkyl group here is preferably an alkyl group having 1 to 5 carbon atoms, and examples of such alkyl groups include methyl group, ethyl group, propyl group, isopropyl group, butyl group, t-butyl group, and pentyl group. Can be mentioned.

The substituted alkyl group is preferably an alkyl group having 1 to 5 carbon atoms substituted with a hydroxyl group or a halogen atom, and examples of such substituted alkyl groups include hydroxymethyl group, oxiranyl group, chloromethyl group, dichloromethyl group, Examples include substituents represented by the following formulas.

I R3 HO-C-C- ] I R1! R4 (wherein R+, R1SR3 and R4 each represent a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, R1, R
e5R3 and R4 may be the same or different. ) The arylalkyl group has 7 carbon atoms.
-10 arylalkyl groups are preferred, and examples of such arylalkyl groups include benzyl, phenylethyl, phenylpropyl, and phenylbutyl groups.

Substituted arylalkyl groups include hydroxyl group, nitro group,
7 carbon atoms substituted with methoxy group, halogen atom, etc.
-11 arylalkyl groups are preferable, and examples of such substituted arylalkyl groups include p-hydroxyphenylethyl group, 0-nitrophenylpropyl group, 0-nitrophenylpropyl group,
-chlorobenzyl group and m-methoxyphenylbutyl group.

As the aryl group, a phenyl group is preferred.

The substituted aryl group is preferably an aryl group having 6 to 9 carbon atoms substituted with a hydroxyl group, a nitro group, a methoxy group, an isopropyl group, a halogen atom, etc. Such a substituted aryl group includes, for example, a p-hydroxyphenyl group. , 0-nitrophenyl group, 0-chlorophenyl group, m-
Examples include methoxyphenyl group and p-isopropylphenyl group.

The aldehyde used as a raw material may be one separated and purified from a reaction solution obtained by a known aldehyde synthesis method, or an aldehyde-containing reaction solution before separation and purification. Furthermore, the aldehyde may be extracted from the reaction solution with an organic solvent and then the solvent is distilled off, or a commercially available aldehyde may be dissolved in water at a temperature and then cooled.

For example, when using hydroxypivalaldehyde as the aldehyde [in the above general formula (i), R1 and R2 are hydrogen atoms and R3 and R4 are methyl groups, aldol condensation of isobutyraldehyde and formaldehyde is used. In addition to hydroxypivalaldehyde separated and purified from the reaction solution, the aldol condensation reaction solution, hydroxypivalaldehyde extracted from this aldol condensation reaction solution with an organic solvent and distilled off the solvent, or commercially available Hydroxypivalaldehyde may be dissolved in water at a warm temperature and then cooled to a predetermined temperature.

In the method for producing optically active cyanohydrin of the present invention, the above-mentioned aldehyde and hydrocyanic acid are reacted in the presence of lipase.

The reaction solvent at this time is water, acetate buffer,
It is preferable to use a buffer such as a citrate buffer or a phosphate buffer, or an alcohol-containing buffer.

Hydrocyanic acid (HCN) may be dissolved as it is in a solvent, but for safety reasons, cyanide salts such as NaCN and KCN and acids such as HCj2 and Ht SOa are dissolved in a solvent. It is preferable to generate and use hydrocyanic acid.

The amount of hydrocyanic acid at this time is preferably equal to or more than the aldehyde used as a raw material.

A variety of lipases are known, including those derived from higher animals, plants, and microorganisms. In the present invention, lipases derived from microorganisms for which industrial mass production technology has been established, such as Achromobacter
cter) genus and Pseudomonas (Pseudomonas)
Phycomyces, a bacterium of the genus Phycomyces
), Rhizopus and Mucor (M
ucor) genus and Aspergillus (Aspergi I
It is particularly preferred to use lipases derived from filamentous fungi of the genus Ius, yeasts of the genus Candida, and the like.

In addition to purified lipase, lipases include bacterial cells or cells that produce lipase, bacterial cell extracts or cell extracts containing lipase, culture supernatants of bacterial cells or cells that produce lipase, etc. A lipase immobilized on a suitable carrier may also be used (the lipase in the present invention is a general term for these).

The carriers used for immobilization include inorganic carriers such as glass, porous glass, silica gel, and alumina, natural polymers and their derivatives such as cellulose, dextran, agarose, and starch, polyacrylamide, polyhydroxyacryl methacrylate, polystyrene, Synthetic polymers such as nylon and various ion exchange resins, and polymers into which functional groups such as amino groups, carboxyl groups, hydroxyl groups, and phenol groups have been introduced, polysaccharides such as agar, alginic acid, carrageenan, and cellulose derivatives, and gelatin. , albumin, collagen, and other proteins.

The reaction is carried out, for example, by adding a predetermined amount of hydrocyanic acid or a cyanide salt to a solvent, and optionally adding a predetermined amount of acid for producing hydrocyanic acid, and then adjusting the pH of the solvent to 3.0 to 7.0, preferably 3.0 to 7.0.
6 to 6.0, add lipase, further add a predetermined amount of raw material aldehyde, and conduct the reaction at a temperature of 4 to 60°C, preferably 15 to 40°C. The reaction format may be a continuous type, a counter-continuous type, or a batch type, and the reaction time is approximately 0.5 to 2 hours.

By reacting aldehyde and hydrocyanic acid in the presence of lipase in this way, the optical activity expressed by the following general formula % formula % ([) (wherein R is the same as the above general formula (I)) cyanohydrin can be produced.

After the reaction is completed, the produced cyanohydrin may be separated and purified if necessary. The method for separating and purifying cyanohydrin is not particularly limited. For example, unreacted hydrocyanic acid remaining in the reaction solution is removed by a vacuum method, blowing in a N2 gas stream, etc., and then the cyanohydrin in the reaction solution is purified by ether or dichloromethane. This can be carried out by extracting with an organic solvent such as, dehydrating the extracted layer with anhydrous sodium sulfate, etc., and then concentrating to dryness.

Next, to explain the method for producing an optically active lactone of the present invention, in this method, the following general formula (i), which is an embodiment of the aldehyde (R-CHO) of the general formula (I), is used.
, Rj R3 HO-C-C-CHO...(i) R1! R4 (wherein Rj, RE, R3 and R4 each represent a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, and Rj
, RE, R3 and R4 may be the same or different. ) is used as a raw material, and this aldehyde and hydrocyanic acid are reacted in the presence of lipase. is the same as the general formula (i) above.) An optically active cyanohydrin represented by the following is obtained.

Specific examples of aldehydes used as raw materials include hydroxypivalaldehyde, 3-hydroxypropionaldehyde, 2,2-diethyl-3-hydroxypropionaldehyde, 2,2-dipropyl-3-hydroxypropionaldehyde, and the like.

In the method for producing an optically active lactone of the present invention, the optically active cyanohydrin of the general formula (11) thus obtained is subjected to acid hydrolysis treatment, and the following general formula (i
il) (In the formula, H+, Rj, R3 and R4 are the same as in the general formula (i) above.) An optically active lactone is obtained.

At this time, acid hydrolysis uses acids such as hydrochloric acid and sulfuric acid.
The reaction may be carried out using a reaction solution containing the optically active cyanohydrin of the general formula (if), or may be carried out using an optically active cyanohydrin separated and purified from the reaction solution. Furthermore, when using a lipase other than purified lipase or when using immobilized lipase, the reaction solution is centrifuged to extract the bacterial cells or cells that produce the lipase and the bacterial cell extract containing the lipase. Alternatively, the assay may be carried out using cell extracts, culture supernatants of microbial cells or cells that produce lipase, or fractions obtained by removing carriers on which these are immobilized. This acid hydrolysis is 60 to 10
It is preferable to carry out the reaction at 0'C for 1 to 3 hours.

In this way, the aldehyde represented by the general formula (i) and hydrocyanic acid are reacted in the presence of lipase, and the aldehyde represented by the general formula (1) is reacted with cyanide in the presence of lipase.
By performing acid hydrolysis after obtaining the optically active cyanohydrin represented by 1), it is possible to produce an optically active 5-membered cyclic lactone, such as pantolactone (when hydroxybivalaldehyde is used as the aldehyde). can.

After acid hydrolysis, the generated lactone is separated as necessary.
May be purified. The method for separating and purifying lactones is not particularly limited, and can be carried out, for example, by extracting the lactones in the acid hydrolysis solution with an organic solvent such as ether or dichloromethane, and then concentrating to dryness. Alternatively, an oil layer can be obtained by adding ammonium sulfate, Glauber's salt, etc. to the acid hydrolysis solution.

[Example] Examples of the present invention will be described below.

Example 1 (Production of optically active cyanohydrin) Using benzaldehyde as a raw material aldehyde, optically active mandelnitrile (benzaldehyde cyanohydrin) was produced in the following manner.

First, 100mM acetate buffer (pH
5, 6), and after putting this acetic acid buffer 3 parts into a 5 parts reaction container, a 10M aqueous solution of NaCN was added to the reaction container to have a concentration of 100 mM, and an equivalent amount of HC was added to the reaction container.
12 was added.

Next, lipase (product name: Lipas) manufactured by Ohno Pharmaceutical ■
Add 50mg of eP "Amano") and adjust the pH of the solution to 5.
A 99% solution of benzaldehyde was adjusted to 10
A reaction solution was prepared by adding it to a concentration of 0 mM.

Thereafter, the reaction was carried out at a reaction temperature of 25° C. for a reaction time of 30 minutes while stirring the reaction solution.

After the reaction is completed, unreacted HCN is removed by blowing a stream of N into the reaction solution, and then the reaction product is extracted with ether, and the ether extracted layer is dehydrated with anhydrous sodium sulfate, and then concentrated to dryness. I got something. Production of manzolonitrile was confirmed by thin layer chromatography. That is, after spotting the concentrated dry product on a Merck plate (product name: RP-18), it was developed with a developing solution (a 1:1 mixture of methanol and water), and after air drying, a wavelength of 254 nm was applied.
The Rf value was determined under an ultraviolet lamp of m and was compared with the Rf value of commercially available manzolonitrile determined in the same manner.

As a result, the Rf value of the reaction product obtained in Example 1 was 0.34 to 0.36, which was similar to the Rf value of commercially available manzolonitrile.

Furthermore, the optical purity of the produced manzolonitrile was determined in the following manner.

First, the above-mentioned concentrated and dried product was dissolved in 6N HCl,
Acid hydrolysis was performed at 0°C for 3 hours to produce mandelic acid, and the mandelic acid in the hydrolysis solution was extracted with ether. The ether extracted layer was dehydrated with anhydrous sodium sulfate, and further concentrated to dryness. A concentrated dry product was obtained.

Next, the obtained concentrated dry product was analyzed by high performance liquid chromatography (manufactured by Waters) using an optical isomer separation column under the following conditions.

Column: Chiralback WH (trade name, manufactured by Daicel Chemical ■, φ4.6 x 250 mm) Flow rate: 1. Om/minute effluent: 0.25mM copper sulfate aqueous solution test 8: Ultraviolet detector (wavelength 254nm) After this, the obtained analysis results are compared with the analysis results of the standard sample analyzed under the same conditions, and the main The optical purity of the manzolonitrile produced in Example 1 was determined. The results are shown in Table-1.

In addition, commercially available (D)-(-)-mandelic acid and (L)-(+)-mandelic acid were used as standards. In addition, the retention times of each sample were as follows.

(D)-(-)-mandelic acid 35.3 minutes (L)-(+
)-mandelic acid 27.9 minutes Examples 2 to 4 (Production of optically active cyanohydrin) Table 1 as lipase
A reaction solution was prepared in the same manner as in Example 1 except that 10 to 100 mg of the lipase shown in was used, and the reaction temperature was 20 to 30°C.
, reaction time was 30 to 60 minutes.

After the reaction was completed, concentrated dry products of the reaction products were obtained in the same manner as in Example 1, and the Rf values of these concentrated and dried products were determined in the same manner as in Example 1. It was confirmed that manzolonitrile was produced.

Furthermore, the optical purity of the manzolonitrile produced in each example was determined in the same manner as in Example 1.

These results are also shown in Table-1.

Example 5 (Production of optically active cyanohydrin) 3-phenylpropionaldehyde 98 as raw material aldehyde
A reaction solution was prepared and the reaction was carried out in the same manner as in Example 1, except that 50 Il 1 g of Amano Pharmaceutical Co., Ltd. lipase (trade name: Neurase F) was used as the lipase.

After the reaction was completed, the reaction product was concentrated to dryness in the same manner as in Example 1, and this concentrated and dried product was subjected to 'H-NMR (CDCβ3
), the following results were obtained.

'H-NMR (CDCj23) analysis result 2.06 ((
1, CH2,2H), 2.75 (t, CH2,2H
), 3.26 (s, OH, LH), 4.29 (t
, cH, LH), 7.15 (s, aroma, H, 5H
) From this result, it was confirmed that the reaction product was 2-hydroxy-4-phenylbutyronitrile.

In addition, the produced 2-hydroxy-4-phenylbutyronitrile was acid-hydrolyzed in the same manner as in Example 1 to produce 2-hydroxy-4-phenylbutyric acid, and concentrated to dryness in the same manner as in Example 1. After obtaining, this concentrated and dried product was prepared in Example 1.
Analyzed by high performance liquid chromatography under the same conditions as
The analysis results obtained were compared with those of a standard sample analyzed under the same conditions. As a result, the optical purity of 2-hydroxy-4-phenylbutyronitrile produced in Example 5 was 10
It was %e,e.

The standard sample was (D,L)-2, which was obtained by generating cyanohydrin from phenylpropionaldehyde and cyanide according to a conventional method, and acid hydrolyzing this cyanohydrin.
-Hydroxy-4-phenylbutyric acid was used. The retention time of the standard under this analytical condition was 42.5 minutes and 53.4 minutes.
It was a minute.

In addition, the specific rotation of the concentrated dry product after acid hydrolysis is [α]D
= +1.3°, and since it is +, it was found that it is a D body.

The results are also shown in Table-1.

Example 6 (Production of optically active cyanohydrin) The same procedure as in Example 1 was carried out except that 2-phenylacetaldehyde was used as the raw material aldehyde and 50 mg of lipase (trade name: Neurase F) manufactured by Ohno Pharmaceutical ■ was used as the lipase. A reaction solution was prepared and the reaction was carried out.

After the reaction was completed, the reaction product was concentrated to dryness in the same manner as in Example 1, and this concentrated and dried product was subjected to IH-NMR (CDCf3
), the following results were obtained.

IH-NMR (CDCβ3) analysis results 3.10 (d, cH2,2H), 3.40 (s,
OH, LH), 4.60 (t, CH-CN, LH),
7.37' (s, aron+, H, 5H) From this result, it was confirmed that the reaction product was 2-hydroxy-3-phenylpropionitrile.

In addition, the produced 2-hydroxy-3-phenyl diobionitrile was acid-hydrolyzed to produce 3-phenyl lactic acid in the same manner as in Example 1, and a concentrated dry product was obtained in the same manner as in Example 1. Detection wavelength of this concentrated dry product is 230nm.
This example was analyzed in the same manner as in Example 1, except that it was analyzed by high performance liquid chromatography under the same conditions as in Example 1. The optical purity of 2-hydroxy-3-phenylfuropionitrile produced in step 6 was determined. This result is also shown in the table.
Shown in 1.

In addition, commercially available (D, L)-3-phenyl lactic acid and (L)-3-phenyl lactic acid were used as standards. Under the present analysis conditions, the retention time of (D)-3-yenyl lactic acid was 47.5 minutes, and the retention time of (L)-3-phenyl lactic acid was 59.6 minutes.

Example 7 (Production of optically active cyanohydrin) The same procedure was carried out except that a 99% solution of n-butyraldehyde was used as the raw material aldehyde, and 50 mg of lipase (trade name: Lipase P, N) manufactured by Wako Pure Chemical Industries Ltd. was used as the lipase. A reaction solution was prepared and the reaction was carried out in the same manner as in Example 1.

After the reaction was completed, the reaction product was concentrated to dryness in the same manner as in Example 1, and this concentrated and dried product was subjected to IH-NMR (CDCj2
3), the following results were obtained.

IH-NMR (CD123) analysis results 1.2 (d,
CHa, 3H), 1.6 (m, cH2,4H), 4
．． 6 (t, CH-CN, LH), 4.0 (s, OH
, LH) From this result, it was confirmed that the reaction product was 2-hydroxybutyronitrile.

In addition, the produced 2-hydroxybutyronitrile was acid-hydrolyzed to produce 2-hydroxypentanoic acid in the same manner as in Example 1, and a concentrated dry product was obtained in the same manner as in Example 1. The dried product was analyzed by high performance liquid chromatography under the same conditions as in Example 1 except that the detection wavelength was 230 nm, and the obtained analysis results were compared with the analysis results of the standard sample analyzed under the same conditions. The optical purity of 2-hydroxybutyronitrile produced in Example 7 was determined in the same manner as in Example 1. As a result, the optical purity of 2-hydroxybutyronitrile produced in Example 7 was 33%e,e.

In addition, commercially available (D, L)-2-hydroxypentanoic acid was used as a standard product. The retention time of the standard under these analysis conditions is 22. 6 minutes, 26.4 minutes.

In addition, the specific rotation of the concentrated dry product after acid hydrolysis is [α]o
= +1.05°, and since it is 10, it was found that it is a D body.

The results are also shown in Table-1.

Example 8 (Production of optically active cyanohydrin) First, lipase used in this example was obtained in the following manner.

Distilled water 1! Glucose (final concentration 2%), olive oil (final concentration 2%), peptone (final concentration 2%), dipotassium monohydrogen phosphate decahydrate (final concentration 0.1%), ammonium sulfate (final concentration 0.1%). 2%) and magnesium sulfate heptahydrate (final concentration 0.1%), adjust the pH to 7.0 and use it as a medium, and add 100 ml of this medium to a 500 d culture flask and sterilize it in an autoclave. After that, a seed culture tooth (Achromobacter lyticus [Achrom
obacter 1yticus (IF O127
26) was inoculated and cultured under shaking conditions at 30°C and 20 Orpm.

The lipase activity of the culture supernatant is sequentially measured, and when the lipase activity reaches the maximum value, the bacterial cells are removed by centrifugation to obtain a culture supernatant, and this supernatant is further filtered using an ultrafiltration membrane. Concentrated. In addition, the removed cultured cells were washed with 10mM phosphate buffer, disrupted under water cooling using an ultrasonic cell disruption device, and unbroken microbial cells were removed by centrifugation, followed by the addition of ethanol at -20°C. , a precipitate fraction containing lipase activity was obtained.

Thereafter, a concentrate of the culture supernatant and a precipitated fraction having lipase activity were mixed to obtain a crude enzyme solution (one of the substances generally referred to as lipase in the present invention).

Next, a reaction solution was prepared and a reaction was carried out in the same manner as in Example 1, except that 0.5 d of the above crude enzyme solution was used as lipase.

After the reaction is complete, transfer the reaction solution to a Merck plate (product name: R
P-18) and analyzed by reverse phase thin layer chromatography using a 1=1 mixture of methanol and water as a developing solution. After air drying, the Rf value was determined under an ultraviolet lamp with a wavelength of 254 nm.

As a result, the Rf value of the reaction product obtained in Example 8 was 0.34 to 0.34, which is similar to the Rf value of commercially available manzolonitrile.
0.36, and it was confirmed that the reaction product obtained in Example 8 was manzolonitrile.

Furthermore, after removing protein and HCN from the reaction solution, the optical purity of the produced manzolonitrile was determined in the same manner as in Example 1. The results are also shown in Table-1.

(The following is a blank space) As is clear from Table 1, the cyanohydrins obtained in Examples 1 to 8 are all optically active cyanohydrins.

Example 9 (Production of optically active lactone) Using hydroxypivalaldehyde as a raw material aldehyde, optically active pantolactone was produced in the following manner.

First, 100mM acetate buffer (pH
5, 6), this acetate buffer 3 component was placed in a 5 component reaction vessel, a 10M aqueous solution of NaCN was added to the reaction vessel to a concentration of 100mM, and an equivalent amount of HC was added to the reaction vessel.
j2 was added.

Next, lipase manufactured by Amano Pharmaceutical Co., Ltd. (product name: Lipas
30 mg of eP r Amano) was added, and the pH of the liquid was adjusted to 5.
6, and then added a 1M aqueous solution of hydroxypivalaldehyde to a concentration of 100mM (10+wg/j2) to prepare a reaction solution.

Next, while stirring the reaction solution, the reaction was carried out at a reaction temperature of 25°C and a reaction time of 30 minutes to form 3゜3-dimethyl-
2,4-dihydroxybutyronitrile was produced.

After the reaction was completed, unreacted HCN was removed by blowing a stream of N2 into the reaction solution, the reaction product was extracted with ether, the ether extracted layer was dehydrated with anhydrous sodium sulfate, and then concentrated to dryness. 6N HC
/ and acid hydrolysis was performed at 60°C for 3 hours.

Thereafter, the reaction product in the hydrolysis solution was extracted with ether, and the ether extract layer was concentrated to dryness. ) was analyzed under the following conditions.

Column: Chiralcel OA (trade name, manufactured by Daicel Chemical ■,
φ4.6 x 250 mm) Flow rate 20.5 m/min Effluent: 9=1 mixture of n-hexane and isopropanol Detection: Ultraviolet detector (wavelength 230 ni) As a result,
The reaction product finally obtained in Example 9 was confirmed to be pantolactone.

Furthermore, this analysis result was compared with the analysis result of a standard sample analyzed under the same conditions, and in the same manner as in Example 1, the optical purity of pantolactone obtained in Example 10 was determined. The results are shown in Table-2.

In addition, commercially available (D)-pantolactone and (L)-pantolactone were used as standards. The retention time of (D)-pantolactone under this analytical condition is 28.
8 minutes, and the retention time of (L)-pantolactone was 25.8 minutes.

Examples 10 to 18 (Production of optically active lactone) 10 to 50 m
A reaction solution was prepared in the same manner as in Example 9 except that g was used,
The reaction was carried out at a reaction temperature of 20 to 25 °C and a reaction time of 30 to 60 minutes to produce 3,3-dimethyl-2,4-dihydroxybutyronitrile, and further acid hydrolysis was performed in the same manner as in Example 9. After that, a concentrated dry product was obtained.

When each of the obtained concentrated and dried products was analyzed in the same manner as in Example 9, it was confirmed that pantolactone was produced in all Examples.

Furthermore, the optical purity of pantolactone produced in each example was determined in the same manner as in Example 9. These results are shown in Table-2.

Example 19 (Production of optically active lactone) First, Pseudomos fluorescens was used as a seed culture tooth.
monas fluorescens (I F 0
A crude enzyme solution was obtained in the same manner as in Example 8, except that 3081)] was used.

Next, 0.0% of this crude enzyme solution was used as lipase. 57111
A reaction solution was prepared and the reaction was carried out in the same manner as in Example 9 except that 3.3-dimethyl-2゜4-dihydroxybutyronitrile was produced. Acid hydrolysis was performed.

Thereafter, the reaction product in the hydrolysis solution was extracted and analyzed in the same manner as in Example 9.

As a result, it was confirmed that the reaction product finally obtained in Example 19 was pantolactone.

Furthermore, after removing protein and HCN from the hydrolysis solution, the optical purity of the produced pantolactone was determined in the same manner as in Example 9. The results are also shown in Table-2.

(Left below) As is clear from Table 2, the lactones obtained in Examples 9 to 19 are all optically active lactones.

[Effects of the Invention] As explained above, according to the present invention, relatively inexpensive
Furthermore, optically active hyaline hydrin and optically active lactone can be obtained using enzymes that can be obtained in a stable quantity.

Therefore, by carrying out the present invention, optically active cyanohydrin and optically active lactone can be supplied industrially more easily and more stably.

Claims (5)

[Claims] (1) The following general formula (I) R-CHO...(I) (wherein R is from the group consisting of an alkyl group, a substituted alkyl group, an arylalkyl group, a substituted arylalkyl group, an aryl group, and a substituted aryl group) The following general formula (II) is characterized by reacting an aldehyde represented by (the selected substituent) with hydrocyanic acid in the presence of lipase ▲There are mathematical formulas, chemical formulas, tables, etc.▼... (II) A method for producing an optically active cyanohydrin represented by the following formula (wherein R is the same as in the above general formula (I)). (2) Using a lipase derived from a microorganism as the lipase,
A method for producing optically active cyanohydrin according to claim (1). (3) The following general formula (i) ▲There are mathematical formulas, chemical formulas, tables, etc.▼... (i) (In the formula, R^1, R^2, R^3 and R^4 are each hydrogen atom or carbon Represents an alkyl group of numbers 1 to 4, R
^1, R^2, R^3 and R^4 may be the same or different. ) is reacted with hydrocyanic acid in the presence of lipase to form the following general formula (
ii) ▲There are mathematical formulas, chemical formulas, tables, etc.▼...(ii) (In the formula, R^1, R^2, R^3 and R^4 are the same as the general formula (i) above.) After obtaining optically active cyanohydrin, this cyanohydrin is subjected to acid hydrolysis treatment. , R^1, R^2, R^3 and R^4 are the same as in the general formula (i) above.) A method for producing an optically active lactone represented by: (4) Pantolactone is obtained as the lactone represented by the general formula (iii) by using hydroxypivalaldehyde as the aldehyde represented by the general formula (i).
3) The method for producing an optically active lactone as described above. (5) Using a lipase derived from a microorganism as the lipase,
A method for producing an optically active lactone according to claim (3) or (4).