WO1999004028A1

WO1999004028A1 - PROCESS FOR PREPARING OPTICALLY ACTIVE α-TRIFLUOROMETHYLLACTIC ACID AND ANTIPODE ESTERS THEREOF AND METHOD OF PURIFICATION THEREOF

Info

Publication number: WO1999004028A1
Application number: PCT/JP1998/003160
Authority: WO
Inventors: Eiji Sato; Kanehiko Enomoto; Toshitaka Uragaki
Original assignee: Mitsubishi Rayon Co., Ltd.
Priority date: 1997-07-15
Filing date: 1998-07-14
Publication date: 1999-01-28
Also published as: JPH1175889A

Abstract

A process for preparing optically active α-trifluoromethyllactic acid and antipode esters thereof, characterized by asymmetrically hydrolyzing a racemic α-trifluoromethyllactic ester represented by formula (I) (wherein R represents a substituted or unsubstituted hydrocarbon group having 1 to 12 carbon atoms) in the presence of an enzyme, an immobilized enzyme, a microorganism, a cell culture, or a treated cell each having the capability of asymmetrically hydrolyzing the ester; and a method for purifying optically active α-trifluoromethyllactic acid, characterized by recrystallizing the optically active α-trifluoromethyllactic acid prepared by the above process or a racemic optically active α-trifluoromethyllactic acid prepared by hydrolyzing the racemic ester prepared by the above process and recovering the formed crystals.

Description

Method for producing and refining optically active tri-fluoromethyllactic acid and its enantiomer ester

The present invention relates to a method for producing optically active α-trifluoromethyl lactic acid and an enantiomer ester thereof useful as a raw material or an intermediate for pharmaceuticals, agricultural chemicals, and the like.

Light

Background technology

It has been reported that racemic α-trifluoromethyl lactic acid and its ester can be conventionally synthesized from 1,1, trifluorotrifluoroacetone and sodium cyanide as raw materials (Journal of Chemical Society, 2329, 1951). In this report, a method for producing optically active α-trifluoromethyl lactic acid from racemic α-trifluoromethyl lactic acid by an optical resolution method using brucine is reported. Further, W093 / 23358 discloses a method for producing optically active α-trifluoromethyl lactic acid from racemic H-trifluoromethyl lactic acid by optical resolution method using (S)-(-)-α-methylbenzylamine. Force ⁵ 'has been reported.

However, both methods require expensive resolving agents to increase the optical purity, resulting in high production costs. Further, in these methods, it is necessary to repeat the recrystallization several times or more in the form of a salt with a resolving agent, and then to perform a de-splitting agent treatment, which makes the operation complicated.

In addition, the above-mentioned literature does not mention at all the purification of optically active α-trifluoromethyllactic acid by recrystallization (improvement of optical purity), and it is not possible to improve the optical purity by recrystallization. It was completely unknown. Disclosure of the invention

An object of the present invention is to provide a method for producing an optically active α-trifluoromethyl lactic acid and its enantiomer ester, which is useful as a raw material or an intermediate for pharmaceuticals and agricultural chemicals, without using an optical resolving agent. An object of the present invention is to provide a method and a simple purification method (a method for improving optical purity) of an optically active substance obtained by the method.

The present inventors have conducted intensive studies to solve the above problems, and as a result, have found an enzyme having an activity of optically selectively hydrolyzing racemic α-trifluoromethyl lactate, and furthermore, have found an optically active α- The inventors have found that recrystallization of trifluoromethyl lactic acid improves its optical purity, and completed the present invention.

That is, the present invention includes the following inventions.

(1) General formula (I) _:

0Η

I

CH ₃ -C-COOR (I) CF ₃

(Wherein, R is a substituted or unsubstituted hydrocarbon group having 1 to 12 carbon atoms.) An enzyme having the ability to asymmetrically hydrolyze an ester, which is a racemic α-trifluoromethyl lactate ester represented by the formula: A process for producing optically active α-trifluoromethyl lactic acid and its enantiomer ester, comprising asymmetric hydrolysis in the presence of an enzyme-immobilized product, a microorganism, a culture of a bacterial cell, or a treated product of a bacterial cell.

(2) Optically active α-trifluoromethyllactic acid obtained by the method described in (1) or enantiomer obtained by the method described in (1) (obtained by hydrolyzing this ester) A method for purifying optically active α-trifluoromethyl lactic acid, comprising recrystallizing the enantiomer optically active _α- trifluoromethyl lactic acid and recovering the crystals.

Hereinafter, the present invention will be described in detail.

In the general formula (I), R is a substituted or unsubstituted hydrocarbon group having 1 to 12 carbon atoms. Specifically, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, π-pentyl, iso-pentyl, n-hexyl, An alkyl group having 1 to 12 carbon atoms such as a heptyl group, an octyl group, a 2-ethylhexyl group, a decyl group, a dodecyl group; a vinyl group, a propenyl group, an isopropyl group, a butenyl group, an isobutenyl group; An alkenyl group having 2 to 12 carbon atoms such as a hexenyl group; a carbon atom such as an ethynyl group, a propynyl group and a butynyl group;

One Alkynyl group having 2 to 2 carbon atoms; cycloalkyl group having 3 to 12 carbon atoms, preferably 3 to 7 carbon atoms such as cyclopentyl group, cyclohexyl group and cycloheptyl group; phenyl group, tolyl group, naphthyl group and the like Aralkyl group; benzyl group, phenyl group and other aralkyl groups. In the hydrocarbon group, the hydrogen atom bonded to the carbon atom may be substituted with a substituent such as halogen.

The racemic α-trifluoromethyl lactate as a raw material can be synthesized by a known method as described in, for example, Journal of Chemical Society, 2329 (1951). That is, an aqueous solution of sodium cyanide is added dropwise to an aqueous solution of 1,1,1-trifluoroacetone under cooling, and then sulfuric acid is added thereto and reacted at room temperature for 24 hours to obtain α-trifluoromethyl lactone. It can be produced by synthesizing tolyl and then hydrolyzing it with a strong acid such as sulfuric acid to synthesize α-trifluoromethyllactic acid, followed by esterification of α-trifluoromethyllactic acid according to a conventional method. The ester asymmetric hydrolase used in the present invention is an ester capable of asymmetrically hydrolyzing racemic α-trifluoromethyl lactate to produce optically active _α- trifluoromethyl lactate and its enantiomer ester. Any asymmetric hydrolase can be used regardless of the type of enzyme and its production source. Among them, enzymes generally called lipases, esterases and proteases are particularly effective.

Examples of the ester asymmetric hydrolase include, for example, those isolated from microorganisms capable of producing optically active α-trifluoromethyl lactate and its enantiomer ester by asymmetric hydrolysis of racemic α-trifluoromethyl lactate. Crude or purified enzymes can be used. Examples of such microorganisms include the genus Bacillus, the genus Aspergillus, the genus Candida, the genus Pseudomonas, the genus Rhizopus, the genus Mucor and the like Humicola. Microorganisms to which the gene belongs.

Bacillus subtilis, Bacillus licheniformus, Bacillus polymixa and the like belonging to the genus Bacillus.

Aspergillus saito i, Aspergillus so.jae and other microorganisms belonging to the genus Candida Candida rugosa, Candida antarcia, Candida utilus and the like, and microorganisms belonging to Pseudomonas II include Pseudomonas fluorescence, Pseudomonas antarcia, Pseudomonas sp.MR-2301 (FER BP-4870) and the like. the that microorganisms, Rhizopus Juponicus. Examples of the microorganisms belonging to the genus Mucor, Mucor juponicus, ucor miehei, and examples of the microorganisms belonging to Humicola genus, Humicola lanuginosa Hitoshiryoku ^s' is illustrated.

As the ester hydrolase used in the present invention, a commercially available ester hydrolase can be used. Representative enzymes produced by microorganisms include, for example, NOVO Alcalase (derived from Bacillus genus), NOVO Dueurazaim (derived from Bacillus genus), NOVO Esperase (derived from Bacillus）), NOVO Savinase (from Bacillus II), NOVO Neutrase (from Bacillus sp.), NOVO Rebonase (from Humicola sp.), NOVO Flavozym (from Aspergillus sp.), Nagase Biochemical Co., Ltd. Oprase conc (from Bacillus genus), Nagase Seikagaku Corporation Denateam AP and AP-15 (both from Aspergi llus us), Nagase Seikagaku Corporation lipase 2 G (Pseudomonas genus), Amano Pharmaceutical lipase P (From Pseudomonas), Amano Pharmaceutical Lipase PS (from Pseudomonas), Amano Pharmaceutical Neulase F (from Rhizopus), Amano Pharmaceutical Lipase MFL, Amano Lipase M (from Mucor), Amano Pharmaceutical Lipase AY (from Candida), Amano Pharmaceutical Lipase A (from Aspergillus), Amano Pharmaceutical Lipase M-AP-10, Asahi Kasei Enzymes such as LP-015-S manufactured by Toshiba Corporation, and the like, and ester hydrolases of animal origin include pancreatin and tribubine derived from pigs or porcines.

Furthermore, not only the crude enzyme or the purified enzyme isolated from the microorganism as described above, but also a culture obtained by culturing the microorganism in a culture medium as it is, or by a cell collection operation such as centrifugation from the culture The asymmetric hydrolysis of racemic α-trifluoromethyl lactic acid ester represented by the general formula (I) in the presence of the obtained culture supernatant, cells, or processed cells results in optically active α-trimethylbenzene. Rifluoromethyl lactic acid and its enantiomer ester can also be produced. Examples of the treated cells include cells treated with acetone, toluene, and the like, crushed cells, cell-free extracts obtained by crushing cells, and the like. In the production method of the present invention, when the ester asymmetric hydrolase is subjected to the reaction, its use form is not particularly limited as long as the enzyme shows activity, and the enzyme is immobilized on a suitable carrier and used. You can also. By immobilizing the enzyme, the separation and recovery of the optically active α-trifluoromethyllactic acid and its enantiomer ester and the enzyme after the reaction is completed, and the enzyme can be reused. In the present invention, the optically selective hydrolysis of the racemic α-trifluoromethyl lactate can be carried out by the following method. A racemic α-trifluoromethyl lactate ester as a substrate is dissolved or suspended in a reaction solvent. Before or after the addition of the substrate to the reaction solvent, an enzyme, enzyme-immobilized product, microorganism, bacterial cell culture solution, or treated cell product having the ability to perform asymmetric hydrolysis as a catalyst is added. Then, the reaction is carried out until about half the amount of α-trifluoromethyl lactate is hydrolyzed while controlling the reaction temperature and, if necessary, ρ of the reaction solution. In some cases, the reaction is interrupted at an early stage of the reaction, or the reaction is excessively performed.

The substrate concentration of the reaction solution is not particularly limited in the range of fl.i to 80% by weight, but is preferably 1 to 50% by weight in consideration of productivity and the like.

The enzyme concentration of the reaction solution is usually 0.01 to 10% by weight, and preferably 0.05 to 5% by weight.

The pH of the reaction solution depends on the optimum pH of the enzyme used, but is generally in the range of pH 4 to II. It is preferable to carry out the reaction at pH 5 to 9 in that a decrease in optical purity and a decrease in yield due to the chemical hydrolysis reaction can be suppressed. In addition, as the reaction proceeds, the pH decreases.In this case, an appropriate neutralizing agent, such as sodium hydroxide or an aqueous solution of a hydroxide power, is added to adjust the pH to the optimum value. Is desirable. The reaction temperature is preferably from 5 to 70 ° C, more preferably from 10 to 50 ° C.

As the reaction solvent, an aqueous medium such as ion-exchanged water or a buffer solution is usually used, but the reaction can be carried out in a system containing an organic solvent. Examples of the organic solvent include alcohol solvents such as methanol, ethanol, propanol, isopropanol, butanol, isobutanol, t-butyl alcohol, t-amyl alcohol, and fats such as pentane, hexane, heptane, octane, etc. Aromatic hydrocarbon solvents, aromatic hydrocarbon solvents such as benzene, toluene, and xylene, methylene chloride, chloroform, tetrasalt Halogenated hydrocarbon solvents such as carbonized carbon and dichloroethane; ether solvents such as getyl ether, diisopropyl ether, tetrahydrofuran, and dioxane; ester solvents such as ethyl acetate, propyl acetate, and butyl acetate; acetone, methyl ethyl ketone, and methyl A ketone solvent such as isobutyl ketone, other acetonitril, リ, Ν-dimethylformamide and the like can be used as appropriate. It is also possible to add two or more of these organic solvents so as to perform the reaction in a two-layer system by adding the organic solvents to the solvent or more. Coexistence of an organic solvent in the reaction system often improves selectivity, conversion, yield, and the like. The reaction time is generally 1 hour to 1 week, preferably 1 to 72 hours, and it is preferable to select a reaction condition under which the reaction is completed.

Regarding the above substrate concentration, enzyme concentration, ΡΗ, temperature, solvent, reaction time and other reaction conditions, the target optically active compound is the most common in consideration of the reaction yield, optical yield, etc. under those conditions. It is desirable to select the conditions that can be collected as appropriate.

By reaction of the racemic alpha - Bok Rifuruoromechiru lactates is asymmetric hydrolysis, optically active alpha - triflate Ruo B methyl lactate force ^s to produce. The remaining substrate is the enantiomeric ester of the optically active α-trifluoromethyllactic acid produced.

Isolation of the resulting optically active α-trifluoromethyl lactic acid and optically active α-trifluoromethyl lactate (ie, the optically active α-trifluoromethyl lactic acid enantiomer ester) from the reaction mixture is performed by extraction and distillation. It can be carried out by a conventional isolation method such as column separation. .

For example, for optically active α-trifluoromethyl lactate, for example, after adjusting ρΗ of the reaction solution to near neutrality, ethers such as getyl ether and diisopropyl ether; esters such as ethyl acetate; It can be extracted and separated using common organic solvents such as hydrocarbons such as xane, octane, benzene, and toluene; and halogenated hydrocarbons such as methylene chloride.

On the other hand, optically active α-trifluoromethyl lactic acid can be extracted and separated with a common organic solvent similar to the above after adding a strong acid such as sulfuric acid or hydrochloric acid to the above-mentioned extraction residue.

Further, the optically active α-trifluoromethyl lactate ester is hydrolyzed by a usual method. By solving the above, α-trifluoromethyl lactic acid can be obtained while maintaining the optical activity. The optically active α-trifluoromethyl lactic acid can be converted into α-trifluoromethyl lactate ester while maintaining the optical activity by esterification by a usual method. Therefore, α-trifluoromethyllactic acid and an ester thereof having an arbitrary configuration can be obtained.

The optically active α-trifluoromethyl lactic acid obtained as described above can be further purified by recrystallization to improve its optical purity. Hereinafter, purification by recrystallization will be described in detail.

In the present invention, the optically active α-trifluoromethyl lactic acid to be subjected to recrystallization may be either R-form or S-form. The optical purity is not particularly limited as long as it is not 0% e.e., that is, as long as one of the R-form and the S-form is contained more than the other, but is preferably 10% e.e. or more. In this specification, the optical purity is represented by an enantiomeric excess (% e.e.).

The optically active α-trifluoromethyllactic acid obtained by asymmetric hydrolysis according to the present invention can be subjected to recrystallization as it is. On the other hand, the enantiomeric ester produced simultaneously with the optically active α-trifluoromethyl lactic acid needs to hydrolyze the ester bond by a usual method before being subjected to recrystallization.

The solvent used for recrystallization of the optically active α-trifluoromethyl lactic acid is not particularly limited as long as it does not react with the optically active α-trifluoromethyl lactic acid. The optical purity of the raw materials used, the desired optical purity, etc. Depending on the situation, the decision can be made appropriately in consideration of the recovery rate of the target product. Examples of the solvent include aliphatic hydrocarbon solvents such as pentane, hexane, heptane, and octane; aromatic hydrocarbon solvents such as benzene, toluene, and xylene; methylene chloride, chloroform, carbon tetrachloride, Halogenated hydrocarbon solvents such as dichloroethane; ether solvents such as getyl ether, isopropyl ether, tetrahydrofuran, and dioxane; ester solvents such as ethyl acetate, propyl acetate, and butyl acetate; acetone, Ketone solvents such as methyl ethyl ketone and methyl isobutyl ketone; and acetate ditolyl, Ν, Ν-dimethylformamide and the like. Among them, toluene, π-hexane, ethyl acetate, methylene chloride, chloroform, isopropyl ether, Acetone and acetate nitrile are preferred. Of these, toluene has a range of freezing point to boiling point (1 atm, 95 ° C to 106 ° C) within an easy-to-handle range. Is particularly preferred because of its wide range. These recrystallization solvents can be used alone or in combination.

In addition, alcoholic solvents such as methanol, ethanol, propanol and butanol may be combined with other solvents as a mixed solvent as long as they do not cause an esterification reaction with optically active α-trifluoromethyllactic acid. Use may be effective in some cases.

The recrystallization operation can be performed according to a general method, and there is no particular limitation. That is, the optically active α-trifluoromethyl lactic acid as a raw material can be dissolved in the above-mentioned recrystallization solvent under heating, and then can be precipitated by cooling. Crystals may be precipitated by adding a poor solvent for optically active α-trifluoromethyl lactic acid. Further, in order to smoothly and efficiently precipitate crystals, seeds of crystals can be seeded. The crystal seed is not particularly limited, but it is preferable to use a crystal having a high optical purity, a racemic crystal, or the like, depending on the purpose.

The temperature conditions for the recrystallization operation can be appropriately determined according to the boiling point and the freezing point of the solvent used. Generally, the raw material is dissolved at room temperature (25 ° C) to the boiling point of the solvent, and is -80 ° C to 50 ° C. Crystals can be precipitated with C. When using toluene (freezing point: -95 °, boiling point: 110.6 ° C under 1 atm) as a solvent, dissolve the raw material at 90: to 110, and at 20 to 50 ° C. The amount of the recrystallization solvent and the amount of the optically active α-trifluoromethyl lactic acid used as the raw material are not particularly limited as long as they are completely dissolved. It can be appropriately determined in consideration of the recovery rate of the target substance according to the purity, the target optical purity, and the like.

Next, the purified optically active α-trifluoromethyl lactic acid can be recovered by a conventional method such as filtration and centrifugation.

The optical purity of these optically active α-trifluoromethyl lactic acids can be easily measured by gas chromatography using a GC capillary column for optical resolution after esterification by a conventional method. The optical purity (enantiomeric excess;% e.e.) Is generally determined by GC using (S) -α-trifluoromethyl lactic acid and) -α-trifluoro It can be calculated from each peak area of fluoromethyl lactic acid by the following formula.

If R> S:

Optical purity of R-form (% e.e.) = (R— S / R + S) 100

For S> R:

Optical purity of S-isomer (% e.e.) = (S-R / R + S) 100

S: (S) -a—Trifluoromethyllactic acid peak area

R: peak area of (R) -α-trifluoromethyl lactic acid

The optically active α-trifluoromethyl lactic acid obtained as described above can be converted into an optically active substance having a higher optical purity by repeating the above-mentioned purification by recrystallization once or more times. it can. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described specifically with reference to Examples, but the scope of the present invention is not limited to the Examples.

[Reference Example 1]

Synthesis of racemic monofluoromethyllactic acid η-butyl ester

60 g of racemic α-trifluoromethyl lactic acid and 1 ml of concentrated sulfuric acid were added to 65 ml of η-butanol, and the mixture was reacted for 15 hours under reflux. Next, excess butanol was removed by distillation. After the remaining liquid was put in ice water, extraction was performed three times with 60 ml of getyl ether. The getyl ether layers obtained by the three extraction operations were combined, distilled under reduced pressure and purified to obtain 64 g of racemic α-trifluoromethyl lactate η-butyl ester.

[Example 1]

To a reactor equipped with a pH controller (1) add 300 ml of J.1M phosphate buffer, 15 g of racemic α-trifluoromethyllactic acid η-butyl ester and 3 g of denazymeΑΡ (manufactured by Nagase Seikagaku Corporation). The reaction was carried out overnight at 30 ° C while adjusting the pH of the reaction solution to 7.0 with an aqueous solution of sodium hydroxide of N.

After the completion of the reaction, extraction was performed three times using 100 ml of diisopropyl ether. I got it. The diisopropyl ether layers obtained by the three extraction operations were combined, dehydrated by adding anhydrous magnesium sulfate, and then the diisopropyl ether was removed by distillation. The α-trifluoromethyllactic acid η-butyl ester (4.8 g) thus obtained was analyzed by capillary gas chromatography equipped with an optical resolution column (Chirasil-DEX CB column manufactured by Chrompack). As a result, it was found to be an optically active form (S form) and had an optical purity of 96.5% ee.

To the optically active ester obtained above, 50 ml of a 5% aqueous sodium hydroxide solution was added, and a hydrolysis reaction was carried out at room temperature for 2 hours. Then, the pH was adjusted to 1.5 by adding sulfuric acid, and extraction was performed three times with 50 ml of ethyl acetate each. The ethyl acetate layers obtained by the three extraction operations were combined, dehydrated with magnesium sulfate, and the solvent was removed. Α-Trifluoromethyl lactic acid (2.3 g) obtained in this manner was esterified with diazomethane and then attached to a capillary gas chromatograph equipped with an optical separation column (Chirasil-DEX CB column manufactured by Chrompack). Analysis by Graphic 1 revealed that it was an optically active form (S form) and had an optical purity of 96.5% ee.

The (S) -a-trifluoromethyllactic acid n-butyl ester extraction residue (aqueous layer) was concentrated to 100 ml. After adding concentrated sulfuric acid to adjust the pH to 1. Q, extraction was performed three times by adding 50 ml each of ethyl acetate. The ethyl acetate layers obtained by the three extraction operations were combined into one, and anhydrous magnesium sulfate was added for dehydration, and then the solvent was removed. Α-Trifluoromethyl lactic acid (3.3 g) obtained in this manner was esterified with diazomethane, and then attached to a gas chromatography column equipped with a fe separation column (Chirasil-DEX CB column manufactured by Chrompack). As a result of analysis in, the product was an optically active form (R form), and the optical purity was 58.9% ee.

[Examples 2 to 45]

In each Example, each of the enzymes shown in Table 1 was added to 0.5 ml of a G.1M phosphate buffer (pH = 7.0) in which 1% by weight of racemic α-trifluoromethyllactic acid η-butyl ester was suspended. After adding 10 mg and shaking at 30 ° C. for 24 hours, 0.5 ml of diisopropyl ether was added and stirred. Gas chromatography equipped with an optical resolution column (Chirasil-DEX CB column manufactured by Chrompack) using the optically active α-trifluoromethyllactic acid η-butyl ester contained in the diisopropyl ether layer in the same manner as in Example 1. Table 1 shows the results of the analysis of the configuration and optical purity measured by

Table 1 Example Enzyme Optical purity

9

Reno, Γ ν ^ ΐΤ¾ ¾τ ¾ \ rseuuornondb i ui) 48% e.e.o · »J, _ ΐ ^ £ ^ = T 丄 ^ ε r cUUUiHuiici j ^ jtU ^ (s) 59% e.e.

¾ A · nD v

i Speigl lb (S) 20% ee

0 · ηΓ4 丄, bycl Ut> i¾

) (s) 10% ee

0D ¾ † _L ^ i / ibptil S oj t j (s) 16% e.e.

7 /. M ΛΓ U 丄丄 ^ ΐ (R) 25% e.e.

Q

0.M U ^ 丄, MU UI_JP¾ tn ^ ^-1 14% e.e.

Q

¾ † ϊ ^ ii¾ † _L ^ ¾ I \ U / · υ; ^ tU t (s) 55% ee inu. % ee

ur / SJ Insect Byeon 丄 UlU Ud tu ^) (R) 15% ee L Lr υ IE LH 丄丄丄 (S) 35% ee yd ydbc V lUM † _L ^ £ cUU Oil (R) 21% ee

1 A4 * IL indQbpc

RyucL a I (S) 100% ee υ ri uc tb j JJc uriviii -L ^ c ^ (R) 10% ee

17 Pr Jn ut

(S) 100% ee

P ιr Ln u ten ΡΑ dςρ i, w wl Q! / (Tf uiWivi / Ai T 丄 ^ ΐ R i ihti 7n

63% ee

1 q Prnt iqp Tvnp XIX TGMA

Aqnprei 1 lus concealed from pej ") (S) 100% ee

20 Protease TVDP XXTTT f from Sigma, AsDersillus sp.) (S) 100% e e.

(S) 100% e e.

Trvn ^ in Tvnp TT f TA i + S! I Pig reason) (R) 6.5% e e.ϋ.I ι η ^ ρ f Fluk i Restriction Cnndi genue) (S) 100% e e.iA . (<;)

No Nore Katana Rino Loano L ALr Z 100% e.

(Meiji Seika Co., Bacillus genus)

25. Lipozyme I 20 (NOVO) (R) 100% e.

26. Lipase M (manufactured by Amano Pharmaceutical Co., derived from Mucor II) (R 48% e e.

27. Protease Type XVI (from Sigma, Bacillus 厲) (S 100 e e.

28. Lipase MF (manufactured by Amano Pharmaceutical Co.) (S 40% e.

29.NOVO Alcalase 2.5L (NOVO, BaciUus 厲) (S 100% e e.

30. NOVO Durazym 16. OL (NOVO, from Bacillus 厲) (S 41 e e. 31. NOVO Esperase 8.0L (NOVO, Bacillus sp.) (S) 75% ee

32. NOVO Savinase 16. OL (manufactured by NOVO, from Bacillus 厲) (S) 100% e.e.

33. NOVO Savinase 6. OT (manufactured by NOVO, from the genus Bacillus) (S) 100% e.e.

34. Bioprase conch (S) 69% e.e.

(Bacillus 化学 from Nagase Seikagaku Corporation)

35. Dena Team AP-15 (S) 100% e.e.

(From Nagase Seikagaku Corporation, Aspergillus sp.)

36. Lipolase 100L (NOVO, Humicola genus) (R) 39% e.e.

37. Flavor zyme MG TypeB (NOVO, Aspergillus genus) (S) 100% e.e.

38. Lipase F-AP15 (manufactured by Amano Pharmaceutical Co.) (S) 29% e.e.

39. Lipase AY30 (manufactured by Amano Pharmaceutical Co., derived from the genus Candida) (R) 77% e.e.

40. Palatase M1000L (manufactured by NOVO) (R) 46% e.e.

41. Lilipase A-10 (manufactured by Nagase Seikagaku Corporation, from the genus Rhizopus) (S) 7.4% e.e.

42. Lipase 2G (Pseudomonas genus, manufactured by Nagase Seikagaku Corporation) (R) 43% e.e.

43. Neutrase 0.5L (manufactured by NOVO, from the genus Bacillus) (S) 100% e.e.

44. Bioprase AL-15FG (S) 100% e.e.

(From Nagase Seikagaku Corporation, Bacillus sp.)

45. Flavorzyme 1000L (manufactured by NOVO, from the genus Aspergillus) (S) 100% e.e.

(Example 46)

Prepare a liquid medium consisting of 10 g of peptone (Diico), 5 g of yeast extract (Difco), 5 g of salt, and 1 L of distilled water, and inject 50 ml of this liquid medium into 300 ml Erlenmeyer flask Then, 12 (steam-sterilized at 3 ° C for 15 minutes. One platinum loop of Pseudomonas sp. MR-230KFERM BP-4870) was inoculated into 5 of the flasks, and cultured with shaking at 30 ° C for 1 day. Next, the cells were collected from the culture solution in each flask by centrifugation, washed with water, and suspended in 10 ml of 50 mM phosphate buffer (pH = 7.0). To 0.5 ml of this cell suspension, add 4.5 ml of 0.1% phosphate buffer (ρΗ = 7.0) in which 2% by weight of racemic α-trifluoromethyllactic acid η-butyl ester is suspended, and add 30 ° C For 24 hours with shaking.

To the reaction solution, 5π diisopropyl ether was added and stirred. The optically active α-trifluoromethyllactic acid η-butyl ester contained in the diisopropyl ether layer was treated in the same manner as in Example 1 by using an optical separation column (Chirasil-DEX CB As a result of analyzing by gas chromatography with ram) and measuring the steric configuration and optical purity, it was R-form and the optical purity was 30% ee.

[Example 4-1]

In the same manner as in Example 2, 10 mg of each of the enzymes shown in Table 2 was added to G.5 ml of 0.1 M phosphate buffer (pH = 7.0) in which 1% by weight of racemic α-trifluoromethyl lactate methyl ester was suspended. Then, after shaking at 30 ° C for 24 hours, 0.5 ml of diisopropyl ether was added and stirred. The optically active α-trifluoromethyl lactate methyl ester contained in the disopropyl ether layer was subjected to an optical resolution column in the same manner as in Example 1.

Table 2 shows the results of analysis by gas chromatography equipped with a (Chromapack Chirasito DEX CB column) and measurement of steric configuration and optical purity.

Table 2

Example Enzyme Optical purity

47. Lipase OF (Meito Sangyo Co., Ltd., Candida genus) (R) 11% &. E.

48. Acylase I (from Aspergillus 製, manufactured by SIGMA) (S) 100% e. E.

49. Protease Type I (manufactured by SIGMA Co., Ltd.) (R) 33% e.e.

50.Lipase (Fluka, Candida genus) (S) 100% e.e.

51. Lipozyme IM 20 (NOVO) (R) 9.5% e.e.

52. NOVO Alcalase 2.5L (NOVO, Bacillus genus) (S) 100% e.e.

53. NOVO Savinase 6.0T (NOVO, Bacillus genus) (S) 100% e. E.

54. Bioprasekonk (S) 100% e.e.

(From Nagase Seikagaku Corporation, Bacillus sp.)

55. Dena Team AP-15 (S) 100% e.e.

(From Nagase Seikagaku Corporation, Aspergillus sp.)

56. Flavor zyme MG TypeB (NOVO, from Aspergillus genus) (S) 100 e. E.

57. Lipase F-AP15 (manufactured by Amano Pharmaceutical Co.) (S) 14% e.e.

58. Lipase AY30 (manufactured by Amano Pharmaceutical Co., Ltd., from Candida 厲) (R) 29% e.e.

59. Palatase 1000L (NOVO) (R) 46 e. E.

60. Lilipase A-10 (manufactured by Nagase Seikagaku Corporation, derived from Rhizopus R) (S) 19% e.e.

61. Neutrase 0.5L (NOVO, from Bacillus 厲) (S) 100% e.e.

(Example 62 10 ml of toluene was added to 1.Og of (S) -α-trifluoromethyllactic acid of 96.5% ee obtained in Example 1, dissolved at 100 ° C., and allowed to stand at room temperature at room temperature. When the precipitated crystals were collected, 0.8 g of (S) -α-trifluoromethyllactic acid having 99% ee or more was obtained.

The optical purity was measured as follows. An ether solution of diazomethane was added to the obtained crystals to methylesterify the solution, and the solution was analyzed by gas chromatography (GC) under the following conditions, and (S) -a-trifluoromethyllactic acid and (R) -Optical purity from the peak area of α-trifluoromethyl lactic acid according to the above formula

(Enantiomeric excess) was calculated.

Column: CP-Chirasil DEX CB 0.25mm x 25m (Chrom Pack)

Column temperature: 70 ° C

Detector: F ID

[Example 63]

50 ml of toluene was added to 1.Og of 58.9% e.e. of (R) -α-trifluoromethyllactic acid obtained in Example 1, dissolved at 100 ° C., and allowed to stand at 30 ° C. for 3 hours. The precipitated crystals were collected to obtain 0.28 g of (R) -α-trifluoromethyl lactic acid with 79% e.e. The optical purity was measured by the method described in Example 62.

[Example 64]

500 ml of η-hexane is added to 1. Og of 58.9% ee (R) -α-trifluromethyl lactic acid obtained in Example 1 and dissolved by heating near the boiling point. It was left still. When the precipitated crystals were collected, 0.4 g of (R) -a-trifluoromethyl lactic acid having 70% e.e. was obtained. The optical purity was measured by the method described in Example 62.

[Example 65]

Except that the reaction time was halved, 64% ee (Stra-trifluoromethyllactic acid 2.Og) obtained in the same manner as in Example 1 was dissolved by heating in a solvent shown in Table 3 and then room temperature. The solvent used, the amount of the solvent, the amount of crystal recovered, and the optical purity of the obtained crystal are shown in Table 3. The optical purity was measured in Example 62. This was performed according to the method described in. Table 3

[Example 66]

Dissolve 64% ee (S) -α-trifluoromethyl lactic acid 2.Og obtained in the same manner as in Example 1 except that the reaction time was reduced to half, and then dissolve it in ethyl acetate or isopropyl ether. After adding n-hexane, the mixture was allowed to stand at 120 ° C for 5 hours to precipitate crystals. Table 4 shows the solvent used, the amount of the solvent, the amount of crystal recovered, and the optical purity of the obtained crystal. The optical purity was measured by the method described in Example 62.

Table 4

[Example 6 7]

Dissolve 64% ee (S) -α-trifluoromethyllactic acid 2.Og in acetonitrile or acetone obtained in the same manner as in Example 1 except that the reaction time was halved, and further cooled to room temperature. After adding toluene, the mixture was allowed to stand at 120 ° C. for 5 hours to precipitate crystals. Table 5 shows the solvent used, the amount of solvent, the amount of crystal recovered, and the optical purity of the obtained crystal. The optical purity was measured by the method described in Example 62.

Table 5 Solvent Solvent Amount Crystal Recovery Amount Optical Purity Acetonitrile z Toluene 2ml / 100ml 0.3g 95% ee Acetone / Toluene 2ml / 100ml 0.05g 97% ee Industrial applicability

According to the present invention, optical activity a useful as a raw material or a synthetic intermediate for pharmaceuticals, agricultural chemicals, etc.

-Trifluoromethyl lactic acid and its esters can be produced without using an optical resolving agent. Further, if the optically active α-trifluoromethyllactic acid obtained by the present invention is recrystallized, purification (improvement of optical purity) by a simple method is possible.

Claims

The scope of the claims

1. The following equation (I):

O H

I

CH ₃ 1 C 1 C〇〇R (I) CF ₃

(Wherein R is a substituted or unsubstituted hydrocarbon group having 1 to 12 carbon atoms.) A racemic α-trifluoromethyl lactate represented by the formula: A process for producing optically active α-trifluoromethyl lactic acid and its enantiomer ester, which comprises asymmetric hydrolysis in the presence of an enzyme, an enzyme-immobilized product, a microorganism, a culture of a bacterial cell or a treated bacterial cell. .

2. The method according to claim 1, wherein the enzyme having an ester asymmetric hydrolysis ability is a lipase, an esterase or a protease.

3. Enzymes, enzyme immobilized substances, microorganisms, cell culture broths or processed cells having an asymmetric ester hydrolysis ability can be used for Bacillus, Aspergillus, Candida, and Pseudomonas. The method according to claim 1, wherein the method is derived from a microorganism belonging to the genus (Pseudomonas), the genus Rhizopus, the genus Mucor or the genus Hmnicola.

4. The method according to claim 1, wherein the enzyme capable of asymmetric hydrolysis of an ester is pancreatin or trypsin derived from pig or pepper.

5. An optically active α-trifluoromethyllactic acid obtained by the method according to claim 1, or a pair obtained by hydrolyzing an enantiomeric ester obtained by the method according to claim 1. A method for purifying optically active α-trifluoromethyllactic acid, comprising recrystallizing the enantiomer optically active α-trifluoromethyllactic acid and recovering the crystal.

6. The enantiomer obtained by hydrolyzing the optically active α-trifluoromethyllactic acid obtained by the method of claim 2 or the enantiomer ester obtained by the method of claim 2. A method for purifying optically active α-trifluoromethyl lactic acid, comprising recrystallizing the optically active α-trifluoromethyl lactic acid and recovering the crystals.

7. The enantiomer obtained by hydrolyzing the optically active α-trifluoromethyllactic acid obtained by the method according to claim 3 or the enantiomer ester obtained by the method according to claim 3. A method for purifying optically active α-trifluoromethyl lactic acid, comprising recrystallizing the optically active α-trifluoromethyl lactic acid and recovering the crystals.

8. An optically active α-trifluoromethyllactic acid obtained by the method according to claim 4, or an enantiomer obtained by hydrolyzing an enantiomeric ester obtained by the method according to claim 4. A method for purifying optically active α-trifluoromethyl lactic acid, comprising recrystallizing optically active α-trifluoromethyl lactic acid and collecting crystals.