WO2007072753A1

WO2007072753A1 - Process for production of optically active 3-(3-hydroxyphenyl)-2-alkoxypropanoic acid or ester thereof

Info

Publication number: WO2007072753A1
Application number: PCT/JP2006/325035
Authority: WO
Inventors: Takashi Miki
Original assignee: Sumitomo Chemical Company, Limited; Eisai R & D Management Co., Ltd.
Priority date: 2005-12-19
Filing date: 2006-12-15
Publication date: 2007-06-28
Also published as: JP2007166908A

Abstract

Disclosed is a process for production of an optically active 3-(3-hydroxyphenyl)-2-alkoxypropanoic acid represented by the formula (2): (2) (wherein R1 represents an alkyl group having 1 to 4 carbon atoms; R2 represents an alkyl group having 1 to 6 carbon atoms; and a carbon atom marked with '*' represents an asymmetric carbon atom.) The process comprises the step of enantioselectively hydrolyzing a mixture of enantiomers of a 3-(3-hydroxyphenyl)-2-alkoxypropanoic acid ester represented by the formula (1): (1) (wherein R1 and R2 are as defined in the formula (2) above) with an enantioselective hydrolysis enzyme derived from a microorganism selected from the group consisting of microorganisms belonging to the genera Penicillium, Pseudomonas, RPhizocumcor and Arthropbacter, a culture of a microorganism capable of producing the enzyme, or a product of the culture given by any treatment, wherein the enantioselective hydrolysis enzyme can hydrolyze an ester group in one of the enentiomers preferentially.

Description

Specification

Method for producing optically active 3_ (3-hydroxyphenyl) _2_alkoxypropanoic acid or ester thereof

Technical field

The present invention relates to a method for producing optically active 3- (3 hydroxyphenol) 2 alkoxypropanoic acid or an ester thereof.

Background art

[0002] As a method for producing optically active 3- (3 hydroxyphenol) -2 alkoxypropanoic acid and 3- (3-hydroxyphenyl) -2-alkoxypropanoic acid ester, for example, Patent Document l (WO No. 02Z100812, Examples 279a to 279d) are known.

However, the above-described method requires the steps of introducing and removing the asymmetric auxiliary group in order to produce the optically active compound, which requires many steps, and the reaction temperature is 75 °. Since it is C, there is a problem that it is not necessarily advantageous as an industrial production method.

Patent Literature l: WO 02Z100812

Disclosure of the invention

Problems to be solved by the invention

[0003] The present invention provides an industrially advantageous process for producing optically active 3- (3 hydroxyphenyl) 2 alkoxypropanoic acid and 3- (3 hydroxyphenol) -2 alkoxypropanoic acid ester. For the purpose.

Means for solving the problem

[0004] As a result of intensive studies to solve the above problems, the present inventor has found that 3- (3 hydroxyphenol) 2-alkoxypropanoic acid ester is asymmetrically hydrolyzed using a certain enzyme. The present inventors have found that the above problems can be solved.

That is, the present invention provides:

1.Formula (1):

[Wherein, R ¹ represents an alkyl group having 1 to 4 carbon atoms, and R ² represents an alkyl group having 1 to 6 carbon atoms. A 3- (3 hydroxyphenol) 2 alkoxypropanoate enantiomeric mixture represented by the formula (2), which has the ability to preferentially hydrolyze the ester group of one enantiomer. Cultivation of an asymmetric hydrolase originating from a microorganism selected from the group consisting of the genus Penicillium, Pseudomonas, Rhizomucor and Arthrobacter, or a microorganism capable of producing the enzyme Formula (2) characterized by asymmetric hydrolysis using nutrients or processed products thereof:

[Wherein R ¹ and R ² are as defined in formula (1), and the carbon atom marked with * represents an asymmetric carbon atom. A process for producing optically active 3- (3 hydroxyphenol) 2-alkoxypropanoic acid represented by the formula:

2. Preferentially hydrolyze the enantiomeric mixture of 3- (3 hydroxyphenol) -2 alkoxypropanoate represented by the formula (1) described in 1 above, and the ester group of one enantiomer. Asymmetric hydrolyzing enzymes originating from microorganisms selected from the group consisting of the genus Penicillium, Pseud omonas, Rhizomucor and Arthrobacter Or after asymmetric hydrolysis using a culture or treated product of a microorganism capable of producing the enzyme, the unreacted formula (3) in the asymmetric hydrolysis reaction solution:

[Wherein, R ¹ represents an alkyl group having 1 to 4 carbon atoms, and IT represents an alkyl group having 1 to 6 carbon atoms. * The carbon atom with a mark represents an asymmetric carbon atom. The residual ester represented by the following formula is isolated from the optically active 3- (3 hydroxyphenyl) 2 alkoxypropanoic acid that is an asymmetric hydrolysis reaction product, and then the unreacted residual ester is isolated. A process for producing an optically active 3- (3 hydroxyphenol) 2 alkoxypropanoate represented by the formula (3);

3. Preferentially hydrolyze the enantiomeric mixture of 3- (3 hydroxyphenol) -2 alkoxypropanoate represented by the formula (1) described in 1 above, and the ester group of one enantiomer. Asymmetric hydrolyzing enzymes originating from microorganisms selected from the group consisting of the genus Penicillium, Pseud omonas, Rhizomucor and Arthrobacter Or asymmetrically hydrolyzed using a culture of microorganisms capable of producing the enzyme or a treated product thereof, and then remaining unreacted in formula (3) in the asymmetric hydrolysis reaction solution The above-mentioned unreacted residual ester is hydrolyzed after separating the ester from the optically active 3- (3 hydroxyphenyl) 2 alkoxypropanoic acid which is an asymmetric hydrolysis reaction product. A process for producing an optically active 3- (3 hydroxyphenyl) 2 alkoxypropanoic acid represented by the formula (2) according to 1;

4. The method for producing optically active 3- (3 hydroxyphenol) 2-alkoxypropanoic acid according to 1 above, wherein R ¹ in formula (1) is an ethyl group;

5. The method for producing an optically active 3- (3 hydroxyphenol) 2-alkoxypropanoic acid ester according to 2 above, wherein R ¹ in formula (1) and formula (3) is an ethyl group;

6. The process for producing optically active 3- (3 hydroxyphenol) 2-alkoxypropanoic acid according to 3 above, wherein R ¹ in formula (1) and formula (3) is an ethyl group;

7. The method for producing an optically active 3- (3 hydroxyphenol) 2 alkoxypropanoic acid according to the above 1 or 4, which is an R ² force isopropyl group in formula (1) and formula (2);

8. The optically active 3- (3 hydroxyphenol) -2 alkoxypropanoic acid ester according to 2 or 5 above, wherein R ² in formula (1), formula (2) and formula (3) is an isopropyl group Production method;

9. The above 3 or 6, wherein R ² in formula (1), formula (2) and formula (3) is an isopropyl group A process for producing optically active 3- (3 hydroxyphenol) 2 alkoxypropanoic acid

10. Enzyme power Asymmetry originating from microorganisms selected from the group also having the powers of Penicillium citrinum, Rhizomucor miehei and Arthrobacter globiformis The method for producing optically active 3- (3 hydroxyphenol) 2 alkoxypropanoic acid according to any one of the above 1, 4 and 7, which is a hydrolase;

11. The enzyme is not derived from a microorganism selected from the group having the ability of Penicillium citrinum, Rhizomucor miehei and Arthrobacter globiformis. A method for producing an optically active 3- (3 hydroxyphenyl) 2 alkoxypropanoate according to any one of the above 2, 5 and 8, which is a simultaneous hydrolase;

12. The enzyme is not derived from a microorganism selected from the group having the ability of Penicillium citrinum, Rhizomucor miehei and Arthrobacter globiformis. The method for producing optically active 3- (3 hydroxyphenyl) 2 alkoxypropanoic acid according to any one of the above 3, 6 and 9, which is a simultaneous hydrolase;

13. The optically active enzyme according to any one of the above 1, 4 and 7, wherein the enzyme is an esterase derived from Arthrobacter globif ormis SC 6-98-28 (FERM BP-3658) — (3 hydroxyphenol) 2 alkoxypropanoic acid production method;

14. The optically active compound according to any one of the above 2, 5 and 8, wherein the enzyme is an esterase derived from Arthrobacter globif ormis SC 6-98-28 strain (FERM BP-3658) — (3 Hydroxyphenol) 2 Alkoxypropanoic acid ester production method;

15. The optically active 3 of any one of the above-mentioned 3, 6 and 9, wherein the enzyme is an esterase derived from Arthrobacter globif ormis SC 6-98-28 strain (FERM BP-3658) — (3 Hydroxyphenol) 2 Alkoxypropanoic acid production method; 16. The optically active 3 according to any one of the above 1, 4 and 7, wherein the enzyme is a mutant enzyme in which one or more specific amino acids in the enzyme according to the above 10 are deleted, added or substituted. — (3 hydroxyphenol) 2 production method of alkoxypropanoic acid;

17. The optical activity according to any one of 2, 5, and 8, wherein the enzyme is a mutant enzyme in which one or more specific amino acids in the enzyme according to 11 or 14 are deleted, added, or substituted. 3- (3 hydroxyphenol) 2 production method of alkoxypropanoic acid ester; and

18. The optical activity according to any one of the above 3, 6 and 9, wherein the enzyme is a mutant enzyme obtained by deleting, adding or substituting one or more specific amino acids in the enzyme described in 12 or 15 above. The present invention provides a method for producing 3- (3 hydroxyphenyl) -2 alkoxypropanoic acid.

The invention's effect

[0006] According to the present invention, there is provided an industrially advantageous process for producing optically active 3- (3 hydroxyphenyl) 2 alkoxypropanoic acid 3- (3 hydroxyphenol) 2 alkoxypropanoic acid ester. Can be provided.

BEST MODE FOR CARRYING OUT THE INVENTION

[0007] Hereinafter, the present invention will be described in detail.

In the present invention, a 3- (3 hydroxyphenol) -2 alkoxybutanoic acid ester represented by formula (1) (hereinafter sometimes referred to as substrate (1)) is, for example, the above-mentioned Patent Document 1 ( According to the method described on page 93 of WO 02Z100812), it can be produced from a starting material of 2-alkoxyacetate ester and 3-benzyloxybenzaldehyde through three steps.

A substrate produced by a method described in a document other than Patent Document 1 may be used as the substrate.

[0008] R ¹ in the substrate (1) represents an alkyl group having 1 to 4 carbon atoms. Examples of the alkyl group include a methyl group, an ethyl group, an n propyl group, an isopropyl group, an n butyl group, an isobutyl group, a sec butyl group, and a tert butyl group.

As R ¹ in the substrate (1), a methyl group or an ethyl group is preferred, and an ethyl group is particularly preferred. preferable.

R ² in the substrate (1) represents a C 16 alkyl group. Examples of the alkyl group include methyl group, ethyl group, n-propyl group, isopropyl group, cyclopropyl group, n butyl group, isobutyl group, sec butyl group, tert butyl group, cyclobutyl group, n-pentyl group, isopentyl group. Group, sec pentyl group, tert pentyl group, cyclopentyl group, n-xyl group, isohexyl group, sec hexyl group, tert-xyl group or cyclohexyl group.

R ² in the substrate (1) is particularly preferably an isopropyl group, preferably an ethyl group or an isopropyl group.

Examples of the substrate (1) include 3- (3 hydroxyphenol) -2-methoxypropanoic acid methyl ester, 3- (3 hydroxyphenol) 2-methoxypropanoic acid ethyl ester 3- (3 hydroxyphenol) ) 2-Methoxypropanoic acid n-propyl ester, 3- (3-hydroxyphenol) 2-Methoxypropanoic acid isopropyl ester, 3- (3 hydroxyphenyl) 2-Methoxypropanoic acid n-butyl ester, 3- ( 3-Hydroxyphenol) —2-Methoxypropanoic acid isobutyl ester, 3-— (3 Hydroxyphenol) —2 — Methoxypropanoic acid sec Butyl ester, 3-— (3 Hydroxyphenol) 2-Methoxypropanoic acid tert— Butyl ester, 3- (3 hydroxyphenol) -2 Ethoxypropanoic acid methyl ester, 3- (3 hydroxyphenol) 2 ethoxypropanoic acid Cyl ester, 3— (3 hydroxyphenol) 2 Ethoxypropanoic acid n-propyl ester, 3— (3 hydroxyphenol) —2 Ethoxypropanoic acid isopropyl ester, 3— (3 hydroxyphenol) 2 Ethoxy Propanoic acid n butyl ester, 3— (3 hydroxyphenyl) 2 Ethoxypropanoic acid isobutyl ester, 3— (3 hydroxyphenol) —2 Ethoxypropanoic acid sec Butyl ester, 3— (3 hydroxyphenol) —2 Ethoxypropanoic acid tert-butyl ester, 3— (3 hydroxyphenol) —2 —n—propoxypropanoic acid methyl ester, 3— (3 hydroxyphenol) 2—n—propoxypropanoic acid ethyl ester, 3— (3-hydroxyphenol) -2-n-propoxypropanoic acid _n- propyl ester, 3- (3-hydroxyphenol) 2-n-propoxypro Panic acid isopropyl ester, 3— (3 hydroxyphenol) 2—n—Provo _N- Butyl ester of xylpropanoic acid, 3- (3 hydroxyphenol) 2-n-propoxypropanoic acid isobutyl ester, 3-(3 hydroxyphenol) 2-n-propoxypropanoic acid _sec butyl ester, 3-(3 hydroxy 2) n-propoxypropanoic acid tert butyl ester, 3- (3 hydroxyphenol) -2 isopropoxypropanoic acid methyl ester, 3- (3-hydroxyphenol) -2-isopropoxypropan Acid ethyl ester, 3- (3 hydroxyphenol) 2 Isopropoxypropanoic acid n —Propyl ester, 3— (3 hydroxyphenol) 2-Isopropoxypropanoic acid isopropyl ester, 3— (3 Hydroxyphenol) ) 2-Isopropoxypropanoic acid n-butyl ester, 3- (3 hydroxyphenol) 2-Isopropoxypropanoic acid isobutyl Ester, 3- (3 Hydroxyphenyl) 2 Isopropoxypropanoic acid sec-Butyl ester, 3- (3 Hydroxyphenyl) 2 Isopropoxypropanoic acid tert-Butyl ester, 3- (3 Hydroxyphenyl) 2 Cyclo Propoxypropanoic acid methyl ester, 3— (3 hydroxyphenyl) —2 Cyclopropoxypropanoic acid ethyl ester, 3— (3 hydroxyphenol) 2 Cyclopropoxypropanoic acid n-propyl ester, 3— (3 Hydroxyphenyl- 2) 2-Cyclopropoxypropanoic acid isopropyl ester, 3— (3 hydroxyphenol) —2 Cyclopropoxypropanoic acid n-butyl ester, 3— (3 Hydroxyphenol) 2 Cyclopropoxypropanoic acid isobutyl ester, 3— ( 3 Hydroxyphenol) 2 Cyclopropoxypropanoic acid sec Butyl Este 3— (3 hydroxyphenol) 2 cyclopropoxypropanoic acid tert-butyl ester, 3— (3 hydroxyphenol) 2—n-butoxypropanoic acid methyl ester, 3— (3 hydroxyphenol) 2— n-butoxypropanoic acid ethyl ester, 3- (3 hydroxyphenyl) 2— n-butoxypropanoic acid n-propyl ester, 3-— (3 hydroxyphenyl) 2— n-butoxypropanoic acid isopropyl ester, 3— ( 3-hydroxyphenyl) 2-n-butoxypropanoic acid n-butyl ester, 3-- (3hydroxyphenyl) 2-n-butoxypropanoic acid isobutyl ester, 3-- (3 hydroxyphenyl) 2- n-butoxy Propanoic acid sec butyl ester, 3- (3 hydroxyphenyl) 2— n-butoxypropanoic acid tert-butyl ester, 3-— (3 hydroxyphenyl) —2— Su Wu butoxy propanoic acid methyl ester, 3- (3-hydroxy Hue - Le) 2-Isobutoxypropanoic acid ethyl ester, 3- (3 hydroxyphenol) —2-Isobutoxypropanoic acid n-propyl ester, 3- (3 hydroxyphenyl) -2 Isobutoxypropanoic acid isopropyl ester, 3— (3 hydroxyphenyl) 2-isobutoxy propanoic acid _n-butyl ester, 3- (3-hydroxy Hue - Le) -2 Isobutokishipuro pan acid isobutyl ester, 3- (3-hydroxy Hue - Le) 2-isobutoxy propanoic acid sec-butyl ester 3— (3 hydroxyphenol) 2 Isobutoxypropanoic acid tert butyl ester, 3— (3 hydroxyphenol) —2— sec Butoxypropanoic acid methyl ester, 3— (3 hydroxyphenol) —2 — Sec Butoxypropanoic acid ethyl ester, 3— (3 hydroxyphenyl) —2— sec Butoxypropanoic acid n-propyl ether Steal, 3— (3 hydroxyphenol) 2—sec Butoxypropanoic acid isopropyl ester, 3-— (3 hydroxyphenol) —2—sec Butoxypropanoic acid n-butyl ester, 3-— (3 hydroxyphenol) 2—sec Butoxypropanoic acid isobutyl ester, 3— (3 hydroxyphenol) —2—sec Butoxypropanoic acid sec butyl ester, 3 -— (3 hydroxyphenol) —2—sec Butoxypropanoic acid tert butyl ester, 3- (3-hydroxyphenol) -2-tert-butoxypropanoic acid methyl ester,

3— (3-hydroxyphenol) —2— tert-butoxypropanoic acid ethyl ester, 3-— (3 —hydroxyphenyl) —2— tert-butoxypropanoic acid n-propyl ester, 3— (3 — hydroxyphenol 2) tert-Butoxypropanoic acid isopropyl ester, 3— (3—hydroxyphenol) —2—tert-butoxypropanoic acid n-butyl ester, 3— (3—hydroxyphenol) 2—tert— Butoxypropanoic acid isobutyl ester, 3— (3 Hydroxyphenyl) —2— tert Butoxypropanoic acid sec butyl ester, 3— (3 Hydroxyphenol) —2— tert Butoxypropanoic acid tert-butyl ester, 3— ( 3—Hydroxyphenol) 2 Cyclobutoxypropanoic acid methyl ester, 3— (3 Hydroxyphenyl) 2 Cyclobutoxypropanoic acid ethyl ester, 3— (3 Hydroxyphenyl) Ethyl) —2 Cyclobutoxypropanoic acid n-propyl ester, 3— (3 Hydroxyphenol) 2 Cyclobutoxypropanoic acid isopropyl ester, 3— (3 Hydroxyphenol) 2 Cyclobutoxypropanoic acid n butyl ester, 3— (3 Hydroxyphenyl) —2 Cyclobutoxypropanoic acid isobutyl ester, 3— (3 Hydroxyphenyl) 2 cyclobutoxypropanoic acid sec butyl ester, 3— (3 hydroxyphenol)

2 cyclobutoxypropanoic acid tert-butyl ester, 3— (3 hydroxyphenol) 2—n-pentyloxypropanoic acid methyl ester, 3— (3 hydroxyphenyl) —2 n pentyloxypropanoic acid ethyl ester, 3— (3 Hydroxyphenol) 2 —n—Pentyloxypropanoic acid n-propyl ester, 3— (3-Hydroxyphenol) — 2— n-Pentyloxypropanoic acid isopropyl ester, 3— (3 Hydroxyphenol) 2-n-pentyloxypropanoic acid n-butyl ester, 3- (3 hydroxyphenol) 2-n-pentyloxypropanoic acid isobutyl ester, 3- (3 hydroxyphenyl) 2 — N pentyloxypropanoic acid sec butyl ester, 3— (3 hydroxyphenyl) 2—n—pentyloxypropanoic acid tert butyl ester, 3— (

3-Hydroxyphenol) -2-Isopentyloxypropanoic acid methyl ester, 3- (3-hydroxyphenyl) -2-isopentyloxypropanoic acid ethyl ester, 3- (3-hydroxyphenyl) —2-—Isopentyloxypropanoic acid n-propyl ester, 3- (3-hydroxyphenol) 2-Isopentyloxypropanoic acid isopropyl ester, 3- (3-hydroxyphenol) 2 isopentyloxy Propanoic acid n-butyl ester, 3— (3 hydroxyphenyl) 2 Isopentyloxypropanoic acid isobutyl ester, 3— (3-hydroxyphenol) —2-Isopentyloxypropanoic acid sec butyl ester, 3 — (3-Hydroxyphenol) —2-Isopentyloxypropanoic acid tert butyl ester, 3 -— (3-hydroxyphenol) 2—sec pentyloxyprop Acid methyl ester, 3- (3 hydroxyphenyl) 2-sec pentyloxypropanoic acid ethyl ester, 3- (3 hydroxyphenol) 2-sec pentyloxypropanoic acid n-propyl ester, 3- (3-hydroxyphenol) —2—sec pentyloxypropanoic acid isopropyl ester, 3-— (3 hydroxyphenol) 2—sec pentyloxypropanoic acid n-butyl ester, 3 -— (3 hydroxyphenol) 2—sec—pentyl Oxypropanoic acid isobutyl ester, 3— (3 hydroxyphenol) 2 sec pentyloxypropanoic acid sec butyl ester, 3— (3-hydroxyphenol) 2—sec pentyloxypropanoic acid tert butyl ester, 3— (3 hydroxyphenyl) —2— tert-pentyloxypropanoic acid methyl ester, 3— (3 hydroxy Phenyl) 2-tert-pentyloxypropanoic acid ethyl ester, 3- (3 hydroxyphenyl) —2-tert-pentyloxypropanoic acid n-propyl ester, 3-— (3-hydroxyphenol) 2— tert-pentyloxypropanoic acid isopropyl ester, 3 mono (3-hydroxyphenol) 2-tert-pentyloxypropanoic acid n-butyl ester, 3- (3 hydroxyphenyl) 2-tert-pentyloxypropanoic acid isobutyl Esters, 3- (3 hydroxyphenol) 2-tert-pentyloxypropanoic acid sec butyl ester, 3- (3-hydroxyphenol) 2-tert-pentyloxypropanoic acid tert-butyl ester, 3- (3 hydroxy Phenyl) 2-cyclopentyloxypropanoic acid methyl ester, 3- (3 hydroxyphenol) -2 cyclopentyloxypropanoic acid ethyl ester Le, 3- (3-hydroxy Hue - Le) 2 Shikuropenchiruo Kishipuropan acid _n-propyl ester, 3- (3-hydroxy Hue - Le) 2 Shikuropen chill O carboxymethyl propanoic acid isopropyl ester, 3- (3-hydroxy Hue - Le) 2 Sik Methyl pentyloxypropanoic acid n-butyl ester, 3- (3 hydroxyphenyl) -2-cyclopentyloxypropanoic acid isobutyl ester, 3- (3-hydroxyphenyl) 1-2 cyclopentyloxypropanoic acid sec butyl ester, 3- (3 hydroxyphenyl) -2 cyclopentyloxypropanoic acid tert-butyl ester, 3- (3-hydroxyphenyl) 2- n-hexyloxypropanoic acid methyl ester, 3- (3 hydroxyphenyl 2) n-Hexyloxypropanoic acid ethyl ester, 3- (3 hydroxyphenyl) 2-n-hexyloxy Propanoic acid n-propyl ester, 3- (3-hydroxyphenyl) 2-n-hexyloxypropanoic acid isopropyl ester, 3- (3 hydroxyphenol) 2-n-hexyloxypropanoic acid n- Butyl ester, 3— (3 hydroxyphenol) 2— n-hexyloxypropanoic acid isobutyl ester, 3— (3 hydroxyphenol) 2— n-hexyloxypropanoic acid sec butyl ester, 3— (3 Hydroxyphenyl) 2— n—Hexyloxypropanoic acid tert —Butyl ester, 3— (3—Hydroxyphenyl) —2-Isohexyloxypropanoic acid methyl ester, 3— (3 —Hydroxyphenol) —2-Isohexyloxypropanoic acid ethyl ester, 3 -— (3 hydroxyphenyl) 2 Isohexyloxypropanoic acid n —Propyl ester, 3— (3 Hydroxyphenyl) - Le) hexyl O carboxymethyl propane to 2-iso Isopropyl ester, 3- (3 hydroxyphenol) 2-Isohexyloxypropanoic acid n butyl ester, 3- (3 hydroxyphenol) 2 Isohexyloxy ester Butanolic acid isobutyl ester, 3- (3 hydroxyphenol- 2) 2-Isohexyloxypropanoic acid _sec butyl ester, 3— (3-hydroxyphenol) —2-Isohexyloxypropanoic acid tert butyl ester, 3— (3 hydroxyphenol) —2—sec hexyl Oxypropanoic acid methyl ester, 3— (3 hydroxyphenyl) —2—sec Hexyloxypropanoic acid ethyl ester, 3 -— (3 hydroxyphenol) 2—sec—Hexyloxypropanoic acid n-propyl Esters, 3— (3 hydroxyphenol) —2— sec Hexyloxypropanoic acid isopropyl ester, 3— (3-hydroxyphenol) 2—sec Xyloxypropanoic acid n-butyl ester, 3- (3 hydroxyphenol) —2—sec hexyloxypropanoic acid isobutyl ester, 3-— (3 hydroxyphenyl) —2—sec hexyloxypropanoic acid sec Butyl ester, 3— (3-hydroxyphenol) —2— sec Hexyloxypropanoic acid tert Butyl ester, 3- (3-hydroxyphenol) —2— tert-Hexyloxypropanoic acid methyl Ester, 3- (3-Hydroxyphenyl) -2-tert-hexyloxypropanoic acid ethyl ester, 3- (3 Hydroxyphenyl) -2-2-tert-hexyloxypropanoic acid n-propyl ester, 3- (3 hydroxyphenol) 2-tert-hexyloxypropanoic acid isopropyl ester, 3- (3-hydroxyphenol) -2-tert-hexyloxypropanoic acid n-butyl ester 3- (3-hydroxy Hue - Le)-2-tert to Kishiruokishi propanoic acid isobutyl ester, 3- (3-hydroxy Hue - Le) hexyl Okishipuropan acid _sec-butyl ester to-2-tert, 3- (3-hydroxy-Hue -L) 2-tert-hexyloxypropanoic acid tert butyl ester, 3- (3 hydroxyphenol) 2 -cyclohexyloxypropanoic acid methyl ester, 3- (3 hydroxyphenol) -2-cyclo Hexyloxypropanoic acid ethyl ester, 3- (3-hydroxyphenyl) 2 cyclohexyloxypropanoic acid n-propyl ester, 3- (3 hydroxyphenyl) 2 cyclohexyloxypropanoic acid isopropyl ester 3- (3 hydroxyphenol) 2 cyclohexyloxypropanoic acid n-butyl ester, 3- (3 hydroxyphenyl) -2-cyclohexyl Okishipuropan acid isobutyl ester, 3- (3- Hydroxyphenyl) 2 cyclohexyloxypropanoic acid sec butyl ester, 3 (3-hydroxyphenyl) 2 cyclohexyloxypropanoic acid tert butyl ester, and the like.

[0009] Among the substrates (1) which are enantiomeric mixtures, enzymes having the ability to preferentially hydrolyze the ester group of one enantiomer include, for example, the genus Penicillium, Pseudomonas And asymmetric hydrolyzing enzymes originating from microorganisms selected from the group consisting of the genus Rhizomucor and the genus Arthrobacter.

Examples of the hydrolytic enzyme include hydrolytic enzymes originating from microorganisms such as Penicillium citrinum, Rhizomucor miehei or Arthrobacter globiformis. Can be mentioned.

Examples of the hydrolase originating from the microorganism include enzymes (esterase or lipase) derived from ALSLOPACTOR 'Grobifolmis SC-6-98-28 strain (FERM BP-3658) and the following commercially available enzymes.

Protease B “Amano” [Penicillium citrinum origin; manufactured by Amano Enzym Co., Ltd.], CH E Amano '2 [Pseudomonas sp. Origin; [Pseudom onas sp. Origin; manufactured by Showa Denko], Lipase 62291 [Rhizomucor miehei derived; manufactured by Fluka], and the like.

[0010] Enzymes etc. derived from the above-mentioned Alslobacter 1 'Grobifolmis SC-6-98-28 strain (FERM BP-36 58) are derived from mutants produced from the above microorganisms by treatment with mutagens or ultraviolet rays. It may be an enzyme or an enzyme produced by a recombinant microorganism transformed by introducing a gene encoding the present enzyme of the microorganism.

Further, it may be a mutant enzyme in which one to several specific amino acids in the amino acid sequence of the enzyme are deleted, added or substituted by genetic engineering techniques.

A method for producing a recombinant microorganism transformed by introducing a gene encoding an enzyme For example, ¾J. Sambrook, EF Fritsch, T. Maniatis; described in Molecular Cloning 2nd edition, Cold Spring Harbour Laboratory, published in 1989, etc. The method according to the usual genetic engineering method can be mentioned. More specifically, the method described in JP-A-7-163364 can be mentioned.

[0011] Examples of a method for producing a mutant enzyme by genetic engineering techniques include the method of Olfert Landt (Gene 96 125-128 1990). More specifically, JP-A No. 2000-78988 discloses a method according to the method described in JP-A-7-213280. Examples of mutant enzymes that can be produced in this way include mutant esterases or mutant lipases produced from esterases derived from ALS lobacter I. globifolumis SC-6-98-28. Can do.

[0012] Any microorganism that produces an enzyme can be liquid-cultured by an ordinary method. As the medium, various mediums appropriately containing carbon sources, nitrogen sources, inorganic substances and the like used in normal microorganism culture can be used.

For example, examples of the carbon source include glucose, glycerin, organic acid and molasses. Nitrogen sources include peptone, yeast extract, malt extract, soy flour, corn steep liquor, cottonseed flour, dry yeast, casamino acid, salty ammonium ammonia, ammonium nitrate, ammonium sulfate Examples include urea.

Examples of the inorganic material include hydrochlorides of metals such as potassium, sodium, magnesium, iron, manganese, and corona zinc, sulfates of the above metals, and phosphates of the above metals. Specifically, potassium chloride, sodium chloride, magnesium sulfate, ferrous sulfate, mangan sulfate, cobalt chloride, zinc sulfate, potassium phosphate, sodium phosphate, and the like can be used. In addition, in order to enhance the asymmetric water decomposing ability of the microorganism, triglyceride such as olive oil or tributyrin or the above-mentioned substrate may be added to the medium as appropriate.

Usually, it is preferable to perform shaking culture or aeration-agitation culture in an aerobic atmosphere. The culture temperature is usually about 20 to 40 ° C, preferably about 25 to 35 ° C, and the pH is preferably about 6 to 8. The culture time is preferably about 1 to 7 days depending on various conditions. In addition, a method for obtaining a microbial cell having an asymmetric water-dissolving ability of the above substrate is also possible. For example, a solid culture method can be employed.

[0013] In order to purify the microbial culture obtained by cultivating the above-mentioned enzyme as described above, it is generally used for the purification of the enzyme according to the method.

For example, first, the cells in the microorganism culture are crushed by a method such as ultrasonic treatment, dynomill treatment or French press treatment. Next, after removing insoluble matter from the obtained crushed liquid by centrifugation or the like, cation exchange column chromatography, anion exchange column chromatography, hydrophobic column chromatography and gel, which are usually used for enzyme purification, are used. The desired enzyme can be purified by appropriately combining one or more of filtration column chromatography and the like. Examples of the carrier used in these column chromatography include DEAE—Sepharose fastflow (Amersham “Falmasia” Biotech), Butyl—Toyopearl650S (Tosoichi Co., Ltd.), and the like.

[0014] The enzyme can be used in various forms such as a purified enzyme, a crude enzyme, a microorganism culture, a microbial cell, and a product obtained by treating these. The above-mentioned treated substances are, for example, freeze-dried microbial cells, acetone-dried microbial cells, microbial cell grinds, microbial self-digested products, sonicated microbial cells, microbial cell extracts, or alkaline treatment of microbial cells. It refers to things. In addition, enzymes having various purities or forms as described above may be included, for example, by adsorption on inorganic carriers such as silica gel and ceramics, cellulose, ion-exchanged resin, polyacrylamide method, and carrageenan gel method. Fix it by a known method such as sulfur polysaccharide gel method, alginic acid gel method, or agar gel method.

[0015] The amount of the enzyme used is appropriately selected so that the reaction time is not delayed and the selectivity is not lowered.

For example, when a purified enzyme or a crude enzyme is used, the amount used is usually about 0.001 to 2 times by weight, preferably about 0.002 to 0.5 times by weight of the above substrate.

In addition, the amount used in the case of using a microbial culture, a microbial cell, and a processed product thereof is usually about 0.01 to 200 times by weight, preferably 0.1 to 50 times by weight with respect to the above-mentioned substrate. It is moderate.

[0016] The water used in the asymmetric hydrolysis reaction may be a buffered aqueous solution! Buffered aqueous solution For example, an aqueous solution of an organic acid salt such as an alkaline acid salt salt solution such as an aqueous solution of an inorganic acid salt such as an aqueous solution of an alkali metal phosphate such as an aqueous solution of sodium phosphate or an aqueous solution of potassium phosphate, or an aqueous solution of an alkali metal salt such as an aqueous solution of sodium acetate or aqueous solution of potassium acetate. Buffer aqueous solution etc. are mentioned. The amount of water used is usually in the range of 0.5 molar to 200 weight times the substrate.

[0017] The asymmetric hydrolysis reaction in the present invention may be performed in the presence of an organic solvent such as a hydrophobic organic solvent or a hydrophilic organic solvent.

Examples of the hydrophobic organic solvent include aliphatic ethers such as tert-butyl methyl ether diisopropyl ether, and hydrocarbons such as toluene, hexane, cyclohexane, heptane, and octane isooctane. .

Examples of the hydrophilic organic solvent include tert-butanol, methanol, ethanol, isopropanol, alcohols such as isobutanol and _n -butanol, alicyclic ethers such as tetrahydrofuran, sulfoxides such as dimethyl sulfoxide, Examples thereof include ketones such as acetone, -tolyls such as acetonitrile, and amides such as N, N-dimethylformamide.

These hydrophobic organic solvents and hydrophilic organic solvents can be used alone or in admixture of two or more. Further, a hydrophobic organic solvent and a hydrophilic organic solvent may be mixed and used.

The amount of the organic solvent used is usually 200 times by weight or less, preferably 0.1 to L00 times the weight of the substrate.

[0018] The asymmetric hydrolysis reaction is performed, for example, by a method of mixing water, a substrate and an enzyme.

In the case of using an organic solvent, the organic solvent, water, substrate and enzyme may be mixed.

The pH of the reaction system is appropriately selected so that the asymmetric hydrolysis reaction by the enzyme proceeds with good selectivity. The pH of the reaction system is usually in the range of about 4 to 10, preferably about 6 to 8. During the reaction, the pH may be adjusted within the above range by adding a base.

Examples of the base include alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, alkali metal carbonates such as sodium carbonate and potassium carbonate, and alkaline earth metal carbonates such as calcium carbonate. Al power such as sodium bicarbonate and potassium bicarbonate Remetal bicarbonate, sodium dihydrogen phosphate, disodium hydrogen phosphate, phosphates such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate, organic bases such as triethylamine and pyridine, and ammonia are used. .

The above bases may be used alone or in combination of two or more. The base is usually added as an aqueous solution, but may be added as a solution in a mixture of an organic solvent and water. The same organic solvent as that used in the reaction can be used as the organic solvent. In addition, the base can be added as a solid or added as a suspension.

If the reaction temperature for asymmetric hydrolysis is too high, the stability of the enzyme tends to decrease, and if it is too low, the reaction rate tends to decrease. The reaction temperature is usually in the range of about 5 to 65 ° C, preferably in the range of about 10 to 50 ° C.

[0019] A reaction solution containing optically active 3- (3 hydroxyphenol) 2 alkoxypropanoic acid represented by formula (2) [hereinafter also referred to as asymmetrically hydrolyzed carboxylic acid] is obtained. In order to separate the asymmetrically hydrolyzed carboxylic acid from the enzyme or buffer used in the reaction, or the remaining substrate represented by the formula (3) [hereinafter also referred to as residual ester] or the like, In addition, post-processing operations may be performed. Examples of the post-treatment operation include a method of separating and purifying using silica gel chromatography after removing the solvent in the reaction solution, and a method of separating and purifying by liquid separation operation.

When separating and purifying by separation operation, if an organic solvent that dissolves in both water and hydrophobic organic solvent is used during the reaction, a solvent that dissolves in both the water and hydrophobic organic solvent must be removed. After removing by distillation, a liquid separation operation may be performed.

In addition, when an insoluble enzyme or an immobilization carrier is present in the liquid containing the asymmetric hydrolysis carboxylic acid, the enzyme or the immobilization carrier may be removed by filtration.

[0020] The residual ester contained in the asymmetric hydrolysis reaction solution may be separated from the aqueous layer after the residual ester is extracted with a hydrophobic organic solvent.

Examples of the hydrophobic organic solvent used in the above extraction include aliphatic ethers such as tert butyl methyl ether and isopropyl ether; hydrocarbons such as toluene, hexane, cyclohexane, heptane, octane and isooctane. Halogenated hydrocarbons such as dichloromethane, dichloroethane, black mouth form, black mouth benzene and orthodichlorobenzene; Examples include esters such as methyl acetate, ethyl acetate and butyl acetate.

When the hydrophobic organic solvent exemplified above is used during the asymmetric hydrolysis reaction, the liquid separation operation can be performed as it is. In addition, if a hydrophobic organic solvent is not used during the asymmetric hydrolysis reaction, or if the hydrophobic organic solvent or water is used in a small amount! A solvent and Z or water may be added as appropriate, and the mixture may be left still for separation.

[0021] The amount of the hydrophobic organic solvent used is not particularly limited, but is usually in the range of about 0.1 to 200 times by weight, preferably about 0.2 to about LOO weight times based on the substrate. It is.

The pH during the above-described extraction or liquid separation operation is usually in the range of about 6 to 12, preferably in the range of about 7 to 10.

[0022] When the asymmetrically hydrolyzed carboxylic acid and the residual ester are separated, an acid or a base can be used as appropriate in order to adjust the pH of the liquid to the above range. Examples of the acid include inorganic acids such as hydrogen chloride, hydrogen bromide, sulfuric acid and phosphoric acid, acidic salts of the inorganic acid and metal, organic acids such as acetic acid, citrate and methanesulfonic acid, and the organic acids. And acid salts of acids and metals. Further, as the base, the same base as that used for pH adjustment during the reaction can be used.

If the separation between the asymmetrically hydrolyzed carboxylic acid and the residual ester is insufficient, the above-described extraction and separation operations may be repeated several times.

The residual ester separated from the carboxylic acid which is an asymmetric hydrolyzate by the above extraction can be isolated by distilling off the organic solvent in the oil layer.

The residual ester isolated by distilling off the organic solvent in the oil layer may be further purified by column chromatography or the like.

[0023] The residual ester obtained by the above operation can be derived into optically active 3- (3 hydroxyphenol) 2 alkoxypropanoic acid, for example, by hydrolysis in the presence of alkali. This optically active 3- (3 hydroxyphenol) 2 alkoxypropanoic acid may be further purified by column chromatography, recrystallization or the like.

[0024] The asymmetrically hydrolyzed carboxylic acid is present in the aqueous layer during the above extraction operation. To separate asymmetrically hydrolyzed carboxylic acid present in the water layer from water-soluble components such as enzymes and buffers The organic layer may be extracted with a hydrophobic organic solvent and then separated from the aqueous layer. As the hydrophobic organic solvent used for the extraction, the same solvent as that used for extracting the residual ester described above can be used. The amount of the hydrophobic organic solvent to be used is usually in the range of about 0.1 to 200 times by weight, preferably in the range of about 0.1 to about LOO weight times based on the substrate.

The pH upon extraction of the asymmetrically hydrolyzed carboxylic acid is usually in the range of about 1-7, preferably in the range of about 2-5. In order to adjust the liquid property during extraction to the above pH range, an acid and a base can be appropriately used. As the acid and base that can be obtained, the same acids and bases used in the liquid separation operation when separating from the above-mentioned residual ester can be used.

When the amount of asymmetrically hydrolyzed carboxylic acid extracted from the aqueous layer is small, the extraction operation and the liquid separation operation may be repeated a plurality of times.

The asymmetrically hydrolyzed carboxylic acid can be isolated by distilling off the hydrophobic organic solvent in the oil layer obtained by the above method. The asymmetrically hydrolyzed carboxylic acid may be further purified by column chromatography or recrystallization.

Examples of the asymmetric hydrolysis carboxylic acid obtained by the present invention include the following compounds.

(S) — 3— (3-hydroxyphenol) 2-methoxypropanoic acid, (S) — 3-— (3-hydroxyphenol) —2 ethoxypropanoic acid, (S) —3— (3 hydroxyphenol 2) n-propoxypropanoic acid, (S) —3— (3 hydroxyphenol) 2 isopropoxy lipanoic acid, (S) — 3— (3 hydroxyphenol) — 2 cyclopropoxypropanoic acid , (S) —3— (3 hydroxyphenol) 2— n-butoxypropanoic acid, (S) —3— (3 hydroxyphenol) —2-isobutoxypropanoic acid, (S) — 3— ( 3—Hydroxyphenol) —2 — sec Butoxypropanoic acid, (S) —3— (3 Hydroxyphenol) 2—tert—Butoxypropanoic acid, (S) —3— (3 Hydroxyphenol) — 2 Cyclobutoxypropanoic acid, (S) —3— (3 Hydroxyphenol) 2— n-Pentyloxypropanoic acid, (S) —3— (3-Hydroxyphenol 2) -Isopentyloxypropanoic acid, (S) —3— (3-hydroxyphenyl) —2—sec pentyloxypropanoic acid, (S) —3— (3 hydroxyphenol) — 2-tert-pentyloxypropanoic acid, (S) -3- (3 hydroxyphenol) —2—Cyclopentyloxypropanoic acid, (S) -3- (3-hydroxyphenol) — 2— n xyloxypropanoic acid, (S) — 3— (3-hydroxyphenol) 2-iso Hexyloxypropanoic acid, (S) 3— (3 Hydroxyphenol) 2 sec Hexyloxypropanoic acid, (S) —3— (3 Hydroxyphenol) 2— tert-Hexyloxypropanoic acid, (S) — 3— (3 hydroxyphenol) 2 cyclohexyloxypropanoic acid, (R) — 3— (3-hydroxyphenol) 2-methoxypropanoic acid, (R) — 3— (3—hydroxyphene- 2) 2-Ethoxypropanoic acid, (R) —3-— (3 Hydroxyphenol) 2— n— Propoxypropanoic acid, (R) — 3— (3-Hydroxyphenol) —2-—Isopropoxypropanoic acid , (R) — 3— (3 hydroxyphenol) — 2 cyclopropoxypropanoic acid, (R) —3— (3 hydroxyphenol 2) n-butoxypropanoic acid, (R) —3— (3 hydroxyphenyl) 2-isobutoxypropanoic acid, (R) — 3— (3 hydroxyphenol) 2 — sec butoxypropane Acid, (R) —3— (3 hydroxyphenol) 2—tert butoxypropanoic acid, (R) —3— (3 hydroxyphenol) —2 cyclobutoxypropanoic acid, (R) —3— (3 Hydroxyphenol) 2— n-pentyloxypropanoic acid, (R) —3— (3 Hydroxyphenol) 2-Isopentyloxypropanoic acid, (R) — 3— (3 Hydroxyphenol) —2— sec pentyloxypropanoic acid, (R) — 3— (3-hydroxyphenol) —2— tert-pentyloxypropanoic acid, (R) —3— (3 hydroxyphenol) —2 Cyclopentyloxypropanoic acid, (R) —3— (3 hydroxyphenol) 2—n Xyloxypropanoic acid, (R) —3— (3-hydride Xyphenyl) 2-isohexyloxypropanoic acid, (R) — 3— (3 hydroxyphenol) 2 sec hexylpropanoic acid, (R) —3— (3 hydroxyphenol) —2— tert— Hexyloxypropanoic acid, (R) — 3— (3 hydroxyphenol) 2 cyclohexyloxypropanoic acid, etc.

According to the present invention, by using an enzyme, or a culture or treated product of a microorganism capable of producing the enzyme, optically active 3- (3 hydroxyphenol) 2 alcoholpropanoic acid, and optical activity 3- (3 Hydroxyphenol) -2 alkoxypropyl panic acid ester can be reacted at a room temperature with fewer steps. Therefore, the present invention is industrially advantageous. Hereinafter, the present invention will be described in more detail with reference to examples. However, it is needless to say that the present invention is not limited to these examples.

[Examples 1 to 6]

[0027] Examples of production of optically active 3- (3 hydroxyphenol) 2 isopropoxypropanoic acid and optically active 3- (3 hydroxyphenol) -2-isopropoxypropanoic acid ethyl ester

An aqueous solution of nofer was prepared by dissolving 1.70 g of disodium hydrogen phosphate and 0.96 g of sodium dihydrogen phosphate in 200 g of water. Separately, 100 ml of a tert butyl methyl ether (hereinafter referred to as MTBE!) Solution containing 10.09 g of 3- (3 hydroxyphenol) 2-isopropoxypropanoic acid ethyl ester was prepared.

Each of the various enzymes shown in Table 1 below was weighed (see Table 2 below for the amount weighed), and 5 ml of buffer solution and 3- (3 hydroxyphenol) 2 isopropoxypropanoic acid. 0.5 ml of an ethyl ester solution of MTBE was added. Next, after stirring at 25 ° C. for 21 hours, MTBE10 ml and 5 ml of 5% phosphoric acid aqueous solution were added and mixed. The oil layer obtained after the separation was analyzed by high performance liquid chromatography (HPLC) [column: Chiralpack AD—H, 4.6 mm × 25 cm (manufactured by Daicel)], and the optically active 3- (3 hydroxyphenol) was analyzed. -L) The conversion to 2-isopropoxypropanoic acid and the enantiomeric excess of the asymmetrically hydrolyzed carboxylic acid and residual ester were determined. The results are shown in Table 2.

[0028] [Table 1]

# · Prepared according to the method described in JP-A-5-56787.

[Table 2] Actual mm conversion Unacceptable Ϊ: Ι: Moisture angle, boric acid remaining rate J rate over imaged body ¾ * ίί over enantiomer overfilled M

Optical isomer. Ratio (% _e e) Optical isomer

1 2 rag 43%> 9 9 (S) body 7 7 (R) body

'2B> 9 9 (S)… body 3 8 (R) J

3 Same as above 1> 9 9 (S) body 4 6 (R)-body

4 Same as above lO 9 9 (S) -body 1 O (R) -body

5 Same as above 15 7 9 (S)-Body 1 4 (R)-J

6! OOrri 74 3 4 () Integrated 9 9 (S)-Body

[Example 7]

[0029] (S) — 3— (3 Hydroxyphenol) —2-Isopropoxypropanoic acid and (R) — 3—

(3 Hydroxyphenol) 2 Production example of isopropoxypropanoic acid ethyl ester

A solution consisting of 4.00 g (0.0159 mol) 3- (3 hydroxyphenol) -2-isopropoxypropanoic acid ethyl ester and 29 g MTBE was prepared.

0.84 g of disodium hydrogen phosphate and 0.48 g of sodium dihydrogen phosphate were dissolved in 96 g of water, and 0.08 g of protease B-amano and an MTBE solution of 3- (3 hydroxyphenol) 2 isopropoxypropanoic acid ethyl ester were added. This was stirred at 25 ° C for 5 hours and further at 40 ° C for 20 hours, washed and extracted twice with MTBE to separate the remaining ester into the MTBE layer, and the aqueous layer was mixed with ethyl acetate, 10% hydrochloric acid, radiolite. The asymmetrically hydrolyzed carboxylic acid was extracted into the oil layer, filtered and separated to obtain an oil layer. The aqueous layer was further extracted with ethyl acetate, and the mixed oil layer was washed and separated with water. The oil layer was concentrated under reduced pressure to remove the solvent. The content of the concentrated residue (1.98 g) of the pale yellow oil was quantified to be 85.0% (yield 47%). Further, when the enantiomer excess was analyzed by HPLC [column: Chiralpack AD-H, 4.6 mm X25 cm (manufactured by Daicel)], it was> 99% ee (S).

When the residual ester content in the MTBE layer was quantified, the yield was 42%. Further, when the enantiomeric excess was analyzed by H PLC [column: Chiralpack AD-H, 4.6 mm × 25 cm (manufactured by Daicel)], it was 94% ee (R).

[Comparative Examples 1 to 5]

[0030] Comparative experiment using another enzyme

An aqueous solution of nofer was prepared by dissolving 1.70 g of disodium hydrogen phosphate and 0.96 g of sodium dihydrogen phosphate in 200 g of water. Separately, 3— (3 hydroxyphenol) 2—Isoprovo 100 ml of MTBE solution containing 10.09 g of xypropanoic acid ethyl ester was prepared. Each of the various enzymes shown in Table 3 below was weighed (see Table 4 below for the amount weighed), and 5 ml of aqueous buffer solution and 3- (3 hydroxyphenol) 2 isopropoxypropanoic acid. 0.5 ml of an ethyl ester solution of MTBE was added. Next, after stirring at 25 ° C. for 21 hours, MTBE10 ml and 5 ml of 5% phosphoric acid aqueous solution were added and mixed. The oil layer obtained after the separation was analyzed by high performance liquid chromatography (HPLC) [column: Chiralpack AD—H, 4.6 mm × 25 cm (manufactured by Daicel)], and the optically active 3- (3 hydroxyphenol) was analyzed. -L) Conversion to 2-isopropoxypropanoic acid and enantiomeric excess were determined. The results are shown in Table 4.

[0031] [Table 3]

[Table 4]

Industrial applicability

[0032] The optically active 3- (3 hydroxyphenyl) -2 alkoxypropanoic acid and the optically active 3- (3 hydroxyphenol) -2 alkoxypropanoic acid ester of the present invention are drugs for treating diabetes It is useful as an intermediate for synthesis or the like (see, for example, WO 02/100812).

Claims

The scope of the claims

Equation (1):

[Wherein R ¹ and R ² are as defined in formula (1), and the carbon atom marked with * represents an asymmetric carbon atom. ] The optically active 3- (3 hydroxyphenol) 2-alkoxypropanoic acid manufacturing method shown by these.

[2] The enantiomeric mixture of 3- (3 hydroxyphenol) 2 alkoxypropanoate represented by the formula (1) according to claim 1 is hydrolyzed preferentially on the ester group of one enantiomer. Asymmetric hydrolyzing enzymes originating from microorganisms selected from the group consisting of the genus Penicillium, Pseud omonas, Rhizomucor and Arthrobacter Or after asymmetric hydrolysis using a culture or treated product of a microorganism capable of producing the enzyme, the unreacted formula (3) in the asymmetric hydrolysis reaction solution:

[Wherein, R ¹ represents an alkyl group having 1 to 4 carbon atoms, and R ² represents an alkyl group having 1 to 6 carbon atoms. A carbon atom marked with * represents an asymmetric carbon atom. The residual ester represented by the following formula is isolated from the optically active 3- (3 hydroxyphenyl) 2 alkoxypropanoic acid that is an asymmetric hydrolysis reaction product, and then the unreacted residual ester is isolated. A method for producing an optically active 3- (3 hydroxyphenol) 2 alkoxypropanoate represented by the formula (3).

[3] The enantiomeric mixture of 3- (3 hydroxyphenol) 2 alkoxypropanoate represented by the formula (1) according to claim 1 is hydrolyzed preferentially on the ester group of one enantiomer. Asymmetric hydrolyzing enzymes originating from microorganisms selected from the group consisting of the genus Penicillium, Pseud omonas, Rhizomucor and Arthrobacter Or after being asymmetrically hydrolyzed using a culture of microorganisms capable of producing the enzyme or a treated product thereof, and represented by the formula (3) according to claim 2, which is unreacted in the asymmetric hydrolysis reaction solution. The residual ester is separated from the optically active 3- (3 hydroxyphenyl) 2-alkoxypropanoic acid, which is an asymmetric hydrolysis reaction product, and then the unreacted residual ester is hydrolyzed. Motomeko optically active 3 represented by 1 formulas described (2) (3-hydroxy Hue - Le) 2-alkoxy manufacturing method of propanoic acid.

[4] The process for producing optically active 3- (3-hydroxyphenyl) 2-alkoxypropanoic acid according to claim 1, wherein R ¹ in the formula (1) is an ethyl group.

5. The optically active 3— (2) according to claim 2, wherein R ¹ in formula (1) and formula (3) is an ethyl group.

3 Hydroxyphenol) 2 A process for producing alkoxypropanoic acid ester.

6. The optically active 3— (3) according to claim 3, wherein R ¹ in the formulas (1) and (3) is an ethyl group.

3 Hydroxyphenol) 2 A process for producing alkoxypropanoic acid.

7. The process for producing optically active 3- (3 hydroxyphenol) -2 alkoxypropanoic acid according to claim 1 or 4, wherein R ² in the formulas (1) and (2) is an isopropyl group.

8. The optically active 3- (3 hydroxyphenol) -2 alkoxypropanoic acid according to claim 2 or 5, wherein R ² in formula (1), formula (2) and formula (3) is an isopropyl group. Esters manufacturing method.

[9] The optically active 3- (3 hydroxyphenol) 2 alkoxypropanoic acid according to claim 3 or 6, wherein R ² in the formula (1), the formula (2) and the formula (3) is an isopropyl group. Production method.

[10] The enzyme is not derived from a microorganism selected from the group that also has the power of Penicillium citrinum, Rhizomucor miehei, and Arthrobacter glo biformis. The method for producing optically active 3- (3 hydroxyphenyl) 2-alkoxypropanoic acid according to any one of claims 1, 4 and 7, which is a simultaneous hydrolase.

[11] Enzymatic power Asymmetry originating from microorganisms selected from the group consisting of Penicillium citrinum, Rhizomucor miehei and Arthrobacter glo biformis The method for producing an optically active 3- (3 hydroxyphenol) 2-alkoxypropanoic acid ester according to any one of claims 2, 5 and 8, which is a hydrolase.

[12] Enzymatic power Asymmetry originating from microorganisms selected from the group consisting of Penicillium citrinum, Rhizomucor miehei and Arthrobacter glo biformis The method for producing optically active 3- (3 hydroxyphenyl) 2-alkoxypropanoic acid according to any one of claims 3, 6 and 9, which is a hydrolase.

[13] Enzyme S, Arthrobacter globiformis SC 6

The method for producing optically active 3- (3 hydroxyphenyl) 2 alkoxypropanoic acid according to any one of claims 1, 4 and 7, which is an esterase derived from 98-28 strain (FERM BP-3658).

[14] Enzyme is Arthrobacter globiformis SC 6

9. The method for producing an optically active 3- (3 hydroxyphenol) 2 alkoxypropanoate according to claim 1, which is an esterase derived from 98-28 strain (FERM BP-3658).

[15] The optically active enzyme according to any one of claims 3, 6 and 9, wherein the enzyme is an esterase derived from Arthrobacter globiformis SC-6 / 98-28 (FERM BP-3658). 3— (3 Hydroxyphenol) 2 A method for producing alkoxypropanoic acid.

[16] The optical according to any one of [1], [4] and [7], wherein the enzyme is a mutant enzyme obtained by deleting, adding or replacing one or more specific amino acids in the enzyme described in 10 above. Process for producing active 3- (3 hydroxyphenol) 2 alkoxypropanoic acid.

[17] The enzyme according to any one of claims 2, 5 and 8, wherein the enzyme is a mutant enzyme in which one or more specific amino acids in the enzyme according to claim 11 or 14 are deleted, added or substituted. Of optically active 3- (3 hydroxyphenol) -2 alkoxypropanoate.

[18] The enzyme according to any one of claims 3, 6 and 9, wherein the enzyme is a mutant enzyme in which one or more specific amino acids in the enzyme according to claim 12 or 15 are deleted, added or substituted. Of optically active 3- (3 hydroxyphenol) -2 alkoxypropanoic acid.