JP2019024494A - Method for asymmetric synthesis of chiral carboxylic acid compounds using thioester bond hydrolase - Google Patents

Abstract

To provide a novel method of asymmetrically synthesizing a chiral carboxylic acid.SOLUTION: The invention relates to a method of asymmetrically synthesizing a chiral carboxylic acid, through an enzymatic hydrolysis reaction mediated by at least one acyl-CoA esterase selected from the group consisting of ACOT7 and ACOT13, from the racemate of a thioester compound having an asymmetric carbon at the α-position and represented by general formula (I) in the figure. [In the formula, R, R, Rand Rrepresent hydrogen or a substituent, or Rand Rmay form a substituted or unsubstituted cyclic structure together with the carbon atom to which they are bonded, provided that R, R, Rand CO-S-Rare different from each other.]SELECTED DRAWING: None

Description

  The present invention relates to a method for asymmetric synthesis of carboxylic acid compounds.

For optically active biological substances, only one enantiomer may be useful or harmful, and it is necessary to synthesize them as chiral substances that do not contain optical isomers. One of the methods is asymmetric synthesis, which has been put into practical use as a method for separately producing optical isomers by stereoselective chemical reaction (for example, Non-Patent Document 1).
On the other hand, in the chemical synthesis industry, there is a demand for a reaction having high reaction selectivity, or a chemical reaction system having high activity in an aqueous solution (without using an organic solvent), and an enzyme having high selectivity and activity in an aqueous solution. Reactions have been developed and put into practical use (for example, Non-Patent Document 2).
In addition, as one of the asymmetric synthesis reactions for synthesizing chiral compounds, there is an enzyme asymmetric synthesis reaction utilizing the stereoselectivity of an enzyme serving as a catalyst. One of them is a method using an ATP-dependent acyl coenzyme A ligase (acyl CoA ligase) as an asymmetric synthesis method of a carboxylic acid compound having an asymmetric carbon at the α-position (hereinafter referred to as carboxylic acid) and its derivative ( For example, Patent Document 1). In this method, when a racemic carboxylic acid is converted into a CoA thioester compound dehydrated and condensed with coenzyme A (CoA) by acyl CoA ligase, one optical isomer of the carboxylic acid reacts selectively, resulting in chirality. New CoA thioester compounds are synthesized. Since this method proceeds in an aqueous solution, there is an advantage that an organic solvent is unnecessary.

  However, this enzymatic reaction requires expensive ATP as a cofactor. In addition, the chiral CoA thioester compound as a reaction product is easily racemized by enolization, and the optical purity may not be maintained in a high state. In such a case, only the carboxylic acid remaining unreacted is chiral and usable, but the recovery rate is inevitably 50% or less. Even when the CoA thioester compound, which is the reaction product, is difficult to racemize and can be used as a chiral compound, the CoA thioester compound is water-soluble, so the separation and purification becomes complicated, requiring a lot of time and money. There is also.

  By combining thioester hydrolase (thioesterase) with acyl CoA ligase enzyme reaction and optical resolution with racemization, so-called Dynamic Kinetic Resolution (DKR) reaction system, theoretically 100% yield There is also a method of synthesizing an optically active carboxylic acid (Non-Patent Document 3 and Non-Patent Document 4). However, since these methods use thioesterase that has no or very little stereoselectivity, it is necessary to repeat the stereoselective enzymatic reaction with acyl CoA ligase, and it is necessary to add a large amount of acyl CoA ligase. . Furthermore, when racemization of the CoA thioester compound as an intermediate is difficult to proceed, it is necessary to add epimerase. In this case, the reaction is performed in the presence of three kinds of enzymes, and the reaction conditions must be optimized and controlled. There is a problem that time and cost are required.

Taichiro Kato, Hiromichi Ota, Kenji Miyamoto, Mikio Baba “Method for producing optically active carboxylic acid using luciferase” JP 2007-29017.

"Enzymes are used to separate enantiomers, creating, the power of kinetic resolution", Furuta Miyu et al., Journal of Biotechnology, 2014, 92 (6), 298-302. Supervised by Satoshi Komiyama: Enzyme utilization technology system: From basics and analysis to modification, high functionality, and industrial use, p.434-438, NTS Publishing (2010). Juri Maeda, Dai-ichiro Kato, Masatoshi Okuda, Masahiro Takeo, Seiji Negoro, Kazunari Arima, Yuji Ito, Kazuki Niwa, "Biosynthesis-inspired deracemizative production of D-luciferin by combining luciferase and thioesterase" BBA-General Subjects 1861, 2112- 2118 (2017) Xiao Qu, Amanda Allan, Grace Chui, Thomas J. Hutchings, Ping Jiao, Lawrence Johnson, Wai Y. Leung, Portia K. Li, Georgina R. Steel, Andrew S. Thompson, Michael D. Threadgill, Timothy J. Woodman, Matthew D. Lloyd, "Hydrolysis of ibuprofenoyl-CoA and other 2-APP-CoA esters by human acyl-CoA thioesterase-1 and -2 and their possible role in the chiral inversion of profens" Biochemical Pharmacology 86, 1621-1625 (2013 )

  The problem to be solved by the present invention is to provide a novel method for asymmetric synthesis of chiral carboxylic acids.

  Under such circumstances, the present inventors use a mixture of optical isomers of a thioester compound having a specific structure containing an asymmetric carbon as a reaction substrate, and subject this to an enzymatic hydrolysis reaction using a specific acyl CoA esterase. It has been found that chiral carboxylic acids can be asymmetrically synthesized. The present invention is based on such novel findings.

［式中、Ｒ¹、Ｒ²、Ｒ³及びＲ⁴は、水素又は置換基を示す。
Ｒ²及びＲ³はこれらが結合する炭素原子と共に置換又は無置換の環構造を形成してもよい。
ただし、Ｒ¹、Ｒ²、Ｒ³及びＣＯ−Ｓ−Ｒ⁴は互いに異なる。]。 Accordingly, the present invention typically provides the invention as defined in the following sections:
Item 1. By an enzymatic hydrolysis reaction with at least one acyl CoA esterase selected from the group consisting of ACOT7 and ACOT13 from a mixture of optical isomers of a thioester compound having an asymmetric carbon at the α-position represented by the following general formula (I) Asymmetric synthesis of chiral carboxylic acids:

[Wherein R ¹ , R ² , R ³ and R ⁴ represent hydrogen or a substituent.
R ² and R ³ may form a substituted or unsubstituted ring structure together with the carbon atom to which they are bonded.
However, R ¹ , R ² , R ³ and CO—S—R ⁴ are different from each other. ].

Item 2. Item 2. The method according to Item 1, wherein R ¹ is hydrogen.

［式中、Ｒ¹⁰及びＲ¹¹は、同一又は異なって、水素又はアルキル基を示す。−Ｏ−Ｒ¹²は、水酸基、リン酸基含有官能基又はスルホン酸基を示す。ｐは１〜６の自然数を示す。ｑは０〜５の整数を示す。ｒは１〜６の自然数を示す。］
で表される基である、項１又は２に記載の方法。 Item 3. R ⁴ represents the following general formula

[Wherein, R ¹⁰ and R ¹¹ are the same or different and each represents hydrogen or an alkyl group. —O—R ¹² represents a hydroxyl group, a phosphate group-containing functional group, or a sulfonic acid group. p shows the natural number of 1-6. q represents an integer of 0 to 5. r represents a natural number of 1-6. ]
Item 3. The method according to Item 1 or 2, which is a group represented by:

Item 4. Item ^4. The method according to any one of Items 1 to 3, wherein R ¹ is hydrogen and —S—R ⁴ is —S—CoA.

［式中、Ｒ⁵は、結合、−Ｏ−、−Ｓ−又は−Ｃ（＝Ｏ）−を示す。Ｒ⁶は置換基を示す。ｍは０〜５の整数を示す。ｍが２以上の場合、Ｒ⁶は同一でも異なってもよい。］
で表される基である、項１〜４のいずれか１項に記載の方法。 Item 5. R ² is the following general formula

[Wherein R ⁵ represents a bond, —O—, —S— or —C (═O) —. R ⁶ represents a substituent. m shows the integer of 0-5. When m is 2 or more, R ⁶ may be the same or different. ]
Item 5. The method according to any one of Items 1 to 4, which is a group represented by:

［式中、Ｒ⁵及びＲ⁷は、同一又は異なって、結合、−Ｏ−、−Ｓ−又は−Ｃ（＝Ｏ）−を示す。Ｒ⁶及びＲ⁸は、同一又は異なって、置換基を示す。ｍ'は１〜５の自然数を示す。ｎは０〜４の整数を示す。ｍ'−１が２以上の場合、Ｒ⁶は同一でも異なってもよい。ｎが２以上の場合、Ｒ⁸は同一でも異なってもよい。］
で表される基である、項５に記載の方法。 Item 6. R ² is the following general formula

[Wherein, R ⁵ and R ⁷ are the same or different and each represents a bond, —O—, —S—, or —C (═O) —. R ⁶ and R ⁸ are the same or different and each represents a substituent. m ′ represents a natural number of 1 to 5. n shows the integer of 0-4. When m′-1 is 2 or more, R ⁶ may be the same or different. When n is 2 or more, R ⁸ may be the same or different. ]
Item 6. The method according to Item 5, which is a group represented by:

［式中、Ｒ⁹は水素又は置換基を示す。］
で示される環構造を形成している、項１〜４のいずれか１項に記載の方法。 Item 7. R ² and R ³ together with the carbon atom to which they are attached have the general formula

[Wherein R ⁹ represents hydrogen or a substituent. ]
Item 5. The method according to any one of Items 1 to 4, which forms a ring structure represented by:

で示される基である、項７に記載の方法。 Item 8. R ⁹ is

Item 8. The method according to Item 7, which is a group represented by:

Item 9. Item 7. The method according to any one of Items 1 to 6, wherein R ³ is an alkyl group having 1 to 6 carbon atoms.

  According to the present invention, a chiral carboxylic acid can be asymmetrically synthesized by subjecting a mixture of optical isomers of a thioester of a carboxylic acid having an asymmetric carbon at the α-position to an enzymatic hydrolysis reaction.

The test result of this-application Example 3 is shown. The test result of this-application Example 4 is shown.

［式中、Ｒ¹、Ｒ²、Ｒ³及びＲ⁴は、水素又は置換基を示す。
Ｒ²及びＲ³はこれらが結合する炭素原子と共に置換又は無置換の環構造を形成してもよい。ただし、Ｒ¹、Ｒ²、Ｒ³及びＣＯ−Ｓ−Ｒ⁴は互いに異なる。]
を提供する。 The present invention relates to an enzyme by at least one acyl CoA esterase selected from the group consisting of ACOT7 and ACOT13 from a mixture of optical isomers of a thioester compound having an asymmetric carbon at the α-position represented by the following general formula (I) Asymmetric synthesis of chiral carboxylic acids by hydrolysis:

[Wherein R ¹ , R ² , R ³ and R ⁴ represent hydrogen or a substituent.
R ² and R ³ may form a substituted or unsubstituted ring structure together with the carbon atom to which they are bonded. However, R ¹ , R ² , R ³ and CO—S—R ⁴ are different from each other. ]
I will provide a.

Thioester Compound In the present invention, the “substituent” is not particularly limited unless otherwise specified in the present specification, and examples thereof include a hydroxyl group, an amino group, alkyl, halogen, aryl, arylcarbonyl, aryloxy, Examples include thioaryl, a substituted or unsubstituted 5- to 10-membered unsaturated heterocyclic group having 1 to 3 heteroatoms, and the like.

Further, the “substituent” represented by R ² is represented by the group represented by the general formula (A1) and the general formula (A2) described above unless otherwise specified in the present specification. Groups and the like are also included. Further, the “substituent” represented by R ⁴ includes the group represented by the general formula (B1) described above.

  In the present invention, examples of the alkyl include a linear or branched alkyl group having 1 to 6 carbon atoms, and more specifically, for example, methyl, ethyl, n-propyl, isopropyl, 2,2- Examples thereof include dimethylpropyl, 1-ethylpropyl, n-butyl, isobutyl, tert-butyl, isopentyl, n-pentyl, n-hexyl group and the like.

  Examples of the halogen include fluorine, chlorine, bromine and iodine.

  Examples of the aryl include aryl groups having 6 to 14 carbon atoms, and more specifically, for example, phenyl, naphthyl, fluorenyl, anthryl, biphenylyl, tetrahydronaphthyl and the like.

  Examples of the arylcarbonyl include an arylcarbonyl group having 7 to 17 carbon atoms having a structure in which an aryl group having 6 to 14 carbon atoms is bonded to the carbonyl group. More specifically, examples of the arylcarbonyl include groups having a structure in which the above-mentioned aryl is bonded to a carbonyl group.

  Examples of aryloxy include aryloxy in which the aryl moiety is an aryl group having 6 to 14 carbon atoms. More specifically, examples of aryloxy include aryloxy in which the aryl moiety is the aryl group listed above.

  Examples of the 5- to 10-membered unsaturated heterocyclic group having 1 to 3 heteroatoms include, for example, at least one heteroatom selected from the group consisting of nitrogen, sulfur and oxygen (preferably nitrogen and / or And 5- to 10-membered unsaturated heterocyclic group having 1 to 3 (preferably 1 to 2, more preferably 2) sulfur. More specifically, examples of the 5- to 10-membered unsaturated heterocyclic group having 1 to 3 heteroatoms include thienyl, pyrrolyl, furyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, isoxazolyl, isothiazolyl, thiadiazolyl, triazolyl , Tetrazolyl, pyridyl, pyrazyl, pyrimidinyl, pyridazinyl, indolyl, isoindolyl, indazolyl, triazolopyridyl, benzoimidazolyl, benzoxazolyl, benzothiazolyl, benzothienyl, benzothiazolyl (eg benzo [d] thiazol-2-yl etc.) quinolyl, And isoquinolyl.

  The 5- to 10-membered unsaturated heterocyclic group having 1 to 3 heteroatoms may have a substituent or may be unsubstituted. When the 5- to 10-membered unsaturated heterocyclic group having 1 to 3 heteroatoms has a substituent, the substituent is not particularly limited, and examples thereof include those described above. Group, alkyl, halogen, aryl, arylcarbonyl, aryloxy, thioaryl, substituted or unsubstituted 5- to 10-membered unsaturated heterocyclic group having 1 to 3 heteroatoms, and the like. A 5- to 10-membered unsaturated heterocyclic group having 1 to 3 heteroatoms is further substituted with a “5- to 10-membered unsaturated heterocyclic group having 1 to 3 heteroatoms having a substituent. In the unsaturated heterocyclic group bonded as a substituent to the unsaturated heterocyclic group, the substituent further includes, among the above-mentioned substituents, 1 to 3 substituted or unsubstituted heteroatoms. Other than the “5- to 10-membered unsaturated heterocyclic group having”.

In the present invention, R ¹ is not particularly limited. For example, hydrogen, an alkyl group having 1 to 6 carbon atoms (preferably 1 to 3), amino group, halogen, aryl, arylcarbonyl, aryloxy, thioaryl, substituted Or an unsubstituted heterocyclic group having 5 to 10 members having 1 to 3 heteroatoms, such as hydrogen, aryl, aryloxy, thioaryl and the like are preferable, and hydrogen, aryl, aryloxy and the like are more preferable. Hydrogen is particularly preferred.

［式中、Ｒ⁵は、結合、−Ｏ−、−Ｓ−又は−Ｃ（＝Ｏ）−を示す。Ｒ⁶は置換基を示す。ｍは０〜５の自然数を示す。ｍが２以上の場合、Ｒ⁶は同一でも異なってもよい。］で表される基であることが好ましい。 In the present invention, as R ² , for example, the following general formula (A1)

[Wherein R ⁵ represents a bond, —O—, —S— or —C (═O) —. R ⁶ represents a substituent. m represents a natural number of 0 to 5. When m is 2 or more, R ⁶ may be the same or different. It is preferable that it is group represented by this.

When R ² is a group represented by the above general formula (A1), R ⁵ is a bond, —O—, —S— or —C (═O) —, preferably a bond, —O—. Or -S-, more preferably a bond or -O-, and still more preferably a bond.

When R ² is a group represented by the above general formula (A1), the substituent represented by R ⁶ is not particularly limited, and examples thereof include a hydroxyl group, an amino group, halogen, aryl, arylcarbonyl, thioaryl and the like. Halogen, aryl, arylcarbonyl, thioaryl and the like are preferable, and a hydroxyl group, an amino group, halogen, aryl, arylcarbonyl and the like are more preferable. In the general formula (A1), when m is 2 or more, R ⁶ is preferably present at least in the para position and 1 or 2 in the meta position. In general formula (A1), m is preferably 2, and one R ⁶ is present at each of the para and meta positions. In general formula (A1), m is 2 and R ⁶ is at the para position. It is more preferable that one each exists at the meta position and R ⁵ is a bond.

［式中、Ｒ⁵は、結合、−Ｏ−、−Ｓ−又は−Ｃ（＝Ｏ）−を示す。Ｒ⁶は置換基を示す。］
で表される基を示す。 In the above embodiment of the present invention, preferably R ² is represented by the following general formula (A-1):

[Wherein R ⁵ represents a bond, —O—, —S— or —C (═O) —. R ⁶ represents a substituent. ]
The group represented by these is shown.

In the case where R ² is a group represented by the general formula (A1-1), examples of R ⁵ include those described above. In this embodiment, R ⁵ is preferably —O—.

In the case where R ² is a group represented by the general formula (A1-1), examples of the substituent represented by R ⁶ include those described above. In this embodiment, R ⁶ is halogen. Preferably there is.

［式中、Ｒ⁵及びＲ⁷は、同一又は異なって、結合、−Ｏ−、−Ｓ−又は−Ｃ（＝Ｏ）−を示す。Ｒ⁶及びＲ⁸は、同一又は異なって、置換基を示す。ｍ'は１〜５の自然数を示す。ｎは０〜４の整数を示す。ｍ'−１が２以上の場合、Ｒ⁶は同一でも異なってもよい。ｎが２以上の場合、Ｒ⁸は同一でも異なってもよい。］
で表される基であってもよい。 As another preferred embodiment in the case where R ² is a group represented by the general formula (A1), R ² is represented by the following general formula (A2).

[Wherein, R ⁵ and R ⁷ are the same or different and each represents a bond, —O—, —S—, or —C (═O) —. R ⁶ and R ⁸ are the same or different and each represents a substituent. m ′ represents a natural number of 1 to 5. n shows the integer of 0-4. When m′-1 is 2 or more, R ⁶ may be the same or different. When n is 2 or more, R ⁸ may be the same or different. ]
The group represented by these may be sufficient.

In the case where R ² is a group represented by the general formula (A2), examples of R ⁵ include those mentioned above for the case where R ² is a group represented by the general formula (A1). In this embodiment, R ⁵ is preferably a bond.

When R ² is a group represented by the general formula (A2), examples of substituents represented by R ^6, a halogen, and the like, in the embodiments, R ⁶ is halogen (e.g., fluorine, etc.) Etc.

When R ² is a group represented by the general formula (A2), R ⁷ represents a bond, —O—, —S— or —C (═O) —, and represents a bond or —C (═O) —. Is preferred.

When R ² is a group represented by the general formula (A2), examples of R ⁸ include a hydroxyl group, amino group, halogen, aryl, arylcarbonyl, thioaryl and the like, and a hydroxyl group and the like are preferable.

When R < ² > is group represented by general formula (A2), m shows the natural number of 1-5, 1-3 are preferable and 1-2 are more preferable.

When R ² is a group represented by the general formula (A2), n represents an integer of 0 to 4, preferably from 0 to 1, more preferably 0.

When R ² is a group represented by the general formula (A2), as a preferable combination of m ′, R ⁶ and R ⁷ , m ′ is 1 (therefore, R ⁶ is not present) and R ⁷ is − Combinations in which C (═O) — are included; combinations in which m ′ is 2 to 3 (preferably 2), R ⁶ is the halogen, and R ⁷ is a bond.

In the present invention, the substituent represented by R ³ is not particularly limited, and examples thereof include an alkyl group having 1 to 6 carbon atoms (preferably 1 to 3 and more preferably 1), an amino group, and the like. An alkyl group or amino group having 1 to 6 (preferably 1 to 3, more preferably 1) is preferable, and an alkyl group having 1 to 6 carbons (preferably 1 to 3, more preferably 1) is more preferable. .

Among the embodiments of the present invention, in the embodiment in which R ² is a group represented by the general formula (A1), as a combination of R ¹ , R ² and R ³ ,
R ¹ is hydrogen, hydroxyl group, amino group, halogen, aryl, arylcarbonyl, or thioaryl;
R ² is a group represented by the general formula (A2),
A combination in which R ³ is an alkyl group having 1 to 6 carbon atoms (preferably 1 to 3, more preferably 1), a hydroxyl group, an amino group, halogen, aryl, arylcarbonyl, or thioaryl is preferable;
R ¹ is hydrogen;
R ² is a group represented by the general formula (A2),
A combination in which R ³ is an alkyl group having 1 to 6 carbon atoms (preferably 1 to 3 and more preferably 1) is more preferable.

［式中、Ｒ⁹は水素又は置換基を示す。］
で示される環構造を形成してもよい。 In another embodiment of the present invention, R ² and R ³ may form a ring structure together with the carbon atom to which they are bonded. Such a ring structure is not particularly limited as long as it has one carbon atom corresponding to the α carbon, and may be a hydrocarbon ring or a heterocyclic ring having at least one carbon atom. More specifically, for example, as such a ring structure, in a hydrocarbon ring of 3 to 6 members such as cyclopentane, cyclobutane, cyclopentane, cyclohexane, cyclohexene, dihydroquinoline, etc., one hydrocarbon ring has A divalent group formed by removing two hydrogen atoms bonded to carbon; having 1 to 3 heteroatoms such as pyrrolidine, tetrahydrofuran, tetrahydrothiophene, thiazoline, pyrrolidine, etc. (corresponding to α carbon) —CH ₂ -5- to 10-membered unsaturated heterocyclic ring containing a structure. In a preferred embodiment, R ² and R ³ together with the carbon atom to which they are attached have the general formula (A3)

[Wherein R ⁹ represents hydrogen or a substituent. ]
May be formed.

In this embodiment, the substituent represented by R ⁹ includes a substituted or unsubstituted 5- to 10-membered unsaturated heterocyclic group having 1 to 3 heteroatoms, and 1 to 6 carbon atoms (preferably 1). To 3) alkyl group, hydroxyl group, amino group, halogen, aryl, arylcarbonyl, thioaryl and the like, and a substituted or unsubstituted 5- to 10-membered unsaturated heterocyclic group having 1 to 3 heteroatoms Etc. are preferred.

When R ⁹ is a 5- to 10-membered unsaturated heterocyclic group having 1 to 3 heteroatoms having a substituent, a 5- to 10-membered unsaturated heterocyclic ring having 1 to 3 heteroatoms Examples of the group include benzothiazole. When the 5- to 10-membered unsaturated heterocyclic group having 1 to 3 hetero atoms further has a substituent, the substituent is not particularly limited, and examples thereof include aryl and hydroxyl groups. Etc. are preferred.

で示される基が挙げられる。 In a preferred embodiment of the present invention, R ⁹ is

The group shown by these is mentioned.

［式中、Ｒ¹⁰及びＲ¹¹は、同一又は異なって、水素又はアルキル基を示す。−Ｏ−Ｒ¹²は、水酸基、リン酸基含有官能基又はスルホン酸基を示す。ｐは１〜６の自然数を示す。ｑは０〜５の整数を示す。ｒは１〜６の自然数を示す。］
で表される基等が挙げられる。 In a preferred embodiment of the present invention, R ⁴ has the following general formula:

[Wherein, R ¹⁰ and R ¹¹ are the same or different and each represents hydrogen or an alkyl group. —O—R ¹² represents a hydroxyl group, a phosphate group-containing functional group, or a sulfonic acid group. p shows the natural number of 1-6. q represents an integer of 0 to 5. r represents a natural number of 1-6. ]
The group etc. which are represented by these are mentioned.

  p represents a natural number of 1 to 6, preferably 1 to 3, and more preferably 2. q represents an integer of 0 to 5, preferably 1 to 3, and more preferably 2. r represents a natural number of 1 to 6, preferably 1 to 3, more preferably 2. When r is 2 or more, a plurality of p may be the same or different. When r is 2 or more, a plurality of q may be the same or different.

In formula (B1), R ¹⁰ and R ¹¹ are the same or different and are hydrogen or an alkyl group (preferably an alkyl group having 1 to 6 carbon atoms, more preferably 1 to 3 carbon atoms, still more preferably 1 carbon atom). Indicates.

下記一般式（Ｃ１）

［式中、ｓは、１〜３の自然数を示す。Ｘは、リン酸基で置換されていてもよいヌクレオシド基を示す。］
で表される基等が挙げられる。 As a phosphate group-containing functional group, a phosphate group

The following general formula (C1)

[Wherein s represents a natural number of 1 to 3. X represents a nucleoside group which may be substituted with a phosphate group. ]
The group etc. which are represented by these are mentioned.

の部分は、当該ヌクレオシド基の５位に結合する。
ヌクレオシド基としては、アデノシル基、グアノシル基、ウリジル基、シチジル基等が挙げられる。 In the present invention, the nucleoside group means a monovalent group formed by removing hydrogen contained in the hydroxyl group at the 5-position of the nucleoside. Therefore, in the group represented by the general formula (C1), when X is a nucleoside group,

Is bonded to the 5-position of the nucleoside group.
Examples of the nucleoside group include an adenosyl group, a guanosyl group, a uridyl group, and a cytidyl group.

  s shows the natural number of 1-3, Preferably it is 1-2, More preferably, it is 2.

  Moreover, these nucleoside groups may be substituted with a phosphate group. Typically, the hydroxyl group at the 2-position of these nucleoside groups may be substituted with a phosphate group.

In the present invention, —S—R ⁴ is preferably —S—CoA, and therefore —S—R ⁴ is preferably a group represented by the following formula:

Enzymatic hydrolysis reaction In the method of the present invention, enzymatic hydrolysis with at least one acyl CoA esterase selected from the group consisting of ACOT7 and ACOT13 is performed by converting a thioester compound having an asymmetric carbon at the α-position represented by the general formula (I). By subjecting to a decomposition reaction, a chiral carboxylic acid can be asymmetrically synthesized from a mixture of optical isomers of the thioester compound.

。 In the present invention, the mixture of optical isomers is a mixture of a predetermined chiral isomer and its optical isomer, and typically the excess of the predetermined chiral isomer is 20% ee or less, for example, 10% Below ee, more typically a racemate. In another embodiment, in the present invention, the predetermined chiral isomer in the mixture of optical isomers has an excess ratio of the predetermined chiral isomer of 0% ee or lower, -10% ee or lower, -20% ee or lower, etc. May be. In the present invention, asymmetric synthesis means that the proportion of one kind of chiral isomer is significantly higher than that of other isomers from a mixture of optical isomers including optical isomers. Means that the excess of a given chiral substance is 50% ee or more, preferably 75% ee or more, more preferably 90% ee or more, and further preferably 99% ee or more. Here, the excess of the chiral body can be calculated from the following formula:

.

ここで、ＡＣＯＴ７は、下記アミノ酸配列（ｉ）の部分に特徴的なモチーフを有することが知られている：
（ｉ）RPDDANVAGN VHGGTILKMI EEAGAIISTR HCNSQNGERC VAALARVERTDFLSPMCIGE VAHVSAEITY TSKHSVEVQV NVMSENI及び（配列番号２）LHGFVHGGVT MKLMDEVAGI VAARHCKTNI VTASVDAINF HDKIRKGCVI TISGRMTFTS NKSMEIEVLV DA（配列番号３）
従って、本発明においては、アシルＣｏＡエステラーゼＡＣＯＴ７として、上記（ｉ）で示されるアミノ酸配列を有するもの等を用いることもできる。また、本発明において、ＡＣＯＴ７には、Genbank Accession No. NP_009205で示されるアミノ酸配列；配列番号１で示されるアミノ酸配列；または配列番号２及び３で示されるアミノ酸配列を有するもの等だけでなく、本発明の効果が得られる範囲において、これらのいずれかのアミノ酸配列において、１個または数個（例えば、１〜５個、１〜３個、１個等）のアミノ酸が欠失、置換、挿入もしくは付加されたアミノ酸配列を含むものも包含される。 Examples of the acyl CoA esterase used in the present invention include acyl-coenzyme A thioesterase 7 (ACOT7). ACOT7 is a kind of human acyl CoA thioesterase and can be prepared by a protein expression system using E. coli, yeast, animal cells, or a cell-free protein expression system. As ACOT7, what has the amino acid sequence shown by Genbank Accession No. NP_009205 is mentioned. More specifically, ACOT7 includes those having the amino acid sequences shown below:

Here, ACOT7 is known to have a characteristic motif in the following amino acid sequence (i):
(I) RPDDANVAGN VHGGTILKMI EEAGAIISTR HCNSQNGERC VAALARVERTDFLSPMCIGE VAHVSAEITY TSKHSVEVQV NVMSENI and (SEQ ID NO: 2) LHGFVHGGVT MKLMDEVAGI VAARHCKTNI VTASVDAINF HDKIRKGCVI TISGRMLVTS NK
Therefore, in this invention, what has the amino acid sequence shown by said (i) etc. can also be used as acyl CoA esterase ACOT7. In the present invention, ACOT7 includes not only an amino acid sequence represented by Genbank Accession No. NP_009205; an amino acid sequence represented by SEQ ID NO: 1; or an amino acid sequence represented by SEQ ID NOs: 2 and 3, etc. As long as the effects of the invention are obtained, one or several (for example, 1 to 5, 1 to 3, 1, etc.) amino acids are deleted, substituted, inserted or inserted in any of these amino acid sequences. Also included are those containing added amino acid sequences.

また、本発明において、ＡＣＯＴ１３には、Genbank Accession No. NP_060943で示されるアミノ酸配列；又は配列番号４で示されるアミノ酸配列を有するもの等だけでなく、本発明の効果が得られる範囲において、これらのいずれかのアミノ酸配列において、１個または数個（例えば、１〜５個、１〜３個、１個等）のアミノ酸が欠失、置換、挿入もしくは付加されたアミノ酸配列を含むものも包含される。これらのチオエステラーゼとしては、ヒト、マウス、ラット等の哺乳動物由来；ゼブラフィッシュ、メダカ等の魚類由来；カイコ、ショウジョウバエ、コクゾウムシ等 昆虫由来等、様々なものを用いることができる。 Examples of the acyl CoA esterase used in the present invention also include acyl-coenzyme A thioesterase 13 (ACOT13). ACOT13 is a kind of human acyl CoA thioesterase and can be prepared by a protein expression system using E. coli. As ACOT13, what has the amino acid sequence shown by Genbank Accession No. NP_060943 is mentioned. More specifically, ACOT13 includes those having the amino acid sequence shown below:

Further, in the present invention, ACOT13 includes not only an amino acid sequence represented by Genbank Accession No. NP_060943; or an amino acid sequence represented by SEQ ID NO: 4, but also those having the effects of the present invention. Any amino acid sequence includes an amino acid sequence in which one or several (for example, 1-5, 1-3, 1, etc.) amino acids have been deleted, substituted, inserted or added. The As these thioesterases, various substances such as those derived from mammals such as humans, mice and rats; derived from fish such as zebrafish and medaka; derived from insects such as silkworms, Drosophila and weevil are usable.

本発明者らは、多種多様な酵素を用いた反応を鋭意検討した結果、ＡＣＯＴシリーズのうち、ＡＣＯＴ７及びＡＣＯＴ１３を用い、反応基質として前述の化合物を用いた場合に、立体選択性が非常に高い反応を実現できることを見出した。
上記表１に示すように、ＡＣＯＴ７及びＡＣＯＴ１３は、いずれも他のＡＣＯＴシリーズに対し相同性が低く、構造的に大きく異なっている。また、本願実施例に示すように、ＡＣＯＴ７及びＡＣＯＴ１３は、いずれも他のＡＣＯＴシリーズと比較して、機能（反応性、立体選択性）の点でも大きく異なる。 Here, 13 types of ACOT 1 to 13 are known as ACOT series proteins. Table 3 below shows the results of calculating the homology of the ACOT series of proteins.

As a result of intensive studies on reactions using a wide variety of enzymes, the inventors of the present invention have very high stereoselectivity when ACOT7 and ACOT13 are used as the reaction substrate in the ACOT series. It was found that the reaction can be realized.
As shown in Table 1 above, both ACOT7 and ACOT13 have low homology with other ACOT series and are structurally different. Further, as shown in the examples of the present application, ACOT7 and ACOT13 are greatly different in function (reactivity, stereoselectivity) as compared with other ACOT series.

  The method of the present invention includes a step of subjecting a thioester compound having an asymmetric carbon at the α-position represented by the following general formula (I) to an enzymatic hydrolysis reaction.

  The reaction is usually performed in a solution in which the thioester compound is dissolved or dispersed in a solvent such as water. The concentration of the thioester compound is not particularly limited, and can be appropriately set within a range of, for example, 0.1 to 1000 mM, preferably 10 to 100 mM. Further, the amount of the enzyme in the reaction system is not particularly limited, but can be appropriately set within a range of, for example, 1 ng / mL to 1 mg / mL, preferably 10 ng / mL to 1 μg / mL. Although the reaction time is not particularly limited, it can be appropriately set within a range of, for example, 5 to 60 minutes, preferably 10 to 30 minutes. The reaction temperature is not particularly limited, but can be appropriately set within a range of, for example, 20 to 40 ° C, preferably 30 to 37 ° C. The pressure during the reaction is not particularly limited, and the reaction may be performed under normal pressure, increased pressure, or reduced pressure.

  By this reaction, the thioester part of the thioester compound represented by the general formula (I) becomes a carboxyl group. Here, in the present invention, the thioester reaction proceeds preferentially with respect to one of the optical isomers contained in the mixture of optical isomers of the thioester compound represented by the general formula (I). Carboxylic acid can be asymmetrically synthesized. Further, in the present invention, since it proceeds preferentially with respect to one of the optical isomers contained in the mixture of optical isomers of the thioester compound, when the reaction proceeds, the ratio of the other thioester compound remaining is relatively Can be expensive.

  Therefore, according to the present invention, a chiral carboxylic acid can be synthesized by stereoselectively hydrolyzing only one optical isomer of a thioester of a carboxylic acid having an asymmetric carbon at the α-position. . The thus obtained chiral carboxylic acid can be used as various chemicals, physiologically active substances, fine chemicals such as chiral liquid crystal materials, or intermediates for producing them. Therefore, the asymmetric synthesis method of the present invention is useful for producing these compounds.

  In the present invention, for example, CoA or a thioester compound having a structure similar to this (sometimes referred to as a CoA thioester compound in this specification) and the like can generally be racemized more easily than carboxylic acid. This is preferable. When the thioester compound represented by the general formula (I) is a compound that is easily racemized, the compound that is less prone to undergo thioester hydrolysis among the optical isomers is converted into the other optical isomer by racemization. Therefore, in this embodiment, since the optical resolution proceeds with racemization, the optical resolution can be performed with higher resolution, which is preferable. In a preferred embodiment of the present invention, racemization of the thioester compound represented by the general formula (I) can be further promoted by epimerase.

  Racemic CoA thioester compounds can be synthesized organically from the original racemic carboxylic acid and CoA, and if this can be stereoselectively hydrolyzed, chiral carboxylic acids can be synthesized without using ATP. it can. If the carboxylic acid produced is water-insoluble, it can be separated from the reaction aqueous solution containing enzyme, CoA, etc. by liquid phase separation, and a cheaper asymmetric synthesis reaction system can be provided. become.

  So far, there has been no report that thioester hydrolase (thioesterase) has stereoselectivity. In addition, CoA thioester compounds can be easily racemized compared to carboxylic acids, and further racemization can be promoted by epimerase. By utilizing these characteristics, stereoselective thioesterase can be used to construct optical resolution with racemization, so-called Dynamic Kinetic Resolution (DKR) reaction system without using stereoselective CoA ligase. It is also possible to synthesize an optically active carboxylic acid with a theoretical yield of 100%. Therefore, the present invention is very useful from this point of view.

Example 1
Add 10 μL of 2 mM flurbiprophenyl-CoA aqueous solution and 100 L of 0.1 M Tris-HCl [pH 8] buffer in a 1.5 mL plastic tube, add 10 μL of 10 μg / mL ACOT7 aqueous solution, Set for 2 hours. ACOT7 uses an entry clone (FLJ32606AAAN) in which the ORF sequence has been cloned and a destination vector for N-terminal GST-tag and FLAG-tag fusion, and in accordance with the method described in Non-Patent Document 1, a wheat germ extract (WEPRO7240G , Cell Free Science) was used for protein synthesis.
Ten times the amount of 2N hydrochloric acid was added to the reaction solution to stop the reaction, and solvent extraction with diethyl ether and drying were performed. The optical purity of the resulting flurbiprofen was analyzed by high performance liquid column chromatography (HPLC) using a chiral column CHIRALCEL OD-H manufactured by Daicel (hexane / i-PrOH / TFA = 95/5 / 0.1%, 1 mL / min, 210 nm). As a control experiment, an enzyme-free solution was analyzed in the same manner. The results are shown in FIG.

  As a result, R-flurbiprofen was selectively produced by the addition of the enzyme ACOT7, and the enantiomeric excess was 100% ee. Flurbiprofen was produced in the same manner as described above except that ACOT2, ACOT4, ACOT8, ACOT9, ACOT11, and ACOT12 were used instead of the enzyme ACOT7, and the optical purity was analyzed. As a result, the enantiomeric excess of ACOT9 was only 46% ee. In addition, the reaction itself did not proceed for ACOT2, ACOT4, ACOT8, ACOT11, and ACOT12.

Example 2
To 0.05 mM luciferyl-CoA (racemic), 10 μg / mL enzyme ACOT13 was added, and the optical purity of the produced luciferin was analyzed by HPLC using a chiral column CHIRALCEL OD-RH manufactured by Daicel (H ₂ O / acetonitrile). / TFA = 73/27 / 0.1%, 1 mL / min, ex330 nm, em510 nm). As a result, the enantiomeric excess of L-luciferin was 100% ee. The enzyme luciferase was prepared by introducing an expression vector into which E. coli-derived genes were introduced into E. coli, and the enzyme ACOT13 was prepared in the same manner as ACOT7 in Example 1. As a control experiment, a solution containing no enzyme ACOT13 was analyzed in the same manner. The results are shown in FIG. Furthermore, luciferin was produced in the same manner as above except that TESB, ACOT2, ACOT4, ACOT7, ACOT8, ACOT9, ACOT11, or ACOT12 was used in place of the enzyme ACOT13, and the optical purity was analyzed. As a result, the enantiomeric excess for ACOT7 reached 77% ee. In contrast, the enantiomeric excess of ACOT9 was only 31% ee. In addition, for ACOT2, ACOT4, ACOT8, ACOT11 and ACOT12, the reaction itself did not proceed.

Claims (9)

By an enzymatic hydrolysis reaction with at least one acyl CoA esterase selected from the group consisting of ACOT7 and ACOT13 from a mixture of optical isomers of a thioester compound having an asymmetric carbon at the α-position represented by the following general formula (I) Asymmetric synthesis of chiral carboxylic acids:

[Wherein R ¹ , R ² , R ³ and R ⁴ represent hydrogen or a substituent.
R ² and R ³ may form a substituted or unsubstituted ring structure together with the carbon atom to which they are bonded.
However, R ¹ , R ² , R ³ and CO—S—R ⁴ are different from each other. ]. The method of claim 1, wherein R ¹ is hydrogen. R ⁴ represents the following general formula

[Wherein, R ¹⁰ and R ¹¹ are the same or different and each represents hydrogen or an alkyl group. —O—R ¹² represents a hydroxyl group, a phosphate group-containing functional group, or a sulfonic acid group. p shows the natural number of 1-6. q represents an integer of 0 to 5. r represents a natural number of 1-6. ]
The method of Claim 1 or 2 which is group represented by these. The method according to claim ¹ , wherein R ¹ is hydrogen and —S—R ⁴ is —S—CoA. R ² is the following general formula

[Wherein R ⁵ represents a bond, —O—, —S— or —C (═O) —. R ⁶ represents a substituent. m shows the integer of 0-5. When m is 2 or more, R ⁶ may be the same or different. ]
The method of any one of Claims 1-4 which is group represented by these. R ² is the following general formula

[Wherein, R ⁵ and R ⁷ are the same or different and each represents a bond, —O—, —S—, or —C (═O) —. R ⁶ and R ⁸ are the same or different and each represents a substituent. m ′ represents a natural number of 1 to 5. n shows the integer of 0-4. When m′-1 is 2 or more, R ⁶ may be the same or different. When n is 2 or more, R ⁸ may be the same or different. ]
The method of Claim 5 which is group represented by these. R ² and R ³ together with the carbon atom to which they are attached have the general formula

[Wherein R ⁹ represents hydrogen or a substituent. ]
The method of any one of Claims 1-4 which forms the ring structure shown by these. R ⁹ is

The method of Claim 7 which is group shown by these. R ³ is an alkyl group having 1 to 6 carbon atoms, The method according to any one of claims 1-6.

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