WO2007148514A1

WO2007148514A1 - Enzymatic synthesis method for 4-hydroxy-2-quinoline

Info

Publication number: WO2007148514A1
Application number: PCT/JP2007/060964
Authority: WO
Inventors: Ikuro Abe; Tsuyoshi Abe; Hiroshi Noguchi
Original assignee: Japan Science And Technology Agency
Priority date: 2006-06-22
Filing date: 2007-05-30
Publication date: 2007-12-27

Abstract

Disclosed is a method for synthesis of a 4-hydroxy-2-quinoline by using an enzyme under clean and mild conditions in a simple manner. More specifically, disclosed is a method for synthesis of a 4-hydroxy-2-quinoline by conducting unimolecular condensation of an anthranyl-CoA derivative and a malonyl-CoA derivative by using, as a catalyst, a benzalacetone synthase which is a type-III polyketide synthase capable of catalyzing the condensation reaction between coumaroyl-CoA and malonyl-CoA and constituting a chalcone synthase super family specific to a plant.

Description

Specification

Enzymatic synthesis of 4-hydroxy-2-quinolinones

Technical field

The present invention relates to a method for synthesizing 4-hydroxy-2-quinolinones using an enzyme.

Background art

[0002] 4-Hydroxy-2-quinolinones are known to have an antagonistic action on NMDA receptor (Non-patent Document 1) and serotonin 5-HT receptor (Non-patent Document 2).

Three

It is also attracting attention from the standpoint of drug development, such as central antiemetics that cross the brain barrier. It is also an important intermediate in the chemical synthesis of a series of alkaloids.

[0003] On the other hand, these quinolinone alkaloids are known to be produced by some Rutaceae plants, but their production is limited, and their supply is mainly from organic synthesis. Recently, a method for synthesizing 4-hydroxy-2-quinolinone by treating an α-norocarboxylic acid ester of an anthrallic acid derivative with sodium telluride in tetrahydrofuran and cyclizing it has been reported ( Non-patent document 3).

[0004] Benzalacetone synthase (BAS) is a type III polyketide synthase that constitutes a superfamily of chalcone synthases that are specific to plants that catalyze the condensation reaction of coumaroyl CoA and malol CoA. This is the first example of a polyketide synthase that condenses only the molecular malol CoA (Non-patent Document 4).

Non-Patent Document 1: Rowley, M. et al, J. Med. Chem. 40, 4053, 1997

Non-Patent Document 2: Hayashi, H. et al "J. Med. Chem. 36, 617, 1993

Non-Patent Document 3: Dittmer, D.C. et al., J. Org. Chem. 70, 4682, 2005

Non-Patent Document 4: Abe, I. et al., Eur. J. Biochem. 268, 3354, 2001

Disclosure of the invention

Problems to be solved by the invention

[0005] Creation of new technologies that efficiently synthesize compounds that are friendly to the global environment while avoiding wasteful use of earth resources is required. The present invention replaces the conventional chemical synthesis with a bio-based It utilizes biocatalysts typified by enzymes and cells that can be called stems.

Accordingly, an object of the present invention is to provide a method for synthesizing 4-hydroxy-2-quinolinones using an enzyme.

Means for solving the problem

[0007] As a result of intensive studies, the present inventor succeeded in solving the above problems by utilizing the wide range of substrate specificity and catalytic ability exhibited by benzalacetone synthase, and completed the present invention.

That is, the present invention relates to the following aspects.

[Aspect 1] Using a benzalacetone synthase as a catalyst, a general formula is obtained by single-molecule condensation of an anthranyl CoA derivative represented by the general formula (II) and a malonyl CoA derivative represented by the general formula (Formula 2). A method for synthesizing 4-hydroxy-2-quinolinones represented by (Chemical Formula 3).

[Chemical 1]

[Chemical 2]

R 1

(In the formula, Rl and R2 each represent hydrogen, a carbon atom, or an organic group having up to 10 carbon atoms and heteroatoms in total)

[Aspect 2] The method according to Aspect 1, wherein R1 and R2 are each an organic group having a total of up to 6 carbon atoms and heteroatoms, each of hydrogen, carbon atoms, and heteroatoms.

[Aspect 3] The method according to Aspect 2, wherein R1 and R2 forces are each hydrogen or a lower alkyl group.

[Aspect 4] The method according to aspect 3, wherein R1 and R2 are hydrogen.

[Aspect 5] The method according to Aspect 3, wherein R1 is hydrogen and R2 is a methyl group.

[Aspect 6] The method according to Aspect 3, wherein R1 is a methyl group and R2 is hydrogen.

[Aspect 7] The method according to Aspect 3, wherein R1 and R2 are carbonyl groups.

[Aspect 8] The method according to any one of Aspects 1 to 7, wherein substantially equal amounts of anthranyl CoA derivative and malonyl CoA derivative are used.

[Aspect 9] The method according to any one of V and Aspects 1 to 8, wherein the reaction is performed at 25 to 35 ° C. for 0.5 to 20 hours in a buffer having a pH of 6 to 8.

[Aspect 1Ο] The method according to Aspect 9, wherein the reaction is carried out in 0.1 M potassium phosphate buffer (pH 7.0) at 30 ° C. for 1 hour.

[Aspect 11] The method according to any one of Aspects 1 to 10, wherein benzalacetone synthase, which is a recombinant enzyme obtained using Escherichia coli as a host, is used.

The invention's effect

According to the present invention, a series of 4-hydroxy-2-quinolinones can be easily synthesized under clean and mild conditions by allowing an artificial substrate to act on an enzyme. Brief Description of Drawings

[0010] [Fig. 1] Condensation reaction of coumaroyl CoA and malol CoA by benzalacetone synthase (BAS) (upper), and an anthral CoA derivative and malonyl CoA derivative in the method of the present invention. Molecular condensation (bottom) is shown.

BEST MODE FOR CARRYING OUT THE INVENTION

[0011] The present invention takes advantage of the broad substrate specificity and catalytic ability of benzalacetone synthase to produce an anthral CoA (or an anthral CoA derivative such as N-methylanthral CoA). In addition, by simultaneously acting with malol CoA (or a malol CoA derivative such as methylmalol CoA), the 4-hydroxy-2-quinolinones can be treated under clean and mild conditions by single molecule condensation of these artificial substrates. Below, it is synthesized efficiently and simply. The synthesis of these quinolinone alkaloids using biocatalysts is a new technology that has never been seen before.

[0012] As described in Non-Patent Document 4, for example, benzalacetone synthase used as a catalyst in the method of the present invention is a plant such as Rheum palmutum, particularly such as Rheum palmutum. It can be obtained and prepared by a method known to a person skilled in the art from a plant of the family Rosaceae and Raspberry (Rubus idaeus).

[0013] In addition, as long as it has substantially the activity as a polyketide synthase that condenses only one molecule of malonyl CoA, it is a protein having an amino acid sequence different from the amino acid sequence of such a natural product-derived benzalacetone synthase. Even if it exists, it belongs to “benzalaceton synthase” in the present invention and can be used in the method of the present invention.

[0014] Accordingly, benzalacetone synthase is prepared from a natural product such as the above-mentioned plant or the like by using a suitable gene recombination technique known to those skilled in the art, and prokaryotic cells such as Escherichia coli, and yeast. It can also be used in the method of the present invention as a so-called “recombinant enzyme” obtained by expressing in a suitable host of eukaryotic cells such as. Furthermore, 1 to several amino acids obtained by expressing a gene obtained by modifying the enzyme gene derived from a natural product by any genetic engineering technique known to those skilled in the art, such as site-directed mutagenesis, are substituted. A benzalacetone synthase having a modified 'altered amino acid sequence such as deletion, insertion, etc. and having substantially the enzyme activity (or enhanced enzyme activity) can also be used in the method of the present invention. Such a recombinant enzyme may be expressed as a fusion protein with an appropriate tag substance known to those skilled in the art, such as a His tag, for the sake of purification. Furthermore, benzalacetone synthase comprising a polypeptide chemically synthesized by any method known to those skilled in the art can also be used in the method of the present invention.

[0015] Furthermore, in the method of the present invention, the use of benzalacetone synthase purified by any method known to those skilled in the art is preferable from the standpoint of reaction efficiency and the like, but natural products (plants ) Or a recombinant product obtained by transformation with the enzyme gene such as Escherichia coli, and prepared as a crude extract containing the enzyme.

[0016] In the anthranyl CoA derivative represented by the general formula (1) and the malonyl CoA derivative represented by the general formula (2), R1 and R2 are each a sum of hydrogen, carbon atoms, or carbon atoms and heteroatoms. Organic groups having up to 10, preferably up to 6 are shown. There are no particular restrictions on the types of such organic groups, for example, oxygen represented by saturated aliphatic hydrocarbon groups, unsaturated aliphatic hydrocarbon groups, alicyclic hydrocarbon groups, aromatic hydrocarbon groups, and heterocyclic groups. And various organic groups containing heteroatoms such as nitrogen or nitrogen atoms. In particular, R1 and R2 are each preferably hydrogen or a lower alkyl group having up to 6 carbon atoms, benzene, or various groups derived from heterocyclic rings such as pyridine, furan and thiophene. Can be mentioned. Of the lower alkyl groups, methyl and ethyl groups are preferred. When R1 and R2 are hydrogen, the forces correspond to anthral CoA and malol CoA, respectively. When R1 and R2 are hydrogen in general formula (3), they correspond to 4-hydroxy-2-quinolinone. The malol CoA derivative is commercially available and can be easily obtained. An anthral CoA derivative can also be synthesized, for example, according to the method described in the literature (Stoeckigt, J. & Zenk, MH, Z. Naturforsch. 30c, 352, 1975). .

[0017] Since the method of the present invention is a single-molecule condensation reaction of an anthranyl CoA derivative and a malol CoA derivative, these substrate molecules are used in substantially equal amounts from the viewpoint of reaction efficiency and economy. Although it is preferable, the addition (use) amount of both does not need to be equal.

[0018] Enzyme reaction conditions can be appropriately selected by those skilled in the art according to various reaction conditions such as the type of substrate molecule, reaction scale, degree of enzyme purification, and the like. For example, benzal acetone synthesis fermentation Element (40 μg / mL) with each substrate molecule (50 μΜ) in a buffer solution of ρΗ 6-8 at a reaction temperature of 25-35 ° C, preferably 25-30 ° C For about 0.5 to 20 hours, preferably about 1 to 20 hours. The reaction is preferably carried out in an appropriate buffer known to those skilled in the art, such as potassium phosphate buffer and Tris-HCl buffer. Furthermore, in the present invention method, the enzyme reaction is continuously performed using any form known to those skilled in the art, for example, a batch system or a column containing the enzyme bound to an appropriate support. It is also possible.

[0019] Furthermore, 4-hydroxy-2-quinolinones synthesized by the method of the present invention can be easily purified by any means and method known to those skilled in the art, such as reverse phase HPLC and silica gel column. It is.

Example

Hereinafter, the present invention will be described in more detail with reference to examples. The technical scope of the present invention is not limited by these descriptions.

[0021] According to the method described in Non-Patent Document 4, heterozygous expression of Rheum palmutum-derived benzalaceton synthase in Escherichia coli as a recombinant enzyme with an N-terminal addition of a His tag was performed using a nickel chelate column. Purified. Using this purified enzyme in 0.1 M potassium phosphate buffer (pH 7.0) and N-methylanthral CoA (50 μΜ) and methylmalol CoA (100 μΜ) as substrates at 30 ° C. When reacted for 1 hour, 4-hydroxy-2-quinolinone was obtained as a single product in high yield (85%). Similarly, high yields (80%) were obtained when anthral CoA and methylmalol CoA were used as substrates. In addition, when N-methylanthral CoA or anthral CoA and malol CoA were used as substrates, the yields were about 10%, respectively (Fig. 1).

[0022] After each enzyme reaction, the enzyme reaction product was extracted with an organic solvent such as ethyl acetate, then concentrated with a rotary evaporator, and the target product was fractionated by HPLC. Using a TSK-gel ODS-8 0Ts (4.6 x 150 mm; Tosoh Corporation) column, elution was performed in the presence of 0.1% TFA for 40 minutes using the following methanol concentration gradient (0-5 min, 30% MeOH; 5-17 min, linear gradient from 30 to 60% MeOH; 17-25 min, 60% MeOH; 25-27 min, linear gradient from 60 to 70% MeOH; 27-35 min, 70% MeOH; 35 -40 min, linear gradient from 70 to 100 % MeOH) ₀ Fractionated every 0.5 minutes at a flow rate of 0.8 ml per minute. Examine the absorption spectra (200 to 400 nm) of each fraction to 224 to 228 nm, 272 to 284 nm, and 312-318 nm. The fraction with the maximum absorption was collected as the 4-hydroxy-2-quinolinone fraction. Elution time: Compound la (18.3 min), Compound 1 b (18.9 min), Compound lc (22.8 min), Compound Id (23.5 min).

Industrial applicability

[0023] By implementing the method of the present invention using biotechnology such as fermentation using transformed Escherichia coli and an artificial substrate, it is expected to open a path to inexpensive and efficient mass production. 4-Hydroxy-2-quinolinones obtained by the present invention can be further subjected to molecular modification, such as the NMDA receptor serotonin 5-HT receptor antagonist described above.

Three

First, drug development becomes possible.

[0024] The contents described in the documents cited in the present specification constitute the disclosure of the present specification as part of the present specification.

Claims

[Claims] Using benzalacetone synthase as a catalyst, one-molecule condensation of an anthral CoA derivative represented by the general formula (II) and a malol CoA derivative represented by the general formula A method for synthesizing 4-hydroxy-2-quinolinones represented by the general formula (Chemical Formula 3).

[Chemical 1]

[Chemical 2]

[Chemical 3] R 1

[2] The method according to claim 1, wherein R1 and R2 are each hydrogen, a carbon atom, or an organic group having up to 6 carbon atoms and heteroatoms.

[3] The method according to claim 2, wherein R1 and R2 forces are each hydrogen or a lower alkyl group.

[4] The method according to claim 3, wherein R1 and R2 are hydrogen.

5. The method according to claim 3, wherein R1 is hydrogen and R2 is a methyl group.

6. The method according to claim 3, wherein R1 is a methyl group and R2 is hydrogen.

[7] The method of claim 3, wherein R1 and R2 are methyl groups.

[8] The method according to any one of [1] to [7], wherein substantially equal amounts of anthranyl CoA derivative and malonyl CoA derivative are used.

[9] The method according to any one of claims 1 to 8, wherein the reaction is carried out in a buffer solution of pH 6 to 8 at 25 to 35 ° C for 0.5 to 20 hours.

[10] The method according to claim 9, wherein the reaction is carried out in 0.1 M potassium phosphate buffer (pH 7.0) at 30 ° C. for 1 hour.

[11] The method according to any one of [1] to [10], wherein benzalacetone synthase, which is a recombinant enzyme obtained using Escherichia coli as a host, is used.