JP2020092674A - Environmental cleaner - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a technique capable of simply and efficiently purifying pollution in the environment such as soil.
An environmental purification agent containing a compound represented by the general formula (1) and at least one selected from the group consisting of cells containing the compound.
[Selection diagram] None

Description

The present invention relates to an environmental purification agent and the like.

In recent years, as environmental awareness has increased, awareness of environmental pollution problems such as soil pollution has increased, and legislation concerning environmental pollution has been developed. For example, in the former sites of factories, power plants, incinerators, gas stations, etc., soil pollution due to hydrocarbon pollutants such as benzene and dioxins is often a problem, and countermeasures are required.

As a soil pollution countermeasure, a method of excavating contaminated soil and treating it off-site is often adopted. However, this method has not only the problem that a large amount of cost is required for excavating the soil, but also the problem that the environmental load is large, and that the treatment facility for excavated contaminated soil is insufficient in recent years.

On the other hand, the method of soil purification by bioremediation has been attracting attention in recent years. This method does not require excavation and removal of soil. However, since microorganisms used for bioremediation are genetically modified organisms or specific species, large-scale preparation thereof may require a large amount of cost. In addition, when a genetically modified organism is used, it is necessary to take measures to prevent its diffusion into the environment.

Japanese Patent Laid-Open No. 2012-024009 JP 2017-071589

An object of the present invention is to provide a technique capable of simply and efficiently purifying pollution in the environment such as soil.

Cytochrome P450, which is an enzyme derived from bacteria, is known to have higher catalytic activity than cytochrome P450 derived from eukaryote. As cytochrome P450s derived from bacteria, cytochrome P450 monooxygenases such as cytochrome P450cam derived from Pseudomonas aeruginosa and cytochrome P450BM3 derived from Bacillus megaterium which is a bacterium belonging to the genus Bacillus are known. Cytochrome P450s derived from these bacteria have high catalytic activity for hydroxylating substrates such as fatty acids. Moreover, since it is not a membrane-bound protein, it is easy to obtain and handle. For these reasons, the bacterial cytochrome P450 monooxygenase is considered to be suitable as a biocatalyst.

Therefore, the present inventor has focused on performing environmental purification using cytochrome P450 monooxygenase. Then, attention was paid to the point that pollutants such as hydrocarbons and dioxins were hydroxylated by cytochrome P450 monooxygenase. If the pollutant is hydroxylated, it will lead to environmental purification such as improved degradability, improved water solubility, and reduced toxicity.

However, wild-type cytochrome P450 monooxygenase does not necessarily have high catalytic activity in hydroxylating hydrocarbons, dioxins, etc. which are problems in environmental pollution. Therefore, although various mutant cytochrome P450s have been proposed, the introduction of gene mutations is complicated. In order to solve these problems, the present inventors incorporated a pseudo substrate (decoy substrate) so as to cause a malfunction of cytochrome P450 monooxygenase to a hydrocarbon that does not exhibit high catalytic activity. It has already been reported that it catalyzes the hydroxylation reaction with high efficiency (Patent Documents 1 and 2).

However, since this technique carries out the hydroxylation reaction in vitro, it was necessary to add NADPH to sustain the enzymatic activity of cytochrome P450 monooxygenase. Therefore, it was considered not suitable for environmental purification from the viewpoint of cost.

As a result of intensive studies under such circumstances, the present inventor has found that the compound represented by the general formula (1) is taken up by cells and functions as a decoy substrate for cytochrome P450 monooxygenase. And, there are usually many bacteria that express cytochrome P450 in the environment such as soil. However, by contacting the compound represented by the general formula (1) with such environment, pollutants in the environment can be reduced. It was found to be hydroxylated. Hydroxylation leads to environmental purification such as decomposition of environmental pollutants, water-soluble environmental pollutants, and reduction of toxicity. The present inventor has completed the present invention as a result of further research based on these findings.

That is, the present invention includes the following aspects.

Item 1. General formula (1):

[In the formula, R ¹ represents an optionally substituted hydrocarbon group. R ² represents a single bond or a linker. R ³ is a single bond or formula (2):

(In the formula, the nitrogen atom is bonded to R ^2. )
Represents a group represented by. R ⁴ and R ⁵ represent the same or different amino acid side chains. R ⁶ represents a hydrogen atom, a hydroxyl group, an aldehyde group, a carboxy group, or a carboxy derivative group. However, R ⁵ and R ⁶ may combine with each other to form a lactone ring. n represents 0 or an integer of 1 or more. ]
An environmental purifying agent containing at least one selected from the group consisting of the compound represented by: and a cell containing the compound.

Item 2. Item 2. The environmental purification agent according to Item 1, wherein R ⁶ is a carboxy group.

Item 3. Item 1 or 2, wherein the amino acid side chain is at least one selected from the group consisting of an optionally substituted alkyl group, alkenyl group, aryl group, aralkyl group, heteroaryl group, and heteroaralkyl group. Environmental purifying agent described.

Item 4. Item 4. The environmental purification agent according to any one of Items 1 to 3, wherein R ¹ is an alkylaralkyl group, R ² and R ³ are single bonds, and n is 0.

Item 5. In any one of items 1 to 3, R ¹ is an alkyl group, R ² is a single bond, R ³ is a group represented by the formula (2), and n is 0. Environmental purifying agent described.

Item 6. The compound has the formula (1D):

Item 6. The environmental cleaning agent according to item 5, which is a compound represented by.

Item 7. Item ^6. The environmental purification agent according to any one of Items 1 to 5, wherein R ⁵ and R ⁶ do not form a lactone ring.

Item 8. Item 6. The environmental purifier according to any one of Items 1 to ^5, wherein R ⁵ and R ⁶ are bonded to each other to form a lactone ring.

Item 9. Item 9. The environmental purification agent according to Item 8, wherein the compound is quarmon.

Item 10. Item 10. The environmental purification agent according to any one of Items 1 to 9, wherein the cells are cells having a cytochrome P450 monooxygenase.

Item 11. Item 11. The environmental purification agent according to any one of Items 1 to 10, wherein the cells are cells into which the compound is exogenously introduced.

Item 12. Item 12. The environmental purification agent according to any one of Items 1 to 11, wherein the environment to be purified is at least one selected from the group consisting of environments in which microorganisms having cytochrome P450 are present.

Item 13. Item 13. The environmental cleaning agent according to any one of Items 1 to 12, which is used for conversion of environmental pollutants.

Item 14. Item 14. The environmental cleaning agent according to any one of Items 1 to 13, wherein the environmental pollutant is at least one selected from the group consisting of hydrocarbon pollutants and dioxins pollutants.

Item 15. Item 16. An environmental purification method comprising a step of bringing the environmental purification agent according to any one of items 1 to 14 into contact with an environment to be purified.

According to the present invention, pollution in the environment such as soil can be easily and efficiently purified.

A schematic diagram of the experimental operation of Example 4 (left) and a photograph of the reaction solution (right) are shown. The calibration curve (Example 4) of the amount of phenols in vermiculite is shown. The production amount of phenol over time using C7-L-Pro-L-Phe (Example 4) is shown. 1 shows a GC chromatogram (Example 5) after a hydroxylation reaction of benzene in a model soil using a giant bacterium.

In this specification, the expressions "containing" and "including" include the concepts of "containing", "including", "consisting essentially of" and "consisting solely of".

The present invention, in some embodiments thereof, comprises an environmental purifying agent containing at least one compound selected from the group consisting of a compound represented by the general formula (1) and a cell containing the compound (specification: May be referred to as the "environmental purifying agent of the present invention") and a step of bringing the environmental purifying agent of the present invention into contact with the environment to be purified (in the present specification, "the environmental purifying agent of the present invention"). "Environmental purification method"). These will be described below.

1. Compound general formula (1):

The compound represented by will be described.

<1-1. R ¹ ＞
R ¹ represents an optionally substituted hydrocarbon group.

The hydrocarbon group represented by R ¹ is not particularly limited, and examples thereof include an alkyl group, an aryl group and the like, and a group formed by arbitrarily combining these (eg, an aralkyl group, an alkylaryl group, an alkylaralkyl group) and the like. Is mentioned.

The alkyl group represented by R ¹ includes any of linear, branched, or cyclic (preferably linear or branched, more preferably linear). The number of carbon atoms of the alkyl group is not particularly limited and is, for example, 1 to 15, preferably 5 to 14, more preferably 6 to 13, still more preferably 6 to 12, still more preferably 6 to 11, still more preferably 7-11, more preferably 7-10, and even more preferably 7-9. Specific examples of the alkyl group include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, tert-butyl group, sec-butyl group, n-pentyl group, neopentyl group, n. Examples include -hexyl group, 3-methylpentyl group, n-heptyl group, n-octyl group, n-nonyl group, n-decyl group, n-undecyl group and n-dodecyl group.

The aryl group represented by R ¹ is not particularly limited, but preferably has 6 to 12 carbon atoms, more preferably 6 to 12 carbon atoms, and further preferably 6 to 8 carbon atoms. The aryl group may be either monocyclic or polycyclic (eg, bicyclic, tricyclic, etc.), but is preferably monocyclic. Specific examples of the aryl group include a phenyl group, a naphthyl group, a biphenyl group, a pentalenyl group, an indenyl group, an anthranyl group, a tetracenyl group, a pentacenyl group, a pyrenyl group, a perylenyl group, a fluorenyl group, and a phenanthryl group. And preferably a phenyl group.

The aralkyl group represented by R ¹ is not particularly limited, but is, for example, a linear or branched alkyl group having 1 to 6 carbon atoms (preferably 1 to 4, more preferably 1 to 3, and further preferably 2). The aralkyl group and the like in which the above hydrogen atom (for example, 1 to 3, preferably 1 hydrogen atom) is substituted with the above aryl group.

The alkylaryl group represented by R ¹ is not particularly limited, but for example, the hydrogen atom (for example, 1 to 3, preferably 1 hydrogen atom) of the above aryl group has a linear or branched carbon number of 1 to Examples thereof include an alkylaryl group which is substituted with 6 (preferably 2 to 6, more preferably 3 to 6, still more preferably 3 to 5, still more preferably 4) alkyl groups.

The alkylaralkyl group represented by R ¹ is not particularly limited, but for example, a hydrogen atom on the aromatic ring of the above aralkyl group (for example, 1 to 3, preferably 1 hydrogen atom) may have a linear or branched chain structure. Examples thereof include an alkylaralkyl group substituted by an alkyl group having 1 to 6 carbon atoms (preferably 2 to 6, more preferably 3 to 6, still more preferably 3 to 5, still more preferably 4).

The substituent that the hydrocarbon group represented by R ¹ may have is not particularly limited. Examples of the substitution include substitution of a hydrocarbon group with an oxygen atom, a sulfur atom, a nitrogen atom, a silicon atom, a carbon atom, a selenium atom, a tellurium atom or the like. More specifically, examples of the substituent include an oxo group and the like. The number of substituents is, for example, 0 to 3, preferably 0 to 1, and more preferably 0.

R ¹ is preferably an alkyl group, an alkylaryl group, an alkylaralkyl group, or the like, more preferably an alkyl group, an alkylaralkyl group, or the like, and further preferably an alkylaralkyl group.

<1-2. R ² ＞
R ² represents a single bond or a linker.

The linker represented by R ² is not particularly limited as long as it is a relatively short divalent group containing a hetero atom on the main chain. Examples of the hetero atom on the main chain include an oxygen atom, a sulfur atom and a nitrogen atom. The number of atoms constituting the main chain of the linker is, for example, 1 to 3. As the linker, for example, -O-, -C(=O)-, -S(=O)-, -S(=O) _2- , -NR- (R is a hydrogen atom or an alkyl group (preferably carbon And an divalent group (for example, -O-C(=O)-, -O-S(=O)-). , -O-S(=O) _2- , -C(=O)-NR-, etc.).

R ² is preferably a single bond.

<1-3. R ³ ＞
R ³ is a single bond or formula (2):

(In the formula, the nitrogen atom is bonded to R ^2. )
Represents a group represented by.

As R ³ , a group represented by the formula (2) is preferable.

When R ² and R ³ are both single bonds, R ² and R ³ together represent a single bond.

<1-4. R ¹ −R ² −R ³ −>
In the prior art (Patent Document 2), R ¹ -R ² -R ³ -was said to be an alkyl group, but the present inventor unexpectedly found that even if it is a group other than an alkyl group, it has high activity. Have been found. From this viewpoint, R ¹ -R ² -R ^3- is preferably a group other than an alkyl group. Among them, R ¹ is an alkylaralkyl group (further, R ² and R ³ are single bonds), or R ³ is a group represented by the formula (2) (further, R ¹ is It is an alkyl group and R ² is a single bond).

<1-5. R ⁴ , R ⁵ ＞
R ⁴ and R ⁵ are the same or different and represent amino acid side chains. Further, when a plurality of R ⁴ are present, R ⁴ independently represents an amino acid side chain at each occurrence.

The amino acid side chain represented by R ⁴ or R ⁵ is not particularly limited as long as it is a monovalent group that can be adopted as the side chain of an amino acid. The term "amino acid" as used herein includes artificial amino acids as well as natural amino acids. Specific examples of the amino acid side chain include an optionally substituted alkyl group, alkenyl group, aryl group, aralkyl group, heteroaryl group, and heteroaralkyl group.

The alkyl group represented by R ⁴ or R ⁵ includes any of linear, branched, or cyclic (preferably linear or branched, more preferably branched) It The carbon number of the alkyl group is not particularly limited and is, for example, 1 to 8, preferably 2 to 6, and more preferably 3 to 5. Specific examples of the alkyl group include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, tert-butyl group, sec-butyl group, n-pentyl group, neopentyl group, n. -Hexyl group, 3-methylpentyl group, n-heptyl group, n-octyl group and the like.

The alkenyl group represented by R ⁴ or R ⁵ is not particularly limited, and has a linear, branched, or cyclic (preferably linear or branched) carbon number of 2 to 8, preferably 2 -6, more preferably 3-5 alkenyl groups are mentioned. Examples of such an alkenyl group include a vinyl group, an allyl group, a 1-propenyl group, an isopropenyl group, a butenyl group, a pentenyl group and a hexenyl group.

The aryl group represented by R ⁴ or R ⁵ is not particularly limited, but preferably has 6 to 12 carbon atoms, more preferably 6 to 12 carbon atoms, and further preferably 6 to 8 carbon atoms. The aryl group may be either monocyclic or polycyclic (eg, bicyclic, tricyclic, etc.), but is preferably monocyclic. Specific examples of the aryl group include a phenyl group, a naphthyl group, a biphenyl group, a pentalenyl group, an indenyl group, an anthranyl group, a tetracenyl group, a pentacenyl group, a pyrenyl group, a perylenyl group, a fluorenyl group, and a phenanthryl group. And preferably a phenyl group.

The aralkyl group represented by R ⁴ or R ⁵ is not particularly limited, but is, for example, a linear or branched carbon number 1 to 6 (preferably 1 to 4, more preferably 1 to 3, and further preferably 1 to An aralkyl group and the like obtained by substituting a hydrogen atom (for example, 1 to 3, preferably 1 hydrogen atom, more preferably a terminal hydrogen atom) of the alkyl group of 2) with the above aryl group can be mentioned.

The heteroaryl group represented by R ⁴ or R ⁵ is not particularly limited, but is preferably a heteroaryl group having 3 to 20, preferably 3 to 12, and more preferably 7 to 11 ring-constituting atoms. Specific examples of the heteroaryl group include a pyrrolyl group, a pyridyl group, a pyrrolidyl group, a piperidyl group, an imidazolyl group, an imidazolyl group, a pyrazolyl group, a pyrazyl group, a pyrimidyl group, a pyridazyl group, a piperazyl group, a triazinyl group, and an oxazolyl group. Group, isoxazolyl group, morpholyl group, thiazolyl group, isothiazolyl group, furanyl group, thiophenyl group, indolyl group, quinolyl group, isoquinolyl group, benzimidazolyl group, quinazolyl group, phthalazyl group, purinyl group, pteridyl group, benzofuranyl group, coumaryl group, A chromonyl group, a benzothiophenyl group, etc. are mentioned, and an indolyl group is preferable.

The heteroaralkyl group represented by R ⁴ or R ⁵ is not particularly limited, but is, for example, a linear or branched carbon number 1 to 6 (preferably 1 to 4, more preferably 1 to 3, and further preferably 1). To 2) a heteroaralkyl group in which a hydrogen atom (for example, 1 to 3, preferably 1 hydrogen atom, more preferably a terminal hydrogen atom) of the alkyl group is substituted with the above heteroaryl group.

The alkyl group represented by R ⁴ or R ⁵ , an alkenyl group, an aryl group, an aralkyl group, a heteroaryl group, the substituent which the heteroaralkyl group may have is not particularly limited, and examples thereof include an amino group and a carboxyl group. , A carbamoyl group, a hydroxyl group, an alkylthio group and the like, and preferably a hydroxyl group, an alkylthio group and the like. The number of substituents is not particularly limited, but is, for example, 0 to 3, preferably 0 to 1.

As the amino acid side chain, preferably, the side chains of hydrophobic amino acids such as alanine, valine, leucine, isoleucine, methionine, tryptophan, and phenylalanine can be mentioned. Among these, side chains such as leucine, methionine, phenylalanine and tryptophan are more preferable, and side chains such as leucine and phenylalanine are more preferable. Further, in the prior art (Patent Document 2), the side chain of a hydrophobic amino acid was said to be preferable, but the present inventor has unexpectedly found that the hydrophilic amino acid side chain also has a high activity. From this viewpoint, hydrophilic amino acid side chains are also preferable as amino acid side chains. The side chain of the hydrophilic amino acid preferably includes side chains such as serine, threonine, asparagine and glutamine, more preferably side chains such as serine and threonine, and further preferably side chains of serine. ..

<1-6. R ⁶ ＞
R ⁶ represents a hydrogen atom, a hydroxyl group, an aldehyde group, a carboxy group, or a carboxy derivative group.

Examples of the carboxyl derivative group include alkyl esters, amides, and anhydrides having about 1 to 4 carbon atoms. Further, examples of the carboxyl derivative group include an aminoacyl group in which one or more amino acids are introduced into the carboxyl group.

R ⁶ is preferably a carboxy group.

<1-7. R ⁵ , R ⁶ ＞
R ⁵ and R ⁶ may combine with each other to form a lactone ring.

The lactone ring is, for example, a 4- to 6-membered ring, preferably a 5- to 6-membered ring, and more preferably a 5-membered ring.

Forming a lactone ring means the following formula in the general formula (1):

The partial structure represented by is, for example, the following formula:

It means that it has a partial structure represented by.

<1-8. n>
n represents 0 or an integer of 1 or more.

n is preferably 0 to 2, more preferably 0 to 1, and even more preferably 0.

<1-9. Preferred General Formula (1) Compound>
In one embodiment of the present invention, the compound represented by the general formula (1) is preferably the general formula (1A):

[In the formula, R ¹ , R ² , R ³ , R ⁵ , and R ⁶ are the same as defined above. ]
And more preferably a compound represented by the general formula (1B):

[In the formula, R ¹ , R ² , R ³ , and R ⁵ are the same as defined above. ]
And more preferably a compound represented by the general formula (1C):

[In the formula, R ¹ , R ³ , and R ⁵ are the same as defined above. ]
The compound represented by

Further, it has a structure similar to that of quorum synthesized by bacteria and the like, and in the use of the present invention described below, from the viewpoint that an endogenous quorum or a lactone ring of quorum can be hydrolyzed and used as a decoy substrate, In the general formula (1), preferably R ⁵ and R ⁶ are bonded to each other to form a 5-membered lactone ring, R ² and R ³ are single bonds, and n=0, or It is preferable that ⁵ is a serine side chain, R ⁶ is a carboxy group, R ² and R ³ are single bonds, and n=0. In these cases, R ¹ is preferably an alkyl group optionally substituted with 1 or 2 (preferably 1) oxo groups.

Examples of quorum included in the general formula (1) include those listed below.

<1-10. Isomers, salts, hydrates, solvates>
The compound represented by the general formula (1) includes stereoisomers and optical isomers, and these are not particularly limited.

The compound represented by the general formula (1) includes a salt form thereof. The salt is not particularly limited. As the salt, either an acidic salt or a basic salt can be adopted. Examples of acidic salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, phosphate; acetate, propionate, tartrate, fumarate, maleate, malic acid. Organic salts such as salts, citrates, methanesulfonates and paratoluenesulfonates can be mentioned, and examples of basic salts include alkali metal salts such as sodium salts and potassium salts; and calcium salts, magnesium salts. Alkaline earth metal salts such as salts; Salts with ammonia; Morpholine, piperidine, pyrrolidine, monoalkylamines, dialkylamines, trialkylamines, mono(hydroxyalkyl)amines, di(hydroxyalkyl)amines, tri(hydroxyalkyl)s Examples thereof include salts with organic amines such as amines.

The compound represented by the general formula (1) includes hydrates and solvates. Examples of the solvent that forms a solvate include organic solvents (eg, ethanol, glycerol, acetic acid, etc.) and the like.

2. Production Method The compound represented by the general formula (1) can be synthesized by various methods. For example, a compound in which n is 0 in the general formula (1) (a compound represented by the general formula (1A)) has the following reaction formula:

[In the formula, R ¹ , R ² , R ³ , R ⁵ , and R ⁶ are the same as defined above. ]
Can be synthesized according to or analogously. Further, a compound in which n is 1 or more in the general formula (1) can also be synthesized by a method according to this.

In this reaction, the compound represented by the general formula (1A) is reacted with the compound represented by the general formula (1b) to obtain the compound represented by the general formula (1A).

From the viewpoint of yield and the like, the amount of the compound represented by the general formula (1b) is usually 0.3 to 3 mol, preferably 0.5 to 2 mol, relative to 1 mol of the compound represented by the general formula (1a). More preferably, it is more preferably 0.8 to 1.2 mol.

This reaction is preferably carried out in the presence of a condensing agent. The condensing agent is not particularly limited and, for example, known condensing agents can be widely used. Specific examples of the condensing agent include 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N,N'-dicyclohexylcarbodiimide (DCC), and preferably EDC. The condensing agents may be used alone or in combination of two or more.

The amount of the condensing agent used varies depending on the kind of the catalyst, but as an example, 0.5 to 3 mol is preferable, and 1 to 2 mol is more preferable, relative to 1 mol of the compound represented by the general formula (1a).

This reaction is preferably carried out in the presence of a base catalyst. The base catalyst is not particularly limited and, for example, known base catalysts can be widely used. Specific examples of the base catalyst include diisopropylethylamine (DIPEA), and preferably DIPEA. The base catalyst may be used alone or in combination of two or more.

The amount of the base catalyst used varies depending on the type of the catalyst, but as an example, 0.8 to 4 mol is preferable, and 1.2 to 2 mol is more preferable, relative to 1 mol of the compound represented by the general formula (1a).

In addition to this, the reaction is preferably performed in the presence of various additives such as 1-hydroxybenzotriazole.

This reaction is usually performed in the presence of a reaction solvent. The reaction solvent is not particularly limited, but examples thereof include N,N-dimethylformamide, dichloromethane, acetonitrile, tetrahydrofuran, acetone, toluene, and the like, and preferably N,N-dimethylformamide. The solvent may be used alone or in combination of two or more.

The reaction can be carried out under heating, at room temperature or under cooling, and is usually preferably carried out at 0 to 50°C (particularly 10 to 30°C). The reaction time is not particularly limited and can be usually 1 hour to 12 hours, particularly 4 hours to 8 hours.

The progress of the reaction can be followed by a usual method such as chromatography. After completion of the reaction, the solvent is distilled off, and the product can be isolated and purified by a usual method such as a chromatography method or a recrystallization method. The structure of the product can be identified by elemental analysis, MS (FD-MS) analysis, IR analysis, ¹ H-NMR, ¹³ C-NMR and the like.

In the reaction, in the case where the compound represented by the general formula (1a) and/or the compound represented by the general formula (1b) has a functional group that inhibits the reaction, instead of these, It is also possible to use compounds in which the functional groups in these compounds are protected. In this case, a deprotection treatment is carried out, if necessary, after the completion of the above reaction.

3. Applications The compound represented by the general formula (1) functions as a cytochrome P450 monooxygenase decoy substrate. This mechanism, though not intended to be a limited interpretation, is considered as follows. At the active center of cytochrome P450 monooxygenase (hereinafter, simply referred to as P450), there is a substrate binding site to which a substrate binds. When the substrate binds to the substrate binding site, water molecules existing in the active center are pushed out from the active center. Hereinafter, this state is referred to as the activation switch being turned on. The active center then catalyzes the hydroxylation of the substrate after being acted on by electrons or oxygen molecules. Therefore, it is considered that the catalytic reaction of P450 does not start unless the activation switch is turned on. As a substrate of wild-type P450, for example, lauric acid, myristic acid and palmitic acid are known. These substrates generally have an end structure attached to the substrate binding site and an alkyl chain. In the compound represented by the general formula (1), and capable of binding sites to substrate binding site of P450 (-C (= O) -NH -CH 2 (-R 5) -R 6), substrates alkyl chains And a part (R ¹ −R ² −R ³ −) corresponding to. Since the compound represented by the general formula (1) has these two sites, it can bind to the substrate binding site of P450 instead of the substrate. On the other hand, since the compound represented by the general formula (1) keeps a sufficient distance from the active center, the activity switch is not turned on. When a substrate such as a hydrocarbon-based substrate or dioxins substrate enters between the compound represented by the general formula (1) and the active center, the water molecule existing near the active center is changed from the active center due to the structural change of P450. It is pushed out and the activation switch is turned on. It is believed that this causes the substrate to be hydroxylated.

It is considered that the compound represented by the general formula (1) enters the cytochrome P450-expressing cells in the environment, functions as a decoy substrate, and converts (hydroxylates) an environmental pollutant into a more degradable substance. This leads to environmental purification such as decomposition of environmental pollutants, water-soluble environmental pollutants, and reduction of toxicity. Therefore, at least one selected from the group consisting of the compound represented by the general formula (1) and cells containing the compound can be used as an active ingredient of the environmental purification agent.

The cell is not particularly limited as long as it can contain the compound represented by the general formula (1). Examples of cells include prokaryotic cells (preferably Gram-positive) such as Gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa; Gram-positive bacteria such as Bacillus and Brevibacillus (particularly Gram-positive endospore-forming bacilli). Fungi), eukaryotic cells such as yeast, plant cells, animal cells and the like can be used.

The method for obtaining the cells containing the compound represented by the general formula (1) is not particularly limited, but as an example, the compound represented by the general formula (1) is added to the cell suspension to intracellularly May be introduced. At this time, when the cell membrane permeability of the compound represented by the general formula (1) used in the cell is low (for example, when R ⁴ and/or R ⁵ is a hydrophilic amino acid side chain), a known method is used. The compound represented by the general formula (1) may be introduced after the cell membrane permeability is increased by treating the cell with an organic solvent or the like according to the above, or a precursor of the compound represented by the general formula (1) ( For example, in the compound represented by the general formula (1) to be used intracellularly, when R ⁵ is a hydrophilic amino acid side chain, R ⁵ and R ⁶ are bonded to each other to form a lactam ring. The compound) may be introduced into the cell, and then the compound represented by the general formula (1) may be produced from the precursor (for example, by hydrolysis of the lactam ring) in the cell. In the latter case, in order to accelerate the production of the compound represented by the general formula (1) from the precursor, it is preferable to express intracellularly a hydrolase such as AHL-lactonase.

When the compound represented by the general formula (1) to be used is a cell-endogenous compound (eg, quorumone etc.), the cell itself containing the quorumon is used or the quorumone is secreted. To obtain a cell containing the compound represented by the general formula (1), by co-culturing with a cell to be treated, or by using a cell into which a quorum biosynthesis gene and a hydrolase gene such as AHL-lactonase are introduced. be able to.

The cell is preferably a cell harboring a cytochrome P450 monooxygenase. The purification effect of the present invention can be exhibited by using such cells. Note that in one embodiment of the present invention, the cell is a cell into which the compound represented by the general formula (1) has been introduced exogenously.

Examples of the mode of exerting the purifying effect of the present invention include a mode of purifying by utilizing wild bacteria (not genetically modified) existing in the environment. This embodiment can be used if it is known that there are bacteria having P450 in the environment. It should be noted that whether or not there are bacteria having P450 in the environment can be determined by metagenomic analysis of soil microorganisms, actual tests using a part of soil samples, and the like. Further, as another example of the above aspect, in an environment in which a recombinant microorganism can be used, an aspect in which the microorganism represented by P450 and the compound represented by the general formula (1) are dispersed in the environment to be purified Is mentioned.

The P450 may include a known cytochrome P450 monooxygenase. P450 is preferably P450 derived from bacteria, more preferably P450 derived from Pseudomonas aeruginosa or P450 derived from genus Bacillus. Examples of such P450s include CYP102 family enzymes such as cytochrome P450BM3 (CYP102A1), and P450s targeting fatty acids (for example, CYP153 family enzymes such as CYP153A1).

P450s also include P450s having an amino acid sequence identity of 40% or more with P450s having known amino acid sequences. For example, P450 (a family enzyme of cytochrome P450BM3) having 40% or more identity with the amino acid sequence of cytochrome P450BM3, or P450 (subfamily of cytochrome P450BM3 (CYP102A etc.) with 55% or more identity with the amino acid sequence of cytochrome P450BM3). The enzyme))) is preferably mentioned. Alternatively, P450 (a family enzyme of cytochrome P450cam) having 40% or more identity with the amino acid sequence of cytochrome P450cam or P450 (subfamily enzyme of cytochrome P450cam) having 55% or more identity with the amino acid sequence of cytochrome P450cam Preferred examples include: The cytochrome P450 BM3 family enzyme or subfamily enzyme is preferably capable of hydroxylating a fatty acid, and the cytochrome P450cam family enzyme or subfamily enzyme is preferably capable of hydroxylating camphor (C ₁₀ H ₁₆ O). ..

Here, the “identity” of amino acid sequences refers to the degree of matching of two or more comparable amino acid sequences with each other. Therefore, the more identical a given amino acid sequence is, the greater the identity or similarity of those sequences. The level of amino acid sequence identity is determined, for example, using FASTA, a tool for sequence analysis, using default parameters. Alternatively, the algorithm BLAST (Karlin S, Altschul SF. “Methods for assessing the statistical significance of molecular sequence features by using general scoringschemes” Proc Natl Acad Sci USA. 87:2264-2268 (1990), Karlin S, Altschul SF. "Applications and statistics for multiple high-scoring segments in molecular sequences." Proc Natl Acad Sci USA. 90:5873-7 (1993)). A program called BLASTX based on such a BLAST algorithm has been developed. Specific methods of these analysis methods are known, and the website (http://www.ncbi.nlm.nih.gov/) of the National Center of Biotechnology Information (NCBI) may be referred to. The “identity” of the nucleotide sequences is also defined according to the above.

The amino acid sequence of such a cytochrome P450BM3 is, for example, J. Biol.chem.264 (19), 10987-10998 (1989) or NCBI beta base (National Center for Biotechnology Information http://www.ncbi.nlm.nih. gov/protein/P14779.2). In addition, the amino acid of cytochrome P450cam is also disclosed in the NCBI database (http://www.ncbi.nlm.nih.gov/protein/YP_714029).

P450 may be a wild type or a mutant type. Preferable examples of the mutant form include a mutant form of P450 derived from bacteria. Examples include mutant forms of cytochrome P450 BM3 and cytochrome P450 cam. For details of these variants and the method of obtaining, for example, special table 2000-508163 publication, special table 2003-517815 publication, special table 2003-521889 publication, CJC Whitehouse, SG Bell, LL Wong, Chem. Soc. Rev. 2012, 41, 1218-1260 (all variants of cytochrome P450BM3), F. Xu, SG Bell, J. Lednik, A. Insley, ZH Rao, LL Wong, Angew. Chem. Int Ed. 2005, 44, 4029-4032, H. Joo, ZL Lin, FH Arnold, Nature 1999, 399, 670-673 (above, mutant form of cytochrome P450cam).

The environment to be purified is not particularly limited as long as it is an environment in which cells having cytochrome P450 can live, and examples thereof include soil, lakes, seas, groundwater, sewage, reservoirs, sludge, rivers, industrial wastewater, and the like. Usually, microorganisms are present in these environments, and there are also microorganisms expressing cytochrome P450 monooxygenase. The environment is preferably an environment in which a microorganism having a cytochrome P450 is present. Examples of such microorganisms existing in the environment include Bacillus bacteria such as Giant Bacteria, Bacillus natto and Bacillus subtilis.

More specifically, the compound represented by the general formula (1) is used for conversion (hydroxylation) of environmental pollutants. Therefore, the environmental cleaning agent of the present invention can be used for conversion of environmental pollutants. The environmental pollutant is not particularly limited as long as it is a substance that can be converted (hydroxylated) by cytochrome P450 in the presence of a decoy substrate. Examples of the environmental pollutants include organic compounds represented by volatile organic compounds such as benzene, dioxins, pesticides, mineral oil, heavy oil, and the like. Among these, hydrocarbon pollutants and dioxins pollutants are preferable. In the present specification, hydroxylation of the environmental pollutant means that the C—H bond of the environmental pollutant is oxidized by an oxidation reaction, and as a result, a hydroxyl group is introduced into the carbon atom.

The hydrocarbon pollutant is not particularly limited, and examples thereof include aromatic compounds, alkanes, alkenes, cycloalkanes, cycloalkenes and the like.

The aromatic compound may be mono-substituted or poly-substituted. Examples of the substituent include alkyl having 1 to 4 carbons, alkenyl having 2 to 4 carbons, hydroxyl group, halogen atom and the like. The alkyl or alkenyl substituent may have a keto group or an aldehyde group. The aromatic compound may be mononuclear or polynuclear. It is preferably mononuclear or dinuclear. Further, the aromatic compound may be condensed with two or more nuclei or may be condensed with a non-aromatic ring. Examples of such aromatic compounds include mononuclear or binuclear aromatic compounds, and more specifically, optionally substituted benzene, naphthalene, indene, and the like. Of these, benzene is preferable.

Examples of the alkane include a linear or branched alkane having about 1 to 15 carbon atoms. For example, methane, ethane, n-propane, n-butane, n-pentane, n-hexane, n-heptane, n-octane, n-nonane, n-decane, n-undecane and n-dodecane and the like Examples include branched chains having the above branches.

Examples of the alkene include the above-mentioned alkanes having one or more unsaturated bonds. Examples include alkenes with 1 unsaturation.

Examples of the cycloalkane and cycloalkene include optionally substituted cycloalkane and cycloalkene each having 4 to 8 carbon atoms. For example, cyclopentane, cyclopentene, cyclohexane, cyclohexene, cycloheptane, cycloheptene and the like can be mentioned. The cycloalkane and cycloalkene may have one or more substituents, and may have, for example, 1 to 5 substituents. As the substituent, the above-mentioned substitution can be applied. Examples of cycloalkanes and cycloalkenes include cyclohexane and cyclohexene which are unsubstituted or have about 1 to 3 substituents.

Examples of dioxins pollutants include halogenated dibenzodioxins, halogenated dibenzofurans, PCBs (particularly coplanar PCBs in which chlorine atoms are substituted at positions other than the ortho positions).

Examples of halogenated dibenzodioxins include 2,3,7,8-tetrachlorodibenzo-P-dioxin, 1,2,3,7,8-pentachlorodibenzo-P-dioxin, 1,2,3, 4,7,8-Hexachlorodibenzo-P-dioxin, 1,2,3,4,6,7,8-heptachlorodibenzo-P-dioxin, 1,2,3,4,6,7,8,9 -Octachlorodibenzo-P-dioxin and the like.

Examples of halogenated dibenzofurans include 2,3,7,8-tetrachlorodibenzofuran, 1,2,3,7,8-pentachlorodibenzofuran, 1,2,3,4,7,8-hexachlorodibenzofuran, Examples include 1,2,3,4,6,7,8-heptachlorodibenzofuran and 1,2,3,4,6,7,8,9-octachlorodibenzofuran.

Examples of PCBs (particularly coplanar PCBs having chlorine atoms substituted at positions other than the ortho positions) include 3,3′,4,4′,5-tetrachlorobiphenyl, 3,3′,4,4′,5 -Pentachlorobiphenyl, 3,3',4,4',5,5'-hexachlorobiphenyl and the like can be mentioned.

The environmental cleaning agent of the present invention may contain components other than the active ingredient. Other components include, for example, a carrier, a fixing agent, a dispersant, an auxiliary agent and the like. Examples of the carrier include solid carriers such as talc, bentonite, clay, kaolin, diatomaceous earth, white carbon, vermiculite, and silica sand; water-soluble polymer compounds (polyvinyl alcohol, polyvinylpyrrolidone, polyacrylic acid, etc.), water, vegetable oil. , And liquid carriers such as liquid animal oil. Examples of the fixing agent include casein, gelatin, gum arabic, alginic acid and the like. Examples of the dispersant include alcohol sulfates and polyoxyethylene glycol ethers. Examples of the auxiliary agent include carboxymethyl cellulose, starch, lactose and the like. The above-mentioned carrier, fixing agent, dispersant and auxiliary agent can be used alone or in appropriate combination depending on the purpose.

The content of the active ingredient in the environmental purification agent of the present invention is not particularly limited and is, for example, 0.001 to 100 mass %.

The dosage form of the environmental purification agent of the present invention is not particularly limited. Examples thereof include granules, powders, powder granules, powders, wettable powders, emulsions, liquids, oils, aerosols, pastes, microcapsules and packs.

The environmental purification agent of the present invention can be used by bringing it into contact with the environment to be purified. The method of contact can be appropriately selected depending on the type of environment. When used for soil purification, for example, a method of spraying, mixing or irrigating the soil can be mentioned. After contact, by leaving it for a certain period of time, conversion (hydroxylation) of environmental pollutants into more degradable substances occurs, and environmental purification is achieved.

More specific modes of use are as follows.

Examples of the method for spraying the environmental purification agent include a method of spraying the compound represented by the general formula (1) to the polluted environment and incorporating it into cytochrome P450 existing in the polluted environment, which is represented by the general formula (1). Examples thereof include a method of spraying the compound-loaded cells to a contaminated soil, and a method of separately spraying the compound represented by the general formula (1) and cells having P450.

Examples of the action method of the environmental purifying agent include a method of spraying in a contaminated environment and bringing it into contact with the contaminated environment by a permeation action, a method of bringing the contaminated environment and the purifying agent into contact with each other by stirring.

Examples of the place of action of the environmental cleaning agent include a contaminated environment site and a place where the contaminated environment (soil, liquid) is collected and brought back.

Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited to these examples.

Synthesis example 1. Nonanoyl-L-alanine (C9-L-Ala)
<Protection of amino acid ester>

In an eggplant-shaped flask, 1.69 g (19 mmol, 1.0 eq) of L-alanine was weighed, and 19 mL of methanol was added and dissolved. The solution was cooled to -20°C (ice/NaCl), and 1.45 mL (2.37 g, 20 mmol, 1.05 eq) of thionyl chloride was added dropwise. The solution was stirred at 0°C for 1 hour and then at room temperature for 16 hours, and the solvent was distilled off with an evaporator. The obtained white solid was washed with diethyl ether and vacuum dried to obtain 2.32 g of a white solid (yield: 88%).

<Condensation of carboxylic acid and amino acid>

Nonanoic acid 949 mg (6 mmol), L-alanine methyl ester hydrochloride 838 mg (6 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC・HCl) 1.44 mg in an eggplant-shaped flask. (7.5 mmol), 1-hydroxybenzotriazole monohydrate (HOBt·H ₂ O) 1.15 g (0.75 mmol) was weighed out, and 30 mL of dehydrated dimethylformamide was added under an argon atmosphere. Then, 1.29 g (10 mmol) of diisopropylethylamine (DIPEA) was added, and the mixture was stirred at room temperature for 6 hours. About 20 mL of ion-exchanged water was added to the reaction solution, extracted three times with ethyl acetate, and washed with saturated aqueous sodium hydrogen carbonate solution and saturated saline. Magnesium sulfate was added, the mixture was dried for 10 minutes, filtered, and then the solvent was distilled off by an evaporator. It was purified by a silica gel column using hexane:ethyl acetate=4:1 as a developing solvent. The corresponding fractions were collected, the solvent was evaporated, and the residue was dried under vacuum to obtain 1.01 g of white solid (yield: 69%).

<Demethylation>

851 mg (3.5 mmol) of C9-L-Ala-Me was weighed in an eggplant-shaped flask, 1 M LiOH aqueous solution/THF = 4:1 was added, and the mixture was stirred at 60°C for 3 hours. The solution was cooled to room temperature, 2M hydrochloric acid was added to adjust the pH to 1-2, and the mixture was extracted 3 times with Et ₂ O. The extracted organic phases were combined, magnesium sulfate was added, and the mixture was dried for 30 minutes, filtered, and then the solvent was distilled off by an evaporator. Recrystallization from hexane-ethyl acetate and vacuum drying gave 658 mg of white crystals (yield: 82%).
¹ H-NMR (DMSO-D ₆ )δ: 12.44 (1H, br), 8.06 (1H, d, J = 7.3 Hz), 4.17 (1H, quin), 2.08 (2H, t, J = 7.6 Hz), 1.47 (2H, s), 1.27-1.23 (13H, m), 0.85 (3H, t, J= 6.6 Hz). ESI-MS: m/z 252.16 ([M+Na] ⁺ ), 481.34 ([2M+ Na] ⁺ ), 503.32 ([2M-H+2Na] ⁺ ), 710.49 ([3M+Na] ⁺ ), 732.50 ([3M-H+2Na] ⁺ ).

Synthesis example 2. Octanoyl-L-phenylalanine (C8-L-Phe)

Synthesized according to Synthesis Example 1 using appropriate materials.
¹ H-NMR (DMSO-D ₆ )δ: 12.66 (1H, s), 8.11 (1H, d, J = 8.3 Hz), 7.28-7.17 (5H, m), 4.41 (1H, td, J = 9.1, 4.4 Hz), 3.05 (1H, dd, J = 13.7, 4.4 Hz), 2.82 (1H, dd, J = 13.7, 10.2 Hz), 2.02 (2H, t, J = 7.3 Hz), 1.37 (2H, quin, J = 7.4 Hz), 1.27-1.08 (8H, m), 0.85 (3H, t, J = 7.1 Hz). ESI-MS: m/z 314.18 ([M+Na] ⁺ ), 605.37 ([2M+Na ] ⁺ ), 896.56 ([3M+Na] ⁺ ), 918.54 ([3M-H+2Na] ⁺ ).

Synthesis example 3. Nonanoyl-L-phenylalanine (C9-L-Phe)

Synthesized according to Synthesis Example 1 using appropriate materials.
¹ H-NMR (DMSO-D ₆ ) δ: 12.66 (1H, s), 8.11 (1H, d, J = 8.3 Hz), 7.28-7.17 (5H, m), 4.42 (1H, td, J = 9.0, 4.2 Hz), 3.05 (1H, dd, J = 13.7, 4.4 Hz), 2.82 (1H, dd, J = 13.7, 10.2 Hz), 2.02 (2H, t, J = 7.3 Hz), 1.37 (2H, quin, J = 7.3 Hz), 1.29-1.10 (10H, m), 0.86 (3H, t, J= 6.8 Hz). ESI-MS: m/z 328.19 ([M+Na] ⁺ ), 350.17 ([M-H +2Na] ⁺ ), 633.40 ([2M+Na] ⁺ ), 655.38 ([2M-H+2Na] ⁺ ), 938.60 ([3M+Na] ⁺ ), 960.58 ([3M-H+2Na] ⁺ ).

Synthesis example 4. Decanoyl-L-phenylalanine (C10-L-Phe)

Synthesized according to Synthesis Example 1 using appropriate materials.
¹ H-NMR (DMSO-D ₆ ) δ: 12.66 (1H, s), 8.11 (1H, d, J = 8.3 Hz), 7.28-7.17 (5H, m), 4.41 (1H, td, J = 9.0, 4.1 Hz), 3.05 (1H, dd, J = 13.9, 4.6 Hz), 2.82 (1H, dd, J = 13.4, 10.0 Hz), 2.02 (2H, t, J = 7.3 Hz), 1.37 (2H, quin, J = 7.3 Hz), 1.27-1.10 (12H, m), 0.86 (3H, t, J= 6.8 Hz). ESI-MS: m/z 342.21 ([M+Na] ⁺ ), 364.19 ((M-H +2Na] ⁺ ), 661.43 ([2M+Na] ⁺ ), 683.41 ([2M-H+2Na] ⁺ ), 980.65 ([3M+Na] ⁺ ), 1002.63 ([3M-H+2Na] ⁺ ).

Synthesis example 5. Undecanoyl-L-phenylalanine (C11-L-Phe)

Synthesized according to Synthesis Example 1 using appropriate materials.
¹ H-NMR (DMSO-D ₆ )δ: 12.66 (1H, s), 8.11 (1H, d, J = 8.3 Hz), 7.28-7.17 (5H, m), 4.41 (1H, td, J = 8.9, 4.4 Hz), 3.05 (1H, dd, J = 13.9, 4.6 Hz), 2.82 (1H, dd, J = 13.7, 10.2 Hz), 2.02 (2H, t, J = 7.1 Hz), 1.41-1.33 (2H, quin, 7.1 Hz), 1.29-1.10 (14H, m), 0.86 (3H, t, J = 6.6 Hz). ESI-MS: m/z 356.23 ([M+Na] ⁺ ), 378.23 ([M-H +2Na] ⁺ ), 689.46 ([2M+Na] ⁺ ), 711.44 ([2M-H+2Na] ⁺ ), 1022.68 ([3M+Na] ⁺ ), 1044.68 ([3M-H+2Na] ⁺ ).

Synthesis example 6. Octanoyl-L-leucine (C8-L-Leu)

Synthesized according to Synthesis Example 1 using appropriate materials.

¹ H-NMR (DMSO-D ₆ ) δ: 12.66 (1H, s), 8.11 (1H, d, J = 8.3 Hz), 7.28-7.17 (5H, m), 4.41 (1H, td, J = 9.1, 4.4 Hz), 3.05 (1H, dd, J= 13.7, 4.4 Hz), 2.82 (1H, dd, J = 13.7, 10.2 Hz), 2.02 (2H, t, J= 7.3 Hz), 1.37 (2H, quin, J = 7.4 Hz), 1.27-1.08 (8H, m), 0.85 (3H, t, J= 7.1 Hz). ESI-MS: m/z 314.18 ([M+Na] ⁺ ), 605.37 ([2M+Na ] ⁺ ), 896.56 ([3M+Na] ⁺ ), 918.54 ([3M-H+2Na] ⁺ ).

Synthesis example 7. Nonanoyl-L-Leucine (C9-L-Leu)

Synthesized according to Synthesis Example 1 using appropriate materials.
¹ H-NMR (DMSO-D ₆ ) δ: 12.45 (1H, s), 8.02 (1H, d, J = 8.3 Hz), 4.20 (1H, td, J = 8.9, 5.0 Hz), 2.15-2.03 (2H , m), 1.67-1.57 (1H, m), 1.55-1.42 (4H, m), 1.29-1.23 (10H, m), 0.89-0.82 (9H, m). ESI-MS: m/z 294.21 (( M+Na] ⁺ ), 565.44 ([2M+Na] ⁺ ), 836.66 ([3M+Na] ⁺ ), 858.64 ([3M-H+2Na] ⁺ ), 1129.87 ([4M-H+2Na] ⁺ ) , 1151.85 ([4M-2H+3Na] ⁺ ).

Synthesis example 8. Nonanoyl-L-methionine (C9-L-Met)

Synthesized according to Synthesis Example 1 using appropriate materials.
¹ H-NMR (DMSO-D ₆ ) δ: 12.56 (1H, s), 8.07 (1H, d, J = 7.8 Hz), 4.29 (1H, td, J = 8.7, 4.6 Hz), 2.48-2.40 (2H , m), 2.10 (2H, t, J = 7.1 Hz), 2.03 (3H, s), 1.97-1.89 (1H, m), 1.86-1.78 (1H, m), 1.51-1.44 (2H, m), 1.29-1.24 (10H, m), 0.85 (3H, t, J = 6.6 Hz). ESI-MS: m/z 312.17 ([M+Na] ⁺ ), 601.36 ([2M+Na] ⁺ ), 890.53 ( [3M-H+2Na] ⁺ ).

Synthesis example 9. Nonanoyl-L-Tryptophan (C9-L-Trp)

Synthesized according to Synthesis Example 1 using appropriate materials.

¹ H-NMR (DMSO-D ₆ ) δ: 12.55 (1H, s), 10.82 (1H, s), 8.03 (1H, d, J = 8.3 Hz), 7.52 (1H, d, J = 7.8 Hz), 7.32 (1H, d, J = 8.3 Hz), 7.12 (1H, d, J = 2.0 Hz), 7.05 (1H, t, J = 7.3 Hz), 6.97 (1H, t, J = 7.3 Hz), 4.46 ( 1H, td, J = 8.3, 4.9 Hz), 3.15 (1H, dd, J = 14.6, 4.9 Hz), 2.98 (1H, dd, J = 14.6, 8.8 Hz), 2.05 (2H, t, J = 7.1 Hz ), 1.40 (2H, quin, J = 7.2 Hz), 1.32-1.08 (10H, m), 0.85 (3H, t, J = 6.8 Hz). ESI-MS: m/z 367.21 ([M+Na] ⁺ ), 711.43 ([2M+Na] ⁺ ), 1055.64 ([3M+Na] ⁺ ).

Synthesis example 10. Nonanoyl-D-phenylalanine (C9-D-Phe)

Synthesized according to Synthesis Example 1 using appropriate materials.
¹ H-NMR (DMSO-D ₆ ) δ: 12.65 (1H, s), 8.09 (1H, d, J = 7.8 Hz), 7.28-7.17 (5H, m), 4.42 (1H, td, J = 9.0, 3.9 Hz), 3.05 (1H, dd, J = 13.9, 4.6 Hz), 2.82 (1H, dd, J = 13.7, 9.8 Hz), 2.02 (2H, t, J = 7.3 Hz), 1.37 (2H, quin, J = 7.3 Hz), 1.27-1.10 (10H, m), 0.86 (3H, t, J= 6.8 Hz). ESI-MS: m/z 328.20 ([M+Na] ⁺ ), 633.41 ([2M+Na ] ⁺ ), 938.61 ([3M+Na] ⁺ ), 960.59 ([3M-H+2Na] ⁺ ).

Synthesis example 11. Nonanoyl-D-Leucine (C9-D-Leu)

Synthesized according to Synthesis Example 1 using appropriate materials.
¹ H-NMR (DMSO-D ₆ ) δ: 12.44 (1H, s), 8.01 (1H, d, J = 7.8 Hz), 4.21 (1H, td, J = 8.9, 5.0 Hz), 2.13-2.05 (2H , m), 1.65-1.58 (1H, m), 1.52-1.46 (4H, m), 1.28-1.23 (10H, m), 0.89-0.82 (9H, m). ESI-MS: m/z 294.21 (( M+Na] ⁺ ), 565.44 ([2M+Na] ⁺ ), 587.41 ([2M-H+2Na] ⁺ ), 836.65 ([3M+Na] ⁺ ), 858.64 ([3M-H+2Na] ⁺ ) , 1129.86 ([4M+Na] ⁺ ), 1151.84 ([4M-H+2Na] ⁺ ).

Synthesis example 12. (2-(4-isobutylphenyl)propanoyl)-L-phenylalanine (R-Ibu-L-Phe)

Synthesized according to Synthesis Example 1 using appropriate materials.
¹ H-NMR (CDCl ₃ ) δ: 7.82 (1H, br s), 7.17-7.08 (7H, m), 6.82 (2H, d, J = 7.3 Hz), 5.76 (1H, d, J = 7.3 Hz) , 4.83 (1H, q, J = 6.2 Hz), 3.51 (1H, q, J = 7.0 Hz), 3.08 (1H, dd, J = 13.9, 5.1 Hz), 2.99 (1H, dd, J = 13.9, 6.1 Hz), 2.48 (2H, d, J = 7.3 Hz), 1.91-1.84 (1H, m), 1.46 (3H, d, J = 6.8 Hz), 0.92 (6H, d, J = 6.8 Hz). ESI- MS: m/z 376.18 ([M+Na] ⁺ ), 729.38 ([2M+Na] ⁺ ), 1082.58 ([3M+Na] ⁺ ), 1104.57 ([3M-H+2Na] ⁺ ).

Synthesis example 13. (2-(4-isobutylphenyl)propanoyl)-L-phenylalanine (S-Ibu-L-Phe)

Synthesized according to Synthesis Example 1 using appropriate materials.
¹ H-NMR (CDCl ₃ )δ: 8.06 (1H, br s), 7.22-7.20 (3H, m), 7.10-7.07 (4H, m), 6.96-6.94 (2H, m), 5.76 (1H, d , J = 6.8 Hz), 4.74 (1H, q, J = 6.3 Hz), 3.54 (1H, q, J = 7.2 Hz), 3.15 (1H, dd, J = 13.8, 5.2 Hz), 3.00 (1H, dd , J = 14.0, 6.7 Hz), 2.46 (2H, d, J = 7.3 Hz), 1.91-1.80 (1H, m), 1.49 (3H, d, J = 7.3 Hz), 0.90 (6H, d, J = 6.6 Hz). ESI-MS: m/z 376.19 ([M+Na] ⁺ ), 729.40 ([2M+Na] ⁺ ), 1082.60 ([3M+Na] ⁺ ), 1104.58 ([3M-H+2Na] ⁺ ).

Synthesis example 14. (Benzyloxy)carbonyl)glycyl-L-phenylalanine (Z-Gly-L-Phe)

Synthesized according to Synthesis Example 1 using appropriate materials.

¹ H-NMR (DMSO-D ₆ ) δ: 12.78 (1H, s), 8.12 (1H, d, J = 7.8 Hz), 7.42-7.20 (11H, m), 5.02 (2H, s), 4.44 (1H , td, J = 8.1, 5.2 Hz), 3.61 (2H, qd, J = 16.7, 6.4 Hz), 3.04 (1H, dd, J = 13.9, 5.1 Hz), 2.89 (1H, dd, J = 13.4, 9.0 Hz). ESI-MS: m/z 379.12 ([M+Na] ⁺ ), 735.26 ([2M+Na] ⁺ ).

Synthesis example 15. N-(6-phenylhexanoyl)-L-phenylalanine (PhC6-L-Phe)

Synthesized according to Synthesis Example 1 using appropriate materials.
¹ H NMR (DMSO-d ₆ , 600MHz at 80°C) δ: 12.30 (1H, brs), 7.75 (1H, d, J = 5.4 Hz), 7.28-7.11 (10H, m), 4.48 (1H, td , J = 9.0, 5.4 Hz), 3.06 (1H, dd, J = 14.4, 5.4 Hz), 2.87 (1H, dd, J = 14.1, 9.6 Hz), 2.52 (2H, t, J = 7.8 Hz), 2.05 (2H, t, J = 7.8 Hz), 1.52 (2H, m, J = 7.8 Hz), 1.45 (2H, m, J = 7.8 Hz), 1.21 (2H, m, J = 7.8 Hz). ESI-MS : m/z 362.17 ([M+Na] ⁺ ), 384.15 ([M-H+2Na] ⁺ ), 701.36 ([2M+Na] ⁺ ), 1040.55 ([3M+Na] ⁺ ).

Reference example 1. N-alkylproline

388 mg (3 mmol) of L-proline methyl ester was weighed in an eggplant-shaped flask and dissolved in 8 mL of acetonitrile. After adding sodium hydrogencarbonate 1.01 g (12 mmol) and 1-bromohexane 743 mg (4.5 mmol), it stirred at 60 degreeC for 48 hours. The solution was cooled to room temperature and extracted 3 times with diethyl ether. The extracted organic phases were combined, washed with brine, magnesium sulfate was added, and the mixture was dried for 10 minutes. After filtration, the solvent was distilled off by an evaporator to obtain 461 mg of a yellow oily substance. (Yield: 72%)
FAB-MS confirmed that L-proline methyl ester and 1-bromohexane did not remain in the product.

Synthesis example 16. (Benzyloxy)carbonyl)-L-prolyl-L-phenylalanine (Z-L-Pro-L-Phe)

According to Synthesis Example 2, instead of nonanoic acid, a proline derivative synthesized in the same manner as in Reference Example 1 was used.
¹ H-NMR (DMSO-D ₆ ) δ: 12.70 (1H, s), 8.17 (1H, dd, J = 39.0, 7.8 Hz), 7.38-7.18 (10H, m), 5.10-4.84 (2H, m) , 4.50-4.39 (1H, m), 4.22 (1H, d, J = 8.3 Hz), 3.45-3.35 (2H, m), 3.07-2.86 (2H, m), 2.09-1.99 (1H, m), 1.77 -1.68 (3H, m). ESI-MS: m/z 419.16 ([M+Na] ⁺ ), 441.14 ([M-H+2Na] ⁺ ), 859.30 ([2M-2H+3Na] ⁺ ).

Synthesis example 17. (N-hexyl-L-prolyl)-L-phenylalanine (C6-L-Pro-L-Phe)

According to Synthesis Example 2, instead of nonanoic acid, a proline derivative synthesized in the same manner as in Reference Example 1 was used.
¹ H-NMR (DMSO-D ₆ ) δ: 12.90 (0.7H, br s), 7.76 (1H, d, J = 6.8 Hz), 7.26-7.14 (5H, m), 4.53-4.48 (1H, m) , 3.10 (1H, dd, J = 13.7, 4.9 Hz), 3.04-2.95 (2H, m), 2.83 (1H, d, J = 7.8 Hz), 2.45-2.40 (1H, m), 2.32-2.26 (1H , m), 2.21-2.16 (1H, m), 2.00-1.90 (1H, m), 1.68-1.62 (1H, m), 1.53-1.42 (2H, m), 1.35-1.18 (8H, m), 0.84 (3H, t, J = 6.8 Hz). ESI-MS: m/z 347.2 ([M+H] ⁺ ). MS calcd for [M+H] ⁺ m/z 347.2, found m/z 347.

Synthesis example 18. (N-heptyl-L-prolyl)-L-phenylalanine (C7-L-Pro-L-Phe)

According to Synthesis Example 2, instead of nonanoic acid, a proline derivative synthesized in the same manner as in Reference Example 1 was used.
¹ H-NMR (DMSO-D ₆ )δ: 12.91 (1H, br s), 7.76 (1H, d, J = 6.8 Hz), 7.28-7.14 (5H, m), 4.53-4.48 (1H, m), 3.10 (1H, dd, J = 13.7, 4.9 Hz), 3.03-2.95 (2H, m), 2.83 (1H, d, J = 7.8 Hz), 2.45-2.40 (1H, m), 2.32-2.26 (1H, m), 2.22-2.16 (1H, m), 1.97-1.92 (1H, m), 1.68-1.62 (1H, m), 1.53-1.44 (2H, m), 1.36-1.19 (10H, m), 0.85 ( 3H, t, J = 6.8 Hz). ESI-MS: m/z 361.26 ([M+H] ⁺ ), 383.24 ([M+Na] ⁺ ), 721.50 ([2M+H] ⁺ ), 743.49 ([ 2M+Na] ⁺ ).

Synthesis Example 19-22. N-acyl homoserine lactone (AHL)

(Synthesis example 19: n=5, synthesis example 20: n=6, synthesis example 21: n=7, synthesis example 22: n=8)
l-Homoserine latone (1.2 mmol, 165 mg) and Triethylamine (1.2 mmol, 170 _L) were dissolved in 20 mL of water, and then 10 mL of acetonitrile was added to EDC-HCl (1.6 mmol, 310 mg) and C7-C10 carboxylic acid ( A solution in which 1.6 mmol, C7 227 μL, C8 254 μL, C9 278 μL, C10 276 mg) was dissolved was added. The mixture was reacted with stirring at room temperature overnight, and then the solvent was distilled off. The solid was dissolved in chloroform and washed with a saturated NaHCO ₃ aqueous solution, a 1 M NaHSO ₄ aqueous solution, and a saturated NaCl aqueous solution. The organic phase was dried over Na ₂ SO ₄ and the solvent was distilled off, followed by recrystallization with a solution of Hexane:EtOAc=2:1. The solid was washed with a small amount of chilled EtOAc to obtain the target compound (yield: C7AHL 45.6%, C8AHL 43.0%, C9AHL 35.5%, C10AHL 33.5%). 6 mg of the compound was dissolved in d6-DMSO and identified by ¹ H NMR. A representative compound (C8AHL) ¹ H NMR chart is shown below.
¹ H-NMR (DMSO-D ₆ , 400 MHz, TMS) δ (ppm): 8.30 (1H, d, J = 8.3 Hz), 4.52 (1H, dt, J = 10.0, 8.8 Hz), 4.33 (1H, td, J = 8.8, 2.0 Hz), 4.21-4.18 (1H, m), 2.39-2.35 (1H, m), 2.18-2.08 (3H, m), 1.50 (2H, quintet, J = 6.8 Hz), 1.28 -1.26 (8H, m), 0.86 (3H, t, J = 6.8 Hz).

Synthesis Example 23-25. N-acyl homoserine (AHS)

(Synthesis example 23: n=6, synthesis example 24: n=7, synthesis example 25: n=8)
4.5 mL of water and 1.0 mL of 1 M LiOH aqueous solution were added to 30 mg of the synthesized C8, C9, and C10AHL, and the mixture was reacted for 22 hours with stirring. Then, 1 mL of a 6 M HCl aqueous solution was added dropwise to the ice-cooled reaction solution. After the reaction solution was ice-cooled for 1 hour, the solution was suction-filtered and the precipitated solid was collected. The solid was washed with a small amount of cooled EtOAc to obtain the target compound (yield: C8AHS 57.4%, C9AHS 56.2%, C10AHS 57.4%). 6 mg of the compound was dissolved in d6-DMSO and identified by ¹ H NMR. A representative compound (C10AHS) ¹ H NMR chart is shown below.
¹ H-NMR (DMSO-D ₆ , 400 MHz, TMS) δ (ppm): 7.99 (1H, d, J = 8.0 Hz), 4.27-4.22 (1H, m), 2.09 (2H, t, J = 6.8) Hz), 1.86-1.80 (1H, m), 1.72-1.65 (1H, m), 1.48-1.46 (2H, m), 1.27-1.24 (12H, m), 0.86 (3H, t, J = 6.8 Hz) .

Example 1. Intracellular production of phenol (benzene hydroxylation reaction)
Using the compound obtained in Synthesis Example as a decoy substrate, phenol was produced intracellularly. Specifically, it was performed as follows.

<Preparation of cell suspension>
Wild-type P450 BM3 Escherichia coli BL21(DE3) strain transformed with the forced expression vector pET28a(+) having the full-length nucleotide sequence inserted was added to LB medium containing 15 μg/mL kanamycin, and cultured at 37°C and 180 rpm. did. 5-aminolevulinic acid was added to a final concentration of 0.5 mM when the OD ₆₀₀ reached 0.4-0.6, and IPTG was added to a final concentration of 1 mM when the OD ₆₀₀ reached 0.8-0.9. The enzyme expression was induced by lowering the temperature at 25°C for 12 hours. After induction, the cells were collected by centrifugation at 7300 g for 6 minutes, and the reaction buffer (NaCl 86 mM, Na ₂ PO ₄ 93 mM, KH ₂ PO ₄ 15 mM, MgSO ₄ 7 mM, CaCl ₂ 0.1 mM, H _{3 BO 3 20 μM, CoCl 2 1.5} μM, CuSO 4 0.5 μM, MnCl 2 4.0 μM, ZnSO 4 0.5 μM, Na 2 MoO 4 1.0 μM, washed twice with pH 7.4), OD ₆₀₀ in the same buffer 7.0 Resuspended so that

<Benzene hydroxylation reaction>
Cell suspension 900 μL (final concentration 25 g/L wet cell weight), 2 M glucose solution 20 μL (final concentration 40 mM), 20 mM decoy substrate DMSO solution 5 μL (final concentration 100 μM), reaction buffer 65 μL and 10 μL of a 1 M benzene DMSO solution (final concentration 10 mM) were added to a 6 mL sample bottle and reacted at 25° C. and 200 rpm for 9 hours. After the reaction, the reaction solution was transferred to a 2 mL Eppendorf tube, frozen by liquid nitrogen to stop the reaction, thawed, and then the cells were disrupted by ultrasonication (Vibra Cell sonics VCX-750, AMPL 25%, 10 min). .. To the disrupted solution, 800 μL of dichloromethane and 10 μL of 40 mM 4-chlorotoluene DMSO solution as an internal standard were added and vortexed, followed by centrifugation at 20000 g for 3 minutes. Then, the organic layer was extracted and analyzed by gas chromatography. GC-2014 with a FID detector manufactured by Shimadzu was used as the gas chromatograph, and Rtx-1 column (internal diameter 0.53 mm, film thickness 3 μm, column length 60 m) manufactured by Restek was used as the column. Separation conditions were set so that the sample inlet temperature and detector temperature were 250°C, and the temperature program of the column oven was maintained at 120°C for 25 minutes, heated to 250°C over 5 minutes, and maintained for 15 minutes. .

<Results>
Table 1 shows the phenol concentration (μM) and the phenol conversion rate (%).

Example 2. Intracellular production of cresol (toluene hydroxylation reaction)
Cresol was produced intracellularly using the compound obtained in the synthesis example as a decoy substrate. Specifically, it was performed as follows.

<Preparation of cell suspension>
The same method as in Example 1 was used.

<Toluene hydroxylation reaction>
Cell suspension 900 μL (final concentration 25 g/L wet cell weight), 4 M glucose solution 10 μL (final concentration 40 mM), 20 mM decoy substrate DMSO solution 10 μL (final concentration 200 μM), reaction buffer 65 μL and 5 μL of a 4 M toluene DMSO solution (final concentration 20 mM) were added to a 6 mL sample bottle and reacted at 25° C. and 200 rpm for 9 hours. After the reaction, the reaction solution was transferred to a 2 mL Eppendorf tube, frozen by liquid nitrogen to stop the reaction, thawed, and then the cells were ultrasonically disrupted (Vibra Cell sonics VCX-750, AMPL 25%, 10 min). .. To the disrupted solution, 400 µL of dichloromethane and 10 µL of 40 mM 4-chlorotoluene DMSO solution as an internal standard were added, vortexed, and centrifuged at 20000 g for 3 minutes. Then, the organic layer was extracted and analyzed by gas chromatography. As the gas chromatograph, GC-2014 having a FID detector manufactured by Shimadzu was used, and as the column, an Rtx-1 column (internal diameter 0.53 mm, film thickness 3 μm, column length 60 m) manufactured by Restek was used. Separation conditions were set to sample inlet temperature and detector temperature of 250°C, and the temperature program of the column oven was maintained at 120°C for 25 minutes, raised to 250°C over 5 minutes, and maintained for 15 minutes. ..

<Results>
Most of the cresols detected were o-cresols. Table 2 shows o-cresol concentration (μM) and o-selectivity (%).

Example 3. In vitro production of phenol (hydroxylation of benzene)
Phenol was produced in vitro using the compound obtained in the synthesis example as a decoy substrate. Specifically, it was performed as follows.

<Preparation of cytochrome P450BM3 (CYP102A1)>
Escherichia coli BL21(DE3) strain, DNA encoding the full length of cytochrome P450BM3 (CYP102A1) (amino acid sequence, SEQ ID NO: 1) (CJC Whitehouse, SG Bell, LL Wong, Chem. Soc. Rev. 2012, 41, 1218-1260. PUCBM3 gene was expressed by incorporating a pUC vector into which a DNA encoding A1 in the alignment result of the CYP102A subfamily disclosed in Fig. 2 of Fig. 2 was inserted. In order to extract and purify the produced P450BM3 from E. coli, the E. coli was disrupted by sonication, the disrupted solution was centrifuged, the supernatant was collected, and this supernatant was once bound to the anion exchange column (DE-52). Elution was performed using a gradient of 0 to 250 mM KCl, and a brown peak fraction indicating P450BM3 was collected.

Check the purity of the eluted peak fractions by SDS-PAGE, mix the highly purified fractions, concentrate with an ultrafiltration kit with a molecular weight cut-off (MWCO) of 30,000, and then use buffer A (20 mM Tris It was desalted by diluting with -HCl (pH 7.4) to make the KCl concentration 5 mM or less. Then, using an anion exchange column (DEAE650S), KCl was subjected to a concentration gradient in the range of 0 to 1000 mM to elute BM3, and the same purification was performed. Finally, Buffer B (20 mM Tris-HCl (pH7.4), 100 mM KCl) was passed through a gel filtration column (Sephacryl S-300HR) to elute P450BM3, and the obtained brown peak fraction was collected, This was used as a wild-type P450BM3 sample in the following hydroxylation reaction.

<Benzene hydroxylation reaction>
Fill 90% or more of the total volume of the reaction solution with a buffer solution (20 mM Tris-HCl (pH7.4), 100 mM KCl) saturated with high-purity oxygen gas and fill it with 500 nM (final concentration) wild-type P450BM3, 10 mM (final concentration). (Concentration) benzene, 100 μM (final concentration) Each component was added so as to become a decoy substrate, and finally 5 mM NADPH was added to start the reaction. Since it was necessary to use a DMSO solution to dissolve the decoy substrate, the reaction solution consequently contained 0.5 v/v% DMSO.

The reaction was performed in a sealed vial, and the reaction solution was allowed to proceed at 25°C or lower while stirring. After reacting for 10 minutes, 0.15 mL of 1M hydrochloric acid was added to stop the reaction, and the mixture was stirred for 10 minutes. The reaction solution was neutralized with 0.15 mL of a 1 M aqueous sodium hydroxide solution, 1.3 mL of acetonitrile was added, and then filtered through a filter and analyzed by HPLC. LC-10ADVP manufactured by Shimadzu was used for HPLC, and a reversed-phase column Inertsil ODS-3 (inner diameter 4.6 mm, column length 250 mm, particle diameter 5 μm) manufactured by GL Sciences was used as the column. Buffer B/acetonitrile=1:1 was used as an eluent, and measurement was performed at a flow rate of 0.5 mL/min for 35 minutes.

In addition, the same operation was performed as a system in which only 0.5 v/v% DMSO was added instead of adding the decoy substrate (control example).

On the HPLC chromatogram of each reaction solution, a new peak not observed in the control example was observed. Since it was confirmed that the retention time of the new peak matches the retention time of phenol, it was found that phenol was produced as a reaction product in this reaction solution. The amount of phenol and the amount of NADPH consumed in each reaction solution were also measured.

<Results>
Table 3 shows the amount of phenol produced per minute (phenol production rate) with respect to the added decoy substrate, and the ratio of the NADPH consumption amount utilized for phenol production (coupling rate).

Example 4. Environmental purification test 1
Using the compound obtained in Synthesis Example as a decoy substrate, an environmental purification test was conducted. Specifically, it was performed as follows.

<Preparation of reaction solution>
E. coli suspension: An E. coli suspension in which P450BM3 was overexpressed by genetic engineering under the same conditions as Reference 1 (M. Karasawa et al., Angew. Chem. Int. Ed. 2018, 57, 12264-12269.). The liquid was prepared so that the amount of cells was constant (OD ₆₀₀ = 1.6, 3.2, 6.3, 12.6, 25.2, concentration at the time of reaction). Then, a glucose solution and a decoy substrate DMSO solution were added to the bacterial cell suspension to prepare an Escherichia coli suspension (glucose 40 mM, decoy substrate 100 μM, concentration at the time of reaction).

Benzene solution: A benzene DMSO solution was added to the buffer used for the reaction to prepare a benzene solution (10 mM, concentration at the time of reaction).

<Hydroxylation of benzene in model soil using decoy substrate and recombinant E. coli>
0.3 g of vermiculite per 1 mL of the reaction solution was added as a model soil to a 6 mL glass sample bottle. The prepared benzene solution was added to a sample bottle containing vermiculite, and then an Escherichia coli suspension was added (FIG. 1, left). At this time, it was confirmed that the reaction solution was adsorbed by vermiculite (Fig. 1, right).

After the reaction, 1000 μL of dichloromethane and 10 μL of 40 mM indan DMSO solution as an internal standard were added and vortexed, followed by centrifugation at 20000 g for 3 minutes. Then, the organic layer was extracted and analyzed by gas chromatography. As the gas chromatograph, GC-2014 having a FID detector manufactured by Shimadzu was used, and as the column, Rtx-1 (internal diameter 0.53 mm, film thickness 3 μm, column length 60 m) manufactured by Restek was used. Separation conditions were set to sample inlet temperature and detector temperature of 250°C, and the temperature program of the column oven was maintained at 120°C for 25 minutes, heated to 250°C over 5 minutes, and maintained for 15 minutes. ..

First, a calibration curve was prepared to quantify phenol in vermiculite. A phenol solution of each concentration was added to vermiculite, and extraction and analysis were performed under the same conditions as during the reaction. The prepared calibration curve showed good linearity, and it became clear that the amount of phenol in this reaction system can be quantified (Fig. 2).

<Result 1>
Hydroxylation of benzene was performed by changing the type of decoy substrate, the amount of benzene in the model soil, and the amount of cells. The results are shown in Tables 4-6.

Phenol was not observed at all under the condition that the decoy substrate was not added, whereas the amount of phenol produced was greatly increased by allowing E. coli to react with the decoy substrate. C7-L-Pro-L-Phe, which showed the highest activity in the reaction in the aqueous phase system, promoted benzene hydroxylation with high efficiency even in the soil model (Table 4). Furthermore, even when the amount of decoy group used was reduced to 1/10, the equivalent phenol conversion rate was obtained.

The conversion rate to phenol was similar regardless of the amount of benzene, suggesting that this system is effective even in the presence of benzene at a low concentration of 1 mM (Table 5).

The rate of conversion to phenol did not change significantly with changes in the cell mass. From this result, it was revealed that benzene in soil can be hydroxylated with a small amount of cells (Table 6).

<Result 2>
Next, the reaction time was changed and the hydroxylation reaction of benzene was performed. The cell amount was OD ₆₀₀ =6.3, the benzene concentration was 10 mM, and C7-Pro-Phe was used as the decoy substrate. Results are shown in FIG.

The conversion rate to phenol greatly increased due to the extension of the reaction time, and the conversion rate to phenol reached 28% in the reaction for 24 hours (Fig. 3).

Example 5. Environmental purification test 2
Hydroxylation of benzene in model soil was carried out using a giant strain standard strain. Specifically, it was performed as follows.

In the same manner as in Example 4, the benzene hydroxylation reaction was carried out in the same manner as in Example 4 except that the wild type Giant Bacteria standard strain ATCC14581 was used in place of the recombinant Escherichia coli introduced with the P450BM3 expression vector. Giant bacteria were cultivated in LB medium at 30° C. and 150 rpm under aerobic conditions, and the bacteria were collected and then resuspended in a reaction solution similar to that of E. coli. The cell amount was OD ₆₀₀ = 14.8 (about 50 g/L wet cell weight), and the reaction was carried out in vermiculite for 44 hours in the presence of benzene 5 mM and C7-Pro-Phe 100 μM. After the reaction, it was similarly extracted with dichloromethane and evaluated by gas chromatography.

The GC chromatograph after the reaction is shown in FIG. No phenol peak was observed in the absence of the decoy substrate, but addition of the decoy substrate resulted in the formation of phenol, reaching a conversion of 6%.

Claims (15)

General formula (1):
[In the formula, R ¹ represents an optionally substituted hydrocarbon group. R ² represents a single bond or a linker. R ³ is a single bond or formula (2):
(In the formula, the nitrogen atom is bonded to R ^2. )
Represents a group represented by. R ⁴ and R ⁵ represent the same or different amino acid side chains. R ⁶ represents a hydrogen atom, a hydroxyl group, an aldehyde group, a carboxy group, or a carboxy derivative group. However, R ⁵ and R ⁶ may combine with each other to form a lactone ring. n represents 0 or an integer of 1 or more. ]
An environmental purifying agent containing at least one selected from the group consisting of the compound represented by: and a cell containing the compound. The environmental cleaning agent according to claim 1, wherein R ⁶ is a carboxy group. The amino acid side chain is at least one selected from the group consisting of an optionally substituted alkyl group, alkenyl group, aryl group, aralkyl group, heteroaryl group, and heteroaralkyl group, 3. Environmental cleaning agent described in. The environmental purification agent according to claim 1, wherein R ¹ is an alkylaralkyl group, R ² and R ³ are single bonds, and n is 0. 5. The R ¹ is an alkyl group, the R ² is a single bond, the R ³ is a group represented by the formula (2), and n is 0, any one of claims 1 to 3. Environmental cleaning agent described in. The compound has the formula (1D):
The environmental cleaning agent according to claim 5, which is a compound represented by: The environmental purification agent according to claim 1, wherein the R ⁵ and the R ⁶ do not form a lactone ring. The environmental purification agent according to claim 1, wherein the R ⁵ and the R ⁶ are bonded to each other to form a lactone ring. The environmental cleaning agent according to claim 8, wherein the compound is quarmon. The environmental purification agent according to claim 1, wherein the cells are cells having cytochrome P450 monooxygenase. The environmental purification agent according to claim 1, wherein the cells are cells into which the compound is exogenously introduced. The environmental purification agent according to any one of claims 1 to 11, wherein the environment to be purified is at least one selected from the group consisting of environments in which microorganisms having cytochrome P450 are present. The environmental cleaning agent according to any one of claims 1 to 12, which is used for conversion of environmental pollutants. 14. The environmental cleaning agent according to claim 1, wherein the environmental pollutant is at least one selected from the group consisting of hydrocarbon pollutants and dioxins pollutants. An environmental purification method comprising a step of bringing the environmental purification agent according to any one of claims 1 to 14 into contact with an environment to be purified.