JP4802036B2 - Composition for alkylation and method for detoxifying harmful compounds using the composition - Google Patents

Description

  The present invention relates to a composition for alkylation and a method for detoxifying harmful compounds using the composition.

  Heavy metals such as arsenic, antimony, and selenium are widely used as industrial materials such as semiconductors. However, since they are toxic to living organisms, there are concerns about their impact on living organisms when they flow into the environment. Has been.

  Conventionally, as a method of removing these heavy metals, a flocculant such as polyaluminum chloride (PAC) is added to waste water containing inorganic arsenic such as toxic arsenous acid, and arsenic is agglomerated and adsorbed to the iron in the flocculant and raw water. Then, after precipitation, a method of removing by filtration, a method of adsorbing an arsenic compound or the like with activated alumina or a cerium-based adsorbent are generally known.

  On the other hand, in the natural world, it has become clear that marine organisms such as seaweed accumulate inorganic arsenic, and that part of the inorganic arsenic is converted into organic arsenic compounds such as dimethylated arsenic by physiological reactions (non- Patent Document 1). These organic arsenic compounds are generally known to exhibit lower toxicity to mammals than inorganic arsenic.

Kaiseet al., 1998, Organomet. Chem., 12 137-143

  However, in the method of removing heavy metals described above using filtration, adsorption, etc., the sludge containing harmful compounds such as inorganic arsenic that remains harmful, and the adsorbent on which the harmful compounds are adsorbed are used. It is necessary to store or landfill after sealing with concrete etc. so that the compound does not leak to the outside, and a large space for storage and landfill is required, so there is a problem that mass processing is difficult. It was.

  Further, even when inorganic arsenic is incorporated into the marine organisms described above, there is a problem that only a part of the incorporated inorganic arsenic becomes an organic arsenic compound, and harmful inorganic arsenic is still accumulated in the marine organisms.

  Therefore, in order to solve the above problems, the present invention provides a composition useful for efficiently and systematically detoxifying harmful compounds including arsenic and the like, and detoxifying harmful compounds using the composition It aims to provide a method.

  In order to achieve the above-mentioned object, the present inventors have obtained a harmful compound containing arsenic or the like by a chemical reaction using an organometallic complex having a cobalt-carbon bond. As a result of intensive studies on methylation, particularly dimethylation, more preferably trimethylation, and intensive studies on the methylation reaction of the harmful compounds, the present invention has been found.

That is, the alkylated composition of the present invention is characterized by containing an organometallic complex having a cobalt-carbon bond and an organic halogen compound .

  In a preferred embodiment of the alkylating composition of the present invention, a harmful compound containing at least one element selected from the group consisting of arsenic, antimony and selenium is alkylated by using the organometallic complex. It is characterized by that.

  In a preferred embodiment of the alkylated composition of the present invention, the composition further comprises a reducing agent that reduces at least one metal selected from the group consisting of arsenic, antimony, and selenium.

  In a preferred embodiment of the alkylation composition of the present invention, the reducing agent is a substance having an SH group.

  In a preferred embodiment of the alkylation composition of the present invention, the substance having an SH group is at least one selected from the group consisting of glutathione, reduced glutathione (GSH), cysteine, S-adenosylcysteine, and sulforaphane. It is characterized by being.

  In a preferred embodiment of the alkylation composition of the present invention, the composition further comprises a methylation additive having an S-Me group.

  In a preferred embodiment of the alkylation composition of the present invention, the methylation additive is at least one selected from the group consisting of methionine and S-adenosylmethionine.

  In a preferred embodiment of the alkylation composition of the present invention, the composition further comprises a buffer.

  In a preferred embodiment of the alkylated composition of the present invention, the pH of the buffer solution is in the range of 5-10.

  In a preferred embodiment of the alkylated composition of the present invention, the organic halogen compound is methyl halide.

In a preferred embodiment of the alkylated composition of the present invention, the methyl halide is at least one selected from the group consisting of methyl iodide, methyl bromide, and methyl chloride.

In a preferred embodiment of the alkylated composition of the present invention, the organic halogen compound is a halogenated acetic acid.
In a preferred embodiment of the alkylation composition of the present invention, the halogenated acetic acid is at least one selected from the group consisting of chloroacetic acid, bromoacetic acid and iodoacetic acid.
In a preferred embodiment of the alkylated composition of the present invention, the organic halogen compound is methyl chloride, methyl bromide, methyl iodide, chloroacetic acid, bromoacetic acid, iodoacetic acid, chloroethanol, bromoethanol, iodoethanol, It is at least one selected from the group consisting of chloropropionic acid, bromopropionic acid, iodopropionic acid, chloroacetic acid ethyl ester, bromoacetic acid ethyl ester, iodoacetic acid ethyl ester.
In a preferred embodiment of the alkylated composition of the present invention, the organic halogen compound is a Grignard reagent represented by the following chemical formula 1.
Chemical formula 1 RMgX
(In Chemical Formula 1, R = Me, CH ₂ COOH, or CH ₂ COOC ₂ H ₅ and X = Cl, Br, or I.)

  The detoxification method of the present invention contains at least one element selected from the group consisting of arsenic, antimony, and selenium in the presence of the composition according to any one of claims 1 to 16. It is characterized by detoxifying harmful compounds by alkylating.

  In a preferred embodiment of the detoxification method of the present invention, the detoxification is performed by setting the valence of the one element to a high oxidation number.

  In a preferred embodiment of the detoxification method of the present invention, at least one bond of the one kind of element is alkylated.

  In a preferred embodiment of the detoxification method of the present invention, the element is arsenic.

In a preferred embodiment of the detoxification method of the present invention, the 50% lethal dose (LD ₅₀ ) of the compound detoxified by the alkylation is 1000 mg / kg or more.

In a preferred embodiment of the detoxification method of the present invention, the compound detoxified by alkylation has a 50% cell growth inhibitory concentration (IC ₅₀ ) of 1000 μM or more.

  Further, in a preferred embodiment of the detoxifying method of the present invention, the harmful compound is arsenous acid, arsenic pentoxide, arsenic trichloride, arsenic pentachloride, arsenic sulfide compound, cyanoarsenic compound, chloroarsenic compound, and other It is selected from the group consisting of arsenic inorganic salts.

  In a preferred embodiment of the detoxification method of the present invention, the alkylation is methylation.

In a preferred embodiment of the detoxification method of the present invention, the harmful compound is converted into a dimethyl compound or a trimethyl compound by the methylation.

  Moreover, in a preferred embodiment of the detoxification method of the present invention, the dimethyl compound is dimethylarsonylethanol (DMAE), dimethylarsonyl acetate (DMAA), dimethylarsinic acid, or arsenosugar. .

  In a preferred embodiment of the detoxification method of the present invention, the trimethyl compound is an arsenocolin, an arsenobetaine, a trimethylarsenosugar or a trimethylarsine oxide.

  The alkylating composition of the present invention has an advantageous effect that it can easily and easily alkylate harmful compounds, particularly harmful compounds containing arsenic, antimony, selenium and the like. Further, according to the method of the present invention, harmful compounds can be made innocuous as much as possible, so that there is an advantageous effect that a large space such as a storage place is not required. In addition, according to the method of the present invention, since the living organism itself is not used as it is, an advantageous effect that unnecessary by-products are not generated is produced. Furthermore, according to the present invention, there is an advantageous effect that harmful inorganic arsenic can be reduced by a simple operation.

The alkylating composition of the present invention contains an organometallic complex having a cobalt-carbon bond. Here, the following can be illustrated as an example of the organometallic complex which has a cobalt-carbon bond. That is, methylcobalamin (or methylated vitamin B12, formal name: Coα- [α-5,6-dimethylbenz-1H-imidazol-1-yl-Coβ-methylcobamide] is preferably used. Also, vitamins such as cyanocobalamin are used. B12, cobalt (II) acetylacetonate, cobalt (III) acetylacetonate, cobalt carbonyl (dicobalt octacarbonyl), cobalt (II) 1, 1, 1, 5, 5, 5-hexafluoroacetylacetonate, cobalt (II) Meso-tetraphenylporphine, bis (pentamethylcyclopentadienyl) cobalt hexafluorophosphate, N, N′-bis (salicylidene) ethylenediaminecobalt (II), bis (2,2,6,6-tetra Methyl-3,5-heptanedionato) cobalt (II), (chlorophthalocyaninato) cobalt (II), chlorotris (triphenylphosphine) cobalt (I), methyl complex of cobalt acetate (II), cobalt (II) benzoate, cobalt (II) cyanide, cobalt (II) cyclohexanebutyrate, 2-ethylhexanoic acid Cobalt (II), meso-tetramethoxyphenylporphyrin cobalt (II), cobalt naphthenate, phthalocyanine cobalt (II), methyl cobalt (III) protoporphyrin IX, cobalt stearate, cobalt (II) sulfamate, (1R, 2R )-(-)-1,2-cyclohexanediamino-N, N'-bis (3,5-di-t-butylsalicylidene) cobalt (II), (1S, 2S)-(+)-1, 2-cyclohexanediamino-N, N'-bis (3,5-di-t-butylsalicylidene) cobalt (II), cyclopentadienylbis (triphenylphosphine) cobalt (I), cyclopentadi At least one selected from enyl cobalt dicarbonyl, dibromobis (triphenylphosphine) cobalt (II), (tetraaminochlorophthalocyaninato) cobalt (II), and (tetra-t-butylphthalocyaninato) cobalt (II) Or a cobalt-methyl complex formed by the coexistence of the cobalt compound and an alkyl halide, particularly a methyl halide, and a harmful compound containing harmful inorganic arsenic can be alkylated relatively easily. From the viewpoint of being able to make an organic substance with low toxicity, methylcobalamin is preferable as the organometallic complex having a cobalt-carbon bond.

  That is, in the alkylating composition of the present invention, by using the organometallic complex, it is possible to alkylate a harmful compound containing at least one element selected from the group consisting of arsenic, antimony, and selenium. It is. Here, in the present specification, the harmful compound means a compound that may flow into the environment and have some adverse effect on the organism when exposed to the organism.

Among the harmful compounds, harmful compounds containing arsenic include arsenous acid, arsenic pentoxide, arsenic trichloride, arsenic pentachloride, arsenic sulfide compounds, cyanoarsenic compounds, chloroarsenic compounds, and other arsenic inorganic salts. Be mentioned. These arsenic has an LD ₅₀ (mg / kg) (50% lethal dose in mice) of 20 or less, and is generally toxic to organisms.

  Examples of harmful compounds containing antimony include antimony trioxide, antimony pentoxide, antimony trichloride, and antimony pentachloride.

  Furthermore, examples of harmful compounds containing selenium include selenium dioxide and selenium trioxide.

  In a preferred embodiment, the composition of the present invention may further contain a reducing agent that reduces at least one metal selected from the group consisting of arsenic, antimony, and selenium. The presence of such a reducing agent can further promote alkylation. It is possible that the ability to reduce arsenic and the methyl transfer reaction may be rate-limiting in the conversion of arsenic to arsenobetaine, but the conversion to arsenobetaine etc. can be promoted by adding a reducing agent. it is conceivable that. Examples of such a reducing agent include substances having an SH group. Specifically, substances having an SH group include glutathione, reduced glutathione (GSH), cysteine, S-adenosylcysteine, Mention may be made of at least one selected from the group consisting of sulforaphanes.

  Moreover, in the preferable embodiment of the alkylation composition of this invention, the methylation addition factor which has S-Me group is contained further. If the methylation additive is present, more alkyl groups can be provided, and more alkylation and detoxification can be achieved. Examples of the methylation additive factor include at least one selected from the group consisting of methionine and S-adenosylmethionine.

  The alkylating composition of the present invention may contain a buffer. As the buffer, those usually used for isolating, purifying, and storing biomaterials can be used, and are not particularly limited. For example, Tris buffer, phosphate buffer, carbonate buffer, Examples of the buffer solution include a borate buffer solution. In addition, the pH of the buffer solution is preferably in the range of 5 to 10 in view of safer detoxification.

  The alkylated composition of the present invention can further contain an organic halogen compound. From the viewpoint of facilitating conversion from a dimethyl compound and / or a trimethyl compound to an arsenobetaine, an example of the organic halogen compound is methyl halide. Examples of the methyl halide include at least one selected from the group consisting of methyl iodide, methyl bromide, and methyl chloride from the viewpoint of high methylation reactivity.

In addition, examples of the organic halogen compound include at least one selected from the group consisting of iodoacetic acid, iodoethanol, bromoacetic acid, bromoethanol, and iodopropionacetic acid from the viewpoint of high alkylation reactivity.
In a preferred embodiment, the organic halogen compound is a halogenated acetic acid. Examples of the halogenated acetic acid include at least one selected from the group consisting of chloroacetic acid, bromoacetic acid, and iodoacetic acid.
In a preferred embodiment, the organic halogen compound includes methyl chloride, methyl bromide, methyl iodide, chloroacetic acid, bromoacetic acid, iodoacetic acid, chloroethanol, bromoethanol, iodoethanol, chloropropionic acid, bromopropionic acid. And at least one selected from the group consisting of iodopropionic acid, chloroacetic acid ethyl ester, bromoacetic acid ethyl ester, and iodoacetic acid ethyl ester.
Furthermore, in the present invention, the organic halogen compound has the following chemical formula 1:
Chemical formula 1 RMgX
(In Chemical Formula 1, R = Me, CH ₂ COOH, or CH ₂ COOC ₂ H ₅ , and X = Cl, Br, or I) may be used.

  Next, the detoxification method of the present invention will be described. That is, the harmful compound detoxification method of the present invention comprises a harmful compound containing at least one element selected from the group consisting of arsenic, antimony, and selenium in the presence of the above-described alkylating composition of the present invention. It is rendered harmless by alkylation. Here, the composition for alkylation and harmful compound of the present invention mean those described above, and can be applied as it is in the detoxification method of the present invention.

In a preferred embodiment of the detoxification method of the present invention, 50% cell growth inhibitory concentration (IC ₅₀ ) or LD ₅₀ is large, and from the viewpoint that more detoxification can be achieved, the value of the one kind of element contained in the harmful compound It is preferable to render the harmful compound harmless by setting the number to a high oxidation number. Specifically, the above-described composition of the present invention can be used as a catalyst for the reaction, and the valence of the one kind of element can be increased to a high oxidation number by alkylation. In the case where the element is arsenic or antimony, it is preferable that the trivalent valence is pentavalent, and in the case of selenium, the tetravalent valence is hexavalent.

  In the present invention, the harmful compound is detoxified by alkylating the harmful compound. Here, detoxification can be achieved by alkylating at least one bond of the one kind of element in the harmful compound.

  Specifically, by performing the reaction using the above-described alkylating composition of the present invention, at least one bond of the one kind of element can be alkylated. Here, examples of the alkyl group added to the one kind of element include a methyl group, an ethyl group, and a propyl group. From the viewpoint of achieving detoxification more efficiently, a methyl group is preferred as the alkyl group.

In the detoxification method of the present invention, from the viewpoint of safety to the living body, 50% lethal dose (LD ₅₀ ) of the compound detoxified by alkylation (oral dose at a drug dose at which 50% of mice die) Toxicity) is preferably 1000 mg / kg or more, and more preferably 5000 mg / kg or more.

In the detoxification method of the present invention, from the viewpoint of safety to the living body, the 50% cell growth inhibitory concentration (IC ₅₀ ) of the compound detoxified by the alkylation or arylation is 1000 μM or more. It is preferable that it is 3000 μM or more. Here, in this specification, 50% cell growth inhibitory concentration (IC ₅₀ ) is a numerical value indicating the concentration of a substance necessary to inhibit or inhibit the growth of 100 cells with a substance by 50%. means. A smaller IC ₅₀ value indicates greater cytotoxicity. IC ₅₀ was calculated from the results of examining the cytotoxicity of plasmid DNA damage under conditions of 37 ° C. and 24 hours.
Here, the IC ₅₀ of each arsenic compound is shown in Table 1.

  From Table 1, Arsenosugar with trivalent arsenic is more cytotoxic than monomethylated arsenic (MMA) and dimethylated arsenic (DMA), but with trivalent arsenic, MMA with trivalent arsenic. It is found that the cytotoxicity is lower than that of DMA and arsenous acid. On the other hand, MMA and DMA having trivalent arsenic have higher cytotoxicity than arsenous acid (trivalent and pentavalent), but from the viewpoint of cytotoxicity, arsenic compounds having pentavalent arsenic are generally used. It can be understood that the safety to living bodies is higher than that of an arsenic compound having trivalent arsenic.

Table 2 shows the LD ₅₀ of each arsenic compound.

  In the detoxification method of the present invention, the biological half-life of the compound detoxified by alkylation is preferably 8 hours or less from the viewpoint of safety to the living body. In the detoxification method of the present invention, it is preferable that the harmful compound is converted to a dimethyl compound or a trimethyl compound by the methylation from the viewpoint of safety and low toxicity. Examples of the dimethyl compound include dimethylarsonyl ethanol (DMAE), dimethylarsonyl acetate (DMAA), dimethylarsinic acid, and arsenosugar. Examples of the trimethyl compound include arsenocholine, arsenobetaine, trimethylarsenosugar, and trimethylarsine oxide.

Examples of the present invention will be described below. However, the following examples do not limit the scope of the present invention.
First, abbreviations used in the examples will be described as follows.
(Abbreviation)
iAs (III): inorganic trivalent arsenic MMA: monomethylarsonic acid DMA: dimethylcinnoic acid TMAO: trimethylarsine oxide AB: arsenobetaine (trimethylarsonium acetic acid)
DMAA: dimethylarsonium acetate MeCo: methylcobalamin GSH: glutathione (reduced form)
iSe (IV): Inorganic selenium (tetravalent)
MIAA: Monoiodoacetic acid

Example 1
[Reaction scheme]
MeCo
iAs (III) → MMA
GSH

(Reaction operation)
To a 1.5 mL Eppendorf tube, 740 μL of reaction buffer (20 mM Tris-HCl (pH 7.6)) was added. To this, 220 μL of 100 mM GSH aqueous solution was added, stirred with Voltex for 30 seconds, and allowed to stand at 37 ° C. for 30 minutes. To this, 20 μL of 100 ppm inorganic arsenic (III) standard solution (for atomic absorption) was added and stirred for 30 seconds. To this, 20 μL of a 7.4 mM methylcobalamin (MeCo) aqueous solution was added (Composition A). This was reacted in a thermostat kept at 37 ° C., and periodically sampled to follow the increase in product.

(Analysis of product)
Qualitative and quantitative analysis is performed by comparing retention times of standard samples and reactive organisms with an inductively coupled plasma ion mass spectrometer (Agilent 7500ce) directly connected to a high performance liquid chromatograph (Agilent 1100). It was.

Example 2
[Reaction scheme]

MeCo
iAs (III) → MMA
GSH, iSe (IV)

(Reaction operation)
The same procedure as in Example 1 was performed except that 20 μL of 1000 ppm inorganic Se (IV) standard solution (for atomic absorption) was added to the composition A of Example 1 (Composition B).

Comparative Example 1
In Example 1, it carried out like Example 1 except not adding MeCo (composition C). Table 3 shows the detoxification of inorganic arsenic to MMA (Example 1) and the detoxification of inorganic arsenic to DMA (Example 2).

実施例１〜２に示したように、比較例に比べて、メチル化ヒ素（ＭＭＡ）が時間の経過とともに生成した。実施例２では、更にメチル化が進行し、ジメチル化ヒ素（ＤＭＡ）の生成も確認した。ＭｅＣｏ存在下で、有毒な無機ヒ素が、毒性の低いメチル化ヒ素に無毒化される顕著な効果を確認した。

As shown in Examples 1 and 2, methylated arsenic (MMA) was produced over time as compared to the comparative example. In Example 2, methylation further progressed, and formation of dimethylated arsenic (DMA) was also confirmed. In the presence of MeCo, toxic inorganic arsenic was confirmed to have a remarkable effect of detoxification to less toxic methylated arsenic.

Example 3
[Reaction scheme]

MeCo
MMA → DMA
GSH

(Reaction operation)
The same procedure as in Example 2 was performed except that 20 μL of 1000 ppm of MMA was added to the composition B of Example 2 (Composition D).

Comparative Example 2
Example 3 was carried out in the same manner as Example 3 except that MeCo was not added.

Example 4
[Reaction scheme]

MeCo
DMA → TMAO
GSH

(Reaction operation)
The same procedure as in Example 2 was performed except that 20 μL of 1000 ppm of DMA was added to the composition B of Example 2 (Composition E).

実施例３−１〜３−３に示したように、ジメチル化ヒ素（DＭＡ）の濃度が時間の経過とともに増大した。比較例２はでは、DMAの生成は観測されなかった。実施例４−１〜４−３に示したように、トリメチル化ヒ素（ＴＭＡO）の濃度が増加しており、ヒ素基質が、最も無害なトリメチル化ヒ素に変換されていることがわかった。比較例３では、トリメチル化ヒ素の生成は観測されなかった。 Comparative Example 3
In Example 4, it carried out like Example 4 except not adding MeCo. Table 2 shows the detoxification of MMA to DMA (Example 3) and the detoxification of DMA to TMAO (Example 4).

As shown in Examples 3-1 to 3-3, the concentration of dimethylated arsenic (DMA) increased with time. In Comparative Example 2, no DMA was observed. As shown in Examples 4-1 to 4-3, the concentration of trimethylated arsenic (TMAO) was increased, and it was found that the arsenic substrate was converted to the most harmless trimethylated arsenic. In Comparative Example 3, formation of trimethylated arsenic was not observed.

Example 5
[Reaction scheme]

MeCo, MIAA
TMAO → AB
GSH
(Reaction operation)
In the composition B of Example 2, it carried out like Example 2 except having added 20 microliters of 1000 ppm TMAO instead of inorganic arsenic (composition F).

Example 6
[Reaction scheme]

MIAA
TMAO → AB
GSH

(Reaction operation)
The same procedure as in Example 5 was performed except that MeCo was not added to the composition F of Example 5 (Composition G).

Example 7
[Reaction scheme]

MeCo, MIAA
DMA → AB
GSH
(Reaction operation)
The same procedure as in Example 5 was performed except that DMA was used in place of TMAO in the composition F of Example 5 (Composition G). Table 3 shows the conversion to AB.

実施例５に示したように、MeCoとMIAA存在下で、ヒ素基質であるTMAOからABの変換が確認された。実施例６に示したように、MIAA存在下のみでもTMAOからABの変換が確認された。実施例７に示したように、MeCoとMIAA存在下でヒ素基質であるDMAからABの変換が確認された。

As shown in Example 5, the conversion of AB from arsenic substrate TMAO was confirmed in the presence of MeCo and MIAA. As shown in Example 6, conversion of TMAO to AB was confirmed only in the presence of MIAA. As shown in Example 7, conversion of DMA as an arsenic substrate to AB was confirmed in the presence of MeCo and MIAA.

Example 8
First, abbreviations used in the following examples will be described as follows.
(Abbreviation)
iAs (III): inorganic trivalent arsenic,
MMA: Monomethylarsonic acid DMA: Dimethylcinnamate TMAO: Trimethylarsine oxide AB: Arsenobetaine (trimethylarsonium acetic acid)
DMAA: dimethylarsonium acetate MeCo: methylcobalamin GSH: glutathione (reduced form)
MIAA: Monoiodoacetic acid AS: Arsenosugar iSe (IV): Inorganic selenium (tetravalent)

(1) Microalgae culture The microalgae Chlorella (Chlorella vulgaris IAM C-629 strain) pre-cultured until the logarithmic growth phase was inoculated into 150 ml of Bold's Basal (BB) Medium at 1 × 10 ⁶ cells / ml, Under a fluorescent lamp irradiation (4000 Lux, 24 hr irradiation), the culture was allowed to stand at a temperature of 25 ° C. Under the present circumstances, the culture medium which added 10 mM glucose or 10 mM sodium acetate as a carbon source was adjusted to the culture solution.

(2) Arsenic uptake test Arsenic uptake test was carried out by adding arsenic acid to 1 ppm as metal arsenic after inoculation and culturing for 284 hours after addition of arsenic.

(3) Arsenic content measurement Inorganic and organic arsenic in algal cells are reacted with a standard sample using an inductively coupled plasma ion mass spectrometer (Agilent 7500ce) directly connected to a high performance liquid chromatograph (Agilent 1100). Qualitative and quantitative analysis was performed by comparing the retention time of sex organisms.

(4) Analytical conditions MMA, DMA, TMAO, TeMA, AB and AC are used as standard samples of organic arsenic compounds, and reagents of Trichemical Laboratories are used as standard samples of inorganic arsenic. ) Wako Pure Chemicals special grade sodium salt. A standard solution of 100 mg / 100 mL of each arsenic compound was prepared by diluting with ultrapure water (Millipore).

ICP-MS equipment conditions

RF forward
power: 1.6kW
RF reflect
power: <1W
Carrier gas flow: Ar 0.75L / min
Sampling 8.5mm
Monitoring mass: m / z = 75 and 35
internal standard m / Z = 71
Dwell time: 0.5 sec
0.01sec 0.1sec
Times of scan: 1 time

HPLC condition eluent: 5 mM nitric acid / 6 mM ammonium nitrate / 1.5 mM pyridinedicarboxylic acid Eluent flow rate: 0.4 mL / min Injection volume: 20 μL
Column: Cation exchange column Shodex RSpak NN-614 (150mm × 4.6mm id)
Column temperature: 40 degrees

[Extraction of arsenic compounds from microalgae incorporating arsenic]
A microalgae extract (Chlorella was extracted with methanol and methanol was removed by evaporation) was prepared. This was diluted by adding pure water to obtain a solution having the concentration shown in the table below. In addition, the components of UN1 (Unknown1) and UN6 were assigned as compounds corresponding to Arsenosugar (FIG. 1). FIG. 1 shows the state of HPLC-ICP-MS analysis (upper: standard sample, lower: sample) of a chlorella extract. Table 6 shows the arsenic compound concentration (ppm) of the chlorella extract.

〔ＡＢへの変換〕
容量１．５ｍＬのエッペンドルフチューブに、反応緩衝液（20mM Tris-HCl ( pH7.6 )）７４０μＬを添加した。これに、２０ｍＭのＧＳＨ水溶液を２００μＬを添加して３０秒間、Ｖｏｌｔｅｘで攪拌して、３０分間３７℃で静置した。前記クロレラ抽出液１００μＬを加え３０秒間攪拌した。これに、７．４ｍＭメチルコバラミン（ＭｅＣｏ）水溶液を１３５μＬを添加した。これに、ＭＩＡＡを６８ｍｇ（０．３５μＭ）加えて溶解した。これを３７℃に保持した恒温槽中で反応し、定期的にサンプリングして生成物の増加量を追跡した。図２に示すように、ＧＳＨ、ＭｅＣｏ、ＭＩＡＡが存在する場合に、ＡＢの生成が確認できた。
図２．クロレラ抽出物のＨＰＬＣ−ＩＣＰ−ＭＳ分析（上：標準サンプル、中：ＧＳＨ添加（NE_14-7）、下：ＭｅＣｏ＋ＧＳＨ＋ＭＩＡＡ添加（NE_15-7））

[Conversion to AB]
To a 1.5 mL Eppendorf tube, 740 μL of reaction buffer (20 mM Tris-HCl (pH 7.6)) was added. To this, 200 μL of 20 mM GSH aqueous solution was added, stirred with Voltex for 30 seconds, and allowed to stand at 37 ° C. for 30 minutes. 100 μL of the chlorella extract was added and stirred for 30 seconds. To this, 135 μL of a 7.4 mM methylcobalamin (MeCo) aqueous solution was added. To this, 68 mg (0.35 μM) of MIAA was added and dissolved. This was reacted in a thermostat kept at 37 ° C., and periodically sampled to follow the increase in product. As shown in FIG. 2, the generation of AB was confirmed when GSH, MeCo, and MIAA existed.
FIG. HPLC-ICP-MS analysis of chlorella extract (top: standard sample, middle: GSH added (NE_14-7), bottom: MeCo + GSH + MIAA added (NE_15-7))

Example 9
Alkaline MeCO, MIAA
AS → DMA → AB
GSH

[Conversion from Arseno Sugar to DMA]
100 μL of the chlorella extract was mixed with 4N NaOH aqueous solution (1 mL) and allowed to stand at 80 ° C. overnight. It was confirmed that DMA was generated and that Arsenosugar was converted to DMA (FIG. 3).

[Conversion from DMA to AB]
Performed in the same manner as described in Examples 1-7. FIG. 3 shows the addition of GSH (NE_14-4) and GSH + MeCo + MIAA (NE_15-4) to the chlorella extract by NaOH treatment (bottom). FIG. 4 shows addition of GSH + MeCo + MIAA (NE_9-4) to DMA.

The composition of the present invention can provide a more practical and industrial method of detoxifying harmful compounds that can contribute to detoxification of harmful compounds including arsenic. Harmful compounds such as alkylated arsenic are converted into more harmless compounds, which are extremely stable and safe, so they are widely used in the fields of industrial waste treatment, sludge and soil environmental protection. Is extremely effective.

FIG. 1 is a diagram showing HPLC-ICP-MS analysis (upper: standard sample, lower: sample) of a chlorella extract. FIG. 2 is a diagram showing HPLC-ICP-MS analysis (upper: standard sample, middle: GSH added (NE — 14-7), lower: MeCo + GSH + MIAA added (NE — 15-7)) of the chlorella extract. FIG. 3 is a view showing a state in which GSH addition (NE_14-4) and GSH + MeCo + MIAA addition (NE_15-4) are treated with NaOH (below) in the chlorella extract. FIG. 4 is a diagram showing a state in which GSH + MeCo + MIAA is added to the DMA (NE_9-4).

Claims (26)

An alkylating composition comprising an organometallic complex having a cobalt-carbon bond and an organic halogen compound .   The composition according to claim 1, wherein a harmful compound containing at least one element selected from the group consisting of arsenic, antimony, and selenium is alkylated by using the organometallic complex.   The composition according to claim 1, further comprising a reducing agent that reduces at least one metal selected from the group consisting of arsenic, antimony, and selenium.   The composition according to claim 3, wherein the reducing agent is a substance having an SH group.   The composition according to claim 4, wherein the substance having an SH group is at least one selected from the group consisting of glutathione, reduced glutathione (GSH), cysteine, S-adenosylcysteine, and sulforaphane.   Furthermore, the composition of any one of Claims 1-5 containing the methylation addition factor which has a S-Me group.   The composition according to claim 6, wherein the methylation additive is at least one selected from the group consisting of methionine and S-adenosylmethionine.   Furthermore, the composition of any one of Claims 1-7 containing a buffer solution.   The composition according to claim 8, wherein the pH of the buffer solution is in the range of 5 to 10. The composition according to claim 1 , wherein the organic halogen compound is methyl halide. The composition according to claim 10 , wherein the methyl halide is at least one selected from the group consisting of methyl iodide, methyl bromide, and methyl chloride. The composition according to claim 1 , wherein the organic halogen compound is a halogenated acetic acid. The composition according to claim 12 , wherein the halogenated acetic acid is at least one selected from the group consisting of chloroacetic acid, bromoacetic acid, and iodoacetic acid. The organic halogen compound is methyl chloride, methyl bromide, methyl iodide, chloroacetic acid, bromoacetic acid, iodoacetic acid, chloroethanol, bromoethanol, iodoethanol, chloropropionic acid, bromopropionic acid, iodopropionic acid, ethyl chloroacetate. The composition according to any one of claims 1 to 13, which is at least one selected from the group consisting of ester, bromoacetic acid ethyl ester, and iodoacetic acid ethyl ester. The composition according to any one of claims 1 to 14 , wherein the organic halogen compound is a Grignard reagent represented by Chemical Formula 1.

Chemical formula 1 RMgX
(In Chemical Formula 1, R = Me, CH ₂ COOH, or CH ₂ COOC ₂ H ₅ and X = Cl, Br, or I.) Harmless by alkylating a harmful compound containing at least one element selected from the group consisting of arsenic, antimony, and selenium in the presence of the composition according to any one of claims 1 to 15. A method for detoxifying the harmful compound. The method according to claim 16 , wherein the valence of the one element is made harmless by setting it to a high oxidation number. The method according to claim 16 or 17, wherein at least one bond of the one element is alkylated. The method according to claim 16 , wherein the element is arsenic. The method according to any one of claims 16 to 19 , wherein a 50% lethal dose (LD ₅₀ ) of the compound detoxified by the alkylation is 1000 mg / kg or more. The method according to any one of claims 16 to 20 , wherein a 50% cell growth inhibitory concentration (IC ₅₀ ) of the compound detoxified by the alkylation is 1000 µM or more. The harmful compound is arsenic trioxide, pentoxide arsenic, arsenic trichloride, five arsenic chloride, arsenic sulfide compound, cyano arsenic compound, chloro arsenic compound, and claim 16 is selected from the group consisting of other arsenic inorganic salts 22. A method according to any one of items 21 . The method according to any one of claims 16 to 22 , wherein the alkylation is methylation. The method according to claim 23 , wherein the harmful compound is converted to a dimethyl compound or a trimethyl compound by the methylation. The method according to claim 24 , wherein the dimethyl compound is dimethylarsonyl ethanol (DMAE), dimethylarsonyl acetate (DMAA), dimethylarsinic acid, or arsenosugar. The method according to claim 24 , wherein the trimethyl compound is arsenocholine, arsenobetaine, trimethylarsenosugar or trimethylarsine oxide.

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