CN102757395A - Preparation method of biochemical detection reagent of chlorinated tetrazolium salts - Google Patents

Abstract

The invention relates to a preparation method of a tetrazolium chloride salt biochemical detection reagent, that is, formazan is used as a reactant and a hydrogen chloride/hydrogen peroxide system is used to oxidize formazan into tetrazolium chloride salt. The preparation method is as follows: 1) dissolving formazan in concentrated hydrochloric acid/methanol mixture, 2) adding hydrogen peroxide solution dropwise under the condition that the dropping temperature ranges from -10°C to 15°C. , stirred at room temperature, 3) Rotary evaporation to remove methanol, water-insoluble organic solvent extraction and drying, adding anhydrous ether to precipitate the crude product, mixed solvent recrystallization to obtain tetrazolium chloride fine product. The method of the invention finally improves the yield, is simple to handle, has no heavy metal residue in the product, and makes the preparation process green and technical.

Description

A kind of preparation method of tetrazolium chloride salt biochemical detection reagent

technical field

The invention relates to a tetrazolium chloride salt compound, which can be used in the fields of cell proliferation detection, dehydrogenase detection and the like.

Background technique

Tetrazolium chloride salt is an organic salt with a five-membered tetrazole ring structure, in which the substituents on the ring are mostly benzene rings and substituted benzene rings, and the anion is chloride ion. These compounds are colorless solids, and their solutions only absorb in the ultraviolet region. Because their redox potential is similar to that in biological systems, they can generate formazan compounds under the action of cellular dehydrogenases. ), resulting in absorption in the visible region. Therefore, it is often used as a coloring agent for biological cells to qualitatively observe and quantitatively detect the activity of microorganisms and cells, and because tetrazolium salts and formazan are non-toxic, they can be directly used in the study of microorganisms or mammalian active tissues. Chemical, histochemical, and clinical analysis and diagnosis have been widely used, such as anti-HIV and anti-tumor drug testing, cell proliferation detection, dehydrogenase detection, plant selection, etc.

Commonly used commercial tetrazolium chloride salt structures can be divided into two categories: single tetrazole ring and double tetrazole ring, as shown in the following structures respectively:

The principle of action is that dehydrogenases can transfer electrons from substrates to oxidizing reagents or electron bodies. Tetrazolium salts are used as hydrogen acceptors. In the reaction, dehydrogenase transfers the hydrogen atom of the substrate to tetrazolium salts to form colored formazan. The schematic diagram is as follows:

It is found through technical retrieval that the prior art for preparing tetrazolium chloride salts is prepared by one-step reaction using formazan as a reactant, and the reactant formazan can be synthesized by conventional methods. The reaction formula is as follows:

The conventional method for preparing tetrazolium chloride salt from formazan one-step reaction has: lead tetraacetate oxidation method [Chem.Revs, 1955 (55): 355-483], this method uses lead tetraacetate as oxidant, formsformazolium in the presence of hydrogen chloride Oxidation of tetrazolium chloride to tetrazolium chloride salt, because lead tetraacetate itself is reduced to divalent lead ion, there is a shortcoming of heavy metal residue in the tetrazolium chloride salt product, which will produce toxicity when used in live cell or tissue detection. And this method produces more lead-containing wastewater.

Isopentyl nitrite oxidation method [Talanta, 44 (1997), 1299-1305], this method usually is to reflux formazan with isopentyl nitrite in acetic acid solvent, and the reaction temperature is high, because formazan is at high temperature Unstable, partially decomposed, resulting in more by-products, thus having the disadvantages of complex post-treatment and poor yield of tetrazolium chloride salt products.

Tert-butyl hypochlorite oxidation method [J Am Chem Soc, 1954, 76 (6): 1695-1695], this method is to dissolve formazan in the mixed solvent of dioxane and glacial acetic acid, reflux after heating, and cool Precipitate jelly, add chloroform to dissolve and then add acetone to precipitate product. The disadvantage of this method is that the mixed solvent of dioxane and glacial acetic acid is used in a large amount, which is difficult to separate and recover. The precipitated jelly sticks to the wall in the kettle, which is difficult to handle. Suitable for industrial production.

Chlorine oxidation method [Chinese Journal of Pharmaceutical Industry, 2006,37 (1): 10-11], this method is to pass into chlorine gas in the methanol solution of formazan to prepare, the shortcoming of this method is that chlorine gas will be in tetrazole in reaction process Aromatic ring chlorinated by-products are produced on the aromatic ring substituents of the salt, and the purification is difficult and the yield is low.

Contents of the invention

Purpose of the invention: the problem to be solved by this invention is to overcome the deficiencies in the existing background technology, and provide a kind of preparation method of new tetrazolium chloride salt, promptly take formazan as reactant, adopt hydrogen chloride/hydrogen peroxide system to formazan Oxidation to tetrazolium chloride salt. Finally, the yield is improved, the treatment is simple, there is no heavy metal residue in the product, and the preparation process is green and technological.

Technical scheme: the preparation method of tetrazolium chloride salt biochemical detection reagent of the present invention is specifically:

1) Dissolve formazan in concentrated hydrochloric acid/methanol mixture,

2) Under the condition that the dropping temperature ranges from -10°C to 15°C, add aqueous hydrogen peroxide solution dropwise, after the dropwise addition, stir at room temperature,

3) Rotary evaporation to remove methanol, water-insoluble organic solvent extraction and drying, adding anhydrous ether to precipitate crude product, mixed solvent recrystallization to obtain tetrazolium chloride fine product.

In the step 1), the weight ratio of formazan to methanol is between 1:10 and 1:20, the volume ratio of concentrated hydrochloric acid to methanol is between 1:15 and 1:30, and the mixture is stirred and dissolved at room temperature.

The weight percentage concentration of the hydrogen peroxide aqueous solution added dropwise in the step 2) is 30%~50%, after the dropwise addition is completed, stir at room temperature for 1~8 hours.

The water-insoluble organic solvent in step 3) is chloroform, and diethyl ether is added dropwise until no precipitation occurs. The mixed solvent is methanol/diethyl ether, the volume ratio is between 1:1 and 1:10, and it is vacuum-dried at 25°C for 24 hours.

Described this method is applicable to the preparation of the tetrazolium chloride salt with following structural formula:

Wherein, R ₁ is phenyl, naphthyl, 4-nitrophenyl, R ₂ is phenyl, 4-nitrophenyl, R ₃ is phenyl, 4-nitrophenyl,

Described this method is applicable to the preparation of following tetrazolium chloride salt, and its structural formula is as follows:

Beneficial effects: the present invention adopts the hydrogen chloride/hydrogen peroxide system, the formazan is completely oxidized, the hydrogen peroxide is reduced to water, and the chloride negative ions of hydrogen chloride are transferred to the structure of the product tetrazolium chloride salt. In the reaction process, no other by-products are produced except tetrazolium chloride salt products, so as to achieve the purpose of green production, and since no by-products are produced, post-reaction treatment is simple and the yield is high. The disadvantages of the prior art, such as many by-products, complicated treatment, low yield and heavy metal residues in the product, are overcome.

Detailed ways

The following examples will help to understand the present invention, but not limit the content of the present invention.

The present invention implements with reference to following route:

Example 1:

Preparation of 2-p-iodophenyl-3-p-nitrophenyl-5-phenyltetrazolium chloride

Put 4.80g of formazan in a 150ml four-neck flask, add 50ml of methanol, then add 2.5ml of concentrated hydrochloric acid, stir to dissolve, and cool to 0°C. In the range of 0°C to 10°C, add 5ml of 30% hydrogen peroxide solution dropwise, after the dropwise addition, stir at room temperature for 3 hours, remove methanol by rotary evaporator, then extract with chloroform (3×30ml), dry over anhydrous magnesium sulfate After that, 30ml of anhydrous diethyl ether was added dropwise to precipitate a crude product, which was then recrystallized with methanol/diethyl ether (1:2), filtered, and vacuum-dried at 25°C for 24 hours to obtain 4.50 g of tetrazolium chloride salt crystals with a yield of 91%.

Example 2:

Preparation of 2,5-diphenyl-3-(1-naphthyl)tetrazolium chloride

Put 3.51g of formazan in a 150ml four-neck flask, add 50ml of methanol, and then add 2.5ml of concentrated hydrochloric acid, stir to dissolve, and cool to 0°C. In the range of 0°C to 10°C, add 5ml of 30% hydrogen peroxide solution dropwise, after the dropwise addition, stir at room temperature for 5 hours, remove methanol by rotary evaporation, then extract with chloroform (3×30ml), dry over anhydrous magnesium sulfate , dropwise added 30ml of anhydrous diethyl ether to precipitate a crude product, then recrystallized the crude product with methanol/diethyl ether (1:2), filtered, and vacuum-dried at 25°C for 24 hours to obtain 3.55 g of tetrazolium chloride salt crystals, with a yield of 93 %.

Example 3:

Preparation of 3,3'-dimethoxy-4,4'-diphenylene-3",3"'-bis(2-p-nitrophenyl-5-phenyltetrazolium chloride)

Put 8.17g of formazan into a 250ml four-necked flask, add 100ml of methanol, and then add 2.5ml of concentrated hydrochloric acid, stir to dissolve, and cool to 0°C. In the range of 0°C to 10°C, add 5ml of 30% hydrogen peroxide solution dropwise, after the dropwise addition, stir at room temperature for 8 hours, remove methanol by rotary evaporation, then extract with chloroform (3×50ml), dry over anhydrous magnesium sulfate , dropwise added 60ml of anhydrous diethyl ether to precipitate a crude product, then recrystallized the crude product with methanol/diethyl ether (1:1.5), filtered, and vacuum-dried at 25°C for 24 hours to obtain 7.11 g of tetrazolium chloride salt crystals, with a yield of 87 %.

Claims (6)

1. the preparation method of a tetrazolium chloride salt biochemistry detection reagent is characterized in that this preparation method is specially:

1) is dissolved in the Jia Za in concentrated hydrochloric acid/methyl alcohol mixed liquor,

2) under the dropping temperature scope is-10 ℃ ~ 15 ℃ condition, drip aqueous hydrogen peroxide solution, after dropwising, stirring at room,

3) rotary evaporation is removed methyl alcohol, and water-insoluble organic solvents is extracted after drying, adds anhydrous diethyl ether and separates out bullion, and the mixed solvent recrystallization obtains tetrazolium chloride salt elaboration.

2. the preparation method of tetrazolium chloride salt biochemistry detection reagent according to claim 1; The weight ratio that it is characterized in that described step 1) Zhong Jia Za and methyl alcohol is between 1:10 ~ 1:20; Concentrated hydrochloric acid and methyl alcohol volume ratio are between 1:15 ~ 1:30, stirring and dissolving under the room temperature.

3. the preparation method of tetrazolium chloride salt biochemistry detection reagent according to claim 1 is characterized in that described step 2) the aqueous hydrogen peroxide solution weight percent concentration that drips is 30% ~ 50%, after dropwising, stirring at room 1 ~ 8 hour.

4. the preparation method of tetrazolium chloride salt biochemistry detection reagent according to claim 1 is characterized in that water-insoluble organic solvents described in the described step 3) is a chloroform, till dropping ether amount is separated out with the nothing deposition.Said mixed solvent is a methanol, and volume ratio is between 1:1 ~ 1:10, and vacuum-drying is at 25 ℃, 24 hours.

5. the preparation method of tetrazolium chloride salt biochemistry detection reagent according to claim 1 is characterized in that described this method is applicable to the preparation of the tetrazolium chloride salt with following general structure:

Wherein, R ₁Be phenyl, naphthyl, 4-nitrophenyl, R ₂Be phenyl, 4-nitrophenyl, R ₃Be phenyl, 4-nitrophenyl,

6. the preparation method of tetrazolium chloride salt biochemistry detection reagent according to claim 1 is characterized in that described this method is applicable to the preparation of following tetrazolium chloride salt, and its structural formula is following: