CN102757395A - Preparation method of biochemical detection reagent of chlorinated tetrazolium salts - Google Patents
Preparation method of biochemical detection reagent of chlorinated tetrazolium salts Download PDFInfo
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- CN102757395A CN102757395A CN2012102278084A CN201210227808A CN102757395A CN 102757395 A CN102757395 A CN 102757395A CN 2012102278084 A CN2012102278084 A CN 2012102278084A CN 201210227808 A CN201210227808 A CN 201210227808A CN 102757395 A CN102757395 A CN 102757395A
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Abstract
The invention relates to a preparation method of a biochemical detection reagent of chlorinated tetrazolium salts. In the preparation method, formazan is used as a reactant and is oxidized into a chlorinated tetrazolium salt by adopting a hydrogen chloride/hydrogen peroxide system. The preparation method specifically comprises the following steps of: 1) dissolving formazan into the concentrated hydrochloric acid/methanol mixed liquid, 2) dropwise adding aqueous hydrogen peroxide solution under the condition that the dropwise adding temperature range is minus 10 DEG C to 15 DEG C, and stirring at the room temperature after dropwise adding, and 3) removing methanol by rotary evaporation, extracting with water-insoluble organic solvent and drying, adding anhydrous ethyl ether to separate out a crude product, and recrystallizing the mixed solvent to obtain the chlorinated tetrazolium salt refined product. The preparation method provided by the invention has the advantages of improving the yield at last, being simple in treatment, having no heavy metal residual in the product and being green in the preparation process.
Description
Technical field
The present invention relates to a kind of tetrazolium chloride salt compounds, this compounds can be used as fields such as cell proliferation detection, desaturase detection.
Background technology
Tetrazolium chloride salt is the organic salt with five yuan of tetrazole ring structures, and wherein the substitution in ring base is phenyl ring and substituted benzene ring mostly, and negatively charged ion is a cl ions.This compounds is colorless solid; Its solution only has absorption in the ultraviolet region; Because their oxidation-reduction potential is close with the oxidation-reduction potential value in the biosystem, under the effect of cell desaturase, can generate Jia Za compounds (formazan), produce absorption at visible region.Therefore often be used as the tinting material of biomass cells; In order to qualitative observation and detection by quantitative mikrobe and cell activity; And because of tetrazolium salts Ji the Jia Za is nontoxic; Can directly be used for the research of mikrobe or mammiferous active mass, in biological chemistry, histological chemistry and clinical analysis diagnosis, obtain application widely, detect, detect desaturase, plant selection etc. like anti-HIV and antitumor drug test, cell proliferation.
Business-like tetrazolium chloride salt structure commonly used can be divided into monotetrazolium ring and two big types of two tetrazole rings, respectively shown in structure:
Its action principle, desaturase can transfer an electron to from substrate on oxidising agent or the electron.Tetrazolium salts is transferred to the substrate Wasserstoffatoms tetrazolium salts and is formed colored first Za by desaturase in reaction as hydrogen acceptor, and synoptic diagram is following:
Find that through the technology retrieval prior art of preparation tetrazolium chloride salt all is that Yi Jia Za is that reactant prepares through single step reaction, and anti-Wu Jia Za of answering can be synthetic by ordinary method.Reaction formula is following:
The ordinary method that Cong Jia Za single step reaction prepares tetrazolium chloride salt has: lead tetra-acetate oxidation style [Chem.Revs; 1955 (55): 355-483]; This method as oxygenant, is oxidized to tetrazolium chloride salt Jiang the Jia Za with lead tetra-acetate in the presence of hydrogenchloride, make the shortcoming that has heavy-metal residual in the tetrazolium chloride product salt owing to lead tetra-acetate itself is reduced to lead; Meeting toxigenicity when being used for viable cell or tissue detection, and this method generation lead waste water is more.
Isopentyl nitrite oxidation style [Talanta, 44 (1997), 1299-1305]; This method normally Jiang the Jia Za with reflux in acetate solvate with Isopentyl nitrite; Temperature of reaction is high, because the Jia Za is at high temperature unstable, part is decomposed; Cause by product more, aftertreatment is complicated, the shortcoming of the poor yields of tetrazolium chloride product salt thereby exist.
T-butyl hypochlorate oxidation style [J Am Chem Soc, 1954,76 (6): 1695-1695]; This method is that the Jia Za is dissolved in dioxane and the Glacial acetic acid min. 99.5 mixed solvent, refluxes in the heating back, and jelly is separated out in cooling; Add and add acetone after the chloroform dissolving and separate out product, this method shortcoming dioxane and Glacial acetic acid min. 99.5 mixed solvent large usage quantity are difficult to Separation and Recovery; Separate out jelly and in still, be bonded on the wall, difficult treatment is not suitable for suitability for industrialized production.
Chlorine oxidation process [Chinese Journal of Pharmaceuticals, 2006,37 (1): 10-11]; This method is in the methanol solution of Jia Za, to feed chlorine to prepare; The shortcoming of this method is that chlorine can produce aromatic ring chloro by product in the reaction process on the aromatic ring substituting group of tetrazolium salts, purification difficult, and yield is lower.
Summary of the invention
Goal of the invention: problem to be solved by this invention is to overcome existing disadvantages of background technology, and a kind of preparation method of new tetrazolium chloride salt is provided, and is reactant with the first Za promptly, adopts hydrogenchloride/hydrogen peroxide system that the first Za is oxidized to tetrazolium chloride salt.Final improve yield, to handle in simple, the product heavy metal free residual and make preparation process friendly processization.
Technical scheme: the preparation method of tetrazolium chloride salt biochemistry detection reagent of the present invention 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.
The weight ratio of described step 1) Zhong Jia Za and methyl alcohol is between 1:10 ~ 1:20, and concentrated hydrochloric acid and methyl alcohol volume ratio are between 1:15 ~ 1:30, stirring and dissolving under the room temperature.
Described step 2) the aqueous hydrogen peroxide solution weight percent concentration that drips is 30% ~ 50%, after dropwising, and stirring at room 1 ~ 8 hour.
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.
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,
Described this method is applicable to the preparation of following tetrazolium chloride salt, and its structural formula is following:
Beneficial effect: the present invention adopts hydrogenchloride/hydrogen peroxide system, and the oxidation of first Za is complete, and hydrogen peroxide is reduced to water, and the chlorine negative ion of hydrogenchloride is then transferred in the structure of product tetrazolium chloride salt.Except that producing the tetrazolium chloride product salt, do not produce other by product in the reaction process, reach the green production purpose, thereby and simple owing to not producing the by product post-reaction treatment, yield is high.Overcome in the prior art that by product is many, dealing with complicated, yield is low and product in shortcoming such as heavy-metal residual is arranged.
Embodiment
Following examples will help to understand the present invention, but be not limited to content of the present invention.
The present invention implements with reference to following route:
Embodiment 1:
2-is to the preparation of iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride
4.80g places the 150ml four-hole bottle Jiang the Jia Za, adds 50ml methyl alcohol, adds the 2.5ml concentrated hydrochloric acid again, after the stirring and dissolving, is chilled to 0 ℃.In 0 ℃ to 10 ℃ scope, drip 30% aqueous hydrogen peroxide solution 5ml, after dropwising; Stirring at room 3 hours, Rotary Evaporators is removed methyl alcohol, then with chloroform extraction (3 * 30ml); Behind the anhydrous magnesium sulfate drying, drip anhydrous diethyl ether 30ml and separate out deposition and obtain bullion, then with bullion with methanol (1:2) recrystallization; Filter, 25 ℃ of vacuum-dryings obtain tetrazolium chloride salt crystal 4 .50 gram, yield 91% after 24 hours.
Embodiment 2:
2, the preparation of 5-phenylbenzene-3-(1-naphthyl) tetrazolium chloride
3.51g places the 150ml four-hole bottle Jiang the Jia Za, adds 50ml methyl alcohol again, adds the 2.5ml concentrated hydrochloric acid again, after the stirring and dissolving, is chilled to 0 ℃.In 0 ℃ to 10 ℃ scope, drip 30% aqueous hydrogen peroxide solution 5ml, after dropwising; Stirring at room 5 hours, rotary evaporation is removed methyl alcohol, then with chloroform extraction (3 * 30ml); Behind the anhydrous magnesium sulfate drying, drip anhydrous diethyl ether 30ml and separate out deposition and obtain bullion, then with bullion with methanol (1:2) recrystallization; Filter, 25 ℃ of vacuum-dryings obtain tetrazolium chloride salt crystal 3 .55 gram, yield 93% after 24 hours.
Embodiment 3:
3,3 '-dimethoxy-4 ', 4 '-two penylenes-3 ", 3 " '-preparation of two (2-p-nitrophenyls-5-phenyl tetrazolium chloride)
Jia Za 8.17g is inserted in the 250ml four-hole bottle, add 100ml methyl alcohol again, add the 2.5ml concentrated hydrochloric acid again, after the stirring and dissolving, be chilled to 0 ℃.In 0 ℃ to 10 ℃ scope, drip 30% aqueous hydrogen peroxide solution 5ml, after dropwising; Stirring at room 8 hours, rotary evaporation is removed methyl alcohol, then with chloroform extraction (3 * 50ml); Behind the anhydrous magnesium sulfate drying, drip anhydrous diethyl ether 60ml and separate out deposition and obtain bullion, then with bullion with methanol (1:1.5) recrystallization; Filter, 25 ℃ of vacuum-dryings obtain tetrazolium chloride salt crystal 7.11 grams, yield 87% after 24 hours.
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:
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110117791A (en) * | 2019-05-23 | 2019-08-13 | 电子科技大学 | A kind of binding force promotor for holes on high density interconnected printed circuit board brownification liquid |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3425241B2 (en) * | 1994-09-16 | 2003-07-14 | 栄研化学株式会社 | Pollution prevention method |
| CN1861584A (en) * | 2005-05-12 | 2006-11-15 | 青岛科技大学 | Process of preparing 2,3-disubstituted-tetrazole-5-thione |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3425241B2 (en) * | 1994-09-16 | 2003-07-14 | 栄研化学株式会社 | Pollution prevention method |
| CN1861584A (en) * | 2005-05-12 | 2006-11-15 | 青岛科技大学 | Process of preparing 2,3-disubstituted-tetrazole-5-thione |
Non-Patent Citations (2)
| Title |
|---|
| 申利群等: "过氧化氢氧化法合成四氯苯醌的研究", 《精细化工中间体》 * |
| 金诚等: "四唑盐细胞活力指示剂研究进展", 《化学试剂》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110117791A (en) * | 2019-05-23 | 2019-08-13 | 电子科技大学 | A kind of binding force promotor for holes on high density interconnected printed circuit board brownification liquid |
| CN110117791B (en) * | 2019-05-23 | 2023-11-10 | 电子科技大学 | Binding force promoter for brown oxide liquid of high-density interconnection printed circuit board |
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Application publication date: 20121031 |