JPS59106476A - Water-soluble tetrazolium compound and a method for determining reductive substance using the same - Google Patents

Abstract

NEW MATERIAL:A water-soluble tetrazolium compound of the formula [at least one of R<1> is nitro and the others are H; R<2> is H, lower alkyl, lower alkoxy; R<3> is H or OR<4>; R<4> is 1-4C aliphatic hydrocarbon having at least one of sulfonic acid groups and/or carboxylic acid groups and having or not hydroxy groups; the number of the solfonic acid groups and carboxylic acid groups in R<3> totalizes 2 or more and one of them forms intramolecular salt with the tetrazole ring]. EXAMPLE:2-(4-Nitrophenyl)3,5-bis(3-sulfopropoxy)-2H-tetrazolium sodium. USE:It is used in determination of reductive substances, especially in determination of components in humor of living bodies and of additives in food. PREPARATION:Nitrophenylhydrazine and sodium sulfopropoxybenzaldehyde are used as starting materials to give a compound of the formula.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel water-soluble tetrazolium compound and its uses. For further details, 2,3.5-l-
To the phenyl group of IJ phenyl-2H-tetrazolium,
A novel tetrazolium compound in which a water-soluble substituent is provided not directly but through oxygen and carbon to make the tetrazolium compound water-soluble, and when it is reduced, a water-soluble formazan is obtained, and the use thereof The present invention relates to a method for measuring a reducing substance characterized by the following.

Tetrazolium compounds function as hydrogen acceptors for reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinic acid amidodinucleotide phosphate (NADPH), or are reduced by superoxide ions, resulting in The amount of NADHXNADPH or superoxide ion can be measured by quantitatively measuring the degree of color development, which is proportional to the amount of formazan produced, using the absorption quadrature measurement method.

Therefore, as is well known, it is possible to measure the activity of dehydrogenase, thereby quantifying the substrate, and also to quantify the substrate that is the target of the action of the oxidase that generates superoxide ions, such as biological fluid components and additives in food. extremely useful for quantifying

These principles can be illustrated using the example of measuring the activity of lactate dehydrogenase (LDH).Since these reactions proceed quantitatively and specifically, the color density of the produced formazan By quantifying LDH, the activity of LDH can be measured. In addition, when measuring biological fluid components using dehydrogenase, the amount of cholesterol can be similarly measured using non-sulphate. Next, by measuring superoxide ions,
To explain the case of quantifying cholesterol, it is cholesterol cholestenone skewered tetrazolium salt formazan, and cholesterol can be quantified in the same way. In this formula, NIA D is the oxidized form of NADH,
indicates halogen.

Tetrazolium compounds conventionally provided for such methods include 2-(4-phenyl iodide)-3-(
4-nitrophenyl)-5-phenyl-2H-tetrazolium salt (INT), 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl-2H-tetrazolium salt (MTT), 22', 5,5'-tetrakis (4
-nitrophenyl)-3゜3'-(3,3'-dimethoxy-4,4'-diphenylene) = 2 H, 2' H-
Ditetrazolium salt (No2-TB), 2.2'-P-
Diphenylene-3,3',5,5'-tetraphenyl-2J(,2'H-ditetrazolium salt (Neo-
TB), but all of them are sparingly soluble in water, and the formazans produced from them are even more sparingly soluble. Therefore, when using it, an organic solvent is used in combination, but this is unfavorable from both measurement and environmental standpoints, and even when used as an aqueous solution at a low concentration, there is a risk that formazan will precipitate and significantly impair measurement accuracy. Both contaminate measurement equipment, limit the use of automatic analyzers, and even when measurements using tetrazolium compounds are desirable, the current situation is that a transition to increasingly more sophisticated methods is being forced.

In contrast, attempts have already been made to make tetrazolium compounds and formazan water-soluble. This was an attempt to directly introduce a sulfonic acid group, a carboxylic acid group, or a side chain containing a quaternary ammonium salt into the phenyl group substituted on the tetrazole ring, and was disclosed in NGK Publication No. 56-38154 and JP-A No. 56-3815.
6-61366, Japanese Patent Application Publication No. 56-61367, Proceedings of the 102nd Annual Meeting of the Pharmaceutical Society of Japan, page 341, pages 4 and 2-4.

However, because of the acid group directly introduced into the phenyl group for the purpose of solubilization, these proposed tetrazolium compounds do not release hydrogen within the optimum pH range where essential enzymatic functions are exhibited in actual measurements. Since it does not produce formazan upon acceptance, it is difficult to use it for practical purposes such as clinical tests, and the quaternary ammonium salt-introduced compound is insufficiently water-soluble.

In order to solve this problem, the present inventors discovered that (1) the tetrazolium compound and its reduced product, formazan, are easily soluble in water; (2) the oxidation-reduction potential is low, and the electron transfer agent in the presence of NADH or NADPH
(1) It is a tetrazolium compound that produces formazan that develops color by 2-peroxide ion or by 2-peroxide ion, and (2) It is a tetrazolium compound that does not dye and contaminate the chemical route of analytical equipment, and the formazan produced is the same. As a result of intensive research, the present invention was completed to satisfy all requirements.

The compound of the present invention has the general formula (R16 is a nitro group, non-nitro group is hydrogen, R2 is hydrogen, lower alkyl group, lower alkoxy group or halogen, R3 is hydrogen or OR4, R4 is a sulfonic acid group) or a salt thereof, or (and) an aliphatic hydrocarbon having 1 to 4 carbon atoms (straight chain or The number of sulfonic acid groups (or salts thereof) or carboxylic acid groups (or salts thereof) contained in R3 is two or more in total, one of which is a tetrazole ring. It forms an inner salt.

) is a water-soluble tetrazolium compound.

The compounds of the present invention can be produced using methods known per se.

For example, nitrophenylhydrazine and sodium sulfopropoxybenotaldehyde are reacted to form a phenylhydrazone compound, then a solution of diazotized sulfopropoxyaniline is reacted, and the resulting formazan is oxidized to form 2-trophenyl. ) -3,5-(sulfopropoxyphenyl)-2H-tetrazolium, the compound of the present invention, is obtained.

The characteristics of the chemical formula that enable the compound of the present invention to meet the above three goals are that at least one of R1 in the general formula is a nitro group, R3 has a sulfonic acid group or a carboxylic acid group, which is a soluble group, and phenyl The groups are not directly substituted, and one of them forms an internal salt with the tetrazole, while the others confer solubility to the compound.

As mentioned above, R2 represents hydrogen, lower alkyl, lower alkyloxy, or halogen;
Methyl, ethyl, propyl, butyl, etc. Examples of R3 include -0CH2Z, -0CH2Z, -0CH2Z, - 0CH2CH2I -0
CH2CH(Z)CH2Z.

-0CH2CH(OH) CH22% -0CH2CH
Examples include (OH)CH(CH3)CH2Z, and a sulfonic acid group and a carboxylic acid group may be mixed.

Four methods for measuring reducing substances using the compound of the present invention are:
Experimental Example 1 shows the measurement of NADH in comparison with a conventional tetrazolium compound.

Example (1) Tetrazolium compound for comparison 2-(4-nitrophenyl)-3,5-bis(3-sulfopropoxy)-2H, one of the compounds of the present invention
-Tetrazolium-sodium (hereinafter referred to as compound prisoner) (B) INT, one of the conventional compounds C) One of the water-soluble tetrazolium compounds previously proposed, Theal 2,5-di(4-sulfophenyl)-3-phenyl-
2H--r t Lasolium dipotassium (2) Same <2-(4-nitrophenyl)'-3-(4
-phenyl iodide)-5-(2-sulfophenyl) -
2H-tetrazolium chloride (Nittokuko Sho 56-313
1.54 Example) (2) Measurement of N A D H 0, 1 M Tris phosphate buffer containing 0.4% of Tril-7X-100 (pH 7,0, 8,'0 and H9 .0
2. Adjustment) 1007 each of the four types of tetrazolium compound anastomosis 2.5 mM and electron transfer agent 1-methoxy7phenanone methosulfate (IVfeO-PMS) Iom, y
To each of those solutions 4.0- to 6-mM NADH1 was added.
Add 0.1 m6 of solution, heat at 37°C for 2 minutes, and immediately measure absorbance.

Uncensored (3) Situation and results during operation Solubility　○ Well dissolved.

× Poor solubility, insoluble matter or precipitates There is.

Color development ○ Good color development in homogeneous liquid.

△ Color develops, but there are insoluble substances.

× No color develops.

As is clear from this, (B) which does not have a water bathing group is
It can act as a hydrogen acceptor for N A D H near neutrality, but
Practically speaking, (C) and (C), which have a low measurement value and in which a water-soluble group is directly substituted with a phenyl group, do not develop color near neutrality, and are insoluble. On the other hand, the compound compound, which is one of the compounds of the present invention, is sufficiently soluble in water, both itself and the formed formazan, and is conveniently soluble in N at around neutrality.
The color development of y-formazan, which is a hydrogen acceptor for ADH, is also good.

As in Experimental Example 1, the combined use of a surfactant has the effect of shifting the coloring wavelength to the longer wavelength side, but also has the effect of increasing the sensitivity. Polyoxyethylene derivatives of aliphatic and aromatic alcohols are used as surfactants. These alkyl-substituted phenyl ethers of polyoxyethylene with a degree of polymerization of usually about 5 to 20, or HitE[
As the nonionic surfactant which is an alkyl ether, those which are commercially available and commonly used in clinical reagents can be used without any problem. Commercial product name is Triton X-100, also 11
Examples include 4, 405, Br1j 35, and 58.

As is well known, colored substances such as bilirubin exist in serum, and this has a considerable influence on the absorption spectrum near the wavelength of 450 nm, causing errors in measurements. The coexistence of an activator causes the coloring wavelength to shift to longer wavelengths.
This is extremely desirable. Next, the effects of using surfactants will be shown.

Example 0,1 M l- containing 4% Triton X-100
Compound (A) of the present invention (A) 2.5] TIM and 310 mg of MeO-PM in lithium phosphate buffer (1) H8,5) 100-
Dissolve and take 4.0 ml of the solution. On the other hand, Torridon X-
Using the same buffer solution that does not contain 100 and having the same formulation (
Step 4: Take Oi. Add NAD H2,5 to both solutions.
Add 0.1 μm of the mM solution and incubate at 37°C for 2 minutes, then measure the absorbance. The absorption curve in the visible region is shown in Figure 1, and when a surfactant is used in combination,
The thickest wavelength shifts from 460 nm to 495 nm, and its absorbance increases by more than 3.5 times.

In addition, the pH dependence of color development by the compound of the present invention was investigated using NA
The measurement of D'H will be taken as an example.

Example 0, IMF containing 0.4 fraction of Triton X-100
Lys phosphate buffer (pH 5.5.6.0., 6.5,
Adjust to 7°o, 7.5, s,o, 8.5)
To each 100 ml, add 2.5 mM of the compound (A) of the present invention and 810 m# of MeO-P M, take 4.0 onions of each solution, and add 1 go of a 1.6 mM solution of NADH to each. was added, heated at 37°C for 25 minutes, and then the absorbance at a wavelength of 495 nm was measured. Plotting the results gives Figure 2. In other words, the optimum pH for color development is around neutrality, and the method of the present invention corresponds to the optimum pH for enzyme activity in the target measurement field.

In this way, 2.3.5-) 1) By remotely imparting a water-soluble group to the phenyl of phenyl-2H-tetrazolium, the present invention dramatically improves the aqueous solution color development using conventional tetrazolium. , the benefits to this industry are enormous. The present invention will be further explained with reference to Examples below, but these Examples are not intended to limit the present invention.

Example 1 15.4 4-nitrophenylhydrazine was suspended in 300 ml of #womeknol, and while stirring while heating on a water bath, 26.6 ml of 4-sodium sulfobropoxybenzaldehyde @ was added, and further 1 After heating and stirring for half an hour, cooled with water and using 500m7 of cold acetone! is added to precipitate crystals, and dried several times to obtain the hydrazone compound 3-(4-(p)
Sodium -nitrophenylhydrazonomethyl)phenoxy)propylsulfonate 282 was obtained. v: ”1
590 cm ” (N-f()+13002' (C
-NO2), 1190cm” (sulfonic acid), 1240
The Rfl value of thin layer chromatography (TLC) using cIrL-SenAr-0-)+ethyl acetate:acetonitrile;acetic acid:water (2:2:1:1) as a developing solvent was 0.63.

Separately, 5% sodium hydroxide solution was added to a solution of 2.32% of 3-(4-aminophenoxy)propylsulfonic acid diazotized in a conventional manner to prepare a neutral diazonium solution.

pE (hydroxide adjusted to 10) Add this diazonium solution to a solution of the above hydrazone compound 3.22 dissolved in IJum sub-solution at 5°C or less, stir briefly at the same temperature, and then Bring to pH 10 with sodium solution. Stir for 1 hour and then leave overnight. After adjusting the pH to 4 with glacial acetic acid and adding Impropatool, the precipitated black-purple crystals were collected and the formazan compound 1,3-bis(F)-(3-sodiumsulfoproboxy)phenyl l-5 5.1 g of -(nitrophenyl)- formazan was obtained. ν: 1590cm” (
N-H), 1330 CIIL” (C-NO2), 118
0cfn~1 (sulfonic acid), 1250cIrL-
1 (Ar-0-), Rf IIF of TLC using the same developing solvent as above was 0.43.

Next, 1.52 of this formazan compound was dissolved in 80 ml of methanol, and after adjusting the pH to 4 with acetic acid, 2.0-tert-butyl hypochlorite was added at room temperature, and the mixture was stirred and reacted at the same temperature for 2 hours. The reddish-purple color disappears from the reaction solution and it becomes pale yellow. The reaction mixture was neutralized with an IJ solution, acetone was added, the precipitated crystals were dried several times, and purified by column chromatography to obtain 1 g of white crystals of compound (5).

IR: V(crn')=1040 (tetrazole).

1180 (sulfonic acid), 1260 (Ar-0-).

1340 (CN02), 1530 (OC=N), visible region maximum absorption (λmax) = 460 nm .

HN Calculation (Direct (CJ (2+NPS20+oNa) 46
．． 80 377 10-95 actual measurement [direct
46.62 3.87 10.91 Examples 2 to 6 The following compounds were obtained in the same manner as in Example 1. All of them are white crystals, and when used for measurement, they may be in the form shown below or in a form in which the acid group other than the one forming the inner salt forms an alkali salt.

Example 7 (Measurement of NADH) Compound (A) 190 m9/dL, IVIeO-PMS
10mg/dt, 1-lido:/X-100 is 0.4y
01M Tris phosphate buffer (
(pH 8.5) to prepare a coloring test solution. N to be measured
Take 0.1ml of the ADH solution and add color reagent 4 to it.
．． 0- and incubate for 5 minutes at 37°C. The absorbance at 490 nm is measured using a reagent blank as a control.

The concentration of N A D H is determined from the calibration curve shown in FIG. 3, which was separately prepared under the same conditions using N A D H solutions of various concentrations.

Example 8 (Quantification of cholesterol by superoxide ion measurement) Compound (A) 1 m IVI/l, 7 x Nor 1
．． 06 m IVI/l.

Reduced glucthione 0.65 m M/l, peroxidase 300 u/dt, cholesterol oxidase 15 u/dt, trito 7X-
100 is dissolved in 0.1 MIJ hydrochloric acid buffer (pH 8,0) to a concentration of 0.1 g/, dt O') to prepare a coloring reagent.

Take 20 μl of serum and use coloring reagent 3. Add Ornl, 3
After isocubating at 7°C for 10 minutes, the absorbance at 490 nrn is measured using reagent black as a control.

Separately, the concentration of cholesterol in serum is determined from a calibration curve created using cholesterol standard solutions of various concentrations under the same conditions.

[Brief explanation of drawings]

FIG. 1 shows a comparison of absorption curves in the visible region of formazan produced when measuring NADH when a surfactant is used and when a surfactant is not used. Figure 2 shows NADH at various pH values in the presence of a surfactant.
This is a plot of the maximum absorption in the visible region of the formed formazan when measured. No. 3121 shows a calibration curve when measuring N A D H using a compound prisoner. Figure 1 Figure 2 H

Claims (1)

[Claims] \/ (R' is at least one nitro group, non-nitro group is hydrogen, R2 is hydrogen, lower alkyl group, lower alkoxy group, or) rogene, R3 is hydrogen or OR', R4 is an aliphatic hydrocarbon having 1 to 4 carbon atoms, which has one or more substituted sulfonic acid groups or salts thereof, or (and) carboxylic acid groups or salts thereof, and has or does not have a substituted hydroxyl group. (having a straight chain or a side chain), and the number of sulfonic acid groups (or salts thereof) or carboxylic acid groups (or salts thereof) contained in R3 is two or more in total, and one of them forms an inner salt with the tetrazole ring. ), a new water-soluble tetrazolium compound. (R1 is at least one nitro group, non-nitro group is hydrogen, R2 is hydrogen, lower alkyl group, lower alkoxy group, or halogen, R3 is hydrogen or OR', R4 is a sulfonic acid group or a salt thereof, or (and) An aliphatic hydrocarbon (having a straight chain or a side chain) having 1 to 4 carbon atoms, which has one or more substituted carboxylic acid groups or salts thereof, and has or does not have a substituted hydroxyl group. The number of sulfonic acid groups (or salts thereof) or carboxylic acid groups (or salts thereof) contained in R3 is two or more in total, and ] among them forms an inner salt with the tetrazole ring. )
A method for measuring a reducing substance in an aqueous solution, characterized by using a tetrazolium compound represented by: (3) The measuring method according to claim 2, wherein a tetrazolium compound is used in combination with a surfactant. (4) The measuring method according to claim 2 or 3, wherein the reducing substance is reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide dinucleotide 911 (NADPH). (5) It is a reducing substance or superoxide ion,
A measuring method according to claim 2 or 3. (6) Claims 2 and 3, which quantify components in a liquid by measuring reducing substances in an aqueous solution;
The measuring method according to item 4 or 5. (7) The measuring method according to claim 6, wherein the component in the liquid is a component in biological fluids.