JPH05168496A - Composition for measurement of hydrolase and measurement of hydrolase by using the same composition - Google Patents

Abstract

PURPOSE:To provide the subject analytic composition containing a specified disulfide compound, a thioester compound and a metallic ion and capable of readily, rapidly, high sensitively and high accurately detecting a hydrolase without using an expensive equipment or a toxic reagent. CONSTITUTION:The objective composition for measurement of a hydrolase is obtained by blending (A) a disulfide compound [e.g. dithiobis(4-methoxy-2- nitrosobenzene)] represented by formula I (R1 to R4 are H, a halogen, an alkyl, an alkoxy, an aryl, nitro, sulfonic acid, carboxyl, amino, etc.) with (B) a thioester compound [e.g. N-(p-toluenesulfonyl)-L-alanylthiobenzene] expressed by formula II (A is an amino acid residue or a peptide composed of 2 to 5 amino acid residues; B is an amino-protecting residue; R'1 to R'5 are H, a halogen, an alkyl, an alkoxyl, hydroxyl, an aryl, an acyl, etc.) and (C) a metallic ion such as Cu, Zn, Ni, Co or Fe.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a disulfide compound,
The present invention relates to a method for measuring a hydrolase using a thioester compound and a metal ion, a composition for measurement, and a measuring instrument.

[0002]

2. Description of the Related Art The amount of white blood cells appearing in urine is a useful index for diagnosing diseases of the kidneys and genitourinary organs. Conventionally, white blood cells have been detected by centrifuging urine and microscopically examining the sediment. But this method is
It has a drawback that it requires a microscope, requires a long measurement time, requires complicated operations, and requires skill, and that it can measure only intact white blood cells. In particular, it has been reported that white blood cells in urine are likely to be decomposed and the quantification is extremely deteriorated in a sample that has been collected for a long time.

In order to solve these drawbacks, a method of detecting a hydrolase in leukocytes and measuring the amount of leukocytes by using the same has been attempted in recent years. Compared with the previous method of counting white blood cells by microscopic examination, this method is simpler in operation, results can be obtained in a shorter time, and even leukocytes that have collapsed can be detected.

As a detection principle in such a method, a method using an enzyme substrate composed of phenoxyamino acid ester (Japanese Patent Publication No. 61-45682) and a method using an enzyme substrate composed of indoxyl-amino acid ester or peptide ester (Japanese Patent Publication No. 59-59- 3475), a method using 5-phenylpyrrole ester (Japanese Patent Publication No. 62-4171).
0) etc. are known. According to these principles, an enzyme substrate is hydrolyzed with an enzyme, and the product thereof is reacted with a diazonium salt to develop a color as an azo dye.

However, these methods have problems that they do not have sufficient sensitivity for detecting a very small amount of white blood cells in urine, and that they lack coloration stability.

[0006]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method and composition for assaying hydrolase or leukocyte with easy operation, quickly, without using expensive equipment, and with high sensitivity and high accuracy. It is to provide objects and measuring instruments.

[0007]

The above object can be achieved by a method for measuring a hydrolase using a disulfide compound represented by the following chemical formula 3, a thioester compound represented by the following chemical formula 4, and a metal ion.

[Chemical 3] (In the formula, R _{1 to} R ₄ each independently represent a group selected from a hydrogen atom, a halogen atom, an alkyl group, an alkoxyl group, an aryl group, a nitro group, a sulfonic acid group, a carboxyl group and an amino group, Two adjacent substituents may form a condensed aromatic ring.)

[Chemical 4] (In the formula, A peptide group consisting of amino acid residues or amino acid residues 2 to 5, B is an amino-protecting group, R _'1 ~R' ₅ represents a hydrogen atom, a halogen atom, an alkyl group, an alkoxyl group, a hydroxyl group , An aryl group, an acyl group, a nitro group, a thioalkyl group and an alkylamino group, and at least two substituents adjacent to each other may form a condensed aromatic ring. ) The present invention is also achieved by a composition for measuring a hydrolase, which comprises the disulfide compound, the thioester compound and a metal salt.

The present invention is further achieved by a measuring instrument for measuring a hydrolase, which comprises the above composition.

According to the present invention, the thiophenol ester type substrate represented by Chemical formula 4 has high specificity for hydrolases, particularly those present in human neutrophils, and the thioester bond is decomposed to be represented by Chemical formula 5 below. This produces a free thiol.

[Chemical 5] The detection of this thiol can be carried out using the disulfide compound shown in Chemical formula 3, as shown in the following formula. According to the present invention, the disulfide compound represented by Chemical formula 3 is
The disulfide bond is selectively cleaved by the thiol to be measured, producing ortho-nitrosothiophenol. This compound itself is colorless or has only yellow color, but forms a chelate represented by the following chemical formula 6 with a certain kind of metal and is colored red to blue.

[Chemical 6] The amount of thiol can be known by measuring the absorbance of this chelate compound or reading it visually. Since the amount of thiol produced is correlated with the amount of hydrolase, the amount of hydrolase can be known by reading the degree of color change. In the disulfide compound represented by Chemical formula 3, the coloration wavelength and the easiness of cleavage of the disulfide bond, that is, the coloration sensitivity are affected by the difference in the substituents represented by R _{1 to} R ₄ .

Hydrogen atom, halogen atom, alkyl group, alkoxyl group, aryl group, nitro group, sulfonic acid group,
A thiol can be measured even if it has a substituent selected from a carboxyl group and an amino group, but each property can be imparted by introducing the following substituents.

For example, the introduction of the electron-withdrawing group stabilizes the thiol anion of the generated ortho-nitrosothiophenol, so that the disulfide bond cleavability is improved.
It also has the function of shifting the coloration wavelength to a higher wavelength and improving the absorption coefficient. Sulfonic acid group, carboxyl group,
The introduction of an ionic group such as an amino group has the effect of making the disulfide compound water-soluble, and is particularly effective for the measurement system of biological components. Furthermore, the use of a condensed ring also leads to a higher wavelength of coloration and an improved absorption coefficient. It has been revealed that the nitrosonaphthalene disulfide compound exhibits a better coloration than the monocyclic compound.

The disulfide compound of the present invention is synthesized by the following method.

That is, after using the corresponding orthohalonitrobenzene derivative as a starting material and reacting it with Na ₂ S ₂ to disulfide the halogen position, the nitro group is reduced to hydroxylamine with a metal such as zinc, and further deuterated. It is oxidized to nitroso group with chromic acid.

In the thioester compound represented by Chemical formula 4, A represents an amino acid residue or a peptide group, and particularly L-alanyl, L-valyl, L-leucinyl and L-
A peptide group consisting of isoleucinyl or the like or an amino acid thereof is preferable. B represents an amino acid protecting group, and examples thereof include an acyl group, a sulfonyl group and a carbamoyl group.
The thioester compound of the present invention is synthesized by the following method.

That is, it is obtained by condensing a corresponding thiol and a protected amino acid. This condensation reaction can be easily performed by a normal ester bond formation reaction.
For example, it can be synthesized using an acid chloride method, a mixed acid anhydride method, a method using a condensing agent such as DCC, or the like.

Any metal can be used in the method for quantifying thiols according to the present invention, so long as it forms a chelate with an orthonitrosothiophenol derivative to form a color and does not inhibit the enzyme activity to be measured. Zinc, nickel, cobalt and iron are preferably used, and they may be used alone or in combination of two or more. Further, when used as a measuring material or measuring instrument, these metal ions are provided in the form of salt. Any salt may be used as long as it is a dissociative salt, but a metal halide is particularly preferable. These metal compounds are preferably used in a ratio of 0.3 to 1 of the disulfide compound.

The actual measurement of hydrolase is carried out by reacting a measuring composition comprising a thioester compound, a disulfide compound and a metal ion with a sample in an appropriate solvent, and then visually observing the change in color tone of the solution or measuring the transmission absorbance. Read by.

Any reaction solvent may be used as long as it can dissolve the sample and all the components, but a buffer solution,
Also, a mixed solvent of a buffer solution and an organic solvent or the like is used.
In some cases, by adding a reaction aid such as a surfactant, an enzyme activator or a stabilizer, higher sensitivity and accurate measurement results may be obtained. For example, when the sample component or the disulfide compound is difficult to dissolve in the reaction solution, a uniform reaction system can be obtained by adding the surfactant.

For the measurement of hydrolase in neutrophils, the use of ether alcohols such as ethylene glycol monohexyl ether as an enzyme activator shows a great improvement in sensitivity.

The reaction is carried out in a solution, and the operation required for the measurement becomes simpler by using a test tool in which components necessary for the reaction are adsorbed on a carrier. In this case, after the test tool is impregnated in the sample solution, the change in color tone of the test tool is read visually or by measuring the reflection absorbance of the surface. This method is effective for routine examinations of urine samples and the like. As the adsorptive carrier, filter paper, cloth, or organic or inorganic porous material is used, and it is produced by dissolving the components necessary for the reaction in a suitable solvent, impregnating the carrier, and then removing the solvent by drying.

The present invention will be described in more detail with reference to the following examples.

[0022]

Example 1 Synthesis of disulfide compound 2,2′-dinitrobenzene disulfide 60.1 g (250 mmol) of Na ₂ S.9H ₂ O and 8.02 g (250 mmol) of sulfur powder were mixed with 15 parts of ethanol.
It was dissolved in 00 ml and heated under reflux for 10 minutes. Besides, o-
Bromonitrobenzene (100 g, 495 mmol) was added, and the mixture was heated under reflux for 1 hour. After the reaction mixture was cooled to room temperature, 100 ml of water was added, the deposited precipitate was filtered, and the precipitate was washed with cold ethanol and water. This solid was dried under reduced pressure to obtain 70.1 g of a product.

2,2'-dinitrosobenzene disulfide 2,2'-dinitrobenzene disulfide 50 g (16
2 mmol) is dissolved in 1000 ml of 80% ethanol, 45 g (688 mmol) of zinc powder is added, and the mixture is vigorously stirred. While continuing the stirring, 400 ml of 10% ammonium chloride aqueous solution is slowly added through the dropping funnel. The reaction mixture is kept at 45 ° C. and left stirring for 2 hours. The reaction mixture is filtered and the residue is washed with a further 500 ml of hot water. 1 kg of ice is added to the combined filtrate and washing solution, and the reaction vessel is cooled in an ice bath. 20% concentrated sulfuric acid with good stirring
ml, and sodium dichromate 50 g (1
A solution of 67 mmol) in 100 ml of water is added rapidly.

After vigorously stirring the mixture for 3 minutes, 1500 ml of ether was added to the filtrate for extraction. The ether layer was washed with 30% sodium bicarbonate and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 23 g of a crude product. This was purified by silica gel column chromatography using methylene chloride as an eluent to obtain 20 g.
I got the object. The structure was confirmed by MS, NMR and IR.

[Spectral data] MS [M / z]: 276 (M ⁺ ), 216 (M ⁺ -2N)
O), 138 (M ⁺ / 2) NMR [ppm]: 7.80-8.80 (aromat)
ic) IR [cm ^-1 ]: 1505 (NO).

Other nitrosobenzene disulfide derivatives are synthesized in exactly the same manner by using the appropriate substituted halonitrobenzene as the starting material. The disulfide compound tried, the yield and the spectrum data are shown below.

Dithiobis (4-methoxy-2-nitrosobenzene) yield: 42%, MS [M / z]: 336 (M ⁺ ), 276 (M ⁺ -2N).
O), 168 (M ⁺ / 2), NMR [ppm]: 3.70 (OCH ₃ ), 7.30-
8.20 (aromatic), IR [cm ^-1 ]: 1505 (NO).

Dithiobis (4-acetyl-2-nitrosobenzene) yield: 38%, MS [M / z]: 360 (M ⁺ ), 300 (M ⁺ -2N)
O), 180 (M ⁺ / 2), NMR [ppm]: 2.40 (COCH ₃ ), 7.50
-8.20 (aromatic), IR [cm ^-1 ]: 1505 (NO), 1730 (C =
O).

2,2'-dithiobis (4-methoxy-
2-nitrosonaphthalene) yield: 53%, MS [M / z]: 376 (M ⁺ ), 316 (M ⁺ -2N).
O), 188 (M ⁺ / 2), NMR [ppm]: 7.70-8.50 (aromat)
ic), IR [cm ^-1 ]: 1505 (NO).

[0030]

Example 2 Synthesis of thioester compound N- (p-toluenesulfonyl) -L-alanylthiobenzene 1.0 g (9.08 mmol) of thiophenol, N-
(P-toluenesulfonyl) -L-alanine 2.44 g
(10.0 mmol) and 676 mg (5.0 mmol) of 1-hydroxyventriazole were dissolved in 30 ml of tetrahydrofuran, and DCC2.17 was stirred at 0 ° C.
g (10.5 mmol) is added.

After stirring for 1 hour at 0 ° C., stirring is continued for another 3 hours at room temperature. After completion of the reaction, the mixture was evaporated to dryness under reduced pressure, 30 ml of ethyl acetate was added to the residue, and the mixture was stirred, suction-filtered to remove the generated urea, and the filtrate was concentrated again under reduced pressure to obtain a crude product. This was purified by silica gel chromatography using ethyl acetate 3 / n-hexane 7 as an elution solvent to obtain 2.81 g of the desired product (yield 92.3%). The structure was confirmed by MS, NMR and IR.

[Spectral data] MS [M / z]: 335 (M ⁺ ), ¹ H-NMR [ppm]: 1.35 (d, Ala.),
2.40 (s, Φ-CH3), 4.20 (m, CH),
7.10-7.85 (aromatic), IR [cm ^-1 ]: 3360 (NH), 1700 (C
O).

Other thioester derivatives can be synthesized in exactly the same manner by using the substituted thiophenol corresponding to the starting material. The thioester compound tried, the yield and the spectrum data are shown below.

4- [N- (p-toluenesulfonyl)
-L-alanylthio] chlorobenzene Yield: 84.2%, MS [M / z]: 369 (M ⁺ ), ^{< 1} > H-NMR [ppm]: 1.35 (d, Ala.),
2.40 (s, Φ-CH ₃ ), 4.20 (m, CH),
7.10-7.85 (aromatic), IR [cm ^-1 ]: 3360 (NH), 1700 (C
O).

4- [N- (p-toluenesulfonyl)
-L-alanylthio] toluene yield: 88.5%, MS [M / z]: 349 (M ⁺ ), ¹ H-NMR [ppm]: 1.35 (d, Ala.),
2.30 (s, Ts), 2.40 (s, toluen
e), 4.15 (m, CH), 7.00-7.85 (a
romantic), IR [cm ^-1 ]: 3360 (NH), 1700 (C
O).

4- [N- (p-toluenesulfonyl)
-L-alanylthio] anisole yield: 79.1%, MS [M / z]: 363 (M ⁺ ), ¹ H-NMR [ppm]: 1.35 (d, Ala.),
2.40 (s, Φ-CH3), 3.80 (s, OC
_{H 3), 4.20 (m,} CH), 6.75-7.90 (a
romantic), IR [cm ^-1 ]: 3360 (NH), 1700 (C
O).

4- [N- (p-toluenesulfonyl)
-L- Araniruchio] nitrobenzene Yield: 73.7%, MS [M / z]: 276 (M +), 1 H-NMR [ppm]: 1.35, (d, Ala.)
2.40 (s, Φ-CH ₃ ), 4.25 (m, CH),
7.10-8.20 (aromatic), IR [cm ^-1 ]: 3360 (NH), 1705 (C
O), 1525/1345 (NO ₂ ).

[0038]

Example 3 Measurement of hydrolase 200 mM borax-hydrochloric acid buffer solution (pH = 6.0) 1
Ethanol 10ml of 15mM disulfide compound of the present invention in a 00ml, 50mM N- (p- toluenesulfonyl) -L- alaninate Lucio benzene ethanol 10ml, 10mMNiCl _l2 solution 10ml
Was added to prepare a reagent solution. Granulocyte fraction isolated from human peripheral blood by a conventional method was added to this reagent solution at 1000 cells / μ.
0.1 ml of a sample solution prepared by suspending in PBS to a concentration of 1 was added, and the mixture was stirred and reacted at 37 ° C. for 2 minutes. The reaction solution was filtered through a membrane filter to remove the suspension, and then the transmission absorbance was measured. The measurement was performed using a spectrophotometer U-2000 (manufactured by Hitachi Ltd.).

As a control, P containing no cells was used.
Similar experiments were performed using BS. The results obtained by using various disulfide compounds are shown in Table 1.

[Table 1] It should be noted that almost the same results were obtained even if other thioester compounds were used.

[0040]

[Example 4] A measuring filter paper (Advantech No. 514A) using a hydrolase measuring test tool was replaced with 1 mM Ni.
200mM containing Cl _l2 borax - impregnated with HCl buffer (pH = 6.0), and air dried for 50 minutes at 60 ° C.. Further, this filter paper was treated with 0.4 mM disulfide compound, 2.0 mM N- (p-toluenesulfonyl) -L.
-Alanyl thiobenzene, impregnated with an acetone solution containing 2% ethylene glycol monohexyl ether, 4
It was air-dried at 0 ° C. for 20 minutes. This was cut into a 5 × 5 mm square and fixed to a 0.2 mm-thick polystyrene sheet using a double-sided tape.

The hydrolyzing enzyme assay test device thus obtained was provided with 100 cells / granulocyte fraction isolated from human peripheral blood by a conventional method.
A sample solution prepared by suspending in PBS so as to have a concentration of μl was added dropwise, and the color change after 1 minute was observed by measuring the reflection absorbance on the surface of the test paper. The measurement was performed using MCPD-200 manufactured by Otsuka Electronics. As a control, the same experiment was performed using PBS containing no cells. The results are shown in Table 2.

[Table 2] A test tool capable of quantifying thiol was obtained by an easy operation, and since the coloration wavelength was in the visible region, it was very easy to visually judge.

[0042]

EFFECTS OF THE INVENTION As shown in each of the examples, a hydrolase can be measured by a composition comprising a disulfide compound represented by Chemical formula 3, a thioester compound represented by Chemical formula 4, and a metal salt. In addition, a measuring instrument using it can measure hydrolase with a simple operation.

That is, the present invention provides a method, a composition, and a method for assaying a hydrolase or leukocyte with a high sensitivity and a high accuracy by an easy operation, quickly and without using expensive equipment.
Provide a measuring instrument.

Claims (5)

[Claims] Claims: (In the formula, R _{1 to} R ₄ each independently represent a group selected from a hydrogen atom, a halogen atom, an alkyl group, an alkoxyl group, an aryl group, a nitro group, a sulfonic acid group, a carboxyl group and an amino group, Two adjacent substituents may form a condensed aromatic ring.) (In the formula, A peptide group consisting of amino acid residues or amino acid residues 2 to 5, B is an amino-protecting group, R _'1 ~R' ₅ represents a hydrogen atom, a halogen atom, an alkyl group, an alkoxyl group, a hydroxyl group At least one selected from the group consisting of an aryl group, an acyl group, a nitro group, a thioalkyl group, and an alkylamino group, and two adjacent substituents each may form a condensed aromatic ring. A composition for measuring a hydrolase containing a disulfide compound represented by the above chemical formula 1, a thioester compound represented by the chemical formula 2, and a metal ion. 2. The composition for measuring a hydrolase according to claim 1, wherein the metal ions are copper, zinc, nickel, cobalt and iron. 3. The disulfide compound according to claim 1,
A method for measuring a hydrolase using a thioester compound and a metal salt. 4. The method for measuring a hydrolase according to claim 3, wherein the metal salt is copper, zinc, nickel, cobalt or iron. 5. A measuring instrument for measuring a hydrolase, which comprises the composition according to claim 1.