JPH05130897A - Reagent composition for determination of thiol compound - Google Patents

Abstract

PURPOSE:To provide the subject composition containing a cobalt(II) salt and a complex formation promoting agent and effective for detecting a thiol compound, a hydrolase and accordingly leukocyte in high sensitivity and accuracy without using expensive instrument, complicate operation, skilled labor, etc. CONSTITUTION:The objective composition contains (A) a cobalt(II) salt [e.g. cobalt(II) acetate and cobalt(II) chloride] and (B) a complex formation promoting agent at a weight ratio (A:B) of preferably (3-15):1. The component B is preferably a combination of a cationic surfactant with a component selected from quinoline compounds (e.g. quinaldine), quinazoline compounds (e.g. quinazoline), sulfanyl compounds (e.g. sulfanyl amide) and stilbazole compounds (e.g. 2- stilbazole).

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a reagent composition for measuring a thiol compound, a method for detecting a thiol compound using the reagent composition, and a method for detecting a hydrolase.

[0002]

2. Description of the Related Art By detecting leukocytes appearing in urine, diseases such as kidneys, genitourinary organs and urinary tract can be diagnosed. It is also known that peritonitis can be detected early by monitoring leukocytes in the peritoneal dialysate. Conventionally, the detection of white blood cells in the test liquid has been performed by counting the number of white blood cells in the liquid with a microscope, this method requires expensive equipment, long measurement time, complicated operation, skill, etc.,
Further, it has a drawback that it can measure only complete white blood cells that have not been disintegrated.

In order to solve the drawbacks of the above-mentioned conventional methods, a detection method has been recently attempted in which a hydrolase in leukocytes is detected and thereby the amount of leukocytes is measured. In this method, the number of leukocytes is counted by microscopic examination. Compared with the above-mentioned conventional methods, there are advantages that the operation is easy, the results can be obtained in a short time, and even the disintegrated leukocytes can be detected. Then, the present inventors previously described the following formula (V);

[0004]

[Chemical 8] (In the formula, A ₁ is an amino acid residue or a peptide group consisting of 2 to 5 amino acid residues, B ₁ is a protecting group for an amino group, and R ′ _{1 to} R ′ ₅ are each independently a hydrogen atom or a halogen atom. ,
It is a group selected from the group consisting of 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 two adjacent substituents each form a condensed aromatic ring. Thioester compound represented by the formula (V
I);

[Chemical 9] (In the formula, A ₂ is an amino acid residue or a peptide residue, B ₂ is a nitrogen protecting group, and R ′ _{6 to} R ′ ₁₁ are each independently a hydrogen atom, a halogen atom, an alkyl group, an alkoxyl group, an aryl group. Table in groups, a group selected from aralkyl and aralkyl alkoxyl group, or shows the two adjacent groups are bonded may also form a condensed ring group of R _'6 ~R' ₁₁₎ And found that an enzyme substrate comprising a thioester compound can be detected, and that the enzyme substrate can be used to detect a hydrolase in leukocytes and thus leukocytes themselves (Japanese Patent Application Nos. 3-191921 and 2). -308689).

The thioester compound represented by the above formulas (V) and (VI) has its thioester bond, that is, S and A in the formulas (V) and (VI), due to the hydrolase contained in leukocytes. The bond between the two is cleaved to give the following formula (VII);

[0007]

[Chemical 10] (In the formula, R ′ _{1 to} R ′ ₅ represent the same groups as described above) and a thiophenol represented by the following formula (VIII);

[0008]

[Chemical 11] (Wherein, R _'6 ~R' ₁₁ has the same group as above) resulting thiophenol represented by, by measuring the presence and amount of these thiophenol, hydrolase hence leukocytes detection measurement can do.

On the other hand, conventional methods have been used to detect thiol compounds having an SH group in biological components and environmental samples, including the thiophenols represented by the above formulas (VII) and (VIII). , The titration method by the formation of silver mercaptide, the titration method by mercury mercaptide, the oxidative titration method by iodine and copper ions, the bromine titration method, etc. have been mainly used, but the operation is complicated and the sensitivity is poor. There was a problem.

Thereafter, as a detection agent for a thiol compound which is highly sensitive and easy to operate and has a high quantitative accuracy, a disulfide compound or a benzhydrol system which directly reacts with a thiol compound to cause a color change. Compounds have come into use. However, in the case of using these compounds, the color of the reaction product with the thiol compound is yellow, making it difficult to distinguish it from the color of the urine itself, making it difficult to perform semi-quantitative determination by visual observation, or as a result of the reaction. There is a problem that the color is easily influenced by other components in the liquid to be inspected, and it is difficult to perform reliable and stable detection.

The cobalt (II) salt is a purple complex with a thiophenol compound such as thiophenol, p-toluenethiol, p-nitrothiophenol and p-chlorothiophenol in a non-aqueous solvent, especially isopropanol. It is known that they are formed, and this property is utilized to detect and measure a thiophenol compound by absorptiometry. However, since the above complex formation reaction hardly progresses in an aqueous solution, it has a drawback that it cannot be applied to urine, body fluid, and other aqueous liquids.

[0012]

From the above point of view, the present inventors have made it possible to include in an aqueous solution with high sensitivity and high accuracy without requiring expensive equipment, long measuring time, complicated operation, skill and the like. Studies have been carried out for the purpose of obtaining a thiol compound, a hydrolyzate enzyme, and a reagent and a detection method capable of detecting leukocytes. As a result, cobalt (II)
When a composition containing a salt and a complex formation accelerator is used, a thiol compound can be detected with high sensitivity and high precision by a simple operation, and the cobalt (II) salt and the complex formation accelerator are represented by the above formula (V). The present invention has been completed by finding that a hydrolase, and thus leukocytes, can be easily and stably detected when used in combination with a thioester compound represented by.

Therefore, the present invention is a reagent composition for measuring a thiol compound, which contains a cobalt (II) salt and a complex formation accelerator. And this invention includes the method of detecting a thiol compound using the said reagent composition. Furthermore, the present invention provides the following formula (V);

[0014]

[Chemical 12] (Wherein A ₁ , B ₁ and R ′ _{1 to} R ′ ₅ represent the same groups as described above) [hereinafter referred to as “thioester compound (V)”] and the following formula (VI) ;

[0015]

[Chemical 13] (Wherein A ₂ , B ₂ and R ′ _{6 to} R ′ ₁₁ represent the same groups as described above), at least one kind of thioester compound [hereinafter referred to as “thioester compound (VI)”], cobalt ( II) A method of detecting a hydrolase using a salt and a complex formation promoter.

The "thiol compound" in the present invention is not limited to thiophenol but is a generic term for compounds having an SH group. Therefore, the thiol compound measuring reagent composition and the thiol compound detecting method of the present invention are as follows. Any compound having an SH group can be used for detection and measurement of both an inorganic thiol compound and an organic thiol compound. Among them, the thiol compound measuring reagent composition and the thiol compound detecting method of the present invention are particularly suitable for detecting and measuring thiol compounds such as thiophenol compounds, thioalcohol compounds, and thioglycol compounds.

In the present invention, any cobalt (II) salt may be used as long as it is a salt of cobalt (II) which is dissociable in an inspection liquid such as an aqueous liquid or an organic liquid.
For example, cobalt (II) acetate, cobalt (II) nitrate, cobalt (II) chloride, cobalt (II) sulfate, and other inorganic cobalt (II) salts and organic cobalt (II) salts can be used.

Further, as the above-mentioned complex formation accelerator used with the cobalt (II) salt, an ionic surfactant, the following formula (I);

[0019]

[Chemical 14] (Wherein R _{1 to} R ₇ each independently represent a group selected from a hydrogen atom, a halogen atom, an alkyl group, an alkoxy group, an aryl group, a nitro group, a sulfonic acid group, a carboxyl group and an amino group). A quinoline compound represented by the following formula (II);

[0020]

[Chemical 15] (In the formula, R _{8 to} R ₁₃ each independently represent a group selected from a hydrogen atom, a halogen atom, an alkyl group, an alkoxy group, an aryl group, a nitro group, a sulfonic acid group, a carboxyl group and an amino group). A quinazoline compound represented by the following formula (III);

[0021]

[Chemical 16] (In the formula, each of R _{14 to} R ₁₇ independently represents a group selected from a hydrogen atom, a halogen atom, an alkyl group, an alkoxy group and an aryl group), and a sulfanilamide compound represented by the following formula (IV _a ) or (IV _b );

[0022]

[Chemical 17]

[0023]

[Chemical 18] (In the formula, each of R _{18 to} R ₂₆ independently represents a group selected from a hydrogen atom, a halogen atom, an alkyl group, an alkoxy group and an aryl group), and a stilbazole compound represented by the formula: Preference is given to using one compound.

As the above-mentioned ionic surfactant, any one can be used as long as it is an anionic surfactant, a cationic surfactant and an amphoteric surfactant, and preferable examples thereof are Examples thereof include alkyl sulfonates (anionic surfactant), laurylpyridinium chloride and the like (cationic surfactant). Among them, the cationic surfactant is more preferable.

Typical examples of quinoline compounds include quinaldine, 3-aminoquinoline, 8-aminoquinoline, 6-methylquinoline and 7-methylquinoline. Furthermore, quinazoline can be mentioned as a typical example of the quinazoline compound. Further, representative examples of the sulfanilamide-based compound include sulfanilamide, sulfanylbenzylamine, sulfapyridine, sulfapyrazine, and the like, and representative examples of the stilbazole-based compound include 2-stilbazole, 4-stilbazole, and the like. it can.

As described above, the complex formation accelerator may be used alone or in combination of two or more, and in particular, quinoline compounds, quinazoline compounds, sulfanilamide compounds and stilbazole. When one kind of the system compounds and the ionic surfactant are used in combination, the complex formation between the cobalt (II) salt and the thiol compound is further promoted, and a clear and stable coloration state can be obtained. ..

The cobalt (II) salt used in the reagent composition for measuring a thiol compound of the present invention may vary depending on the kind of the cobalt (II) salt used, the kind of the complex formation accelerator, the kind of the thiol compound to be detected, and the like. The ratio of salt to complexation promoter used is generally preferably in the range of cobalt (II) salt: complexation promoter = about 3: 1 to about 15: 1 by weight.

When the thiol compound is detected and measured, the cobalt (II) salt and the complex formation accelerator may be added individually or simultaneously to the liquid to be inspected, or may be added beforehand with cobalt (II). The salt and the complex formation accelerator may be mixed and then the mixture may be added. Among them, a cobalt (II) salt and a complex formation accelerator are mixed in advance in a solid state (powder state) in a dry state to give a dry reagent composition which can be stored for a long time, and when the thiol compound is tested, It is convenient to use the required amount of the reagent composition. When the reagent composition is added to the liquid to be inspected, it may be directly added in a solid state, but it is dissolved in water or an appropriate organic solvent (eg alcohol) to be added in a solution form for quick detection. It can be done conveniently. When adding in solution, cobalt
(II) It is preferable to add it as a solution having a salt concentration of about 0.2 to 2% by weight. Further, the cobalt (II) salt and the complex formation accelerator may be carried on a carrier made of filter paper or other material by an appropriate method to prepare a color test paper or a test tool, and a detection test may be conducted.

The thiol compound measuring reagent composition containing a cobalt (II) salt and a complex formation accelerator may further contain other components such as a buffer and a stabilizer, if necessary.

This thiol compound measuring reagent composition is
It can be effectively used for all inspected substances that require detection and measurement of thiol compounds such as clinical samples, industrial samples, food samples, research samples and natural samples. When a thiol compound is contained in the inspection object, the complex formation reaction between the thiol compound and cobalt (II) is promoted by the action of the complex formation accelerator, and a red having a maximum absorption wavelength at 532 nm is usually obtained. Since a vivid purple color is developed,
By measuring the absorbance at 532 nm, the presence or absence and the concentration of the thiol compound can be detected and measured.

When the cobalt (II) salt and the complex formation accelerator are further used in combination with at least one of the above thioester compound (V) and thioester compound (VI), as described above, urine and other The thioester compound (V) or the thioester compound (VI) is hydrolyzed by the hydrolase contained in the sample to produce the thiophenol represented by the above formula (VII) or (VIII). Phenol forms a complex with cobalt (II) in the presence of a complex formation promoter that exhibits a bright red-purple color with a maximum absorption wavelength at 532 nm, so the presence or absence and concentration of hydrolase in the sample It can detect and measure.

Therefore, in the present invention, as described above, at least one of the thioester compound (V) and the thioester compound (VI), the cobalt (II) salt and the above-mentioned complex formation accelerator are used to hydrolyze the enzyme, and thus the hydrolase. It includes a method for detecting and measuring white blood cells.

The thioester compound (V) and the thioester compound (VI) can be produced by the method described in the above-mentioned Japanese Patent Application No. 3-191921 and Japanese Patent Application No. 2-308669 and any other method.

In the thioester compounds (V) and (VI), the amino acid residue or peptide group A ₁ or A ₂ is
Any amino acid residue can be used as long as it forms a thioester bond which can be hydrolyzed by a hydrolase, and amino acid residues from L-type, D-type and racemic type, especially L-type alanine, valine, leucine, isoleucine and phenylalanine, Alternatively, a peptide group consisting of 2 to 5 of those amino acids is preferable. The amino protecting group B may be any amino protecting group commonly used in amino acids and peptides, and examples thereof include an acyl group, an oxycarbamoyl group, a phosphoryl group, a carbamoyl group, a thiocarbamoyl group, a sulfonyl group, a sulfenyl group and a vinyl group. , Cyclohexenyl group and the like.

Further, the group R _'1 ~R' _5, hydrogen; fluorine, chlorine, bromine and iodine, especially chlorine and bromine; -C 1-5 alkyl group; a phenyl group, a naphthyl group; a benzyl group the hydroxyl group, the group R _'6 ~R' _11, hydrogen; nitro group; an aryl group such as fluorine, chlorine, bromine and iodine, especially chlorine and bromine; number of carbon atoms 1
Five alkyl groups; phenyl group, naphthyl group; aryl group such as benzyl group; alkoxyl group such as methoxy group, ethoxy group; aralkyl group such as phenylethyl group, phenylpropyl group; alkoxyl group such as benzyloxy group A nitro group; a hydroxyl group and the like.

Specific examples of the thioester compound (V) and the thioester compound (VI) are as follows, but of course the invention is not limited thereto. ○ N- (p-toluenesulfonyl) -L-alanylthiobenzene ○ 4- [N- (p-toluenesulfonyl) -L-alanylthio] chlorobenzene ○ 4- [N- (p-toluenesulfonyl) -L-alanylthio] Toluene o 4- [N- (p-toluenesulfonyl) -L-alanylthio] anisole o 4- [N- (p-toluenesulfonyl) -L-alanylthio] nitrobenzene o 8- (Np-toluenesulfonyl-L- Alanylthio) quinoline 5-chloro-8- (Np-toluenesulfonyl-
L-alanylthio) quinoline 4-methyl-8- (Np-toluenesulfonyl-
L-alanylthio) quinoline 5-methoxy-8- (Np-toluenesulfonyl-L-alanylthio) quinoline o- (N-carbobenzyloxy-L-alanylthio) quinoline o- (Nt-butoxycarbonyl) -L-alanylthio) quinoline

When detecting hydrolase and / or white blood cells in the liquid to be tested, a thioester compound is used.
(V) and at least one of thioester compound (VI),
The cobalt (II) salt and the complex formation accelerator are added to the liquid to be tested individually or simultaneously, or they are mixed in advance to form a reagent composition for detecting a hydrolase or leukocyte in advance. Then, the reagent composition may be used at the time of inspection. Furthermore, at least one of thioester compound (V) and thioester compound (VI), cobalt (I
I) Coloring by supporting salt and complex formation accelerator on a suitable carrier such as filter paper, fiber fleece or cloth made of natural or synthetic fiber, natural or synthetic inorganic or organic foam or porous body by a suitable method A test paper or a color tester may be formed in advance, and a test for detecting a hydrolase or a white blood cell may be performed using the test paper or the test tool.

The thioester compound (V) and the thioester compound (VI) used, the type of cobalt (II) salt, the type of complex formation accelerator, etc. may vary, but in detecting and measuring hydrolase and leukocytes. Is generally in a molar ratio of at least one of thioester compound (V) and thioester compound (VI): cobalt (II) salt: complexation accelerator = about 40: 3: 1 to about 20: 10: 3. Preference is given to using. When detecting the hydrolase, it is preferable to use about 0.2 to 20 mmol of at least one of the thioester compound (V) and the thioester compound (VI) per liter of the liquid to be detected. Next, the present invention will be specifically described with reference to examples, but the present invention is not limited thereto.

[0039]

【Example】

Example 1 A test tube was charged with 1.2 ml of a 1% by weight cobalt (II) acetate aqueous solution and a 0.2 M borate buffer solution (pH 8.0) 4
0 μl and 40 μl of a 1% by weight aqueous solution of laurylpyridinium chloride were added. Next, 20 μl of an ethanol solution of thiophenol having a concentration of 10 mM was added to the solution to give a solution of 532 nm.
The time-dependent change of the absorbance in (1) was measured by using an absorptiometer (U-2000 type manufactured by Hitachi, Ltd.) to examine the complex formation state between cobalt (II) and thiophenol. The results are shown in Table 1 below.

Example 2 A test tube was charged with 1.2 ml of a 1 wt% cobalt (II) acetate aqueous solution and a 0.2 M borate buffer solution (p.
40 μl of H8.0) and 40 μl of a 1% by weight solution of 3-aminoquinoline in ethanol. Next, concentration 10m
20 μl of an ethanol solution of M thiophenol was added, and the change with time of the absorbance of the solution at 532 nm was measured in the same manner as in Example 1. The results are shown in Table 1 below.

Example 3 A test tube was charged with 1.2 ml of a 1 wt% cobalt (II) acetate aqueous solution and a 0.2 M borate buffer solution (p.
40 μl of H8.0) and 40 μl of a 1% by weight solution of 6-methylquinoline in ethanol. Next, concentration 10m
20 μl of an ethanol solution of M thiophenol was added, and the change with time of the absorbance of the solution at 532 nm was measured in the same manner as in Example 1. The results are shown in Table 1 below.

Example 4 A test tube was charged with 1.2 ml of a 1 wt% cobalt (II) acetate aqueous solution and a 0.2 M borate buffer solution (p.
40 μl of H8.0) and 40 μl of 1% by weight quinazoline in ethanol. Next, 20 μl of an ethanol solution of thiophenol having a concentration of 10 mM was added, and
The change with time in absorbance at 32 nm was measured in the same manner as in Example 1. The results are shown in Table 1 below.

Example 5 A test tube was charged with 1.2 ml of a 1 wt% cobalt (II) acetate aqueous solution and a 0.2 M borate buffer solution (p.
40 μl of H8.0) and 40 μl of a 1% by weight solution of sulfanilamide in ethanol were added. Next, concentration 10m
20 μl of an ethanol solution of M thiophenol was added, and the change with time of the absorbance of the solution at 532 nm was measured in the same manner as in Example 1. The results are shown in Table 1 below.

Example 6 A test tube was charged with 1.2 ml of a 1 wt% cobalt (II) acetate aqueous solution and a 0.2 M borate buffer solution (p.
40 μl of H8.0) and 40 μl of 1 wt% 2-stilbazole in ethanol. Next, concentration 10m
20 μl of an ethanol solution of M thiophenol was added, and the change with time of the absorbance of the solution at 532 nm was measured in the same manner as in Example 1. The results are shown in Table 1 below.

Example 7 A test tube was charged with 1.2 ml of a 1 wt% cobalt (II) acetate aqueous solution and a 0.2 M borate buffer solution (p.
40 μl of H8.0) and 40 μl of an aqueous solution of 1% by weight sodium sulfosuccinate. Next, concentration 10 mM
20 μl of thiophenol ethanol solution
The change with time of the absorbance of the liquid at 532 nm was measured in the same manner as in Example 1. The results are shown in Table 1 below.

Comparative Example 1 1.2 ml of 1% by weight cobalt (II) acetate aqueous solution and 0.2 M borate buffer solution (p
40 μl of H8.0) and 40 μl of ethanol were added. Next, 20 μl of an ethanol solution of thiophenol having a concentration of 10 mM was added, and the change with time of the absorbance of the solution at 532 nm was measured in the same manner as in Example 1. The results are shown in Table 1 below.

[0047]

[Table 1] Example of absorbance at 532 nm Initially 1 minute later 2 minutes later 3 minutes later 4 minutes later 5 minutes later Example 1 0.189 0.242 0.331 0.404 0.459 0.503 Example 2 0.158 0.336 0.421 0.452 0.482 0.488 Example 3 0.180 0.372 0.471 0.554 0.590 0.591 Example 4 0.280 0.510 0.636 0.701 0.736 0.748 Example 5 0.184 0.354 0.495 0.589 0.649 0.685 Example 6 0.121 0.196 0.254 0.312 0.379 0.433 Example 7 0.304 0.416 0.524 0.605 0.659 0.699 Comparative Example 1 0.002 0.046 0.083 0.113 0.134 0.153

From the results shown in Table 1, in Examples 1 to 7 of the present invention in which the complex formation promoter was used together with the cobalt (II) salt, the absorbance at a wavelength of 532 nm was greatly increased by 5 minutes after the reaction, and The complex formation reaction between (II) and thiophenol is promoted. On the contrary, in Comparative Example 1 in which the complex formation accelerator is not added, the reaction is 5 even after 5 minutes of reaction.
It can be seen that the absorbance at 32 nm was not so different from the initial value, that the complex formation between cobalt (II) and thiophenol was scarcely performed, and thiophenol could not be detected.

[0049]

EFFECTS OF THE INVENTION Conventionally, it was almost impossible to detect a thiol compound contained in an aqueous solution with a cobalt (II) salt, but the reagent composition of the present invention containing a cobalt (II) salt and a complex formation accelerator. Expensive equipment, if used
The thiol compound contained in the aqueous solution can be specifically detected and measured with high sensitivity and in a short time without requiring long measurement time, complicated operation, skill, and the like. Furthermore, in the present invention, by using at least one of the thioester compound (V) and the thioester compound (VI), the cobalt (II) salt and the complex formation accelerator, the hydrolase, and thus the leukocyte in the test liquid can be Presence / absence and concentration can be detected and measured easily, stably and highly sensitively and specifically.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location G01N 33/49 A 7055-2J 33/50 K 7055-2J

Claims (4)

[Claims] 1. A reagent composition for measuring a thiol compound, which contains a cobalt (II) salt and a complex formation accelerator. 2. The complex formation accelerator is an ionic surfactant, the following formula (I); (Wherein R _{1 to} R ₇ each independently represent a group selected from a hydrogen atom, a halogen atom, an alkyl group, an alkoxy group, an aryl group, a nitro group, a sulfonic acid group, a carboxyl group and an amino group). A quinoline compound represented by the following formula (II); (In the formula, R _{8 to} R ₁₃ each independently represent a group selected from a hydrogen atom, a halogen atom, an alkyl group, an alkoxy group, an aryl group, a nitro group, a sulfonic acid group, a carboxyl group and an amino group). Quinazoline compounds,
The following formula (III); (In the formula, each of R _{14 to} R ₁₇ independently represents a group selected from a hydrogen atom, a halogen atom, an alkyl group, an alkoxy group and an aryl group), and a sulfanilamide compound represented by the following formula (IV _a ) or (IV _b ); [Chemical 5] (In the formula, each of R _{18 to} R ₂₆ independently represents a group selected from a hydrogen atom, a halogen atom, an alkyl group, an alkoxy group and an aryl group), and at least one selected from the group consisting of stilbazole compounds. The reagent composition of claim 1 which is a compound of a species. 3. A method for detecting a thiol compound using the reagent composition according to claim 1. 4. The following formula (V); (In the formula, A ₁ is an amino acid residue or a peptide group consisting of 2 to 5 amino acid residues, B ₁ is a protecting group for an amino group, and R ′ _{1 to} R ′ ₅ are each independently a hydrogen atom or a halogen atom. ,
It is a group selected from the group consisting of 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 two adjacent substituents each form a condensed aromatic ring. Thioester compound represented by the formula (V
I); (In the formula, A ₂ is an amino acid residue or a peptide residue, B ₂ is a nitrogen protecting group, and R ′ _{6 to} R ′ ₁₁ are each independently a hydrogen atom, a halogen atom, an alkyl group, an alkoxyl group, an aryl group. Table in groups, a group selected from aralkyl and aralkyl alkoxyl group, or shows the two adjacent groups are bonded may also form a condensed ring group of R _'6 ~R' ₁₁₎ At least one of the thioester compounds
A method for detecting a hydrolase using a seed, a cobalt (II) salt, and a complex formation promoter.