JP2583440B2 - Substrate for measuring enzyme activity and method for measuring enzyme activity using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to an amide compound or a salt thereof, and a peptidase in a test solution using the amide compound or a salt thereof as a substrate, γ-
The present invention relates to a novel method for measuring the activities of glutamyl transpeptidase and leucine aminopeptidase.

[Prior Art] Peptidase has long been generally known as a general term for an enzyme that acts on an amide bond of a peptide to cleave from the amino terminus to release an amino acid or a lower peptide. For example, γ-glutamyl transpeptidase,
Enzymes such as leucine aminopeptidase, allyl amidase, cistyl aminopeptidase, proline iminopeptidase, arginine aminopeptidase, and alanine aminopeptidase are well known.

Among the above enzymes, particularly γ-glutamyl transpeptidase (hereinafter, referred to as γ-GTP), and leucine aminopeptidase, allyl amidase, cistyl aminopeptidase (hereinafter, these three enzymes are collectively referred to as leucine aminopeptidase [LAP]) Is widely distributed in tissues of living organisms, is present in serum, and is increased by pathological conditions. Therefore, it has a large diagnostic value and is an important enzyme activity measurement item in clinical tests.

γ-GTP is an enzyme whose presence was confirmed by Hanes in 1950, and is a membrane-bound enzyme that has the function of hydrolyzing γ-glutamyl peptide and transferring γ-glutamyl group to other peptides and amino acids. is there. Clinically
Since 1960, Orlowski, Goldbarg et al. Have begun to attract attention by measuring this enzyme using a synthetic substrate and reporting increased activity in liver and biliary tract diseases and pancreatic cancer. The diagnostic significance of this enzyme includes chronic hepatitis, cirrhosis, biliary tract disease, liver cancer,
Its usefulness has been demonstrated in alcoholic liver injury, myocardial infarction, etc., and has great diagnostic value and is an important measurement item in clinical tests.

LAP is an enzyme that hydrolyzes the N-terminal of a peptide to release amino acids such as leucine. As widely as γ-GTP, it is widely distributed in various tissues in a living body, and is also present in serum. Clinically, Goldbarg et al. Measured this enzyme using leucyl-β-naphthylamide, clinically applied it, and became widely used. The diagnostic significance of this enzyme is
Its usefulness has been shown in acute hepatitis, liver cancer, metastatic liver cancer, cirrhosis, and biliary tract disease, and has great diagnostic value and is an important measurement item in clinical tests.

Hitherto, many methods for measuring γ-GTP activity have been reported. Most of them are colorimetrically determining the amine compound generated by γ-GTP from the synthetic substrate, or by deriving the generated amine compound to another compound and colorimetrically determining the γ-GTP activity value. It is a method of seeking.
As a synthetic substrate, γ-L-glutamyl-p-nitroanilide is generally used, and p-produced by γ-GTP is used.
-A method of colorimetrically determining the yellow color of nitroaniline at 410 nm can be mentioned, but the solubility of the substrate is low, and it is necessary to take some means of solubilization. Usually, this disadvantage is solved by adding dilute hydrochloric acid, a surfactant, an organic solvent or the like to the dissolution of the substrate [Japanese Patent Laid-Open No. 52-29999]. However, even in this case, the stability of the substrate after dissolving the substrate is poor, and there is a problem in the measurement operation due to bubbles generated by the addition of the surfactant.
In recent years, there have been reports of using modified cyclodextrins (Japanese Patent Application Laid-Open No. 60-160896) and crown ethers (Japanese Patent Application Laid-Open No. 60-16599) to improve the solubility of this substrate, but these are expensive. Is not economical.

Furthermore, this method has an overlap between the absorption spectra of the substrate and the generated p-nitroaniline,
It was impossible to measure at the maximum wavelength of p-nitroaniline, and it had to be measured at the shoulder of the absorption spectrum. In addition, the effects of bilirubin, which is a coexisting substance in serum, and hemolysis could not be avoided.

In order to improve the solubility of γ-L-glutamyl-p-nitroanilide, γ-L-glutamyl-3-carboxy-4-nitroanilide (JP-A-49-86338) was developed, and the solubility of the substrate was improved. Has improved, but the effect of coexisting substances has not been eliminated.

There is also a method of colorimetrically determining the generated p-nitroaniline by condensing it with p-dimethylaminocinnamaldehyde. However, the effect of temperature on color development sensitivity is large, and there is a problem in reproducibility of measured values. There is also a method of diazotizing the produced p-nitroaniline and condensing it with m-xylenol to determine the colorimetrically, but there is a problem that the number of operation steps is large and the method is not simple.

In recent years, synthetic substrates such as γ-L-glutamyl-3-carboxy-4-hydroxyanilide and γ-L-glutamyl-3,5-dibromo-4-hydroxyanilide have been developed,
A method has been developed for the colorimetric determination of the dye formed by oxidative condensation of an aniline compound formed by γ-GTP with p-xylenol or the like, but there is a problem in operability due to the use of strong alkali and metaperiodate. There is.

On the other hand, many methods for measuring LAP activity have been reported. Most of them, like γ-GTP, are either colorimetrically determining amine compounds generated by LAP from synthetic substrates, or deriving the generated amine compounds to further compounds and colorimetrically determining LAP. This is a method for determining the activity value.

For example, there is a method using L-leucyl-β-naphthylamide as a synthetic substrate, and β-produced by LAP is used.
Naphthylamine is diazotized with sodium nitrite to give N-
This is a method of colorimetrically determining a dye produced by coupling to (1-naphthyl) -ethylenediamine or condensing p-dimethylaminocinnamaldehyde, but has the same disadvantages as γ-GTP, and Generate β-
Naphthylamine has the drawbacks of being extremely toxic and carcinogenic.

In addition, there is a method using L-leucyl-p-nitroanilide as a synthetic substrate, and a method for colorimetrically determining the yellow color of p-nitroaniline generated by LAP. It has drawbacks in that it must be colorimetrically determined on the basis of the nature and absorption of the generated p-nitroaniline, and the effect of coexisting substances in serum cannot be avoided.

[Problems to be Solved by the Invention] The present inventors have found that conventional γ-GTP and L
As a result of intensive studies to improve the method for measuring AP activity, a novel synthetic substrate having excellent solubility was found. In addition, a method for quantitatively determining a substance produced by the action of γ-GTP or LAP using a synthetic substrate using completely new oxygen was developed, and the present invention was completed as an excellent method for measuring peptidase activity.

[Means for Solving the Problems] That is, the present invention provides a compound of the general formula (I) useful as a peptidase synthetic substrate: (Wherein, R ₁ is a γ-L-glutamyl group, an L-leucyl group,
L-alanyl group, cistyl group, L-prolyl group or L-
Represents an arginyl group, R ₂ and R ₃ are each a hydrogen atom, a halogen atom, a lower alkyl group, a lower alkoxy group, an amino group, a substituted amino group, a hydroxyl group, a carboxyl group, a sulfonic acid group, a nitro group, a hydroxyalkyl group, It represents an alkyl group or a sulfoalkyl group.

However, when R ₁ is a γ-L-glutamyl group, R _{2 and} R ₃ are not both hydrogen atoms, and one of R ₂ and R ₃ is not a hydrogen atom and the other is not a hydroxyl group. ) Or a salt thereof.

Further, the present invention provides a compound represented by the general formula (II) (Wherein, R ₁ is a γ-L-glutamyl group, an L-leucyl group,
L-alanyl group, cistyl group, L-prolyl group or L-
Represents an arginyl group, R ₂ and R ₃ are each a hydrogen atom, a halogen atom, a lower alkyl group, a lower alkoxy group, an amino group, a substituted amino group, a hydroxyl group, a carboxyl group, a sulfonic acid group, a nitro group, a hydroxyalkyl group, It represents an alkyl group or a sulfoalkyl group. The peptidase contained in the test solution is allowed to act on the amide compound represented by the formula (Wherein R ₂ and R ₃ are each a hydrogen atom, a halogen atom, a lower alkyl group, a lower alkoxy group, an amino group, a substituted amino group, a hydroxyl group, a carboxyl group, a sulfonic acid group, a nitro group, a hydroxyalkyl group, a carboxyalkyl A 4-aminobenzoic acid hydroxylase and a reduced coenzyme are allowed to act on aminobenzoic acid or a derivative thereof represented by the following formula: A method for measuring peptidase activity, comprising measuring consumption of an amount.

Further, the present invention provides a compound represented by the general formula (IV) (Wherein, R ₁ represents a γ-L-glutamyl group, R ₂ and R ₃ each represent a hydrogen atom, a halogen atom, a lower alkyl group, a lower alkoxy group, an amino group, a substituted amino group, a hydroxyl group, a carboxyl group, a sulfone An amide compound represented by an acid group, a nitro group, a hydroxyalkyl group, a carboxyalkyl group or a sulfoalkyl group) or a salt thereof is reacted with γ-glutamyl transpeptidase contained in a test solution to liberate the compound. (III) (Wherein R ₂ and R ₃ are each a hydrogen atom, a halogen atom, a lower alkyl group, a lower alkoxy group, an amino group, a substituted amino group, a hydroxyl group, a carboxyl group, a sulfonic acid group, a nitro group, a hydroxyalkyl group, a carboxyalkyl A 4-aminobenzoic acid hydroxylase and a reduced coenzyme are allowed to act on aminobenzoic acid or a derivative thereof represented by the following formula: A method for measuring γ-glutamyl transpeptidase activity, which comprises measuring consumption of an amount.

Further, the present invention relates to a compound represented by the general formula (V): (Wherein, R ₁ is a γ-L-glutamyl group, an L-leucyl group,
L-alanyl group, cistyl group, L-prolyl group or L-
Represents an arginyl group, R ₂ and R ₃ are each a hydrogen atom, a halogen atom, a lower alkyl group, a lower alkoxy group, an amino group, a substituted amino group, a hydroxyl group, a carboxyl group, a sulfonic acid group, a nitro group, a hydroxyalkyl group, It represents an alkyl group or a sulfoalkyl group. ) The leucine aminopeptidase contained in the test solution is allowed to act on the amide compound represented by the formula (Wherein R ₂ and R ₃ are each a hydrogen atom, a halogen atom, a lower alkyl group, a lower alkoxy group, an amino group, a substituted amino group, a hydroxyl group, a carboxyl group, a sulfonic acid group, a nitro group, a hydroxyalkyl group, a carboxyalkyl A 4-aminobenzoic acid hydroxylase and a reduced coenzyme are allowed to act on aminobenzoic acid or a derivative thereof represented by the following formula: A method for measuring leucine aminopeptidase activity, which comprises measuring consumption of an amount.

In the present invention, the amide compound represented by the general formula (I) or a salt thereof, the amide compound represented by the general formula (II) or a salt thereof, the amide compound represented by the general formula (IV) or a salt thereof and the general formula (V The amide compound represented by the formula (1) or a salt thereof can be produced by a conventional method. That is, it is obtained by a condensation reaction between the γ-carboxyl group of L-glutamic acid or the α-carboxyl group of L-leucine and an aniline derivative. Upon the above condensation reaction, the functional groups (-NH _2, -COOH, etc.) that should not be involved in the pre-reaction it is preferable to protect at conventional protecting group.

For example, it is preferable to protect the α-amino group and α-carboxyl group of L-glutamic acid and the α-amino group of L-leucine. As the protecting group for the amino group, a protecting group usually used for peptide synthesis is used. For example, phthalyl,
Examples include t-butyloxycarbonyl, carbobenzoxy, benzyl, and formyl groups. As the protecting group for the carboxyl group, a protecting group usually used for peptide synthesis is used. For example, methyl ester, ethyl ester,
t-butyl ester, benzyl ester, p-nitrobenzyl ester and the like.

The above condensation reaction can be carried out according to a conventional method after protecting a functional group which should not participate in the reaction in advance. Examples of the condensation reaction include acid chloride, azide, acid anhydride, mixed acid anhydride, carbodiimide, active ester, isocyanate, phosphazo, and phosphite methods.

Removal of the protecting group after the condensation reaction can be carried out according to a conventional method. For example, phthalyl group which is a protecting group for amino group, hydrazine for formyl group, hydrogen chloride for t-butyloxycarbonyl group, carbobenzoxy, catalytic hydrogen reduction for benzyl group, carboxyl group protecting group. In the case of certain methyl ester and ethyl ester, saponification is used, in the case of t-butyl ester, hydrogen chloride, benzyl ester, and in the case of p-nitrobenzyl ester, catalytic hydrogen reduction is used.

The above condensation reaction can be carried out in an appropriate solvent, for example, in an inert organic solvent such as dimethylformamide, dimethylacetamide, dimethylsulfoxide, tetrahydrofuran, dioxane, benzene, and chloroform. Determined by the reaction.

In the present invention, the amide compound represented by the general formula (I) or a salt thereof, the amide compound represented by the general formula (II) or a salt thereof, the amide compound represented by the general formula (IV) or a salt thereof, and the general formula (V) The amide compound represented by or a salt thereof can form a salt with an inorganic acid such as hydrochloric acid, sulfuric acid or nitric acid, or a salt with an alkali metal such as sodium, potassium or lithium, if necessary.

Examples of the aniline derivative used in the condensation reaction include 4-aminobenzoic acid, 4-amino-2-chlorobenzoic acid, 4-aminosalicylic acid, 3,4-diaminobenzoic acid, 4-aminophthalic acid, and 4-amino- 2-iodobenzoic acid, 4-amino-2-bromobenzoic acid, 4-amino-
2-methylbenzoic acid, 4-amino-2,6-dimethylbenzoic acid, 4-amino-2-hydroxymethylbenzoic acid, and the like.

The amide compound represented by the general formula (I) or its salt, the amide compound represented by the general formula (II) or its salt, the amide compound represented by the general formula (IV) or its salt, and the general formula (V) of the present invention The amide compound represented by or a salt thereof can be obtained by the above-mentioned production method. For example, when γ-L-glutamyl-3-chloro-4-carboxyanilide is produced, 4-amino -2-Chlorobenzoic acid and N, N-phthalyl-L-glutamic anhydride are reacted in dimethylformamide.After the reaction, dimethylformamide is distilled off under reduced pressure, and hydrazine is added in methanol to remove a phthalyl group as a protecting group. Upon desorption, the desired product is obtained. When producing L-leucyl-4-carboxyanilide, 4-aminobenzoic acid is reacted with t-butyloxycarbonyl-L-leucine-N-hydroxysuccinimide ester, and 2N hydrochloric acid is added in acetic acid to protect the compound. When the t-butyloxycarbonyl group as a group is eliminated, the desired product is obtained.

Further, the present invention provides a novel method for measuring peptidase activity using the amide compound represented by the general formula (II) or a salt thereof as a substrate, and a novel γ using the amide compound represented by the general formula (IV) or a salt thereof as a substrate. To provide a novel method for measuring glutamyl transpeptidase (γ-GTP) activity and a novel leucine aminopeptidase (LAP) activity using an amide compound represented by the general formula (V) or a salt thereof as a substrate.

The present invention relates to a method in which an amide compound represented by the general formula (II) or a salt thereof is allowed to act on a peptidase contained in a test solution, or γ-glutamyl transpeptidase (γ-GTP) contained in the test solution. To the amide compound represented by the general formula (IV) or a salt thereof,
Alternatively, an amide compound represented by the general formula (V) or a salt thereof is allowed to act on leucine aminopeptidase (LAP) contained in a test solution, and the aminobenzoic acid represented by the general formula (III) or a salt thereof is liberated thereby. A new peptidase activity, γ-GTP activity, in which the derivative is subjected to monoatomic oxygenation with 4-aminobenzoic acid hydroxylase and reduced coenzyme, and the absorbance of reduced coenzyme reduction or consumption of dissolved oxygen is measured. Or a method for measuring LAP activity. or,
At this time, aminophenol, its derivative,
It is also possible to measure carbon dioxide.

 The reaction formula of the method of the present invention is shown below.

(Wherein R ₂ and R ₃ are each a hydrogen atom, a halogen atom, a lower alkyl group, a lower alkoxy group, an amino group, a substituted amino group, a hydroxyl group, a carboxyl group, a sulfonic acid group, a nitro group, a hydroxyalkyl group, a carboxyalkyl Represents an amino group or a sulfoalkyl group.) 4-Aminobenzoic acid hydroxylase is an intermolecular electron donor-required monoatomic oxygenase and is an enzyme that catalyzes the following reaction. (H.Tsuji, T.Ogawa, N.Bando, K.Sasaoka,
Journal of Biological Chemistry, 261 , (28), p.1
3203,1986].

(Wherein R ₂ and R ₃ are each a hydrogen atom, a halogen atom, a lower alkyl group, a lower alkoxy group, an amino group, a substituted amino group, a hydroxyl group, a carboxyl group, a sulfonic acid group, a nitro group, a hydroxyalkyl group, a carboxyalkyl The enzyme is obtained especially from food-grown mushrooms, such as Agaricus bisporus. In practicing the present invention, 4-aminobenzoic acid hydroxylase is
Any source (origin) may be used.
It may be present at least 01 units / ml, preferably 0.05 to 5
Unit / ml is good.

Further, flavin adenine dinucleotide (FAD) may be added to the enzyme if necessary. FAD may be a salt thereof, and the concentration used in the final test solution is preferably 0.0001 mM.
That is all.

FMN, riboflavin, riboflavin-4,5 instead of FAD
-Cyclic phosphate, flavin peptide, riboflavin glucoside, liposoluble riboflavin derivative may be used.

If necessary, albumin or the like may be added as a stabilizer for 4-aminobenzoic acid hydroxylase. Albumin may be derived from any source, for example, bovine serum albumin (BSA), human serum albumin (HSA) and the like.

The reduced coenzyme used in the present invention is reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH). NADH or NADPH may be present in the final test solution at a concentration of 0.05 mM or more, but is preferably 0.1 to 0.4 mM. NADH or NADP
H may be its salt.

As a method of measuring aminobenzoic acid or a derivative thereof by reducing the reduced coenzyme, a decrease in the absorbance of NAD (P) H is continuously measured by a change in absorbance per unit time (ΔA / mi
n) Measurement (rate assay method), measurement after a fixed time, or measurement after stopping the reaction by adding a reaction inhibitor after a fixed time (endpoint method)
can do. In addition, fluorescence, luminescence, immunoassay,
An electrode method or the like is used.

As a method of measuring the consumption of dissolved oxygen, it can be measured by an oxygen electrode or the like. As a method for measuring aminophenol or its dielectric substance, indophenol is prepared using phenol, naphthol, or the like, and it can be measured from its absorbance.

The reaction temperature is about 10 to 45 ° C, preferably 20 to 40 ° C. The pH may be such that the peptidase activity, γ-GTP activity or LAP activity in the test solution is high and within a stable pH range. Preferably, pH 5 to 9 is good. The test liquid (sample) to be measured may be any biochemical substance, and in particular, serum, urine and the like are used. Next, measurement of γ-GTP activity will be described in more detail. The activity of γ-GTP is determined by adding the amide compound represented by the general formula (IV) or a salt thereof to γ-GTP in the test solution.
The aminobenzoic acid represented by the general formula (III) or a derivative thereof is allowed to act in the presence of NAD (P) H and 4-aminobenzoic acid hydroxylase to give NAD (P) H Can be measured as a decrease in absorbance or by measuring the consumption of dissolved oxygen. In the reaction of γ-GTP, an appropriate amount of an amino acid or peptide such as glycylglycine is used as a receptor.

The pH is between 6.5 and 9.0, preferably between 7 and 8. As the buffer, phosphoric acid, organic acid, glycine, glycylglycine, Tris, and others widely used in the field of biochemistry may be used. The concentration may be about 0.01 to 0.5M.

Next, measurement of LAP activity will be described in more detail. L
The activity of AP is determined by reacting LAP in a test solution with an amide compound represented by the general formula (V) or a salt thereof, and releasing the aminobenzoic acid represented by the general formula (III) or a derivative thereof by γ- Measure the decrease in NAD (P) H as a decrease in absorbance in the presence of NAD (P) H and 4-aminobenzoic acid hydroxylase as in the case of GTP, or measure the consumption of dissolved oxygen Can be performed.

The pH is preferably from 6 to 8. As the buffer, the above γ-GTP
Is the same as

[Effect of Action] The amide compound represented by the general formula (I) or a salt thereof of the present invention useful as a synthetic substrate for peptidase has extremely high solubility, and means for improving the solubility like conventional synthetic substrates. Is not required.

Further, the peptidase, γ-GTP, and LAP of the present invention
Is a novel method for measuring activity, which uses no color reaction system and reduces the change in absorbance of NAD (P) H in comparison with the conventional method, so that peptidase, γ-GT
The activity of P or LAP can be measured. Further, since the maximum absorption wavelength of the reduced coenzyme is measured, there is no influence derived from the wavelength accuracy of the spectrophotometer and the slit width.

Furthermore, since the change in the absorbance of NAD (P) H is assayed, there is no influence from blood coexisting substances such as bilirubin and hemolysis.

Further, it has advantages such as being suitable for an automatic analyzer capable of processing a large number of samples, and being capable of simple and rapid activity measurement.

Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited thereto.

Example 1 Synthesis of potassium γ-L-glutamyl-3-chloro-4-carboxyanilide (γ-glu-PAClBK) 7.78 g (30 mm) of N, N-phthalyl-L-glutamic anhydride
ol) and 4.63 g of 4-amino-2-chlorobenzoic acid (27 mmo
l) in 30 ml of dimethylformamide (DMF)
For 1 hour. After the reaction, DMF was distilled off under reduced pressure, and the residue was dissolved by adding 50 ml of methanol, adjusted to pH 9.0 with 80% hydrazine-hydrate, and allowed to stand at room temperature for 2 days. The deposited precipitate was washed with methanol, suspended in 1N hydrochloric acid (75 ml), stirred at room temperature for about 30 minutes, filtered, and the filtrate was adjusted to pH 3.5 with 4N potassium hydroxide. The resulting precipitate was collected by filtration, washed with pure water, ethanol, and ether, and suspended by adding 30 ml of pure water, and adjusted to pH 8.0 with 2N potassium hydroxide. This solution was added with stirring to acetonitrile 1 and stirred at room temperature for about 1 hour. The resulting white precipitate was collected by filtration, washed with acetonitrile, ethanol and ether, and dried under reduced pressure. By the above operation, γ-L-glutamyl-3-chloro-4-
3.6 g of potassium 4-carboxyanilide was obtained (white powder).

Yield: 39%, melting point: 193 to 195 ° C. (decomposition) Silica gel TLC: Rf = 0.41 (n-butanol: acetic acid: water =
4: 1: 1) IR chart (KBr method): As shown in FIG.

Elemental analysis: C ₁₂ H ₁₂ N ₂ O ₅ ClK.H ₂ O (Molecular weight 356.79) Specific light intensity ▲ [α] ²² _D ▼: +22.5 (C = 1.0, 1N HCl) ▲ [α] ²⁸ _D ▼: +5.6 (C = 1.0, H ₂ O) ¹³ C NMR: Table 1 and As shown in FIG.

Example 2 L-leucyl-4-carboxyanilide (L-Leu-PAB
A) Synthesis of 4-aminobenzoic acid 5.49 g (40 mmol) and triethylamine 9.11 g (90 mmol) in dimethylformamide (DMF)
Dissolve in 80 ml, add t-butyloxycarbonyl-L
-Leucine-N-hydroxysuccinimide ester 1
A solution obtained by dissolving 4.77 g (45 mmol) in 120 ml of tetrahydrofuran was added dropwise at −10 ° C. over about 30 minutes and stirred for 1 hour.
Thereafter, the reaction was carried out at room temperature for 22 hours. After the reaction, the solvent was distilled off under reduced pressure, and the residue was dissolved by adding 400 ml of ethyl acetate.
After washing with 1% sodium bicarbonate, water, 1N hydrochloric acid and saturated saline, and drying over anhydrous magnesium sulfate, ethyl acetate was distilled off. 36 ml of 2.4N hydrochloric acid / acetic acid was added, and the mixture was stirred at 15 to 20 ° C. for 2 hours. A white precipitate formed upon addition to 500 ml of dry ether. The precipitate was collected by filtration, dissolved in 30 ml of water, adjusted to pH 7 with an aqueous sodium hydroxide solution, and after removing insolubles, concentrated to dryness under reduced pressure. The resulting white powder was purified by silica gel column chromatography (eluent, chloroform: methanol: acetic acid: water = 14: 6: 0.5: 1) to give the desired product, L-leucyl-4-carboxyanilide (L-Leu). −P
(ABA) 1 g (white powder).

Yield: 10%, melting point: 213 to 215 ° C (decomposition) Silica gel TLC: Rf = 0.42 (chloroform: methanol:
Acetic acid: water = 14: 6: 1: 1) IR chart (KBr method): as shown in FIG.

Elemental _{analysis: C 13 H 18 N 2 O} 3 · 2H 2 O ( molecular weight 286.30) ¹³ C NMR: as shown in Table 2 and FIG.

Example 3 Synthesis of L-leucyl-3-chloro-4-carboxyanilide (L-Leu-PAClBA) Synthesis was performed in the same manner as in Example 2 using 6.86 g (40 mmol) of 4-amino-2-chlorobenzoic acid. , L-leucyl-3
-Chloro-4-carboxyanilide (L-Leu-PAClB
A) 1 g was obtained (white powder).

Yield: 9%, melting point: decomposed at 120 ° C. or higher Silica gel TLC: Rf = 0.30 (chloroform: methanol:
Acetic acid: water = 14: 6: 1: 1) IR chart (KBr method): as shown in FIG.

Elemental _{analysis: C 13 H 17 N 2 O} 3 Cl · 3H 2 O ( molecular weight 338.76) ¹³ C NMR: as shown in Table 3 and FIG.

Example 4 γ-GTP using γ-L-glutamyl-3-chloro-4-carboxyanilide potassium salt (γ-glu-PAClBK)
Activity measurement (1) Preparation of reagent Prepare 100 ml of 50 mM phosphate-potassium-potassium hydroxide buffer (pH 7.0) containing RI 100 mM glycylglycine 0.28 mM NADH.

R-II 100 mM glycylglycine 50 mM γ-glu-PAClBK 2.5 U / ml 4-aminobenzoic acid hydroxylase 100 μM FAD 0.1% BSA containing 50 mM phosphate-potassium-potassium hydroxide buffer (pH 7.0) 100 ml Prepare.

(2) Measurement procedure Take 400 μ of RI and 50 μ of serum, heat at 37 ° C. for 5 minutes, add 100 μ of R-II, and immediately measure the decrease in absorbance per unit time at 340 nm at 37 ° C. γ-
The GTP activity value is calculated by the following equation. (Reagent blank uses pure water instead of serum.) ΔA340 / min is the change in absorbance at 340 nm per minute. The molecular extinction coefficient of NADH is 6.22 × 10 ^3. It was shown to. The results correlated well with the conventional method.

FIG. 7 shows the results obtained by serially diluting the serum containing a high unit of γ-GTP and performing the same procedure as described above. A linear relationship passing through the origin was obtained, indicating that serum γ-GTP was accurately measured.

Example 5 Measurement of γ-GTP activity using γ-L-glutamyl-4-carboxyanilide potassium salt (γ-glu-PABK) (1) Preparation of reagent RI Same as in Example 4.

R-II Instead of 50 mM γ-glu-PAClBK of R-II of Example 4, 50 mM γ-glu-PABK, 2.5 U / ml 4-aminobenzoic acid hydroxylase is used at 1.7 U / ml. Others are the same.

(2) Measurement operation method Same as in Example 4. The results obtained are shown in Table 5 in comparison with the conventional γ-glutamyl-p-nitroanilide method. The results correlated well with the conventional method.

FIG. 8 shows the results obtained by serially diluting the serum containing a high unit of γ-GTP and performing the same procedure as described above. The result that the serum γ-GTP was measured well was obtained.

Example 6 L-leucyl-4-carboxyanilide (L-Leu-PAB
Measurement of LAP activity using A) (1) Preparation of reagent Prepare 100 ml of 50 mM phosphate-potassium-potassium hydroxide buffer (pH 7.0) containing RI 0.28 mM NADH.

R-II 15 mM L-Leu-PABA 2.0 U / ml 4-Aminobenzoic acid hydroxylase Prepare 100 ml of 50 mM phosphate-potassium-potassium hydroxide buffer (pH 7.0) containing 100 μM FAD 0.1% BSA.

(2) Measuring procedure Take 400 μ of RI and 50 μ of serum, heat at 37 ° C. for 3 minutes, add 100 μ of R-II, and immediately measure the decrease in absorbance per unit time at 340 nm at 37 ° C. LAP
The activity value is calculated by the following equation. (Reagent blank uses pure water instead of serum.) ΔA340 / min is the change in absorbance at 340 nm per minute. The molecular extinction coefficient of NADH was measured by serially diluting 6.22 × 10 ³ LAP high units of serum, and the results are shown in FIG. The result that the serum LAP was measured well was obtained.

Example 7 LAP activity measurement using L-leucyl-3-chloro-4-carboxyanilide (L-Leu-PAClBA) (1) Preparation of reagent RI Same as in Example 6.

R-II Instead of 15 mM L-Leu-PABA of R-II of Example 6, 15 mM L-Leu-PAClBA, 2.0 U / ml 4-aminobenzoic acid hydroxylase is used at 3.5 U / ml. Others are the same.

(2) Measurement operation method Same as in Example 6.

Serially dilute the LAP high unit serum and measure the results.
This is shown in FIG. The result that the serum LAP was measured well was obtained.

Example 8 Measurement of γ-GTP Activity Using an Oxygen Sensor Using γ-glu-PAClBK (1) Preparation of Reagent The same as in Example 4.

(2) Measuring operation method RI: R-II: Mix at a ratio of 4: 1 to obtain a measurement solution. The measurement solution is heated to 37 ° C., introduced into a cell in contact with the oxygen sensor, and 100 μl of serum is added. The consumption of oxygen concentration at this time is measured by an oxygen sensor (Stat Glucose Analyzer S-80: manufactured by AIC).

(3) γ-GTP activity unit conversion 100 μm of serum (standard serum) with a known unit was operated in the same manner as the above-mentioned measurement method, and expressed as a ratio with the measured value.

FIG. 11 shows the measurement results obtained by serially diluting serum containing a high unit of γ-GTP. The result that the serum γ-GTP was measured well was obtained.

Example 9 LAP activity measurement by oxygen sensor using L-Leu-PAClBA (1) Preparation of reagent The same as in Example 7.

(2) Measurement operation method The same as in Example 8.

(3) Conversion to LAP activity value unit Same as in Example 8.

Serially dilute the LAP high unit serum and measure the results.
It is shown in Figure 2. The result that the serum LAP was measured well was obtained.

[Brief description of the drawings]

FIG. 1 is an IR spectrum of potassium γ-L-glutamyl-3-chloro-4-carboxyanilide, and FIG.
¹³ C NMR spectrum of -L-glutamyl-3-chloro-4-carboxyanilide potassium salt, FIG. 3 shows the IR spectrum of L-leucyl-4-carboxyanilide, FIG.
The figure shows ¹³ C NMR of L-leucyl-4-carboxyanilide.
Spectrum, FIG. 5 shows L-leucyl-3-chloro-4-
IR spectrum of carboxyanilide. FIG. 6 shows ¹³ C NMR of L-leucyl-3-chloro-4-carboxyanilide.
FIG. 7 is a calibration curve in Example 4, FIG. 8 is a calibration curve in Example 5, FIG. 9 is a calibration curve in Example 6, FIG. 10 is a calibration curve in Example 7, and FIG. FIG. 12 shows a calibration curve in Example 8, and FIG. 12 shows a calibration curve in Example 9.

Continuation of the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical indication location C12Q 1/37 C12Q 1/37 1/48 7823-4B 1/48 A (56) References JP-A-54- 157691 (JP, A) JP-A-55-69549 (JP, A) JP-A-56-158745 (JP, A)

Claims (4)

(57) [Claims] 1. The compound of the general formula (I) (Wherein, R ₁ is a γ-L-glutamyl group, an L-leucyl group,
L-alanyl group, cistyl group, L-prolyl group or L-
Represents an arginyl group, R ₂ and R ₃ are each a hydrogen atom, a halogen atom, a lower alkyl group, a lower alkoxy group, an amino group, a substituted amino group, a hydroxyl group, a carboxyl group, a sulfonic acid group, a nitro group, a hydroxyalkyl group, It represents an alkyl group or a sulfoalkyl group. However, when R ₁ is a γ-L-glutamyl group, R _{2 and}
R ₃ is not both a hydrogen atom, and one of R ₂ and R ₃ is not a hydrogen atom and the other is not a hydroxyl group. Or a salt thereof. 2. A compound of the general formula (II) (Wherein, R ₁ is a γ-L-glutamyl group, an L-leucyl group,
L-alanyl group, cistyl group, L-prolyl group or L-
Represents an arginyl group, R ₂ and R ₃ are each a hydrogen atom, a halogen atom, a lower alkyl group, a lower alkoxy group, an amino group, a substituted amino group, a hydroxyl group, a carboxyl group, a sulfonic acid group, a nitro group, a hydroxyalkyl group, It represents an alkyl group or a sulfoalkyl group. The peptidase contained in the test solution is allowed to act on the amide compound represented by the formula (Wherein R ₂ and R ₃ are each a hydrogen atom, a halogen atom, a lower alkyl group, a lower alkoxy group, an amino group, a substituted amino group, a hydroxyl group, a carboxyl group, a sulfonic acid group, a nitro group, a hydroxyalkyl group, a carboxyalkyl A 4-aminobenzoic acid hydroxylase and a reduced coenzyme are allowed to act on aminobenzoic acid or a derivative thereof represented by the following formula: A method for measuring peptidase activity, comprising measuring consumption of an amount. 3. A compound of the general formula (IV) (Wherein, R ₁ represents a γ-L-glutamyl group, R ₂ and R ₃ each represent a hydrogen atom, a halogen atom, a lower alkyl group, a lower alkoxy group, an amino group, a substituted amino group, a hydroxyl group, a carboxyl group, a sulfone An amide compound represented by an acid group, a nitro group, a hydroxyalkyl group, a carboxyalkyl group or a sulfoalkyl group) or a salt thereof is reacted with γ-glutamyl transpeptidase contained in a test solution to liberate the compound. (III) (Wherein R ₂ and R ₃ are each a hydrogen atom, a halogen atom, a lower alkyl group, a lower alkoxy group, an amino group, a substituted amino group, a hydroxyl group, a carboxyl group, a sulfonic acid group, a nitro group, a hydroxyalkyl group, a carboxyalkyl A 4-aminobenzoic acid hydroxylase and a reduced coenzyme are allowed to act on aminobenzoic acid or a derivative thereof represented by the following formula: A method for measuring γ-glutamyl transpeptidase activity, comprising measuring consumption of an amount. 4. The formula (V) (Wherein, R ₁ is a γ-L-glutamyl group, an L-leucyl group,
L-alanyl group, cistyl group, L-prolyl group or L-
Represents an arginyl group, R ₂ and R ₃ are each a hydrogen atom, a halogen atom, a lower alkyl group, a lower alkoxy group, an amino group, a substituted amino group, a hydroxyl group, a carboxyl group, a sulfonic acid group, a nitro group, a hydroxyalkyl group, It represents an alkyl group or a sulfoalkyl group. ) The leucine aminopeptidase contained in the test solution is allowed to act on the amide compound represented by the formula (Wherein R ₂ and R ₃ are each a hydrogen atom, a halogen atom, a lower alkyl group, a lower alkoxy group, an amino group, a substituted amino group, a hydroxyl group, a carboxyl group, a sulfonic acid group, a nitro group, a hydroxyalkyl group, a carboxyalkyl A 4-aminobenzoic acid hydroxylase and a reduced coenzyme are allowed to act on aminobenzoic acid or a derivative thereof represented by the following formula: A method for measuring leucine aminopeptidase activity, which comprises measuring consumption of an amount.