JPH0627111B2 - Novel .GAMMA.-L-glutamyl-4-nitroanilide derivative and method for measuring .GAMMA.-GTP activity using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention is a novel γ-L-glutamyl-useful substrate for measuring γ-glutamyl transpeptidase (hereinafter abbreviated as γ-GTP) activity. The present invention relates to a 4-nitroanilide derivative and a γ-GTP activity measuring method using the same.

BACKGROUND OF THE INVENTION γ-GTP hydrolyzes γ-glutamyl peptides to give γ
-A membrane-bound enzyme that acts to transfer a glutamyl group to another peptide or amino acid.

The measurement of γ-GTP activity is widely used for diagnosis of liver / biliary tract diseases, screening for alcoholism and the like.
Recently, γ-G specifically appearing in the serum of patients with hepatocellular carcinoma
The diagnostic value of the activity measurement of γ-GTP is renewed attention, such as the discovery of TP isozyme and the report of the relationship between γ-GTP in urine and pathological conditions.

As a method for measuring γ-GTP activity, various methods have been proposed and put into practical use so far, but a reaction rate measurement method using γ-L-glutamyl-p-nitroanilide as a substrate (late assay method) Is the most common and is still popular today.

However, the problem that the substrate γ-L-glutamyl-p-nitroanilide is poorly soluble in the optimum pH range for measuring the activity of γ-GTP, and is unstable after dissolution in the pH range where solubility is obtained to some extent Have a point.

On the other hand, as a water-soluble substrate with these points improved, γ-L-
Glutamyl-3-carboxy-4-nitroanilide and γ
There are -L-glutamyl-3-sulfo-4-nitroanilide and the like (Japanese Patent Publication No. 54-7781). These water-soluble substrates are far superior in solubility and stability after dissolution as compared with the above-mentioned γ-L-glutamyl-p-nitroanilide, but are still not satisfactory in terms of stability after dissolution. It is not stable enough and there is room for improvement.

When measuring the activity of γ-GTP in human serum,
Serum containing γ-GTP of known concentration (control serum)
Is often used as a standard or an index for quality control, and γ added to such control serum
As GTP, those derived from bovine kidney or pig kidney are mainly used. That is, there is a high possibility that serum hepatitis virus is present in human serum having a high γ-GTP content, and there is a sanitary problem in purifying γ-GTP from this, and a large amount of γ-GTP is required. Γ-GT derived from bovine kidney or swine kidney as a substitute because human tissues included in
That is, P is used. However, since the water-soluble substrate as described above has a stronger polar group (water-soluble group) than γ-L-glutamyl-p-nitroanilide, the charge state of γ-GTP, especially the charge state in the vicinity of the active site. The reactivity as a substrate easily changes depending on the
Reactivity as a substrate is considerably different for each γ-GTP having different origins such as bovine kidney and pig kidney. That is, a sample prepared by dissolving various γ-GTPs as described above in a predetermined molar concentration is used as a sample, and the water-soluble substrate as described above is used to
When the activity value of GTP [international unit (IU) / l] is measured, the difference between the activity values is considerably large. Therefore, when a water-soluble substrate as described above is used in the measurement of γ-GTP in human serum using normal control serum as a standard or an index for quality control, conventional γ
As compared with the assay method using -L-glutamyl-p-nitroanilide as a substrate, there is a drawback in that control serum needs to be managed more strictly.

[Object of the Invention] The present invention has been made in view of the above situation, and is water-soluble, has good stability after dissolution, and has a different γ
An object of the present invention is to provide a novel substrate for measuring γ-GTP activity with a small difference in reactivity with GTP and a method for measuring γ-GTP activity using the same.

[Structure of the Invention] The present invention has the general formula (In the formula, R represents a lower hydroxyalkyl group which may have two or more hydroxyl groups.) Γ-L-
It is an invention of a glutamyl-4-nitroanilide derivative and a method for measuring γ-GTP activity using the same as a substrate.

That is, the present inventors have introduced a novel γ-L- group in which a hydroxyalkyl group having a remarkably weak polarity, unlike a carboxyl group or a sulfonic acid group, is introduced at the ortho position of a nitro group as a water-soluble group.
If a glutamyl-4-nitroanilide derivative is created and used as a substrate for γ-GTP activity measurement, the above problems will be greatly solved, and it becomes possible to assemble an unprecedented excellent γ-GTP activity measurement method. As a result of thought and earnest research,
The present invention has been completed.

The compound of the present invention represented by the above general formula is a novel compound which has not been published in the literature.

The feature of the novel compound of the present invention is that the water-soluble group bonded to the ortho position of the nitro group is a hydroxyalkyl group having extremely weak polarity unlike the carboxyl group and the sulfonic acid group.

In the compound of the present invention represented by the above general formula, examples of the lower hydroxyalkyl group represented by R, which may have two or more water-soluble groups, include hydroxymethyl group and 1
-Hydroxyethyl group, 2-hydroxyethyl group, 1-
Hydroxypropyl group, 2-hydroxypropyl group, 3
-Hydroxypropyl group, 1,2-dihydroxypropyl group, 1,3-dihydroxypropyl group, 2,3-dihydroxypropyl group, 1,4-dihydroxybutyl group, 2,3-dihydroxybutyl group, 3,4-dihydroxy group Examples thereof include a lower hydroxyalkyl group having 1 to 4 carbon atoms such as a butyl group.

The compound of the present invention comprises a corresponding 4-nitroaniline derivative and N-protected-L-glutamic acid having a carboxyl group activated such as N-phthaloyl-L-glutamic anhydride.
It can be easily synthesized by reacting in an organic solvent such as dioxane or dimethyl sulfoxide, and then removing the amino protecting group with hydrazine or the like.

The 4-nitroanilide derivative as an intermediate raw material is obtained by acylating a corresponding aniline derivative and then nitrating to isolate an N-acyl derivative in which a desired position is nitrated, and then hydrolyzing this to remove an acyl group. Can be easily synthesized.

Since the γ-L-glutamyl-4-nitroanilide derivative of the present invention has a hydroxyl group at the ortho position of the nitro group via a lower hydrocarbon group, it is excellent in water solubility and stability after dissolution, and Unlike the carboxyl group and the sulfonic acid group, the hydroxyalkyl group is a remarkably weak polar group.
Highly reactive with GTP and γ-derived from various animals
It has excellent properties as a substrate such as a small difference in reactivity with GTP, and is an excellent substrate for measuring γ-GTP activity.

The γ-GTP activity measuring method of the present invention uses the γ-L of the present invention as a substrate.
It is sufficient to carry out this according to a known γ-GTP activity measuring method using γ-glutamyl-p-nitroanilide as a substrate except that a -glutamyl-4-nitroanilide derivative is used. That is, γ-L-glutamyl-of the present invention
Using a 4-nitroanilide derivative as a substrate, in the presence of a glutamate receptor such as glycylglycine, a sample such as human serum or human urine and the substrate are added to an appropriate buffer to cause a reaction, and γ -The amount of the 4-nitroaniline derivative produced by the enzymatic action of GTP can be measured, for example, by an initial rate measuring method (rate assay method or the like) in which its absorbance is directly measured, or by adding p-dialkylaminobenzaldehyde or p-
It may be measured by a method of adding dialkylaminocinnamaldehyde or the like to develop a color and measuring the absorbance, and the measuring method itself is arbitrary. In order to equalize the inhibition caused by amino acids in serum, it is optional to add amino acids such as glutamic acid to the reagent in advance. Further, the measurement conditions such as the buffer solution, the reaction solution pH, the amount of the substrate and the acceptor, the measurement temperature, the measurement wavelength and the like may be according to the conventional method.

The substrate of the present invention has been conventionally used as a standard substrate for measuring γ-GTP activity, γ-L-glutamyl-p-nitroanilide and γ-L-glutamyl-3-carboxy-4-.
Nitroanilide has various characteristics that could not be used alone, and it has excellent properties in all aspects of water solubility, stability after dissolution, and reactivity with γ-GTP, which is extremely high than ever before. As an excellent substrate for γ-GTP activity measurement, its effect is fully demonstrated.

Examples will be shown below, but the present invention is not limited by these examples.

[Example] Example 1. Synthesis of γ-L-glutamyl-3-hydroxymethyl-4-nitroanilide 3aminobenzyl alcohol 24.6 g and acetic anhydride 81.6 g
And was refluxed for 3 hours. After completion of the reaction, post-treatment was carried out according to a conventional method to obtain 34.6 g of crystals of 3-acetoxymethylacetanilide.

mp 81-83 ° C.

56 g of concentrated nitric acid (d = 1.42) was added to 300 ml of acetic anhydride, and 3-acetoxymethylacetanilide 3 obtained above was added to this.
2.0 g was added and dissolved, and reacted at 14 to 16 ° C for 2 hours.
After completion of the reaction, the target product was extracted according to the usual method, and the extracted oil
7.9 g was purified by column chromatography and then crystallized to obtain 9.7 g of 3-acetoxymethyl-4-nitroacetanilide crystals.

mp 110 ° C.

The obtained 3-acetoxymethyl-4-nitroacetanilide (9.7 g) was dissolved in 100 ml of 2N-hydrochloric acid with heating, and the mixture was refluxed for 0.5 hours and then post-treated according to a conventional method to give 3-hydroxymethyl-4-nitroanilide. 5.4 g of crystals were obtained.

mp 143-145 ° C.

UV λ _max : 382 nm, ε = 1.28 × 10 ⁴ (H ₂ O).

IR υ cm ^-1 (KBr): 3460, 3360 (NH ₂ )), 3250 (O
_{H), 1340 (NO 2)} .

NMR δ ppm (Acetone-d ₆ ): 2.95 (2H, s, -C H ₂ OH), 4.50
(1H, d, CH ₂ O H ), 6.00 (2H, br.,-N H ₂ ), 6.50 to 6.90 (1H, dd, 7.05 ~ 7.35 (1H, d, 7.90-8.20 (1H, d, 11.8 g of N-phthaloyl-L-glutamic anhydride was added to the total amount of 3-hydroxymethyl-4-nitroaniline obtained.
And 30 ml of dioxane were added and dissolved by heating, and the mixture was refluxed for 7 hours. After completion of the reaction, post-treatment was carried out according to a conventional method to obtain 11.8 g of crystals of N-phthaloyl-γ-L-glutamyl-3-hydroxymethyl-4-nitroanilide.

mp 211-213 ° C.

To the total amount of the obtained N-phthaloyl-γ-L-glutamyl-3-hydroxymethyl-4-nitroanilide, 140 ml of methanol was added and dissolved, 4.6 g of 80% hydrazine hydrate was added, and the mixture was heated at 17 to 20 ° C. for 5 minutes. After stirring for an hour, it was left overnight. After completion of the reaction, post-treatment was carried out according to a conventional method to give γ-L-glutamyl-3.
5.7 g of crystals of hydroxymethyl-4-nitroanilide
Got

mp 156 ° C (dec).

UV λ _max : 317 nm, ε = 1.01 × 10 ⁴ (H ₂ O).

IR υcm ^-1 (KBr): 3400 (NH, NH ₂ ), 3230 (OH), 16
80 (CO), 1620 (COOH), 1320,1500 (NO ₂ ).

NMR δ ppm (DMSO-d ₆ ): 1.7 to 2.3 (2H, br., -CHC H ₂ CH
_2- ), 2.3 to 3.0 (2H, br.,-CHCH ₂ C H _2- ), 3.1 to 3.7 (1H, br.,
-C H CH ₂ CH _2- ), 4.8 (2H, s, -C H ₂ OH), 5.5 ~ 8.0 (4H, br., N
_{H 2, CON H, O H} ), 7.5~8.2 (3H, m, phenyl), 10.5~11.5 (1H,
br.,-COO H ).

Example 2. Synthesis of γ-L-glutamyl-3- (α-hydroxyethyl) -4-nitroanilide 12.3 g of 3- (α-hydroxyethyl) aniline was dissolved in 35 ml of acetic anhydride and reacted at 105 to 110 ° C for 1.5 hours. . After completion of the reaction, the target product was extracted according to a conventional method to obtain 20 g of an oil containing 3- (α-acetoxyethyl) acetanilide.

100 ml of acetic anhydride was added to 19 g of the obtained oil, 21 ml of concentrated nitric acid (d = 1.42) was added thereto at 0 to 10 ° C., and the reaction was carried out at the same temperature for 4 hours. After completion of the reaction, the desired product was extracted by a conventional method, and 1.8 g of 3- (α-acetoxyethyl) -4-nitroacetanilide oil was obtained from 19 g of the extracted oil by column chromatography.

To the total amount of the obtained 3- (α-acetoxyethyl) -4-nitroacetanilide, 10 ml of 2N-hydrochloric acid was added and dissolved by heating, and the mixture was reacted at 99 to 101 ° C for 1 hour. After completion of the reaction, the desired product was extracted by a conventional method to obtain 1.1 g of 3- (α-hydroxyethyl) -4-nitroaniline oil.

UV λ _max : 380 nm, ε = 0.96 × 10 ⁴ (H ₂ O).

NMR δ ppm (DMSO-d ₆ ): 1.55 (3H, d, -C H ₃ ), 5.0 (1H, m,
-O H ), 5.3 to 5.7 (1H, m, 5.8 ~ 6.3 (2H, m, -N H ₂ ), 6.5 (1H, dd, J = 18Hz, 3Hz), 7.2 (1H, d, J = 3Hz), 7.8 (1H, d, J = 18Hz).

To 1.1 g of the obtained 3- (α-hydroxyethyl) -4-nitroaniline, 1.9 g of N-phthaloyl-L-glutamic anhydride and 20 ml of dioxane were added and dissolved by heating to 90-9
The reaction was carried out at 5 ° C for 3.5 hours. After completion of the reaction, post-treatment was performed according to a conventional method to give N-phthaloyl-γ-glutamyl-3- (α-
Viscous oil of hydroxyethyl) -4-nitroanilide 2.
7 g was obtained.

Obtained N-phthaloyl-γ-L-glutamyl-3-
To the whole amount of (α-hydroxyethyl) -4-nitroanilide, 30 ml of methanol was added and dissolved, 0.5 ml of 80% hydrazine hydrate was added, and the mixture was stirred at 18 to 20 ° C. for 4 hours and then left overnight. After completion of the reaction, post-treatment was carried out according to a conventional method to obtain 0.5 g of crystals of γ-L-glutamyl-3- (α-hydroxyethyl) -4-nitroanilide.

mp 160 ° C (dec).

UV λ _max : 306 nm, ε = 0.52 × 10 ⁴ (H ₂ O).

NMR δ ppm (DMSO-d ₆ ): 1.35 (3H, d, -C H ₃ ), 1.5 to 3.0
(4H, m, 3.0 to 3.5 (1H, m, 4.5 ~ 6.2 (6H, br.,-COO H , -N H ₂ , 7.8-8.2 (3H, m, phenyl).

Example 3. Synthesis of γ-L-glutamyl-3- (β-hydroxyethyl) -4-nitroanilide 10.3 g of 3- (β-hydroxyethyl) aniline was dissolved in 30 ml of acetic anhydride and reacted at 109-112 ° C for 0.5 hours. . After completion of the reaction, the target product was extracted by a conventional method to obtain 17.2 g of an oil containing 3- (β-acetoxyethyl) acetanilide.

To 140 ml of acetic anhydride was added 27 g of concentrated nitric acid (d = 1.42), and to this was added a solution of 17.0 g of 3- (β-acetoxyethyl) acetanilide obtained above in 15 ml of acetic anhydride.
Reacted for 2 hours at -16 ° C. After completion of the reaction, the target product was extracted by a conventional method, and 18 g of the extracted oil was purified and crystallized by column chromatography to obtain 1.7 g of 3- (β-acetoxyethyl) -4-nitroacetanilide crystals.

mp 137-139 ° C.

1.6 g of the obtained 3- (β-acetoxyethyl) -4-nitroacetanilide was added to 16 ml of 2N-hydrochloric acid and dissolved by heating, and reacted at 99 to 101 ° C. for 0.5 hours. After completion of the reaction, the desired product was extracted and purified by a conventional method to obtain 0.88 g of crystals of 3- (β-hydroxyethyl) -4-nitroanilide.

mp 95-96 ° C.

UV λ _max : 381 nm, ε = 1.09 × 10 ⁴ (H ₂ O).

IR υcm ^-1 (KBr): 3460 (OH), 3230, 3350 (NH ₂ ),
1305 (NO ₂ ).

NMR δ ppm (CDCl ₃ + DMSO-d ₆ ): 3.2 (2H, t, -C H ₂ OH), 3.
8 (1H, br.,-O H ), 3.9 (2H, t, -CH ₂ C H ₂ OH), 5.3 (2H, br.,-N H
₂ ), 6.4-8.1 (3H, m, phenyl). 0.80 g of the obtained 3- (β-hydroxyethyl) -4-nitroaniline was added to 1.59 g of N-phthaloyl-L-glutamic anhydride and dioxane.
15 ml was added and dissolved by heating and reacted at 95 ° C. for 4 hours. After completion of the reaction, the product was purified by column chromatography according to a conventional method and then crystallized to give N-phthaloyl-γ-L-
1.4 g of crystals of glutamyl-3- (β-hydroxyethyl) -4-nitroanilide were obtained.

mp 70-95 ° C.

Obtained N-phthaloyl-γ-L-glutamyl-3-
(Β-hydroxyethyl) -4-nitroanilide 1.3 g
15 ml of methanol was added to and dissolved, 0.5 g of 80% hydrazine hydrate was added, and the mixture was stirred at 24-26 ° C for 5 hours and then left overnight. After completion of the reaction, post-treatment was carried out according to a conventional method to obtain 0.52 g of crystals of γ-L-glutamyl-3- (β-hydroxyethyl) -4-nitroanilide.

mp 164-167 ° C.

UV λ _max : 309 nm, ε = 0.816 × 10 ⁴ (H ₂ O).

IR υ cm ^-1 (KBr): 3450 (OH), 3320,3250 (NH ₂ ),
1680 (CONH), 1620 (COOH), 1345, 1505 (NO ₂ ).

NMR δ ppm (DMSO-d ₆ ): 1.8 to 2.4 (2H, br., -CHC H ₂ CH
_2- ), 2.4 to 2.9 (2H, br.,-CHCH ₂ C H _2- ), 2.9 to 3.3 (2H, t, -C
H ₂ CH ₂ OH), 3.3 to 3.9 (3H, m, -CH ₂ C H ₂ OH, -C H CH ₂ CH _2- ), 5.5
~ 8.0 (5H, br., N H ₂ , -COO H , -CON H -,-CH ₂ CH ₂ O H ), 7.5 ~ 8.1
(3H, m, phenyl).

Example 4. Synthesis of γ-L-glutamyl-3-hydroxymethyl-4-nitroanilide 3aminobenzyl alcohol 24.6 g and acetic anhydride 81.6 g
And was refluxed for 3 hours. After completion of the reaction, post-treatment was carried out according to a conventional method to obtain 34.6 g of crystals of 3-acetoxymethylacetanilide.

A mixture of 150 ml of glacial acetic acid and 150 ml of concentrated sulfuric acid was added to concentrated nitric acid (d = 1.4
2) 56 g was added, and 32.0 g of 3-acetoxymethylacetanilide obtained above was added to the solution and dissolved,
The reaction was carried out at 0 ° C for 2 hours. After completion of the reaction, the target product was extracted by a conventional method, and 57.5 g of the extracted oil was recrystallized from 700 ml of toluene to obtain 17.5 g of crystals of 3-acetoxymethyl-4-nitroacetanilide.

17.5 g of the obtained 3-acetoxymethyl-4-nitroacetanilide was dissolved in 180 ml of 2N-hydrochloric acid with heating, and the mixture was refluxed for 0.5 hours and then subjected to post-treatment according to a conventional method to give 3-hydroxymethyl-4-nitroanilide. 9.7 g of crystals were obtained.

21.3 g of N-phthaloyl-L-glutamyl anhydride was added to the total amount of 3-hydroxymethyl-4-nitroanilide obtained.
And 55 ml of dioxane were added and dissolved by heating, and the mixture was refluxed for 7 hours. After completion of the reaction, post-treatment was carried out according to a conventional method to obtain 21.3 g of N-phthaloyl-γ-L-glutamyl-3-hydroxymethyl-4-nitroanilide crystals.

To the total amount of the obtained N-phthaloyl-γ-L-glutamyl-3-hydroxymethyl-4-nitroanilide, 250 ml of methanol was added and dissolved, and 8.3 g of 80% hydrazine hydrate was added, and the mixture was heated at 17 to 20 ° C. for 5 minutes. After stirring for an hour, it was left overnight. After completion of the reaction, post-treatment was carried out by a conventional method to give γ-L-glutamyl-3.
-Hydroxymethyl-4-nitroanilide crystals 10.3 g
Got

The physical properties of the obtained compounds were the same as those obtained in Example 1.

Example 5. Measurement of γ-GTP activity (Preparation of reagents) Buffer solution Tris (hydroxymethyl) aminomethane 100 mmol and glycylglycine 60 mmol were added to purified water 800 ml, and an appropriate amount of hydrochloric acid was added thereto to adjust pH to 8.2 (at 25 ° C). The total volume was adjusted to 1 liter with purified water.

Substrate solution 15 mmol of γ-L-glutamyl-3-hydroxymethyl-4-nitroanilide obtained in Example 1 was dissolved in purified water to make a total amount of 1 liter.

(Sample) Human serum and a 7% bovine albumin aqueous solution to which γ-GTP derived from pig kidney or bovine kidney was added (hereinafter, abbreviated as pig sample and bovine sample, respectively) were used as samples.

(Measurement method) Add 2.0 ml of buffer solution to 50 μl of sample, preheat at 37 ° C for 3 minutes, add 0.5 ml of substrate solution, mix well and leave for 1 minute, then measure the rate of increase in absorbance at 410 nm do it,
From the molecular extinction coefficient (7.6 × 10 ⁶ cm ² / mol) of the produced 3-hydroxymethyl-4-nitroaniline, γ-G in the sample
The TP activity value (IU / 1) was calculated.

Example 6 Measurement of γ-GTP activity (Preparation of test solution) Buffer solution Tris (hydroxymethyl) aminomethane 100 mmol and glycylglycine 60 mmol were added to purified water 800 ml, and hydrochloric acid was added thereto to adjust pH to 8.2 (at 25 ° C.). After that, the total volume was adjusted to 1 liter with purified water.

Substrate solution 15 mmol of γ-L-glutamyl-3- (β-hydroxyethyl) -4-nitroanilide obtained in Example 3 was dissolved in purified water to make a total amount of 1 liter.

(Sample) The same sample as in Example 5 was used.

(Measurement method) 2.0 ml of buffer solution was added to 50 μl of sample and pre-heated at 37 ° C. for 3 minutes, 0.5 ml of substrate solution was added thereto, and mixed well 1
After leaving for a minute, measure the increase rate of absorbance at 410 nm,
The γ-GTP activity value (IU / l) in the sample was calculated from the molecular extinction coefficient (7.3 × 10 ⁶ cm ² / mol) of the produced 3- (β-hydroxyethyl) -4-nitroaniline.

Comparative Example 1. Measurement of γ-GTP activity (Preparation of sample solution) Buffer solution Tris (hydroxymethyl) aminomethane 100 mmol and glycylglycine 60 mmol were added to purified water 800 ml, and hydrochloric acid was added to this to adjust the pH to 8.4 (at 25 ° C), and then purified. The total volume was 1 liter with water.

Substrate solution γ-L-glutamyl-p-nitroanilide 20 mmol 1N
-Dissolved in 100 ml of sulfuric acid and made up to a total volume of 1 l with purified water.

The composition of the above-mentioned test solution was in accordance with the composition of each test solution of γ-GTP B-ARII (trade name of Wako Pure Chemical Industries, Ltd.).

(Sample) The same sample as in Example 5 was used.

(Measurement method) 2.0 ml of buffer solution was added to 50 μl of sample and pre-heated at 37 ° C. for 3 minutes, 0.5 ml of substrate solution was added thereto, and mixed well 1
After standing for a minute, the rate of increase in absorbance at 410 nm was measured, and the molecular extinction coefficient (8.8 of the p-nitroaniline produced was measured.
Γ-GTP activity value (IU) in the sample from × 10 ⁶ cm ² / mol)
/ L) was calculated.

Comparative example 2. Measurement of γ-GTP activity (Preparation of test solution) Buffer solution Add 100 mmol of tris (hydroxymethyl) aminomethane and 100 mmol of glycylglycine to 800 ml of purified water, and add hydrochloric acid to adjust the pH to 8.2 (at 25 ° C) and purify. The total volume was 1 liter with water.

Substrate solution 14.5 mmol of γ-L-glutamyl-3-carboxy-4-nitroanilide ammonium salt was dissolved in 100 mM glycylglycine buffer solution (pH 8.2) to make a total volume of 1 liter.

The composition of the reagent solution was in accordance with the compositions of γ-GT reagents 1 and 2 (trade name of Boehringer Mannheim Yamanouchi Co., Ltd.).

(Sample) The same sample as in Example 5 was used.

(Measurement method) 2.0 ml of buffer solution was added to 50 μl of sample and pre-heated at 37 ° C. for 3 minutes, 0.5 ml of substrate solution was added thereto, and mixed well 1
After leaving for a minute, the rate of increase in absorbance at 410 nm was measured, and the molecular extinction coefficient (7.9 × 10 ⁶ cm ² / mol) of 3-carboxy-4-nitroaniline produced was used to determine γ-GT in the sample.
The P activity value (IU / l) was calculated.

The results obtained in Examples 5 and 6 and Comparative Examples 1 and 2 are shown in Table 1.

The numerical values in the table are the γ-GTP activity values (IU / l) in each case calculated based on the change in absorbance (ΔE / 5 min) at 410 nm for 5 minutes after the reaction was started. It is shown as a relative value when each is set to 100. Further, the difference in the relative activity value is the difference between the maximum value and the minimum value of the relative activity value in each sample for each substrate.

Next, a measurement reagent solution for the substrate for measuring γ-GTP activity used in Examples 5 and 6 and Comparative Examples 1 and 2 (a reagent solution obtained by mixing the buffer solution and the substrate solution in each case at a ratio of 4: 1). Table 2 shows the results of the solubility test in () and the stability test of the substrate solution in each case.

In addition, the Km value of γ-L-glutamyl-3-hydroxymethyl-4-nitroanilide, which is the compound of the present invention, for each γ-GTP derived from human serum, porcine kidney, and bovine kidney is shown by a conventional method. 3 shows.

For reference, Table 3 shows the presentation of Leslie M. Shaw (C
linical Chemistry, 23 , 79, 1977), γ-L-glutamyl-3-carboxy-4-nitroanilide and γ.
The Km value of -L-glutamyl-p-nitroanilide is also shown.

As is clear from Tables 1, 2 and 3, the compounds of the present invention are
It can be seen that the reactivity with γ-GTP as a substrate is good, and the difference in reactivity with γ-GTP from different origin is quite small.

In addition, from the results in Table 3, the reactivity with γ-GTP of different origin was found to be higher in the case of γ-L-glutamyl-3-carboxy-4-nitroanilide having a relatively polar substrate having a water-soluble group. However, γ-L-glutamyl-having no water-soluble group
It can be seen that the difference due to the origin of γ-GTP is larger than that of the substrate such as the compound of the present invention having p-nitroanilide or a water-soluble group (hydroxyalkyl group) having weak polarity. It is presumed that this is because when the substrate and the enzyme react with each other, they are greatly influenced by the mutual charge states. That is,
It is considered that the reactivity with each enzyme is greatly affected in the case of a substrate having a highly polar water-soluble group because the charge state of the enzyme, especially the charge state near the active site, differs depending on its origin. .

The solubility of the substrate of the present invention in water is significantly higher than the practically required solubility of 5 mmol or more, and the substrate solution is stored at room temperature (20 to 25 ° C) or refrigerated (5 ° C) as compared with the conventional substrate. It is much more stable and can be used practically without any problem for at least 3 months in the case of refrigerated storage.

Example 7. (Preparation of reagent solution) Buffer solution (R1) The one prepared in Example 5 was used.

Substrate solution (R2) 10 mmol of γ-L-glutamyl-3-hydroxymethyl-4-nitroanilide obtained in Example 4 was dissolved in purified water to make a total amount of 1 liter.

(Sample) 50 samples of human serum were used as samples.

(Measurement method) Hitachi automatic analyzer 7150 was used under the conditions shown in Table 4 for measurement.

(Measurement Results) Table 5 shows the results of examining the reproducibility of measured values using two types of samples.

As is clear from the results in Table 5, good reproducibility was exhibited.

Moreover, when a calibration curve was prepared using a 7% bovine albumin aqueous solution to which γ-GTP derived from pig kidney was added, a calibration curve of about 6400 was obtained.
It was found that good linearity was obtained up to 1 U / l.

Comparative Example 3. (Preparation of Reagent) Buffer (R1) The one prepared in Comparative Example 2 was used.

 Substrate solution (R2) The one prepared in Comparative Example 2 was used.

(Sample) The same sample as in Example 7 was used.

(Measurement method) Of CHEMISTRY PARAMETERS in Table 4, set K FACTOR to 7123 (ε
= 9.5), the measurement was performed under the same conditions as in Example 7.

Comparative Example 4. (Test solution) In accordance with the Scandinavian Society of Clinical Chemistry Recommendation Method (SSCC method),
The following were used:

Buffer solution (R1) 0.125 M Tris-hydrochloric acid buffer solution (pH 7.8, at 37 ° C, containing 96 mM glycylglycine and 13 mM magnesium chloride).

Substrate solution (R2) A 0.1N hydrochloric acid solution containing 20 mM γ-L-glutamyl-4-nitroanilide.

(Sample) The same sample as in Example 7 was used.

(Measuring method) Of CHEMISTRY PARAMETERS in Table 4, K FACTOR is 6835
The measurement was performed under the same conditions as in Example 7 except that (ε = 9.90) was changed.

FIG. 1 shows the correlation between the measured value of each person's serum obtained in Example 7 and that obtained in Comparative Example 3. The correlation coefficient, regression line equation, etc. obtained by the statistical processing are shown below.

Number of samples n = 50.

Correlation coefficient γ = 0.9998.

Regression linear equation (X: Comparative Example 3, Y: Example 7) Y = 1.087X-0.038 Average value: Comparative Example 3 = 106.72, Example 7 = 115.94.

FIG. 2 shows the correlation between the measured value of each person's serum obtained in Example 7 and that obtained in Comparative Example 4. The correlation coefficient, regression line equation, etc. obtained by the statistical processing are shown below.

Number of samples n = 50.

Correlation coefficient γ = 0.9998.

Regression linear equation (X: Comparative example 4, Y: Example 7) Y = 0.985X + 2.603 Average value: Comparative example 4 = 115.02, Example 7 = 115.94.

As is clear from FIGS. 1 and 2, the measured values obtained by the γ-GTP measuring reagent using the compound of the present invention as the substrate are the SSCC method using γ-L-glutamyl-p-nitroanilide as the substrate and It can be seen that a good correlation can be obtained with the measured value obtained by the conventional method using γ-L-glutamyl-3-carboxy-4-nitroanilide as a substrate.

EFFECTS OF THE INVENTION The present invention provides a novel γ-L-glutamyl-4-nitroanilide derivative useful as a substrate for γ-GTP activity measurement, and a γ-GTP activity measurement method using the same. The substrate for γ-GTP activity measurement comprising the compound of the invention is water-soluble and has good stability after dissolution, and γ-GT as a substrate
It has a remarkable effect in that it has excellent properties as a substrate such as good reactivity with P and small difference in reactivity with γ-GTP of different origin.

[Brief description of drawings]

FIG. 1 shows the correlation between the measured value of each person's serum obtained in Example 7 and that obtained in Comparative Example 3, in which the horizontal axis represents the value obtained in Comparative Example 3 and the vertical axis represents Indicates the values obtained in Example 7, respectively. FIG. 2 shows the correlation between the measured value of each human serum obtained in Example 7 and that obtained in Comparative Example 4, in which the horizontal axis represents the value obtained in Comparative Example 4 and the vertical axis represents Indicates the values obtained in Example 7, respectively.

Claims (2)

[Claims] 1. A general formula (In the formula, R represents a lower hydroxyalkyl group which may have two or more hydroxyl groups.) Γ-L-
Glutamyl-4-nitroanilide derivative. 2. General formula (In the formula, R represents a lower hydroxyalkyl group which may have two or more hydroxyl groups.) Γ-L-
A method for measuring γ-glutamyl transpeptidase activity using a glutamyl-4-nitroanilide derivative as a substrate.