JPS6327467A - Gamma-glutamyl-4-nitroanilide derivative and determination of gamma-glutamyl transpeptidase activity using same - Google Patents

Abstract

NEW MATERIAL:A compound of formula I [R is (CH2)mSO3M or (CH2CH2O)nR' (m and n are 1-4; M is H or alkali metal; R' is H or lower alkyl)]. EXAMPLE:gamma-Glutamyl-4-nitro-3-(3-Na-sulfopropoxy)anilide. USE:Determination of gamma-glutamyl transpeptidase activity. The determination is useful for the diagnosis of hepatocholepathy and screening of alcoholics. The compound has high water-solubility and stability. PREPARATION:The compound of formula I can be produced by reacting the compound of formula II with hydrazine.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a new water-soluble substrate for measuring r-glutamyl transpeptidase activity and a new method for measuring γ-glutamyl transpeptidase activity using the same.

[Background of the invention]

γ-glumemyltranspeptidase (hereinafter referred to as γ-GT
It is abbreviated as P. ) Measurement of enzyme activity is widely used clinically for diagnosis of hepatobiliary tract diseases, screening of alcohol drinkers, etc., and various methods have been published.
The reaction rate measurement method using -L-glutamyl-p-nitroanilide as a substrate is the most common method, and is still widely used.

However, the substrate γ-L-glutamyl-p-nitroanilide c will hereinafter be referred to as the present substrate. ) has extremely poor solubility near neutrality (pH = approximately 8.3), which is the optimal temperature for substrate stabilization and enzyme reaction. A common method of use is an acid dissolution method in which the compound is dissolved and mixed with a buffer solution around neutrality (pH=about 8.5) before use.

Therefore, this substrate undergoes hydrolysis by the dissolved strong acid, and the blank value gradually increases, and the shelf life of the preparation is about 5 hours after preparation.

Therefore, various attempts have been made to improve the solubility of this substrate.
The following methods (1) to (3) have been proposed and put into practical use.

(1) A method of improving the solubility of a substrate in water by adding a cationic surfactant or anionic surfactant, and alleviating the enzymatic reaction inhibition effect of these ionic surfactants with a nonionic surfactant.

(2) -CO2H at the ortho position of the -NO□ group of this substrate
A method of improving the solubility of a substrate in water by adding a water-soluble group such as a -5o3H group or a -5o3H group.

(3) A method of improving the solubility of a substrate in water by utilizing the inclusion power of cyclodextrin.

However, these conventional methods also have various drawbacks.

For example, in the method (1) using a surfactant, the ionic surfactant has a very strong enzyme inhibiting power, and even if a nonionic surfactant is added, the recovery rate is about 70 to 80%. Above, the addition of surfactant changes the absorption of hemoglobin over time, and the absorption of hemoglobin at 410 nm, which tracks the production rate of p-nitroaniline, decreases over time, resulting in r- Gives a negative error to the measurement of GTP enzyme activity. In addition, in the method (2) of adding a water-soluble group, the measured value of γ-GTPO enzyme activity is high, and the substrate changes in quality within a few days after preparing the substrate agent, resulting in γ-GTP
A decrease in the measured value of O enzyme activity is observed, and in some cases, problems such as precipitation of precipitates due to deterioration occur.

Furthermore, in the method (3) that utilizes the inclusion power of cyclodextrin, there is no problem with the effects of hemoglobin or fluctuations in the measured value of γ-GTP enzyme activity, but the important improvement in solubility in water is not sufficient. In particular, the limit is to formulate a concentrated liquid that is about twice as thick as a sheep. That is, none of the methods (1) to (3) can be said to be a sufficiently improved and satisfactory measuring method for the lateral base.

[Purpose of the invention]

The present invention was made to solve the above problems in the r-GTP activity measurement method using r-L-glutamyl-p-nitroanilide as a substrate. γ- with excellent solution stability
A new synthetic substrate for measuring GTP activity and γ-
The purpose of this invention is to provide a method for measuring GTP activity.

[Summary of the invention]

The present invention is based on the general formula [11 [wherein R is ÷CH2+m503M or ÷CH2CH2
O+rlR' (where m r n is an integer of 1 to 4, M is hydrogen, an alkali metal or NH4, and R' is hydrogen or a lower alkyl group, respectively). This is an invention of an r-L-glutamyl-4-nitroanilide derivative represented by the following and a method for measuring γ-GTP activity using the same.

The present inventors have conducted extensive research to solve the above-mentioned problems in the γ-GTP activity measurement method using r-L,-glutamyl-p-nitroanilide as a substrate. An r-L-glutamyl-4-nitroanilide derivative in which a specific water-soluble atomic group is substituted via oxygen at the ortho position of the nitro group solves the above problems, and also surprisingly improves its substrate solution. The present invention was completed based on the discovery that the stability of the compound was also significantly superior to that of conventional products.

γ-L-gourmetyl of the present invention represented by general formula [I]
In the 4-nitroanilide derivative, M in the water-soluble group ÷ cH2 + m503M represented by R is hydrogen, for example Na
, K, represents an alkali metal such as Li or NH4, and m represents an integer of 1 to 4. Further, the water-soluble group ÷cH2cH2o+rIR'noR' similarly represented by Rf represents hydrogen or a lower alkyl group such as a methyl group or an ethyl group, and n represents an integer of 1 to 4.

The synthesis process of the γ-L-glutamyl-4-nitroanilide derivative according to the present invention is roughly as follows.

That is, for example, when 3-acetamidophenol is first nitrated with a nitration reagent (e.g., concentrated nitric acid-acetic anhydride) in a room source, a nitro group is introduced into the ortho position of the hydroxyl group, and a nitro group is introduced into the para position of the hydroxyl group. Approximately 1:
Since it is produced at a ratio of 1:1, for example, sodium
5-acetamido-2-ditrophenol Na salt, which is sparingly soluble in methanol, is isolated as crystals by the action of an alkali such as tyrate. Next, the Na salt is reacted with the corresponding sultone or halide to obtain a 4-nitroacetanilide compound in which the atomic group R is introduced into the 3-position of the benzene nucleus via oxygen. When this is deacetylated to obtain the corresponding aniline compound and reacted with phthaloyl anhydride glutamic acid,
Since the desired amide is obtained, this is reacted with hydrazine to obtain the desired γ-L-glutamyl-4-nitroanili P derivative. The obtained derivative has excellent water solubility and stability, and also shows good sensitivity to r-GTP enzyme activity. That is, the solubility of the γ-L-glutamyl-4-nitroanilide derivative in water according to the present invention is extremely high, and it dissolves very well in water even at around neutrality, and its solution stability is as high as 40 hours in an aqueous solution. is so large that no change is noticeable. Furthermore, when this is used as a substrate for measuring r-GTP activity, it shows good sensitivity for the enzyme activity.

The method for measuring r-GTP activity of the present invention uses the γ- of the present invention as a substrate.
Except for using the L-glutamyl-4-nitroanilide derivative, it is sufficient to carry out this according to a known method for measuring γ-GTP activity using γ-L-glutamyl-p-nitroanilide as a substrate. That is, using the γ-L-glutamyl-4-nitroanilide derivative of the present invention as a substrate, a reaction is carried out in an appropriate buffer in the presence of a glutamate receptor such as glycylglycine, and the enzymatic action of r-GTP is The absorbance of the 4-nitroaniline derivative that is produced is directly measured by an initial rate measurement method (-point measurement method, late assay method, etc.), or by adding p-dialkylaminobenzaldehyde, p-noalkylaminocinnamaldehyde, etc. The measurement method itself is arbitrary, such as adding a color to the color and measuring its absorbance. Note that it is optional to use an r-GTP activator such as glutamic acid in the γ-GTP enzyme reaction system. Further, the measurement conditions such as buffer solution, reaction solution-1 substrate and receptor amount, measurement temperature, measurement wavelength, etc. may be determined according to conventional methods. However, since the water-soluble substrate of the present invention has extremely excellent water solubility and can be used at high concentrations, and its aqueous solution can be stored stably for a long time, this point should be taken into consideration in the measurement method of the present invention. It is of course possible to assemble the loaded reagents.

Examples are shown below, but the present invention is not limited in any way by these examples.

〔Example〕

Example 1 (Synthesis of 1-5-acetamido-2-ditrophenol) Mix 12QmA of acetic anhydride and 3Qml of concentrated nitric acid,
40 g of 3-acetamidophenol was added to this and reacted for 30 minutes at 15 to 30° C. with stirring. Pour the reaction solution into ice water for 50 minutes.
0. Inject into F, remove the precipitate, wash with water, and dry to obtain crystal 4.
I got 0J. Dissolve this 26& in 1 oom/methanol and add 25% of a 28% methanol solution of sodium methylate
．． 59 was added, and after stirring at 20°C for 30 minutes, the precipitated crystals were removed.
Crystals were obtained by drying.

Dissolve this in 200mJ of water, then 10% hydrochloric acid solution
0rrLt was added to precipitate crystals, which were collected and dried to obtain crystals 141, which were the desired product. m, p, 219-
220℃ [Literature value: m-p, 217-218℃ (J
, Am , Chem.

Soc, rdan, 4947-4948 (1964
))).

'H-NMR (DMSO-d,): δppm2.17
(s, 3H, -COCH3), 7.10 (d
d, I H, J = 9Hz, J = 21 (z, ar
omatic-H (4th position)), 7.65(d
, IH,J=2Hz.

aromatic-H (6th position) + 8.05 (d
+ LH+ J=9Hz, aromatic-H (3rd position)) s 10.4 (broad+ IH+ -0
H) + 11.0 (broad +I H, -NH
CO-) IR(KBr): v cln-'3600(-
OH), 3310(-NH-), 1688(-
co-), 1525(-No2) (2) 4-nitro-3-(3-Na sulfopropoquine)
5-acetami P-2-nitrophenol obtained in synthesis (1) of acetanilide (6.09) was dissolved in 55 ml of ethanol, then 1,19 ml of sodium hydroxide was added, and to this was added 3.81 ml of propane sultone at 70°C. was added and reacted at the same temperature for 6.5 hours.

After cooling, the precipitated crystals were collected and dried to obtain the desired crystal 6.29.

'H-NMR (DMSO-d,): δppm2.1 (
s, 3H, -COCH3) + 2.7(m, 2
H, -CH2CH2CH2-).

3.4 (m, 2H, -0-CH2CH2-) #
4.2 (m, 2H, -CH2CH2CH2-3
O3Na) + 7.3 (d+L I H, aroma
atic-H (6th position)), 7.6 (d.

IH, aromatic-H (2nd place) + 7.9
(d r IH* aromatic-H (5th position)) IR (KBr), White 1685 (-Co-), 1560 (-No□) (3) 4
-Synthesis of nitro-3-(3-sulfopropoquine)aniline 4-nitro-3-(3-Nasulfopropoquine)acetanilide obtained in (2) 2. O,! 20 ml of 10% salt aqueous solution was added to i', and the mixture was refluxed for 3 hours. After cooling, the precipitated product was collected by F and dried to obtain 0.8J of crystals of the target product.

m, p, 245°C (dec).

'H-NMR (DMSO-d, ): δppm2.2
(m #2 Hz -CH2CH2CH2-) 1
2.9 (ml2Hl-0-CH2CH2-)
+4.2 (m, 2H, -CH2CH23-3o3e)
16.0 (broad, 3H.

-NH5) e 6.3 (dd, IH, aromat
ic-H (6th position)), 6.4 (d.

IH, aromatic-H (2nd place)), 7.8
5 (d, IH+ aromatic-H (5th position)) IR (KBr), να ■ 3430 (-Nl (3), 1520 (-No.),
1205 (-0-) (4) γ-L-glutamyl-4-
Synthesis of nitro-3-(3-Na-sulfopropoquine)anilide Dissolve 1.2I of 4-nitro-3-(3-sulfopropoquine)aniline in 20ml of methanol and add 1rnl of a 28% methanol solution of sodium methylate. After distilling off the methanol, 1.0 g of phthaloyl glutamic anhydride and 15 mg of DMF were added to the solution.
The reaction was carried out at 0 to 145°C for 12 hours. After the reaction is complete, DM
F′ was distilled off, the residue was dissolved in 20rnl of methanol,
This includes 80 hydrazone hydrates, 2m7! 6 at room temperature by adding
After reacting for an hour, the device was set up overnight. Insoluble materials were removed from the reaction solution, methanol was distilled off, acetone was added, and the resulting crystals were subjected to column chromatography (Wakodal C-200 (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.); methanol).
The desired fraction was obtained. This was concentrated, treated with activated carbon, the solvent was distilled off, and dried in vacuum.
Nitro 3-(3-Na sulfopropoxy)anilyrO
091 was obtained.

'H-NMR (DMSO-d, ): δppm5.7
(broad, 4H, -COOH, -NH2, -N
H-), 7.4 (dd, IH.

aromatic-H (6th position) ) + 7.7 (
d, IH, aromatic-H (2nd position)).

7.9 (d + IH, aromatic-H (5th place)
) Example 2゜(1) Synthesis of 3-(2-hydroxyethoxy)-4-nitroacetanili P 5-acetamido- obtained in the same manner as in Example 1 (1)
30% of 2-bromoethanol was added to 17 g of Na salt of 2-ditrophenol, and the mixture was reacted at 80 to 85°C for 7 hours. After the reaction was completed, the reaction solution was neutralized with a 5% aqueous sodium hydroxide solution and extracted with ethyl acetate. The extract was concentrated, and the precipitated crystals were collected and dried to obtain 9.9 g of the desired product. m, p,
131-134°C.

(m, 2 H-D 0-CH2-), 485 (m
, IH1-OH), 7.25(d+L IHe
J=10Hz + J=2Hz+ aromatic-
H (6th place) ), 7.65 (d r IHe J=
2Hz, aromatic-H (2nd place)), 7.
95 (d, IH.

J=10Hz + aromatic -H (5th place)
), 10.4 (broad, IH.

-NH-) (2) Synthesis of 3-(2-hydroxyethoxy)-4-nitroaniline Add 30vtl of 2N-hydrochloric acid to 7.2I of 3-(2-hydroxyethoxy)-4-nitroacetanilide obtained in (1). The mixture was added and reacted at 80 to 85°C for 1 hour. After the reaction is complete,
A 5% aqueous sodium hydroxide solution was added to the reaction solution, and the precipitated crystals were removed and dried to obtain the desired product 6. I got Ol. m, p, 90~
93℃.

'H-NMR (DMSO-d, ): δppm3.6
5(m, 2I(,-3-OH), 4.10(m, 2H
(n0-(3-).

6.25 (dd, IHr J=2Hz r J=1
0Hz, aromatic-H (6th place)).

6.30 (d, IHy J=2Hz, aroma
tic-H(2nd position)) + 6.5(broad.

2H, -NH2), 7.80 (d r IH,J
=10Hz, aromatic-H (5th place)) IR (KBr) Rainbow m 3360, 3450 (-NH2), 1360 (-No□
)(3) Synthesis of γ-L-glutamyl-3-(2-hydroxyethoxy)-4-nitroanilide (2) 3-
(2-hydroxyethoxy)-4-nitroaniline 5g
Phthaloyl glutamic anhydride 7.5,! i', 50 ml of dioxane was added, and the mixture was reacted at 80 to 85°C for 5 hours. After the reaction was completed, the precipitated crystals were collected and dried to obtain 10 das of N-7taloyl r-L-glutamyl-3-(2-hydroxyethoxy)-4-nitroanilide. m, p, 2
25-227°C. 79 of this was suspended in 50 mJ of methanol, and then 90 parts of hydrazine hydrate were added to 1.1
After adding rnl and reacting at room temperature for 3 hours, the reactor was incubated overnight. Methanol was distilled off from the reaction solution, 100 d of water was added and dissolved, and the precipitate was removed with glacial acetic acid.
After treatment with 5 g of activated carbon and evaporation of the solvent, the resulting oily residue was crystallized from methanol to obtain the desired product 3. Om got Ol
, p, 174-175°C.

-C-Q-,-OH) + 7.4 (dd, IH,
aromatic-H (6th position)), 7.8 (d r
IHy aromatic-H (2nd place) ) + 7
．． 9 (d r IH+aromatic-H (5th place)
) Example 3゜(1) Synthesis of 3-(2-(2-hydroxyethoxy)ethoxy)-4-nitroaniline 5-acetamido-2-ditrophenol Na salt 10!
29 ml of 2-(2-hydroxyethoxy)ethyl chloride was added to j, and the mixture was reacted at 110 to 120°C for 5 hours. After the reaction is complete, add 3Qm7 of water to the reaction solution! was added, extracted with ethyl acetate, and the ethyl acetate was distilled off to obtain an oily residue 10
．． 9 by column chromatography (Wakoglu C-2
00; ethyl acetate) to obtain the desired fraction. After evaporating the solvent, hexane was added, and the precipitated crystals were collected and dried to obtain the desired product 2.311. m, p, 85-87°C.

'H-NMR (DMSO-d, ): δppm3.5
~3.3 (m, 8H, -0(CH2CH20)2
H), 4.5 (broad, IH, -0H)
.

6.1-6.3 (m, 2H, aromatic
-H (2nd, 6th) ), 6.5 (broad.

2 H+ −Nl2 ) * 7.8 (d + I
H+ J =9Hz + aromatic-H(
5th place)) IR (KBr) Niji□-1 3420, 3340 (-Nl2), 1575.135
2(-No□), 1260(2) γ-L-glutamyl-3-(2-(2-hydroxyethoxy)ethoxy)
-1.33 of 3-[2-(2-hydroxyethoxy)ethoxy)-4-nitroaniline obtained in synthesis (1) of -4-nitroanilide and 1.4.9 of phthaloylglutamic anhydride. 15rnl of dioxane was added and reacted at 80-85°C for 5 hours. After the reaction was completed, dioxane was distilled off from the reaction solution and dissolved in 20 ml of methanol. 0.4 rIdl of 90% hydrazine hydrate was added, and the mixture was stirred and reacted at room temperature for 2 hours. After the reaction was completed, the precipitate was removed from the furnace, methanol was distilled off, dissolved in 20 g of water, the pH was adjusted to 5 with acetic acid, and the precipitate was removed again. After solvent distillation, cation exchange resin (Amberly) IR120B)
After distilling off the water, the product was crystallized from acetone to obtain the desired product 1.0Ii. m, p, 113°C (dec).

H2N-CH-CH2-), 3.5 (m, 4
H, -CI (2c!!fO-CH,,CH2-0H)
.

3.7-4.3 (m, 4H, -3CH2-0-
CH2(3-OH), 4.5-5.8 (broa
d.

5H, -Nl2--C0OI(, -NHCO-, -O
H), 7.4 (m, I H.

aromatic-H (2nd place)), 7.8-8.
0 (m, 2H, aromatic-H (5th place,
6th place)) IR (KBr) crn"-' 1670 (-CONH-), 1560, 133
0 (-No□) Example 4゜(1) 3-(2-(2-methoxyethoxy)ethoxy)
-Synthesis of 4-nitroaniline 5-acetamibe 2-nitrophenol Na salt 1
0,'lK2-(2-chloroethoxy)ethoxymethane 35g was added, dissolved in DMSO20ml, and 120~
The reaction was carried out at 125°C for 2 hours. After the reaction was completed, 5 QmJ of water was added to the reaction solution, and the mixture was extracted with chloroform.

The chloroform was distilled off to give an oily residue 9,9 which was crystallized from ethyl acetate to give 5.5 g of 3-(2-(2-methoxyethoxy)ethoxy)-4-nitroacetanilide.
I got it. m, p, 80°C. Add 3Qm of 2N-hydrochloric acid to this total amount.
A! was added and reacted for 1 hour at 80°C with stirring, then 5 g of sodium hydroxide was added to the reaction solution, and the precipitated crystals were collected by F, dried, and recrystallized from toluene-methanol to obtain the desired product 3.
．． 9g was obtained. m, p.

72-73℃.

'I(-NMR(CDCl2): δppm3.35(
s, 3H, -0CH3), 3.45-4.0(
m, 6H.

-0-CH2 Relaxation → CH3), 4.15 (
t, 2H, Qwo-c horse-).

4.50 (broad, 2H, -Nl2) 16.
20 (m, 2H, aromatic-H (2nd and 6th positions)) m 7.85 (t, IH, aromat
ic-H (5th position)) IR (KBr) Niji crn'' 3460.3360 (-Nl2), 1340 (-No□
), 11oo(-o-)(2) γ-L-glutamyl-3
- (2-(2-methoxyethoxy)ethoxy)-4-
Synthesis of nitroanilide Add 3.8.9% of phthaloylglutamic anhydride and 4QrnJ of dioxane to 3.31% of 3-(2-(2-methoxyethoxy)ethoxy)-4-nitroaniline obtained in step (1) at 75-78°C. The reaction was carried out for 5 hours. After the reaction, dioxane is distilled off from the reaction solution and crystallized from ethyl acetate to obtain N-phthaloyl-γ-L-glutamyl-3-(2-C
4.6 g of 2-methoxyethoxy)ethoxy)-4-nitroanilide was obtained.

m, p, 135-139°C. Of these, take 4.4I,
Methanol 30tA! 190T hydrazine hydrate 1.2I
was added and reacted at room temperature for 7 hours. - Precipitated product after standing overnight 6.2. ! i' was obtained, and after dissolving it in water, it was diluted with acetic acid.
I gave it a 5. After removing the precipitate, the solvent was distilled off, and the residue was crystallized from ethanol to obtain the desired product 2.29.

m, p, 162-163°C.

'H-NMR (DMSO-d,): δppm2.10
(broad, 2H, -CHCH2-) -2,
60 (broad, 2H, -CHCoNH-).

H2N-CH-CFL2- ) -5,5~8.0 (
b ro ad , 4 )(z -cOOH1-NH
2+-NH-) e 7. :2~8.1 (m*3
H, aromatic-H (2nd, 5th, 6th)) I
R (KBr) Niji G 1695 (-CONH-), 1600 (-COOH)
, 1550 , 1340' (-No□), 1100
(-0-) Example 5゜(1) 3-1: Synthesis of 2-(2-(2-methoxyethoxy)ethoxy)ethoxy-4-nitroacetanilide 5-acetamido-2-ditrophenol Na salt 10.
6F, 2- (2-(2-chloroethoxy)ethoxy)
Ethoxymethane 30! ! was dissolved in 20 ml of DMSo and reacted at 120°C for 2 hours. After the reaction is complete, add 100mA of water to the reaction solution! was added and extracted with chloroform. The chloroform was distilled off to obtain an oily residue of 10.9%, which was crystallized from ethyl acetate-n-hexane and dried for 3 hours.
7.3 g of -[2-(2-(2-methoxyethoxy)ethoxy)ethoxy-4-nitroacetanilide was obtained.

m, p, 79-82°C.

'H-NMR (CDCl2): δ, P2.20 (s
, 3H1COCH3), 3.33(s, 3H1
OCRρ, 3.4-4.15 (m + 12Hr −
0(CH2CH20)3-)+6.95 (ddJ
H* aromatic-H (6th place)) #7.6
6 (d*IH, aromatic-H (2nd place))
, 7.85 (d r IH, aromatic -H (
5th place) ) -8,65 (broad, LH, -N
et-)IR(KBr)', vcIn170
0(-CONH-), 1545, 1325(-N
o□), 1090(-0-)(2) Synthesis of N-phthaloyl-γ-L-glutamyl-3-[2-(2-(2-methoxyethoxy)ethoxy)ethoxy-4-nitroanilide.

30 rnl of 2N-hydrochloric acid was added to 7.09 g of 3-(:2-(2-(2-methoxyethoxy)ethoxy)ethoxy-2-4-nitroacetanilide obtained in (1), and the mixture was reacted at 80°C for 1.5 hours. After the reaction was completed, 2.51 grams of sodium hydroxide was added to the reaction solution, extracted with chloroform, and the chloroform was distilled off to give 3-(:2-(2-(2-methoxyethoxy)ethoxy)ethoxy]-4- Nitroaniline 6, Ol was obtained. m, p, 43-46°C. Of these, 5
I and 9, phthaloylglutamic anhydride 6.3.9. 50 ml of dioxane was added and reacted at 80°C for 2.5 hours. After completion of the reaction, dioxane was distilled off from the reaction solution and crystallized from methanol to obtain N-7talloyl-γ-L-glutamyl-3-C2-(2-(2-methoxyethoxy)ethoxy)ethoxy]-4-nitroanilibi. 4.5g was obtained.

m, p, 102-105°C. 4.2 g of this was removed, 4 Qrnl of methanol was added, 1.21 g of 90 g of hydrazine hydrate was added, and the mixture was allowed to react at room temperature for 7 hours. - After standing overnight, methanol was distilled off, water was added and the pH was adjusted to 5 with acetic acid, and the precipitate was sweated off. The lacrimal fluid was treated with activated carbon, and after the solvent was distilled off, it was crystallized from ethanol to obtain the desired product 1. I got III. m, p, foaming at around 120°C, melting at 139°C.

'I(-NMR(DMSO-d, ): δppm1.
8 (m, 2H, -CI(-CH-), 2.2
(m, 2H, -CH2-CH, -CONH-)
.

3.2 (s, 3H, -0CHs), 3.4~
4.2 (m, 13H, -0(CH2CH2)3
.

COOH H2N-CH-CH2-) #5.0~7.0 (b
road, 4H, -NH2, -Cool(.

-NH-) + 7.3 (broad, IH, ar
omatic-H (6th place)), 7.7 (broa
d + 2H aromatic-H (2nd, 5th)
) IR (KBr) Niji-1700 (-CONH-), 1340 (-No2) Example. , , -1-guar, γ-G by mi7゜-3-(3-7゜hopropoxy)-4-nitroanilide substrate
TP activity measurement (preparation of test solution) ■ Buffer 0, 1 M) Dissolve glycylglycine at a concentration of 40 mmol/l and glutamic acid at a concentration of 0.015 in Lis-HCl buffer (pH 8,5). did.

(2) Substrate Solution An aqueous solution of γ-L-glutamyl-3-(3-Na sulfopropoxy)-4-nitroanilide at a concentration of 40 mmol/l was prepared.

(Measurement method) Take 50 μl of serum, add 2.0 ml of buffer, and
Changes in absorbance (ΔE15min) for 5 minutes at a wavelength of 410 nm in a constant temperature bath were measured (reagent blind control).

Comparative example 1. Measurement of r-GTP activity using γ-L-glutamyl-4-nitroanilide substrate (preparation of test solution) ■ Buffer 0, 1 M) Glycylglycine in 40 mmol/L-HCl buffer (pi (B, 4)) l, glutamic acid was dissolved to a concentration of 0.015 l.

■ 1 mmol of substrate solution γ-L-glutamyl-4-nitroanilide
(!:D, After dissolving with 0.5 N sulfuric acid IQmA!, the total volume was made up to 53 m/m with ion-exchanged water.

(Measurement method) Table 1.2 shows the results of comparing the ΔE15min of the same serum and the presence or absence of crystal precipitation after leaving the substrate solution at 4°C for 43 hours according to Example 6 and Comparative Example 1. .

Table 1 (ΔE15min) Table 2 (Substrate W after standing at 4°C/43 hours) Increase in this sample blank = (Reagent blank absorbance after standing at 4°C for 743 hours) - (Reagent using substrate solution immediately after preparation) Blank Absorbance) As is clear from Table 1, Example 6 using the substrate of the present invention is almost the same as Comparative Example 1 using the conventional substrate in terms of sensitivity, but as shown in Table 2. It can be seen that the solubility of the substrate in water and the stability of the substrate solution over time are significantly superior to Comparative Example 1 using the conventional method.

Example 7. Measurement of γ-GTP activity using γ-L-glutamyl-3-[2-(2-methoxyethoxy)ethoxy]-4-nitroanilide substrate (preparation of test solution) ■ Buffer 0, 1 M Tris-HCl buffer (pH 8,5 ) contains 40 mm of glycylglycine and 0.0 mm of 11 glutamic acid.
It was dissolved to a concentration of 15%.

■ Substrate solution 40 m mo of γ-L-glutamyl-3-(2-(2-methoxyethoxy)ethoxyi-4-nitroanilide)
An aqueous solution of l/l concentration was prepared.

■ Coloring reagent p-noethylaminobenzaldehyde 22.6mm0
Take 1 ml of 1x hydrochloric acid and add ethanol to the total volume of 100rn.
It was set as l.

(Measurement method) Take 50 μl of serum and add buffer 2. Add OWLt to 37
After heating in a constant temperature bath at 37°C, 0.5 ml of substrate solution was added, and after further heating at 37°C for 10 minutes, 2.5 ml of color reagent solution was added, and the absorbance at a wavelength of 470 nm was measured. Next, a Kanazawa series was prepared using TF-irradiated standard serum (1-GTP 240 mU/M), and a calibration curve was created from the absorbance measured using each of them (Figure 1). As shown, the calibration curve was a straight line passing through the origin.Also, the substrate solution was heated at 4℃.
/No crystal precipitation was observed when stored for 43 hours,
Moreover, there was no increase in reagent blind values.

Example 8. γ-GTP activity measurement using 1-L-glutamyl-3-(2-(2-hydroxyethoxy)ethoxy]-4-nitroanilide substrate (preparation of test solution) ■ Buffer Same as Example 7.

■ Substrate solution 40 m m of γ-L-glutamyl-3-C2-C2-hydroxyethoxy)ethoxy-4-nitroanilide
An aqueous solution of ol/l concentration was prepared.

■ Coloring test solution Same as Example 7°.

(Measurement method) Same as Example 7.

A dilution series was created using standard serum (r-GTP 240 mU/ml), and a calibration curve was created from the absorbance measured using each dilution series (Figure 2). As is clear from FIG. 2, the calibration curve was a straight line passing through the origin.

Further, when the substrate solution was stored at 4°C for 743 hours, no crystal precipitation was observed, and there was no increase in the reagent blind value.

〔Effect of the invention〕

The present invention provides a novel water-soluble substrate for measuring γ-GTP activity and a method for measuring γ-GTP activity using the same. In particular, the substrate of the present invention has excellent solubility in water and is neutral. It has a special effect in that it is a stable water-soluble substrate that dissolves very well in water even in the vicinity and is difficult to be hydrolyzed in an aqueous solution.

[Brief explanation of the drawing]

FIG. 1 shows the calibration curve created in Example 7, and FIG. 2 shows the calibration curve created in Example 8. In Figures 1 and 2, the horizontal axis indicates γ-GTP enzyme activity (γ-
GTP mU7fnl), and the vertical axis represents absorbance. Patent Applicant: Wako Pure Chemical Industries, Ltd. Figure 1: r-CqTp (Vnvsu) Figure 2: γ-GTP (mUiζυ Procedural Amendment October 6, 1985)

Claims (2)

[Claims] (1) General formula [I] ▲There are mathematical formulas, chemical formulas, tables, etc.▼[I] [In the formula, R is -(CH_2)-_mSO_3M or -(
CH_2CH_2O)-_nR' (however, m,
n is an integer of 1 to 4, M is hydrogen, alkali metal or NH
_4, R' represents hydrogen or a lower alkyl group, respectively. ). ] A γ-L-glutamyl-4-nitroanilide derivative. (2) General formula [I] ▲There are mathematical formulas, chemical formulas, tables, etc.▼[I] (In the formula, R is -(CH_2)-_mSO_3M or -(
CH_2CH_2O)-_nR' (however, m,
n is an integer of 1 to 4, M is hydrogen, alkali metal or NH
_4, R' represents hydrogen or a lower alkyl group, respectively. ). A method for measuring γ-glutamyl transpeptidase activity, which comprises using a γ-L-glutamyl-4-nitroanilide derivative represented by the following as a substrate.