JPS6143360B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides an amino acid derivative represented by the formula (),
The present invention relates to a method for producing the compound and a method for measuring enzyme activity using the compound as a substrate.

In the formula, R represents a naphthyl group.

The substance of the present invention () is a novel compound and is a compound useful as a test drug for measuring enzyme activity.

The substance of the present invention () is formed into an ester body represented by the formula () by a normal dehydration condensation reaction between a glycyrrhizine derivative () and naphthol, and then ω
-Can be produced by removing the amino group protecting group.

In the formula, R represents a naphthyl group. R′ represents an amino group-protecting group.

In carrying out the present invention, the ester compound () can be obtained by a conventional dehydration condensation reaction between the glycyrrhizine derivative () and naphthol. That is, a glycyrrhizine derivative () is dissolved in a suitable solvent, an ester activator such as DCC (dicyclohexylcarbodiimide), DPPA (diphenylphosphoryl azide), or alkyl chlorocarbonate is added thereto, and phenol or naphthol is added thereto. It can be produced by adding a base such as triethylamine if necessary and stirring. It can also be produced by the well-known acid chloride method or by using a well-known naphthylating agent (Ber. 49 , 2339, 1916) as a sulfite form of naphthol.

Usable solvents include commonly used solvents such as chloroform, dichloromethane, dimethylformamide, and tetrahydrofuran, as long as they can dissolve the raw material. The reaction temperature may be 0° to 40°C.

After the reaction is completed, the ester () can be obtained from the reaction solution by a commonly used treatment method.
That is, for example, when DCC is produced as a condensing agent, precipitated dicyclohexyl urea is removed by filtration, washed with an aqueous hydrochloric acid solution, an aqueous NaOH solution, and a saturated saline solution, dried with a drying agent such as anhydrous magnesium sulfate, and then the solvent is removed. It can be obtained by distillation. The ester () may be recrystallized if desired.
It can be purified by chromatography or the like.

The target compound () can be obtained from the ester () by removing the amino group-protecting group in a normal reaction. That is, for example, when the amino group-protecting group is a carbobenzyloxy group, () is dissolved in a suitable solvent and reductively removed using a catalyst such as palladium on carbon, or () is added to a hydrobromide-acetic acid solution and precipitated. It can be obtained by removing the hydrobromide salt of the target compound.

The substance of the present invention () acts as a substrate when brought into contact with an enzyme, and after a certain period of time, it is hydrolyzed by the enzyme and liberated naphthol is measured, thereby making it possible to measure the activity of the enzyme. Measuring enzyme activity is very important for purposes such as standardization of enzyme preparations, diagnosis by measuring blood enzyme patterns, and measurement of blood enzyme concentration.

Conventionally, various methods are known for measuring enzyme activity. One method is to use an alkyl ester of an amino acid as a substrate, contact it with an enzyme, and measure the activity based on the degree of hydrolysis of the ester. For example, a method well known as the Hesterin method is one of them.
This is done by bringing an enzyme into contact with an alkyl ester of an amino acid, converting the remaining ester portion after a certain period of time into hydroxamic acid using hydroxyamine, reacting with ferric chloride to develop a color, and measuring the color development as absorbance. Based on the results, This method measures the ester water-degrading ability of an enzyme, that is, the activity of the enzyme.

Other methods that use a substrate and use the water-dissolving ability of esters as an index include the chromotropic acid method.
These methods require a certain amount of enzyme and are difficult to measure when the enzyme is at a low concentration or has low activity. Therefore, the inventor searched for a compound as a substrate that had affinity for the enzyme, was easy to quantify, and had high detection sensitivity. We were able to discover an excellent compound and its method.

In carrying out the present invention, the activity of the enzyme is measured by bringing the enzyme and a certain amount of the compound () into contact with each other in a suitable buffer solution, and measuring the liberated naphthol after a certain period of time at a certain temperature. can do.

The buffer may be a suitable buffer having the optimum pH of the enzyme. Further, the reaction temperature and reaction time may be set under any suitable constant conditions, but it is preferable to measure after 30 minutes at 25 to 37°C.

Conventional methods for measuring naphthol include physicochemical methods such as well-known gas chromatography and thin layer chromatography, ferric chloride reaction, diazo coupling reaction, or FVB (first violet B salt) method. Although any of the above chemical methods may be used, a method in which FVB is added after the reaction to develop a color and measured as absorbance using a spectrophotometer is a more preferable method in terms of simplicity and detection sensitivity. This method can be used not only for measurements in a single enzyme system but also for measurements involving various enzymes. That is, for example, when measuring the enzyme activity contained in serum, the serum is added to an appropriate preparation, the enzymes are decomposed by electrophoresis, etc., and this is immersed in a solution of the substance of the present invention, and after an appropriate time, further By adding the above coloring reagent,
Enzyme patterns in the blood that were previously impossible to see can be seen. According to this method, changes in enzyme patterns caused by various pathological conditions can be observed.

The substance of the present invention acts as an excellent substrate for various enzymes such as trypsin, plasmin, kallikrein, urokinase, C1 esterase, and thrombin, and in particular when enzyme activity is measured using acetylglycyrrhizine naphthyl ester as a substrate, 3 compared to when measured by the hesterin method using acetylglycyrrhizine methyl ester and tosylarginine methyl ester.
The detection sensitivity was between 550 times and 550 times higher.

Next, examples will be given and explained in more detail.

Example 1 Synthesis of N-acetylglycyrrhizine naphthyl ester Acetylglycyl-ε-carbobenzyloxylysine methyl ester (Biophys Acta., 132,
Dissolve 7.6 g of 104-114 (1967) in 100 ml of methanol, add 30 ml of N-NaOH, and stir at room temperature for 2 hours.
Acetate and water are added to the reaction solution, shaken, and the aqueous layer is acidified with 10% hydrochloric acid solution, and the precipitated oil is extracted four times with acetate. After washing the acetate ester solution with saturated saline, drying with magnesium sulfate, and concentrating under reduced pressure, acetylglycyl-ε
- Carbobenzyloxylysine is obtained as a crystalline powder.

Yield 5.8g Yield 75% Melting point 112~4゜IR (KBr, cm ^-1 ) 3375, 3275, 3250 (NH), 1750
(CO), 1690 (NHCOO), 1640 (NHCO) Elemental analysis C ₁₈ H ₂₅ N ₃ O ₆ (MW379.40) Theoretical value C, 56.98, H, 6.64, N, 11.08 Actual value C, 56.41, H, 6.60, N, 10.80 Take 3.6 g of acetylglycyl-ε-carbobenzyloxylidine, dissolve it in 20 ml of N,N dimethylformamide, and stir with 1.4 g of α-naphthol, 2.4 g of N,N' dicyclohexylcarbodiimide, and 1.4 ml of triethylamine for one day and night. Add acetate to the reaction solution, wash sequentially with 10% citric acid solution, saturated deuterated water, and saturated brine, dry over magnesium sulfate, concentrate under reduced pressure, collect the precipitated powder, and recrystallize from acetate. N-acetylglycyl-
ε-carbobenzyloxylysine-α-naphthyl ester is obtained.

Yield 2.5g Yield 52% Melting point 103-50 IR (KBr, cm ^-1 ) 3325 (NH) 1755 (CO) 1680,
1640 (NHCO) Elemental analysis C ₂₈ H ₃₁ N ₃ O ₆ (MW505.55) Theoretical value C, 66.52, H, 6.18, N, 8.31 Actual value C, 66.06, H, 6, 36, N, 7.96 N acetyl 2.5 g of glycyl-ε-carbobenzyloxylysine-α-naphthyl ester was added to N,
Dissolve in 25 ml of N dimethylformamide and add 3 g of hydrochloric acid gas-dioxane solution (0.06527 g HClgas/
g) Add 500 mg of 10% palladium on carbon and pass hydrogen gas through for 2 hours. After the reaction, the palladium on carbon was removed by filtration, and anhydrous ether was added to the mother liquor to collect the precipitated oil, which was further washed with acetic acid ester to obtain N-acetylglycyrrhizine naphthyl ester hydrochloride as a colorless oil, yield 1.0 g.

IR (ueat, cm ^-1 ) 3250 (NH), 2950 (NH ₃ ⁺ ) 1750
(COO) 1630 (NHCO) Example 2 Thrombin activity measurement method using N-acetylglycyrrhizine naphthyl ester as a substrate N-acetylglycyrrhizine naphthyl ester solution (0.5 μmole/
Add 0.5 ml of 5% DMSO) and incubate at 25°C for 30 minutes. Add 0.1 ml of 1% first violet B salt (FVB) to this, leave it for 30 minutes at 0°C, then add 1 ml of glacial acetic acid, and measure the color development as absorbance (515 nm) using an absorptiometer. Quantitate liberated naphthol. The amount of liberated naphthol is the activity of the enzyme.

Reference example 1 Thrombin activity measurement method using N-acetylglycyrrhizine methyl ester as a substrate (Hesterin method) N-acetylglycyrrhizine methyl ester solution (10 μM/0.4 ml 5%) in the thrombin diluted solution
DMSO) 0.4ml, and phosphate buffer (PH7.4) 0.1ml
Add. After incubation at 37°C for 30 min, 1.5 ml of hydroxylamine solution (equal mixture of 2M-NH ₂ OH and 3.5M NaOH) was added and the solution was incubated at room temperature for 15 min.
Leave for a minute. Add 1 ml of 18% trichloroacetic acid to this.
Add 1 ml of 4N hydrochloric acid and 1 ml of 10% ferric chloride, stir thoroughly, and centrifuge at 3000 rpm for 10 minutes. The color development of the supernatant liquid is measured as absorbance (530 nm) using a spectrophotometer. This value correlates to the amount of substrate remaining without being hydrolyzed by thrombin, and the enzyme activity corresponds to the value obtained by subtracting the value obtained after performing the enzyme reaction from the value for substrate only (control). The results at each concentration level of thrombin measured in Example 3 and Reference Example 1 are shown in FIG. 1, and it can be seen that the former method is 50 times more sensitive than the latter method. Similarly, when urokinase was measured, it was 11 times more effective than when measured using N-acetylglycyrrhizine methyl ester as a substrate and using the method of Reference Example 1. It was 555 times more sensitive than the conventional method.

[Brief explanation of the drawing]

FIG. 1 shows the effects of the compounds of the present invention using thrombin activity as an index. AGLNE represents acetylglycyrrhizine naphthyl ester, and AGLME represents acetylglycyrrhizine methyl ester.

Claims (1)

[Claims] 1 Formula () Amino acid derivative represented by. (In the formula, R represents a naphthyl group) 2 Formula () By subjecting the glycyrrhizine derivative shown by the formula () to a normal dehydration condensation reaction with naphthol, the formula () is obtained. Formula () (in the formula, R
is a naphthyl group, R′ is an amino protecting group) A method for producing an amino acid derivative represented by 3 formula () A method for measuring enzyme activity, which comprises bringing an amino acid derivative represented by the formula (R represents a naphthyl group) into contact with an enzyme as a substrate. 4. The method of measuring enzyme activity according to claim 3, characterized in that the amino acid derivative represented by the formula () is brought into contact with an enzyme as a substrate, and after a certain period of time, the naphthol liberated by hydrolysis by the enzyme is measured. Measurement method.