JPS6112898B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a tyrosine 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 phenyl 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 acetyltyrosine () It can be produced by a conventional dehydration condensation reaction between phenol and phenol.

In carrying out the present invention, the inventive substance (2) can be obtained by a conventional dehydration condensation reaction between the acetyltyrosine (2) and phenol.
In this method, the hydroxyl group of acetyltyrosine may be protected with a commonly used hydroxyl-protecting group such as a carbobenzyloxy group. When the group is a carbobenzyloxy group, the substance of the present invention () can be obtained by catalytic reduction with palladium on carbon or the like, or decomposition reaction with a hydrobromide-acetic acid solution.

To carry out the dehydration condensation reaction, the raw material is dissolved in a suitable solvent, an ester activator such as DCC (dicyclohexylcarbodiimide), DPPA (diphenylphosphoryl azide), or alkyl chlorocarbonate is added. It can be produced by adding phenol to the solution, adding a base such as triethylamine if necessary, and stirring. In addition, the well-known acid chloride method and the well-known phenylating agent (Ber. 49 ,
2339, 1916), but the method using the above-mentioned ester activator is preferred. Solvents that can be used include chloroform,
Any commonly used dichloromethane, dimethylformamide, tetrahydrofuran, etc. may be used as long as it dissolves the raw material. Reaction temperature is 0°~
40℃ is good. After the reaction is completed, the target compound can be obtained from the reaction solution by a commonly used treatment method. That is, for example, when DCC is produced as an ester activator, precipitated dicyclohexyl urea is removed, washed with an aqueous hydrochloric acid solution, an aqueous NaOH solution, and saturated saline, dried with a drying agent such as anhydrous magnesium sulfate, and then washed with a solvent. It can be obtained by distilling off. The target compound can be purified by recrystallization, chromatography, etc., if desired.

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 the liberated phenol 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 hydroxylamine, reacting with iron perchloride to develop a color, and measuring the color development as absorbance. This method measures the ester water-degrading ability of the enzyme, that is, the activity of the enzyme. Other methods that use a substrate and use the water-degrading ability of esters as an indicator include the chromotropic acid method, but these methods require a certain amount of enzyme and are difficult to use when the enzyme is at a low concentration or has low activity. Measuring enzymes was difficult. 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 a compound that is far superior to conventional ones.

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 an appropriate buffer solution, and measuring the released phenol 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 appropriate constant conditions, but it is preferable to measure after 30 minutes at 25 to 37°C. Conventional methods for measuring phenol include well-known physicochemical methods such as gas chromatography or thin layer chromatography, perchloriron reaction, diazo coupling reaction,
Either the FVB (first violet B salt) method or a chemical method such as the 4-aminoantipyrine method may be used, but in the case of measuring phenol, 4-aminoantipyrine is added to the reaction solution to develop color. A method of measuring absorbance using a spectrophotometer is a more preferable method due to its 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 a suitable preparation, the enzymes are separated from this by electrophoresis, etc., this is immersed in a solution of the substance of the present invention, and after an appropriate time, further By adding the above-mentioned coloring reagent, it is possible to see enzyme patterns in the blood that were previously impossible to see. 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 C1 esterase or chymotrypsin, which is well known as an inflammatory enzyme among various enzymes, and C1 esterase was measured using acetyl tyrosine phenyl ester as a substrate. In the case, conventionally,
The detection sensitivity was higher than that measured by the hesterin method using acetyl tyrosine ethyl ester or tosyl arginine methyl ester, which are known enzyme substrates.

Next, examples of the present invention will be given and explained in more detail.

Example 1 Synthesis of N-acetyltyrosine phenyl ester Dissolve 22.3g of N-acetyltyrosine in 125ml of N・N'-dimethylformamide and add 10.0ml of phenol.
g, add 16 ml of triethylamine and stir under ice cooling with N.
After adding 26.7 g of N'-dicyclohexylcarbodiimide and stirring for 1 hour, the mixture was returned to room temperature and further stirred overnight. After the reaction, N-N'-dicyclohexylurea precipitated was removed by filtration, ethyl acetate was added to the mother liquor, and the mixture was washed with 10% citric acid, saturated aqueous sodium chloride, and saturated brine, and dried over anhydrous magnesium sulfate. The solvent was distilled off and the residue was adsorbed on silica gel.
Collect the fractions eluted with chloroform containing % methanol, add ether and collect the precipitated crystals.

Yield 9.0g, yield 30%, melting point 116-118℃ IR (KBr) cm ^-1 ; 3250, 1750, 1640 MS; m/e299M+ Elemental analysis C ₁₇ H ₁₇ NO ₄ (299.31) Theoretical value C, 68.21 H , 5.73 N, 4.68 Actual value C, 68.33 H, 5.88 N, 4.66 Example 2 Method for measuring the activity of C1 esterase using N-acetyl tyrosine phenyl ester as a substrate Add N-acetyl tyrosine phenyl to 0.5 ml of a diluted solution of C1 esterase. Ester solution (14.9mg/10ml
Add 0.5 ml of H ₂ O) and 2 ml of 4-aminoantipyrine solution (134 mg/200 ml phosphate buffer) and heat at 37℃.
Incubate for 30 minutes. Furthermore, add 2 ml of potassium ferricyanide solution (500 mg/200 ml citrate buffer).
After incubation at ℃ for 30 minutes, color development is measured as absorbance (500 nm) using a spectrophotometer to quantify the amount of phenol released by hydrolysis by the enzyme.
The amount of released phenol indicates the activity of the enzyme.

Reference Example 1 A method for measuring the activity of C1 esterase using N-acetyltyrosine ethyl ester as a substrate. (Hesterin method) Add N-acetyl tyrosine ethyl ester solution (10 μmoles/0.4 ml) to 0.5 ml of C1 esterase diluted solution.
5% DMSO) 0.4ml, and phosphate buffer (PH7.4)
Add 0.1ml. After incubation at 37°C for 30 minutes, hydroxylamine solution (2M−
Equivalent mixture of NH ₂ OH HCl and 3.5M NaOH)
Add 1.5ml and leave at room temperature for 15 minutes. To this, 1 ml of 18% trichloroacetic acid, 1 ml of 4N hydrochloric acid, and 1 ml of 10% ferric chloride were added, stirred thoroughly, and then heated at 3000 r.p.
Centrifuge for 10 min at m. 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 C1 esterase, and the enzyme activity is equivalent to the value obtained by subtracting the value obtained after carrying out the enzyme reaction from the value of substrate only (control). do. Example 2
The results at each concentration level of C1 esterase measured in Reference Example 1 are shown in the figure, and it can be seen that the former method has about 10 times the sensitivity than the latter method. In the drawings, ○ marks indicate the standard curve obtained by the method of Example 2, and × marks indicate the standard curve obtained by the method of Reference Example 1.

[Brief explanation of the drawing]

The figure shows a standard curve for C1 esrase activity.

Claims (1)

[Claims] 1 Formula () (In the formula, R represents a phenyl group.) An acetyltyrosine derivative represented by the following. 2 formula () Formula () characterized by a normal dehydration condensation reaction of acetyltyrosine and phenol shown by
expression () A method for producing an acetyltyrosine derivative represented by the formula (wherein R represents a phenyl group). 3 formula () A method for measuring enzyme activity, which comprises contacting an acetyltyrosine derivative represented by the formula (wherein R represents a phenyl group) with an enzyme as a substrate. 4. Enzyme activity according to claim 3, characterized in that the acetyltyrosine derivative represented by formula () is brought into contact with an enzyme as a substrate, and after a certain period of time, the phenol released by hydrolysis by the enzyme is measured. How to measure.