JPS5850986B2 - Lysine derivative - Google Patents

Description

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

In the formula, R represents a phenyl group or an α-naphthyl group.

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

The substance (I) of the present invention is formed into an ester body represented by the formula (I) by a conventional dehydration condensation reaction between the N-tosyllisine derivative (II) and phenol or α-naphthol, and then ω-
It can be produced by removing the amino group protecting group.

In the formula, R represents a phenyl group or an α-naphthyl group.

R' represents an amino group-protecting group. In carrying out the present invention, the ester (II) can be obtained by a conventional dehydration condensation reaction between the above N-tosyllysine derivative (II) and phenol or α-naphthol.

That is, an N-tosyl lysine derivative is dissolved in a suitable solvent, DCC (dicyclohexylcarposiimide), DP
It can be produced by adding an ester activator such as PA (diphenylphosphorylaceto) or alkyl chlorocarbonate, adding phenol or α-naphthol thereto, adding a base such as triethylamine if necessary, and stirring.

In addition, the well-known acid chloride method, phenol or α
- Phenylating agent or α-naphthylating agent (Ber, 49, 2339), which is well known as the sulfite form of naphthol.
, 1916).

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 (l[) 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,
It can be obtained by removing the precipitated dicyclohexyl urea, washing with an aqueous hydrochloric acid solution, an aqueous NaOH solution, and a saturated saline solution, drying with a desiccant such as anhydrous magnesium sulfate, and then distilling off the solvent.

Ester (1) can be purified by recrystallization, chromatography, etc., if desired.

It can be obtained by a usual reaction in which the amino group protecting group is removed when producing the target compound (1) from the ester (I).

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

The substance (I) of the present invention acts as a substrate when brought into contact with an enzyme, and the activity of the enzyme can be measured by measuring the phenol or α-naphthol released after being hydrolyzed by enzyme removal for a certain period of time.

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 thawing 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 moiety after a certain period of time into hydroxamic acid with hydroxylamine, reacting with ferrous iron to develop a color, and measuring the color development as absorbance. Based on the results, This method measures the ester ice-melting ability of an enzyme, that is, the activity of the enzyme.

Other methods that use a substrate and use the ice-melting ability of esters as an indicator include the chromotrobic 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. There were difficulties in measuring and determining enzymes.

Therefore, the inventor has an affinity for enzymes,
Furthermore, by searching for compounds that can serve as substrates that meet the following three conditions: simple quantitative method and high detection sensitivity, we were able to find a compound that is far superior to conventional methods.

In carrying out the present invention, an enzyme and a certain amount of a compound (
Enzyme activity can be measured by contacting 1) in an appropriate buffer solution and measuring liberated phenol or α-naphthol after a certain period of time at a certain temperature.

The buffer may be any suitable buffer having a pH below that 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 or α-naphthol are:
Either the well-known physicochemical methods such as gas chromatography or thin layer chromatography, or chemical methods such as loriron reaction, diazo coupling reaction, FVB (first violet B salt) method or 4-amino antipyrine method. However, in the case of measuring phenol, 4-aminoantipyrine is added to the reaction solution to develop a color, and the absorbance is measured using a spectrophotometer.
- In the case of naphthol, a method in which FVB is added to the reaction solution to develop a color and measured as absorbance using a spectrophotometer is a more preferable method in terms of simplicity and detection sensitivity.

This property 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 enzyme is separated 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-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 substances of the present invention are trypsin, plasmin, kallikrein,
It acts as an excellent substrate for various enzymes such as urokinase, C1 esterase, and thrombin.
When enzyme activity is measured using naphthyl ester as a substrate, it is 14 to 25 times more active than when measured using the hesterin method using tosyl lysine methyl ester and tosyl arginine methyl ester, which are conventionally known as enzyme substrates.
It had a detection sensitivity of 0 times.

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

Example l Synthesis of Na-tosyl-lysine-α-naphthyl ester Na-tosyl-N5-carbobenzyloxylysine (J
, C, S, , 48.30-34.1957) was dissolved in 10rrll of pyridine, and 1,1'-dinaphthylsulfite (Ber, 49, 2339, 1916) 3
．． Add 5g and stir at room temperature for 3 days.

After the reaction was completed, ethyl acetate was added, diluted hydrochloric acid, saturated deuterated water,
After washing with saturated brine, drying over anhydrous magnesium sulfate and distilling off the solvent.

The precipitated crystals are recrystallized from ether to obtain Na-1-silylN'-nicarbobenzyloxylysine-α-naphthyl ester.

Yield 5.1 g, yield 91% This was dissolved in a 30% hydrobromic acid acetic acid solution and stirred at room temperature for 30 minutes.

Anhydrous ether is added to the reaction solution, a crystalline powder is collected, and recrystallized from a mixed solvent of ethanol and ether to obtain N-tosyllisine α-naphthyl ester hydrobromide.

Yield 3.6g, yield 50% Melting point 195-197℃IR
(KBr)CrrL-': 3250, 2950, 175
0 elemental analysis C23H27N2045Br (507,
45) Theoretical value C, 5, 4, 43H, 5, 36N, 5.5
2 Actual value C, 54,61H, 5,38N, 5.64 Example 2 Synthesis of Na-1-silurisine-phenyl ester Na-
) Suspend 2.1 g of sil-N-carbobenzyloxylysine in 10 ml of anhydrous ether and add 1.2 g of phosphorus pentachloride.
Add g and stir at room temperature for 1 hour.

The reaction solution is filtered, and the mother liquor is dried under reduced pressure at a temperature below 40°C.

The residue was dissolved in 1011 liters of anhydrous tetrahydrofuran, 0.47 g of phenol and 2.1 ml of triethylamine were added, and the mixture was stirred for 1 hour while cooling with water, then brought to room temperature and stirred for 1 day and night.

Ethyl acetate was added to the reaction mixture, washed with 10% citric acid, saturated aqueous sodium chloride, and saturated brine, and then dried over anhydrous magnesium sulfate.

When the solvent was distilled off and the residue was recrystallized from ether, Na-
1-Syl-N5-carbobenzyloxe-717 diphenyl ester is obtained.

Yield i 0-9g1 Yield 38%, melting point 111-113° Dissolve the garlic in 5Tll of DMF, dioxane hydrochloric acid solution (0
,065gHCl19) 0.8ml and 10%
Add Pd-c250IQ and pass hydrogen gas through for 3 hours at room temperature.

After the reaction is complete, filter and add ether to the mother liquor to remove N.
-t-silurizine phenyl ester hydrochloride is obtained as colorless needles.

Yield 0.7g, yield 88%, melting point 117-120℃IR
(KBr)crfL';3400,3150.1740
Elemental analysis C19H25N204SC1 (400,8
9) Theoretical value C, 56,92H, 6,29N, 6.99 Actual value C, 56,68H, 6,31N, 6.80 Example 3 Activity measurement method N-)sillysine-α-naphthyl ester solution (0.5 μmoles
10.5m Add 0.5 ml of 5% DMSO
Incubate for 30 minutes at °C.

Add 1% First Violet B Salt (FVB) to this.
Add 0.1d and leave it at 0℃ for 30 minutes, then add glacial acetic acid ITL.
1 and the absorbance (515 nm
), and the α-
Determine naphthol.

The amount of liberated naphthol is the activity of the enzyme.

Reference example l Trypsin activity measurement method using Na-tosylurisine methyl ester as a substrate (Hesterin method) Trypsin diluted solution (0.5d) with N-) syllysine methyl ester solution (10 μmoles 10.4 mA'5% DMSO)
0.4TrLl, and phosphate buffer (pH 7,4) 0
．． Add 1ml.

After incubation for 30 minutes at 37°C, hydroxylamine solution (2M-NH2OH-HCI and 3.
5. Add 1.5 TLl of M NaOH and leave to stand at room temperature for 15 minutes.

Add to this 1 ml of 18% trichloroacetic acid and 1 mA' of 4N hydrochloric acid.
, and 1 ml of 10% ferric chloride were added, stirred thoroughly, and centrifuged at 3000 rJ) m for 10 minutes.

The color development of the supernatant was measured by absorbance (530 nm) using a spectrophotometer.
Measure as.

This value correlates to the amount of substrate remaining without being hydrolyzed by trypsin, and the enzyme activity is the value of substrate only (control)
It corresponds to the value obtained by subtracting the value obtained after carrying out the enzyme reaction.

The results at each concentration level of trypsin measured in Example 3 and Reference Example 1 are shown in FIG. 1, and it can be seen that the former method is about 250 times more sensitive than the latter method.

In FIG. 1, ○ marks indicate the standard curve obtained by the method of Example 3, and X marks indicate the standard curve obtained by the method of Reference Example 1.

Example 4 Thrombin activity measurement method using Na-tosyllisine phenyl ester as a substrate N-tosyllisine phenyl ester solution (0.5 μmoles / 0.5 ml 5%
Add 0.5 ml of DMSO) and 2 ml of 4-aminoantipyrine solution (134ml/200ml phosphate buffer) and react at 37°C for 30 minutes.

Furthermore, potassium ferricyanide solution (500m9/20
2rILl (0rfLl citrate buffer) is added and incubated at 37°C for 30 minutes, and the color development is measured as absorbance (500 nm) using a spectrophotometer to quantify the phenol released by hydrolysis by the enzyme.

The amount of released phenol indicates the activity of the enzyme.

Example 5 A method for measuring blood enzyme patterns by electrophoresis.

A polyacrylamide gel column is prepared using a glass tube with four internal diameters and a length of 7 CIrL1.

After adding 40 μl of serum to this and electrophoresing at 2 mA for about 50 minutes, the gel is taken out.

This gel was mixed with a substrate solution (N-tosyllysine-α-naphthyl ester 0.5 μrnoles/yrtl) 10
ml, incubated at 25°C for 30 minutes, cooled to 0°C,
Add 1 rnl of solution (salt) and leave at 0°C for 30 minutes to stain.

By the above method, the blood enzyme pattern can be observed as a pink to purple band.

Figure 2 shows the results when normal human serum was used.

[Brief explanation of the drawing]

Figure 1 shows the standard curve of trypsin activity and Figure 2 shows the blood enzyme pattern.

Claims (1)

[Claims] 1. An N-1-silurisine derivative represented by formula (I). (In the formula, R represents a phenyl group or an α-naphthyl group) 2 By subjecting the N tosyl-lysine derivative represented by the formula (Il) and phenol or α-naphthol to a normal dehydration condensation reaction, a compound represented by the formula (I[) is obtained. N3 represented by the formula (I) (wherein R is a phenyl group or an α-naphthyl group, and R' is an amine protecting group) Formula (1) Method for producing tosyl lysine derivative. (In the formula, R represents a phenyl group or an α-naphthyl group.) A method for measuring enzyme activity, which comprises bringing a lysine derivative represented by the following formula into contact with oxygen as a substrate. 4. Claim 3, characterized in that the lysine derivative represented by formula (I) is brought into contact with an enzyme as a substrate, and after a certain period of time, phenol or α-naphthol released by hydrolysis by the enzyme is measured. The method for measuring enzyme activity described in .