JPH0358999A - Peptide derivative and determination of enzyme activity using the same - Google Patents

Abstract

NEW MATERIAL:D-phenylalanyl-prolyl-arginyl-3-tert.-butyloxycarbonyl-4- nitroanilide or its salt represented by formula I. USE:A color-developing synthetic substrate for determining thrombin activity having high reactivity, specificity and stability. PREPARATION:For example, the reaction of N<alpha>-t-butyloxycarbonyl-arginine with t-butyl 5-amino-2-nitrobenzoate in the presence of a condensing agent is followed by treatment of the product with hydrochloric acid-acetic acid- dimethylformamide to remove only t-butyloxycarbonyl group selectively. Then, the product is allowed to react with t-butyloxycarbonyl-D-phenylalanine-proline in the presence of a condensing agent to give the compound of formula I.

Description

[Detailed description of the invention] 1! TECHNICAL FIELD The present invention relates to a peptide derivative and a method for measuring enzyme activity using the same. More specifically, the present invention relates to D-phenylalanyl-prolyl-arginyl-3-tert-butyloxycarbonyl-4-nitroanilide represented by the structural formula (I) and its acid addition salt.

(2) The thrombin activity in the sample is measured by mixing a sample containing thrombin with the compound according to claim 1 and determining the reaction rate between thrombin and the compound or the amount of reaction product. Thrombin activity measurement method.

(3) D-phenylalanyl-prolyl-arginyl, which is obtained by reacting a sample containing antithrombin III with a certain excess amount of thrombin, and determining the remaining thrombin activity by the measuring method described in claim 2. -3-t
The present invention relates to ert-butyloxycarbonyl-4-nitroanilide and its acid addition salt, and a method for measuring thrombin and antithrombin In activity in serum and plasma using the same as a substrate.

Thrombin is a type of proteolytic enzyme involved in blood coagulation, and has the effect of converting fibrinogen into fibrin and promoting blood coagulation. Antithrombin III is a substance that broadly inhibits proteolytic enzymes related to blood coagulation, and particularly strongly inhibits thrombin and factor Xa in the presence of acidic mucopolysaccharide.It is produced in the liver and exists in large amounts in the blood.

As a method for quantifying thrombin activity in human plasma, there is a method using a substrate for enzyme activity measurement that releases a chromogenic compound or a fluorescent compound by the action of thrombin. In such measurement methods, the absorbance of a color-forming compound or the fluorescence intensity of a fluorescent compound released from a substrate for measuring enzyme activity by an enzyme reaction is measured, or the absorbance of a dye produced by a color reaction of the released compound is measured. and quantify enzyme activity.

There are various methods for measuring antithrombin III activity, such as coagulation methods and immunological methods.
Measurement methods using enzyme activity measurement substrates that release color-forming compounds have become mainstream because of their ease of automation.

In this measurement method, a certain excess amount of thrombin, which is inhibited by antithrombin III, is added to a sample containing antithrombin III, and after the reaction between thrombin and antithrombin III in the sample is completed, the remaining thrombin activity is used to release a color-forming compound. Antithrombin III activity in a sample is measured in the same manner as in the above-mentioned thrombin activity measurement using a substrate for enzyme activity measurement [Thromb, Res, 5.621-63.
2 (1974)].

In the thrombin and antithrombin nr activity measurement method described above, the enzyme activity measurement substrate used has high reactivity and specificity for thrombin, high detectability of decomposed products such as liberated chromogenic compounds, and is resistant to water or buffer solutions. Good solubility in all, and stability in all, is required.

Another important requirement is that thrombin activity can be measured by a reaction rate method that is not affected by coexisting substances such as dyes, that is, it is possible to measure thrombin activity by determining the reaction rate between thrombin and a substrate for enzyme activity measurement. It is.

Conventionally, substrates for measuring thrombin activity that can be measured by the reaction rate method include the following Japanese Patent Publication No. 56-15239,
56-22519, JP-A-57-2253, 59-
There are varanitroanilide (pNA) derivatives described in specifications such as No. 106446.

The substrate shown in Japanese Patent Publication No. 56-15239 has the following formula (II
)HD-Phe-A-Arg-pNA
(II) (wherein A represents Pro or Pip)
When A is Pro, such a substrate has the advantage of high reactivity to thrombin, but has a drawback in enzyme specificity. When A is Pip, specificity is high but reactivity is low.

The substrate shown in Japanese Patent Publication No. 56-22519 has the following formula (II
I) Na-Tos-Gly-Pro-Arg-
It is represented by pNA (III) (in the formula, Tos represents a tosyl group), and such a substrate has high reactivity, but has drawbacks in specificity and solubility.

JP-A-59-106446 discloses substrates represented by the following formulas (IV) and (V) (R represents an alkyloxy group, an alkylamino group, or an amino acid residue). Among the substrates represented by formula (IV), examples of substrates having an ester residue in which R is an alkyloxy group include primary or secondary ester residues such as methyl, ethyl, isopropyl, n-butyl, 2-pentyl, and benzyl.
Substrates having ester residues of alcohols are disclosed. These substrates have the disadvantage that they do not have sufficient reactivity with thrombin, and are easily hydrolyzed and unstable in the alkaline range used for measuring thrombin activity.

In the above-mentioned Japanese Patent Application Laid-open No. 59-106446, the third
Substrates having ester residues of alcohols are not specifically disclosed. Tertiary alcohol esters are primary
Compared to esters of primary or secondary alcohols, it has the unique property of being easily hydrolyzed in acidic conditions, but stable in alkaline conditions. Therefore, if a substrate containing a tertiary alcohol ester residue could be synthesized, the above-mentioned drawback of instability of the substrate would be overcome for enzymes whose activity is measured in the alkaline side, such as thrombin. .

However, it is not possible to synthesize a substrate having an ester residue of a tertiary alcohol by the method described in JP-A-59-106446. Japanese Patent Publication No. 59-106
According to the specification of No. 446, the ester-type substrate represented by formula (IV) is synthesized by the following method.

(υ) ulJLJR, that is, reaction of BOC (tert-butyloxycarbonyl)-protected dipeptide (A) and arginyl-3-alkyloxycarbonyl-4-nitroanilide (B) (1)
After condensation, the protecting group BOC is removed from the reaction product (C) with acetic acid and hydrochloric acid (reaction (2)).
, to obtain the desired substrate (D). However, the third
Since esters of alcohols are easily decomposed by acids, the reaction (
In 2), if -COOR of compound (C) is an ester of a tertiary alcohol, even the ester bond is cleaved. Therefore, a substrate having a tertiary alcohol ester residue cannot be synthesized by the method described in JP-A-59-106446.

The substrate represented by the formula (V) described in JP-A-59-106446 has good specificity for thrombin, but has the drawbacks of low reactivity and poor solubility.

On the other hand, antithrombin A I activity measurement is carried out by reacting antithrombin III with a certain excess amount of thrombin, and measuring the remaining thrombin activity using the various substrates for measuring thrombin activity described above. be able to.

An example of such a method for measuring antithrombin III activity is a method using, for example, D-phenyl-Pro-Arg-pNA as a substrate for measuring thrombin activity (Japanese Patent Laid-Open No. 102400/1983). This substrate also does not have satisfactory characteristics in terms of reactivity and specificity for thrombin, and it cannot be said that it is suitable for measuring antithrombin III activity. In addition, the above-mentioned Japanese Patent Application Laid-Open No. 59-10644
There is a method for measuring antithrombin III activity using a substrate represented by formula (IV) described in the specification of No. 6 (Japanese Patent Laid-Open No. 62-61600). Since the substrate represented by formula (IV) has the above-mentioned drawbacks, this method for measuring antithrombin III activity cannot be said to be a satisfactory method.

Problems to be Solved by the Invention Therefore, an object of the present invention is to overcome the drawbacks of conventional substrates for measuring thrombin, and to provide high reactivity to thrombin, high specificity, high solubility in water or buffer solutions, and the like. It is an object of the present invention to provide a color-forming synthetic substrate that has high stability and that can be measured by a reaction rate method using an automatic analyzer without being affected by sample components during measurement.

Another object of the present invention is to provide a method for measuring thrombin activity with high sensitivity, high precision, and high accuracy using the above-mentioned chromogenic synthetic substrate.

Yet another object of the present invention is to provide a highly sensitive, highly accurate, and highly accurate method for measuring antithrombin III activity using the above-mentioned chromogenic synthetic substrate.

Means for Solving the Problems Under these circumstances, the present inventors conducted intensive research to solve the drawbacks of the conventional methods, and as a result, the above-mentioned structural formula (I) having an ester residue of a tertiary alcohol was found. We have discovered that the compound solves the above problems and makes it possible to measure thrombin activity and antithrombin III activity using an automatic analyzer using a reaction rate method with high sensitivity, high precision, and high accuracy. It has been completed.

That is, the present invention provides D-phenylalanyl-prolyl-arginyl-3-tert-butyloxycarbonyl-4-nitroanilide represented by the structural formula (1) and its acid addition salt; ) and measuring the thrombin activity in the sample by determining the reaction rate or the amount of the reaction product between thrombin and the compound; and antithrombin III. This antithrombin III activity measuring method is characterized in that the antithrombin III activity in the sample is measured by reacting the sample with a certain excess amount of thrombin and determining the remaining thrombin activity by the above-mentioned thrombin activity measuring method.

The abbreviations used in this specification are as follows.

Abbreviations Phe: Phenylalanine Pro Nibroline Pip: Pipecolic acid Arg: Arginine Boc: tert-butyloxycarbonyl pNA
: p-nitroaniline HCI : Tos hydrochloride: p-)luenesulfonic acid G1y nigericin The compound represented by the formula (I) of the present invention can be easily obtained under specific conditions as shown in the following reaction formula and Example 1.

C(CH3)5 (a) C(CH3)3 (c) (d) + Boa-D-Phe-Pr.

(e) That is, first, Nα-butyloxycarbonyl-arginine hydrochloride-hydrate (a) and 5-amino-2-
Nitrobenzoic acid 7-butyl ester (b) is dehydrated and condensed by the carbodiimide method, which is a commonly used method in peptide chemistry, to obtain Nα-t-butyloxycarbonyl-arginyl-3-t-butyloxycarbonyl-4-nitro-anilide. Obtain (C).

Next, the commonly used method of removing the t-butyloxycarbonyl group using 1 to 2N hydrochloric acid-acetic acid also cleaves the t-butyl ester, so the present inventors developed a hydrochloric acid-acetic acid method as a result of intensive research. Only the t-butyloxycarbonyl group is selectively removed under mild conditions using -dimethylformamide to obtain arginyl-3-t-butyloxycarbonyl-4-nitroanilide dihydrochloride (d). Next, t-butyloxycarbonyl-D-phenylalanyl-proline (e) and arginyl-3-tert-
Butyloxycarbonyl-4-nitroanilide (d) is condensed by a mixed acid anhydride method, which is a method commonly used in peptide chemistry, preferably a carbodiimide method with the addition of hydroxybenzotriazole, and t-butyloxycarbonyl-D
-phenylalanyl-prolyl-arginyl-3-carboxy-4-nitroanilide monohydrochloride (0 is obtained.Finally, t-butyloxy The carbonyl group is eliminated to obtain D-phenylalanyl-prolyl-arginyl-3-butyloxycarbonyl-4-nitroanilide (I).

The compound of formula (I) thus obtained has a t-butyloxycarbonyl group which is an ester residue of a tertiary alcohol, is stable under alkaline conditions, has high solubility in water, and is resistant to thrombin.

It has excellent reactivity and specificity. The compound of formula (I) may be an acid addition salt, such as:
Examples include inorganic acid salts such as hydrochloride, sulfate, phosphate, nitrate, and hydrobromic acid; organic acid salts such as malate, citrate, methanesulfonate, and p-)luenesulfonate. It will be done.

To measure thrombin activity using a compound of formula (I), a sample containing thrombin and a compound of formula (I) are mixed in a suitable buffer, and the rate of reaction between thrombin and formula (I), i.e. The hydrolysis reaction rate of the compound of formula (1) is measured by a colorimetric method, or the chromogenic compound 5- is liberated from the compound of formula (I) by the reaction of thrombin with formula (I).
This is carried out by measuring the amount of amino-2-nitrobenzoic acid t-butyl ester by a colorimetric method.

Antithrombin III activity can be measured by reacting a sample containing antithrombin III with a certain excess amount of thrombin in an appropriate buffer, and then measuring the remaining thrombin activity by the same method as above. . In addition, when actually performing measurements,
The measurement can also be carried out by mixing a sample containing antithrombin II with the compound of formula (1) and adding a quantitative excess of thrombin.

Next, an example of a method for measuring antithrombin III activity will be explained. Antithrombin III activity measurement according to the invention is performed using 0.05 to 1 preferably 0.5 U/me of beparin, θ to 0.5 mole/e of Tris, Bis-Trispropane, HEPPS, HEPPSO, Tricine, Glycinamide, Glycine. Silglycine, preferably Tris 0.03-0.1 mole#, pH 8-9 preferably pH 8,4-8.6.0-0.5 mole/
e, the sample is added to a reagent containing a neutral salt or sodium chloride, preferably 0.1 to 0.2 mole/mole, and antithrombin III and thrombin are reacted;
By reacting residual thrombin with 0.1 to 5 mmole/e, preferably 1.0 to 1.5 mmole/e of compound (I), which is a substrate, and measuring the hydrolysis reaction rate of substrate (I). It is done.

EXAMPLES A detailed method for synthesizing compound (I) will be described below as Example 1.

Measurement of concentration of antithrombin III using substrate <1) in Example 2, measurement of thrombin activity using substrate (I) in Example 3, Km and Vmax of thrombin when using substrate (I) in Example 4 In Example 5, the solubility of the substrate (I) and in Example 6, the stability in an alkaline aqueous solution will be specifically explained.

It goes without saying that these examples represent one aspect of the present invention, and that the present invention is not limited thereto.

27.41 g (83.4 mmol) of NG-t-butyloxycarbonyl-arginine hydrochloride hydrate and 5-
Amino-2-nitro-benzoic acid t-butyl ester 1
After dissolving 9.87 g (83.4 mmol) in 167 m/m of anhydrous pyridine, the solution was cooled to -5°C and stirred with N-N.
'-Dicyclohexylcarbodiimide 37.86 g
A solution of 83 me (183.5 mmol) in pyridine was added dropwise, and the mixture was stirred and reacted overnight at room temperature. After the reaction was completed, 333m<' of ethyl acetate was added to the reaction solution, the precipitated dicyclohexylurea was filtered off, the solvent was distilled off under reduced pressure, and 750me of ethyl acetate was added thereto and the insoluble materials were collected by filtration. 37.32 g (yield 8
4.3%) of Not-t-butyloxycarbonyl-arginyl-3-1-butyloxycarbonyl-4-nitro-anilide was obtained.

Not-t-butyloxycarbonyl-arginyl-3-t-butyloxycarbonyl-4-nitro- obtained here
7.97 g (15 mmol) of anilide was added to DMF9 me.
and 3 m of acetic acid (dissolved in 2 N hydrochloric acid-acetic acid 6 m) under water-cooling and stirring.
Add Nα
Selective removal of protecting groups was performed. After the reaction was completed, 36me of ethyl acetate was added to the reaction solution, and the solution was poured into 2.5e of ether to obtain a precipitate, which was collected by filtration and dried under reduced pressure to obtain 6.48 g of crude crystals. Furthermore, the crystal is Dowe
x 2 x 8 (acetic acid type) [Dow Chemical
alginyl-3-t-butyloxycarbonyl-4-nitroanilide, dihydrochloride, was purified by column (methanol eluent), added with an equivalent amount of 0, IN hydrochloric acid-methanol, and precipitated with ether. crystal 5
．． 68 g (yield 81.0%) was obtained.

Melting point: 65-95°C (decomposed) Specific optical rotation: [α]D = +41.5° (C = 1, water) This crystal was analyzed by silica gel thin layer chromatography (n-butanol:acetic acid:water = 4:1: 2) with a single spot (Rf=
0.48).

Elemental analysis value C1□H28N605Ce2・1/2H
CHN as 20 Actual value (%) 42.77 6.20 17
．． 60 Theoretical value (%) 42.86 6.14
17.64 Salt t-butyloxycarbonyl-D-phenylalanyl-
Proline 14.83 g (40.9 mmol), arginyl-3-t-butyloxycarbonyl-4-nitroanilide, dihydrochloride 21.03 g (45 mmol)
and 6.26 g of hydroxybenzotriazole (
40.9 mmol) in 100 m dimethylformamide (
5.7 me of N-ethyl-morpholine followed by 8.63 g of water-soluble carbodiimide 45 mmole
was added and reacted at 0°C for 1 hour, and then at room temperature overnight. After the reaction, the solvent was distilled off, dissolved in ethyl acetate:n-butanol (1:1), cooled 5% hydrochloric acid, saturated brine,
After washing with saturated aqueous sodium hydrogen carbonate solution and saturated brine, it was dehydrated and dried over anhydrous magnesium sulfate. After drying, the solvent was distilled off and Sephadex LH-20 (Pharma
CIA) column [eluent dimethanol], 26.12 g (82.4%) of home-like t
-butyloxy-D-phenylalanyl-prolyl-arginyl-3-t-butyloxycarbonyl-4-nitro-anilide was obtained.

Specific optical rotation = [α]D = -32.5° (C = 0.1 methanol) This compound was analyzed by silica gel thin layer chromatography (chloroform: methanol: acetic acid: water = 80:20
:2.5:5) with a single spot (Rf = 0.40
) was given.

Elemental analysis value C36H51N8o9Ce+315H2
CHN Actual value (%) 54.93 6.72 14
．． 53 Theoretical value (%) 55.00 6.69
14.25 t-Butyloxycarbonyl-D-phenylalanyl-prolyl-arginyl-3-t-butyloxycarbonyl-4-nitro-anilide hydrochloride 25.
12 g (32.4 mmol) was dissolved in dimethylformamide (17.2 me) and stirred under water cooling. ON hydrochloric acid-acetic acid (256 rne) was added and the mixture was reacted at 200C for 60 minutes to selectively eliminate the Nt-butyloxycarbonyl group, and the mixture was poured into ether and the crude crystals were collected by filtration.

The crude crystals were collected on a Toyopearl HW40F column (eluent: 3
Purified with 0% acetic acid) and lyophilized to 13.22 g (5
7.3%) of D-7enylalanylprolylarginyl-3-1-butyloxycarbonyl-4-nitroanilide was obtained.

Melting point = 115-125 °C Resolution Specific rotation: [α] D = -128 ° (C = 0.5 water) This compound was purified by silica gel thin layer chromatography eluent (n
-butanol:acetic acid:water=4:1:2) to give a single spot (Rf=0.48).

Elemental analysis value C3□H44N8o7Ce2+2H20
As CHN Actual value (%) 49.40 6.12 15
．． 06 Theoretical value (%) 49.80 6.47
14.99 (1) Reagent ■ First reagent (hereinafter abbreviated as R-1) pH 8,6 (2
Sodium chloride (150 mmole/l!) in 75 mmole/e) Lys buffer at 5 °C),
Thrombin reagent consisting of bovine thrombin (0,45 NIH/me) and heparin (0,5 U/me).

■ Second reagent (hereinafter abbreviated as R-2) 6 mmole
/ e 1 (Compound (1)) aqueous solution.

(2) Creation of a calibration curve Normal plasma containing a certain amount of antithrombin III was diluted 6 times with physiological saline, 100%, and 0% physiological saline.
25.50.75.100.125.150 as %.
175% was adjusted and used as standard plasma, and 5% of standard plasma and R-
1400% was incubated at 37°C for 5 minutes, R-2 was added while maintaining the temperature at 37°C, and the reaction rate (ΔE/mi) was increased at 415 mm.
n and ΔE (change in absorbance) were measured, and a calibration curve shown in FIG. 1 was created.

(1) Reagent measurement reagent is a commercially available measurement kit (Daiichi Kagaku Test Team A
Adjustments were made using T-III・2) according to the instructions for use.

(2) Creation of a calibration curve The calibration curve shown in FIG. 1 was created using the attached standard plasma according to the instruction manual.

A calibration curve for measuring the substrate shown in FIG. 1 was prepared in the same manner as in 1 above except that 1 was used instead of the second reagent in 1 above.

Comparing the straight lines of the three calibration curves shown in FIG. 1, the slope of the calibration curve when compound (I) according to the present invention is used as a substrate is It can be seen that the slope is 1.8 times that when 18 is used as a substrate, and 1.5 times as much as when 18 is used as a substrate, and the measurement sensitivity is greatly improved.

Example 3 Group specificity for enzymatic reactions The specificity of the substrates according to the present invention and known substrates for enzymatic reactions was investigated by the following method.

(1) Substrate solution: Each substrate solution is dissolved in water, 2mM and 1
It was used as 0mM.

(2) Buffer: Buffer-like, NaC6, and their concentrations and pH (25°C) were as follows depending on the enzyme.

(3) Enzyme used (6) Measurement results (4) Reaction stop solution = 10% acetic acid aqueous solution (5) Measurement method Buffer solution 0.5 me and substrate solution 0.1 me in silicon-treated hard glass test tube or plastic Collect into a test tube and prewarm for 10 minutes in a 37°C constant temperature bath.

Next, 0.05 ml of enzyme reagent was added to incubate the enzyme reaction for 3
Perform for 10 minutes at 7°C.

After exactly 10 minutes, add 2.5 m of reaction stop solution to stop the enzyme reaction, leave it at 37°C for 10 minutes, and then
Measure the absorbance of

The measurement results in the case of mM are shown.

(Compound (I)) As described above, the substrate of the present invention, Compound (I), has a specificity for thrombin equivalent to or higher than that of conventional substrates. Regarding d and e, substrate inhibition against thrombin was observed at high concentrations, and the substrate concentration within the measurement system could not be increased.

Example 4 Measurement of Vmax and Km of the substrate The Vmax/Km value for thrombin of the substrate according to the present invention was determined and compared with that of a commercially available substrate.

(1) Substrate solution: Each substrate solution is dissolved in water and has a concentration of 0.25 to 5mM.
It was used as a solution.

(2) Buffer Nidris 100 mM, NaC (15
0mM, pH 8,2 (3) Thrombin: 0.258 NIHU/me
(4) Solution 2: mixture of assay buffer and thrombin aqueous solution at a ratio of 2:1. Orn, e is collected in a silicon-treated hard glass test tube or a plastic test tube, and prewarmed for 5 minutes in a 37°C constant temperature bath.

Next, 0.5 me of the substrate solution was added to carry out the enzymatic reaction, and after a lag time of 15 seconds, the dissociation rate of the chromogen was determined, and Km,
Vmax was calculated.

a: 2HCe-H-D-Phe-Pip
-Arg-pNAb': Compound (1) From the above, Vmax / Km of compound (I) of the present invention
The value is twice that of a known substrate, indicating that compound (I) has a high affinity and high reactivity for thrombin.

Example 5 The substrates a, d, f, and g used in Example 3 were mixed at 2 mM and 1
It was dissolved in water at a concentration of 0mM to check its solubility.

○: Completely dissolved x 2 Partially insoluble △ Knee Dissolved after standing overnight From the above, it can be seen that the compound (I) of the present invention has high solubility in water.

3-tert-butyloxycarbonyl-4-nitroaniline and 3-n-butyloxycarbonyl-4- which are chromogens (color-forming compounds) in the synthetic substrate (I) of the present invention
Nitroanilide 0. It was dissolved in an aqueous IN NaOH solution and left at 37°C for 24 hours, and the stability of tertiary alcohol esters and primary alcohol esters in alkaline solutions was confirmed using thin layer chromatography. As a result, 3-tert-butyloxycarbonyl-4-
While no decomposition was observed for nitroaniline, 3-n
-butyloxycarbonyl-4-nitroaniline is 90
It was confirmed that 3-carboxy-4-nitroanilide (sodium salt) was produced as a decomposition product.

This suggests that with conventional substrates containing primary and secondary ester residues, the ester moiety of the chromogen is hydrolyzed during long-term storage in the measurement system and solution, causing errors in measured values. In contrast, the substrate of the present invention having a tertiary alcohol ester residue is extremely stable.

A feature of the present invention is that the compound represented by formula (I) is useful as a substrate for measuring thrombin activity, is very stable in an alkaline region compared to conventional synthetic substrates, and has sufficient It has good specificity while maintaining good solubility, and furthermore, the reactivity of the substrate to thrombin has been greatly improved. Alternatively, the compound of formula (I) may be used as antithrombin II.
By using this method for I measurement, the sensitivity is greatly improved compared to conventional measurement methods.

For example, 1! The substrates having ester residues of Although stable, tert-butyl ester, which is the ester moiety of the compound of formula (I) of the present invention, has such stability in an alkaline range that it is not hydrolyzed even in an aqueous sodium hydroxide solution.

The formula (III
) and the substrate of formula (V) shown in JP-A-59-106446 have poor solubility, but the substrate of formula (I) of the present invention
) has sufficient solubility.

Furthermore, the substrates of formula (I) of the present invention have thrombin specificity comparable to or greater than conventional substrates. And, HD-Phe-Pip-Arg-pNA (Special Publication 5
When comparing the Km/Vmax for thrombin between the substrate of formula (I) of the present invention and the substrate of formula (I) of the present invention, the Km/Vmax of formula (I) was improved twice.

Therefore, the method for measuring thrombin and antithrombin III activity using the compound of formula (I) of the present invention is extremely effective in diagnosing disseminated angiocoagulant syndrome (DIC) and liver disease.

[Brief explanation of drawings]

FIG. 1 shows a calibration curve used to measure antithrombin III activity using the compound of formula (I) of the present invention and a known substrate.

Claims (3)

[Claims] (1) D-phenylalanyl-prolyl-arginyl-3-tert-butyloxycarbonyl-4-nitroanilide and its acid addition salt represented by the structural formula (I) ▲Mathematical formula, chemical formula, table, etc.▼(I). (2) The thrombin activity in the sample is measured by mixing a sample containing thrombin with the compound according to claim 1 and determining the reaction rate between thrombin and the compound or the amount of reaction product. Thrombin activity measurement method. (3) The antithrombin III activity in the sample is measured by reacting a sample containing antithrombin III with a certain excess amount of thrombin, and determining the remaining thrombin activity by the measuring method described in claim 2. Antithrombin III activity measurement method.