JPH0366319B2 - - Google Patents

Description

[Detailed description of the invention]

[Field of Application of the Invention] The present invention relates to a novel peptide derivative useful as a plastid substrate for measuring specific enzyme activity. [Background of the Invention] In order to measure the action and potency of various enzymes, a method is usually used in which the enzyme is allowed to act on a substance that is specifically affected by the enzyme, and the conditions before and after the action are compared. Substances that act specifically with the enzyme used in this case, ie, naturally occurring substances and various synthetic peptide derivatives as substrates, have been developed and reported. A method for detecting trace amounts of endotoxin has been developed and put into practical use, based on the phenomenon in which horseshoe crab blood cell extract (amebocyte lysate) reacts with trace amounts of bacterial endotoxin and turns into a gel. A detection method based on this principle is based on the US Pharmacopeia Act.
XX.888 (1980)], there are methods for visually determining gel hardness, turbidity measurement, clot protein quantification, etc., but all of them are based on the gelation phenomenon and are highly accurate. do not have. Also, “Novel plastid enzyme substrate” (Japanese Patent Application Laid-Open No. 51-83535
The invention of R ₁ −A ₁ −A ₂ −Gly−Arg−
This method uses a plastid substrate represented by NH-R ₂ to measure the chromophore liberated through enzymatic action using a spectrophotometer, but this method has the disadvantage of being easily interfered with by pigment components in the blood sample. There is. [Object of the Invention] The present invention was made in view of the above-mentioned circumstances, and allows the activity of a specific enzyme (protease) to be measured easily and with high sensitivity, while also eliminating the influence of pigment components in blood samples. The aim is to provide a plastid matrix that is refractory. [Structure of the invention] The present invention is based on the general formula [] (In the formula, R ¹ represents a leucyl group, a valyl group, or a valyl-leucyl group having a protecting group at the N-terminus.Also, even if the guanidino group of the arginine residue in the formula has a protecting group, (The invention may be in the form of an acid addition salt.) This invention relates to a peptide derivative or an acid addition salt thereof. That is, the present inventors sought a plastid substrate that allows the activity of a specific enzyme (protease) to be measured easily and with high sensitivity, and that is not easily affected by pigment components in blood samples. As a result of extensive research, 4-amino-N-ethyl-, which produces a dye with maximum absorption (λmax) on the long wavelength side, is produced by oxidative condensation with phenol, naphthol, etc. on the C-terminal side of an arginine residue. N-
A peptide derivative represented by the general formula [ ] to which (β-hydroxyethyl)aniline or 4-amino-3-methyl-N-ethyl-N-(β-hydroxyethyl)aniline is bonded can achieve this purpose. This discovery led to the completion of the present invention. That is, when the peptide derivative represented by the general formula [ ] is used as a substrate for a specific enzyme (protease), for example, an amidase-like enzyme produced as a result of the reaction of horseshoe crab amebocyte lysate with bacterial endotoxin, the activity of the enzyme can be inhibited. It can be measured simply and with high sensitivity, and is hardly affected by pigment components in the blood sample. The N-terminal protecting group for R ¹ in the general formula [ ] is not particularly limited and may be any group commonly used as an N-terminal protecting group of amino acids or peptides, but examples include: Examples include acyl groups such as acetyl group and benzoyl group, carbobenzoxy group, tertiary alkyloxycarbonyl group, tosyl group, and glutaryl group. In addition, as a protecting group for the guanidino group of the arginine residue of the general formula [], for example, a nitro group, a tosyl group, a p-methoxybenzenesulfonyl group, a 4-
Examples include those commonly used as N-guanidino protecting groups such as methoxy-2,6-dimethylbenzenesulfonyl group. The peptide derivative represented by the general formula [] may be an acid addition salt. Examples of such acid addition salts include inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, and phosphoric acid;
Examples include acid addition salts of organic enzymes such as acetic acid, oxalic acid, tartaric acid, succinic acid, citric acid, and toluenesulfonic acid. The peptide derivative of the present invention can be synthesized, for example, as follows. That is, the general formula [ ] (In the formula, R represents a hydrogen atom or an N-terminal protecting group of an amino acid.) An arginine derivative (usually one in which the N-terminus of an amino acid is protected with an appropriate protecting group) is used. It is dissolved in a solvent commonly used in the synthesis of peptide derivatives, such as dimethylformamide (DMF), dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), water, or a mixture thereof, and then heated in a nitrogen atmosphere. , glycine, leucyl-glycine, baryl-glycine, baryl-leucyl-glycine, or active esters thereof [e.g., N-hydroxysuccinimide ester, N-hydroxy-5- Norbornene-2,3-dicarboximide ester (HONB), 1-hydroxybenzotriazole ester, p-nitrophenyl ester, etc. are bonded to the C-terminal side of the above-mentioned amino acids or peptides. ] and a condensing agent commonly used for peptide synthesis, such as dicyclohexylcarbodiimide (DCCD), and then react at room temperature, if necessary with heating, for an appropriate time. After the reaction is complete, concentrate the reaction solution. The product of the present invention can be obtained by drying, and then purifying it by a method commonly used for purifying peptide derivatives, such as silica gel column chromatography, ion exchange column chromatography, etc., and deprotecting it if necessary. Peptide derivatives are easily obtained. The peptide derivatives of the present invention thus obtained are useful as substrates for specific enzymes (proteases), such as amidase-like enzymes produced as a result of the reaction of horseshoe crab amebocyte lysate with bacterial endotoxins. That is, the peptide derivative of the present invention thus obtained has the property that the peptide bond on the C-terminal side of the arginine residue is easily hydrolyzed by the action of an amidase-like enzyme; 4-amino-N-ethyl-N-(β-hydroxyethylethyl)aniline or 4-amino-3-methyl-N- released
By oxidative condensation of ethyl-N-(β-hydroxyethyl)aniline with phenol, naphthol, etc. in the presence of an appropriate oxidizing agent, and measuring the change in absorbance caused by the blue indophenol dye produced, amidase In other words, the amount of bacterial endotoxin in a sample can be quantitatively detected (see cascade below).

[Table] The arginine derivative represented by the general formula [], which is a raw material for the synthesis of the peptide derivative of the present invention, can be easily synthesized, for example, as follows, so it is sufficient to use it. That is, arginine whose amino group is protected with an appropriate protecting group or arginine whose amino group and guanidino group are protected with an appropriate protecting group is mixed with a solvent commonly used in the synthesis of peptide derivatives, such as dimethylformamide (DMF). ), dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), water, or a mixture thereof, and 4-amino-N-ethyl-N-(β-hydroxyethyl)aniline was dissolved in this under a room atmosphere. or 4
-amino-3-methyl-N-ethyl-N-(β-
After adding hydroxyethyl)aniline and a condensing agent commonly used in peptide synthesis, such as dicyclohexylcarbodiimide (DCCD), the mixture is allowed to react at room temperature, with heating if necessary, for an appropriate period of time. After the reaction is completed, the reaction solution is concentrated to dryness, and then purified by a method commonly used for purifying peptide derivatives.
For example, purification by silica gel column chromatography, ion exchange column chromatography, etc.
By carrying out deprotection treatment if necessary, the arginine derivative represented by the general formula [] can be easily obtained. EXAMPLES Below, the present invention will be explained in more detail with reference to Examples. [Example] Reference example 1 (a) Synthesis of carbobenzoxy-L-leucyl-glycine ethyl ester A solution of 26 g of carbobenzoxy-L-leucine and 14 g of glycine ethyl ester hydrochloride dissolved in 400 ml of THF was cooled in an ice bath. Lower triethylamine 15
ml, 18 g of HONB and 22 g of DCCD were added. Next, this was stirred and reacted at room temperature for 20 hours, and then the precipitate was filtered off and the solvent was distilled off. The resulting residue was dissolved in 500 ml of ethyl acetate, washed successively with saturated aqueous sodium bicarbonate solution, 1N hydrochloric acid, and water, and then the organic layer was dried over anhydrous sodium sulfate. After filtering out the desiccant,
The organic layer was concentrated to dryness under reduced pressure, and petroleum benzine was added to the resulting residue to obtain a gel-like solid, which was further recrystallized from ethyl acetate/petroleum benzine to obtain carboxy-L-leucyl-glycine ethyl ester. Ta. Yield: 30.1g (83%). Melting point 114-115℃. Specific optical rotation [α] ²¹ _D = -26.1 (C = 1.05, ethanol). Elemental analysis values (as C ₁₈ H ₂₆ N ₂ O ₅ ) Calculated values (%): C61.70, H7.48, N7.99, Actual values (%): C62.07, H7.50, N7.91. (b) t-butyloxycarbonyl-L-valyl-
Synthesis of L-leucyl-glycine 10.5 g of carbobenzoxy-L-leucyl-glycine ethyl ester obtained in (a) and 5.7 g of p-toluenesulfonic acid were dissolved in 200 ml of ethyl alcohol.
To this was added 5 g of 5% palladium black catalyst, and the mixture was stirred and reacted at room temperature for 3 hours while passing hydrogen gas.
After the reaction was completed, the catalyst was filtered off from the reaction solution, and then the solvent was distilled off under reduced pressure. The obtained oil and 6.5 g of t-butyloxycarbonyl-L-valine and
Dissolve 5.4 g of HONB in 300 ml of THF, and add 4.2 ml of triethylamine and 7.4 g of DCCD to this while cooling in an ice bath.
was added, and the mixture was stirred and reacted at room temperature for 20 hours. After the reaction was completed, insoluble materials were filtered off and the solvent was distilled off. The resulting residue was dissolved in 500 ml of ethyl acetate, washed successively with saturated aqueous sodium bicarbonate solution, 10% citric acid, and water, and then the organic layer was dried over anhydrous sodium sulfate. After filtering off the desiccant, the organic layer was concentrated to dryness under reduced pressure. Petroleum ether was added to the residue to solidify it, which was further recrystallized from ethyl acetate/petroleum ether to obtain t-butyloxycarbonyl-L-valyl-L-leucyl-glycine ethyl ester. Yield 10g (83%). Melting point 113-114℃. Specific optical rotation [α] ²¹ _D = 56.3 (C = 0.95, ethanol). Elemental analysis values (as C ₂₀ H ₃₇ N ₃ O ₆ ) Calculated values (%): C57.81, H8.98, N10.11, Actual values (%): C57.97, H8.91, N 9.92. Then, this t-butyloxycarbonyl-L-
Valyl-L-leucyl-glycine ethyl ester
4.1 g was dissolved in 30 ml of methyl alcohol, and while cooling in an ice bath, 20 ml of 1N aqueous sodium hydroxide solution was added, and the reaction was stirred for 2 hours. After the reaction is complete, add
After neutralizing by adding 18 ml of 1N hydrochloric acid and concentrating under reduced pressure,
Add 2 ml of 1N hydrochloric acid and add 300 ml of ethyl acetate.
Extracted in ml. The extract was dried over anhydrous sodium sulfate, the desiccant was filtered off, and the organic layer was concentrated to dryness under reduced pressure. After adding petroleum ether to the residue and solidifying it,
This was recrystallized from ethyl acetate/petroleum ether,
t-Butyloxycarbonyl-L-valyl-L-
Leucyl-glycine was obtained. Yield 3.6g (93)%. Melting point 104-108℃. Specific optical rotation [α] ²¹ _D = -54.6 (C = 0.99, ethanol). (c) t-butyloxycarbonyl-L-valyl-
L-leucyl-glycine-N-hydroxy-5
- Synthesis of norbornene-2,3-dicarboximide ester 1.5 g of t-butyloxycarbonyl-L-valyl-L-leucyl-glycine obtained in (b) was added to 50 ml of THF.
0.84 g of HONB and
1.0 g of DCCD was added and the mixture was stirred and reacted at room temperature for 20 hours. After the reaction, the precipitate was filtered off and the solvent was distilled off to give t-butyloxycarbonyl-L-valyl-L-leucyl-glycine-N-hydroxy-5.
-Norbornene-2,3-dicarboximide ester was obtained. Yield 1.9g (90%). Reference Example 2 Synthesis of 4-[N〓-carbobenzoxy-L-arginine)amino]-N-ethyl-N-(β-hydroxyethyl)aniline 3.0 g of N〓-carbobenzoxy-L-arginine
Dissolve it in 80 ml of DMF under heat, add it to this in a nitrogen stream,
4-Amino-N-ethyl-N-(β
-Hydroxyethyl)aniline 2.75g and DCCD4.0
g was added thereto, and the mixture was stirred and reacted at room temperature for 20 hours. After the reaction, insoluble materials were filtered off, the solvent was distilled off, and the resulting residue was subjected to silica gel column chromatography (column: 15 x 5 cm, eluent: ethyl acetate: pyridine: water: acetic acid = 60: 20:10:5.), and the concentrated dry matter of the main eluate was dissolved in water and lyophilized to obtain 4-[(N〓-carbobenzoxy-L-arginyl)amino]-N -ethyl-N-
(β-hydroxyethyl)aniline diacetate was obtained. Yield 1.45g (25%). Specific optical rotation [α] ²¹ _D = -17.2 (C = 0.95, methanol). Elemental analysis value (as _C24H35N6O4・_2CH3COOH ) Calculated _value (%): C56.83, H7.33, N14.20, Actual _value ( _% ): C57.02, H7.05, N14.14. Reference example 3 4-[(N〓-carbobenzoxy-L-
Synthesis of N-carbobenzoxy-L-arginine 2.0 g
Dissolve it in 20 ml of DMF under heat, add it to this in a nitrogen stream,
1.8 g of 4-amino-3-methyl-N-ethyl-N-(β-hydroxyethyl)aniline under ice bath cooling.
and 1.4 g of DCCD were added, and the mixture was stirred and reacted at room temperature for 20 hours. After the reaction, the precipitate was filtered off, the solvent was distilled off, and the resulting residue was subjected to silica gel column chromatography (column: 40 x 2 cm, eluent: ethyl acetate: pyridine: water: acetic acid = 120:20). :10:5.)
The concentrated and dried main eluate was dissolved in water and lyophilized to give 4-[N〓-carbobenzoxy-L-arginyl)amino]-3-methyl-N-ethyl-N. -(β-hydroxyethyl)aniline diacetate was obtained. Yield 0.86g (22%). Specific optical rotation [α] ²¹ _D = -10.1 (C = 0.88, methanol). Elemental analysis value (as _C25H37N6O4・_2CH3COOH ) Calculated _value (%): C57.51, H7.49, N13.87, Actual _value ( _% ): C57.22, H7.18, N13.65. Example 1 4-[(t-butyloxycarbonyl-
Synthesis of L-valyl-L-leucyl-glycyl-L-arginyl)amino]-N-ethyl-N-(β-hydroxyethyl)aniline 4-[(N〓-carbobenzoxy- L-arginyl)amino]-N-ethyl-
N-(β-hydroxyethyl)aniline. Add t-butyl obtained in Reference Example 1(c) to 30 ml of THF solution of 1.45 g of diacetate and catalytic reduction product 4-[(L-arginyl)amino]-N-ethyl-N-(β-hydroxyethyl)aniline. Oxycarbonyl-L-valyl-L
30 ml of a DMF solution containing 0.95 g of -leucyl-glycine-N-hydroxy-5-norbornene-2,3-dicarboximide ester was added, and the mixture was stirred and reacted at room temperature for 72 hours. After the reaction was completed, the reaction solution was concentrated to dryness, and the resulting residue was dissolved in 120 ml of water, passed through Amberlite IRA-410 (acetic acid form), and subjected to ion exchange column chromatography (CM cellulose, 0.2M
ammonium acetate), and the main eluate was lyophilized to give 4-[t-butyloxycarbonyl-L-valyl-L-leucyl-glycine-L-arginyl)amino]-N-ethyl-N-( β-hydroxyethyl)aniline diacetate 1/2 hydrate was obtained. Yield 110mg (11%). Melting point 115-122℃. Specific optical rotation [α] ²¹ _D = -37.1 (C = 0.42, methanol). Elemental analysis value (as C ₃₄ H ₅₉ N ₉ O ₇・2CH ₃ COOH・1/2H ₂ O) Calculated value (%): C54.66, H8.21, N15.09, Actual value (%): C54. 54, H8.02, N15.54. Example 2 4-[(N-carbobenzoxy-L-leucyl-glycine-L-arginyl)amino]
-N-ethyl-N-(β-hydroxyethyl)
Synthesis of aniline N-carbobenzoxy obtained in Reference Example 1(a)
Dissolve 4.1 g of L-leucyl-glycine ethyl ester in 30 ml of methyl alcohol, and add 20 ml of 1N aqueous sodium hydroxide solution under ice bath cooling.
The reaction was stirred for hours. After the reaction is complete, add 1N to the reaction solution.
- Neutralize by adding 18 ml of hydrochloric acid, concentrate under reduced pressure, add 2 ml of 1N hydrochloric acid, and add 300 ml of ethyl acetate.
Extracted with. Dry the extract with anhydrous sodium sulfate,
After filtering off the desiccant, the organic layer was concentrated to dryness under reduced pressure. After adding petroleum ether to the residue and solidifying it, this was further recrystallized from ethyl acetate/petroleum ether to give N-
Carbobenzoxy-L-leucyl-glycine was obtained. Using this N-carbobenzoxy-L-leucyl-glycine as a raw material, the same reaction and operation as in Reference Example 1(c) were performed to obtain N-carbobenzoxy-L-leucyl-glycine-N-hydroxy-5-norbornene. -2,3-dicarboximide ester 0.85g
I got it. 4-[(N〓-carbobenzoxy-L-arginyl)amino]-N-ethyl-
Add 30 ml of a THF solution of 4-[(L-arginyl)amino]-N-ethyl-N-(β-hydroxyethyl)aniline, a catalytic reduction product of 1.5 g of N-(β-hydroxyethyl)aniline diacetate, 72 at room temperature
The reaction was stirred for hours. After the reaction was completed, the reaction solution was concentrated to dryness, and the resulting residue was dissolved in 120 ml of water, passed through Amberlite IRA-410 (acetic acid type), and subjected to ion exchange column chromatography (CM cellulose,
The main eluate was lyophilized to give 4-[(N-carbobenzoxy-L-leucyl-glycine-L-arginyl)amino]-N-ethyl-N-(β -hydroxyethyl)aniline diacetate was obtained. Yield 311 mg (15%). Melting point 119℃. Specific optical rotation [α] ²¹ _D = -12.2 (C = 0.41, ethanol). Elemental analysis value (as _C32H48N8O6・_2CH3COOH ) Calculated _value (%): C56.82, H7.42, N14.70, Actual _value ( _% ): C56.50, H7.11, N14.50. Example 3 4-[(t-butyloxycarbonyl-
Synthesis of L-valyl-glycyl-L-arginyl)amino]-N-ethyl-N-(β-hydroxyethyl)aniline 26 g of t-butyloxycarbonyl-L-valine
and glycine ethyl ester hydrochloride 14g in THF400
15 ml of triethylamine, 18 g of HONB, and 22 g of DCCD were added thereto under cooling in an ice bath, and the mixture was stirred and reacted at room temperature for 20 hours. After the reaction, the precipitate was filtered off, the solvent was distilled off, the resulting residue was dissolved in 500 ml of ethyl acetate, and a saturated aqueous sodium bicarbonate solution,
After washing in this order with 1N citric acid and water, the organic layer was dried over anhydrous sodium sulfate. After filtering off the desiccant, the organic layer was concentrated to dryness under reduced pressure, and petroleum benzine was added to the residue to obtain a gel-like solid. This was recrystallized from ethyl acetate/petroleum benzine to obtain t-butyloxycarbonyl-L-valyl-glycine ethyl ester. Yield 18.1g (50%). Melting point 98-99℃. 4.1 g of t-butyloxycarbonyl-L-valyl-glycine ethyl ester obtained above was dissolved in 30 ml of methyl alcohol, and 1N-
20 ml of aqueous sodium hydroxide solution was added and the reaction was stirred for 2 hours. After the reaction is complete, add 1N hydrochloric acid 18 to the reaction solution.
ml to neutralize, concentrate under reduced pressure, and then add 1N-
2 ml of hydrochloric acid was added, and this was extracted with 300 ml of ethyl acetate. The extract was dried over anhydrous sodium sulfate, the desiccant was filtered off, and the organic layer was concentrated to dryness under reduced pressure. After adding petroleum ether to the residue and solidifying it, this was recrystallized from ethyl acetate/petroleum ether to obtain t-butyloxycarbonyl-L-valyl-glycine. Using this t-butyloxycarbonyl-L-valyl-glycine as a raw material, the same reaction and operation as in Reference Example 1(c) were carried out to produce t-butyloxycarbonyl-L-valyl-glycine.
L-valyl-glycine-N-hydroxy-5-norbornene 2,3-dicarboximide ester
0.95g was obtained. Reference example 2 is added to this DMF 20ml solution.
Catalytic reduction product of 1.5 g of 4-[(N〓-carbobenzoxy-L-arginyl)amino]-ethyl-N-(β-hydroxyethyl)aniline diacetate obtained by the same method as 4-[ (L-arginyl)
After adding 50 ml of a THF solution of [amino]-N-ethyl-N-(β-hydroxyethyl)aniline, the mixture was stirred and reacted at room temperature for 72 hours. After the reaction was completed, the reaction solution was concentrated to dryness, and the resulting residue was dissolved in 120 ml of water, passed through Amberlite IRA-410 (acetic acid form), and subjected to ion exchange column chromatography (CM cellulose, 0.2M ammonium acetate). The main eluate was lyophilized to give 4-[(t-butyloxycarbonyl-L-valyl-glycine-L-arginyl)amino]-N-ethyl-N-(β-hydroxyethyl)aniline. Diacetate was obtained. Yield 387 mg (12%). Melting point 101-102℃. Specific optical rotation [α] ²¹ _D = -0.7 (C = 0.35, DMF). Elemental analysis value (as _C28H48N8O6・_2CH3COOH ) Calculated _value (%): C53.92, H7.92, N15.71, Actual _value ( _% ): C53.72, H7.82, N15.46. Reference Example 4 Using the peptide derivative obtained in Example 1, 2, or 3 as a substrate, endotoxin concentration was measured using the following test solution. (Test solution) (1) LAL solution Limulus amebosite lysate (LAL)
(lyophilized product, for 5 ml) was dissolved in 5 ml of distilled water for injection to prepare the LAL solution. (2) Standard endotoxin solution Standard endotoxin (lyophilized product, 0.5μg/
vial) was dissolved in distilled water for injection to a predetermined concentration and used as a standard endotoxin solution. (3) Substrate buffer 0.1M tris(hydroxymethyl)amino containing potassium 1-naphthol-2-sulfonate (0.2mM) and the peptide derivative of the present invention obtained in Example 1, 2 or 3 (0.2mM) Methane/hydrochloric acid buffer (PH8.26, containing 0.03M _MgCl2 , sterilized in an autoclave) was used as the substrate buffer. (4) Coloring liquid A solution containing 0.2% periodic acid and 0.3M boric acid was used as the coloring liquid. (Measurement procedure) Add 0.05ml of standard endotoxin solution of specified concentration.
The mixture was mixed with 0.1 ml of LAL solution and incubated at 37°C for 10 minutes, then 2.0 ml of substrate buffer was added thereto, and further incubated at 37°C for 15 minutes. After adding 1.0 ml of coloring solution to this solution to develop color, the absorbance at the maximum absorption wavelength (660 nm) was measured. (Results) The results are shown in Table 1. Comparative Examples 1 and 2. In place of the peptide derivative of the present invention obtained in Example 1, 2 or 3, 4-[(t-butyloxycarbonyl-L), an existing peptide derivative having a similar structure to this, was used. -valyl-L-leucyl-glycyl-L-arginyl)amino]-N,N-diethylaniline diacetate or 4-[(t-butyloxycarbonyl-L-valyl-L-leucyl-glycyl-L-arginyl) ) Amino]-N,N-diethyl-m-toluidine diacetate was used as the substrate, but the same reagents and reagents as in Reference Example 4 were used, and the same procedures were performed to measure endotoxin. , are also shown in Table 1. In addition, the absorbance measurement is performed at each maximum absorption wavelength (675 nm or
690nm).

[Table] As is clear from Table 1, when the endotoxin concentration was measured using the peptide derivative of the present invention obtained in Example 1, 2, or 3 as a substrate, when the existing similar peptide derivative was used. The sensitivity is clearly higher than that of I understand that. [Effects of the Invention] As described above, the present invention produces a dye having maximum absorption (λmax) on the long wavelength side by oxidative condensation with phenol, naphthol, etc. on the C-terminal side of an arginine residue. -Amino-
N-ethyl-N-(β-hydroxyethyl)aniline or 4-amino-3-methyl-N-ethyl-
It provides a peptide derivative to which N-(β-hydroxyethyl)aniline is bonded, and when used as a substrate for a specific enzyme (protease), the activity of the enzyme can be easily and highly sensitively measured. Moreover, this invention has a remarkable effect in that it enables measurements to be carried out almost without being influenced by pigment components in blood samples, and it is a great invention that contributes to this industry.

Claims (1)

[Claims] 1. General formula [] (In the formula, R ¹ represents a leucyl group, a valyl group, or a valyl-leucyl group having a protecting group at the N-terminus.Also, even if the guanidino group of the arginine residue in the formula has a protecting group, ) or an acid addition salt thereof. 2. Claim 1, wherein the N-terminal protecting group is an acyl group, a carbobenzoxy group, a tertiary alkyloxycarbonyl group, a tosyl group, or a glutaryl group.
The peptide derivative or acid addition salt thereof as described in 2.