JPH03227967A - Arginine derivative - Google Patents

Abstract

NEW MATERIAL:An arginine derivative expressed by the formula (R is H or N-end protecting group of amino acid; R<1> is H or methyl) or acid addition salt thereof. EXAMPLE:4-[(N<alpha>-Carbobenzoxy-L-arginyl)amino]-N-ethyl-N-(beta-hyd roxyethyl) aniline. USE:Capable of simply measuring activity of a specific enzyme (protease) in high sensitivity and useful as an intermediate for various kind of peptide derivatives for synthesizing a dye substrate hardly influenced by dye ingredient in blood sample. PREPARATION:For example, arginine in which an amino acid is protected by a proper protecting group is reacted with 4-amino-N-ethyl-N-(beta-hydroxyethyl) aniline in the presence of a condensing agent in a solvent under nitrogen stream at ambient temperature to heated temperature and as necessary subjected to deprotecting treatment to provided the compound expressed by the formula.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Application of the Invention] The present invention relates to an arginine derivative useful as a synthetic raw material for 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.

Horseshoe crab blood cell extract (amebocyte lysade)
A method for detecting trace amounts of endotoxin has been developed and put into practical use, based on the phenomenon in which bacteria react with a trace amount of bacterial endotoxin and turn into gel.

The detection method based on this principle is based on the United States Pharmacopoeia Act [U.
S, Pharmacopeia XX, 888 (198
There are methods to visually determine the hardness of gels, such as the method used in [0)], turbidity measurement, clot protein determination, etc., but all of them are based on gelation and are not very accurate. .

Also, “Novel plastid enzyme substrate” (Japanese Patent Application Laid-Open No. 51-8353
The invention of R1-AI-A2-Gly-Ar
It is characterized by using a plastid substrate represented by g-NH-R2 and measuring the chromophore liberated by enzymatic action using a spectrophotometer, but it has the disadvantage that water droplets are susceptible to interference by pigment components in blood samples. There is.

[Purpose 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 purpose is to provide an intermediate raw material for synthesizing difficult-to-receive plastid substrates.

[Structure of the Invention] The present invention is based on the general formula [IcoNH C=NI (NH2 (wherein, R represents a hydrogen atom or an N-terminal protecting group of an amino acid, and R1 represents a hydrogen atom or a methyl group). The present invention is an arginine derivative or an acid addition salt thereof represented by:

That is, the present inventors have found that the activity of a specific enzyme (protease) can be measured easily and with high sensitivity, and that it is not easily affected by pigment components in a blood sample.
As a result of extensive research in search of a plastid substrate, we found that by oxidative condensation with phenol, naphthol, etc. on the C-terminal side of the arginine residue, maximum absorption (λmax) was achieved on the long wavelength side.
4-Amino-N-ethyl-N-
(β-hydroxyethyl)aniline is 4-amino-3
-Methyl-N-ethyl-N-(β-hydroxyethyl)
The present inventors have discovered that a peptide derivative obtained from an arginine derivative represented by the general formula ['I] to which aniline is bonded can achieve the above object, and have completed the present invention.

That is, for example, when a peptide derivative obtained from the arginine derivative represented by the general formula [1] 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. In this way, the activity of the enzyme can be measured simply and with high sensitivity, and is hardly affected by the pigment components in the blood sample.

The N-terminal protecting group for the amino acid in R in general formula [I] is not particularly limited and may include any group commonly used as an N-terminal protecting group for amino acids and peptides. For example, preferred examples include acyl groups such as acetyl group and benzoyl group, carbobenzoxy group, tertiary alkyloxycarbonyl group, tosyl group, and glutaryl group.

In addition, the guanidino group of the arginine residue of general formula [1] can be used in peptide synthesis, for example, a nitro group,
N such as tosyl group, p-methoxybenzenesulfonyl group, 4-methoxy-2,6-synthylbenzenesulfonyl group, etc.
- May be protected by a guanidino protecting group.

The arginine derivative represented by the general formula [I] may also be in the form of an acid addition salt. Examples of such acid addition salts include inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, and phosphoric acid, such as acetic acid and sulfuric acid. Acid, tartaric acid.

Examples include acid addition salts of organic acids such as succinic acid, citric acid, and toluenesulfonic acid.

The arginine derivative of the present invention can be synthesized, for example, as follows.

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), aqueous solution, etc. are dissolved in a mixture thereof, and 4-amino-N-ethyl-N-(β-hydroxyethyl)aniline or 4-amino-N-ethyl-N-(β-hydroxyethyl)aniline or After adding 4-amino-3-methyl-N-ethyl-N-(β-hydroxyethyl)aniline and a condensing agent commonly used in peptide synthesis such as dicyclohexylcarbodiimide (DCCD), the mixture was heated at room temperature for as long as necessary. The mixture is reacted under heating 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, such as silica gel column chromatography, ion exchange column chromatography, etc., and if necessary, deprotection is performed. By performing this, the arginine derivative of the present invention is obtained. In addition, in order to deprotect the protecting group of the obtained arginine derivative, a method commonly used in ordinary peptide synthesis can be used, for example, when a carbobenzoxy group or a benzyl group is used as a protecting group, for example, a method such as alcohol or the like can be used. This can be carried out by hydrogenation in a solvent, or when a tertiary-butyloxycarbonyl group is used as a protecting group, by reacting it with toluenesulfonic acid in a solvent such as acetic acid.

The arginine derivative of the present invention is useful as an intermediate raw material for various peptide derivatives. An example of a method for synthesizing a peptide derivative using the arginine derivative of the present invention as an intermediate raw material is as follows.

That is, the arginine derivative of the present invention obtained by the above method (usually used is one in which the N-terminus of the amino acid is protected with a suitable protecting group) is used in the synthesis of peptide derivatives. Solvents such as dimethylformamide (DMF), dimethylsulfoxide (1)M
Glycine, leucyl-glycine, baryl-glycine, whose N-terminus is protected with an appropriate protecting group, dissolved in SO), tetrahydrofuran (THF), water, or a mixture thereof, under a nitrogen atmosphere. Valyl-leucyl-glycine or active varnishes and chilled forms 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 in peptide synthesis, such as glycine hexylcarbodiimide (DCCD), at room temperature.
In short, the reaction only needs to be carried out under heating for an appropriate period of time. After the reaction is complete, the reaction solution is concentrated to dryness, and then purified using methods commonly used to purify peptide derivatives, such as silica gel column chromatography, ion chromatography, etc. The desired peptide derivative can be easily obtained by purification by exchange column chromatography or the like, followed by deprotection if necessary.

In addition, in order to deprotect the protecting group of the obtained peptide derivative, a method commonly used in ordinary peptide synthesis can be used, for example, when a carbobenzoxy group or a benzyl group is used as a protecting group, for example, using an alcohol such as A method of hydrogenation treatment in a solvent, for example, when a tertiary butyloxycarbonyl group is used as a protecting group, hydrogenation treatment is carried out in a solvent such as #acid.
It is sufficient to carry out this by a method of reacting with ~luenesulfonic acid, etc. The peptide derivatives thus obtained are useful as substrates for amidase-like enzymes produced as a result of the reaction of horseshoe crab amebocyte lysates with bacterial endotoxins. That is, the peptide derivative thus obtained has the property that the peptide bond at the C-terminal side of the arginine residue is easily hydrolyzed by the action of an amidase-like enzyme, and as a result, it is liberated. 4-amino-
Oxidation of N-ethyl-N-(β-hydroxyethyl)aniline or 4-amino-3-methyl-N-ethyl-N-(β-hydroxyethyl)aniline with phenol, naphthol, etc. in the presence of a suitable oxidizing agent. By condensing and measuring the change in absorbance caused by the blue indophenol dye produced, it is possible to quantitatively detect the amount of amidase-like enzyme, in other words, the amount of bacterial endotoxin in the sample. (See cascade below.

).

(In the cascade, R' represents a leucyl group, a valyl group, or a valyl-leucyl group, which may have a protecting group at the N-terminus.) The present invention will be explained in more detail with reference to Examples below.

[Example] Reference Example 1゜(a) Synthesis of carbobenzoxy-isoacyl-glycine ethyl ester In a solution of 26 g of carbobenzoxy L-leucine and 14 g of glycine ethyl ester hydrochloride dissolved in 400 ml of THF. , 15 ml of triethylamine under cooling in a water bath,
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 diluted with 50% ethyl acetate.
After dissolving in 0 ml of the solution and washing with saturated aqueous sodium bicarbonate solution, IN-hydrochloric acid, and water in this order, the organic layer was dried over anhydrous sodium sulfate. After removing the desiccant by filtration, 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-1, -Glycine ethyl ester was obtained.

Yield 30. ], g (83%).

Melting point: 114-115°C.

Specific optical rotation [α cog' = -26,1 (C= ], 0
5. Ethanol).

Elemental analysis value (as CI8H26N205) total X (MO
ζ): C61,70,)(7,48,N
7.99, Actual value (%): C62,07, ) (7,5
0, N 7.91° (b) Synthesis of t-butyloxycarbonyl-L-valyl-I7-leucyl-glycine Carbobenzoxy-silyl-glycine 1 Synethyl ester obtained in (a) 10.5 g and p-) Dissolve 5.7 g of luenesulfonic acid 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 is complete,
The catalyst was filtered off from the reaction solution, and then the solvent was distilled off under reduced pressure. The obtained oil was mixed with 6.5 g of monobutyloxycarbonyl-valine and 5.4 g of HONB in THF 3
00 ml, and to this was added 4.2 ml of triethylamine and 7.4 g of DCCD while cooling in a water bath, and the mixture was stirred and reacted at room temperature for 20 hours. After the reaction was completed, insoluble matter was filtered off, and the solvent was distilled off. The resulting residual liquid was diluted with 500 ml of ethyl acetate.
The organic layer was washed with saturated aqueous sodium bicarbonate solution, 10% citric acid, and water in this order, and the organic layer was dried over anhydrous sodium sulfate. After removing the desiccant by filtration, the organic layer was concentrated to dryness under reduced pressure.

Petroleum ether was added to the residual solution to solidify it, and this was further recrystallized from ethyl #acid/petroleum ether to obtain t-butyloxycarbonyl-I,-valyl-L-isoyl-glycine ethyl ester.

Yield: 10g (83%).

2 Melting point: 113-114°C.

Specific optical rotation [α]g' = -56,3 (C = 0.95
, ethanol).

Elemental analysis value (as C2OH37N 306) Calculation α
! (%): C57,8], H8,98,
N 1.0.11, actual value (%): C57,97
, H8,91,N 9.92° Then, 4.1 g of this t-butyloxycarbonyl-shi-valyl-shi-isoyl-glycine ethyl ester was added to 30 ml of methyl alcohol.
While cooling in a water bath, 20 ml of 1N aqueous sodium hydroxide solution was added, and the mixture was stirred and reacted for 2 hours. After the reaction is complete,
The reaction solution was neutralized by adding 18 ml of IN-hydrochloric acid and concentrated under reduced pressure. Then, 2 ml of IN-hydrochloric acid was further added, and this was extracted with 300 ml of ethyl #acid.

The extract was dried over anhydrous sodium sulfate, the desiccant was removed by filtration, and the organic layer was concentrated to dryness under reduced pressure. After adding petroleum ether to the residual liquid and solidifying it, this was recrystallized from ethyl acetate/petroleum ether to obtain t-butyloxycarbonyl-L-valyl-L.
-Glycine was obtained.

Yield: 3.6g (93%).

Melting point: 104-108°C.

Specific optical rotation [α]g' = -54,6 (C = 0.99, ethanol).

(c) t-butyloxycarbonyl-L-valyl-L-
Synthesis of leucyl-glycine-N-hydroxy-5-norbornene-2,3-dicarboximide ester (b)
1-butyloxycarbonyl-17-valyl-L obtained in
-1.5 g of leucyl-glycine was dissolved in 50 ml of THF, and 0.84 g of HONB and DC were added to this under cooling in a water bath.
0 g of CDl was added, and the mixture was stirred and reacted at room temperature for 20 hours. After the reaction is completed, the precipitate is filtered off, the solvent is distilled off, and t-
Butyloxycarbonyl-L-valyl-L-leucyl-
Glycine-N-hydroxy-5-norbornene-2,3
-Dicarboximide ester was obtained.

Yield L9g (90%).

Example 1, 4-[(N'-carbobenzoxy-I, -
Synthesis of arginyl)amino]-N=ethyl-N-(β-hydroxyethyl)aniline
Dissolve in 80 ml of MF while hot, add 2.75 g of 4-amino-N-ethyl=N~(β-hydroxyethyl)aniline and 4.0 g of DCCD under water bath cooling in a nitrogen stream, and stir at room temperature for 20 hours. Made it react. After the reaction was completed, insoluble materials were filtered off, the solvent was distilled off, and the resulting residue was subjected to silica gel column chromatography (column: 15% 5 cm, eluent: ethyl acetate: pyridine: water: acetic acid: = 60:20).
4-[(N'-
Carbobenzoxy-arginyl)aminocol N-ethyl-N-(β-hydroxyethyl)aniline 2# acid salt was obtained.

Amount taken: 1.45g (25%).

Specific optical rotation [α cog' = -17,2 (C = 0.95
, methanol).

Elemental analysis value (C24H35N60.1・2C83COO
11) Calculated value (%): C56,83, H7,
33, N 1.4.20, Actual value (%): C57
, 02, H7, 05, N 14.14° Example 2.4
Synthesis of -[:(N'-carbobenzoxy-L-arginyl)aminoco-3-methyl-N-ethyl-N-(β-hydroxyethyl)aniline p(R-carbobenzoxy-L-arginine 2.Og was dissolved hot in 20 ml of DMF, and 4-amino-3-methyl-N-ethyl-N-(β
-hydroxyethyl)aniline 1.8 g and DCCDl,
4g of the mixture was added, and the mixture was stirred and reacted at room temperature for 20 hours. After the reaction is complete,
The precipitate was filtered off, the solvent was distilled off, and the resulting residue was subjected to silica gel column chromatography C column: 40% 2 cm
, eluent di'#ethyl acid:pyridine:water:#acid=120
4-[
(N'-carbobenzoxy-L-arginyl)aminoco-3-methyl-N=ethyl-N-(β-hydroxyethyl)aniline 2# acid salt was obtained.

Yield: 0.86g (22%).

Specific optical rotation [αg' = -1,0,1 (C = 0.88
, methanol).

Elemental analysis value (C2SH37N604・2CI13COO
Calculated value (%): C57,51, H7,4
9, N 13.87, Actual value (%): C57,2
2, H7,18, N 13.65° Reference Example 2.4-[
(t-butyloxycarbonyl-f, -valyl-1,-
leucyl-glycyl-L-arginyl)aminocol N-
Synthesis of ethyl-N-(β-hydroxyethyl)aniline 4,-[(N'-carbobenzoxy-L-arginyl)aminocol N-ethyl-N~(β-
Catalytic reduction product of 1.45 g of hydro6xyethyl)aniline diacetate 4-C(L-arginyl)aminocol N-ethyl-N
-(βhydroxyethyl)aniline THF solution 30ml
1 obtained in Reference Example 1(c)=Butyloxycarbonyl-L-valyl-L-isoyl-glycine-N-hydroxy-5-norbornene-2,3-dicarboximide ester 0.95g in DMF solution 30ml was added, and the mixture was stirred and reacted at room temperature for 72 hours. After the completion of the reaction, the reaction solution was concentrated to dryness, and the resulting residue was dissolved in 120+ nl of water, passed through Amberley IRA-410 (acetic acid type), and subjected to ion exchange column chromatography (CM cellulose, 0.0 mL).
The main eluate was lyophilized to give 4-[(t-butyloxycarbonyl-
Valyl-L-glycyl-L-arginyl)aminocol N-ethyl-N-(β-hydroxyethyl)aniline diacetate 1/2 hydrate was obtained.

Yield: 110 mg (11%).

Melting point: 115-122°C.

Specific optical rotation [α cog' = −3,7,,], (C=0
．． 42, methanol).

Elemental analysis value (C34H59N907・2CH3C00H・1/21
-120) Calculated value (%): C54,66,H
8,2], N 15.09, Actual value (%):
C54,54, H8,02, N' 15.54° Reference Example 3.4-[(N-carbobenzoxy-L-leucylglycyl-s-arginyl)amino]-N-ethyl-N-
Synthesis of (β-hydroxyethyl)aniline 461 g of N-carbobenzoxy-L-leucyl-glycine ethyl ester obtained in Reference Example 1(a) was dissolved in 30 ml of methyl alcohol, and aqueous IN sodium hydroxide solution was added to the solution under cooling in a water bath. 20 ml was added and the reaction was stirred for 2 hours. After the reaction was completed, the reaction solution was neutralized by adding 18 ml of IN-hydrochloric acid, concentrated under reduced pressure, further added with 2 ml of IN-hydrochloric acid, and extracted with 300 ml of ethyl #acid.

The extract was dried over anhydrous sodium sulfate, the desiccant was removed by filtration, and 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 #acid/petroleum ether to obtain N-carbobenzoxy-L-isoyl-glycine.

Using this N-carbobenzoxy-L-leucyl-glycine as a raw material, the same reaction and operation as in Reference Example 1(c) were carried out to obtain N-carbobenzoxy-L-leucyl-glycine-N.
-Hydroxy-5-norbornene-2,3-dicarboximide ester O-85gt! It's 8. This DMF30
m] 4-obtained by the same method as Example 1 was added to the solution.
[(N'-Carbobenzoxy-1,-arginyl)amino]-N-ethyl-N-(β-hydroxyethyl)aniline-2i! #acid salt],, 5g of catalytic reduction product 4-[(L
-Arginyl)amino]-N-ethyl-N-(β-hydroxyethyl)aniline in THF solution (30 mL) 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.
Through Amberlite I RA-410 (# acid type),
It was purified by ion exchange column chromatography (CM cellulose-0.2M ammonium acid), and the main eluate was freeze-dried to obtain 4-[(N-carbobenzoxy-L-
acyl-glycyl-L-arginyl)ami, /:]-
]N-ethyl-N-β-hydroxyethyl)aniline・
Diacetate was obtained.

Yield: 31.1 mg (15%).

Melting point 1]9°C0 Specific rotation [α]g' = -12,2 (C=0.41
, ethanol).

9 Elemental analysis value (C32H4BN806・2CH3COOH
) Kotojukan I M (%): C56,82,H
7,42,N], 4.70, Actual value (%):
C56,50, H7,11, N 14-50-Reference Example 4.4-[(t-Butyloxycarbonyl-L-valyl-glycyl-L-arginyl)amino]-N-ethyl N-(β-hydroxyethyl ) Synthesis of aniline 26 g of t-butyloxycarbonyl-L-valine and 14 g of glycine ethyl ester hydrochloride were dissolved in 400 ml of THF, and 15 ml of triethylamine and H
18 g of ONB and 22 g of DCCD were added and reacted with stirring at room temperature for 20 hours. After the reaction was completed, the precipitate was filtered off, the solvent was distilled off, the resulting residue was dissolved in 500 ml of ethyl acetate, and washed with a saturated aqueous sodium bicarbonate solution, IN-citric acid, and water in this order, and the organic layer was dissolved. was dried with anhydrous sodium sulfate. After removing the desiccant by filtration, the organic layer was concentrated to dryness under reduced pressure, and petroleum benzine was added to the residual liquid to obtain a gel-like solid. This was recrystallized from ethyl #acid/petroleum benzine to obtain t-butyloxycarbonyl-Lvalyl-glycine ethyl ester.

J82 amount], 88.1 g (50%).

0 Melting point: 98-99°C.

4.1 g of monobutyloxycarbonyl-monovalylglycine ethyl ester obtained above was mixed with 30 ml of methyl alcohol.
1 and 20 mL of IN-sodium hydroxide aqueous solution was added thereto while cooling in a water bath, and the mixture was stirred and reacted for 2 hours. After the reaction was completed, 18 ml of IN-hydrochloric acid was added to the reaction solution to neutralize it, and the mixture was concentrated under reduced pressure. Two 1 parts of IN-hydrochloric acid were further 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 L-butyloxycarbonyl-L-valyluglycine.

Using this t-butyloxycarbonyl-valyl-glycine as a raw material, the same reaction and operation as in Reference Example 1(C) were carried out. 0.95 g of -2,3-dicarboximide ester was obtained. This D
4-[(N'-carbobenzoxy-[,-arginyl)amino]-N-ethyl-N-(β-hydroxyethyl)aniline obtained by the same method as Example 1 was added to 20 ml of MF solution. Catalytic reduction product of 1.5 g of diacetate 4
-[(L-arginyl)aminoco-N-ethyl-N-(
β-Hydroxyethyl]aniline THF solution 50ml
After adding, 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 mixed with 120 ml of water.
Dissolved in Amberlite I RA-410 (#acid type)
The main eluate was lyophilized to give 4-[(t-butyloxycarbonyl-valyl-glycyl-L-arginyl). Amino]-N-ethyl=N-(β-hydroxyethyl)aniline 2# acid salt was obtained.

Yield: 387 mg (12%).

Melting point: 101-102°C.

Specific optical rotation [α]g' = -0,7<C=0.35,
DMF).

Elemental analysis value (C28H48N806・2CH3COOI
(as) Calculated value (%): C53,92, H7,9
2, N 15.71, Actual value (%): C53.7
2, H7,82, N 15-46°Reference Example 5°The endotoxin concentration was measured using the peptide derivative obtained in Reference Example 2 as a substrate and the following test solution.

(Test solution) (1) LAL solution Limulus amebosite lysate (LAL) (lyophilized product, for 5 mJ) was dissolved in 5 ml of distilled water for injection to prepare an LAL solution.

(2) Standard endotoxin solution Standard endotoxin (lyophilized product, 0.5 μg/via
1) was dissolved in distilled water for injection to a predetermined concentration and used as a standard endotoxin solution.

(3) Substrate buffer 1-naphthol-2-potassium sulfonate (0.2mM
) and the peptide derivative obtained in Reference Example 2 (0.2mM
) including 0. IM) Lis(hydroxymethyl)aminomethane @ acid buffer (pH 8,26,0,03M Hg
Contains C32, sterilized in an autoclave. ) was used as the substrate buffer.

(4) Coloring liquid 3 A solution containing 0.2z periodic acid and 0.3M boric acid was used as a coloring liquid.

(Measurement procedure) Standard endotoxin solution of predetermined concentration 0.05 m] and LA
The mixture was mixed with 0.1 ml of L solution and incubated at 37°C for 10 minutes, and 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.

(result) The results are shown in Table 1.

Comparative Example 1゜In place of the peptide derivative obtained in Reference Example 2, 4-mouth (
7-Butyloxycarbonyl-L-leucyl-glycyl-L-arginyl)amino]-N,N diethylaniline diacetate was used as the substrate, but the same reagents and reagents as in Reference Example 5 were used, and the same operation was performed. was performed to measure endotoxin. The results are also shown in Table 1. The absorbance was measured at the maximum absorption wavelength (675 nm).

Table 1 As is clear from Table 1, when the endotoxin concentration was measured using the peptide derivative obtained in Reference Example 2 as a substrate, the sensitivity was more than twice as high as when using existing similar peptide derivatives. It turns out 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-3methyl-N-ethyl-N-(β-hydroxyethyl)aniline is bonded to an arginine derivative. When a peptide derivative obtained from this as a raw material is used as a substrate for a specific enzyme (protease), the activity of the enzyme can be measured easily and with high sensitivity, and with almost no influence from the pigment components in the blood sample. This is an invention that has a remarkable effect in that it makes it possible to do so, and it is a great invention in that it contributes to this industry.

Claims (2)

[Claims] (1) General formula [I] ▲Mathematical formulas, chemical formulas, tables, etc.▼[I] (In the formula, R represents a hydrogen atom or an N-terminal protecting group of an amino acid, and R^1 represents a hydrogen atom or a methyl group. ) An arginine derivative or an acid addition salt thereof. (2) The arginine derivative or acid addition salt thereof according to claim 1, wherein the N-terminal protecting group of the amino acid is an acyl group, carbobenzoxy group, tertiary alkyloxycarbonyl group, tosyl group, or glutaryl group. .