JPH01186861A - Peroxy ester compound, production thereof and test composition and test tool using said compound - Google Patents

Abstract

NEW MATERIAL:To obtain a compound expressed by formula I (R<1> and R<2> are alkyl, aryl or aralkyl which may be substituted, e.g., R<1>CO-represents a peptide group consisting of natural L-amino acid and D- and DL-type thereof or two - five numbers of amino acids thereof and R<2> represents 1,1-dimethyl-5- phenylpentyl group etc.). EXAMPLE:Hydrocinnamic acid-tert.-butylhydroperoxy ester. USE:A test composition for measuring asterase or protease. PREPARATION:A compound expressed by formula II (X is OH, halogen, alkylcarbonyloxy, alkoxycarbonyloxy, imidazole, etc.) and compound expressed by formula III are dissolved in an organic solvent such as CH2Cl2 and N,N'- dicyclohexylcarbodiimide and 4-dimethylaminopyridine are added into the solution and the mixture is subjected to condensation reaction to provide the compound expressed by formula I.

Description

[Detailed description of the invention] [Industrial application field].

The present invention relates to a novel peracid ester compound, a method for producing the same, a test composition for measuring esterase or protease using the compound, and a test device carrying the same.

The compositions and test devices provided by the present invention are useful for detecting esterases or proteases such as leukocytes.

If leukocytes or esterase are contained in the urine or dialysate, it can be assumed that a disease in the kidneys and genitourinary tract or inflammation is progressing at the dialysis site. Therefore, in order to promptly diagnose and treat these diseases, it is important to accurately detect leukocytes or esterases in the urine, dialysate, etc.

The peracid ester compound of the present invention is suitably used as a reagent for testing such esterases.

[Conventional technology]

Conventional esterase or protease detection test devices are
Ester compounds, diazonium salt compounds.

It consists of a carrier impregnated with a buffer and an activator, and when esterase or protease is present during the test, the ester compound is hydrolyzed to produce a highly reactive hydroxyl-containing compound, which is coupled with the diazonium salt compound. Develops color. As an ester compound, 3-(
Totoluenesulfonyl-mono-alanyloxy)-indole (Patent Publication No. 59-3475) and 3-(N-)-toluenesulfonyl-mono-alanyloxy)-5-phenylbrole (Japanese Unexamined Patent Publication No. 60-260558) are known. It has been put into practical use. In addition, Nuttall AS-D chloroacetate,
α-naphthol butyrate and the like are known. All of these are compounds having an ester group.

[Purpose of the invention]

An object of the present invention is to provide a novel peracid ester compound, a method for producing the same, a test composition for measuring esterase or protease using the compound, and a test device supporting the same.

[Structure of the invention]

The novel compound according to the present invention has the general formula (1) general formula (I) group, aryl group or aralkyl group, but these are hydroxyl group, alkoxy group,
Halogen atom, amino group.

It may be substituted with an alkylamino group, dialkylamino group, acylamino group, sulfonialamino group, nitro group, arylsulfone group, alkylsulfone group, carboxy group, amide group or alkoxycarbonyl group.

] It is a filtered acid ester compound.

According to the present invention, the general formula (I [) indicates an alkyl group, an aryl group, or an aralkyl group, and these are a hydroxyl group or an alkoxy group.

It may be substituted with a halogen atom, an amino group, an alkylamino group, a dialkylamino group, an acylamino group, a sulfonylamino group, a nitro group, an arylsulfone group, an alkylsulfone group, a carboxy group, an amide group, or an alkoxycarbonyl group. ] and a compound represented by the general formula (I[[)% formula%() [wherein R1 represents an alkyl group, an aryl group, or an aralkyl group, these include a hydroxyl group, an alkoxy group, a halogen atom, It may be substituted with an amino group, an alkylamino group, a dialkylamino group, an acylamino group, a sulfonylamino group, a nitro group, an arylsulfone group, an alkylsulfone group, a carboxy group, an amide group, or an alkoxycarbonyl group. ] A manufacturing method for obtaining the peracid ester compound by condensing the compound represented by the above is provided.

Further, according to the present invention, there is provided a test composition for measuring esterase or protease, which contains the above peracid ester compound, peroxidase, and an oxidized color indicator.

Furthermore, according to the present invention, there is provided a test device for measuring esterase or protease, which comprises the composition supported on a carrier.

The novel test composition and test device of the present invention are completely different from the above-mentioned conventional method, in which a decomposed product of an ester compound and a diazonium salt are coupled to form a color, and a decomposed product of a peracid ester is used. A new method is adopted in which the oxidized color indicator is colored by the nascent oxygen produced by reaction with peroxidase.

A typical compound of the filter acid ester compound represented by formula (I) of the present invention includes N-phthaloyl-thi-phenylalanine-1,1-dimethyl-2-phenylethyl hydroperoxy ester.

The filtrate ester compound represented by the formula (I) of the present invention is a new compound, which is obtained by condensing a carboxylic acid derivative represented by the formula (II) with a hydroperoxide compound represented by the formula (III). Manufactured by. Preferably, the carboxylic acid compound (n) and the hydroperoxide compound (I[[) are dissolved in a suitable organic solvent such as dichloromethane, and N is added to the solution.

No-dicyclohexylcarbodiimide and 4-dimethylaminopyridine are added and reacted. After completion of the reaction, the desired product is collected from the reaction mixture according to a conventional method.

For example, the desired product can be obtained by filtering the reaction mixture through a glass filter to remove the formed precipitate, distilling off the solvent from the filtrate, and purifying the residue by column chromatography or the like.

As described above, the peracid ester compound (1) of the present invention is used as a substrate in the measurement of esterase or protease, and is particularly advantageously used in the detection of leukocytes in urine and dialysate.

Such a test device for detecting esterase or protease consists of a carrier impregnated with a composition consisting of the peracid ester (I) of the present invention, peroxidase, a coloring indicator, and optionally a buffer, an activator, a stabilizer, a wetting agent, and a solvent. .

As indicators, so-called oxidation indicators are used, which change color when oxidized. Examples include a combination of 2,4-dichlorophenol and 4-aminoantipyrine, and toethyl-N-(2-hydroxy-3-sulfonate). Examples include a combination of propylline and orthotolidine.

Buffers are used to maintain a constant pH value directly above the test;
For example, tris-(hydroxylmethyl)-aminomethane buffer, phosphate buffer. Borate buffer.

Examples include barbiturate buffers and amino acid buffers, and those that can maintain the pH in the range of 6 to 1O when the test device is immersed in the sample are preferred. This composition can be used by directly dropping it into a test liquid such as urine, or it can be used by dropping a test liquid such as urine into this composition.

Furthermore, the test device for measuring esterase or protease of the present invention has the above composition supported on a carrier.
Filter paper and glass fiber are used as carriers.

A nonwoven fabric made of plastic material is suitable, as long as it does not dissolve or react with the solvent and absorbs the above composition.

The amounts of the peracid ester compound and other reagents used in the above test composition and test device are not particularly important and are determined as appropriate. That is, an amount sufficient to react with the esterase or protease to be detected and cause a color reaction is selected.

Next, the present invention will be explained in more detail with reference to Examples and Test Examples.

(Example 1) Under an argon atmosphere, 3.84 g of hydrocinnamic acid (25,
6+wmol) and t-butyl hydroperoxide 2
．． A solution of 31 g (25,6a + 5ol) in dry dichloromethane (25all) contains 6.33 g (30,7 mmol) of N,N'-dicyclohexylcarbodiimide and 4
-dimethylaminopyridine 0.78g (6,40in
ol) was added and reacted at room temperature for 4 hours.

The reaction solution was filtered through a glass filter, concentrated under reduced pressure, and the resulting residue was separated and purified by silica gel chromatography. Hydrocinnamic acid t-butyl hydroperoxy ester was obtained by elution with dichloromethane.

NMR (PPm.CDcj! 3) 7.20 (S,
5H) 3,15 - 2.40 (m.i), 1.23 (S
．． 9H)H(v.-'+CHclt3)1765
(Example 2) Hydrocinnamic acid 4.32 g (2
8.8 mmol) and 2,5-dimethylhexane-2
．． 5-dihydroperoxide5. 13 g (28.
8 mmol) of dry dichloromethane (3(ld)), N.N-dicyclohexylcarbodiimide 7,14
g (34.6 smol) and 0.88 g (7.20TIIIO1) of 4-dimethylaminopyridine were added, and the mixture was reacted at room temperature for 18 hours. The reaction solution was filtered through a glass filter, concentrated under reduced pressure, and the resulting residue was separated and purified using silica gel column chromatography. By elution with dichloromethane, 6.05 g (20.7 mmol) of bishydrocinnamic acid-2.5-dimethyl-2.5-dihydroperoxyester was obtained.

NMR (PPm.CDcj!s) 7.22 (S.
1011) 3, 18-2.43 (m.81), 1.62
(5.48), 1.17 (3.128) IR(νcm-
' , CHc j! s) 1770 (Example 3) 2.86 g of todosyl-shi-alanine under argon atmosphere
(11.8+nol) and t-butyl hydroperoxide 2.66 g (29.5 mm+ol) in dry dichloromethane (30 msol) was added N,N'-dicyclohexylcarbodiimide 2.93 g (14.2 msol)
) and 0.36 g of 4-dimethylaminopyridine (
2.95 inol) was added at 0°C and reacted for 16 hours. The reaction solution was filtered through a glass filter, concentrated under reduced pressure, and the resulting residue was separated and purified using silica gel column chromatography. Ethyl acetate-hexane (2
: By eluting with 3), N-)',/L
-Alanine-t-butylhydroperoxyester2.
64 g (8.38+nol) was obtained.

NMR (PPm.CDcj!s+DMso-di)
7.67 (d, J-8Hz.2H) and 7.20 (d
．． J-8Hz. 2H), 5. 87 (d.
J=8Hz. 18), 5. 48-4.97 (m
, IH), 2.39 (S, 3H), 1.38 (d, J=
6Hz, 3B).

1,01(S,9H) IR(S,,-',KBr) 3250.1600 (Example 4) 3.86 g of N-phthaloylglycine under argon atmosphere
N, N'-
Dicyclohexyccarbodiimide 4,66 g (22
．． 6 msol) and 4-dimethylaminopyridine 0.
57 g (4.70 inol) was added at 0°C and reacted for 4 hours. The reaction solution was filtered through a glass filter, concentrated under reduced pressure, and the resulting residue was separated and purified by silica gel column chromatography.

By elution with dichloromethane, N-phthaloylglycine-t-butylhydroperoxyester 4.0
8 g (14.7 wmol) was obtained.

O' NMR (PPm, CDcj!s+DMso-di) 7
．． 87 (S, 4H) 4.69 (S, 2fl), 1.02
(S,9H)IR(y cm-', KBr) 17
95,1765,1720 (Example 5) Under an argon atmosphere, 3.00 g (2
0,0+wnol) of dry tetrahydrofuran (20 m
Add 2.81'l of methylmagnesium bromide to the solution.
An ether solution (28.5d, 79.9s+mol) was added to 0°C and reacted for 18 hours, then a saturated aqueous ammonium chloride solution was added and extracted with ethyl acetate. After washing the organic layer with water, it was concentrated under reduced pressure, and the resulting residue was separated and purified using silica gel column chromatography. 2.82 g (18,8 mm) of 1,1-dimethyl-2-phenylethyl alcohol by elution with dichloromethane
ol) was obtained.

Ether 51d to 2.82 g (18.8 m-ol) of the above compound.

20 d of a 50% aqueous hydrogen peroxide solution and 0.50 d of concentrated sulfuric acid were added, and condensate that had been reacted at room temperature for 200 hours was added, followed by extraction with ethyl acetate. The organic layer was washed with water, concentrated under reduced pressure, and the resulting residue was separated and purified by silica gel column chromatography. Elution with dichloromethane gave 2.38 g (14.3 ma+ol) of 1,1-dimethyl-2-phenylethyl hydroperoxide.

2.38 g (14,3+u+ol) of the above peroxide and 4.22 g of N-phthaloyl-L-phenylalanine
g (14,3wnol) of dry dichloromethane (20
m) 3.55 g of N,N'-dicyclohexylcarbodiimide (17,2 ms+ol) and 0.44 g of 4-dimethylaminopyridine (3,58 mmo
1) was added at 0°C and reacted for 6 hours. The reaction solution was filtered through a glass filter, concentrated under reduced pressure, and the resulting residue was separated and purified by silica gel column chromatography. N-phthaloyl-phenylalanine-1,1-dimethyl- by elution with dichloromethane
2-phenylethyl hydroperoxy ester 5.14
g (11 Jmsol) was obtained.

NMR (PPm, CDcj!s+DMsO-da)
7.63 (S, 4H) 7.20 (S, 5H), 7.0
7(S, 5H), 5.02(t, H-7Hz, 11)3
．． 51 (d, H = 7Hz, 2H), 2.84 (S, 2H
), 1.17(S,6H)IR(v c--', KB
r) 1790,1765.1720 (Example 6) Ethyl undecanoate 3.00 under argon atmosphere
g (14,0 ma+ol) of methylmagnesium bromide in dry tetrahydrofuran (20 m).
8H ether solution (20d, 56.0 mmol)
After adding at ℃ and reacting for 16 hours, saturated ammonium chloride aqueous solution was added and extraction was performed with ethyl acetate. After washing the organic layer with water, it was concentrated under reduced pressure, and the resulting residue was separated and purified using silica gel column chromatography. Elution with dichloromethane gave 2.65 g (13,2 malo) of 1,1-dimethylundecyl alcohol.

2.65 g (13.2 mmol) of the above compound and 5 m of ether.

After adding 20 ml of a 50% aqueous hydrogen peroxide solution and 0.50 d of concentrated sulfuric acid and reacting at room temperature for 19 hours, water was added and extraction was performed with ethyl acetate. The organic layer was washed with water, concentrated under reduced pressure, and the resulting residue was separated and purified by silica gel column chromatography. By elution with dichloromethane, 8d of thionyl chloride was added to 1.96 g (12.9 mmol) of 1,1-dimethylundecylhydroperoxide phenoxyacetic acid, and the mixture was stirred at 50°C for 2 hours.
When the reaction solution was added to hexane cooled to 0°C, 1.65 g of phenoxyacetyl chloride (9.67 ms
+ol) was obtained.

1,1-dimethylundecyl hydroperoxide 2,2
After adding 1.28 g (12.6 mmol) of triethylamine to a solution of 7 g (10.5 + u + ol) and 1.65 g (9.67 gmol) of phenoxyacetyl chloride in dry dichloromethane (15I11) and reacting at room temperature for 5 hours, Water was added and extraction was performed with ethyl acetate. The organic layer was washed with water, concentrated under reduced pressure, and the resulting residue was separated and purified by silica gel column chromatography. Elution with dichloromethane gave 2.94 g (8,38R11IO1) of phenoxyacetic acid-1,1-dimethylundecyl hydroperoxyester.

NMR (PPm, CDcl, +DMSO-da)
7.45-6.64 (s, 5H).

4.54 (S, 2H), 1.93-0.65 (+m,
27H) IR (C-', KBr) 1765 (Example 7) Production example of one composition - Solution I Peroxidase (horseradish) 200■0.2M T
ris-Hcj! Buffer (p)l-7,5) 10
0 m and solution ■ 2.4-dichlorophenol 1.9 g 4-aminoantipyrine 2.4 g N-phthaloyl-
Shi-phenylalanine-1,1-dimethyl-2-phenylhydroperoxyester molecular weight X0.2X10-"
g Acetone-decanol (98:2) 100jd
A test composition for measuring esterase or protease according to the present invention was obtained.

(Example 8) Example of manufacturing a test strip - Solution I Beroxidase (horseradish extract: manufactured by SIGMA) 200■ 0.2M Tris-Hcj! Buffer solution (PH-7,
5) Wet a 100ad filter paper sufficiently with the solution
Dry in a drying oven at °C for 50 minutes.

Solution ■ 2.4-dichlorophenol 1.9 g 4-aminoantipyrine 2.4 g Acetone-decanol (98:2) 100 m Solution I Solution The dried filter paper was thoroughly wetted with solution ■ and dried in a drying oven at 40°C for 10 minutes. , a test paper according to the present invention was obtained.

(Test Example) The test paper obtained in Example 8 above is immersed in urine for 1 second. The color development of the test paper is visually read over time.

As a result, the color of the test paper changed from white to red in about 4 minutes for urine with 500 G of leukocytes/μl and in about 10 minutes for urine with 1000 G of leukocytes/μl.

〔Effect of the invention〕

According to the present invention, a novel peracid ester compound is provided.

Furthermore, the present invention provides an advantageous method for producing such peracid ester compounds.

Furthermore, the peracid ester compound of the present invention is effectively used for detecting esterase or protease, particularly leukocytes.

That is, a test composition and a test device are provided that utilize a new detection method that is completely different from conventional methods for measuring esterases or proteases containing esters and diazonium salts as main components.

As described above, the peracid ester compound of the present invention has excellent properties as a substrate for measuring esterase or protease.

Claims (10)

[Claims] (1) General formula (I) ▲Mathematical formulas, chemical formulas, tables, etc.▼(I) [In the formula, R^1 and R^2 may be the same or different, and each represents an alkyl group, an aryl group, or an aralkyl group. These include hydroxyl group, alkoxy group, halogen atom, amino group, alkylamino group, dialkylamino group, acylamino group, sulfonylamino group, nitro group, arylsulfone group, alkylsulfone group, carboxy group, and amide group. Alternatively, it may be substituted with an alkoxycarbonyl group. ] A peracid ester compound represented by (2) In the above formula (I), ▲ has a mathematical formula, a chemical formula, a table, etc. ▼ represents a natural L-amino acid, its D- and DL-type, or a peptide group consisting of 2 to 5 of these amino acids; A compound according to claim 1, wherein the group is protected. (3) In (I) above, R^2 is a t-butyl group 1
, 1-dimethylundecyl group, 1,1-dimethyl-5-
The compound according to claim 1, which is a phenylpentyl group, a 1,1-dimethyl-2-phenylethyl group, or a group represented by ▲a numerical formula, a chemical formula, a table, etc.▼. (4) The peracid ester compound of the above formula (I) is hydrocinnamic acid-t-butylhydroperoxyester, bishydrocinnamic acid-2,5-dimethylhexane-2,5-dihydroperoxyester, N-tosyl- L-alanine-t-butylhydroperoxyester, N-phthaloylglycine-t-butylhydroperoxyester, N
-phthaloyl-L-phenylalanine-1,1-dimethyl-2-phenylethyl hydroperoxyester or phenoxyacetic acid-1,1-dimethylundecyl hydroperoxyester. (5) General formula (II) ▲ Numerical formula, chemical formula, table, etc. ▼ (II) [In the formula, X represents a hydroxy group, halogen group, alkylcarbonyloxy group, alkoxycarbonyloxy group, or imidazole group, etc. 1 represents an alkyl group, an aryl group, or an aralkyl group; , an alkylsulfone group, a carboxy group, an amide group, or an alkoxycarbonyl group. ] A compound represented by the general formula (III) H-O-O-R^2(III) [In the formula, R^2 represents an alkyl group, an aryl group, or an aralkyl group, but these are hydroxyl groups. , an alkoxy group, a halogen atom, an amino group, an alkylamino group, a dialkylamino group, an acylamino group, a sulfonylamino group, a nitro group, an arylsulfone group, an alkylsulfone group, a carboxy group, an amide group, or an alkoxycarbonyl group. Good too. ] General formula (I) characterized by condensation with a compound represented by They may be the same or different, and each represents an alkyl group, an aryl group, or an aralkyl group; , a nitro group, an arylsulfone group, an alkylsulfone group, a carboxy group, an amide group, or an alkoxycarbonyl group. ] A method for producing a peracid ester compound represented by (6) General formula (I) ▲Mathematical formula, chemical formula, table, etc.▼(I) [In the formula, R^1 and R^2 may be the same or different, and each represents an alkyl group, an aryl group, or an aralkyl group. These include hydroxyl group, alkoxy group, halogen atom, amino group, alkylamino group, dialkylamino group, acylamino group, sulfonialamino group, nitro group, arylsulfone group, alkylsulfone group, carboxy group, amide group. Alternatively, it may be substituted with an alkoxycarbonyl group. ] A test composition for measuring asterase or protease containing a peracid ester compound represented by the above, peroxidase, and an oxidized color indicator. (7) The oxidation color indicator is phenol, 2,4-dichlorophenol N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine, N,N-dimethylaniline, 2-hydroxy-3, 7. The composition according to claim 6, comprising a combination of one compound of 5-dichlorobenzenesulfonic acid and 4-aminoantipyrine. (8) The composition according to claim 6, wherein the oxidized color indicator is orthotolidine, benzidine or leucomalachide green. (9) General formula (I) ▲Mathematical formula, chemical formula, table, etc.▼(I) [In the formula, R^1 and R^2 may be the same or different, and each represents an alkyl group, an aryl group, or an aralkyl group. These include hydroxyl group, alkoxy group, halogen atom, amino group, alkylamino group, dialkylamino group, acylamino group, sulfonialamino group, nitro group, arylsulfone group, alkylsulfone group, carboxy group, amide group. or an alkoxycarbonyl group. ] A test device for measuring esterase or protease, comprising a carrier supporting a composition containing a peracid ester compound represented by the following formula, peroxidase, and an oxidized color indicator. (10) The test device according to claim 9, wherein the carrier is a nonwoven fabric made of filter paper, glass fiber, or a plastic material.