JPH02306991A - Cyclic acetal derivative of indophenyl-alpha-glycoside, its production and utilization thereof as reagent for measuring alpha-amylase activity - Google Patents

Abstract

NEW MATERIAL:A compound expressed by formula I (X1 to X6 are H, halogen, nitro, cyano, azide, acyl, sulfonic acid, nitroso, sulfonyl, sulfoxyl, thiocyano, isothiocyano, isonitrile, imino, azo, diazo, alkyl, allyl or aryl or X3 and X4 and/or X5 and X6 may be linked to form condensed aromatic ring; R1 and R2 are H, lower alkyl or phenyl or R1 and R2 together may form cyclohexane ring or cyclopentane ring; n is 0-8). EXAMPLE:Isopropylidine-phenolindophenyl-alpha-pentaoside. USE:A reagent for measuring alpha-amylase activity. PREPARATION:A cyclic acetal derivative of a 4-aminophenylglycoside expressed by formula II is reacted with a quinone derivative expressed by formula III.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a cyclic acetal derivative of indophenyl glycoside useful for measuring the activity of a polysaccharide hydrolase, such as amylase, and a method for producing the same.

[Conventional technology]

Various diseases are diagnosed by measuring the activity of α-amylase contained in body fluids such as pancreas and urine. The amylase activity measurement method uses a method in which a phenol derivative is bound to the reducing end of an oligosaccharide, such as p-nitrophenylmaltopentaoside (Japanese Patent Publication No. 57-53079), p-nitrophenylmaltohexaoside (Japanese Patent Publication No. 57-53079),
7-53079), p-nitrophenylmaltohebutaoside (Japanese Unexamined Patent Publication No. 54-51892), p
-Nitrophenyl maltohebutaoside (JP-A-54-5
1892), 2,4-dichlorophenylmaltobentaoside (Japanese Unexamined Patent Publication No. 56-35998), and the like are used as substrates.

These substrates are hydrolyzed by amylase in body fluids, and the resulting fragments are further hydrolyzed by α- or β-glucosidases, e.g.
-Ditrophenol comes at a distance of '1JiL. This p-
By optically measuring nitrophenol, α-
Amylase activity can be easily measured. However, when measuring α-amylase activity using such a substrate, it is necessary to measure light absorption at a fairly short wavelength around 410 nm. When a substance is present, it is easily susceptible to interference.

On the other hand, a method using indoxyl maltodextrin is known as a method free from such problems (Japanese Unexamined Patent Publication No. 103199/1983).

[Problem to be solved by the invention]

However, indoxyl maltodextrin cannot be said to be an excellent substrate because it is difficult to produce and the production of indico dyes by oxidizing agents is slow. Furthermore, since the pre-contained α or β-glucosidase acts on the substrate, albeit slightly, there is a drawback that the blank value increases. In order to overcome these drawbacks, a method has been proposed that uses a type of substrate in which the hydroxyl group at the 4- or 6-position of the glucose at the non-reducing end of the malto-oligo is modified (for example, Japanese Patent Application Laid-open No. 1986-60-1). Publication No. 237998, JP-A-61-6
It is disclosed in JP-A No. 3299, JP-A-63-17895, JP-A-63-17895, and JP-A-63-7797. ), However, even when these substrates are used, there is a problem that the peak of the absorption wavelength is around 410 nm, so the light absorption of serum is large and the measurement error becomes large.

The purpose of the present invention is to reduce pigments in blood in α-amylase,
For example, it is an object of the present invention to provide a means that is resistant to interference by bilirubin and hemoglobin and substrate decomposition by pre-contained α- or β-glucosidase, and that enables measurement with simple operation and high detection sensitivity.

[Means to solve the problem]

The cyclic acetal derivative of indophenyl glycoside suitable as a reagent for measuring the activity of α-amylase of the present invention has the non-reducing terminal glucose at the 4- and 6-positions modified with an acetal derivative, and the reducing terminal modified with an indophenyl derivative. Compound (1) modified with [1] (wherein, It means a sulfoxyl group, thiocyano group, isothiocyano group, isonitrile group, imino group, azo group, diazo group, alkyl group, allyl group or aryl group, and X3 and X4 and/or X and X6 are connected to form a fused aromatic ring. R1 or R2 may be the same or different and represent a hydrogen atom, a lower alkyl group or a phenyl group, and examples of the lower alkyl group include methyl, ethyl,
Propyl, isopropyl, butyl, etc. or R8 and R2 may jointly form a cyclohexane ring or a cyclopenkune ring. ) is an indophenyl glycoside represented by

Further, it is particularly preferable that n is 3.4 or 5.

The lower alkyl group of R7 and Rt is a methyl group, ethyl group, propyl group, isopropyl group, butyl group, etc., and n is 3
．． 4 or 5 is particularly preferred.

Examples of cyclic acetal derivatives of indophenyl glycosides include the following compounds.

However, in the following formula, Q is (n represents 3 or 5).

The cyclic acetal derivative of indophenyl glycoside of formula i is represented by the following formula. ) is produced by reacting a quinone derivative represented by the following formula 2 (wherein X and Xh have the above-mentioned meanings) with a cyclic acetal derivative of 4-aminophenyl-ligated body represented by be able to.

For example, compound
The non-reducing terminal hydroxyl groups at the 4 and 6 positions of 4-nitrophenyl-α-maltopentaoside are protected with benzylidene, and the nitro group is removed by catalytic reduction using Raney nickel or palladium-carbon as a catalyst according to a conventional method. ■ can be synthesized by converting into an amine group (
Can, J. Chem, vol. 38, p. 363, 1960).

Examples of compound H include 4-aminophenyl-α-maltopentaose, 4-aminophenyl-2-chloro-β-
Examples include cyclic acetal derivatives such as maltoheptaose, 4-amino-2,6-difluoro-β-maltohexaose, and 4-amino-2-bromophenyl-β-heptaose, and more specifically, on page 8 ( Examples include the compounds shown in 1) to (6).

Examples of compound ① include P-quinone, 2-chloro-p-
Quinone, 2,6-dichloro-p-quinone, 2,5-dibromo-p-quinone, 2.3,5,6-tetrachloro-p
-quinone (chloranil), 1,4-naphthoquinone, anthraquinone and the like.

To produce compound ■, which is the target substance, compound ■ is dissolved in a neutral organic solvent under anhydrous conditions, a neutral dehydrating agent is coexisted, a catalytic amount of strong acid is added, and compound ■ is allowed to act. It is preferable.

The amount of Compound (1) to be used is 10 to 50 times the molar equivalent, preferably 15 to 25 times the molar equivalent of Compound (2).

Examples of neutral organic solvents include ethers such as diethyl ether, THF, dioxane, etc., hydrocarbons such as n-hexane, benzene, toluene, etc., halogenated hydrocarbons such as dichloromethane, chloroform, dichloroethane, carbon tetrachloride, etc., dimethyl Formamide (DM
F), dimethylacetamide (DMA), hexaphosphoramide (HMPA), dimethyl sulfoxide (DM
SO), etc., and these may be used in combination.

The amount of the solvent is about 10 to 100 times, preferably 40 to 60 times, the weight of compound (1).

As a neutral dehydrating agent, molecular sieve, AhOr, Si
ng, anhydrous Na, SOa, anhydrous MgSO4, etc., and these may be used in combination. The amount is 0.5 to 5 times the weight of compound (1), preferably 0.8 to 1.5 times the weight of compound (2).
It's double. Strong acids include trifluoroacetic acid, trichloroacetic acid, P-)luenesulfonic acid, methanosulfonic acid, fuming sulfuric acid, AlCl3, BFff/Et, 0, TiCl4
, FeClff, etc., and these may be used in combination. The amount thereof is 0.01 to 0.1 times the molar equivalent, preferably 0.02 to 0.04 times the molar equivalent of Compound (1).

The reaction temperature and reaction time vary depending on the type of compound ①, compound ②, solvent, and strong acid, but the reaction temperature is usually 15 to 15 minutes.
The reaction temperature is 25°C, and the reaction is completed by continuing the reaction for 0.5 to 4 hours.

After the reaction is completed, an alkali is added to the reaction solution to neutralize it, and water is added to transfer Compound I to the aqueous layer. By separating the neutral organic solvent layer, unreacted quinones contained therein are removed. Alcohol is added to the separated aqueous layer to precipitate the compound, and the compound can be purified by recrystallization. Furthermore, it can be separated as a single compound by using gel chromatography, adsorption chromatography, etc.

Compound (1) of the present invention is useful as a substrate for the detection and quantitative measurement of α-amylase.

As a measurement method, an aqueous solution containing this compound 1 and α- or β-glucosidase is prepared. This aqueous solution may further contain a pH-adjusting agent, a glucosidase activator, a stabilizer, and the like. Although glucosidase may be used in a separate solution, it is convenient for the measurement procedure to use the same solution as compound ①. A sample solution is added to a solution containing Compound 1 and glucosidase, and heated under certain conditions to cause an enzyme reaction. Thereafter, the change in absorbance at the characteristic absorption wavelength of the indophenol compound produced by the enzymatic reaction is measured, and α-amylase can be quantified using the M line.

(Action) The cyclic acetal derivative of indophenyl glycoside of the present invention consists of a glucose chain part and an indophenyl part, and the glucose chain part is first cleaved by α-amylase, and one or two molecules of glucose remain in the indophenyl part. Compounds are produced.

Next, alpha or beta-glucosidase acts to cleave off the remaining glucose molecules, liberating the indophenol compound. As a result, the hydroxyl group of the phenyl group dissociates and the absorption wavelength changes. By measuring its absorbance, α−
Quantify amylase.

The compounds of the present invention are present at the non-reducing terminal glucose positions 4 and 6.
Since the position is modified with an acetal derivative and the reducing end is also bonded with an indophenyl group, the glucose chain portion is not cleaved by α- or β-glucosidase at all.

〔Example〕

Isopropylidene-phenolindo-phenyl-α-
Production of pentaoside and measurement of α-amylase using the same (1) Isopropylidene-4-nitrophenyl-α-pentaoside (synthesized with reference to the method described in JP-A-61-63299)5. OOg to methanol 2
00d, 1.0 g of 5% palladium on carbon was added, and the mixture was stirred for 24 hours under normal pressure while introducing hydrogen gas.Next, palladium-carbon was filtered off, the solvent was distilled off, and recrystallized from isopropanol. Then, isopropylidene-4-aminophenyl-α-pentaoside is 285
g was obtained.

Melting point ('C); 192-195 (decomposition) (2) 18.0 g of p-benzoquinone was dissolved in 100 Id of 1,4-dioxane.
Further, 0.5 g of trifluoroacetic acid and 15 g of molecular sheep (4A) were added and mixed. To this was added OOg of isopropylidene-4-aminophenyl-α-pentaoside 2 obtained in (1) above, and the mixture was stirred at room temperature for 30 minutes. Next, the reaction solution was neutralized with sodium methoxide. 200ml of water was added and excess benzoquinone was removed with chloroform. The aqueous layer was freeze-dried to obtain a yellow substance. Purification with Biogel (P-2) and recrystallization of the eluted fraction from Improper Tool yields isopropylidene-
Phenolindo-phenyl-α-pentaoside 0.
95 g was obtained.

Melting point ('C); 181-183 Ultraviolet/visible absorption spectrum; Swax (nrm) = 468.265 Infrared absorption spectrum (CII+-1): 1640.1620.14
91.1253.1082.1044.997.877 (3) Next, α or β, which is a problem when measuring amylase.
-The presence or absence of shadows caused by glucosidase was investigated.

Phenolindo-phenyl-α-pentaoside and isopropylidene-phenolindo-phenyl-α-pentaoside were dissolved in 50mM Gud buffer at pl+6.7 to a concentration of 4/- each, and the two substrate solutions (A), CB) was prepared. On the other hand, the same buffer as above contains α-glucosidase at 1500/ml! , β-glucosidase was added and dissolved at a concentration of 200/d to make enzyme solution 1.
Seeds were prepared.

Enzyme solution 0.5J! f! 0.5 d of substrate solutions (A) and (B) were added to the solution and mixed, and the mixture was left at 37°C. After standing for a predetermined time, a 10+H potassium periodate aqueous solution was added, and the absorbance at 610na+ was measured. The results are shown in Table 1.

Table 1 As is clear from Table 1, α-glucosidase and β-glucosidase for phenolindo-phenyl α-pentaoxide
- Glucosidase acted slightly and the absorbance at 610 nm increased by 0.360 in 1 hour, whereas isopropylidene-phenolindo-phenyl-α-pentaoside is hardly affected by α-glucosidase etc.
The absorbance at 10 nm increased by only 0.010.

As is clear from the above results, since the compound represented by the general formula (Il) has almost no effect on α-glucosidase or θ-glucosidase, it is possible to use the reagent for measuring amylase in one solution, which is very advantageous in terms of measurement operation. In addition, we were able to make a significant contribution to improving measurement accuracy.

(4) Measurement of α-amylase activity (20 mmof of the compound obtained in step 2), 10 mmof of calcium chloride, and 500 units of α-glucosidase were dissolved in purified water, and added with sodium hydroxide to pl+6.
9, and the total amount was 207.

After adding 100 pE of sample serum to the above solution 2- and heating it to 37°C, the change in absorbance of this reaction solution at a wavelength of 610 nm was measured.

Separately, using a standard specimen with known α-amylase activity, the same procedure as above was performed to determine the calibration relationship, and the α-amylase activity of the specimen was determined from this calibration curve.

At this time, α-amylase activity (Somogyi units/dl) and wavelength of 610 nm at each dilution stage of the standard sample
FIG. 1 shows the relationship between the increase in absorbance N (ΔA) per minute and the increase in absorbance per minute.

As is clear from Figure 1, α-amylase activity (Soa
The calibration curve connecting the increase in absorbance (ΔA) in pairs/units to +ogyi units/d) is a straight line passing through the origin, and the calibration curve shows good quantitative properties.

As is clear from FIG. 1, amylase activity in control serum could be measured with high accuracy using the analytical element of the present invention.

〔Effect of the invention〕

When the cyclic acetal derivative of indophenyl glycoside of the present invention is used as a substrate in α-amylase, it is difficult to prevent interference by pigments in the blood, such as bilirubin and hemoglobin, and inhibition of the substrate by pre-contained α or β-glucosidase. Resistant to decomposition (can perform measurements with simple operation and high detection sensitivity).

[Brief explanation of drawings]

Figure 1 shows the calibration curve obtained in the example, and each α on the horizontal axis
- The absorbance increase (ΔA) obtained for amylase activity (Somogyi units/di) is plotted along the horizontal axis and the points are connected. Patent applicant: Fuji Photo Film Co., Ltd. Agent
Patent attorney Kuninaka Politics 1 person Figure 1

Claims (1)

[Claims] 1. General formula [I] [I] ▲ Numerical formulas, chemical formulas, tables, etc.▼ (In the formula, X_1 to X_6 are hydrogen atoms, halogen atoms, nitro groups, cyano groups, azido groups, acyl Group - means a sulfonic acid group, nitroso group, sulfonyl group, sulfoxyl group, thiocyano group, isothiocyano group, isonitrile group, imino group, azo group, diazo group, alkyl group, allyl group or aryl group, X_3 and X_4 and/ Or X_5 and X_6 may be connected to form a condensed aromatic ring.R_1
, R_2 are the same or different and mean a hydrogen atom, a lower alkyl group, or a phenyl group, R_1 and R_2 may jointly form a cyclohexane ring or a cyclopentane ring, and n is any one from 0 to 8. Represents an integer. ) Cyclic acetal derivative of indophenyl glycoside 2
, the following formula [II] [II] ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (wherein R_1, R_2, Cyclic acetal derivatives of saccharides include the following formula [III] ▲ Numerical formulas, chemical formulas, tables, etc. ▼ [III] (In the formula, X_3 to X_6 are the same as those described in claim 1). A method 3 for producing a cyclic acetal derivative of an indophenyl glycoside according to claim 1, characterized in that the indophenyl glycoside according to claim 1 is used as a substrate for detection and quantitative measurement of amylase Use of cyclic acetal derivatives 4, detection and measurement of α and/or β as auxiliary enzymes
- The use according to claim 3, which is carried out in the presence of glucosidase.