JPH02258794A - Glycoside capable of forming dyestuff and production thereof - Google Patents

Abstract

NEW MATERIAL:A compound shown by formula I [A is 1,4-naphthylene bonded, at the 1-position, to oligosaccharide; R<1> is (substituted) lower alkyl; n is 0-8]. USE:An enzyme for hydrolyzing polysaccharides, useful for measuring activity of amylase, slightly being hindered by a dyestuff in blood such as bilirubin and hemoglobin. PREPARATION:An oligosaccharide shown by formula II is treated with an organic acid anhydride shown by the formula (RCO)2O (end is lower alkyl) and a prepared compound shown by formula III (Y is COR) is halogenated to give a compound shown by formula IV (X is halogen). Then this compound is treated with a compound shown by the formula HO-A-OR<1> to give a compound shown by formula V. Then this compound is deacylated.

Description

Detailed Description of the Invention [Field of the Invention] The present invention relates to a glycoside useful for measuring the activity of a polysaccharide hydrolase, such as amylase, a method for producing the same, a use thereof, and a method for enzyme detection or activity measurement using the same. Regarding.

Prior Art and Its Disadvantages] Oligosaccharide derivatives in which an aromatic ring having characteristic absorption in a specific wavelength range of ultraviolet or visible light, such as phenol, p-nitrophenol, etc., are bonded to the reducing end of a glucose unit are, for example, Nature, Vol, 182. p, 5
25-526 (1958); Journal or
Biochemistry (Tokyo), Vo
l, 62. No. 4.439-446 (1967)
;Carbohydrate Research, V
ol, 2. No, 5.418-420 (198B)
; JP-A-53-12381, JP-A-54-51892
It is known by the number etc. These substrates are useful for measuring the activity of polysaccharide hydrolases such as maltase, glucoamylase, amylase, etc. in biological fluids such as blood. 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. If there is a substance present, it is likely to be interfered with, and in order to remove the interference, it is necessary to perform a so-called sample blind test, which makes the analysis operation complicated.

Indoxyl maltodextrin, described in JP-A-56-103199, is a substrate that can be hydrolyzed to produce an indigo dye that absorbs at long wavelengths (requires the coexistence of an oxidizing agent). Although it is not interfered with by substances that absorb at short wavelengths, such as bilirubin, which exist in .

"Technical Problems to be Solved" The present invention enables the measurement of the activity of polysaccharide hydrolase, such as α-amylase, to be less susceptible to interference by pigments in the blood, such as bilirubin and hemoglobin, and with simple analytical procedures.
Moreover, it is a technical problem to provide a compound useful as a self-developing substrate and a method for producing the same, which enables measurement with high detection sensitivity.

[Means for Solving Technical Problems] The first of the above problems has been solved by a compound represented by the following maximum [1].

[I] However, in the formula, R1 represents a lower alkyl group (which may be substituted), A represents a 1,4-naphthylene group, n represents ot or a positive integer up to 8, n is 3,4 or 5 is preferable.

-a The alkyl group represented by R1 in formula [1] preferably has 1 to 5 carbon atoms, and is preferably an unsubstituted alkyl group, but may have a substituent. Examples of substituents is a halogen atom (e.g. chlorine atom), a nitro group, a cyano group,
Alkanesulfonyl group (preferably 4 or less carbon atoms)
, an alkanesulfonamide group or an alkanesulfamoyl group (preferably having 1 to 7 carbon atoms), an alkoxy group or an alkylthio group (preferably having 4 or less carbon atoms, and may be further substituted with an alkoxy group, etc.)
, phenoxy group (which may have a substituent such as halogen), phenylthio group, etc. A preferred substituent is an alkoxy group.

-The 1,4-naphthylene group represented by A of maximum [1] is 5
．． 6. One or more of the 7th and 8th positions may have a substituent.

Substituents include, for example, halogen atoms (e.g. chlorine atoms), alkyl groups (preferably 4 or less carbon atoms), alkoxy groups or alkylthio groups (preferably 4 or less carbon atoms, and may be further substituted with an alkoxy group, etc.) , an alkanesulfonamide group or an alkanesulfamoyl group (preferably having 7 or less carbon atoms), a carbonamide group or carbamoyl group (preferably having 8 or less carbon atoms), or a hydroxy group.

These include an amine group, a hepano group, a di-alkylamino group, and the like.

The second problem mentioned above was solved by the following method. That is, the oligosaccharide represented by - maximum [Ial = [I
al (However, in the formula, A is 1, 4 where the 1st position is bonded to the hydroxyl group)
-represents a naphthylene group, R1 represents a lower alkyl group (which may be substituted), obtained by reacting with an organic acid anhydride represented by the formula (RCO)20 (R represents a lower alkyl group) By halogenating the compound represented by the following formula [Ib]: [I
b) By acting on this a compound represented by the following formula [Ial:
[Ial HO-A-OR (wherein A is 1, 4 where the 1st position is bonded to the hydroxy group)
~represents a naphthylene group, R+ represents a lower alkyl group (optionally substituted)) A compound represented by the following formula [Iel is obtained: [Iel (where Y represents COR) In the following formula [1c] The problem was solved by deacylating the compound represented by: [Ial (where X represents a halogen).

-i The compound represented by formula [1] differs from p-nitrophenylpentaose or p-nitrophenylheptaose, which are substrates widely used in the measurement of α-amylase activity, in that it is a free 4-alkoxynaphthol. Since the maximum absorption of pigments derived from chromophores is at long wavelengths, they are less likely to be interfered with by bilirubin, hemoglobin, etc. in the blood.

-i Specific examples of the compound represented by formula [I] are shown below.

Each reaction in the method for producing the compound of the present invention will be explained in detail below.

Acylation reaction of hydroxyl group Acylation of oligosaccharides is carried out by a known method, for example, by heat treatment in an organic acid anhydride as a reactant, preferably in the presence of a catalyst such as an alkali metal salt of an anhydrous organic acid. be able to.

The organic acid anhydride represented by (RCO)20 is, for example, acetic anhydride, propionic anhydride, butyric anhydride, and the like. As the catalyst, alkali metal salts such as sodium salts and potassium salts of anhydrous organic acids, pyridine, picoline, etc. are used.

In order to facilitate reaction control or purification of the target product after the reaction, a non-aqueous solvent such as chloroform, dichloromethane, etc. may be added to the reaction solution.

The amount of organic acid anhydride used in the reaction L is 5 to 50 times, preferably 7 to 15 times the weight of the oligosaccharide, and when an alkali metal salt of an anhydrous organic acid is used as a catalyst, The amount is 0.5 to 3 times, preferably 0.5 to 1.5 times the amount of oligosaccharide superposition.

The reaction temperature is conventionally about 90-140°C, preferably 100-140°C.
One reaction time at 110° C. depends on the reaction temperature, but is about 2 to 4 hours under preferred reaction temperature conditions. The reaction mixture is cooled to 0 to 5° C. in a conventional manner, and the precipitated solids are separated, washed with water, and then dried. The obtained solid product can be recrystallized using solvents such as alcohols such as ethanol and methanol, ketones such as methyl ethyl ketone and acetone, and ethers such as dimethyl ether and diethyl ether, alone or in combination. However, the solid product can also be sufficiently dried and used as it is in the reaction.

Halogenation of oligosaccharide reducing end: Halogenation is carried out using anhydrous hydrogen halide, aluminum chloride and phosphorus pentachloride, titanium tetrachloride, or stannic chloride, but the yield of the product and related Particularly preferred is a method in which PA is treated with anhydrous titanium tetrahalide in a low polar nonaqueous solvent such as chloroform or dichloromethane from the viewpoint of suppressing side reactions and facilitating purification of the target product.

The anhydrous titanium tetrahalide includes titanium tetrachloride,
Titanium tetrabromide, titanium tetraiodide, etc. can be used, and the amount of anhydrous titanium tetrahalide relative to heptadecaacylmaltopentaose may be usually 1 to 20 times the molar amount,
3 to 81 tomole is preferred.

This halogenation reaction is carried out under normal pressure between room temperature and the boiling point of the solvent used, but it is particularly preferable to carry out the reaction without refluxing at the boiling point of the solvent.The reaction time depends on the reaction temperature, but When reacting near the boiling point of
9. Cool the reaction mixture in a conventional manner, add an organic solvent such as chloroform, dichloromethane, ethyl acetate, etc.
The organic solvent layer is separated, washed several times with water and a 5-saturated sodium bicarbonate aqueous solution, and then dried to dryness.

The obtained solid product is separated and purified by conventional methods such as silica gel chromatography, and then treated with solvents such as alcohols such as ethanol and methanol, ketones such as methyl ethyl getone and acetone, and ethers such as dimethyl ether and diethyl ether. These can be used alone or in combination for recrystallization. It can also be used in the next reaction after being sufficiently dried as a dry solid.

Substitution reaction: The anomeric halogen group of the halogen compound [Iel,
Substitution with a compound of the general formula [Idl gives [Iel.

The amount of 4-alkoxy-1-naphthol used in this reaction is 1 to 20 times by mole, preferably 1.2 to 3.0 times by mole. 4-alkoxy-1-naphthol needs to be dissociated in the form of a salt in the reaction solvent in order to accelerate the reaction, and for this reason it may be added with an inorganic salt, such as a sodium salt, a potassium salt, a barium salt, or an organic salt, such as triethylamine. It is used as salt, tributylamine salt, pyridine salt, picoline salt, etc. Two or more types of these salts can be used in combination. Alternatively, an inorganic base or an organic base may be added to the reaction solution without preparing the salt in advance. The base may be added to the reaction solvent or an organic base may be used directly as a reaction solvent.The amount of base added is preferably an amount necessary to maintain the liquid at a neutral or alkaline level until the reaction is completed.

This reaction is usually preferably carried out in the presence of a solvent.

The solvent is not particularly limited as long as it does not participate in this reaction, but halogen [ic] and 4-alkoxy-
1-naphthol [Idl or its salt has high solubility,
Preferably, the solvent increases the reactivity. For example, the following solvents are used. amides such as dimethylformamide and dimethylacetamide, nitrites such as acetonitrile, benzonitrile, dimethyl sulfoxide, organic bases such as trialkylamines, pyridine, lutidine, etc.
Aromatic hydrocarbons such as benzene, toluene, and mixtures of 116 or more thereof.

This reaction generally proceeds at about -5 to 100°C, but the reaction temperature is usually preferably 10 to 50°C. The reaction time varies depending on the type of base as a reaction aid and the reaction temperature, but is usually 5 to 50°C. After the completion of the 20-hour reaction, the reaction mixture is poured into ice water and the precipitated solid matter is filtered, or
Or by extracting the target product with an appropriate organic solvent, drying, and then solidifying to dryness.

Obtain solids. The compound [Iel is obtained as a solid.

This can be purified by conventional methods such as column chromatography using alumina, silica gel, etc., crystallization using an organic solvent, and the like in an appropriate combination.

Deacylation reaction: Removal of the acyl group from the compound (Iel) can be carried out by known methods, for example in the presence of a catalyst such as an alkali metal alkoxide in dehydrated methanol or a methanolic solution of anhydrous ammonia. For example, sodium methoxide, potassium methoxide
oxide, potassium-L-butoxide, etc. can be used.

In order to facilitate the purification of the target product after the reaction, it is preferable to add a low polar non-aqueous solvent such as chloroform or dichloromethane sugar to dehydrated methanol. 4 produced without inhibiting
- Since it is necessary for the alkoxynaphthol glycoside to precipitate from the reaction system, the amount varies depending on the solvent, but
The amount of dehydrated methanol used is preferably from 0.5 to 2f&.

The deacylation reaction is completed within 6 to 24 hours at a temperature of 0 to 30°C. In the reaction system of dehydrated methanol solvent,
After the reaction is complete, methanol is removed under reduced pressure, and the resulting solid is neutralized with basic substances mixed in using an acidic ion exchange resin or inorganic acid. Compounds are then separated by thin-layer chromatography, column chromatography, etc. refine.

In the case of a reaction system in which a low polar solvent is added, the target product precipitates from the reaction solution, and is separated and purified.

The compound of general formula [1] of the present invention is hydrolyzed to liberate a compound of formula -a Ndi. A compound of the general formula [Idl is oxidized with a suitable oxidizing agent, and as a result of self-coupling, a dye is produced.For example, the compound of the above specific example (1) produces a dye of the following formula.

Pigments with corresponding structures are also produced for other compounds.

The compound of the present invention can be used to measure amylase activity, for example, by the reaction shown below.

1> 4-methoxy-1-naphtholmaltopentaose-1-8,0 amylase → 4-methoxy to 1-naphtholmaltoglucoside ten maltotriose 1) 4-methoxy-1-f phtholmaltoglucoside +
H20 glucosidase 4-methoxy-1-naphtho-fluoryl glucose oxidation iii>4-methoxy-]-naphtho-1-→ Dimeric pigment, i.e., amylase, hydrolyzes the substrate to produce 4-methoxy-1- It becomes naphthol maltoglucoside and is further hydrolyzed by a second enzyme (α- or β-glucosidase).

The liberated 4-methoxy-I-naphthol undergoes self-coupling in the presence of an oxidizing agent to produce a blue pigment,
The rate of pigment production corresponds to enzyme activity. Suitable oxidizing agents are, for example, ferric compounds such as ferric thiocyanate, ferric stannate, potassium ferricyanide, or transition metal compounds with high valence.

Examples of methods for producing typical compounds of the present invention are shown below, and other compounds can be easily synthesized according to the examples.

[Example 1] Process for producing compound PA (1) (A) Synthesis of heptadecaacetyl maltopentaose 20 g of maltopentaose, 11.260 sj of acid anhydride! A mixture of 20 g of anhydrous sodium acetate was stirred at 100 to 110° C. for 5 hours, poured into 600 mff1 of ice water, and stirred for 10 hours. The crystals were cooled to below 5° C. to crystallize, P removed, washed with water, and dried. The obtained crystals were recrystallized twice from ethanol to obtain 35 g of the target product corresponding to the following structural formula.

Melting point 123 to 127°C <However, Y does not represent COCH3> (B) Synthesis of hexadecaacetylmaltopentaosyl chloride Heptadecaacetylmaltopentaose Log obtained in the above (A), dehydrated chloroform 50-1 and 6 g of titanium chloride was reacted with reflux stirring for 1#. After the reaction, 300 chloroform was added, washed 3 times with 100 ml of water, the chloroform phase was separated, and then 30 g of anhydrous sodium sulfate was added to dehydrate. , anhydrous sodium 1ijE acid was removed by filtration. After concentrating and drying under reduced pressure, the mixture was heated and dissolved in 20 ml of methanol, cooled with water, and crystallized.

Remove methanol by decantation and ethanol Lo
When the mixture of a1 and n-hexane (20 ml) is heated and dissolved, and the resulting solution is cooled with water using a stirrer, white crystals are precipitated. The crystals were collected, washed with n-hexane, and dried. 8.1 g of white crystals corresponding to the following structural formula were obtained. Melting point 1
23-129℃ (Y represents COCH3) Elemental valve N value: C5zHli (CHC1 as L+CJ Theoretical value (%) 49.00 5.56 2.23 Actual value (%) 4B, 70 5.40 2.18 ( C
) Synthesis of Compound Example (1) 2 g of hexadecaacetyl maltopentao silchloride obtained in (B) above, 8.7 g of 4-methoxy-1-naphthol, and 5.05 g of triethylamine were dissolved in 30 ml of dehydrated dimethylformamide. After the reaction was completed, 200 ml of ethyl acetate was added, washed with saturated brine, and dehydrated with magnesium sulfate.
Silica gel column chromatography, evaporating to dryness under reduced pressure (eluent: 1:5 mixture of ethyl acetate and n-hexane)
Purification was performed to obtain 1.5 g of a pale yellow product.

1 g of this crude product was dissolved in a mixture of 2 m of dehydrated methanol and 7 m of dehydrated dichloromethane, and further added 0.0 m of this crude product.
Add 5N sodium methoxide, stir at room temperature, and react for 10 hours. After the reaction, 1 of the precipitate that did not precipitate was collected, and a mixture of dehydrated methanol and dichloromethane (1:1
), and then dichloromethane, and dried under reduced pressure to obtain 0.31 g of 11I crystals of the target compound. This crude product was mixed with water and Bio-Rad Laboratories.
It was purified by column chromatography using B1o-Ge1 P-2 manufactured by Atories Ltd. to obtain 0.28 g of the target compound (t).

Melting point: 193-195°C Next, an example of a multilayer analytical element and a method for measuring amylase activity using the above azo dye glycoside will be shown.

[Example 3] An aqueous solution having the following composition (a) was applied onto a 180 μm thick colorless transparent smooth film of polyethylene terephthalate that was undercoated with gelatin so that the thickness after drying was 7 μm and dried.

(a) Gelatin 300g Surfactant 5g (Olin Co., Ltd. 5ur4actanL 10G) Bolico (
Styrene-tomethylmorpholinium (methylstyrene-divinylbenzene) Polymerization ratio 55.43:2 15% latex solution 280g Potassium ferricyanide 5g Water
2150g
(The pH was adjusted to 7.0 with a dilute NaOH solution.) Next, an aqueous solution having the following composition (b) was applied onto the gelatin layer so that the thickness after drying was 5 μm, and then dried.

(b) Gelatin 20
0g surfactant 5g (
Orin 5ur4acLant 10 G > β-glucosidase 3.5 million I [J water
2600 g (adjust p+-1 to 7.0 with dilute NaOH solution) Next, an aqueous solution having the following composition (c) was applied onto the gelatin layer so that the thickness after drying was 3 μm, and dried.

(c) Gelatin 30g surfactant
4g (Olin 5urfac
tant IOG) Titanium oxide (anatase type)
20g water 950
g (Adjust the pH to 7.0 with dilute NaOH solution) Next, water is uniformly supplied to the entire surface at a rate of about 30 g/m2 on top of the titanium oxide/gelatin layer to moisten it, and then Tricot knits (40 gauge polyester) were laminated with light pressure and allowed to dry.

Next, 150 cc of an aqueous solution of the following composition (d) was applied to this cloth.
/m'' and dried to produce a multilayer analytical element for measuring amylase.

(d) Compound example (1) 5g water
1600g potassium phosphate 60g polyvinylpyrrolidone
140g (average molecular weight 700,000) (adjust pH to 7.3 with dilute NaOH solution) 1st
As is clear from Table 1, amylase activity in control serum could be measured with high accuracy using the analytical element of the present invention.

Claims (1)

[Claims] (1) A compound represented by the following general formula [I]: [I] ▲There are mathematical formulas, chemical formulas, tables, etc.▼ {However, in the formula, A is 1, where the 1st position is bonded to an oligosaccharide, 4-
It represents a naphthylene group, and R^1 represents a lower alkyl group (which may be substituted). n represents an integer up to 0 or 8. } (2) The compound according to claim (1), wherein n in general formula (I) represents 3, 4 or 5. (3) The oligosaccharide represented by the following general formula [Ia] [
I a] ▲Mathematical formulas, chemical formulas, tables, etc. By halogenating the compound that is
b] ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (However, Y represents COR) As a compound represented by the following formula [I c], [I c] ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (However, X is (Represents halogen) This is treated with a compound represented by the following formula [I d] to form [I d] HO-A-OR^1 {However, in the formula, A is 1,4 where the 1-position is bonded to the hydroxy group.
- represents a naphthylene group, and R^1 represents a lower alkyl group (which may be substituted). . } A compound represented by the following formula [ I e] is obtained, [ I e] ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ The general formula [ I
] A method for producing a compound represented by. [I] ▲There are mathematical formulas, chemical formulas, tables, etc.▼ {However, in each formula, n represents an integer up to 0 or 8. }
(4) Claim (1) or (4) as a substrate for amylase
2) Use of the compound. (5) A method for detecting or measuring amylase activity using the compound of claim (1) or (2) as a substrate. (6) The detection or activity measurement method according to claim (5), which is carried out in the presence of α-glucosidase and/or β-glucosidase. (7) Claim (5), wherein a blue dye produced from a 4-alkoxynaphthol compound is optically detected in the presence of an oxidizing agent.
Detection or activity measurement method.