JP3029925B2 - Maltooligosaccharide derivatives and their production - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel maltooligosaccharide derivative, a method for producing the same, and a reagent for measuring α-amylase activity using the maltooligosaccharide derivative as a substrate.

[0002]

2. Description of the Related Art Various diseases are diagnosed by measuring the activity of α-amylase contained in body fluids such as pancreatic juice and urine. As a method for measuring the activity of α-amylase, for example, there is a method using maltooligosaccharide (maltotetraose, maltopentaose, maltohexaose, etc.) as a substrate. In this method, the maltooligosaccharide and α-glucosidase are allowed to act on a sample containing α-amylase to release glucose from a substrate, and the amount of glucose is measured to determine the activity value of α-amylase. The produced glucose is measured, for example, by a quantitative method using a glucose oxidase / peroxidase / dye system: a measuring method using a hexokinase / phosphoglucomutase / glucose-6-phosphate dehydrogenase / NADH system.

[0003] α-Glucosidase hardly acts on oligosaccharides such as maltopentaose or other tetrasaccharides and works well on oligosaccharides such as maltose or maltotriose such as trisaccharides or less. By measuring, the activity of α-amylase can be measured. However, since α-glucosidase acts on maltopentaose, which is a substrate, albeit slightly, the blank value of the measurement increases, and as a result, the error of the measurement value increases. Due to the decomposing action of α-glucosidase on the substrate, it is not preferable to combine α-glucosidase and the substrate in one solution because the stability as a reagent is impaired.

[0004] A method using a substrate in which a phenyl group, a naphthyl group, or a derivative thereof is bonded as an aglycone to the reducing end of maltooligosaccharide has also been proposed. For example, p-nitrophenylmaltopentaoside (JP-B-57-53079), p-nitrophenylmaltohexaoside (JP-B-57-53079), and p-nitrophenylmaltoheptaoside (JP-B-57-53079) are used as substrates. JP-B-62-50119), 2,4-dichlorophenylmaltopentaoside (JP-B-59-13199)
Etc. are used. When these substrates are used, aglycone is released, and the activity of α-amylase can be easily measured by optically measuring the released aglycone, for example, p-nitrophenol. Also in the above method, since α-glucosidase slightly acts on the substrate, there is a disadvantage that the blank value increases.
Due to the substrate-decomposing action of α-glucosidase, it is difficult to make α-glucosidase and the substrate into one liquid as in the method of measuring glucose.

[0005] In order to solve such disadvantages, there has been proposed a method using a type of substrate in which the hydroxyl group at the 6-position of glucose at the non-reducing terminal of maltooligosaccharide is modified. For example in JP 60-237998 JP OH group at the 6-position of non-reducing terminal glucose, for example, a halogen _{atom, -OR, -OCOR, -OSO 2 R} -, - N
HR (R is an alkyl group, phenyl group, pyridyl group, etc.)
And a substituted or unsubstituted phenyl group bonded to a reducing terminal glucose as an aglycone is disclosed as a substrate. When the OH group at the 6-position of the non-reducing terminal glucose is substituted, the decomposition of the substrate by α-glucosidase does not occur. However, such a substrate having a substituent introduced only at the 6-position has the drawback that chemical synthesis is difficult and the yield is poor.

JP-A-60-54395 discloses that the OH groups at the 4- and 6-positions of glucose at the non-reducing terminal are substituted with an alkyl group, an alcoyl group or a phenyl group, and aglycone is added to glucose at the reducing terminal. The use of bound maltooligosaccharides as substrates is disclosed. If the OH groups at the 4- and 6-positions of the non-reducing terminal glucose are substituted, the decomposition of the substrate by α-glucosidase is unlikely to occur. However, since such a substrate is substituted with two OH groups, it has poor water solubility, making it impossible to prepare a high-concentration solution, and cannot dissolve a sufficient concentration for α-amylase activity measurement. There is.

[0007] To improve this disadvantage, 3-ketobutylidene group, 2-ketobutylidene group, 2-ketopropylidene group, 4-ketopentylidene group, methylsulfinylethylidene group, ethylsulfinylethylidene group, methanesulfonylethylidene group, ethanesulfonylethylidene group A method using a substrate having a highly water-soluble modifying group introduced into a non-reducing terminal glucose such as described above has been proposed (JP-A-1-157996). However, although these substrates are excellent in water solubility, they have a drawback that they do not reflect the mode of action of α-amylase which recognizes glucose chains such as starch and amylose and cleaves the bonds.

On the other hand, a method using a substrate in which galactose is β-linked to non-reducing terminal glucose of p-nitrophenyl α-maltopentaoside has been reported (Abstracts of the Japanese Society of Agricultural Chemistry, Vol. 65, No. No. 3, 117
1991, and JP-A-3-264596). However, when this substrate is used, the molar extinction coefficient (hereinafter referred to as ε) of the α-amylase activity of the p-nitrophenol used as the color-forming group near the measurement range (pH 7.0) is only about half of the maximum ε, In addition, ε greatly changes due to slight pH fluctuation, temperature,
When the amount of sodium chloride or albumin changes, ε also fluctuates, so that there is a problem that a measurement error occurs, and there is a disadvantage that the sensitivity when used as a reagent for measuring amylase activity is not sufficient.

[0009]

SUMMARY OF THE INVENTION The present invention relates to the conventional α
-It is intended to solve the drawbacks of the amylase activity measurement substrate, the conventional α-amylase activity measurement reagent, and the like. A novel maltooligosaccharide derivative as a substrate which is not affected by an enzyme, is easily synthesized, has excellent water solubility, and reflects the mode of action of amylase more purely, and a method for producing the same. An object of the present invention is to provide a reagent for accurately measuring α-amylase activity in a body fluid by a simple operation using a maltooligosaccharide derivative as a substrate.

[0010]

Means for Solving the Problems The present invention has the following gist.

(1) General formula (I)

[0012]

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(Wherein R ₁ represents hydrogen, R ₂ represents a β -galactopyranosyl group, n represents 2 , and a wavy line represents a β bond ). .

(2) General formula (I):

[0015]

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(Wherein R ₁ represents hydrogen, R ₂ represents a β -galactopyranosyl group, n represents 2 and a wavy line represents a β bond ). And a reagent for measuring α-amylase activity.

(3) General formula (II):

[0018]

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Wherein n represents an integer of 1 to 7; and a maltooligosaccharide derivative represented by the following general formula (I) is reacted with lactose in the presence of β-galactosidase:

[0020]

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(Wherein, R ₁ represents hydrogen, R ₂ represents a β -galactopyranosyl group, n represents 2, and a wavy line represents a β bond ).
A method for producing a maltooligosaccharide derivative represented by the formula:

(4) General formula (III) or general formula (IV):

[0023]

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[0024]

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In the method for producing a maltooligosaccharide derivative represented by the formula:
(V) or general formula (VI):

[0026]

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[0027]

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(Wherein n represents an integer of 1 to 7), and the acetylation is carried out by heating together with an alkali and acetic anhydride in the presence of an organic medium or in the absence of a solvent in the presence of an organic medium. Process, the resulting general formula (VI
I) or general formula (VIII):

[0029]

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[0030]

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(Wherein, n represents an integer of 1 to 7; Ac represents an acetyl group). An acetylated galactosyl maltooligosaccharide and 2-chloro-4-nitrophenol in an organic solvent are acid-catalyzed. And a step of removing an acetyl group of the obtained reaction product, followed by heating.

(5) General formula (III) or general formula (IV):

[0033]

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[0034]

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(Wherein, n represents an integer of 1 to 7), wherein maltooligosaccharide and lactose are reacted in the presence of β-galactosidase. (V) or the general formula (V
I):

[0036]

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[0037]

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(Wherein n represents an integer of 1 to 7), and acetylated by heating together with alkali and acetic anhydride in the presence of an organic medium or in the absence of a solvent. Process, the resulting general formula (VI
I) or general formula (VIII):

[0039]

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[0040]

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(Wherein n represents an integer of 1 to 7; Ac represents an acetyl group). An acetylated galactosylmaltooligosaccharide represented by the following formula: and 2-chloro-4-nitrophenol are acid-catalyzed in an organic solvent. And a step of removing an acetyl group of the obtained reaction product, followed by heating.

In the present specification, maltooligosaccharide derivative (I) [hereinafter referred to as maltooligosaccharide derivative (I) including maltooligosaccharide derivatives represented by general formulas (III) and (IV)]. The maltooligosaccharide as the skeleton is 3 to
It is formed from 10 sugars (corresponding to n = 1 to 7 in formula (I)). Examples of the maltooligosaccharide include maltotriose, maltotetraose, maltopentaose, maltohexaose, and maltoheptaose, and maltotetraose, maltopentaose, maltohexaose, and maltoheptaose are particularly preferable. Among these, maltooligosaccharides having a maltotetraose skeleton, pancreatic α- amylase, water-decomposable cut part by saliva from α- amylase is one place, the reaction mechanism is clear, pancreatic α- amylase, salivary There is no difference in the substrate hydrolyzing action of the derived α-amylase.

Galactose, which is a modifying group of the non-reducing terminal glucose of maltooligosaccharide, is bound to the hydroxyl group at the 4- or 6-position of the non-reducing terminal glucose in β form.

The maltooligosaccharide derivative (I) of the present invention is a novel compound, and the maltooligosaccharide derivative (II) whose reducing end is substituted by 2-chloro-4-nitrophenol
And lactose in the presence of β-galactosidase. The reaction is carried out at a pH of 3 to 10, preferably 5 to 8 and a temperature of 0 to 80 ° C, preferably 30 to 50 ° C.
C. for 1 minute or more, preferably for 20 to 120 minutes. Next, after stopping the enzymatic reaction, the reaction solution is fractionated by preparative HPLC or the like, and purified to obtain the maltooligosaccharide derivative (I) of the present invention. The origin of β-galactosidase used here is not particularly limited, and any of those obtained from animals, plants, microorganisms and the like can be used.

The maltooligosaccharide derivative (I) of the present invention
Can also be synthesized by reacting a maltooligosaccharide derivative whose non-reducing terminal glucose is galactose-modified with 2-chloro-4-nitrophenol.

In the reaction of galactosyl maltooligosaccharide whose non-reducing terminal is modified with galactose and 2-chloro-4-nitrophenol, the galactosyl maltooligosaccharide is first acetylated by a conventional method, and the acetylated galactosyl maltooligosaccharide is reacted with 2 The reaction can be carried out by reacting -chloro-4-nitrophenol with, for example, zinc chloride in a boiling water bath for about 1 hour.
Thereafter, the mixture is cooled, extracted with benzene, washed with water, 5% sodium hydroxide and water in that order, dried, and recrystallized with anhydrous alcohol. Then, it is dissolved in anhydrous methanol, and sodium methoxide is added thereto. The mixture is de-acetylated by boiling it slightly, so that the solvent is distilled off and recrystallized from hot water to obtain the maltooligosaccharide derivative (I) of the present invention. .

Specifically, the following method can be used. The galactosyl maltooligosaccharide represented by the general formula (V) or the general formula (VI) is acetylated by heating with an alkali and acetic anhydride in the presence or absence of an organic medium. By the reaction, the compound represented by the general formula (VII) or (VII
An acetylated galactosyl maltooligosaccharide represented by I) is obtained.

As the organic medium in this case, methylene chloride, chloroform, benzene, toluene, ether, ethyl acetate, DMF, DMSO and the like are used. As the alkali, sodium acetate, triethylamine, pyridine and the like are used. The conditions of the acetylation reaction are room temperature to 150 ° C., preferably when sodium acetate is used, 90 to 120 ° C., when pyridine is used,
Room temperature to 60 ° C.

Next, the obtained general formula (VII) or general formula
The acetylated galactosyl maltooligosaccharide represented by (VIII) is heated together with 2-chloro-4-nitrophenol in an organic solvent in the presence of an acid catalyst. By the reaction, 2-
A reaction product in which a chloro-4-nitrophenyl group is α- or β-linked to reducing terminal glucose (2-chloro-nitrophenyl group)
It is also referred to as a protected 4-nitrophenyl (CNP-modified) galactosylmaltooligosaccharide. ) Is obtained.

In this case, methylene chloride, chloroform, benzene, toluene, ether, ethyl acetate and the like are used as the organic solvent. As the acid catalyst,
Lewis acids such as ZnCl ₂ , SnCl ₄ and TiCl ₄ or organic sulfonic acids such as p-toluenesulfonic acid and methanesulfonic acid are used. The above reaction conditions are room temperature to 200 ° C, preferably 30 to 120 ° C. The 2-chloro-4-nitrophenyl group can be α-bonded or β-bonded depending on the reaction conditions.

In the present invention, the acetyl group of the reaction product obtained in the above step is further eliminated by a known deprotection reaction with a catalytic amount of sodium methylate in methanol to give a compound represented by the general formula (III) or ( A maltooligosaccharide derivative represented by IV) is obtained. As another elimination reaction, a method using triethylamine in anhydrous methanol, an anion exchange resin or anhydrous methanol is exemplified.

The galactosyl maltooligosaccharide represented by the general formula (V) or (VI) used in the above reaction is, for example, a maltooligosaccharide having 2 to 9 glucoses such as maltotetraose and maltopentaose. Raw materials,
It can be obtained by introducing a galactosyl group that is cleaved as a result of a β-galactosidase reaction using lactose as a substrate at the 4- or 6-position of the non-reducing terminal glucose of maltooligosaccharide.

The conditions for using β-galactosidase are as follows:
In a buffer of pH 5 to 9, preferably in a buffer of pH 6 to 8, the temperature is 15 to 60 ° C, and the reaction time is about 10 to 100 hours. Depending on the reaction conditions, β-
A galactosyl group can be introduced.

The reagent for measuring α-amylase activity of the present invention contains the above maltooligosaccharide derivative (I) as a substrate. Further, usually, the reagent may further contain an enzyme system (following enzyme system) in which α-glucosidase, glucoamylase and β-glucosidase are appropriately combined, and if necessary, other additives.

In the reagent for measuring α-amylase activity according to the present invention, the glycosidic bond between the 2-chloro-4-nitrophenyl group and the glucose chain may be either α-type or β-type. Beta-linked bonds are more advantageous in formulating a substrate because they are more soluble in water.

The origin of α-glucosidase used in the reagent for measuring α-amylase activity of the present invention is not particularly limited. Α- obtained from animals, plants, microorganisms, etc.
Glucosidase may be utilized. In particular, α-
Glucosidase can be suitably used because it acts well on maltotrioside and lower glycosides, hardly acts on maltotetraoside and higher glycosides, and has a wide specificity of aglycone.

The origin of β-glucosidase and glucoamylase used in the reagent for measuring α-amylase activity of the present invention is not particularly limited. For example, β-glucosidase obtained from almond and glucoamylase obtained from Rhiz. Delemar can be suitably used.

The measurement of α-amylase activity using the reagent for measuring α-amylase activity of the present invention is performed, for example, as follows. That is, a sample containing α-amylase is allowed to act on the reagent for measuring α-amylase activity.

The following enzyme systems include α-glucosidase and / or glucoamylase and, if necessary, β-glucosidase.
Glucosidase is used.

The reaction formula for the decomposition of the substrate in the measuring method using the reagent for measuring α-amylase activity of the present invention is represented by 2-chloro-4-nitrophenyl 4-O-β-D-galactopyranosyl-β-malto The case where pentaoside is used as a substrate will be described as an example.

[0061]

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The activity of α-amylase can be measured by measuring 2-chloro-4-nitrophenol released by the above reaction by an appropriate means. If the released 2-chloro-4-nitrophenol exhibits a different spectral absorption than the substrate, the spectrum of the reaction mixture is measured directly.

As a method for measuring 2-chloro-4-nitrophenol, both a rate method for continuously following the reaction of α-amylase and an end point method for measuring after stopping the reaction after reacting for a certain period of time. Can be used.

During the enzymatic reaction, glucoamylase has almost the same function as α-glucosidase. However, α-glucosidase works well on low molecular weight glycosides below maltotrioside, but hardly works on glycosides above maltotetraoside, whereas glucoamylase also works on glycosides above maltotetraoside. Works. For example, when a high molecular glycoside of maltoheptaoside or higher is used as a substrate, maltotetraoside or higher glycoside may be produced by the action of α-glucosidase. Such glycosides of maltotetraoside or higher are not easily decomposed by α-glucosidase, but are easily decomposed into glucose units by using glucoamylase. For this reason, there is an advantage that the sensitivity of the measurement system is increased. Therefore, a follower enzyme system in which α-glucosidase and glucoamylase coexist and β-glucosidase is used together if necessary is preferably used.

[0065]

According to the reagent of the present invention, α- or β-maltooligoside in which the OH group at the 4- or 6-position of the non-reducing terminal glucose is modified with galactose, that is, the maltooligosaccharide derivative (I) is used as a substrate. Α-
Even if a reagent in which the following enzyme system such as glucosidase or glucoamylase and the substrate are liquefied is prepared and stored, these enzymes hardly decompose the substrate. Therefore, an increase in the reagent blank value is suppressed, and the α-amylase activity can be accurately measured. The maltooligosaccharide derivative (I), which is a substrate of the α-amylase activity measuring reagent of the present invention, has a water-soluble group in which galactose bonded to the hydroxyl group at the 4- or 6-position of non-reducing terminal glucose,
Excellent solubility in water. In addition, the substrate of the present invention can be provided at low cost because it can be selectively synthesized with high yield using an enzyme. Furthermore, since the maltooligosaccharide derivative (I) which is a substrate of the reagent for measuring α-amylase activity of the present invention has a non-reducing terminal modifying group of galactose, the mode of action of α-amylase on the derivative is starch or Maltooligosaccharide derivatives in which the α-amylase which recognizes a glucose chain such as amylose and cleaves the bond is reflected in a state close to the original mode of action, in which the OH group at the 4-position of the non-reducing terminal glucose is modified with galactose. When is used as a substrate, α-amylase has a higher reactivity than a maltooligosaccharide derivative in which the OH group at the 6-position is modified with galactose, which is preferable. Further, the color-forming group released from the maltooligosaccharide derivative (I) of the present invention is 2-chloro-4-nitrophenol, which is an α-amylase measurement region, pH7, which has been a problem with p-nitrophenol generated from a conventional substrate. Problems such as measurement errors due to fluctuations in sensitivity, pH, sodium chloride concentration, albumin concentration, etc. around 0.0 are eliminated.

According to the reagent of the present invention, continuous measurement using an automatic analyzer is possible, and α-amylase activity can be measured simply and inexpensively.

In the method for producing the maltooligosaccharide derivative (I), galactosylmaltooligosaccharide is synthesized by using an enzyme reaction of β-galactosidase using lactose as a substrate as described in the present invention, using p-nitrophenylmaltopendaoside as a starting material. The side reaction in which two galactosyl groups are introduced is far less than that of the conventional reaction. Further, the yield of the resulting galactosylmaltooligosaccharide is also greatly increased, and it is easy to separate and purify it from by-products, unlike the conventional method.
Furthermore, in the subsequent acetylation reaction, there are few side reactions,
The resulting 2-chloro-4-nitrophenyl of the present invention (C
Since the protected (NP-modified) galactosylmaltooligosaccharide is separated in polarity from the unreacted raw material, it is easily purified and sufficiently purified by one silica gel column chromatography. In the present invention, a maltooligosaccharide in which a galactosyl group is β-linked to the 4- or 6-position of the non-reducing terminal glucose
By using (V) or (VI) as a raw material, the yield of the maltooligosaccharide derivative (I) was increased, and the difficulty of purification due to mixing of by-products or unreacted raw materials was eliminated.

That is, the maltooligosaccharide derivative (I) obtained according to the present invention comprises a raw material (V) or (VI) obtained by introducing a galactosyl group at the 4- or 6-position of the non-reducing terminal glucose of maltooligosaccharide with β-galactosidase. A method in which a hydroxyl group is protected by an acetyl group in a first step, and 2-chloro-4-nitrophenol is allowed to act in a second step, and then the acetyl group is eliminated to obtain a target product, This is a production method with a high yield with few by-products difficult to purify and remove, which has been a problem in the conventional method.

[0069]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples, but it goes without saying that the present invention is not limited to these Examples.

Example 1 (Synthesis example) Production example of 2-chloro-4-nitrophenyl 4-O-β-D-galactopyranosyl-β-maltopentaoside:
1 g of 2-chloro-4-nitrophenyl-β-maltopentaoside and 1 g of lactose were dissolved in 5 ml of 50 mM acetate buffer (pH 6.0). Bacilluscirculan
s-derived β-galactosidase solution (65 u / ml) 25
0 μl was added, and the mixture was incubated at 40 ° C. for 90 minutes. The enzyme reaction was stopped by treating at 100 ° C. for 10 minutes. This enzyme reaction solution was purified by preparative HPLC. FIG. 1 is a chart of analytical HPLC before purification of the enzyme reaction solution. Purification by preparative HPLC was performed by collecting fractions corresponding to the peak at a retention time of 27.24 minutes in analytical HPLC in FIG. 1, distilling off methanol under reduced pressure with a rotary evaporator, and freeze-drying the remaining aqueous solution. A colorless or pale yellow powder was obtained.

The conditions for preparative and analytical HPLC are shown below. (Preparation conditions) Column: SH-345-5 S-5 120A AQ (22 × 500 mm) Elution solvent: methanol / water = 20/80 Flow rate: 7 ml / min Column temperature: 45 ° C. (Analysis conditions) Column: ODS- AQ (4.6 × 250mm) Elution solvent: methanol / water = 20/80 Flow rate: 0.5 ml / min Column temperature: 45 ° C.

500 MHz ¹ H-NMR; δ value (split type, relative proton number) 3.5 to 4.0 (multiple line, 36) 4.453 (double line, 1) Note: Anomer hydrogen of β-galactosyl group 5.343 (double line , 1) Note: Anomer hydrogen of β-glucosyl group 5.385 (double line, 1) Note: Anomer hydrogen of α-glucosyl group 5.389 (double line, 1) Note: Anomer hydrogen of α-glucosyl group 5.393 (double Line, 1) Note: Anomer hydrogen of α-glucosyl group 5.445 (double line, 1) Note: Anomer hydrogen of α-glucosyl group 7.42 (double line, 1) 8.24 (double double line, 1) 8.43 (double line) Heavy line, 1)

Example 2 (Synthesis Example) Production Example of 2-chloro-4-nitrophenyl 6-O-β-D-galactopyranosyl-β-maltopentaoside:
1 g of 2-chloro-4-nitrophenyl-β-maltopentaoside and 1 g of lactose were dissolved in 5 ml of 50 mM acetate buffer (pH 6.0). Bacilluscirculan
β-galactosidase solution (195u / ml) 2 derived from s
50 μl was added, and the mixture was incubated at 40 ° C. for 90 minutes. The enzyme reaction was stopped by treating at 100 ° C. for 10 minutes.
This enzyme reaction solution was purified by preparative HPLC. FIG.
It is a chart of the analytical HPLC before refinement | purification of the said enzyme reaction liquid. Purification by preparative HPLC was carried out by collecting fractions corresponding to the peak at a retention time of 24.29 min in analytical HPLC in FIG. 2, distilling off methanol under reduced pressure using a rotary evaporator, and freeze-drying the remaining aqueous solution. A colorless or pale yellow powder was obtained.

The conditions for preparative and analytical HPLC are shown below. (Preparation conditions) Column: SH-345-5 S-5 120A AQ (22 × 500 mm) Elution solvent: methanol / water = 20/80 Flow rate: 7 ml / min Column temperature: 45 ° C. (Analysis conditions) Column: ODS- AQ (4.6 × 250mm) Elution solvent: methanol / water = 20/80 Flow rate: 0.6 ml / min Column temperature: 45 ° C.

500 MHz ¹ H-NMR; δ value (split type, relative proton number) 3.5 to 4.0 (multiple line, 35) 4.20 (double double line, 1) Note: 6th position due to geminal coupling Since it is one of the hydrogen atoms and has a lower magnetic field shift than usual, it can be proved that β (1-6) bond is formed. The other hydrogen at the 6-position was assigned to a δ value of 3.88 from a COSY spectrum experiment. 4.424 (double line, 1) Note: Anomer hydrogen of β-galactosyl group 5.342 (double line, 1) Note: Anomer hydrogen of β-glucosyl group 5.366 (double line, 1) Note: α-glucosyl group Anomer hydrogen 5.373 (double line, 1) Note: Anomer hydrogen of α-glucosyl group 5.389 (double line, 1) Note: Anomer hydrogen of α-glucosyl group 5.445 (double line, 1) Note: α-glucosyl group Anomer of hydrogen 7.42 (double line, 1) 8.24 (double double line, 1) 8.43 (double line, 1)

Example 3 Using the maltooligosaccharide derivative (described in Table 1) obtained in Examples 1 and 2 as a substrate, an α having the following composition
-Each amylase activity measuring reagent was prepared. Reagent composition A: 50 mM Good buffer (pH 7.0) α-glucosidase 90 u / ml β-glucosidase 12 u / ml CaCl ₂ 1 mM substrate 2 mM Reagent composition B: 50 mM good buffer (pH 7.0) glucoamylase 2 u / ml α-glucosidase 90 u / Ml β-glucosidase 12u / ml CaCl ₂ 1mM substrate 2mM The same reagent as described above was prepared using the substrates shown in Table 1 as Comparative Examples 1, 2 and 3.

[0077]

[Table 1]

Experimental Example 1 Three kinds of serums 1, 2 and 3 were added to 3 ml of each reagent shown in Table 1 in an amount of 0.25 ml, and the mixture was allowed to stand at 37 ° C. for 3 minutes. Then, the change in absorbance at 415 nm was measured. The change in absorbance per minute was calculated. The results are shown in Table 2. Table 2 also shows the change in absorbance per minute of the reagent blank.

[0079]

[Table 2]

As is evident from the results shown in Table 2, the reagent of the present invention has a very low change in absorbance per minute in the reagent blank as compared with the comparative examples, particularly comparative examples 1a and 1b, and the maltooligosaccharide of the present invention. It can be seen that the non-reducing end of the derivative is blocked and thus is not affected by the following enzyme. Further, since the measurement sensitivity of the reagent of the present invention is higher than that of Comparative Examples 2a and 2b, the maltooligosaccharide derivative of the present invention is 3-ketobutylidene 2-
It can be seen that the reactivity to amylase is higher than that of chloro-4-nitrophenyl β-maltopentaoside,
This is because the non-reducing end of the maltooligosaccharide derivative of the present invention is galactose closer to the more natural substrate,
-It is believed that it more purely reflects the mode of action of amylase.

Experimental Example 2 To examine the suitability of preparing a substrate formulation, the water solubility of each substrate shown in Table 3 below was examined. In the reagent composition A shown in Example 3, each substrate in Table 3 was dissolved so that the concentration of the substrate was 100 mM. Table 3 shows the results.

[0082]

[Table 3]

From the above results, it is understood that the maltooligosaccharide derivative of the present invention has excellent water solubility and is suitable for preparing a substrate.

In the following Example 4, a compound in which β-galactose is 1,4-linked is shown, but a compound in which 1,6-linkage is obtained depending on the reaction conditions.

Example 4 (A) Production of β- (1,4) -galactosylmaltotetraose 1 g of maltotetraose and 1 g of lactose were mixed with 50 m
It was dissolved in 5 ml of M acetate buffer (pH 6.0). 250 μl of a β-galactosidase solution (65 units / ml) derived from Bacillus circulans was added, and the mixture was incubated at 40 ° C. for 90 minutes. The enzyme reaction was stopped by treating at 100 ° C. for 10 minutes. This enzyme reaction solution is
After purification on a Toyopearl HW-40S column (eluent:
(Water, flow rate: 1.4 ml / min) Lyophilized to obtain 250 mg of a white powder. The reaction product obtained is β- (1,4)
-Galactosyl maltotetraose (general formula (VI), n =
2,100%). FIG. 3 shows the results of HPLC analysis of the obtained β- (1,4) -galactosylmaltotetraose. FIG. 3 shows that the reducing terminal glucose is α-D-
A compound that is glucopyranose and a compound that is β-D-glucopyranose are shown, and it is clear that there are no two galactosyl groups. The analysis conditions were as follows: column: YMC-PACK AQ-303 4.6 × 25 cm, eluent: water, flow rate:
1.0 ml / min, temperature: room temperature, detector: RI.

(B) Production of protected β- (1,4) -galactosylmaltotetraose 1 g of β- (1,4) -galactosylmaltotetraoside and 200 mg of sodium acetate obtained in the above step were treated with acetic anhydride. And acetylated by heating at 100 ° C to 110 ° C for 5 hours. After cooling, it was poured into cold water and left overnight. The precipitated crystals were filtered off with suction and dried well under vacuum. Yield about 100%. 200 MHz ¹ H-NMR: δCDCl ₃ (split type, relative proton number) 1.95 to 2.35 (multiple line, 51H) Note: acetyl group 3.68 to 5.5 (multiple line, 34H) 5.76 ( Double line, 1H) Note: J = 8.1 Hz Anomer hydrogen at the non-reducing end The reaction product obtained by the reaction is β- (1,4)-
Protected galactosyl maltotetraose (general formula VIII,
n = 2, 100%).

(C) Production of protected 2-chloro-4-nitrophenyl-β (1,4) -galactosylmaltotetraose Protected β- (1,4) -galactosylmaltotetraose obtained in the above step 1 g was dissolved in 5 ml of methylene chloride, and 600 mg of SnCl ₄ was added. 2-chloro-4-
Add 500 mg of nitrophenol, and add 1
Refluxed for 0 hours. After cooling, the mixture was extracted with trichloromethane and concentrated to dryness, and then purified by silica gel column chromatography using hexane / ethyl acetate (6: 4) as an eluent to obtain 350 mg of a white powder. 200 MHz ¹ H-NMR: δ (split type, relative proton number) 1.95 to 2.20 (multiple line, 48H) Note: acetyl group 3.68 to 5.50 (multiple line, 35H) 7.29 (two Note: J = 8.1 Hz Ring proton of CNP 8.17 (double double line, 1H) Same as above 8.31 (double line, 1H) Same as above The product obtained by the reaction was 2 -Chloro-4-nitrophenyl-β (1,4) -galactosylmaltotetraose (CNP-protected galactosylmalto-oligosaccharide, β-bonded with CNP group, 100%).
The CNP-protected galactosylmaltooligosaccharide had few side reactions and was separated from unreacted raw materials in polarity, so that it was easy to purify and one purification by silica gel column chromatography was sufficient.

(D) Preparation of 2-chloro-4-nitrophenyl-β (1,4) -galactosylmaltotetraose 2-chloro-4-nitrophenyl-β obtained above
(1,4) -galactosylmaltotetraose protected 1
g was dissolved in 10 ml of anhydrous methanol. 5 mg of sodium methylate was added and left overnight. 1 ml of acetic acid was added for neutralization, and the mixture was concentrated using a rotary evaporator. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / methanol / water = 8: 2: 1) and freeze-dried to obtain 620 mg of a white powder. 500 MHz ¹ H-NMR: δCDCl _{3 PPM} (split type, relative proton number) 3.5-4.0 (multiple line, 28H) 4.45 (double line, 1H) Note: Anomer hydrogen of β-galactosyl group 5 .34 (double line, 1H) Note: Anomer hydrogen of β-glucosyl group 5.38 (double line, 1H) Note: Anomer hydrogen of α-glucosyl group 5.39 (double line, 1H) Note: α -Anomer hydrogen of glucosyl group 5.45 (double line, 1H) Note: Anomer hydrogen of α-glucosyl group 7.42 (double line, 1H) Ring proton of CNP 8.24 (double double line, 1H) Same as above 8.43 (double line, 1H) Same as above 2-chloro-4-nitrophenyl-β (1,
4) -Galactosyl maltotetraose (general formula (IV):
n = 2, CNP group β-bonded, 100%) HP
The LC analysis is shown in FIG. The analysis conditions are column: TS
K-GEL, NH ₂ -60, solvent: acetonitrile - water (7: 3), flow rate: 1 ml / min, Detector: UV280n
m. FIG. 5 shows a comparison with other maltooligosaccharide derivatives. In FIG. 5, (a) shows 2-chloro-4-nitrophenylmaltotetraose and (b) shows 2-chloro-4-nitrophenylmaltopentaose.

From the above results, according to the production method of the present invention,
Galactosylmaltooligosaccharides were produced with few side reactions in each step, easy purification, and high yield.

Example 5 and Comparative Example 4 Measurement of α-amylase using 2-chloro-4-nitrophenyl-β (1,4) -galactosylmaltotetraose Reagent composition A (Example 5) 50 mM Good buffer Solution (pH 7.0) α-glucosidase 90 u / ml β-glucosidase 12 u / ml CaCl ₂ 1 mM 2-chloro-4-nitrophenyl β (1,4) -galactosylmaltotetraose (hereinafter β (1,4)- 2 mM Reagent Composition B (Comparative Example 4) 50 mM Good Buffer Solution (pH 7.0) α-glucosidase 90 u / ml β-glucosidase 12 u / ml CaCl ₂ 1 mM 3-ketobutylidene-2-chloro-4 -Nitrophenyl-β-maltopene taoside (hereinafter abbreviated as 3-KB-G5-CNP) 2 mM The sample 0.25ml was added to the reagent 3mM, 3
After leaving at 7 ° C. for 3 minutes, the change in absorbance at 415 nm was measured, and the α-amylase activity was calculated from the change in absorbance per minute. In addition, P / S (P-type amylase), which is a ratio of measured values of amylase derived from saliva (hereinafter referred to as P-type amylase) and amylase derived from pancreatic juice (hereinafter referred to as S-type amylase), is used. / S-type amylase).

[0091]

[Table 4]

From the initial absorbance, 3KB-G5-CN
It is clear that the stability is good because a product having a higher purity than P can be produced and the rise of the blank is low.
In addition, the sensitivity of the serum measurement value is good, and the P / S ratio is 3 KB-G
While the value measured using 5-CNP is 0.86,
When β (1,4) -Gal-G4-CNP is used,
0.92, which is close to 1, and is considered to more purely reflect the mode of action of α-amylase.

Example 6 For each of the substrates shown in Table 5, human pancreatic α-
The mechanism of water hydrolysis by amylase and α-amylase derived from human salivary gland was examined under the following conditions.

[0094]

[Table 5]

[Reaction conditions] Substrate 2 mg / ml MES 0.05 M CaCl ₂ 0.4 mM α-amylase 20 U / ml [Analysis conditions] HPLC column TSKgel NH2-60 Eluate CH ₃ CN: water = 7: 3 Flow rate 1 ml / Min Detection wavelength 280nm

The reaction solution 30 minutes after the start of the reaction was added to the column. The residual ratio of the reaction product in the reaction solution was determined from the obtained chromatographic data, and from the result, it was further calculated which portion of each substrate was hydrolyzed by α-amylase and at what ratio. The result is shown in FIG.
Shown in As is clear from the results shown in FIG. 6, the substrate of the comparative example has a plurality of hydrolyzed sites by α-amylase, whereas the substrate of the present invention has one hydrolyzed site. For this reason, the reaction mechanism is simplified, and it has a more reasonable form.

[Brief description of the drawings]

FIG. 1 is an analytical HPLC chart before purification of an enzyme reaction solution in a synthesis example of Example 1.

FIG. 2 is an analytical HPLC chart before purification of an enzyme reaction solution in a synthesis example of Example 2.

FIG. 3 shows the results of HPLC analysis of β (1,4) -galactosylmaltotetraose.

FIG. 4. 2-Chloro-4-nitrophenyl β- (1,
4) shows the HPLC analysis of -galactosylmaltotetraose.

FIG. 5 shows the results of HPLC analysis of other maltooligosaccharide derivatives. (A) shows 2-chloro-4-nitrophenylmaltotetraose and (b) shows 2-chloro-4-nitrophenylmaltopentaose.

FIG. 6 shows the mechanism of water degradation by human pancreatic α-amylase and human salivary gland α-amylase using the maltooligosaccharide derivatives shown in Table 5 as substrates.

──────────────────────────────────────────────────の Continuing on the front page (72) Inventor Nobuhiro Kuwahara 2493-10 Fujisawa, Fujisawa-shi, Kanagawa D-801 Fujisawa D-801 (72) Inventor Takateru Fujita 2-2-1 Konandai, Konan-ku, Yokohama, Kanagawa 72-1006 (72) Inventor Koji Hara Room 2-6-23, Uemachi 2-chome, Izumisano-shi, Osaka (72) Inventor Hajiichi Majima 10-24 Toyo-cho, Tsuruga-shi, Fukui Toyo Boseki Tsuruga Enzyme Plant (72) Invention Person Yoshio Hamada 10-24 Toyo-cho, Tsuruga-shi, Fukui Prefecture Toyobo Co., Ltd., Tsuruga Enzyme Plant (72) Inventor Shinichi Teshima 10-24 Toyo-cho, Tsuruga City, Fukui Prefecture Toyobo Co., Ltd., Tsuruga Enzyme Plant (72) Invention Yuzo Hayashi 2-8-8 Dojimahama, Kita-ku, Osaka-shi Toyo Spinning Co., Ltd. (56) References JP-A-60-2199 (JP, A) JP-A 60-62999 (JP, A) Agriculturalization Proceedings of the Annual Meeting of the Society of Japan, Vol. 65, No. 3, page 117 “2Wa8” (58) Fields surveyed (Int. Cl. ⁷ , DB name) C07H 15/203 C08B 33/04 C12Q 1/40 CA (STN) REGISTRY (STN)

Claims (2)

(57) [Claims] 1. A compound of the general formula (I) (Wherein, R ₁ represents hydrogen, R ₂ represents a β -galactopyranosyl group, n represents 2 , and a wavy line represents a β bond ). 2. A compound of the general formula (I) (Wherein, R ₁ represents hydrogen, R ₂ represents a β -galactopyranosyl group, n represents 2 , and a wavy line represents a β bond ). Reagent for measuring α-amylase activity.