JPH0630602B2 - A novel method for producing non-reducing end-modified oligosaccharide derivatives - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel method for producing a non-reducing end-modified oligosaccharide derivative useful as a substrate for measuring α-amylase activity.

Background of the Invention Samples, especially α-in saliva, pancreatic juice, blood and urine in human organisms
Measurement of amylase activity is important in medical diagnostics. For example, in pancreatitis, pancreatic cancer, and parotitis, the α-amylase activity in blood or urine shows a marked increase as compared with the usual value.

Regarding the method for measuring the α-amylase activity, various methods have been published so far.
It can be divided into two types: a method using a long-chain natural product such as amylose and amylopectin and a modified product thereof, and a method using an oligosaccharide having 4 to 7 glucose residues and a derivative thereof.

However, recently, for example, maltotetraose,
A method using maltopentaose, maltohexaose, maltoheptaose, and other oligosaccharides as substrates (Japanese Patent Laid-Open Nos. 50-56998 and 53-37096) and p.
-A method using a uniform and well-defined substrate, such as a method using an oligosaccharide in which a primary color such as nitrophenol is bound to the reducing end (JP-A-54-51892), has been widely used until now. It is becoming the mainstream of the amylase assay instead of the method used.

In these methods, α-glucosidase (EC3.2.1.20; α-D-glucoside glucohydrolase) or glucoamylase (EC3.2.1.3;
1,4-α-D-glucan glucohydrolase), or β
-Requires glucosidase (EC 3.2.1.21; β-D-glucoside glucohydrolase).

These coupling enzymes are exotype enzymes that hydrolyze the α-1,4-glucoside bond from the non-reducing end of the sugar chain having the α-1,4-glucoside bond, regardless of the α-amylase reaction. It has the drawback of degrading the substrate. Therefore, in the above-mentioned measuring method using these conjugated enzymes, the measuring reagent solution was unstable, and the reagent blind test value was extremely high, which significantly deteriorated the measuring accuracy. Furthermore, a sufficient amount of glucoamylase or α-glucosidase could not be used for measurement, and it was difficult to assemble an accurate and highly accurate measurement method.

In order to solve such a problem, the present inventors have applied for a patent for a method of synthesizing several kinds of novel modified oligosaccharides and using them to measure an α-amylase activity.

For example, when measuring α-amylase activity, the primary alcohol (-CH ₂ OH) at the 6-position of the non-reducing terminal glucose of a linear oligosaccharide consisting of 4 to 7 glucose is represented by the general formula -CH ₂ R. There is a method for measuring α-amylase activity which uses as a substrate an oligosaccharide derivative having the following structural formula substituted with a group represented by JP-A-59-51800.
issue).

(In the formula, the glucose unit at the right end is a reducing group, and k is 2-5.
And R represents, for example, a 2-pyridylamino group. ) These substrates are characterized in that they are uniform and have a clear structure and do not serve as substrates for α-glucosidase, β-glucosidase or glucoamylase. However, in order to measure α-amylase activity using these substrates, high performance liquid chromatography method or α-amylase method can be used.
Glucosidase, β-glucosidase, or glucoamylase must be used as a coupling enzyme to measure glucose produced, and the former requires special equipment, the latter is affected by glucose contained in the sample. There was a problem with the points.

Therefore, the present inventors have conducted further research and found a method for measuring α-amylase activity that does not have such a problem, and applied for a patent for it (Japanese Patent Laid-Open No. 61-83195).

That is, the primary alcohol (-CH ₂ OH) at the 6-position of the non-reducing terminal glucose of a linear oligosaccharide consisting of 4 to 7 glucose is substituted with a group represented by -CH ₂ R ₀ ¹ , and further,
The following structural formula [1], in which the 1-position hydroxyl group of the reducing terminal glucose is substituted with a phenoxy group, a substituted phenoxy group or an umbelliferyl group: [Wherein n ₀ is an integer of 2 to 5, R ₀ ¹ represents an organic residue, and R ₀ ² is Represents (However, R ₀ ^{3 to} R ₀ ⁶ are hydrogen, a lower alkyl group,
Lower alkoxy group, a nitro group, a carboxyl group, a sulfonic acid group or a halogen, may be different even severally same, R ₀ ⁷ represents hydrogen, a lower alkoxy group, a halogen or nitro group. Also, R ₀ ⁸ represents hydrogen, a methyl group or a trifluoromethyl group. )] The method for measuring α-amylase activity using the oligosaccharide derivative represented by This measurement method is an excellent measurement method that solves all the various problems of the conventional α-amylase activity measurement method, but the non-reducing end-modified oligosaccharide used as a substrate is a conventional measurement method using dextrin or amylose as a starting material. However, the production method of the above method can be obtained only in an extremely low yield, and the production method has a long reaction step and is a complicated production method, and thus a problem remains in practical application (commercialization). On the other hand, cyclomaltodextrin glucanotransferase (EC2.4.1.19)
However, it has been known for a long time that it acts on cyclodextrin and an acceptor to generate a linear oligosaccharide having an acceptor bound to the reducing end (J. Am. Chem.
Soc. 72, 1202-1205, 1949). However, it is not known whether or not a linear oligosaccharide having an acceptor bound to the reducing end is similarly obtained when the modified cyclodextrin and the acceptor are allowed to act on the acceptor, and JP-A-60-237998 discloses the method. There is a description suggesting the possibility of this, but even if this is carried out according to the method disclosed in the publication, the target product is not actually obtained.

[Object of the Invention]

An object of the present invention is to provide a production method by which a non-reducing end-modified oligosaccharide useful as a substrate for measuring α-amylase activity can be easily obtained in high yield.

[Structure of Invention]

The present invention provides one modified glucose (ie,
The modified cyclodextrin containing glucose in which the hydroxyl group at the 6-position is modified or glucose in which the 6-position is replaced by a carboxyl group, glucose, maltose, maltotriose as an acceptor, or the hydroxyl group at the 1-position of these reducing terminal glucose In the presence of a substituted derivative,
Non-reducing, in which the 6-position of the non-reducing terminal glucose is substituted and an acceptor is bound to the reducing terminal, which is characterized by acting cyclomaltodextrin glucanotransferase and then acting glucoamylase or α-glucosidase It is an invention of a method for producing an end-modified oligosaccharide derivative.

The modified cyclodextrin used in the production method of the present invention includes one containing one modified glucose per molecule.

The modified glucose has a hydroxyl group at the 6-position, for example, 2
A fluorescent substituent such as a pyridylamino group and a 3-pyridylamino group, an anilino group, a methylanilino group,
Substituents having UV absorption such as hydroxyanilino group and carboxyphenylamino group, alkoxy groups such as methoxy group and ethoxy group, carboxymethoxy group, hydroxyethoxy group, benzyloxy group, phenethyloxy group,
Substituted alkoxy group such as pyridylmethyloxy group, chlorine,
Examples thereof include glucose substituted with a halogen atom such as bromine, a hydrazono group, a phenylhydrazono group or an amino group, or glucuronic acid in which the -CH ₂ OH group at the 6-position of glucose is replaced with a -COOH group.

Examples of the acceptor used in the production method of the present invention include glucose, maltose, maltotriose, and derivatives thereof in which the hydroxyl group at the 1-position of glucose at the reducing terminal is substituted. Oligosaccharides of maltotetraose or higher are not preferable because the reaction is slow and side reactions occur. On the other hand, when the glucose chain becomes long, the glucose chain of the main product at the initial stage of the reaction also becomes long. Therefore, according to the glucose chain length of the target substance, the glucose chain lengths of 1 to 3 are
The acceptor may be appropriately selected and used.

Examples of the substituent of the hydroxyl group at the 1-position of the reducing terminal glucose of the derivative of glucose, maltose or maltotriose used as an acceptor include (However, R ^{3 to} R ⁶ represent hydrogen, a lower alkyl group, a lower alkoxy group, a nitro group, a carboxyl group, a sulfonic acid group or halogen, and they may be the same or different, and R ³ and R ³ R ⁵ or R ⁴ and R ⁶ may combine to form an aromatic ring, R ⁷ represents hydrogen, a lower alkoxy group, a halogen or a nitro group, and R ⁸ represents hydrogen, a methyl group or a tri group. Represents a fluoromethyl group, R ⁹
Represents hydrogen or halogen. ), A phenoxy group which may have a substituent, a naphthoxy group which may have a substituent, an umbelliferyl group which may have a substituent or a substituent which has a substituent. And an indoxyl group and the like. Further, specific examples of the substituent of the 1-position hydroxyl group include, for example, p-nitrophenoxy group, m
-Nitrophenoxy group, o-chlorophenoxy group, p-
Chlorophenoxy group, 2,6-dichlorophenoxy group, o
-Methoxyphenoxy group, p-methoxyphenoxy group,
o-methylphenoxy group, o-carboxyphenoxy group, o-sulfophenoxy group, 1-naphthoxy group, 2-
Examples thereof include, but are not limited to, a sulfo-1-naphthoxy group, a 2-carboxy-1-naphthoxy group, an umbelliferyl group, a 4-methylumbelliferyl group, and an indoxyl group.

Cyclomaltodextrin glucanotransferase (hereinafter abbreviated as CGTase) used in the production method of the present invention.
There is no particular limitation on the origin and origin of the bacterium, for example, those derived from Bacillus macerans, those derived from Bacillus megaterium, those derived from Klepschella pneumoniae, those derived from alkaline bacteria, and the like.

Further, the origin and origin of glucoamylase or α-glucosidase used in the production method of the present invention are not particularly limited.

The modified cyclodextrin used in the production method of the present invention uses α, β or γ-cyclodextrin as a raw material, and various modifications described in, for example, Method in Carbohydrate Chemistry I (1962) to V (1965) Academic Press. It can be easily obtained by reacting this with various reaction reagents for introducing a target modifying group according to the method for producing glucose. [alpha], [beta], or [gamma] -cyclodextrin may be selected arbitrarily, and one having the highest yield can be appropriately selected according to the glucose chain length of the target modified oligosaccharide derivative.

CGTase on modified cyclodextrin in the presence of acceptor
The pH at the time of reacting by reacting with CGTase used
Although it differs slightly depending on the origin, it is usually 6 to 8. The buffer used to maintain the same pH may be any as long as it does not inhibit the enzyme reaction, and examples thereof include Good's buffer, ammonium acetate, carbonate, phosphate and the like.

The concentration of the modified cyclodextrin used in the production method of the present invention is usually 1 to 50 mmol / mol, and the concentration of the acceptor is usually 5 mmol / mol or higher, up to the solubility limit.
It is desirable that the molar ratio of the modified cyclodextrin to the acceptor is 1 or more for the former and 5 or more for the latter. The amount of CGTase used in the production method of the present invention is usually 50 to 50.
00 U / m, and the reaction is usually performed at 20 to 50 ° C. After completion of the enzymatic reaction with CGTase, it is inactivated by heating (for example, 90 ° C. or higher for 10 minutes or longer) or the pH is changed (for example, pH 4.0 or lower) to lose its action.

The reaction with glucoamylase or α-glucosidase is usually performed at its optimum pH (usually 4 to 6), and the amount used is usually 5 to 100 U / m.

According to the production method of the present invention, a non-reducing end-modified oligosaccharide useful as a substrate for measuring α-amylase activity or as a substrate for separately measuring each isozyme activity of human α-amylase is collected by a simple reaction operation. You can get it efficiently.

The measuring principle of the α-amylase activity measuring method using the non-reducing end-modified oligosaccharide derivative according to the present invention as a substrate is roughly as follows.

(In the formula, G represents a glucose unit, R ¹ represents a substituent at the 6-position of non-reducing terminal glucose, m ¹ and m ² represent an integer of 1 or more whose sum is 2 to 5, OR ² Is a phenoxy group optionally substituted at the 1-position of the reducing terminal glucose, a naphthoxy group optionally having a substituent, an umbelliferyl group optionally having a substituent or a substituent Represents an indoxyl group which may have a group.) That is, first, the α-amylase in the sample acts on the non-reducing end-modified oligosaccharide derivative according to the present invention to give 6-fold of non-reducing end glucose. -Grade primary alcohol (-CH
₂ OH) was substituted with the group R ¹ . And a phenoxy group which may have a substituent at the 1-position of the reducing terminal glucose, a naphthoxy group which may have a substituent, an umbelliferyl group which may have a substituent or a substituent G _m with an indoxyl group which may have
^2- G-OR ² is produced, and then a coupling enzyme such as glucoamylase, α-glucosidase or β-glucosidase acts on this G _m ² -G-OR ² to give (m ² +1) G and R. ^2- OH is produced. The R ² -OH, for example, in the case of such nitrophenols R ² -OH is p- nitrophenol, by the absorption spectrum directly (e.g. absorbance at 405 nm) measurement, also, R ² -OH is a phenol or naphthol having no nitro group (which may have a nitro group) such as phenol, o-chlorophenol, 2,6-dichlorophenol, p-methoxyphenol, etc. Is produced by reacting oxidases such as catechol oxidase, laccase, tyrosinase or monophenol oxidase or oxidants such as iodic acid and periodate, or by allowing peroxidase and hydrogen peroxide to act. Coupling (oxidative condensation) with couplers such as aminoantipyrine, 3-methyl-2-benzothiazolinone hydrazone (MBTH), By measuring the absorption spectrum of the dye to be formed, or R ² -OH umbelliferone, in the case of compounds having a fluorescence as 4-methylumbelliferone, by measuring the fluorescence intensity,
Further, when R ² —OH is indoxyl, the α-amylase activity in each sample can be determined by measuring the absorption spectrum of the indigo dye produced by oxidation.

In the method for measuring α-amylase activity using the non-reducing end-modified oligosaccharide derivative according to the present invention as a substrate,
The concentration of the non-reducing end-modified oligosaccharide derivative according to the present invention used as a substrate is not particularly limited, but usually about 0.1 to 10 mM is preferably used.

The sample to be measured in the α-amylase activity measuring method using the non-reducing end-modified oligosaccharide derivative according to the present invention as a substrate may be any sample containing α-amylase, for example, as a biological component. Examples include blood, serum and urine.

The conjugating enzyme glucoamylase, α-glucosidase or β-glucosidase is not particularly limited, but, for example, those derived from animals, plants and microorganisms can be used, and they can be used alone or in combination. The amount of these coupling enzymes used is usually 0.5 to 50 units / m, preferably 2 to 20 units / m.

Further, as the measurement conditions for carrying out the present invention, the reaction temperature is not particularly limited, but is preferably about 25 to 40 ° C,
The reaction time can be freely selected according to the purpose.

The optimum pH is not particularly limited, but a pH of about 6 to 8 is a preferred example. The buffer that maintains the optimum pH can be freely selected, and for example, phosphate, trishydroxymethylaminomethane-hydrochloric acid, Good's buffer and the like can be arbitrarily selected.

Further, as an activator of α-amylase, for example, sodium chloride, calcium chloride, potassium chloride or the like is used.

As couplers for coupling (oxidative condensation) with phenols or naphthols released by the action of a coupling enzyme, 4-aminoantipyrine, 3-methyl-2-benzothiazolinonehydrazone (MBTH), p-amino-N, N-
Examples thereof include, but are not limited to, diethylaniline and the like. Examples of the oxidase for coupling the phenols or naphthols with the coupler (oxidative condensation) include laccase, catechol oxidase, tyrosinase, monophenol oxidase, and the like. Any of these can be used, and it is usually used in the range of 0.2 to 10 units / m, preferably 0.5 to 4 units / m. Examples of the oxidizing agent for coupling (oxidative condensation) include, but are not limited to, iodic acid or / and its salt, periodic acid or / and its salt, and the like.

The non-reducing end-modified oligosaccharide derivative according to the present invention is a primary alcohol at the 6-position of the non-reducing end glucose.
Since (-CH ₂ OH) is substituted with various substituents, it does not become a substrate for glucoamylase, α-glucosidase or β-glucosidase as it is, and is easily soluble in water, α-
It has a good affinity for amylase, and thus is a good specific substrate for α-amylase. Therefore, in the assay method using the non-reducing end-modified oligosaccharide derivative according to the present invention as a substrate, a side reaction does not occur, the reagent blind value is extremely small, and the assay reagent solution is extremely stable. Further, since a single compound is used as a substrate, the stoichiometry of the reaction is established, and the kinetic detection of α-amylase becomes possible.

In addition, in the α-amylase activity measuring method using the non-reducing end-modified oligosaccharide derivative according to the present invention as a substrate, glucoamylase, α-glucosidase, isomaltase or β-glucosidase or the like is sufficiently used as a coupling enzyme. Therefore, the reaction rate after the α-amylase reaction is fast, and more accurate and accurate measurement of the α-amylase activity can be performed.

Furthermore, in the assay method according to the present invention, the absorption spectrum of nitrophenols or indigo dyes that liberate detection is measured, or phenols or naphthols that liberate 4-aminoantipyrine, MBTH
Oxidative coupling with, etc., to measure the absorption spectrum of the dye, or because it is carried out by measuring the fluorescence intensity of umbelliferone released, glucose coexisting in the sample, sugars such as maltose, It is hardly affected by reducing substances such as ascorbic acid and bilirubin.

The method for measuring amylase activity according to the present invention may be a late assay for measuring the reaction rate under a fixed condition or an end point assay using a reaction terminator, and any measuring method can be carried out.

Further, the measuring method according to the present invention has good adaptability to an automatic analyzer, and can be carried out by a manual method or an automatic analysis as required.

Furthermore, when the substrate of the present invention is used, the coloration of the dye can be measured by a so-called colorimetric method. Therefore, a simple test strip method or a multilayer analysis sheet containing a reaction reagent can be used. It can also be applied to a so-called dry quantitative method using (multilayer integrated quantitative analysis film).

The non-reducing end-modified oligosaccharide derivative according to the present invention is also disclosed in Japanese Patent Application No. 61-
No. 181564, a method for fractionating salivary gland-derived α-amylase and pancreatic-derived α-amylase, that is, a decomposition product produced by the hydrolysis of α-amylase, which has two or more different substrate specificities. An effective substrate for the differential measurement method of α-amylase isozymes, which performs a differential measurement of α-amylase derived from human pancreas and α-amylase derived from human salivary gland by reacting a coupling enzyme and measuring the resulting product. Can be used as

Examples and reference examples are shown below, but the present invention is not limited to these examples and reference examples.

〔Example〕

Example 1. p-nitrophenyl O-6-deoxy-6
-[(2-Pyridyl) amino] -α-D-glucopyranosyl- (1 → 4) -O-α-D-glucopyranosyl-
(1 → 4) -O-α-D-glucopyranosyl- (1 →
4) -O-α-D-glucopyranosyl- (1 → 4) -α
Synthesis of -D-glucopyranoside (hereinafter abbreviated as FG5P) (1) Mono-6-deoxy-6 [(2-pyridyl) amino] -β-cyclodextrin (hereinafter abbreviated as F-β-CD) 20 g of β-cyclodextrin and 30.4 g of DCC (dicyclohexylcarbodiimide) were dissolved in 120 m of DMSO (dimethylsulfoxide), 2.6 m of dichloroacetic acid was added, and the mixture was stirred at room temperature for 30 minutes. What melt | dissolved oxalic acid 12.8g in methanol 50m was added to this, and it was set to pH 9 by 2M sodium carbonate aqueous solution. A solution prepared by dissolving 1.6 g of 2-aminopyridine in 200 m of water was added thereto, 3.6 g of pyridineborane was further added, and the mixture was reacted at 65 to 70 ° C. for 2 hours. 700m of water was added to remove insoluble matter, and pH was adjusted to 7 with hydrochloric acid.
After that, add 2.4 g of sodium borohydride and
Reacted for hours. Hydrochloric acid was added to adjust the pH to 3, and excess sodium borohydride was decomposed, and then aqueous ammonia was added to adjust the pH to 7.
It was set to 0. Acetone 2 was added and the deposited precipitate was collected to obtain 3.0 g of F-β-CD.

Elemental analysis C ₄₇ H ₇₄ O ₃₄ N ₂ (2) Synthesis of FG5P 10 g of F-β-CD and 10 g of p-nitrophenyl α-glucoside were added to 10 mM ammonium acetate buffer (pH 6.5) 1
It was dissolved in and added with 1000 kU of CGTase and reacted at 37 ° C. for 5 hours. Next, after adjusting the pH to 4.0 with acetic acid, add 20 kU of glucoamylase (from Rhizopus niveus),
And reacted for 10 hours. The reaction solution was freeze-dried and then purified with a column (30 x 1500 mm) packed with Bio-Gel P-2 (Bio-Rad) equilibrated with 50 mM acetic acid to obtain 1.1 g of FG5P.

p-nitrophenyl α-glucoside 10 g instead of p
The same reaction and post-treatment were carried out using 10 g of -nitrophenyl β-glucoside to obtain 2.5 g of β form of FG5P.

The structure was confirmed according to the confirmation method of the same compound described in JP-A-61-83195.

Example 2. p-nitrophenyl O- (2-O-carboxymethyl) -α-D-glucopyranosyl- (1 → 4)
-O-α-D-glucopyranosyl- (1 → 4) -O-α
Synthesis of -D-glucopyranosyl- (1 → 4) -O-α-D-glucopyranosyl- (1 → 4) -α-D-glucopyranoside (hereinafter abbreviated as CMG5P) (1) Mono-O-carboxymethyl- Synthesis of β-cyclodextrin (hereinafter abbreviated as CM-β-CD) β-cyclodextrin 15.0 g, sodium hydroxide 13.5
g was dissolved in 120 m of water, 90 m of a 10% monochloroacetic acid aqueous solution was added, and the mixture was stirred at 25 ° C for 5 hours. After adjusting the pH to 7.0 with 6N hydrochloric acid, 600 m of acetone was added and the precipitated crystals were collected to obtain 12 g of CM-β-CD.

Elemental analysis C ₄₄ H ₇₅ O ₃₇ N MW1210 (NH ₄ salt) (2) Synthesis of CMG5P CM-β-CD 10 g, p-nitrophenyl glucoside 10
g was dissolved in 10 mM ammonium acetate buffer (pH 6.5) 1, CGTase (1000 kU) was added, and the mixture was reacted at 37 ° C. for 5 hours. Then, the pH was adjusted to 4.0 with acetic acid, 20 kU of glucoamylase was added, and the mixture was reacted at 37 ° C for 10 hours. The reaction solution was freeze-dried and then equilibrated with 50 mM acetic acid Bio-Gel P-2 (Bio-R
Purification was performed using a column (30 × 1500 mm) packed with ad company to obtain 3 g of CMG5P.

Example 3. Phenyl O- (α-D-glucopyranosyl uronic acid)-(1 → 4) -O-α-D-glucopyranosyl- (1 → 4) -O-α-D-glucopyranosyl-
(1 → 4) -O-α-D-glucopyranosyl- (1 →
4) -α-D-glucopyranoside (hereinafter, carboxyl
Abbreviated as G5P. )) (1) Synthesis of monocarboxyl-β-cyclodextrin (hereinafter abbreviated as carboxyl-β-CD) 50 g of β-cyclodextrin is dissolved in water 1 and platinum-carbon (10%) 5 g, isopropanol 2.5
m and add 50 m / min of air at 70 ° C under stirring.
Flow rate of. 1M sodium hydrogen carbonate solution was added dropwise, and the reaction was carried out for 2.5 hours while keeping the pH neutral, then, the solution was subjected to ion exchange chromatography (Dowex I).
Then, 10.5 g of carboxyl-β-CD was obtained.

Elemental analysis C ₄₂ H ₇₁ O ₃₆ N MW1165 (NH ₄ salt) (2) Synthesis of carboxyl G5P 10 g of carboxyl-β-CD and 10 g of phenyl α-glucoside were added to 10 mM ammonium acetate buffer (pH 6.5) 1
Was dissolved in the mixture, 1000 gU of CGTase was added, and the mixture was reacted at 37 ° C for 1 hour. Next, the pH was adjusted to 4.0 with acetic acid, 20 kU of glucoamylase was added, and the mixture was reacted at 37 ° C for 10 hours. The reaction solution was lyophilized and then equilibrated with 20 mM acetic acid Bio-Gel P-2 (Bio
Purification was carried out using a column (30 × 2400 mm) packed with Rad) to obtain 1.2 g of carboxyl G5P.

The number of glucose residues with respect to the phenyl group in the obtained carboxyl G5P was measured as follows. Carboxyl G5P was subjected to methanolysis with 1.4N hydrochloric acid-methanol at 90 ° C. for 2 hours. After concentration, it is converted to trimethylsilyl and 2% OV-1
Using a 7 (0.4 x 200 cm) column, 110 ° C to 250 ° C
The amount of glucose was quantified using a heating program up to 4 ° C / min. In addition, the phenyl group is 2 in 0.1M acetic acid.
It quantified from the absorbance at 65 nm. As a result Carboxyl G5
The glucose / phenol ratio of P was 3.9.

From the results obtained above and the fact that carboxyl G5P is not affected by glucoamylase, the structure of carboxyl G5P is considered to be as follows.

The mass spectrum of this compound is shown in FIG.

Example 4. O-6-deoxy-6-[(2-pyridyl)
Amino] -α-D-glucopyranosyl- (1 → 4) -O
-Α-D-glucopyranosyl- (1 → 4) -O-α-D
Synthesis of -glucopyranosyl- (1 → 4) -O-α-D-glucopyranosyl- (1 → 4) -O-α-D-glucopyranosyl- (1 → 4) -D-glucitol (hereinafter abbreviated as FG6R). F-β-CD (10 g) and maltotriitol (50 g) obtained in the same manner as in (1) of Example 1 were dissolved in 10 mM ammonium acetate buffer (pH 6.5) 1, CGTase (1000 kU) was added, and the mixture was reacted at 37 ° C. for 5 hours. Let Next, the pH was adjusted to 4.0 with acetic acid, 20 kU of glucoamylase was added, and the mixture was reacted at 37 ° C for 10 hours. The reaction solution was freeze-dried and then packed with a column (30 × 15) packed with Bio-Gel P-2 (Bio-Rad) equilibrated with 50 mM acetic acid.
(00 mm) to obtain 1.2 g of FG6R.

Example 5. Phenyl O- (6-amino-6-deoxy) -α-D-glucopyranosyl- (1 → 4) -O-α
-D-glucopyranosyl- (1 → 4) -O-α-D-glucopyranosyl- (1 → 4) -O-α-D-glucopyranosyl- (1 → 4) -α-D-glucopyranoside (hereinafter referred to as amino G5P and (1) Synthesis of mono-6-O-p-toluenesulfonyl β-cyclodextrin 5.0 g of β-cyclodextrin was dissolved in 100 m of pyridine, and 0.8 g of p-toluenesulfonyl chloride was added to add 4 g.
Stirred at 15 ° C for 15 hours. After concentration under reduced pressure, the column was charged with activated carbon (5 × 60 cm) and eluted with water, 30% ethanol, 25% n-propanol, 3 each.
25% n-propanol fractions were collected and concentrated under reduced pressure to give 6-O-
0.7 g of p-toluenesulfonyl-cyclodextrin was obtained.

(2) Synthesis of mono-6-azido-6-deoxy-β-cyclodextrin 6-Op-toluenesulfonyl β-cyclodextrin (1.0 g) was dissolved in 80 m of water, and sodium nitride 1 was added.
g was added and reacted at 95 ° C. for 90 minutes. After the reaction, the reaction solution was concentrated under reduced pressure and separated and purified by gel filtration (Bio-Gel P-2) to obtain 0.7 g of mono-6-azido-6-deoxy-β-cyclodextrin.

(3) Synthesis of amino G5P Mono-6-azido-6-deoxy-β-cyclodextrin 0.5 g, phenyl α-D-glucopyranoside 0.5 g
Was dissolved in 50 mM of 10 mM ammonium acetate buffer (pH 6.5), 50 kU of CGTase was added, and the mixture was reacted at 37 ° C. for 2 hours. Then adjust the pH to 4.0 with acetic acid and then use 1 kU of glucoamylase
Was added and reacted at 37 ° C. for 10 hours. 10 in the reaction solution
% -Palladium-Carbon (0.1 g) was added, and hydrogen gas was blown thereinto at 25 ° C. for 8 hours with stirring. After freeze-drying the reaction mixture, it was separated and purified by gel filtration (Bio-Gel P-2) and amino G5P80m
got g.

The number of glucose residues with respect to the phenyl group in the obtained amino G5P was measured as follows. Amino G5P 1.4N
Methanolysis was performed with hydrochloric acid-methanol at 90 ° C. for 2 hours.
After concentration, it is converted to trimethylsilyl and 2% OV-17 (0.4 x 20
0 ° C) column, 110 ° C to 250 ° C, 4 ° C
The amount of glucose was quantified using a heating program per minute. The phenyl group was quantified from the absorbance at 265 nm in 0.1 M acetic acid. As a result, the glucose / phenol ratio of amino G5P was 3.6.

Based on the results obtained above and the fact that amino G5P was not affected by glucoamylase and the color development by ninhydrin was observed on the TLC plate, the structure of amino G5P is considered to be as follows.

The mass spectrum of this compound is shown in FIG.

Example 6. p-Nilothophenyl O- (6-O-benzyl) -α-D-glucopyranosyl- (1 → 4) -O-α
-D-glucopyranosyl- (1 → 4) -O-α-D-glucopyranosyl- (1 → 4) -O-α-D-glucopyranosyl- (1 → 4) -α-D-glucopyranoside (hereinafter abbreviated as BG5P) .) (1) Synthesis of mono-O-benzyl-β-cyclodextrin (hereinafter abbreviated as benzyl-β-CD) β-cyclodextrin 15 g, sodium hydroxide 13.5
g was dissolved in 120 m of water, 15 m of benzyl chloride was added, and the mixture was reacted with stirring at 10 ° C for 2 hours. After reaction with 6N hydrochloric acid
After adjusting the pH to 7.0, acetone 1 was added and the deposited precipitate was collected to obtain 5 g of benzyl-β-CD (2nd, 3rd, 6th).
Mixture of position substitutions).

(2) Synthesis of BG5P Benzyl-β-CD10g, p-nitrophenyl α-D-
Glucopyranoside (10 g) was dissolved in 10 mM ammonium acetate buffer (pH 6.5) 1 and CGTase (1000 kU) was added to the solution.
The mixture was reacted with stirring at 7 ° C. for 5 hours. Next, after adjusting the pH to 4.0 with acetic acid, add 20 kU of glucoamylase,
Reacted for hours. The reaction solution was freeze-dried and then packed with a column (30) packed with Bio-Gel P-2 (Bio-Rad) equilibrated with 50 mM acetic acid.
(1500 mm) to obtain 1.6 g of crude BG5P (mixture of 2-, 3- and 6-position substitution products). This was separated and purified by reverse phase high performance liquid chromatography to give 6-position substitution product 180 mg.
Got (2-position substitution product 700 mg, 3-position substitution product 650 mg) HPLC: content 96% [column: packing material silica gel Z-OD
S, 5C ₁₈ (Wako Pure Chemical Industries, Ltd. product name, 10 × 300 m
m), eluate 10% CH ₃ CN-0.1% AcOH and 90% CH ₃ CN-0.1
Linear gradient with% AcOH, flow rate 3m / min, 305
Measured in nm] IR: As shown in FIG. ¹ H-NMR: As shown in FIG.

Also, the glucose / p-nitrophenol ratio of BG5P determined in the same manner as in Examples 3 and 5 was 3.8.

From the results obtained above and the fact that BG5P is not affected by glucoamylase, the structure of BG5P is considered to be as follows.

Example 7. p-nitrophenyl O- (6-O-methyl) -α-D-glucopyranosyl- (1 → 4) -O-α
-D-glucopyranosyl- (1 → 4) -O-α-D-glucopyranosyl- (1 → 4) -O-α-D-glucopyranosyl- (1 → 4) -α-D-glucopyranoside (hereinafter abbreviated as MG5P) (1) Synthesis of mono-O-methyl-β-cyclodextrin (hereinafter abbreviated as methyl-β-CD) β-cyclodextrin 10 g, sodium hydroxide 13.5
g was dissolved in 120 m of water, 2.7 g of dimethylsulfate was added, and the mixture was stirred and reacted at 10 ° C. for 2 hours. After the reaction, pH with 6N hydrochloric acid
After adjusting to 7.0, acetone 1 was added and the deposited precipitate was removed to obtain 2 g of methyl-β-CD.

(2) Synthesis of MG5P Methyl-β-CD10g, p-nitrophenyl α-D-
Glucopyranoside (10 g) was dissolved in 10 mM ammonium acetate buffer (pH 6.5) 1 and CGTase (1000 kU) was added to the solution.
The mixture was reacted with stirring at 7 ° C. for 5 hours. Next, after adjusting the pH to 4.0 with acetic acid, add 20 kU of glucoamylase,
Reacted for hours. The reaction solution was freeze-dried and then packed with a column (30) packed with Bio-Gel P-2 (Bio-Rad) equilibrated with 50 mM acetic acid.
X 1500 mm) to obtain 1.5 g of crude MG5P (mixture of 2-, 3- and 6-position substitution products). This was separated and purified by reverse phase high performance liquid chromatography to obtain 160 mg of the 6-position substitution product. (680 mg of 2-position substitution product, 560 mg of 3-position substitution product).

HPLC: content 93% (measurement conditions are the same as in Example 6).

The glucose / p-nitrophenol ratio of MG5P determined in the same manner as in Examples 3 and 5 was 3.9.

From the above results and the fact that MG5P is not affected by glucoamylase, the structure of MG5P is considered to be as follows.

Example 8. p-nitrophenyl O- (6-bromo-6
-Deoxy) -α-D-glucopyranosyl- (1 → 4)
-O-α-D-glucopyranosyl- (1 → 4) -O-α
Synthesis of -D-glucopyranosyl- (1 → 4) -O-α-D-glucopyranosyl- (1 → 4) -α-D-glucopyranoside (hereinafter abbreviated as BrG5P) (1) Mono-6-bromo-6 Synthesis of -deoxy-β-cyclodextrin (hereinafter abbreviated as Br-β-CD) Mono-6-O- synthesized according to the method of Example 1 (1).
5 g of p-toluenesulfonyl β-cyclodextrin
Was dissolved in 250 m of DMF, 15 g of lithium bromide was added, and the mixture was refluxed for 2 hours. The reaction mixture was concentrated under reduced pressure, 500 m of acetone was added, and the deposited precipitate was removed to remove mono-6.
-Bromo-6-deoxy-β-cyclodextrin 2.3
g was obtained.

(2) Synthesis of BrG5P Br-β-CD (1 g) and p-nitrophenyl α-D-glucopyranoside (1 g) were added to 10 mM ammonium acetate buffer (pH).
6.5) Dissolve in 100m and add 100kU of CGTase to 37 ℃
And reacted with stirring for 5 hours. Then add acetic acid to bring the pH to 4.0, add 2000 U of glucoamylase, and add 10
The reaction was allowed to stir for an hour. The reaction solution was concentrated and a column (30 ×) packed with Bio-Gel P-2 (Bio-Rad) equilibrated with 50 mM acetic acid.
Purification was performed at 1500 mm) to obtain 0.16 g of BrG5P.

HPLC: content 97% (measurement conditions are the same as in Example 6). ¹ H-NMR: As shown in FIG.

The glucose / p-nitrophenol ratio of BrG5P determined in the same manner as in Examples 3 and 5 was 3.6.

From the above results and the fact that BrG5P is not affected by glucoamylase, the structure of BrG5P is considered to be as follows.

Reference example 1. Measurement of α-amylase activity [Measurement test solution] 30 mg of BG5P obtained in Example 6 was used as 400 units of glucoamylase, 300 units of α-glucosidase and 20 mmol /
50 mMol / 2- (N-morpholino) ethanesulfonic acid (MES) -NaOH buffer containing calcium chloride (pH 6.9) 3
It was dissolved in 0 m and used as a measurement reagent solution.

[Measurement operation]

100 μl of sample serum was added to 2 m of the test reagent solution and heated to 37 ° C., and the change in absorbance of this reaction solution at a wavelength of 405 nm was measured.

Separately, using a standard sample of known α-amylase activity, the same procedure as above was performed to determine the calibration relationship, and the α-amylase activity of the sample was determined from this calibration curve. FIG. 6 shows the relationship between the α-amylase activity (Somogyi unit / d) at each dilution step of the standard sample and the increase in absorbance (ΔA) per minute at a wavelength of 405 nm at this time.

As is clear from FIG. 6, α-amylase activity (Somogy
Absorbance increase (Δ)
The calibration curve connecting (A / min) is a straight line passing through the origin, and the calibration curve shows good quantification.

Reference example 2. Measurement of α-amylase activity [Measurement test solution] 36 mg of BG6P obtained in the same manner as in Example 6 was analyzed by 400 units of glucoamylase, 300 units of α-glucosidase and 20 mm.
It was dissolved in 50 mMol / MES-NaOH buffer solution (pH 6.9) 30 m containing ol / calcium chloride to give a measurement test solution.

[Measurement operation]

100 μl of sample serum was added to 2 m of the test reagent solution and heated to 37 ° C., and the change in absorbance of this reaction solution at a wavelength of 405 nm was measured.

Separately, using a standard sample of known α-amylase activity, the same procedure as above was performed to determine the calibration relationship, and the α-amylase activity of the sample was determined from this calibration curve. FIG. 7 shows the relationship between the α-amylase activity (Somogyi unit / d) at each dilution step of the standard sample and the increase in absorbance (ΔA) per minute at a wavelength of 405 nm at this time.

As is clear from FIG. 7, α-amylase activity (Somogy
Absorbance increase (Δ)
The calibration curve connecting (A / min) is a straight line passing through the origin, and the calibration curve shows good quantification.

Reference example 3. Measurement of α-amylase activity [Measurement reagent] 30 mg of BrG5P obtained in Example 8 was added to glucoamylase 400
Unit, 300 units of α-glucosidase and 20 mmol /
It was dissolved in 50 mMol / MES-NaOH buffer solution (pH 6.9) 30 m containing calcium chloride to obtain a test solution.

[Measurement operation]

100 μl of sample serum was added to 2 m of the test reagent solution and heated to 37 ° C., and the change in absorbance at 405 nm wavelength of this reaction solution was measured.

Separately, using a standard sample of known α-amylase activity, the same procedure as above was performed to determine the calibration relationship, and the α-amylase activity of the sample was determined from this calibration curve. FIG. 8 shows the relationship between the α-amylase activity (Somogyi unit / d) at each dilution step of the standard sample and the increase in absorbance (ΔA) per minute at a wavelength of 405 nm at this time.

As is clear from FIG. 8, α-amylase activity (Somogy
Absorbance increase (Δ)
The calibration curve connecting A) is a straight line passing through the origin, and the calibration curve shows good quantification.

〔The invention's effect〕

The present invention provides a novel and effective method for producing a non-reducing end-modified oligosaccharide useful as a substrate for measuring α-amylase activity or as a substrate for separately measuring each isozyme activity of human α-amylase. According to the method of the present invention, non-reducing end-modified oligosaccharides can also be obtained in a high yield by a simple reaction procedure, and therefore, α-amylase activity assay using these as substrates or fractionation of the isozymes This is a great point that contributes to the practical application (commercialization) of the measurement method. And the non-reducing end-modified oligosaccharide derivative according to the present invention does not have any problems of the conventional substrate for measuring α-amylase activity, and is easy to synthesize and can be obtained in good yield, so that it can be put to practical use. The point that it can provide an excellent method for measuring α-amylase activity that can be (commercialized).

It has a remarkable effect on.

[Brief description of drawings]

1 and 2 show FAB-mass spectra of the non-reducing end-modified oligosaccharide derivative according to the present invention obtained in Examples 3 and 4, respectively (apparatus Jeol HX-100). FIG. 3 shows an IR chart of the non-reducing end-modified oligosaccharide derivative according to the present invention obtained in Example 6. FIG. 4 and FIG. 5 show ¹ H-NMR charts of the non-reducing end-modified oligosaccharide derivative according to the present invention obtained in Example 6 and Example 8, respectively. 6, FIG. 7 and FIG. 8 show Reference Example 1 and Reference Example 2, respectively.
And the calibration curve obtained in Reference Example 3, where each α on the horizontal axis
-The points where the amount of increase in absorbance (ΔA / min) obtained for amylase activity (Somogyi units / d) is plotted along the vertical axis are connected.

Claims (2)

[Claims] 1. A modified cyclodextrin containing one modified glucose per molecule (that is, glucose in which the hydroxyl group at the 6-position is modified or glucose in which the 6-position is replaced with a carboxyl group), and glucose as an acceptor,
In the presence of maltose, maltotriose or a derivative in which the hydroxyl group at the 1-position of glucose at the reducing end thereof is substituted, cyclomaltodextrin glucanotransferase is allowed to act, and then glucoamylase or α-glucosidase is allowed to act. A method for producing a non-reducing end-modified oligosaccharide derivative in which the 6-position of glucose at the non-reducing end is substituted and an acceptor is bonded to the reducing end. 2. A modified glucose has a fluorescent group such as a 2-pyridylamino group or a 3-pyridylamino group in which the hydroxyl group at the 6-position has fluorescence, an U group such as an anilino group, a methylanilino group, a hydroxyanilino group or a carboxyphenylamino group.
Substituent having V absorption, methoxy group, alkoxy group such as ethoxy group, carboxymethoxy group, hydroxyethoxy group, benzyloxy group, phenethyloxy group, substituted alkoxy group such as pyridylmethyloxy group, halogen atom such as chlorine and bromine The method according to claim 1, wherein the glucose is hydrazono group, phenylhydrazono group or amino group-substituted glucose, or glucuronic acid.