JPH07126281A - Production of non-reducing end modified oligosaccharide derivative - Google Patents

Abstract

PURPOSE:To obtain the compound useful as a substrate for measuring alpha-amylase activity in a high yield by a simple operation by a new method using a specific modified saccharide and a prescribed oligosaccharide derivative as raw materials. CONSTITUTION:First, (A) a saccharide which contains one of hydroxyl groups replaced with an SR (R is a lower alkyl or allyl) and the residual functional groups protected with acyl groups is condensed with (B) an oligosaccharide derivative of formula I (R<1> is an acyl; R<2> is optically a detectable group or an acyl; (n) is 0-5) prepared by protecting hydroxyl groups except at the 4- and the 6-positions of a nonreducing end glucose in an organic solvent not containing a hydroxyl group (e.g. dichloromethane) preferably at <=0 deg.C. Then the reaction product is deacylated, for example, by a method for treating with 0.01-1N sodium methoxide.methanol solution to give the compound of formula II (R<3> and R<4> are each a saccharide residue or H; with the proviso that a case wherein both R<3> and R<4> are H at the same time is omitted).

Description

Detailed Description of the Invention

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.

[0002]

BACKGROUND OF THE INVENTION The determination of α-amylase activity in samples, especially saliva, pancreatic juice, blood and urine in human organisms is important in medical diagnosis. 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.

Various methods have been published so far for the measurement of α-amylase activity. Broadly speaking, there are methods using a long-chain natural product such as starch, amylose and amylopectin and a modified product thereof. , Oligosaccharides having 4 to 7 glucose residues and derivatives thereof can be divided into two types.

Among these methods, recently, a substrate having a uniform and well-defined structure, that is, having a chromophore at the reducing end,
A coupled enzyme method using a maltooligosaccharide derivative modified at the 6-position or 4,6-position of non-reducing terminal glucose as a substrate is widely used. These substrates do not serve as a substrate for a coupling enzyme such as α-glucosidase, β-glucosidase, and glucoamylase, and furthermore, the detection can be carried out by measuring the amount of the chromogenic substance that liberates. -It is particularly superior as a substrate for measuring amylase activity. As one of such substrates for measuring α-amylase activity, there is a modified maltooligosaccharide derivative having a sugar residue introduced at the non-reducing end. For example, Japanese Patent Laid-Open No. 2-11
N- at the C4 position of the non-reducing terminal glucose described in Japanese Patent No. 595.
Derivatives containing an acetylglucosamine residue,
It is a derivative in which a galactose residue is introduced at the C4 position or C6 position of non-reducing terminal glucose described in JP-A-3-264596. However, since all of these modified oligosaccharide derivatives are synthesized by a synthetic method using an enzyme, the yield is extremely low, and a problem remains in practical application (commercialization).

Therefore, in order to put a modified maltooligosaccharide derivative having a sugar residue introduced into the non-reducing end into practical use as a substrate for measuring α-amylase activity, the non-reducing end modification is performed at a much higher yield than the existing ones. The advent of manufacturing methods capable of synthesizing oligosaccharide derivatives is absolutely essential.

[0006]

SUMMARY OF THE INVENTION The present invention has been made in view of the above situation, and provides a modified maltooligosaccharide derivative having a sugar residue introduced at the non-reducing end, which can be an excellent substrate for measuring α-amylase activity. It is an object of the present invention to provide a novel method for producing the non-reducing end-modified oligosaccharide derivative that can be synthesized in a yield.

[0007]

In the present invention, one of the hydroxyl groups is --SR (R
Represents a lower alkyl group or an allyl group. ), The remaining functional group is protected with an acyl group, and the following general formula [1] in which hydroxyl groups other than the 4th and 6th positions of non-reducing terminal glucose are protected with an acyl group:

[0008]

[Chemical 3]

[In the formula, R ¹ represents an acyl group, R ² represents an optically detectable group or an acyl group, and n is 0-5.
Represents the integer. ] The oligosaccharide derivative represented by the formula [6] is subjected to a condensation reaction in an organic solvent having no hydroxyl group, followed by deacylation.
The following general formula [2] in which sugar is introduced at the 4-position and / or 4-position

[0010]

[Chemical 4]

[In the formula, R ³ and R ⁴ each independently represent a sugar residue or a hydrogen atom (provided that R ³ and R ⁴ are both hydrogen atoms), and R ⁵ is the following general formula: When R ^{2 of the} formula [1] is an optically detectable group, it also represents an optically detectable group, and when R ² is an acyl group, it represents a hydrogen atom. n is the same as above. ] It is invention of the manufacturing method of the oligosaccharide derivative shown by these.

That is, the present inventors can synthesize an oligosaccharide derivative having a sugar residue introduced into non-reducing terminal glucose, which can be a substrate for measuring α-amylase activity, in a higher yield than existing methods. As a result of intensive studies for a new method for producing the oligosaccharide derivative, a oligosaccharide derivative represented by the above general formula [2] can be obtained by introducing a sugar residue into non-reducing terminal glucose by a chemical method. The manufacturing method of the present invention for manufacturing has been reached. Incidentally, all of the existing methods are methods for producing an oligosaccharide derivative in which a sugar residue is introduced into a non-reducing terminal glucose by an enzymatic method, and the yield is low, and further, the sugar residue that can be introduced is limited. However, there is no conventional method for producing a non-reducing end-modified oligosaccharide derivative that can be synthesized in a high yield by a chemical synthesis method like the oligosaccharide derivative of the present invention and the sugar that can be introduced is not limited. Met.

In the production method of the present invention, a sugar used as a raw material in which one of the hydroxyl groups is substituted with -SR (R is the same as above) and the remaining functional group is protected with an acyl group (hereinafter, It may be abbreviated as "modified sugar according to the present invention".)
As one of the hydroxyl groups is substituted with -SR and the remaining functional group is protected with an acyl group, for example, glucose, mannose, galactose, rhamnose, ribose, xylose, arabinose, glucosamine, galactosamine,
Examples include monosaccharides such as sialic acid.

As R of the substituent -SR of the modified sugar according to the present invention, for example, a lower alkyl group such as methyl group, ethyl group, propyl group, butyl group, pentyl group, etc., such as phenyl group, tolyl group, naphthyl group, etc. The allyl group of is mentioned.

In the modified sugar of the present invention, examples of the acyl group protecting the functional group include an acetyl group, a propionyl group, a butyryl group and a benzoyl group.

The modified sugar according to the present invention is prepared by using, as a raw material, a monosaccharide having functional groups such as hydroxyl group, amino group and carboxyl group protected by an acyl group as described above. For example, methylthiotrimethylsilane (TMSSMe is abbreviated as this). ), Trimethylsilyl trifluoromethanesulfonate (TM)
It can be easily synthesized by the action of SOTf). The modified sugar according to the present invention also uses, as a raw material, a halogen-substituted product of the monohydroxyl group of the monosaccharide as described above, according to the method described in JP-A-63-41494 or EP Publication 0557021, and the like, It can also be synthesized by introducing a halogen moiety into -SM (M is an alkali metal molecule) and then reacting this with an alkyl halide or an allyl halide.

In the oligosaccharide derivative represented by the general formula [1], examples of the acyl group represented by R ¹ and R ² include acetyl group, propionyl group, butyryl group,
Examples thereof include a benzoyl group, and the optically detectable group represented by R ² includes glucoamylase [EC3.2.1.
3.], α-glucosidase [EC3.2.1.20], β-glucosidase [EC3.2.1.21.], Isomaltase [EC3.2.1.20].
10.] or β-amylase [EC 3.2.1.2.] And the like which can be hydrolyzed by the action of a coupling enzyme, and further,
After hydrolysis, it will have absorption itself in the visible part or ultraviolet like nitrophenol, or it will have fluorescence itself like umbelliferone, catechol oxidase, laccase, tyrosinase or Any one which is bound to a coupler to give a dye by the action of an oxidase such as monophenol oxidase, or is bound to an coupler by an oxidizing agent to give a dye Good. Such an optically measurable group is already clear to those skilled in the art, and a detailed description thereof is not necessary. For example, phenyl group, 1-naphthyl group, 2-methylphenyl group, 2-methyl-1-naphthyl group. Aryl groups such as groups such as 4-nitrophenyl group, 3-nitrophenyl group, 2-chlorophenyl group, 4-chlorophenyl group, 2,6-dichlorophenyl group, 2-chloro-4-nitrophenyl group, 2-methoxyphenyl Group, 4-methoxyphenyl group, 2-carboxyphenyl group, 2-sulfophenyl group, 2-
Substituted aryl group such as sulfo-1-naphthyl group and 2-carboxy-1-naphthyl group, umbelliferyl group, for example, substituted umbelliferyl group such as 4-methylumbelliferyl group and 4-trifluoromethylumbelliferyl group Typical examples thereof include groups and indoxyl groups, for example, substituted indoxyl groups such as 5-bromoindoxyl group and 4-chloro-3-bromoindoxyl group. Further, as the optically detectable group, in addition to these,

[0018]

[Chemical 5]

The fructose residue represented by ## STR1 ## is also included. In this case, after hydrolysis with a conjugating enzyme, the degree of decrease in NADH produced by the action of mannitol dehydrogenase and NADH is optically measured to obtain α-. Amylase activity can be measured.

As a method for synthesizing the oligosaccharide derivative represented by the general formula [1], the method described in JP-A-2-49796 and Japanese Patent Application No. 3-131880, that is, the non-reducing terminal of the oligosaccharide derivative is used. After protecting the glucose at the 4- and 6-positions by cyclic borate esterification, the remaining hydroxyl groups are protected with an acyl group, and then the 4- and 6-positions are deprotected, and the method described in JP-B-62-51960, That is, it can be easily synthesized by a method via an oligoglucoside derivative in which 4,6-positions of non-reducing terminal glucose are benzylidene-bridged.

In the oligosaccharide derivative represented by the general formula [2], the sugar residues represented by R ³ and R ⁴ include glucose, mannose, galactose, rhamnose, ribose, xylose, arabinose, and the like. Examples include sugar residues of monosaccharides such as glucosamine, galactosamine, and sialic acid. Further, as the optically detectable group represented by R ⁵ , the same groups as the above R ² can be mentioned.

In the production method of the present invention, examples of the organic solvent having no hydroxyl group used as the reaction solvent include dichloromethane, dichloroethane, diethyl ether, acetonitrile, propionitrile, butyronitrile, nitromethane and the like.

The manufacturing method of the present invention can be carried out as follows. That is, first, the oligosaccharide derivative represented by the general formula [1] and the modified sugar according to the present invention in an amount of 1 to 10 times equivalent thereto (that is, one of the hydroxyl groups is replaced with -SR, and the remaining functional group is Is an acyl-protected glucose, galactose, ribose, glucosamine, sialic acid, etc.) and an organic solvent having no hydroxyl group (eg, dichloromethane, dichloroethane, acetonitrile, etc.), such as N-iodosuccinimide / trifluoromethanesulfone. The condensation reaction is carried out in the presence of a Lewis acid such as acid, methyl triflate (methyl trifluoromethanesulfonate), trimethylsilyl triflate (trimethylsilyl trifluoromethanesulfonate), dimethyl (dithio) sulfonium triflate. The reaction temperature is usually room temperature or lower, preferably 0 ° C. or lower, and the reaction time itself varies depending on the reaction temperature, solvent and other conditions, but it usually takes several hours to several tens of hours, and the reaction progresses by TLC, etc. You can track it with. During the reaction, the yield can be further improved by using a dehydrating agent such as molecular sieves or drylite (CaSO ₄ ).

Depending on the type of monosaccharide, it is also possible to appropriately adjust the introduction position of the oligosaccharide derivative into the non-reducing terminal glucose by adjusting the equivalent ratio.

Then, a deacylation reaction is carried out. The deacylation reaction is carried out according to a conventional method, for example, 0.01 to 1.0.
The target oligosaccharide derivative represented by the general formula [2] is obtained by treating with an alkaline solution such as sodium N-methoxide / methanol solution for several hours to several tens of hours at room temperature to slightly heating (or ice cooling). Easily obtained.

The post-treatment after the reaction may be carried out by a conventional method, and the final product may be purified by a conventional method such as column chromatography.

According to the production method of the present invention, a non-reducing end-modified oligosaccharide derivative useful as a substrate for measuring α-amylase activity or a substrate for measuring each isozyme fraction of human α-amylase is collected by a simple operation. Since it can be obtained efficiently, it will be a great contribution to this industry.

Α-Using an oligosaccharide derivative obtained by the production method of the present invention (hereinafter abbreviated as “oligosaccharide derivative according to the present invention”) as a substrate
An example of the measuring principle of the amylase activity measuring method is as follows.

[0029]

[Formula 1]

(Wherein G represents a glucose unit, and R represents
Represents a sugar introduced at the 6-position of non-reducing terminal glucose, m ¹ and m ² represent an integer of 0 or more whose sum is 1 to 6, and R ′ is substituted at the 1-position of reducing terminal glucose. , Represents an optically detectable group. )

That is, first, the α-amylase in the sample acts on the oligosaccharide derivative according to the present invention to introduce the above-mentioned compound [A] in which a sugar is introduced into the 6-position of non-reducing terminal glucose, and a reducing terminal. The above compound [B] having an optically detectable group at the 1-position of glucose is produced, and then
Coupling enzymes such as glucoamylase, α-glucosidase, β-glucosidase, or isomaltase act on the compound [B] in an amount of 1 to 3 to produce m ² G and R′-OH.
R'-OH can be measured by directly measuring its absorption spectrum (for example, the absorbance at 405 nm) in the case where R'-OH is a nitrophenol such as p-nitrophenol. -OH is, for example, phenol, o-chlorophenol, 2,6-dichlorophenol, p-
In the case of phenols or naphthols having no nitro group such as methoxyphenol (which may have a nitro group), oxidative enzymes such as catechol oxidase, laccase, tyrosinase or monophenol oxidase or iodic acid, By reacting an oxidizing agent such as periodic acid or peroxidase with hydrogen peroxide, coupling with a coupler such as 4-aminoantipyrine, 3-methyl-2-benzothiazolinone hydrazone (MBTH) (oxidative condensation), Further, by measuring the absorption spectrum of the produced dye, or in the case where R′-OH is a compound having fluorescence such as umbelliferone and 4-methylumbelliferone, its fluorescence intensity is further measured. Is R '
When -OH is indoxyl, by measuring the absorption spectrum of the indigo dye formed by oxidation,
The α-amylase activity in each sample can be determined.

As another example of the method for measuring α-amylase activity using the oligosaccharide derivative according to the present invention as a substrate, hydrogen azide or azide (as described in JP-A-63-183595, etc. In the presence of an activator such as an alkali metal salt of hydrogen azide or an alkaline earth metal salt),
There is a method of directly producing a dye without using a coupling enzyme. Other activators include thiocyanates (eg KSCN, NaSCN, etc.) and cyanates (eg KCNO).
Etc.) can also be effectively used.

As described in JP-A-63-183595, when an oligosaccharide derivative in which a sugar is not introduced into a non-reducing terminal glucose is used as a substrate, a glycosyl transfer reaction by α-amylase occurs and various glucose numbers There is a problem that a substrate is produced and only a part of the hydrolysis reaction of α-amylase can be measured, and various substrates are produced, and the reaction system becomes complicated. However, the oligosaccharide derivative according to the present invention When using as a substrate, all of these problems are solved.

A method for measuring α-amylase activity using the oligosaccharide derivative according to the present invention as a substrate (hereinafter, referred to as
It is abbreviated as "the measuring method according to the present invention". In (), the concentration of the oligosaccharide derivative used as the substrate is not particularly limited, but usually about 0.1 to 30 mM is preferably used.

The sample to be measured in the measuring method according to the present invention may be any sample containing α-amylase, for example saliva, pancreatic juice, blood, serum, etc. as biological components.
Examples include urine. Coupling enzyme glucoamylase, α-
The glucosidase, β-glucosidase, or isomaltase is not particularly limited, but those derived from animals, plants, and microorganisms can be used, and they can be used alone or in appropriate combination. The amount of these coupling enzymes used is usually 0.
It is 5 to 1000 U / ml, preferably 2 to 500 U / ml.

As the oligosaccharide derivative according to the present invention used for carrying out the assay method without using a coupling enzyme, those in which n in the general formula [2] is 0 to 2 are preferable, and n = 1 in the above. Or 2 is more preferable. In this case, it is usual to add an azide, thiocyanate or the like, which is known as an activator of α-amylase, but the present inventors have also added sodium chloride, potassium chloride and the like. Alkali metal salts and cyanates (eg KCNO
It has been found that the same effect can be obtained by adding the above).
The concentration of these activators used varies depending on the kind of the activator, but usually about 50 mmol / l to 10 mol / l is preferably used.

The reaction temperature is not particularly limited as the measuring conditions for carrying out the measuring method according to the present invention, but it is preferably about
The reaction time is 25 to 40 ° C, and the reaction time can be freely selected depending on 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 the activator of α-amylase, when an oligosaccharide derivative in which n is 3 or more in the general formula [2] is used as a substrate, for example, sodium chloride, calcium chloride, potassium chloride, etc. are used,
When an oligosaccharide derivative having n of 2 or less is used as a substrate, as described above, an azide, thiocyanate, cyanate or the like, or an alkali metal salt such as sodium chloride or potassium chloride or the former one is used. A combination of either of them and the latter is mentioned.

As couplers for coupling (oxidative condensation) with phenols or naphthols released by the action of a coupling enzyme, 4-aminoantipyrine and 3-methyl-2-
Benzothiazolinone hydrazone (MBTH), p-amino-N, N-
Examples thereof include, but are not limited to, diethylaniline and the like. Examples of oxidases for coupling (oxidative condensation) phenols or naphthols with couplers include laccase, catechol oxidase, tyrosinase, monophenol oxidase, and the like. Any one can be used, usually 0.2-10 U / ml, preferably 0.5
Used in the range of ~ 4U / ml. 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 hydrogen peroxide.

The oligosaccharide derivative according to the present invention has a sugar residue introduced at the 6-position and / or the 4-position of the non-reducing terminal glucose, and therefore, as it is, glucoamylase, α-glucosidase, β-glucosidase, or isoform. Since it does not serve as a substrate for maltase, and many of them are easily soluble in water, they have excellent affinity with α-amylase.
It becomes a good specific substrate for α-amylase. Therefore, in the assay method using the oligosaccharide derivative according to the present invention as a substrate, 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.

Further, in the assay method according to the present invention, a coupling enzyme such as glucoamylase, α-glucosidase, β-glucosidase or isomaltase can be sufficiently used, so that the reaction after the α-amylase reaction can be performed. A faster, more accurate and sensitive measurement of α-amylase activity can be performed.

Further, in the measuring method according to the present invention, the absorption spectrum of nitrophenols or indigo dyes liberated by detection is measured or phenols or naphthols liberated by 4-amino are measured. Oxidative coupling with antipyrine, MBTH, etc., or by measuring the absorption spectrum of the dye, or by measuring the fluorescence intensity of the free umbelliferone, etc., glucose coexisting in the sample, such as maltose It is hardly affected by sugars and reducing substances such as ascorbic acid and bilirubin.

The measuring method 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 a good adaptability to an automatic analyzer, and can be carried out by any of a manual method and an automatic analysis as required.

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

The oligosaccharide derivative according to the present invention is also a method for differentially measuring α-amylase derived from human salivary gland and α-amylase derived from human pancreas described in, for example, JP-A-63-39600, that is, α-amylase. Degradation products produced by the hydrolysis of amylase have different substrate specificities.
By performing a coupling enzyme of more than one species and measuring the resulting product, the fractional measurement of human pancreas-derived α-amylase and human salivary gland-derived α-amylase is performed. It can be sufficiently used as an effective substrate.

When an oligosaccharide derivative of the general formula [2] where n is 1 or 2 is used as a substrate, the α-amylase isoenzyme can be fractionally measured even with one type of conjugated enzyme.

That is, for example, when the case where n is 1 will be described as an example, the measurement principle is roughly as follows.

[0050]

[Formula 2]

(In the formula, G, R and R'are the same as above.)

That is, when α-amylase in the sample acts on the oligosaccharide derivative according to the present invention in which n = 1 in the general formula [2], the above two reactions (reaction (1) and reaction
(2)) occurs at a certain ratio according to the type of sugar introduced, and R'OH is produced by the reaction (2). Then reaction
When one or more coupling enzymes such as α-glucosidase, β-glucosidase and isomaltase act on the product of (1), R'OH is produced from G-OR 'by the reaction (3).

Therefore, the ratio of the amount of R'OH produced by the reaction (2) (per unit time) to the total amount of R'OH produced by the reaction (2) and the reaction (3) (per unit time) is calculated as follows. The α-amylase isozyme may be fractionally measured according to the method described in JP-A-61-181564, based on the obtained ratio.

In this case, it is desirable to add the above-mentioned α-amylase activator such as azide, thiocyanate, cyanate, sodium chloride and potassium chloride.

The oligosaccharide derivative according to the present invention further comprises human salivary gland-derived α-amylase (saliva-type α).
-Amylase) In the coexistence of an inhibitory substance, it can also be used as a substrate for pancreatic α-amylase activity measurement for specifically measuring human pancreatic α-amylase (pancreatic α-amylase) activity. At this time, if necessary, 1 to 3 kinds of the above-mentioned coupling enzymes are used, and if necessary, the above-mentioned α-amylase activity of azide, thiocyanate, cyanate, sodium chloride, potassium chloride and the like. Addition of an agent and the like are carried out in the usual manner.

Examples of salivary α-amylase inhibitors include:
For example, Clin. Chem 28/7 1525 to 1527 (1982) described wheat germ-derived inhibitor, may be used a monoclonal antibody that specifically suppresses salivary α-amylase, such a monoclonal antibody, for example, , JP Sho
58-183098, JP60-155134A, JP61
-164161, JP 61-194365, JP 63-
Examples thereof include those described in Japanese Patent No. 2600, but are not limited to these.

When the monoclonal antibodies are used, it is optional to use two or more kinds of monoclonal antibodies having different activities in combination. Examples will be given below, but the present invention is not limited to these examples.

[0058]

【Example】

Example 1. p-nitrophenyl O-β-D-glucopyranosyl
-(1 → 6) -O-α-D-Glucopyranosyl- (1 → 4) -tris [O-α
-D-glucopyranosyl- (1 → 4)]-α-D-glucopyranoside (hereinafter abbreviated as compound 1)

[0059]

[Chemical 6] Synthesis of

(1) Methyl 2,3,4,6-tetra-O-acetyl-1-
Thio-β-D-glucopyranoside (hereinafter abbreviated as compound a)

[0061]

[Chemical 7] Synthesis of

2.56 mmol of 2,3,4,6-tetra-O-acetylglucose (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in 6 ml of 1,2-dichloroethane, and methylthiotrimethylsilane was stirred at 0 ° C. (Hereinafter abbreviated as TMSSMe) 7.68 mmol and trimethylsilyl triflate (hereinafter abbreviated as TMSOTf) 0.76
8 mmol was added, and the mixture was reacted with stirring at 50 ° C. overnight. After the reaction was completed, the reaction solution was extracted with dichloromethane, and 1M Na ₂ CO ₃ was added.
The solution was washed with an aqueous solution and water in this order, and dried over anhydrous sodium sulfate. The syrup obtained by filtering off the desiccant and concentrating the filtrate under reduced pressure was subjected to column chromatography [Wakogel C-200
(Wako Pure Chemical Industries, Ltd. product name); Eluent (a) 1: 6 (b) 1: 5
(c) 1: 4 (ethyl acetate-hexane)], and the eluate (c)
Thus, 770 mg of Compound a (yield 79%) was obtained.

(2) Methyl 2,3,4,6-tetra-O-benzoyl-
1-thio-β-D-glucopyranoside (hereinafter abbreviated as compound b)

[0064]

[Chemical 8] Synthesis of

0.529 mmol of the compound a obtained in (1) was dissolved in 2 ml of methanol, 0.03 ml of a methanol solution containing about 28% sodium methoxide was added, and the mixture was allowed to stand at 25 ° C. for 3 hours to carry out the deacetylation reaction. I went. After completion of the reaction, the reaction solution was neutralized with ion exchange resin Amberlite IR-120 (H ⁺ ). The resin was filtered off, washed with methanol, and the filtrate and washings were combined and concentrated under reduced pressure. Dissolve the obtained syrup in dichloromethane and add 0.5 ml of pyridine and 3.45 mm of benzoyl chloride.
ol was added, and the mixture was reacted at 25 ° C. overnight with stirring. After the reaction was completed, 2 ml of methanol was added to decompose excess reagents, and the reaction solution was concentrated under reduced pressure. The residue is extracted with dichloromethane and 2M
It was washed with hydrochloric acid and water in this order, and dried over anhydrous sodium sulfate. The drying agent was filtered off, and the syrup obtained by concentrating the filtrate under reduced pressure was subjected to column chromatography [Wakogel C-200; eluent (a) 1:30 (b) 1:20 (acetone-hexane)], From the eluent (b), 367 mg of compound b was quantitatively obtained.

(3) p-Nitrophenyl O- (2,3,4,6-tetra-O-benzoyl-β-D-glucopyranosyl)-(1 → 6) -O-
(2,3-di-O-acetyl-α-D-glucopyranosyl)-(1 → 4)-
Tris [O- (2,3,6-tri-O-acetyl-α-D-glucopyranosyl)-(1 → 4)]-2,3,6-tri-O-acetyl-α-D-glucopyranoside (hereinafter , Abbreviated as compound c.)

[0067]

[Chemical 9] Synthesis of

Compound b (0.13 mmol) synthesized in (2) and p-nitrophenyl O- (2,3-di-O-acetyl-α-D-glucopyranosyl)-(1 → 4) -tris [O- (2 , 3,6-Tri-O-acetyl-α-
D-glucopyranosyl)-(1 → 4)]-2,3,6-tri-O-acetyl-
0.065 mmol of α-D-glucopyranoside (hereinafter abbreviated as 4,6-free-acetyl G5P) was dissolved in 1 ml of dichloromethane, 200 mg of molecular sieves 4A was added, and the mixture was stirred at 25 ° C. for 5 hours under a nitrogen stream. Cool to 35 ℃, N
-Iodosuccinimide (hereinafter abbreviated as NIS) 0.26 mmol
And trifluoromethanesulfonic acid (hereinafter abbreviated as TfOH)
0.052 mmol was added, and the mixture was reacted with stirring at -35 ° C for 3 hours. After the reaction was completed, the precipitate was filtered off through Celite, the filtrate was extracted with dichloromethane, and the solution was extracted with 1M Na ₂ CO ₃ aqueous solution and 1M Na ₂
It was washed with an aqueous solution of SO ₃ in this order and then dried over anhydrous sodium sulfate. The syrup obtained by filtering off the desiccant and concentrating the filtrate under reduced pressure was subjected to column chromatography [Wakogel C-20
0; eluate (a) 1: 0 (b) 150: 1 (c) 50: 1 (dichloromethane-methanol)], and the compound c 118m from the eluate (c)
g (86% yield) was obtained.

(4) Synthesis of Compound 1 0.025 mmol of the compound c synthesized in (3) was dissolved in 5 ml of methanol, 0.03 ml of a methanol solution containing 28% sodium methoxide was added, and the mixture was allowed to stand at 25 ° C. overnight. Then, deacetylation reaction was performed. After completion of the reaction, the reaction solution was neutralized with ion exchange resin Amberlite IR-120 (H ⁺ ). The resin is filtered off,
The syrup obtained by concentrating the filtrate under reduced pressure was subjected to column chromatography [Sephadex LH-20 (Pharmacia, trade name); eluent methanol, Sap-Pak (ENV. _T C ₁₈ , manufactured by Waters); eluate (a ) 1: 0 (b) 10: 1 (water-methanol)], and 28 mg of Compound 1 was quantitatively obtained from the eluate (b). The results of optical rotation [α] _D , IR and ¹ H NMR of Compound 1 are shown below.

[Α] _D + 258 ° (C = 0.23, CH ₃ OH) IRνcm ^-1 (KBr): 3400 (OH), 2940 (CH), 1520,1350 (N
O ₂ ), 870 (phenyl). ¹ H NMR δ ppm (CD ₃ OD): 4.12 (t, 1H, J = 9.1 Hz), 4.30
(D, 1H, J = 7.7Hz), 5.09 (d, 1H, J = 4.0Hz), 5.13,5.16
(2d, 2H, J = 3.8Hz), 5.22 (d, 1H, J = 4.0Hz), 5.67 (d, 1
H, J = 3.9Hz), 7.32 (2d, 2H, J = 9.3Hz), 8.23 (2d, 2H, J =
9.3Hz).

Example 2. p-nitrophenyl O-β-D-ribofuranosyl- (1 → 6) -O-α-D-glucopyranosyl- (1 → 4)
-Tris [O-α-D-glucopyranosyl- (1 → 4)]-α-D-glucopyranoside (hereinafter abbreviated as compound 2)

[0072]

[Chemical 10] Synthesis of

(1) Methyl 2,3,5-tri-O-benzoyl-1-thio-β-D-ribofuranoside (hereinafter abbreviated as compound d)

[0074]

[Chemical 11] Synthesis of

2.96 mmol of 2,3,5-tri-O-benzoyl-D-ribose (manufactured by Aldrich) was added to 40 ml of 1,2-dichloromethane.
Dissolve in, and stir at 0 ° C with TMSSMe 11.9 mmol and TMS
1.19 mmol of OTf was added, and the mixture was stirred and reacted at 50 ° C. for 1 hour.
After the reaction is completed, the reaction solution is extracted with dichloromethane and the reaction mixture is adjusted to 1M.
It was washed with an aqueous Na ₂ CO ₃ solution and water in this order and then dried over anhydrous sodium sulfate. The desiccant was filtered off, the filtrate was concentrated under reduced pressure, and the resulting syrup was subjected to column chromatography [Wakoge
l C-200; eluate (a) 1:30 (b) 1:25 (c) 1:20 (acetone-hexane)], and compound d 1.07 g from the eluate (b)
(Yield 87%) was obtained.

(2) p-nitrophenyl O- (2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)-(1 → 6) -O- (2,3-di-O-acetyl -α-D-glucopyranosyl- (1 → 4) -tris [O- (2,3,6-
Tri-O-acetyl-α-D-glucopyranosyl)-(1 → 4)]-2,
3,6-tri-O-acetyl-α-D-glucopyranoside (hereinafter,
It is abbreviated as compound e. )

[0077]

[Chemical 12] Synthesis of

Compound d 0.159 mmol synthesized in (1) and 4,6-
Free-Acetyl G5P 0.133mmol dichloromethane 1m
Dissolve in l, add 300 mg of molecular sieves 4A, stir at 25 ° C for 5 hours under nitrogen stream, cool to -40 ° C, add NIS 0.311mmol and TfOH 0.0311mmol, stir at -40 ° C for 2 hours, It was made to react. After completion of the reaction, (3) of Example 1
Post-treatment was carried out in the same manner as in the above to obtain compound e 214 mg (yield 82
%) Was obtained.

(3) Synthesis of Compound 2 0.108 mmol of the compound e synthesized in (2) was dissolved in 10 ml of methanol, cooled to 0 ° C., and 0.06 ml of a methanol solution containing about 28% sodium methoxide was added, The reaction was allowed to stir overnight at 7 ° C. After completion of the reaction, post treatment was carried out in the same manner as in (4) of Example 1 to quantitatively obtain 128 mg of Compound 2. still,
The results of optical rotation [α] _D , IR and ¹ H NMR of Compound 2 are shown below.

[Α] _D + 138 ° (C = 0.39, CH ₃ OH) IRνcm ^-1 (KBr): 3400,1020 (OH), 2940 (CH), 1510,1340
(NO ₂ ), 860 (phenyl). ¹ H NMR δppm (CD ₃ OD): 3.22 (t, 1H, J = 9.4Hz), 4.11
(D, 1H, J = 9.7Hz), 4.12 (dd, 1H, J = 4.8Hz and 6.8H
z), 4.89 (s, 1H), 5.07 (d, 1H, J = 3.7Hz), 5.14,5.15
(2d, 2H, J = 4.2Hz), 5.22 (d, 1H, J = 3.7Hz), 5.66 (d,
1H, J = 3.7Hz), 7.32 (2d, 2H, J = 9.2Hz), 8.22 (2d, 2H, J
= 9.2Hz).

Example 3. p-nitrophenyl O-β-D-ribofuranosyl- (1 → 6)-[O-β-D-ribofuranosyl- (1 → 4)]-O
-α-D-glucopyranosyl- (1 → 4) -tris [O-α-D-glucopyranosyl- (1 → 4)]-α-D-glucopyranoside (hereinafter abbreviated as compound 3)

[0082]

[Chemical 13] Synthesis of

(1) p-Nitrophenyl O- (2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)-(1 → 6)-[O- (2,3,5-tri- O-benzoyl-β-D-ribofuranosyl)-(1 → 4)] O- (2,3
-Di-O-acetyl-α-D-glucopyranosyl- (1 → 4) -tris
[O- (2,3,6-tri-O-acetyl-α-OD-glucopyranosyl)
-(1 → 4)]-2,3,6-Tri-O-acetyl-α-D-glucopyranoside (hereinafter abbreviated as compound f)

[0084]

[Chemical 14] Synthesis of

Compound d 0.790 m synthesized in (1) of Example 2
mol and 4,6-free-acetyl G5P 0.130mmol are dissolved in dichloromethane 2ml, and molecular sieves 4A 50 is dissolved.
After adding 0 mg and stirring under a nitrogen stream at 25 ° C for 5 hours, -4
Cool to 0 ° C., add NIS 1.38 mmol and TfOH 0.138 mmol,
The mixture was stirred and reacted at -40 ° C overnight. After completion of the reaction, Example 1
Post-treatment was carried out in the same manner as in (3) above to obtain 279 mg of compound f (yield 88%).

(2) Synthesis of Compound 3 0.115 mmol of the compound f synthesized in (1) was dissolved in 28 ml of methanol, cooled to 0 ° C., and 0.09 ml of a methanol solution containing about 28% sodium methoxide was added, The mixture was stirred and reacted at 7 ° C. for 33 hours. After completion of the reaction, post treatment was carried out in the same manner as in (4) of Example 1 to obtain 136 mg of compound 3 (yield 98%). The results of optical rotation [α] _D , IR and ¹ H NMR of Compound 2 are shown below.

[Α] _D + 117 ° (C = 0.53, CH ₃ OH) IRνcm ⁻¹ (KBr): 3400,1020 (OH), 2940 (CH), 1510,1340
(NO ₂ ), 860 (phenyl). ¹ H NMR δppm (CD ₃ OD): 4.11 (d, 1H, J = 8.8Hz), 4.13
(Dd, 1H, J = 4.6Hz and 6.8Hz), 4.27 (dd, 1H, J = 4.6Hz a
nd 7.3Hz), 4.91, 4.92 (2s, 2H), 5.11 (d, 1H, J = 3.7H
z), 5.15,5.16 (2d, 2H, J = 3.8Hz and 3.7Hz), 5.23
(D, 1H, J = 3.7Hz), 5.67 (d, 1H, J = 3.7Hz), 7.32 (2d, 2
H, J = 9.3Hz), 8.22 (2d, 2H, J = 9.3Hz).

Example 4. p-nitrophenyl O-β-D-ribofuranosyl- (1 → 4) -O-α-D-glucopyranosyl- (1 → 4)-
Tris [O-α-D-glucopyranosyl- (1 → 4)]-α-D-glucopyranoside (hereinafter abbreviated as compound 4)

[0089]

[Chemical 15] Synthesis of

(1) p-nitrophenyl O- (2,3-di-O-acetyl-6-O-benzoyl-α-D-glucopyranosyl)-(1 → 4) -tris [O- (2,3 , 6-Tri-O-acetyl-α-OD-glucopyranosyl)-(1 → 4)]-2,3,6-tri-O-acetyl-α-D-glucopyranoside (hereinafter referred to as 4-free-acyl G5P (Abbreviated.)

[0091]

[Chemical 16] Synthesis of

0.130 mmol of 4,6-free-acetyl G5P was dissolved in 20 ml of dichloromethane and then cooled to 0 ° C., 3.90 mmol of pyridine and 0.390 mmol of benzoyl chloride were added, and the mixture was stirred and reacted at 25 ° C. overnight. After completion of the reaction, the reaction solution was extracted with dichloromethane, washed with 2M hydrochloric acid and water in this order, and then dried over anhydrous sodium sulfate. The syrup obtained by filtering off the desiccant and concentrating the filtrate under reduced pressure was subjected to column chromatography [Wakogel C-200; eluent (a) 100: 1 (b).
50: 1 (dichloromethane-methanol)], and 185 mg (yield 87%) of 4-free-acyl G5P was obtained from the eluate (b).

(2) p-Nitrophenyl O- (2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)-(1 → 4) -O- (2,3-di-O-
Acetyl-6-O-benzoyl-α-D-glucopyranosyl)-(1
→ 4) -Tris [O- (2,3,6-tri-O-acetyl-α-D-glucopyranosyl)-(1 → 4)]-2,3,6-Tri-O-acetyl-α-D -Glucopyranoside (hereinafter abbreviated as compound g)

[0094]

[Chemical 17] Synthesis of

Compound d 0.274m synthesized in (1) of Example 2
mol and 4-free-acyl G5P 0.0609 synthesized in (1) above
Dissolve mmol in dichloromethane, add 150 mg of Molecular Sieves 4A, stir overnight at 25 ° C under nitrogen flow, then cool to -35 ° C. NIS 0.548 mmol and TfOH 0.0548 mmol.
Was added, and the mixture was stirred and reacted at -35 ° C overnight. After the reaction,
Post-treatment was carried out in the same manner as in (3) of Example 1 to give compound g
87 mg (yield 73%) was obtained.

(3) Synthesis of Compound 4 0.044 mmol of the compound g synthesized in (2) was dissolved in 10 ml of methanol, cooled to 0 ° C., and 0.03 ml of a methanol solution containing about 28% sodium methoxide was added, The mixture was stirred and reacted at 7 ° C. overnight. After completion of the reaction, post treatment was carried out in the same manner as in (4) of Example 1 to obtain 44 mg of compound 4 (yield 94%). The results of optical rotation [α] _D , IR and ¹ H NMR of compound 4 are shown below.

[Α] _D + 146 ° (C = 0.88, CH ₃ OH) IRνcm ^-1 (KBr): 3300 (OH), 2940 (CH), 1520,1350 (N
_{O 2), 850 (phenyl)} . ¹ H NMR δppm (CD ₃ OD): 4.11 (t, 1H, J = 9.2Hz), 4.27
(Dd, 1H, J = 4.6Hz and 7.3Hz), 4.97 (s, 1H), 5.14,5.
15,5.17 (3d, 3H, J = 3.5Hz, 3.8Hz and 4.0Hz), 5.23 (d,
1H, J = 3.9Hz), 5.67 (d, 1H, J = 3.7Hz), 7.32 (2d, 2H, J =
9.3Hz), 8.22 (2d, 2H, J = 9.3Hz).

As is clear from the results of Examples 1 to 4, according to the production method of the present invention, the target non-reducing end-modified oligosaccharide derivative can be obtained by a simple operation and in a high yield (for example, The yield of glucose introduction was 43% in the König-Kunner reaction, whereas it was 86% in the method of the present invention.)

The ribose introduction positions are the compounds d and 4,
It is also possible to adjust by the equivalent ratio of 6-free-acetyl G5P.

Reference Example 1. Measurement of Hydrolysis Rate and Km Value by α-Amylase of Various Oligosaccharide Derivative Substrates

(Procedure) Using various oligosaccharide derivatives of compounds 1 to 4 as substrates, 50 mM BES [N, N-bis
(2-Hydroxyethyl) -2-aminoethanesulfonic acid] -N
In an aOH buffer solution (pH 7.6, containing ₂₀ mM NaCl and 2 mM CaCl ₂ ), the Km value (mM) and the hydrolysis rate (Vmax) were determined by the usual methods.

(Results) Table 1 shows the measurement results. The rate of hydrolysis is (p-nitrophenyl) maltopentaoside (G5P).
It was shown by the relative value which made the water-dissolution rate with respect to 1.

Reference Example 2. Retrieval of Hydrolysis Position of Various Oligosaccharide Derivative Substrates by α-Amylase The hydrolysis position of each oligosaccharide derivative substrate used in Reference Example 1 was examined.

(Procedure) 50 mM BES [N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid] -NaOH buffer (p
H7.6, 20mM NaCl and 2mM CaCl ₂ containing) substrate 1.5mM solution 50μl, human pancreatic α-amylase (HPA) or human salivary gland α-amylase (HSA) 5μl mixed, 37 Warm at 5 ° C for 5-10 minutes. The enzyme reaction was stopped by adding 100 μl of 7.5% acetic acid solution to the reaction mixture. The amount of hydrolyzed product due to α-amylase in the sample was determined by HPLC.

(Results) Table 1 also shows the measurement results.

[0106]

[Table 1]

As is clear from Table 1, the series of oligosaccharide derivatives according to the present invention, as a substrate for measuring α-amylase activity, (1) have a high hydrolysis rate (all except compound 3 are faster than G5P), (2 ) It can be used as a substrate having a better fractionation than the substrate FG5P in the fractionation measurement method described in JP-A-63-39600 (especially compound 2 is FG5P).
It has about 1.69 times more distinction than that of).

Reference Example 3. Measurement of α-amylase activity (measurement reagent solution) Compound 4. In addition, 90 mg each of (p-nitrophenyl) maltopentaoside (G5P) was added to 400 units of glucoamylase, 300 units of α-glucosidase, 20 mmol / l NaCl and 2 mm.
50 mmol / l BES-NaOH buffer containing ol / l CaCl ₂ (pH 7.6) 30 m
It was dissolved in 1 l and used as a measurement test solution.

(Measurement method) 10 ml of sample serum was added to 2 ml of the measurement reagent solution.
0 μl was added and the mixture was 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. The relationship between the α-amylase activity (Somogyi unit / dl) at each dilution step of the standard sample and the increase in absorbance (ΔA) per minute at a wavelength of 405 nm is shown in FIG. 1.

In FIG. 1, − ◯ − shows the results when Compound 4 was used, and −x− shows the results when G5P was used.

As is clear from FIG. 1, the calibration curve connecting the absorbance increase amount (ΔA / min) plotted against the α-amylase activity (Somogyi unit / dl) is a straight line passing through the origin in both cases, and the calibration curve Shows good quantification,
Compound 4 was about 1.7 times more sensitive than G5P.

Reference Example 4. Fractional measurement of α-amylase activity (Test solution) 1st solution Compound 2 and p-nitrophenyl O- [6-deoxy-6-{(2-
Pyridyl) amino} -α-D-glucopyranosyl]-(1 → 4) -tris {O-α-D-glucopyranosyl- (1 → 4)}-α-D-glucopyranoside (FG5P) 300 mg each and isomaltase 25 units 50m
l 50 mM BES-NaOH buffer (pH 7.6, 20 mmol NaCl and 2 mmol CaCl
Including ₂ . ) And were used as the first liquid. Second solution 300 mg each of compound 2 and FG5P and 1150 units of α-glucosidase were added to 50 ml of 50 mM BES-NaOH buffer solution (pH 7.6, 20 mmol NaCl and 2 mmo).
Contains lCaCl ₂ . ) And were used as the second liquids. Enzyme sample solution Human salivary α-amylase (manufactured by Sigma) and human pancreatic α-amylase prepared from human pancreatic juice were adjusted to 5.5 units / l, and then mixed at a ratio of 3: 1, 1: 1, 1: 3. did.

(Measurement method) To 300 μl of the first or second liquid preheated to 30 ° C., 50 μl of the enzyme sample solution was added, mixed well, and kept at 30 ° C., and the absorbance at 400 nm of this reaction solution was measured. When using the 1st liquid, change the
When 50 μl was added and mixed well, and when the second solution was used, 50 μl of purified water was added to the second solution and well mixed, and the results were measured as a blind test. Then, the rate of change in absorbance (ΔE ₁ / min) in the first solution and the rate of change in absorbance (ΔE ₂ / min) in the second solution were calculated to calculate ΔE ₁ / min / ΔE ₂ / min. . The relationship between ΔE ₁ / min / ΔE ₂ / min and the activity ratio of human pancreatic α-amylase in the sample solution is shown in FIG. In FIG. 2,-○-indicates the result when Compound 2 was used, and -X-
-Indicates the results when FG5P was used. As is clear from FIG. 2, a good linear relationship was obtained between ΔE ₁ / min / ΔE ₂ / min and the activity ratio of human pancreatic α-amylase in both cases, but in the case of compound 2, It is about 1.69 times more discriminating than FG5P.

[0114]

INDUSTRIAL APPLICABILITY The present invention provides a novel method for producing a modified maltooligosaccharide derivative in which a sugar residue is introduced into non-reducing terminal glucose, which can be an excellent substrate for measuring α-amylase activity. According to the production method of the invention, (1) the modified malto-oligosaccharide derivative can be produced with a much higher yield than that of the existing enzymatic method, and (2) the sugar residue that can be introduced into the non-reducing terminal glucose. It is a great invention that has a remarkable effect in that the group is not limited and contributes to the related art.

[0115]

[Brief description of drawings]

FIG. 1 shows the relationship between the α-amylase activity (Somogyi unit / dl) and the increase in absorbance per minute (ΔA) at a wavelength of 405 nm obtained in Reference Example 3.

FIG. 2 ΔE ₁ / min / ΔE ₂ / min obtained in Reference Example 4
The relationship between the ratio of the rate of change in absorbance in the first solution and the rate of change in absorbance in the second solution and the activity ratio of human pancreatic α-amylase in the sample solution is shown.

[Explanation of symbols]

In FIG. 1, − ◯ − shows the results when the compound 4 of the present invention was used, and −x− shows the results when the existing G5P was used. In FIG. 2,-○-shows the results when the compound 2 of the present invention was used, and -x- shows the results when the existing FG5P was used.

Claims (1)

[Claims] 1. A sugar in which one of the hydroxyl groups is substituted with —SR (R represents a lower alkyl group or an allyl group) and the remaining functional groups are protected with an acyl group, and a non-reducing terminal glucose of 4, The following general formula [1] in which hydroxyl groups other than the 6-position are protected by an acyl group: [In the formula, R ¹ represents an acyl group, R ² represents an optically detectable group or an acyl group, and n represents an integer of 0 to 5. ] The sugar is introduced at the 6-position and / or the 4-position of the non-reducing terminal, which is characterized by conducting a condensation reaction with an oligosaccharide derivative represented by The following general formula [2] [In the formula, R ³ and R ⁴ each independently represent a sugar residue or a hydrogen atom (provided that R ³ and R ⁴ are both hydrogen atoms), and R ⁵ is a general formula [1]. When R ^{2 of} is an optically detectable group, it also represents an optically detectable group, and R ²
Is a hydrogen atom. n is the same as above. ] The manufacturing method of the oligosaccharide derivative shown by these. [0001]