JPH0686474B2 - 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]

Α-in samples, especially saliva, pancreatic juice, blood and urine in human body
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,
Methods using oligosaccharides such as maltopentaose, maltohexaose and maltoheptaose as substrates (JP-A-50-56998, JP-A-53-37096) and chromogens such as p-nitrophenol A method using a substrate having a uniform and well-defined structure, such as a method using an oligosaccharide bound to the reducing end (Japanese Patent Laid-Open No. 54-51892), replaces the method using starch, which has been widely used, with amylase measurement. The law is becoming mainstream.

In these methods, usually, α-glucosidase (EC3.2.1.20; α-D-glucoside glucohydralolase) 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, and they are substrates regardless of the α-amylase reaction. Has the drawback of decomposing. 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, a primary alcohol (-CH ₂ O) at the 6-position of non-reducing terminal glucose of a linear oligosaccharide consisting of 4 to 7 glucose is used.
There is a method for measuring α-amylase activity using an oligosaccharide derivative having the following structural formula in which H) is substituted with a group represented by the general formula —CH ₂ R (JP-A-59-59).
No. 51800).

(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 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, to measure α-amylase activity using these substrates, high performance liquid chromatography method or α
-Glucosidase, β-glucosidase or glucoamylase must be a method for measuring glucose produced using a coupling enzyme, the former requires special equipment, the latter is affected by glucose contained in the sample There was a problem in receiving it.

Therefore, the present inventors further conducted research and found an α-amylase activity measuring method which does not have such a problem, and applied for a patent thereof (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 reduction is performed. The following structural formula [A] in which the 1-position of the 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 (Wherein R ₀ ^{3 to} R ₀ ⁶ represent hydrogen, a lower alkyl group, a lower alkoxy group, a nitro group, a carboxyl group, a sulfone group or a halogen, which may be the same or different, and R ₀ ⁷ represents hydrogen, a lower alkoxy group, a halogen or a nitro group. Further, R ₀ ⁸ represents a hydrogen or a methyl group.). ] The method for measuring α-amylase activity using the oligosaccharide derivative represented by This assay method is an excellent assay method that solves all the various problems of the conventional α-amylase activity assay method, but is practically used because the synthetic yield of the substrate used is extremely low. ) Remains a problem. Therefore, α-
Under the present circumstances, the advent of a novel and effective production method capable of obtaining a non-reducing end-modified oligosaccharide derivative which can be an excellent substrate for measuring amylase activity in a higher yield is awaited.

[Object of the Invention]

The present invention has been made in view of the above situation, and α-
It is an object of the present invention to provide a production method by which a non-reducing end-modified oligosaccharide derivative useful as a substrate for measuring amylase activity can be easily obtained in high yield.

[Structure of Invention]

The present invention, the 1-position hydroxyl group of the reducing terminal glucose may have a phenoxy group which may have a substituent, a naphthoxy group which may have a substituent, an umbelliferyl group which may have a substituent, Non-reducing terminal glucose of a modified oligosaccharide substituted with an indoxyl group which may have a substituent or a fructose residue is reacted with aldehydes, ketones, acetals or ketals to give 4,6-O.
-Cyclic acetal or 4,6-O-cyclic ketal, which is then reduced to give the general formula [I] [In the formula, R ¹ is a benzyl group, a substituted benzyl group (the substituent is
It represents a lower alkyl group, a lower alkoxy group, an alkyl-substituted amino group, a carboxyl group, a nitro group or a halogen atom. ), 2-, 3- or 4-pyridylmethyl group, carbon number 1
~ 6 linear, branched or cyclic alkyl group or alkenyl group having 1 to 6 carbon atoms, R ² is (However, R ^{3 to} R ⁶ represent a hydrogen atom, a lower alkyl group, a lower alkoxy group, a nitro group, a carboxyl group, a sulfonic acid group or a halogen atom, which may be the same or different, and R 3 ³ and R ⁵ or R ⁴ and R ⁶ may combine to form an aromatic ring, R ⁷ represents a hydrogen atom, a lower alkoxy group, a halogen atom or a nitro group, and R ⁸ represents a hydrogen atom. , A methyl group or a trifluoromethyl group, and R ⁹ represents a hydrogen atom or a halogen atom) or And n represents an integer of 2 to 5. ] It is a manufacturing method of the oligosaccharide derivative shown by these.

The production method of the present invention has the formula [II] in which the 1-position hydroxyl group of the reducing terminal glucose is substituted with the group represented by R ² above. It is usually carried out as follows using the reducing end-modified oligosaccharide derivative represented by

That is, first, an aldehyde, a ketone, an acetal or a ketal is allowed to act on the oligosaccharide derivative represented by the formula [II] to form a cyclic acetal group between the 4-position and the 6-position of non-reducing terminal glucose. . As the oligosaccharide derivative represented by the formula [II], a commercially available product may be used as it is, or it may be synthesized according to the method described in, for example, JP-A-54-51892. Examples of aldehydes used in the reaction include aromatic aldehydes such as benzaldehyde and tolylaldehyde, and aliphatic aldehydes such as acetaldehyde, propionaldehyde and butyraldehyde, and ketones such as acetone and acetone. Examples thereof include methyl ethyl ketone, examples of acetals include methylal, benzaldehyde dimethyl acetal, acetaldehyde dimethyl acetal, acetaldehyde diethyl acetal, and the like, and examples of ketals include methyl ethyl ketone ethylene ketal, cyclohexanone dimethyl ketal, and the like. , But not limited to these, cyclic acetalization or 4 or 6-position of the non-reducing terminal glucose of the oligosaccharide derivative represented by the formula [II] or Aldehydes can Jo ketalized, ketones,
Any acetal or ketal may be used. The molar amount used is usually about 1 to 100 times the molar amount of the oligosaccharide derivative. The reaction is usually performed in the presence of a Lewis acid catalyst such as p-toluenesulfonic acid and zinc chloride in a suitable solvent such as dimethylformamide (DMF). The reaction temperature is usually room temperature to reflux temperature, and the reaction time is Usually, it is about 0.5 to 12 hours, though it depends on the reaction temperature.

The cyclic acetal compound thus obtained is usually subjected to a reduction step after modifying other hydroxyl groups by acylation.

For the acylation reaction, for example, acetic anhydride, acetyl chloride, benzoyl chloride or the like may be used as an acylating agent in the presence of a base such as pyridine, and this may be performed according to a conventional method.

The acylation reaction is not essential, but it is preferable in the sense of increasing the solubility in the reaction solvent in the next step (reduction step).

In the reduction step, sodium cyanoborohydride, sodium borohydride, lithium aluminum hydride, polyzine borane, dimethylamine borane, trimethylamine borane, tert-butylamine borane, diborane, etc. are usually used as a reducing agent, and aluminum chloride,
In the presence of an acid catalyst such as boron trifluoride, zinc chloride, p-toluenesulfonic acid, methanesulfonic acid, hydrogen chloride gas, etc. in a suitable solvent (for example, tetrahydrofuran (THF) etc.),
Usually, the reaction is carried out at 0 to 50 ° C for several tens of minutes to several hours. The amount of the reducing agent used is usually 1 to 1000 times the mol of the oligosaccharide derivative, and the amount of the acid catalyst used is usually about 0.5 to 5 mol of the reducing agent.

In the case of being acyl-protected, after the reduction, deacylation may be carried out by a conventional method, for example, by treating with 0.01 to 1.0 N sodium methylate / methanol solution for several hours to several tens hours at room temperature or under slight heating. Thus, the desired non-reducing end-modified oligosaccharide derivative can be easily obtained. Post-treatment after the reaction in each step may be carried out in accordance with a conventional method,
Further, the purification of the final product may be performed according to a conventional method such as column chromatography.

The α-1,4 bond of oligosaccharides is hydrolyzed under acidic conditions and is subjected to methanolysis under anhydrous methanol-HCl conditions, or because of glycoside bond (reducing terminal glucose and glucose) under acidic conditions. Bond with the group represented by R ² )
It is unclear whether the oligosaccharide derivative derived from the oligosaccharide derivative represented by the formula [II] can withstand such a reduction treatment because the stability of is unknown. Moreover, the present inventors have for the first time found that the cyclic acetal portion can be opened without any inconvenience by the above reduction treatment, and have completed the present invention.

In the oligosaccharide derivative represented by the general formula [I] that can be produced by the method of the present invention, R ¹ of —CH ₂ OR ¹ at 6-position of non-reducing terminal glucose is benzyl group or substituted benzyl group (substituent Are, for example, methyl group, ethyl group,
Lower alkyl groups such as propyl group and butyl group, eg lower alkoxy groups such as methoxy group, ethoxy group, propoxy group, butoxy group, eg dimethylamino group, diethylamino group, N-ethyl-N- (β-hydroxyethyl) amino Examples thereof include substituted amino groups such as groups, carboxyl groups, nitro groups, and halogen atoms such as chlorine, bromine, and iodine. ), 2-, 3- or 4-pyridylmethyl group such as methyl group, ethyl group, iso-propyl group, n-butyl group, t
ert-butyl group, n-pentyl group, iso-amyl group, n-hexyl group, cyclopentyl group, cyclohexyl group and the like, straight-chain, branched or cyclic alkyl group having 1 to 6 carbon atoms, such as ethenyl group, 2 -Propenyl group, 2-butenyl group and other carbon atoms 1
~ 6 alkenyl groups and the like. In addition, in -R ² at the 1-position of the reducing terminal glucose of the general formula [I], As the phenoxy group which may have a substituent or the naphthoxy group which may have a substituent,
Glucoamylase [EC
3.2.1.3.], Α-glucosidase [EC 3.2.1.20.],
β-glucosidase [EC 3.2.1.21.], isomaltase [EC 3.2.1.10.] 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 hydrolyzing, those which themselves have absorption in the visible part, such as nitrophenols, or catechol oxidase, laccase, tyrosinase or Any one may be used as long as it is coupled with a coupler to give a dye under the action of an oxidase such as monophenol oxidase, or is coupled with a coupler with an oxidizing agent to give a dye. Examples of R ² satisfying such conditions include, for example, p-nitrophenoxy group, m-nitrophenoxy group, o-chlorophenoxy group, p-chlorophenoxy group, 2,6-dichlorophenoxy group, 2 -Chloro-4-nitrophenoxy group, o-methoxyphenoxy group, p-methoxyphenoxy group, o-methylphenoxy group, o-carboxyphenoxy group, o-sulfophenoxy group, 1-naphthoxy group, 2-sulfo-1 Examples thereof include, but are not limited to, a -naphthoxy group and a 2-carboxy-1-naphthoxy group.

Also, As a specific example of an umbelliferyl group which may have a substituent, R ⁸ is an umbelliferyl group having a hydrogen atom or R ⁸ is a 4-methylumbelliferyl group having a methyl group or R ⁸ is 4-trifluoromethyl umbelliferyl group etc. of a trifluoromethyl group are mentioned.

Furthermore, Specific examples of the indoxyl group which may have a substituent represented by are, for example, an indoxyl group, a 5-bromoindoxyl group, a 4-chloro-3-bromoindoxyl group and the like.

In addition to these as R ² , The fructose residue represented by, for example, can be used to measure amylase activity by measuring the degree of NADH reduction that occurs when NADH is reacted with mannitol dehydrogenase after hydrolysis with a coupling enzyme. .

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.

Examples will be shown below, but the present invention is not limited to these examples.

〔Example〕

Example 1. p-Nitrophenyl 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 BF5P) .) Synthesis of p-nitrophenyl-α-D-maltopentaoside 3 g
After dissolving in 50 ml of DMF, add 15 ml of benzaldehyde dimethyl acetal and 1 g of p-toluenesulfonic acid (anhydrous),
The reaction was allowed to stir for 3 hours at ° C. After the reaction, 100 ml of pyridine was added, and 60 ml of benzoyl chloride was added dropwise under ice-cooling to react, and the reaction solution was poured into 200 ml of a saturated sodium hydrogen carbonate solution to stop the reaction. After extraction with 200 ml of chloroform and washing twice with saturated saline, the solvent was distilled off. The obtained syrup was transferred to a 14-neck Korben, dissolved in 250 ml of THF, and then dissolved by adding 3 g of dimethylamine borane. To this solution, 10 ml of a solution prepared by adding hydrochloric acid gas to diethyl ether to 0.5 N was added dropwise with stirring, and the reaction solution was poured into 200 ml of a saturated sodium hydrogen carbonate solution to stop the reaction.
extracted with l. After washing twice with saturated saline, the solvent was distilled off,
500 ml of methanol solution containing 0.1N sodium methylate
Was added and the mixture was stirred at 25 ° C. for 4 hours, and acetic acid was added to neutralize. After the solvent was distilled off, 20 ml of a 50 mM acetic acid solution was added, and the mixture was charged on a Biogel P-2 (Bio-Rad, 2 cmφ × 150 cm) column equilibrated with 50 mM acetic acid, and the BG5P fraction was collected and freeze-dried.

Yield 450 mg.

IR chart: As shown in Figure 1. ¹ H-NMR: As shown in FIG.

HPLC: content 95% [column: packing = silica gel Z-ODS, 5
C ₁₈ (Wako Pure Chemical Industries, Ltd. product name, 4 × 150 mm), eluent: 10% to 80% of acetonitrile aqueous solution containing 0.1% acetic acid
Gradient, measured at 305 nm. ].

GC composition analysis: glucose / 6-O-benzyl-glucose = 3.8: 1.

Example 2. p-Nitrophenyl 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) -O-α-D-glucopyranosyl-
Synthesis of (1 → 4) -α-D-glucopyranoside (hereinafter abbreviated as BF6P) 3 g of p-nitrophenyl α-D-maltohexaoside
After dissolving in 50 ml of DMF, add 15 ml of benzaldehyde dimethyl acetal and 1 g of p-toluenesulfonic acid (anhydrous),
The reaction was allowed to stir for 3 hours at ° C. After the reaction, 100 ml of pyridine was added and 80 ml of benzoyl chloride was added dropwise under ice cooling to react.
The reaction solution was poured into 300 ml of a saturated sodium hydrogen carbonate solution to stop the reaction. The mixture was extracted with 200 ml of chloroform, washed twice with saturated saline and the solvent was distilled off. The obtained syrup was transferred to a 14-neck Korben, dissolved in 250 ml of THF, and then dissolved by adding 3 g of dimethylamine borane. To this solution, 10 ml of a 0.5 N solution of hydrochloric acid blown into diethyl ether was added dropwise with stirring, the reaction solution was poured into 300 ml of a saturated sodium hydrogen carbonate solution to stop the reaction, and then 200 m of chloroform was added.
extracted with l. After washing twice with saturated saline, the solvent was distilled off,
500 ml of methanol solution containing 0.1N sodium methylate
Was added, and the mixture was stirred at 25 ° C. for 4 hours and then acetic acid was added to neutralize. After distilling off the solvent, 20 ml of a 50 mM acetic acid solution was added, and the column was charged on a Biogel P-2 (Bio-Rad, 2 cmφ × 150 cm) column equilibrated with 50 mM acetic acid, and the BG6P fraction was collected and freeze-dried.

Yield 500mg.

HPLC: content 94% (measurement conditions are the same as in Example 1).

GC composition analysis: glucose / 6-O-benzyl-glucose = 4.9: 1.

Example 3. p-Nitrophenyl 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 BF5P) .) Synthesis of 5 g of p-nitrophenyl α-D-maltopentaoside
After dissolving in 40 ml of DMF, 50 ml of methylal and 1 g of p-toluenesulfonic acid (anhydrous) were added, and the mixture was reacted with stirring at 50 ° C. for 3 hours. After the reaction was concentrated in vacuo, pyridine (300 ml) and acetic anhydride (100) were added.
After adding 12 ml to the reaction solution at room temperature for 12 hours, 500 ml of a saturated sodium hydrogen carbonate solution was added to the reaction solution to stop the reaction. Chloroform (100 ml) was added for extraction, the extract was washed twice with saturated saline and the solvent was distilled off. 14 syrups obtained
Transfer to cervical Kolben and dissolve in 250 ml of THF, then use pyridine borane.
4 g was added and dissolved. To this solution, 10 ml of a 0.5 M p-toluenesulfonic acid THF solution was added dropwise with stirring, the reaction solution was poured into 200 ml of a saturated sodium hydrogen carbonate solution to stop the reaction, and then extracted with 200 ml of chloroform. After washing twice with a saturated saline solution, the solvent was distilled off, and 700 ml of a methanol solution containing 0.1N sodium methylate was added to the solution. After reacting at 25 ° C. for 4 hours, acetic acid was added to neutralize the solution. After the solvent was distilled off, Biogel P-2 (Bio-Rad, 2 cmφ × 150) equilibrated with 50 mM acetic acid was used.
cm) column and the BG5P fraction was collected and freeze-dried.

Yield 730mg.

HPLC: content 93% (measurement conditions are the same as in Example 1).

〔The invention's effect〕

The present invention provides a novel and effective method for producing a non-reducing end-modified oligosaccharide derivative useful as a substrate for measuring α-amylase activity or a substrate for separately measuring each isozyme activity of human α-amylase. According to the method of the present invention, a non-reducing end-modified oligosaccharide derivative can be obtained with a simple reaction procedure and in good yield, and therefore, an α-amylase activity measuring method or isozyme of the same using these as a substrate can be used. Contribution to the practical application (commercialization) of the differential measurement method has a remarkable effect on the very large point.

[Brief description of drawings]

1 shows an IR chart of the non-reducing end-modified oligosaccharide derivative obtained in Example 1, and FIG. 2 shows Example 1
Of the non-reducing end-modified oligosaccharide derivative obtained in
An NMR chart is shown.

Claims (2)

[Claims] 1. A phenoxy group which may have a substituent on the 1-position hydroxyl group of reducing terminal glucose, a naphthoxy group which may have a substituent, and an umbelliferyl group which may have a substituent. , A non-reducing terminal glucose of a modified oligosaccharide substituted with an optionally substituted indoxyl group or a fructose residue is reacted with an aldehyde, a ketone, an acetal or a ketal, 4,6-O
-Cyclic acetal or 4,6-O-cyclic ketal, which is then reduced to give the general formula [I] [In the formula, R ¹ is a benzyl group, a substituted benzyl group (the substituent is
It represents a lower alkyl group, a lower alkoxy group, an alkyl-substituted amino group, a carboxyl group, a nitro group or a halogen atom. ), 2-, 3- or 4-pyridylmethyl group, carbon number 1
~ 6 linear, branched or cyclic alkyl group or alkenyl group having 1 to 6 carbon atoms, R ² is (However, R ^{3 to} R ⁶ represent a hydrogen atom, a lower alkyl group, a lower alkoxy group, a nitro group, a carboxyl group, a sulfonic acid group or a halogen atom, which may be the same or different, and R 3 ³ and R ⁵ or R ⁴ and R ⁶ may combine to form an aromatic ring, R ⁷ represents a hydrogen atom, a lower alkoxy group, a halogen atom or a nitro group, and R ⁸ represents a hydrogen atom. , A methyl group or a trifluoromethyl group, and R ⁹ represents a hydrogen atom or a halogen atom) or And n represents an integer of 2 to 5. ] The manufacturing method of the oligosaccharide derivative shown by these. 2. A non-reducing terminal glucose of a reducing end-modified oligosaccharide is reacted with an aldehyde, a ketone, an acetal or a ketal to give a 4,6-O-cyclic acetal or a 4,6-O-cyclic ketal. The method according to claim 1, wherein another hydroxyl group is subsequently modified by acylation and then this is reduced.