JPS63301892A - Novel production of nonreducing end modified oligosaccharide derivative - Google Patents

Abstract

PURPOSE:To obtain the titled compound useful as a substrate for measuring alpha-amylase activity industrially and advantageously, by reacting a nonreducing end glucose of modified oligosaccharide wherein a hydroxyl group at the 1-position of the reducing end glucose is substituted, with an aldehyde and reducing. CONSTITUTION:A nonreducing end glucose of modified oligosaccharide wherein a hydroxyl group at the 1-position of the reducing end glucose is substituted with a (substituted)phenoxy group, a (substituted)umbelliferyl group, a (substituted)indoxyl group or fructose residue is reacted with an aldehyde, a ketone, an acetal or a ketal to give a 4,6-O-cylic acetal or 4,6-O-cyclic ketal, which is reduced to give the aimed compound sown by formula I [R<1> is (substituted)benzyl or pyridylmethyl; R<2> is group shown by formula II [R<3>-R<6> are H, lower alkyl, nitro or halogen; R' is H or lower alkoxy); n is 2-5 integer)].

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application 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 in human saliva, pancreatic juice, blood, and urine
Measuring amylase activity is important in medical diagnosis. For example, in pancreatitis, pancreatic cancer, and parotitis, α-amylase activity in blood and urine shows a marked increase compared to normal values.

Various methods have been published for measuring α-amylase activity, but they can be roughly divided into starch, starch,
There are two types of methods: methods using long-chain natural products such as amylose and amylopectin and modified products thereof, and methods using oligosaccharide P having 4 to 7 glucose residues and its derivatives.

However, recently, maltotetraose,
A method using oligosaccharides such as maltopentaose, maltohexaose, and maltoheptaose as a substrate (
JP-A-50-56998, JP-A-53-3709
6) and a method using a substrate with a uniform and clear structure, such as a method using oligosaccharide P with a chromogen such as p-nitrophenol bound to the reducing end (Japanese Patent Application Laid-Open No. 54-51892). However, the amylase measurement method is becoming the mainstream, replacing the method using starch that has been used extensively up until now.

These methods usually use α-glucosidase (E, C, 3, 2, 1, 20; (7-D-glucoside glucohyplorase) or glucoamin 5-ase) as the coupled enzyme for measurement.
E, C, 3,2,1,3; 1.4-α-D-glucan glucosidase), or β-glucosidase (E,
c.

3.2.1.21; β-D-glucoside glucohydrolase).

These conjugated enzymes are exotype enzymes that hydrolyze α-1,4-glucosidic bonds from the non-reducing end of sugar chains that have α-1,4-glucosidic bonds, and they are able to hydrolyze substrates regardless of the α-amylase reaction. It has the disadvantage that it decomposes. Therefore, in the above measurement method using these coupled enzymes,
The measurement reagent solution was unstable, and the reagent blind value was extremely high, which significantly degraded measurement accuracy.Furthermore, it was not possible to use a sufficient amount of glucoamylase or α-glucosidase for measurement, making it difficult to obtain accurate and precise measurements. It was difficult to assemble a measurement method with high

In order to solve these problems, the present inventors have so far synthesized several types of novel modified oligosaccharide P, and have filed a patent application for a method for measuring α-amylase activity using these.

For example, when measuring α-amylase activity, the primary alcohol (-CH2
There is a method for measuring α-amylase activity using as a substrate an oligosaccharide derivative having the following structural formula in which 0H) is substituted with a group represented by the general formula -CF3H (Japanese Patent Laid-Open No. 59-51800).

(In the formula, the rightmost glucose unit is a reducing group, k is 2 to 5
is an integer, 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, in order to measure α-amylase activity using these substrates, it is necessary to use high-performance liquid chromatography or α-amylase activity.
A method must be used to measure glucose produced using glucosidase, β-glucosidase, or glucoamylase as a coupled enzyme; the former requires special equipment, and the latter is affected by the glucose contained in the sample. There was a problem with points etc.

Therefore, the present inventors conducted further research and found a method for measuring α-amylase activity that does not have such problems, and has filed a patent application for this method (Japanese Patent Application Laid-Open No. 83195/1983).

That is, the primary alcohol (-CH20H) at the 6th position of the non-reducing terminal glucose of a linear oligosaccharide consisting of 4 to 7 glucose atoms is substituted with a group represented by -CH2Ro, and the 1st-position of the reducing terminal glucose is substituted with a group represented by -CH2Ro. represents the following structural formula [A], in which is substituted with a 7-enoxy group, a substituted phenoxy group, or an umbelliferyl group (where 〜〜马 is hydrogen, a lower alkyl group, a lower alkoxy group, a nitro group, a carzexyl group, It represents a sulfone group or a rogen, which may be the same or different, and each represents hydrogen, a lower alkoxy group, a rogen or a nitro group, and - represents hydrogen or a methyl group. ). ] is the α-
This is the amylase activity measurement method.

Although this measurement method is an excellent measurement method that solves all the problems of the conventional α-amylase activity measurement method, it has not been put into practical use (commercialization) because the synthesis yield of the substrates used is very low. ), a problem remains. Therefore, the emergence of a new and effective production method that can obtain non-reducing end-modified oligo-9 saccharide derivatives with higher yields that can serve as excellent substrates for measuring α-amylase activity is currently being awaited.

[Purpose of the invention]

The present invention was made in view of the above-mentioned situation, and
The object of the present invention is to provide a manufacturing method that allows easy and high-yield production of non-reducing end-modified oligosaccharide P derivatives useful as substrates for measuring amylase activity.

[Structure of the invention]

The present invention provides a phenoxy group which may have a substituent at the 1-position hydroxyl group of the reducing end glucose, a naphthoxy group which may have a substituent, an umbelliferyl group which may have a substituent, 4.6-0 by reacting aldehydes, ketones, acetals or ketals with the non-reducing terminal glucose of a modified oligosaccharide substituted with an indoxyl group or fructose residue which may have a substituent.
- a cyclic acetal or a 4.6-0-cyclic ketal;
This is then reduced to form a compound of the general formula [1] [wherein R1 is a benzyl group, a substituted benzyl group (the substituent is a lower alkyl group, a lower alkoxy group, an alkyl-substituted amino group, a carzexyl group, a nitro represents a group or a halogen atom), 2-. It represents a 3- or 4-pyridylmethyl group, a linear, branched or cyclic alkyl group having 1 to 6 carbon atoms, or an alkenyl group having 1 to 6 carbon atoms, and R2 is (however, R3-R6 are hydrogen atoms , represents a lower alkyl group, a lower alkoxy group, a nitro group, a carxyl group, a sulfonic acid group, or a norogen atom, and each may be the same or different, and R and R or R4 and R6 are bonded. to form an aromatic ring.R7 is a hydrogen atom,
Represents a lower alkoxy group, a nitrogen atom or a nitro group. Further, R represents a hydrogen atom, a methyl group or a trifluoromethyl group, and R9 represents a hydrogen atom or a hydrogen atom. ) or H, and n represents an integer of 2 to 5. ] This is a method for producing an oligosaccharide derivative.

The production method of the present invention is usually carried out as follows using as a raw material the reducing end-modified oligosaccharide derivative represented by a] in which the 1-position hydroxyl group of the reducing end glucose is substituted with the group represented by R2 above. .

That is, first, an aldehyde, a ketone, an acetal, or a ketal is reacted on the oligosaccharide derivative represented by the formula CIII to convert the 4- and 6-positions of the non-reducing terminal glucose.
A cyclic acetal group is formed between the two positions. As the oligosaccharide derivative represented by the formula [■], a commercially available product may be used as it is, or it may be synthesized, for example, according to the method described in JP-A-54-51892. Examples of aldehydes used in the reaction include aromatic aldehydes such as benzaldehyde and tolylaldehyde, acetaldehyde, and probionaldehyde P.

Examples include aliphatic aldehydes such as butyraldehyde, and examples of ketones include acetone.

Examples of the acetals include methylal, penzaldehy P dimethyl acetal, acetaldehyde diethyl acetal, and acetaldehyde diethyl acetal. Examples of the ketals include &f.

Examples include, but are not limited to, methyl ethyl ketone ethylene ketal, cyclohexanone dimethyl ketal, etc., and cyclic acetalization or cyclic ketal formation at the 4 and 6 positions of the non-reducing terminal glucose of the oligosaccharide derivative represented by formula [11] aldehydes that can be converted into
Any ketones, acetals, or ketals are fine. The molar ratio used is usually about 1 to 100 times the molar amount of the oligosaccharide derivative. The reaction is usually carried out in the presence of a Lewis acid catalyst such as p-toluenesulfonic acid or zinc chloride in a suitable solvent such as dimethylformamide (DMF), and the reaction temperature is usually room temperature to reflux temperature, and the reaction time is Although the time depends on the reaction temperature, it is usually about 0.5 to 12 hours.

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

The acylation reaction may be carried out in the presence of a base such as pyridine using acetic anhydride, acetyl chloride, benzoyl chloride, or the like as an acylating agent according to a conventional method.

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

The reduction process is usually carried out as a reducing agent) IJ umcyano zero hydride, sodium zero hydride, lithium aluminum ramino hydride, pyrinone gelane, -
) Methylamine gellan, trimethylamine gelan, t
Using art-butylamine, diborane, aluminum chloride, boron trifluoride, etc.

Usually, 0
The reaction is carried out at ~50°C for several tens of minutes to several hours. The amount of the reducing agent used is usually 1 to 1000 times the mole of the oligosaccharide derivative, and the amount of the acid catalyst used is usually about 0.5 to 5 moles relative to the reducing agent.

In the case of acyl protection, after reduction, for example, 0.01 to 1. The desired non-reducing end-modified oligosaccharide derivative can be easily obtained by deacylation by treating with ON sodium methylate/methanol solution for several hours to several tens of hours at room temperature or with slight heating. Post-treatments after the reaction in each step can be carried out according to conventional methods (and purification of the final product can also be carried out according to conventional methods such as column chromatography.

The α-1,4 bond of oligosaccharide P is hydrolyzed by acid under acidic conditions, and is metathulysed under conditions of anhydrous methanol-I (Ct). Since the stability of the bond between the reducing terminal glucose and the group represented by R2 is unknown, it is possible that the oligosaccharide derivative derived from the oligosaccharide derivative represented by the formula [] will withstand such reduction treatment. Until now, it was unclear whether or not the cyclic acetal moiety could be obtained, but surprisingly, the present inventors discovered for the first time that it was possible to open the cyclic acetal moiety without any inconvenience.
The present invention has now been completed.

In the oligosaccharide derivative represented by the general formula C1) that can be produced by the method of the present invention, R1 of -CH20R1 at the 6-position of the non-reducing terminal glucose is a benzyl group,
Substituted benzyl group (substituents include, for example, lower alkyl groups such as methyl, ethyl, propyl, and butyl; lower alkoxy groups such as methoxy, ethoxy, propoxy, and butoxy; for example, dimethylamino group; Substituted amino groups such as diethylamine/group, N-ethyl-N-(β-hyproxethyl)amine group, carxyl group, nitro group, or halogen atoms such as chlorine, bromine, and iodine, etc.), 2 -23- or 4-pyridylmethyl group, such as methyl group, ethyl group.

1so-propyl group, n-butyl group, tert-butyl group, n-pentyl group, 1so-amyl group, n-hexyl group, cyclopentyl group, cyclohexyl group Straight chain monobranched or cyclic having 1 to 6 ring carbon atoms Examples include alkenyl groups having 1 to 6 carbon atoms such as evaethynyl group, 2-probenyl group, and +1412-1-butenyl group. In addition, the phenoxy group or naphthoxy group which may have a substituent in the general formula [1] has a good reducing terminal glucolysis, and the ori is bonded to the reducing terminal of the sugar, and glucoamylase (E, C, 3
, 2, 1, 3,), α-glucosidase [:E, C, 3
, 2, 1, 20, ], ] β-glucosidase: E, C,
3,2,1,21,], isomaltase (E, c, 3.
z, t, 1o,] or] β-amylase E, C, 3,2
, 1, 2,: 1, etc., and after thawing the ice, it can be hydrolyzed by the action of conjugated enzymes such as nitrophenols, or catechol oxidase, Any substance that produces a dye by coupling with a coupler under the action of an oxidizing enzyme such as laccase, tyrosinase or monophenol oxidase, or which produces a dye by coupling with a coupler with an oxidizing agent may be used. . Specific examples of R2 satisfying such conditions include, for example, ri-di)lophenoxy group, m-nitrophenoxy group, 0-chlorophenoxy group, p-chlorophenoxy group, 2,6-dichlorophenoxy group, 2-chloro-4-nitrophenoxy group, 0-methoxyphenoxy group, p-methoxyphenoxy group, 0-
Methylphenoxy group, 0-calcexyphenoxy group, 0
Examples include, but are not limited to, -sulfophenoxy group, 1-naphthoxy group, 2-sulfo-1-naphthoxy group, 2-calzexy-1-naphthoxy group, and the like.

Specific examples of the umbelliferyl group that may be
Specific examples of the umbelliferyl group where R8 is a hydrogen atom, the 4-methylumbelliferyl group where R8 is a methyl group, or the in-xyl group which may be 4-trifluoromethane where R8 is a trifluoromethyl group include, for example. , in-xyl group, and 5-bromoindoxy, after deicing with a conjugated enzyme, the degree of decrease in NADH caused by the action of mannitol dehydrogenase and NADH was measured. For example, a non-reducing end-modified oligosaccharide useful as a substrate for measuring α-amylase activity or as a substrate for differentially measuring the activity of each isodeme of human α-amylase can be obtained in good yield by simple reaction operations. .

Examples are shown below, but the present invention is not limited to these Examples in any way.

〔Example〕

Example 1. p-Nitrophenyl 0-(6-0-benzyl)-α-D-glucopyrine-(1→4)-〇-α-D-glucopyranosyl-(1→4)-〇-α-D
-Glucopyranosyl(1→4)-〇-α-D-Glucopyranosyl(1→4)-α--D-Glucopyranosyl P
Synthesis of p-nitrophenyl-α-D-maltopentaoside 32 (hereinafter abbreviated as BG5P)
After dissolving in DMF 50 d, 15 m of penzaldehyr dimethyl acetal and 14 m of p-toluenesulfonic acid (anhydride) were added, and the mixture was stirred and reacted at 50°C for 3 hours. After the reaction, 100 mA' of pyridine was added, and 60 mA' of penzoyl chloride was added dropwise under water cooling.
The reaction was stopped by pouring into 0.00 trtl of saturated sodium bicarbonate solution. After extraction with 200 d of chloroform and washing twice with saturated brine, the solvent was distilled off. Transfer the resulting syrup to a 114-necked kolben and add THF 250 tn.
After dissolving in 1 liter of water, 3 t of dimethylamine gellan was added and dissolved. To this solution, add hydrochloric acid gas to diethyl ether.
5N solution 10++tA! The reaction solution was poured into 200 d of saturated hydrogen carbonate solution to stop the reaction, and then extracted with 200 d of chloroform. After washing twice with saturated saline, the solvent was distilled off to give a 0. After adding 500 d of a methanol solution containing IN sodium methylate and stirring at 25°C for 4 hours, acetic acid was added to neutralize.

After evaporation of the solvent, 50mM acetic acid solution 20- was added and the biogel P-2 equilibrated with 50mM acetic acid (Bio-Rad
Co., Ltd., 2 crnφ x 150 m) column and charge it with BG.
Both parts of 5P were collected and freeze-dried.

Yield: 450■.

IR chart: As shown in Figure 1.

'H-NMR: As shown in Figure 2.

HPLC: Content 95% [Column: Filling material = Silicage #
Z-ODS, 5C18 (Wako Pure Chemical Industries, Ltd. trade name, 4 x 150 m), eluent 20, a 10-80% gradient of acetonitrile aqueous solution containing 1% acetic acid.

Measured at 305 nm. ].

GCm component plate: /'lucose/6-0-benzyluglucose = 3.8:1° Example 2. p-Nitrophenyl O-(6-0-benzyl)-α-D-glucopyra, nosyru(1→4)-
〇-α-D-glucopyranosyl (1→4)-〇-α-
D-glucopyranosyl (1→4)-〇-α-D-glucopyranosyl (1→4)-〇-α-D-glucopyranosyl (1→4)-α-D-glucopyranosyl P (below,
It is abbreviated as BG6P. ) Synthesis of p-nitrophenyl α-D-maltohexaoside 3f
After dissolving in DMF 50 at, penzaldehy Y-)
1 t of l-tylacetal, 1 t of p-)luenesulfonic acid (anhydride) was added, and the mixture was stirred and reacted at 50°C for 3 hours.

After the reaction, 100 mA' of pyridine was added and 80 ml of benzoyl chloride was added dropwise under water cooling to react.
The reaction was stopped by pouring into 1 liter of saturated sodium bicarbonate solution. The mixture was extracted with 200 mA' of chloroform, washed twice with saturated brine, and then the solvent was distilled off. 1 of the resulting syrup
The mixture was transferred to a No. 14-necked container, dissolved in 250 rrtl of THF, and then 3 f of dimethylamine gellan was added and dissolved. Hydrochloric acid gas was bubbled into diethyl ether into this solution and 0.5
1゜rug of N solution was added dropwise under stirring, and the reaction solution was diluted with 3.
00m7? The reaction was stopped by pouring into saturated sodium bicarbonate solution, and then extracted with 200 ml of chloroform. After washing twice with saturated saline, the solvent was distilled off to 0.1N). 500 d of methanol solution containing IJ methylate.
After stirring at 25° C. for 4 hours, acetic acid was added to neutralize the mixture.

After evaporating the solvent, add 20mA' of 50mM acetic acid solution and
Biogel P-2 (Bio-R
ad Co., 2crnφX150I:rIK) column was charged and both portions of BG6P were collected and freeze-dried.

Yield: 500■.

HPLC: Content: 94 h (measurement conditions are the same as in Example 1).

).

GC composition analysis: Cucrose/6-0-benzyl-glucose = 4.9:1° Example 3. 9-nitrophenyl 0-(6-0-methyl)-α-D-glucopyranosyl = (1→4)-〇-α
-D-glucopyranosyl (1 → 4) -〇-α-D-glucopyranosyl (1 → 4) -〇-α-D-glucopyranosyl (1 → 4) -α-D-glucopyranoside Synthesis of p-nitrophenyl α-D-maltopentaoside 5f (hereinafter abbreviated as M05P)
After dissolving in DMF 40 rttl, methylal 50r
at, p-) luenesulfonic acid (anhydride) IIJD, t,
The reaction was stirred at 50° C. for 3 hours. After concentrating the reaction solution in vacuo,
After adding 300 m of pyrinone and 100 m of acetic anhydride and reacting at room temperature for 12 hours, add saturated 4-hydrogen carbonate to the reaction solution.
The reaction was stopped by adding 500 rnl of IJum solution. The extract was extracted with 100 mJ of chloroform, washed twice with saturated brine, and then the solvent was distilled off.

Transfer the resulting syrup to 114 ml of THF.
After dissolving in 50 tttl, pyridine gellan 41 was added and dissolved. To this solution, a solution of 0.5M p-toluenesulfonic acid in THF (10) was added dropwise with stirring, and the reaction solution was stirred at 200 rpm.
After pouring into a saturated sodium bicarbonate solution of IL1 to stop the reaction, the mixture was extracted with 200 d of chloroform. After washing twice with saturated brine, the solvent was distilled off, and 0. 700 t/methanol solution containing IN sodium methylate was added, and after reacting at 25°C for 4 hours, acetic acid was added to neutralize. After evaporation of the solvent, biogel P-2 equilibrated with 50:acetic acid (
The MG5P fraction was collected by charging a column (Bio-Rad, 2tMφX150cm) and freeze-dried.

Yield: 730■.

HPLC: Content: 93 h (measurement conditions are the same as in Example 1).

).

〔Effect of the invention〕

The present invention provides a novel and effective method for producing non-reducing end-modified oligosaccharide P derivatives that are useful as substrates for measuring α-amylase activity or as substrates for differentially measuring the activities of each isodime of human α-amylase. According to the method of the present invention, non-reducing end-modified oligosaccharide derivatives can be obtained in high yield with simple reaction operations, and therefore, α-amylase activity assay methods using these as substrates or This has had a significant effect in contributing to the practical application (commercialization) of the method for separately measuring the isozyme.

[Brief explanation of the drawing]

Figure 1 shows the IR chart of the non-reducing end-modified oligosaccharide P derivative obtained in Example 1, and Figure 2 shows the IR chart of the non-reducing end-modified oligosaccharide r derivative obtained in Example 1. show.

Claims (2)

[Claims] (1) A phenoxy group which may have a substituent on the 1-position hydroxyl group of the reducing terminal glucose, a naphthoxy group which may have a substituent, an umbelliferyl group which may have a substituent, a substituent The non-reducing terminal glucose of a modified oligosaccharide substituted with an indoxyl group or a fructose residue, which may have an optional group, is reacted with an aldehyde, a ketone, an acetal, or a ketal to form a 4,6-O-cyclic The general formula [I]▲ is characterized by forming an acetal or a 4,6-O-cyclic ketal and then reducing this.
There are mathematical formulas, chemical formulas, tables, etc. represents a halogen atom), a 2-, 3- or 4-pyridylmethyl group, a linear, branched or cyclic alkyl group having 1 to 6 carbon atoms, or an alkenyl group having 1 to 6 carbon atoms, R ^2 has ▲mathematical formulas, chemical formulas, tables, etc.▼, ▲mathematical formulas, chemical formulas,
There are tables, etc. ▼, ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (However, R^3 to R^6 are hydrogen atoms, lower alkyl groups,
Represents a lower alkoxy group, nitro group, carboxyl group, sulfonic acid group or halogen atom, which may be the same or different, and R^3 and R^5 or R^4
and R^6 may be combined to form an aromatic ring. R
^7 represents a hydrogen atom, a lower alkoxy group, a halogen atom or a nitro group. Further, 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 ▲There are mathematical formulas, chemical formulas, tables, etc.▼, and n represents an integer from 2 to 5. ] A method for producing an oligosaccharide derivative represented by. (2) After reacting the non-reducing end glucose of the reducing end-modified oligosaccharide with aldehydes, ketones, acetals, or ketals to form a 4,6-O-cyclic acetal or a 4,6-O-cyclic ketal, 2. The production method according to claim 1, wherein other hydroxyl groups are modified by acylation and then reduced.