JPH03200801A - Anhydromaltooligoside derivative, reagent for assaying alpha-amylase activity containing the same as effective component, and assay of alpha-amylase activity - Google Patents

Abstract

PURPOSE:To obtain a 3,6-anhydromaltooligoside derivative having a specified structural formula and being desirable as a reagent for assaying alpha-amylase activity effectively and exactly. CONSTITUTION:A 3,6-anhydromaltooligoside derivative represented by formula I (wherein R is an aromatic color-forming group; and (n) is an integer of 1-6). Examples of the group R include a group of formula II (wherein R<1> to R<5> which may be the same or different are each a hydrogen atom, an alkyl group, an allyl group, an aryl group, an acyl group, a nitro group, an amino group, a sulfonic group or the like) and a group of formula III (wherein R<6> is a hydrogen atom or an alkyl group). An alpha-amylase activity can be assayed by, e.g. subjecting a sample containing alpha-amylase to an enzymatic reaction by adding thereto an alpha-anomer of a 3,6-anhydromaltooligoside derivative of formula I, alpha-glucosidase and/or glucoamylase and determining the released aromatic color- forming compound.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a novel 3,6-anhydromaltooligosite derivative, a reagent for measuring α-amylase activity containing the derivative as an active ingredient, and a method for measuring σ-amylase activity using the derivative. The present invention relates to a method for efficiently and accurately measuring amylase activity.

Conventional technology Conventionally, α has been used to target body fluids such as serum, urine, pancreatic juice, and saliva.
- Measuring amylase activity is extremely important for clinical diagnosis, and is an essential measurement item in the differential diagnosis of acute and chronic hepatitis, pancreatic cancer, mumps, and the like.

Various methods have been used to measure this α-amylase activity, such as (1) using starch or dye-bound starch as a substrate and measuring reducing power or absorbance, (2) maltotetraose, maltopentaose, etc. A method for quantifying maltose, glucose or glucose-6-phosphate produced by using a series of maltooligosaccharides as substrates, cleaving them with α-amylase, and then acting on a coupled enzyme system, (3) various substituted phenyl maltooligosaccharides. Using cytosine as a substrate, it is cleaved by σ-amylase, then acted on with a coupled enzyme system, and the resulting substituted phenols are produced as is, after changing the pH as necessary, or after a condensation reaction. Method for color determination, (4) Utilizing various substituted phenyl malto-oligosites modified with an aryl group, alkyl group, etc. at the 6-position and/or 4-position of the non-reducing terminal glucose as a substrate, (
Similar to 3), a method of colorimetric determination is known.

However, in method (1), the measured values vary depending on the quality of the starch used as the substrate, and there are many enzyme cleavage sites, so the a-amylase reaction is measured as a truly stoichiometric reaction. It has drawbacks such as being unable to do so. On the other hand, method (2) uses a uniform substrate (this can compensate for the drawback of (1) above, but it requires completely eliminating carbohydrates such as maltose and glucose from the sample in advance). In addition, when measuring glucose produced in an enzymatic reaction using glucose oxidase, peroxidase, or a chromogen system, it is necessary to correct for the influence of glucose in the sample, and a large amount of glucose oxidase is required. ,
Furthermore, it has drawbacks such as being affected by reducing substances such as ascorbic acid and bilirubin present in the sample.

-4, method (3), especially the method using 2-chloro-4-nitrophenyl-β-maltopentaoside as a substrate, is currently widely used as an excellent method;
Since the substrate is decomposed into the conjugated enzyme, positive errors tend to occur, and reducing the amount of the conjugated enzyme increases the lag time. Therefore, in order to improve the drawback of method (3), the above-mentioned (
Method 4) has been developed.

However, these substrates have many drawbacks, such as low solubility in water, low affinity for α-amylase, low rate of degradation by α-amylase, and chemical instability that prevents long-term storage. have.

Problems to be Solved by the Invention The present invention overcomes the drawbacks of such conventional α-amylase activity measurement reagents and measurement methods using the same, and
The purpose of this work is to provide a novel compound suitable as a reagent for efficiently and accurately measuring α-amylase activity, and to provide a novel method for measuring α-amylase activity using this compound as a reagent. be.

Means for Solving the Problems In order to achieve the above object, the present inventors have conducted various studies and found that a specific new 3,6-anthromalto-oligoside derivative is extremely suitable as a reagent for measuring a-amylase activity. It was discovered that the purpose could be achieved by measuring α-amylase activity using this method,
Based on this knowledge, we have completed the present invention.

That is, the present invention provides a 3,6-anhytromaltooligoside derivative represented by the general formula (R in the formula is an aromatic chromogenic group, and n is an integer of 1 to 6), the general formula (I) A reagent for measuring σ-amylase activity containing a compound as an active ingredient, and a sample containing α-amylase,
a- Anomer of the compound of general formula (I) and σ-glucosidase and/or glucoamylase are added to perform an enzymatic reaction and the liberated aromatic chromogenic compound is quantified.
A method for measuring amylase activity, and a sample containing α-amylase containing β-anomer or a mixture of α-anomer and β-anomer of the compound of general formula (1), α-glucosidase and/or glucoamylase, and β-glucosidase. The present invention provides a method for measuring α-amylase activity, which is characterized by adding an enzyme, causing an enzymatic reaction, and quantifying the liberated aromatic chromogenic compound.

The present invention will be explained in detail below.

The 3,6-anhydromaltooligosaccharide moiety in the 3,6-anhydromaltooligoside derivative of the general formula (I) of the present invention is, for example, a- or β-3'',6''-anhydro-D-maltotrigosaccharide moiety. α or β-3' from ose,
All compounds corresponding to 6'-anhydro-D-maltooctaose can be used. Among these, especially 3',
6'-Anhydro-D-maltopentaose, 38.6
Low anhydro D-maltohexaose and 3',6
'-Anhydro-D-maltoheptaose is preferred. In addition, the symbols 3", 6"-13', 6s-13', 6', 3', 6 added before anhydro in the above compound
'-3', 6'-, etc. are intramolecularly located at the 3rd and 6th positions of glucose on the non-reducing end side of the 3, 5, 6, 7, and 8 glucose units constituting the maltooligosaccharide, respectively. This means that it forms an ether.

In the 3,6-anthromaltooligoside derivative represented by the general formula (1), 1 of the reducing end glucose
R substituted with the hydroxyl group at position is an aromatic color-forming group, and examples of such groups include the following.

(RI-R6 is a hydrogen atom, an alkyl group, an allyl group, an aryl group, an acyl group, a carboxyl group, a cyano group, a formyl group, an alkoxy group, a nitro group, a nitroso group, an amino group, an azide group, a sulfonic acid group, a sulfoxyl group) , a sulfonyl group, or a halogen atom, which may be the same or different, and R1 and R2,
Alternatively, R2 and R3 may be combined to form a fused aromatic ring) (R' is a hydrogen atom or an alkyl group) (R7 is a hydrogen atom or a halogen atom) (R8-R16 are hydrogen atoms , alkyl group, allyl group, aryl group, acyl group, carboxyl group, cyano group, formyl group, alkoxy group,
A nitro group, a nitroso group, an amino group, an azide group, a sulfonic acid group, a sulfoxyl group, a sulfonyl group, or a halogen atom, each of which may be the same or different from each other, and Ra and R'' , and/or 1?I
o and R11 may be combined to form a fused aromatic ring,
Furthermore, R9 and RIG and/or Rlm and R14 may form a fused ether ring by forming a common oxygen atom,
(Z is a nitrogen atom or N→0) The 3,6-anthromaltooligoside derivative represented by the general formula (I) may be either a σ-anomer or a β-anomer.

Therefore, as the compound represented by the general formula (I), for example, 2-chloro-4-nitrophenyl = 3%,
5m-Anhydro-β-D-maltopentaocyto, 4-
Nitrophenyl = 3', 6'-anhydro-α-D-maltopentaoside, phenolindo-3'-chlorophenyl = 3', 6'-anhydro-β-D-maltopentaoside, 4-nitrophenyl = 3',6'-Anhydro-β-D-maltoheptaoside, 2-chloro-4-nitrophenyl-3',6'-7-Fo-β-D-maltoheptaoside, methylumbellifero= Lou 3', 6
Examples include '-anhydro-β-D-maltopentaoside, rezathrinil 3', 6'-anhydro-β-D-maltoheptaoside.

The 3,6-anthromaltooligond derivative represented by the general formula (I) of the present invention is a novel compound that has not been published in any literature, for example, the following methods (A) and (B).
It can be manufactured by the method.

Method (A) First, a maltooligosite derivative represented by the general formula (R and n in the formula have the same meanings as above), such as 2-chloro-4-nitrophenyl = β-D-maltohentaoside, 4-nitrophenylloα-D-maltoheptaoside, phenolinindophenyl Cβ-D-maltopentaoside, etc.
By reacting benzaldehyde, benzaldehyde dimethyl acetal, etc., a 4,6-0-benzylidene malto-oligoside derivative represented by the general formula (R and n in the formula have the same meanings as above), such as 2-chloro-4- Nitrophenyl-4',65
-0-Benzylidene-β-D-maltopentaoside, 4
-nitrophenyl=4',67-〇-benzylidene-α
-D-maltoheptaoside, phenolindo-3'-
Chlorophenyl = 4',6'-0-benzylidene-β-
D-maltotetraoside and the like are obtained.

The reaction of the maltooligosite derivative represented by the general formula (II) with benzaldehyde, benzaldehyde dimethyl acetal, etc. can be carried out using, for example, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO
), sulfuric acid, hydrogen chloride, p-+ in an aprotic polar solvent such as ethylene glycol dimethyl ether.
-luenesulfonic acid, the 4,6-0-benzylidene malto-oligoside derivative represented by the general formula ([1)] thus obtained is acylated to produce acyl-4,
6-0-Benzylidene malto-oligoside derivatives, such as 2-chloro-4-nitrophenyl-tetrade--〇-acetyl-45,6'-0-benzylidene-β-D-maltopentaoside, 4-nitrophenyl -Eicosa-0-
Benzoyl-4'+6'-o-benzylidene-α-D-
Maltoheptaondo, phenolindo-3'-chlorophenyl-undeca-O-butyryl-44,134-o
-Benzylidene-β-D-maltotetraoside and the like are obtained. At this time, examples of the acylating agent include acetic acid, monochloroacetic acid, propionic acid, σ-chloropropionic acid, and β-chloropropionic acid.
-Chloropropionic acid, n-butyric acid, benzoic acid, etc., and reactive derivatives thereof such as acid anhydrides, acid chlorides, and esters are used. There are no particular limitations on the conditions for the acylation reaction, and conditions conventionally used in acylation can be used.

Next, the acyl-4,6-0-benzylidene maltooligoside derivative thus obtained is subjected to an oxidative bromination reaction using N-bromosuccinimide (NBS) to obtain a general formula tetraoside and the like.

The oxidative bromination reaction using NBS is carried out using a hydrogen bromide removal agent such as barium sulfate under reflux of a nonpolar solvent such as carbon tetrachloride, 1.1,2.2-tetrachloroethane, benzene, or toluene. carried out in the presence of

Next, this acyl-4-0-benzoyl-6-bromo malto-oligoside derivative is treated with an alkali to cause an intramolecular ether formation reaction, and the general formula (R1'' in the formula is an acyl group, B2 is a benzoyl group) , R and n have the same meanings as above), e.g. %-o-benzoyl-6'-bromo-β-D-maltopentaoside, 4-nitrophenyl-heneicosa-0-benzoyl-6-promoa-D-maltoheptaoside, resorufinyl-undeca-7' Tylyl-44-O-benzoyl-64-bromo-β-D-malto (wherein R1,
B2, R and n have the same meanings as above) Acyl-3,6-anhydro-4-0-benzoyl-maltooligoside derivatives, such as 2-chloro-4-nitrophenyl-trideca-〇-acetyl-31
',6'-anhydro-46-〇-benzoyl-β-D
-maltopentaoside, 4-nitrophenyl-eicosa O-benzoyl-3',6'-anhydro-α-D-
Maltoheptaoside, phenolindo-3', 5'-
Difluorophenyl decar〇-7'tyryl-3',6
'-Anhydro-44-O-benzoyl-β-ri-maltotetraoside and the like are obtained.

'There are no particular restrictions on the conditions for the above intramolecular ether formation reaction, but examples include hexamethylphosphoric triamide (HMPA), DMSO, DMF, acetone,
Potassium carbonate, NaBH at a temperature of 50-80 °C in an aprotic polar solvent such as acetonitrile, nitromethane, etc.
An intramolecular ether formation reaction is carried out by reacting with an alkali such as a, NaBH, or CN at a ratio of 10 to 50 times the mole.

Finally, the acyl-3,6-anhydro-4-0-benzoylmaltooligoside derivative is deacylated to obtain the target compound 3,6-anhytromaltooligoside derivative represented by the general formula (I).

There are no particular restrictions on the conditions for this deacylation reaction, but for example, when the acyl group is an acetyl group, 0.5 to 3 times the mole of potassium carbonate is added in an alcoholic solution such as methanol, and the temperature is heated at 20 to 40°C. A method of reacting for 1 to 10 hours is used [Journal of American Chemical Society (J, Am, Chem, Soc,
) Volume 94, page 8613 (1972)].

In addition, using the benzaldehyde, benzaldehyde dimethyl acetal acetal and acetaldehyde, respectively,
Corresponding inpropylidene malto-oligoside derivatives and ethylidene malto-oligoside derivatives were obtained in the same manner as above, and then acylation, oxidative bromination, intramolecular ether formation, and deacylation were carried out in the same manner to obtain the general formula (I ) can be obtained.

(B) Method A known aryl glucoside derivative represented by the general formula (R in the formula has the same meaning as above) is added to 3 represented by the general formula (n' in the formula is an integer from 4 to 6) , 6-anhydrocyclodextrin is added, a known enzyme cyclodextrin glucosyltrans 7-erase (CGT-ase) is applied to perform a coupling-ring cleavage reaction, and then exo-type saccharifying enzymes are applied thereto. When the glucose residue on the non-reducing end side is hydrolyzed so that the 3,6-anhydroglucose residue becomes the non-reducing end, the desired 3,6-anhydroglucose represented by the general formula CI) is produced. A human tromaltooligoside derivative is obtained.

Examples of the aryl glucoside derivative represented by the general formula (Vl) include 2-chloro-4-nitrophenyl=β-D-curcoside, 4-nitrophenyl and a-D-
Examples include glucoside, phenolindo-3', 5'-dichlorophenyl β-D-glucoside.

These can be obtained as commercial products, and can be easily manufactured by known methods.

The cyclodextrin represented by the general formula (■) is
Cyclodextrins having a nclodextrin skeleton, including derivatives such as branched products and modified products, such as commercially available α-1β-1γ-cyclodextrin (each has a degree of glucose polymerization of 6.7.8), can be obtained by a known method. Obtainable. For example, cyclodextrin is dissolved in a solvent such as pyridine, 7 to 14 times the mole of tosyl chloride is added, reacted at 15 to 30°C for 4 to 6 hours to tosylate, and if necessary, purified by a conventional method. After obtaining 6-tosylcyclodextrin, dissolve it in water, add 2 to 10 times the mole of alkali such as sodium hydroxide or potassium carbonate, and react at 40 to 60°C for about 5 to 10 hours. 3,6-Anhydrocyclodextrin can be obtained by intramolecular ether formation and, if necessary, purification by conventional methods [for example, "Chemistry Letters"
Chem.

Lett, ) J, 198B Hayabusa, pp. 705-708].

Further, the coupling-cleavage reaction using CGT-ase is usually carried out in an aqueous medium in the presence of a buffer. The reaction conditions at this time are as follows:
The operating pH and temperature are selected appropriately depending on the origin of -ase, but usually the pH is 5.0 to 7.5 and the temperature is 35 to 45°.
C1 reaction time is selected in the range of 0.5 to 10 hours.

The concentration of the compound represented by the general formula (Vl) at this time is usually selected within the range of 0.01=ly/raQ, preferably from 0.02 to 0.2 g/II+12; The concentration of 3,6-anhydrocyclodextrins represented by (ii) is usually 1-200199/
m12, preferably selected in the range of 100 to 200 mg/WIQ. There is no particular restriction on the origin of CGT-ase, but those obtained from, for example, Bacillus macerans, Bacillus megaterium, Bacillus circulans, etc. are preferred. Its concentration is usually 0.3
~3 units/mQ.

It is preferably selected within the range of 0.5 to 1.5 units/mQ.

Further, as the exo-type saccharifying enzymes, for example, known a-glucosidase, glucoamylase, etc. may be used alone or in combination.

Although the exo-type saccharifying enzymes may coexist with the above-mentioned CGT-ase to carry out the enzymatic reaction simultaneously, it is preferable that the exo-type saccharifying enzymes be allowed to act further after the CGT-ase acts on the starting material. Particularly preferred is C in the starting material.
For example, when the product reaches the maximum level by the action of GT-ase, the reaction is temporarily stopped by acid or heat treatment, and further, for example, octadecylated silica gel (005
) This is a method in which exo-type saccharifying enzymes are applied after purification treatment such as passing liquid through a column and adsorbing and removing unreacted raw materials. When CGT-ase and exo-type glycosylases coexist, it is necessary to appropriately select a temperature range of 9Hs common to both enzymes, but usually pH 5.0 to 7.5.35 to 45. ℃, 0.5~
48 hour conditions are used.

Further, the reaction conditions for the action of exo-type saccharifying enzymes after the action of CGT-ase are appropriately selected from the action pH and temperature range of the enzyme used, but usually pH 4.0 to 7. 5.35-45℃, 0.5-4
8 hour conditions are used. There is no particular restriction on the amount of exo-type saccharifying enzymes used in this case, but it is usually in the range of 10 to 100 units/g based on 3,6-anhydrocyclodextrin. This enzymatic reaction is stopped, for example, by acid or heat treatment.

Next, in order to obtain the target compound 3,6-anhytromalto-oligoside derivative represented by the general formula (1) from the 3,6-anhytromalto-oligoside derivative-containing reaction solution obtained in this way, Conventional oligosaccharide separation method,
For example, unreacted 3,6-anhydrocyclodextrin is removed from the reaction solution, and then separated and purified by fraction collection using activated carbon column chromatography, silica gel column chromatography, thin layer chromatography, or the like. Further, unreacted 3,6-anhydrocyclodextrin can be removed by known methods such as cooling treatment, organic solvent addition treatment, and adsorption column treatment.

The general formula (I) obtained by various production methods as described above
The 3,6-anthromaltooligoside derivative represented by is extremely useful for measuring α-amylase activity,
α-Amylase activity can be measured using this 3,6-anthromaltooligoside derivative.

As mentioned above, the 3,6-anthromaltooligoside derivative represented by the general formula CI) contains an α-anomer and a β-anomer.
- anomer exists, but when using only the σ-anomer when measuring a-amylase activity, it is necessary to use α-glucosidase and/or glucoamylase as a coupled enzyme system, and only the β-anomer Alternatively, when using a mixture of σ-anomer and β-anomer, it is necessary to use β-glucosidase in addition to α-glucosidase and/or glucoamylase. Further, β-amylase may be used as the coupling enzyme if necessary.

Advantageous systems for measuring α-amylase activity include, for example, α- and/or β-
3,6-Anhytromaltooligoside derivative l~20m
Contains 2 to 100 mM of M and a buffer, and 15 to 150 units/mQ of α-glucosidase and/or glucoamylase, respectively, as a coupled enzyme, and 3 to 150 units/mQ of β-glucosidase when the derivative contains β-anomer. 3
A system having a pH of 4 to 10 and containing 0 units/IIIQ is mentioned. Buffers used in this system include, for example, phosphate, acetate, carbonate, tris-(hydroxymethyl)-aminomethane, borate, citrate, dimethylglutarate, and the like. The a-glucosidase may be of any origin, such as animal, plant, or microbial, but yeast-derived a-glucosidase is particularly preferred from the viewpoint of substrate specificity. Furthermore, the glucoamylase may be of any origin, but for example, glucoamylase of the genus Rhizopus (Rhizopus) may be used.
sp) etc. are preferred.

Furthermore, β-glucosidase may be of any origin, for example, one obtained from almond seeds. Furthermore, β-amylase of any origin may be used, and for example, β-amylase derived from bacteria is preferred.

In the present invention, such a system includes, in addition to the above-mentioned components,
If necessary, various conventional additives such as glycerin, bovine serum albumin, α- or β-cyclodextrin, Triton May be added. Furthermore, as a σ-amylase activator, NaCQ,
MHCO3, Mg5O,.

Cff- ions used in the form of CaCQ, CaCl22.2H7O, Ca"+ ions, Mg"+ ions, etc. may be added. These additive components may be used alone or in combination of two or more, and
It may be added at any appropriate stage of the system preparation.

The reagent of the present invention may be used in dry or dissolved form, or may be impregnated into a thin film carrier such as a sheet or impregnated paper. By using such a reagent of the present invention, α-amylase activity contained in various samples can be measured accurately and with high sensitivity through simple operations.

Next, to explain one preferred example of the method for measuring α-amylase activity of the present invention, first, 15 to 150 units of Nobichi glucondase and/or glucoamylase are added to a sample containing α-amylase as a conjugate enzyme. /mQ
, preferably 30-70 units/mQ, and the general formula (
When the 3,6-anthromaltooligoside derivative represented by I) contains a β-anomer, 3 to 30 units/mQ, preferably 5 to 15 units/mQ of β-glucosidase is further added to the derivative simultaneously or sequentially. After adding l~20mM, preferably 2~5mM and buffer, temperature 2
Enzymatic reaction is performed at 5 to 50°C and pH 4 to 10 for 1 minute or more, preferably 2 to 60 minutes, and then the resulting aromatic color-forming compound is left as it is or the pH is changed as necessary by a conventional method. Alternatively, after performing the condensation reaction, measure the absorbance value continuously or intermittently at an appropriate absorption wavelength, and calculate the α-amylase activity in the sample by comparing it with the absorbance value of the α-amylase sample measured in advance. do.

The α-amylase-containing sample used in the present invention may be any sample containing α-amylase and is not particularly limited, but specifically, microorganism culture fluid, plant extract, or animal body fluid. Tissues and their extracts can be used.

If the σ-amylase-containing sample is a solid, it is preferably dissolved or suspended in a buffer solution. Also, if necessary,
Insoluble materials may be removed by filtration.

Examples of the buffer include phosphate, acetate, carbonate, tris-(hydroxymethyl)-aminomethane, borate, citrate, dimethylglutarate, and the like.

Effects of the Invention The 3,6-anthromaltooligoside derivative represented by the general formula (1) of the present invention is a novel compound and is extremely useful as a reagent for measuring α-amylase activity. As a result, α-amylase activity can be measured easily and accurately in a short time using automated analysis methods or manual methods without being affected by glucose, maltose, bilirubin, hemoglobin, etc. contained in the sample. Moreover, it has the advantage of being able to maintain a stable state for a long period of time even in the coexistence of a conjugated enzyme.

Example Next, the present invention will be explained in more detail with reference to Examples.

Commercially available 2-chloro-4-nitrophenyl β-D-maltopentaoside (manufactured by Daiichi Chemical Co., Ltd.) 15. h(15
, 2 mmol) in anhydrous DMF 225+++ (2), and dissolved benzaldehyde dimethyl acetal 11.4 mQ (
76,0 mmol) and tosylic acid 2.25 g (11,4 mmol)
mol) was added and reacted at 40°C for 4 hours without stirring. Next, this reaction solution was slowly added dropwise to 1.2 Q of ice water while stirring.

A saturated aqueous sodium bicarbonate solution was slowly added to the mixture while stirring under water cooling for neutralization, and the mixture was washed three times with 300 mQ of dichloromethane, and the aqueous layer was concentrated to remove DMF and water. The residue was then purified by ODS column chromatography, and the target fraction eluted with an ethanol-water mixture (volume ratio 2:3) was concentrated and recrystallized from inpropanol-methanol to give 2-chloro-
4-nitrophenyl-4',65-benzylidene-β-
D-maltopentaondo 11.2g (10.4mmol
, yield 68.3%).

Melting point ('O): 196.0-200.0 ultraviolet
Visible absorption spectrum: Maximum absorption wavelength [λ:″”:','] (nm) =
292 (logt = 3.90) Infrared absorption spectrum (cm-'): 3410.29
40.1630°1586, 1520.1486.13
50.1274.1152.1074.1028.

930.896 Nuclear magnetic resonance spectrum (200MHz) ppm (DzO
) :3.20~5-5-70(,5,05(2H,d
, J-3,4Hz), 5.12(IH,d, J-4,4
Hz), 5.13 (l H, d, J = 4.4 Hz).

5.25 (l H, d, J = 7.6Hz), 5.5
6 (l H, s), 7.33-7.55 (5 H, m),
7.35 (l H, d, J = 9.0Hz), 8.1
4 (IH.

dd, J -9,0Hz, 2.7Hz), 8-29(l
H, d, J = 2.7 Hz) High performance liquid chromatography [TSKgel Am1de-8 manufactured by Takun Co., Ltd.
0 column (4,6mm IDX 250+++m).

UV2aO, Detection, Eluent Niacetonitrile/Water-
3/1 (V/V), flow rate: 1. OmQ/min] :
13.3 g (12.4 mmol) of 2-chloro-4-nitronenyl-45.65-benzylidene-β-D-maltopentaoside obtained in R = 4.8 m1n (1) was dissolved in 200 mQ of pyridine, and anhydrous Acetic acid 100+++<1
(1,28mo+) was added and allowed to react at room temperature for 2 days.

Next, this reaction solution was concentrated under reduced pressure to distill off pyridine, acetic anhydride, and acetic acid. The residue was purified by silica gel column chromatography, and the target fraction was eluted with a methanol-dichloromethane mixture (volume ratio 2:98). It was concentrated and recrystallized from ethanol to give 18.1 g of 2-chloro-4-nitrophenyl-tetrazokar-0-acetyl-4'', 6s-benzylidene-β-D-maltopentaoside (
10.9 mmol, yield 87.9%) was obtained.

Melting point (°O): 130.0-135.0 Ultraviolet/visible absorption spectrum: Maximum absorption wavelength [λ:”:f,'] (nm) = 292
(109t = 3.90) Infrared absorption spectrum (cm-'): 3410,29
40,1630°1586.1520.1486.13
50,1274,1152.1074°1028.93
0,896 Nuclear magnetic resonance spectrum (200MHz) ppm (CDC
Q, ): 2.0O-2-21 (42H, each s
), 3.55-4.90 (m).

5.15-5.50 (m), 7.30 (IH, a, J
= 9.2Hz).

7.26-7.41 (5H, m), 8.17 (IH, d
d, J = 9.2Hz.

2.7Hz), 8.30 (IH, d, J= 2.7Hz
) High performance liquid chromatography [Nacalai Tesque C Co., Ltd.]
C05MO5I LCIa column C4,6rnm I
DX 15f)mm).

UV... 1. Detection, eluent niacetonitrile/water = 3
/l (V/V), flow rate: 1. Om+2/m1nl :'
R=7.7m1n Specific optical rotation ([α]”): (c O
, 25,1,4-dioxane); 2-chloro-4-nitrophenyl-tetrazocar 〇-acetyl-4',6'-benzylidene-β- obtained at +84° (2)
D-maltopentaoside 2.999 (1,80 mmo
1) was dissolved in a mixed solvent of 120 m of carbon tetrachloride (2 and 120 m of 1.1,2.2-tetrachloroethane), and 3.569 (18.0 mmol) of barium carbonate was added.
The mixture was heated to reflux while stirring, and then 427 mg (2,
40 mmol) and reacted for 1 hour while refluxing. After cooling this reaction solution to room temperature, insoluble matter was filtered off, and carbon tetrachloride and 1.1, 2.2
-Tetrachloroethane was distilled off under reduced pressure.

Next, this residue was purified by silica gel column chromatography, and the target fraction eluted with a dichloromethane-ethyl acetate-methanol mixture (volume ratio 99:25:l) was concentrated to yield 2-chloro-4-nitrophenyl tetrazocar O- 2.71 g (1,56
mmol, yield 86.5%) was obtained. The properties of this thing are as follows.

Melting point (°C): 121.5-123.5 Ultraviolet/visible absorption spectrum: Maximum absorption wavelength [λtei',' ] (nm) = 280 (l
og+=4.02), 229(Iogc=4.37
), 213(sh), 203(log<=4.33
) Infrared absorption spectrum Ccm-1): 3470,175
6,1532°1372, +234.1034,89
4,714,602 nuclear magnetic resonance spectrum (200MH
z) ppm (CDCI2.): 1.90-2.21 (
428, each s), 3.41 (IH, dd, J=
12.0Hz, 5.5Hz), 3.51 (IH, dd,
J = 12.0Hz.

2.3Hz), 3.88-4.95(m), 5.18-
5.61 (m), 7.29 (LH, d, J = 9.0Hz
), 7.46 (2H, dd, J = 7.51 (z, 7.5
Hz), 7.60 (IH, dd, J= 7.5Hz, 7
．． 5Hz), 8.00 (2H.

brd, J=7.5Hz), 8.17(IH, dd, J
=9.0Hz, 2.8Hz), 8', 30(l)l, d
, J=2.7Hz) High performance liquid chromatography [CO5MOS I LCr a column (4.6 mm 1DX 150 mm) manufactured by Decalai Tesque Co., Ltd.

UV28゜nm detection, eluent niacetonitrile/water = 7
:3 (v/v), flow rate: 1. Om4/m1n) :'R
=16.3m1n ratio light intensity C[a]”): (c O,
50,1,4-dioxane); + 75.4° 2-chloro-4-nitrophenyl tetrazocar 0-acetyl-46-O-benzoyl-65-bromo-β-D-malto obtained in (3) Pentaoside 2.719 (1,5
6 mmol) was dissolved in 100 ml of HMPA, and NaB
Add 1.82 g (29.0 mmol) of HsCN,
The reaction was allowed to proceed at 0°C for 3 hours with stirring, and then the reaction solution was slowly dropped into 500 mQ of toluene, and the mixed solution was washed three times with 300 m12 of water. Next, the toluene layer was dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure to distill off the toluene.
This residue was purified by silica gel column chromatography, and the target fraction eluted with a dichloromethane-ethyl acetate-methanol mixture (volume ratio 99:25:l) was concentrated.
2.43 g of 〇-acetyl-35,6'-anhydro-4s-0-benzoyl-β-D-maltopentaoside (
1.50 mmol, yield 96.5%) was obtained. The properties of this thing are as follows.

Melting point ('C): 109.0 to 111.0 Ultraviolet/visible absorption spectrum: Maximum absorption wavelength [λ: center''] (nffi) -281 (l
ogε=3.99), 229(log t=4.3
1) Infrared absorption spectrum (cm-'): 3470.17
52.1530°1370.1234.1030.89
8,792,738,714.600 Nuclear Magnetic Resonance Spectrum (200MHz) ppm (CDCffs): 2.0
0-2.21 (39H, m), 3.60-3.63 (2
H, m), 3.85-4.95 (m), 5.05-5.
62 (m), 7.28 (IH, d, J-9,0Hz),
7.46 (2H, dd, J= 7.6Hz, 7.6Hz
), 7.60 (LH.

dd, J = 7.6Hz, 7.6Hz), 8.00 (2
H, d, J = 7.6Hz).

8.17 (IH, dd, J = 9.0Hz, 2.7Hz
), 8.30 (IH, d, J = 2.7 Hz) High performance liquid chromatography [Decalai Tesque Co., Ltd. CO5MOS I LCr a column (4.6 mm 1D
x 150mm).

Uvz,. nm detection, eluent niacetonitrile/water = 7
: 3 (v/v), flow rate: 1. Om (L/m1n):
'R=15.3m1n specific light intensity ([α]"): (c O
, 52,1,4-dioxane); + 70.2° 2-chloro-4-nitrophenyl trideca-〇-acetyl-3',6'-anhydro-4S-0
-Benzoyl-β-D-maltopentaoside 2.22g
(1,37 mmol) was dissolved in 70 mQ of methanol,
After adding 380+ng (2.75 mmol) of potassium carbonate and reacting at 25°C for 5 hours with stirring,
The reaction solution was concentrated under reduced pressure, and the methanol contained therein was distilled off. The residue was then purified by ODS column chromatography and mixed with an ethanol-water mixture (volume ratio l:3).
), the target fraction eluted with 2-chloro-4-
Nitrophenyl=3',6'-anhydro-β-D-maltopentaoside is 938+IIg (0,971 mmo
1, yield 70.9%).

The properties of this thing are as follows.

Melting point (°C): 185-187 (decomposed) Ultraviolet/visible absorption spectrum: Maximum absorption wavelength [λ::g'] (nm) -291 (log
ε-3,96), 228(sh), 209(logε=
4.16) Infrared absorption spectrum (cm-'): 34
20.2920.1628°1586.1520.14
84.1348.1274.1150,1024 Nuclear magnetic resonance spectrum (200MHz) ppm (DzO): 3
．． 41-4.15 (m), 5.29 (IH, d, J=
7.6Hz), 5.34-5.38 (3H, m), 5.
41 (IH, d, J -3,9Hz), 7.39 (IH
, d, J = 9.3 Hz), 8.21 (IH, dd, J
= 9.3Hz, 2.7Hz), 8.37(IH, d
, J=2.7Hz) High performance liquid chromatography [TSKg manufactured by Tosoh Corporation
el Amlde-80 column (4,6mm 1Dx 2
50mm).

UV28゜nm detection, eluent niacetonitrile/water =
3: l (v/v), flow rate: 1. OmQ/m1n)
:'R=7.5m1n Elemental analysis value: Cs*HszC
H as QNO2r Theoretical value (%') 44.75 5.42 1.45
Actual measurement value (%) 44.77 5.48 1.44 Relative light intensity ([α]”): (c O, 508, H, 0); +
92.4゜Commercially available 210 rho 4-nitrophenyl san β-
D-maltoheptaoside (manufactured by Kanto Kagaku Co., Ltd.) 5-Og
(3.82 mmol) was dissolved in anhydrous DMF75111Q, and 2.30 ml of benzaldehyde dimethyl acetal was added.
2 (15,3 mmol) and tosylic acid 625+++ g (
After adding 3.63 mmol) and reacting at 50°C for 4 hours with stirring, the reaction solution was slowly dropped into 400 mQ of ice water while stirring.

A saturated aqueous sodium bicarbonate solution was slowly added to this solution while stirring under water cooling to neutralize it, and then this mixture was washed three times with 100Ila of dichloromethane, and the aqueous layer was concentrated to remove the DMF and water contained therein. Distilled away. This residue was purified by ODS column chromatography,
The target fraction eluted with an ethanol-water mixture (volume ratio 3 nia) was concentrated and recrystallized from impropanol-methanol to yield 2-chloro-4-nitrophenyl-4t,6y.
-Benzyritine-β-D-maltoheptaoside is 2.3
51? (1.68 mmol, yield 44.0%) was obtained. The physical properties of this material are as follows.

Melting point (°C): 201-202.5 Ultraviolet/visible absorption spectrum: Maximum absorption wavelength [λ:: (18] (nm) -295 (
log t = 3.98), 229 (sh), 208 (
log t = 4.36) infrared absorption spectrum (cm
-→: 3420, 2930, 1630° 1586.15
22.14g6.1348,1276.1150,10
78.1026 nuclear magnetic resonance spectrum (200MHz)
ppm (D, O): 3.50-4.45 (m), 5.1
9-5.35 (7H, m), 5.62 (lH, s).

7.29 (IH, d, J -9,3Hz), 7.41-
7.46 (5H, m).

8.15 (IH, dd, J=9.3Hz, 2.7Hz)
, 8.27 (IH, d, J = 2.7 Hz) High performance liquid chromatography [TSKg manufactured by Tosoh Corporation
el Amlde-80 column (4,6mm 1DX 2
50+u+).

UV2. . nm detection, eluent niacetonitrile/water = 3
: l (v/v), flow rate: 1. OmQ/m1n) :'
2.20 g (1,58 mmol) of 2-chloro-4-nitrophenyl-4',6'-benzylidene-β-D-maltoheptaoside obtained in R-5,8m1n (1) was dissolved in pyridine 80- and acetic anhydride 40 + aK512mmo
After adding 1) and reacting at room temperature for 2 days, the reaction solution was concentrated under reduced pressure, and the pyridine, acetic anhydride, and acetic acid contained therein were distilled off. The residue was purified by silica gel column chromatography, and methanol - Concentrate the target fraction eluted with a dichloromethane mixture (volume ratio 2:98) and recrystallize it from ethanol.
Benzylidene-β-D-maltoheptaoside is 3.48
9 (1.55 mmol, yield 98.8%) was obtained. The properties of this thing are as follows.

Melting point (°C): 134.0-136.0 Ultraviolet/visible absorption spectrum: Maximum absorption wavelength [λ hand', N] (nm) -283 (log
t -3,98), 228 (sh), 207 (log
t-4,39) Infrared absorption spectrum (cm""): 3
460.2950.1752゜1586.1528.1
484.1430.1370.1348.1236.1
034゜900.750.702 Nuclear magnetic resonance spectrum (200MHz) ppm (CDC
a,): 1.98-2.20 (60H, each s)
, 3.56-5.55 (m), 7.26-7.45 (6
H, m), 8.16 (IH, dd, J = 9.1Hz
, 2.7Hz).

8.29 (IH, d, J = 2.7 Hz) High performance liquid chromatography [C05M05I manufactured by Decalai Tesque Co., Ltd.
L(+s column (150 IDX questions between 4 and 6).

UV... nm detection, eluent niacetonitrile/water = 3
: l (v/v), flow rate: 1. Os+12/m1n)
:R-10,6m1n ratio light intensity ([α]”): (c 1
．． 03.1.4-dioxane); + 109.6° (2) 2-chloro-4-nitrophenyl-Eifcer〇-acetyl-4',6'-benzylidene-β-D
- Maltoheptaoside 3.389 (1,51 mmol
), carbon tetrachloride 140+1172 and 1.1,2.2-
It was dissolved in a mixed solvent with 140 mff of tetrachloroethane, 2.99 g (15.1 mmol) of barium carbonate was added, and the mixture was heated to reflux while stirring, and then 351 m9 of N-bromosuccinimide (NBS) was added to the reflux solution.
(2.27 mmo4) was added, and the mixture was allowed to react for 1 hour under reflux. After cooling this reaction solution to room temperature, insoluble matter was filtered out, and carbon tetrachloride contained in the filtrate and 1.1,
2.2-Tetrachloroethane was distilled off under reduced pressure.

Next, without isolating 2-chloro-4-nitrophenyl-eicosa O-acetyl-47-O-pensoyl-6fubromo β-D-maltoheptaoside contained in this residue, HMPAIO (h++f2 Dissolved, add 90 g (30.3 mmol) of NaBHsCNl, and react at 70°C for 3 hours with stirring. Next, this reaction solution was slowly added dropwise into 500 mQ of toluene, and the mixed solution was added 3 times with 300 mQ of water. The toluene layer was then dried with anhydrous sodium sulfate.
After filtration, the filtrate was concentrated under reduced pressure and toluene was distilled off. 2-Chloro-4-nitrophenyl-nonadecar〇-acetyl-3',6'-anhydro-47-O-benzoyl-β-D-maltoheptaoside contained in this residue was not isolated and was used as is. methanol 60rtr
Dissolved in Q, potassium carbonate 84.6+++9 (0,61
After adding 3 mmol) and reacting at 25°C for 5 hours with stirring, the reaction solution was concentrated under reduced pressure to distill off the methanol contained therein. The residue was then purified by ODS column chromatography, and the target fraction eluted with an ethanol-water mixture (volume ratio 1:3) was concentrated to yield 2-chloro-4-nitrophenyl-3',6'-7'.
human o-β-D-maltoheptaoside is 517m5
(0,401 mmol, yield 26.5%) was obtained.

The properties of this thing are as follows.

Melting point ('O): 208-210 (decomposition) Ultraviolet/visible absorption spectrum: Maximum absorption wavelength [λ::C:H] (nm) = 292 (lo
gε=3.96), 229 (sh), 209 (log
t-4,17) Infrared absorption spectrum (cm-'): 3
400.2920,1584゜1520.1484,1
348.1272.1150,1022 Nuclear magnetic resonance spectrum (200MHz) ppm (DzO): 3.4
5-4.20 (m), 5.30-5.43 (7H, m)
, 7.40 (IH, d, J = 9.3 Hz), 8-22
(In, dd, J = 9-3Hz, 2.4Hz), 8
．． 39 (IH, d, J = 2.4Hz) High performance liquid chromatography [TSKg manufactured by Tosoh Corporation
el Am1de-80 column (4,6+n+++ID
X 150++m).

UV2sonm detection, eluent niacetonitrile/water = 3
: 1 (v/v), flow rate: 1. O+n12/m1n)
:'R=20.6m1n Elemental analysis value: C,,H,□C
HN as 12NO,, Theoretical value (%) 44.67 5.62 1.09 Actual value (%) 44.59 5.68 1.07 2-chloro-4-nitrophenyl obtained in Example 8 Take 100 mg of 3S65-anhydro-β-D-maltoheptaoside and add it to 50 mM phosphate buffer (pH
=7.0) to make a total volume of 25ml to prepare a substrate solution.

(2) Preparation of conjugated enzyme solution 50mM phosphate buffer containing 50mM Nacl (pH=
7.0), commercially available yeast-derived Nobiichi glucosidase and commercially available almond-derived β-glucosidase were added to
A conjugated enzyme solution was prepared by adding 700U and 1370u to make a total volume of 100ml.

(3) Preparation of reagent solution The substrate solution and conjugated enzyme solution were mixed well at a volume ratio of 2:1 to prepare a reagent solution.

(4) Preparation of standard a-amylase solution Commercially available human-derived σ-amylase (P:S=1:1) was mixed with 50 mM phosphate buffer containing 50 mM NaCl (pH=
7.0), as well as 0125.50.100.150.
Prepare standard amylase solution by dissolving it to a concentration of 200.300U/Q.

(5) Preparation of sample liquid When the sample for α-amylase activity measurement was a liquid, it was used as a sample liquid as it was. In the case of a solid, accurately weigh 500 mg of the sample, add 50 mM phosphate buffer containing 50 mM NaCl (
A sample solution was prepared by adding pH=7.0) to a total volume of 5 m(t).

(6) Creation of calibration curve Reagent solution 3. After heating OmQ at 37 °C for 3 minutes, add 300 μQ of standard α-amylase solution and stir, and then measure the increase in absorbance at 400 nm for 2 minutes after heating at 37 °C for 2 minutes. A calibration curve was created from the relationship between the standard a-amylase solution activity and the increase in absorbance. Standard σ-amylase manufactured by Kokusai Reagent Co., Ltd. (Cabrizyme A)
MY), the equation of the calibration curve is U = 3.63 ΔAX102 + 1.0O (U: enzyme activity U/Q, ΔA: increase in absorbance). The graph is shown in FIG.

(7) Reagent solution for measuring α-amylase activity in sample solution 3.
After heating OmQ at 37°C for 3 minutes, the sample solution
Add μQ, stir, and heat at 37℃ for 2 minutes.
The increase in absorbance at 400 nm per minute was measured.

The a-amylase activity in the sample solution can be measured by calculating from this measured value and the calibration curve created in (6). Note that the enzyme activity value in the sample solution falls within the applicable range of the calibration curve (0 to
If it exceeds 300 U/ff), repeat the measurement after 5 times containing 50 mM NacI.

Example 4 In order to demonstrate that the compound of the present invention exists stably within the measurement system, a reaction with a conjugated enzyme solution was performed using a non-reducing end-unmodified compound as a control according to the test method described below.

[Test method] 2-chloro-4-nitrophenyl-3' obtained in Example 1
,65-anhydro-β-D-maltopentaoside 10
0 mg was taken, and 50 mM phosphate buffer (pH=7.0) containing 50 mM NaCl was added to make the total volume 25 mQ to prepare substrate solution (2).

(2) Preparation of substrate solution (control substrate) Commercially available (7) 2-chloro-4-nitrophenyl β-D-
Take 100 mg of maltopentaoside and add 50 mM
Substrate solution (2) was prepared by adding acI-containing 50 mM phosphate buffer (pH=7.0) to make a total volume of 25++12.

(3) Preparation of conjugated enzyme solution 50mM phosphate buffer containing 50mM Nacl (pH
= 7.0), commercially available yeast-derived a-glucosidase,
A conjugated enzyme solution was prepared by adding 11,700 U and 137 U of commercially available almond-derived β-glucosidase, respectively, and using the total amount as lomQ.

(4) After heating the conjugated enzyme reaction substrate solution ■ and the conjugated enzyme solution at 37°C for 3 minutes, prepare the substrate solutions ■ and ■ respectively at a ratio of substrate solution:conjugated enzyme solution=2:l (volume ratio). Mix and further incubate at 37°C for 2
After heating for 5 minutes, the increase in absorbance at 400 nm (ΔA) was measured.

The results are shown in FIG. In FIG. 2, the marks are for the substrate solution, and the △ marks are for the substrate solution ■.

From FIG. 2, it can be seen that the substrate of the present invention does not react with the conjugated enzyme and exists stably within the measurement system.

Example 5 Measurement reagent (1) Reagent composition Reagent A and reagent B were prepared by dissolving the following components in purified water at the following concentrations.

Anhydro-β-D-maltopentaoside 2.0mM
σ-glucosidase 600/++
+I2β-glucosidase 100
/m12β-glycerophosphate buffer (pH=7.0)
10mM NaCl
40mM bovine serum albumin
0.05% component Soji's black reagent B: β-glycerophosphate buffer (pH = 7.0)
10mM bovine serum albumin 0
．． 05%NaCl
40mM (2) Measurement method When the measurement sample was a liquid, it was used as a sample solution as it was. In the case of a solid, sample 50k was accurately weighed, and reagent B was added to make a total volume of 5 mQ, which was used as a sample solution. Next, reagent A3. Om
After heating Q at 37°C for 3 minutes, 300 μa of sample solution was added.
was added and stirred, and the increase in absorbance at 400 nm was measured for 2 minutes after further heating at 37°0 for 2 minutes. The a-amylase activity in the sample solution can be measured by calculating from this measured value and a calibration curve prepared in advance. Note that if the value of enzyme activity in the sample solution exceeds the applicable range of the calibration curve (0 to 300 U/ff), remeasurement is performed after diluting with reagent B to the corresponding multiple.

[Brief explanation of drawings]

FIG. 1 is a graph showing a calibration curve used for measuring σ-amylase activity, and FIG. 2 is a graph showing the stability of the substrate of the present invention and a control substrate in the measurement system. Figure 1 Increase in absorbance (JA) Figure 2

Claims (1)

[Claims] 1 3,6- represented by the general formula ▲ Numerical formulas, chemical formulas, tables, etc. ▼ (R in the formula is an aromatic chromogenic group, and n is an integer from 1 to 6) Anhydromaltooligoside derivatives. 2. A reagent for measuring α-amylase activity, which contains the 3,6-anhydromaltooligoside derivative according to claim 1 as an active ingredient. 3. 3 and 6 according to claim 1 in the α-amylase-containing sample.
- Determining α-amylase activity by adding α-anomer of anhydromalto-oligoside derivative and α-glucosidase and/or glucoamylase to perform an enzymatic reaction and quantifying the liberated aromatic chromogenic compound. Measuring method. 4. 3 and 6 according to claim 1 in the α-amylase-containing sample.
- An aromatic color is released by adding β-anomer or a mixture of α-anomer and β-anomer of the anhydromalto-oligoside derivative and α-glucosidase and/or glucoamylase and β-glucosidase to perform an enzymatic reaction. 1. A method for measuring α-amylase activity, which comprises quantifying a chemical compound.