JPH0532687A - Alkoxymethylidene maltooligosaccharide derivative, reagent for determination of alpha-amylase activity containing the derivative as active component and determination of alpha-amylase activity - Google Patents

Abstract

PURPOSE:To provide the subject new derivative enabling accurate and efficient determination of alpha-amylase activity and useful as a reagent for the determination of alpha-amylase activity. CONSTITUTION:The derivative of formula I (X<1> is methoxy or ethoxy; X<2> is H, methoxy, ethoxy, etc.; R is H, aromatic chromogenic group, etc.; n is 2-6), e.g. 2-chloro-4-nitrophenyl:4<5>,6<5>-0-dimethoxymethylidene-beta-D-maltope ntoxide. The compound of formula I can be produced e.g. by adding 2-100mol of a compound of formula III, formula TV (X<3> and X<4> are 1-4C alkyl), their acetal or ketal to 1mol of D-maltooligosaccharide of formula II or its derivative and reacting in an aprotic polar solvent at 20-100 deg.C for 1-20hr.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel alkoxymethylidene maltooligosaccharide derivative, an α-amylase activity measuring reagent containing the derivative as an active ingredient, and an α-amylase activity using the derivative efficiently and It relates to an accurate measurement method.

[0002]

2. Description of the Related Art Conventionally, the measurement of α-amylase activity in body fluids such as serum, urine, pancreatic juice and saliva is extremely important in clinical diagnosis, and particularly acute and chronic hepatitis, pancreatitis, pancreatic cancer, It is an essential measurement item in the differential diagnosis of mumps.

Various methods have been heretofore known for measuring this α-amylase activity. In recent years, various substituted phenylmalto-oligosides have been prepared by modifying the non-substituted terminal glucose with various substituents. Characteristic of having resistance (stability) to the enzyme system] as a substrate,
A method for colorimetric determination is widely used, in which the substituted phenols produced are cleaved with α-amylase and then the coupled enzyme system is allowed to act to produce the substituted phenols as they are, or after changing the pH as necessary, or after condensation. It has become possible to be.

However, these substrates have insufficient resistance to the coupling enzyme, low solubility in water, low affinity for α-amylase (high km value), and low decomposition rate by α-amylase. However, it has various drawbacks such that it is chemically and biochemically unstable and cannot be stored for a long period of time.

[0005]

DISCLOSURE OF THE INVENTION The present invention overcomes the drawbacks of the conventional reagents for measuring α-amylase activity and the measuring methods using the same, and efficiently and accurately determines α-amylase activity. The present invention has been made for the purpose of providing a novel compound suitable as a measurable reagent and a novel method for measuring α-amylase activity using this as a reagent.

[0006]

As a result of various studies conducted by the present inventors to achieve the above object, a specific alkoxymethylidene maltooligosaccharide derivative is extremely suitable as a reagent for measuring α-amylase activity. The inventors have found that the objective can be achieved by measuring α-amylase activity using the same, and have completed the present invention based on this finding.

That is, the present invention has the general formula

[Chemical 2] (In the formula, X ¹ is a methoxy group or an ethoxy group, X ² is a hydrogen atom, a methoxy group, an ethoxy group or a substituted or unsubstituted hydrocarbon group, R is a hydrogen atom, an aromatic color-forming group or a monosaccharide other than glucose. Residue, n is an integer of 2 to 6)
Alkoxymethylidene maltooligosaccharide derivative represented by :, a reagent for measuring α-amylase activity containing the alkoxymethylidene maltooligosaccharide derivative as an active ingredient, and α
-The amylase-containing sample is characterized in that the alkoxymethylidene maltooligosaccharide derivative and the conjugated enzyme for measuring α-amylase activity are added to carry out an enzymatic reaction to quantify the liberated aromatic color-forming compound or monosaccharide. The present invention provides a method for measuring α-amylase activity.

The maltooligosaccharide moiety in the alkoxymethylidene maltooligosaccharide derivative of the general formula (I) of the present invention corresponds to α- and β-D-maltotetraose to α- and β-D-maltooctaose. Everything can be used. Among these, D-maltopentaose, D-maltohexaose and D-maltoheptaose are preferable from the viewpoint of the properties of the final substrate.

In the alkoxymethylidene maltooligosaccharide derivative represented by the general formula (I), X ¹ is a methoxy group or an ethoxy group, and X ² is a hydrogen atom, a methoxy group, an ethoxy group or a substituted or unsubstituted group. It is a hydrocarbon group. Examples of this hydrocarbon group include linear, branched, saturated or unsaturated aliphatic hydrocarbon groups such as methyl group, ethyl group, isopropyl group, butyl group, allyl group and cyclohexyl group, and benzyl. Group, phenyl group, toluyl group, naphthyl group, aromatic hydrocarbon group such as biphenyl group, and the like. These hydrocarbon groups are alkoxy groups, acyl groups, carboxyl groups,
It may be substituted with a nitro group, an alkylsilyl group, a sulfonyl group, a halogen atom or the like.

The alkoxymethylidene maltooligosaccharide derivative represented by the general formula (I) of the present invention has X ¹
When at least one of X ² and X ² is a methoxy group or an ethoxy group, the water solubility is significantly improved, and the effectiveness as a substrate for α-amylase activity measurement is enhanced. From this viewpoint, it is desirable that X ² in the general formula (I) is also a methoxy group or an ethoxy group.

Further, R in the alkoxymethylidene maltooligosaccharide derivative represented by the above general formula (I) is a hydrogen atom, an aromatic color-forming group or a residue of a monosaccharide other than glucose. Group chromophoric groups are preferred.

The aromatic chromophoric group is not particularly limited as long as it can be detected spectroscopically.

[Chemical 3] (In the formula, R ¹ to R ⁵ are a hydrogen atom, a halogen atom, a nitro group, an alkyl group, an aryl group, an aralkyl group, an amino group, a sulfonic acid group or a carboxyl group, and they may be the same or different. R may
¹ and R ² , R ² and R ³ are bonded to each other,
May form a fused aromatic ring)

[0013]

[Chemical 4] (R ⁶ in the formula is a hydrogen atom or an alkyl group)

[Chemical 5] (R ⁷ in the formula is a hydrogen atom or a halogen atom)

[Chemical 6] (Wherein R ⁸ to R ¹⁵ are hydrogen atom, halogen atom,
A nitro group, an alkyl group, an aryl group, an aralkyl group, an amino group, a sulfonic acid group or a carboxyl group, which may be the same or different, and R ⁸ and R ⁹ , R ¹⁰ and R ¹¹ and R may be bonded to each other to form a condensed aromatic ring, and R ⁹ and R
¹⁰ and / or R ¹³ and R ¹⁴ may be a common oxygen atom to form a condensed ether ring, and Z is preferably a nitrogen atom or a group represented by N → O).

The residue of a monosaccharide other than glucose includes a residue of a broadly defined monosaccharide or a derivative thereof, for example, a residue of fructose, inositol, glucitol, glucose-6-phosphate or the like.

The alkoxymethylidene maltooligosaccharide derivative represented by the general formula (I) is an α-anomer (α-
It may be either a glycoside) or a β-anomer (β-glycoside). Representative examples of such alkoxymethanamine dust Den malto oligosaccharide derivative represented by the above general formula (I), 2-chloro-4-nitrophenyl 4 ⁵ 6 ⁵
-O- dimethoxy methylidene-beta-D-maltopentaoside, ^{2-chloro-4-nitrophenyl} 4 5 ^{6 5} -
O-(1-methoxy) ethylidene-beta-D-maltopentaoside, ^{2-chloro-4-nitrophenyl 4} 5, 6
⁵ -O- (1-ethoxy) methylidene-beta-D-maltopentaoside, 4-nitrophenyl ⁼ 4 ^5, 6 ⁵ -O-
Dimethoxymethylidene-α-D-maltopentaoside,
2-chloro-4-nitrophenyl = 4 ⁵ , 6 ⁵ -O-
(1-ethoxy) benzylidene-beta-D-maltopentaoside, ^{2-chloro-4-nitrophenyl} 4 7 ^{6 7}
-O- (1-methoxy) ethylidene-beta-D-maltoheptaoside, phenol India 3'-chlorophenyl ⁼ 4 ^5, 6 ⁵ -O- dimethoxy methylidene-beta-D-maltopentaoside, 4- Methylumbelliferonil = 4
^{5, 6 5} -O-1- ethoxy-3- Ketobuchiriden -α
-D- maltopentaoside, Rezazuriniru ⁼ 4 6, ^{6 6}
-O-1-methoxy-methylidene-.alpha.-D-maltohexaoside, Rushiferiniru ⁼ 4 ^4, 6 4 -O- dimethoxy methylidene-beta-D-maltotetraosyl ^glucoside, 4 5, ^{6 5}
-O- dimethoxy methylidene -D- ^{maltopentaose, 4 7, 6 7 -O- (} 1- methoxy) ethylidene -D
- maltopentaose, fructosyl ^{= 4 4,} 6 4 -O
-Dimethoxymethylidene-α-D-maltotetraoside and the like.

In the above, the symbols 6 ^5- , 6
⁷ - 4 5 ^- 4 7 ^- etc., shown from the reducing end side of glycose units constituting the maltooligosaccharides, 5 th, 6 of 7 th glycose, that the 4-position of the hydroxyl groups are substituted.

The alkoxymethylidene maltooligosaccharide derivative represented by the above general formula (I) of the present invention is a novel compound which has not been described in any literature, and its production method is not particularly limited, and any method can be used. However, it can be produced, for example, by the following method.

That is, as a starting material, a commercially available product or a general formula which can be obtained by a known production method is used.

[Chemical 7] D-malto-oligosaccharide represented by the formula (R and n have the same meanings as described above) or a derivative thereof, for example, 2-chloro-4-nitrophenyl = β-D-maltopentaoside, 4-nitrophenyl = α-D-maltoheptaoside, phenol indo-3'-chlorophenyl = β-D
-Maltopentaoside, D-Maltopentaose, D-
General formula using maltoheptaose, fructosyl-β-D-maltotetraose, etc.

[Chemical 8] (Wherein X ¹ and X ² have the same meanings as described above) or

[Chemical 9] (Wherein X ¹ and X ² have the same meanings as described above, X ³ and X ⁴ are each an alkyl group having 1 to 4 carbon atoms,
They may be the same or different)
By reacting with the carbonyl compound represented by or its acetal or ketal, the alkoxymethylidene maltooligosaccharide derivative represented by the above general formula (I) can be obtained.

Examples of the carbonyl compound represented by the general formulas (III) and (IV) or the acetal or ketal thereof include tetramethoxymethane, tetraethoxyethane, triethyl orthoacetate, trimethyl orthoacetate, α, α, α-tri Methoxytoluene and the like can be mentioned.

The alkoxymethylidene maltooligosaccharide derivative represented by the general formula (I) can be obtained by, for example, adding the D-maltooligosaccharide represented by the general formula (II) or a derivative thereof to the above general formulas (III) and (IV). The represented carbonyl compound or its acetal or ketal is added in an amount of about 2 to 100 times, preferably about 2 to 10 times, and usually in an aprotic polar solvent in the presence of a catalyst, at 20 to 100 ° C., preferably 25 to It can be obtained by reacting while stirring at a temperature in the range of 50 ° C. for about 1 to 20 hours, preferably for about 2 to 5 hours.

As the aprotic polar solvent used in this case, for example, N, N-dimethylformamide (DM
F), N, N-dimethylacetamide (DMA), dimethyl sulfoxide (DMSO), hexamethylphosphoric triamide (HMPA) and the like, and the catalyst includes, for example, p-toluenesulfonic acid, hydrogen chloride, sulfuric acid,
Examples thereof include anhydrous zinc chloride and strongly acidic ion exchange resins.

In this reaction, a desiccant such as molecular sieves, drylite or phosphorus pentoxide may coexist in order to remove water, or the reaction is carried out under reduced pressure while distilling off water, alcohol and the like. May be.

Then, the reaction solution can be purified by a conventional method such as column chromatography or thin layer chromatography to obtain the desired compound. The alkoxymethylidene maltooligosaccharide derivative represented by the general formula (I) thus obtained is extremely useful for the measurement of α-amylase activity, and the alkoxymethylidene maltooligosaccharide derivative is used to measure α-amylase activity. It can be measured efficiently.

When measuring the α-amylase activity, the alkoxymethylidene maltooligosaccharide derivative represented by the general formula (I), which is α-amylase and the substrate, in the presence of a coupled enzyme for measuring α-amylase activity is used in a conventional method. To act. There is no particular limitation on the relationship between the substrate and the coupling enzyme used, and a conventional method may be used.

Further, as described above, the alkoxymethylidene maltooligosaccharide derivative represented by the general formula (I) has α-anomer and β-anomer, and in this case, as a conjugated enzyme for measuring α-amylase activity, Is, for example, (1) in the case of a compound (α-anomer and / or β-anomer) in which R in the general formula (I) is a hydrogen atom, α-
Glucosidase and / or glucoamylase (2)
A compound in which R is an aromatic color-forming group or a monosaccharide residue other than glucose, and (a) α-anomer in the case of only α-anomer
Glucosidase and / or glucoamylase
In the case of the β-anomer alone or a mixture of the α-anomer and the β-anomer, a combination of α-glucosidase and / or glucoamylase and β-glucosidase is used. In any of the above cases, β-amylase can be used in combination if necessary.

An advantageous system for measuring α-amylase activity is, for example, an alkoxymethylidene maltooligosaccharide derivative of the general formula (I) 0.1-10 mM.
And a buffer solution of 2 to 300 mM, and each of α-glucosidase and / or glucoamylase as a coupling enzyme is 5 to 1000 units / ml, and when β-glucosidase is used, it is 0.5 to 30 units / ml. Examples include systems having a pH of 4 to 10. Examples of the buffer used in this system include phosphate, acetate, carbonate, Go
od's buffer, borate, citrate, dimethyl glutarate and the like.

The α-glucosidase may be of any origin such as animals, plants and microorganisms, but is preferably yeast-derived. In addition, glucoamylase may be derived from any origin, but is preferably derived from Rhizopus sp., For example. Furthermore, β-glucosidase of any origin may be used, for example, those obtained from almond seeds are used.

The β-amylase may be of any origin, and for example, those of bacterial or plant origin can be used. In such a system, in addition to the above-mentioned components, within a range that does not impair the object of the present invention, and if necessary, various conventional addition components such as a solubilizing agent and a stabilizer, glycerin, bovine serum albumin, α- or β-cyclodextrin, Triton X-100, etc. can be added, and as an α-amylase activator, NaC
1, MgCl ₂ , MgSO ₄ , CaCl ₂ , CaCl ₂
・ Cl ⁻ ions used in the form of H ₂ O, Ca ²⁺
Ions, Mg ²⁺ ions and the like may be added. These additive components may be used alone or in combination of two or more, and may be added at an appropriate stage of the preparation of the above system.

Further, in the alkoxymethylidene maltooligosaccharide derivative represented by the general formula (I), when R is a hydrogen atom or a substrate which is a residue of a monosaccharide other than glucose, a monosaccharide such as glucose produced by an enzymatic reaction is used. , Fructose, or other monosaccharides by NAD → NADH
Alternatively, enzymes normally used in a photometric change measurement system associated with oxidation-reduction reaction such as production of quinone-based substance, that is, glucose-6-phosphate dehydrogenase (eg Leuc
derived from onostoc mesentercides, etc.), maltose phosphorylase (eg La
Ctobacillus brevis and the like), hexokinase (for example, yeast and the like), β-phosphomutase [for example, rabbit muscle (rabbi
derived from tmuscle, etc.], sorbitol dehydrogenase [eg sheep liver]
r)], glucose oxidase (eg, Aspergillus niger, etc.), peroxidase (eg, wasabi, etc.), NAD, ATP, and the like.

When a substrate in which R is an aromatic chromophoric group is used, as described above, in addition to the conjugated enzyme system involved in the α-amylase reaction, the absorbance without the need for an enzyme involved in the light absorption system. It is more preferable because the method can be applied.

The reagent of the present invention may be used in a dried or dissolved form, or may be used by impregnating it with a thin film carrier such as a sheet or an impregnable paper. By using such a reagent of the present invention, the α-amylase activity contained in various samples can be accurately measured with a simple operation and with high sensitivity.

Next, a preferred embodiment of the method of the present invention will be described. First, to a sample containing α-amylase, 5 to 1 of α-glucosidase or glucoamylase or both of them as a coupling enzyme for measuring α-amylase activity
000 units / ml, preferably 10-500 units / ml
In addition, when the alkoxymethylidene maltooligosaccharide derivative represented by the general formula (I) contains a β-anomer, β-glucosidase is further added in an amount of 0.5 to 30 units / m 2.
1, preferably 1 to 15 units / ml, and simultaneously with or after this, the alkoxymethylidene maltooligosaccharide derivative 0.1 to 10 mM, preferably 0.3 to 5 mM
Is added together with a buffer, the temperature is 25-45 ° C, preferably 35-40 ° C, pH 4-10, preferably 6-
After carrying out an enzymatic reaction for at least 1 minute, preferably 2 to 10 minutes under the conditions of 8, the resulting aromatic color-forming compound is subjected to a conventional method as it is or after adjusting the pH as necessary, or after performing a condensation reaction. In addition, the amount of change in absorbance is measured continuously or intermittently at an appropriate absorption wavelength, and the amount of change in absorbance of the α-amylase preparation measured in advance is compared to calculate the α-amylase activity in the sample. It can also be calculated from the molecular extinction coefficient of the produced aromatic color-forming compound.

When R of the compound represented by the general formula (I) is a hydrogen atom or a residue of a monosaccharide other than glucose, an enzyme relating to the absorption system and other necessary components are appropriately added so that R is It can be performed in the same manner as in the case of an aromatic color-forming group.

The α-amylase-containing sample used in the present invention is not particularly limited as long as it has α-amylase activity, and specifically, it is a microbial culture solution, a plant extract, or Body fluids and tissues of animals and extracts thereof can be used. When the α-amylase-containing sample is a solid, it may be dissolved or suspended in purified water or the above-mentioned buffer. Also,
If necessary, insoluble matter may be removed by an operation such as filtration.

[0035]

EFFECT OF THE INVENTION The alkoxymethylidene maltooligosaccharide derivative represented by the general formula (I) of the present invention is a novel compound and is extremely useful as a reagent for measuring α-amylase activity. The α-amylase activity can be easily measured with high accuracy and in a short time by an automatic analysis method, a manual method, etc. without being affected by glucose, maltose, bilirubin, hemoglobin and the like contained in the sample.

Further, since the alkoxymethylidene group of the compound of the general formula (I) of the present invention has an ether structure in the modifying group, its water solubility is extremely higher than that of the conventional modifying group, and the compound is formulated. , The preparation of a reagent is easy, and since the alkoxymethylidene group is a functional group that is chemically and biochemically stable, even if a reagent is dissolved to prepare a reagent and a coupling enzyme coexists, There is an advantage that the initial state can be maintained for a long period of time.

Further, the compound of the above-mentioned general formula (I) of the present invention is a non-reducing terminal glycose which is effective as a substrate for measuring α-amylase activity. It is also useful as an intermediate for producing a converted maltooligosaccharide derivative. For example, from the alkoxymethylidene maltooligosaccharide derivative of the present invention, 4,6-O
-Alkoxymethylidene-derived acylmaltooligosaccharide derivative, which is dealkoxymethylatedylidene to give the corresponding partially acylated maltooligosaccharide derivative. Alternatively, an acylated maltooligosaccharide derivative in which two positions are modified or converted with various groups is obtained, and then deacylation is performed to modify one or two positions at the 6-position, 5-position, and 4-position of the non-reducing terminal glycose. Alternatively, a converted maltooligosaccharide derivative or the like can be produced.

The alkoxymethylidene maltooligosaccharide derivative represented by the general formula (I) used as the intermediate is excellent in the alkoxymethylidene group in the 4,6-O-alkoxymethylidene acylmaltooligosaccharide derivative. Conditions required for dealkylation or arylation reaction in the case of conventional 4,6-O-alkyl- or arylmethylidene-acylated malto-oligosaccharide derivatives due to their high reactivity due to different polarities (water solubility) It is possible to carry out dealkoxymethylidene formation under much weaker conditions, that is, at a lower temperature, in a shorter time, and with a weaker acid catalyst, and as a result, the decomposition of R substituted with the hydrogen atom of the hydroxyl group of the reducing terminal glycose. And side reactions such as sugar chain cleavage can be prevented, and therefore the final target compound It has the advantage that can be obtained in high yield.

[0039]

The present invention will be described in more detail by way of examples, which should not be construed as limiting the invention thereto. The maximum absorption wavelength in each example is a value measured in methanol and the specific optical rotation is a value measured by the D line at 25 ° C., unless otherwise specified.

[0040] Example 1 2-Chloro-4-nitrophenyl ⁼ 4 ^5, 6 ⁵ -O- dimethoxy methylidene-beta-D-
Preparation of Maltopentaoside 1.50 g (1.52 mmol) of commercially available 2-chloro-4-nitrophenyl = β-D-maltopentaoside was dissolved in 7.5 ml of anhydrous DMF to prepare tetramethoxymethane.1.
5 ml (11.3 mmol) and amberlyst (15
E) 0.75 g was added, and the reaction was carried out while stirring at 35 ° C. for 4 hours. Then, this reaction solution was slowly added dropwise to 2.0 l of 100 mM phosphate buffer (pH = 7.0) under ice cooling while stirring. This mixture was purified by ODS (octadecyl silica gel) column chromatography, and the target fraction eluted with an acetonitrile-water mixture (volume ratio 3: 7) was concentrated and recrystallized from isopropanol-methanol to give 2-chloro-4- Nitrophenyl =
4 ^{5, 6 5} -O- dimethoxy methylidene-beta-D-maltopentaoside is 1.07g (1.01mmol, 66.5% yield) were obtained.

Melting point (° C.): 93.0 to 95.0 (decomposition) UV / visible absorption spectrum: absorption maximum wavelength [λmax] (nm) = 295 (log ε)
= 3.95), 227 (sh), 209 (log ε =
4.17) Infrared absorption spectrum (cm ^-1 ): 3420, 294
0,1648,1588,1524,1490,135
2,1276,1246,1154,1082,105
0,1026,930,898 Nuclear magnetic resonance spectrum (200 MHz) ppm (DMS
O-d ₆ ): 3.25 to 3.85 (m), 3.23 (3
H, s), 3.30 (3H, s), 3.89 (1H,
d, J = 3.9 Hz), 4.30 to 4.70 (m).
5.04 (2H, d, J = 3.2Hz), 5.10 (1
H, d, J = 3.7 Hz), 5.12 (1H, d, J =
3.4 Hz), 5.27 (1H, d, J = 7.6H
z), 5.25 to 5.70 (m), 7.47 (1H,
d, J = 9.3 Hz), 8.19 (1H, dd, J =
9.3 Hz, 2.7 Hz), 8.31 (1H, d, J =
2.7Hz) High performance liquid chromatography [C from Nacalai Tesque, Inc.
OSMOSILC ₁₈ column (4.6 mm ID x 250
mm), UV ₂₈₀ nm detection, eluent: acetonitrile / water = 1: 4 v / v, flow rate: 1.0 ml / min]: R
^t = 10.2 min Specific rotation [α]: (c 0.50,50 mM phosphate bu
+ 86.7 ° Elemental analysis: C ₃₉ H ₅₈ ClNO ₃₀ CH N theoretical value (%) 44.35 5.53 1.33 Found value (%) 44.55 5.43 1.34

[0042] Example 2 2-Chloro-4-nitrophenyl ^{= 4 5, 6 5 -O- (} 1- ethoxy) ethylidene -β
Except for using -D- manufacturing triethyl orthoacetate maltopentaoside the reagent, by performing the same operation as in Example 1, 2-chloro-4-nitrophenyl 4 5 ^{purpose, 5} - O- (1-ethoxy) ethylidene-β-D-maltopentaoside 509
mg (0.483 mmol, yield 31.6%) was obtained. Melting point (° C): 220 to 222 (decomposition) Ultraviolet / visible part absorption spectrum: Absorption maximum wavelength [λmax] (nm) = 289 (log ε)
= 3.97), 227 (log ε = 3.96), 209
(Log ε = 4.15) Infrared absorption spectrum (cm ⁻¹ ): 3400, 293
0,1648,1584,1520,1486,135
0, 1274, 1152, 1124, 1080, 105
0,1022,932,892 Nuclear magnetic resonance spectrum (200 MHz) ppm (DMS
O-d ₆ ): 1.16 (3H, t, d = 6.9 Hz),
1.38 (3H, s), 3.15 to 3.80 (m),
4.35 to 4.65 (m), 5.03 (2H, d, J =
3.7 Hz), 5.08 (1H, d, J = 2.9H
z), 5.10 (1H, d, J = 3.2 Hz), 5.2
7 (1H, d, J = 7.6 Hz), 5.25 to 5.70
(M), 7.47 (1H, d, J = 9.1 Hz), 8.
18 (1H, dd, J = 9.1Hz, 2.8Hz),
8.31 (1H, d, J = 2.8Hz) High performance liquid chromatography [TSKge manufactured by Tosoh Corporation]
l Amide-80 column (4.6 mm ID x 250
mm), UV ₂₈₀ nm detection, eluent: acetonitrile / water = 3: 1 v / v, flow rate: 1.0 ml / min]: R
^t = 4.8 min Specific rotation [α]: (c 0.416, methanol); +
85.2 ° Elemental analysis: C ₄₀ H ₆₀ ClNO ₂₉ C H N theoretical value (%) 45.57 5.74 1.33 Actual value (%) 45.13 5.89 1.28

[0043] Example 3 2-Chloro-4-nitrophenyl ^{= 4 5, 6 5 -O- (} 1- methoxy) ethylidene -β
Except for using -D- manufacturing orthoacetate of maltopentaoside the reagent, by performing the same operation as in Example 1, 2-chloro-4-nitrophenyl 4 5 ^{purpose, 5} - O- (1-methoxy) ethylidene-β-D-maltopentaoside 315
mg (0.303 mmol, yield 19.9%) was obtained. Melting point (° C): 85.0 to 87.0 (decomposition) Ultraviolet / visible absorption spectrum: absorption maximum wavelength [λmax] (nm) = 295 (log ε)
= 3.95), 227 (log ε = 3.91), 209
(Log ε = 4.15) Infrared absorption spectrum (cm ⁻¹ ): 3410,293
0,1630,1586,1524,1488,135
0, 1276, 1152, 1080, 1044, 102
2,934,886 Nuclear magnetic resonance spectrum (200 MHz) ppm (DMS
O-d ₆ ): 1.16 (3H, t, d = 6.9 Hz),
1.38 (3H, s), 3.15 to 3.80 (m),
4.35 to 4.65 (m), 5.03 (2H, d, J =
3.7 Hz), 5.08 (1H, d, J = 2.9H
z), 5.10 (1H, d, J = 3.2 Hz), 5.2
7 (1H, d, J = 7.6 Hz), 5.25 to 5.70
(M), 7.47 (1H, d, J = 9.1 Hz), 8.
18 (1H, dd, J = 9.1Hz, 2.8Hz),
8.31 (1H, d, J = 2.8Hz) High performance liquid chromatography [TSKge manufactured by Tosoh Corporation]
l Amide-80 column (4.6 mm ID x 250
mm), UV ₂₈₀ nm detection, eluent: acetonitrile / water = 3: 1 v / v, flow rate: 1.0 ml / min]: R
^t = 6.0 min Specific rotation [α]: (c 0.508, 50 mM phosphate b
); + 91.3 ° Elemental analysis: C ₃₉ H ₅₈ ClNO ₂₉ C H N theoretical value (%) 45.03 5.62 1.35 actual value (%) 44.89 5.51 1.15

[0044] Example 4 4-nitrophenyl ^{= 4} 7, 6
^7- O- (1-ethoxy) ethylidene-α-D-maltoheptaoside commercially available 4-nitrophenyl = α-D-maltoheptaoside is used as a raw material, and triethyl orthoacetate is used as a reagent. except that the, by performing the same operation as in example 1, the purpose of 4-nitrophenyl ⁼ 4 ^7, 6 ⁷ -O-
(1-Ethoxy) ethylidene-α-D-maltoheptaoside 511 mg (0.380 mmol, yield 32.3
%)was gotten. Ultraviolet / visible absorption spectrum: absorption maximum wavelength [λmax] (nm) = 298 (log ε)
= 4.01), 227 (sh), 209 (log ε =
4.25) Infrared absorption spectrum (cm- ¹ ): 3410,294
0,1612,1592,1518,1500,134
6,1252,1154,1082,1022,93
4,876,852 Nuclear magnetic resonance spectrum (200 MHz) ppm (DMS
O-d ₆ ): 1.15 (3H, t, d = 7.1 Hz),
1.39 (3H, s), 3.20 to 3.80 (m),
4.35 to 4.65 (m), 5.03 (4H, d, J =
3.2 Hz), 5.09 (1H, d, J = 2.7H
z), 5.11 (1H, d, J = 3.2 Hz), 5.2
0 (1H, d, J = 4.2Hz), 5.25 to 5.70
(M), 7.23 (2H, d, J = 9.2Hz), 8.
23 (2H, d, J = 9.2 Hz) High performance liquid chromatography [TSKge manufactured by Tosoh Corporation]
l Amide-80 column (4.6 mm ID x 250
mm), UV ₂₈₀ nm detection, eluent: acetonitrile / water = 3: 1 v / v, flow rate: 1.0 ml / min]: R
^t = 6.9 min Elemental analysis: C ₅₂ H ₈₁ NO ₃₉ C H N theoretical value (%) 46.46 6.07 1.04 Actual value (%) 46.22 5.90 1.18

Example 5 Measurement of α-amylase activity (1) (1) Preparation of substrate solution 2-chloro-4-nitrophenyl obtained in Example 3
4 ^{5, 6 5} -O- (1- methoxy) ethylidene-beta-D
-Maltopentaoside (Mw1040) at a concentration of 3.0 mM, 40 mM-NaCl and 2 mM-M
50 mM phosphate buffer containing gCl ₂ (pH = 7.
It was dissolved in 0).

(2) Preparation of coupled enzyme solution Commercially available α-glucosidase derived from yeast and β-glucosidase derived from almond were used at 117 u / ml and 13%, respectively.
40 mM NaCl and 2 to make u / ml concentration
50 mM phosphate buffer containing mM-MgCl ₂ (p
H = 7.0) and dissolved. As these commercially available α- and β-glucosidases, those manufactured by Toyobo Co., Ltd. were used.

(3) Preparation of standard α-amylase solution Purified water was added to commercially available human α-amylase (P: S = 1: 1) to prepare 0,154,310,459,591 IU / l.
The solution was dissolved in the solution to give a standard α-amylase solution. In addition,
As this commercially available human α-amylase, Calibzyme AMY manufactured by International Reagents Co., Ltd. was used. In addition, the activity of α-amylase was 1 μmol of 2-chloro-
The amount of enzyme degrading 4-nitrophenyl = β-D-maltopentaoside (commercially available product) was defined as 1 international unit (IU).

(4) Preparation of sample liquid When the sample for measuring α-amylase activity was a liquid, it was used as it was. In the case of a solid, generally, 500 mg of a sample was accurately weighed, purified water was added to make a total amount of 5 ml, and a sample solution was prepared.

(5) Preparation of calibration curve To 250 μl of the standard α-amylase solution, 1.0 m of the conjugate enzyme solution
After adding 1 and stirring and heating at 37 ° C. for 1 minute, 2.0 ml of the substrate solution was added and stirring, and the amount of change in absorbance at 400 nm for 2 minutes after heating at 37 ° C. for 2 minutes was measured. A calibration curve was prepared from the relationship between the activity of each standard α-amylase solution and the amount of change in absorbance. as a result,
The formula of the calibration curve is U = 10.1 · ΔA × 10 ³ −28.5.
[U; enzyme activity (IU / l), ΔA; amount of change in absorbance]
Became. The graph is shown in FIG.

(6) Measurement of α-amylase activity in the sample solution To 250 μl of the sample solution, 1.0 ml of the coupled enzyme solution was added and stirred, and after heating at 37 ° C. for 1 minute, 2.0 ml of the substrate solution was added.
Was stirred, and the amount of change in absorbance at 400 nm for 2 minutes after heating at 37 ° C. for 2 minutes was measured. The α-amylase activity in the sample solution can be measured by calculating from this measured value and the calibration curve prepared in (5). When the value of the enzyme activity in the sample solution exceeds the applicable range of the calibration curve (0 to 547 IU / l), it is diluted again with purified water by a corresponding multiple and then remeasured.

Example 6 Measurement of α-amylase activity (2) (1) Preparation of substrate solution 4-nitrophenyl obtained in Example 4 = 4 ⁷ , 6 ⁷ -O-
(1-Ethoxy) ethylidene-α-D-maltoheptaoside (Mw1344) at a concentration of 3.0 mM, 50 mM phosphate buffer (pH = 7.40 mM) containing 40 mM NaCl and 2 mM MgCl ₂ . It was dissolved in 0).

(2) Preparation of coupled enzyme solution A coupled enzyme solution was prepared in the same manner as in (2) of Example 5 except that β-glucosidase was not added. (3) Preparation of standard α-amylase solution (4) Preparation of sample solution

(5) Preparation of calibration curve In the same manner as in (3) to (5) of Example 5, a standard α-amylase solution, a sample solution and a calibration curve were prepared. As a result, the formula of the calibration curve is U = 4.04 · ΔA × 10
⁴ -6.8 became [U;; enzyme activity (IU / l), ΔA absorbance variation. The graph is shown in FIG.

(6) Measurement of α-amylase activity in sample solution The α-amylase activity in the sample solution was measured by the same operation as in (6) of Example 5.

Example 7 Coupling enzyme resistance test (1) (1) Preparation of substrate solution (a) 2-chloro-4-nitrophenyl obtained in Example 1
4 ^{5, 6 5} -O- dimethoxy methylidene-beta-D-maltopentaoside and (Mw1056) (hereinafter referred to as the present invention the substrate) to a concentration of 3.0 mM, 40 mM-Na
It was dissolved in 50 mM phosphate buffer (pH = 7.0) containing Cl and 2 mM-MgCl ₂ .

(2) Preparation of substrate solution (a) Commercially available 2-chloro-4-nitrophenyl = β-D-maltopentaoside (Mw984) (hereinafter referred to as control substrate)
40 mM-NaCl to a concentration of 3.0 mM
And 50 mM phosphate buffer (pH = 7.0) containing ₂ mM-MgCl ₂ . (3) Preparation of coupled enzyme solution Commercially available α-glucosidase derived from yeast and β-glucosidase derived from almond were 1100 u / ml and 1 respectively.
40 mM NaCl to a concentration of 5.5 u / ml
And 50 mM phosphate buffer (pH = 7.0) containing ₂ mM-MgCl ₂ were mixed and dissolved. As these commercially available α- and β-glucosidases, those manufactured by Toyobo Co., Ltd. were used.

(4) Coupling enzyme reaction After heating 1.0 ml of the coupling enzyme solution at 37 ° C. for 5 minutes,
2.0 ml each of the substrate solution of the present invention or the control substrate solution was added and mixed well, and after heating at 37 ° C. for 3 minutes, 5 minutes, 40 minutes
The amount of change in absorbance at 0 nm was measured. The result is shown in FIG. In FIG. 3, the symbol ⋄ indicates the substrate liquid (a), and the symbol □ indicates the substrate liquid (a). From FIG. 3, it can be seen that the substrate of the present invention is stable in the assay system without reacting with the coupling enzyme.

Example 8 Coupling enzyme resistance test (2) 2-chloro-4-nitrophenyl obtained in Example 3
4 ^{5, 6 5} -O- (1- methoxy) ethylidene-beta-D
-Maltopentaoside (Mw1040) as substrate solution (a)
The same operation as in Example 7 was carried out except that the above was used. The result is shown in FIG. In FIG. 4, the symbol ⋄ indicates the substrate liquid (a), and the symbol □ indicates the substrate liquid (a).
From FIG. 4, it can be seen that the substrate of the present invention does not react with the coupling enzyme,
It can be seen that it exists stably in the measurement system.

Example 9 Measurement Reagent (1) Preparation of Reagent A reagent was prepared by dissolving the following components in purified water at the concentrations shown below.

[0060] Ingredient Concentration 2-chloro-4-nitrophenyl ^{= 4 5, 6 5 -O- (} 1- methoxy) ethylidene-beta-D-Marutopenta oside 1.50 mm alpha-glucosidase 40 [mu / ml beta-glucosidase 5. 0 μ / ml β-glycerophosphate buffer (pH = 7.0) 20 mM Bovine serum albumin 0.05%

(2) Measuring method When the measuring sample is a liquid, the sample liquid is used as it is. In the case of a solid, 500 mg of a sample was accurately weighed, purified water was added to make the total volume 5.0 ml, and this was used as a sample solution. To 250 μl of the sample solution, 3.0 ml of a reagent previously heated at 37 ° C. for 2 minutes was added and stirred, and after heating at 37 ° C. for 2 minutes, the amount of change in absorbance at 400 nm for 2 minutes was measured. The α-amylase activity in the sample solution can be measured by calculating from this measured value and a calibration curve prepared in advance. When the value of the enzyme activity in the sample solution exceeds the applicable range of the calibration curve (0 to 547 IU / l), it is diluted again with purified water by a corresponding multiple and then remeasured.

[Brief description of drawings]

FIG. 1 is a graph showing a calibration curve used for measuring α-amylase activity in Example 5.

FIG. 2 is a graph showing a calibration curve used for measuring α-amylase activity in Example 6.

FIG. 3 is a graph showing the stability of the substrate of the present invention and the control substrate in Example 7 in the measurement system.

FIG. 4 is a graph showing the stability of the substrate of the present invention and the control substrate in Example 8 in the measurement system.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Tadashi Tomikura             339 Noda, Noda City, Chiba Prefecture Kitsukoman             Within the corporation (72) Inventor Kazuo Otani             First 5-5-12, Nairairi, Sumida-ku, Tokyo             Gakuyaku Co., Ltd. Tokyo Technical Center (72) Inventor Kazunori Saito             First 5-5-12, Nairairi, Sumida-ku, Tokyo             Gakuyaku Co., Ltd. Tokyo Technical Center (72) Inventor Koichiro Tobe             339 Noda, Noda City, Chiba Prefecture Susumu Pharmaceutical Co., Ltd.             In the company

Claims (3)

[Claims] 1. A general formula: (In the formula, X ¹ is a methoxy group or an ethoxy group, X ² is a hydrogen atom, a methoxy group, an ethoxy group or a substituted or unsubstituted hydrocarbon group, R is a hydrogen atom, an aromatic color-forming group or a monosaccharide other than glucose. Residue, n is an integer of 2 to 6)
An alkoxymethylidene maltooligosaccharide derivative represented by. 2. A reagent for measuring α-amylase activity, which comprises the alkoxymethylidene maltooligosaccharide derivative according to claim 1 as an active ingredient. 3. The alkoxymethylidene maltooligosaccharide derivative according to claim 1 and α-amylase-containing sample and α-amylase
A method for measuring α-amylase activity, which comprises adding a coupled enzyme for measuring amylase activity and causing an enzymatic reaction to quantify the released aromatic chromophoric compound or monosaccharide.