JPH0390095A - Deoximaltooligocide derivative, alpha-amylase activating reagent containing same derivative as active ingredient and determination of alpha-amylase activity using same reagent - Google Patents

Abstract

NEW MATERIAL:A compound expressed by formula I (R is aromatic chromophoric group; n is 2-6). EXAMPLE:2-chloro-4-nitrophenyl-6<5>-deoxy-beta-D-maltopentaoside. USE:Used as a reagent for alpha-amylase activation. PREPARATION:6-deoxycyclodextrin expressed by formula II (n is 4-6) is affected with cyclodextrinase to afford a reacted solution containing a mixture of 6- deoxymaltooligosaccharide expressed by formula III (one of X is H and other n+1 pieces are OH) having 6-deoxyglucose residues at various positions as principal ingredient.

Description

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

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. Using a series of malto-oligosaccharides as a substrate, cleavage with α-amylase;
Rawhide maltose, glucose or glucose-6-
Method for quantifying phosphoric acid (3) Utilizing various substituted phenyl malto-oligosites as substrates, cleaving them with α-amylase, then acting on a coupled enzyme system, and converting the raw substituted phenols as they are or as needed to pH. (4) Various substituted phenylmaltooligosites in which the 6-position and/or 4-position of the non-reducing terminal glucose is modified with an aryl group, an alkyl group, etc. using 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 for the substrate. Furthermore, since there are many enzymatic cleavage sites, it has drawbacks such as the inability to measure the α-amylase reaction as a truly stoichiometric reaction.

On the other hand, method (2) uses a homogeneous substrate, so it can compensate for the drawback of (1), but it requires completely eliminating carbohydrates such as maltose and glucose in the sample in advance. In addition, when measuring glucose from the raw skin 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.

On the other hand, method (3), especially the method using 2-chloro-4-nitrophenyl-β-maltopentaoside as a substrate, is currently widely used as the best 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 drawbacks of the method (3), the method (4) above, which is not degraded by the conjugated enzyme, 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 are chemically unstable and cannot be stored for long periods of time. have.

Problems to be Solved by the Invention The present invention overcomes the drawbacks of such conventional σ-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 The present inventors have conducted various studies to achieve the above-mentioned object, and have found that a specific new 6-deoxymalto-oligoside derivative is extremely suitable as a reagent for measuring α-amylase activity. By measuring α-amylase activity using this, it was discovered that the purpose was distantly related, and based on this knowledge, the present invention was completed.

That is, the present invention provides a 6-deoxymaltooligoside derivative represented by the general formula (R in the formula is an aromatic chromogenic group, and n is an integer of 2 to 6), a compound of the general formula (1). The compound of general formula (I), a-glucosidase and/or glucoamylase, and optionally β-glucosidase are added to the reagent for measuring a-amylase activity as an active ingredient and the α-amylase-containing sample to carry out an enzymatic reaction. The present invention provides a method for measuring α-amylase activity, which is characterized in that the aromatic chromogenic compound liberated is quantified.

The present invention will be explained in detail below.

The 6-dioxymalto-oligosaccharide moiety in the 6-deoxymalto-oligoside derivative of the general formula (I) of the present invention includes, for example, σ- and/or β-64-deoxy-D
-maltotetraose to a- and/or β-61-deoxy-D-maltooctaose can all be used. Among these, 65-deoxy
D-maltopentaose, 6@-deoxy-D-maltohexaose, and 6-fudeoxy-D-maltoheptaose are preferred. Note that the symbols 6", 6'-6, -, etc. added before deoxy in the above compounds refer to the 6th position of the 4th, 5th, and 6th glucose from the reducing end side of the glucose unit constituting the maltooligosaccharide, respectively. This means that the hydroxyl group in is substituted with a hydrogen atom.

In the 6-deoxymalto-oligoside derivative represented by the general formula (I), R substituted with the hydroxyl group at the 1-position of the reducing terminal glucose is an aromatic color-forming group, and examples of such groups include the following: Examples include:

(Rl #RI is a hydrogen atom, an alkyl group, an allyl group,
Aryl group, acyl group, carboxyl group, cyano group, formyl group, alkoxy group, nitro group, nitroso group, amino group, azido group, sulfonic acid group, sulfoxyl group, sulfonyl group, or halogen atom, each of which is the same. or may be different, and R1 and R', or R2 and R3 may be bonded to form a condensed aromatic ring.

However, RIM/R1 are not hydrogen atoms at the same time) 6 (R1 is a hydrogen atom or an alkyl group) (R7 is a hydrogen atom or a halogen atom) (R8 to RI5 are hydrogen atoms, alkyl groups, allyl groups, Aryl group, acyl group, carboxyl group, cyano group, formyl group, alkoxy group, nitro group, nitroso group, amino group, azido group, sulfonic acid group, sulfoxyl group, sulfonyl group, or halogen atom, each of which is the same. or may be different, and R
8 and R9, and/or RIO and R11 may be combined to form a condensed aromatic ring, and further R1 and Rle, and/
Alternatively, R13 and R14 may be a common oxygen atom to form a condensed ether ring, and Z is a nitrogen atom or N-4
0) and 6- represented by the general formula CI)
Deoxymaltooligoside derivatives are a-anomer or β
- Any anomer may be used.

Therefore, the compound represented by the general formula (1), for example, 2-chloro4-nitrophenyl-62-deoxy-β-D-maltopentaoside, 4-nitrophenyl-6s-deoxy- σ-D-maltopentaoside,
Phenolindo-3'-chlorophenyl-6s-deoxy-β-D-maltopentaoside, 4-nitrophenyl-6'-thioxy-β-D-maltoheptaoside, 2
-chloro4-nitrophenyl-67-deoxy-β-
D-maltoheptaoside, methyllambelliferonyl-
Examples thereof include 65-deoxy-β-D-maltopentaoside, resazurinyl-6fudeoxy-β-D-maltoheptaoside, and the like.

The 6-dioxymaltooligoside derivatives represented by the general formula (I) of the present invention are all new compounds that have not been published in any literature, and can be produced, for example, by the following various methods (A-D methods). can.

Method A: By allowing a certain type of cyclodextrinase to act on 6-deoxycyclodextrin represented by the general formula (wherein is an integer of 4 to 6),
6-dioxyglucose residues are present at various positions represented by the general formula (in the formula, it'll is a hydrogen atom, the other n+1 are hydroxyl groups, and n is an integer from 4 to 6). 6 with
- Obtain a reaction solution containing a mixture of dioxymalto-oligosaccharides as a main component.

The starting material, 6-deoxycyclodextrin represented by the general formula (n), is a cyclodextrin having a cyclodextrin skeleton including derivatives such as branched products and modified products, such as commercially available α-1β-1γ. -Cyclodextrin (glucose polymerization degree is 6 and 7.8 respectively)
) etc. by a known method. For example, dissolve cyclodextrin in a solvent such as pyridine, add 7 to 14 times the mole of tosyl chloride, and heat at 15 to 30°C.
The mixture is reacted for 4 to 6 hours to undergo tosylation, and if necessary, purified by a conventional method to obtain 6-tosylcyclodextrin. Next, dissolve this in an organic solvent such as dimethyl sulfoxide (DMSO) or dimethyl formamide (DMF),
Add 10 to 30 times the mole of a reducing agent such as sodium borohydride (NaBH*) and reduce by reacting at 40 to 60°C for 10 to 20 hours. If necessary, purify by a conventional method to obtain 6-deoxycyclo Obtain dextrin [for example, “Carbohydrate Research (Carbohydrate, Res.
) J, Volume 18, Pages 29-37 (1971)
reference〕.

In this way, a suitable starting material such as 6-dioxy-α-16-zooxy-β-16-dioxy-twocyclodextrin can be obtained. Among them, 6-dioxy-β-cyclodextrin is particularly preferred from the viewpoint of enzyme reaction rate.

The cyclodextrinase used in this method is specified by the following physical and chemical properties (a) to (g) (see Japanese Patent Application No. 1-146891) 4 (a)
Function: It has the effect of cleaving rainbow cyclodextrin and producing Mar-L oligosaccharide derived from the degree of glucose polymerization of the cyclodextrin.

(b) Substrate specificity The ice-melting rate or affinity for dichlorodextrin has greater substrate specificity than polysaccharides or linear oligosaccharides having the same degree of polymerization as cyclodextrin.

Tables 1 and 2 show specific examples of substrate specificity and reaction rate parameters for cyclodextrins and maltooligosaccharides, respectively.

Table 1 Table (c) Optimal pt+ and stable pH range: When β-cyclodextrin is used as a substrate, N p) (8
, has an optimum pH near 0, and has a stable pH range of 5.5.
~9.5.

Figure 1 shows 0.48 mQ, 2% (v/
v) β-cyclodextrin solution at a concentration of 0.50+mO
A graph showing the relationship between pl (and relative activity) is obtained by mixing 0.02 mQ of enzyme solution [substrate β-cyclodextrin concentration 1% (v/v)] and performing a reaction at 40'0 for 1 hour. In this figure, the dashed line is the case using acetate buffer, the solid line is the case using phosphate buffer, and the dotted line is the case using borate buffer.From this figure 1, the optimum pH is around 8.0.
It turns out that it has H.

FIG. 2 shows acetate buffer, phosphate buffer and borate buffer at 100mM concentration O-0-2O and enzyme solution 0.0-2O, respectively.
0.05m12 were mixed, each pu was treated at 25°C for 24 hours, and this treatment solution was mixed with 0.05m12. lO+mff, 100
mM concentration of phosphate buffer (pH 7,5) 0.40+++
It is a graph showing the relationship between treatment pH and relative activity when Q and 2% (w/v) concentration β-cyclodextrin solution 0-50d were mixed and a reaction was performed at 40° C. for 1 hour. In this figure, the broken line is the case using acetate buffer, the solid line is the case using phosphate buffer, and the dotted line is the case using borate buffer.

From FIG. 2, it can be seen that the stable pH range is 5.5 to 9.5.

(d) Suitable temperature for action: It has an optimal temperature of action around 40°C.

Figure 3 shows 0.50ml of 2% (w/v) concentration of β-cyclodextrin solution, 0.4h+2 of 100mM phosphate buffer (pH 7,5), and enzyme solution Q, Q2ml.
This is a graph showing the relationship between temperature and relative activity in the case of mixing and reacting for 10 minutes at each temperature. This third
From the figure, it can be seen that the optimum temperature for action is around 40'O.

(e) Deactivation: It has the property of being deactivated by treatment at a temperature of 50° C. or higher for 15 minutes.

Figure 4 shows that after treating 100mM phosphate buffer (pl(7,5)0.05+*ff) containing enzyme at each temperature for 15 minutes, 0.02ml of this treatment solution, LOOm
0.48 mQ of phosphate buffer (pH 7.5) at a concentration of M and 0.48 mQ of a 2% (w/v) concentration of β-cyclodextrin solution.
5 is a graph showing the relationship between treatment temperature and relative activity when 50 mα is mixed and reacted at 40'O for 1 hour. From this Figure 4, it can be seen that the phosphate buffer (p
When treated in H7,5) for 15 minutes, the activity is stable up to 45°C, but it is found that it is almost inactivated at temperatures above 50°C.

(f) Inhibition and activation: H14, Cu'', Zn'', Ni'' and Fe''1 inhibited by more than 90%, Ca'''' and Mg2+ inhibited 1
It has the property of being activated by 0-30%.

Table 3 shows the influence of metal ions on enzyme activity.

Table 3 From Table 3, we can see that the divalent metal ion H1ゝCu”
, Zn", Ni" and Fe", and activated by 10 to 30% by Ca2+ and M1+.

(g) Molecular weight: The molecular weight is 144,000 according to the Niger filtration method, and the SDS
The molecular weight determined by PAGE method is 72.000. That is, the enzyme is a dimer consisting of subunits with a molecular weight of 72,000.

The titer of the enzyme was calculated using 500 μα of a 2% (w/v) concentration of β-cyclodextrin solution and 500 μα of a loOmM phosphate buffer (pH 7.5) containing an appropriate amount of the enzyme.
After mixing 00μQ and reacting at a temperature of 40°C for an appropriate time, the reaction was stopped by boiling for 10 minutes, and maltoheptaose was extracted by high performance liquid chromatography (hereinafter referred to as HPLC). It was determined by quantitative determination. In addition, when the amount of enzyme was small, the reducing power was determined by quantifying the reducing power using the Somogyi-Nelson method using glucose as a standard.

Regarding the enzyme unit of the cough enzyme, 1 unit was defined as the amount of enzyme that produced 1 micromole of maltoheptaose per minute.

Next, a method for producing the cyclodextrinase used in the present invention will be explained. The microorganism that produces this enzyme is not particularly limited as long as it belongs to the genus Bacillus and produces the enzyme. For example, Bacillus sphaericus
) E-244 strain can be mentioned.

This Bacillus sphaericus E-244 strain is a wild strain obtained from soil and has the following mycological properties.

Mycological characteristics of Bacillus sphaericus E-244 strain (a) Morphology (1) Cell shape and size = 0.6-0.8X 1.6
~4.0 μm rod.

(2) Presence or absence of cell polyplasia: Not observed.

(3) Motility: Has periflagella and is motile.

(4) Presence or absence of spores: Yes Spore sac: Swelling size: 0.8-0.9X 1.1" 1.2μ
Rtr shape: Oval Position: Sub-end standing (5) Durham staining: Positive (6) Anti-acidity: Negative (b) Growth status in each medium (1) Broth agar plate II: Colorless diffusive colonies. It has a strong shape, its colonies are smooth with smooth edges, and no pigment production is observed.

(2) Meat juice agar slant culture: The fungal moss is smooth with smooth edges, and no pigment production is observed.

(3) Meat juice liquid culture: Growth is observed throughout the medium, but no precipitate is observed.

(4) Meat juice gelatin puncture culture: It grows only on the top of the medium, and no liquefaction is observed.

(5) Lidmus milk: No coagulation is observed, and no acid or alkali production is observed.

(c) Physiological properties (1) Nitrate reduction: No reduction (2) Denitrification reaction: None 3) MR test: Negative (4) VP test - Negative (5) Indole formation: No formation (6) Sulfurization Production of hydrogen: Not produced (7) Hydrolysis of starch: Not decomposed (8) Utilization of citric acid: Not used (9) Utilization of inorganic nitrogen source: Not used (10) Production of pigment: Not produced (11) ) Urease: Negative (12) Oxidase: Positive (13) Catalase: Positive (14) Growth range Temperature = 13-38°C pH: 6
~10.5 (15) Attitude towards oxygen: Aerobic (16) O-F test: Negative (no acid production observed)
(17) Attitude towards sugars2 L-arabinose, D-xylose, D-glucose,
Neither acid production nor gas production from D-mannose, D-7 lactose, D-galactose, maltose, sucrose, lactose, trehalose, D-sorbit, D-mannite, inosit, glycerin, and starch is observed.

(d) Phenylalanine deamination reaction: Positive This Bacillus sphaericus strain E-244 was identified as a bacterium belonging to the genus Bacillus because it is a Gram-positive bacillus that forms spores. Furthermore, since no acid or gas generation from sugar was observed, the pH of the VP broth was 7.0 or higher, and phenylalanine deamination reaction was observed, Purge Aids Manual of Systemic Bata Theriology, Volume 2 (198
4), it was identified as a bacterium belonging to the Bacillus sphaericus species.

In addition, Bacillus sphaericus E-244 was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology, as part of the Microbiological Research Institute No. 2458 (
FERM BP-2458).

The cultivation of this strain is in principle the same as the method used for aerobic cultivation of general microorganisms, but usually a shaking culture method using a liquid medium or an aeration agitation culture method is used. . The medium used includes a suitable nitrogen source, carbon source, vitamins, minerals, etc., and cyclodextrin, which is an inducing substrate for the enzyme. The pH is
Any region is acceptable as long as it is in the p)l region where the seedlings grow,
Usually, a range of 6 to 8 is preferred.

Cultivation is usually carried out at a temperature in the range of 20-40°C for 16
This is carried out by shaking culture or aerated agitation culture for about 4 hours to 4 days.

In order to obtain the desired enzyme from the culture, for example, first collect the bacteria by centrifugation or membrane concentration, then crush the bacterial cells by ultrasonication or surfactant treatment, and then Remove the residue by centrifugation to obtain a crude enzyme solution.
This crude enzyme solution was then subjected to ion exchange chromatography,
By performing appropriate combinations of column chromatography such as hydrophobic chromatography and gel filtration,
A method for obtaining a purified product of the enzyme can be used.

Table 4 shows the differences in physicochemical properties between the cyclodextrinase according to the present invention thus obtained and conventionally known cyclodextrinases.

Next, as the substrate concentration of 6-dioxycyclodextrin during the enzyme reaction, it is preferable to use a concentration equal to or higher than the Km value for the cyclodextrinase substrate.

The reaction conditions for causing the cyclodextrinase to act on 6-dioxycyclodextrin are appropriately selected within the action pH and temperature range of the cyclodextrinase, but usually pH 7.0 to 9.0. And the temperature is 35-45℃
range is used. At this time, organic solvents such as ethanol and dimethyl sulfoxide can also be used if necessary. The reaction time varies depending on the stability of the reaction product, but is usually about 30 minutes to 48 hours. There is no particular restriction on the amount of enzyme, and it is sufficient to add the necessary amount that maximizes the product within the reaction time, but usually 0.5 to 5 units/g is added to 6-dioxycyclodextrin. used.

In this way, a mixture of various 6-dioxyoligosaccharides represented by the general formula (1![) is obtained as described above, but when the starting material is 6-dioxy-β cyclodextrin, The main component is a mixture of ose, 6@-deoxymaltoheptaose, 6s-deoxymaltoheptaose, 64-deoxymaltoheptaose, 63-deoxymaltoheptaose, 6'-deoxymaltoheptaose, and 6-deoxymaltoheptaose. In the case of 6-dioxy-α-cyclodextrin, 61-deoxymaltohexaose, 6'-deoxymaltohexaose, 64-deoxymaltohexaose, 63-deoxymaltohexaose, etc. A substance containing a mixture of oose, 6'-deoxymaltohexaose, and 6-deoxymaltohexaose as a main component is obtained.

Next, an exo-type saccharifying enzyme is applied to this to hydrolyze the glucose residue on the non-reducing end side so that the 6-dioxyglucose residue becomes the non-reducing end. As the exo-type saccharifying enzymes in this case, for example, known a-glucosidase, glucosidase, etc. may be used alone or in combination. Note that β-amylase may be allowed to act, if necessary.

Through this enzymatic reaction, various 6-deoxy-maltooligosaccharides (i.e., 61-deoxymaltotetraose, 6'-deoxymaltopentaose, 6'-deoxymaltopentaose, 6'-deoxymaltopentaose, A mixture of 63-deoxymaltotriose, 6'-deoxymaltose, 6-dioxyglucose and glucose is obtained.

Using β-amylase as an exo-type saccharifying enzyme;
In some cases, 6-deoxymaltose is also produced in addition to the above compound. These exo-type saccharifying enzymes may coexist with the above-mentioned cyclodextrinase to carry out the enzymatic reaction simultaneously, but after the cyclodextrinase has acted on the starting material, it may be allowed to act further. is preferred. Particularly preferred is to allow cyclodextrinase to act on the starting material, for example, when the product reaches the maximum, to temporarily stop the reaction by acid or heat treatment, and then to pass it through, for example, an octadecylated silica gel (005) column. This is a method in which exo-type saccharifying enzymes are applied after purification treatment such as liquidization and adsorption removal of unreacted raw materials. When cyclodextrinase and exo-type saccharifying enzymes coexist, the reaction conditions are as follows:
Although it is necessary to select the temperature range appropriately, it is usually pH 7.
, 0-9, 0.35-45°C, and 0.5-48 hours.

In addition, the reaction conditions for the action of exo-type saccharifying enzymes after the action of the cyclodextrinase are appropriately selected from the action pH and temperature range of the enzyme used, but usually pH 4.0 - 7.5. At 35-45°C, 0.
Conditions of 5 to 48 hours are used.

There is no particular limit to the amount of exo-type saccharifying enzymes used in this printing, but it is usually to
A range of -100 units/g is used. This enzymatic reaction is stopped, for example, by acid or heat treatment.

Next, in order to obtain the 6-deoxymaltooligosaccharide represented by formula (1), which is a 9-type compound, from the 6-dioxymaltooligosaccharide-containing reaction solution obtained in this way, a normal oligosaccharide separation method is used. , for example, unreacted 6 from the reaction solution
-Deoxycyclodextrin is removed, and the product is further separated and purified by a method of fraction collection using activated carbon column chromatography, silica gel column chromatography, thin layer chromatography, etc. In addition, unreacted 6.
Deoxycyclodextrin can be removed by known methods such as cooling treatment, organic solvent addition treatment, and adsorption column treatment.

If the hydroxyl group of the 6-dioxymalto-oligosaccharide represented by formula (IV) thus obtained is acetylated by a known method, - formula (wherein is an integer from 2 to 6) ) acetyl 6-dioxymaltooligosaccharide,
For example, hexadeca-O-acetyl 6S-deoxymaltopentaose, nonadeca-〇-acetyl-6 rhodeoxymaltohexaose, docosa-O-acetyl-6
Foodeoxymaltoheptaose and the like are obtained.

For this acetylation method, for example, 100 to 200 times the mole of acetic anhydride or acetyl halide is added to 6-zoxymaltooligosaccharide in the presence of an organic base such as pyridine or triethylamine, and the mixture is heated at 10 to 40°C for 10 to 200 times. 80
A method of time reaction can be mentioned [Journal of Molecular Biology (J, Mo1.
Biol-) Vol. 72, p. 219 (1972)).

Next, the 1-position of the reducing terminal glucose of this product is brominated by a known method to produce acetyl-6.deoxymaltooligosyl bromide (e.g., a-pentadeca.0.acetyl-6s-deoxymaltobentaosyl bromide, α- octadeca-0, acetyl, 6', deoxymaltohexaosyl bromide, σ-heneifucer〇-acetyl-6
(butaosyl bromide, etc.) is obtained.

This bromination method involves, for example, adding phosphorus tribromide or titanium tribromide to acetyl-6-deoxymalto-oligosaccharide in a nonpolar solvent such as dichloromethane, optionally in the presence of a catalytic amount of water. Add 0.5 to 3 times the mole, 10 to
A method of reacting at 40° C. for 2 to 50 hours is used (see, for example, Japanese Patent Application Laid-Open No. 60-202893).

Next, this product is treated with the aromatic compound having an aromatic chromogenic group by a known method to glucosylate the 1-position of the reducing terminal glucose to obtain an acetyl 6-deo, ximaltooligosite derivative. Examples of this derivative include 2-chloro.4-nitrophenylpentadeca-O-acetyl-6S-deoxy-β-D-maltopentaoside,
Phenolindo-3'-chlorophenylbentadecar O-acetyl-6'-deoxy β-D-? rutoheptaoside, 4, nitrophenylheneicosa, 0-acetyl-6 fudeoxy, β-D-maltoheptaoside, 2-
Examples include chloro-4-nitrophenylheneicosa-〇-acetyl-67-deoxy-β-D-maltoheptaoside.

For this glucosylation method, for example, acetyl-6-deoxymaltooligosyl bromide, acetonitrile,
In an aprotic solvent such as nitromethane, optionally in the presence of a silver compound such as silver oxide or silver carbonate, 2 to 10 times the mole of the aromatic compound or its alkali metal salt is added, and the mixture is heated at 20 to 50°. A method of reacting with C for 5 to 50 hours is used (see, for example, Japanese Patent Publication No. 62-283989).

Next, the above derivative is deacetylated by a known method to obtain the 6-deoxymaltooligoside derivative represented by the above formula (1). For this deacetylation reaction, for example, 100 to 200 times the mole of ammonia water is added to the acetyl 6-dioxymaltooligoside derivative in an alcohol such as methanol, and 5 to 50 times the amount of ammonia water is added at 20 to 50°C.
A time reaction method is used [Canadian Journal of Chemistry (Can, J, Ch.
Em,) J, Volume 49, Page 493 (1971)
reference〕.

Method B Maltooligosite derivatives represented by the general formula (R in the formula has the same meaning as above, and n is an integer of 2 to 6), such as 2-chloro・4-nitrophenyl・β-D・Maltopentaoside, 4-nitrophenyl-a-D-maltoheptaoside, phenolindophenyl-β-D-maltopentaoside, etc., have the general formula (R14 and R17 in the formula are hydrogen atoms or hydrocarbons) (which may be combined with each other to form a ring) or its acetal is reacted to form alkylidene or alkylidene maltooligosite derivatives represented by
-nitrophenyl, impropylidene-a-〇-maltoheptaoside, phenolindo-3'-chlorophenyl, ethylidene-β.D-maltotetraoside, etc. are obtained.

The reaction between the maltooligosite derivative represented by formula (Vl) and the carbonyl compound represented by formula (■) or its acetal can be carried out using an aprotic polar solvent such as dimethylformamide, dimethyl sulfoxide, or ethylene glycol dimethyl ether. The reaction is carried out in the presence of a catalyst such as sulfuric acid, hydrogen chloride, p-toluenesulfonic acid, or anhydrous zinc chloride. The thus obtained alkylidene or arylidean maltooligoside derivative represented by formula (■) is acylated to produce an acyl alkylidene or alkylidene deamaltooligoside derivative, such as 2-chloro-4-nitrophenyl. , tetradeca O acetyl-benzylidene-β-D
-maltopentaoside, 4-nitrophenyl, eicosa-O-penzoyl-inglobylidene-α-D-maltoheptaoside, phenolindo-3'-chlorophenyl, undec-0-butyryl-ethylidene-β-D - Obtain maltotetraoside, etc. At this time, examples of the acylating agent include acetic acid, monochloroacetic acid, propionic acid, σ
-chloropropionic acid, β-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 thus obtained acyl alkylidene or acyl arylidea maltooligoside derivative is subjected to a dealkylidene or dealylidene reaction to obtain the general formula (wherein R1 is an acyl group, R and n are the above-mentioned partially acylated maltooligosite derivatives represented by
di-O-acetyl-σ-D-glucopyranosyl) (1
-4) Tris[0・(2,3,6-tri〇・acetyl-σ-D-glucopyranosyl)-(1-4))・2.3
．． 6-tri〇・acetyl-β-D-glucopyranoside, 4・nitrophenyl-〇・(2,3-di・0・benzoyl・VD-glucopyranosyl)-(1→4)-pentakis(0-(2,3 ,6-tree〇-benzoyl a
-D-glucopyranosyl)-(1-4)) -2,3,
6-tri0-benzoyl-α・D-glucopyranoside and the like are obtained.

There are no particular restrictions on the conditions for the above dealkylideneation reaction or dealylideneation reaction, and known methods such as the method of reacting with acetic acid or formic acid [for example, "Journal.
of the American Chemical Society (J,A
84, p. 430 (1962)].

Next, the partially acylated maltooligosite derivative represented by the formula (ff) thus obtained is added to the partially acylated maltooligosite derivative represented by the formula (ff), where RIg to R13 represent a hydrogen atom, an alkyl group, a nitro group, or a halogen atom, They may be the same or different from each other, or R11 and R10 or Rto and Ra+ may combine with each other to form a condensed aromatic ring.Y represents a halogen atom) 6 of the non-reducing terminal glucose in formula (ff) by reacting the arylsulfonyl halide represented by
The hydroxyl group at the position is arylsulfonated to produce acylarylsulfonyl malto-oligosaccharides, such as 2-chloro-4-nitro-7-manyl, tetrazocar〇-acetyl-63-deoxy-p-toluenesulfonyl-β-D-maltopentaoside, 4- Nitrophenyl, eicosa-0, benzoyl-57+ thioxy-β-naphthalenesulfonyl-α,
D-maltoheptaoside, phenolindo・3'-fluorophenyl, undeca-〇-butyryl・61-deoxy-2,4,6-trimethylbenzenesulfonyl・β
-D.maltotetraoside and the like are obtained.

Examples of the arylsulfonyl halide represented by formula (X) include I)-toluenesulfonyl chloride, β-naphthalenesulfonyl chloride, 2,4-dinitrobenzenesulfonylbromi F, 2,4,6-trimethylbenzenesulfonyl chloride, etc. can be mentioned. There are no particular restrictions on the conditions for this arylsulfonylation reaction, but it is usually carried out in pyridine at room temperature by reacting 2 to 20 times the molar amount of arylsulfonylation. Next, the above acylarylsulfonyl malto-oligosaccharide is reductively dealylsulfonated to form the above formula (
In II), the hydroxyl group at the 6-position of the non-reducing terminal glucose is substituted with a hydrogen atom I; partially acylated 6-dioxymaltooligoside derivatives, such as 2-chloro-4-nitrophenyl, tetrazocar-acetyl-6-rhodeoxy-β・D-maltopentaoside, 4-nitrophenyl, eicosa-0-benzoyl-6fudeoxy-α-〇-maltoheptaoside, phenolindo-3' chlorophenyl, undec-O-butyryl 64-deoxy-β
-D-maltotetraoside and the like are obtained.

There are no particular restrictions on the conditions for this reductive desulfonylation reaction, but it is usually carried out in an aprotic polar solvent such as dimethyl sulfoxide or dimethyl formamide at 40-60%
NaBH, or NaBH, CN, etc. at a temperature of 2O-1
The reaction is carried out at a ratio of 00 times the molar ratio.

Finally, the partially acylated 6-dioxymaltooligoside derivative is deacylated to obtain the target compound 6-dioxymaltooligoside derivative represented by the above-mentioned formula (I).

There are no particular limitations on the conditions for this deacylation reaction, but for example, the deacetylation reaction in Method A above can be used as is.

The above formula (II) is added to a known aryl glucoside derivative represented by the general formula Q (in which R has the same meaning as above).
Add 6-dioxycyclodextrin represented by
A known enzyme, cyclodextrin glucosyltransferase, is applied to produce the formula (in which one X is a hydrogen atom and n are hydroxyl groups,
R and n have the same meanings as above) 6.
A mixture of deoxymaltooligoside derivatives is obtained, and then the exo-type saccharifying enzymes are allowed to act on the mixture to obtain the desired 6-deoxymaltooligoside derivative represented by the above-mentioned formula (I).

The aryl glucoside derivative represented by formula (n) is, for example, 2-chloro-4-nitrophenyl-
Examples thereof include β.D-glucoside, 4.nitrophenyl.alpha.-D-glucoside, and phenolindo-3',5'-dichlorophenyl.beta..D-glucoside.

This reaction is carried out in aqueous solution and in the presence of a buffer. At this time, the concentration of the compound represented by the above formula (■) is:
Usually 0.01 to 19/llI2, preferably 0.02 to
The concentration of 6-dioxycyclodextrins is usually 1 to 200 mg/m2.
Q, preferably 100-200m! 9/ rtrQ(1
) selected within the range. There are no particular restrictions on the origin of cyclodextrin glucosyltransferase, but those obtained from, for example, Bacillus macerans, Bacillus megaterium, Bacillus circulans, etc. are preferred. Its concentration is usually 0.3 to 3 units/tribute Q1
It is preferably selected within the range of 0.5 to 1.5 units/mQ.

Method D: A known aryl glucoside derivative represented by the formula (n) above and the formula (■) (where n is 1 to
6-dioxy-malto-oligosaccharide represented by the integer 3) is mixed, and known enzymes such as maltotetraose-generating amylase and maltopentaose-generating amylase are suitably selected in relation to the target compound and allowed to act on the mixture. By carrying out the rearrangement reaction, the desired 6-deoxymaltooligoside derivative can be obtained.

There are no particular restrictions on the conditions for these transfer reactions, but
Appropriate reaction conditions can be obtained by selecting temperature, concentration, liquid properties, and reaction time suitable for each maltooligosaccharide-producing enzyme.

The above formula (1) obtained by various manufacturing methods as described above.
The 6-deoxymaltooligoside derivative represented by
This 6-amylase activity is extremely useful for measuring a-amylase activity.
- σ-amylase activity can be measured using deoxymaltooligoside derivatives.

As mentioned above, there are α-anomer and β-anomer in the 6-deoxymalto-oligoside derivative represented by formula (1), but when only α-anomer is used when measuring α-amylase activity. It is necessary to use σ-glucosidase and/or glucoamylase as a coupled enzyme system, and when using only β-anomer or a mixture of σ-anomer and β-anomer, it is necessary to use α-glucosidase and/or glucoamylase. It is necessary to use β-glucosidase in addition to amylase.

Advantageous systems for measuring α-amylase activity include, for example: α- and/or β-amylases of the formula (I);
Contains 1-20mM of 6-deoxymalto-oligoside derivative and 2-100mM of buffer, and a as a conjugated enzyme.
- 15-150 units/mQ of glucosidase and/or glucoamylase, respectively.

When the derivative contains a β-anomer, pt+4 to approximately contains 5 to 50 units of β-glucosidase/mQ.
An example is the IO system. Buffers used in this system include, for example, phosphate, acetate, carbonate, tris-(hydroxymethyl)-aminomethane, borate, citrate, dimethylglutarate, and the like. Although α-glucosidase may be derived from any origin, such as animal, plant, or microbial, α-glucosidase derived from yeast is particularly preferred from the viewpoint of substrate specificity. Furthermore, glucoamylase of any origin may be used, but for example, glucoamylase of the genus Rhizopus (Rh)
Izopus sp) and the like are preferred.

Furthermore, β-glucosidase may be of any origin, for example, one obtained from almond seeds.

In the present invention, such a system includes, in addition to the above-mentioned components,
If necessary, various conventional additive components such as glycerin, bovine serum albumin, a- or β-cyclodextrin, Triton May be added. Furthermore, as an a-amylase activator, NaCl
,MgC1,,111g5o,,CaCIzlCaCl
, j 2H20, etc. CI-ion,
C1+ ions, lJg''+ ions, etc. may be added.

These additive components may be used alone or in combination of two or more, or may be added at an 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 σ-glucosidase and/or glucoamylase as a conjugate enzyme are added to a sample containing α-amylase. /mQ
, preferably 30 to 70 units/lla, and when the 6-zoxymaltooligoside derivative represented by formula (I) contains a β-anomer, further β-glucondase, preferably 5 to 50 units/mQ, is added. is 5 to 15 units/
Add mQ, simultaneously or sequentially add 1 to 20 mM of the derivative, preferably 2 to 6IIIM, and a buffer; then, at a temperature of 25 to 50°C, 84 to 1O for at least 1 minute, preferably 2 to 60 minutes. After enzymatic reaction, the raw aromatic color-forming compound is subjected to a conventional method as it is or the pH is changed if necessary; or after a condensation reaction, the absorbance value is measured continuously or intermittently at an appropriate absorption wavelength. is measured, and the α-amylase activity in the sample is calculated by comparing it with the absorbance value of the α-amylase standard measured in advance.

The σ-amylase-containing sample used in the present invention is not particularly limited as long as it contains α-amylase, but specifically, it may be a microorganism culture solution, a plant extract, or an animal body fluid. Tissues and their extracts can be used.

a. If the amylase-containing sample is a solid, it is preferably dissolved or suspended in a buffer solution. Examples of the buffer include phosphate, acetate, carbonate, 1-lis(hydroxymethyl)-aminomethane, borate, citrate, dimethylglutarate, and the like.

Effects of the Invention The 6-dioxymalto-oligoside derivative represented by formula (I) of the present invention is a novel compound, and has a σ-
It is extremely useful as a reagent for measuring amylase activity, and by using this reagent, α-amylase activity can be measured by automatic analysis without being affected by glucose, maltose, bilirubin, hemoglobin, etc. contained in the sample.
It has the advantage that it can be easily measured with high accuracy and in a short time using manual techniques, and that it can maintain a stable state for a long period of time even in the presence of a conjugated enzyme.

Example Next, this onset period will be explained in more detail using examples.

(1) Synthesis of 6-tosyl-β-cyclodextrin Commercially available β-cyclodextrin [manufactured by Wako Pure Chemical Industries, Ltd.]
5.009 (4.41 mmol) in 50 mQ of pyridine
10.0 g (5.0 g) of tosyl chloride was dissolved in this solution.
2.4 mmol) was added thereto, and the mixture was stirred and reacted at room temperature for 5 hours. Then, after distilling off the pyridine in the reaction solution under reduced pressure, 100 mff of water and 15 mff of benzene were added to the residue.
Add 0 mQ while stirring to precipitate solid matter;
.

Next, this solid substance was filtered through a glass filter, washed twice with 50 mQ of acetone, and the filtered material was purified by ODS column chromatography and eluted with an ethanol-water mixture (volume ratio 1:9). By concentrating the fractions and recrystallizing from water, 6-tosyl-β-cyclodextrin 1.639 (1,26 mmol, yield 28
．． 6%) was obtained.

The melting point, infrared absorption spectrum and nuclear magnetic resonance spectrum of this product are shown below.

Melting point ('O); 172.0-174.0 (decomposition)
Infrared absorption spectrum (cm'-'): 3400,2
930,1642゜1632,1600.1424.1
360.1300.1178°1156.1078.1
028 Nuclear magnetic resonance spectrum (200ML) ppm: (D
MSO-ds) 2.44 (3H, s), 3.1! r4.
45 (m), 4.76 (2H, br, s), 4.85 (
5H, br, s), 7.44 (IHd, J -8,88
Z), 7.75 (IH, d, J = 8.88Z) (2
) 6-deoxy β-cyclodextrin synthesis method,
After dissolving 1.27 g (0,985 mmol) of the 6-tosyl-β-cyclodextrin thus obtained in 20 mQ of dimethyl sulfoxide (DMSO), 3 mL of sodium borohydride (NaBH*) was added to the solution.
84m + 9 (add 10.2 mm, 1 at 50°C
The reaction was allowed to proceed for 2 hours. Next, 10,100 ml of water was added to this reaction solution.
By adding O + and removing DMSO by ODS column chromatography, purification was performed, and the target fraction eluted with an ethanol-water mixture (volume ratio 1:9) was concentrated and recrystallized from methanol. 6-dioxy-β-
Cyclodextrin 839.6 mg (0,750 mmo
1, yield 76.1%) was obtained.

The melting point, infrared absorption spectrum, nuclear magnetic resonance spectrum, and elemental analysis values of this product are shown below.

Melting point ('(:l): 280.0-281.0 (decomposition)
Infrared absorption spectrum (c+++-→: 3370,2
920.11521080.1020 Nuclear magnetic resonance spectrum (200MHz) ppm (DMS
O-d, ): 1.20 (3H, d, J = 6.1H =
), 2.80=4.05(m)4.84(7H,br,
s) Elemental analysis value: C*2t(H as yoo3* Theoretical value (%) 45.08 6.31 Actual value (%
) 44.99 6.45 (3) Preparation of cyclodextrinase 1% (w/v) β-cyclodextrin, 1% (w/v
) peptone, 0.5% (w/v) NaCI2 and 0.1
% (w/v) liquid medium made from yeast extract (tap water used, pH 7,0) 1 () Omff for 500 m! Pour into a 2-volume slope flask and sterilize at 12,000 for 20 minutes. In addition, 1 from the preserved slant of Bacillus sphaericus E-244 strain (FERM BP-2458)
A platinum loop was inoculated and cultured with shaking at 30°C for 1 day. 50 m+2 of this culture solution was prepared using the same medium composition and sterilization conditions as above. 3000m containing 2000mQ medium
Inoculate into Q volume mini jars at 30°C, 1 vvm.

Aerated agitation culture was carried out for 2 days under the conditions of 35 Or, p, m, and after culturing, 8000 r, p, m, 2 from this culture solution.
Bacterial cells were separated by centrifugation for 0 minutes, and 2% (w
/v) The bacterial cells were suspended in 10mM phosphate buffer (pH 7.0) 500XQ12 containing Triton X-100 and stirred at 25°C for 1 day. From the suspension, 1200 or
After removing the flue body residue by centrifugation treatment for 20 minutes at
0) for 16 hours. The obtained dialysate was
Centrifuge at 000r, p, m for 20 minutes to remove insoluble matter, and close the upper groove with the crude enzyme solution (1).

Next, this crude enzyme solution (1) has a concentration of about 500+Q (total activity 20
0 units, protein amount 2083 mg, specific activity 0.1. p
DEAE Sepharose packed column (-34X) equilibrated with 10mM phosphate buffer (pH 7,0)
170 mm) to adsorb the enzyme, and then 0 to 0.
Elution was performed with a 5M NaCQ gradient gradient.

The elution pattern of this DEAE Sepharose column chromatography is shown in FIG. In Figure 5, ◎ mark is activity (U/mQ), ○ mark is protein fn (mg/mQ
). In addition, the elution conditions at this time are as follows. Elution conditions: Eluent + 10mM phosphate buffer (pH 7,0)
OM-0,5M NaC! Amount of fraction: L2mQ/Fraction flow rate: 6m+2/min Temperature: Room temperature Collect the active fractions thus obtained and prepare crude enzyme solution (2) 105m+2 (governing property: 145 units, specific activity: 0)
．． 58, yield 72.5%).

Next, 20 mQ of this crude enzyme solution (2) (governing property 31 units, protein amount 29 mg) was added to IM sodium sulfate containing 1
The enzyme was applied to an ether 5PW packed column (121.5×150 questions) equilibrated with 00 mM phosphate buffer (pH 7.0) to adsorb the enzyme, and then eluted with a gradient gradient of IM-0M sodium sulfate. This elution pattern is shown in FIG. In Figure 6, ◎ marks are active (U
/mQ), and the circle mark is the protein amount (m*/ma). Moreover, the elution conditions at this time are as follows.

Elution conditions Eluent: 100mM phosphate buffer (pl (7,0) Elution: LM-Ohl Na2SO4 (1hr) Amount of fraction: 5mQ/fraction flow rate: 5ml/min Pressure Crab 35-50 hit/cm Temperature: At room temperature, the active fractions thus obtained were collected and mixed into 50 ml of crude enzyme solution (3) (governing property: 72 units, specific activity: 2.9
3, yield 36%).

Next, this crude enzyme solution (3) was divided into 1 portion using a collodion bag.
．． After ultraconcentration to 5m12, 0.2mQ (1
m activity 8 units, protein j 11.2 mg) TSK g
el G3000SW (Column I 7.5X 600m
mX 2 ) and 0.2M Na
Elution was performed with 100 mM phosphate buffer (pH 7,0) containing CQ. This elution pattern is shown in FIG.

In FIG. 7, the ◎ mark indicates the activity (07m12), and the O mark indicates the protein amount (Cmg/ma). Moreover, the elution conditions at this time are as follows.

Eluent: 100 medium M phosphate buffer (pH 7,0) +0
．． 2M NaCQ Amount of fraction: 1m+2/Fraction flow rate: 0.7mQ/min Pressure: Crab approx. 70kg/cm" Temperature: Room temperature Collect the active 7 fractions obtained in this way and prepare the purified enzyme solution 1.4+++Ql>, activity 2 .2 units, protein fl) 0.24+++g, specific activity 9.17, yield 1%)
I got it. FIG. 8 shows the results of SOS PAGE of this enzyme. As can be seen from this figure, the enzyme is SDS P
Single in terms of AGE -t:.

(4) Amount of 6'-deoxy-D-maltopentaose 15 g of 6-deoxy/β-cyclodextrif obtained in the same manner as in (2) above was added to 100 mM phosphate buffer (pH 7).
After dissolving in ゜0) 100 (h++Q), about 24 units of the crude enzyme solution obtained in (3) above was added, and the enzymatic reaction was carried out at 40°C for 48 hours to obtain a reaction solution.

The reaction was stopped by adding hydrochloric acid to the reaction solution to bring the pH to about 2.0, and then neutralized by adding sodium hydroxide solution. The reacted 6-dioxy-β-cyclodextrin was adsorbed to obtain a liquid fraction. Repeat this operation 4 times,
A total of 63 g of 6.deoxy-β.cyclodextrin was treated.

This permeable fraction was mixed with 1/10 of the liquid volume of 100 mM acetate buffer (pH 4, 5), and the PL was further adjusted to 4.5 with 100 mM acetic acid. After adding the unit and carrying out the enzymatic reaction at 40 °C for 8 hours, the reaction was stopped by adding hydrochloric acid to the reaction solution to make the pH about 2.0, and then adding a sodium hydroxide solution. It was neutralized.

Next, after passing this solution through an activated carbon column,
% ethanol gradient, and then the approximately 25% ethanol elution fraction was lyophilized to yield 65-deoxy-
Approximately 2.5 y of D-maltopentaose was obtained.

The infrared absorption spectrum, nuclear magnetic resonance spectrum, high performance liquid chromatography, and elemental analysis values of this product are shown below.

Infrared absorption spectrum (am""): 3420.29
25,1628°1412.136B, 1150°1080.1040 Nuclear 1i1fi resonance spectrum (200MHz) ppm (
D20): 1.27 (3H, d, J = 6.3Hz)
, 3.16 (IH, t.

J=9.0Hz), 3.29(IH,t, J-9,0H
z).

3.50-4.10 (m), 4.65 (0.5H, d
, J-8,1Hz, a-H), 5.23(0,5H
, d, J = 3.4 Hz, β-H), 5.27 (IH,
d, J = 3.2Hz).

5.35 (3H, d, J -3,9Hz) high performance liquid chromatography [manufactured by Tosoh Corporation] TSK get A
m1de-80 column (4,6m+n1DX 250m
), R1 detection, eluent niacetonitrile/water-3/2 (
V/V), flow rate: 1. om4/m1n): '
R-8.7m1n elemental analysis value 2C1°Hs, CH as 0□ Theoretical value (%) 44.34 6.45 Actual value (%
)44.45 6.45 65-deoxymaltopentaose 821 obtained in (4)
Dissolve m9 (1,0 in+ol) in pyridine 30rnQ, add 15 m12 (0,159 mol) of acetic anhydride, and react at room temperature for 2 days. Next, pyridine in the reaction solution,
After distilling off acetic anhydride and acetic acid, the afterflame was purified by silica gel column chromatography, and the target fraction eluted with a methanol-dichloromethane mixture (volume ratio 1.5:98.5) was concentrated and purified with hexadeca-〇-acetyl. 6'-
Deoxymaltopentaose (α form: β form - about i:
l) 1.369g (0,923mM, yield 92.3%)
I got it.

Infrared absorption spectrum (am-'): 3480.2
970,1754°1372,1236,1038,9
46.898 nuclear magnetic resonance spectrum (2001JHz)
ppm (CDCIs): 1, 15 (3f(, d, J
-6,1)1z), 1.75~2.25 (48H,
each), 3.70-5.10(m), 5
．． 76 (ca, o, 5H, d,, J -8, 1Hz,
a-H), 6.24 (ca, 0.5H, dJ-3
, 5Hz, β-H) High performance liquid chromatography [CO5MOS I LCr a column (4,6mm 1DX 150mm) manufactured by Gyudon Chemical Co., Ltd., R1 detection, eluent Niacetonitrile/water-3/2 (V/V) flow rate :
1. om(1/min): 'R=9.4m1n(6
) α-pentadeca-O-acetyl-6s-deoximer (
Hexadeca-〇-acetyl 6s-deoxymaltopentaose obtained in 5) 1.321? (0,889mol)
Dissolve in dichloromethane □+Q to make 3JL! J7
84-5 μa (0,892 mo1) and water 35.2 μα (
1.96 mol) was added thereto, and the mixture was reacted at room temperature for 22 hours with stirring. Next, 860% of anhydrous potassium carbonate was added to the reaction solution.
++19 was added and reacted at room temperature for 15 minutes with stirring. Insoluble materials were filtered off using a glass filter, washed three times with 20 mQ of dichloromethane, the filtrate and washing liquid were combined, and dichloromethane was distilled off. The afterflame was then purified by silica gel column chromatography, and the target fraction eluted with a dichloromethane-ethyl acetate mixture (volume ratio 7:3) was concentrated and recrystallized from diethyl ether to obtain a-
604 mg (0,401 mol, yield 45.1%) of pentadeca.O-acetyl.65-deoxymaltopentaosyl bromide was obtained.

Melting point ('C'): 118.0-120.0 Infrared absorption spectrum (cm-'): 1756, 1372, 12
40゜1.042.946.902 Nuclear magnetic resonance spectrum (200MHz) ppm (CDC
I, ): 1.15 (3H, d, J = 6.4Hz),
1.85-2.40 (45H, each), 3.70
~5.70 (m), 6.51 (l) l, d, J・3.9
Hz) 574 mg (0,3
81rsol) was dissolved in acetonitrile 1Orn12, and 199mg of 2-chloro-4-nitrophenol (1,
After adding silver oxide (Ag20)
266 mg (L, 14 mol) was added and reacted at 35°C for 17 hours with stirring. Next, the reaction solution was filtered through a glass filter, and dichloromethane (20 mQ) was added to the solution.
After washing twice, the filtrate and washing liquid were combined and concentrated under reduced pressure to remove acetonitrile and dichloromethane. Next, dichloromethane lOOmQ was added to the afterflame, filtered through a cotton plug, and then 0
．． Wash with 50 m of 5N aqueous sodium hydroxide solution (1 time and 3 times with 50 mQ of saturated saline, then add 5 g of anhydrous sodium sulfate, dry, filter through a cotton plug, and then concentrate under reduced pressure to remove dichloromethane. The afterflame was then purified by silica gel column chromatography and dichloromethane-methanol solution (volume ratio 98.5:1.5)
The target fraction eluted with
0-acetyl-6%-deoxy-β・D-maltopentaoside 494m9 (0,309mol, yield 81.0%
) was obtained.

Melting point ('O): 126.0-128.0 Ultraviolet/visible absorption spectrum: "A-harvesting large wavelength [J(lX(3CN)](nm)-280
(109tax = 3.97), 227 (sh), 20B (log+'
-4,21) Infrared absorption spectrum (am”): 34
70.2960.1754゜1530, 1372.13
50.1234.1038.946.898 Nuclear magnetic resonance spectrum (200MHz) ppm (CDC1a):
1.15 (38, d, J-6, 1Hz), 1.90-2
．． 30 (45H.

each s), 3.70-5.50(m), 7-29
(I H, d, J=9.0Hz), 8.16 (
I H, dd, J-9,002゜2.7) 1z), 8
．． 30 (I H, d, J = 2.7 Hz) high performance liquid chromatography [CO3MOS I L manufactured by Gyudon Chemical Co., Ltd.
C+ a column (4,6mm ID x 150mm)
.

UVzaass detection, solution 1liII solution Niacetonitrile/water-7/3 (V/V), flow rate: 1. OmQ,/m
1nl: 'R=9.1m1n Specific optical rotation (Aa 1 zero): (CO,50,1,4-dioxane) knee 1.
5゜(7) 460 mg (0,288 mol) of 2-chloro-4-nitrophenylpentadecaro-acetyl-6s-deoxy-β-D-maltopentaoside was dissolved in 1 methanol (3 mQ). 28% ammonia water gm1
2 and water 4rnQ were added, and the mixture was reacted at 35° C. with stirring for 20 hours.

The reaction solution was then concentrated under reduced pressure to remove water and methanol. The afterflame was then purified by ODS gel column chromatography, and the target fraction eluted with an ethanol-water mixture (volume ratio 1:4) was concentrated and recrystallized in water to give 2-chloro-4-nitrophenyl-65- 196 mg (0,203 mol, yield 68.3%) of deoxy-β-D-maltopentaoside was obtained.

Melting point ('C): 187.5-190.5 Ultraviolet/visible absorption spectrum: Maximum absorption wavelength [JMo"l (nm) - 288 (Io9g
ax = 4.02), 228 (sh), 207 (logt
-4,27) Infrared absorption spectrum (cm-t: 343)
0,2940,1644°1588.1522.148
8,1352,1276.1252.1154,108
2゜1046.1026 Nuclear magnetic resonance spectrum (2001JHz) ppm (Dz
O): 1.28 (3H, d, J-5, 9Hz), 3.
14 (IH, t, J=8.6Hz).

3.50-4. OO (m), 5.28 (18, dj=3
．． 4Hz), 5.35 (3H, m), 5.43 (18,
d, J -6,8Hz), 7.40 (IH, d, J-9
, 2Hz), 8.22 (IH, dd, J=9.2Hz,
2.7Hz), 8.40 (IH, d, J-2,7Hz) High performance liquid chromatography [TSK manufactured by Tosoh Corporation
gel Amlde-30 column (4,6m+ IDx
250mm).

Uv,. , 1 detection, eluent niacetonitrile/water-3/
l (V/V), flow rate: 1. Om+2/m1nl 2'R
-9.2m1n elemental analysis value: Cxalfs4C12N
CHN as Ozy Theoretical value (%) 44.66 5.62 1.45 air M
+I/1V(01)AA (OM 71'l
l AA specific optical rotation ([a 1”): (c
O, 37, HzO): +0.95° The same operations as in (1) to (4) of Example 1 were performed, except that about 29% ethanol elution fraction was collected by the ethanol gradient, and the purity was 99. Approximately 2.7 g of 5% 6-fudeoxymaltohebutaose was obtained.

Infrared absorption spectrum (cm-'): 3420.2
930.1628°1412, 1364.1150,1
078.1022 nuclear magnetic resonance spectrum (200MHz
) ppm (D, O): 1.25 (3H, d, J-6,
4Hz), 3.16 (IH,t,J-8,7Hz).

3.27 (IH, t, J = 8.7Hz), 3.45
-4.10 (m), 4.64 (0.5H, d, J -8
,0Hz, a-H), 5.20(0,5H,d, J
-3,5 Hz, β-H), 5.26 (LH, d, J
-3.1Hz), 5.35 (5H.

a, J-3,7Hz) TSK manufactured by Tosoh Corporation by high performance liquid chromatography
gel Am1de-80 column (4,6zm lDX
250 mm), R1 detection, eluent niacetonitrile/water = 3/2 (V/V).

? ! Mushroom,in'mn/,,1in1"D-1
9.1min elemental analysis value: C+JyiOza as C
)1 Theoretical value (%) 44.37 6.38 Actual value (%) 44.40 6.35 1140 my(1,
oOmol) was subjected to the same operation as in (5) of Example 1 to obtain 1208 mg of dococer-O acetyl-6 fudeoxymaltoheptaose (α form: β form, approximately 1:1) (
0,586 mol, yield 58.6%) was obtained.

Infrared absorption spectrum (cm""): 3490.29
70,1754°1430,1374.1238.11
36.1038.946,898 Nuclear Magnetic Resonance Spectrum (200MHz) ppm (CDC1,): 1-15(
3H, d, J□6.1H2), 1.80-2.40 (6
6H, each), 3.70-5.55 (m), 5.
75 (ca, 0.5H, d, J-8,0Hz, a-
H), 6.24 (Ca, 0.5H, d, J = 3.2H
z, β-H) High performance liquid chromatography [C05M0SILC+ m column (4.6 mm
IDX 150 mm), RI detection, eluent niacetonitrile/water-7/3 (V/V).

Flow rate +1.0m12/min]: 'R-5,4m1
Docosa-O-acetyl-6 fudeoxymaltohebutaose obtained in n(2) 1177 mg (0,571 mol)
Then, the same operation as in Example 1 (6) was carried out to obtain 401 mg (0,193 mol, yield 3
3.8%).

Melting point (O): 120.0-121.5 Infrared absorption spectrum (cm-'): 3510.2990.17
56°1432, 1372.1240.1042,94
8,898 nuclear magnetic resonance spectrum (200MHz)pp
m(CDC1,) :1.15(3H,d,J=5.9
Hz), 1.90-2.30 (63H.

each s), 3.70-5.70(i), 6.50
(l H, d, J-3,9Hz) High performance liquid chromatography [CO5 manufactured by Hanbo Kagaku Yakuhin Co., Ltd.
MOS I LC+ m column (4,6mm 10X
150 mm), RI detection, eluent niacetonitrile/
Water - 7/3 (V/V).

Flow rate: 1.0+++12/min]: 'R=6.5
α-heneicosa-O-acetyl-6 foodoxymaltohebutaosyl bromide 4 obtained in (3) of Example 2
01 mg (0,193 mol) was subjected to the same operation as in Example (7) and recrystallized from methanol to obtain 2-chloro-4-nitrophenyl-heneicosa 10-acetyl-6'-thioxy -β-D-y lutoheptaoside 365
m1? (0,168 mol.

A yield of 87.0%) was obtained.

Melting point ('O) 7125.0-127.0 Ultraviolet/visible absorption spectrum: Maximum absorption wavelength [λ = 1 (nm): 282 (log
+ -4,00), 228 (sh), 207 (log+
= 4.25) Infrared absorption spectrum (am-→: 3
470,2950.1746°1582,1526.1
482.1426.1368.1346.1230.1
032 nuclear magnetic resonance spectrum (200tJHz) ppm
(CDC1x) +1.15 (3H, d, J=6.1H
z), 1.98-2.20 (63H, eachs), 3
．． 70-5.50 (m), 7.28 ((IH, d, J
”9.0Hz).

7.16 (IH, dd, J=9.()Hz, 2.7Hz
), 8.29 (IH, d, J = 2.7 Hz) High performance liquid chromatography F Gyudon Chemical Co., Ltd. C05
MO3I LCr m column (4,6mm IDX 1
50mm).

UV... 0. , detection, eluent niacetonitrile/water-
7/3 (V/V), flow rate: 1. OmL'm1nl
: 'R-14,2m1n specific optical rotation ([αl”j)
: (c 0-51.1.4-dioxane): +1.2
° Obtained in Example 2 (4); 2-chloro-4-nitrophenylheneicosa O-acetyl-6-feuteoxy-β
-D-maltoheptaoside 336m9 (0,155mo
1) was subjected to the same operation as in Example 1 (8) to obtain 2-chloro-4-nitrophenyl-6fudeoxy β-D-
148 m9 (0,115 mol, yield 74.1%) of maltoheptaoside was obtained.

Melting point ('O): 209.0-210.0 (decomposition) Ultraviolet/visible absorption spectrum: Maximum absorption wavelength [λ'max] (n"') -282
(' o g t =4.00), 228 (sh), 2
07 (logε-4,25) infrared absorption spectrum (cm
-'): 3400, 2930, 1634.

1584.1518.1484,1348,1276.
1254.11541Q80.1042,1024 Nuclear magnetic resonance spectrum (200MHz) ppm (D20
): 1.27 (3H, d, J=5.9Hz), 3.1
5 (IH, t, J -8,5Hz), 3.55-4. l
O(m), 5.30 (IH, brs).

5.38 (5H, brs), 5.43 (IH, d, J=
6.8Hz).

7.39 (LH, d, J -9,0Hz), 8.22 (
1H, ddj -9.0Hz, 2.5Hz), 8.39
(IH, d, J = 2.5 Hz) High performance liquid chromatography [T'SK get Am1d manufactured by Tosoh Corporation
e-80 column (4,6mm IDX 250mm).

UV2. . , □Detection, Eluent Niacetonidrile/Water-7
/3 (V/U, flow rate: 1.omQ/m1nl: '
R-11,0m1n elemental analysis value: C+5HxCQNO
CHN as 37 Theoretical value (%) 44.60 5.77 1.08 Actual value (%) 44.45 5.80 1.11 Specific optical rotation CCa 1 ”d): (CO, 48, H2O):
+ 1.2゜Example 3 6-dioxymaltotrioside derivatives are hardly affected by a-amylase, whereas 6-dioxymaltotrioside derivatives in the present invention
The -dioxymalto-oligoside derivatives were very well affected by the effect, and this point was measured using the following test method.

Test method (1) 2-chloro-4-nitrophenyl- obtained from Fukkasa Iwao Q Nidanri lH'lJl
6S-deoxy-β-D-maltopentaoside (hereinafter referred to as the substrate of the present invention) 50. hg, lOIIIM phosphate buffer containing 0.05% bovine serum albumin (pH-7,0
) was added to make the total amount 251RQ to prepare substrate solution (2).

(2) 2-chloro-4-nitrophenyl-6 obtained by the method described below
! -Deoxy-β-D-maltotriocytate (hereinafter referred to as control substrate) 50.0+19 was added with 10mM phosphate buffer (pH-7-0) containing 0.05% bovine serum albumin to make a total volume of 25mQ. Substrate solution (2) was prepared.

(3) Preparation of conjugated enzyme solution Add 2340 U each of commercially available yeast-derived α-glucosidase and commercially available Arnmod-derived β-glucosidase to 10 mM phosphate buffer (pH 7,0) containing 0.05% bovine serum albumin, and 260U plus all ff120m
A conjugated enzyme solution was prepared in step 4.

(4) Preparation of reagent solution The substrate solution and conjugated enzyme solution were mixed well at a volume ratio of 1.5:1 to prepare a reagent solution.

(5) Preparation of σ-amylase solution Commercially available human-derived α-amylase (manufactured by Kokusai Reagent Co., Ltd., Cablizyme AMY (Pusyoi: 1)) was mixed with IQmM phosphate buffer containing 0.05% bovine serum albumin (pH 7,
0) and dissolved it to a concentration of 0.150.450 U/I to prepare an α-amylase solution.

(6) After heating 2.5 m+2 of α-amylase hydrolysis reaction reagent solution at 37°C for 5 minutes,
- Add 100μa of amylase solution and stir, 37°0
After heating for 2 minutes, the increase in absorbance at 400 nm was measured for 2 minutes.

The results are shown in Table 5.

Table ft, Control M quality 240 rho 4-nitrophenyl-6
3-deoxy-β-D-maltotriosite was obtained by the following method.

(1) to (4) of Example 1 except that about 21% ethanol elution fraction was collected by the ethanol gradient.
), approximately 1.1 g of α-63-deoxymaltotriose with a purity of 98.6% was obtained. Next, 401 mg (0,842 mol) of this compound was subjected to the same operation as in (5) of Example 1, and deca-acetyl-63
-Deoxymaltotriose (α form - β form - about 1:
1) 604mi+(0,665mol, yield 81.0
%) was obtained.

Next, 555 mg (0,611 mol) of this compound was subjected to the same operation as in (6) of Example 1 to obtain a-nona.
-Acetyl-61-deoxymaltotriosylbromide 173m9 (0,186 moi, yield 30.4%) was obtained. Then, 150 m9 (0,161 mol) of the compound
Then, the same operation as in Example 1 (7) was performed to recrystallize from methanol to obtain 140 mg (0 , 137 mol.

Yield 85.0%) was obtained. Furthermore, this compound 108
1+1g (0,106 mol) was subjected to the same operation as in (8) of Example 1 to obtain 2-chloro-4-nitronenyl-a3-thio-t-cy-β-D-maltotriosyto42.
8u (0,665 mol, yield 63.1%) was obtained.

Melting point (°C): 150.0-152.0 Ultraviolet/visible absorption spectrum: Maximum absorption wavelength [λM00H] (rull) = 289
(log t -ax 3.94), 228 (sh), 207 (logg -4
, 17) Infrared absorption spectrum (cm-'): 342
0.2940.1590°1524, 1490.141
4.1354.1282.1256.1156°105
0.924,896 Nuclear magnetic resonance spectrum (200MHz) ppm (DtO
): 1.28 (3H, d, J-6, 4Hz), 3.1
8 (lH, t, J-9, 4Hz), 3.45-4.00
(m), 5.28 (IH, d, J=3.1Hz), 5
．． 32 (1B, d, J = 3.8Hz), 5.42 (
IH, d, J = 5.9Hz), 7.40 (IH, d,
J = 9.0Hz), 8.21 (IH.

dd, J = 9.0Hz, 2.7Hz), 8.40(
LH, d, J□2.7Hz) High performance liquid chromatography [TSK get Am1de-80 manufactured by Tosoh Corporation
Column (4,6mm IDX 250mm).

UV2@Osm detection, eluent niacetonitrile/water-3
/l (V/V), flow rate: 1. om(2/min):
'R-4,3m1n elemental analysis value: C2+Hs+CQ
NO+r and shite CHN Theoretical value (%) 44.76 5.32 2. i8 Actual value (%) 44°68 5,35 2.18 Specific optical rotation ((cr) 5): (c 0-27.8zO)
: +0-52゜Commercially available (7)2-') Clo4-nitrophenyl-β-D-maltopentaoside (manufactured by Daiichi Chemical Co., Ltd.) 15.0g (15.2m01) was added to anhydrous DMF2.
11.4 ml (76.0 mol) of benzaldehyde dimethyl acetal and 2.0 mol of tosylic acid were dissolved in 25 ml of dimethyl acetal.
259 (11.4 mol) was added and reacted at 40°C for 4 hours with stirring. Next, this reaction solution was poured into ice water 1.
It was slowly dripped into 2Q while stirring. A saturated aqueous sodium bicarbonate solution was slowly added to this while stirring under water cooling to neutralize it, and then this mixture was washed three times with 300 ml of dichloromethane, and the aqueous layer was concentrated.
DMF and water were distilled off. Next, the afterflame was 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',6''-benzylidene-β-D-
11.2 g (10.4 mci, yield 68.3%) of maltopentaoside was obtained.

Melting point ('(ri): 196.0-200.0 Ultraviolet/visible absorption spectrum: Maximum absorption wavelength [λ (nm) = 292 (+09ε)
-3,90) Infrared absorption spectrum (c++-'): 3410,29
40.1630°1586.1520,1486,13
50.1274, 1152, 1074, 1028930
．． 896 Nuclear magnetic resonance spectrum (200MHz) ppm (CDC
1x): 3.20-5.70(m), 5.05(
2H,d, J-3,4Hz), 5.12(l
H, d, J = 4.4 Hz), 5-13 (L H, d
, J = 4.4Hz).

5.25 (I H, d, J = 7.6Hz), 5.5
6 (l H, s), 7.33-7.55 (5H, +n)
, 7.35 (l H, d, J -9,0Hz), 8.1
4(lH.

dd, J -9,0Hz, 2.7Hz), 8.29(l
H, d, J -2,7Hz) High performance liquid chromatography [TSK gel Am1de-8 manufactured by Tosoh Corporation
0 column (4,6mra IDX 250++++++
).

UV2. . , detection, eluent niacetonitrile/water-3/
1 (V/V), flow rate: 1. Om(2/m1nl:
'2-chloro-4-nitrophenyl-4s,6s-benzylidene-β-D-maltopentaoside obtained from R-4,8m1n (1)! 3.3y (12,4 mol) was dissolved in 200 m4 of pyridine, and 100 mf of acetic anhydride (1,2
8 mol) was added thereto, and the mixture was 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 eluted with a methanol-dichloromethane mixture (volume ratio 2:98) was purified. Concentrate and recrystallize from ethanol to obtain 2-chloro-4-nitrophenyltetrazocar〇-acetyl-45,+31-
Benzylidene β-D-maltopentaoside 18-1g
(10.9 mol, yield 87.9%) was obtained.

Melting point (O): 130.Om135.0 Ultraviolet/visible absorption spectrum: Maximum absorption wavelength [λ5nm)”292 (xotg -3,
90) Infrared absorption spectrum (cm-'): 34L0
．． 294Q, 1630°1586.1520.1486
．． 1350.1274. l! 52.1074°1028
．． 930.896 Nuclear magnetic resonance spectrum (200MHz) ppm (CDC
! s): 2, OO~2.21 (42H, each s
), 3.55-4.90 (rn).

5.15 = 5.50 (m), 7.30 (lH, d, J-
9.2Hz).

7.26”7.41 (5H, m), 8.17 (IH, d
d, J −9,2Hz.

2.7) 1z), 8.30 (11(, d, J-2, 7Hz) High performance liquid chromatography [Gyudon Chemical Co., Ltd. CO5
MOS I LCr a column (4,6mm IDx
150mm).

UV... 1. Detection, eluent niacetonitrile/water-3
/l (V/V), flow rate: 1. Om(2/m1nl :'
R-7.7m1n Specific optical rotation ([α]￥p: (c 0
-25.1.4-dioxane)? +0.84'2-chloro-4.nitrophenyltetradeca.0.acetyl.4',6'.benzylidene-β-D obtained in (2)
-Maltopentaoside! , 669 (1,00 mol)
was dissolved in 50+++ (l) of acetic acid, 12.5 m (2) of water was added, and the mixture was reacted at 30°C with stirring for 3 days.Then, this reaction solution was slowly dropped into 30011 rl of ice water while stirring, and the mixture was dichloromethane 3
The dichloromethane layer was then washed three times with 300 mf2 of water, and the dichloromethane layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to remove dichloromethane. The residue was then purified by silica gel column chromatography, and a mixture of ethyl acetate-methanol-dichloromethane (volume ratio 6
The target fraction eluted at 6:2.5:33) was concentrated to give 2-chloro4-nitrophenyl-0-(2,3
-D-0-acetyl α-D-glucopyranosyl) (
1→4)・Tris[0-(2,3,6・Tory〇・acetyl・α-D・glucopyranosyl)-(1→4)]・2
．． 3.6.tri-0-acetyl-β-D-glucopyranoside 1.04g (0,661mol, yield 66.1%)
I got it.

Melting point ('O): 126.0-130.0 Ultraviolet/visible absorption spectrum: Maximum absorption wavelength [Ae5 (nm) -282 (logs
-3,94) Infrared absorption spectrum (cm-t: 348)
0.2970.1752°1588, 1530.148
6.1432.1372.1350, 1236.10
30°944.898 Nuclear magnetic resonance spectrum (200MHz) ppm (CDC
I2. ): 1.18-2.12 (42H, each
s), 3.50-4.74 (m).

5.05 (m), 7.22 (18, d, J-9,0Hz
), 8.09 (IH, dd.

J = 9.0Hz, 2.7)1z), 8.22(IH
, dj -2,7Hz) High performance liquid chromatography E
C05M0SILC+ a column (4
, hv+ IDx 1,50mm).

UV. , Detection, Eluent: Acetyl nitrile/water-7/
3 (V/V), flow rate: 1.0m12/mlnl:
'R=4.2m1n Specific optical rotation ([a]): (c
0.26.1.4-dioxane): +0.88' Noside synthetic leather 2.chloro-4.nitrophenyl.0-(2,3-di-0-acetyl- α-D-glucopyranosyl)-(1-4)-1-! J ;t, [o-
(2,3,6-tri〇-acetyl-α・D-glucopyranosyl)-(1-4)]・2. '3.6- Tri〇-acetyl-β-D-glucopyranoside 1.169 (0
, 738 mol) was dissolved in 30 mQ of pyridine, 2.119 (11, omol) of tosyl chloride was added, and the mixture was reacted with stirring at room temperature for 5 hours. Next, pyridine in the reaction solution was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography, and purified with ethyl acetate.
Methanol-dichloromethane mixture (volume ratio 40:1:2
The target fraction eluted in step 0) was concentrated to give 2-chloro-4-
Nitrophenyl 0-(2,3-di-0-acetyl 6
-〇-Tosyl a-D glucopyranosyl)・(1-4
)-1-Lis[0-(2,3,6-tri-O-acetyl-α-D-glucopyranosyl)-(1→4)]-2,3
, 6-tri.0-acetyl-β.D-glucopyranoside 643B (0,372 mol, yield 50.5%) was obtained.

Melting point ('C): 109.0-113.5 ultraviolet
Visible absorption spectrum: Maximum absorption wavelength [λma”] (nm) - 281 (logt
= 3.95), 272 (sh) Infrared absorption spectrum Ccya-'>: 3490.2
970.1752°1586, 1528.1486.1
430.1372.1350.1240°1178, 1
034.942 Nuclear magnetic resonance spectrum (200MHz) ppm (CDC
L): 1.99-2.17 (42H, each s)
, 3.50-4.80 (+).

5, LO ~ 5.50 (m), 7.32 (2H, d, J
-9,0Hz).

7.35 (2H, d, J-8, 5Hz), 7.79 (I
H, d, J-8,5Hz), 8.15 (IH, dd, J
-9.0Hz, 2.7Hz), 8.29(IHz, d
, J -2,7Hz) High performance liquid chromatography [CO5 manufactured by Hanshi Chemical Co., Ltd.
MOS [LC, column (4,6mm IDX 150
mm).

Uvo. 0. Detection, eluent Niacetonitrile/Water-7/
3 (V/V), flow rate: 1.0 mff/ml: '
R-10,0m1n specific optical rotation ([α]”7): (
c 0-65.1.4-dioxane): +0.88° 2-chloro-4-nitrophenyl-〇-(2,3-di-0-acetyl-6- obtained in (4) of Example 4) 0-1-Syl-a-D-glucopyranosyl)-(l→4)-tris[0-(2,3,6-tri・O-acetyl・α-D-
glucopyranosyl)-(1-4)]-2,3,6-tri〇-acetyl-β-D-glucopyranoside 500 u (
0-290 mol) in DMSOl Om (l,
Sodium borohydride (NaBJ) 110m9 (2,
91 mol) and reacted at 50°C for 13 hours. Next, this reaction solution was slowly dropped into 500 ml of ice water while stirring. Next, add 200ml of benzene to this mixture.
The benzene layer was dried over anhydrous sodium sulfate and then filtered through a cotton plug. This filtrate is concentrated and the benzene contained therein is distilled off. Without purification, dissolve this residue in 16mα of methanol and add 8mQ of 28% aqueous ammonia.
Then, 4 ml of water was added, and the mixture was reacted at 35°C for 20 hours with stirring. Next, the reaction solution is concentrated under reduced pressure, and the water and methanol contained therein are distilled off. Then the residue was
Purified by DS column chromatography, the target fraction eluted with an ethanol-water mixture (volume ratio 1:4) was concentrated and recrystallized from water to obtain 2-chloro-4-nitrophenyl 65-deoxymaltopentao. Sid is 177mg (
0,183mol.

A yield of 63.1%) was obtained. The 2-chloro-
The physical properties of 4.nitrophenyl.6-rhodeoxymaltopentaoside were all consistent with those of 2.chloro-4-nitrophenyl-6'-deoxymaltopentaoside obtained in Example 1.

Example 5 Method for measuring σ-amylase activity (1) Preparation of substrate solution Example m 2-chloro4-nitrophenyl-6s obtained in Example i
-deoxy-β-D-maltopentaoside 50.0mg
A substrate solution was prepared by adding J, OmM phosphate buffer (pH 7.0) containing 0.05% bovine serum albumin to make a total volume of 25 mf2.

(2) Preparation of conjugated enzyme solution Add 2340 U each of commercially available yeast-derived α-glucosidase and commercially available almond-derived β-glucosidase to 10 mM phosphate buffer (pH 7.0) containing 0.05% bovine serum albumin. and 260 U to make the total amount 20mQ
A conjugated enzyme solution was prepared as follows.

(3) Preparation of reagent solution A reagent solution was prepared by thoroughly mixing the substrate solution and the conjugated enzyme solution at a volume ratio of i and s: 1, respectively.

(4) Preparation of vIA product α-amylase solution Commercially available human-derived α-amylase (P:S=l:1) was mixed with 10mM phosphate buffer (pH 7,
0) plus 0.25.50, l'oo, 150.3
Dissolve the standard α- at a concentration of 00.450.600U/Q.
An amylase solution was prepared.

(5) Preparation of sucrose solution When the sample for α-amylase activity measurement was a liquid, it was used as a sample solution as it was. In the case of a solid, accurately weigh 500 μ of the sample and prepare a sample containing 0.05% bovine serum albumin.
Add mMIJ acid buffer (pH 7,0) to bring the total volume to 5 m
A sample solution was prepared as Q.

(6) Creating a calibration curve After heating 2.5 mQ of the reagent solution at 37°C for 5 minutes, add standard α/amylase solution lOOμα and stir.
The increase in absorbance at 400 nm was measured for 2 minutes after heating at C for 2 minutes. At this time, a calibration curve was created from the relationship between the standard σ-amylase solution activity and the increase in absorbance. Standard α-amylase manufactured by Kokusai Reagent Co., Ltd. (Cabrizyme AM)
Y), the calibration curve formula is U-5,875-△A
X to' -3,37 [U: Enzyme activity U/Q, △A
;Increase in absorbance]. The graph is shown in FIG.

(7) Reagent solution for measuring σ-amylase activity in sample solution 2.
After heating 5m12 at 37℃ for 5 minutes, 100ml of sample solution
Add μQ, stir, and heat at 37℃ for 2 minutes.
The increase in absorbance at 400 nm over a period of minutes 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 value of enzyme activity in the sample solution falls within the applicable range of the calibration curve (O ~
If it exceeds 600 U/+2), perform the re-measurement after diluting the sample to the corresponding multiple using 10 mM phosphate buffer (pH 7,0) containing 0.05% bovine serum albumin.

Example 6 In order to demonstrate that the compound of the present invention exists stably within the measurement system, a reaction with a conjugated enzyme was performed according to the following test method using a non-reducing end-unmodified compound as a control.

Test method (1) 2.chloro-4-nitrophenyl-65-deoxy.β.D-maltopentaoside (hereinafter referred to as the substrate of the present invention) obtained from Misato Q Ushitan Hoshimi 2i111 (hereinafter referred to as the substrate of the present invention) 50. Substrate solution (2) was prepared by adding 10 mM phosphate buffer (pH-7.0) containing 0.05% bovine serum albumin to hg to make the total volume 2511IQ.

(2) 50.0111 commercially available 2-chloro-4-nitrophenyl-β-〇-maltopentaoside (hereinafter referred to as control substrate)
9 and 10 mM phosphate buffer (pH-7.0) containing 0.05% bovine serum albumin to make a total volume of 25 mf2 to prepare substrate solution (2).

(3) Preparation of conjugated enzyme solution Conjugated enzyme solution A was prepared by adding commercially available yeast-derived a-glucosidase 11700L+ to 10mM phosphate buffer (pH 7.0) containing 0.05% bovine serum albumin to make a total volume of 5ml. . Similarly, conjugated enzyme solution B was prepared by adding 182 U of commercially available almond-derived β-glucosidase to 10 mM phosphate buffer (pH 7.0) containing 0.05% bovine serum albumin to make a total volume of 5 mQ.

(4) Conjugated enzyme reaction Substrate solution ■, substrate solution ■, and conjugated enzyme solutions A and B are heated at 37°C for 5 minutes, and then substrate solution ■ and substrate solution Conjugate liquid-2, l: 0.5
(volume ratio), mix well, and after 2 minutes, apply 400n for 5 minutes.
Measure the increase in absorbance at m.I:.

The results are shown in FIG. The seals in the figure are substrate liquid■,
The Δ mark is due to the substrate solution ■. This shows that the substrate of the present invention does not react with the conjugated enzyme and exists stably within the measurement system.

Example 7 Measurement reagent (1) Preparation of reagent Reagent A was prepared by dissolving the following components in purified water at the following concentrations.

Component 2-chloro-4-nitrophenyl-6s-deoxy-β
-D-maltopentaoside σ-glucosidase β-glucosidase β-glycerophosphate buffer (pH 7.0) Bovine serum albumin concentration 2.0mM 6QU#nQ 10U/mQ 0mM 0905% Add the following ingredients to purified water. Reagent B was prepared by dissolving at concentrations.

Ingredients
-Glycerophosphate buffer (pH 7,0) 10m
M bovine serum albumin 0.05% (
2) If the sample for Sōtō measurement was a liquid, it was used as the sample liquid as it was. In the case of a solid, 500 layer sp of the sample was accurately weighed, and reagent B was added to make a total volume of 5 m12, which was used as a sample liquid. Then reagent A
After heating 3.0+mα at 37°C for 5 minutes, sample solution 1
After adding 00 μa and stirring, the mixture was heated at 37° C. for 2 minutes, and then the increase in absorbance at 400 nm was measured for 2 minutes. The α-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, remeasurement is performed after diluting the sample solution by the corresponding multiple using reagent B.

[Brief explanation of drawings]

FIG. 1 is a graph showing the optimum pn of the cyclodextrinase used in the present invention, FIG. 2 is a graph showing the stability pH,
Figure 3 is a graph showing the action temperature, Figure 4 is a graph showing the conditions for deactivation due to temperature, and Figure 5 [3! fI is a graph showing the results of column chromatography using DEAE Sepharose, Figure 6 is a graph showing the results of HPLC using TSK gel ether 5PW, and Figure 7 is a graph showing the results of HPLC using TSK gel ether 5PW.
t A graph showing the results of HPLC using G3000SW, FIG. 8 is a graph showing the results of SDS PAGE, FIG. 9 is a graph showing the calibration curve used for measuring σ-amylase activity, and FIG. 1O is a graph showing the substrate of the present invention and a control substrate. It is a graph showing the stability within the measurement system.

Claims (1)

[Claims] 1. 6-deoxymalto-oligo 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 2 to 6) Sid derivative. 2. A reagent for measuring α-amylase activity, which contains the 6-deoxymaltooligoside derivative according to claim 1 as an active ingredient. 3. The α-anomer of the 6-deoxymalto-oligoside derivative according to claim 1 and α-
1. A method for measuring α-amylase activity, which comprises adding glucosidase and/or glucoamylase to perform an enzymatic reaction, and quantifying an aromatic chromogenic compound liberated. 4. The β-anomer or α-amylase-containing sample of the 6-deoxymalto-oligoside derivative according to claim 1 is added to the α-amylase-containing sample.
α-amylase, characterized in that an enzyme reaction is carried out by adding a mixture of an anomer and a β-anomer, α-glucosidase and/or glucoamylase, and β-glucosidase, and the liberated aromatic chromogenic compound is quantified. How to measure activity.