JPS6323199B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to novel amylase substrates. Maltooligosaccharides with well-defined structures, such as maltotetraose, maltopentaose, and maltohexaose, are increasingly being used as substrates for measuring amylase activity in human body fluids.
Taking up and explaining the case where maltopentaose is used as a substrate as a typical example of these, the measurement format can be expressed as follows. (1) Maltopentaose α-amylase――――――――→ Maltose + maltotriose (2) Maltose + maltotriose α-glucosidase――――――――→ Glucose produced here Glucose is calibrated using known methods, such as the glucose oxidase/peroxidase/dye system or the hexokinase/phosphoglucomutase/glucose-6-phosphate dehydrogenase/NADH system. However, this measurement system has the drawback that α-glucosidase acts, albeit slightly, on the substrate (maltooligosaccharide). This drawback manifests itself as an increase in the blank value, and this phenomenon gives unfavorable results whether measured by the rate method or the end point method. On the other hand, there is a phenyl group at the reducing end of the maltooligosaccharide,
Reagents with naphthyl groups or analogs thereof are being synthesized, and amylase measurement reagents using the following as substrates have been proposed. p-Nitrophenylmaltopentaoside (Japanese Patent Publication No. 57-53079) p-Nitrophenylmaltohexaoside (Japanese Patent Publication No. 57-53079) p-Nitrophenylmaltoheptaoside (Japanese Patent Publication No. 54-51892) 2 , 4-dichlorophenyl maltopentaoside (JP-A-56-35998) The following is an example of a method for measuring amylase activity using these compounds as substrates. In the case of p-nitrophenyl-α-maltopentaside (1) p-nitrophenyl-α-maltopentaoside α-amylase――――――――→ p-nitrophenyl-α-
Maltoside + maltotriose (2) p-nitrophenyl-α-maltoside + maltotriose α-glucosidase――――――――――→ p-nitrophenol + glucose In this system, both the rate method and the endpoint method However, in this case as well, p-nitrophenyl-
α-glucosidase acts slightly on α-maltopentaoside, increasing the blank value, which is also not desirable in terms of measurement. In the case of 2,4-dichlorophenyl-β-maltopentaoside (1) 2,4-dichlorophenyl-β-maltopentaoside α-amylase――――――――→ 2,4-dichloro Phenyl-β-maltoside + maltotriose (2-a) 2,4-dichlorophenyl-β-maltoside + maltotriose α-glucosidase――――――――――→
2,4-dichlorophenyl-β-glucoside + glucose (2-b) 2,4-dichlorophenyl-β-glucoside β-glucosidase――――――――――→ 2,4-dichloro Phenol + glucose (2-b-1) 2,4-dichlorophenyl + 4-aminoantipyrine KIO ₄ ---→ Quinone dye This system is measured by the end point method, but in this case also the substrate 2,4 -Dichlorophenyl-β-maltopentaoside is slightly affected by α-glucosidase, resulting in an undesirable increase in the blank value. In the end, the conventional measurement systems using substrates have a common drawback in that it is difficult to use a single-liquid reagent system. As a result of various studies, the present inventors have discovered a substrate that is not affected by α-glucosidase or β-glucosidase, and it has become possible to use a single-component measurement system. That is, the present invention provides an amylase substrate having the above characteristics, a method for producing the same, and a method for measuring amylase activity using the substrate. The new substrate has the general formula (In the formula, R ¹ and R ² are the same or different hydrogen atoms,
means lower alkyl group or phenyl group, R ¹
and R ² may jointly form a cyclohexane ring. One of R ³ and R ⁴ means a halogen atom, and the other means a hydrogen atom, a hydroxy group, a lower alkyl group, a halogen atom, or a nitro group. n is an integer from 0 to 6) is a cyclic acetal derivative of α,β-nucleo-substituted phenyl malto-oligoside, and the method for producing the above substrate is as follows: α denoted by (in the formula, R ³ and R ⁴ have the same meanings as before),
β-nucleus substituted phenyl malto-oligoside has the general formula

[expression] or

[Formula] (In the formula, R ¹ and R ² have the same meanings as before, and R ⁵ and R ⁶ are the same or different and mean a lower alkyl group)
The gist is that the carbonyl compound or acetal compound shown in is reacted,
Furthermore, the method for measuring amylase activity using the above substrate is as follows: (In the formula, R ¹ and R ² are the same or different hydrogen atoms,
means lower alkyl group or phenyl group, R ¹
and R ² may jointly form a cyclohexane ring. One of R ³ and R ⁴ means a halogen atom, and the other means a hydrogen atom, a hydroxy group, lower alkyl, a halogen atom group, or a nitro group. n means an integer from 0 to 6), α-glucosidase and/or β - A method for measuring amylase activity, characterized in that the amylase activity in the sample is determined by allowing glucosidase to act and measuring the phenol compound liberated. In the above compounds (), () and (),
The lower alkyl groups represented by R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, t-butyl,
Examples include pentyl and hexyl groups. Alternatively, in the compounds () and (), the substituents represented by R ³ and R ⁴ can be substituted at any position and in any mutual relationship of ortho-, meta-, and para-. Further, the halogen atoms represented by R ³ and R ⁴ may be any of fluorine, chlorine, bromine, and iodine. In compounds () and (), n is 0 to 6
The maltooligosaccharide used in these maltooligosides can be anything from maltose to maltooctaose. Examples include heptaose. Among these, particularly preferred are maltotetraose, maltopentaose, maltohexaose, and maltoheptaose. The phenolic compound bound to the above maltooligosaccharide as an aglycone can be α- or β-linked to the reducing end of the maltooligosaccharide and is easily subjected to the action of α-glucosidase or β-glucosidase. It is something that is liberated. As the nuclear-substituted phenol, 2,4-dichlorophenol, 2-chloro-4-nitrophenol, 4-chloro-2-nitrophenol, 4-chlorophenol, etc. can be used, but of course these are just examples. It is not intended to limit the invention. The phenolic compound as exemplified here can be α- or β-linked to the reducing end of the maltooligosaccharide. Next, the substrate () of the present invention will be further explained using specific examples. Examples include the following. In addition, in [ ] after each compound name, abbreviations that may be used in this specification are added. (1) Isopropylidene-2,4-dichlorophenyl-β-maltopentaoside (2,4-dichlorophenyl 4,6-O-isopropylidene-O-α-D-glucopyranosyl-{(1→4 )
-O-α-D-glucopyranosyl-} ₃ -O-β
-D-glucopyranoside) [isopropylidene-β-G5DCP] (2) Benzylidene-2,4-dichlorophenyl-
β-maltopentaoside (2,4-dichlorophenyl 4,6-O-benzylidene-O-α
-D-glucopyranosyl-{(1→4)-O-α
-D-glucopyranosyl} ₃ -O-β-D-glucopyranoside) [Benzylidene-β-G5DCP] (3) Isopropylidene-2,4-dichlorophenyl-β-maltoside (2,4-dichlorophenyl 4, 6-O-isopropylidene-O-α
-D-glucopyranosyl-(1→4)-O-β-
D-glucopyranoside) [isopropylidene-
β-G2DCP] (4) Isopropylidene-2-chloro-4-nitrophenyl-β-maltopentaoside (2-chloro-4-nitrophenyl 4,6-O-isopropylidene-O-α-D-glucopyranosyl -{(1→4)-O-α-D-glucopyranosyl-} ₃ -O-β-D-glucopyranoside) [isopropylidene-β-G5CHP] (5) Cyclohexylidene-2-chloro-4-nitrophenyl -β-maltopentaoside (2-
Chloro-4-nitrophenyl 4,6-O-cyclohexylidene-O-α-D-glucopyranosyl-{(1→4)-O-α-D-glucopyranosyl-} ₃ -O-β-D-glucopyranoside)
[Cyclohexylidene-β-G5CNP] (6) Isopropylidene-2-chloro-4-nitrophenyl-β-maltohexaoside (2-chloro-4-nitrophenyl 4,6-O-isopropylidene-O-α- D-glucopyranosyl-{(1→4)-O-α-D-glucopyranosyl-} ₄ -O-β-D-glucopyranoside) [isopropylidene-β-G6-CNP] (7) Benzylidene-2-4-nitrophenyl −α
-maltoheptaoside (2-chloro-4-nitrophenyl 4,6-O-benzylidene-O
-α-D-glucopyranosyl-{(1→4)-O
-α-D-glucopyranosyl} ₅ -O-α-D-
glucopyranoside) [benzylidene-α-
F7PNP] The substrate ( ) of the present invention is a novel compound, and can be produced by reacting the compound ( ) with a carbonyl compound or its acetal ( ). The reaction is generally carried out in a non-reactive solvent, and examples of the non-reactive solvent include dimethylformamide, dimethyl sulfoxide, ethylene glycol dimethyl ether, etc. The above compound () itself may be used as a solvent if necessary. The reaction temperature is not particularly limited, but is usually 0°C.
It is recommended to carry out the reaction at a temperature ranging from 40 to 100° C. to the boiling point of the solvent. Furthermore, it is also effective to carry out the reaction under reflux under reduced pressure, or to carry out the reaction while removing by-product alcohols by evaporation. A catalyst such as a small amount of pure sulfuric acid, hydrochloric acid gas, p-toluenesulfonic acid or anhydrous zinc chloride may be used to accelerate the reaction. According to the production method of the present invention, it is possible to selectively bond a cyclic acetal protecting group to a non-reducing terminal glucose residue of a maltooligosaccharide. According to Hasegawa et al.'s research report on sugar cyclic acetals [Carbohyd.Res29 209 (1973)] and other research reports, the monosaccharide glucose 4,
A method for selectively bonding a cyclic acetal to the 6-position has been announced. However, the method of selectively bonding a cyclic acetal to the 4- and 6-OH positions of non-reducing terminal glucose residues in higher molecular weight nuclear-substituted phenyl malto-oligosides is a completely new invention and has great utility. It is a raw material. Nucleically substituted phenyl malto-oligoside () can be produced, for example, by referring to the method of Konig-Knorr [Angew.Chem. 69 438 (1957), etc.]. That is, for example, in the presence of a catalytic amount of anhydrous zinc chloride, peracetyl malto-oligoside is added to a large excess of nuclear-substituted phenol which also serves as a solvent, and the mixture is heated to about 100° C. under reduced pressure. The generated nuclear-substituted phenyl peracetyl malto-oligoside is deacetylated in anhydrous methanol at room temperature using sodium methoxide as a catalyst, and purified by chromatography using octadecylsilane or Cephadex as an adsorbent to obtain the above raw material ( ) is obtained. Next, a method for measuring amylase activity using the above substrate (2) will be described. The basics of this method are reaction mixtures (e.g., glucosides, maltosides, maltotrioxides, etc. whose aglycones are phenyl compounds) produced by the action of amylase on the substrate ().
The purpose of this method is to release a phenolic compound by reacting α-glucosidase or β-glucosidase, or both of them, and to measure this phenolic compound. For this measurement, for example, there is a method in which the color of the liberated halogenated phenol compound is directly measured by absorbance (preferably by the rate method). In addition, if direct absorbance measurement is not possible,
For example, there are methods using color reagents such as 4-aminoantipyrine as disclosed in JP-A-56-35998, titration methods using bromine solutions, and colorimetric analysis methods using 2,6-dibromoquinone chlorimide. . In the latter case, since it is a substrate for maltooligosaccharide amylase with a halogenated phenyl group attached to the reducing end, it has the advantage of not being affected by glucose, maltose, etc. that may be present in the sample. However, the analytical method itself is dependent on the end point, and it has the disadvantage that it cannot correspond to rate assays that accompany advances in clinical analytical chemistry. On the other hand, one of the applicants found out that it was possible to perform analysis by the rate method by adding a step in which phenol oxidase was applied.
We have previously applied for a patent (patent application 184359/1984).
(No.), the method can be used as is in the present invention. That is, the key point of the method disclosed in the application is to react the liberated phenolic compound with phenol oxidase, if necessary in the presence of a coupler, and measure the amount of oxygen consumed or the pigment produced, thereby removing the phenolic compound from the sample. The amylase activity of Coupler is a substance that forms a pigment by oxidative condensation with phenols, and in addition to 4-aminoantipyrine, p-amino-N,N-dimethylaniline, p-amino-N,N-diethylaniline, p-amino-N,N-diethylaniline, p-amino-N,N-diethylaniline, -amino-N-ethyl-N-hydroxyethylaniline, 3-methyl-4-amino-
Examples include N,N-diethylaniline. Quinones formed in the absence of coupler can also be measured. When oxidized as a coupler, for example, with 4-aminoantipyrine, extremely high color intensity is obtained, which is extremely advantageous for measuring amylase activity. On the other hand, α-glucosidase and β used in the present invention
- Glucosidase may be of animal origin, plant origin, microbial origin, etc., but to explain what is particularly preferable, α-glucosidase is of yeast origin from the viewpoint of substrate specificity. things are advantageous. In other words, α-glucosidase originating from yeast generally has a wide range of substrate specificity, is less affected by differences in aglycones, and acts well on glucosides below maltotrioside, but not on glycosides above maltotetraoside. This is advantageous because it is compatible with the purpose of the present invention, which is to measure amylase activity. On the other hand, regarding β-glucosidase, from the same point of view, for example, those derived from almonds are strongly recommended. The ratio when using these α-glucosidase and β-glucosidase in combination may be determined by taking into consideration the type of substrate and the abundance ratio of α-bonds and β-bonds in the substrate, but in general, α−
Any ratio is sufficient as long as it can very quickly decompose maltotriose and nuclear-substituted phenyl maltoside produced by endohydrolysis by amylase, and usually a large amount of α-glucosidase is blended. α-glucosidase and β-glucosidase may be allowed to act after the amylase-containing sample is made to act, or they may be made to act simultaneously. Rather, it can be said that the ability to act simultaneously is the most characteristic effect of the present invention. Once the phenol compound is liberated in this way, the phenol oxidase is activated to quantify the phenol compound. Any type of compound that can be colored by oxidative condensation can be applied to the present invention. Particularly preferred examples include laccase and tyrosinase obtained from basidiomycetes. Next, α-
The superiority of the substrate () of the present invention will be demonstrated by presenting an experimental example in which the presence or absence of influence by (or β-)glucosidase was investigated. Experimental example 1 2,4-dichlorophenyl-β-maltopentaoside (abbreviated as β-G5DCP) and isopropylidene-2,4-dichlorophenyl-β-maltopentaoside (isopropylidene-β-G5DCP)
) was dissolved in 50mM, pH 6.7 Gutsud buffer to a concentration of 4mg/ml each, and the two substrate solutions were prepared.
(A) and (B) were prepared. On the other hand, an enzyme solution was prepared by adding and dissolving α-glucosidase at 150 U/ml and β-glucosidase at 20 U/ml in the same buffer as above. Add substrate solution (A) and (B) to 0.5ml of enzyme solution separately.
Add 0.5 ml each, mix, and leave at 37°C. After standing for a specified time, 10mM potassium periodate aqueous solution was added, and 20μ of 4-aminochinpyrine aqueous solution (0.5g of aminoantipyrine was added and dissolved in 10ml of distilled water) was added, and the mixture was left at 37°C for 5 minutes. rear
Absorbance at 500 nm was measured. The results are shown in Table 1.

[Table] As is clear from Table 1, α-glucosidase and β-glucosidase act slightly on β-G5DCP, reducing the absorbance at 500 nm in 1 hour.
0.305, while isopropylidene
Since β-G5DCP is hardly affected by α-glucosidase etc., the absorbance at 500 nm increased by only 0.012. Example 2 An experiment was conducted in exactly the same manner as in Example 1 except that the standing temperature was 4° C., and the results shown in Table 2 were obtained.

[Table] As is clear from the above results, the compound represented by the general formula () has almost no effect on α-glucosidase or β-glucosidase, so it is possible to use the amylase measurement reagent in a single solution, which is extremely difficult for measurement operations. This was advantageous for the purpose of the present invention, and could greatly contribute to improving measurement accuracy. Next, examples of the present invention will be described. Example 1 75U/ml of α-glucosidase and β-glucosidase in 1.0ml of 50mM glucosidase at pH 6.7
10U/ml, isopropylidene-β-G5DCP2mg,
1.0 mg of 4-aminoantipyrine was added and dissolved, and the mixture was poured into a 20 ml test tube and left at 37° C. for about 5 minutes. Commercial controlled serum “Huumylase H”
50 μ of was added to start the enzyme reaction. exactly 10
After a minute reaction, 10mM potassium periodate aqueous solution 2.0
ml was added, and the absorbance was measured at 500 nm after being left at room temperature for 5 minutes. A blank was obtained by using the same amount of distilled water instead of the control serum. The results shown in Table 3 are expressed as a value (ΔOD) obtained by subtracting the absorbance of the blank from the absorbance when "Hyumylase H" was added. For comparison, β-G5DCP was used as a substrate.
Since the measurements were carried out under exactly the same conditions, the results are also shown in Table 3.

[Table] According to these results, isopropylidene-β-
The analytical sensitivity of G5DCP is slightly reduced to 80% compared to that without isopropylidene conversion, but since the analytical sensitivity is originally high when isopropylidene-β-G5DCP is used as a substrate, there is no problem in practical use. . Example 2 A measurement test was conducted according to Example 1, except that a diluted human serum strain was used as the amylase sample. That is, the following dilution series was prepared by adding 5% bovine serum albumin aqueous solution to human serum.

[Table] Using these as samples, the same reaction as in Example 1 was carried out, and the results shown in Table 5 and FIG. 1 were obtained.

[Table] As shown in this result, a comparative relationship is observed between enzyme activity and ΔOD500. Therefore, it was found that isopropylidene-β-G5DCP can be used as a substrate for measuring amylase activity. Example 3 1. Reagents The following reagents were prepared using the substrates shown in Table 6 below. PIPES buffer (PH7.0) 50mM α-glucosidase 150U/ml β-glucosidase 20U/ml Calcium chloride 1mM Substrate 2mM

[Table] The absorbance of this drug at 400 nm was measured immediately after the preparation of the reagent, 10 minutes later, 20 minutes later, and 30 minutes later (Reagent 3
ml: 37°C), and the results are shown in Table 7.

【table】

[Table] From Table 7, it can be seen that the reagent containing a substrate with a modified non-reducing end used in the method of the present invention has a lower blank value than reagents containing other substrates, and the degree of increase over time It can be seen that it is also extremely small. Example 4 A reagent similar to Example 3 was prepared. Next, 3ml of the above reagent was added to 50μ of sample serum, and 3ml was added at 37°C.
After heating for ~4 minutes, the change in absorbance per minute at 400 nm (which is an indicator of amylase activity) was calculated. Table 8 shows the change in absorbance of the reagent blank and the change in absorbance of the sample serum (values obtained by subtracting the change in absorbance of the blank).

[Table] As is clear from the above results, the reagent containing a substrate with a non-reducing end modified used in the present invention has a higher value than the reagent blank containing a substrate with no non-reducing end modification. The change in absorbance is also small. Next, an example of manufacturing the substrate () according to the present invention will be explained. Production example 1 β-G5DCP10.4g in a 200ml flask
(11mM), N,N-dimethylformamide 100ml,
0.1 g of p-toluenesulfonic acid was charged and stirred to form a uniform solution. While keeping at 20-25℃ 2.
After 3.4 g (33 mM) of 2-dimethoxypropane was added dropwise over 2 hours, the reaction was continued for an additional 1.5 hours. Weakly basic ion exchange resin (Diaion WA
-30) 20g was added, stirred for 30 minutes, filtered, and the filtrate was concentrated to dryness to obtain 10.0g of white crystals. This was dissolved in 20 ml of distilled water and loaded onto a column with an inner diameter of 40 mm packed with 300 ml of activated carbon for chromatography (manufactured by Wako Pure Chemical Industries, Ltd.).
Develop and elute using 20% acetonitrile aqueous solution,
2.4 g of white crystals of β-G5DCP acetal were isolated. Melting point 190℃ NMR; δ value (splitting type, relative proton number) 1.5 (1
Multiplet, 3), 1.6 (singlet, 3), 2.7-4.4 (multiplet, 31), 4.7 (singlet, 14), 5-5.4 (multiplet,
4), 7 to 7.5 (multiplet, 3) The two independent peaks at δ1.5 (3H) and 1.6 (3H) are absorptions based on acetal groups, and β-
One G5DCP acetal exists in one molecule. Production example 2 5 g of 2,4-dichlorophenyl-β-maltoside (abbreviated as β-G2DCP) in a 100 ml flask
(10mM), N,N-dimethylformamide 70ml,
90 mg of p-toluenesulfonic acid was charged and stirred to achieve uniform dissolution. 2,2 while keeping at 20-25℃
After 3.1 g (30 mM) of -dimethoxypropane was added dropwise over 2 hours, the reaction was continued for an additional hour. After adding 15g of Diaion WA-30 and stirring for 30 minutes,
It was filtered and concentrated to dryness to obtain 4.9 g of white crystals.
The β-
1.8 g of white crystals of G2DCP acetal were obtained. melting point
108℃ NMR; δ value (splitting type, relative proton number) 1.5 (singlet, 3), 1.6 (singlet, 3), 3.6-4
(Multiplet, 10), 4.8 (Singlet, 5), 5-5.2 (Multiplet, 2), 5.4-5.5 (Multiplet 2), 7.1-7.6 (Multiplet) δ1.5 (3H), 1.6 The two independent peaks of (3H) are absorption based on acetal groups, and β-
One G2DCP acetal exists in one molecule. Production Example 3 α- instead of β-G5DCP in Production Example 1
Using G5DCP10.4g, α-
3.8 g of white crystals of G5DCP acetal were obtained. melting point
191℃ It is known that cyclic acetalization is carried out to selectively protect the 4- and 6-OH positions of monosaccharides [H.
M.Flowers, RWJeanlog, J.Org.Chem. 28
1277 (1963) / A. Hasegawa, H. Fletcher,
Carbohyd.Res. 29 209 (1973)/ANde Belder.
Advance in Carbohydrate Chemistry 34 179
(1977)] It is presumed that the compounds of Production Examples 1, 2, and 3 are cyclic acetalized at the 4 and 6 positions of the non-reducing terminal. Table 9 shows the results of testing the effects of α,β-glucosidase and α-amylase on the compounds of Production Examples 1, 2, and 3. The results were obtained by looking at the color development with 4-aminoantipyrine after 1 day, and the compound protected by cyclic acetal is not affected by α-glucosidase, while the G5-α,β-DCP acetal is affected by α-amylase. A structure in which the 4th and 6th positions of the non-reducing terminal are cyclically acetalized is supported.

[Table] Production Example 4 2-chloro-4-nitrophenyl-β-maltopentoxide (β-
10.8 g (abbreviated as G5CNP) (11 mM), 100 ml of N,N-dimethylformamide, and 0.1 g of p-toluenesulfonic acid were added and stirred to form a homogeneous solution.
After dropping 3.4g (33mM) of 2,2-dimethoxypropane over 2 hours while maintaining the temperature at 20-25℃,
The reaction continued overnight. After adding 20 g of a weakly basic ion exchange resin (Diaion WA-30) and stirring for 1 hour, the mixture was filtered and the filtrate was concentrated to dryness to obtain 10.4 g of pale yellow crystals. This was dissolved in 20 ml of distilled water, loaded onto a column with an inner diameter of 40 mm packed with 300 ml of activated carbon for chromatography (manufactured by Wako Pure Chemical Industries, Ltd.), and developed and eluted using a 15% acetonitrile aqueous solution to produce 2.1 g of white crystals of β-G5CNP acetal. was separated. NMR; δ value (split type, relative proton number) 1.5 (singlet, 3) 1.6 (singlet, 3) 2.8-4.1 (multiplet, 31) 4.8 (singlet, 14) 5-5.4 (multiplet Line, 4) 7.4 (Double line, J=9Hz, 1) 8.18 (Double line, J=3Hz, J=9Hz, 1) 8.24 (Double line, J=3Hz, 1) δ1.5 (3H ), 1.6 (3H) are absorptions based on acetal groups, and β-
One G5CNP acetal exists in one molecule. Production example 5 10.8 mg of β-G5CNP (11 m
M), N,N-dimethylformamide 100ml, p-
0.1 g of toluenesulfonic acid was charged and stirred to form a homogeneous solution. 1,1 while keeping at 20~25℃
-4.8g (33mM) of dimethoxychlorohexane
After dripping over time, the reaction was continued overnight. Weakly basic ion exchange resin (Diaion WA
-30) was added, and after stirring for 1 hour, it was filtered and the filtrate was concentrated to dryness to obtain 13.9 g of pale yellow crystals. This was dissolved in 30 ml of distilled water, loaded onto a column with an inner diameter of 40 mm packed with 300 ml of activated carbon for chromatography (manufactured by Wako Pure Chemical Industries, Ltd.), and developed and eluted using a 30% acetonitrile aqueous solution. 3.8 g of crystals were separated. NMR; δ value (split type, relative proton number) 1-2.1 (multiplet, 10) 2.8-4.1 (multiplet, 31) 4.8 (singlet, 14) 5-5.4 (multiplet, 4) 7.4 (2 Double line, J = 9Hz, 1) 8.18 (Double line, J = 3Hz, J = 9Hz, 1) 8.24 (Double line, J = 3Hz, 1) The 10H minute peak from δ1 to 2.1 is in the cyclohexane ring. This is the absorption based on β-G5CNP cyclohexylidene ketal, and there is one in one molecule of β-G5CNP cyclohexylidene ketal.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the relationship between amylase activity and absorbance at 500 nm.

Claims (1)

[Claims] 1. General formula (In the formula, R ¹ and R ² are the same or different hydrogen atoms,
means lower alkyl group or phenyl group, R ¹
and R ² may jointly form a cyclohexane ring. One of R ³ and R ⁴ means a halogen atom, and the other means a hydrogen atom, a hydroxy group, a lower alkyl group, a halogen atom, or a nitro group. n is an integer from 0 to 6). 2 General formula (In the formula, R ¹ and R ² are the same or different hydrogen atoms,
means lower alkyl group or phenyl group, R ¹
and R ² may jointly form a cyclohexane ring. One of R ³ and R ⁴ means a halogen atom, and the other means a hydrogen atom, a hydroxy group, a lower alkyl group, a halogen atom, or a nitro group. In producing a cyclic acetal derivative of α,β-nucleo-substituted phenyl malto-oligoside represented by (n means an integer from 0 to 6), the general formula α denoted by (in the formula, R ³ and R ⁴ have the same meanings as before),
β-nucleus substituted phenyl malto-oligoside has the general formula [formula] or [formula] (in the formula, R ¹ and R ² have the same meanings as above, and R ⁵ and R ⁶ are the same or different and mean a lower alkyl group. do)
1. A method for producing a cyclic acetal derivative of α,β-nucleo-substituted phenyl malto-oligoside, which comprises reacting a carbonyl compound or an acetal compound represented by: 3 General formula (In the formula, R ¹ and R ² are the same or different hydrogen atoms,
means lower alkyl group or phenyl group, R ¹
and R ² may jointly form a cyclohexane ring. One of R ³ and R ⁴ means a halogen atom, and the other means a hydrogen atom, a hydroxy group, a lower alkyl group, a halogen atom, or a nitro group. n means an integer from 0 to 6), α-glucosidase and/or β - A method for measuring amylase activity, which comprises determining the amylase activity in the sample by applying glucosidase and measuring the phenol compound liberated.