KR850000475B1 - Method of preparing for the glycocyl moranoline derivative - Google Patents

Abstract

Glucosyl moranoline derivs. of formula (III) are prepd. from moranoline compds. of formula (I), compds. of formula (III) are prepd. from moranoline compds. of formula (I), compds. of formula (III), and α-1,4-glucanglucohydrolase with addn. of an acid cation exchanger. In the formulas, R is H or lower alkyl, and n is 0-20. (III) are used to control blood sugar levels.

Description

Preparation of Glucosyl Moranoline Derivatives

FIG. 1 shows the percent decomposition of each compound when α-1,4-glucanglucohydrolase is acted on methyl oligo glycosyl moranoline.

In the figure,-●-is maltose, -x- is 4- (α-D-glycosyl) -N-methylmoranoline,-▲-is 4- (α-D-maltyl) -N- Methylmoranoline, -Δ- is 4- (α-D-maltraosyl) -N-methylmoranoline,-○-is 4- (α-D-manttetraoyl) -N-methylmora Mark the target.

FIG. 2 and FIG. 3 show the results of high-speed liquid chromatography (type ALC / GPC-244 manufactured by Waters) before and after reacting α-1,4-glucaglycohydrolase in Example 2. FIG.

Using an M-Bonderpack-NH ₂ column, the mixture was developed with acetonitrile: water: 70: 30, 1.5 ml / min, and analyzed by Shimadzu Zekmartpak C-RIA type. The vertical axis represents the parallax refractive index, the horizontal axis represents the development time, the number in the drawing is the retention time in minutes, S is the sorbent, A, B, C, D, E, F, G, H, and I are N-methyl Oligo Glycosyl Moranoline is indicated.

The present invention is the following general formula (II)

4- (α-D-glycosyl) -moranoline and 4- (α-D-glycosyl) -N-lower alkyl represented by the formula (wherein R represents hydrogen or a lower alkyl group) It relates to a production method of moranorin.

MEANS TO SOLVE THE PROBLEM The present inventors earnestly researched in order to develop a safe and effective diabetes therapeutic drug, and 4-((alpha) -D-glucosyl) -monolarin and 4-((alpha) -D- which are represented as General formula (II). It has been patented to invent that glycosyl) -N-lower alkylmoranoline has an excellent glycemic inhibitory effect upon sugar loading and is extremely useful as a medicine, for example, a diabetes drug. (Special elements 54-159417, and special elements 55-76838), that is, the following general formula (III)

Cyclodextrin glycosyltransferase (EC 2. 4. 1. 19. cyclodextrin glycosyl) in an aqueous solution containing a molarin or N-lower alkyl molarin and a cyclodextrin or soluble starch represented by (R is as described above). transferase), the following general formula (I)

(R is the same as above. N represents an integer of 0 to 20.) A mixture of compounds is produced. Typically this reaction is unreacted or mono- or lower alkyl moranoline, 4- (α-D-glycosyl) -moranoline or 4- (α-D-glycosyl) -N- of n = 0. Lower alkylmoranorin, n = 1 4- (α-D-mantsilyl) -moranoline or 4- (α-D-maltyl) -N-lower alkylmoranoline, n = 2 4- (α- D-Malttrioxyl) -Moranoline or 4-([alpha] -D-Malttrioxyl) -N-Lower alkylmoranoline, n = 3 4-([alpha] -D-Malttetraoxyyl) -Moranoline, or 4- (α-D-maltetraoxyl) -N-lower alkylmoranoline, n = 4 4- (α-D-malpentaosyl) -moranoline, or 4- (α-D-malpenta It is obtained as a mixture of an oxyyl) -N-lower alkylmoranoline, an oligo glucosyl monarine, n = 5 or more, or N-lower alkyl oligo glucosyl monarine. The production rate of each component varies depending on the reaction conditions, but in general, n = 0, 1, 2, 3 are large, and as n increases, the ratio decreases. For practical use as a medicament, 4- (α-D-glycosyl) -moranoline or 4- (α-D-gnorcosyl) -N-lower alkylmoranoline of n = 0, its activity and preparation It is most advantageous in terms of ease of use. Sepadex 4- (α-D-glycosyl) -moranoline or 4- (α-D-glycosyl) -N-lower alkylmoranoline than the mixture is required for use as a medicament. Molecular weight fractionation is needed to isolate and isolate. Therefore, if the mixture can be formed into 4-(?-D-glycosyl) -moranoline or 4-(?-D-glycosyl) -N-lower alkyl moranoline by a simple reaction, It can be very advantageous and at the same time can change other products to 4- (α-D-glycosyl) -monolarin, or 4- (α-D-glycosyl) -N-lower alkylmoranoline. Yield is also significantly increased, which is very advantageous in production. The present inventors pay attention to the advantages, and as a result of intensive studies, it is a very simple treatment to act α-1,4-glucan glucohydrolase (EC 3.2.1.3.α-1.4-Glucanglucohydrolase) in the mixed solution, n = 1 or more oligo glucosyl moranorin or N-lower alkyloligo glucosyl monarine, respectively, with 4- (α-D-glucosyl) -monolarin of n = 0, or 4- (α-D-glo The present invention has been accomplished by discovering that cosyl) -N-lower alkyl molarine can be changed in extremely good quantities. Α-1,4-glycanglucohydrolase used in the present invention is Resus. Rhizopus niveus, Rezos. Produced mainly by filamentous fungi such as Rhizopus Delemar, Aspergillus niger, Aspergillus awamori, and also known as Amyloglucosidase γ Amylase, Taka. Amylase B and the like.

In order to carry out the present invention, lyase and coenzyme residues. A culture solution containing the present enzymes such as Niveus and the like can be used, and other carbohydrate enzymes such as α-amylase β-amylase may coexist.

The action of α-1,4-glucan glucohydrolase is to break down the glycos unit at the non-reducing end of the starch. Of course, oligosaccharides bound with maltose, malttriose, maltetraose, and more α-1,4 are also decomposed to glucose. In other words, starch + nH ₂ O → n · Glucose GG--G + mH ₂ O → mG (where n and m are integers, G is glucose and GG is α-1,4 bond) ) Can be displayed. It is obvious that the origin of the enzyme used in the present invention is not limited to filamentous fungi.

A first feature of the invention is structural formula (IV)

Maltose represented by is rapidly decomposed by α-1,4-glucanoglucohydrolase into glucose, but 4- (α-D-glycosyl) -moranoline represented by the formula (II) and 4- (α-D-glycosyl) -N-lower alkyl moranoline is an oligo that is hardly degraded by α-1.4 glucanoglucohydrolase and excludes n = 0 in compounds of formula (I) It is clear from the novel fact that the prior art that glycosylmoranorin and N-lower alkyloligoglucosyylmonanorin is not as fast as maltose but degrades at substantially sufficient rate. The following will be described in detail again.

Herein, G is a G, moranorin or N-lower alkyl moranorin M _R , m is an integer, α-1,4-glucan glucohydrolase G · A, the bond is α-1.4, K _m , K _m-1 ,... , K ₂ , K ₁ is the reaction rate constant of each step, the reaction of the present invention is generally

Can be displayed as The actual reaction is a mixture of different glucose numbers (m) and the reaction of each step of the common sense proceeds simultaneously. Therefore, in order to accumulate GM _R of the present invention in the reaction solution, K _m , K _m-1 . K ₂ should be large enough and K ₁ should be small enough for it. Figure 1 makes this clear. 1 is described below.

A fixed amount of N-methyl oligoglugosyl moranoline was adsorbed to 125 ml of a triangular flask, and then adsorbed to 1 g of Dow's 50W × 2 (H ⁺ ), suspended in 50 ml of distilled water, and α-1,4-glycanglucohydro 10 mg of LaA tablets are added, incubated at 40 ° C, and 500 µl is sampled over time to quantify glucose. When the substrate is malose, the mixture is incubated at 40 ° C with 1 g of Dow's 50 W x 2 (H ⁺ ) mixed.

Quantification of glucose: 1 ml of 0.3NBa (OH) ₂ 1ml 5% ZnSO ₄ was added to 500µl of the reaction solution and centrifuged at 2000g for 10 minutes. To 30 µl of clear supernatant, 3 ml of a commercially available glucos chromophore (Beringer. Mannheim Co., Sinbread. Sugar. Test) was added thereto, stirred well, and the absorbance was measured at 420 nm after standing for 35 minutes at room temperature. Separately, the same procedure was followed for treating Glucose Standard Reagent (9.1 mg / dl), and the absorbance was measured at 420 nm to quantify the amount of Glucose produced in the reaction solution.

Enzyme: About 22 units / mg of α-1,4-glucan glucohydrolase (crystal) from Biochemical Industry Co., Ltd.

Enzymatic activity: 1 ml of enzyme solution is mixed with 5 ml of 1.0% starch solution and 4 ml of 0.05 M acetate buffer (pH 4.5) and incubated at 40 ° C to reduce the reducing sugar (glucos). Reheman. The screen is quantified by the Fehling-Lehman-Schoorl Method.

One unit of enzyme produces 10mg of glucos in 30 minutes. Base mass adsorbed on substrate and resin:

Maltose 100 mg

4- (α-D-glycosyl) -N-methylmoranoline 100

4- (α-D-maltyl) -N-methylmoranoline 50

4- (α-D-maltrioxyl) -N-methylmoranoline 50

4- (α-D-maltetraosyl) -N-methylmoranoline 50

The test results are shown in FIG. The horizontal side is reaction time, and the vertical axis | shaft is represented by the ratio with respect to the decomposition | decomposition of the produced | generated glucos. However, since the powder of N-methylglycosyl moranorin is very small as shown in FIG. 1, in the case of N-methyl oligo glycosyl moranorin, it is compared with maltose decomposition rate, respectively. When it decomposed | disassembled to ilmonorine and N-methylglyco seal | monoral morin, it was expressed as% as 100. After the determination of glucose, the reaction solution was filtered to collect ion exchange resin (Daxes 50W × 2), washed with water sufficiently, eluted with 0.5N ammonia water, concentrated to dryness under reduced pressure, and then dissolved in distilled water. Through chromatography, (ALC / GPC-244 type from Waters, M Bundock Pack-NH ₂ column, Acetnittil: water = 70: 30 1.5C / min), Shimadzu Corporation Zecromart Pack C- Analysis by type RIA confirmed the reaction product. As a result, the production of N-methylmoranoline is 4- (α-D-glycosyl-N-methylmonolinine, 4- (α-D-maltyl) -N-methylmonolinine, 4- (α- In the case of D-maltiooxyl) -N-methylmoranoline 4- (α-D-maltetraoxyl) -N-methylmonanoline, the concentration was about 3%, 2%, 2%, 1%, and 2%, respectively. The same facts have been confirmed in the case of oligoglucosyl moranorin, which is faster in the case of oligoglucosyl moranorin than in the case of N-methyl oligoglucoyl sylmonarine, and faster in N-methyl. Maltose was almost completely decomposed with reaction time of 1 hour under the same reaction conditions as oligo-glycosyl moranorin, but degradation of 4- (α-D-glycosyl) -monanoline was about 3% and 4- (α-D -Malt seal) -Moranoline was 53%, 4-((alpha) -D-Malttrioxyl) -Moranoline was 50% (The meaning of% decomposition is the same as for N-methyloligoglycosyl moranoline) ).

In addition, although the adsorption | suction (mixing in case of maltose) is strongly adsorbed to 50 Wx2 of strong acidic ion exchange resin dachsh by the above experiment, this reason is demonstrated in detail later. In addition, coexistence of excess strong acidic ion exchange resin inhibits the enzymatic reaction, but up to about 4 g in 100 ml of the reaction solution is practically no problem. In addition, if the resin is saturated with moranoline, N-lower alkylmoranine, oligoglosyl moranoline, and N-lower alkyl oligoglucosyylmonarine, the reaction is not inhibited even if more resin is added.

Usually, α-1.4-glucan glucohydrolase is carried out in a buffer solution, but there is no problem that the activity is slightly reduced even when reacted in distilled water.

The second feature of the present invention is that by adsorbing the mixture to an acid carion ion exchanger and reacting α-1,4-glucanglycohydrolase, the concentration of the reaction mixture can be significantly increased, It is not influenced by the state, and does not suffer from product innibition due to the small amount of the mono- or mono-or lower alkylolanoline, which is very advantageous in production.

This will be described in more detail below.

Moranoline N-lower alkylmoranoline, oligo glucoyl moranorin, N-lower alkyloligo glycosyl moranoline inhibits α-1,4-glycanglucohydrolase.

The intensity of the inhibition is summarized in the first table.

Measurement of inhibition is as follows.

Enzyme: Resulfs, manufactured by Biochemical Industries, Ltd. Glucoamylase (crystal) derived from Niveus is dissolved in distilled water so as to be 50 µg / ml and used.

Substrate: Soluble starch is dissolved in buffer solution when hot to 1.4% and used after natural cooling.

Buffer solution: 0.1M phosphate buffer (pH 5.7)

Reaction solution:

Method: After the reaction solution was incubated at 40 ° C. for 10 minutes, 1 ml of 0.3N Ba (OH) ₂ aqueous solution and 1 ml of 5% ZnSO ₄ aqueous solution were added thereto, and centrifuged at 2000 g for 10 minutes. 3 ml of Beringer, Mannheim company, Sinbread, Sugar test) is added, and it is left to stand at room temperature for 35 minutes, and the absorbance is measured at 420 nm.

C: absorbance of the control

T: absorbance of the test

B ': absorbance of the blank (Ⅰ)

B: absorbance of the blank (II)

The percent inhibition of the dilution series is determined and the concentration at which 50% inhibition is obtained (IC ₅₀ ) is obtained. When the inhibition is weak in the first table, it is expressed as the percent inhibition at the concentration indicated in ().

[Table 1]

As shown in the first table, the inhibition of molarine and N-lower alkyl molarine is very strong, and the inhibition generally decreases when glucosylated. Therefore, when the reaction is carried out at low concentration, the reaction proceeds, but the reaction is substantially inhibited at a concentration of usually 10 to 100 (占 // ml). This concentration varies depending on the mixing ratio of each component in the mixture and the type of lower alkyl group, as evident from the first table, but in particular, the molar, the N-lower alkyl molar, is in the range of 100 to 1000 Since pear inhibition is strong, it differs remarkably depending on the amount of monarine and N-lower alkyl monarine. In actual production, however, it is very advantageous in terms of production cost to carry out as high a concentration as possible, and the reaction should not be affected by the state of the mixture. As a result of intensive research on the advantages of the present inventors, the reaction proceeds at a concentration of at least 1000 (占 // ml) when the enzyme is actuated while the mixture is adsorbed on the acid cationic ion exchanger. The present invention was completed by discovering a surprising new fact that the reaction is not affected. The reactions of Examples 1, 2, 3, and 5 did not proceed at all even when the enzyme was reacted without adsorption to the strong acid ion exchange resin.

It demonstrates more concretely according to an Example below.

Example 1

neutralized liver digest) 0.1%, 한천(寒天) 1.5%의 사면배지상에서 충분히 생육하고 있는 바칠루스. 마세란스 IFO 3490주(株)를 3백금이(白金耳) 접종하여 37℃로 3일간 배양한다. 이 배양액 300ml을 같은 배지조성 9ℓ의 발효기(내용량 15ℓ)에 접종하여, 37℃에서 3일간 충분히 통기교반하면서 배양하면 원심 맑은액 약 130∼150단위(단위의 정의는 다음에 표시한다)의 효소액을 얻는다.Bacillus. Cultivation of Maseran: To 150 ml of culture solution containing 1% of conspicier, 1% of soluble starch, 0.5% of (NH ₄ ) ₂ SO _4, 0.5% of CaCO ₃ and pH 7 was added to 500 ml of 3 square flasks. Heat sterilize in minutes. Peptone 1%, yeast 0.5%, glucose 0.3%, glycerol 1.5%, NaCl 0.3%, lever delivery (OXOID

neutralized liver digest) A bacillus that is fully grown on slope media of 0.1% and 1.5% agar. 3490 strains of maserans IFO were inoculated with platinum and incubated at 37 ° C. for 3 days. 300 ml of this culture solution was inoculated into a fermenter (15 liters capacity) of 9 L in the same medium composition, and the culture solution was incubated at 37 ° C. for 3 days with sufficient aeration for about 130 to 150 units of centrifuged clear solution (the definition of the unit is shown next). Get

Unit of Activity of Cyclodextrin Glycosyl Transferase: Dissolve 0.7% of Soluble Starch (for Hansen Chemical, Biochemical Research) in 0.05 M Acetate Buffer pH 5.5 to prepare a substrate solution. 50 µl of the enzyme solution is added to 950 µl of the substrate solution, and the reaction is stopped by adding 40 ml of 0.5 N acetic acid for 10 minutes. 100 µl of the reaction solution is taken, 0.8 ml of iodine solution in which iodine is dissolved in a 0.25 MKI aqueous solution and 3 ml of water are added, and after stirring, 660 nm is measured by absorbance. In the same manner as in (A _T ), 950 µl of the substrate solution, 50 µl of water, and 100 µl of 0.5 ml of 0.5N acetic acid were added to the solution, and iodine solution was added to measure absorbance at 660 nm. (A _R )

At this time

This is an activity in which 1 ml of enzyme solution causes a decrease in absorbance of 1% at 40 ° C. for 1 minute.

Preparation of coenzyme solution: Bacillus. Centrifuge the culture of Maserans IFO 3490 to obtain a clear supernatant. This is freeze-dried and dissolved in a small amount of water to obtain a concentrated solution of the enzyme. Distilled water is sufficiently dialyzed in the external solution at 5 ° C to remove low molecules, and an internal solution is used as the crude enzyme solution. Next, 6.5 g of moranoline is dissolved in a small amount of water, and the pH is adjusted to 5.7 with 3N hydrochloric acid. (32.5 ml after adjustment) 26 g of (alpha)-cyclodextrins are melt | dissolved in 1300 ml of co-enzyme liquids of 460 units / ml of cyclodextrin glycosyltransferases, aqueous solution of moranoline is added to this, and pH is adjusted to 5.67. Shake for 3 days at 39 ° C to make a semitone. The reaction solution is centrifuged and the clear upper water is passed through a dopants 50W × 2 (H ⁺ ) lump (50 ml of resin) to adsorb the basic substance.

After sufficiently washing with water, 4 parts (about 2 ml) of the resin was taken out, eluted with 0.5 N ammonia water, and the eluate was concentrated to dryness under reduced pressure. The obtained powder was dissolved in a small amount of water and subjected to Water Liquid Chromatography (ALC / GPC-244 type, μ-Bonderpack-NH ₂ column, acetonitrile: water = 70: 30, 1.5 ml / min). And Shimazu Corporation chromamart pack (C-RIA type). (Composition of the mixture before reacting α-1.4-glucano glucohydrolase) Approximately 48 ml of the remaining resin is suspended in 1200 ml of water to form α-1,4-glucanes derived from Reusph niebeus. 120 mg of locohydrolase (approximately 22 units / mg) was added, incubated at 40 ° C, sampled over time to quantify the glucose generated in the reaction solution, and the reaction progress was tracked. Glucose increased rapidly after the reaction, reaching the end point 89% in 5 hours. The reaction was continued again, and the reaction was stopped at 25 hrs. After the reaction, the resin was filtered and washed with water sufficiently, eluted with 0.5 N ammonia water, the eluate was concentrated under reduced pressure, and the resultant powder was dried. Analyzed. This result is shown below.

Example 2

2 g of the mixture shown in FIG. 2 was adsorbed onto 10 ml of Dow's 50 W × 2 (H ⁺ ), suspended in 50 ml of water, and 50 mg of α-1,4-glucaglycohydrolase (about 22 units / mg) was obtained. Add. After 17 hours of reaction at 40 DEG C, the reaction solution was filtered to collect the resin, eluted with 0.5 N ammonia water, and the eluate was concentrated to dryness under reduced pressure, followed by high performance liquid chromatography (ALC / GPC-244 type) manufactured by Wotaz Corporation. And Shimazu Klomart Pack (C-RIA type). It is shown in figure 3.

The mixing ratio of each component in FIG. 2, FIG. 3 was shown below.

Example 3

The reaction was carried out at 40 ° C. for 26 hours under the conditions of Example 2.

As a result, monarine 5.8% and 4-((alpha) -D-glycosyl) -monarine 93.2%, and the other component was not seen.

Example 4

200 mg of the mixture represented in FIG. 2 is dissolved in 4 L of water, and the pH is adjusted to 5.7 with 0.5 N HCl, and 200 mg of α-1,4-glycanglucohydrolase (about 22 units / mg) is added. After reacting at 40 ° C for 41 hours, the reaction solution is washed with water through a column of 50 Wx 2 (H ⁺ ) 10 ml of Dax, and then eluted with 0.5N ammonia water. The eluate is concentrated to dryness under reduced pressure, dissolved in a small amount of water, and passed through high performance liquid chromatography under the same conditions as in Example 1. As a result, 6% of the molarin and 93.4% of 4-((alpha) -D-glycosyl) -moranoline did not show other components.

Example 5

)하여 반응시킨다. 반응액을 원심분리하여 맑은 윗물을 다웩스 50W×2(H⁺)의 컬럼(수지량 8ml)에 통과시켜서, 염기성물질을 흡착시킨다. 충분히 수세후, 수지의 일부(약 2ml)을 취하여, 0.5N 암모니아수로 용출하여 감압하에 농축건조하여, 실시예 1과 동일조건으로 액체크로마토그래피에 통과시켜서 클로마트팩으로 분석한다. (α-1,4-글르칸글르코 하이드로라아제를 반응시키기 전의 혼합물의 조성)나머지 수지 약 6ml을 물 150ml에 현탁하여, α-1,4-글르칸글르코하이드로라아제(약 22유닛/mg) 90mg을 첨가하여 40℃에서 48시간 반응시켜서 반응액을 여과하여 수지를 모아서, 충분히 수세한 후, 0.5N암모니아수로 용출하여, 용출액을 감압하에 농축건고하여 액체크로마트그래피에 통과시켜서 분석한다.A small amount of 1 g of N-methyl moranoline is dissolved in water, and the pH is adjusted to 5.7 with 1 N hydrochloric acid. (10 ml after adjustment) 16 g of α-cyclodextrin is dissolved in 790 ml of cyclodextrin glycosyl transferase coenzyme solution of 336 units / ml separately. After mixing the nutrient solution to adjust the pH to 5.6, shake for 2 days at 39 ℃

To react. The reaction solution is centrifuged and the clear upper water is passed through a column of Dowex 50 W × 2 (H ⁺ ) (8 ml of resin) to adsorb the basic substance. After sufficiently washing with water, a portion of the resin (about 2 ml) was taken out, eluted with 0.5 N ammonia water, concentrated to dryness under reduced pressure, and passed through liquid chromatography under the same conditions as in Example 1 to be analyzed by clomatpack. (Composition of the mixture before reacting α-1,4-glucagonglucose hydrolysis) Approximately 6 ml of the remaining resin was suspended in 150 ml of water, and α-1,4-glucagonglucohydrolase (about 22 units / mg ) 90 mg is added and reacted at 40 ° C. for 48 hours. The reaction solution is filtered, the resin is collected, washed with water sufficiently, eluted with 0.5 N ammonia water, the eluate is concentrated to dryness under reduced pressure, and passed through liquid chromatography for analysis.

This result is shown below.

Example 6

A small amount of 1 g of N-propylmonaranoline is dissolved in water and the pH is adjusted to 5.7 with 1 N hydrochloric acid. (20 ml after adjustment) Separately, 16 g of α-cyclodextrin is dissolved in 760 ml of 260 μ / ml coenzyme solution. The nutrient solution is mixed and the pH is adjusted to 5.7, followed by shaking for 3 days at 39 ° C. The reaction solution is centrifuged and the clear upper water is passed through a column of Dowex 50 W × 2 (H ⁺ ) (8 ml of resin) to adsorb the basic substance. After sufficiently washing with water, a part of the resin (about 2 ml) was taken out, eluted with 0.5 N ammonia water, concentrated to dryness under reduced pressure, and analyzed by chromatographic pack through liquid chromatography under the same conditions as in Example 1. (Composition of the mixture before reacting α-1,4-glycanglucohydrolase) Approximately 6 ml of the remaining resin was suspended in 150 ml of water, and α-1,4-glycanglycohydrolase (about 22 units / mg ) 15 mg is added and reacted at 40 ° C. for 26 hours. The reaction solution is filtered, the resin is collected, washed with water sufficiently, eluted with 0.5 N ammonia water, the eluate is concentrated to dryness under reduced pressure, and analyzed by gromatpack through liquid chromatography. This result is shown below.

Claims (1)

General formula (Ⅰ)

(R represents hydrogen or lower alkyl group and n represents an integer.) Among the compounds which can be represented by n is 0 to 20 and the following general formula (III)

Acid cationic exchange directly or as needed with a solution containing a mixture containing the compound represented by (R is the same as above) except that only the compound of n = 0 is contained in (I). After addition of a sieve, the following general formula (II) characterized in that α-1,4-glycanglucohydrolase is reacted

(R is the same as the above) A method for producing a glycosyl moranoline derivative.

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