JPH0675510B2 - Method for producing moranolin derivative - Google Patents

Description

Detailed Description of the Invention [Industrial applications]

The present invention relates to an efficient and inexpensive method for obtaining an N-substituted moranolin derivative represented by the following general formula [I], which is useful as a medicine. Here, R represents hydrogen or lower alkyl. Material R is hydrogen in formula (I), initially as Moranorin, herbal mulberry white usefulness as extracted pharmaceuticals than skin is one that was confirmed (Yagi et al., "Japan Agricultural Chemistry Journal" [50, 571, (1976)]. The compound of formula (I) in which R is lower alkyl is a compound which has been publicly known as, for example, Japanese Patent Publication No. 59-43459 (Patent No. 1268030) together with a pharmacological action such as suppression of blood glucose elevation. The compound according to the present invention was subsequently confirmed to have an antiviral effect (Proc.Natl.Acad.Sci.USA, 9229P 1
988), its importance as a drug is extremely high.

[Prior art]

A method for obtaining moranolin by culturing a moranolin-producing bacterium has been established (Japanese Patent Publication No. 56-099195 etc.). In the general formula [I], R is lower alkyl:
Moranoline is reacted by an ordinary method, for example, a method of reacting an appropriate alkylating agent (for example, an alkyl halide or the like), a method of N-acylating and then reducing it to an N-alkyl compound, and an action of an alkylaldehyde and a reducing agent. It was possible to obtain it at any time by the method of making it happen. By the way, in order to apply the above-mentioned methods, it was essential to obtain moranolin in a pure form. The present inventors have carried out research to obtain a strain having a high ability to produce moranolin, and have made many achievements.In addition, moranolin, which is a target substance, and 1,5 It was found to contain a plurality of low molecular weight basic water-soluble substances such as -dideoxy-1,5-imino-D-mannitol and 2-amino-2-deoxy-D-mannitol. Up to now, in order to obtain moranolin from the culture solution, for example, ion exchange method, activated carbon adsorption method, column chromatography fractionation of Sephadex, cellulose, silica gel and the like have been applied for purification. Although these methods were appropriate as methods for isolating moranolin, it became clear that moranolin is an extremely important substance as a drug or a raw material for drugs, as it is today. With the desire for an efficient method, and the discovery of a strain having a high productivity, the method for isolating the target moranolin from the culture medium has attracted attention as an extremely important technical problem.

[Problems to be Solved by the Invention]

Therefore, the object of the present invention was to obtain the compound represented by the general formula [I] by an efficient method.

[Means for Solving the Problems]

As a result of intensive studies to solve the above problems, the present inventors have found that the transglycosylation reaction by cyclodextrin glycosyltransferase (CGTase) shows that moranolin and N
-It reacts with lower alkylmoranolin but does not react with a mixed basic substance, and the glucose oligomers of moranolin and the N-substituted moranolin derivative obtained by the above reaction are obtained by the method disclosed by the present inventors ( JP-B-60-2038), which can be easily converted into glucosyl molanolin and glucosyl-N-lower alkylmoranolin, glucosyl molanolin and glucosyl-
N-lower alkylmoranolin can be easily isolated by the method disclosed by the present inventors (JP-A-62-242691), glucosylmoranolin and glucosyl-N-.
Focusing on the fact that lower alkylmoranolin can be induced to moranolin and N-lower alkylmoranolin by acid or glucoamylase (GA), and the like, if these processes are carried out, the target product can be obtained in high yield from the culture solution. Conceived that you can get. Furthermore, although surprisingly surprising, while studying the above-mentioned process, the present inventors have conducted a glycosyl transfer reaction with moranolin or the like by CGTase, and thereafter, glucose oligomers such as moranolin with GA to glucosylmoranolin or the like. We have encountered the fact that the enzyme activity is greatly stabilized by the presence of the substrate, moranolin, etc. This will be described in more detail later. The present invention has been completed for the first time by discovering the above new findings. The constitution of the present invention will be described in more detail below. In the present invention, first, a bacterium that produces moranolin is cultured. Examples of such a bacterium include actinomycetes. For example, the present inventors can use the bacterium disclosed in Japanese Examined Patent Publication No. 56-9919 (Microtechnology Research Institute, No. 4301). In the culture solution, in addition to the target product moranolin, as described above, a plurality of low concentrations of 1,5-dideoxy-1,5-imino-D-mannitol, 2-amino-2-deoxy-D-mannitol, and the like are contained. The basic water-soluble substance of the molecule is mixed. When it is desired to obtain N-lower alkylmoranolin as the final target product, this culture solution is subjected to an alkylation reaction. In the present invention, lower alkyl means, for example, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso.
-Butyl, tert-butyl and the like can be mentioned. Alkylation can be performed, for example, with a suitable alkylating agent (eg,
A method of reacting an alkyl halide) can be applied. In this state, in addition to N-lower alkylmoranolin, N-lower alkyl-1,5-dideoxy-1,5-
This is where imino-D-mannitol and N-lower alkyl-2-amino-2-deoxy-D-mannitol are mixed. In the present invention, a glucose donor such as starch, dextrin or cyclodextrin is then added to the culture medium. Then, using cyclodextrin glucosyltransferase (CGTase), the general formula [II] (Wherein R is the same as above, n is an integer from 0 to 24), and then converted into a glucose oligomer by a glucoamylase (GA), if necessary, in the general formula [II]. Where n is 0 (glucosyl form)
Convert to. For compounds of the general formula [II] where R is hydrogen, GA
Is best done. The general formula [I
This is because the compound (glucosylmoranolin) in which R is hydrogen and n is 0 in I] has a specific property that it can form a molecular compound with methanol and can be fractionally crystallized. When it is not necessary to act on GA, the GA reaction can be omitted for the purpose of increasing yield and convenience of operation. In this process, mixed 1,5-dideoxy-1,5-imino-D-mannitol and 2-amino-2-deoxy-
D-mannitol and these N-lower alkyl compounds are not affected by CGTase and the like. The above-mentioned reaction based on CGTase and GA is an enzymatic reaction,
One of the important points of the present invention lies in the establishment of a method capable of advantageously executing this enzymatic reaction as described later. The compound represented by [II] obtained above is produced only for the target compound of the present invention. [II] can be isolated very efficiently as compared with the compound represented by the general formula [I], for example, by an economical operation of fractional crystallization. This intends to utilize the physicochemical properties of [II] being the glucose oligomer of [I]. For example, glucosylmoranolin can be crystallized very efficiently because it forms a molecular compound with methanol by stirring in water-containing methanol, and with respect to N-lower alkylmoranolin,
Aryl sulphonic acids can likewise be crystallized very efficiently. In addition, in this reaction, even after recovering the [II] compound from the mother liquor, the unrecovered compound [II] can be easily recovered by repeating the same operation using the residue as a raw material again. Recovery rate can be increased. As described above, the purpose of the present invention is to efficiently isolate the contaminants, and the reason for inducing [II] is to increase the yield of the isolation. It will be demonstrated in. As the above-mentioned isolation method, for example, the method described in JP-A-61-115093 related to the invention of the present inventors can be applied. The [II] obtained as described above is further heated, for example, in an acidic aqueous solution such as a hydrochloric acid aqueous solution to be hydrolyzed, or by causing excess GA to act, so that moranolin or N-lower alkylmoranolin and glucose are added. Can be decomposed into Since glucose is a neutral substance and the target substance is basic, for example, only the target substance can be obtained by treating it with a strongly acidic ion exchange resin and treating it in a conventional manner. In carrying out these, the methods disclosed in JP-A-62-242691 and JP-A-62-242692 related to the present inventors can be applied. In this case, if the final target product is subjected to a reaction for inducing another compound, it is not always necessary to crystallize it. After the compound [II] is hydrolyzed, the desired product is practically pure enough to be used in the synthesis reaction, and therefore can be directly applied to the synthesis reaction. Now, the enzymatic reaction according to the present invention, which is an important subject matter of the present invention, will be described below. Recent technological advances in enzyme reaction technology are remarkable, and there is a sense of distant life.In particular, the immobilized enzyme method applying chitosan beads is compared with conventional methods such as enzyme reaction in solution. It was extremely effective in that the amount of enzyme used was small and the reaction was efficient. Several patent applications have been made for the immobilized enzyme method using chitosan beads.For example, Japanese Patent Laid-Open No. 63-196290 discloses that after immobilizing cyclodextrin glucanotransferase on porous chitosan beads. Further, a technique of crosslinking treatment with a crosslinking agent is disclosed. Also in the present invention, the immobilized enzyme method can be applied. Exemplifying the method for producing an immobilized enzyme applied in the present invention, a crosslinking agent is first reacted with chitosan beads,
Crosslinks are formed on amino groups on the surface of chitosan beads. Then, the CGTase or GA according to the present invention is fixed to the end of the cross-linking agent. As the cross-linking agent according to the present invention, for example, glutaraldehyde or the like can be used. As the chitosan beads according to the present invention, for example, Chitopearl (Fuji Spinning Co., Ltd.) BCW-1000 series and the like can be used. In order to produce the immobilized enzyme according to the present invention, for example, the above chitosan beads are added to an appropriate buffer solution (eg, 0.1 M acetic acid buffer of pH 6.0) at an appropriate temperature (eg, room temperature) and gently added. Shake to appropriate concentration (eg 25
(W / V%) glutaraldehyde is added and gently shaken for an appropriate time (eg, 1 to 48 hours), filtered, washed with water, and then added to an appropriate buffer (eg, 0.025M acetic acid with pH 6.0). In a buffer), a CGTase solution having an appropriate concentration (Example 1 to 500 mg / ml) is added, gently shaken for an appropriate time (Example 1 to 48 hours), filtered and washed with water. The above method is a method first established by the present inventors, whereby the immobilized enzyme method can be carried out without using the technique disclosed in JP-A-63-196290. it can. In the following, important new findings regarding the implementation of the present invention will be described in detail. In the process of completing the present invention, the present inventors have found that, in the enzyme reaction according to the present invention described above, the presence of the moranolin or the N-substituted moranolin derivative according to the present invention or a glucose oligomer thereof stabilizes the enzyme reaction. Turned into
We have found a surprising new finding that the enzyme activity does not decay even after a certain period of time. For example, immobilized CGTase can retain 90% or more of its initial activity even after 366 days at 55 ° C.
In GA, 80% or more of the initial activity was 50% or more even after 43 days at 55 ° C.
It can hold 80% or more at 175 ° C for 175 days and 90% or more for 133 days at 40 ° C. It is not always clear why this fact occurs, but since the substrate of the enzyme reaction acts as an inhibitor to the enzyme reaction, it is considered that some function is exerted in relation to this inhibitory effect. You can guess. The above findings are the facts disclosed for the first time in this specification. This makes the immobilized enzyme extremely stable and provides advantageous conditions for application to the present invention.

【Example】

Hereinafter, the present invention will be described in more detail with reference to Reference Examples and Examples according to the present invention. Reference Example 1 Immobilization of CGTase (1) 10 l of Fuji Spinning Co., Ltd. Chitopearl BCW-3010 was added to 40 l of 0.1 M acetate buffer (pH 6.0) containing 2.5 w / v% glutaraldehyde.
After being immersed for 24 hours at room temperature, it was thoroughly washed with ion-exchanged water. Then, CGTase (manufactured by Hayashibara Institute of Biochemistry, from Bacillus stearothermophilus) equivalent to 20 mg of protein per 1 ml of chitopearl was added, and the final concentration of acetate buffer (pH 6.0) was 0.025M. The total amount of the solution was adjusted to 30 L. Immersion was continued at room temperature for 24 hours, followed by thorough washing with deionized water to obtain immobilized CGTase. Reference Example 2 Immobilization of CGTase (2) In the same manner as in Reference Example 1, protein adsorption amount 5 mg / ml (beads).
So that the Bacillus sterothermophilus (Baci
llus stearothermophilus) -derived CGTase was immobilized. Reference Example 3 Immobilization of GA 60-65 after dialysis of Amano Pharmaceutical Co. glucozyme NL-3
A protein concentration of mg / ml was used as the GA aqueous solution. Fuji Spinning Co., Ltd. Chitopearl BCW-3010-1 was added with 2.5l / 50% glutaraldehyde in 50mM acetate buffer (pH 5.0) 3l
After soaking at room temperature for 24 hours, it was thoroughly washed with ion-exchanged water. Then, a GA aqueous solution corresponding to 100 mg of protein per 1 ml of chitopearl was added, and further water was added to make the total liquid volume 3 l. Immersion was carried out for 24 hours at room temperature as it was, followed by thorough washing with deionized water to obtain immobilized GA. The protein adsorption amount of the obtained beads was 12.8 mg / ml (beads). Reference Example 4 Stabilization of immobilized CGTase (saccharyl transfer reaction of moranolin) Moranolin 2.5 w / v%, Amycol No. 6L [manufactured by Nippon Starch Chemical Co., Ltd.] 25 w / v%, pH of unadjusted aqueous solution 100 ml, Reference Example 1 10 ml of the immobilized CGTase obtained in 1. was added, and rotation and shaking was continued at 55 ° C.
Beads (immobilized CG
(Tase) is filtered, washed thoroughly with water, and then immobilized as follows.
The enzyme activity of CGTase was measured. (Method of measuring enzyme activity of immobilized CGTase) Moranolin 2.5w / v%, Amycol No.6L25w / v%, 1ml of beads (washed and immobilized CGTase after a certain period of time at 55 ° C) was added to 10ml of pH-unadjusted aqueous solution. The mixture was reacted at 55 ° C for 24 hours.
3 ml of the reaction solution was added to the strongly acidic ion exchange resin Dowex 50W x 2
(H ⁺ ) 7 ml, wash thoroughly with water, elute with 0.5N ammonia water, concentrate the eluate to dryness under reduced pressure, dissolve in 3 ml of water, and inject 10 μl thereof into high performance liquid chromatography. Analysis was performed to determine the concentration of unreacted moranolin. The analytical conditions for high performance liquid chromatography are:
eosil NH ₂ , 5 μm, 4 mm id × 25 cm), developing solvent (acetonitrile-water = 70: 30), detector (Hitachi 655A-30, RI)
Detector), data processor (Hitachi D-2000). When the reaction progress rate at the time of starting the rotary shaking at 55 ° C. was set to 100, the relative reaction progress rate after a certain period of time was calculated by the above formula, and this value was defined as the enzyme activity retention rate.
The results are shown in Table 1. The fact that the enzyme activity of the immobilized CGTase is stabilized by the moranolin according to the present invention is clear. Reference Example 5 Stabilization of immobilized CGTase (sucrose transfer reaction of N-methylmoranolin) A jacketed column having a diameter of 1 cm was filled with 20 ml of the immobilized CGTase obtained in Reference Example 1, and N-methylmoranolin, 1.5 was added. w
/ v%, soluble starch 9w / v%, pH unadjusted solution were continuously passed through at a passage rate of 160 ml / day at 55 ° C, and the reaction progress rate of the passed solution was determined with time. The reaction progress rate and activity retention rate were determined in the same manner as in Reference Example 4. As a result, it was found that almost 100% of the activity was retained even after 140 days. Reference Example 6 Stabilization of immobilized CGTase (moranolin, glucose oligomer of moranolin) 1 ml of the immobilized CGTase obtained in Reference Example 2 was supplemented with moranolin (2.5 w /
v%), glucose oligomers of moranolin (mixture)
(5.0 w / v%) was added, and the whole was shaken at 55 ° C in a total volume of 10 ml. For the glucose oligomer of moranolin, the transglycosylation reaction solution obtained in Example 7 was used as a strongly acidic ion exchange resin Dowex 50W × 2.
This is a base fraction obtained by treating with (H ⁺ ), washing thoroughly with water, eluting with 0.5N aqueous ammonia, and concentrating and drying after elution. Table 2 shows the residual enzyme activity retention rate (%) when the enzyme activity at the time of adjustment was set to 100. The method for measuring the enzyme activity and the like were all the same as in Reference Example 4. CGTa on Moranolin, Glucose Oligomer of Moranolin
It is clear that it has the effect of retaining the activity of se. Reference Example 7 Stabilization of immobilized enzyme CGTase (N-substituted moranolin, N-
Substituted Glucosyl Moranolin) 1 ml of the immobilized CGTase obtained in Reference Example 1 was added with N-substituted moranolin (0.5w / v%) and N-substituted glucosyl moranolin (0.5w).
/ v%) was added, the mixture was shaken in a total volume of 10 ml at 55 ° C for 14 days, and the residual enzyme activity was measured. Table 3 shows the residual enzyme activity rate when the initial enzyme activity was set to 100. The method for measuring the enzyme activity is the same as in Reference Example 4. It is clear that the compounds according to the invention show a good stabilizing effect. Reference Example 8 Stabilization of immobilized GA (morranoline transglycosylation reaction solution) Unreacted moranolin produced by the method of Example 7,
A reaction solution containing unreacted Amycol No. 6L and oligoglycosyl moranolin was adjusted to pH 5.2 with sulfuric acid, and 480 ml was applied to a jacketed column with a diameter of 1 cm filled with 20 ml of fixed GA obtained in Reference Example 3. The reaction was continuously carried out at 50 ° C at a passage rate of / day. After reacting for a certain period of time, the immobilized GA in the column was taken, the enzyme activity was measured by the following method, and the activity retention rate (stability) was determined when the enzyme activity at the start of the reaction was 100%. The results are shown in Table 4. (Fixed GA
Activity measurement method) A fixed amount (wet weight 30-80mg) of immobilized GA is taken on a glass filter of G-2, lightly sucked to filter water, and then the immobilized GA on the glass filter is placed on a lens paper. After removing the excess water by placing it on, the sample was placed in a sample bottle with a stopper, and the wet weight of the immobilized GA was determined. Next, immobilized GA 10
It was added to ml of 5% maltose solution (0.05M acetate buffer, pH 4.6) and incubated at 37 ° C for 20 minutes. 100 of the reaction solution
The reaction was stopped by adding μl to 0.05 N sodium hydroxide. Glucose was quantified using 10 μl of this reaction stop solution. The quantification of glucose was performed using Diacolor GC (manufactured by Ono Pharmaceutical Co., Ltd.). The activity was defined as follows. The enzyme activity for producing 1 μM glucose per minute was defined as 1 unit. As a blank, 0.05N sodium hydroxide mixed with the substrate in the same volume was used instead of the reaction solution. At this time, the activity of immobilized GA is 5.56 × OD _SA / OD _STD / wet weight of immobilized GA. Here OD _SA is the sample (immobilized GA)
Absorbance at 500 nm, OD _STD means the absorbance at 500 nm of a 1 mg / ml glucose aqueous solution. In Table 4, the value of the activity retention varies, but this is a measurement error. The stabilizing effect of the compound of the present invention on immobilized GA is clear. Reference Example 9 Stabilization of immobilized GA (molanolin transglycosylation reaction solution) An experiment was conducted at 40 ° C in the same manner as in Reference Example 8. The results are shown in Table 5. Reference Example 10 Stabilization with GA (Aqueous Solution) (Moranolin, N-Methylmoranolin) Moranolin and N-methylmoranolin inhibit the enzymatic activity of glucoamylase, so the stabilizing effect on solution glucoamylase is as follows. I examined it. The enzyme is Rhizopus niveu manufactured by Seikagaku Corporation.
s) derived reagent glucoamylase was used. 5 mg of enzyme was dissolved in 1 ml of 0.1 M acetate buffer (pH 5.0), and 6000 μg / ml of moranolin and N-methylmoranolin
The resulting solution was dissolved in the following manner, placed in a screw cap test tube, and incubated at 50 ° C. Each enzyme solution was 0.
Dilute 100 times with 1M acetate buffer (pH 5.0) and add 100 μl.
Take 5% maltose solution (0.05M acetate buffer, pH 4.6)
Add 400 μl and incubate at 40 ° C. for 20 minutes. 100 μl of the reaction solution is taken and 100 μl of 0.05N sodium hydroxide is added to stop the reaction. Take 100 μl of this solution, add 3.0 ml of the glucose measurement reagent Diacolor GC, let stand at 37 ° C for 30 minutes to develop color, and then cool with ice to 500n.
The absorbance was measured at m to measure the amount of glucose. This glucose production amount was defined as the enzyme activity in the presence of the inhibitor. The relative enzyme activity was calculated when the initial enzyme activity (glucose production) was set to 100. The results are shown in Fig. 1. The enzyme stabilizing effect of the compound of the present invention is clear. Example 1 Cultivation of Moranolin-Producing Bacteria Starch 2%, soybean flour 1%, potassium chloride 0.05%, magnesium sulfate 0.05%, salt 0.2%, calcium carbonate 0.35% (p
H7.2) medium 200ml in a 500ml Erlenmeyer flask, sterilized in the usual manner, on the slope of Streptomyces lavendulae SEN-158 strain (Microtechnology Research Institute No. 4301) Several platinum ear spores were inoculated from the culture and cultured at 27 ° C. for 3 days with shaking at 200 rpm to obtain a seed culture. Starch 8%, soybean flour 3%, yeast extract 1.5%, potassium chloride 0.05%, magnesium sulfate heptahydrate 0.05%, salt 0.1
%, Calcium carbonate 0.15% (pH 7.2) 250l medium
Was placed in a 420-liter jar fermenter and sterilized in the usual manner, and 2.4 l of seed culture was inoculated. 100 revolutions / minute,
The cells were cultured at 27 ° C with an aeration rate of 125 l / min for 10 days. In the same manner as in the method described in Reference Example 4, the result of quantifying moranolin in the culture solution by high performance liquid chromatography was about 3500.
It contained μg / ml of moranolin. Example 2 Partial Purification of Culture Solution Containing Moranolin (1) The culture solution obtained by the method of Example 1 was used as an ultrafiltration membrane (UF Modul).
e; MU-6303-HG; manufactured by Kuraray Co., Ltd.), and the passing liquid was further applied to a reverse osmosis membrane (HR-5155 F1; hollow fiber type; manufactured by Toyobo Co., Ltd.), and the concentrated non-passage liquid was used as a partially purified product. Example 3 Partial Purification of Moranolin-Containing Culture Solution (2) The culture filtrate obtained by filtering the culture solution obtained by the method of Example 1 with a press filter was used as a strongly acidic ion exchange resin DIAION SK-
It was applied to a column of 104 (H ⁺ ) 70 l, washed thoroughly with water, and then eluted with 1N ammonia water. After the eluate was concentrated under reduced pressure, it was applied to a column of 35 L of strongly basic ion exchange resin Diaion SA-11A (OH ⁻ ) and eluted with water. The eluate was concentrated under reduced pressure to give a partially purified product. Example 4 N-Methylation of Moranolin-Containing Culture Solution To 1000 ml of the culture solution obtained in Example 1, 100 g of High Flow Supercell was added and filtered to obtain 850 ml of a culture filtrate. 30 ml of formalin, about 15 ml of Raney nickel catalyst for commercial use
Was added and catalytically reduced at room temperature and atmospheric pressure. After completion of the reaction, the catalyst was filtered off, and the filtrate was applied to a column of strongly acidic ion exchange resin Dowex 50W × 2 (H ⁺ ) 500 ml, washed thoroughly with water, and eluted with 1N ammonia water. The eluate was concentrated under reduced pressure to remove ammonia. In this way, a partially purified product of the culture solution containing N-methylmoranolin that could be used in the next reaction was obtained. Example 5 N- (n-butyl) ation of Moranolin-Containing Culture Solution (1) 200 ml of the culture solution obtained in Example 1 was mixed with High Flow Supercell 1
00 g was added and filtered to obtain 1800 ml of culture filtrate. This was concentrated to dryness under reduced pressure, then N, N-dimethylformamide 200
ml was added, and the mixture was stirred and filtered. It was further washed with 100 ml of N, N-dimethylformamide. The filtrate and washings were combined, and 16.1 g of n-butyl bromide and 21.8 g of potassium carbonate were added to this, and
The reaction was carried out at 0 ° C for 7 hours. After the reaction, the solution was filtered and the solvent was removed under reduced pressure to obtain a partially purified product of the N- (n-butyl) -ized culture solution that could be used in the next reaction. Example 6 N- (n-Butyl) ation of Moranolin-Containing Culture Solution (2) The partially purified product obtained by treating 1000 ml of the culture filtrate obtained in Example 1 by the method of Example 3 was concentrated to dryness under reduced pressure. To this, 50 ml of methanol was added and stirred under ice cooling. 20.5 ml of n-butyraldehyde, 12.0 ml of 5% hydrochloric acid methanol solution and 2.5 g of sodium cyanoborohydride were added, and after stirring for 30 minutes, at room temperature, 16
Reacted for hours. The solvent was distilled off under reduced pressure, the residue was dissolved in 25 ml of water, and the mixture was distributed 3 times with 15 ml of chloroform. The aqueous layer was applied to a column of 100 ml of strongly acidic ion exchange resin DIAION SK-104 (H ⁺ ), washed thoroughly with water, and then eluted with 0.5N aqueous ammonia. The eluate was concentrated under reduced pressure to obtain an N- (n-butyl) -modified partially purified product of the culture liquid which could be used in the next reaction. Example 7 Glycosylation Reaction of Partially Purified Product (1) of Moranolin-Containing Culture Solution A culture solution (2000 l) cultured in the same manner as in Example 1 was
A partially purified product (150 l; containing about 7 kg of moranolin) treated in the same manner as in Example 2 was treated with a 420 l jar fermenter and 5
A 0 liter column was added to the jar fermenter of the connected reactor. Further, 70 kg of Amycol No. 6L was added and dissolved by heating. After adjusting the pH to 9.0 with 6N sodium hydroxide, water was added to bring the total volume to 280 l. Immobilized CGTase produced by the method of Reference Example 1
Fill the column with 30 l and circulate at a flow rate of 3 l / min 55
The reaction was carried out at ℃ for 48 hours to obtain a transglycosylation solution of moranolin. Example 8 Glycosylation Reaction of Partially Purified Product (2) of Moranolin-Containing Culture Solution A partially purified product (100 liters) obtained by treating the culture solution (2000 l) cultured in the same manner as in Example 1 with the same method as in Example 3 was used. Approximately 7 kg of moranoline) 420 l jar fermenter and 50 l
The column was added to the jar fermenter of the reactor connected. Further, 70 kg of Amycol No. 6L was added and dissolved by heating. Water was added without adjusting the pH to make the total volume 300 l. Immobilized CGTase 30l produced by the method of Reference Example 1 was packed in a column and reacted at 55 ° C for 72 hours while circulating at a flow rate of 3l / min to obtain a moranolin transglycosylation reaction solution. Example 9 Glycosylation Reaction of N-Methylmoranolin-Containing Culture Solution To the N-methylmoranolin-containing culture solution (50 ml; containing about 3 g of N-methylmoranolin) produced by the method of Example 4, 12 g of soluble starch was added. After warm dissolution, water was added to bring the total volume to 100 ml. 10 ml of immobilized CGTase produced by the method of Reference Example 1 was added, and the mixture was reacted at 55 ° C. for 48 hours and then filtered through a glass filter to obtain a N-methylmoranolin transglycosylation reaction solution. Example 10 Glycosylation reaction of N- (n-butyl) moranolin-containing culture solution (1) Part of partially purified product of culture solution containing N- (n-butyl) moranolin produced by the method of Example 5 [18.3 g; about this
4.0 g is N- (n-butyl) moranolin], water 10
30 ml of Amycol No.6L was dissolved in 0 ml by heating, and water was added to make the total volume 200 ml. Add 10 ml of immobilized CGTase produced by the method of Reference Example 2,
After reacting at ℃ for 48 hours, it is filtered with a glass filter and N-
A transglycosylation reaction solution of (n-butyl) moranolin was obtained. Example 11 Glycosylation reaction of N- (n-butyl) moranolin-containing culture solution (2) Part of partially purified product of culture solution containing N- (n-butyl) moranolin produced by the method of Example 6 [ 10.0g; about this
8.0 g is N- (n-butyl) moranolin] 80 ml of water
80 g of Amycol No.6L was dissolved by heating, and water was added to make a total volume of 160 ml. Add 20 ml of immobilized CGTase produced by the method of Reference Example 1,
After reacting at ℃ for 48 hours, it is filtered with a glass filter and N-
A transglycosylation reaction solution of (n-butyl) moranolin was obtained. Example 12 GA reaction (moranolin) according to Example 7 In a reactor in which a 420 l jar fermenter and a 50 l column were frozen, the 50 l column was packed with 15 l of immobilized GA prepared by the method of Reference Example 3, For the jar fermenter, 280 liters of the transglycosylation reaction solution of moranolin produced by the method of Example 7 (the transglycosylation reaction solution in which about 7 kg of moranolin was reacted)
Was added and the pH was adjusted to 5.2 with concentrated sulfuric acid, and then reacted at 50 ° C. for 19 hours while circulating at a flow rate of 3 l / min. Example 13 GA reaction (moranolin) according to Example 8 In a reactor in which a 420 l jar fermenter and a 50 l column were connected, the 50 l column was packed with 15 l of immobilized GA prepared by the method of Reference Example 3, For the jar fermenter, 300 liters of the transglycosylation reaction solution of moranolin produced by the method of Example 8 (transglycosylation reaction solution in which about 7 kg of moranolin was reacted)
Was added and the pH was adjusted to 5.2 with concentrated sulfuric acid, and then reacted at 40 ° C. for 18 hours while circulating at a flow rate of 3 l / min. Example 14 GA reaction according to Example 9 (N-methylmoranolin) 100 ml of the sugar transfer reaction solution of N-methylmoranolin produced by the method of Example 9 (before the sugar transfer reaction, N-methylmoranolin was After adjusting the pH to 5.2 with concentrated sulfuric acid)
Add 10 ml of immobilized GA prepared by the method of Reference Example 3 to 50 ° C
And reacted for 20 hours. The immobilized GA was removed by filtration with a glass filter to obtain a GA reaction solution. Example 15 GA reaction according to Example 10 (N- (n-butyl) moranolin) 200 ml of a sugar transfer reaction solution of N- (n-butyl) moranolin produced by the method of Example 10 [before the sugar transfer reaction N-
(N-butyl) moranolin (about 3 g)] with concentrated sulfuric acid
Immobilized G prepared by the method of Reference Example 3 after adjusting to 5.2
A 20 ml was added and the reaction was carried out at 50 ° C. for 20 hours. The GA reaction was obtained by removing the immobilized GA by filtration with a glass filter. Example 16 GA reaction according to Example 11 (N- (n-butyl) moranolin) 160 ml of the sugar transfer reaction solution of N- (n-butyl) moranolin produced by the method of Example 11 [before the sugar transfer reaction N- (n
-Butyl) moranoline (about 8 g) was added, and the pH was adjusted to 5.
After adjusting to 2, immobilized GA 20 produced by the method of Reference Example 3
ml was added and reacted at 50 ° C. for 20 hours. The immobilized GA was removed by filtration with a glass filter to obtain a GA reaction solution. Example 17 Purification of Glucosylmoranolin (1) GA reaction solution 280 l produced by the method of Example 12 was treated with a decolorizing resin.
It was passed through a column (120 l) of HS (Hokuetsu Carbon Co., Ltd.) and washed with water. After the passage and the washing solution were combined, they were passed through a column of 180 liters of strongly acidic ion exchange resin Diaion SK-104 (H ⁺ ). After thoroughly washing with water, elution was performed with 1N ammonia water. Ammonia was removed while concentrating the eluate under reduced pressure. The concentrated solution was passed through a column of strongly basic ion exchange resin SA-11A (OH ⁻ ) 180 l and eluted with water. The eluate was concentrated to about 18 l, 320 l of methanol was added with stirring, and the mixture was stirred overnight. The resulting results were collected by filtration and dried to give 7.9 kg of the adduct of glucosylmoranolin with methanol. This product was analyzed by high performance liquid chromatography under the same conditions as those described in Reference Example 4, and as a result, it contained about 3% of moranolin, but virtually no other impurities were observed. Example 18 Purification of glucosylmoranolin (2) 300 l of the GA reaction solution produced by the method of Example 13 was passed through a column of 180 l of strongly acidic ion exchange resin DIAION SK-104 (H ⁺ ). After thoroughly washing with water, elution was performed with 1N ammonia water. Ammonia was removed while concentrating the eluate under reduced pressure. Column ^- strongly basic ion exchange concentrate resin SA-11A (OH)
It was passed through 50 l and eluted with water. The eluate was concentrated to about 20 l, 350 l of methanol was added with stirring, and the mixture was stirred overnight. The generated crystals were collected by filtration and dried to obtain 7.3 kg of a methanol adduct of glucosylmoranolin. This product was analyzed by high performance liquid chromatography under the same conditions as those described in Reference Example 4, and as a result, it contained about 0.5% of moranolin, but virtually no other impurities were observed. Example 19 Purification of glucosyl-N-methylmoranolin 100 ml of the GA reaction solution prepared by the method of Example 14 was passed through 100 ml of a column of strongly acidic ion exchange resin Dowex 50 × 2 (H ⁺ ). After thoroughly washing with water, elution was performed with 0.5N ammonia water. Ammonia was removed while concentrating the eluate under reduced pressure. The concentrated solution was passed through a column of 100 ml of strongly basic ion exchange resin SA-11A (OH ⁻ ) and eluted with water. The passing solution and the eluate were combined, concentrated to dryness under reduced pressure, dissolved in 50 ml of methanol, added with 4.8 g of p-toluenesulfonic acid (-hydrate), and allowed to stand at room temperature overnight. The crystals formed were collected by filtration, dried and p- of glucosyl-N-methylmoranolin was collected.
4.4 g of toluenesulfonate was obtained. A part of this product was taken as a basic fraction and analyzed by high performance liquid chromatography under the same conditions as described in Reference Example 4 to find that it contained about 0.5% of N-methylmoranolin. , Practically no other impurities were found. Example 20 Purification of glucosyl-N- (n-butyl) moranolin (1) 200 ml of the GA reaction solution prepared by the method of Example 15 was passed through 200 ml of a strongly acidic ion exchange resin Dowex 50 × 2 (H ⁺ ) column. Let After thoroughly washing with water, elution was performed with 0.5N ammonia water. Ammonia was removed while concentrating the eluate under reduced pressure. The concentrated solution was passed through a column of 100 ml of strongly basic ion exchange resin SA-11A (OH ⁻ ) and eluted with water. The passing solution and the eluate were combined, concentrated to dryness under reduced pressure, dissolved in 50 ml of methanol, added with 5.2 g of P-toluenesulfonic acid (-hydrate), and allowed to stand at room temperature overnight. The formed crystals were collected by filtration and dried to obtain 5.5 g of p-toluenesulfonate of glucosyl-N- (n-butyl) moranolin. After a part of this was taken as a basic fraction and analyzed by high performance liquid chromatography under the same conditions as described in Reference Example 4, about 1.5% of N- (n-
Butyl) moranolin, but virtually no other impurities. Example 21 Purification of glucosyl-N- (n-butyl) moranolin (2) 160 ml of GA reaction solution prepared by the method of Example 16 was passed through 200 ml column of strongly acidic ion exchange resin Dowex 50 × 2 (H ⁺ ). Let After thoroughly washing with water, elution was performed with 0.5N ammonia water. Ammonia was removed while concentrating the eluate under reduced pressure. The concentrated solution was passed through a column of 100 ml of strongly basic ion exchange resin SA-11A (OH ⁻ ) and eluted with water. The passing solution and the eluate were combined, concentrated to dryness under reduced pressure, dissolved in 50 ml of methanol, added with 5.2 g of P-toluenesulfonic acid (-hydrate), and allowed to stand at room temperature overnight. The resulting crystals were collected by filtration and dried to obtain 10.0 g of glucosyl-N- (n-butyl) moranolin p-toluenesulfonate. A part of this product was taken as a basic fraction and analyzed by high performance liquid chromatography under the same conditions as described in Reference Example 4, and as a result, about 0.5% of N- (n-
Butyl) moranolin, but virtually no other impurities. Example 22 Glucosylmoranolin Glycosylation reaction using purified mother liquor residue during purification, GA reaction and purification of moranolin Obtained when the solvent (methanol) of the mother liquor fractionated by methanol in Example 17 was recovered and removed. Residue (reacted in the same manner as in Example 7 and Example 12
The same operation as in Example 7, Example 12, and Example 17 was performed using 3 lots of the recovery residue of 3 lots treated in the same manner as in Example 17 as a raw material, and as a result, 9.0 kg of a glucosylmoranolin methanol adduct was obtained. Obtained. Example 23 Purification of Moranolin 1 kg of a methanol adduct of glucosylmoranolin purified by the method of Example 17 was dissolved in 1N hydrochloric acid (5 l) and reacted at 90 ° C for 3 hours. The reaction solution weakly basic ion exchange resin Diaion WA-20 (OH ^-) subjected to a column of 10l, and sufficiently washed with water. The passing solution and the washing solution were combined, applied to a column of 5 L of strongly acidic ion exchange resin DIAION SK-104 (H ⁺ ), thoroughly washed with water, and then eluted with 1N ammonia water. The eluate was concentrated to dryness under reduced pressure to obtain 468 g of moranolin. This product has a high purity even if it is not crystallized, and can be sufficiently used as a raw material for synthesis as it is. Example 24 Purification of N-methylmoranolin 2 g of p-toluenesulfonate of glucosyl-N-methylmoranolin produced by the method of Example 19 was treated with 1N hydrochloric acid (10 m
It was dissolved in l) and reacted at 90 ° C. for 3 hours. The reaction solution weakly basic ion exchange resin Diaion WA-20 (OH ^-) subjected to a column of 100 ml, was thoroughly washed with water. Combine the passing solution and the washing solution,
Strongly acidic ion exchange resin Diaion SK-104 (H ⁺ ) 50 ml
The column was washed with water, and then eluted with 0.5N ammonia water. The eluate was concentrated to dryness under reduced pressure and then recrystallized from ethanol to obtain 590 mg of N-methylmoranolin. Example 25 Purification of N- (n-butyl) moranolin (1) 3 g of p-toluenesulfonate of glucosyl-N- (n-butyl) moranolin produced by the method of Example 20 was dissolved in 1N hydrochloric acid (30 ml). The mixture was reacted at 90 ° C for 3 hours. The reaction solution weakly basic ion exchange resin Diaion WA-20 (OH ^-) 2
It was applied to a 00 ml column and washed thoroughly with water. The passing solution and the washing solution are combined, and strongly acidic ion exchange resin Dowex 50 x 2 (H ⁺ )
It was applied to a 100 ml column, washed thoroughly with water, and then eluted with 0.5N ammonia water. The eluate was concentrated to dryness under reduced pressure and then recrystallized from ethanol to obtain 850 mg of N- (n-butyl) moranolin. Example 26 Purification of N- (n-butyl) moranolin (2) 5 g of p-toluenesulfonate of glucosyl-N- (n-butyl) moranolin produced by the method of Example 21 was dissolved in water and adjusted to pH with 1N hydrochloric acid. Adjusted to 5.2. After adjusting the total volume to 1000 ml, 50 ml of immobilized GA produced by the method of Reference Example 3 was added and reacted at 50 ° C. for 24 hours. And filtered through a glass filter except immobilized GA, filtrate, weakly basic ion exchange resin Diaion WA-20 (OH ^-) subjected to a column of 100 ml, was thoroughly washed with water. The passing solution and the washing solution were combined, applied to a column of 100 ml of strongly acidic ion exchange resin Dowex 50 × 2 (H ⁺ ), thoroughly washed with water, and then eluted with 0.5N ammonia water. The eluate was concentrated to dryness under reduced pressure and then recrystallized from ethanol to give N
1.6 g of-(n-butyl) moranolin was obtained.

[Brief description of drawings]

FIG. 1 illustrates the stabilizing effect of GA in Reference Example 10. The horizontal axis represents time (days) and the vertical axis represents relative enzyme activity. ○ indicates control, ● indicates 6 mg / ml of moranolin, △
Represents 6 mg / ml of N-methylmoranolin, respectively.

Claims (2)

[Claims] 1. The following general formula [I]: (Wherein R represents hydrogen or lower alkyl), in order to obtain the N-substituted moranolin derivative, (1) the culture solution in which the moranolin-producing bacterium is cultivated,
Alternatively, it is subjected to an alkylation reaction, and (2) after adding a glucose donor, a cyclodextrin glycosyl transferase (CGTase) is allowed to act on it to give the following general formula [II] (Wherein R is the same as above, n represents an integer from 0 to 24), and an oligoglycosyl-N-substituted moranolin derivative is produced, and glucoamylase (G
A) is caused to act to produce a compound in which n is 0 in the general formula [II], (3) a fractionation operation is performed to isolate the compound represented by the general formula [II], and (4) isolation Hydrolyzing the compound represented by the formula [II] to obtain the N-substituted moranolin derivative represented by the general formula [I], and sequentially performing the series of operations from (1) to (4) above. A method for producing an N-substituted moranolin derivative represented by the general formula [I]. 2. The following general formula [I]: (Wherein R represents hydrogen or lower alkyl), in order to obtain the N-substituted moranolin derivative, (1) the culture solution in which the moranolin-producing bacterium is cultivated,
Alternatively, it is subjected to an alkylation reaction, and (2) after adding a glucose donor, a cyclodextrin glycosyl transferase (CGTase) is allowed to act on it to give the following general formula [II] (Wherein R is the same as above, n is an integer from 0 to 24), and an oligoglycosyl-N-substituted moranolin derivative is produced, and (3) glucoamylase (GA) is allowed to act, A glucosyl N-substituted moranolin derivative in which n is 0 in the general formula [II] is produced, (4) a glucosyl N-substituted moranolin derivative is isolated by performing a fractionation operation, and (5) the isolated glucosyl N-substituted moranolin The derivative is hydrolyzed to obtain an N-substituted moranolin derivative represented by the general formula [I], and the series of operations (1) to (5) are sequentially performed, and the general formula [I ] The manufacturing method of the N- substituted moranolin derivative represented by these.