AT392288B - METHOD FOR PRODUCING MORANOLINE DERIVATIVES - Google Patents

Description

AT 392 288 B

The invention relates to a new process for the preparation of oligoglucosylmoranolin derivatives of the general formula

wherein R is hydrogen or a lower alkyl group, which may have one or more hydroxyl groups, and n is an integer from zero to 15.

A mixture of the compounds of the formula (I) in which n = zero to 15 can be obtained in high yield by treatment, for example, with glucoamylase (al, 4-glucan glucohydrolase EC 3.2.1.3) (cf. JP-OS 57/058890) into a compound of the general formula

wherein R has the above meaning, are converted or the compound (III) can be obtained in high yield from a mixture of (I) using fractional crystallization with a polar solvent (cf. JP-OS 59/237326). Otherwise (III) is a compound (I) in which n is zero.

The compound (III) has an excellent inhibitory effect against the rise in the blood sugar level when sugar is loaded and is a very useful medicine for the treatment of diabetes mellitus (JP-OS 56/081595). Accordingly, the present invention provides a process for the preparation of starting materials for the production of useful drugs.

For the preparation of the compounds of formula (I) it is known to cyclodextrin glycosyltransferase (EC 2.4.1.19) on an aqueous, cyclodextrin or soluble starch and a moranolin derivative of the general formula -2-

AT 392 288 B

CH20H R

OH in which R has the above meaning to allow the solution containing it to act, a mixture of oligoglucosylmoranolin derivatives of the formula (I) being obtained.

However, this cyclodextrin glycosyltransfeiase (hereinafter referred to as " CGTase ") is very expensive, so there is a need for a method by which the enzyme can be recovered and recovered by recycling. In addition, since CGTase is an amphoteric substance, many pretreatments are required to separate the enzyme in order to obtain pure (I) (which is a basic substance) from the reaction mixture by the enzymatic reaction. In addition, when it comes to large-scale production, a process that can be used repeatedly over a long period of time is essential, but the use of enzymes that are unstable for this purpose is extremely difficult from a technical point of view.

Based on the prior art, thorough investigations were carried out according to the invention, and it was found that the above problem can be solved by setting the CGTase.

The process according to the invention is characterized in that cyclodextrin glycosyl transfer asc is used in enclosed or bound form.

The expression '' specified " CGTase refers to both the enclosed and the bound form (see " Immobilized Enzyme " published by Ichiro Chibata, published by Kodansha).

If the enclosed form is used in carrying out the method according to the invention, for example CGTase can be enclosed on a carrier. A polymeric gel, for example synthetic high-molecular polyacrylamide gel and polyvinyl alcohol gel, is preferably used as the carrier for enclosed CGTase. The CGTase can also be included in polysaccharide that swells to become a gel (such as K-carrageenan). If necessary, the carrier can be physically reinforced by adding glutaraldehyde or hexamethylene diamine. If the method according to the invention is carried out after such a physical amplification, the contact with the reaction solution proceeds in a blue manner without hindering the enzymatic reaction, so that this procedure is preferred. For the carrier material, spherical bodies, for example with a diameter of 1 mm to 1 cm, are produced and filled into a column through which the reaction solution is circulated, whereby the cyclodextrin or soluble starch and (II) -containing solution are obtained with (I) the CGTase is brought into contact.

As for the included form, the crude enzyme solution can be used as such without purification of the CGTase, and therefore such a method can be even more advantageous.

When using bound forms in the process according to the invention, for example, covalent bonding, ion binding or physical adsorption can be used. When the covalent bond is used in the process according to the invention, a covalent bond is produced with an insoluble carrier which has a group which, for example, can form a covalent bond with the amino or carboxyl group, whereupon the bond to the carrier takes place. For example, after purification, CGTase is reacted with, for example, polysaccharide, such as CNBr-cephalose, the bound product obtained is suspended in aqueous solution and brought into contact with an aqueous solution of (II) and a solution containing cyclodextrin or soluble starch, where (I) is obtained.

Even in this case, according to the present invention, any carrier that can bind CGTase will be referred to by " fixing " includes.

In the repeated and continuous use of enzymes, a person skilled in the art could have come up with the idea of determining these enzymes. According to the invention, however, the idea was first taken up of defining CGTase and applying it to the compounds according to the invention. In addition, it was found for the first time that the specified CGTase has a stable enzymatic activity for a long period of time, such as more than 120 days, as will be explained later.

The invention is described in detail below with the aid of examples. -3-

AT 392 288 B

Example

Maturation of Bacillus macerans 100 ml of a culture medium (pH 7) containing 1% corn steep, 1% soluble starch, 0.5% ammonium sulfate and 0.5% calcium carbonate were placed in a 500 ml Erlenmeyer flask and heated for 15 minutes Sterilized at 120 ° C. Three platinum eyelets B. macerans ΠΌ-3490 strain based on 1% peptone, 0.5% yeast, 0.3% glucose, 1.5% glycerol, 0.3% sodium chloride, 2.5% liver powder (Oxoid, Trademark) and the slant medium containing 1.5% agar had been well grown, were inoculated and subjected to the shaking culture at 37 ° C. for 3 days. The cultured liquid (600 ml) was inoculated into a shaker fermenter (contents 301) containing 18 l of the medium with the same composition and cultured for 3 days at 37 ° C with sufficient shaking and aeration, with a 130 to 150 after centrifugation Unit-containing enzyme solution was obtained as a supernatant liquid.

Unit of cyclodextringlvcosvltransferase activity

Soluble starch (quality for biochemical research; manufactured by Nakarai Kagaku KK) was dissolved in 0.05 M acetate buffer (pH 5.5) to produce a substrate solution in a proportion of 0.7%. 50 μl of enzyme solution were added to 950 μl of this substrate solution; it was reacted for 10 min at 40 ° C. and the reaction was stopped by adding 0.5 ml of 0.5N acetic acid. To 100 μΐ of the reaction solution, 0.8 ml of iodine solution (0.01 M iodine dissolved in 0.25 M aqueous potassium iodide solution) and 3 ml of water were added and the mixture was stirred, and the absorbance at 660 nm was measured, whereby an AT value was obtained. In the same way, 50 μl of water and 0.5 ml of 0.5N acetic acid were added to 950 μl of substrate solution, iodine solution was added to 100 μl of this solution and the absorbance at 660 nm was measured, an AR value being obtained.

AR-AT 1 unit = -x 100 x 2

AR 1 unit means the activity which causes a 1% / min decrease in the absorbance of 1 ml of enzyme solution at 40 ° C.

Preparation of the raw enzyme solution

The cultivated solution of B. macerans IFO 3490 was centrifuged, the supernatant solution was freeze-dried and then dissolved in a small amount of water to obtain a concentrated enzyme solution. This was dialyzed well against water at 5 ° C. to remove low-molecular substances, and the inner solution was used as the crude enzyme solution. If necessary, it is freeze-dried and the powder obtained is used

Production of the Enzvm

The crude enzyme solution was added to a saturation of 0.25 to ammonium sulfate, filtered with Celite and the filtrate adsorbed on corn starch. It was eluted with 0.033 M sodium hydrogen phosphate and the eluate was salted out with ammonium sulfate (0 to 0.6 saturation). The mixture was then dialyzed, the dialyzed solution was subjected to column chromatography with DEAE cellulose, washed with water and eluted. The eluate was concentrated and subjected to column chromatography with Sephadex G-75, whereby purified enzyme was obtained (see S. Kitahata et al., Arg. Biol. Chem., 38, 387-393, 1974).

Example 1: (1) K-Carrageenan was dissolved in a 0.9% aqueous sodium chloride solution at 65 ° C. to produce a 5% solution (mass / volume). This solution (100 ml) was heated to 65 ° C. 33 g of lyophilized crude cyclodextrin glucosyl transferase (4150 units / g) were dissolved in 400 ml of water and the solution was heated to 30 ° C. Both solutions were combined, added dropwise to 4000 ml of a 0.3 M aqueous solution of CaC ^ with stirring and the spherical fixed cyclodextrin glucosyltransfeiase obtained was left overnight at 5 ° C. This was passed through a coarse sieve to remove liquid, and particles with circular and elliptical sides of a few mm in diameter were obtained. They were added to 4000 ml of a 2.5% aqueous glutaraldehyde solution and the mixture was gently stirred for 40 minutes. Then it was added to 4000 ml of a 0.5% aqueous hexamethylenediamine solution, followed by the same treatment for 30 minutes. It was passed through a coarse sieve to remove liquid and washed with water to give a granular solid. -4-

AT 392 288 B (2) 50 g of the above granular solid (wet mass) were placed in a column with an inner diameter of 2.5 cm and a height of 30 cm. A solution containing 3 g of moranolin and 12 g of α-cyclodextrin in 200 ml of water and adjusted to pH 5.7 was circulated through the column at 40 ° C. for 72 h at a rate of 20 ml / h. The reaction solution was collected, treated with a strongly acidic ion exchange resin (Dowex 50W x 2; H +, 50 ml), washed thoroughly with water, eluted with 1N ammonia water and the eluate concentrated to dryness in vacuo to give 8.7 g of powder were. This was analyzed by high performance liquid chromatography, and it was found that it was a mixture of 11% moranolin and 89% oligoglucosylmoranolin.

The conditions in high performance liquid chromatography were as follows:

Sumipax R741 (Nucleosil 5 NH2.5 mm, 4 mm inner diameter x 25 mm height); Developer: Acetonitrile

Water (65:35); Flow rate: 1 ml / min; RI detection (ERC-7510, manufactured by Eimer Optical Co.); Data processing: Model 655-60, manufactured by Hitachi Ltd.

Example 2: (1) 105 mg of cyclodextrin glucosyltransferase purified by Sephadex G-75 column chromatography was dissolved in 30 ml of 0.1 M aqueous sodium bicarbonate solution containing 0.5 M sodium chloride and activated carrier material (corresponding to 3 g of the lyophilized product) of CNBr-cephalose 4B included. The CNBr-cephalose 4B, which was used as the activated carrier material, was prepared by placing 3 g of lyophilized CNBr-cephalose 4B (commercially available) on a glass filter and then washing repeatedly and swelling with 600 ml of ImM hydrochloric acid. The product was reacted by shaking at 25 ° C. for 3 h and then the powder obtained was washed with water and treated with 30 ml of 0.5 M ethanolamine at room temperature for 2 h. The product was washed well with 0.1 M aqueous sodium monohydrogen phosphate solution containing 0.5 M sodium chloride and then thoroughly washed with water to obtain a powdery solid. (2) 18 g of soluble starch was dissolved in 200 ml of hot water, 3 g of moranolin was dissolved therein, and the mixture was adjusted to pH 5.7. Then 1 g of the powder obtained previously was added and the mixture was stirred at 40 ° C. for 3 days. The reaction solution was filtered off, passed through 50 ml of a strongly acidic ion exchange resin (Dowex 50W x 1; H +), the column was washed thoroughly with water, eluted with 1N ammonia water and the eluate was evaporated to dryness in vacuo, 6.9 g of powder were obtained.

This was analyzed by high performance liquid chromatography in the manner described in Example 1, and was found to consist of a mixture of 18% unreacted moranolin and 82% oligoglucosylmoranolin.

Example 3: 50 g (wet mass) of the particles obtained in the manner described in Example 1 (1) were placed in a column (2.5 cm inside diameter x 30 cm high) and a solution (pH 5.7) of 1.4 g of N-methylmoranolin and 22.4 g of α-cyclodextrin in 200 ml of water were circulated through the column at 40 ° C. at a flow rate of 20 ml / h for 100 h. The reaction solution was collected by 50 ml of a strongly acidic one

Ion exchange resin (Dowex 50W x 2; H +), the column was washed thoroughly with water and eluted with 1N ammonia water and the eluate was evaporated to dryness in vacuo to give 3.5 g of powder.

This was analyzed by high performance liquid chromatography in the manner set forth in Example 1, and was found to consist of a mixture of 15% unreacted N-methylmoranolin and 85% oligoglucosyl-N-methylmoranolin.

Example 4: 18 g of soluble starch were dissolved in 200 ml of hot water, then 3 g of N-methylmoranolin were dissolved in it and the solution was adjusted to pH 5.7. 1 g of the powder obtained in the manner given in Example 2 (2) was added and the mixture was shaken at 40 ° C. for 3 days. The reaction solution was filtered, treated with a strongly acidic ion exchange resin (Dowex 50W x 2, H +, 50 ml), the resin was washed thoroughly with water, eluted with 1N ammonia water and the eluate was evaporated to dryness in vacuo, 6.3 g Powder were obtained.

This was analyzed in the manner described in Example 1 by high performance liquid chromatography, and was found to consist of a mixture of 21% N-methylmoranolin and 79% oligoglucosyl-N-methylmoranolin. -5-

Claims (1)

AT 392 288 B Example 5: A solution (pH 5.7) obtained by dissolving 3 g of moranolin and 12 g of α-cyclodextrin in 200 ml of water was allowed to pass through at a flow rate of 20 ml / h at 40 ° C. for 3 days Rotate column (2.5 cm inner diameter x 30 cm height), in which there were 50 g (wet mass) of the particles according to Example 1 (1), as used in Example 1 (2). The reaction solution was collected, passed through a strongly acidic ion exchange resin (Dowex 50 W x 2; ff * "), the resin washed thoroughly with water, eluted with 1N ammonia water and the eluate evaporated to dryness in vacuo, 8.7 g of powder were obtained. This was analyzed by high performance liquid chromatography and was found to consist of a mixture of 11% moranolin and 89% oligoglucosylmoranolin. This process was repeated 40 times in total over a total of 120 days. The powder obtained after the 40th operation was analyzed by high performance liquid chromatography to find that it consisted of a mixture of 11% moranolin and 89% oligoglucosylmoranolin. Example 6: 50 g (wet mass) of the particles obtained in the manner described in Example 1 (1) were filled into a column (2.5 cm inner diameter x 30 cm high) and then a solution was left by dissolving 2 g N- (2-hydroxyethyl) moranolin and 32 g of α-cyclodextrin in 300 ml of water and subsequent adjustment to pH 5.7 had been obtained, circulating for 100 h at 40 ° C. at a flow rate of 20 ml / h. The reaction solution was collected, passed through a strongly acidic ion exchange resin (Dowex 50W x 2; H +, 50 ml), the resin washed thoroughly with water, eluted with 1N ammonia water and the eluate concentrated to dryness in vacuo, 4.8 g Powder were obtained. This was analyzed in the manner described in Example 1 by high performance liquid chromatography, and it was found that it consisted of a mixture of 17% unreacted N- (2-hydroxyethyl) moranolin and 83% 01igoglucosyl-N- (2-hydroxyethyl) moranolin . Example 7: 50 g (wet mass) of the particles obtained in the manner described in Example 1 (1) were filled into a column with an inner diameter of 2.5 cm and a height of 30 cm, and one by dissolving 2 g of N- ( 2,3-Dihydroxypropyl) moranolin and 32 g of α-cyclodextrin in 300 ml of water were allowed to circulate through the column at 40 ° C at a flow rate of 20 ml / h after adjusting the pH to 5.7 for 100 h . The reaction solution was collected, passed through a strongly acidic ion exchange resin (Dowex 50W x 2; H +, 50 ml), the resin washed thoroughly with water, eluted with 1N ammonia water and the eluate concentrated to dryness in vacuo, 4.8 g Powder were obtained. This was analyzed in the manner described in Example 1 by high performance liquid chromatography, whereby it was found that it consisted of a mixture of 20% unreacted N- (2,3-dihydroxypropyl) moranolin and 80% 01igoglucosyl-N- (2,3- dihydroxypropyl) moranolin. PATENT CLAIM Process for the preparation of oligoglucosylmoranolin derivatives of the general formula -6- 5 AT 392 288 B wherein R is hydrogen or a lower alkyl group, which may have one or more hydroxyl groups, and n is an integer from zero to 15, where moranolin of the general formula 10 15 , (Π) 20 wherein R has the meaning given above, and an aqueous solution containing cyclodextrin or soluble starch can act on fixed cyclodextrin glycosyltransferase, characterized in that cyclodextrin glycosyltransferase is used in enclosed or bound form. -7-