JPH0759591A - Production of deacetylcephalosporanic acid derivative - Google Patents

Abstract

PURPOSE:To obtain a deacetylcephalosporanic acid derivative useful as a production intermediate for a cephalosporanic acid-type antibiotic substance in high conversion by reacting a cephalosporanic acid derivative with a microorganism of the genus Rhodotorula under specific condition. CONSTITUTION:A cephalosporanic acid derivative of the formula (R is H, an R<1>-CO or an R<2>-SO2; R<1> is H, amino, etc.; R<2> is a 1-3C alkyl, etc.) is reacted with a microorganism of the genus Rhodotorula such as Rhodotorula glutinis 38B1 (FERM P-13039). The conversion activity against a deacetylcephalosporanic acid derivative corresponding to the cephalosporanic acid derivative of the formula can be improved by culturing the microorganism of the genus Rhodotorula in the presence of a fatty acid ester compound. The cephalosporanic acid derivative may be reacted with processed materials of the microorganisms in place of the microorganism itself.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel method for producing a deacetylcephalosporanic acid derivative which is a useful intermediate in the production of cephalosporin antibiotics.

[0002]

2. Description of the Related Art As a method for producing deacetylcephalosporanic acid by removing an acetyl group at the 3-position from a cephalosporanic acid derivative, a chemical method is economically disadvantageous as compared with an enzymatic method. As the enzymatic method, a method using acetylhydrolase which is an esterase is used. Deacetyl-7 using acetylhydrolase
As a method for producing aminocephalosporanic acid, JP-A-49-132294, JP-A-61-70999, JP-A-4-53499, JP-B-39-9894, JP-B-42-
7553, JP-B-49-35993, JP-B-54-43
598, JP-B-56-46830, JP-B-61-116
00 and the like. In the same manner, deacetyl cephalosporin C using acetylhydrolase
The method for producing the compound is disclosed in JP-A-55-39735 and JP-A-5-35735.
7-39798, JP-A-59-108790, JP-A-2
-49580, JP-A-1-47158, JP-B-1-54
998 and the like. Further, in the same manner, JP-A-61-67489 is disclosed as a method for producing deacetyl-7-β-acylamide cephalosporanic acid using acetylhydrolase.

[0003]

It takes a long time to convert a cephalosporanic acid derivative into a deacetylcephalosporanic acid derivative using the conventional acetylhydrolase as shown above, and it is necessary to industrialize it. It wasn't economical enough.

[0004]

[Means for Solving the Problems] In order to solve the above problems, the present inventors have proceeded with the search for a microorganism having a strong acetylhydrolase activity. As a result, a microorganism having a strong ability to convert the cephalosporanic acid derivative represented by the chemical formula 1 into the deacetylcephalosporanic acid derivative represented by the chemical formula 2 was found, and further, a culture for increasing this conversion activity to an industrial level. The conditions were discovered and the present invention was completed. That is, the present invention is a method for producing a deacetylcephalosporanic acid derivative represented by the formula 2 by allowing a microorganism belonging to the genus Rhodotorula or a preparation thereof to act on the cephalosporanic acid derivative represented by the formula 1.
R in the chemical formulas 1 and 2 is a hydrogen atom, R ¹ −
Represents a CO group or an R ² —SO ₂ group, R ¹ is a hydrogen atom,
A linear or branched alkyl group having 1 to 6 carbon atoms, substituted by one or more amino groups, carbonyl groups, and carboxyl groups, halogen atom, cyano atom, alkoxy group having 1 to 3 carbon atoms, substituted Or an unsubstituted aryloxy group, imino group, alkyl group having 1 to 4 carbon atoms substituted with an alkylidene group having 1 to 4 carbon atoms, or a straight chain or branched chain having 1 to 4 carbon atoms An alkoxy group, a substituted or unsubstituted aroxy group, R ² represents an alkyl group having 1 to 3 carbon atoms, a halogen-substituted alkyl group having 1 to 3 carbon atoms, or a substituted or unsubstituted aryl group.

More specifically, examples of the alkyl group represented by R ¹ include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, a t-butyl group, a pentyl group and a hexyl group. As the substituted alkyl group,
For example, chloromethyl group, 2,2,2-trichloroethyl group, cyanomethyl group, cyanoethyl group, methoxyethyl group, ethoxyethyl group, methoxypropyl group, phenoxymethyl group, tolyloxymethyl group, p-chlorophenoxy group, p -Nitrophenoxy group, (2-thienyl)
Methyl group, (1- (1H) -tetrazole) methyl group,
(2-amino-4-thiazolyl) methyl group, (2-furyl) methyl group, (2-amino-4-thiazolyl) methoxyiminomethyl group, (2-amino-4-thiazolyl) propylidenemethyl group and the like. . As an alkoxy group,
There are methoxy group, ethoxy group, propoxy group, isopropoxy group, butoxy group, t-butoxy group and the like. Examples of the araloxy group include a benzyloxy group and a p-nitrobenzyloxy group. Examples of the alkyl group of R ² include a methyl group, an ethyl group and a propyl group. Examples of the halogen-substituted alkyl group include a trifluoromethyl group and the like.

The microorganism used in the present invention is a microorganism belonging to the genus Rhodotorula. Specifically, Rhodotorula glutini (Rhodotorula glutini)
nis) 38B1 (Microtechnology Research Institute, No. 13039) can be used. This fungus was isolated from soil in Kyoto Prefecture and has been deposited at the Institute of Microbial Science and Technology with the above number. The mycological properties of this bacterium are as follows. 38B1 strain: morphology in YM medium; liquid medium; (3-5) x (4-6) in 48-hour culture
Present in a single cell with a spherical or oval shape of μm. After 21 days of culture, a non-hairy precipitate was formed, which was cream colored. Agar medium: (3-5) x (4-8) after 48 hours of culture
μm spherical or long oval, present in single cells and multiple pairs of cells. After 21 days of culture, it became orange, the surface was smooth and slightly glossy. Pseudohyphae and true hyphae: On cornmeal agar and potato dextrose agar, no pseudomycelia and true hyphae were found under aerobic and anaerobic conditions. About spores: ejected spores, segmented spores, endospores,
Neither chlamydospores were observed. Regarding sexual spores: No sexual spores are observed on Gorodkowa medium and corn meal agar medium. Fermentability: D-glucose, D-galactose, sucrose, maltose, cellobiose, α, α-trehalose, lactose, melibiose, raffinose, melezitose, inulin, soluble starch, methyl α-D
-No fermentative capacity for glucopyranoside.

Assimilation ability; D-glucose + D-galactose + L-sorbose-sucrose + maltose + cellobiose-α, α-trehalose + lactose-mellibiose-raffinose ± melezitose + inulin + soluble starch-xylose + L-arabinose + D-arabinose + D-ribose + L-rhamnose-ethanol + glycerol + erythritol-ribitol + galactitol-D-mannitol + D-glucitol-methyl α-D-glucopyranoside + salicin + lactic acid + succinic acid + myo- Inositol-D-glucono-1,5-lactone + D-glucosamine-methanol-xylitol + ammonium sulfate + potassium nitrate + ethylamine-cadaba Phosphorus + L-lysine +

Other characteristics: 0.01% cycloheximide growth + 0.1% cycloheximide growth + 50% glucose growth-1% acetic acid growth-oil and fat degradability-bioacidity-37 ° C growth + arbutin hydrolysis-urease activity -Starch Productivity-NaCl Tolerance-The above mycological properties are described by Iizuka and Goto in "Yeast classification and identification method, 2nd edition (1973)" and J. By Rodder, “The Yeasts, A Taxonic
Study, 2nd ed. , (1970) ”, the 38B1 strain was identified as Rhodotorula glutinis.

In the reaction method of the present invention, a hydrolysis reaction is carried out by bringing a microorganism or its preparation into contact with the cephalosporanic acid derivative represented by the chemical formula 1,
A deacetylcephalosporanic acid derivative represented by the formula is obtained. The microorganism or its preparation is specifically a culture obtained by culturing the above-mentioned microorganism, a treated bacterial cell collected from the microorganism (for example, a disrupted bacterial cell or an enzyme separated and extracted from the bacterial cell), and a bacterial cell. Alternatively, a treated product of cells is immobilized by an appropriate method, for example, an ion-exchange resin on which an enzyme is immobilized with glutaraldehyde. The culture of the microorganism used in the present invention can be carried out according to a known method. As the medium to be used, those known as nutrient sources for general yeasts can be used, glucose, fructose, ethanol, sucrose, maltose, acetic acid, carbon sources such as ethyl oleate, nitric acid, ammonium sulfate, ammonium chloride, nitrogen such as ammonia. Source, organic extract such as yeast extract, malt extract, peptone, meat extract, potato, L-
Amino acids such as valine and L-glutamic acid, and inorganic nutrient sources such as phosphoric acid, magnesium, potassium, iron, cobalt and manganese can be appropriately combined and used.

It is necessary to add various fatty acid ester compounds to the medium in order to sufficiently induce the activity of the microorganism used in the present invention for converting the compound represented by formula 1 into the compound represented by formula 2. That is, a fatty acid ester compound such as methyl oleate is added to the medium in an amount of 0.01 to 10
%, Preferably 0.1 to 2.0%. The pH of the medium may be selected in the range of 3 to 10, and the culture temperature is 15 ° C to 40 ° C, preferably 25 ° C to 32 ° C.
℃. The number of days of culture may be 1 to 10 days until the activity becomes maximum. In addition, Japanese Examined Japanese Patent Publication No. 54-43
598, a method for producing deacetyl-7-aminocephalosporanic acid using the same Rhodotorula genus as in the present invention is shown. Among them, cephalosporin C, cephalosin and salts thereof are added to a medium as an inducer of enzyme activity. Is described. Then, when compared with the activity under the culture condition of the present invention, it was found that the addition of the fatty acid ester, which is the culture condition of the present invention, had a higher activity (illustrated in Comparative Examples). From an industrial point of view, it is clear that it is advantageous to use an inexpensive fatty acid ester.

The reaction conditions in the present invention will be described below.
The reaction generally proceeds conveniently in the presence of an aqueous solvent.
The aqueous solvent is a buffer solution containing water and various salts (for example,
A buffer solution containing phosphoric acid and boric acid), and an alcohol solvent such as methanol and a ketone solvent such as acetone,
Two-phase consisting of an ether solvent such as 1,4-dioxane, a mixed solvent with a water-soluble organic solvent such as a polar aprotic solvent such as dimethyl sulfoxide, and a mixed solvent of an aqueous solvent and a non-water-soluble organic solvent It means a system solvent and the like. The reaction temperature is not necessarily constant depending on the type of raw material, the type of reaction solvent, and other conditions, but it is usually about 0 to 80 ° C., preferably 4 to 50 ° C. The reaction concentration is between about 0.001 and 70% by weight, preferably between 0.1 and 40% by weight. Reaction pH is 3-1
It is 1, and preferably selected from 5 to 9. Ammonia, sodium hydroxide or the like may be appropriately added to adjust the pH during the reaction. When using bacterial cells in the reaction, the bacterial cell concentration is usually 0.05% by weight to 20% by weight.
Choose between The reaction time is selected between 0.5 and 100 hours. The cephalosporanic acid derivative represented by the chemical formula 1 consumed by the reaction may be supplemented continuously or intermittently and added so that the concentration in the reaction solution is maintained within the above range.

From the reaction mixture thus obtained,
To recover the target compound, first adjust the pH to 7-8, remove insolubles such as microorganisms by centrifugation or filtration, acidify with diluted sulfuric acid or diluted hydrochloric acid, and add 0-1.
The mixture is allowed to stand at 0 ° C. for 6 to 48 hours, and the produced crystals are collected by filtration. The desired compound can be highly purified by recrystallization, washing with alcohols such as methanol, or purification by column chromatography using an adsorption resin or an ion exchange resin, if necessary.

[0013]

EXAMPLES Next, the present invention will be described in more detail by way of examples. However, these examples do not limit the scope of the present invention. (Example 1) Glucose 2%, peptone 1%, yeast extract 1%, 1 potassium phosphate 0.5%, polypropylene glycol 0.1% and various fatty acid ester 1%, p
One platinum loop of the slant-cultured Rhodotorula glutinis 38B1 strain was transplanted into 50 ml of a sterilizing medium (H 500) (using a 500-ml Erlenmeyer flask) and cultured at 25 ° C. for 72 hours. After culturing, the culture solution itself is used as an enzyme solution,
mM7-aminocephalosporanic acid (R in the chemical formula 1
Is a hydrogen atom) and reacted for 30 minutes in a potassium phosphate buffer at 25 ° C. and pH 7.5 to determine the activity. The results are shown in Table 1. The activity of 1 Unit was 25 ° C, pH 7.
5 shows the ability to decompose 1 μmole of 7-aminocephalosporanic acid in 1 minute.

[Table 1]

Comparative Example Glucose 2%, Peptone 1
%, Yeast extract 1%, potassium phosphate 1% 0.5%, polypropylene glycol 0.1%, and cephalosporin C
Or 50 ml of sterilized medium containing 0.1% of cephalosin and adjusted to pH 6.0 (500 ml Erlenmeyer flask is used)
, Slant-cultured Rhodotorula glutinis 38B
One strain was transplanted with one platinum loop and cultured at 25 ° C. for 72 hours. After culturing, the culture solution was used as an enzyme solution and the activity was measured in the same manner as in Example 1. The results are shown in Table 2.

[Table 2]

Example 2 Glucose 4%, Peptone 2
%, Yeast extract 2%, potassium phosphate 1% 0.5%, polypropylene glycol 0.1%, methyl oleate 1%
2% of Rhodotorula glutinis 38B1 strain previously cultured in the same sterilization medium 12 liter (using a 20 liter fermenter) containing pH 6.0 was transplanted to
Culturing was carried out for 72 hours under conditions of ° C, 250 rpm, and 1 vvm. Growth after culture (OD ₆₁₀ ) is 50.0, activity is 38
Units / ml (culture medium). This culture solution 30
The cells were collected by centrifuging 0 ml and washed once with physiological saline. 300 g of 7- (2-chloroacetyl) cephalosporanic acid (R ¹ in the chemical formula ¹ is a chloromethyl group) was put in 2000 ml of distilled water, and about 15 m of ammonia water was added.
1 to adjust the pH to 6.0, add washed cells,
At 25 ° C., the reaction was completed in 130 minutes while adding aqueous ammonia so that the pH was 6.0. After removing the cells from the reaction-completed solution by centrifugation, 4 liters of ethyl acetate was added, the pH was adjusted to 2.0 with concentrated hydrochloric acid, and the target product was extracted with a solvent. The extraction solvent was washed with 1200 ml of a saturated sodium chloride solution and then diluted with 13 ml of diisopropylamine.
0 ml was added, and the mixture was concentrated to dryness with a rotary evaporator. This is further washed with ether (350 ml x 2
And then dried to give 7- (2-chloroacetyl)-
305 g of diisopropylamine deacetylcephalosporanic acid salt was obtained. The compound was identified by comparing it with the chromatographic data of the standard product and the spectral data of NMR, IR, UV and the like.

(Example 3) 0.1 M mixed with bacterial cells (about 100 mg-dcw) cultured in the same manner as in Example 2
To 100 ml of potassium phosphate buffer (pH 7.8), add 7
-2.72 g of aminocephalosporanic acid (R in the chemical formula 1 is a hydrogen atom) was added and reacted at 30 ° C for 2 hours. However, 1N sodium hydroxide was appropriately added to adjust the pH during the reaction. After the reaction was completed, the reaction solution was centrifuged to remove the cells, and the pH was adjusted to 4 with diluted sulfuric acid.
The mixture was allowed to stand at 24 ° C. for 24 hours, and the produced crystals were collected by filtration. This was washed with water and methanol, dried, and deacetyl-7.
2.09 g of aminocephalosporanic acid was obtained. The compound was identified by the chromatographic data of the standard and NMR, IR,
It was performed by comparing with spectral data such as UV.

(Example 4) In the same manner as in Example 3, in 100 ml of a buffer solution in which cells were suspended, 7- (5-amino-5-carboxy-1-oxopentyl) cephalosporanic acid (in the formula 1) R ¹ is 4-amino-4-carboxybutyl group: cephalosporan C)
Then, the reaction was carried out for 2 hours while adjusting the pH to 7.8 ± 0.2 with ammonia. After completion of the reaction, the reaction solution was centrifuged to remove the cells, the pH was adjusted to 2.0 to 3.0 with dilute sulfuric acid, and the mixture was allowed to stand at 2 ° C. for 24 hours, and the generated crystals were collected by filtration.
This was washed with water and methanol and then dried to give 7- (5
4.21 g of -amino-5-carboxy-1-oxopentyl) -deacetylcephalosporanic acid was obtained. The compound was identified by the chromatographic data of the standard and NMR, IR,
It was performed by comparing with spectral data such as UV.

(Example 5) As in Example 3, 5 g of 7-benzyloxycarbonylcephalosporanic acid (R ¹ in the chemical formula ¹ is a benzyloxy group) was added to 100 ml of a buffer solution in which cells were suspended. The reaction and crystallization were performed in the same manner as in Example 3 to obtain 4.30 g of 7-benzyloxycarbonyl-deacetylcephalosporanic acid. The compound was identified by comparing it with the chromatographic data of the standard product and the spectral data of NMR, IR, UV and the like.

(Example 6) In the same manner as in Example 3, in 100 ml of a buffer solution in which cells were suspended, 7β-[(Z) -2- (2
-Aminothiazol-4-yl) -2-methoxyiminoacetyl] cephalosporanic acid [R in the chemical formula 1 is (2-amino-4-thiazolyl) methoxyiminoacetyl group] 5 g was added, and the same as in Example 3. React and crystallize to give 7β-[(Z) -2- (2-aminothiazole-4
-Yl) -2-methoxyiminoacetyl] deacetylcephalosporanic acid 4.39 g was obtained. The compound identification is
It was performed by comparing with the chromatographic data of the standard product and the spectral data of NMR, IR, UV and the like.

[0020]

INDUSTRIAL APPLICABILITY By utilizing the present invention, a deacetylcephalosporanic acid derivative, which is an intermediate for the production of cephalosporin antibiotics, can be prepared from a corresponding cephalosporanic acid derivative using a microorganism at room temperature and atmospheric pressure. It is economically very advantageous because it can be produced under a reaction condition in a short time at a high reaction rate. Although the present invention has been described in detail, and particularly in its embodiment, with examples, various changes and modifications can be made within the scope of the present invention without departing from the spirit and scope of the present invention. Will be apparent to those of skill in the art.

Claims (1)

[Claims] 1. A method for converting a cephalosporanic acid derivative represented by the following chemical formula 1 into a deacetylcephalosporanic acid derivative represented by the following chemical formula 2 by allowing a microorganism belonging to the genus Rhodotorula or a preparation thereof to act on the cephalosporanic acid derivative. A method for producing a deacetylcephalosporanic acid derivative, which comprises culturing the microorganism in the presence of a fatty acid ester compound to enhance the conversion activity. [Chemical 1] (In the formula, R is a hydrogen atom, R ¹ —CO group or R ² —SO
Represents _two groups, R ¹ is a hydrogen atom, an amino group, a carbonyl group, a linear or branched alkyl group having 1 to 6 carbon atoms substituted by one or more with a carboxyl group, a halogen atom, a cyano atom An alkoxy group having 1 to 3 carbon atoms, a substituted or unsubstituted aryloxy group, an imino group,
An alkyl group having 1 to 3 carbon atoms substituted by an alkylidene group having 1 to 4 carbon atoms or a heterocyclic group, a linear or branched alkoxy group having 1 to 4 carbon atoms, a substituted or unsubstituted aroxy group, R ² represents an alkyl group having 1 to 3 carbon atoms, a halogen-substituted alkyl group having 1 to 3 carbon atoms, or a substituted or unsubstituted aryl group. ) [Chemical 2] (In the formula, R is the same as that in the above chemical formula 1.)