JPS5920360B2 - Method for producing 7-aminocephalosporanic acid and its derivatives - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel method for producing 7-aminocephalosporanic acid and its derivatives.

More specifically, a cephalosporin derivative represented by the general formula (wherein R represents a hydrogen atom, a hydroxyl group, an acetoxyl group, or a nucleophilic residue) and its salt are added to a cephalosporin derivative belonging to the genus Bacillus in an aqueous medium. The general formula (in the formula , R represents the same group as above) and its derivatives or salts thereof.

7-Aminocephalosporanic acid has traditionally been chemically deacylated from cephalosporin C (UK% 1,041
, 985 ), is an important intermediate that can be further reacylated with other acyl groups to provide various antibacterial substances.

Furthermore, by variously modifying the 3-position of 7-aminocephalosporanic acid, it is possible to provide an intermediate for an excellent antibacterial substance.

7-Amino-3-methylcephalosporanic acid, in which R in formula (1) is a hydrogen atom, is an important intermediate for cephalexin, and it is provided by chemical ring expansion of penidilines. However, it can also be produced by chemically deacylating desacetoxycephalosporin C produced by a mutant strain of cephalosporin C-producing bacteria.

However, attempts to deacylate cephalosporin C or desacetoxycephalosporin C by enzymatic means have been difficult because the acyl group is a D-α-aminoadipyl group, and no success has been seen.

The present inventors previously discovered that the GK-340 strain (Japanese Patent Application No. 5O-147467) belonging to the genus Gliocladium, which was isolated from the soil collected on Yoron Island, was also isolated from IFO-6617.
(Unexamined Japanese Patent Application No. 50-113608.

found that the D-α-aminoadipyl group was converted to a 5'-ketoadipyl group through oxidative membrane amination, and further converted to a glitalyl group in the absence of catalase, and was used for enzymatic deacylation. Established a manufacturing method for an important intermediate.

Briefly, by contacting cephalosporin C with an oxidative membrane amine enzyme produced by Gliocratylum, 3-acetoxymethyl-7-(5-carboxy-5-
oxopentanamide) cef-3-emu-4-carboxylic acid, and in the absence of catalase, 3-acetoxymethyl-7-(4-carboxybutanamide) cef-
They found that 3-emu-4-carboxylic acid can be produced.

The present inventors discovered that among the intermediates based on these previously obtained findings, the final product produced by D-amino acid oxidase (
The corresponding glutarylamide cephalosporanic acid, namely 3-acetoxymethyl-7-(4-carboxybutanamide) cef-3-emu-4-carboxylic acid, is enzymatically (deacylated) to form 7-amine cephalosporanic acid or As a result of conducting research for the purpose of producing derivatives, two strains of the genus Bacillus (CA-78) were isolated from soil in Tojinbo, Fukui Prefecture, and one strain (CA-78) of the genus Arthrobacterium isolated from soil in Toyonaka City, Osaka Prefecture. One strain (CA-218) was
Deacylating the compound represented by general formula (II),
It was discovered that the present invention has an acylase action to produce 7-aminocephalosporanic acid and its derivatives, and this invention was made.

The mycological properties of these soil isolates are as follows.

CA-78 strain with the above mycological properties and CA-
The taxonomic position of the 218 strains is determined by The Genera.
The Gene-ra of Bacteria
Bacteria) 2nd edition and the Bersey's Manual of Determinative Bacteriology 8th edition, and as a result of comparison with type cultures recognized as being closely related to this strain, various morphological and Physiological characteristics showed that strain CA-78 is closely related to Bacillus firmmus species.

However, this strain CA-78 does not produce catalase, produces hydrogen sulfide, is resistant to 8% salt, and has the ability to cleave the acid amide bond of the compound represented by general formula (n). In this respect, it is different from Bacillus Farmus IFO-3330.

Based on the above results, this bacterial species was identified as a new bacterial species belonging to Bacillus firmus, and was given the name Bacillus firmus CA-78.

In addition, the CA-218 strain is a strain of Arthrobacter globiformis.
is) was found to be closely related to the species.

However, this strain CA-218 has the ability to cleave the acid amide bond of the compound represented by the general formula (II), and is therefore similar to Arthrobacter globiformis IFO-21.
There is a difference from 062.

Based on the above results, this bacterial species was identified as a new bacterial species belonging to Arthrobafter globiformis, and was given the name Arthrobafter globiformis CA-218.

This strain has been submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology, with the microbial accession number ``Feikoken Bacteria Collection No. 3466 J (CA-78)''.
, [has been deposited as Microtechnical Research Institute No. 3467 J (CA-218).

The enzyme produced by this strain that decomposes the acid amide bond of the compound represented by general formula (II) is present in the cells of both strains.

Therefore, in order to use enzymes, it is necessary to collect bacteria by centrifuging the booth after culturing, to separate the bacteria by adding a polymer flocculant and using a filter aid (dicalide, etc.), or to convert it into an immobilized enzyme form. .

In addition, through ultrasonic treatment, chemical denaturation treatment of cell membranes, etc.
The enzyme can also be extracted and solubilized and then converted into known forms for use.

To culture these enzyme-producing strains, aerobic conditions are desirable and liquid aeration culture is commonly used.

The medium composition is the usual bacterial medium components, such as peptone, meat extract, yeast extract, peanut glaze, cottonseed meal, etc. as a nitrogen source, glucose, glycerin, sodium acetate, and molasses as a carbon source, and salt as an inorganic salt. A nutrient medium appropriately containing ferric chloride, phosphate, biotin, calcium pantothenate, nicotinic acid amide, etc. as a growth promoting substance is used.

In addition, surfactants (Nonipol 100, Adekanol L
G-126, etc.), glutaric acid can be added to the medium to increase enzyme activity.

Desirable culture conditions are pH 7.5-8.5 and temperature 2.
5 to 35°C, and the time is usually 2 to 5 days, but the culture should be terminated when the acylase activity reaches its maximum.Measurement of acylase activity: For convenience, acylase activity is defined as follows.

That is, a fixed amount of bacterial cells at the same concentration as in the culture process deacylates 1 μmole of substrate per hour and produces the equivalent 7-
One unit is the ability to produce aminocephalosporanic acid or its derivatives.

In fact, a certain amount of the culture was centrifuged to collect the bacteria, and 3-acetoxymethyl-7-(4-carboxybutanamide) cef-3-emu-4-carboxylic acid was added at 5 μ/- as a substrate.
The generated 7-
Marrel method of aminocephalosporanic acid
li).

Specifically, after incubation, centrifuge and remove the supernatant 2.
Take rIll, add 0.8N caustic soda solution of O,l rnl formic acid and 1-binary citric acid and 0.5 rnl.
1 of 5% ninhydrin in methyl cellosolve solution was added to develop a color, and then the absorbance at 410 nm was measured.

The amount of 7-aminocephalosporanic acid is calculated from a separately determined calibration curve.

It is preferable to stop the culture by measuring the acylase activity using the method described above during the culture and stopping the culture at the point when the acylase activity is the highest.

The culture thus obtained is collected as is or by conventional means.

This I bacterial cell can deacylate the substrate by contact with the substrate without any treatment, and can withstand long-term storage by freezing the bacterial mass at -20°C.

Furthermore, the enzyme can be used repeatedly by immobilizing the bacterial cells in an acrylamide gel, or the enzyme can be extracted from inside the cells by conventional means and used.

A desired reaction is carried out using the thus prepared microbial cells, immobilized microbial cells, extracted and immobilized enzymes, etc. (This is usually carried out in an aqueous medium.

The aqueous medium here refers to a substrate for the compound represented by general formula (n) dissolved in an appropriate buffer solution, or a culture broth for cephalosporins treated with D-amino acid oxidase to form a non-sterile substance. The reaction solution that produced the removed compound represented by general formula (n) is shown.

The pH of the acylase reaction solution is 6.5 to 8.5, preferably 7.5 to 8.0.

The reaction temperature can be from 20 to 40°C, preferably from 35 to 37°C.

The amount of enzyme to be used depends on the acylase activity, but is generally the same concentration as the culture broth or 2 to 3 times the concentration of the culture broth, regardless of the type of use.

The amount of substrate is also determined by acylase activity, but usually o,
i~3%, reaction time is 2~IO hours.

The compound represented by the general formula (n) is produced by reacting cephalosporin C and a modified compound at the 3-position thereof (with D-amino acid oxidase).

The present inventors previously discovered that this D-amino acid oxidase exists in a strain belonging to the genus Gliocladium and completed the invention (Unexamined Patent Application No. 5O-147467
).

A compound in which R1 is C0C0OH of the compound represented by the general formula (C) can be used with the enzyme produced by strain GK-340 or IFO-6617 belonging to the genus Gliocladium in an aqueous medium containing cephalosporin C.
H7.5-8.0, produced by contacting under aerobic conditions.

In addition, a compound in which R Alternatively, the catalase of a strain belonging to the genus Gliocladium is brought into aerobic contact with bacteria in which the catalase has been previously inactivated.

The aqueous medium containing cephalosporin C mentioned here specifically refers to cephalosporin C dissolved in an appropriate buffer solution, or cephalosporin C culture broth (
Reaction inhibitors have been removed).

The generated compound of general formula (n) is not isolated from the oxidase reaction system, but is brought into contact with an enzyme produced by a strain belonging to the genus Bacillus or Arthrobacterium to form 7-aminocephalosporanic acid or 7-aminocephalosporan. A 3-position derivative of the acid can be obtained.

In order to isolate 7-aminocephalosporanic acid and its derivatives from the above-mentioned acylase reaction system, known methods such as isoelectric focusing precipitation, column chromatography, ion exchange resin method, etc. are used.

More specifically, after removing bacterial cells or insoluble substances from the reaction solution by centrifugation or f-filtration, the supernatant is concentrated if necessary, and then the pH of the concentrated solution is adjusted to the specific pH of 7-aminocephalosporanic acid and its derivatives. If the isoelectric point is set to , the target 7-aminocephalosporanic acid and its derivatives will precipitate as crystals.

Alternatively, correct the pH of the centrifugation supernatant from 6.0 to 6.5 and use an anion exchange resin (e.g. Amberlite IRA-4).
01, Diaion PA-306, Diamondion 5A-
20, etc.), elute with an appropriate eluent (e.g. ammonium chloride, ammonium acetate, etc.), concentrate the eluate if necessary, and bring it to a specific isoelectric point to precipitate it as a crystal. I can do it.

EXAMPLES Next, the present invention will be specifically explained with reference to Examples, but the present invention is not limited thereto.

Example 1 Glucose 1%, sodium acetate 2%, cottonseed meal 1.6%
, salt 0.5%, K2HP0,0.3%, KCl010
Dispense 60ml of a medium with a composition of 5% FeCl3"6H200.05% into five 500rnl flasks, sterilize it at 115°C for 30 minutes, adjust the pH to 7.4, and inoculate it with Arthrobacter strain CA-218. , and shaking culture was performed at 28°C for 5 days.

After culturing, apply 300 mA of the culture solution to 5.00 Or, p
, rn and centrifuged to obtain a bacterial suspension of 35 m7! ! I got it.

The enzyme activity of this suspension was 0.307 units.

235 μl of 3-acetoxymethyl-7-(4-carboxybutanamide)cef-3-em-4-carboxylic acid was added to 0.
The mixture was dissolved in 35-ml of 2M phosphate buffer (pH 7,4), added with 35 rnl of the above enzyme solution, and incubated at 37°C for 10 hours.

After the reaction, the reaction solution was centrifuged at 7.00 Or, p, m to remove insoluble matter, and the amount of 7-aminocephalosporanic acid in the supernatant was measured using the Marelli method*.
62%).

This supernatant liquid was mixed with strong basic anion exchange resin Amberly 1.
-IRA-401 acetic acid type 30rnl column Oko5
V-3 to adsorb 7-aminocephalosporanic acid, then washed with 200 ml of distilled water, and washed with 0.2 Mu ammonium fat solution.

40ml of the eluate was collected and concentrated to 10rr11.

The concentrated solution had a pH of 3.7, and the resulting precipitate was collected and dried under reduced pressure to obtain 87.3 ml of 7-aminocephalosporanic acid (yield: 52.7%).

This material was subjected to thin layer chromatography using TLC plate silica gel 60F254 (manufactured by Merck & Co., Ltd.), and n
- After developing with butanol/acetic acid/water (3:l:l), ninhydrin and '[JV absorption (from this, spots were confirmed).

The Rf value was 0.34. This Rf value is 7
-Concordant with aminocephalosporanic acid.

In addition, the production of the target product was confirmed by bioautography using paper chromatography as follows.

That is, the centrifuged supernatant of the reaction solution was spotted on Toyo P Paper 2, developed with a solvent system: n-butanol/acetic acid/water (3:1:1), and activated by spraying an acetone solution of phenylacetyl chloride. After conversion, Bacillus subtilis ATCC663
Microbial assay was performed in step 3.

At this time, the Rf value of the substrate 3-acetoxymethyl-7-(4-carboxybutanamide) cef-3-em-4-carboxylic acid was 0750. The Rf value of 7-aminocephalosporanic acid was 0.525. Ta.

Example 2 150 ml of 3-hydroxymethyl-7-(4-carboxybutanamide) cef-3-emu-4-carboxylic acid was added to 0.
The mixture was dissolved in 2M phosphate buffer (pH 7.4) and combined with 25yd of a cell suspension of CA-218 strain obtained by the same culture method as in Example 1, and incubated at 37°C for 6 hours.

After the reaction, the reaction solution was centrifuged at 7,0 OOr, p, m to remove small particles, and the supernatant was measured by the Marelli method* to give a 3- Human dimethyl-7-amitsef-3-M-4-carboxylic acid was produced.

When this product was subjected to TLC in the same manner as in Example 1, the Rf value was 0.25, which coincided with the Rf value of the standard product.

Example 3 150 μl of 3-methyl-7-(4-carboxybutanamide) cef-3-em-4-carboxylic acid was dissolved in 0.2 M phosphate buffer (pH 7,4) 25 μm, and Example 1 25 ml of cell suspension of CA-218 strain obtained by the same culture method as
．． Incubation was performed at 37°C for 6 hours.

After the reaction, the reaction solution was centrifuged at 7.00 Or, p, m to remove small particles, and the supernatant was measured using the Marelli method to obtain 44 mg (yield 45%) of 3-methyl-7. -Aminocef-3-em-4-carboxylic acid was produced.

When this product was subjected to TLC in the same manner as in Example 1, the Rf value was 0.33, which coincided with the Rf value of the standard product.

Example 4 7-(4-carboxybutanamide)-3-(1,2,
3-triazol-4-ylthiomethyl)cef-3-emu-4-carboxylic acid 150WII! was dissolved in 25ydG of 0.2M phosphate buffer (pH 7.4), combined with 25d of bacterial cell suspension of CA-218 strain obtained by the same culture method as in Example 1, and incubated at 37°C for 6 hours. .

After the reaction, the reaction solution was centrifuged at 7.00 Or, p, m to remove insoluble matter, and the supernatant was measured by Marelli method 1 to give a yield of 42.2 mm (yield 38.4%). 7-Amino-
3-(L2,3-triazol-4-ylthiomethyl)
Cef-3-emu-4-carboxylic acid was produced.

When this product was subjected to TLC in the same manner as in Example 1, the Rf value was 0.37, which coincided with the Rf value of the standard product.

Example 5 150 μl of 7-(4-carboxybutanamide)-3-(l-carboxymethyltetrazol-5-yl-thiomethyl)cef-3-emu-4-carboxylic acid was added to 0.2M phosphate buffer (pH 7,4 ) 25-, and combined with 25d of bacterial cell suspension of CA-218 strain obtained by the rotary culture method as in Example 1, and incubated at 37°C for 6 hours.

After the reaction, the reaction solution was centrifuged at 7.00 Or, p, m to remove insoluble materials, and the supernatant was measured using the Marelli method, yielding 55.5 ■ (yield 48.3%). 7-amino-3-(1-carboxymethyltetrazol-5-yl-thiomethyl)cef-3-em-4-carphonic acid was produced.

When this product was subjected to TLC in the same manner as in Example 1, the R,f value was 0.17, which matched the Rf value of the standard product.

Example 6 Activated cultured cells of the GK-340 strain belonging to the genus Gliocladium containing D-amino acid oxidase were added to cephalosporin C culture broth IA (containing 875v as cephalosporin C). Add compressed product 837, sodium azide 1.57 and 35% hydrogen peroxide solution 4.2 ml, and heat at 33°C while maintaining pli at 8.0 to 8.3.
The mixture was allowed to react for 2 hours under ventilation.

After the reaction, the pH of the reaction solution was adjusted to 6.0, filtered, and 3-
Floss containing acetoxymethyl-7-(4-carboxybutanamide) cef-3-emu-4-carboxylic acid 1
．． Obtained 15 tons.

This reaction broth was prepared using the U measurement method described below (3-acetoxymethyl-7-(4-carboxybutanamide)).
Cef-3-M-4-carboxylic acid 6.17f (yield 76
It was confirmed that it contains

That is, 3-acetoxymethyl-7-(4
The content of cef-3-em-4-carboxylic acid (carboxybutanamide) is obtained by solvent extraction of a certain amount of the reaction broth in an acidic state, and then inversion with water in an alkaline state;
It was determined by measuring the amount of V absorption.

(More specifically, take the reaction broth 1-, lower the pH to 2.0, and then shake and extract with ethyl acetate 8- for 5 minutes.

Take 5rnl of the ethyl acetate layer and 5rnl of 4N baking soda solution.
1 and shake again for 5 minutes to reverse the baking soda layer.

Take 3rnl of the baking soda bath solution and add 1rnl of 2N hydrochloric acid,
The absorbance at 260 nm was measured, and the amount of 3-acetoxymethyl-7-(4-carboxybutanamide) cef-3-emu-4-carboxylic acid in the reaction broth was calculated from the standard curve.

The pH of the reaction solution for this oxidation reaction was again adjusted to 7.5, and the C
Add 600ml of A-218 cell suspension and incubate for 10 minutes at 37°C.
Allowed time to react.

After the reaction, the reaction solution was centrifuged at 7.00 Or, p, m to obtain 1.62 t of supernatant.

When this supernatant was measured by the Marelli method*, it was found that 2.52f of 7-aminocephalosporanic acid (yield 58%) was produced.

This supernatant liquid was mixed with a strong basic anion exchange resin Amberly I.
The solution was passed through an acetic acid type 500 column of IRA-401 and washed with 2 tons of water, followed by a 0.5M acetic acid solution of ammonium chloride.

The eluate 450- was concentrated to 50 yd, and its pH was adjusted to 3.7.
7-aminocephalosporanic acid was precipitated.

The precipitated crystals were collected and dried under reduced pressure to obtain 2.09f of 7-aminocephalosporanic acid (yield: 48.1%).

This product was confirmed in accordance with Example 1, and the same crystals were obtained.

Example 7 Culture broth of CA-218 strain cultured by the same method as in Example 1: 350 TIl at 5.00 Or, p, m.

The cells were collected by centrifugation and diluted with 0.2M phosphate buffer (pH
7,4) Resuspend in 25 ml.

This bacterial cell suspension (this acrylamide 3751 and N, N'
- After adding and dissolving 0.25f of methylenebisacrylamide, 5% β-dimethylaminopropionic I-I in water cooling
Add 25 rnl of J.L. liquid and 25 ml of 2.5% potassium persulfate liquid, leave for 20 minutes to gel,
．． A 1 ft acrylamide gel containing bacterial cells was obtained.

3-acetoxymethyl-7-(4-carboxybutanamide) cef-3-em-4-carboxylic acid 500R? 0
．． After dissolving 100rnlt of 2M phosphate buffer, adding the above gel and incubating at 37°C for 10 hours, the reaction solution was filtered, and 7-amine cephalosporanic acid in the solution was measured by Marelli method*. Cephalosporanic acid 183
~(yield 52%) was produced.

Example 8 254 grams of acrylamide gel containing bacterial cells obtained in the same manner as in Example 7 was suspended in 02M phosphate buffer (pH 7,4) 507, and packed in a column with a tube (l x 35 cm).
Example: Warm water at 37°C was circulated through the tube to equilibrate it.

A 0.2M phosphate buffer (pH 7,
4) 50mi! The enzyme reaction was carried out by passing through the tube at a flow rate of 0.2 rrIl per minute.

The passed-through liquid was separated into 5 portions and 7-aminocephalosporanic acid produced by the Marelli method* was measured.

As a result, 19.5 ■ of 7-aminocephalosporanic acid (
It was found that a yield of 55.3%) was produced.

Example 9 1 ton of culture broth of CA-218 strain cultured in the same manner as in Example 1 was filtered through Toyo Sawa paper & 101, and the liquid was centrifuged at 5.00 Or, p, m to collect bacteria. did.

The bacterial cells were placed in a porcelain mortar and crushed with 50 g of dry ice, and when they were completely powdered, the mortar was immersed in warm water at 37°C to rapidly vaporize the dry ice, thereby dissolving the bacterial cells.

This freezing and thawing operation was performed three times, and finally 20 yd of 0.2 M phosphate buffer (pH 7,4) was added, and the mixture was left at 5° C. overnight.

Thereafter, this freeze-thaw solution was heated to 7,0 OOr, p, m. Centrifuge the liquid, collect the supernatant, and remove the supernatant.

Centrifugation was performed at r,p,m to obtain 20rnl of cell-free extract.

90μ of 3-acetoxymethyl-7 was added to this cell-free extract.
-(4-carboxybutanamide)cef-3-emu 4
-Add 60- of 0.2M phosphate buffer containing carbonic acid,
The reaction was carried out at 37°C for 5 hours.

After the reaction, as measured by the Marelli method*, the yield was 46.3■ (yield 73
%) of 7-aminocephalosporanic acid was produced.

Example IO 2% glycerin, 2% sodium acetate, 0.9 dry yeast
%, salt 0.5%, K2HPO40.3%, KCl09
05%, F e Cl 3 ” 6H20,0,05%
, biotin lOmI? 0.5% glutaric acid and 0.01% nonipol were added to a medium with a composition of 1/l, and after sterilization,
Bacillus CA-78 strain was inoculated and cultured at 28°C for 6 days.

After incubation, the broth was incubated at 5,0 OOr, p, m. Bacteria were collected by centrifugation.

40 μ of 3-acetoxymethyl-7-(4-carboxybutanamide) Cef-3 was added to 20rr11 of this bacterial suspension.
-0.2M phosphate buffer containing emu-4-carboxylic acid 20
- was added thereto, incubated at 37°C for 6 hours, and measured by the Marelli method. As a result, 7-amine cephalosporanic acid of 14.9 TnfI (yield 53%) was observed.

References “L., P., Marrelli.

J, Pharmacol, Sci, 57,217
2 (1968).

Claims (1)

[Scope of Claims] 1. A compound represented by the general formula (wherein R represents a hydrogen atom, a hydroxyl group, an acetoxyl group, or a nucleophilic residue) and a salt thereof in an aqueous medium are prepared from Bacillus spp. The general formula 7-aminocephalosporanic acid, derivatives thereof, or salts thereof, represented by the following formula (indicates the same group as above).